Sample records for activity cytochrome p4501a

  1. Membrane Phospholipid Augments Cytochrome P4501a Enzymatic Activity by Modulating Structural Conformation during Detoxification of Xenobiotics

    PubMed Central

    Ghosh, Manik C.; Ray, Arun K.

    2013-01-01

    Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment. PMID:23469105

  2. Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

    PubMed

    Ghosh, Manik C; Ray, Arun K

    2013-01-01

    Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD) in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

  3. Tributyltin potentiates 3,3',4,4',5-pentachlorobiphenyl-induced cytochrome P-4501A-related activity.

    PubMed

    DeLong, G T; Rice, C D

    1997-06-06

    Induction of cytochrome P-4501A protein and induction of related enzyme activity are hallmark physiological responses following exposure to planar halogenated aromatic hydrocarbons (HAHs) such as 3,3',4,4',5-pentachlorobiphenyl (PCB 126; PeCB). Environments contaminated by HAHs are often contaminated by mixtures of anthropogenic contaminants, including organometallic compounds. Both HAHs and organometallics easily bioconcentrate and bioaccumulate in aquatic food chains that may ultimately be linked to humans through seafood consumption. Tributyltin (TBT), a marine biocide, has been detected in many aquatic environments due to its primary use as a marine antifoulant agent. Exposure to TBT, as well as several PCBs, has been associated with immunotoxicity, neurotoxicity, and endocrine disruption. Recently TBT has been shown to inhibit cytochrome P-4501A activity in vitro, but information concerning these effects in vivo and in combination with classical inducers of P-4501A, such PeCB, is lacking. We exposed female B6C3F1 mice to 0.01, 0.1, and 1.0 mg/kg PeCB, TBT, or both in combination, with corn oil (CO) serving as a carrier control. Cytochrome P-4501A protein levels and related benzo[a]pyrene hydroxylation (BaP-OHase) activity were measured following a single acute intraperitoneal (ip) dose or seven daily injections. Body, thymus, and liver weights were used to monitor general physiological responses following exposure. P-4501A levels and BaP-OHase activity were significantly elevated in mice exposed to PeCB alone. This effect was enhanced by coexposure to low levels of TBT; PeCB-induced P-4501A-related activity was potentiated at the low range of each. The highest dose of TBT, however, inhibited these activities when given in combination with PeCB. Thymic atrophy was evident only in mice exposed daily to 0:1 and 1.0 mg/kg PeCB alone, or to a combination of the lowest and highest dose of PeCB and TBT, respectively. Because environmental levels of TBT are not

  4. Cytochrome P4501A immunoassay in freshwater turtles and exposure to PCBs and environmental pollutants

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yawetz, A.; Benedek-Segal, M.; Woodin, B.

    1997-09-01

    This is the result of a comparative study of cytochrome P4501A (CYP1A) induction in liver microsomes from three species of freshwater turtles. CYP1A induction in turtle hepatic microsomes was compared to CYP1A induction in microsomes from the alligator. Alligator mississippiensis. Treatment of two species of freshwater turtles with four consecutive intraperitoneal injections of 100 mg/kg Aroclor 1254 caused a four- to five-fold increase in P4501A in hepatic microsomes of Chrysemys picta picta and Chrysemys picta elegans. The same treatment administered to another freshwater turtle, Mauremys caspica rivulata, resulted in a very low but significant (p < 0.01) induction of P4501Amore » in hepatic microsomes. Specimens of M. caspica rivulata collected from an organic waste oxidation pond near the petrochemical industry area of the city of Ashdod exhibited normal levels of total hepatic microsomal cytochrome P450 but no detectable level of induction of cytochrome P4501A. The lack of P4501A1 induction could have resulted from two possible reasons. The first possibility is that the turtles were not exposed to residues of petrochemical waste in the pond. More likely, the apparent lack of induction resulted from the low response to CYP1A inducers found in this species. Induction of cytochrome P4501A was evaluated immunohistochemically in liver tissue of C. picta picta pretreated with Aroclor 1254 or 3,3{prime},4,4{prime}-tetrachlorobiphenyl. The most intensive staining was exhibited by sections of liver from a 3,3{prime},4,4{prime}-tetrachlorobiphenyl-treated turtle. Staining of P4501A in liver sections from Aroclor 1254-treated turtles was relatively moderate. In induced turtles, staining of the hepatocytes concentrated near the cell membranes and nuclear membranes, but stained granules were observed throughout the cytoplasm. The presence of inducible CYP1A enzymes in turtles is of importance from an evolutionary point of view and has potential ecological relevance.« less

  5. Reduced cytochrome P4501A activity and recovery from oxidative stress during subchronic benzo[a]pyrene and benzo[e]pyrene treatment of rainbow trout

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Curtis, Lawrence R., E-mail: larry.curtis@oregonstate.edu; Garzon, Claudia B.; Arkoosh, Mary

    2011-07-01

    This study assessed the role of aryl hydrocarbon receptor (AHR) affinity, and cytochrome P4501A (CYP1A) protein and activity in polyaromatic hydrocarbon (PAH)-induced oxidative stress. In the 1-100 nM concentration range benzo[a]pyrene (BaP) but not benzo[e]pyrene (BeP) competitively displaced 2 nM [{sup 3}H]2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin from rainbow trout AHR2{alpha}. Based on appearance of fluorescent aromatic compounds in bile over 3, 7, 14, 28 or 50 days of feeding 3 {mu}g of BaP or BeP/g fish/day, rainbow trout liver readily excreted these polyaromatic hydrocarbons (PAHs) and their metabolites at near steady state rates. CYP1A proteins catalyzed more than 98% of ethoxyresorufin-O-deethylasemore » (EROD) activity in rainbow trout hepatic microsomes. EROD activity of hepatic microsomes initially increased and then decreased to control activities after 50 days of feeding both PAHs. Immunohistochemistry of liver confirmed CYP1A protein increased in fish fed both PAHs after 3 days and remained elevated for up to 28 days. Neither BaP nor BeP increased hepatic DNA adduct concentrations at any time up to 50 days of feeding these PAHs. Comet assays of blood cells demonstrated marked DNA damage after 14 days of feeding both PAHs that was not significant after 50 days. There was a strong positive correlation between hepatic EROD activity and DNA damage in blood cells over time for both PAHs. Neither CYP1A protein nor 3-nitrotyrosine (a biomarker for oxidative stress) immunostaining in trunk kidney were significantly altered by BaP or BeP after 3, 7, 14, or 28 days. There was no clear association between AHR2{alpha} affinity and BaP and BeP-induced oxidative stress. - Highlights: > No direct association between aryl hydrocarbon receptor affinity and polyaromatic hydrocarbon induced oxidative stress. > There was a strong correlation between cytochrome P4501A activity and oxidative stress as measured with the comet assay. > There was no correlation between

  6. Maternal obesity alters feto-placental Cytochrome P4501A1 activity

    PubMed Central

    DuBois, Barent N.; O’Tierney, Perrie; Pearson, Jacob; Friedman, Jacob E.; Thornburg, Kent; Cherala, Ganesh

    2012-01-01

    Cytochrome P4501A1 (CYP1A1), an important drug metabolizing enzyme, is expressed in human placenta throughout gestation as well as in fetal liver. Obesity, a chronic inflammatory condition, is known to alter CYP enzyme expression in non-placental tissues. In the present study, we test the hypothesis that maternal obesity alters the distribution of CYP1A1 activity in feto-placental unit. Placentas were collected from non-obese (BMI<30) and obese (BMI>30) women at term. Livers were collected from gestation day 130 fetuses of non-human primates fed either control diet or high-fat diet (HFD). Cytosol and microsomes were collected using differential centrifugation, and incubated with 7-Ethoxyresorufin. The CYP1A1 specific activity (pmoles of resorufin formed/min/mg of protein) was measured at excitation/emission wavelength of 530/590nm. Placentas of obese women had significantly reduced microsomal CYP1A1 activity compared to non-obese women (0.046 vs. 0.082; p<0.05); however no such effect was observed on cytosolic activity. Similarly, fetal liver from HFD fed mothers had significantly reduced microsomal CYP1A1 activity (0.44±0.04 vs. 0.20±0.10; p<0.05), with no significant difference in cytosolic CYP1A1 activity (control, 1.23±0.20; HFD, 0.80±0.40). Interestingly, multiple linear regression analyses of placental efficiency indicates cytosolic CYP1A1 activity is a main effect (5.67±2.32 (β±SEM); p=0.022) along with BMI (−0.57±0.26; p=0.037), fetal gender (1.07±0.26; p<0.001), and maternal age (0.07±0.03; p=0.011). In summary, while maternal obesity affects microsomal CYP1A1 activity alone, cytosolic activity along with maternal BMI is an important determinant of placental efficiency. Together, these data suggest that maternal lifestyle could have a significant impact on CYP1A1 activity, and hints at a possible role for CYP1A1 in feto-placental growth and thereby well-being of fetus. PMID:23046808

  7. Modulation of the Rat Hepatic Cytochrome P4501A Subfamily Using Biotin Supplementation

    PubMed Central

    Ronquillo-Sánchez, M. D.; Camacho-Carranza, R.; Fernandez-Mejia, C.; Hernández-Ojeda, S.; Elinos-Baez, M.; Espinosa-Aguirre, J. J.

    2013-01-01

    Studies have found that biotin favors glucose and lipid metabolism, and medications containing biotin have been developed. Despite the use of biotin as a pharmacological agent, few studies have addressed toxicity aspects including the possible interaction with cytochrome P450 enzyme family. This study analyzed the effects of pharmacological doses of biotin on the expression and activity of the cytochrome P4501A subfamily involved in the metabolism of xenobiotics. Wistar rats were treated daily with biotin (2 mg/kg, i.p.), while the control groups were treated with saline. All of the rats were sacrificed by cervical dislocation after 1, 3, 5, or 7 days of treatment. CYP1A1 and CYP1A2 mRNAs were modified by biotin while enzyme activity and protein concentration were not affected. The lack of an effect of biotin on CYP1A activity was confirmed using other experimental strategies, including (i) cotreatment of the animals with biotin and a known CYP1A inducer; (ii) the addition of biotin to the reaction mixtures for the measurement of CYP1A1 and CYP1A2 activities; and (iii) the use of an S9 mixture that was prepared from control and biotin-treated rats to analyze the activation of benzo[a]pyrene (BaP) into mutagenic metabolites using the Ames test. The results suggest that biotin does not influence the CYP1A-mediated metabolism of xenobiotics. PMID:23984390

  8. HALOGENATED AROMATIC HYDROCARBON-MEDIATED PORPHYRIN ACCUMULATION AND INDUCTION OF CYTOCHROME P4501A IN CHICKEN EMBRYO HEPATOCYTES. (R823889)

    EPA Science Inventory

    Concentration-dependent induction of cytochrome P4501A (CYP1A) and intracellular porphyrin accumulation were observed following treatment of chicken embryo hepatocyte (CEH) cultures with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4'...

  9. Highly purified hexachlorobenzene induces cytochrome P4501A in primary cultures of chicken embryo hepatocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mundy, Lukas J.; Environment Canada, National Wildlife Research Centre, Ottawa, Ontario; Jones, Stephanie P.

    Some uncertainty exists regarding the purity of hexachlorobenzene (HCB) used in past toxicity studies. It has been suggested that reported toxic and biochemical effects initially attributed to HCB exposure may have actually been elicited by contamination of HCB by polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs). Herein, primary cultures of chicken embryo hepatocytes (CEH) were used to compare the potencies of two lots of reagent-grade hexachlorobenzene (HCB-old [HCB-O] and HCB-new [HCB-N]), highly purified HCB (HCB-P) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A4 (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. The study also compared themore » EROD- and CYP1A4/5 mRNA-inducing potencies of HCB to the potencies of two mono-ortho substituted polychlorinated biphenyls (PCBs), 2,3,3',4,4'-pentachlorobiphenyl (PCB 105) and 2,3'4,4',5-pentachlorobiphenyl (PCB 118). HCB-O, HCB-N and HCB-P all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNAs. Induction was not caused by contamination of HCB with PCDDs or PCDFs. Based upon a comparison of the EC{sub 50} and EC{sub threshold} values for EROD and CYP1A4/5 mRNA concentration-response curves, the potency of HCB relative to the potency of TCDD was 0.0001, and was similar to that of PCB 105 and PCB 118. The maximal EROD activity and CYP1A4/5 mRNA expression differed greatly between HCB and TCDD, and may contribute to an overestimation of the ReP value calculated for highly purified HCB.« less

  10. Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

    PubMed

    Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

    2001-06-01

    1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

  11. Induced cytochrome P450 1A activity in cichlid fishes from Guandu River and Jacarepaguá Lake, Rio de Janeiro, Brazil.

    PubMed

    Parente, Thiago E M; De-Oliveira, Ana C A X; Paumgartten, Francisco J R

    2008-03-01

    The induction of cytochrome P4501A-mediated activity (e.g. ethoxyresorufin-O-deethylation, EROD) has been used as a biomarker for monitoring fish exposure to AhR-receptor ligands such as polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and polychlorinated dibenzo-dioxins/furans (PCDD/Fs). In this study we found that hepatic EROD is induced in fish ("Nile tilapia", Oreochromis niloticus and "acará", Geophagus brasiliensis) from the Guandu River (7-17-fold) and Jacarepaguá Lake (7-fold), Rio de Janeiro, Brazil. Since both cichlid fish are consumed by the local population and the Guandu River is the main source of the drinking water supply for the greater Rio de Janeiro metropolitan area, pollution by cytochrome P4501A-inducing chemicals is a cause for concern and should be further investigated in sediments, water and biota. We additionally showed that EROD activity in the fish liver post-mitochondrial supernatant-simpler, cheaper and less time consuming to prepare than the microsomal fraction-is sufficiently sensitive for monitoring purposes.

  12. Quantitative evaluation of hepatic cytochrome P4501A transcript, protein, and catalytic activity in the striped sea bream (Lithognathus mormyrus).

    PubMed

    Tom, Moshe; Shmul, Merav; Shefer, Edna; Chen, Nir; Slor, Hanoch; Rinkevich, Baruch; Herut, Barak

    2003-09-01

    Hepatic cytochrome P4501A (CYP1A) expression was partially characterized in the striped sea bream (Lithognathus mormyrus) from the Mediterranean coast of Israel as part of the process of establishing the CYP1A gene as an environmental biomarker. Reverse transcription-competitive polymerase chain reaction, competitive enzyme-linked immunosorbent assay, and ethoxyresorufin O-deethylase (EROD) assay were used for evaluating transcript, protein, and catalytic activity levels, respectively, in absolute units. Highest elucidated transcript, protein, and catalytic activity levels were 0.264 +/- SD 0.084 fmol/microg total RNA, 0.88 +/- 0.52 pmol/microg total protein, and 1.11 +/- 0.52 pmol resorufin/min/microg total protein, respectively, and the lower levels were 0.009 +/- 0.007 fmol/microg total RNA, 0.17 +/- 0.08 pmol/microg total protein, and 0.11 +/- 0.06 pmol resorufin/min/microg total protein, respectively, demonstrating substantial induction potential. All alternate pairs of seven examined field samples, revealing a transcript-level ratio higher than 1.7, also demonstrated a significant difference between their transcript levels, indicating a potential to detect relatively small biomarker changes (1.7-fold) caused by environmental effects. Simultaneous triple measurements of transcript, protein, and catalytic activity were carried out in individuals from two field samples and during a 318-d decay experiment. Fish from the field samples revealed significant alternate bivariate correlation between transcript, protein, and enzymatic activity. Conflicting results were found when analyzing the decay experiment, in which both protein and catalytic activity levels decreased significantly to basal levels, in contrast to no significant change in transcript levels throughout the experiment. No significant difference was observed between males and females regarding the levels of CYP1A transcript, protein, and EROD.

  13. Changes in cytochrome P4501A activity during development in common tern chicks fed polychlorinated biphenyls, as measured by the caffeine breath test

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Feyk, L.A.; Giesy, J.P.; Bosveld, A.T.C.

    2000-03-01

    Cytochrome P4501A (CYPIA) activity is often used as a biomarker of exposure of wildlife to polyhalogenated diaromatic hydrocarbons and is usually measured ex vivo in liver tissue. A caffeine breath test (CBT) with radiolabeled substrate ({sup 14}C-caffeine) was used to measure in vivo CYP1A activity twice during development in 14 common tern (Sterna hirundo) chicks treated with polyhalogenated diaromatic hydrocarbons. Tern hatchlings were fed fish spiked with 3,3{prime}, 4,4{prime},5-pentachlorobiphenyl (PCB 126) and 2,2{prime},4,4{prime},5,5{prime}-hexachlorobiphenyl (PCB 153) such that the diet contained an average of 23, 99, or 561 pg of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents per gram of fish for 21 d. Sixteen additionalmore » common tern chicks were similarly dosed with polyhalogenated diaromatic hydrocarbons but were not subjected to the CBT procedure. In weeks 1 and 2, caffeine N-demethylation and ethoxyresorufin-O-deethylation activity on day 21 were elevated in birds that received the greatest PCB dose. There was less constitutive and greater induction of ethoxyresorufin-O-deethylation activity than caffeine N-demethylation. The {sup 14}C-CBT was less invasive than the ethoxyresorufin-O-deethylase assay. Only one morphological parameter differed significantly between CBT subjects and no-CBT subjects fed the same level of PCBs. Bursa weight was significantly less in control CBT subjects than in control no-CBT subjects, but bursa weights did not differ among CBT and no-CBT birds from the two PCB treatment groups. No alterations of survival or growth occurred in CBT subjects compared with no-CBT subjects.« less

  14. 2,3,7,8-Tetrachlorodibenzo-p-dioxin induces apoptotic cell death and cytochrome P4501A expression in developing Fundulus heteroclitus embryos

    USGS Publications Warehouse

    Toomey, B.H.; Bello, S.; Hahn, M.E.; Cantrell, S.; Wright, P.; Tillitt, D.E.; Di Giulio, R.T.

    2001-01-01

    Fundulus heteroclitus embryos were exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) during early development using nanoinjection or water bath exposure. TCDD caused developmental abnormalities that included hemorrhaging, loss of vascular integrity, edema, stunted development and death. The LC50 and LD50 of TCDD for Fundulus embryos were ???19.7??9.5 pg TCDD/??l (water bath) and 0.25??0.09 ng TCDD/g embryo (nanoinjection). To identify a possible cause for these developmental abnormalities we analyzed the effects of TCDD on apoptotic cell death and cytochrome P4501A (CYP1A) expression in the embryos. TCDD exposure increased apoptotic cell death in several tissues including brain, eye, gill, kidney, tail, intestine, heart, and vascular tissue. CYP1A expression was also increased in the TCDD-exposed embryos predominantly in liver, kidney, gill, heart, intestine, and in vascular tissues throughout the embryo. There was co-occurrence of TCDD-induced apoptosis and CYP1A expression in some, but not all, cell types. In addition the dose response relationships for apoptosis and mortality were similar, while CYP1A expression appeared more sensitive to TCDD induction. Copyright ?? 2001 Elsevier Science B.V.

  15. Relationship between polychlorinated biphenyl 126 treatment and cytochrome P4501A activity in chickens, as measured by in vivo caffeine and ex vivo ethoxyresorufin metabolism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Feyk, L.A.; Giesy, J.P.; Lambert, G.H.

    1999-09-01

    Cytochrome P4501A (CYPIA) activity is often used as a biomarker of exposure of wildlife to polyhalogenated diaromatic hydrocarbons (PHDHs) and is usually measured ex vivo in liver tissue. A caffeine breath test with radiolabeled substrate ({sup 14}C-CBT) has been developed to measure in vivo avian CYPIA activity. Research goals were to develop stable isotope methods ({sup 13}C-CBT), determine dose-response relationships between caffeine N-demethylation (CNDM) and PHDH exposure, and assess the relative utility of the CBT and ex vivo ethoxyresorufin-O-deethylase (EROD) assay. The {sup 13}C-CBT methods were developed with 20 chickens (Gallus domesticus). Chickens received three intraperitoneal injections of 0, 1,more » 5, or 50 {micro}g 3,3{prime},4,4{prime},5-pentachlorobiphenyl (PCB 126)/kg body weight, and CNDM was quantified by measurement of {sup 13}CO{sub 2}/{sup 12}CO{sub 2} in expired breath. The {sup 13}C-CBT was not as sensitive or specific as the EROD assay as an indicator of PHDH exposure and effect in birds. Constitutive CNDM of great interindividual variability was observed, and the magnitude of induction was greater for EROD activity than for CNDM (approximately 1,000- and 2-fold, respectively). Variability associated with baseline {sup 13}CO{sub 2}/{sup 12}CO{sub 2} ratios in expired breath reduced the sensitivity of the {sup 13}C-CBT method.« less

  16. Cessation of oil exposure in harlequin ducks after the Exxon Valdez oil spill: Cytochrome P4501A biomarker evidence

    USGS Publications Warehouse

    Esler, Daniel N.; Ballachey, Brenda E.; Bowen, Lizabeth; Miles, A. Keith; Dickson, Rian D.; Henderson, John D.

    2017-01-01

    The authors quantified hepatic hydrocarbon-inducible cytochrome P4501A (CYP1A) expression, as ethoxyresorufin-O-deethylase (EROD) activity, in wintering harlequin ducks (Histrionicus histrionicus) captured in Prince William Sound, Alaska (USA), during 2011, 2013, and 2014 (22–25 yr following the 1989 Exxon Valdez oil spill). Average EROD activity was compared between birds from areas oiled by the spill and those from nearby unoiled areas. The present study replicated studies conducted from 1998 to 2009 demonstrating that harlequin ducks using areas oiled in 1989 had elevated EROD activity, indicative of oil exposure, up to 2 decades post spill. In the present study, it was found that average EROD activity during March 2011 was significantly higher in wintering harlequin ducks captured in oiled areas relative to unoiled areas, which the authors interpret to indicate that harlequin ducks continued to be exposed to residual Exxon Valdez oil up to 22 yr after the original spill. However, the 2011 results also indicated reductions in exposure relative to previous years. Average EROD activity in birds from oiled areas was approximately 2 times that in birds from unoiled areas in 2011, compared with observations from 2005 to 2009, in which EROD activity was 3 to 5 times higher in oiled areas. It was also found that average EROD activity during March 2013 and March 2014 was not elevated in wintering harlequin ducks from oiled areas. The authors interpret these findings to indicate that exposure of harlequin ducks to residual Exxon Valdez oil abated within 24 yr after the original spill. The present study finalizes a timeline of exposure, extending over 2 decades, for a bird species thought to be particularly vulnerable to oil contamination in marine environments

  17. Cytochrome P4501A induction, benzo[a]pyrene metabolism, and nucleotide adduct formation in fish hepatoma cells: Effect of preexposure to 3,3',4,4',5-pentachlorobiphenyl

    USGS Publications Warehouse

    Smeets, J.M.W.; Voormolen, A.; Tillitt, D.E.; Everaarts, J.M.; Seinen, W.; Vanden Berg, M.D.

    1999-01-01

    In PLHC-1 hepatoma cells, benzo[a]pyrene (B[a]P) caused a maximum induction of cytochrome P4501A (CYP1A) activity, measured as ethoxyresorufin O-deethylation (EROD), after 4 to 8 h of exposure, depending on the B[a]P concentration. The decline of EROD activity at longer exposure times was probably caused by the rapid metabolism of B[a]P in this system (57% metabolism within 4 h incubation). In subsequent experiments, PLHC-1 cells were preinduced with PCB 126 for 24 h and then received a dose of 10, 100, or 1,000 nM 3H-B[a]P. A 1-nM concentration of PCB 126 caused an 80-fold induction of CYP1A activity, resulting in an increase in B[a]P metabolism of less than 10%, except at the highest concentration of B[a]P (1,000 nM), where a 50% increase was observed. In another experiment, an 80-fold induction of CYP1A activity caused a 20% increase in the metabolism of B[a]P (100 nM), and RNA adduct formation was increased approximately twofold. These results indicate that, at exposure concentrations up to 100 nM B[a]P, CYP1A activity is not rate limiting for B[a]P metabolism. Furthermore, CYP1A seems to also he specifically involved in B[a]P activation in PLHC-1 cells. However, CYP1A induction causes only a relatively small increase in activation, probably because of the action of other enzymes involved in B[a]P activation and deactivation.

  18. Influence of cytochrome P450 oxidoreductase genetic polymorphisms on CYP1A2 activity and inducibility by smoking.

    PubMed

    Dobrinas, Maria; Cornuz, Jacques; Pedrido, Leticia; Eap, Chin B

    2012-02-01

    Cytochrome P4501A2 (CYP1A2) presents a high interindividual variability in its activity and also in its inducibility by smoking. Cytochrome P450 oxidoreductase (POR) is an electron transfer protein that catalyzes the activity of several cytochromes P450. We aimed to study the influence of POR genetic polymorphisms on CYP1A2 activity while smoking and after smoking cessation, as well as on CYP1A2 inducibility. CYP1A2 activity was determined by the paraxanthine/caffeine ratio in 184 smokers and in 113 of these smokers who were abstinent during a 4-week period. Participants were genotyped for POR rs17148944G>A, rs10239977C>T, rs3815455C>T, rs2286823G>A, rs2302429G>A, and rs1057868C>T (POR*28) polymorphisms. While smoking, none of the tested POR polymorphisms showed a significant influence on CYP1A2 activity. After smoking cessation, significantly higher CYP1A2 activity was found in POR rs2302429A carriers (P=0.038) and in carriers of rs17148944G-rs10239977C-rs3815455T-rs2286823G-rs2302429A-rs1057868T haplotype (P=0.038), whereas carriers of POR rs2286823A (P=0.031) and of the rs17148944G-rs10239977C-rs3815455C-rs2286823A-rs2302429G-rs1057868C haplotype (P=0.031) had decreased CYP1A2 activity. In the complete regression model, only POR rs2302429G>A showed a significant effect (P=0.017). No influence of POR genotypes or haplotypes was observed on the inducibility of CYP1A2. POR genetic polymorphisms influence CYP1A2 basal but not induced activity and do not seem to influence CYP1A2 inducibility. Future work is warranted to identify other clinical and genetic factors that may explain the variability in CYP1A2 activity and inducibility by smoking.

  19. Cytochrome P4501A biomarker indication of oil exposure in harlequin ducks up to 20 years after the Exxon Valdez oil spill.

    PubMed

    Esler, Daniel; Trust, Kimberly A; Ballachey, Brenda E; Iverson, Samuel A; Lewis, Tyler L; Rizzolo, Daniel J; Mulcahy, Daniel M; Miles, A Keith; Woodin, Bruce R; Stegeman, John J; Henderson, John D; Wilson, Barry W

    2010-05-01

    Hydrocarbon-inducible cytochrome P4501A (CYP1A) expression was measured, as ethoxyresorufin-O-deethylase (EROD) activity, in livers of wintering harlequin ducks (Histrionicus histrionicus) captured in areas of Prince William Sound, Alaska, USA, oiled by the 1989 Exxon Valdez spill and in birds from nearby unoiled areas, during 2005 to 2009 (up to 20 years following the spill). The present work repeated studies conducted in 1998 that demonstrated that in harlequin ducks using areas that received Exxon Valdez oil, EROD activity was elevated nearly a decade after the spill. The present findings strongly supported the conclusion that average levels of hepatic EROD activity were higher in ducks from oiled areas than those from unoiled areas during 2005 to 2009. This result was consistent across four sampling periods; furthermore, results generated from two independent laboratories using paired liver samples from one of the sampling periods were similar. The EROD activity did not vary in relation to age, sex, or body mass of individuals, nor did it vary strongly by season in birds collected early and late in the winter of 2006 to 2007, indicating that these factors did not confound inferences about observed differences between oiled and unoiled areas. We interpret these results to indicate that harlequin ducks continued to be exposed to residual Exxon Valdez oil up to 20 years after the original spill. This adds to a growing body of literature suggesting that oil spills have the potential to affect wildlife for much longer time frames than previously assumed. Copyright (c) 2010 SETAC.

  20. Cytochrome P4501A biomarker indication of oil exposure in harlequin ducks up to 20 years after the Exxon Valdez oil spill

    USGS Publications Warehouse

    Esler, Daniel N.; Trust, Kimberly A.; Ballachey, Brenda E.; Iverson, Samuel A.; Lewis, Tyler L.; Rizzolo, Daniel; Mulcahy, Daniel M.; Miles, A. Keith; Woodin, Bruce R.; Stegeman, John J.; Henderson, John D.; Wilson, Barry W.

    2010-01-01

    Hydrocarbon-inducible cytochrome P4501A (CYP1A) expression was measured, as ethoxyresorufin-O-deethylase (EROD) activity, in livers of wintering harlequin ducks (Histrionicus histrionicus) captured in areas of Prince William Sound, Alaska, USA, oiled by the 1989 Exxon Valdez spill and in birds from nearby unoiled areas, during 2005 to 2009 (up to 20 years following the spill). The present work repeated studies conducted in 1998 that demonstrated that in harlequin ducks using areas that received Exxon Valdez oil, EROD activity was elevated nearly a decade after the spill. The present findings strongly supported the conclusion that average levels of hepatic EROD activity were higher in ducks from oiled areas than those from unoiled areas during 2005 to 2009. This result was consistent across four sampling periods; furthermore, results generated from two independent laboratories using paired liver samples from one of the sampling periods were similar. The EROD activity did not vary in relation to age, sex, or body mass of individuals, nor did it vary strongly by season in birds collected early and late in the winter of 2006 to 2007, indicating that these factors did not confound inferences about observed differences between oiled and unoiled areas. We interpret these results to indicate that harlequin ducks continued to be exposed to residual Exxon Valdez oil up to 20 years after the original spill. This adds to a growing body of literature suggesting that oil spills have the potential to affect wildlife for much longer time frames than previously assumed.

  1. Cessation of oil exposure in harlequin ducks after the Exxon Valdez oil spill: Cytochrome P4501A biomarker evidence.

    PubMed

    Esler, Daniel; Ballachey, Brenda E; Bowen, Lizabeth; Miles, A Keith; Dickson, Rian D; Henderson, John D

    2017-05-01

    The authors quantified hepatic hydrocarbon-inducible cytochrome P4501A (CYP1A) expression, as ethoxyresorufin-O-deethylase (EROD) activity, in wintering harlequin ducks (Histrionicus histrionicus) captured in Prince William Sound, Alaska (USA), during 2011, 2013, and 2014 (22-25 yr following the 1989 Exxon Valdez oil spill). Average EROD activity was compared between birds from areas oiled by the spill and those from nearby unoiled areas. The present study replicated studies conducted from 1998 to 2009 demonstrating that harlequin ducks using areas oiled in 1989 had elevated EROD activity, indicative of oil exposure, up to 2 decades post spill. In the present study, it was found that average EROD activity during March 2011 was significantly higher in wintering harlequin ducks captured in oiled areas relative to unoiled areas, which the authors interpret to indicate that harlequin ducks continued to be exposed to residual Exxon Valdez oil up to 22 yr after the original spill. However, the 2011 results also indicated reductions in exposure relative to previous years. Average EROD activity in birds from oiled areas was approximately 2 times that in birds from unoiled areas in 2011, compared with observations from 2005 to 2009, in which EROD activity was 3 to 5 times higher in oiled areas. It was also found that average EROD activity during March 2013 and March 2014 was not elevated in wintering harlequin ducks from oiled areas. The authors interpret these findings to indicate that exposure of harlequin ducks to residual Exxon Valdez oil abated within 24 yr after the original spill. The present study finalizes a timeline of exposure, extending over 2 decades, for a bird species thought to be particularly vulnerable to oil contamination in marine environments. Environ Toxicol Chem 2017;36:1294-1300. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America

  2. Prenatal administration of the cytochrome P4501A inducer, {Beta}-naphthoflavone (BNF), attenuates hyperoxic lung injury in newborn mice: Implications for bronchopulmonary dysplasia (BPD) in premature infants

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Couroucli, Xanthi I., E-mail: xanthic@bcm.edu; Liang Yanhong Wei; Jiang Weiwu

    Supplemental oxygen contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. In this investigation, we tested the hypothesis that prenatal treatment of pregnant mice (C57BL/6J) with the cytochrome P450 (CYP)1A1 inducer, ss-napthoflavone (BNF), will lead to attenuation of lung injury in newborns (delivered from these dams) exposed to hyperoxia by mechanisms entailing transplacental induction of hepatic and pulmonary CYP1A enzymes. Pregnant mice were administered the vehicle corn oil (CO) or BNF (40 mg/kg), i.p., once daily for 3 days on gestational days (17-19), and newborns delivered from the mothers were either maintained in room air or exposed tomore » hyperoxia (> 95% O{sub 2}) for 1-5 days. After 3-5 days of hyperoxia, the lungs of CO-treated mice showed neutrophil infiltration, pulmonary edema, and perivascular inflammation. On the other hand, BNF-pretreated neonatal mice showed decreased susceptibility to hyperoxic lung injury. These mice displayed marked induction of ethoxyresorufin O-deethylase (EROD) (CYP1A1) and methoxyresorufin O-demethylase (MROD) (CYP1A2) activities, and levels of the corresponding apoproteins and mRNA levels until PND 3 in liver, while CYP1A1 expression alone was augmented in the lung. Prenatal BNF did not significantly alter gene expression of pulmonary NAD(P)H quinone reductase (NQO1). Hyperoxia for 24-72 h resulted in increased pulmonary levels of the F{sub 2}-isoprostane 8-iso-PGF{sub 2{alpha}}, whose levels were decreased in mice prenatally exposed to BNF. In conclusion, our results suggest that prenatal BNF protects newborns against hyperoxic lung injury, presumably by detoxification of lipid hydroperoxides by CYP1A enzymes, a phenomenon that has implications for prevention of BPD in infants. - Highlights: > Supplemental oxygen is routinely administered to premature infants. > Hyperoxia causes lung injury in experimental animals. > Prenatal treatment of mice with beta-naphthoflavone attenuates

  3. 5 CFR 4501.103 - Prior approval for certain outside activities.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... activities. 4501.103 Section 4501.103 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT SUPPLEMENTAL STANDARDS OF ETHICAL CONDUCT FOR EMPLOYEES OF THE OFFICE OF PERSONNEL MANAGEMENT § 4501.103 Prior approval... specialized skills or the same educational background as performance of the employee's official duties; (2...

  4. ELEVATED LEVELS OF MULTIPLE CYTOCHROME P450 FORMS IN TILAPIA FROM BILLINGS RESERVOIR-SAO PAULO, BRAZIL. (R827102)

    EPA Science Inventory

    Cytochrome P4501A (CYP1A) levels in tissues of fish inhabiting polluted areas have been used extensively in biomonitoring studies in Europe and North America. However, little information is available about the extent of CYP1A expression in fish from South American waters, nor on ...

  5. Dietary Omega-3 Polyunsaturated Fatty Acids Prevent Vascular Dysfunction and Attenuate Cytochrome P4501A1 Expression by 2,3,7,8-Tetrachlorodibenzo-P-Dioxin.

    PubMed

    Wiest, Elani F; Walsh-Wilcox, Mary T; Rothe, Michael; Schunck, Wolf-Hagen; Walker, Mary K

    2016-11-01

    Omega-3 polyunsaturated fatty acids (n-3 PUFAs) found in fish protect against cardiovascular morbidity and mortality; however, many individuals avoid fish consumption due to concerns about pollutants. We tested the hypothesis that n-3 PUFAs would prevent vascular dysfunction induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). C57Bl/6 male mice were fed a chow or n-3 PUFA diet for 10 weeks and were exposed to vehicle or 300 ng/kg/d TCDD during the final 2 weeks on each diet. Aortic vasoconstriction mediated by arachidonic acid (AA) ± SKF525 (P450 inhibitor) or SQ29548 (thromboxane/prostanoid [TP] receptor antagonist) was assessed. RBC fatty acids and expression of n-3 and n-6 PUFA metabolites were analyzed. Cytochrome P4501A1 (CYP1A1), CYP1B1, and aryl hydrocarbon receptor (AHR) expression was measured. TCDD significantly increased AA-mediated vasoconstriction on a chow diet by increasing the contribution of P450s and TP receptor to the constriction response. In contrast, the n-3 PUFA diet prevented the TCDD-induced increase in AA vasoconstriction and normalized the contribution of P450s and TP receptor. Although TCDD increased the levels of AA vasoconstrictors on the chow diet, this increase was prevent by the n-3 PUFA diet. Additionally, the n-3 PUFA diet significantly increased the levels of n-3 PUFA-derived vasodilators and TCDD increased these levels further. Interestingly, the n-3 PUFA diet significantly attenuated CYP1A1 induction by TCDD without a significant effect on AHR expression. These data suggest that n-3 PUFAs can prevent TCDD-induced vascular dysfunction by decreasing vasoconstrictors, increasing vasodilators, and attenuating CYP1A1 induction, which has been shown previously to contribute to TCDD-induced vascular dysfunction. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Expression of a ripening-related cytochrome P450 cDNA in Cavendish banana (Musa acuminata cv. Williams).

    PubMed

    Pua, Eng-Chong; Lee, Yi-Chuan

    2003-02-13

    As part of a study to understand the molecular basis of fruit ripening, this study reports the isolation and characterization of a banana cytochrome P450 (P450) cDNA, designated as MAP450-1, which was associated with fruit ripening of banana. MAP450-1 encoded a single polypeptide of 507 amino acid residues that shared an overall identity of 27-45% with that of several plant P450s, among which MAP450-1 was most related phylogenetically to the avocado P450 CYP71A1. The polypeptide that possessed residue domains conserved in all P450s was classified as CYP71N1. Expression of CYP71N1 varied greatly between banana organs. Transcripts were detected only in peel and pulp of the ripening fruit and not in unripe fruit tissues at all developmental stages or other organs (root, leaf, ovary and flower). During ripening, transcripts were barely detectable in pre-climacteric and climacteric fruits but, as ripening progressed, they began to accumulate and reached a maximum in post-climacteric fruits. CYP71N1 expression in pre-climacteric fruit could be upregulated by exogenous application of ethylene (1-5 ppm) and treatment of overripe fruit with exogenous sucrose (50-300 mM) but not glucose downregulated the expression. These results indicate that P450s may not play a role in fruit development and its expression is associated with ripening, which may be regulated, in part, by ethylene and/or sucrose, at the transcript level.

  7. Elevated blood pressure in cytochrome P4501A1 knockout mice is associated with reduced vasodilation to omega − 3 polyunsaturated fatty acids

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Agbor, Larry N.; Walsh, Mary T.; Boberg, Jason R.

    In vitro cytochrome P4501A1 (CYP1A1) metabolizes omega − 3 polyunsaturated fatty acids (n − 3 PUFAs); eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), primarily to 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP), respectively. These metabolites have been shown to mediate vasodilation via increases in nitric oxide (NO) and activation of potassium channels. We hypothesized that genetic deletion of CYP1A1 would reduce vasodilatory responses to n − 3 PUFAs, but not the metabolites, and increase blood pressure (BP) due to decreases in NO. We assessed BP by radiotelemetry in CYP1A1 wildtype (WT) and knockout (KO) mice ± NO synthase (NOS) inhibitor.more » We also assessed vasodilation to acetylcholine (ACh), EPA, DHA, 17,18-EEQ and 19,20-EDP in aorta and mesenteric arterioles. Further, we assessed vasodilation to an NO donor and to DHA ± inhibitors of potassium channels. CYP1A1 KO mice were hypertensive, compared to WT, (mean BP in mm Hg, WT 103 ± 1, KO 116 ± 1, n = 5/genotype, p < 0.05), and exhibited a reduced heart rate (beats per minute, WT 575 ± 5; KO 530 ± 7; p < 0.05). However, BP responses to NOS inhibition and vasorelaxation responses to ACh and an NO donor were normal in CYP1A1 KO mice, suggesting that NO bioavailability was not reduced. In contrast, CYP1A1 KO mice exhibited significantly attenuated vasorelaxation responses to EPA and DHA in both the aorta and mesenteric arterioles, but normal vasorelaxation responses to the CYP1A1 metabolites, 17,18-EEQ and 19,20-EDP, and normal responses to potassium channel inhibition. Taken together these data suggest that CYP1A1 metabolizes n − 3 PUFAs to vasodilators in vivo and the loss of these vasodilators may lead to increases in BP. -- Highlights: ► CYP1A1 KO mice are hypertensive. ► CYP1A1 KO mice exhibit reduced vasodilation responses to n-3 PUFAs. ► Constitutive CYP1A1 expression regulates blood pressure and vascular function.« less

  8. Evaluating cytochrome p450 in lesser scaup (Aythya affinis) and tree swallow (Tachycineta bicolor) by monooxygenase activity and immunohistochemistry: Possible nonlethal assessment by skin immunohistochemistry

    USGS Publications Warehouse

    Melancon, M.J.; Kutay, A.L.; Woodin, Bruce R.; Stegeman, John J.

    2006-01-01

    Six-month-old lesser scaup (Aythya affinis) and nestling tree swallows (Tachycineta bicolor) were injected intraperitoneally with beta-naphthoflavone (BNF) in corn oil or in vehicle alone. Liver samples were taken and stored at -80 degrees C until microsome preparation and monooxygenase assay. Skin samples were placed in buffered formalin for subsequent immunohistochemical (IHC) analysis for cytochrome P4501A (CYP1A). Lesser scaup treated with BNF at 20 or 100 mg/kg body weight showed approximately 6- to 18-fold increases in four monooxygenases (benzyloxyresorufin-O-dealkylase, ethoxyresorufin-O-dealkylase, methoxyresorufin-O-dealkylase, and pentoxyresorufin-O-dealkylase). No IHC response was observed for CYP1A in the skin of vehicle-injected ducks, whereas in the skin from BNF-treated ducks, the positive IHC response was of similar magnitude for both dose levels of BNF. Tree swallows injected with BNF at 100 mg/kg, but not at. 20 mg/kg, showed significant increases (approximately fivefold) in hepatic microsomal O-dealkylase activities. Cytochrome P4501A was undetectable by IHC response in skin from corn oil-treated swallows, but positive IHC responses were observed in the skin of one of five swallows at 20 mg/kg and four of five swallows at 100 mg/kg. Although these data do not allow construction of significant dose-response curves, the IHC responses for CYP1A in skin support the possible use of this nonlethal approach for biomonitoring contaminant exposure of birds. In addition, the CYP1A signal observed at the bases of emerging feathers suggest that these might provide less invasive sampling sites for IHC analysis of CYP1A.

  9. Cytochrome P450 3A4 activity after surgical stress.

    PubMed

    Haas, Curtis E; Kaufman, David C; Jones, Carolyn E; Burstein, Aaron H; Reiss, William

    2003-05-01

    To evaluate the relationship between the acute inflammatory response after surgical trauma and changes in hepatic cytochrome P450 3A4 activity, compare changes in cytochrome P450 3A4 activity after procedures with varying degrees of surgical stress, and to explore the time course of any potential drug-cytokine interaction after surgery. Prospective, open-label study with each patient serving as his or her own control. University-affiliated, acute care, general hospital. A total of 16 patients scheduled for elective repair of an abdominal aortic aneurysm (n = 5), complete or partial colectomy (n = 6), or peripheral vascular surgery with graft (n = 5). Cytochrome P450 3A4 activity was estimated using the carbon-14 [14C]erythromycin breath test (ERMBT) before surgery and 24, 48, and 72 hrs after surgery. Abdominal aortic aneurysm and colectomy patients also had an ERMBT performed at discharge. Blood samples were obtained before surgery, immediately after surgery, and 6, 24, 32, 48, and 72 hrs after surgery for determination of plasma concentrations of interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha. Clinical markers of surgical stress that were collected included duration of surgery, estimated blood loss, and volume of fluids administered in the operating room. ERMBT results significantly declined in all three surgical groups, with the lowest value at the time of the 72-hr study in all three groups. There was a trend toward differences in ERMBT results among groups that did not reach statistical significance (p =.06). The nadir ERMBT result was significantly and negatively correlated with both peak interleukin-6 concentration (r(s) = -.541, p =.03) and log interleukin-6 area under the curve from 0 to 72 hrs (r(s) = -.597, p =.014). Subjects with a peak interleukin-6 of >100 pg/mL had a significantly lower nadir ERMBT compared with subjects with a peak interleukin-6 of <100 pg/mL (35.5% +/- 5.2% vs. 74.7% +/- 5.1%, p <.001). Acute inflammation after

  10. Enhanced hepatic and kidney cytochrome p-450 activities in nandrolone decanoate treated albino mice.

    PubMed

    Acharjee, B K; Mahanta, R

    2009-04-01

    Anabolic androgenic steroids are the xenobiotic substrates that are metabolized in the body by the protective enzyme systems. Mixed function oxygenase enzymes include a group of enzymes which play an essential role in the metabolism of a broad range of xenobiotics including endogenous and exogenous substrates. Cytochrome P-450, a member of mixed function oxygenase enzymes, plays an important role in oxidative metabolism of drugs and xenobiotics entering human body. Various anabolic steroids are found either to increase or decrease the activity of cytochrome P-450. However, effect of nandrolone decanoate, most commonly abused anabolic steroid, on cytochrome P-450 activity is still fragmentary. In the present study, albino mice were administered intramuscular 2.5 mg of nandrolone decanoate injection at 15 days interval. Cytochrome P-450 activity is determined by following the method of Omura and Sato (1964) in liver and kidney tissues of both normal and experimental groups upto 90 days. Investigation shows a significant (p <0.01) increase of cytochrome P-450 (nmol/mg) activity in liver tissue as compared to that of kidney tissues. A tissue specific and dose specific increase of cytochrome P-450 activity is observed. Mean cytochrome P-450 is found highest in liver tissue on 45(th) day whereas the activity in kidney tissue is noticed on 90(th) day of treatment. From the above observation, nandrolone decanoate can be suggested as a potent inducer of cytochrome P-450 activity like other anabolic steroids.

  11. Blarina brevicauda as a biological monitor of polychlorinated biphenyls: Evaluation of hepatic cytochrome p450 induction

    USGS Publications Warehouse

    Russell, J.S.; Halbrook, R.S.; Woolf, A.; French, J.B.; Melancon, M.J.

    2004-01-01

    We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with $-naphthoflavone ($NF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received $NF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.

  12. Blarina brevicauda as a biological monitor of polychlorinated biphenyls: evaluation of hepatic cytochrome P450 induction.

    PubMed

    Russell, Julie S; Halbrook, Richard S; Woolf, Alan; French, John B; Melancon, Mark J

    2004-08-01

    We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with beta-naphthoflavone (betaNF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received betaNF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.

  13. Significance of Cytochrome P450 System Responses and Levels of Bile Fluorescent Aromatic Compounds in Marine Wildlife Following Oil Spills

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Richard F.; Anderson, Jack W.

    2005-07-01

    The relationships among cytochrome P450 induction in marine wildlife species, levels of fluorescent aromatic compounds (FAC) in their bile, the chemical composition of the inducing compounds, the significance of the exposure pathway, and any resulting injury, as a consequence of exposure to crude oil following a spill, are reviewed. Fish collected after oil spills often show increases in cytochrome P450 system activity, cytochrome P4501A (CYP1A) and bile fluorescent aromatic compounds (FAC), that are correlated with exposure to polycyclic aromatic hydrocarbons (PAH) in the oil. There is also some evidence for increases in bile FAC and induction of cytochrome P450 inmore » marine birds and mammals after oil spills. However, when observed, increases in these exposure indicators are transitory and generally decrease to background levels within one year after the exposure. Laboratory studies have shown induction of cytochrome P450 systems occurs after exposure of fish to crude oil in water, sediment or food. Most of the PAH found in crude oil (dominantly 2- and 3-ring PAH) are not strong inducers of cytochrome P450. Exposure to the 4-ring chrysenes or the photooxidized products of the PAH may account for the cytochrome P450 responses in fish collected from oil-spill sites. The contribution of non-spill background PAH, particularly combustion-derived (pyrogenic) PAH, to bile FAC and cytochrome P450 system responses can be confounding and needs to be considered when evaluating oil spill effects. The ubiquity of pyrogenic PAH makes it important to fully characterize all sources of PAH, including PAH from natural resources, e.g. retene, in oil spill studies. In addition, such parameters as species, sex, age, ambient temperature and season need to be taken into account. While increases in fish bile FAC and cytochrome P450 system responses, can together, be sensitive general indicators of PAH exposure after an oil spill, there is little unequivocal evidence to suggest a

  14. Cytochrome P4501A1 expression in blubber biopsies of endangered false killer whales (Pseudorca crassidens) and nine other odontocete species from Hawai'i.

    PubMed

    Foltz, Kerry M; Baird, Robin W; Ylitalo, Gina M; Jensen, Brenda A

    2014-11-01

    Odontocetes (toothed whales) are considered sentinel species in the marine environment because of their high trophic position, long life spans, and blubber that accumulates lipophilic contaminants. Cytochrome P4501A1 (CYP1A1) is a biomarker of exposure and molecular effects of certain persistent organic pollutants. Immunohistochemistry was used to visualize CYP1A1 expression in blubber biopsies collected by non-lethal sampling methods from 10 species of free-ranging Hawaiian odontocetes: short-finned pilot whale, melon-headed whale, pygmy killer whale, common bottlenose dolphin, rough-toothed dolphin, pantropical spotted dolphin, Blainville's beaked whale, Cuvier's beaked whale, sperm whale, and endangered main Hawaiian Islands insular false killer whale. Significantly higher levels of CYP1A1 were observed in false killer whales and rough-toothed dolphins compared to melon-headed whales, and in general, trophic position appears to influence CYP1A1 expression patterns in particular species groups. No significant differences in CYP1A1 were found based on age class or sex across all samples. However, within male false killer whales, juveniles expressed significantly higher levels of CYP1A1 when compared to adults. Total polychlorinated biphenyl (∑PCBs) concentrations in 84% of false killer whales exceeded proposed threshold levels for health effects, and ∑PCBs correlated with CYP1A1 expression. There was no significant relationship between PCB toxic equivalent quotient and CYP1A1 expression, suggesting that this response may be influenced by agonists other than the dioxin-like PCBs measured in this study. No significant differences were found for CYP1A1 expression among social clusters of false killer whales. This work provides a foundation for future health monitoring of the endangered stock of false killer whales and other Hawaiian odontocetes.

  15. Cytochrome P450 Activity in Ex Vivo Cornea Models and a Human Cornea Construct.

    PubMed

    Kölln, Christian; Reichl, Stephan

    2016-07-01

    The pharmacokinetic behaviors of novel ophthalmic drugs are often preliminarily investigated in preclinical studies using ex vivo animal cornea or corneal cell culture models. During transcorneal passage, topically applied drugs may be affected by drug metabolizing enzymes. The knowledge regarding the functional expression of metabolic enzymes in corneal tissue is marginal; thus, the aim of this study was to investigate cytochrome P450 activity in an organotypic three-dimensional human cornea construct and to compare it with porcine and rabbit corneas, which are commonly used ex vivo cornea models. The total cytochrome P450 activity was determined by measuring the transformation of 7-ethoxycoumarin. Furthermore, the expression of the cytochrome P450 enzyme 2D6 (CYP2D6) was investigated at the protein level using immunohistochemistry and western blotting. CYP2D6 activity measurements were performed using a d-luciferin-based assay. In summary, similar levels of the total cytochrome P450 activity were identified in all 3 cornea models. The protein expression of CYP2D6 was confirmed in the human cornea construct and porcine cornea, whereas the signals in the rabbit cornea were weak. The analysis of the CYP2D6 activity indicated similar values for the human cornea construct and porcine cornea; however, a distinctly lower activity was observed in the rabbit cornea. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  16. 1-Ethynylpyrene, a suicide inhibitor of cytochrome P-450 dependent benzo(a)pyrene hydroxylase activity in liver microsomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gan, L.S.L.; Acebo, A.L.; Alworth, W.L.

    The preparation of 1-ethynylpyrene (EP) by incubation of EP with liver microsomes in the presence of NADPH yields fluorescent products briefly. Addition of microsomes restores the original rate. The metabolism of EP is initially more rapid in microsomes from 5,6-benzoflavone- (BF) pretreated rats than in those from phenobarbital (PB) pretreated rats or controls. Ep inhibits the hydroxylation of benzo(a)pyrene (BP) by liver microsomes. Ep more effectively inhibits the oxidation of BP in liver microsomes from BF rats than from PB rats or from controls. The inhibition of BP hydroxylation activity due to EP is dependent upon NADPH and is apparentlymore » irreversible. Kinetic analyses show that the inhibition of BP hydroxylation is due to loss of the activity by a process that is first order in EP and that reaches a limiting value at infinite EP concentrations. A self-catalyzed inhibition of the cytochrome P-450 dependent BP hydroxylation may occur in the presence of EP. Incubation with EP under conditions that result in loss of BP hydroxylase activity in microsomes from BF rats and 66% of the activity from PB rats causes the loss of 6 and 12% of the cytochrome P-450, respectively. Thus the loss of P-450 content is an insensitive measure of the effect of this inhibitor upon this cytochrome P-450 dependent enzyme activity. Selectivity of the loss of P-450 due to the incubation of the different microsomal preparations with EP is observed to be different than the selectivity for loss of BP hydroxylase activity. It is proposed that the inhibition of cytochrome P-450 dependent enzymes by alkynes need not involve heme alkylation and a resulting loss of P-450 content. In vivo EP does not cause a significant change in the cytochrome P-450 content in the microsomes isolated, or result in the change in BP hydroxylation.« less

  17. RELATIVE POTENCY VALUES FOR POLYCHLORINATED DIBENZO-P-DIOXIN, DIBENZOFURAN AND BIPHENYL CONGENERS TO INDUCE CYTOCHROME P4501A MRNA IN A ZEBRAFISH LIVER CELL LINE (ZF-L)

    EPA Science Inventory

    Induction of cytochrome P450 (CYPIA) mRNA by polychlorinated dibenzo-p-dioxin (PCDD), polychlorinated dibenzofuran (PCDF) and polychlorinated biphenyl (PCB) congeners was measured in a zebrafish liver cell line (ZF-L). ZF-L cells were far less sensitive to PCDD, PCDF and PCB cong...

  18. HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay for Cytochrome P450 1A2 Activity

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Fletke, Kyle J.

    2007-01-01

    Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among…

  19. 48 CFR 4.501 - [Reserved

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false [Reserved] 4.501 Section 4.501 Federal Acquisition Regulations System FEDERAL ACQUISITION REGULATION GENERAL ADMINISTRATIVE MATTERS Electronic Commerce in Contracting 4.501 [Reserved] ...

  20. 5 CFR 4501.102 - Examination information.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 5 Administrative Personnel 3 2013-01-01 2013-01-01 false Examination information. 4501.102 Section 4501.102 Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT SUPPLEMENTAL STANDARDS OF ETHICAL CONDUCT FOR EMPLOYEES OF THE OFFICE OF PERSONNEL MANAGEMENT § 4501.102 Examination information. (a) An...

  1. Cytochrome P450-mediated metabolism of vitamin D

    PubMed Central

    Jones, Glenville; Prosser, David E.; Kaufmann, Martin

    2014-01-01

    The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1α,25-dihydroxyvitamin D3, which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression described in companion articles in this review series. This review updates our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1α-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease, and hyperproliferative diseases, such as psoriasis and cancer; as well as explore potential future developments in the field. PMID:23564710

  2. Sustained induction of cytochrome P4501A1 in human hepatoma cells by co-exposure to benzo[a]pyrene and 7H-dibenzo[c,g]carbazole underlies the synergistic effects on DNA adduct formation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gábelová, Alena, E-mail: alena.gabelova@savba.sk; Poláková, Veronika; Prochazka, Gabriela

    To gain a deeper insight into the potential interactions between individual aromatic hydrocarbons in a mixture, several benzo[a]pyrene (B[a]P) and 7H-dibenzo[c,g]carbazole (DBC) binary mixtures were studied. The biological activity of the binary mixtures was investigated in the HepG2 and WB-F344 liver cell lines and the Chinese hamster V79 cell line that stably expresses the human cytochrome P4501A1 (hCYP1A1). In the V79 cells, binary mixtures, in contrast to individual carcinogens, caused a significant decrease in the levels of micronuclei, DNA adducts and gene mutations, but not in cell survival. Similarly, a lower frequency of micronuclei and levels of DNA adducts weremore » found in rat liver WB-F344 cells treated with a binary mixture, regardless of the exposure time. The observed antagonism between B[a]P and DBC may be due to an inhibition of Cyp1a1 expression because cells exposed to B[a]P:DBC showed a decrease in Cyp1a1 mRNA levels. In human liver HepG2 cells exposed to binary mixtures for 2 h, a reduction in micronuclei frequency was also found. However, after a 24 h treatment, synergism between B[a]P and DBC was determined based on DNA adduct formation. Accordingly, the up-regulation of CYP1A1 expression was detected in HepG2 cells exposed to B[a]P:DBC. Our results show significant differences in the response of human and rat cells to B[a]P:DBC mixtures and stress the need to use multiple experimental systems when evaluating the potential risk of environmental pollutants. Our data also indicate that an increased expression of CYP1A1 results in a synergistic effect of B[a]P and DBC in human cells. As humans are exposed to a plethora of noxious chemicals, our results have important implications for human carcinogenesis. - Highlights: • B[a]P:DBC mixtures were less genotoxic in V79MZh1A1 cells than B[a]P and DBC alone. • An antagonism between B[a]P and DBC was determined in rat liver WB-F344 cells. • The inhibition of CYP1a1 expression by B[a]P

  3. 30 CFR 56.4501 - Fuel lines.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Fuel lines. 56.4501 Section 56.4501 Mineral... Installation/construction/maintenance § 56.4501 Fuel lines. Fuel lines shall be equipped with valves capable of stopping the flow of fuel at the source and shall be located and maintained to minimize fire hazards. This...

  4. 30 CFR 56.4501 - Fuel lines.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Fuel lines. 56.4501 Section 56.4501 Mineral... Installation/construction/maintenance § 56.4501 Fuel lines. Fuel lines shall be equipped with valves capable of stopping the flow of fuel at the source and shall be located and maintained to minimize fire hazards. This...

  5. 30 CFR 56.4501 - Fuel lines.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Fuel lines. 56.4501 Section 56.4501 Mineral... Installation/construction/maintenance § 56.4501 Fuel lines. Fuel lines shall be equipped with valves capable of stopping the flow of fuel at the source and shall be located and maintained to minimize fire hazards. This...

  6. 30 CFR 57.4501 - Fuel lines.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 30 Mineral Resources 1 2014-07-01 2014-07-01 false Fuel lines. 57.4501 Section 57.4501 Mineral... Installation/construction/maintenance § 57.4501 Fuel lines. Fuel lines shall be equipped with valves capable of stopping the flow of fuel at the source and shall be located and maintained to minimize fire hazards. This...

  7. 30 CFR 56.4501 - Fuel lines.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Fuel lines. 56.4501 Section 56.4501 Mineral... Installation/construction/maintenance § 56.4501 Fuel lines. Fuel lines shall be equipped with valves capable of stopping the flow of fuel at the source and shall be located and maintained to minimize fire hazards. This...

  8. 30 CFR 57.4501 - Fuel lines.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 30 Mineral Resources 1 2011-07-01 2011-07-01 false Fuel lines. 57.4501 Section 57.4501 Mineral... Installation/construction/maintenance § 57.4501 Fuel lines. Fuel lines shall be equipped with valves capable of stopping the flow of fuel at the source and shall be located and maintained to minimize fire hazards. This...

  9. 30 CFR 57.4501 - Fuel lines.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 30 Mineral Resources 1 2013-07-01 2013-07-01 false Fuel lines. 57.4501 Section 57.4501 Mineral... Installation/construction/maintenance § 57.4501 Fuel lines. Fuel lines shall be equipped with valves capable of stopping the flow of fuel at the source and shall be located and maintained to minimize fire hazards. This...

  10. 30 CFR 57.4501 - Fuel lines.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 30 Mineral Resources 1 2012-07-01 2012-07-01 false Fuel lines. 57.4501 Section 57.4501 Mineral... Installation/construction/maintenance § 57.4501 Fuel lines. Fuel lines shall be equipped with valves capable of stopping the flow of fuel at the source and shall be located and maintained to minimize fire hazards. This...

  11. Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: Comparisons with AHR1-mediated reporter gene activity and in ovo toxicity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Manning, Gillian E., E-mail: gmann017@uottawa.ca; Environment Canada, National Wildlife Research Centre, Ottawa, ON, Canada K1A 0H3; Mundy, Lukas J., E-mail: lukas.mundy@ec.gc.ca

    2013-01-01

    Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, inmore » combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species. -- Highlights: ► The chicken isn't the most sensitive species to CYP1A induction by PCB 105 and 118. ► The relative potency of PCBs differs between avian species. ► EROD activity was correlated with luciferase activity from the LRG assay. ► EROD activity was a better predictor of toxicity

  12. A world of cytochrome P450s

    PubMed Central

    Nelson, David R.

    2013-01-01

    The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450. PMID:23297353

  13. Albendazole sulfonation by rat liver cytochrome P-450c.

    PubMed

    Souhaili-El Amri, H; Mothe, O; Totis, M; Masson, C; Batt, A M; Delatour, P; Siest, G

    1988-08-01

    The metabolism of albendazole (ABZ) was studied in perfused livers from control and ABZ-treated rats (10.6 mg/kg, per os, each day for 10 days). In the perfusion fluid, the concentration of ABZ-sulfoxide (SO-ABZ) remained unchanged in treated, as compared to control animals, whereas ABZ-sulfone (SO2-ABZ) was increased in treated animals. In bile, only SO-ABZ was present. The transformation kinetics of SO-ABZ to SO2-ABZ in microsomes from rats treated with ABZ, 3-methylcholanthrene, Aroclor and isosafrole were biphasic. This suggests that enzyme activity was a consequence of two enzyme systems, one characterized by low affinity and high capacity, the other by high affinity and low capacity, the latter could be induced by 3-methylcholanthrene, ABZ, Aroclor and isosafrole. Cytochrome P-450c was induced potently in vivo by ABZ as proven by increased monooxygenase (7-ethoxyresorufin and 7-ethoxycoumarin-O-deethylase) activities and by Elisa test (a 5-fold increase in hemoprotein concentration was observed). Purified and reconstituted cytochrome P-450c from 3-methylcholanthrene or ABZ-treated rat liver were able to produce SO2-ABZ (2.01 and 1.70 nmol/mg/15 min, respectively, whereas cytochrome P-450b produced 10 times less SO2-ABZ). Immunological assays, as well as activity measurements showed a relationship between cytochrome P-450c-3-methylcholanthrene and cytochrome P-450c-ABZ. We conclude that induction of cytochrome P-450c by ABZ is the probable explanation for the enhanced formation of SO2-ABZ in vivo.

  14. Relation among cytochrome P450, AH-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

    USGS Publications Warehouse

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

    1994-01-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). Hepatic cytochrome P-450-associated monooxygenases and cytochrome P-450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r-2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  15. Engineering of a functional human NADH-dependent cytochrome P450 system

    PubMed Central

    Döhr, Olaf; Paine, Mark J. I.; Friedberg, Thomas; Roberts, Gordon C. K.; Wolf, C. Roland

    2001-01-01

    A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent kcat of W676A is equivalent (90%) to the NADPH-dependent kcat of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent KMNADPH and KMNADH values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2′-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system. PMID:11136248

  16. Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

    PubMed

    Papa, S; Lorusso, M; Izzo, G; Capuano, F

    1981-02-15

    component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562. This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes. Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes.

  17. Metformin inhibits 7,12-dimethylbenz[a]anthracene-induced breast carcinogenesis and adduct formation in human breast cells by inhibiting the cytochrome P4501A1/aryl hydrocarbon receptor signaling pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Maayah, Zaid H.; Ghebeh, Hazem; Alhaider, Abdulqader A.

    2015-04-15

    Recent studies have established that metformin (MET), an oral anti-diabetic drug, possesses antioxidant activity and is effective against different types of cancer in several carcinogen-induced animal models and cell lines. However, whether MET can protect against breast cancer has not been reported before. Therefore, the overall objectives of the present study are to elucidate the potential chemopreventive effect of MET in non-cancerous human breast MCF10A cells and explore the underlying mechanism involved, specifically the role of cytochrome P4501A1 (CYP1A1)/aryl hydrocarbon receptor (AhR) pathway. Transformation of the MCF10A cells into initiated breast cancer cells with DNA adduct formation was conducted usingmore » 7,12-dimethylbenz[a]anthracene (DMBA), an AhR ligand. The chemopreventive effect of MET against DMBA-induced breast carcinogenesis was evidenced by the capability of MET to restore the induction of the mRNA levels of basic excision repair genes, 8-oxoguanine DNA glycosylase (OGG1) and apurinic/apyrimidinic endonuclease1 (APE1), and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG). Interestingly, the inhibition of DMBA-induced DNA adduct formation was associated with proportional decrease in CYP1A1 and in NAD(P)H:quinone oxidoreductase 1 (NQO1) gene expression. Mechanistically, the involvements of AhR and nuclear factor erythroid 2-related factor-2 (Nrf2) in the MET-mediated inhibition of DMBA-induced CYP1A1 and NQO1 gene expression were evidenced by the ability of MET to inhibit DMBA-induced xenobiotic responsive element and antioxidant responsive element luciferase reporter gene expression which suggests an AhR- and Nrf2-dependent transcriptional control. However, the inability of MET to bind to AhR suggests that MET is not an AhR ligand. In conclusion, the present work shows a strong evidence that MET inhibits the DMBA-mediated carcinogenicity and adduct formation by inhibiting the expression of CYP1A1 through an AhR ligand

  18. Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Reed, James R., E-mail: rreed@lsuhsc.edu; The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112; Cawley, George F.

    2014-06-01

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of severalmore » P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is

  19. Effects of membrane curvature and pH on proton pumping activity of single cytochrome bo3 enzymes.

    PubMed

    Li, Mengqiu; Khan, Sanobar; Rong, Honglin; Tuma, Roman; Hatzakis, Nikos S; Jeuken, Lars J C

    2017-09-01

    The molecular mechanism of proton pumping by heme-copper oxidases (HCO) has intrigued the scientific community since it was first proposed. We have recently reported a novel technology that enables the continuous characterisation of proton transport activity of a HCO and ubiquinol oxidase from Escherichia coli, cytochrome bo 3 , for hundreds of seconds on the single enzyme level (Li et al. J Am Chem Soc 137 (2015) 16055-16063). Here, we have extended these studies by additional experiments and analyses of the proton transfer rate as a function of proteoliposome size and pH at the N- and P-side of single HCOs. Proton transport activity of cytochrome bo 3 was found to decrease with increased curvature of the membrane. Furthermore, proton uptake at the N-side (proton entrance) was insensitive to pH between pH6.4-8.4, while proton release at the P-side had an optimum pH of ~7.4, suggesting that the pH optimum is related to proton release from the proton exit site. Our previous single-enzyme experiments identified rare, long-lived conformation states of cytochrome bo 3 where protons leak back under turn-over conditions. Here, we analyzed and found that ~23% of cytochrome bo 3 proteoliposomes show ΔpH half-lives below 50s after stopping turnover, while only ~5% of the proteoliposomes containing a non-pumping mutant, E286C cytochrome bo 3 exhibit such fast decays. These single-enzyme results confirm our model in which HCO exhibit heterogeneous pumping rates and can adopt rare leak states in which protons are able to rapidly flow back. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Hepatic cytochrome P450 activity, abundance, and expression throughout human development

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo M.

    Cytochrome P450s are Phase I metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes can vary considerably throughout human development, especially when comparing fetal development to neonates, children, and adults. In an effort to develop a more comprehensive understanding of the ontogeny of P450 expression and activity we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. To quantify the functional activity of individual P450s we employ activity-based protein profiling, which uses modified mechanism-based inhibitors of P450s as chemical probes, in tandem with proteomicmore » analyses to quantify activity. Our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. The results were used to distribute P450s into three general classes based upon developmental stage of expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that our ontogeny results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.« less

  1. Olfactory cytochrome P-450. Studies with suicide substrates of the haemoprotein.

    PubMed Central

    Reed, C J; Lock, E A; De Matteis, F

    1988-01-01

    1. The olfactory epithelium of male hamsters has been found to be extremely active in the cumene hydroperoxide-supported oxidation of tetramethylphenylenediamine, and this peroxidase activity has been shown to be cytochrome P-450-dependent. 2. The interaction of a series of suicide substrates of cytochrome P-450 with the hepatic and olfactory mono-oxygenase systems has been assessed by determination of peroxidase, 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) activities after treatment in vivo with these compounds. Chloramphenicol, OOS-trimethylphosphorothiolate and two dihydropyridines [DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) and 4-ethyl DDC (3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine)] all caused similar percentage inhibitions of hepatic and olfactory activities, but the absolute amounts of enzymic activity lost were considerably greater in the latter tissue. In contrast, halothane had little effect upon hepatic cytochrome P-450-dependent reactions, whereas it severely inhibited those of the olfactory epithelium. 3. The time course of loss and recovery of hepatic and olfactory peroxidase, ECOD and EROD activities after a single dose of 4-ethyl DDC was studied. The rates of loss of activity observed were very similar, irrespective of tissue or reaction examined. In the olfactory epithelium, all three activities recovered concurrently and at a rate similar to that of the hepatic peroxidase activity. In contrast, the hepatic de-ethylation of 7-ethoxycoumarin and 7-ethoxy-resorufin recovered significantly more rapidly. 4. It is suggested that this behaviour is due to 4-ethyl DDC acting not only as a suicidal inhibitor but also as an inducer of certain forms of cytochrome P-450 in the liver; in the olfactory epithelium, however, inactivation, but not induction, occurs. Classical inducing agents were reported to have no effect upon olfactory cytochrome P-450, and in the present study neither phenobarbitone

  2. Structural and Kinetic Basis of Steroid 17α,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2*

    PubMed Central

    Pallan, Pradeep S.; Nagy, Leslie D.; Lei, Li; Gonzalez, Eric; Kramlinger, Valerie M.; Azumaya, Caleigh M.; Wawrzak, Zdzislaw; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

    2015-01-01

    Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17α-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116–119) showed only a few differences near the active site, despite only ∼50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity. PMID:25533464

  3. Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase.

    PubMed

    Henderson, Colin J; Otto, Diana M E; Carrie, Dianne; Magnuson, Mark A; McLaren, Aileen W; Rosewell, Ian; Wolf, C Roland

    2003-04-11

    Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.

  4. Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions.

    PubMed

    Ismail, Hanafy M; O'Neill, Paul M; Hong, David W; Finn, Robert D; Henderson, Colin J; Wright, Aaron T; Cravatt, Benjamin F; Hemingway, Janet; Paine, Mark J I

    2013-12-03

    Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or "pyrethrome." Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of unique tools for disease control.

  5. Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions

    PubMed Central

    Ismail, Hanafy M.; O’Neill, Paul M.; Hong, David W.; Finn, Robert D.; Henderson, Colin J.; Wright, Aaron T.; Cravatt, Benjamin F.; Hemingway, Janet; Paine, Mark J. I.

    2013-01-01

    Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or “pyrethrome.” Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450–insecticide interactions and aiding the development of unique tools for disease control. PMID:24248381

  6. Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

    PubMed

    Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

    1976-03-01

    Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

  7. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

    PubMed Central

    Doehmer, J; Dogra, S; Friedberg, T; Monier, S; Adesnik, M; Glatt, H; Oesch, F

    1988-01-01

    V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme. Images PMID:3137560

  8. [Construction of thr461 --> Asn461 and Ile462 --> Val462 mutation vector of P4501A1 gene].

    PubMed

    Wei, Qing; Liu, Yi-Min; Wang, Hui; Zhao, Xiao-Lin; Ren, Tie-ling; Xiao, Yong-mei

    2006-09-01

    To construct Thr461 --> Asn461 and Ile462 --> Val462 mutation vector of P4501A1 gene and to provide scientific base for deeply researching on the function of cytochrome 1A1 gene (CYP1A1) and the mechanism of carcinogenesis. According to cDNA sequence of human CYP1A1 gene, universal primers (Pm3/Pm4) and mutant primers (Pt15/Pt16 and Pt17/Pt18) containing restriction enzyme site and mutation site were designed. The first set of primers involving Pm3/Pt16 and Pm3/Pt18 amplified a forward 1.5kb fragment from pGEM-T-CYP1A1 plasmid. The second set of primers involving Pt15/Pm4 and Pt17/Pm4 amplified a reverse 177-bp fragment from 10ng pGEM-T-CYP1A1 plasmid. The third set of primers involving Pm3/Pm4 amplified a 1.5kb fragment from the fomer PCR amplifications. The third PCR products were separated, purified and recovered from 1% agarose gel, then inserted into pMD-T vector. Subsequently the conjunct products were transformed into E. coil strain DH-5alpha., then the single clone was screened out and plasmids were extracted from such clone finally verified by restriction endonuclease analysis and sequencing. A 1.5kb fragment of tricycle PCR amplifications were digested by restriction endonucleases (BamHI and SailI) and sequenced bidirectionally by universal primers(T7p and SP6). The results verified that the cloned fragment including Asn461 and Val462 mutant site had 99.9% homology with the human cDNA of CYP1A1 gene in Genebank. The objective fragment containing Asn461 and Va462 mutant site with cDNA of the CYP1A1 gene has been successfully constructed in this experiment.

  9. Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

    PubMed

    Szczesna-Skorupa, E; Chen, C D; Rogers, S; Kemper, B

    1998-12-08

    Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

  10. Regulation of cytochrome P-450 4A activity by peroxisome proliferator-activated receptors in the rat kidney.

    PubMed

    Ishizuka, Tsuneo; Ito, Osamu; Tan, Liping; Ogawa, Susumu; Kohzuki, Masahiro; Omata, Ken; Takeuchi, Kazuhisa; Ito, Sadayoshi

    2003-11-01

    The localization of cytochrome P-450 4A, peroxisome proliferator-activated receptor (PPAR) alpha, and PPARgamma proteins, and the inducibility of P-450 4A expression and activity by PPAR agonists were determined in the rat kidney. The expressions of these proteins in isolated nephron segments were evaluated by immunoblot analysis, and the production of 20-hydroxyeicosatetraenoic acid (20-HETE) was measured as P-450 4A activity. P-450 4A proteins were expressed predominantly in the proximal tubule (PT), with lower expression in the preglomerular arteriole (Art), glomerulus (Glm), and medullary thick ascending limb (mTAL), but their expression was not detected in the inner medullary collecting duct (IMCD). PPARalpha protein was expressed in the PT and mTAL, and PPARgamma protein was expressed in the IMCD and mTAL. Treatment with clofibrate, the PPARalpha agonist, increased P-450 4A protein levels and the production of 20-HETE in microsomes prepared from the renal cortex, whereas treatment with pioglitazone, the PPARgamma agonist, affected neither of them. These results indicate that PPARalpha and PPARgamma proteins are localized in different nephron segments and the inducibility of P-450 4A expression and activity by the PPAR agonists correlates with the nephron-specific localization of the respective PPAR isoforms.

  11. The cytochrome p450 homepage.

    PubMed

    Nelson, David R

    2009-10-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  12. Methoxyflurane acts at the substrate binding site of cytochrome P450 LM2 to induce a dependence on cytochrome b5.

    PubMed

    Lipka, J J; Waskell, L A

    1989-01-01

    Rabbit cytochrome P450 isozyme 2 requires cytochrome b5 to metabolize the volatile anesthetic methoxyflurane but not the substrate benzphetamine [E. Canova-Davis and L. Waskell (1984) J. Biol. Chem. 259, 2541-2546]. To determine whether the requirement for cytochrome b5 for methoxyflurane oxidation is mediated by an allosteric effect on cytochrome P450 LM2 or cytochrome P450 reductase, we have investigated whether this anesthetic can induce a role for cytochrome b5 in benzphetamine metabolism. Using rabbit liver microsomes and antibodies raised in guinea pigs against rabbit cytochrome b5, we found that methoxyflurane did not create a cytochrome b5 requirement for benzphetamine metabolism. Methoxyflurane also failed to induce a role for cytochrome b5 in benzphetamine metabolism in the purified, reconstituted mixed function oxidase system. Studies of the reaction kinetics established that in the absence of cytochrome b5, methoxyflurane and benzphetamine are competitive inhibitors, and that in the presence of cytochrome b5, benzphetamine and methoxyflurane are two alternate substrates in competition for a single site on the same enzyme. These results all indicate that the methoxyflurane-induced cytochrome b5 dependence of the mixed function oxidase cytochrome P450 LM2 system is a direct result of the interaction between methoxyflurane and the substrate binding site of cytochrome P450 LM2 and suggest the focus of future studies of this question.

  13. The Cytochrome P450 Homepage

    PubMed Central

    2009-01-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 (CYP) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described. PMID:19951895

  14. Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats.

    PubMed

    Murray, M

    1988-05-01

    Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16 beta- and 6 beta-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7 alpha-hydroxylase activity was decreased to 0.8-fold of control and 16 alpha-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite-P-450 complex revealed an even greater induction of 16 beta- and 6 beta-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16 alpha-hydroxylase activity increased 2-fold after ferricyanide but 7 alpha-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT-A decreased androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16 alpha-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT-A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB-B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT-A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT-F, which is active in steroid 7 alpha-hydroxylation, does not appear

  15. Effects of interferon-tau and steroids on cytochrome P450 activity in bovine endometrial epithelial cells

    USDA-ARS?s Scientific Manuscript database

    The objective of the current study was to examine cyclooxygenase (COX), cytochrome P450 1A (CYP1A) and 2C (CYP2C) activity in bovine endometrial cell cultures following exposure to oxytocin (OT), interferon-t (IFN), estradiol (E2) and/or progesterone (P4). Bovine endometrial epithelial cells were tr...

  16. Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

    PubMed

    Quester, Katrin; Juarez-Moreno, Karla; Secundino, Isamel; Roseinstein, Yvonne; Alejo, Karla P; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

    2017-05-01

    Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Cancer Activation and Polymorphisms of Human Cytochrome P450 1B1

    PubMed Central

    Chun, Young-Jin; Kim, Donghak

    2016-01-01

    Human cytochrome P450 enzymes (P450s, CYPs) are major oxidative catalysts that metabolize various xenobiotic and endogenous compounds. Many carcinogens induce cancer only after metabolic activation and P450 enzymes play an important role in this phenomenon. P450 1B1 mediates bioactivation of many procarcinogenic chemicals and carcinogenic estrogen. It catalyzes the oxidation reaction of polycyclic aromatic carbons, heterocyclic and aromatic amines, and the 4-hydroxylation reaction of 17β-estradiol. Enhanced expression of P450 1B1 promotes cancer cell proliferation and metastasis. There are at least 25 polymorphic variants of P450 1B1 and some of these have been reported to be associated with eye diseases. In addition, P450 1B1 polymorphisms can greatly affect the metabolic activation of many procarcinogenic compounds. It is necessary to understand the relationship between metabolic activation of such substances and P450 1B1 polymorphisms in order to develop rational strategies for the prevention of its toxic effect on human health. PMID:27123158

  18. High-Fat Diets Alter the Modulatory Effects of Xenobiotics on Cytochrome P450 Activities

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sadler, Natalie C.; Webb-Robertson, Bobbie-Jo M.; Clauss, Therese R.

    Cytochrome P450 monooxygenases (P450) are key to the metabolism of myriad endogenous chemicals and xenobiotics, including the majority of therapeutic drugs. Dysregulated P450 activities can lead to altered drug metabolism and toxicity, oxidative stress, and inflammation; all physiological states frequently charged as the impetus for various chronic pathologies. We characterized the impact of common xenobiotic exposures, specifically high-fat diet and active or passive cigarette smoke, on the functional capacity of hepatic and pulmonary P450s. We employed an activity-based protein profiling approach to characterize the identity and activity level of measured individual P450 isoforms. Our results confirm expectations of significant alterationsmore » in pulmonary P450s due to cigarette smoke, but now reveal the repressive impact of high-fat diet-induced obesity on many hepatic P450s activities, and the dynamic alterations due to concomitant diet and smoke exposures on liver and lung P450 activities impacting drug metabolism and pathways of inflammation.« less

  19. Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots.

    PubMed

    Bosilkovska, M; Samer, C F; Déglon, J; Rebsamen, M; Staub, C; Dayer, P; Walder, B; Desmeules, J A; Daali, Y

    2014-09-01

    The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session.

  20. FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE

    EPA Science Inventory

    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  1. Beluga whale liver microsomal cytochrome P4501A (CYP1A) enzymes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bullock, P.L.; Addison, R.; Lockhart, L.

    1995-12-31

    Beluga whale (Delphinapterus leucas) liver from the Canadian arctic was analyzed for the presence of CYP1A enzymes, as part of current studies on biomarkers for environmental contamination. CYP1A1-associated 7-ethoxyresorufin O-dealkylase activity (EROD) varied 13 fold among sixteen male whale liver microsomal samples and 31 fold among five females. Similarly, the rate of 7-methoxyresorufin O-dealkylation (MROD) varied 7 fold and 3 fold in microsomal samples from males and females, respectively. Furthermore, 7-pentoxyresorufin O-dealkylase activity (PROD) varied 10 fold in both sexes. None of these enzyme activities were sexually differentiated, and EROD and MROD were inhibited by {alpha}-naphthoflavone. There was very goodmore » correlation between EROD and MROD (r{sup 2} = .894), EROD and PROD (r{sup 2} = .909), but MROD and PROD were not as well correlated (r{sup 2} = 785). On Western immunoblots, a single band was recognized in Beluga whale liver microsomes by a polygonal antibody raised against an oligopeptide related to trout CYP1A1. This antibody also recognized purified rat CYP1A1 (56 kDa) and stained only one band (56 kDa) in liver microsomes isolated from male rats treated with {beta}-naphthoflavone. The interindividual variation in EROD paralleled differences in the amount of whale liver microsomal protein that cross-reacted with the anti-peptide antibody. The results suggest that Beluga whale liver contains at least one CYP1A enzyme which catalyzes the 0-dealkylation of 7-ethoxy, 7-methoxy and 7-pentoxyresorufin and has a molecular weight less than that of rat CYP1A1, but similar to rat CYP1A2 (52 kDa).« less

  2. Construction and engineering of a thermostable self-sufficient cytochrome P450

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp

    2009-06-19

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C.more » Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.« less

  3. Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, Bruce R.; Stegeman, John J.

    1993-01-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O- dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black- crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross- reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p'-DDE were detected in embryos from Cat Island. Cytochrome P450- associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

  4. Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, Bruce R.; Stegeman, John J.

    1993-01-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu-g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than five-fold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O-dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p, p'-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

  5. Effect of zolpidem on human cytochrome P450 activity, and on transport mediated by P-glycoprotein.

    PubMed

    von Moltke, Lisa L; Weemhoff, James L; Perloff, Michael D; Hesse, Leah M; Harmatz, Jerold S; Roth-Schechter, Barbara F; Greenblatt, David J

    2002-12-01

    The influence of high concentrations of zolpidem (100 microM, corresponding to approximately 200 times maximum therapeutic concentrations) on the activity of six human Cytochrome P450 (CYP) enzymes was evaluated in a model system using human liver microsomes. Zolpidem produced negligible or weak inhibition of human CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A. Transport of rhodamine 123, presumed to be mediated mainly by the energy-dependent efflux transport protein P-glycoprotein, was studied in a cell culture system using a human intestinal cell line. High concentrations of zolpidem (100 microM), exceeding the usual therapeutic range by more than 100-fold, produced only modest impairment of rhodamine 123 transport. The findings indicate that zolpidem is very unlikely to cause clinical drug interactions attributable to impairment of CYP activity or P-gp mediated transport. Copyright 2002 John Wiley & Sons, Ltd.

  6. Geneva Cocktail for Cytochrome P450 and P-Glycoprotein Activity Assessment Using Dried Blood Spots

    PubMed Central

    Bosilkovska, M; Samer, C F; Déglon, J; Rebsamen, M; Staub, C; Dayer, P; Walder, B; Desmeules, J A; Daali, Y

    2014-01-01

    The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography–tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session. PMID:24722393

  7. Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Donnelly, Mark

    2006-08-01

    The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

  8. A human cytochrome P-450 is recognized by anti-liver/kidney microsome antibodies in autoimmune chronic hepatitis.

    PubMed

    Kiffel, L; Loeper, J; Homberg, J C; Leroux, J P

    1989-02-28

    1- Anti-liver/kidney microsome autoantibodies type 1 (anti-LKM1), observed in some children with chronic active hepatitis, were used to isolate their antigen in human liver microsomes. A protein, called P-LKM1 was thus purified. This protein was recognized by a rabbit antiserum directed against the related human cytochromes P-450 bufI and P-450 bufII. 2- A human liver microsomal protein immunoprecipitated with anti-LKM1 sera was also recognized by anti cytochromes P-450 bufI/II antibodies. 3- Anti-LKM1 antibodies potently inhibited microsomal bufuralol 1'-hydroxylation. These results displayed the possible identity between cytochrome P-450 bufI/II and LKM1 antigen.

  9. Iron(IV)hydroxide pK(a) and the role of thiolate ligation in C-H bond activation by cytochrome P450.

    PubMed

    Yosca, Timothy H; Rittle, Jonathan; Krest, Courtney M; Onderko, Elizabeth L; Silakov, Alexey; Calixto, Julio C; Behan, Rachel K; Green, Michael T

    2013-11-15

    Cytochrome P450 enzymes activate oxygen at heme iron centers to oxidize relatively inert substrate carbon-hydrogen bonds. Cysteine thiolate coordination to iron is posited to increase the pK(a) (where K(a) is the acid dissociation constant) of compound II, an iron(IV)hydroxide complex, correspondingly lowering the one-electron reduction potential of compound I, the active catalytic intermediate, and decreasing the driving force for deleterious auto-oxidation of tyrosine and tryptophan residues in the enzyme's framework. Here, we report on the preparation of an iron(IV)hydroxide complex in a P450 enzyme (CYP158) in ≥90% yield. Using rapid mixing technologies in conjunction with Mössbauer, ultraviolet/visible, and x-ray absorption spectroscopies, we determine a pK(a) value for this compound of 11.9. Marcus theory analysis indicates that this elevated pK(a) results in a >10,000-fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

  10. In vitro modulation of cytochrome P450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b(5) and methylene blue.

    PubMed

    Pearson, Josh T; Siu, Sophia; Meininger, David P; Wienkers, Larry C; Rock, Dan A

    2010-03-30

    Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).

  11. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

    PubMed Central

    Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

    1988-01-01

    In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative. Images PMID:3186722

  12. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

    PubMed

    Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

    1988-11-01

    In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative.

  13. Degradation of Morpholine by an Environmental Mycobacterium Strain Involves a Cytochrome P-450

    PubMed Central

    Poupin, P.; Truffaut, N.; Combourieu, B.; Besse, P.; Sancelme, M.; Veschambre, H.; Delort, A. M.

    1998-01-01

    A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ 1H nuclear magnetic resonance (1H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and 1H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. 1H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C—N bond of the morpholine ring. PMID:9435074

  14. Comparison of intrinsic dynamics of cytochrome p450 proteins using normal mode analysis

    PubMed Central

    Dorner, Mariah E; McMunn, Ryan D; Bartholow, Thomas G; Calhoon, Brecken E; Conlon, Michelle R; Dulli, Jessica M; Fehling, Samuel C; Fisher, Cody R; Hodgson, Shane W; Keenan, Shawn W; Kruger, Alyssa N; Mabin, Justin W; Mazula, Daniel L; Monte, Christopher A; Olthafer, Augustus; Sexton, Ashley E; Soderholm, Beatrice R; Strom, Alexander M; Hati, Sanchita

    2015-01-01

    Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide-range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but < 25% sequence identity. Based on their electron transfer protein partner, cytochrome P450 proteins are classified into six broad classes. Traditional methods of pro are based on the canonical paradigm that attributes proteins' function to their three-dimensional structure, which is determined by their primary structure that is the amino acid sequence. It is increasingly recognized that protein dynamics play an important role in molecular recognition and catalytic activity. As the mobility of a protein is an intrinsic property that is encrypted in its primary structure, we examined if different classes of cytochrome P450 enzymes display any unique patterns of intrinsic mobility. Normal mode analysis was performed to characterize the intrinsic dynamics of five classes of cytochrome P450 proteins. The present study revealed that cytochrome P450 enzymes share a strong dynamic similarity (root mean squared inner product > 55% and Bhattacharyya coefficient > 80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in Cα atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes. PMID:26130403

  15. Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

    PubMed Central

    Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

    2016-01-01

    Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

  16. Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

    PubMed Central

    Kumar, Santosh; Jin, Mengyao; Weemhoff, James L

    2013-01-01

    There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation. PMID:25298920

  17. Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

    PubMed Central

    Lamb, David C.; Maspahy, Segula; Kelly, Diane E.; Manning, Nigel J.; Geber, Antonia; Bennett, John E.; Kelly, Steven L.

    1999-01-01

    Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell. PMID:10390230

  18. Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4.

    PubMed

    Hildebrandt, P; Greinert, R; Stier, A; Taniguchi, H

    1989-12-08

    The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form

  19. [Effect of bemethyl on cytochrome P-450-dependent monoxygenases in the human liver and lymphocytes].

    PubMed

    Sorokina, E A; Sibiriak, S V; Sergeeva, S A

    2002-01-01

    Effects of the actoprotector bemithyl (50 mg/kg, p.o.) upon a single or five-fold administration on the cytochrome P-450 and b5 content and the isoform-specific and nonspecific monooxygenase activity [aminopyrine-N-demethylase, aniline-p-hydroxylase, 4-nitroanisole-o-demethylase,2,5-diphenyloxazole-p-hydroxylase, 7-ethoxyresorufin-o-deethylase (EROD), benzyloxyresorufin-o-debenzylase (BROD)] in rat liver were evaluated. In addition, the influence of bemithyl (0.(1)-100 microM) on the development of EROD and BROD activity was studied on the mitogen-stimulated human lymphocytes in vitro. Administered in rats, bemithyl exhibited the properties of a cytochrome P-450 inductor of the mixed type, which was manifested by an increase in the total cytochrome P-450 content in liver microsomes and in the monooxygenase activity related to both Ah-receptor-dependent and -independent isoforms (except for the aniline-p-hydroxylase activity). The induction of the monooxygenase activity realized by Ah-receptor-dependent isoforms (4-nitroanisole-o-demethylase, 2,5-diphenyloxazole-p-hydroxylase, and EROD activity) was more pronounced, reaching maximum upon a single drug administration. Acting upon the human lymphocytes in vitro, high concentrations of bemithyl increased expression of the EROD activity, while low drug concentrations stimulated the BROD activity.

  20. Cytochrome P450 (CYP450) Tests

    MedlinePlus

    ... P450 (CYP450) tests Overview Your doctor may use cytochrome P450 (CYP450) tests to help determine how your body processes (metabolizes) a drug. The human body contains P450 enzymes to process medications. Because of inherited (genetic) traits ...

  1. Versatility of non-native forms of human cytochrome c: pH and micellar concentration dependence.

    PubMed

    Simon, Matthieu; Metzinger-Le Meuth, Valérie; Chevance, Soizic; Delalande, Olivier; Bondon, Arnaud

    2013-01-01

    In addition to its electron transfer activity, cytochrome c is now known to trigger apoptosis via peroxidase activity. This new function is related to a structural modification of the cytochrome upon association with anionic lipids, particularly cardiolipin present in the mitochondrial membrane. However, the exact nature of the non-native state induced by this interaction remains an active subject of debate. In this work, using human cytochromes c (native and two single-histidine mutants and the corresponding double mutant) and micelles as a hydrophobic medium, we succeeded, through UV-visible spectroscopy, circular dichroism spectroscopy and NMR spectroscopy, in fully characterizing the nature of the sixth ligand replacing the native methionine. Furthermore, careful pH titrations permitted the identification of the amino acids involved in the iron binding over a range of pH values. Replacement of the methionine by lysine was only observed at pH above 8.5, whereas histidine binding is dependent on both pH and micelle concentration. The pH variation range for histidine protonation is relatively narrow and is consistent with the mitochondrial intermembrane pH changes occurring during apoptosis. These results allow us to rule out lysine as the sixth ligand at pH values close to neutrality and reinforce the role of histidines (preferentially His33 vs. His26) as the main candidate to replace methionine in the non-native cytochrome c. Finally, on the basis of these results and molecular dynamics simulations, we propose a 3D model for non-native cytochrome c in a micellar environment.

  2. Cytochrome P460 Genes from the Methanotroph Methylococcus capsulatus Bath†

    PubMed Central

    Bergmann, David J.; Zahn, James A.; Hooper, Alan B.; DiSpirito, Alan A.

    1998-01-01

    P460 cytochromes catalyze the oxidation of hydroxylamine to nitrite. They have been isolated from the ammonia-oxidizing bacterium Nitrosomonas europaea (R. H. Erickson and A. B. Hooper, Biochim. Biophys. Acta 275:231–244, 1972) and the methane-oxidizing bacterium Methylococcus capsulatus Bath (J. A. Zahn et al., J. Bacteriol. 176:5879–5887, 1994). A degenerate oligonucleotide probe was synthesized based on the N-terminal amino acid sequence of cytochrome P460 and used to identify a DNA fragment from M. capsulatus Bath that contains cyp, the gene encoding cytochrome P460. cyp is part of a gene cluster that contains three open reading frames (ORFs), the first predicted to encode a 59,000-Da membrane-bound polypeptide, the second predicted to encode a 12,000-Da periplasmic protein, and the third (cyp) encoding cytochrome P460. The products of the first two ORFs have no apparent similarity to any proteins in the GenBank database. The overall sequence similarity of the P460 cytochromes from M. capsulatus Bath and N. europaea was low (24.3% of residues identical), although short regions of conserved residues are present in the two proteins. Both cytochromes have a C-terminal, c-heme binding motif (CXXCH) and a conserved lysine residue (K61) that may provide an additional covalent cross-link to the heme (D. M. Arciero and A. B. Hooper, FEBS Lett. 410:457–460, 1997). Gene probing using cyp indicated that a cytochrome P460 similar to that from M. capsulatus Bath may be present in the type II methanotrophs Methylosinus trichosporium OB3b and Methylocystis parvus OBBP but not in the type I methanotrophs Methylobacter marinus A45, Methylomicrobium albus BG8, and Methylomonas sp. strains MN and MM2. Immunoblot analysis with antibodies against cytochrome P460 from M. capsulatus Bath indicated that the expression level of cytochrome P460 was not affected either by expression of the two different methane monooxygenases or by addition of ammonia to the culture medium. PMID

  3. Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.

    PubMed

    Dodhia, Vikash Rajnikant; Fantuzzi, Andrea; Gilardi, Gianfranco

    2006-10-01

    The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

  4. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jan, Yi-Hua; Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu; Baker, Angela A.

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling,more » a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.« less

  5. Synthesis of ¹³C-lidocaine as a probe of breath test for the evaluation of cytochrome P450 activity.

    PubMed

    Mitome, Hidemichi; Sugiyama, Erika; Sato, Hitoshi; Akira, Kazuki

    2014-01-01

    (13)C-Labeled lidocaine, 2-di[1-(13)C]ethylamino-N-(2,6-dimethylphenyl)acetamide (1), was synthesized from [1-(13)C]acetic acid in six steps, as a probe for a breath test to evaluate in vivo cytochrome P450 activity. The measurement of (13)CO2 in breath was successfully performed following oral administration of (13)C-lidocaine 1 to mice.

  6. Cytochrome P450 2D6 polymorphism and character traits.

    PubMed

    Suzuki, Eiji; Kitao, Yoshie; Ono, Yutaka; Iijima, Yoshimi; Inada, Toshiya

    2003-06-01

    It has been suggested that cytochrome P450 2D6 (CYP2D6) is involved in dopamine metabolism within the brain. The dopamine system is suggested to play a role in determining normal character. The purpose of this study was to examine whether character traits are dependent on cytochrome P450 2D6 activity. We investigated the association between temperament and CYP2D6 gene polymorphism. The subjects were all Japanese and the polymorphism genotyped in the present study was CYP2D6*10. Character traits were assessed using the Temperament and Character Inventory. There was no overall or specific association between personality traits and the CYP2D6*10 allele and genotype frequencies. The present results do not support the hypothesis that CYP2D6 activity affects temperament and character.

  7. Evaluating cytochrome P450 in birds by monooxygenases and immunohistochemistry: possible nonlethal assessment by skin immunohistochemistry

    USGS Publications Warehouse

    Melancon, M.J.; Kutay, A.L.; Woodin, Bruce R.; Stegeman, John J.

    2000-01-01

    Six month old Lesser Scaup and nestling Tree Swallows were injected intraperitoneally with beta-naphthoflavone (BNF) or vehicle. Nestling Tree Swallows were also collected from five sites with differing levels of contaminants. Liver samples were taken and stored at -80C until microsome preparation and monooxygenase (MO) assay. Skin and heart samples were placed in buffered formalin until immunohistochemical (IMHC) analysis for cytochrome P4501A (CYP1A). Scaup treated with BNF at 20 or 100 mg/kg body weight showed approximately 20- to 65-fold increases in four MOs. Responses of two of the four MOs were as high at 20 mg/kg as at 100mg/kg. There was no IMHC response in the vehicle-injected ducks, while in skin the IMHC response was the same for both dose levels of BNF and in heart there was response in two of four samples at 20 mg/kg and in all five samples at 100mg/kg. Tree Swallows injected with BNF at 100, but not at 20 mg/kg showed significant increases (ca.5-fold) in two MO activities. There was no IMHC response in control swallows. In skin and heart there were IMHC responses in one of five swallows at 20 mg/kg and four of five swallows at 100mg/kg. There was poor correlation between individual skin IMHC responses and MO activities and PCB concentrations in 47 field-collected Tree Swallow samples, but 14 of the 16 skin samples with positive IMHC responses were from the location with the highest MO activities and PCB concentrations. Although present data do not allow construction of significant dose response curves, the responses in skin make it well worth continuing study on this potential nonlethal technique for biomonitoring contaminant exposure of birds.

  8. Luminogenic cytochrome P450 assays.

    PubMed

    Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

    2006-08-01

    Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery.

  9. Relationships among Ergot Alkaloids, Cytochrome P450 Activity, and Beef Steer Growth

    NASA Astrophysics Data System (ADS)

    Rosenkrans, Charles; Ezell, Nicholas

    2015-03-01

    Determining a grazing animal’s susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega™ P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids [0, 20, and 40 μM of ergotamine (ET), dihydroergotamine (DHET), and ergonovine (EN)] on human CYP3A4 using the P450-Glo assay (Promega™ V9800). With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of in vitro cytochrome P450 activity was affected (P < 0.001) by an interaction between alkaloids and concentration. That interaction resulted in no concentration effect of EN, but within ET and DHET 20 and 40 µM concentrations inhibited CYP450 activity when compared with controls. In experiment 2, urine was collected from Angus-sired crossbred steers (n = 39; 216 ± 2.6 d of age; 203 ± 1.7 kg) after grazing tall fescue pastures for 105 d. Non-diluted urine was added to the Promega™ P450-Glo assay, and observed inhibition (3.7 % ± 2.7 of control). Urine content of total ergot alkaloids (331.1 ng/mg of creatinine ± 325.7) was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated (r = -0.31; P < 0.05). Steers were genotyped at CYP450 single nucleotide polymorphism, C994G. Steer genotype affected (P < 0.03) inhibition of CYP450 activity by urine; heterozygous steers had the least amount of CYP450 inhibition suggesting that genotyping cattle may be a method of identifying animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega™ P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins.

  10. Electrochemistry of cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17).

    PubMed

    Martin, Lisandra L; Kubeil, Clemens; Simonov, Alexandr N; Kuznetsov, Vladimir L; Corbin, C Jo; Auchus, Richard J; Conley, Alan J; Bond, Alan M; Rodgers, Raymond J

    2017-02-05

    Within the superfamily of cytochrome P450 enzymes (P450s), there is a small class which is functionally employed for steroid biosynthesis. The enzymes in this class appear to have a small active site to accommodate the steroid substrates specifically and snuggly, prior to the redox transformation or hydroxylation to form a product. Cytochrome P450c17 is one of these and is also a multi-functional P450, with two activities, the first 17α-hydroxylation of pregnenolone is followed by a subsequent 17,20-lyase transformation to dehydroepiandrosterone (DHEA) as the dominant pathways to cortisol precursors or androgens in humans, respectively. How P450c17 regulates these two redox reactions is of special interest. There is a paucity of direct electrochemical studies on steroidogenic P450s, and in this mini-review we provide an overview of these studies with P450c17. Historical consideration as to the difficulties in obtaining reliable electrochemistry due to issues of handling proteins on an electrode, together with advances in the electrochemical techniques are addressed. Recent work using Fourier transformed alternating current voltammetry is highlighted as this technique can provide both catalytic information simultaneously with the underlying redox transfer with the P450 haem. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. 49 CFR 450.1 - Purpose.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... APPROVAL OF CARGO CONTAINERS GENERAL General Provisions § 450.1 Purpose. This subchapter establishes requirements and procedures for safety approval and periodic examination of cargo containers used in international transport, as defined in the International Safe Container Act. [45 FR 37213, June 2, 1980] ...

  12. 49 CFR 450.1 - Purpose.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... APPROVAL OF CARGO CONTAINERS GENERAL General Provisions § 450.1 Purpose. This subchapter establishes requirements and procedures for safety approval and periodic examination of cargo containers used in international transport, as defined in the International Safe Container Act. [45 FR 37213, June 2, 1980] ...

  13. 49 CFR 450.1 - Purpose.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... APPROVAL OF CARGO CONTAINERS GENERAL General Provisions § 450.1 Purpose. This subchapter establishes requirements and procedures for safety approval and periodic examination of cargo containers used in international transport, as defined in the International Safe Container Act. [45 FR 37213, June 2, 1980] ...

  14. 49 CFR 450.1 - Purpose.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... APPROVAL OF CARGO CONTAINERS GENERAL General Provisions § 450.1 Purpose. This subchapter establishes requirements and procedures for safety approval and periodic examination of cargo containers used in international transport, as defined in the International Safe Container Act. [45 FR 37213, June 2, 1980] ...

  15. 49 CFR 450.1 - Purpose.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... APPROVAL OF CARGO CONTAINERS GENERAL General Provisions § 450.1 Purpose. This subchapter establishes requirements and procedures for safety approval and periodic examination of cargo containers used in international transport, as defined in the International Safe Container Act. [45 FR 37213, June 2, 1980] ...

  16. EXOGENOUS CYTOCHROME C RESTORES MYOCARDIAL CYTOCHROME OXIDASE ACTIVITY INTO THE LATE PHASE OF SEPSIS

    PubMed Central

    Piel, David A.; Deutschman, Clifford S.; Levy, Richard J.

    2009-01-01

    Mitochondrial dysfunction is thought to play a role in the pathogenesis of a variety of disease states, including sepsis. An acquired defect in oxidative phosphorylation potentially causes sepsis-induced organ dysfunction. Cytochrome oxidase (CcOX), the terminal oxidase of the respiratory chain, is competitively inhibited early in sepsis and progresses, becoming noncompetitive during the late phase. We have previously demonstrated that exogenous cytochrome c can overcome myocardial CcOX competitive inhibition and improve cardiac function during murine sepsis at the 24-h point. Here, we evaluate the effect of exogenous cytochrome c on CcOX activity and survival in mice at the later time points. Exogenous cytochrome c (800 μg) or saline was intravenously injected 24 h after cecal ligation and puncture (CLP) or sham operation. Steady-state mitochondrial cytochrome c levels and heme c content increased significantly 48 h post-CLP and remained elevated at 72 h in cytochrome c-injected mice compared with saline injection. Cecal ligation and puncture inhibited CcOX at 48 h in saline-injected mice. However, cytochrome c injection abrogated this inhibition and restored CcOX kinetic activity to sham values at 48 h. Survival after CLP to 96 h after cytochrome c injection approached 50% compared with only 15% after saline injection. Thus, a single injection of exogenous cytochrome c 24 h post-CLP repletes mitochondrial substrate levels for up to 72 h, restores myocardial COX activity, and significantly improves survival. PMID:18414235

  17. Kinetic study of cytochrome P450 3A4 activity on warfarin by capillary electrophoresis with fluorescence detection.

    PubMed

    Zhang, Jie; Ha, Pham Thi Thanh; Lou, Yijia; Hoogmartens, Jos a; Van Schepdael, Ann

    2005-08-05

    The use of capillary electrophoresis (CE) for the determination of cytochrome P450 3A4 (CYP3A4) activity with R-warfarin as a substrate was investigated. CYP3A4 activity was determined by the quantitation of the product, 10-hydroxywarfarin, based on separation by CE. The separation conditions were as follows: capillary, 80.5 cm (75 microm i.d., 60 cm effective length); 50 mM sodium phosphate buffer (pH 6.5); 23 kV (90 microA) applied voltage; fluorescence detection, excitation wavelength, 310 nm, emission wavelength, 418 nm; capillary temperature, 37 degrees C. With the developed CYP3A4 activity assay and the Lineweaver-Burk equation, the Michaelis-Menten parameters Km and Vmax for formation of 10-hydroxywarfarin from R-warfarin in the presence of CYP3A4 were calculated to be 166 +/- 12 microM and 713 +/- 14 pmol/min/nmol (or 91.4 pmol/min/mg) CYP3A4, respectively.

  18. Tumour suppressor protein p53 regulates the stress activated bilirubin oxidase cytochrome P450 2A6

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hu, Hao, E-mail: hao.hu1@uqconnect.edu.au; Yu, Ting, E-mail: t.yu2@uq.edu.au; Arpiainen, Satu, E-mail: Satu.Juhila@orion.fi

    2015-11-15

    Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular defence against chemical-induced oxidative stress. The encoding gene is regulated by various stress activated transcription factors. This paper demonstrates that p53 is a novel transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six putative p53 binding sites in a 3 kb proximate promoter region. The site closest to transcription start site (TSS) is highly homologous with the p53 consensus sequence. Transfection with various stepwise deletions of CYP2A6-5′-Luc constructs – down to − 160 bp from the TSS – showed p53 responsivenessmore » in p53 overexpressed C3A cells. However, a further deletion from − 160 to − 74 bp, including the putative p53 binding site, totally abolished the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the putative binding site showed specific binding of p53. A point mutation at the binding site abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation of the endogenous p53 with benzo[α]pyrene – a well-known p53 activator – increased the expression of the p53 responsive positive control and the CYP2A6-5′-Luc construct containing the intact p53 binding site but not the mutated CYP2A6-5′-Luc construct. Finally, inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6 gene in C3A cells and further support the putative cytoprotective role of CYP2A6. - Highlights: • CYP2A6 is an immediate target gene of p53. • Six putative p53REs located on 3 kb proximate CYP2A6 promoter region. • The region − 160 bp from TSS is highly homologous with the p53 consensus sequence. • P53 specifically bind to the p53RE on the − 160 bp region.

  19. Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pechurskaya, Tatiana A.; Harnastai, Ivan N.; Grabovec, Irina P.

    2007-02-16

    The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major rolemore » in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human.« less

  20. In vitro activity of commercial valerian root extracts against human cytochrome P450 3A4.

    PubMed

    Lefebvre, Tania; Foster, Brian C; Drouin, Cathy E; Krantis, Anthony; Livesey, John F; Jordan, Scott A

    2004-08-12

    Valerian root ( Valeriana officinalis L.) has been used since antiquity as a medicinal herb. Recent studies have found that certain herbal products used concomitantly with conventional therapeutic products can markedly affect drug disposition. The in vitro effect of aliquots from 14 commercially available single-entity and blended products containing valerian root on cytochrome P450 CYP3A4-mediated metabolism and P-glycoprotein transport has been determined with aqueous, ethanol and acetonitrile extracts. Hydroxyvalerenic acid, acetoxyvalerenic acid and valerenic acid content was analyzed and wide variation was found between samples and compared to the concentrations noted on the product labels. Valerian extracts from the products tested also exhibited a marked capacity to inhibit cytochrome P450 3A4-mediated metabolism and P-glycoprotein transport based upon the ATPase assay. There is wide variation between commercially available samples of valerian root. The findings from this study suggest that valerian root may have an initial inhibitory effect when taken with therapeutic products. Further work is warranted to determine whether valerian root can affect other CYP450 isozymes and how the results of this in vitro investigation can be extrapolated to in vivo situations.

  1. Seasonal changes in the activity of cytochrome P450(C17) from the testis of Bufo arenarum.

    PubMed

    Solari, J J F; Pozzi, A G; Ceballos, N R

    2002-12-01

    In Bufo arenarum, the biosynthesis of testosterone and 5alpha-dihydrotestosterone takes place through a complete 5-ene pathway, 5-androsten-3beta,17beta-diol being the immediate precursor of testosterone. Besides androgens, testes are able to synthesise 5alpha-pregnan-3,20-dione and several 3alpha and 20alpha reduced derivatives. During the breeding season, steroid biosynthesis turns from androgen to C21-steroid production. As a consequence, the cytochrome P450 17-hydroxylase, C17,20-lyase (CypP450(c17)) could be a key enzyme in that metabolic shift. The present study demonstrates that in testes of B. arenarum, CypP450(c17) co-localises with glucose-6-phosphatase in the microsomal fraction. CypP450(c17) possesses more affinity for pregnenolone than for progesterone in both non-reproductive (Km = 43.76 +/- 4.63 nM and 2,170 +/- 630 nM, respectively) and reproductive (Km = 37.46 +/- 4.19 nM and 3,060 +/- 190 nM, respectively) seasons. These results could explain the predominance of the 5-ene pathway for testosterone biosynthesis. Toad CypP450(c17) activity is higher in the non-reproductive period than the reproductive period, suggesting that this enzyme is an important factor in toad steroidogenic changes. Animals in reproductive conditions showed a significant reduction in circulating androgens. This is in agreement with the decrease in Vmax of cytochrome P450 17-hydroxylase activity, enhancing the physiological relevance of these in vitro results.

  2. Plant Expression of a Bacterial Cytochrome P450 That Catalyzes Activation of a Sulfonylurea Pro-Herbicide.

    PubMed Central

    O'Keefe, D. P.; Tepperman, J. M.; Dean, C.; Leto, K. J.; Erbes, D. L.; Odell, J. T.

    1994-01-01

    The Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450SU1 (CYP105A1) was expressed in transgenic tobacco (Nicotiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450SU1 antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450SU1-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-1, 2-benzoisothiazole- 7-sulfonamide-1,1-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450SU1 are susceptible to injury from R7402 treatment that is harmless to plants without P450SU1. Thus, P450SU1 expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450SU1 from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450SU1 from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent. PMID:12232216

  3. Pungent ginger components modulates human cytochrome P450 enzymes in vitro

    PubMed Central

    Li, Mian; Chen, Pei-zhan; Yue, Qing-xi; Li, Jing-quan; Chu, Rui-ai; Zhang, Wei; Wang, Hui

    2013-01-01

    Aim: Ginger rhizome is used worldwide as a spicy flavor agent. This study was designed to explore the potential effects of pungent ginger components, 6-, 8-, and 10-gingerol, on human cytochrome P450 (CYP450) enzymes that are responsible for the metabolism of many prescription drugs. Methods: The activities of human CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were analyzed using Vivid P450 assay kits. The mRNA expression of CYP3A4 in human hepatocellular carcinoma cell line HepG2 was measured using quantitative real-time PCR assay. Results: All three gingerols potently inhibited CYP2C9 activity, exerted moderate inhibition on CYP2C19 and CYP3A4, and weak inhibion on CYP2D6. 8-Gingerol was the most potent in inhibition of P450 enzymes with IC50 values of 6.8, 12.5, 8.7, and 42.7 μmol/L for CYP2C9, CYP2C19, CYP3A4, and CYP2D6, respectively. By comparing the effects of gingerols on CYP3A4 with three different fluorescent substrate probes, it was demonstrated that the inhibition of gingerols on CYP3A4 had no substrate-dependence. In HepG2 cells, 8-gingerol and 10-gingerol inhibited, but 6-gingerol induced mRNA expression of CYP3A4. Conclusion: 6-, 8-, and 10-gingerol suppress human cytochrome P450 activity, while 8- and 10-gingerol inhibit CYP3A4 expression. The results may have an implication for the use of ginger or ginger products when combined with therapeutic drugs that are metabolized by cytochrome P450 enzymes. PMID:23770984

  4. Influence of Panax ginseng on Cytochrome P450 (CYP)3A and P-glycoprotein (Pgp) Activity in Healthy Subjects

    PubMed Central

    Malati, Christine Y.; Robertson, Sarah M.; Hunt, Jennifer D.; Chairez, Cheryl; Alfaro, Raul M.; Kovacs, Joseph A.; Penzak, Scott R.

    2012-01-01

    A number of herbal preparations have been shown to interact with prescription medications secondary to modulation of cytochrome P450 (CYP) and/or P-glycoprotein (P-gp). The purpose of this study was to determine the influence of Panax ginseng on CYP3A and P-gp function using the probe substrates midazolam and fexofenadine, respectively. Twelve healthy subjects (8 males) completed this open label, single sequence pharmacokinetic study. Healthy volunteers received single oral doses of midazolam 8 mg and fexofenadine 120 mg, before and after 28 days of P. ginseng 500 mg twice daily. Midazolam and fexofenadine pharmacokinetic parameter values were calculated and compared pre-and post P. ginseng administration. Geometric mean ratios (post-ginseng/pre-ginseng) for midazolam area under the concentration vs. time curve from zero to infinity (AUC0-∞), half life (T1/2), and maximum concentration (Cmax) were significantly reduced at 0.66 (0.55 – 0.78), 0.71 (0.53 – 0.90), and 0.74 (0.56 – 0.93), respectively. Conversely, fexofenadine pharmacokinetics were unaltered by P. ginseng administration. Based on these results, Panax ginseng appeared to induce CYP3A activity in the liver and possibly the gastrointestinal tract. Patients taking Panax ginseng in combination with CYP3A substrates with narrow therapeutic ranges should be monitored closely for adequate therapeutic response to the substrate medication. PMID:21646440

  5. Thermodynamics of camphor migration in cytochrome P450cam by atomistic simulations.

    PubMed

    Rydzewski, J; Nowak, W

    2017-08-10

    Understanding the mechanisms of ligand binding to enzymes is of paramount importance for the design of new drugs. Here, we report on the use of a novel biased molecular dynamics (MD) methodology to study the mechanism of camphor binding to cytochrome P450cam. Microsecond-long MD simulations allowed us to observe reaction coordinates characterizing ligand diffusion from the active site of cytochrome P450cam to solvent via three egress routes. These atomistic simulations were used to estimate thermodynamic quantities along the reaction coordinates and indicate diverse binding configurations. The results suggest that the diffusion of camphor along the pathway near the substrate recognition site (SRS) is thermodynamically preferred. In addition, we show that the diffusion near the SRS is triggered by a transition from a heterogeneous collection of closed ligand-bound conformers to the basin comprising the open conformations of cytochrome P450cam. The conformational change accompanying this switch is characterized by the retraction of the F and G helices and the disorder of the B' helix. These results are corroborated by experimental studies and provide detailed insight into ligand binding and conformational behavior of the cytochrome family. The presented methodology is general and can be applied to other ligand-protein systems.

  6. Cytochrome P450-2D6 Screening Among Elderly Using Antidepressants (CYSCE)

    ClinicalTrials.gov

    2017-08-15

    Depression; Depressive Disorder; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Intermediate Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Ultrarapid Metabolizer Due to Cytochrome P450 CYP2D6 Variant

  7. 5 CFR 4501.101 - General.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Administrative Personnel OFFICE OF PERSONNEL MANAGEMENT SUPPLEMENTAL STANDARDS OF ETHICAL CONDUCT FOR EMPLOYEES OF THE OFFICE OF PERSONNEL MANAGEMENT § 4501.101 General. In accordance with 5 CFR 2635.105, the regulations in this part apply to employees of the Office of Personnel Management (OPM) and supplement the...

  8. Inactivation of Cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a Thiophene-Containing Anticancer DrugS⃞

    PubMed Central

    Lin, Hsia-lien; Zhang, Haoming; Medower, Christine; Johnson, William W.

    2011-01-01

    An investigational anticancer agent that contains a thiophene moiety, 3-[(quinolin-4-ylmethyl)-amino]-N-[4-trifluoromethox)phenyl] thiophene-2-carboxamide (OSI-930), was tested to investigate its ability to modulate the activities of several cytochrome P450 enzymes. Results showed that OSI-930 inactivated purified, recombinant cytochrome P450 (P450) 3A4 in the reconstituted system in a mechanism-based manner. The inactivation was dependent on cytochrome b5 and required NADPH. Catalase did not protect against the inactivation. No inactivation was observed in studies with human 2B6, 2D6, or 3A5 either in the presence or in the absence of b5. The inactivation of 3A4 by OSI-930 was time- and concentration-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl)coumarin catalytic activity of 3A4 was characterized by a KI of 24 μM and a kinact of 0.04 min−1. This KI is significantly greater than the clinical OSI-930 Cmax of 1.7 μM at the maximum tolerated dose, indicating that clinical drug interactions of OSI-930 via this pathway are not likely. Spectral analysis of the inactivated protein indicated that the decrease in the reduced CO spectrum at 450 nm was comparable to the amount of inactivation, thereby suggesting that the inactivation was primarily due to modification of the heme. High-pressure liquid chromatography (HPLC) analysis with detection at 400 nm showed a loss of heme comparable to the activity loss, but a modified heme was not detected. This result suggests either that the heme must have been modified enough so as not to be observed in a HPLC chromatograph or, possibly, that it was destroyed. The partition ratio for the inactivation of P450 3A4 was approximately 23, suggesting that this P450 3A4-mediated pathway occurs with approximately 4% frequency during the metabolism of OSI-930. Modeling studies on the binding of OSI-930 to the active site of the P450 3A4 indicated that OSI-930 would be oriented properly in the active site for oxidation

  9. Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

    PubMed

    Hidaka, Muneaki; Fujita, Ken-ichi; Ogikubo, Tetsuya; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Arimori, Kazuhiko

    2004-06-01

    There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro.

  10. Defining the in Vivo Role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5.

    PubMed

    Finn, Robert D; McLaughlin, Lesley A; Ronseaux, Sebastien; Rosewell, Ian; Houston, J Brian; Henderson, Colin J; Wolf, C Roland

    2008-11-14

    In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50-90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner.

  11. Cytochrome P450 humanised mice

    PubMed Central

    2004-01-01

    Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s). These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach. PMID:15588489

  12. Cytochromes P450

    PubMed Central

    Bak, Søren; Beisson, Fred; Bishop, Gerard; Hamberger, Björn; Höfer, René; Paquette, Suzanne; Werck-Reichhart, Danièle

    2011-01-01

    There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

  13. Genetic Polymorphisms of Cytochrome P4501A1 (CYP1A1) and Glutathione S-Transferase P1 (GSTP1) and Risk of Hepatocellular Carcinoma Among Chronic Hepatitis C Patients in Egypt.

    PubMed

    Abo-Hashem, Ekbal M; El-Emshaty, Wafaa M; Farag, Raghda El Sayed; Zakaria, Sahar; Abd El-Aziz, Mohammed; Ghonaim, Azza

    2016-10-01

    Cytochrome P450 1A1 (CYP1A1) and Glutathione S-transferase P1 (GSTP1) genes are involved in the metabolism of many carcinogens. Polymorphisms in these genes with altered enzyme activity have been reported. The present study evaluated the synergistic effect between CYP1A1 and GSTP1 gene polymorphisms and smoking on development of HCV-related liver disease and hepatocellular carcinoma (HCC). The patients group comprised 40 patients with HCC and 40 patients with liver cirrhosis. The control group comprised 40 healthy subjects having no history of malignancy. The genetic polymorphisms were studied using polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) technique on blood samples. The number of current or former smoker among HCC and cirrhotic patients as well as the median Pack/year of cigarette smoked were significantly higher in HCC and liver cirrhotic patients than in control group. Subjects with CYP1A1 gene variants (m1 and m3) had no significant risk to develop cirrhosis or HCC compared to control group. Individuals carrying the Ile/Val genotype of GSTP1 had a significant increased risk of HCC (OR of 2.2, 95 % CI 1.143-4.261) and had larger tumor size. No significant risk was observed on combining both genes variants or on combining smoking with variants of both genes. In conclusion, the GSTP1 Ile/Val genotype and Val allele are associated with an increased risk of HCC. CYP1A1 and GSTP1 genes variants interaction did not increase the risk of HCC.

  14. Molecular modeling of cytochrome P450 3A4

    NASA Astrophysics Data System (ADS)

    Szklarz, Grazyna D.; Halpert, James R.

    1997-05-01

    The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B' helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6β-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.

  15. Cytochrome P450-inhibitory activity of parabens and phthalates used in consumer products.

    PubMed

    Ozaki, Hitomi; Sugihara, Kazumi; Watanabe, Yoko; Ohta, Shigeru; Kitamura, Shigeyuki

    2016-01-01

    The in vitro cytochrome P450 (CYP)-inhibitory effects of 11 parabens and 7 phthalates used in consumer products, as well as their hydrolytic metabolites, were investigated, using rat liver microsomes as an enzyme source. The effects on individual CYP isozymes were evaluated by assaying inhibition of activities towards specific substrates, i.e., ethoxyresorufin O-dealkylase (EROD), methoxyresorufin O-dealkylase (MROD), pentoxyresorufin O-dealkylase (PROD), 7-benzyloxy-4-trifluoromethylcoumarin dealkylase (BFCD), 7-methoxy-4-trifluoromethylcoumarin dealkylase (MFCD) and 7-ethoxy-4-trifluoromethylcoumarin dealkylase (EFCD) activities. These activities were dose-dependently inhibited, most potently by medium-side-chain parabens (C6-9) and phthalates (C4-6), and less potently by shorter- and longer-side-chain esters. The hydrolytic product of parabens, 4-hydroxybenzoic acid, was not inhibitory, while those of phthalates, phthalic acid monoesters, showed lower inhibitory activity than the parent phthalates. Parabens showed relatively potent inhibition of MFCD activity, considered to be mainly due to CYP2C, and phthalates showed relatively potent inhibition of PROD activity, considered to be mainly due to CYP2B.

  16. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication.

    PubMed

    Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2015-10-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Limitations of in silico predictability of specificity of co-immobilised cytochromes P450 and mimics in food-bioprocessing.

    PubMed

    Wiseman, Alan

    2003-04-01

    Cytochromes P450 (EC 1.14.14.1) are mixed function oxidases (oxygenases) that can catalyse redox bioconversions of food components. Also, efficacious removal of undesirable components can be achieved using solid-support immobilised enzyme (IME) of a selection from 2700 isoforms of cytochromes P450 (CYP). Cytochromes P450 co-immobilised with other enzymes, or protein receptors, may be used to confer a secondary order of regio- or stereo-specificity of chiral bioconversion: these can be predictable in silico by utilisation of QSARs (quantitative structure/activity relationships).

  18. Cytochrome P450 Initiates Degradation of cis-Dichloroethene by Polaromonas sp. Strain JS666

    PubMed Central

    Nishino, Shirley F.; Shin, Kwanghee A.; Gossett, James M.

    2013-01-01

    Polaromonas sp. strain JS666 grows on cis-1,2-dichoroethene (cDCE) as the sole carbon and energy source under aerobic conditions, but the degradation mechanism and the enzymes involved are unknown. In this study, we established the complete pathway for cDCE degradation through heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. Several lines of evidence indicate that a cytochrome P450 monooxygenase catalyzes the initial step of cDCE degradation. Both the transient accumulation of dichloroacetaldehyde in cDCE-degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 support a pathway for degradation of cDCE through dichloroacetaldehyde. The mechanism minimizes the formation of cDCE epoxide. The molecular phylogeny of the cytochrome P450 gene and the organization of neighboring genes suggest that the cDCE degradation pathway recently evolved in a progenitor capable of degrading 1,2-dichloroethane either by the recruitment of the cytochrome P450 monooxygenase gene from an alkane catabolic pathway or by selection for variants of the P450 in a preexisting 1,2-dichloroethane catabolic pathway. The results presented here add yet another role to the broad array of productive reactions catalyzed by cytochrome P450 enzymes. PMID:23354711

  19. Cytochrome c-553 is not required for photosynthetic activity in the cyanobacterium Synechococcus.

    PubMed Central

    Laudenbach, D E; Herbert, S K; McDowell, C; Fork, D C; Grossman, A R; Straus, N A

    1990-01-01

    In cyanobacteria, the water-soluble cytochrome c-553 functions as a mobile carrier of electrons between the membrane-bound cytochrome b6-f complex and P-700 reaction centers of Photosystem I. The structural gene for cytochrome c-553 (designated cytA) of the cyanobacterium Synechococcus sp. PCC 7942 was cloned, and the deduced amino acid sequence was shown to be similar to known cyanobacterial cytochrome c-553 proteins. A deletion mutant was constructed that had no detectable cytochrome c-553 based on spectral analyses and tetramethylbenzidine-hydrogen peroxide staining of proteins resolved by polyacrylamide gel electrophoresis. The mutant strain was not impaired in overall photosynthetic activity. However, this mutant exhibited a decreased efficiency of cytochrome f oxidation. These results indicate that cytochrome c-553 is not an absolute requirement for reducing Photosystem I reaction centers in Synechococcus sp. PCC 7942. PMID:1967057

  20. NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

    PubMed Central

    Ding, Xinxin; Wolf, C. Roland; Porter, Todd D.; Pandey, Amit V.; Zhang, Qing-Yu; Gu, Jun; Finn, Robert D.; Ronseaux, Sebastien; McLaughlin, Lesley A.; Henderson, Colin J.; Zou, Ling; Flück, Christa E.

    2013-01-01

    This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

  1. Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.

    PubMed Central

    Zahn, J A; Duncan, C; DiSpirito, A A

    1994-01-01

    An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline. Images PMID:7928947

  2. Cytochrome P450 monooxygenases: perspectives for synthetic application.

    PubMed

    Urlacher, Vlada B; Eiben, Sabine

    2006-07-01

    Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.

  3. In vitro effect of important herbal active constituents on human cytochrome P450 1A2 (CYP1A2) activity.

    PubMed

    Pan, Yan; Tiong, Kai Hung; Abd-Rashid, Badrul Amini; Ismail, Zakiah; Ismail, Rusli; Mak, Joon Wah; Ong, Chin Eng

    2014-10-15

    This study was designed to investigate eight herbal active constituents (andrographolide, asiaticoside, asiatic acid, madecassic acid, eupatorin, sinensetin, caffeic acid, and rosmarinic acid) on their potential inhibitory effects on human cytochrome P450 1A2 (CYP1A2) activity. A fluorescence-based enzyme assay was performed by co-incubating human cDNA-expressed CYP1A2 with its selective probe substrate, 3-cyano-7-ethoxycoumarin (CEC), in the absence or presence of various concentrations of herbal active constituents. The metabolite (cyano-hydroxycoumarin) formed was subsequently measured in order to obtain IC50 values. The results indicated that only eupatorin and sinensetin moderately inhibited CYP1A2 with IC50 values of 50.8 and 40.2 μM, while the other active compounds did not significantly affect CYP1A2 activity with IC50 values more than 100 μM. Ki values further determined for eupatorin and sinensetin were 46.4 and 35.2 μM, respectively. Our data indicated that most of the investigated herbal constituents have negligible CYP1A2 inhibitory effect. In vivo studies however may be warranted to ascertain the inhibitory effect of eupatorin and sinensetin on CYP1A2 activity in clinical situations. Copyright © 2014 Elsevier GmbH. All rights reserved.

  4. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Iyanagi, Takashi

    2005-12-09

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form canmore » function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.« less

  5. Fungal lactone ring opening of 6', 7'-dihydroxybergamottin diminishes cytochrome P450 3A4 inhibitory activity

    USDA-ARS?s Scientific Manuscript database

    Furanocoumarins (FCs) are a class of aromatic compounds in grapefruit that inhibit human intestinal cytochrome P450 3A4 (CYP3A4). Since fungi metabolize polycyclic aromatic hydrocarbons, we hypothesized that certain fungi might also metabolize FCs into forms that may be inactive as CYP3A4 inhibitors...

  6. Oleamide synthesizing activity from rat kidney: identification as cytochrome c.

    PubMed

    Driscoll, William J; Chaturvedi, Shalini; Mueller, Gregory P

    2007-08-03

    Oleamide (cis-9-octadecenamide) is the prototype member of an emerging class of lipid signaling molecules collectively known as the primary fatty acid amides. Current evidence suggests that oleamide participates in the biochemical mechanisms underlying the drive to sleep, thermoregulation, and antinociception. Despite the potential importance of oleamide in these physiologic processes, the biochemical pathway for its synthesis in vivo has not been established. We report here the discovery of an oleamide synthetase found in rat tissues using [(14)C]oleoyl-CoA and ammonium ion. Hydrogen peroxide was subsequently found to be a required cofactor. The enzyme displayed temperature and pH optima in the physiologic range, a remarkable resistance to proteolysis, and specificity for long-chain acyl-CoA substrates. The reaction demonstrated Michaelis-Menten kinetics with a K(m) for oleoyl-CoA of 21 microm. Proteomic, biochemical, and immunologic analyses were used to identify the source of the oleamide synthesizing activity as cytochrome c. This identification was based upon peptide mass fingerprinting of isolated synthase protein, a tight correlation between enzymatic activity and immunoreactivity for cytochrome c, and identical functional properties shared by the tissue-derived synthetase and commercially obtained cytochrome c. The ability of cytochrome c to catalyze the formation of oleamide experimentally raises the possibility that cytochrome c may mediate oleamide biosynthesis in vivo.

  7. Selective aliphatic carbon-hydrogen bond activation of protected alcohol substrates by cytochrome P450 enzymes.

    PubMed

    Bell, Stephen G; Spence, Justin T J; Liu, Shenglan; George, Jonathan H; Wong, Luet-Lok

    2014-04-21

    Protected cyclohexanol and cyclohex-2-enol substrates, containing benzyl ether and benzoate ester moieties, were designed to fit into the active site of the Tyr96Ala mutant of cytochrome P450cam. The protected cyclohexanol substrates were efficiently and selectively hydroxylated by the mutant enzyme at the trans C-H bond of C-4 on the cyclohexyl ring. The selectivity of oxidation of the benzoate ester protected cyclohexanol could be altered by making alternative amino acid substitutions in the P450cam active site. The addition of the double bond in the cyclohexyl ring of the benzoate ester protected cyclohex-2-enol has a debilitative effect on the activity of the Tyr96Ala mutant with this substrate. However, the Phe87Ala/Tyr96Phe double mutant, which introduces space at a different location in the active site than the Tyr96Ala mutant, was able to efficiently hydroxylate the C-H bonds of 1-cyclohex-2-enyl benzoate at the allylic C-4 position. Mutations at Phe87 improved the selectivity of the oxidation of 1-phenyl-1-cyclohexylethylene to trans-4-phenyl-ethenylcyclohexanol (92%) when compared to single mutants at Tyr96 of P450cam.

  8. Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition.

    PubMed

    Ishihara, Yasuhiro; Shiba, Dai; Shimamoto, Norio

    2006-07-15

    Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as alpha-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.

  9. ISOLATION OF A CYTOCHROME P-450 STRUCTURAL GENE FROM SACCHAROMYCES CEREVISIAE

    EPA Science Inventory

    We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14-demethyl...

  10. A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants.

    PubMed

    Rong Tan, Li; Chen Lu, Yi; Jing Zhang, Jing; Luo, Fang; Yang, Hong

    2015-09-01

    Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. The Effects of Milk Thistle (Silybum marianum) on Human Cytochrome P450 Activity

    PubMed Central

    Kawaguchi-Suzuki, Marina; Frye, Reginald F.; Zhu, Hao-Jie; Brinda, Bryan J.; Chavin, Kenneth D.; Bernstein, Hilary J.

    2014-01-01

    Milk thistle (Silybum marianum) extracts are widely used as a complementary and alternative treatment of various hepatic conditions and a host of other diseases/disorders. The active constituents of milk thistle supplements are believed to be the flavonolignans contained within the extracts. In vitro studies have suggested that some milk thistle components may significantly inhibit specific cytochrome P450 (P450) enzymes. However, determining the potential for clinically significant drug interactions with milk thistle products has been complicated by inconsistencies between in vitro and in vivo study results. The aim of the present study was to determine the effect of a standardized milk thistle supplement on major P450 drug-metabolizing enzymes after a 14-day exposure period. CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5 activities were measured by simultaneously administering the four probe drugs, caffeine, tolbutamide, dextromethorphan, and midazolam, to nine healthy volunteers before and after exposure to a standardized milk thistle extract given thrice daily for 14 days. The three most abundant falvonolignans found in plasma, following exposure to milk thistle extracts, were silybin A, silybin B, and isosilybin B. The concentrations of these three major constituents were individually measured in study subjects as potential perpetrators. The peak concentrations and areas under the time-concentration curves of the four probe drugs were determined with the milk thistle administration. Exposure to milk thistle extract produced no significant influence on CYP1A2, CYP2C9, CYP2D6, or CYP3A4/5 activities. PMID:25028567

  12. Cytochrome P450 3A expression and activity in the rabbit lacrimal gland: glucocorticoid modulation and the impact on androgen metabolism.

    PubMed

    Attar, Mayssa; Ling, Kah-Hiing John; Tang-Liu, Diane D-S; Neamati, Nouri; Lee, Vincent H L

    2005-12-01

    Cytochrome P450 3A (CYP3A) is an enzyme of paramount importance to drug metabolism. The expression and activity of CYP3A, an enzyme responsible for active androgen clearance, was investigated in the rabbit lacrimal gland. Analysis of CYP3A expression and activity was performed on lacrimal gland tissues obtained from naïve untreated and treated New Zealand White rabbits. For 5 days, treated rabbits received daily administration of vehicle or 0.1% or 1.0% dexamethasone, in the lower cul-de-sac of each eye. Changes in mRNA expression were monitored by real-time RT-PCR. Protein expression was confirmed by Western blot. Functional activity was measured by monitoring the metabolism of CYP3A probe substrates-namely, 7-benzyloxyquinoline (BQ) and [3H]testosterone. Cytochrome P450 heme protein was detected at a concentration of 44.6 picomoles/mg protein, along with its redox partner NADPH reductase and specifically CYP3A6 in the naïve rabbit lacrimal gland. Genes encoding CYP3A6, in addition to the pregnane-X-receptor (PXR) and P-glycoprotein (P-gp) were expressed in the untreated tissue. BQ dealkylation was measured in the naïve rabbit lacrimal gland at a rate of 14 +/- 7 picomoles/mg protein per minute. Changes in CYP3A6, P-gp, and androgen receptor mRNA expression levels were detected after dexamethasone treatment. In addition, dexamethasone treatment resulted in significant increases in BQ dealkylation and CYP3A6-mediated [3H]testosterone metabolism. Concomitant increases in CYP3A6-mediated hydroxylated testosterone metabolites were observed in the treated rabbits. Furthermore, ketoconazole, all-trans retinoic acid, and cyclosporine inhibited CYP3A6 mediated [3H]testosterone 6beta hydroxylation in a concentration-dependent manner, with IC50 ranging from 3.73 to 435 microM. The results demonstrate, for the first time, the expression and activity of CYP3A6 in the rabbit lacrimal gland. In addition, this pathway was shown to be subject to modulation by a commonly

  13. Studying the Inhibitory Effect of Quercetin and Thymoquinone on Human Cytochrome P450 Enzyme Activities.

    PubMed

    Elbarbry, Fawzy; Ung, Aimy; Abdelkawy, Khaled

    2018-01-01

    Quercetin (QR) and thymoquinone (TQ) are herbal remedies that are currently extensively used by the general population to prevent and treat various chronic conditions. Therefore, investigating the potential of pharmacokinetic interactions caused by the concomitant use of these herbal remedies and conventional medicine is warranted to ensure patient safety. This study was conducted to determine the inhibitory effect of QR and TQ, two commonly used remedies, on the activities of selected cytochrome P450 (CYP) enzymes that play an important role in drug metabolism and/or toxicology. The in vitro studies were conducted using fluorescence-based high throughput assays using human c-DNA baculovirus expressed CYP enzymes. For measuring CYP2E1 activity, a validated High-performance liquid chromatography (HPLC) assay was utilized to measure the formation of 6-hydroxychlorzoxazone. The obtained half-maximum inhibitory concentration values with known positive control inhibitors of this study were comparable to the published values indicating accurate experimental techniques. Although QR did not show any significant effect on CYP1A2 and CYP2E1, it exhibited a strong inhibitory effect against CYP2D6 and a moderate effect against CYP2C19 and CYP3A4. On the other hand, TQ demonstrated a strong and a moderate inhibitory effect against CYP3A4 and CYP2C19, respectively. The findings of this study may indicate that consumption of QR or TQ, in the form of food or dietary supplements, with drugs that are metabolized by CYP2C19, CYP2D6, or CYP3A4 may cause significant herb-drug interactions. Neither QR nor TQ has any significant inhibitory effect on the activity of CYP1A2 or CYP2E1 enzymesBoth QR and TQ have a moderate to strong inhibitory effect on CYP3A4 activityQR has a moderate inhibitory effect on CYP2C19 and a strong inhibitory effect on CYP2D6Both QR and TQ are moderate inhibitors of the CYP2C9 activity. Abbreviations used: ABT: Aminobenztriazole, BZF: 7,8 Benzoflavone, CYP

  14. The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Abdulla, Dalya; Goralski, Kerry B.; College of Pharmacy, Burbidge Building, Dalhousie University, Halifax, Nova Scotia, B3H 3J5

    2006-10-01

    It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo,more » an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 {mu}g of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.« less

  15. Comparison of basal and induced cytochromes P450 in 6 species of waterfowl

    USGS Publications Warehouse

    Melancon, M.J.; Rattner, B.A.; Hoffman, D.J.; Beeman, D.; Day, D.; Custer, T.

    1999-01-01

    Cytochrome P450-associated monooxygenase activities were measured in control and prototype inducer-treated mallard duck, black duck, wood duck, lesser scaup, Canada goose and mute swan. Ages of the birds ranged from pipping embryos (that were treated approximately 3 days before pipping) to adults. Three or more of the following hepatic microsomal monooxygenases were assayed in each species: Benzyloxyresorufin-O-dealkylase (BROD), Ethoxyresorufin-O-dealkylase (EROD), methoxyresorufin-O-dealkylase (MROD), and pentoxyresorufin-O-dealkylase (PROD). Baseline activities differed between species, but because of differences in ages, sources of the eggs or birds, and diets, these cannot be viewed as absolute differences. The cytochrome P450 inducers utilized were beta-naphthoflavone (BNF), 3-methylcholanthrene (3MC) and phenobarbital (PB). In general, there was little response to PB; only lesser scaup were induced to greater than three times control level and most species were well under this. Responses to BNF and 3MC occurred in each species studied, but differed in which of the monooxygenases was most induced (absolute values and ratios to control values) and in relative induction between species. BROD frequently had an induction ratio EROD. Overall, lesser scaup were the most responsive, canada geese the least responsive, and the other species intermediate in responsiveness to the cytochrome P450 inducers studied.

  16. Gender and Gonadal Status Differences in Zona Reticularis Expression in Marmoset Monkey Adrenals: Cytochrome b5 Localization With Respect To Cytochrome P450 17,20-Lyase Activity.

    PubMed Central

    Pattison, J Christina; Saltzman, Wendy; Abbott, David H; Hogan, Brynn K; Nguyen, Ann D; Husen, Bettina; Einspanier, Almuth; Conley, Alan J; Bird., Ian M

    2007-01-01

    Neonatal marmosets express an adrenal fetal zone comparable to humans. While adult males fail to express a functional ZR, with barely detectable blood DHEA levels, females produce higher levels of DHEA than males in adulthood. We investigated the presence of a putative functional ZR in adult female marmosets. In contrast to males, immunohistochemical analysis showed the ZR marker cytochrome b5 was elevated in the innermost zone in cycling females (compared to testis-intact males), further elevated in the adrenals from anovulatory females, and substantially elevated and continuous in ovariectomized females. As a functional test in vivo, following overnight dexamethasone treatment, cycling and anovulatory females showed higher levels of DHEA relative to males, but DHEA failed to increase in response to ACTH. In direct contrast, while ovariectomized females exhibited lower initial DHEA levels, clear increases were detectable after ACTH administration (p<0.05), suggesting an adrenal origin. The apparent differences in cytochrome b5 expression between groups were also further verified by western blotting of adrenal microsomes, and compared to 17,20-lyase activity; the two parameters were positively correlated (p<0.01) across multiple treatment groups. We conclude that the cycling female marmoset expresses a rudimentary ZR with at least a capacity for DHEA production that becomes significantly ACTH-responsive after anovulation. Expression of cytochrome b5 in this region may be directly or indirectly controlled by gonadal function, and is, at least in part, a critical determinant in the development of an adrenal ZR that is more defined and significantly ACTH-responsive. PMID:17222503

  17. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    PubMed Central

    Miao, Yinglong; Baudry, Jerome

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site. PMID:21943431

  18. Peroxisome Proliferator-Activated Receptor α Activation Suppresses Cytochrome P450 Induction Potential in Mice Treated with Gemfibrozil.

    PubMed

    Shi, Cunzhong; Min, Luo; Yang, Julin; Dai, Manyun; Song, Danjun; Hua, Huiying; Xu, Gangming; Gonzalez, Frank J; Liu, Aiming

    2017-09-01

    Gemfibrozil, a peroxisome proliferator-activated receptor α (PPARα) agonist, is widely used for hypertriglyceridaemia and mixed hyperlipidaemia. Drug-drug interaction of gemfibrozil and other PPARα agonists has been reported. However, the role of PPARα in cytochrome P450 (CYP) induction by fibrates is not well known. In this study, wild-type mice were first fed gemfibrozil-containing diets (0.375%, 0.75% and 1.5%) for 14 days to establish a dose-response relationship for CYP induction. Then, wild-type mice and Pparα-null mice were treated with a 0.75% gemfibrozil-containing diet for 7 days. CYP3a, CYP2b and CYP2c were induced in a dose-dependent manner by gemfibrozil. In Pparα-null mice, their mRNA level, protein level and activity were induced more than those in wild-type mice. So, gemfibrozil induced CYP, and this action was inhibited by activated PPARα. These data suggested that the induction potential of CYPs was suppressed by activated PPARα, showing a potential role of this receptor in drug-drug interactions and metabolic diseases treated with fibrates. © 2017 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  19. Cytochrome P450 peroxidase/peroxygenase mediated xenobiotic metabolic activation and cytotoxicity in isolated hepatocytes.

    PubMed

    Anari, M R; Khan, S; Liu, Z C; O'Brien, P J

    1995-12-01

    Cytochrome P450 (P450) can utilize organic hydroperoxides and peracids to support hydroxylation and dealkylation of various P450 substrates. However, the biological significance of this P450 peroxygenase/peroxidase activity in the bioactivation of xenobiotics in intact cells has not been demonstrated. We have shown that tert-butyl hydroperoxide (tBHP) markedly enhances 3-20-fold the cytotoxicity of various aromatic hydrocarbons and their phenolic metabolites. The tBHP-enhanced hepatocyte cytotoxicity of 4-nitroanisole (4-NA) and 4-hydroxyanisole (4-HA) was also accompanied by an increase in the hepatocyte O-demethylation of 4-NA and 4-HA up to 7.5- and 21-fold, respectively. Hepatocyte GSH conjugation by 4-HA was also markedly increased by tBHP. An LC/MS analysis of the GSH conjugates identified hydroquinone-GSH and 4-methoxy-catechol:GSH conjugates as the predominant adducts. Pretreatment of hepatocytes with P450 inhibitors, e.g., phenylimidazole, prevented tBHP-enhanced 4-HA metabolism, GSH depletion, and cytotoxicity. In conclusion, hydroperoxides can therefore be used by intact cells to support the bioactivation of xenobiotics through the P450 peroxidase/peroxygenase system.

  20. Active sites of the cytochrome p450cam (CYP101) F87W and F87A mutants. Evidence for significant structural reorganization without alteration of catalytic regiospecificity.

    PubMed

    Tuck, S F; Graham-Lorence, S; Peterson, J A; Ortiz de Montellano, P R

    1993-01-05

    Ferricyanide oxidation of the aryl-iron complexes formed by the reaction of cytochrome P450 enzymes with arylhydrazines causes in situ migration of the aryl group from the iron to the porphyrin nitrogen atoms. The regiochemistry of this migration, defined by the ratio of the four possible N-arylprotoporphyrin IX isomers, provides a method for mapping the topologies of cytochrome P450 active sites. The method has been validated by using it to examine the active site of cytochrome P450cam (CYP101), for which a crystal structure is available. In agreement with the crystal structure, reaction with phenylhydrazine gives a 5:25:70 ratio of the NA:NC:ND (subscript indicates pyrrole ring) N-phenylprotoporphyrin IX isomers. Naphthylhydrazine, however, yields exclusively the NC regioisomer and 4-(phenyl)phenylhydrazine the NA:NC:ND isomers in a 14:40:46 ratio. These isomer ratio differences are readily explained by topological differences between the upper and lower reaches of the active site. Having validated the aryl-iron shift as a topological probe, we used it to investigate the structural changes caused by mutation of Phe-87, a residue that provides the ceiling over pyrrole ring D in the crystal structure of cytochrome P450cam. Mutation of Phe-87 to a tryptophan causes no detectable change in the regiochemistry of camphor hydroxylation and only minor changes in the N-aryl isomer ratios. However, mutation of Phe-87 to an alanine, which was expected to open up the region above pyrrole ring D, severely decreased the proportion of the ND in favor of the NA isomer. Less rather than more space is therefore available over pyrrole ring D in the F87A mutant despite the fact that the regiochemistry of camphor hydroxylation remains unchanged. These results provide evidence for significant structural reorganization in the upper regions of the substrate binding site without alteration of the camphor hydroxylation regiospecificity in the F87A mutant.

  1. Fluorescence labelling of NADPH-cytochrome P-450 reductase with the monobromomethyl derivative of syn-9,10-dioxabimane.

    PubMed Central

    Vogel, F; Lumper, L

    1983-01-01

    The kinetics of thiol-group alkylation in NADPH-cytochrome P-450 reductase during its inactivation by monobromobimane has been studied using the fluorimetric determination of S-bimane-L-cysteine by high-performance liquid chromatography. Loss of activity during the reaction of NADPH-cytochrome P-450 reductase with monobromobimane is caused by the alkylation of one single critical cysteine residue, which can be protected against thiol-specific reagents by NADP(H). The chemical stability of the bimane group allows the digestion of bimane-labelled NADPH-cytochrome P-450 reductase by CNBr. The critical cysteine residue could be located in a CNBr-cleaved peptide purified to homogeneity with Mr 10 500 +/- 1 000 and valine as N-terminus. Images Fig. 2. PMID:6414464

  2. Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling.

    PubMed

    Arlt, Volker M; Poirier, Miriam C; Sykes, Sarah E; John, Kaarthik; Moserova, Michaela; Stiborova, Marie; Wolf, C Roland; Henderson, Colin J; Phillips, David H

    2012-09-03

    Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  3. Active sites of two orthologous cytochromes P450 2E1: Differences revealed by spectroscopic methods

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anzenbacherova, Eva; Hudecek, Jiri; Murgida, Daniel

    2005-12-09

    Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry.more » In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics.« less

  4. The effects of milk thistle (Silybum marianum) on human cytochrome P450 activity.

    PubMed

    Kawaguchi-Suzuki, Marina; Frye, Reginald F; Zhu, Hao-Jie; Brinda, Bryan J; Chavin, Kenneth D; Bernstein, Hilary J; Markowitz, John S

    2014-10-01

    Milk thistle (Silybum marianum) extracts are widely used as a complementary and alternative treatment of various hepatic conditions and a host of other diseases/disorders. The active constituents of milk thistle supplements are believed to be the flavonolignans contained within the extracts. In vitro studies have suggested that some milk thistle components may significantly inhibit specific cytochrome P450 (P450) enzymes. However, determining the potential for clinically significant drug interactions with milk thistle products has been complicated by inconsistencies between in vitro and in vivo study results. The aim of the present study was to determine the effect of a standardized milk thistle supplement on major P450 drug-metabolizing enzymes after a 14-day exposure period. CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5 activities were measured by simultaneously administering the four probe drugs, caffeine, tolbutamide, dextromethorphan, and midazolam, to nine healthy volunteers before and after exposure to a standardized milk thistle extract given thrice daily for 14 days. The three most abundant falvonolignans found in plasma, following exposure to milk thistle extracts, were silybin A, silybin B, and isosilybin B. The concentrations of these three major constituents were individually measured in study subjects as potential perpetrators. The peak concentrations and areas under the time-concentration curves of the four probe drugs were determined with the milk thistle administration. Exposure to milk thistle extract produced no significant influence on CYP1A2, CYP2C9, CYP2D6, or CYP3A4/5 activities. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  5. Modeling of Anopheles minimus Mosquito NADPH-Cytochrome P450 Oxidoreductase (CYPOR) and Mutagenesis Analysis

    PubMed Central

    Sarapusit, Songklod; Lertkiatmongkol, Panida; Duangkaew, Panida; Rongnoparut, Pornpimol

    2013-01-01

    Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two-to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs. PMID:23325047

  6. Scutellarin inhibits cytochrome P450 isoenzyme 1A2 (CYP1A2) in rats.

    PubMed

    Jian, Tun-Yu; He, Jian-Chang; He, Gong-Hao; Feng, En-Fu; Li, Hong-Liang; Bai, Min; Xu, Gui-Li

    2012-08-01

    Scutellarin is the most important flavone glycoside in the herbal drug Erigeron breviscapus (Vant.) Hand.-Mazz. It is used frequently in the clinic to treat ischemic vascular diseases in China. However, the direct relationship between scutellarin and cytochrome P450 (CYP450) is unclear. The present study investigated the in vitro and in vivo effects of scutellarin on cytochrome P450 1A2 (CYP 1A2) metabolism. According to in vitro experiments, scutellarin (10-250 µM) decreased the formation of 4-acetamidophenol in a concentration-dependent manner, with an IC₅₀ value of 108.20 ± 0.657 µM. Furthermore, scutellarin exhibited a weak mixed-type inhibition against the activity of CYP1A2 in rat liver microsomes, with a K(i) value of 95.2 µM. Whereas in whole animal studies, scutellarin treatment for 7 days (at 5, 15, 30 mg/kg, i.p.) decreased the clearance (CL), and increased the T(1/2) (at 15, 30 mg/kg, i.p.), it did not affect the V(d) of phenacetin. Scutellarin treatment (at 5, 15, 30 mg/kg, i.p.) increased the AUC(0-∞) by 14.3%, 67.3% and 159.2%, respectively. Scutellarin at 30 mg/kg also weakly inhibited CYP1A2 activity, in accordance with our in vitro study. Thus, the results indicate that CYP1A2 is inhibited directly, but weakly, by scutellarin in vivo, and provide useful information on the safe and effective use of scutellarin in clinical practice. Copyright © 2012 John Wiley & Sons, Ltd.

  7. Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

    PubMed Central

    Pallan, Pradeep S.; Wang, Chunxue; Lei, Li; Yoshimoto, Francis K.; Auchus, Richard J.; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

    2015-01-01

    Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 107 m−1 s−1) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 107 m−1 s−1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. PMID:25855791

  8. Inter-relation of cytochrome P450 and contaminants burdens in sibling heron embryos and nestlings

    USGS Publications Warehouse

    Rattner, B.; Melancon, M.; Custer, T.; Hothem, R.

    1995-01-01

    Hepatic cytochrome P450-associated monooxygenase activities were measured in 11-day-old nestling black-crowned night-herons (Nycticorax nycticorax) collected from a reference site (next to the Chincoteague National Wildlife Refuge, Virginia) and three polluted sites (Cat Island, Green Bay, Lake Michigan, Wisconsin; Bair Island, San Francisco Bay, California; West Marin Island, San Francisco Bay, California). Activities of arylhydrocarbon hydroxylase (AHH) and benzyl-oxyresorufin-O-dealkylase (BROD) weremodestly elevated (p,p?DDE, other organochlorinepesticides and total PCBs in nestlings were greatest at contaminated sites, although much lower than found in concurrently collected eggs and pipping embryos. At these low pollutant concentrations there was little correlation between monooxygenase activity and contaminant levels in nestlings. These observations markedly contrast the pronounced monooxygenase induction (up to 85-fold) and its significant correlation with total PCBs, arylhydrocarbon receptor-active PCB congeners and toxic equivalents in concurrently collected night-heron embryos that were often siblings of the nestlings. The present findings suggest that cytochrome P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.

  9. Key Elements of the Chemistry of Cytochrome P-450: The Oxygen Rebound Mechanism.

    ERIC Educational Resources Information Center

    Groves, John T.

    1985-01-01

    Discusses the structure and function of the liver protein cytochrome P-450, an important catalyst for a variety of detoxification reactions. Diagnostic substracts for this heme-containing monooxygenase, synthetic modes of the active site, and oxidations with synthetic metalloporphyrins are the major topic areas considered. (JN)

  10. An Isotopic Labelling Strategy to Study Cytochrome P450 Oxidations of Terpenes.

    PubMed

    Rinkel, Jan; Litzenburger, Martin; Bernhardt, Rita; Dickschat, Jeroen Sidney

    2018-04-26

    The cytochrome P450 monooxygenase CYP267B1 from Sorangium cellulosum was applied for enzymatic oxidation of the sesquiterpene alcohols T-muurolol and isodauc-8-en-11-ol. Various isotopically labelled geranyl and farnesyl diphosphates were used for product identification from micro-scale reactions, for determination of the absolute configurations of unknown compounds, to follow the stereochemical course of a cytochrome P450-catalysed hydroxylation step, and to investigate kinetic isotope effects. Overall, this study demonstrates that isotopically labelled terpene precursors are highly useful to follow cytochrome P450 dependent oxidations of terpenes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2

    EPA Science Inventory

    EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2. T M Ross1, B P Anderson1, G Zhao2, R A Pegram1 and J W Allis1. 1U.S. EPA, ORD, NHEERL, Research Triangle Park, NC; 2University of North Carolina, Chapel Hill, NC.
    Sponsor: H Barton

    Bromodichlorometh...

  12. Evaluation of first-pass cytochrome P4503A (CYP3A) and P-glycoprotein activities using alfentanil and fexofenadine in combination.

    PubMed

    Kharasch, Evan D; Walker, Alysa; Hoffer, Christine; Sheffels, Pamela

    2005-01-01

    Cytochrome P4503A (CYP3A) and P-glycoprotein (P-gp) are major determinants of oral bioavailability. Development of in vivo probe(s), for both CYP3A and P-gp, which could be administered in combination, is a current goal. Nevertheless, there is considerable overlap in CYP3A and P-gp substrate selectivities; there are few discrete probes. Alfentanil is a selective CYP3A probe but not a P-gp substrate. Fexofenadine is a P-gp probe but not a CYP3A substrate. This investigation tested the hypothesis that alfentanil and fexofenadine could be administered in combination to probe first-pass CYP3A and P-gp activities in humans. Two 3-way crossover studies were conducted in healthy volunteers. In the first protocol, subjects received oral alfentanil alone, fexofenadine alone, or fexofenadine 1 hour after alfentanil. In the second protocol, subjects abstained from citrus and apple products for 5 days and received fexofenadine alone, fexofenadine 1 hour after alfentanil, or alfentanil 4 hours after fexofenadine. An assay using solid-phase extraction and electrospray liquid chromatography/mass spectrometry was developed for the simultaneous quantification of plasma alfentanil and fexofenadine. In both protocols, alfentanil plasma concentrations and area under the concentration versus time curve (AUC) were unaffected by fexofenadine or meal composition. Fexofenadine given 1 hour after alfentanil and followed 1 hour later by a meal containing orange or apple juice had a somewhat lower AUC compared with fexofenadine alone (geometric mean ratio with and without the interacting drug = 0.73, 90% confidence interval [CI] = 0.59-1.04). Fexofenadine given 1 hour after alfentanil and followed 2 hours later by a meal not containing citrus or apple products had an AUC that was unchanged compared with fexofenadine alone (ratio = 0.91, 90% CI = 0.70-1.35). These results show that alfentanil disposition was not affected by fexofenadine. A dosing regimen was identified in which fexofenadine

  13. Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

    PubMed

    Kärenlampi, S O; Marin, E; Hänninen, O O

    1981-02-15

    The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.

  14. Formation of cytochrome P-450 containing haem or cobalt-protoporphyrin in liver homogenates of rats treated with phenobarbital and allylisopropylacetamide.

    PubMed Central

    Bonkovsky, H L; Sinclair, J F; Healey, J F; Sinclair, P R; Smith, E L

    1984-01-01

    The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem

  15. Effect of gamma-oryzanol on cytochrome P450 activities in human liver microsomes.

    PubMed

    Umehara, Ken; Shimokawa, Yoshihiko; Miyamoto, Gohachiro

    2004-07-01

    The effects of gamma-oryzanol, a drug mainly used for the treatment of hyperlipidaemia, on several cytochrome P450 (CYP) specific reactions in human liver microsomes were investigated to predict drug interactions with gamma-oryzanol in vivo from in vitro data. The following eight CYP catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated bufuralol 1'-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6beta-hydroxylation. gamma-Oryzanol had little inhibitory effects on CYP activities, indicating that this compound would not be expected to cause clinically significant interactions with other CYP-metabolized drugs at expected therapeutic concentrations.

  16. Development of cytochromes P450 in avian species as a biomarker for environmental contaminant exposure and effect: Procedures and baseline values

    USGS Publications Warehouse

    Melancon, M.J.; Bengston, David A.; Henshel, Diane S.

    1996-01-01

    As in mammals and fish, birds respond to many environmental contaminants with induction of hepatic cytochromes P450. In order to monitor cytchromes P450 in specific avian species, for assessing the status of that species or the habitat it utilizes, it is necessary to have background information on the appropriate assay conditions and the responsiveness of cytochrome P450 induction in that species. Assay of four monooxygenases which give resorufin as product using a fluorescence microwell plate scanner has proven to be an effective approach. Information is provided on the incubation conditions and baseline activity for twenty avian species at ages ranging from pipping embryo to adult. Induction responsiveness is presented for sixteen of them. This information can serve as a guide for those who wish to utilize cytochrome P450 as a biomarker for contaminant exposure and effect to aid in selection of appropriate species, age, and monooxygenase assay(s).

  17. Role of Ca2+-independent phospholipase A2 and cytochrome P-450 in store-operated calcium entry in 3T6 fibroblasts.

    PubMed

    Martínez, Javier; Moreno, Juan J

    2005-09-01

    Store-operated calcium (SOC) channels and capacitative Ca2+ entry play a key role in cellular functions, but their mechanism of activation remains unclear. Here, we show that thapsigargin induces [3H] arachidonic acid (AA) release, 45Ca2+ influx and a subsequent enhancement of intracellular calcium concentration ([Ca2+]i. Thapsigargin-induced elevation of [Ca2+]i was inhibited by cytochrome P-450 inhibitors and by cytochrome P-450 epoxygenase inhibitor and was reverted by 11,12 EET addition. However, cyclooxygenase and lipoxygenase inhibitors have no effect. Moreover, we observed that four EETs were able to induce 45Ca2+ influx. Finally, we reported that the effect of 11,12 EET on 45Ca2+ influx was sensible to receptor-operated Ca2+ channel blockers (NiCl2, LaCl3) but not to voltage-dependent Ca2+ channel blocker as verapamil. Thus, AA released by Ca2+-independent phospholipase A2 and AA metabolism through cytochrome P-450 pathway may be crucial molecular determinant in thapsigargin activation of SOC channels and store-operated Ca2+ entry pathway in 3T6 fibroblasts. Moreover, EETs, the main cytochrome P-450 epoxygenase metabolites of AA, are involved in thapsigargin-stimulated Ca2+ influx. In summary, our results suggest that EETs are components of calcium influx factor(s).

  18. Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis.

    PubMed

    Pinto, L F; Moraes, E; Albano, R M; Silva, M C; Godoy, W; Glisovic, T; Lang, M A

    2001-11-01

    N-nitrosodiethylamine (NDEA) is able to induce tumours in the rat oesophagus. It has been suggested that this could be due to tissue specific expression of NDEA activating cytochrome P450 enzymes. We investigated this by characterizing the oesophageal monooxygenase complex of male Wistar rats and comparing it with that of the liver. Total amount of cytochrome P450, NADPH P450 reductase, cytochrome b5 and cytochrome b5 reductase of the oesophageal mucosa was approximately 7% of what was found in the liver. In addition, major differences were found in the cytochrome P450 isoenzyme composition between these organs: CYP 2B1/2B2 and CYP3A were found only in the liver, whereas CYP1A1 was constitutively expressed only in the oesophagus. Of the two well-known nitrosamine metabolizing enzymes, CYP2A3 was found only in the oesophagus whereas CYP2E1 was exclusively expressed in the liver. Catalytic studies, western blotting and RT-PCR analyses confirmed the expression of CYP2A3 in the oesophagus. CYP2A enzymes are known to be good catalysts of NDEA metabolism. Oesophageal microsomes had a K(m) for NDEA metabolism, which was about one-third of that of hepatic microsomes, but they showed similar activities when compared per nmol of total P450. NDEA activity in the oesophagus was significantly increased by coumarin (CO), which also induced oesophageal CYP2A3. Immunoinhibition of the microsomal NDEA activity showed that up to 70% of this reaction is catalysed by CYP2A3 in the oesophagus, whereas no inhibition of the hepatic NDEA activity could be achieved by the anti-CYP2A5 antibody. NDEA, but not N-nitrosodimethylamine (NDMA) inhibited the oesophageal metabolism of CO. The results of the present investigation show major differences in the enzyme composition of the oesophageal and hepatic monooxygenase complexes, and are in accordance with the hypothesis that the NDEA organotropism could, to a large extent, be due to the tissue specific expression of the activating enzymes.

  19. Effects of several pyrethroids on hepatic cytochrome P450 activities in rats.

    PubMed

    Abdou, Rania; Sasaki, Kazuaki; Khalil, Waleed; Shah, Syed; Murasawa, Youhei; Shimoda, Minoru

    2010-04-01

    Four commonly used pyrethroids (permethrin, bifenthrin, ethofenprox, and fenpropathrin) were orally administered to Sprague-Dawley rats for 5 days to study their effects on the liver cytochrome P450 (CYP) activities. Also Michaelis-Menten kinetics of the metabolic reactions catalyzed by liver CYPs were examined after adding these pyrethroids to the assay system to investigate their possible inhibitory effects on liver CYPs activities. These reactions included ethoxyresorufin O-deethylation, tolbutamide hydroxylation, bufuralol 1'-hydroxylation, and midazolam 4-hydroxylation, for CYP1A, 2C, 2D, and 3A activities, respectively. Results showed that oral administration of bifenthrin and ethofenprox highly induced CYP1A. The most potent inhibitors for CYP1A were fenpropathrin and cis-permethrin with K(i) values of 3.71 & 3.87 microM, respectively. CYP2D was slightly inhibited by both of fenpropathrin and cis-permethrin (K(i) values were 307.32 & 632.23 microM, respectively). On the other hand, none of CYP2C or 3A was inhibited by the tested pyrethroids. Since CYP1A may relate to biotransformation of many chemicals to reactive metabolites, bifenthrin and ethofenprox may potentiate mutagenicity of the chemicals through their inducing effects on CYP 1A. As permethrin and fenpropathrin were potent inhibitor for CYP1A, they may result in substantial accumulation of some chemicals. The resultant accumulation may lead to fatal toxicities in some case.

  20. Identification of human cytochrome P450s as autoantigens.

    PubMed

    Manns, M P; Johnson, E F

    1991-01-01

    Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies share these characteristics with other autoantibodies, for example, antinuclear antibodies in systemic lupus erythematosus. The identification of P450s as human autoantigens is clinically important. Diagnostic tests will be developed on the basis of cloned antigen, facilitating a better diagnosis of drug-induced and idiopathic autoimmune hepatitis. It is unknown what triggers autoantibody production against cytochrome P450 proteins. Furthermore, their pathogenetic role and thus their involvement in tissue destruction is unclear. In this context LKM1 autoantibodies may serve as a model. Although LKM1 antibodies are inhibitory, all LKM1 antibody-positive patients tested so far are extensive metabolizers for drug metabolism mediated by P450IID6 and express this protein in their livers. Thus, the inhibitory LKM1 autoantibody does not sufficiently penetrate through the intact liver cell membrane to inhibit enzyme function in vivo. Presumably, tissue destruction in autoimmune hepatitis is mediated by liver-infiltrating T lymphocytes. T lymphocytes have been cloned from liver tissue that specifically proliferate in the presence of recombinant cytochrome P450IID6. The construction of overlapping cDNA subclones is also valuable to identify immunodominant B cell as well as relevant T cell epitopes.

  1. Cytochrome P450s and molecular epidemiology

    NASA Astrophysics Data System (ADS)

    Gonzalez, Frank J.; Gelboin, Harry V.

    1993-03-01

    Cytochrome P450 (P450) represent a superfamily of heme-containing monooxygenases that are found throughout the animal and plant kingdoms and in many microorganisms. A number of these enzymes are involved in biosynthetic pathways of steroid synthesis but in mammals the vast majority of P450s function to metabolize foreign chemicals or xenobiotics. In the classical phase I reactions on the latter, a membrane-bound P450 will hydroxylate a compound, usually hydrophobic in nature, and the hydroxyl group will serve as a substrate for the various transferases or phase II enzymes that attach hydrophilic substituents such as glutathione, sulfate or glucuronic acid. Some chemicals, however, are metabolically-activated by P450s to electrophiles capable of reacting with cellular macromolecules. The cellular concentrations of the chemical and P450, reactivity of the active metabolite with nucleic acid and the repairability of the resultant adducts, in addition to the nature of the cell type, likely determines whether a chemical will be toxic and kill the cell or will transform the cell. Immunocorrelative and cDNA-directed expression have been used to define the substrate specificities of numerous human P450s. Levels of expression of different human P450 forms have been measured by both in vivo and in vitro methodologies leading to the realization that a large degree of interindividual differences occur in P450 expression. Reliable procedures for measuring P450 expression in healthy and diseased subjects will lead to prospective and case- cohort studies to determine whether interindividual differences in levels of P450 are associated with susceptibility or resistance to environmentally-based disease.

  2. Cytochrome P450 Organization and Function Are Modulated by Endoplasmic Reticulum Phospholipid Heterogeneity.

    PubMed

    Brignac-Huber, Lauren M; Park, Ji Won; Reed, James R; Backes, Wayne L

    2016-12-01

    Cytochrome P450s (P450s) comprise a superfamily of proteins that catalyze numerous monooxygenase reactions in animals, plants, and bacteria. In eukaryotic organisms, these proteins not only carry out reactions necessary for the metabolism of endogenous compounds, but they are also important in the oxidation of exogenous drugs and other foreign compounds. Eukaryotic P450 system proteins generally reside in membranes, primarily the endoplasmic reticulum or the mitochondrial membrane. These membranes provide a scaffold for the P450 system proteins that facilitate interactions with their redox partners as well as other P450s. This review focuses on the ability of specific lipid components to influence P450 activities, as well as the role of the membrane in P450 function. These studies have shown that P450s and NADPH-cytochrome P450 reductase appear to selectively associate with specific phospholipids and that these lipid-protein interactions influence P450 activities. Finally, because of the heterogeneous nature of the endoplasmic reticulum as well as other biologic membranes, the phospholipids are not arranged randomly but associate to generate lipid microdomains. Together, these characteristics can affect P450 function by 1) altering the conformation of the proteins, 2) influencing the P450 interactions with their redox partners, and 3) affecting the localization of the proteins into specific membrane microdomains. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Chunxue; Pallan, Pradeep S.; Zhang, Wei

    Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12more » variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37–13,000-fold higher than for WT P450 21A2. Cytochrome b5, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (≥ 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3-progesterone, indicating that C–H bond breaking is a rate-limiting step over a 104-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.« less

  4. Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

    PubMed Central

    Berthier, Sylvie; Hograindleur, Marc-André; Paclet, Marie-Hélène; Polack, Benoît; Morel, Françoise

    2012-01-01

    S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b 558; and (iii) to determine the S100A8 consensus site involved in cytochrome b 558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b 558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase

  5. Cytochrome oxidase assembly does not require catalytically active cytochrome C.

    PubMed

    Barrientos, Antoni; Pierre, Danielle; Lee, Johnson; Tzagoloff, Alexander

    2003-03-14

    Cytochrome c oxidase (COX), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen. COX assembly requires the coming together of nuclear- and mitochondrial-encoded subunits and the assistance of a large number of nuclear gene products acting at different stages of maturation of the enzyme. In Saccharomyces cerevisiae, expression of cytochrome c, encoded by CYC1 and CYC7, is required not only for electron transfer but also for COX assembly through a still unknown mechanism. We have attempted to distinguish between a functional and structural requirement of cytochrome c in COX assembly. A cyc1/cyc7 double null mutant strain was transformed with the cyc1-166 mutant gene (Schweingruber, M. E., Stewart, J. W., and Sherman, F. (1979) J. Biol. Chem. 254, 4132-4143) that expresses stable but catalytically inactive iso-1-cytochrome c. The COX content of the cyc1/cyc7 double mutant strain harboring non-functional iso-1-cytochrome c has been characterized spectrally, functionally, and immunochemically. The results of these studies demonstrate that cytochrome c plays a structural rather than functional role in assembly of cytochrome c oxidase. In addition to its requirement for COX assembly, cytochrome c also affects turnover of the enzyme. Mutants containing wild type apocytochrome c in mitochondria lack COX, suggesting that only the folded and mature protein is able to promote COX assembly.

  6. PROPICONAZOLE-INDUCED CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RAT AND MOUSE LIVER

    EPA Science Inventory

    Conazoles are N-substituted azole antifungal agents used as both pesticides and drugs. Some of these compounds are hepatocarcinogenic in mice and some can induce thyroid tumors in rats. Many of these compounds are able to induce and/or inhibit mammalian hepatic cytochrome P450s t...

  7. Cytochrome P450 systems--biological variations of electron transport chains.

    PubMed

    Hannemann, Frank; Bichet, Andreas; Ewen, Kerstin M; Bernhardt, Rita

    2007-03-01

    Cytochromes P450 (P450) are hemoproteins encoded by a superfamily of genes nearly ubiquitously distributed in different organisms from all biological kingdoms. The reactions carried out by P450s are extremely diverse and contribute to the biotransformation of drugs, the bioconversion of xenobiotics, the bioactivation of chemical carcinogens, the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins and bile acids, the conversion of alkanes, terpenes and aromatic compounds as well as the degradation of herbicides and insecticides. Cytochromes P450 belong to the group of external monooxygenases and thus receive the necessary electrons for oxygen cleavage and substrate hydroxylation from different redox partners. The classical as well as the recently discovered P450 redox systems are compiled in this paper and classified according to their composition.

  8. Humanlike substitutions to Ω-loop D of yeast iso-1-cytochrome c only modestly affect dynamics and peroxidase activity.

    PubMed

    Lei, Haotian; Bowler, Bruce E

    2018-06-01

    Structural studies of yeast iso-1-cytochrome c (L.J. McClelland, T.-C. Mou, M.E. Jeakins-Cooley, S.R. Sprang, B.E. Bowler, Proc. Natl. Acad. Sci. U.S.A. 111 (2014) 6648-6653) show that modest movement of Ω-loop D (residues 70-85, average RMSD versus the native structure: 0.81 Å) permits loss of Met80-heme ligation creating an available coordination site to catalyze the peroxidase activity mediated by cytochrome c early in apoptosis. However, Ala81 and Gly83 move significantly (RMSDs of 2.18 and 1.26 Å, respectively). Ala81 and Gly83 evolve to Ile and Val, respectively, in human cytochrome c and peroxidase activity decreases 25-fold relative to the yeast protein at pH 7. To test the hypothesis that these residues evolved to restrict the peroxidase activity of cytochrome c, A81I and G83V variants of yeast iso-1-cytochrome c were prepared. For both variants, the apparent pK a of the alkaline transition increases by 0.2 to 0.3 relative to the wild type (WT) protein and the rate of opening the heme crevice is slowed. The cooperativity of acid unfolding is decreased for the G83V variant. At pH 7 and 8, the catalytic rate constant, k cat , for the peroxidase activity of both variants decreases relative to WT, consistent with the effects on alkaline isomerization. Below pH 7, the loss in the cooperativity of acid unfolding causes k cat for peroxidase activity to increase for the G83V variant relative to WT. Neither variant decreases k cat to the level of the human protein, indicating that other residues also contribute to the low peroxidase activity of human cytochrome c. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. A complex of cardiac cytochrome c1 and cytochrome c.

    PubMed

    Chiang, Y L; Kaminsky, L S; King, T E

    1976-01-10

    The interactions of cytochrome c1 and cytochrome c from bovine cardiac mitochondria were investigated. Cytochrome c1 and cytochrome c formed a 1:1 molecular complex in aqueous solutions of low ionic strength. The complex was stable to Sephadex G-75 chromatography. The formation and stability of the complex were independent of the oxidation state of the cytochrome components as far as those reactions studied were concerned. The complex was dissociated in solutions of ionic strength higher than 0.07 or pH exceeding 10 and only partially dissociated in 8 M urea. No complexation occurred when cytochrome c was acetylated on 64% of its lysine residues or photooxidized on its 2 methionine residues. Complexes with molecular ratios of less than 1:1 (i.e. more cytochrome c) were obtained when polymerized cytochrome c, or cytochrome c with all lysine residues guanidinated, or a "1-65 heme peptide" from cyanogen bromide cleavage of cytochrome c was used. These results were interpreted to imply that the complex was predominantly maintained by ionic interactions probably involving some of the lysine residues of cytochrome c but with major stabilization dependent on the native conformations of both cytochromes. The reduced complex was autooxidizable with biphasic kinetics with first order rate constants of 6 X 10(-5) and 5 X U0(-5) s-1 but did not react with carbon monoxide. The complex reacted with cyanide and was reduced by ascorbate at about 32% and 40% respectively, of the rates of reaction with cytochrome c alone. The complex was less photoreducible than cytochrome c1 alone. The complex exhibited remarkably different circular dichroic behavior from that of the summation of cytochrome c1 plus cytochrome c. We concluded that when cytochromes c1 and c interacted they underwent dramatic conformational changes resulting in weakening of their heme crevices. All results available would indicate that in the complex cytochrome c1 was bound at the entrance to the heme crevice of

  10. Mechanism-based inactivation of cytochrome P-450 dependent benzo(a)pyrene hydroxylase activity by acetylenic and olefinic polycyclic arylhydrocarbons

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gan, L.S.

    A series of aryl acetylenes and aryl olefins have been examined as substrates and inhibitors of cytochrome P-450 dependent monooxygenases in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene (EP), 3-ethynylperylene (EPL), cis- and trans-1-(2-bromo-vinyl)pyrene (c-BVP and t-BVP), and 1-allylpyrene (AP) serve as mechanism-based irreversible inactivators (suicide inhibitors) of benzo(a)pyrene (BP) hydroxylase, while 1-vinyl-pyrene (VP) and phenyl 1-pyrenyl acetylene (PPA) do not cause a detectable suicide inhibition of the BP hydroxylase. The mechanism-based loss of BP hydroxylase activity caused by the aryl acetylenes is not accompanied by a corresponding loss of the P-450 content of the microsomes. In themore » presence of NADPH, /sup 3/H-labeled EP covalently attached to P-450 isozymes with a measured stoichiometry of one mole of EP per mole of the P-450 heme. The results of the effects of these aryl derivatives in the mammalian cell-mediated mutagenesis assay and toxicity assay show that none of the compounds examined nor any of the their metabolites produced in the incubation system are cytotoxic to V79 cells.« less

  11. Status of Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Neonicotinoids in Iran and Detoxification by Cytochrome P450-Dependent Monooxygenases.

    PubMed

    Basij, M; Talebi, K; Ghadamyari, M; Hosseininaveh, V; Salami, S A

    2017-02-01

    Nine Bemisia tabaci (Gennadius) populations were collected from different regions of Iran. In all nine populations, only one biotype (B biotype) was detected. Susceptibilities of these populations to imidacloprid and acetamiprid were assayed. The lethal concentration 50 values (LC 50 ) for different populations showed a significant discrepancy in the susceptibility of B. tabaci to imidacloprid (3.76 to 772.06 mg l -1 ) and acetamiprid (4.96 to 865 mg l -1 ). The resistance ratio of the populations ranged from 9.72 to 205.20 for imidacloprid and 6.38 to 174.57 for acetamiprid. The synergistic effects of piperonylbutoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) were evaluated for the susceptible (RF) and resistant (JR) populations for the determination of the involvement of cytochrome P450-dependent monooxygenase and carboxylesterase, respectively, in their resistance mechanisms. The results showed that PBO overcame the resistance of the JR population to both imidacloprid and acetamiprid, with synergistic ratios of 72.7 and 106.9, respectively. Carboxylesterase, glutathione S-transferase and cytochrome P450-dependent monooxygenase were studied biochemically, for the purpose of measuring the activity of the metabolizing enzymes in order to determine which enzymes are directly involved in neonicotinoid resistance. There was an increase in the activity of cytochrome P450-dependent monooxygenase up to 17-fold in the resistant JR population (RR = 205.20). The most plausible activity of cytochrome P450-dependent monooxygenase correlated with the resistances of imidacloprid and acetamiprid, and this suggests that cytochrome P450-dependent monooxygenase is the only enzyme system responsible for neonicotinoid resistance in the nine populations of B. tabaci.

  12. KINETICS OF BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P450 ISOENZYMES IN HUMAN LIVER MICROSOMES

    EPA Science Inventory

    Kinetics of Bromodichloromethane Metabolism by
    Cytochrome P450 Isoenzymes in Human Liver Microsomes

    Guangyu Zhao and John W. Allis

    ABSTRACT
    The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have ...

  13. Impact of visceral leishmaniasis and curative chemotherapy on cytochrome P450 activity in Brazilian patients.

    PubMed

    Lanchote, Vera Lucia; Almeida, Roque; Barral, Aldina; Barral-Netto, Manoel; Marques, Maria Paula; Moraes, Natália V; da Silva, Angela M; Souza, Tania M V; Suarez-Kurtz, Guilherme

    2015-11-01

    The aim of the present study was to investigate the impact of human visceral leishmaniasis (VL) and curative chemotherapy on the activity of cytochrome P450 (CYP) 3A, CYP2C9 and CYP2C19 in patients from an endemic region in Brazil. Adult patients with parasitologically confirmed VL were given a CYP phenotyping cocktail, comprising midazolam, omeprazole and losartan, immediately before (Study phase 1), 2-3 days (phase 2) and 3-6 months (phase 3) after curative VL chemotherapy. CYP activity was assessed by the apparent clearance of midazolam (CYP3A), omeprazole/5-hydroxyomeprazol ratio in plasma (CYP2C19) and losartan/E3174 ratio in urine (CYP2C9). Mean values (95% confidence interval) in phases 1, 2 and 3 were, respectively: log apparent midazolam clearance, 1.21 (1.10-1.31), 1.45 (1.32-1.57) and 1.35 (1.26-1.44) ml min(-1)  kg(-1) ; omeprazole/5-hydroxyomeprazole ratio, 0.78 (0.61-0.94), 0.45 (0.27-0.63) and 0.37 (0.20-0.55); losartan/E3174 ratio, 0.66 (0.39-0.92), 0.35 (0.20-0.50) and 0.35 (0.16-0.53). Analysis of variance revealed significant differences in CYP3A (P = 0.018) and CYP2C19 (P = 0.008), but not CYP2C9 (P = 0.11) phenotypic activity, across the three study phases. The phenotypic activities of CYP3A4 and CYP2C19 were significantly reduced during acute VL compared with post-chemotherapy. We propose that increased plasma concentrations of proinflammatory cytokines during active disease account for the suppression of CYP activity. The failure to detect significant changes in CYP2C9 activity in the overall cohort may reflect differential effects of the inflammatory process on the expression of CYP isoforms, although the possibility of insufficient statistical power cannot be dismissed. © 2015 The British Pharmacological Society.

  14. Impact of visceral leishmaniasis and curative chemotherapy on cytochrome P450 activity in Brazilian patients

    PubMed Central

    Lanchote, Vera Lucia; Almeida, Roque; Barral, Aldina; Barral-Netto, Manoel; Marques, Maria Paula; Moraes, Natália V; da Silva, Angela M; Souza, Tania M V; Suarez-Kurtz, Guilherme

    2015-01-01

    Aims The aim of the present study was to investigate the impact of human visceral leishmaniasis (VL) and curative chemotherapy on the activity of cytochrome P450 (CYP) 3A, CYP2C9 and CYP2C19 in patients from an endemic region in Brazil. Methods Adult patients with parasitologically confirmed VL were given a CYP phenotyping cocktail, comprising midazolam, omeprazole and losartan, immediately before (Study phase 1), 2–3 days (phase 2) and 3–6 months (phase 3) after curative VL chemotherapy. CYP activity was assessed by the apparent clearance of midazolam (CYP3A), omeprazole/5-hydroxyomeprazol ratio in plasma (CYP2C19) and losartan/E3174 ratio in urine (CYP2C9). Results Mean values (95% confidence interval) in phases 1, 2 and 3 were, respectively: log apparent midazolam clearance, 1.21 (1.10–1.31), 1.45 (1.32–1.57) and 1.35 (1.26–1.44) ml min–1 kg–1; omeprazole/5-hydroxyomeprazole ratio, 0.78 (0.61–0.94), 0.45 (0.27–0.63) and 0.37 (0.20-0.55); losartan/E3174 ratio, 0.66 (0.39–0.92), 0.35 (0.20–0.50) and 0.35 (0.16–0.53). Analysis of variance revealed significant differences in CYP3A (P = 0.018) and CYP2C19 (P = 0.008), but not CYP2C9 (P = 0.11) phenotypic activity, across the three study phases. Conclusion The phenotypic activities of CYP3A4 and CYP2C19 were significantly reduced during acute VL compared with post-chemotherapy. We propose that increased plasma concentrations of proinflammatory cytokines during active disease account for the suppression of CYP activity. The failure to detect significant changes in CYP2C9 activity in the overall cohort may reflect differential effects of the inflammatory process on the expression of CYP isoforms, although the possibility of insufficient statistical power cannot be dismissed. PMID:25940755

  15. Androgen receptor antagonism drives cytochrome P450 17A1 inhibitor efficacy in prostate cancer

    PubMed Central

    Norris, John D.; Ellison, Stephanie J.; Baker, Jennifer G.; Stagg, David B.; Wardell, Suzanne E.; Park, Sunghee; Alley, Holly M.; Baldi, Robert M.; Yllanes, Alexander; Andreano, Kaitlyn J.; Stice, James P.; Lawrence, Scott A.; Eisner, Joel R.; Price, Douglas K.; Moore, William R.; Figg, William D.; McDonnell, Donald P.

    2017-01-01

    The clinical utility of inhibiting cytochrome P450 17A1 (CYP17), a cytochrome p450 enzyme that is required for the production of androgens, has been exemplified by the approval of abiraterone for the treatment of castration-resistant prostate cancer (CRPC). Recently, however, it has been reported that CYP17 inhibitors can interact directly with the androgen receptor (AR). A phase I study recently reported that seviteronel, a CYP17 lyase–selective inhibitor, ædemonstrated a sustained reduction in prostate-specific antigen in a patient with CRPC, and another study showed seviteronel’s direct effects on AR function. This suggested that seviteronel may have therapeutically relevant activities in addition to its ability to inhibit androgen production. Here, we have demonstrated that CYP17 inhibitors, with the exception of orteronel, can function as competitive AR antagonists. Conformational profiling revealed that the CYP17 inhibitor–bound AR adopted a conformation that resembled the unliganded AR (apo-AR), precluding nuclear localization and DNA binding. Further, we observed that seviteronel and abiraterone inhibited the growth of tumor xenografts expressing the clinically relevant mutation AR-F876L and that this activity could be attributed entirely to competitive AR antagonism. The results of this study suggest that the ability of CYP17 inhibitors to directly antagonize the AR may contribute to their clinical efficacy in CRPC. PMID:28463227

  16. Effects of thalidomide, cytochrome P-450 and TNF-alpha on angiogenesis in a three-dimensional collagen gel-culture.

    PubMed

    Fujita, Keiko; Asami, Yoshiko; Murata, Eiko; Akita, Masumi; Kaneko, Katsuji

    2002-10-01

    The anti-angiogenic effects of thalidomide were examined in mouse aortae grown in a three-dimensional collagen gel-culture. In our in vitro model, (+/-)-thalidomide and (-)-thalidomide exhibited no anti-angiogenic effects. On the other hand, when the culture was treated with thalidomide plus cytochrome P-450, both types of thalidomides significantly inhibited angiogenesis. Co-administration of 100 microg/ml thalidomide plus 200 microg/ml cytochrome P-450 inhibited angiogenesis more strongly than thalidomide plus cytochrome P-450 at other concentrations (10 microg/ml + 200 microg/ml and 100 microg/ml + 20 microg/ml). To study the relation between the anti-angiogenic effect and TNF-alpha, we also evaluated the concentration of TNF-alpha in the culture medium. We found that the concentration of TNF-alpha was correlated to the strength of the anti-angiogenic effect. The inhibition of angiogenesis by thalidomide and cytochrome P-450 takes place through a suppression of TNF-alpha and involves the metabolism of the thalidomide.

  17. Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach.

    PubMed

    Otey, Christopher R; Silberg, Jonathan J; Voigt, Christopher A; Endelman, Jeffrey B; Bandara, Geethani; Arnold, Frances H

    2004-03-01

    Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome p450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded p450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse p450s for exploring function evolution within the p450 structural framework.

  18. Pharmacogenetics of CYP1A2 activity and inducibility in smokers and exsmokers.

    PubMed

    Dobrinas, Maria; Cornuz, Jacques; Eap, Chin B

    2013-05-01

    There is a high interindividual variability in cytochrome P4501A2 (CYP1A2) activity and in its inducibility by smoking, only poorly explained by known CYP1A2 polymorphisms. We aimed to study the contribution of other regulatory pathways, including transcription factors and nuclear receptors, toward this variability. CYP1A2 activity was determined by the paraxanthine/caffeine ratio in 184 smokers and in 113 of them who were abstinent for 4 weeks. Participants were genotyped for 22 polymorphisms in 12 genes. A significant influence on CYP1A2 inducibility was observed for the NR1I3 rs2502815 (P=0.0026), rs4073054 (P=0.029), NR2B1 rs3818740 (P=0.0045), rs3132297 (P=0.036), AhR rs2282885 (P=0.040), rs2066853 (P=0.019), NR1I1 rs2228570 (P=0.037), and NR1I2 rs1523130 (P=0.044) polymorphisms. Among these, the NR1I3 rs2502815 (P=0.0045), rs4073054 (P=0.048), and NR2B1 rs3818740 (P=0.031) also influenced CYP1A2 basal activity. This is the first in-vivo demonstration of the influence of genes involved in CYP1A2 regulatory pathways on its basal activity and inducibility by smoking. These results need to be confirmed by other studies.

  19. Evidence for involvement of multiple forms of cytochrome P-450 in aflatoxin B sup 1 metabolism in human liver

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Forrester, L.M.; Wolf, C.R.; Neal, G.E.

    Liver cancer is a major cause of premature death in many areas of Africa and Asia and its incidence is strongly correlated with exposure to aflatoxin B{sub 1} (AFB{sub 1}). Because AFB{sub 1} requires metabolic activation to achieve a biological response, there is a need for detailed knowledge of the mechanism of activation to assess individual risk. The authors carried out an extensive study using a total of 19 human liver samples to determine the individual variability in the metabolism of the toxin to mutagenic or detoxification products and to identify the specific cytochrome P-450 forms involved in these processes.more » Metabolism to the toxic 8,9-epoxide or to products mutagenic in the Ames test was found to exhibit very large individual variation. These data demonstrate that, although P450IIIA probably plays an important role in AFB{sub 1} activation, several other cytochrome P-450 forms have the capacity to activate the toxin. Similar considerations apply to detoxifying metabolism to aflatoxin Q{sub 1} and aflatoxin M{sub 1}. The levels of expression of many of the forms of cytochrome P-450 involved in AFB{sub 1} metabolism are known to be highly sensitive to environmental factors. This indicates that such factors will be an important determinant in individual susceptibility to the tumorigenic action of AFB{sub 1}.« less

  20. Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.

    2011-08-05

    Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides themore » reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.« less

  1. Menadione Suppresses Benzo(α)pyrene-Induced Activation of Cytochromes P450 1A: Insights into a Possible Molecular Mechanism.

    PubMed

    Sidorova, Yulia A; Perepechaeva, Maria L; Pivovarova, Elena N; Markel, Arkady L; Lyakhovich, Vyacheslav V; Grishanova, Alevtina Y

    2016-01-01

    Oxidative reactions that are catalyzed by cytochromes P450 1A (CYP1A) lead to formation of carcinogenic derivatives of arylamines and polycyclic aromatic hydrocarbons (PAHs), such as the widespread environmental pollutant benzo(α)pyrene (BP). These compounds upregulate CYP1A at the transcriptional level via an arylhydrocarbon receptor (AhR)-dependent signaling pathway. Because of the involvement of AhR-dependent genes in chemically induced carcinogenesis, suppression of this signaling pathway could prevent tumor formation and/or progression. Here we show that menadione (a water-soluble analog of vitamin K3) inhibits BP-induced expression and enzymatic activity of both CYP1A1 and CYP1A2 in vivo (in the rat liver) and BP-induced activity of CYP1A1 in vitro. Coadministration of BP and menadione reduced DNA-binding activity of AhR and increased DNA-binding activity of transcription factors Oct-1 and CCAAT/enhancer binding protein (C/EBP), which are known to be involved in negative regulation of AhR-dependent genes, in vivo. Expression of another factor involved in downregulation of CYP1A-pAhR repressor (AhRR)-was lower in the liver of the rats treated with BP and menadione, indicating that the inhibitory effect of menadione on CYP1A is not mediated by this protein. Furthermore, menadione was well tolerated by the animals: no signs of acute toxicity were detected by visual examination or by assessment of weight gain dynamics or liver function. Taken together, our results suggest that menadione can be used in further studies on animal models of chemically induced carcinogenesis because menadione may suppress tumor formation and possibly progression.

  2. Cytochrome P450 diversity in the tree of life.

    PubMed

    Nelson, David R

    2018-01-01

    Sequencing in all areas of the tree of life has produced >300,000 cytochrome P450 (CYP) sequences that have been mined and collected. Nomenclature has been assigned to >41,000 CYP sequences and the majority of the remainder has been sorted by BLAST searches into clans, families and subfamilies in preparation for naming. The P450 sequence space is being systematically explored and filled in. Well-studied groups like vertebrates are covered in greater depth while new insights are being added into uncharted territories like horseshoe crab (Limulus polyphemus), tardigrades (Hypsibius dujardini), velvet worm (Euperipatoides_rowelli), and basal land plants like hornworts, liverworts and mosses. CYPs from the fungi, one of the most diverse groups, are being explored and organized as nearly 800 fungal species are now sequenced. The CYP clan structure in fungi is emerging with 805 CYP families sorting into 32 CYP clans. >3000 bacterial sequences are named, mostly from terrestrial or freshwater sources. Of 18,379 bacterial sequences downloaded from the CYPED database, all are >43% identical to named CYPs. Therefore, they fit in the 602 named P450 prokaryotic families. Diversity in this group is becoming saturated, however 25% of 3305 seawater bacterial P450s did not match known P450 families, indicating marine bacterial CYPs are not as well sampled as land/freshwater based bacterial CYPs. Future sequencing plans of the Genome 10K project, i5k and GIGA (Global Invertebrate Genomics Alliance) are expected to produce more than one million cytochrome P450 sequences by 2020. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. TRIBUTYLTIN POTENTIATES 3,3',4,4',5-PENTACHLOROBIPHENYL-INDUCED CYTOCHROME P4501A ACTIVITY. (R823881)

    EPA Science Inventory

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  4. Suicide inactivation of cytochrome P-450 by methoxsalen. Evidence for the covalent binding of a reactive intermediate to the protein moiety

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Labbe, G.; Descatoire, V.; Beaune, P.

    Incubation of rat liver microsomes with (3H)methoxsalen and NADPH resulted in the covalent binding of a methoxsalen intermediate to proteins comigrating with cytochromes P-450 UT-A, PB-B/D, ISF-G and PCN-E. Binding was increased by pretreatments with phenobarbital, beta-naphthoflavone (beta NF) and dexamethasone. Such pretreatments also increased the loss of CO-binding capacity either after administration of methoxsalen, or after incubation of hepatic microsomes with methoxsalen and NADPH. Immunoprecipitation of the methoxsalen metabolite-protein adducts in phenobarbital-induced microsomes was moderate with anti-UT-A antibodies, but marked with anti-PB-B/D and anti-PCN-E antibodies. Immunoprecipitation was observed also with anti-ISF-G (anti-beta NF-B) antibodies in beta NF-induced microsomes. Methoxsalenmore » (0.25 mM) inhibited markedly the benzphetamine demethylase activity of phenobarbital-induced microsomes and the erythromycin demethylase activity of dexamethasone-induced microsomes. Whereas methoxsalen itself did not produce any binding spectrum, in contrast either in vivo administration of methoxsalen or incubation in vitro with methoxsalen and NADPH resulted in a low-to-high spin conversion of cytochrome P-450 as suggested by the appearance of a spectrum analogous to a type I binding spectrum. This low-to-high spin conversion was apparently due to a methoxsalen intermediate (probably, covalently bound to the protein and preventing partial sixth ligation of the iron). We conclude that suicide inactivation of cytochrome P-450 by methoxsalen is related to the covalent binding of a methoxsalen intermediate to the protein moiety of several cytochrome P-450 isoenzymes (including UT-A, PB-B/D, PCN-E as well as ISF-G and/or beta NF-B).« less

  5. Use of high doses of quetiapine in bipolar disorder episodes are not linked to high activity of cytochrome P4503A4 and/or cytochrome P4502D6.

    PubMed

    Khazaal, Yasser; Preisig, Martin; Chatton, Anne; Kaufmann, Nadine; Bilancioni, Romain; Eap, Chin B

    2013-09-01

    The use of quetiapine for treatment of bipolar disorders at a higher dosage than the licensed range is not unusual in clinical practice. Quetiapine is predominantly metabolised by cytochrome P450 3A4 (CYP3A4) and to a lesser extent by CYP2D6. The large interindividual variability of those isozyme activities could contribute to the variability observed in quetiapine dosage. The aim of the present study is to evaluate if the use of high dosages of quetiapine in some patients, as compared to patients treated with a dosage in the licensed range (up to 800 mg/day), could be explained by a high activity of CYP3A4 and/or of CYP2D6. CYP3A4 activities were determined using the midazolam metabolic ratio in 21 bipolar and schizoaffective bipolar patients genotyped for CYP2D6. 9 patients were treated with a high quetiapine dosage (mean ± SD, median; range: 1467 ± 625, 1200; 1000-3000 mg/day) and 11 with a normal quetiapine dosage (433 ± 274, 350; 100-800 mg/day). One patient in the high dose and one patient in the normal dose groups were genotyped as CYP2D6 ultrarapid metabolizers. CYP3A4 activities were not significantly different between the two groups (midazolam metabolic ratio: 9.4 ± 8.2; 6.2; 1.7-26.8 vs 3.9 ± 2.3; 3.8; 1.5-7.6, in the normal dose group as compared to the high dose group, respectively, NS). The use of high quetiapine dosage for the patients included in the present study cannot be explained by variations in pharmacokinetics parameters such as a high activity of CYP3A4 and/or of CYP2D6.

  6. Microsomal P-450 induction by some secondary products from thermal oxidation of dietary lipids: epidermal hyperplasia, mutagenicity and cytochrome P-450 activities.

    PubMed

    Crawford, L; Wheeler, E L

    1983-12-01

    Distillable secondary products from roasted fowl were found to be cytotoxic but not mutagenic when assayed with Salmonella typhimurium strains TA98, TA100 and TA1537. A crudely separated fraction of the volatiles produced focal hyperplasia and damage to the epidermis of the backs of mice. The volatiles also caused an apparent synthesis of non-constitutive forms of rat hepatic cytochromes P-450 which metabolize benzo[a]pyrene B [a]P differently from the constitutive P-450.

  7. Cytochrome P450 genes from the aquatic midge Chironomus tentans: Atrazine-induced up-regulation of CtCYP6EX3 contributing to oxidative activation of chlorpyrifos

    USDA-ARS?s Scientific Manuscript database

    The open reading frames of 19 cytochrome P450 monooxygenase (CYP) genes were sequenced from Chironomus tentans, a commonly used freshwater invertebrate model. Functional analysis of CtCYP6EX3 confirmed its atrazine-induced oxidative activation for chlorpyrifos by using a nanoparticle-based RNA inter...

  8. Docking and QSAR comparative studies of polycyclic aromatic hydrocarbons and other procarcinogen interactions with cytochromes P450 1A1 and 1B1.

    PubMed

    Gonzalez, J; Marchand-Geneste, N; Giraudel, J L; Shimada, T

    2012-01-01

    To obtain chemical clues on the process of bioactivation by cytochromes P450 1A1 and 1B1, some QSAR studies were carried out based on cellular experiments of the metabolic activation of polycyclic aromatic hydrocarbons and heterocyclic aromatic compounds by those enzymes. Firstly, the 3D structures of cytochromes 1A1 and 1B1 were built using homology modelling with a cytochrome 1A2 template. Using these structures, 32 ligands including heterocyclic aromatic compounds, polycyclic aromatic hydrocarbons and corresponding diols, were docked with LigandFit and CDOCKER algorithms. Binding mode analysis highlighted the importance of hydrophobic interactions and the hydrogen bonding network between cytochrome amino acids and docked molecules. Finally, for each enzyme, multilinear regression and artificial neural network QSAR models were developed and compared. These statistical models highlighted the importance of electronic, structural and energetic descriptors in metabolic activation process, and could be used for virtual screening of ligand databases. In the case of P450 1A1, the best model was obtained with artificial neural network analysis and gave an r (2) of 0.66 and an external prediction [Formula: see text] of 0.73. Concerning P450 1B1, artificial neural network analysis gave a much more robust model, associated with an r (2) value of 0.73 and an external prediction [Formula: see text] of 0.59.

  9. Catalysis by cytochrome P-450 of an oxidative reaction in xenobiotic aldehyde metabolism: deformylation with olefin formation.

    PubMed Central

    Roberts, E S; Vaz, A D; Coon, M J

    1991-01-01

    As we have briefly described elsewhere, cytochrome P-450 catalyzes the oxidative deformylation of cyclohexane carboxaldehyde to yield cyclohexene and formic acid in a reaction believed to involve a peroxyhemiacetal-like adduct formed between the substrate and molecular oxygen-derived hydrogen peroxide. This reaction is a useful model for the demethylation reactions catalyzed by the steroidogenic P-450s, aromatase, and lanosterol demethylase. In the present study, the cytochrome P-450-catalyzed formation of olefinic products from a series of xenobiotic aldehydes has been demonstrated. Isobutyraldehyde and trimethylacetaldehyde, but not propionaldehyde, are converted to the predicted olefinic products, suggesting a requirement for branching at the alpha carbon. In addition, the four C5 aldehydes of similar hydrophobicity were compared for their ability to undergo the reaction. The straight-chain valeraldehyde gave no olefinic products with five different rabbit liver microsomal P-450 isozymes. However, increasing activity was seen with the other isomers in the order of isovaleraldehyde, 2-methylbutyraldehyde, and trimethylacetaldehyde, with all of the P-450 cytochromes. The catalytic rate with trimethylacetaldehyde is highest with antibiotic-inducible P-450 form 3A6, followed by phenobarbital-inducible form 2B4 and ethanol-inducible form 2E1. Citronellal, a beta-branched aldehyde that is found in many essential oils and is widely used as an odorant and a flavorant, was found to undergo the oxidative deformylation reaction to yield 2,6-dimethyl-1,5-heptadiene, but only with P-450 2B4. The oxidative cleavage reaction with olefin formation appears to be widespread, as judged by the variety of aldehydes that serve as substrates and of P-450 cytochromes that serve as catalysts. PMID:1924356

  10. [Immunomodulators with an 8-azasteroid structure as inducers of liver cytochrome P-450].

    PubMed

    Kuz'mitskiĭ, B B; Dad'kov, I G; Mashkovich, A E; Stoma, O V; Slepneva, L M

    1990-01-01

    Two structural analogues of D-homo-8-azasteroids, both an immunostimulant and an immunodepressant, are inductors of the liver cytochrome P-450 in animals. This capability was shown by means of both a decrease of the hexenal sleep duration in the pharmacological test and an increase of the quantity of cytochrome P-450 and the rate of N-demethylation of aminopyrine in the biochemical assays.

  11. Electron transfer between cytochrome. alpha. and copper A in cytochrome c oxidase: A perturbed equilibrium study

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Morgan, J.E.; Li, P.M.; Jang, D.J.

    1989-08-22

    Intramolecular electron transfer in partially reduced cytochrome c oxidase has been studied by the perturbed equilibrium method. The authors have prepared a three-electron-reduced, CO-inhibited form of the enzyme in which cytochrome a and copper A are partially reduced and in an intramolecular redox equilibrium. When these samples were irradiated with a nitrogen laser to photodissociate the bound CO, changes in absorbance at 598 and 830 nm were observed which were consistent with a fast electron transfer from cytochrome a to copper A. The absorbance changes at 598 nm gave an apparent rate of 17,000 {plus minus} 2,000 s{sup {minus}1} (1more » {sigma}), at pH 7.0 and 25.5{degree}C. These changes were not observed in either the CO mixed-valence or the CO-inhibited fully reduced forms of the enzyme. The rate was fastest at about pH 8.0, falling off toward both lower and higher pHs. There was a small but clear temperature dependence. The process was also observed in the cytochrome c-cytochrome c oxidase high-affinity complex. The electron equilibration measured between cytochrome {alpha} and copper A is far faster than any rate measured or inferred previously for this process.« less

  12. Inhibitory effects of spirulina platensis on carcinogen-activating cytochrome P450 isozymes and potential for drug interactions.

    PubMed

    Savranoglu, Seda; Tumer, Tugba Boyunegmez

    2013-01-01

    Spirulina platensis (SP) has been considered as potential food source of 21st century due to its remarkable nutrient profile and therapeutic benefits. However, the cytochrome P450 (CYP)-mediated drug/chemical interaction potential of SP has not yet been pursued. We investigated the effects of SP on the expressions and enzymatic activities of main CYP isozymes. After the rats were orally administered with SP daily for 5 consecutive weeks, there were significant downregulations in hepatic expression levels and inhibition in enzymatic activities of CYP1A2 and CYP2E1 compared to controls. In addition, a significant decrease was observed in CYP2C6-associated enzyme activity with no remarkable changes in messenger RNA (mRNA)/protein levels. The SP application resulted in significant increases in mRNA/protein levels of both CYP2B1 and CYP3A1 without a significant change in enzyme activities. These findings partly explain the chemopreventive properties of SP toward various organ toxicities, mutagenesis, and carcinogenesis; however, its coadministration with some CYP substrates may lead to undesirable drug interactions.

  13. Immunohistochemical study of temporal variations in cytochrome P-450 isozymes in rat testis and their modifications by the inductive effects of cadinenes

    NASA Astrophysics Data System (ADS)

    Kobayashi, Yasuhito; Motohashi, Yutaka; Miyazaki, Yoshifumi; Yatagai, Mitsuyoshi; Takano, Takehito

    1991-12-01

    Temporal variations in cytochrome P-450 isozymes of rat testis, PB-P-450 (forms of cytochrome P-450 strongly induced by phenobarbital) and MC-P-448 (forms of cytochrome P-450 strongly induced by 3-methylcholanthrene), were investigated immunohistochemically by the avidin-biotin-complex method using specific antibodies against PB-P-450 and MC-P-448 isozymes. Immunoreactivity to both PB-P-450 and MC-P-448 isozymes was observed in Leydig cells. The number of PB-P-450 positive Leydig cells was found to undergo significant time-of-day variation with a peak time of 0000 hours (light phase from 0800 to 2000 hours). Injection of cadinenes (300 mg/kg per day intraperitoneally at 48 and 96 h before sacrifice) induced PB-P-450 isozyme but did not induce MC-P-448 isozyme. The induction of PB-P-450 isozyme by cadinenes was time dependent, and the early dark phase (2000 and 0000 hours) was most sensitive. These results suggest that temporal variation of cytochrome P-450 isozymes is one of the important physiological variations in detoxification and activation of various xenobiotics and chemicals in the testis.

  14. Inhibitory effects of cytostatically active 6-aminobenzo[c]phenanthridines on cytochrome P450 enzymes in human hepatic microsomes.

    PubMed

    Zebothsen, Inga; Kunze, Thomas; Clement, Bernd

    2006-07-01

    Besides assays for the evaluation of efficacy new drug candidates have to undergo extensive testings for enhancement of pharmaceutical drug safety and optimization of application. The objective of the present work was to investigate the pharmacokinetic drug drug interaction potential for the cytostatically active 6-aminobenzo[c]phenanthridines BP-11 (6-amino-11,12-dihydro-11-(4-hydroxy-3,5-dimethoxyphenyl)benzo[c]phenanthridine) and BP-D7 (6-amino-11-(3,4,5-trimethoxyphenyl)benzo[c]phenanthridine) in vitro through incubation with human hepatic microsomes and marker substrates. For these studies the cytochrome P-450 isoenzymes and corresponding marker substrates recommended by the EMEA (The European Agency for the Evaluation of Medicinal Products) were chosen. In detail these selective substrates were caffeine (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), S-(+)-mephenytoin (CYP2C19), dextromethorphane (CYP2D6), chlorzoxazone (CYP2E1) and testosterone (CYP3A4). Incubations with each substrate were carried out without a possible inhibitor and in the presence of a benzo[c]phenanthridine or a selective inhibitor at varying concentrations. Marker activities were determined by HPLC (high performance liquid chromatography). For the isoenzymes showing more than 50% inhibition by the addition of 20 microM BP-11 or BP-D7 additional concentrations of substrate and inhibitor were tested for a characterization of the inhibition. The studies showed a moderate risk for BP-11 for interactions with the cytochrome P-450 isoenzymes CYP1A2, CYP2C9, CYP2D6 and CYP3A4. BP-D7, the compound with the highest cytotstatic efficacy, showed only a moderate risk for interactions with drugs, also metabolized by CYP3A4.

  15. Cytochrome P450 1A2 (CYP1A2) activity, mammographic density, and oxidative stress: a cross-sectional study

    PubMed Central

    Hong, Chi-Chen; Tang, Bing-Kou; Rao, Venketeshwer; Agarwal, Sanjiv; Martin, Lisa; Tritchler, David; Yaffe, Martin; Boyd, Norman F

    2004-01-01

    Introduction Mammographically dense breast tissue is a strong predictor of breast cancer risk, and is influenced by both mitogens and mutagens. One enzyme that is able to affect both the mitogenic and mutagenic characteristics of estrogens is cytochrome P450 1A2 (CYP1A2), which is principally responsible for the metabolism of 17β-estradiol. Methods In a cross-sectional study of 146 premenopausal and 149 postmenopausal women, we examined the relationships between CYP1A2 activity, malondialdehyde (MDA) levels, and mammographic density. In vivo CYP1A2 activity was assessed by measuring caffeine metabolites in urine. Levels of serum and urinary MDA, and MDA–deoxyguanosine adducts in DNA were measured. Mammograms were digitized and measured using a computer-assisted method. Results CYP1A2 activity in postmenopausal women, but not in premenopausal women, was positively associated with mammographic density, suggesting that increased CYP1A2 activity after the menopause is a risk factor for breast cancer. In premenopausal women, but not in postmenopausal women, CYP1A2 activity was positively associated with serum and urinary MDA levels; there was also some evidence that CYP1A2 activity was more positively associated with percentage breast density when MDA levels were high, and more negatively associated with percentage breast density when MDA levels were low. Conclusion These findings provide further evidence that variation in the activity level of enzymes involved in estrogen metabolism is related to levels of mammographic density and potentially to breast cancer risk. PMID:15217501

  16. Evidence that ligand formation is a mechanism underlying the maintenance of cytochrome P-450 in rat liver cell culture. Potent maintenance by metyrapone.

    PubMed Central

    Paine, A J; Villa, P; Hockin, L J

    1980-01-01

    The loss of cytochrome P-450 in cultured rat hepatocytes can be prevented by substituted pyridines, especially isonicotinamide, 3-hydroxypyridine and metyrapone. The effect of these compounds is independent of protein synthesis, suggesting that they maintain pre-existing cytochrome P-450. The efficiency of pyridines at maintaining cytochrome P-450 in hepatocyte culture is highly correlated with their ability to bind to this cytochrome, suggesting that ligand formation with cytochrome P-450 prevents its accelerated turnover in liver cell culture. PMID:7470047

  17. A role for cytochrome b5 in the in vivo disposition of anti-cancer and cytochrome P450 probe drugs in mice

    PubMed Central

    Henderson, Colin J.; McLaughlin, Lesley A.; Finn, Robert D.; Ronseaux, Sebastien; Kapelyukh, Yury; Wolf, C. Roland

    2014-01-01

    The role of microsomal cytochrome b5 (Cyb5) in defining the rate of drug metabolism and disposition has been intensely debated for several decades. Recently we described mouse models involving the hepatic or global deletion of Cyb5, demonstrating its central role in in vivo drug disposition. We have now used the cytochrome b5 complete null (BCN) model to determine the role of Cyb5 in the metabolism of ten pharmaceuticals metabolised by a range of cytochrome P450s, including five anti-cancer drugs, in vivo and in vitro. The extent to which metabolism was significantly affected by the absence of Cyb5 was substrate-dependent, with AUC increased (75-245%), and clearance decreased (35-72%), for phenacetin, metoprolol and chlorzoxazone. Tolbutamide disposition was not significantly altered by Cyb5 deletion, while for midazolam clearance was decreased by 66%. The absence of Cyb5 had no effect on gefitinib and paclitaxel disposition, while significant changes in the in vivo pharmacokinetics of cyclophosphamide were measured (Cmax and terminal half-life increased 55% and 40%, respectively), tamoxifen (AUClast and Cmax increased 370% and 233%, respectively) and anastrozole (AUC and terminal half-life increased 125% and 62%, respectively; clearance down 80%). These data from provide strong evidence that both hepatic and extra-hepatic Cyb5 levels are an important determinant of in vivo drug disposition catalysed by a range of cytochrome P450s, including currently-prescribed anti-cancer agents, and that individuality in Cyb5 expression could be a significant determinant in rates of drug disposition in man. PMID:24115751

  18. Identification of two new cytochrome P450 genes and RNA interference to evaluate their roles in detoxification of commonly used insecticides in Locusta migratoria.

    PubMed

    Guo, Yanqiong; Zhang, Jianzhen; Yu, Rongrong; Zhu, Kun Yan; Guo, Yaping; Ma, Enbo

    2012-05-01

    Cytochrome P450 monooxygenases (cytochrome P450s), found in virtually all living organisms, play an important role in the metabolism of xenobiotics such as drugs, pesticides, and plant toxins. We have previously evaluated the responses of the oriental migratory locust (Locusta migratoria) to the pyrethroid insecticide deltamethrin and revealed that increased cytochrome P450 enzyme activity was due to increased transcription of multiple cytochrome P450 genes. In this study, we identified for the first time two new cytochrome P450 genes, which belong to two novel cytochrome P450 gene families. CYP409A1 belongs to CYP409 family whereas CYP408B1 belongs to CYP408 family. Our molecular analysis indicated that CYP409A1 was mainly expressed in fatbodies, midgut, gastric caecum, foregut and Malpighian tubules of the third- and fourth-instar nymphs, whereas CYP408B1 was mainly expressed in foregut, hindgut and muscle of the insects at all developmental stages examined. The expression of these two cytochrome P450 genes were differentially affected by three representative insecticides, including carbaryl (carbamate), malathion (organophosphate) and deltamethrin (pyrethroid). The exposure of the locust to carbaryl, malathion and deltamethrin resulted in reduced, moderately increased and significantly increased transcript levels, respectively, of the two cytochrome P450 genes. Our further analysis of their detoxification roles by using RNA interference followed by deltamethrin bioassay showed increased nymph mortalities by 21.1% and 16.7%, respectively, after CYP409A1 and CYP408B1 were silenced. These results strongly support our notion that these two new cytochrome P450 genes play an important role in deltamethrin detoxification in the locust. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Cytochrome P450, CYP93A1, as a defense marker in soybean

    USDA-ARS?s Scientific Manuscript database

    CYP93A1 is a cytochrome P450 that is involved in the synthesis of the phytoalexin glyceollin in soybean (Glycine max L. Merr). The gene encoding CYP93A1 has been used as a defense marker in soybean cell cultures, however, little is known regarding how this gene is expressed in the intact plant. To f...

  20. Menadione Suppresses Benzo(α)pyrene-Induced Activation of Cytochromes P450 1A: Insights into a Possible Molecular Mechanism

    PubMed Central

    Pivovarova, Elena N.; Markel, Arkady L.; Lyakhovich, Vyacheslav V.; Grishanova, Alevtina Y.

    2016-01-01

    Oxidative reactions that are catalyzed by cytochromes P450 1A (CYP1A) lead to formation of carcinogenic derivatives of arylamines and polycyclic aromatic hydrocarbons (PAHs), such as the widespread environmental pollutant benzo(α)pyrene (BP). These compounds upregulate CYP1A at the transcriptional level via an arylhydrocarbon receptor (AhR)-dependent signaling pathway. Because of the involvement of AhR-dependent genes in chemically induced carcinogenesis, suppression of this signaling pathway could prevent tumor formation and/or progression. Here we show that menadione (a water-soluble analog of vitamin K3) inhibits BP-induced expression and enzymatic activity of both CYP1A1 and CYP1A2 in vivo (in the rat liver) and BP-induced activity of CYP1A1 in vitro. Coadministration of BP and menadione reduced DNA-binding activity of AhR and increased DNA-binding activity of transcription factors Oct-1 and CCAAT/enhancer binding protein (C/EBP), which are known to be involved in negative regulation of AhR-dependent genes, in vivo. Expression of another factor involved in downregulation of CYP1A—pAhR repressor (AhRR)—was lower in the liver of the rats treated with BP and menadione, indicating that the inhibitory effect of menadione on CYP1A is not mediated by this protein. Furthermore, menadione was well tolerated by the animals: no signs of acute toxicity were detected by visual examination or by assessment of weight gain dynamics or liver function. Taken together, our results suggest that menadione can be used in further studies on animal models of chemically induced carcinogenesis because menadione may suppress tumor formation and possibly progression. PMID:27167070

  1. Promising Tools in Prostate Cancer Research: Selective Non-Steroidal Cytochrome P450 17A1 Inhibitors

    PubMed Central

    Bonomo, Silvia; Hansen, Cecilie H.; Petrunak, Elyse M.; Scott, Emily E.; Styrishave, Bjarne; Jørgensen, Flemming Steen; Olsen, Lars

    2016-01-01

    Cytochrome P450 17A1 (CYP17A1) is an important target in the treatment of prostate cancer because it produces androgens required for tumour growth. The FDA has approved only one CYP17A1 inhibitor, abiraterone, which contains a steroidal scaffold similar to the endogenous CYP17A1 substrates. Abiraterone is structurally similar to the substrates of other cytochrome P450 enzymes involved in steroidogenesis, and interference can pose a liability in terms of side effects. Using non-steroidal scaffolds is expected to enable the design of compounds that interact more selectively with CYP17A1. Therefore, we combined a structure-based virtual screening approach with density functional theory (DFT) calculations to suggest non-steroidal compounds selective for CYP17A1. In vitro assays demonstrated that two such compounds selectively inhibited CYP17A1 17α-hydroxylase and 17,20-lyase activities with IC50 values in the nanomolar range, without affinity for the major drug-metabolizing CYP2D6 and CYP3A4 enzymes and CYP21A2, with the latter result confirmed in human H295R cells. PMID:27406023

  2. CYTOCHROME P450 17A1 STRUCTURES WITH PROSTATE CANCER DRUGS ABIRATERONE AND TOK-001

    PubMed Central

    DeVore, Natasha M.; Scott, Emily E.

    2011-01-01

    Cytochrome P450 17A1 (P450c17) catalyzes the biosynthesis of androgens in humans1. Since prostate cancer cells proliferate in response to androgen steroids2,3, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer4, but drug development has been hampered by the lack of a CYP17A1 structure. Here we report the only known structures of CYP17A1, which contain either abiraterone, a first-in-class steroidal inhibitor recently approved by the FDA for late-stage prostate cancer5, or TOK-001, another inhibitor in clinical trials4,6. Both bind the heme iron forming a 60° angle above the heme plane, packing against the central I helix with the 3β-OH interacting with N202 in the F helix. Importantly, this binding mode differs substantially from those predicted by homology models or from steroids in other cytochrome P450 enzymes with known structures, with some features more similar to steroid receptors. While the overall CYP17A1 structure provides a rationale for understanding many mutations found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate better understanding of the enzyme’s dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers. PMID:22266943

  3. INDUCTION OF CYTOCHROME P450 ISOFORMS IN RAT LIVER BY TWO CONAZOLES, TRIADIMEFON AND MYCLOBUTANIL

    EPA Science Inventory

    1. This study was undertaken to examine the inductive effects of two triazole antifungal agents, myclobutanil and triadimefon on the expression of hepatic cytochrome P450 (CYP) genes and on the activities of CYP enzymes in male Sprague-Dawley rats. Rats were dosed by gavage for 1...

  4. Structure of bovine cytochrome c oxidase crystallized at a neutral pH using a fluorinated detergent.

    PubMed

    Luo, Fangjia; Shinzawa-Itoh, Kyoko; Hagimoto, Kaede; Shimada, Atsuhiro; Shimada, Satoru; Yamashita, Eiki; Yoshikawa, Shinya; Tsukihara, Tomitake

    2017-07-01

    Cytochrome c oxidase (CcO) couples proton pumping to O 2 reduction. Its enzymatic activity depends sensitively on pH over a wide range. However, owing to difficulty in crystallizing this protein, X-ray structure analyses of bovine CcO aimed at understanding its reaction mechanism have been conducted using crystals prepared at pH 5.7, which is significantly lower than that in the cell. Here, oxidized CcO at pH 7.3 was crystallized using a fluorinated octyl-maltoside derivative, and the structure was determined at 1.77 Å resolution. No structural differences between crystals obtained at the neutral pH and the acidic pH were detected within the molecules. On the other hand, some differences in intermolecular interactions were detected between the two types of crystal. The influence of pH on the molecular surface is likely to contribute to the pH dependency of the aerobic oxidation of ferrocytochrome c.

  5. Molecular cloning and functional characterization of multiple NADPH-cytochrome P450 reductases from Andrographis paniculata.

    PubMed

    Lin, Huixin; Wang, Jian; Qi, Mengdie; Guo, Juan; Rong, Qixian; Tang, Jinfu; Wu, Yisheng; Ma, Xiaojing; Huang, Luqi

    2017-09-01

    Andrographis paniculata (Burm.f.) Wall. ex Nees is widely used as medicinal herb in Southern and Southeastern Asia and andrographolide is its main medicinal constituent. Based on the structure of andrographolide, it has been proposed that cytochrome P450 enzymes play vital roles on its biosynthesis. NADPH:cytochrome P450 reductase (CPR) is the most important redox partner of multiple P450s. In this study, three CPRs were identified in the genomic data of A. paniculata (namely ApCPR1, ApCPR2, and ApCPR3), and their coding regions were cloned. They varied from 62% to 70% identities to each other at the amino acid sequence level. ApCPR1 belongs to Class I of dicotyledonous CPR while both ApCPR2 and ApCPR3 are grouped to Class II. The recombinant enzymes ApCPR1 and ApCPR2 reduced cytochrome c and ferricyanide in an NADPH-dependent manner. In yeast, they supported the activity of CYP76AH1, a ferruginol-forming enzyme. However, ApCPR3 did not show any enzymatic activities either in vitro or in vivo. Quantitative real-time PCR analysis showed that both ApCPR1 and ApCPR2 expressed in all tissues examined, but ApCPR2 showed higher expression in leaves. Expression of ApCPR2 was inducible by MeJA and its pattern matched with andrographolide accumulation. Present investigation suggested ApCPR2 involves in the biosynthesis of secondary metabolites including andrographolide. Copyright © 2017. Published by Elsevier B.V.

  6. Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

    PubMed Central

    Bloomer, J C; Baldwin, S J; Smith, G J; Ayrton, A D; Clarke, S E; Chenery, R J

    1994-01-01

    1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation. PMID:7888294

  7. Cytochrome P450IA mRNA expression in feral Hudson River tomcod

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kreamer, G.L.; Squibb, K.; Gioeli, D.

    1991-06-01

    The authors sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, they found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached bymore » 5 days. Intraperitoneal injection of {beta}-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers.« less

  8. Flexible docking-based molecular dynamics/steered molecular dynamics calculations of protein-protein contacts in a complex of cytochrome P450 1A2 with cytochrome b5.

    PubMed

    Jeřábek, Petr; Florián, Jan; Stiborová, Marie; Martínek, Václav

    2014-10-28

    Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2-cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2-cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450-cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.

  9. Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

    PubMed

    Shak, S; Goldstein, I M

    1985-09-01

    Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.

  10. Assessment of toxic potency of complex mixtures of PAHs from Lincoln Creek, Milwaukee, WI

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Villeneuve, D.; Crunkilton, R.; DeVita, W.

    1995-12-31

    An assay of cytochrome P4501A catalytic activity in PLHC-1 fish hepatoma cells was used to evaluate the toxic potency of dialysates from triolein filled semipermeable polymeric membrane devices (SPMDS) exposed for variable durations and under various flow regimes to water from Lincoln Creek. Toxic potency was expressed as 2,3,7,8 tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) calculated from bioassay results. Dose-dependent responses in measured ethoxyresorufin-o-deethylase (EROD) activity of PLHC-1 cells exposed to SPMD dialysates were shown. Toxic potency of dialysates, expressed as bioassay derived TCDD equivalents, increased with duration of SPMD exposure in Lincoln Creek from 2.0 pg/uL for a 2 day exposure tomore » 19.5 pg/uL for a 30 day exposure. This corresponded to an increase in dialysate polycyclic aromatic hydrocarbon (PAH) concentration from 8.82 ug/g after a 2 day exposure to 24.14 ug/g after 30 days. Dialysates from SPMDs exposed to Lincoln Creek stormflow had higher toxic potencies and total PAH concentrations than those exposed to baseflow only, These results suggest that levels of PAH contamination, particularly those associated with stormflow, in Lincoln Creek have potential to accumulate in fish to levels significant enough to elicit a measurable biological response (cytochrome P4501 A induction) at a potency level approaching 0.08% that of TCDD.« less

  11. Binding Specificity Determines the Cytochrome P450 3A4 Mediated Enantioselective Metabolism of Metconazole.

    PubMed

    Zhuang, Shulin; Zhang, Leili; Zhan, Tingjie; Lu, Liping; Zhao, Lu; Wang, Haifei; Morrone, Joseph A; Liu, Weiping; Zhou, Ruhong

    2018-01-25

    Cytochrome P450 3A4 (CYP3A4) is a promiscuous enzyme, mediating the biotransformations of ∼50% of clinically used drugs, many of which are chiral molecules. Probing the interactions between CYP3A4 and chiral chemicals is thus essential for the elucidation of molecular mechanisms of enantioselective metabolism. We developed a stepwise-restrained-molecular-dynamics (MD) method to model human CYP3A4 in a complex with cis-metconazole (MEZ) isomers and performed conventional MD simulations with a total simulation time of 2.2 μs to probe the molecular interactions. Our current study, which employs a combined experimental and theoretical approach, reports for the first time on the distinct conformational changes of CYP3A4 that are induced by the enantioselective binding of cis-MEZ enantiomers. CYP3A4 preferably metabolizes cis-RS MEZ over the cis-SR isomer, with the resultant enantiomer fraction for cis-MEZ increasing rapidly from 0.5 to 0.82. cis-RS MEZ adopts a more extended structure in the active pocket with its Cl atom exposed to the solvent, whereas cis-SR MEZ sits within the hydrophobic core of the active pocket. Free-energy-perturbation calculations indicate that unfavorable van der Waals interactions between the cis-MEZ isomers and the CYP3A4 binding pocket predominantly contribute to their binding-affinity differences. These results demonstrate that binding specificity determines the cytochrome P450 3A4 mediated enantioselective metabolism of cis-MEZ.

  12. Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme

    DOEpatents

    Marrone, Babetta L.; Simpson, Daniel J.; Unkefer, Clifford J.; Whaley, Thomas W.

    1992-01-01

    An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

  13. Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme

    DOEpatents

    Marrone, Babetta L.; Simpson, Daniel J.; Unkefer, Clifford J.; Whaley, Thomas W.

    1993-01-01

    An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

  14. Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B sub 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aoyama, Toshifumi; Yamano, Shigeru; Gelboin, H.V.

    Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B{sub 1} to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B{sub 1} to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B{sub 1} to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cellsmore » expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, and IIIA5, and IVB1, did not significantly activate aflatoxin B{sub 1} as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B{sub 1} activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B{sub 1} in human liver involves the contribution of multiple forms of P450.« less

  15. Functional characterization of NADPH-cytochrome P450 reductase from Bactrocera dorsalis: Possible involvement in susceptibility to malathion

    PubMed Central

    Huang, Yong; Lu, Xue-Ping; Wang, Luo-Luo; Wei, Dong; Feng, Zi-Jiao; Zhang, Qi; Xiao, Lin-Fan; Dou, Wei; Wang, Jin-Jun

    2015-01-01

    NADPH cytochrome P450 reductase (CPR) is essential for cytochrome P450 catalysis, which is important in the detoxification and activation of xenobiotics. In this study, two transcripts of Bactrocera dorsalis CPR (BdCPR) were cloned, and the deduced amino-acid sequence had an N-terminus membrane anchor for BdCPR-X1 and three conserved binding domains (FMN, FAD, and NADP), as well as an FAD binding motif and catalytic residues for both BdCPR-X1 and BdCPR-X2. BdCPR-X1 was detected to have the high expression levels in adults and in Malpighian tubules, fat bodies, and midguts of adults, but BdCPR-X2 expressed lowly in B. dorsalis. The levels of BdCPRs were similar in malathion-resistant strain compared to susceptible strain. However, injecting adults with double-stranded RNA against BdCPR significantly reduced the transcript levels of the mRNA, and knockdown of BdCPR increased adult susceptibility to malathion. Expressing complete BdCPR-X1 cDNA in Sf9 cells resulted in high activity determined by cytochrome c reduction and these cells had higher viability after exposure to malathion than control. The results suggest that BdCPR could affect the susceptibility of B. dorsalis to malathion and eukaryotic expression of BdCPR would lay a solid foundation for further investigation of P450 in B. dorsalis. PMID:26681597

  16. Cytochrome P450 CYP716A254 catalyzes the formation of oleanolic acid from β-amyrin during oleanane-type triterpenoid saponins biosynthesis in Anemone flaccida.

    PubMed

    Zhan, Chuansong; Ahmed, Shakeel; Hu, Sheng; Dong, Shuang; Cai, Qian; Yang, Tewu; Wang, Xuekui; Li, Xiaohua; Hu, Xuebo

    2018-01-01

    Anemone flaccida Fr. Shmidt (Ranunculaceae), known as 'Di Wu' in China, is a perennial herb which has long been used to treat arthritis. The rhizome of A. flaccida contains pharmacologically active components i.e. oleanane-type triterpenoid saponins. Oleanolic acid is natural triterpenoid in plants with diverse biological activities. The biosynthesis of oleanolic acid involves cyclization of 2,3-oxidosqualene to the oleanane-type triterpenoid skeleton, followed by a series of oxidation reactions catalyzed by cytochrome P450 monooxygenase (CYP450). Previously, we identified four possible cytochrome P450 genes belonging to CYP716A subfamily from the transcriptome of A. flaccida. In this study, we identified one of those genes "CYP716A254" encoding a cytochrome P450 monooxygenase from A. flaccida that catalyzes the conversion of the β-amyrin into oleanolic acid. The heterologous expression of CYP716A254 in yeast resulted in oxidation of β-amyrin at the C-18 position to oleanolic acid production. These results provide an important basis for further studies of oleanane-type triterpenoid saponins synthesis in A. flaccida. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Drug-enhanced carbon monoxide production from heme by cytochrome P450 reductase.

    PubMed

    Vukomanovic, Dragic; Rahman, Mona N; Jia, Zongchao; Nakatsu, Kanji

    2017-01-01

    Carbon monoxide (CO) formed endogenously is considered to be cytoprotective, and the vast majority of CO formation is attributed to the degradation of heme by heme oxygenases-1 and -2 (HO-1, HO-2). Previously, we observed that brain microsomes containing HO-2 produced many-fold more CO in the presence of menadione and its congeners; herein we explored these observations further. We determined the effects of various drugs on CO production of rat brain microsomes and recombinant human cytochrome P450 reductase (CPR); CO was measured by gas chromatography with reductive detection. Brain microsomes of Sprague-Dawley rats or recombinant human cytochrome P450 reductase (CPR) were incubated with NADPH and various drugs in closed vials in phosphate buffer at pH 7.4 and 37°C. After 15 minutes, the reaction was stopped by cooling in dry ice, and the headspace gas was analyzed for CO production using gas chromatography with reductive (mercuric oxide) detection. We observed drug-enhanced CO production in the presence of both microsomes and recombinant CPR alone; the presence of HO was not required. A range of structurally diverse drugs were capable of amplifying this CO formation; these molecules had structures consistent with redox cycling capability. The addition of catalase to a reaction mixture, that contained activating drugs, inhibited the production of CO. Drug-enhanced CO formation can be catalyzed by CPR. The mechanism of CPR activation was not through classical drug-receptor mediation. Redox cycling may be involved in the drug-induced amplification of CO production by CPR through the production of reactive oxygen species.

  18. Differences in activity of cytochrome C oxidase in brain between sleep and wakefulness.

    PubMed

    Nikonova, Elena V; Vijayasarathy, Camasamudram; Zhang, Lin; Cater, Jacqueline R; Galante, Raymond J; Ward, Stephen E; Avadhani, Narayan G; Pack, Allan I

    2005-01-01

    Increased mRNA level of subunit 1 cytochrome c oxidase (COXI) during wakefulness and after short-term sleep deprivation has been described in brain. We hypothesized that this might contribute to increased activity of cytochrome oxidase (COX) enzyme during wakefulness, as part of the mechanisms to provide sufficient amounts of adenosine triphosphate to meet increased neuronal energy demands. COX activity was measured in isolated mitochondria from different brain regions in groups of rats with 3 hours of spontaneous sleep, 3 hours of spontaneous wake, and 3 hours of sleep deprivation. The group with 3 hours of spontaneous wake was added to delineate the circadian component of changes in the enzyme activity. Northern blot analysis was performed to examine the mRNA levels of 2 subunits of the enzyme COXI and COXIV, encoded by mitochondrial and nuclear DNA, respectively. Laboratory of Biochemistry, Department of Animal Biology, and Center for Sleep and Respiratory Neurobiology, University of Pennsylvania. 2-month-old male Fischer rats (N = 21) implanted for polygraphic recording. For COX activity, there was a main effect by analysis of variance of experimental group (P < .0001) with significant increases in COX activity in wake and sleep-deprived groups as compared to the sleep group. A main effect of brain region was also significant (P < .001). There was no difference between brain regions in the degree of increase in enzyme activity in wakefulness. Both COXI and COXIV mRNA were increased with wakefulness as compared to sleep. There is an increase in COX activity after both 3 hours of spontaneous wake and 3 hours of sleep deprivation as compared with 3 hours of spontaneous sleep in diverse brain regions, which could be, in part, explained by the increased levels of bigenomic transcripts of the enzyme. This likely contributes to increased adenosine triphosphate production during wakefulness. ADP, adenosine diphosphate; ATP, adenosine triphosphate; COXI, cytochrome c

  19. Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1

    PubMed Central

    Pflugmacher, Stephan; Sandermann, Heinrich

    1998-01-01

    The metabolism of xenobiotics has mainly been investigated in higher plant species. We studied them in various marine macroalgae of the phyla Chlorophyta, Chromophyta, and Rhodophyta. Microsomes contained high oxidative activities for known cytochrome (Cyt) P450 substrates (fatty acids, cinnamic acid, 3- and 4-chlorobiphenyl, 2,3-dichlorobiphenyl, and isoproturon; up to 54 pkat/mg protein). The presence of Cyt P450 (approximately 50 pmol/mg protein) in microsomes of the three algal families was demonstrated by CO-difference absorption spectra. Intact algal tissue converted 3-chlorobiphenyl to the same monohydroxy-metabolite formed in vitro. This conversion was 5-fold stimulated upon addition of phenobarbital, and was abolished by the known P450 inhibitor, 1-aminobenzotriazole. It is concluded that marine macroalgae contain active species of Cyt P450 and could act as a metabolic sink for marine pollutants. PMID:9576781

  20. High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.

    PubMed

    Tsotsou, Georgia Eleni; Cass, Anthony Edward George; Gilardi, Gianfranco

    2002-01-01

    A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts.

  1. Functional Characterization of the Small Regulatory Subunit PetP from the Cytochrome b6f Complex in Thermosynechococcus elongatus[C][W

    PubMed Central

    Rexroth, Sascha; Rexroth, Dorothea; Veit, Sebastian; Plohnke, Nicole; Cormann, Kai U.; Nowaczyk, Marc M.; Rögner, Matthias

    2014-01-01

    The cyanobacterial cytochrome b6f complex is central for the coordination of photosynthetic and respiratory electron transport and also for the balance between linear and cyclic electron transport. The development of a purification strategy for a highly active dimeric b6f complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 enabled characterization of the structural and functional role of the small subunit PetP in this complex. Moreover, the efficient transformability of this strain allowed the generation of a ΔpetP mutant. Analysis on the whole-cell level by growth curves, photosystem II light saturation curves, and P700+ reduction kinetics indicate a strong decrease in the linear electron transport in the mutant strain versus the wild type, while the cyclic electron transport via photosystem I and cytochrome b6f is largely unaffected. This reduction in linear electron transport is accompanied by a strongly decreased stability and activity of the isolated ΔpetP complex in comparison with the dimeric wild-type complex, which binds two PetP subunits. The distinct behavior of linear and cyclic electron transport may suggest the presence of two distinguishable pools of cytochrome b6f complexes with different functions that might be correlated with supercomplex formation. PMID:25139006

  2. Construction and application of a functional library of cytochrome P450 monooxygenases from the filamentous fungus Aspergillus oryzae.

    PubMed

    Nazir, K H M Nazmul Hussain; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2011-05-01

    A functional library of cytochrome P450 monooxygenases from Aspergillus oryzae (AoCYPs) was constructed in which 121 isoforms were coexpressed with yeast NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae. Using this functional library, novel catalytic functions of AoCYPs, such as catalytic potentials of CYP57B3 against genistein, were elucidated for the first time. Comprehensive functional screening promises rapid characterization of catalytic potentials and utility of AoCYPs.

  3. Inhibition of Cytochrome P450 (CYP3A4) Activity by Extracts from 57 Plants Used in Traditional Chinese Medicine (TCM)

    PubMed Central

    Ashour, Mohamed L; Youssef, Fadia S; Gad, Haidy A; Wink, Michael

    2017-01-01

    Background: Herbal medicine is widely used all over the world for treating various health disorders. It is employed either alone or in combination with synthetic drugs or plants to be more effective. Objective: The assessment of the effect of both water and methanol extracts of 57 widely used plants from Traditional Chinese Medicine (TCM) against the main phase I metabolizing enzyme CYP3A4 in vitro for the first time. Materials and Methods: The inhibition of cytochrome P450 activity was evaluated using a luminescence assay. The principal component analysis (PCA) was used to correlate the inhibitory activity with the main secondary metabolites present in the plant extracts. Molecular modeling studies on CYP3A4 (PDB ID 4NY4) were carried out with 38 major compounds present in the most active plant extracts to validate the observed inhibitory effect. Results: Aqueous extracts of Acacia catechu, Andrographis paniculata, Arctium lappa, Areca catechu, Bupleurum marginatum, Chrysanthemum indicum, Dysosma versipellis, and Spatholobus suberectus inhibited CYP3A4 is more than 85% (at a dose of 100 μg/mL). The corresponding methanol extracts of A. catechu, A. paniculata, A. catechu, Mahonia bealei, and Sanguisorba officinalis inhibited the enzyme by more than 50%. Molecular modeling studies revealed that two polyphenols, namely hesperidin and rutin, revealed the highest fitting scores in the active sites of the CYP3A4 with binding energies equal to -74.09 and -71.34 kcal/mol, respectively. Conclusion: These results provide evidence that many TCM plants can inhibit CYP3A4, which might cause a potential interference with the metabolism of other concomitantly administered herbs or drugs. SUMMARY In this study, the inhibitory activity of the aqueous and methanol extracts of 57 widely used plants from Traditional Chinese Medicine (TCM) against the main phase I metabolizing enzyme CYP3A4 was tested in vitro for the first time.Aqueous extracts of Acacia catechu, Andrographis

  4. Nanoscale Electron Transport Measurements of Immobilized Cytochrome P450 Proteins

    PubMed Central

    Bostick, Christopher D.; Flora, Darcy R.; Gannett, Peter M.; Tracy, Timothy S.; Lederman, David

    2015-01-01

    Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of electron transport processes in the enzyme, in addition to occupying the active site. PMID:25804257

  5. Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu

    2013-02-15

    The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomicmore » approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed

  6. A Prenylated p47phox-p67phox-Rac1 Chimera Is a Quintessential NADPH Oxidase Activator

    PubMed Central

    Mizrahi, Ariel; Berdichevsky, Yevgeny; Casey, Patrick J.; Pick, Edgar

    2010-01-01

    The superoxide-generating NADPH oxidase complex of resting phagocytes includes cytochrome b559, a membrane-associated heterodimer composed of two subunits (Nox2 and p22phox), and four cytosolic proteins (p47phox, p67phox, Rac, and p40phox). Upon stimulation, the cytosolic components translocate to the membrane, as the result of a series of interactions among the cytosolic components and among the cytosolic components and cytochrome b559 and its phospholipid environment. We described the construction of a tripartite chimera (trimera) consisting of strategic domains of p47phox, p67phox, and Rac1, in which interactions among cytosolic components were replaced by fusion (Berdichevsky, Y., Mizrahi, A., Ugolev, Y., Molshanski-Mor, S., and Pick, E. (2007) J. Biol. Chem. 282, 22122–22139). We now fused green fluorescent protein (GFP) to the N terminus of the trimera and found the following. 1) The GFP-p47phox-p67phox-Rac1 trimera activates the oxidase in amphiphile-dependent and -independent (anionic phospholipid-enriched membrane) cell-free systems. 2) Geranylgeranylation of the GFP-trimera makes it a potent oxidase activator in unmodified (native) membranes and in the absence of amphiphile. 3) Prenylated GFP-trimera binds spontaneously to native membranes (as assessed by gel filtration and in-line fluorometry), forming a tight complex capable of NADPH-dependent, activator-independent superoxide production at rates similar to those measured in canonical cell-free systems. 4) Prenylation of the GFP-trimera supersedes completely the dependence of oxidase activation on the p47phox phox homology domain and, partially, on the Rac1 polybasic domain, but the requirement for Trp193 in p47phox persists. Prenylated GFP-p47phox-p67phox-Rac1 trimera acts as a quintessential single molecule oxidase activator of potential use in high throughput screening of inhibitors. PMID:20529851

  7. An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications.

    PubMed

    Brixius-Anderko, Simone; Hannemann, Frank; Ringle, Michael; Khatri, Yogan; Bernhardt, Rita

    2017-05-01

    Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500 μM, which is within the physiological concentration range occurring during cultivation of E. coli in complex medium. Biotransformations with C43(DE3)_∆tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  8. Mutational analysis of the Saccharomyces cerevisiae cytochrome c oxidase assembly protein Cox11p.

    PubMed

    Banting, Graham S; Glerum, D Moira

    2006-03-01

    Cox11p is an integral protein of the inner mitochondrial membrane that is essential for cytochrome c oxidase assembly. The bulk of the protein is located in the intermembrane space and displays high levels of evolutionary conservation. We have analyzed a collection of site-directed and random cox11 mutants in an effort to further define essential portions of the molecule. Of the alleles studied, more than half had no apparent effect on Cox11p function. Among the respiration deficiency-encoding alleles, we identified three distinct phenotypes, which included a set of mutants with a misassembled or partially assembled cytochrome oxidase, as indicated by a blue-shifted cytochrome aa(3) peak. In addition to the shifted spectral signal, these mutants also display a specific reduction in the levels of subunit 1 (Cox1p). Two of these mutations are likely to occlude a surface pocket behind the copper-binding domain in Cox11p, based on analogy with the Sinorhizobium meliloti Cox11 solution structure, thereby suggesting that this pocket is crucial for Cox11p function. Sequential deletions of the matrix portion of Cox11p suggest that this domain is not functional beyond the residues involved in mitochondrial targeting and membrane insertion. In addition, our studies indicate that Deltacox11, like Deltasco1, displays a specific hypersensitivity to hydrogen peroxide. Our studies provide the first evidence at the level of the cytochrome oxidase holoenzyme that Cox1p is the in vivo target for Cox11p and suggest that Cox11p may also have a role in the response to hydrogen peroxide exposure.

  9. Flower colour and cytochromes P450.

    PubMed

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-02-19

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

  10. Cytochrome P450-derived eicosanoids: the neglected pathway in cancer

    PubMed Central

    Kaipainen, Arja; Greene, Emily R.; Huang, Sui

    2010-01-01

    Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed. PMID:20941528

  11. Toxic responses of cytochrome P450 sub-enzyme activities to heavy metals exposure in soil and correlation with their bioaccumulation in Eisenia fetida.

    PubMed

    Cao, Xiufeng; Bi, Ran; Song, Yufang

    2017-10-01

    The dose- and time- dependent responses of cytochrome P450 (CYP) sub-enzyme activities to heavy metals in soil, and the relationships between biomarker responses and metal bioaccumulation in Eisenia fetida were evaluated. Earthworms were exposed to soils spiked with increasing doses of Cd, Cu, Pb or Zn for 21 d. Results demonstrated that EROD and CYP3A4 activities responded significantly with increasing dose and exposure duration. EROD activity significantly (P < 0.05) correlated with CYP3A4 activity exposed to Pb and Cu. The earthworm metal burdens had significant correlation with the total metal concentrations in soil (P < 0.01). The bioaccumulation factor (BAF) decreased with the increasing metal concentration in soil. The order of metal bioavailability to E. fetida was Cd > Zn > Cu > Pb. CYP3A4 activity in Pb-exposed earthworms had a significant correlation with the accumulated metal (P < 0.05). Both EROD and CYP3A4 activities in Cu-exposed worms negatively correlated with BAF (P < 0.05). Based on Discriminant Analysis (DA), CYPs activities were sensitive biomarkers of heavy metals exposure, and we also concluded that different biomarkers with multiple durations could be conducted in the eco-toxicological diagnosis of soil pollution. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. N-Heterocyclic Carbene Capture by Cytochrome P450 3A4

    PubMed Central

    Jennings, Gareth K.; Ritchie, Caroline M.; Shock, Lisa S.; Lyons, Charles E.

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the dominant P450 enzyme involved in human drug metabolism, and its inhibition may result in adverse interactions or, conversely, favorably reduce the systemic elimination rates of poorly bioavailable drugs. Herein we describe a spectroscopic investigation of the interaction of CYP3A4 with N-methylritonavir, an analog of ritonavir, widely used as a pharmacoenhancer. In contrast to ritonavir, the binding affinity of N-methylritonavir for CYP3A4 is pH-dependent. At pH <7.4, the spectra are definitively type I, whereas at pH ≥7.4 the spectra have split Soret bands, including a red-shifted component characteristic of a P450-carbene complex. Variable-pH UV-visible spectroscopy binding studies with molecular fragments narrows the source of this pH dependence to its N-methylthiazolium fragment. The C2 proton of this group is acidic, and variable-pH resonance Raman spectroscopy tentatively assigns it a pKa of 7.4. Hence, this fragment of N-methylritonavir is expected to be readily deprotonated under physiologic conditions to yield a thiazol-2-ylidene, which is an N-heterocyclic carbene that has high-affinity for and is presumed to be subsequently captured by the heme iron. This mechanism is supported by time-dependent density functional theory with an active site model that accurately reproduces distinguishing features of the experimental UV-visible spectra of N-methylritonavir bound to CYP3A4. Finally, density functional theory calculations support that this novel interaction is as strong as the tightest-binding azaheterocycles found in P450 inhibitors and could offer new avenues for inhibitor development. PMID:27126611

  13. Molecular characterization and functional analysis of three pathogenesis-related cytochrome P450 genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea).

    PubMed

    Xu, Xiao-Lu; Wu, Xiao-Qin; Ye, Jian-Ren; Huang, Lin

    2015-03-06

    Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.

  14. Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

    USGS Publications Warehouse

    Stolz, J.F.; Gugliuzza, T.; Switzer, Blum J.; Oremland, R.; Martinez, Murillo F.

    1997-01-01

    The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.

  15. Cytochrome P450 CYP1A1: wider roles in cancer progression and prevention

    PubMed Central

    2009-01-01

    CYP1A1 is one of the main cytochrome P450 enzymes, examined extensively for its capacity to activate compounds with carcinogenic properties. Continuous exposure to inhalation chemicals and environmental carcinogens is thought to increase the level of CYP1A1 expression in extrahepatic tissues, through the aryl hydrocarbon receptor (AhR). Although the latter has long been recognized as a ligand-induced transcription factor, which is responsible for the xenobiotic activating pathway of several phase I and phase II metabolizing enzymes, recent evidence suggests that the AhR is involved in various cell signaling pathways critical to cell cycle regulation and normal homeostasis. Disregulation of these pathways is implicated in tumor progression. In addition, it is becoming increasingly evident that CYP1A1 plays an important role in the detoxication of environmental carcinogens, as well as in the metabolic activation of dietary compounds with cancer preventative activity. Ultimately the contribution of CYP1A1 to cancer progression or prevention may depend on the balance of procarcinogen activation/detoxication and dietary natural product extrahepatic metabolism. PMID:19531241

  16. Inhibition of human cytochrome P450 2E1 and 2A6 by aldehydes: structure and activity relationships.

    PubMed

    Kandagatla, Suneel K; Mack, Todd; Simpson, Sean; Sollenberger, Jill; Helton, Eric; Raner, Gregory M

    2014-08-05

    The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes. For saturated aldehydes the optimum chain length for inhibition of 2E1 was 9 carbons (KI=7.8 ± 0.3 μM), whereas for 2A6 heptanal was most potent (KI=15.8 ± 1.1 μM). A double bond in the 2-position of the aldehyde significantly decreased the observed KI relative to the corresponding saturated compound in most cases. A clear difference in the effect of the double bond was observed between the two isoforms. With 2E1, the double bond appeared to remove steric constraints on aldehyde binding with KI values for the 5-12 carbon compounds ranging between 2.6 ± 0.1 μM and 12.8 ± 0.5 μM, whereas steric effects remained the dominant factor in the binding of the unsaturated aldehydes to 2A6 (observed KI values between 7.0 ± 0.5 μM and >1000 μM). The aldehyde function was essential for effective inhibition, as the corresponding carboxylic acids had very little effect on enzyme activity over the same range of concentrations, and branching at the 3-position of the aldehydes increased the corresponding KI value in all cases examined. The results suggest that a conjugated π-system may be a key structural determinant in the binding of these compounds to both enzymes, and may also be an important feature for the expansion of the active site volume in 2E1. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. Inhibition of human Cytochrome P450 2E1 and 2A6 by aldehydes: Structure and activity relationships

    PubMed Central

    Kandagatla, Suneel K.; Mack, Todd; Simpson, Sean; Sollenberger, Jill; Helton, Eric; Raner, Gregory M.

    2014-01-01

    The purpose of this study was to probe active site structure and dynamics of human cytochrome P4502E1 and P4502A6 using a series of related short chain fatty aldehydes. Binding efficiency of the aldehydes was monitored via their ability to inhibit the binding and activation of the probe substrates p-nitrophenol (2E1) and coumarin (2A6). Oxidation of the aldehydes was observed in reactions with individually expressed 2E1, but not 2A6, suggesting alternate binding modes. For saturated aldehydes the optimum chain length for inhibition of 2E1 was 9 carbons (KI=7.8 ±0.3 μM), whereas for 2A6 heptanal was most potent (KI=15.8 ±1.1 μM). A double bond in the 2-position of the aldehyde significantly decreased the observed KI relative to the corresponding saturated compound in most cases. A clear difference in the effect of the double bond was observed between the two isoforms. With 2E1, the double bond appeared to remove steric constraints on aldehyde binding with KI values for the 5–12 carbon compounds ranging between 2.6 ± 0.1 μM and 12.8± 0.5 μM, whereas steric effects remained the dominant factor in the binding of the unsaturated aldehydes to 2A6 (observed KI values between 7.0± 0.5 μM and >1000 μM). The aldehyde function was essential for effective inhibition, as the corresponding carboxylic acids had very little effect on enzyme activity over the same range of concentrations, and branching at the 3-position of the aldehydes increased the corresponding KI value in all cases examined. The results suggest that a conjugated π-system may be a key structural determinant in the binding of these compounds to both enzymes, and may also be an important feature for the expansion of the active site volume in 2E1. PMID:24924949

  18. Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race.

    PubMed

    Tsou, Chung-Yau; Matsunaga, Shigeki; Okada, Shigeru

    2018-01-01

    The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46 kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30-37°C. For steady state kinetic parameters, the recombinant enzyme had a k m for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4 μM, and a k cat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11 μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  19. Model studies in cytochrome P-450-mediated toxicity of halogenated compounds: radical processes involving iron porphyrins.

    PubMed Central

    Brault, D

    1985-01-01

    Haloalkane toxicity originates from attack on biological targets by reactive intermediates derived from haloalkane metabolism by a hemoprotein, cytochrome P-450. Carbon-centered radicals and their peroxyl derivatives are most likely involved. The reactions of iron porphyrin--a model for cytochrome P-450--with various carbon-centered and peroxyl radicals generated by pulse radiolysis are examined. Competition between iron porphyrin and unsaturated fatty acids for attack by peroxyl radicals is pointed out. These kinetic data are used to derive a model for toxicity of haloalkanes with particular attention to carbon tetrachloride and halothane. The importance of local oxygen concentration and structural arrangement of fatty acids around cytochrome P-450 is emphasized. PMID:3007100

  20. Cytochrome P450 responses and PCB congeners in pipping heron embryos from Virginia, the Great Lakes and San Francisco Bay

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Tillett, D.E.; Woodin, Bruce R.; Stegeman, John J.

    1992-01-01

    Pipping black-crowned night-heron (Nvcticorax nvcticorax) embryos were collected from undisturbed (Chincoteague National Wildlife Refuge VA; CNWR) and industrialized (Cat Island, Green Bay WI and San Francisco Bay, CA; SFB) locations. Hepatic monooxygenases (AHH, EROD, BROD, ECOD) were induced up to 100-fold, and were correlated (r=0.50 to 0.72) with total PCB burdens (N =61 embryos). A subset of 30 embryos have now been analyzed by GC/MS for 12 AHH-active PCB congeners and by Western blot for cytochromes P450lA and P450llB. At Cat Island, concentrations of 8 congeners were greater (P <0.05) than at CNWR. P450lA and P450llB were detected in 44% and 100% of the Cat Island embryos compared to 8% and 33% of the CNWR + SFB embryos. Cytochrome P450 parameters were correlated with the total PCBs (r =0.44 to 0.67) and with at least 9 PCB congeners (r =0.39 to 0.77). Since P450 responses might be affected by other contaminants, sample extract potency in the H411E rat hepatoma bioassay is being determined to study relationships among dioxin equivalents and cytochrome P450 parameters.

  1. Evaluation of Memory Enhancing Clinically Available Standardized Extract of Bacopa monniera on P-Glycoprotein and Cytochrome P450 3A in Sprague-Dawley Rats

    PubMed Central

    Singh, Rajbir; Panduri, Jagadeesh; Kumar, Devendra; Kumar, Deepak; Chandsana, Hardik; Ramakrishna, Rachumallu; Bhatta, Rabi Sankar

    2013-01-01

    Bacopa monniera is a traditional Ayurvedic herbal medicine used to treat various mental ailments from ancient times. Recently, chemically standardized alcoholic extract of Bacopa monniera (BM) has been developed and currently available as over the counter herbal remedy for memory enhancement in children and adults. However, the consumption of herbal drugs has been reported to alter the expression of drug metabolizing enzymes and membrane transporters. Present study in male Sprague-Dawley rat was performed to evaluate the effect of memory enhancing standardized extract of BM on hepatic and intestinal cytochrome P450 3A and P-glycoprotein expression and activity. The BM (31 mg/kg/day) was orally administered for one week in BM pre-treated group while the control group received the same amount of vehicle for the same time period. The BM treatment decreased the cytochrome P450 3A (CYP3A) mediated testosterone 6β-hydroxylation activity of the liver and intestine by 2 and 1.5 fold, respectively compared to vehicle treated control. Similarly pretreatment with BM extract decreased the expression of intestinal P-glycoprotein (Pgp) as confirmed by Western blot analysis but did not alter the expression of hepatic Pgp. To investigate whether this BM pretreatment mediated decrease in activity of CYP3A and Pgp would account for the alteration of respective substrate or not, pharmacokinetic study with carbamazepine and digoxin was performed in BM pre-treated rats and vehicle treated rats. Carbamazepine and digoxin were used as CYP3A and Pgp probe drugs, respectively. Significant increase in AUC and Cmax of carbamazepine (4 and 1.8 fold) and digoxin (1.3 and 1.2 fold), respectively following the BM pre-treatment confirmed the down regulation of CYP3A and Pgp. PMID:24015255

  2. Evaluation of memory enhancing clinically available standardized extract of Bacopa monniera on P-glycoprotein and cytochrome P450 3A in Sprague-Dawley rats.

    PubMed

    Singh, Rajbir; Panduri, Jagadeesh; Kumar, Devendra; Kumar, Deepak; Chandsana, Hardik; Ramakrishna, Rachumallu; Bhatta, Rabi Sankar

    2013-01-01

    Bacopa monniera is a traditional Ayurvedic herbal medicine used to treat various mental ailments from ancient times. Recently, chemically standardized alcoholic extract of Bacopa monniera (BM) has been developed and currently available as over the counter herbal remedy for memory enhancement in children and adults. However, the consumption of herbal drugs has been reported to alter the expression of drug metabolizing enzymes and membrane transporters. Present study in male Sprague-Dawley rat was performed to evaluate the effect of memory enhancing standardized extract of BM on hepatic and intestinal cytochrome P450 3A and P-glycoprotein expression and activity. The BM (31 mg/kg/day) was orally administered for one week in BM pre-treated group while the control group received the same amount of vehicle for the same time period. The BM treatment decreased the cytochrome P450 3A (CYP3A) mediated testosterone 6β-hydroxylation activity of the liver and intestine by 2 and 1.5 fold, respectively compared to vehicle treated control. Similarly pretreatment with BM extract decreased the expression of intestinal P-glycoprotein (Pgp) as confirmed by Western blot analysis but did not alter the expression of hepatic Pgp. To investigate whether this BM pretreatment mediated decrease in activity of CYP3A and Pgp would account for the alteration of respective substrate or not, pharmacokinetic study with carbamazepine and digoxin was performed in BM pre-treated rats and vehicle treated rats. Carbamazepine and digoxin were used as CYP3A and Pgp probe drugs, respectively. Significant increase in AUC and Cmax of carbamazepine (4 and 1.8 fold) and digoxin (1.3 and 1.2 fold), respectively following the BM pre-treatment confirmed the down regulation of CYP3A and Pgp.

  3. Overexpression of cytochrome P450 CYP6BG1 may contribute to chlorantraniliprole resistance in Plutella xylostella (L.).

    PubMed

    Li, Xiuxia; Li, Ran; Zhu, Bin; Gao, Xiwu; Liang, Pei

    2018-06-01

    The diamondback moth Plutella xylostella (L.) is the most widely distributed pest of cruciferous crops and has developed resistance to most commonly used insecticides, including chlorantraniliprole. Resistance to chlorantraniliprole is likely caused by mutations of the target, the ryanodine receptor, and/or mediated by an increase in detoxification enzyme activities. Although target-site resistance is documented in detail, resistance mediated by increased metabolism has rarely been reported. The activity of cytochrome P450 was significantly higher in two resistant P. xylostella populations than in a susceptible one. Among ten detected cytochrome P450 genes, CYP6BG1 was significantly overexpressed (over 80-fold) in a field-resistant population compared with expression in a susceptible one. Knockdown of CYP6BG1 by RNA interference dramatically reduced the 7-ethoxycoumarin-O-deethylase (7-ECOD) activity of P450 by 45.5% and increased the toxicity of chlorantraniliprole toward P. xylostella by 26.8% at 48 h postinjection of double-stranded RNA. By contrast, overexpression of CYP6BG1 in a transgenic Drosophila melanogaster line significantly decreased the toxicity of the insecticide to the transgenic flies. Overexpression of CYP6BG1 may contribute to chlorantraniliprole resistance in P. xylostella. Our findings will provide new insights into the mechanisms of resistance to diamide insecticides in other insect pests. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  4. Molecular cloning of a family of xenobiotic-inducible drosophilid cytochrome P450s: Evidence for involvement in host-plant allelochemical resistance

    PubMed Central

    Danielson, Phillip B.; MacIntyre, Ross J.; Fogleman, James C.

    1997-01-01

    Cytochrome P450s constitute a superfamily of genes encoding mostly microsomal hemoproteins that play a dominant role in the metabolism of a wide variety of both endogenous and foreign compounds. In insects, xenobiotic metabolism (i.e., metabolism of insecticides and toxic natural plant compounds) is known to involve members of the CYP6 family of cytochrome P450s. Use of a 3′ RACE (rapid amplification of cDNA ends) strategy with a degenerate primer based on the conserved cytochrome P450 heme-binding decapeptide loop resulted in the amplification of four cDNA sequences representing another family of cytochrome P450 genes (CYP28) from two species of isoquinoline alkaloid-resistant Drosophila and the cosmopolitan species Drosophila hydei. The CYP28 family forms a monophyletic clade with strong regional homologies to the vertebrate CYP3 family and the insect CYP6 family (both of which are involved in xenobiotic metabolism) and to the insect CYP9 family (of unknown function). Induction of mRNA levels for three of the CYP28 cytochrome P450s by toxic host-plant allelochemicals (up to 11.5-fold) and phenobarbital (up to 49-fold) corroborates previous in vitro metabolism studies and suggests a potentially important role for the CYP28 family in determining patterns of insect–host-plant relationships through xenobiotic detoxification. PMID:9380713

  5. Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation.

    PubMed

    Hosoi, Yoshio; Uno, Yasuhiro; Murayama, Norie; Fujino, Hideki; Shukuya, Mitsunori; Iwasaki, Kazuhide; Shimizu, Makiko; Utoh, Masahiro; Yamazaki, Hiroshi

    2012-12-15

    Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V(max) and low K(m) values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with V(max) and V(max)/K(m) values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Reed, James R., E-mail: rreed@lsuhsc.edu; Cruz, Albert Leo N. dela, E-mail: adelac2@tigers.lsu.edu; Lomnicki, Slawo M., E-mail: slomni1@lsu.edu

    Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductasemore » and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2–CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • Particulate matter (PM) competitively inhibited CYP1A2 activity. • EPFRs were much more potent CYP1A2 inhibitors than other types of PM. • PM interacts differently with different forms of P450.

  7. CYTOCHROME P450-DEPENDENT METABOLISM OF TRICHLOROETHYLENE IN THE RAT KIDNEY

    EPA Science Inventory

    The metabolism of trichloroethylene (Tri) by cytochrome P450 (P450) was studied in microsomes from liver and kidney homogenates and from isolated renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. Chloral hydrate (CH) was the only metabolite con...

  8. A novel alkaloid, evodiamine causes nuclear localization of cytochrome-c and induces apoptosis independent of p53 in human lung cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mohan, Vijay; Agarwal, Rajesh; Singh, Rana P., E-mail: ranaps@hotmail.com

    Lung cancer is the most frequently diagnosed malignancy that contributes to high proportion of deaths globally among patients who die due to cancer. Chemotherapy remains the common mode of treatment for lung cancer patients though with limited success. We assessed the biological effects and associated molecular changes of evodiamine, a plant alkaloid, on human lung cancer A549 and H1299 cells along with other epithelial cancer and normal lung SAEC cells. Our data showed that 20–40 μM evodiamine treatment for 24–48 h strongly (up to 73%, P < 0.001) reduced the growth and survival of these cancer cells. However, it also moderately inhibited growth and survivalmore » of SAEC cells. A strong inhibition (P < 0.001) was observed on clonogenicity of A549 cells. Further, evodiamine increased (4-fold) mitochondrial membrane depolarization with 6-fold increase in apoptosis and a slight increase in Bax/Bcl-2 ratio. It increased the cytochrome-c release from mitochondria into the cytosol as well as nucleus. Cytosolic cytochrome-c activated cascade of caspase-9 and caspase-3 intrinsic pathway, however, DR5 and caspase-8 extrinsic pathway was also activated which could be due to nuclear cytochrome-c. Pan-caspase inhibitor (z-VAD.fmk) partially reversed evodiamine induced apoptosis. An increase in p53 as well as its serine 15 phosphorylation was also observed. Pifithrin-α, a p53 inhibitor, slightly inhibited growth of A549 cells and under p53 inhibitory condition evodiamine-induced apoptosis could not be reversed. Together these findings suggest that evodiamine is a strong inducer of apoptosis in lung epithelial cancer cells independent of their p53 status and that could involve both intrinsic as well as extrinsic pathway of apoptosis. Thus evodiamine could be a potential anticancer agent against lung cancer. - Highlights: • Evodiamine, a novel plant alkaloid, relatively selectively inhibited growth and survival of human lung cancer cells. • Increased cancer

  9. Suppressive effects of caraway (Carum carvi) extracts on 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin-dependent gene expression of cytochrome P450 1A1 in the rat H4IIE cells.

    PubMed

    Naderi-Kalali, B; Allameh, A; Rasaee, M J; Bach, H-J; Behechti, A; Doods, K; Kettrup, A; Schramm, K-W

    2005-04-01

    Cytochrome P450 1A1 (CYP1A1) is among the cytochrome P450 classes known to convert xenobiotics and endogenous compounds to toxic and/or carcinogenic metabolites. Suppression of CYP1A1 over expression by certain compounds is implicated in prevention of cancer caused by chemical carcinogens. Chemopreventive agents containing high levels of flavonoids and steroids-like compounds are known to suppress CYP1A1. This study was carried out for assessment of the genomic and proteomic effects of caraway (Carum carvi) extracts containing high levels of both flavonoids and steroid-like substances on ethoxy resorufin dealkylation (EROD) activity and CYP1A1 at mRNA levels. Rat hepatoma cells co-treated with a CYP1A1 inducer i.e. TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) and different preparations of caraway extracts at concentrations of 0, 0.13, 1.3, and 13 microM in culture medium. After incubation (37 degrees C and 7% CO2 for 20 h), changes in EROD specific activity recorded and compared in cells under different treatments. The results show that caraway seed extract prepared in three different organic solvents suppressed the enzyme activity in hepatoma cells in a dose-dependent manner. The extracts added above 0.13 microM could significantly inhibit EROD activity and higher levels of each extract (1.3 and 13 microM) caused approximately 10-fold suppression in the enzyme activity. Accordingly, data obtained from the RT-PCR (TaqMan) clearly showed the suppressive effects of plant extract on CYP1A1-related mRNA expression. These data clearly show that substances in caraway seeds extractable in organic solvents can potentially reverse the TCDD-dependent induction in cytochrome P450 1A1.

  10. Cytochrome P450-mediated hepatic metabolism of new fluorescent substrates in cats and dogs.

    PubMed

    van Beusekom, C D; Schipper, L; Fink-Gremmels, J

    2010-12-01

    This study aimed to investigate the biotransformation of cat liver microsomes in comparison to dogs and humans using a high throughput method with fluorescent substrates and classical inhibitors specific for certain isozymes of the human cytochrome P450 (CYP) enzyme family. The metabolic activities associated with CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A were measured. Cat liver microsomes metabolized all substrates selected for the assessment of cytochrome P450 activity. The activities associated with CYP3A and CYP2B were higher than the activities of the other measured CYPs. Substrate selectivity could be demonstrated by inhibition studies with α-naphthoflavone (CYP1A), tranylcypromine/quercetine (CYP2C), quinidine (CYP2D), diethyldithiocarbamic acid (CYP2E) and ketoconazole (CYP3A) respectively. Other prototypical inhibitors used for characterization of human CYP activities such as furafylline (CYP1A), tranylcypromine (CYP2B) and sulfaphenazole (CYP2C) did not show significant effects in cat and dog liver microsomes. Moreover, IC50-values of cat CYPs differed from dog and human CYPs underlining the interspecies differences. Gender differences were observed in the oxidation of 7-ethoxy-4-trifluoromethylcoumarin (CYP2B) and 3-[2-(N, N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (CYP2D), which were significantly higher in male cats than in females. Conversely, oxidation of the substrates dibenzylfluorescein (CYP2C) and 7-methoxy-4-trifluoromethylcoumarin (CYP2E) showed significant higher activities in females than in male cats. Overall CYP-activities in cat liver microsomes were lower than in those from dogs or humans, except for CYP2B. The presented difference between feline and canine CYP-activities are useful to establish dose corrections for feline patients of intensively metabolized drugs licensed for dogs or humans. © 2010 Blackwell Publishing Ltd.

  11. Insecticide resistance and cytochrome-P450 activation in unfed and blood-fed laboratory and field populations of Culex pipiens pallens.

    PubMed

    Chang, Kyu-Sik; Kim, Heung-Chul; Klein, Terry A; Ju, Young Ran

    2017-01-01

    Understanding the mechanisms of insecticide resistance to vector mosquitoes is critical for the implementation of effective control measures. A nulliparous susceptible Culex pipiens pallens (KSCP) laboratory colony and two field strains from Paju (PAJ) and Jeonju (JEO) Korea were evaluated for susceptibility to five pesticides by microapplication techniques. Unfed PAJ and JEO females demonstrated increased resistance compared to unfed KSCP females, respectively. While blood-fed KSCP females demonstrated <10-fold decreased susceptibility to pesticides compared to unfed KSCP females, blood-fed PAJ and JEO females demonstrated 25.0-50.0- and 16.0-38.6-fold increased resistance compared to unfed PAJ and JEO females, respectively. Unfed and blood-fed groups were assayed for α- and β-esterase, glutathione S -transferases, and cytochrome P-450 (P450) enzyme activity assays. P450 activity was 58.8- and 72.8-fold higher for unfed PAJ and JEO females, respectively, than unfed KSCP females. P450 enzyme activity of KSCP females assayed 1 and 7 days after a blood meal increased by 14.5- and 11.8-fold, respectively, compared to unfed KSCP females, while PAJ and JEO females demonstrated 164.9- and 148.5- and 170.7- and 160.4-fold increased activity, respectively, compared to unfed females of each population. However, other three resistance-related metabolic enzymes showed low activation at <10-fold after a blood meal. The data demonstrate that P450 acts on elevated insecticide resistance after blood meals in resistant field populations. Our findings might reveal that suppressing of the P450 protein by artificial gene mutation increases insecticidal susceptibility of Cx . pipiens and will promise effective vector mosquito control.

  12. Prediction of Cytochrome P450 Profiles of Environmental Chemicals with QSAR Models Built from Drug-like Molecules

    EPA Science Inventory

    The human cytochrome P450 (CYP450) enzyme family is involved in the biotransformation of many environmental chemicals. As part of the U.S. Tox21 effort, we profiled the CYP450 activity of ~2800 chemicals predominantly of environmental concern against CYP1A2, CYP2C19, CYP2C9, CYP2...

  13. Novel approaches to mitigating parathion toxicity: targeting cytochrome P450-mediated metabolism with menadione.

    PubMed

    Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2016-08-01

    Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase. We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH-cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the Food and Drug Administration for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity. © 2016 New York Academy of Sciences.

  14. Glucocorticoid receptor contributes to the altered expression of hepatic cytochrome P450 upon cigarette smoking.

    PubMed

    Li, Xue; Yan, Zhongfang; Wu, Qi; Sun, Xin; Li, Fan; Zhang, Subei; Li, Kuan; Li, Li; Wu, Junping; Xu, Long; Feng, Jing; Ning, Wen; Liu, Zhixue; Chen, Huaiyong

    2016-12-01

    Cigarette smoking has been shown to cause pathological alterations in the liver. However, how hepatic metabolism is altered during cigarette smoking‑induced inflammation remains to be fully elucidated. In the present study, a rat model of smoking was established to examine the effects of cigarette smoking on inflammation, autophagy activity, and the expression of nuclear receptor and CYP in the liver. Elevated expression of interleukin 1β and activation of autophagy in the liver were observed upon smoking exposure in rats. Cigarette smoking induced a significant reduction in the mRNA expression levels of cytochromes, including cytochrome P450 (Cyp)1A2, Cyp2D4 and Cyp3A2. Accordingly, a decrease was also observed in glucocorticoid receptor (GR), a regulator of the expression of Cyp. Activation of the GR signal in human hepatic LO2 cells did not affect autophagic genes, however, it led to the upregulation of hCYP1A2, hCYP2C19 and hCYP3A4, and the downregulation of hCYP2C9. The GR antagonist, RU486, eliminated this effect, suggesting the importance of GR in liver metabolism upon cigarette smoking.

  15. A current review of cytochrome P450 interactions of psychotropic drugs.

    PubMed

    Madhusoodanan, Subramoniam; Velama, Umamaheswararao; Parmar, Jeniel; Goia, Diana; Brenner, Ronald

    2014-05-01

    The number of psychotropic drugs has expanded tremendously over the past few decades with a proportional increase in drug-drug interactions. The majority of psychotropic agents are biotransformed by hepatic enzymes, which can lead to significant drug-drug interactions. Most drug-drug interactions of psychotropics occur at metabolic level involving the hepatic cytochrome P450 enzyme system. We searched the National Library of Medicine, PsycINFO, and Cochrane reviews from 1981 to 2012 for original studies including clinical trials, double-blind, placebo-controlled studies, and randomized controlled trials. In addition, case reports, books, review articles, and hand-selected journals were utilized to supplement this review. Based on the clinical intensity of outcome, cytochrome interactions can be classified as severe, moderate, and mild. Severe interactions include effects that might be acutely life threatening. They are mainly inhibitory interactions with cardiovascular drugs. Moderate interactions include efficacy issues. Mild interactions include nonserious side effects, such as somnolence. Psychotropic drugs may interact with other prescribed medications used to treat concomitant medical illnesses. A thorough understanding of the most prescribed medications and patient education will help reduce the likelihood of potentially fatal drug-drug interactions.

  16. DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE

    EPA Science Inventory

    Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

  17. Molecular cloning of a defense-response-related cytochrome P450 gene from tobacco.

    PubMed

    Takemoto, D; Hayashi, M; Doke, N; Nishimura, M; Kawakita, K

    1999-12-01

    Plant defenses against pathogen attack involve a series of inducible responses that contribute to resistance. Tobacco leaves injected with HWC (hyphal wall components prepared from Phytophthora infestans) elicitor showed typical defense responses, including the induction of localized necrosis and the accumulation of pathogenesis-related proteins. In order to elucidate the molecular mechanisms by which plant defense systems are activated, we screened tobacco plants for genes differentially expressed in response to HWC. We performed differential screening by RT-PCR with random primers and obtained PCR products specific to HWC-treated leaf RNA. Northern hybridization using the PCR products as probes confirmed that one transcript was actually induced by HWC treatment. As the deduced amino acid sequence of this clone showed the highest degree of similarity to elicitor-induced soybean cytochrome P450 CYP82A4, it was designated CYP82E1. The expression of CYP82E1 was strongly induced in tobacco by the soybean pathogen Pseudomonas syringae pv. glycinea (nonpathogenic on tobacco), but it was activated only slightly and in a delayed fashion by the tobacco pathogen P. syringae pv. tabaci (pathogenic on tobacco), implying that the product of CYP82E1 may be involved in disease resistance in tobacco.

  18. Evaluation of six proton pump inhibitors as inhibitors of various human cytochromes P450: focus on cytochrome P450 2C19.

    PubMed

    Zvyaga, Tatyana; Chang, Shu-Ying; Chen, Cliff; Yang, Zheng; Vuppugalla, Ragini; Hurley, Jeremy; Thorndike, Denise; Wagner, Andrew; Chimalakonda, Anjaneya; Rodrigues, A David

    2012-09-01

    Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -2B6, -2D6, -2C9, -2C8, and -1A2) in human liver microsomes. In most cases, IC₅₀ values were greater than 40 μM, except for dexlansoprazole and lansoprazole with CYP1A2 (IC₅₀ = ∼8 μM) and esomeprazole with CYP2C8 (IC₅₀ = 31 μM). With the exception of CYP2C19 inhibition by omeprazole and esomeprazole (IC₅₀ ratio, 2.5 to 5.9), there was no evidence for a marked time-dependent shift in IC₅₀ (IC₅₀ ratio, ≤ 2) after a 30-min preincubation with NADPH. In the absence of preincubation, lansoprazole (IC₅₀ = 0.73 μM) and esomeprazole (IC₅₀ = 3.7 μM) were the most potent CYP2C19 inhibitors, followed by dexlansoprazole and omeprazole (IC₅₀ = ∼7.0 μM). Rabeprazole and pantoprazole (IC₅₀ = ≥ 25 μM) were the weakest. A similar ranking was obtained with recombinant CYP2C19. Despite the IC₅₀ ranking, after consideration of plasma levels (static and dynamic), protein binding, and metabolism-dependent inhibition, it is concluded that omeprazole and esomeprazole are the most potent CYP2C19 inhibitors. This was confirmed after the incubation of the individual PPIs with human primary hepatocytes (in the presence of human serum) and by monitoring their impact on diazepam N-demethylase activity at a low concentration of diazepam (2 μM). Data described herein are consistent with reports that PPIs are mostly weak inhibitors of cytochromes P450 in vivo. However, two members of the PPI class (esomeprazole and omeprazole) are more likely to serve as clinically relevant inhibitors of CYP2C19.

  19. Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.

    2012-03-15

    NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure ofmore » human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.« less

  20. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    NASA Astrophysics Data System (ADS)

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-02-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

  1. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    PubMed Central

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-01-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification. PMID:26899743

  2. Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Osawa, Y.; Coon, M.J.

    1987-08-01

    In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, (1,2,6,7,16,17-3H)progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting (1,7,16-3H)progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of /sup 3/H/submore » 2/O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of (1,7,16-/sup 3/H)progesterone and (4-14C)progesterone was employed.« less

  3. Dose-dependent induction of cytochrome P450 (CYP) 3A4 and activation of pregnane X receptor by topiramate.

    PubMed

    Nallani, Srikanth C; Glauser, Tracy A; Hariparsad, Niresh; Setchell, Kenneth; Buckley, Donna J; Buckley, Arthur R; Desai, Pankaj B

    2003-12-01

    In clinical studies, topiramate (TPM) was shown to cause a dose-dependent increase in the clearance of ethinyl estradiol. We hypothesized that this interaction results from induction of hepatic cytochrome P450 (CYP) 3A4 by TPM. Accordingly, we investigated whether TPM induces CYP3A4 in primary human hepatocytes and activates the human pregnane X receptor (hPXR), a nuclear receptor that serves as a regulator of CYP3A4 transcription. Human hepatocytes were treated for 72 h with TPM (10, 25, 50, 100, 250, and 500 microM) and known inducers, phenobarbital (PB; 2 mM), and rifampicin (10 microM). The rate of testosterone 6beta-hydroxylation by hepatocytes served as a marker for CYP3A4 activity. The CYP3A4-specific protein and mRNA levels were determined by using Western and Northern blot analyses, respectively. The hPXR activation was assessed with cell-based reporter gene assay. Compared with controls, TPM (50-500 microM)-treated hepatocytes exhibited a considerable increase in the CYP3A4 activity (1. 6- to 8.2-fold), protein levels (4.6- to 17.3-fold), and mRNA levels (1.9- to 13.3-fold). Comparatively, rifampicin (10 microM) effected 14.5-, 25.3-, and a 20.3-fold increase in CYP3A4 activity, immunoreactive protein levels, and mRNA levels, respectively. TPM (50-500 microM) caused 1.3- to 3-fold activation of the hPXR, whereas rifampicin (10 microM) caused a 6-fold activation. The observed induction of CYP3A4 by TPM, especially at the higher concentrations, provides a potential mechanistic explanation of the reported increase in the ethinyl estradiol clearance by TPM. It also is suggestive of other potential interactions when high-dose TPM therapy is used.

  4. The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5.

    PubMed

    Canova-Davis, E; Waskell, L

    1984-02-25

    Methoxyflurane is an anesthetic whose metabolism by cytochrome P-450LM2 has been shown to be dependent upon a heat-stable microsomal protein (Canova-Davis, E., and Waskell, L. A. (1982) Biochem. Biophys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modifies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflurane by purified cytochrome P-450LM2. This protein factor has been identified as cytochrome b5 by demonstrating that cytochrome b5 and the heat-stable factor coelute during cytochrome b5 purification. Neither ferriheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome b5 reconstituted from apocytochrome b5 and heme exhibited an activity similar to that of native b5. Examination of the cytochrome b5 molecule by computer graphics suggested that diethylpyrocarbonate did not inactivate b5 by reacting with the anionic surface of the cytochrome b5 molecule. Maximal rates of methoxyflurane metabolism were obtained at a ratio of 1:1:1 of the three proteins, cytochrome P-450LM2:reductase:cytochrome b5. In summary, it has been demonstrated that the heat-stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM2. These data also suggest that cytochrome b5 may be acting as an electron donor to P-450LM2 in the O-demethylation of methoxyflurane.

  5. Localization of cytochrome P4501A in liver and extrahepatic organs of the pilot whale, Globiocephala melaena

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stegeman, J.; Miller, C.; Moore, M.

    1994-12-31

    Marine mammals may be important indicators of effects of contaminants that are globally distributed. Recently the authors described apparent environmental induction of hepatic CYPLA in beluga whales. Here they describe the localization and extent of CYPLA expression in organs of the pilot whale. Tissues from 18 pilot whales stranded on Cape Cod in 1990/91 were frozen in liquid N2 or fixed in formalin and embedded. Liver microsomal EROD activity were comparable to results with other cetaceans. Immunohistochemical analysis showed a periportal localization of CYPLA in liver parenchyma, and staining in the endothelium. Renal staining was strong in brush border andmore » endothelium. Testis, ovary, and spleen showed CYPLA staining only in endothelium. Adrenal zona fasciculata and zona reticularis stained more weakly than did endothelium. In lung there was mild staining of bronchiolar epithelium and strong staining of endothelium. The results indicate that active concentrations of inducer have penetrated throughout the body. CYPLA stained in dermal endothelium, indicating that analysis of skin biopsies could allow nondestructive analysis of CYPLA induction in marine mammals. CYPLA expression in these whales was surprisingly strong, suggesting the possibility of chemical effects related to CYPLA induction.« less

  6. Isoform-specific regulation of cytochrome P450 expression and activity by estradiol in female rats

    PubMed Central

    Choi, Su-Young; Fischer, Liam; Yang, Kyunghee; Chung, Hyejin; Jeong, Hyunyoung

    2011-01-01

    Estradiol (E2) is the major endogenous estrogen, and its plasma concentration increases up to 100-fold during pregnancy in humans. Accumulating evidence suggests that an elevated level of E2 may influence hepatic drug metabolism, potentially being responsible for altered drug metabolism during pregnancy. We characterized effects of E2 on expression and activities of cytochrome P450 enzymes (CYPs) in an in vivo system using rats. To this end, female rats were treated with estradiol benzoate (EB) or known CYP inducers. Liver tissues were obtained after 5 days of treatment, and mRNA and protein expression levels as well as activities of major hepatic CYPs were determined by qRT-PCR, immunoblot, and microsomal assay. E2 increased CYP1A2 expression and activity to a smaller extent than β-naphthoflavone did. E2 also enhanced CYP2C expression (CYP2C6, CYP2C7, and CYP2C12) to levels comparable to those observed by phenobarbital. E2 upregulated CYP3A9 expression, while expression of CYP3A1 was downregulated. Expression of hepatic nuclear receptors (PXR and CAR) and the obligate redox partner of CYPs (POR) was downregulated in EB-treated rats, suggesting their potential involvement in regulation of CYP expression and activity by E2. In summary, in female rats E2 regulates expression of hepatic CYPs in a CYP isoform-specific manner although the directional changes are different from those clinically observed during human pregnancy. Further study is warranted to determine whether the changes in drug metabolism during human pregnancy are attributable to involvement of hormones other than E2. PMID:21219883

  7. A possible role of NADPH-dependent cytochrome P450nor isozyme in glycolysis under denitrifying conditions.

    PubMed

    Watsuji, Tomo-o; Takaya, Naoki; Nakamura, Akira; Shoun, Hirofumi

    2003-05-01

    The denitrifying fungus Cylindrocarpon tonkinense contains two isozymes of cytochrome P450nor. One isozyme, P450nor1, uses NADH specifically as its electron donor whereas the other isozyme P450nor2 prefers NADPH to NADH. Here we show that P450nor1 is localized in both cytosol and mitochondria, like P450nor of Fusarium oxysporum, while P450nor2 is exclusively in cytosol. We also found that the addition of glucose as a carbon source to the culture media leads to the production of much more P450nor2 in the fungal cells than a non-fermentable substrate (glycerol or acetate) does. These results suggest that the NADP-dependent pentose phosphate cycle acts predominantly in C. tonkinense as the glycolysis pathway under the denitrifying conditions, which was confirmed by the observation that glucose induced enzyme activities involved in the cycle. These results showed that P450nor2 should act as the electron sink under anaerobic, denitrifying conditions to regenerate NADP+ for the pentose phosphate cycle.

  8. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  9. Electrochemistry of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: An alternative to animal testing in drug discovery.

    PubMed

    Rua, Francesco; Sadeghi, Sheila J; Castrignanò, Silvia; Valetti, Francesca; Gilardi, Gianfranco

    2015-10-01

    This work reports for the first time the direct electron transfer of the Canis familiaris cytochrome P450 2D15 on glassy carbon electrodes to provide an analytical tool as an alternative to P450 animal testing in the drug discovery process. Cytochrome P450 2D15, that corresponds to the human homologue P450 2D6, was recombinantly expressed in Escherichia coli and entrapped on glassy carbon electrodes (GC) either with the cationic polymer polydiallyldimethylammonium chloride (PDDA) or in the presence of gold nanoparticles (AuNPs). Reversible electrochemical signals of P450 2D15 were observed with calculated midpoint potentials (E1/2) of −191 ± 5 and −233 ± 4 mV vs. Ag/AgCl for GC/PDDA/2D15 and GC/AuNPs/2D15, respectively. These experiments were then followed by the electro-catalytic activity of the immobilized enzyme in the presence of metoprolol. The latter drug is a beta-blocker used for the treatment of hypertension and is a specific marker of the human P450 2D6 activity. Electrocatalysis data showed that only in the presence of AuNps the expected α-hydroxy-metoprolol product was present as shown by HPLC. The successful immobilization of the electroactive C. familiaris cytochrome P450 2D15 on electrode surfaces addresses the ever increasing demand of developing alternative in vitromethods for amore detailed study of animal P450 enzymes' metabolism, reducing the number of animals sacrificed in preclinical tests.

  10. Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes

    PubMed Central

    2012-01-01

    Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems. Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. Conclusions Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared

  11. Thermodynamics of interactions between mammalian cytochromes P450 and b5.

    PubMed

    Yablokov, Evgeny; Florinskaya, Anna; Medvedev, Alexei; Sergeev, Gennady; Strushkevich, Natallia; Luschik, Alexander; Shkel, Tatsiana; Haidukevich, Irina; Gilep, Andrei; Usanov, Sergey; Ivanov, Alexis

    2017-04-01

    Cytochromes P450 (CYPs) play an important role in the metabolism of xenobiotics and various endogenous substrates. Being a crucial component of the microsomal monooxygenase system, CYPs are involved in numerous protein-protein interactions. However, mechanisms underlying molecular interactions between components of the monooxygenase system still need better characterization. In this study thermodynamic parameters of paired interactions between mammalian CYPs and cytochromes b5 (CYB5) have been evaluated using a Surface Plasmon Resonance (SPR) based biosensor Biacore 3000. Analysis of 18 pairs of CYB5-CYP complexes formed by nine different isoforms of mammalian CYPs and two isoforms of human CYB5 has shown that thermodynamically these complexes can be subdivided into enthalpy-driven and entropy-driven groups. Formation of the enthalpy-driven complexes was observed in the case of microsomal CYPs allosterically regulated by CYB5 (CYB5A-CYP3A4, CYB5A-CYP3A5, CYB5A-CYP17A1). The entropy-driven complexes were formed when CYB5 had no effect on the CYP activity (CYB5A-CYP51A1, CYB5A-CYP1B1, CYB5B-CYP11A1). Results of this study suggest that such interactions determining protein clustering are indirectly linked to the monooxygenase functioning. Positive ΔH values typical for such interactions may be associated with displacement of the solvation shells of proteins upon clustering. CYB5-CYP complex formation accompanied by allosteric regulation of CYP activity by CYB5 is enthalpy-dependent. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Relationships between Endogenous Plasma Biomarkers of Constitutive Cytochrome P450 3A Activity and Single-Time-Point Oral Midazolam Microdose Phenotype in Healthy Subjects.

    PubMed

    Woolsey, Sarah J; Beaton, Melanie D; Choi, Yun-Hee; Dresser, George K; Gryn, Steven E; Kim, Richard B; Tirona, Rommel G

    2016-04-01

    Due to high basal interindividual variation in cytochrome P450 3A (CYP3A) activity and susceptibility to drug interactions, there has been interest in the application of efficient probe drug phenotyping strategies, as well as endogenous biomarkers for assessment of in vivo CYP3A activity. The biomarkers 4β-hydroxycholesterol (4βHC) and 6β-hydroxycortisol (6βHCL) are sensitive to CYP3A induction and inhibition. However, their utility for the assessment of constitutive CYP3A activity remains uncertain. We investigated whether endogenous plasma biomarkers (4βHC and 6βHCL) are associated with basal CYP3A metabolic activity in healthy subjects assessed by a convenient single-time-point oral midazolam (MDZ) phenotyping strategy. Plasma 4βHC and 6βHCL metabolic ratios (MRs) were analysed in 51 healthy adult participants. CYP3A activity was determined after administration of an oral MDZ microdose (100 μg). Simple linear and multiple linear regression analyses were performed to assess relationships between MDZ oral clearance, biomarkers and subject covariates. Among study subjects, basal MDZ oral clearance, 4βHC and 6βHCL MRs ranged 6.5-, 10- and 13-fold, respectively. Participant age and alcohol consumption were negatively associated with MDZ oral clearance (p = 0.03 and p = 0.045, respectively), while weight and female sex were associated with lower plasma 4βHC MR (p = 0.0003 and p = 0.032, respectively). Neither 4βHC nor 6βHCL MRs were associated with MDZ oral clearance. Plasma 4βHC and 6βHCL MRs do not relate to MDZ single-time-point metabolic phenotype in the assessment of constitutive CYP3A activity among healthy individuals. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  13. Study on the apoptosis mediated by cytochrome c and factors that affect the activation of bovine longissimus muscle during postmortem aging.

    PubMed

    Zhang, Jiaying; Yu, Qunli; Han, Ling; Chen, Cheng; Li, Hang; Han, Guangxing

    2017-06-01

    This study investigates whether bovine longissimus muscle cell apoptosis occurs during postmortem aging and whether apoptosis is dependent on the mitochondria pathway. This study also determines the apoptosis process mediated by cytochrome c after its release from mitochondria and the factors that affect the activation processes. Results indicate that apoptotic nuclei were detected at 12 h postmortem. Cytochrome c release from the mitochondria to the cytoplasm activated the caspase-9 and caspase-3 at early postmortem aging and the activation of caspase-9 occurs before the activation of caspase-3. The pH level decreased during the first 48 h postmortem, whereas the mitochondria membrane permeability increased from 6 to 12 h. Results demonstrate that an apoptosis process of bovine muscle occurred during postmortem aging. Apoptosis was dependent on the mitochondria pathway and occurred at early postmortem aging. Increased mitochondria membrane permeability and low pH are necessary conditions for the release of cytochrome c during postmortem aging.

  14. Nitrite reduction in paracoccus halodenitrificans: Evidence for the role of a cd-type cytochrome in ammonia formation

    NASA Technical Reports Server (NTRS)

    Hochstein, L. I.; Cronin, S. E.

    1984-01-01

    Cell-free extracts prepared from Paracoccus halodenitrificans catalyzed the reduction of nitrate to ammonia in the presence of dithionite and methyl viologen. Enzyme activity was located in the soluble fraction and was associated with a cytochrome whose spectral properties resembled those of a cd-type cytochrome. Unlike the sissimilatory cd-cytochrome nitrate reductase associated with the membrane fraction of P. halodenitrificans, this soluble cd-cytochrome did not reduce nitrite to nitrous oxide.

  15. Antiepileptic drugs affect neuronal androgen signaling via a cytochrome P450-dependent pathway.

    PubMed

    Gehlhaus, Marcel; Schmitt, Nina; Volk, Benedikt; Meyer, Ralf P

    2007-08-01

    Recent data imply an important role for brain cytochrome P450 (P450) in endocrine signaling. In epileptic patients, treatment with P450 inducers led to reproductive disorders; in mouse hippocampus, phenytoin treatment caused concomitant up-regulation of CYP3A11 and androgen receptor (AR). In the present study, we established specific in vitro models to examine whether CYP3A isoforms cause enhanced AR expression and activation. Murine Hepa1c1c7 cells and neuronal-type rat PC-12 cells were used to investigate P450 regulation and its effects on AR after phenytoin and phenobarbital administration. In both cell lines, treatment with antiepileptic drugs (AEDs) led to concomitant up-regulation of CYP3A (CYP3A11 in Hepa1c1c7 and CYP3A2 in PC-12) and AR mRNA and protein. Inhibition of CYP3A expression and activity by the CYP3A inhibitor ketoconazole or by CYP3A11-specific short interfering RNA molecules reduced AR expression to basal levels. The initial up-regulation of AR signal transduction, measured by an androgen-responsive element chloramphenicol-acetyltransferase reporter gene assay, was completely reversed after specific inhibition of CYP3A11. Withdrawal of the CYP3A11 substrate testosterone prevented AR activation, whereas AR mRNA expression remained up-regulated. In addition, recombinant CYP3A11 was expressed heterologously in PC-12 cells, thereby eliminating any direct drug influence on the AR. Again, the initial up-regulation of AR mRNA and activity was reduced to basal levels after silencing of CYP3A11. In conclusion, we show here that CYP3A2 and CYP3A11 are crucial mediators of AR expression and signaling after AED application. These findings point to an important and novel function of P450 in regulation of steroid hormones and their receptors in endocrine tissues such as liver and brain.

  16. Identification of novel cytochrome P450s in the Acari

    USDA-ARS?s Scientific Manuscript database

    Cytochrome P450s are the major phase I drug metabolising enzymes found in most organisms, including arthropods. Much of the work within the area of xenobiotic metabolism in this group of animals has centered around mosquito species, e.g. Anopheles gambiae and Culex quinquefasciatus, due to their rol...

  17. A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

    PubMed Central

    Sansom, Mark S. P.; Mulholland, Adrian J.

    2014-01-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes. PMID:25033460

  18. A multiscale approach to modelling drug metabolism by membrane-bound cytochrome P450 enzymes.

    PubMed

    Lonsdale, Richard; Rouse, Sarah L; Sansom, Mark S P; Mulholland, Adrian J

    2014-07-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes.

  19. [Consumption of vitamin A in intact and castrated rats. Relation to tocopherol and cytochrome P 450].

    PubMed

    Ferrando, R; Truhaut, R; Fourlon, C

    1979-05-07

    There are significant differences between castrated and non castrated male Rats in regard to vitamine A hepatic storage and plasma level. Cytochrom P 450 levels are the same in both groups. Those results are discussed.

  20. Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle

    Highlights: Black-Right-Pointing-Pointer Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k{sub cat} {approx} 25 min{sup -1}). Black-Right-Pointing-Pointer Reduction is a direct hydride transfer from R-NADP{sup 2}H to the carbonyl moiety. Black-Right-Pointing-Pointer P450 domain variants enhance reduction through potential allosteric/redox interactions. Black-Right-Pointing-Pointer Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k{sub cat} of {approx}25 min{sup -1} was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. Themore » reduction was caused by a direct hydride transfer from preferentially R-NADP{sup 2}H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP{sup 2}H but not D{sub 2}O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.« less

  1. Identification and heterologous expression of the cytochrome P450 oxidoreductase from the white-rot basidiomycete Coriolus versicolor.

    PubMed

    Ichinose, H; Wariishi, H; Tanaka, H

    2002-09-01

    A cDNA encoding cytochrome P450 oxidoreductase (CPR) from the lignin-degrading basidiomycete Coriolus versicolor was identified using RT-PCR. The full-length cDNA consisted of 2,484 nucleotides with a poly(A) tail, and contained an open reading frame. The G+C content of the cDNA isolated was 60%. A deduced protein contained 730 amino acid residues with a calculated molecular weight of 80.7 kDa. The conserved amino acid residues involved in functional domains such as FAD-, FMN-, and NADPH-binding domains, were all found in the deduced protein. A phylogenetic analysis demonstrated that C. versicolor CPR is significantly similar to CPR of the basidiomycete Phanerochaete chrysosporium and that they share the same major branch in the fungal cluster. A recombinant CPR protein was expressed using a pET/ Escherichia coli system. The recombinant CPR protein migrated at 81 kDa on SDS polyacrylamide gel electrophoresis. It exhibited an NADPH-dependent cytochrome c reducing activity.

  2. Flower colour and cytochromes P450†

    PubMed Central

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-01-01

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) and thus they play a crucial role in the determination of flower colour. F3′H and F3′5′H mostly belong to CYP75B and CYP75A, respectively, except for the F3′5′Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3′5′H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3′5′H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3′5′H and F3′H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones. PMID:23297355

  3. Biogenesis of the bacterial cbb3 cytochrome c oxidase: Active subcomplexes support a sequential assembly model.

    PubMed

    Durand, Anne; Bourbon, Marie-Line; Steunou, Anne-Soisig; Khalfaoui-Hassani, Bahia; Legrand, Camille; Guitton, Audrey; Astier, Chantal; Ouchane, Soufian

    2018-01-19

    The cbb 3 oxidase has a high affinity for oxygen and is required for growth of bacteria, including pathogens, in oxygen-limited environments. However, the assembly of this oxidase is poorly understood. Most cbb 3 are composed of four subunits: the catalytic CcoN subunit, the two cytochrome c subunits (CcoO and CcoP) involved in electron transfer, and the small CcoQ subunit with an unclear function. Here, we address the role of these four subunits in cbb 3 biogenesis in the purple bacterium Rubrivivax gelatinosus Analyses of membrane proteins from different mutants revealed the presence of active CcoNQO and CcoNO subcomplexes and also showed that the CcoP subunit is not essential for their assembly. However, CcoP was required for the oxygen reduction activity in the absence of CcoQ. We also found that CcoQ is dispensable for forming an active CcoNOP subcomplex in membranes. CcoNOP exhibited oxygen reductase activity, indicating that the cofactors (hemes b and copper for CcoN and cytochromes c for CcoO and CcoP) were present within the subunits. Finally, we discovered the presence of a CcoNQ subcomplex and showed that CcoN is the required anchor for the assembly of the full CcoNQOP complex. On the basis of these findings, we propose a sequential assembly model in which the CcoQ subunit is required for the early maturation step: CcoQ first associates with CcoN before the CcoNQ-CcoO interaction. CcoP associates to CcoNQO subcomplex in the late maturation step, and once the CcoNQOP complex is fully formed, CcoQ is released for degradation by the FtsH protease. This model could be conserved in other bacteria, including the pathogenic bacteria lacking the assembly factor CcoH as in R. gelatinosus . © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Effect of atrazine and chlorpyrifos exposure on cytochrome P450 contents and enzyme activities in common carp gills.

    PubMed

    Fu, Yao; Li, Ming; Liu, Ci; Qu, Jian-Ping; Zhu, Wen-Jun; Xing, Hou-Juan; Xu, Shi-Wen; Li, Shu

    2013-08-01

    Chlorpyrifos (CPF) and atrazine (ATR) are the most widely used organophosphate insecticides and triazine herbicides, respectively, worldwide. This study aimed at investigating the effects of ATR, CPF and mixture on common carp gills following 40-d exposure and 40-d recovery experiments. Cytochrome P450 content, activities of aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND) and the mRNA levels of the CYP1 family (CYP1A, CYP1B, and CYP1C) were determined. In total, 220 common carps were divided into eleven groups, and each group was treated with a specific concentration of ATR (4.28, 42.8 and 428 μg/L), CPF (1.16, 11.6 and 116 μg/L) or ATR-CPF mixture (1.13, 11.3 and 113 μg/L). The results showed that P450 content and activities of APND and ERND in fish exposed to ATR and mixture were significantly higher than those in the control group. After the 40-d recovery treatment (i.e., depuration), the P450 content and the activities of APND and ERND in fish decreased to the background levels. A similar tendency was also found in the mRNA levels of the CYP1 family (CYP1A, CYP1B, and CYP1C) in common carp gills. The CPF-treated fish showed no significant difference from the control groups, except for a significant CYP1C induction. These results indicated that CYP enzyme levels are induced by ATR but were only slightly affected by CPF in common carp gills. In addition, the ATR and CPF exposure showed an antagonistic effect on P450 enzymes in common carp gills. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites

    PubMed Central

    Di Nardo, Giovanna; Gilardi, Gianfranco

    2012-01-01

    Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may be oxidized by more than one enzyme. As a result, multiple products may be obtained from the same drug, and as the metabolites can be biologically active and may cause adverse drug reactions (ADRs), the metabolic profile of a new drug has to be known before this can be commercialized. Therefore, the metabolites of a certain drug must be identified, synthesized and tested for toxicity. Their synthesis must be in sufficient quantities to be used for metabolic tests. This review focuses on the progresses done in the field of the optimization of a bacterial self-sufficient and efficient cytochrome P450, P450 BM3 from Bacillus megaterium, used for the production of metabolites of human enzymes. The progress made in the improvement of its catalytic performance towards drugs, the substitution of the costly NADPH cofactor and its immobilization and scale-up of the process for industrial application are reported. PMID:23443101

  6. Influence of amino acid residues near the active site of cytochrome P450 from Bacillus megaterium on the selectivity of n-octane oxidation to octanol regioisomers

    NASA Astrophysics Data System (ADS)

    Miyaji, Akimitsu; Baba, Toshihide

    2017-09-01

    A mutant of cytochrome P450 from Bacillus megaterium (CYP450BM-3) was prepared by replacing two alanine residues around active site of the enzyme, alanine 328 and alanine 82, with leucine and tryptophan, respectively. The CYP450BM-3 mutant produced 2-octanol selectively from n-octane under atmospheric temperature and pressure; its selectivity was 74%. Furthermore, the mutant produced 1-octanol, which is not produced by wild-type enzyme.

  7. Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

    PubMed

    Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás

    2015-02-01

    Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

  8. Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.).

    PubMed

    Yu, Liying; Tang, Weiqi; He, Weiyi; Ma, Xiaoli; Vasseur, Liette; Baxter, Simon W; Yang, Guang; Huang, Shiguo; Song, Fengqin; You, Minsheng

    2015-03-10

    Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification.

  9. Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.)

    PubMed Central

    Yu, Liying; Tang, Weiqi; He, Weiyi; Ma, Xiaoli; Vasseur, Liette; Baxter, Simon W.; Yang, Guang; Huang, Shiguo; Song, Fengqin; You, Minsheng

    2015-01-01

    Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification. PMID:25752830

  10. Cytochrome P4501A1 is Required for Vascular Dysfunction and Hypertension Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin

    USDA-ARS?s Scientific Manuscript database

    National Health and Nutrition Examination Survey data show an association between hypertension and exposure to dioxin-like halogenated aromatic hydrocarbons (HAH). Further, chronic exposure of mice to the prototypical HAH, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), induces reactive oxygen species (...

  11. In Silico Prediction of Cytochrome P450-Mediated Biotransformations of Xenobiotics: A Case Study of Epoxidation.

    PubMed

    Zhang, Jing; Ji, Li; Liu, Weiping

    2015-08-17

    Predicting the biotransformation of xenobiotics is important in toxicology; however, as more compounds are synthesized than can be investigated experimentally, powerful computational methods are urgently needed to prescreen potentially useful candidates. Cytochrome P450 enzymes (P450s) are the major enzymes involved in xenobiotic metabolism, and many substances are bioactivated by P450s to form active compounds. An example is the conversion of olefinic substrates to epoxides, which are intermediates in the metabolic activation of many known or suspected carcinogens. We have calculated the activation energies for epoxidation by the active species of P450 enzymes (an iron-oxo porphyrin cation radical oxidant, compound I) for a diverse set of 36 olefinic substrates with state-of-the-art density functional theory (DFT) methods. Activation energies can be estimated by the computationally less demanding method of calculating the ionization potentials of the substrates, which provides a useful and simple predictive model based on the reaction mechanism; however, the preclassification of these diverse substrates into weakly polar and strongly polar groups is a prerequisite for the construction of specific predictive models with good predictability for P450 epoxidation. This approach has been supported by both internal and external validations. Furthermore, the relation between the activation energies for the regioselective epoxidation and hydroxylation reactions of P450s and experimental data has been investigated. The results show that the computational method used in this work, single-point energy calculations with the B3LYP functional including zero-point energy and solvation and dispersion corrections based on B3LYP-optimized geometries, performs well in reproducing the experimental trends of the epoxidation and hydroxylation reactions.

  12. Novel approaches to mitigating parathion toxicity: targeting cytochrome P450–mediated metabolism with menadione

    PubMed Central

    Jan, Yi-Hua; Richardson, Jason R.; Baker, Angela A.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2016-01-01

    Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase (AChE). We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH–cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the FDA for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity. PMID:27441453

  13. Characterization and identification of an indirect cytochrome P-450-initiated denitrosation of 2,6-dichloro-4-nitroaniline in rat hepatic microsomes.

    PubMed

    Myers, L A; Witmer, C M; Gallo, M A

    1988-08-01

    The metabolism of 2,6-dichloro-4-nitroaniline (DCNA) to a unique denitrosated product, 3,5-dichloro-p-aminophenol (DCAP), was investigated in rat hepatic microsomes using an HPLC system containing a reverse-phase column and an electrochemical detector. The parent compound appears to induce its own metabolism. The characterization of this induction was studied by polyacrylamide gel electrophoresis, catalytic enzymatic activity, and immunochemistry. The in vitro microsomal aerobic production of DCAP was increased 4- to 6.5-fold with respect to controls after animals were treated with DCNA. The microsomal production of DCAP can be inhibited by the addition of specific antibodies to cytochrome P-450d, thus indicating that the removal of the nitro group and subsequent replacement with a hydroxyl group was initiated by cytochrome P-450d in the mixed-function oxidase system. Finally, it was demonstrated by the addition of H218O to the assay that this hydroxyl group came from H2O and not molecular oxygen. It is concluded that cytochrome P-450 initiated this novel reaction by the formation of an N-hydroxylamine, followed by a non-P-450-mediated attack of water causing the removal of nitrous acid and the formation of the phenol.

  14. Cytochrome P450 drug interactions with statin therapy.

    PubMed

    Goh, Ivanna Xin Wei; How, Choon How; Tavintharan, Subramaniam

    2013-03-01

    Statins are commonly used in the treatment of hyperlipidaemia. Although the benefits of statins are well-documented, they have the potential to cause myopathy and rhabdomyolysis due to the complex interactions of drugs, comorbidities and genetics. The cytochrome P450 family consists of major enzymes involved in drug metabolism and bioactivation. This article aims to highlight drug interactions involving statins, as well as provide updated recommendations and approaches regarding the safe and appropriate use of statins in the primary care setting.

  15. Effect of thalidomide on endotoxin-induced decreases in activity and expression of hepatic cytochrome P450 3A2.

    PubMed

    Ueyama, Jun; Nadai, Masayuki; Zhao, Ying Lan; Kanazawa, Hiroaki; Takagi, Kenji; Kondo, Takaaki; Takagi, Kenzo; Wakusawa, Shinya; Abe, Fumie; Saito, Hiroko; Miyamoto, Ken-Ichi; Hasegawa, Takaaki

    2008-08-01

    Thalidomide has been reported to inhibit the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) that are involved in the down-regulation of hepatic cytochrome P450 (CYP) induced by endotoxin. In the present study, we investigated the effects of thalidomide on endotoxin-induced decreases in the activity and expression of hepatic CYP3A2 in rats. Thalidomide (50 mg/kg) was administered orally 22 h and 2 h before intraperitoneal injection of endotoxin (1 mg/kg). Twenty-four hours after the injection of endotoxin, antipyrine clearance experiments were conducted, in which the rats were sacrificed and protein levels of hepatic CYP3A2 were measured. There were no significant differences in the histopathological changes in the liver between the endotoxin-treated and endotoxin plus thalidomide-treated rats. Thalidomide had no effect on the systemic clearance of antipyrine, which is a proper indicator for hepatic CYP3A2 activity, whereas it enhanced endotoxin-induced decrease in the systemic clearance of antipyrine. Western blot analysis revealed that thalidomide had no effect on the protein levels of hepatic CYP3A2, whereas it enhanced the down-regulation of hepatic CYP3A2 by endotoxin. However, there were no significant differences in the concentrations of TNF-alpha and NO in plasma between the endotoxin-treated and endotoxin plus thalidomide-treated rats. The present findings suggest that thalidomide enhances endotoxin-induced decreases in the activity and expression of hepatic CYP3A2.

  16. Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450.

    PubMed

    Jung, Christiane; Schünemann, Volker; Lendzian, Friedhelm; Trautwein, Alfred X; Contzen, Jörg; Galander, Marcus; Böttger, Lars H; Richter, Matthias; Barra, Anne-Laure

    2005-10-01

    From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and Mössbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I.

  17. Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure

    USGS Publications Warehouse

    Whyte, J.J.; Jung, R.E.; Schmitt, C.J.; Tillitt, D.E.

    2000-01-01

    This review compiles and evaluates existing scientific information on the use, limitations, and procedural considerations for EROD activity (a catalytic measurement of cytochrome P4501A induction) as a biomarker in fish. A multitude of chemicals induce EROD activity in a variety of fish species, the most potent inducers being structural analogs of 2,3,7,8-tetracholordibenzo-p-dioxin. Although certain chemicals may inhibit EROD induction/activity, this interference is generally not a drawback to the use of EROD induction as a biomarker. The various methods of EROD analysis currently in use yield comparable results, particularly when data are expressed as relative rates of EROD activity. EROD induction in fish is well characterized, the most important modifying factors being fish species, reproductive status and age, all of which can be controlled through proper study design. Good candidate species for biomonitoring should have a wide range between basal and induced EROD activity (e.g., common carp, channel catfish, and mummichog). EROD activity has proven value as a biomarker in a number of field investigations of bleached kraft mill and industrial effluents, contaminated sediments, and chemical spills. Research on mechanisms of CYP1A-induced toxicity suggests that EROD activity may not only indicate chemical exposure, but also may also precede effects at various levels of biological organization. A current research need is the development of chemical exposure-response relationships for EROD activity in fish. In addition, routine reporting in the literature of EROD activity in standard positive and negative control material will enhance confidence in comparing results from different studies using this biomarker.

  18. Multiple doses of saw palmetto (Serenoa repens) did not alter cytochrome P450 2D6 and 3A4 activity in normal volunteers.

    PubMed

    Markowitz, John S; Donovan, Jennifer L; Devane, C Lindsay; Taylor, Robin M; Ruan, Ying; Wang, Jun-Sheng; Chavin, Kenneth D

    2003-12-01

    Saw palmetto (Serenoa repens) is the most commonly used herbal preparation in the treatment of benign prostatic hyperplasia. The objective of this study was to determine whether a characterized saw palmetto product affects the activity of cytochrome P450 (CYP) 2D6 or 3A4 in healthy volunteers (6 men and 6 women). The probe substrates dextromethorphan (CYP2D6 activity) and alprazolam (CYP3A4 activity) were administered orally at baseline and again after exposure to saw palmetto (320-mg capsule once daily) for 14 days. Dextromethorphan metabolic ratios and alprazolam pharmacokinetics were determined at baseline and after saw palmetto treatment. The mean ratio of dextromethorphan to its metabolite was 0.038 +/- 0.044 at baseline and 0.048 +/- 0.080 after 14 days of saw palmetto administration (P =.704, not significant [NS]), indicating a lack of effect on CYP2D6 activity. The area under the plasma alprazolam concentration versus time curve was 476 +/- 178 h. ng. mL(-1) at baseline and 479 +/- 125 h. ng. mL(-1) after saw palmetto treatment (P =.923, NS), indicating a lack of effect on CYP3A4 activity. The elimination half-life of alprazolam was 11.4 +/- 3.1 hours at baseline and 11.6 +/- 2.7 hours after saw palmetto treatment (P =.770, NS), also indicating a lack of effect on CYP3A4 activity. Our results indicate that extracts of saw palmetto at generally recommended doses are unlikely to alter the disposition of coadministered medications primarily dependent on the CYP2D6 or CYP3A4 pathways for elimination. These conclusions must be weighed in the context of the study's limited assessments and regarded as only the initial investigation into the drug interaction potential of saw palmetto.

  19. PRIMARY STRUCTURE OF THE CYTOCHROME P450 LANOSTEROL 14A-DEMETHYLASE GENE FROM CANDIDA TROPICALIS

    EPA Science Inventory

    We report the nucleotide sequence of the gene and flanking DNA for the cytochrome P450 lanosterol 14 alpha-demethylase (14DM) from the yeast Candida tropicalis ATCC750. An open reading frame (ORF) of 528 codons encoding a 60.9-kD protein is identified. This ORF includes a charact...

  20. Comparative evaluation of 12 immature citrus fruit extracts for the inhibition of cytochrome P450 isoform activities.

    PubMed

    Fujita, Tadashi; Kawase, Atsushi; Niwa, Toshiro; Tomohiro, Norimichi; Masuda, Megumi; Matsuda, Hideaki; Iwaki, Masahiro

    2008-05-01

    In a previous study we found that 50% ethanol extracts of immature fruits of Citrus unshiu (satsuma mandarin) have anti-allergic effects against the Type I, II and IV allergic reactions. However, many adverse interactions between citrus fruit, especially grapefruit juice, and drugs have been reported due to the inhibition of cytochrome P450 (CYP) activities. The purpose of this study was to examine the competitive inhibitory effects of extracts from immature citrus fruit on CYP activity. Extracts were prepared from 12 citrus species or cultivars, and were tested against three kinds of major CYPs, CYP2C9, CYP2D6 and CYP3A4, in human liver microsomes. We also estimated the amounts of flavonoids (narirutin, hesperidin, naringin and neohesperidin) and furanocoumarins (bergapten, 6',7'-dihydroxybergamottin and bergamottin) in each extract using HPLC. Citrus paradisi (grapefruit) showed the greatest inhibition of CYP activities, while Citrus unshiu which has an antiallergic effect, showed relatively weak inhibitory effects. Extracts having relatively strong inhibitory effects for CYP3A4 tended to contain higher amounts of naringin, bergamottin and 6',7'-dihydroxybergamottin. These results, providing comparative information on the inhibitory effects of citrus extracts on CYP isoforms, suggest that citrus extracts containing high levels of narirutin and hesperidin and lower levels of furanocoumarins such as C. unshiu are favorable as antiallergic functional ingredients.

  1. Cyanide-insensitive quinol oxidase (CIO) from Gluconobacter oxydans is a unique terminal oxidase subfamily of cytochrome bd.

    PubMed

    Miura, Hiroshi; Mogi, Tatsushi; Ano, Yoshitaka; Migita, Catharina T; Matsutani, Minenosuke; Yakushi, Toshiharu; Kita, Kiyoshi; Matsushita, Kazunobu

    2013-06-01

    Cyanide-insensitive terminal quinol oxidase (CIO) is a subfamily of cytochrome bd present in bacterial respiratory chain. We purified CIO from the Gluconobacter oxydans membranes and characterized its properties. The air-oxidized CIO showed some or weak peaks of reduced haemes b and of oxygenated and ferric haeme d, differing from cytochrome bd. CO- and NO-binding difference spectra suggested that haeme d serves as the ligand-binding site of CIO. Notably, the purified CIO showed an extraordinary high ubiquinol-1 oxidase activity with the pH optimum of pH 5-6. The apparent Vmax value of CIO was 17-fold higher than that of G. oxydans cytochrome bo3. In addition, compared with Escherichia coli cytochrome bd, the quinol oxidase activity of CIO was much more resistant to cyanide, but sensitive to azide. The Km value for O2 of CIO was 7- to 10-fold larger than that of G. oxydans cytochrome bo3 or E. coli cytochrome bd. Our results suggest that CIO has unique features attributable to the structure and properties of the O2-binding site, and thus forms a new sub-group distinct from cytochrome bd. Furthermore, CIO of acetic acid bacteria may play some specific role for rapid oxidation of substrates under acidic growth conditions.

  2. Cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiology.

    PubMed

    Jennings, Brett L; Sahan-Firat, Seyhan; Estes, Anne M; Das, Kanak; Farjana, Nasreen; Fang, Xiao R; Gonzalez, Frank J; Malik, Kafait U

    2010-10-01

    Hypertension is the leading cause of cardiovascular diseases, and angiotensin II is one of the major components of the mechanisms that contribute to the development of hypertension. However, the precise mechanisms for the development of hypertension are unknown. Our recent study showing that angiotensin II-induced vascular smooth muscle cell growth depends on cytochrome P450 1B1 led us to investigate its contribution to hypertension caused by this peptide. Angiotensin II was infused via miniosmotic pump into rats (150 ng/kg per minute) or mice (1000 μg/kg per day) for 13 days resulting in increased blood pressure, increased cardiac and vascular hypertrophy, increased vascular reactivity to vasoconstrictor agents, increased vascular reactive oxygen species production, and endothelial dysfunction in both species. The increase in blood pressure and associated pathophysiological changes were minimized by the cytochrome P450 1B1 inhibitor 2,3',4,5'-tetramethoxystilbene in both species and was markedly reduced in Cyp1b1(-/-) mice. These data suggest that cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiological changes. Moreover, 2,3',4,5'-tetramethoxystilbene, which prevents both cytochrome P450 1B1-dependent and -independent components of angiotensin II-induced hypertension and inhibits associated pathophysiological changes could be clinically useful in the treatment of hypertension and associated cardiovascular and inflammatory diseases.

  3. CYTOCHROME P450 1B1 CONTRIBUTES TO ANGIOTENSIN II-INDUCED HYPERTENSION AND ASSOCIATED PATHOPHYSIOLOGY

    PubMed Central

    Jennings, Brett L.; Sahan-Firat, Seyhan; Estes, Anne M.; Das, Kanak; Farjana, Nasreen; Fang, Xiao R.; Gonzalez, Frank J.; Malik, Kafait U.

    2010-01-01

    Hypertension is the leading cause of cardiovascular diseases, and angiotensin II is one of the major components of the mechanisms that contribute to the development of hypertension. However, the precise mechanisms for the development of hypertension are unknown. Our recent study that angiotensin II-induced vascular smooth muscle cell growth is dependent on cytochrome P450 1B1 led us to investigate its contribution to hypertension caused by this peptide. Angiotensin II was infused via miniosmotic pump into rats (150 ng/kg/min) or mice (1000 μg/kg/day) for 13 days resulting in increased blood pressure, increased cardiac and vascular hypertrophy, increased vascular reactivity to vasoconstrictor agents, increased reactive oxygen species production, and endothelial dysfunction in both species. The increase in blood pressure and associated pathophysiological changes were minimized by the cytochrome P450 1B1 inhibitor, 2,3′,4,5′-tetramethoxystilbene in both species and was markedly reduced in Cyp1b1-/- mice. These data suggest that cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiological changes. Moreover, 2,3′,4,5′-tetramethoxystilbene which prevents both cytochrome P450 1B1-dependent and independent components of angiotensin II-induced hypertension and inhibits associated pathophysiological changes could be clinically useful in the treatment of hypertension and associated cardiovascular and inflammatory diseases. PMID:20805442

  4. Inkjet-printed selective microfluidic biosensor using CNTs functionalized by cytochrome P450 enzyme

    NASA Astrophysics Data System (ADS)

    Krivec, Matic; Leitner, Raimund; Überall, Florian; Hochleitner, Johannes

    2017-05-01

    An additive manufacturing concept, consisting of 3D photopolymer printing and Ag nanoparticle printing, was investigated for the construction of a microfluidic biosensor based on immobilized cytochrome P450 enzyme. An acylate-type microfluidic chamber composed of two parts, i.e. chamber-housing and chamber-lid was printed with a polyjet 3D printer. A 3-electrode sensor structure was inkjet-printed on the lid using a combination of Ag and graphene printing. The working electrode was covered with carbon nanotubes by drop-casting and immobilized with cytochrome P450 2D6 enzyme. The microfluidic sensor shows a significant response to a test xenobiotic, i.e. dextromethorphan; the cyclic voltammetrical measurements show a corresponding oxidation peak at 0.4 V with around 5 μM detection limit.

  5. Differentiation‐associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic‐metabolizing activity of “luminal” muscle‐invasive bladder cancers

    PubMed Central

    Arlt, Volker M.; Indra, Radek; Joel, Madeleine; Stiborová, Marie; Eardley, Ian; Ahmad, Niaz; Otto, Wolfgang; Burger, Maximilian; Rubenwolf, Peter; Phillips, David H.; Southgate, Jennifer

    2018-01-01

    Extra‐hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self‐defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro‐carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier‐forming differentiated states in vitro. However, ethoxyresorufin O‐deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP‐DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain‐of‐function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle‐invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into “luminal” and “basal” groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over‐expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1‐activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over‐expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP‐function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies. PMID:29323757

  6. An in vitro evaluation of cytochrome P450 inhibition and P-glycoprotein interaction with goldenseal, Ginkgo biloba, grape seed, milk thistle, and ginseng extracts and their constituents.

    PubMed

    Etheridge, Amy S; Black, Sherry R; Patel, Purvi R; So, James; Mathews, James M

    2007-07-01

    Drug-herb interactions can result from the modulation of the activities of cytochrome P450 (P450) and/or drug transporters. The effect of extracts and individual constituents of goldenseal, Ginkgo biloba (and its hydrolyzate), grape seed, milk thistle, and ginseng on the activities of cytochrome P450 enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in human liver microsomes were determined using enzyme-selective probe substrates, and their effect on human P-glycoprotein (Pgp) was determined using a baculovirus expression system by measuring the verapamil-stimulated, vanadate-sensitive ATPase activity. Extracts were analyzed by HPLC to standardize their concentration(s) of constituents associated with the pharmacological activity, and to allow comparison of their effects on P450 and Pgp with literature values. Many of the extracts/constituents exerted > or = 50 % inhibition of P450 activity. These include those from goldenseal (normalized to alkaloid content) inhibiting CYP2C8, CYP2D6, and CYP3A4 at 20 microM, ginkgo inhibiting CYP2C8 at 10 microM, grape seed inhibiting CYP2C9 and CYP3A4 at 10 microM, milk thistle inhibiting CYP2C8 at 10 microM, and ginsenosides F1 and Rh1 (but not ginseng extract) inhibiting CYP3A4 at 10 microM. Goldenseal extracts/constituents (20 microM, particularly hydrastine) and ginsenoside Rh1 stimulated ATPase at about half of the activity of the model substrate, verapamil (20 microM). The data suggest that the clearance of a variety of drugs may be diminished by concomitant use of these herbs via inhibition of P450 enzymes, but less so by Pgp-mediated effects.

  7. [Effects of vitamins deficiency on the cytochrome P450 inducibility in rats].

    PubMed

    Trusov, N V; Guseva, G V; Beketova, N A; Aksenov, I V; Avrent'eva, L I; Kravchenko, L V

    2014-01-01

    The purpose of the study was to determine multi-vitamin deficiency effects on the inducibility of main isoforms of cytochrome P450 in the rat liver. The study was carried out on 4 groups of Wistar rats. Rats of the 1st and 3rd group received semi-synthetic diets containing adequate (100% of recommended vitamin level) level of vitamins, the 2nd and 4th--the semi-synthetic diet containing vitamins in the amount of 20% from adequate level. The duration of the experiment was 4 weeks. During the last week indole-3-carbinol (I-3-C) in dose of 20 mg/kg body weight was added to the diet of the 3rd and 4th group of rats. Vitamin E content in liver and blood serum declined by 59 and 34%, respectively in rats which were fed vitamin-deficient diet (2nd group); vitamin A level decreased by 5 times in the liver, but was not changed in blood serum. Multi-vitamin deficiency in the diet led to the increase in the liver ethoxyresorufin O-dealkylase (EROD) activity of CYP1A1, methoxyresorufin O-dealkylase (MROD) activity of CYP1A2 and testosteron 6beta-hydroxylase (6beta-TG) activity of CYP3A by 11, 80 and 53%, respectively, and gene expression of CYP1A1, CYP1A2, CYP3A and AhR by 8,5; 1,6; 2,4 and 3,6 fold. In rats fed diet with adequate levels of vitamins (3rd group) I-3-C increased activity of EROD and MROD by 4,4 and 5,5 fold, and the expression of CYP1A1, CYP1A2 and AhR genes by 148; 3 and 3,5 fold compared to the parameters of the 1st group (without I-3-C). Multi-vitamin deficiency increased I-3-C-related induction of EROD activity and expression of CYP1A1 and CYP1A2 genes, but decreased I-3-C-related induction of the MROD activity. Thus, 5-fold reducing of vitamin content in rat diet lead to significant changes in activity and inducibility of cytochrome P450 of CYP1A and 3A family, which play a key role in the detoxification and metabolism of drugs.

  8. THE DIFFERENTIAL HEPATOTOXICITY AND CYTOCHROME P450 RESPONSE OF F344 RATS TO THE THREE ISOMERS OF DICHLOROBENZENE

    EPA Science Inventory

    The acute hepatotoxicity and response of hepatic cytochrome P450 to treatment with the three isomers of dichlorobenzene (DCB) have been investigated. The objectives were to estimate toxic thresholds and to further e1ucidate the role of cytochrome P450 in the metabolism and toxici...

  9. Constituents of Indonesian medicinal plant Averrhoa bilimbi and their cytochrome P450 3A4 and 2D6 inhibitory activities.

    PubMed

    Auw, Lidyawati; Subehan; Sukrasno; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2015-01-01

    As constituents of Averrhoa bilimbi leaves we identified three new compounds (1-3) together with 12 known ones (4-15); their inhibitory activities on cytochrome P450 3A4 (CYP3A4) and 2D6 (CYP2D6) were examined. Among the isolated compounds, the mixture of 1 and 2, and compounds 4 and 9 showed strong inhibition on CYP3A4, but mild or no inhibition on CYP2D6. These compounds revealed the characteristics of 1) time- and concentration-dependent inhibition, 2) requirement of NADPH for the inhibition, 3) no protection by nucleophiles, and 4) suppression of the inhibition by competitive inhibitor. Thus, they are suggested to be mechanism-based inactivators of CYP3A4 and CYP2D6. The kinetic parameters for the inactivation (k(inact) and K(I)) were 0.19 min(-1) and 36.7 μM for the mixture of 1 and 2, 0.126 min(-1) and 10.5 μM for 4, and 0.29 min(-1) and 23.4 μM for 9.

  10. Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

    PubMed Central

    Wang, Ming; Roberts, David L.; Paschke, Rosemary; Shea, Thomas M.; Masters, Bettie Sue Siler; Kim, Jung-Ja P.

    1997-01-01

    Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase. PMID:9237990

  11. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12.

    PubMed

    Morishima, Nobuhiro; Nakanishi, Keiko; Takenouchi, Hiromi; Shibata, Takehiko; Yasuhiko, Yukuto

    2002-09-13

    Activation of caspase-12 from procaspase-12 is specifically induced by insult to the endoplasmic reticulum (ER) (Nakagawa, T., Zhu, H., Morishima, N., Li, E., Xu, J., Yankner, B. A., and Yuan, J. (2000) Nature 403, 98-103), yet the functional consequences of caspase-12 activation have been unclear. We have shown that recombinant caspase-12 specifically cleaves and activates procaspase-9 in cytosolic extracts. The activated caspase-9 catalyzes cleavage of procaspase-3, which is inhibitable by a caspase-9-specific inhibitor. Although cytochrome c released from mitochondria has been believed to be required for caspase-9 activation during apoptosis (Zou, H., Henzel, W. J., Liu, X., Lutschg, A., and Wang, X. (1997) Cell 90, 405-413, Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. (1997) Cell 91, 479-489), caspase-9 as well as caspase-12 and -3 are activated in cytochrome c-free cytosols in murine myoblast cells under ER stress. These results suggest that caspase-12 can activate caspase-9 without involvement of cytochrome c. To examine the role of caspase-12 in the activation of downstream caspases, we used a caspase-12-binding protein, which we identified in a yeast two-hybrid screen, for regulation of caspase-12 activation. The binding protein protects procaspase-12 from processing in vitro. Stable expression of the binding protein renders procaspase-12 insensitive to ER stress, thereby suppressing apoptosis and the activation of caspase-9 and -3. These data suggest that procaspase-9 is a substrate of caspase-12 and that ER stress triggers a specific cascade involving caspase-12, -9, and -3 in a cytochrome c-independent manner.

  12. Activation of caspase-dependent apoptosis by intracellular delivery of cytochrome c-based nanoparticles

    PubMed Central

    2014-01-01

    Background Cytochrome c is an essential mediator of apoptosis when it is released from the mitochondria to the cytoplasm. This process normally takes place in response to DNA damage, but in many cancer cells (i.e., cancer stem cells) it is disabled due to various mechanisms. However, it has been demonstrated that the targeted delivery of Cytochrome c directly to the cytoplasm of cancer cells selective initiates apoptosis in many cancer cells. In this work we designed a novel nano-sized smart Cytochrome c drug delivery system to induce apoptosis in cancer cells upon delivery. Results Cytochrome c was precipitated with a solvent-displacement method to obtain protein nanoparticles. The size of the Cytochrome c nanoparticles obtained was 100-300 nm in diameter depending on the conditions used, indicating good potential to passively target tumors by the Enhanced Permeability and Retention effect. The surface of Cytochrome c nanoparticles was decorated with poly (lactic-co-glycolic) acid-SH via the linker succinimidyl 3-(2-pyridyldithio) propionate to prevent premature dissolution during delivery. The linker connecting the polymer to the protein nanoparticle contained a disulfide bond thus allowing polymer shedding and subsequent Cytochrome c release under intracellular reducing conditions. A cell-free caspase-3 assay revealed more than 80% of relative caspase activation by Cytochrome c after nanoprecipitation and polymer modification when compared to native Cytochrome c. Incubation of HeLa cells with the Cytochrome c based-nanoparticles showed significant reduction in cell viability after 6 hours while native Cytochrome c showed none. Confocal microscopy confirmed the induction of apoptosis in HeLa cells when they were stained with 4’,6-diamidino-2-phenylindole and propidium iodide after incubation with the Cytochrome c-based nanoparticles. Conclusions Our results demonstrate that the coating with a hydrophobic polymer stabilizes Cytochrome c nanoparticles allowing

  13. Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48

    PubMed Central

    Moreno-Beltrán, Blas; Guerra-Castellano, Alejandra; Del Conte, Rebecca; García-Mauriño, Sofía M.; Díaz-Moreno, Sofía; González-Arzola, Katiuska; Santos-Ocaña, Carlos; Velázquez-Campoy, Adrián; De la Rosa, Miguel A.; Turano, Paola; Díaz-Moreno, Irene

    2017-01-01

    Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation—in particular, at tyrosine 48—is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-l-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects. PMID:28348229

  14. [Cytochrome P-450 and the response to antimalarial drugs].

    PubMed

    Guzmán, Valentina; Carmona-Fonseca, Jaime

    2006-01-01

    To assess the relationship between the genetic and phenotypic factors linked to the cytochrome P-450 enzyme system and the response to the antimalarial drugs chloroquine, amodiaquine, mefloquine, and proguanil, as well as to determine how certain biological and social factors of the host influence the behavior of this enzymatic complex. We performed a systematic review of the medical bibliographic databases PubMed, Excerpta Medica, LILACS, and SciELO by using the following Spanish and English descriptors: "CYP-450" and "citocromo P-450" in combination with "proguanil" (and with "mefloquina," "cloroquina," and "amodiaquina"), "farmacocinética de proguanil" (and the same using "mefloquina," "cloroquina," and "amodiaquina"), "resistencia a proguanil" (and the same using "mefloquina," "cloroquina," and "amodiaquina"), "metabolismo," "farmacogenética," "enfermedad," "inflamación," "infección," "enfermedad hepática," "malaria," "nutrición," and "desnutrición." The same terms were used in English. The search included only articles published in Spanish, English, and Portuguese on or before 30 June 2005 that dealt with only four antimalarial drugs: amodiaquine, chloroquine, mefloquine, and proguanil. Some genetic factors linked to human cytochrome P-450 (mainly its polymorphism), as well as other biological and social factors (the presence of disease itself, or of inflammation and infection, the use of antimalarials in their various combinations, and the patient's nutritional status) influence the behavior of this complex enzymatic system. It has only been in the last decade that the genetics of the cytochromes has been explored and that the mechanisms underlying some therapeutic interactions and aspects of drug metabolism have been uncovered, making it possible to characterize the biotransformation pathway of amodiaquine and chloroquine. Hopefully new research will help answer the questions that still remain, some of which pertain to the metabolism of other

  15. Limited value of the urinary phenytoin metabolic ratio for the assessment of cytochrome P4502C9 activity in vivo

    PubMed Central

    TASSANEEYAKUL, WICHITTRA; BIRKETT, DONALD J.; PASS, MICHAEL C.; MINERS, JOHN O.

    1996-01-01

    Relationships between the ratio of p-hydroxyphenytoin (p-HPPH), the major metabolite of phenytoin, to unchanged phenytoin excreted in urine (the urinary metabolic ratio or MR) were compared with a number of indices of the metabolic clearances of phenytoin and tolbutamide published previously for seventeen subjects separately administered these known cytochrome P4502C9 (CYP2C9) substrates. Significant correlations (rs=0.50–0.60, P<0.05) were observed between the phenytoin MR, derived from either 0–24 or 24–48 h urine collections, and inverse areas under the plasma unbound concentration-time curves (measured over various time intervals) of phenytoin and with plasma unbound tolbutamide clearance. Significant correlations (rs =0.59–0.74) were also observed between the phenytoin MRs and metabolic unbound clearances for p-hydroxyphenytoin formation. Despite the significant correlations, variability in tolbutamide and phenytoin metabolic clearance parameters tended to account for <50% of the variability in phenytoin MR. Correlations between the renal clearance of phenytoin and the phenytoin MRs suggest that variability in the renal clearance of unchanged drug limits the usefulness of the phenytoin MR for the investigation of factors influencing CYP2C9 activity in vivo. PMID:8971435

  16. Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

    PubMed Central

    Arai, Yuka; Kino, Kuniki

    2012-01-01

    Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound. PMID:22729547

  17. Anti-inflammatory effect and modulation of cytochrome P450 activities by Artemisia annua tea infusions in human intestinal Caco-2 cells.

    PubMed

    Melillo de Magalhães, Pedro; Dupont, Isabelle; Hendrickx, Aurélie; Joly, Aurélie; Raas, Thomas; Dessy, Stéphanie; Sergent, Thérèse; Schneider, Yves-Jacques

    2012-09-15

    In an attempt to understand the beneficial health effects of Artemisia annua other than its anti-malaria properties, extracts from different cultivars prepared as tea infusions were investigated using Caco-2 cells on the intestinal inflammation and cytochrome P450 (CYP) activities. The characterisation of their phenolic compound (PC) profile revealed rosmarinic and chlorogenic acids as the main PCs. The extracts, assayed on Caco-2 cells at a plausible intestinal concentration, significantly decreased the secretion of pro-inflammatory cytokines, IL-8 and IL-6. This effect could be attributable at least to their content in rosmarinic acid, detected as a potent anti-inflammatory compound. The extracts also inhibited the activity of CYP3A4, whose expression was induced by 1,25-dihydroxyvitamin D(3), and of CYP1A1, induced by benzo(a)pyrene. Our results highlight the advantage of drinking A. annua infusions for their potent anti-inflammatory effect, linked to PC content, which could synergise their antimalarial activity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  18. Immobilized Cytochrome P450 2C9 (CYP2C9): Applications for Metabolite Generation, Monitoring Protein-Protein Interactions, and Improving In-vivo Predictions Using Enhanced In-vitro Models

    NASA Astrophysics Data System (ADS)

    Wollenberg, Lance A.

    Cytochrome P450 (P450) enzymes are a family of oxoferroreductase enzymes containing a heme moiety and are well known to be involved in the metabolism of a wide variety of endogenous and xenobiotic materials. It is estimated that roughly 75% of all pharmaceutical compounds are metabolized by these enzymes. Traditional reconstituted in-vitro incubation studies using recombinant P450 enzymes are often used to predict in-vivo kinetic parameters of a drug early in development. However, in many cases, these reconstituted incubations are prone to aggregation which has been shown to affect the catalytic activity of an enzyme. Moreover, the presence of other isoforms of P450 enzymes present in a metabolic incubation, as is the case with microsomal systems, may affect the catalytic activity of an enzyme through isoform-specific protein-protein interactions. Both of these effects may result in inaccurate prediction of in-vivo drug metabolism using in-vitro experiments. Here we described the development of immobilized P450 constructs designed to elucidate the effects of aggregation and protein-protein interactions between P450 isoforms on catalytic activities. The long term objective of this project is to develop a system to control the oligomeric state of Cytochrome P450 enzymes to accurately elucidate discrepancies between in vitro reconstituted systems and actual in vivo drug metabolism for the precise prediction of metabolic activity. This approach will serve as a system to better draw correlations between in-vivo and in-vitro drug metabolism data. The central hypothesis is that Cytochrome P450 enzymes catalytic activity can be altered by protein-protein interactions occurring between Cytochrome P450 enzymes involved in drug metabolism, and is dependent on varying states of protein aggregation. This dissertation explains the details of the construction and characterization of a nanostructure device designed to control the state of aggregation of a P450 enzyme. Moreover

  19. Feed-drug interaction of orally applied butyrate and phenobarbital on hepatic cytochrome P450 activity in chickens.

    PubMed

    Mátis, G; Kulcsár, A; Petrilla, J; Hermándy-Berencz, K; Neogrády, Zs

    2016-08-01

    The expression of hepatic drug-metabolizing cytochrome P450 (CYP) enzymes may be affected by several nutrition-derived compounds, such as by the commonly applied feed additive butyrate, possibly leading to feed-drug interactions. The aim of this study was to provide some evidence if butyrate can alter the activity of hepatic CYPs in chickens exposed to CYP-inducing xenobiotics, monitoring for the first time the possibility of such interaction. Ross 308 chickens in the grower phase were treated with daily intracoelomal phenobarbital (PB) injection (80 mg/kg BW), applied as a non-specific CYP-inducer, simultaneously with two different doses of intra-ingluvial sodium butyrate boluses (0.25 and 1.25 g/kg BW) for 5 days. Activity of CYP2H and CYP3A subfamilies was assessed by specific enzyme assays from isolated liver microsomes. According to our results, the lower dose of orally administered butyrate significantly attenuated the PB-triggered elevation of both hepatic CYP2H and CYP3A activities, which might be in association with the partly common signalling pathways of butyrate and CYP-inducing drugs, such as that of PB. Based on these data, butyrate may take part in pharmacoepigenetic interactions with simultaneously applied drugs or other CYP-inducing xenobiotics, with possible consequences for food safety and pharmacotherapy. Butyrate was found to be capable to maintain physiological CYP activity by attenuating CYP induction, underlining the safety of butyrate application in poultry nutrition. Journal of Animal Physiology and Animal Nutrition © 2015 Blackwell Verlag GmbH.

  20. Molecular LEGO by domain-imprinting of cytochrome P450 BM3.

    PubMed

    Jetzschmann, K J; Yarman, A; Rustam, L; Kielb, P; Urlacher, V B; Fischer, A; Weidinger, I M; Wollenberger, U; Scheller, F W

    2018-04-01

    Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his 6 -tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his 6 -tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode. Copyright © 2018. Published by Elsevier B.V.

  1. Effects of 3G cell phone exposure on the structure and function of the human cytochrome P450 reductase.

    PubMed

    Tanvir, Shazia; Thuróczy, György; Selmaoui, Brahim; Silva Pires Antonietti, Viviane; Sonnet, Pascal; Arnaud-Cormos, Delia; Lévêque, Philippe; Pulvin, Sylviane; de Seze, René

    2016-10-01

    Cell phones increase exposure to radiofrequency (RF) electromagnetic fields (EMFs). Whether EMFs exert specific effects on biological systems remains debatable. This study investigated the effect of cell phone exposure on the structure and function of human NADPH-cytochrome P450 reductase (CPR). CPR plays a key role in the electron transfer to cytochrome P450, which takes part in a wide range of oxidative metabolic reactions in various organisms from microbes to humans. Human CPR was exposed for 60min to 1966-MHz RF inside a transverse electromagnetic cell (TEM-cell) placed in an incubator. The specific absorption rate (SAR) was 5W·kg(-1). Conformation changes have been detected through fluorescent spectroscopy of flavin and tryptophan residues, and investigated through circular dichroism, dynamic light scattering and microelectrophoresis. These showed that CPR was narrowed. By using cytochrome C reductase activity to assess the electron flux through the CPR, the Michaelis Menten constant (Km) and the maximum initial velocity (Vmax) decreased by 22% as compared with controls. This change was due to small changes in the tertiary and secondary structures of the protein at 37°C. The relevance of these findings to an actual RF exposure scenario demands further biochemical and in-vivo confirmation. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. [Participation of nitric oxide and arachidonic acid metabolites via cytochrome - P450 in the regulation of arterial blood pressure].

    PubMed

    Sánchez-Mendoza, M Alicia; Martínez-Ayala, Sonia O; Hernández-Hernández, José A; Zúñiga-Sosa, Leonor; Pastelín-Hernández, Gustavo; Escalante-Acosta, Bruno A

    2003-01-01

    Nitric oxide and cytochrome P450 arachidonic acid metabolites participate in blood pressure regulation. The synthesis of these autacoids leads to arterial hypertension. However, it is not known whether there is an interaction between them. Therefore, we studied the modulatory effect of nitric oxide and cytochrome P450-arachidonic acid metabolites, their interaction on blood pressure, and the renal content of cytochrome P450. Male Wistar rats were divided: 1) control, 2) L-NAME (100 mg/kg/d p.o.), 3) L-NAME + SnCl2 (10 mg/kg/d i.p.), and 4) L-NAME + dexamethasone (1 mg/kg/d s.c.). We measured blood pressure and collected urine and blood for nitric oxide measurement. NO2 was quantified by HPLC. Blood pressure was: control, 97 +/- 7 mmHg; L-NAME, 151 +/- 4.6 mmHg; L-NAME + SnCl2, 133 +/- 3 mmHg, and L-NAME + dexamethasone 152 +/- 4.5 mmHg. Urine nitrite concentration was: 1) 1.832 +/- 0.32, 2) 1.031 +/- 0.23, 3) 1.616 +/- 0.33, and 4) 1.244 +/- 0.33 mumol/mL, while the concentration in blood was: 1) 0.293 +/- 0.06, 2) 0.150 +/- 0.05, 3) 0.373 +/- 0.13, and 4) 0.373 +/- 0.07 mumol/mL. L-NAME + SnCl2 decreased cytochrome P450 renal content, and L-NAME + dexamethasone showed a similar response. In conclusion, both, nitric oxide and CYP-arachidonic acid metabolites play a role in the regulation of blood pressure. Nitric oxide also partially regulates renal cytochrome P450 content.

  3. Role of brain cytochrome P450 mono-oxygenases in bilirubin oxidation-specific induction and activity.

    PubMed

    Gambaro, Sabrina E; Robert, Maria C; Tiribelli, Claudio; Gazzin, Silvia

    2016-02-01

    In the Crigler-Najjar type I syndrome, the genetic absence of efficient hepatic glucuronidation of unconjugated bilirubin (UCB) by the uridine 5'-diphospho-glucuronosyltransferase1A1 (UGT1A1) enzyme produces the rise of UCB level in blood. Its entry to central nervous system could generate toxicity and neurological damage, and even death. In the past years, a compensatory mechanism to liver glucuronidation has been indicated in the hepatic cytochromes P450 enzymes (Cyps) which are able to oxidize bilirubin. Cyps are expressed also in the central nervous system, the target of bilirubin toxicity, thus making them theoretically important to confer a protective activity toward bilirubin accumulation and neurotoxicity. We therefore investigated the functional induction (mRNA, EROD/MROD) and the ability to oxidize bilirubin of Cyp1A1, 1A2, and 2A3 in primary astrocytes cultures obtained from two rat brain region (cortex: Cx and cerebellum: Cll). We observed that Cyp1A1 was the Cyp isoform more easily induced by beta-naphtoflavone (βNF) in both Cx and Cll astrocytes, but oxidized bilirubin only after uncoupling by 3, 4,3',4'-tetrachlorobiphenyl (TCB). On the contrary, Cyp1A2 was the most active Cyp in bilirubin clearance without uncoupling, but its induction was confined only in Cx cells. Brain Cyp2A3 was not inducible. In conclusion, the exposure of astrocytes to βNF plus TCB significantly enhanced Cyp1A1 mediating bilirubin clearance, improving cell viability in both regions. These results may be a relevant groundwork for the manipulation of brain Cyps as a therapeutic approach in reducing bilirubin-induced neurological damage.

  4. The effect of lycopene on cytochrome P450 isoenzymes and P-glycoprotein by using human liver microsomes and Caco-2 cell monolayer model.

    PubMed

    Kong, Lingti; Song, Chunli; Ye, Linhu; Xu, Jian; Guo, Daohua; Shi, Qingping

    2018-01-11

    Lycopene is widely used as a dietary supplement. However, the effects of lycopene on cytochrome P450 (CYP) enzymes or P-glycoprotein (P-gp) are not comprehensive. The present study was performed to investigate the effects of lycopene on the CYP enzymes and P-gp activity. A cocktail method was used to evaluate the activities of CYP3A4, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. Caco-2 cell monolayer model was carried out to assay lycopene on P-gp activity. The results indicated that lycopene had a moderate inhibitory effect on CYP2E1, with IC50 value of 43.65 μM, whereas no inhibitory effects on CYP3A4, CYP2C19, CYP2D6 and CYP2E1, with IC50 values all over 100 μM. In addition, lycopene showed almost no inhibitory effect on rhodamine-123 efflux and uptake (p > .05), indicated no effects on P-gp activity. In conclusion, there should be required attention when lycopene are coadministered with other drugs that are metabolised by CYP2E1.

  5. Evidence for induction of cytochrome P-450I in patients with tropical chronic pancreatitis.

    PubMed

    Chaloner, C; Sandle, L N; Mohan, V; Snehalatha, C; Viswanathan, M; Braganza, J M

    1990-06-01

    Theophylline kinetics, as an in vivo probe for the potentially toxic cytochrome P-450I pathway of drug metabolism, were studied in 11 healthy volunteers and 11 patients with calcific chronic pancreatitis at Madras, South India. Theophylline clearance was faster in the patients than controls [median 69 (range 39-114) vs 45 (33-56) ml h-1 kg-1, p = 0.003]. In keeping with this finding, detailed social histories identified a higher exposure level in the patients to xenobiotics that are inducers of cytochrome P-450I and/or yield reactive metabolites upon processing thereby (score 7, 4-11 vs 3, 2-9, p = 0.002). However, the concentration of D-glucaric acid in urine, as a marker of phase II conjugating pathways of drug metabolism, was similar in patients and controls. This pattern of drug metabolism could predispose to oxidant stress: hence micronutrient antioxidant supplements may have therapeutic (or even prophylactic) value in tropical chronic pancreatitis.

  6. Differential effects of traumatic brain injury on the cytochrome p450 system: a perspective into hepatic and renal drug metabolism.

    PubMed

    Kalsotra, Auinash; Turman, Cheri M; Dash, Pramod K; Strobel, Henry W

    2003-12-01

    Traumatic brain injury is known to cause several secondary effects, one of which is altered drug clearance. Given the fact that patients who sustain TBI are subsequently treated with a variety of pharmacological agents for the purpose of either neuroprotection or physiological support, it is imperative to clarify changes in expression and/or activities of enzymes involved in clearing drugs. The mixed function oxidase system, which consists of cytochrome P450 and cytochrome P450 reductase, plays a vital role in phase I drug metabolism. This paper addresses the issue as to what extent TBI affects the levels and activity of various rat CYP450 subfamilies. Our results show that TBI induces tissue-specific and time-dependent alterations. Total hepatic CYP450 content showed a biphasic response with a decrease seen at 24 h followed by an increase at 2 weeks. CYP450 reductase, in contrast, showed an opposite temporal profile. Immunoblot analyses and marker substrate metabolism demonstrated a clear decrease in hepatic CYP1A levels while a significant increase in kidney was seen at both 24 h and 2 weeks. A dramatic induction of CYP3A was evident at 2 weeks in liver, while no changes were noticed in CYP2B or CYP2D subfamilies. CYP4F subfamily showed induction in kidney only. Collectively, the data reveal the differential effects of TBI on hepatic and renal drug metabolism.

  7. Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development.

    PubMed

    Baston, Eckhard; Leroux, Frédéric R

    2007-01-01

    Cytochrome P450's are enzymes which catalyze a large number of biological reactions, for example hydroxylation, N-, O-, S- dealkylation, epoxidation or desamination. Their substrates include fatty acids, steroids or prostaglandins. In addition, a high number of various xenobiotics are metabolized by these enzymes. The enzyme 17alpha-hydroxylase-C17,20-lyase (P450(17), CYP 17, androgen synthase), a cytochrome P450 monooxygenase, is the key enzyme for androgen biosynthesis. It catalyzes the last step of the androgen biosynthesis in the testes and adrenal glands and produces androstenedione and dehydroepiandrosterone from progesterone and pregnenolone. The microsomal enzyme aromatase (CYP19) transforms these androgens to estrone and estradiol. Estrogens stimulate tumor growth in hormone dependent breast cancer. In addition, about 80 percent of prostate cancers are androgen dependent. Selective inhibitors of these enzymes are thus important alternatives to treatment options like antiandrogens or antiestrogens. The present article deals with recent patents (focus on publications from 2000 - 2006) concerning P450 inhibitor design where steroidal substrates are involved. In this context a special focus is provided for CYP17 and CYP19. Mechanisms of action will also be discussed. Inhibitors of CYP11B2 (aldosterone synthase) will also be dealt with.

  8. Summary of Information on the Effects of Ionizing and Non-ionizing Radiation on Cytochrome P450 and Other Drug Metabolizing Enzymes and Transporters

    PubMed Central

    Rendic, Slobodan; Guengerich, F. Peter

    2014-01-01

    The present paper is an update of data on the effects of ionizing radiation (γ-rays, X-rays, high energy UV, fast neutron) caused by environmental pollution or clinical treatments and the effects of non-ionizing radiation (low energy UV) on the expression and/or activity of drug metabolism (e.g., cytochrome P450,, glutathione transferase), enzymes involved in oxidative stress (e.g., peroxidases, catalase,, aconitase, superoxide dismutase), and transporters. The data are presented in tabular form (Tables 1–3) and are a continuation of previously published summaries on the effects of drugs and other chemicals on cytochrome P450 enzymes (Rendic, S.; Di Carlo, F. Drug Metab. Rev., 1997, 29 (1–2), 413–580, Rendic, S. Drug Metab. Rev., 2002, 34 (1–2), 83–448) and of the data on the effects of diseases and environmental factors on the expression and/or activity of human cytochrome P450 enzymes and transporters (Guengerich, F.P.; Rendic, S. Curr. Drug Metab., 2010, 11(1), 1–3, Rendic, S.; Guengerich, F.P. Curr. Drug Metab., 2010, 11 (1), 4–84). The collective information is as presented by the cited author(s) in cases where several references are cited the latest published information is included. Remarks and conclusions suggesting clinically important impacts are highlighted, followed by discussion of the major findings. The searchable database is available as an Excel file (for information about file availability contact the corresponding author). PMID:22571481

  9. The interaction of representative members from two classes of antimycotics--the azoles and the allylamines--with cytochromes P-450 in steroidogenic tissues and liver.

    PubMed

    Schuster, I

    1985-06-01

    Spectrophotometric studies with ketoconazole, clotrimazole and miconazole show strong type-II interactions with several cytochromes P-450, particularly (Ks greater than 10(7)M-1; pH7.4; 25 degrees C) with the 11 beta-hydroxylase of adrenal mitochondria, with the 17 alpha/20 lyase of testis microsomes and with some forms of cytochromes P-450 of liver. A tight binding of the azoles also occurs to the reduced cytochromes, giving rise to an impeded CO binding to the haem iron. The binding of the azoles to 11 beta-hydroxylase and 17 alpha/20 lyase is much tighter than the binding of endogenous substrates, and consequently inhibition of steroidogenesis will occur at these sites. The metabolism of xenobiotic substrates by the cytochromes P-450 of liver will also be severely impeded. In contrast, the allylamines naftifine and SF 86-327 are type-I substrates for a small portion of cytochromes P-450 of liver microsomes only and there is no spectral evidence for binding to the cytochromes P-450 involved in steroid biosynthesis.

  10. Chemoprotective potentials of homoisoflavonoids and chalcones of Dracaena cinnabari: modulations of drug-metabolizing enzymes and antioxidant activity.

    PubMed

    Machala, M; Kubínová, R; Horavová, P; Suchý, V

    2001-03-01

    A series of homoisoflavonoids and chalcones, isolated from the endemic tropical plant Dracaena cinnabari Balf. (Agavaceae), were tested for their potential to inhibit cytochrome P4501A (CYP1A) enzymes and Fe-enhanced in vitro peroxidation of microsomal lipids in C57B1/6 mouse liver. The effects of the polyphenolic compounds were compared with those of prototypal flavonoid modulators of CYP1A and the well-known antioxidant, butylated hydroxytoluene. 2-Hydroxychalcone and partly 4,6-dihydroxychalcone were found to be strong inhibitors of CYP1A-dependent 7-ethoxyresorufin O-deethylase (EROD) activity in vitro comparable to the effects of quercetin and chrysin. The first screening of flavonoids and chalcones of Dracaena cinnabari for antioxidant activity was done in an in vitro microsomal peroxidation assay. While chalcones were shown to be poor antioxidants, 7,8-methylenedioxy-3(4-hydroxybenzyl) chromane, as one of the tested homoisoflavonoids, exhibited a strong antioxidant activity comparable to that of the strongest flavonol antioxidant, quercetin. Copyright 2001 John Wiley & Sons, Ltd.

  11. Curcumin induces Apaf-1-dependent, p21-mediated caspase activation and apoptosis

    PubMed Central

    Zhang, Honghao; Jones, Anthony; Verone, Alissa; Pitarresi, Jason; Jandhyam, Sirisha; Prabhu, Varun; Black, Jennifer D

    2011-01-01

    Previous studies have demonstrated that curcumin induces mitochondria-mediated apoptosis. However, understanding of the molecular mechanisms underlying curcumin-induced cell death remains limited. In this study, we demonstrate that curcumin treatment of cancer cells caused dose- and time-dependent caspase 3 activation, which is required for apoptosis as confirmed using the pan-caspase inhibitor, z-VAD. Knockdown experiments and knockout cells excluded a role for caspase 8 in curcumin-induced caspase 3 activation. In contrast, Apaf-1 deficiency or silencing inhibited the activity of caspase 3, pointing to a requisite role of Apaf-1 in curcumin-induced apoptotic cell death. Curcumin treatment led to Apaf-1 upregulation, both at the protein and mRNA levels. Cytochrome c release from mitochondria to the cytosol in curcumin-treated cells was associated with upregulation of pro-apoptotic proteins, such as Bax, Bak, Bid and Bim. Cross-linking experiments demonstrated Bax oligomerization during curcumin-induced apoptosis, suggesting that induced expression of Bax, Bid and Bim causes Bax channel formation on the mitochondrial membrane. The release of cytochrome c was unaltered in p53-deficient cells, whereas absence of p21 blocked cytochrome c release, caspase activation and apoptosis. Importantly, p21 deficiency resulted in reduced expression of Apaf-1 during curcumin treatment, indicating a requirement for p21 in Apaf-1-dependent caspase activation and apoptosis. Together, our findings identify Apaf-1, Bax and p21 as novel potential targets for curcumin or curcumin-based anticancer agents. PMID:22101335

  12. Human Liver Cytochrome P450 3A4 Ubiquitination

    PubMed Central

    Wang, YongQiang; Kim, Sung-Mi; Trnka, Michael J.; Liu, Yi; Burlingame, A. L.; Correia, Maria Almira

    2015-01-01

    CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate

  13. Evaluation of peppermint oil and ascorbyl palmitate as inhibitors of cytochrome P4503A4 activity in vitro and in vivo.

    PubMed

    Dresser, George K; Wacher, Vincent; Wong, Susan; Wong, Harrison T; Bailey, David G

    2002-09-01

    Our study was designed to determine the effect of peppermint oil and ascorbyl palmitate on cytochrome P4503A4 (CYP3A4) activity in vitro and oral bioavailability of felodipine in humans. Reversible and mechanism-based inhibitions of nifedipine oxidation were studied in human liver microsomes. The oral pharmacokinetics of felodipine and its dehydrofelodipine metabolite were determined in 12 healthy volunteers after administration of felodipine, 10-mg extended-release tablet, with grapefruit juice (300 mL), peppermint oil (600 mg), ascorbyl palmitate (500 mg), or water in a randomized 4-way crossover study. Peppermint oil (inhibition constant [K(i)] = 35.9 +/- 3.3 microg/mL, mean +/- SEM) and 2 constituents, menthol (K(i) = 87.0 +/- 7.0 micromol/L), and menthyl acetate (K(i) = 124.0 +/- 7.0 micromol/L), produced reversible inhibition of nifedipine oxidation. Ascorbyl palmitate was more potent (K(i) = 12.3 +/- 0.5 micromol/L). None of these substances were mechanism-based inhibitors. Grapefruit juice and peppermint oil increased the area under the curve (AUC) values of felodipine to 173% (range, 94%-280%; P <.01) and 140% (range, 77%-262%; P <.05), respectively, of those with water. They augmented the peak plasma concentration (C(max)) of felodipine and the AUC and C(max) of dehydrofelodipine but did not alter the half-life (t(1/2)) of either substance. Grapefruit juice decreased the dehydrofelodipine/felodipine AUC ratio, but peppermint oil did not. Ascorbyl palmitate did not change the pharmacokinetics of felodipine or dehydrofelodipine compared with water. Peppermint oil, menthol, menthyl acetate, and ascorbyl palmitate were moderately potent reversible inhibitors of in vitro CYP3A4 activity. Grapefruit juice increased the oral bioavailability of felodipine by inhibition of CYP3A4-mediated presystemic drug metabolism. Peppermint oil may also have acted by this mechanism. However, this requires further investigation. Ascorbyl palmitate did not inhibit CYP3A4

  14. Polycyclic aromatic hydrocarbons and cytochrome P450 in HIV pathogenesis

    PubMed Central

    Rao, P. S. S.; Kumar, Santosh

    2015-01-01

    High prevalence of cigarette smoking in HIV patients is associated with increased HIV pathogenesis and disease progression. While the effect of smoking on the occurrence of lung cancer has been studied extensively, the association between smoking and HIV pathogenesis is poorly studied. We have recently shown the possible role of cytochrome P450 (CYP) in smoking/nicotine-mediated viral replication. In this review, we focus on the potential role of CYP pathway in polycyclic aromatic hydrocarbons (PAH), important constituents of cigarette smoke, mediated HIV pathogenesis. More specifically, we will discuss the role of CYP1A1 and CYP1B1, which are the major PAH-activating CYP enzymes. Our results have shown that treatment with cigarette smoke condensate (CSC) increases viral replication in HIV-infected macrophages. CSC contains PAH, which are known to be activated by CYP1A1 and CYP1B1 into procarcinogens/toxic metabolites. The expression of these CYPs is regulated by aryl hydrocarbon receptors (AHR), the cellular target of PAH, and an important player in various diseases including cancer. We propose that PAH/AHR-mediated CYP pathway is a novel target to develop new interventions for HIV positive smokers. PMID:26082767

  15. Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor: terminal hydroxylation of branched hydrocarbon chains.

    PubMed

    Jensen, Kenneth; Johnston, Jonathan B; de Montellano, Paul R Ortiz; Møller, Birger Lindberg

    2012-02-01

    The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multifunctional electron carrier that in plants accepts electrons from photosystem I (PSI) and facilitates photoreduction of NADP(+) to NADPH mediated by ferredoxin-NAD(P)H oxidoreductase (FdR). In bacteria, the electron flow is reversed and Fdx accepts electrons from NADPH via FdR and serves as the direct electron donor to bacterial P450s. By combining the two systems, we demonstrate that irradiation of PSI can drive the activity of a bacterial P450, CYP124 from Mycobacterium tuberculosis. The substitution of the costly cofactor NADPH with sunlight illustrates the potential of the light-driven hydroxylation system for biotechnology applications.

  16. Two azole fungicides (carcinogenic triadimefon and non-carcinogenic myclobutanil) exhibit different hepatic cytochrome P450 activities in medaka fish.

    PubMed

    Lin, Chun-Hung; Chou, Pei-Hsin; Chen, Pei-Jen

    2014-07-30

    Conazoles are a class of imidazole- or triazole-containing drugs commonly used as fungicides in agriculture and medicine. The broad application of azole drugs has led to the contamination of surface aquifers receiving the effluent of municipal or hospital wastewater or agricultural runoff. Several triazoles are rodent carcinogens; azole pollution is a concern to environmental safety and human health. However, the carcinogenic mechanisms associated with cytochrome P450 enzymes (CYPs) of conazoles remain unclear. We exposed adult medaka fish (Oryzias latipes) to continuous aqueous solutions of carcinogenic triadimefon and non-carcinogenic myclobutanil for 7 to 20 days at sub-lethal or environmentally relevant concentrations and assessed hepatic CYP activity and gene expression associated with CYP-mediated toxicity. Both triadimefon and myclobutanil induced hepatic CYP3A activity, but only triadimefon enhanced CYP1A activity. The gene expression of cyp3a38, cyp3a40, pregnane x receptor (pxr), cyp26b, retinoid acid receptor γ1 (rarγ1) and p53 was higher with triadimefon than myclobutanil. As well, yeast-based reporter gene assay revealed that 4 tested conazoles were weak agonists of aryl hydrocarbon receptor (AhR). We reveal differential CYP gene expression with carcinogenic and non-carcinogenic conazoles in a lower vertebrate, medaka fish. Liver CYP-enzyme induction may be a key event in conazole-induced tumorigenesis. This information is essential to evaluate the potential threat of conazoles to human health and fish populations in the aquatic environment. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Dependence of microsomal methoxyflurane O-demethylation on cytochrome P-450 reductase and the stoichiometry of fluoride ion and formaldehyde release.

    PubMed

    Waskell, L; Gonzales, J

    1982-07-01

    In order to characterize further the in vitro liver microsomal O-demethylation and defluorination of the volatile anesthetic methoxyflurane, and obtain additional information regarding the participation of cytochrome P-450 in the oxidation, the stoichiometry of the reaction was determined and the effect of antibody to cytochrome P-450 reductase on this unique biotransformation was examined. Liver microsomes were isolated from rabbits and rats in which enzyme induction had previously been produced by phenobarbital. The O-demethylation of methoxyflurane by phenobarbital-induced microsomes results in the production of 1 mol of formaldehyde for every 2 mol of fluoride ion produced. Dichloroacetic acid is also a product of methoxyflurane O-demethylation. Antibody to cytochrome P-450 reductase inhibits by 85% the amount of fluoride ion produced by the microsomal metabolism of methoxyflurane. Thus critical indirect supportive data are contributed to the hypothesis that at least one, but perhaps more, cytochrome P-450 is indeed responsible for methoxyflurane O-demethylation and defluorination.

  18. Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

    PubMed

    Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

    1995-04-04

    A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

  19. Hepatotoxicity of Herbal Supplements Mediated by Modulation of Cytochrome P450

    PubMed Central

    Chen, Taosheng

    2017-01-01

    Herbal supplements are a significant source of drug-drug interactions (DDIs), herb-drug interactions, and hepatotoxicity. Cytochrome P450 (CYP450) enzymes metabolize a large number of FDA-approved pharmaceuticals and herbal supplements. This metabolism of pharmaceuticals and supplements can be augmented by concomitant use of either pharmaceuticals or supplements. The xenobiotic receptors constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) can respond to xenobiotics by increasing the expression of a large number of genes that are involved in the metabolism of xenobiotics, including CYP450s. Conversely, but not exclusively, many xenobiotics can inhibit the activity of CYP450s. Induction of the expression or inhibition of the activity of CYP450s can result in DDIs and toxicity. Currently, the United States (US) Food and Drug Administration does not require the investigation of the interactions of herbal supplements and CYP450s. This review provides a summary of herbal supplements that inhibit CYP450s, induce the expression of CYP450s, and/or whose toxicity is mediated by CYP450s. PMID:29117101

  20. Isolation of the heme-thiolate enzyme cytochrome P-450TYR, which catalyzes the committed step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.

    PubMed Central

    Sibbesen, O; Koch, B; Halkier, B A; Møller, B L

    1994-01-01

    The cytochrome P-450 enzyme (hemethiolate enzyme) that catalyzes the N-hydroxylation of L-tyrosine to N-hydroxytyrosine, the committed step in the biosynthesis of the cyanogenic glucoside dhurrin, has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The cytochrome P-450 enzyme was solubilized with the detergents Renex 690, reduced Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and isolated by ion-exchange (DEAE-Sepharose) and dye (Cibacron blue and reactive red 120) column chromatography. To prevent irreversible aggregation of the cytochrome P-450 enzyme, the isolation procedure was designed without any concentration step--i.e., with dilution of the ion-exchange gel with gel filtration material. The isolated enzyme, which we designate the cytochrome P-450TYR enzyme, gives rise to the specific formation of a type I substrate binding spectrum in the presence of L-tyrosine. The microsomal preparation contains 0.2 nmol of total cytochrome P-450/mg of protein. The cytochrome P-450TYR enzyme is estimated to constitute approximately 20% of the total cytochrome P-450 content of the microsomal membranes and about 0.2% of their total protein content. The apparent molecular mass of the cytochrome P-450TYR enzyme is 57 kDa, and the N-terminal amino acid sequence is ATMEVEAAAATVLAAP. A polyclonal antibody raised against the isolated cytochrome P-450TYR enzyme is specific as monitored by Western blot analysis and inhibits the in vitro conversion of L-tyrosine to p-hydroxymandelonitrile catalyzed by the microsomal system. The cytochrome P-450TYR enzyme exhibits high substrate specificity and acts as an N-hydroxylase on a single endogenous substrate. The reported isolation procedure based on dye columns constitutes a gentle isolation method for cytochrome P-450 enzymes and is of general use as indicated by its ability to separate cytochrome P-450TYR from the cytochrome P-450 enzyme catalyzing the C

  1. Cytochrome P450 and P-Glycoprotein-Mediated Interactions Involving African Herbs Indicated for Common Noncommunicable Diseases

    PubMed Central

    Kikete, Siambi; Liang, Rongjia; Wang, Lili

    2017-01-01

    Herbal remedies are regularly used to complement conventional therapies in the treatment of various illnesses in Africa. This may be because they are relatively cheap and easily accessible and are believed by many to be safe, cause fewer side effects, and are less likely to cause dependency. On the contrary, many herbs have been shown to alter the pharmacokinetics of coadministered allopathic medicines and can either synergize or antagonize therapeutic effects as well as altering the toxicity profiles of these drugs. Current disease burden data point towards epidemiological transitions characterised by increasing urbanization and changing lifestyles, risk factors for chronic diseases like hypertension, diabetes, and cancer which often present as multimorbidities. As a result, we highlight African herb-drug interactions (HDIs) modulated via cytochrome P450 enzyme family (CYP) and P-glycoprotein (P-gp) and the consequences thereof in relation to antihypertensive, antidiabetic, and anticancer drugs. CYPs are enzymes which account for to up to 70% of drug metabolism while P-gp is an efflux pump that extrudes drug substrates out of cells. Consequently, regulation of the relative activity of both CYP and P-gp by African herbs influences the effective drug concentration at the site of action and modifies therapeutic outcomes. PMID:28250793

  2. Cycle affects imidacloprid efficiency by mediating cytochrome P450 expression in the brown planthopper Nilaparvata lugens.

    PubMed

    Kang, K; Yang, P; Pang, R; Yue, L; Zhang, W

    2017-10-01

    Circadian clocks influence most behaviours and physiological activities in animals, including daily fluctuations in metabolism. However, how the clock gene cycle influences insects' responses to pesticides has rarely been reported. Here, we provide evidence that cycle affects imidacloprid efficacy by mediating the expression of cytochrome P450 genes in the brown planthopper (BPH) Nilaparvata lugens, a serious insect pest of rice. Survival bioassays showed that the susceptibility of BPH adults to imidacloprid differed significantly between the two time points tested [Zeitgeber Time 8 (ZT8) and ZT4]. After cloning the cycle gene in the BPH (Nlcycle), we found that Nlcycle was expressed at higher levels in the fat body and midgut, and its expression was rhythmic with two peaks. Knockdown of Nlcycle affected the expression levels and rhythms of cytochrome P450 genes as well as susceptibility to imidacloprid. The survival rates of BPH adults after treatment with imidacloprid did not significantly differ between ZT4 and ZT8 after double-stranded Nlcycle treatment. These findings can be used to improve pesticide use and increase pesticide efficiency in the field. © 2017 The Royal Entomological Society.

  3. Comparison of inhibitory effects of the proton pump-inhibiting drugs omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole on human cytochrome P450 activities.

    PubMed

    Li, Xue-Qing; Andersson, Tommy B; Ahlström, Marie; Weidolf, Lars

    2004-08-01

    The human clearance of proton pump inhibitors (PPIs) of the substituted benzimidazole class is conducted primarily by the hepatic cytochrome P450 (P450) system. To compare the potency and specificity of the currently used PPIs (i.e., omeprazole, esomeprazole, lansoprazole, pantoprazole, and rabeprazole) as inhibitors of four cytochrome P450 enzymes (CYP2C9, 2C19, 2D6, and 3A4), we performed in vitro studies using human liver microsomal preparations and recombinant CYP2C19. Sample analysis was done using selected reaction monitoring liquid chromatography/tandem mass spectometry. With several systems for CYP2C19 activity (two marker reactions, S-mephenytoin 4'-hydroxylation and R-omeprazole 5-hydroxylation, tested in either human liver microsomes or recombinant CYP2C19), the five PPIs showed competitive inhibition of CYP2C19 activity with K(i) of 0.4 to 1.5 microM for lansoprazole, 2 to 6 microM for omeprazole, approximately 8 microM for esomeprazole, 14 to 69 microM for pantoprazole, and 17 to 21 microM for rabeprazole. Pantoprazole was a competitive inhibitor of both CYP2C9-catalyzed diclofenac 4'-hydroxylation and CYP3A4-catalyzed midazolam 1'-hydroxylation (K(i) of 6 and 22 microM, respectively), which were at least 2 times more potent than the other PPIs. All PPIs were poor inhibitors of CYP2D6-mediated bufuralol 1'-hydroxylation with IC(50) > 200 microM. The inhibitory potency of a nonenzymatically formed product of rabeprazole, rabeprazole thioether, was also investigated and showed potent, competitive inhibition with K(i) values of 6 microM for CYP2C9, 2 to 8 microM for CYP2C19, 12 microM for CYP2D6, and 15 microM for CYP3A4. The inhibitory potency of R-omeprazole on the four studied P450 enzymes was also studied and showed higher inhibitory potency than its S-isomer on CYP2C9 and 2C19 activities. Our data suggest that, although the inhibitory profiles of the five studied PPIs were similar, lansoprazole and pantoprazole are the most potent in vitro inhibitors

  4. Cytochrome P450 1A1 expression in cetacean skin biopsies from the Indian Ocean.

    PubMed

    Jauniaux, Thierry; Farnir, Frédéric; Fontaine, Michaël; Kiszka, Jeremy; Sarlet, Michael; Coignoul, Freddy

    2011-06-01

    The study describes cytochrome P450 1A1 (CYPA1) expression in the skin of different cetacean species (Megaptera novaeangliae, n=15; Stenella attenuata, n=7 and Stenella longirostris, n=24) from the Mozambique Channel island of Mayotte. Immunohistochemical examination was performed with a monoclonal antibody against scup cytochrome CYPA1. The sex was determined using a molecular approach consisting in the genotyping sex-specific genes. CYPA1 was detected at the junction between epidermis and blubber on dolphins only, mostly in the endothelial cells. Similar observation was obtained in the dermis of one M. novaeangliae. Immunohistochemical slides were scored to evaluate the expression of the CYPA1 and a higher expression was observed in S. longirostris, suggesting a higher exposure to pollutants for this species. The difference of expression between sexes was not significant. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Effect of feeding quandong (Santalum acuminatum) oil to rats on tissue lipids, hepatic cytochrome P-450 and tissue histology.

    PubMed

    Jones, G P; Birkett, A; Sanigorski, A; Sinclair, A J; Hooper, P T; Watson, T; Rieger, V

    1994-06-01

    Quandong kernels are a traditional Aboriginal food item; they are rich in oil and contain large amounts of an unusual fatty acid, trans-11-octadecen-9-ynoic acid (santalbic acid), but it is not known whether this acid is absorbed and/or metabolized. The oil was fed at 12.6% of total energy content in semi-synthetic diets to groups of male Sprague-Dawley rats for 10 and 20 days. Santalbic acid was found in the lipids of plasma, adipose tissue, skeletal muscle, kidney, heart and liver but not in brain. Hepatic microsomal cytochrome P-450 activity in animals fed for 20 days was significantly higher (P < 0.05) than in controls. Histopathological examination did not reveal any lesions in the tissues of any animal fed quandong oil. The fact that santalbic acid was readily absorbed, widely distributed in tissues and was associated with an elevated level of hepatic cytochrome P-450 indicates that further studies are required to investigate whether or not there is a hazard associated with the human practice of consuming quandong kernels.

  6. Abnormal kinetic behavior of cytochrome oxidase in a case of Leigh disease.

    PubMed Central

    Glerum, M; Robinson, B H; Spratt, C; Wilson, J; Patrick, D

    1987-01-01

    Cultured skin fibroblasts from a child with fatal lacticacidemia displayed an abnormally high lactate:pyruvate ratio of 77:1, compared with control values of 22:1-27:1. When protease-treated isolated mitochondria were used, activity of the respiratory-chain enzymes was found to be approximately 60% of normal, and adenosine triphosphate synthesis was found to be normal with all substrates tested. In mitochondria prepared by means of digitonin treatment, adenosine triphosphate synthesis was depressed with all substrates tested, suggesting a defect in the operation of the cytochrome oxidase complex. In disrupted whole cells from the patient, cytochrome oxidase activity was 56% of the activity in the control cell line with the lowest activity. In the presence of a twofold excess of oxidized cytochrome c, patient cells showed 31% of the activity in controls. Cytochrome oxidase activity in both sonicated whole-cell preparations and in sonicated mitochondria displayed abnormal kinetics with regard to the substrate-reduced cytochrome c, which was particularly evident in the presence of excess oxidized cytochrome c. We believe that kinetically abnormal cytochrome oxidase complex is responsible for the biochemical and clinical abnormalities present in this patient. PMID:2821802

  7. Crystal structure of a cytochrome P450 2B6 genetic variant in complex with the inhibitor 4-(4-chlorophenyl)imidazole at 2.0-A resolution.

    PubMed

    Gay, Sean C; Shah, Manish B; Talakad, Jyothi C; Maekawa, Keiko; Roberts, Arthur G; Wilderman, P Ross; Sun, Ling; Yang, Jane Y; Huelga, Stephanie C; Hong, Wen-Xu; Zhang, Qinghai; Stout, C David; Halpert, James R

    2010-04-01

    The structure of the K262R genetic variant of human cytochrome P450 2B6 in complex with the inhibitor 4-(4-chlorophenyl)imidazole (4-CPI) has been determined using X-ray crystallography to 2.0-A resolution. Production of diffraction quality crystals was enabled through a combination of protein engineering, chaperone coexpression, modifications to the purification protocol, and the use of unique facial amphiphiles during crystallization. The 2B6-4-CPI complex is virtually identical to the rabbit 2B4 structure bound to the same inhibitor with respect to the arrangement of secondary structural elements and the placement of active site residues. The structure supports prior P450 2B6 homology models based on other mammalian cytochromes P450 and is consistent with the limited site-directed mutagenesis studies on 2B6 and extensive studies on P450 2B4 and 2B1. Although the K262R genetic variant shows unaltered binding of 4-CPI, altered binding affinity, kinetics, and/or product profiles have been previously shown with several other ligands. On the basis of new P450 2B6 crystal structure and previous 2B4 structures, substitutions at residue 262 affect a hydrogen-bonding network connecting the G and H helices, where subtle differences could be transduced to the active site. Docking experiments indicate that the closed protein conformation allows smaller ligands such as ticlopidine to bind to the 2B6 active site in the expected orientation. However, it is unknown whether 2B6 undergoes structural reorganization to accommodate bulkier molecules, as previously inferred from multiple P450 2B4 crystal structures.

  8. Preliminary characterization of a murine model for 1-bromopropane neurotoxicity: Role of cytochrome P450.

    PubMed

    Zong, Cai; Garner, C Edwin; Huang, Chinyen; Zhang, Xiao; Zhang, Lingyi; Chang, Jie; Toyokuni, Shinya; Ito, Hidenori; Kato, Masashi; Sakurai, Toshihiro; Ichihara, Sahoko; Ichihara, Gaku

    2016-09-06

    Neurotoxicity of 1-bromopropane (1-BP) has been reported in both human cases and animal studies. To date, neurotoxicity of 1-BP has been induced in rats but not in mice due to the lethal hepatotoxicity of 1-BP. Oxidization by cytochromes P450 and conjugation with glutathione (GSH) are two critical metabolism pathways of 1-BP and play important roles in toxicity of 1-BP. The aim of the present study was to establish a murine model of 1-BP neurotoxicity, by reducing the hepatotoxicity of 1-BP with 1-aminobenzotriazole (1-ABT); a commonly used nonspecific P450s inhibitor. The results showed that subcutaneous or intraperitoneal injection of 1-ABT at 50mg/kg body weight BID (100mg/kg BW/day) for 3days, inhibited about 92-96% of hepatic microsomal CYP2E1 activity, but only inhibited about 62-64% of CYP2E1 activity in brain microsomes. Mice treated with 1-ABT survived even after exposure to 1200ppm 1-BP for 4 weeks and histopathological studies showed that treatment with 1-ABT protected mice from 1-BP-induced hepatic necrosis, hepatocyte degeneration, and hemorrhage. After 4-week exposure to 1-BP, the brain weight of 1-ABT(+)/1200ppm 1-BP group was decreased significantly. In 1-ABT-treated groups, expression of hippocampal Ran protein and cerebral cortical GRP78 was dose-dependently increased by exposure to 1-BP. We conclude that the control of hepatic P450 activity allows the observation of effects of 1-BP on the murine brain at a higher concentration by reduction of hepatotoxicity. The study suggests that further experiments with liver-specific control of P450 activity using gene technology might provide better murine models for 1-bromopropane-induced neurotoxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  9. Effects of soy containing diet and isoflavones on cytochrome P450 enzyme expression and activity.

    PubMed

    Ronis, Martin J J

    2016-08-01

    Cytochromes P450 (CYPs) play an important role in metabolism and clearance of most clinically utilized drugs and other xenobiotics. They are important in metabolism of endogenous compounds including fatty acids, sterols, steroids and lipid-soluble vitamins. Dietary factors such as phytochemicals are capable of affecting CYP expression and activity, which may be important in diet-drug interactions and in the development of fatty liver disease, cardiovascular disease and cancer. One important diet-CYP interaction is with diets containing plant proteins, particularly soy protein. Soy diets are traditionally consumed in Asian countries and are linked to lower incidence of several cancers and of cardiovascular disease in Asian populations. Soy is also an important protein source in vegetarian and vegan diets and the sole protein source in soy infant formulas. Recent studies suggest that consumption of soy can inhibit induction of CY1 enzymes by polycyclic aromatic hydrocarbons (PAHs) which may contribute to cancer prevention. In addition, there are data to suggest that soy components promiscuously activate several nuclear receptors including PXR, PPAR and LXR resulting in increased expression of CYP3As, CYP4As and CYPs involved in metabolism of cholesterol to bile acids. Such soy-CYP interactions may alter drug pharmacokinetics and therapeutic efficacy and are associated with improved lipid homeostasis and reduced risk of cardiovascular disease. The current review summarizes results from in vitro; in vivo and clinical studies of soy-CYP interactions and examines the evidence linking the effects of soy diets on CYP expression to isoflavone phytoestrogens, particularly, genistein and daidzein that are associated with soy protein.

  10. [Cytochrome b-559 photooxidation in the presence of carbonyl cyanide p-trifluorometh-oxyphenylhydrazone and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone or p-benzoquinone in three non-photosynthetic mutants of Chlamydomonas reinhardti (author's transl)].

    PubMed

    Maroc, J; Garnier, J

    1975-04-14

    Studies of absorbance related to the cytochrome b-559 photooxidation induced by FCCP, with and without addition of 3-p-chlorophenyl-1, 1-dimethylurea (CMU), DBMIB or p-benzoquinone, in whole cells and in chloroplast fragments of Chlamydomonas reinhardti, were carried out. In addition to the wild type, three strains of non-photosynthetic mutants were used: Fl 5, which lacks P 700; Fl 9 and Fl 15, which are deficient in bound cytochrome c-553 and in cytochrome b-563. In the presence of FCCP, whole cells and chloroplast fragments of the four strains showed a System II-dependent photooxidation of cytochrome b-559. This photooxidation was inhibited by CMU but it occurred again in presence of FCCP, CMU and DBMIB. In chloroplast fragments, cytochrome b-559 photooxidation was also inhibited by an excess of FCCP; it was recovered, likewise, by addition of DBMIB. In whole cells, the highest measured redox changes were: 1 mu mol oxidized cytochrome b-559 per 1 mmol chlorophyll, corresponding approximately to about one seventh (wild type, Fl5) or one fifth (Fl 9, Fl 15) of the total amount of this cytochrome. Another kind of cytochrome b-559 photooxidation, CMU-insensitive, also occurred in the mutants Fl 9 and Fl 15 and in the wild type, but not in the mutant Fl 5. This latter kind of photooxidation was observed with chloroplast fragments in the presence of FCCP and CMU and also with whole cells in the presence of FCCP, CMU and p-benzoquinone. These reactions can be attributed to the Photosystem I; they do not require the intervention of the cytochrome c-553. A high-potential form of cytochrome b-559, hydroquinone-reducible, was involved in these two kinds of photooxidation. In addition, a lower potential form, reducible only by ascorbate, appeared to be able to interfere also. An interpretation is attempted, taking into consideration the various effects of FCCP and DBMIB, at different concentrations, on photosynthetic electron transport.

  11. Cytochrome P450 isozyme protein verified in the skin of southern hemisphere humpback whales (Megaptera novaeangliae): implications for biochemical biomarker assessment.

    PubMed

    Waugh, Courtney A; Huston, Wilhelmina M; Noad, Michael J; Bengtson Nash, Susan

    2011-04-01

    Large mysticete whales represent a unique challenge for chemical risk assessment. Few epidemiological investigations are possible due to the low incidence of adult stranding events. Similarly their often extreme life-history adaptations of prolonged migration and fasting challenge exposure assumptions. Molecular biomarkers offer the potential to complement information yielded through tissue chemical analysis, as well as providing evidence of a molecular response to chemical exposure. In this study we confirm the presence of cytochrome P450 reductase (CPR) and cytochrome P450 isoenzyme 1A1 (CYP1A1) in epidermal tissue of southern hemisphere humpback whales (Megaptera novaeangliae). The detection of CYP1A1 in the integument of the humpback whale affords the opportunity for further quantitative non-destructive investigations of enzyme activity as a function of chemical stress. Copyright © 2011 Elsevier Ltd. All rights reserved.

  12. Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes

    PubMed Central

    Jungmann, Volker; Molnár, István; Hammer, Philip E.; Hill, D. Steven; Zirkle, Ross; Buckel, Thomas G.; Buckel, Dagmar; Ligon, James M.; Pachlatko, J. Paul

    2005-01-01

    4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined. PMID:16269732

  13. Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

    PubMed

    Hrycay, E G; Bandiera, S M

    2009-12-01

    The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

  14. Aromatic Hydroxylation of Salicylic Acid and Aspirin by Human Cytochromes P450

    PubMed Central

    Bojić, Mirza; Sedgeman, Carl A.; Nagy, Leslie D.; Guengerich, F. Peter

    2015-01-01

    Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids—salicyluric acid and gentisuric acid—and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1. PMID:25840124

  15. Gene structure of CYP3A4, an adult-specific form of cytochrome P450 in human livers, and its transcriptional control.

    PubMed

    Hashimoto, H; Toide, K; Kitamura, R; Fujita, M; Tagawa, S; Itoh, S; Kamataki, T

    1993-12-01

    CYP3 A4 is the adult-specific form of cytochrome P450 in human livers [Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. & Kamataki, T. (1990) Biochemistry 29, 4430-4433]. The sequences of three genomic clones for CYP3A4 were analyzed for all exons, exon-intron junctions and the 5'-flanking region from the major transcription site to nucleotide position -1105, and compared with those of the CYP3A7 gene, a fetal-specific form of cytochrome P450 in humans. The results showed that the identity of 5'-flanking sequences between CYP3A4 and CYP3A7 genes was 91%, and that each 5'-flanking region had characteristic sequences termed as NFSE (P450NF-specific element) and HFLaSE (P450HFLa specific element), respectively. A basic transcription element (BTE) also lay in the 5'-flanking region of the CYP3A4 gene as seen in many CYP genes [Yanagida, A., Sogawa, K., Yasumoto, K. & Fujii-Kuriyama, Y. (1990) Mol. Cell. Biol. 10, 1470-1475]. The BTE binding factor (BTEB) was present in both adult and fetal human livers. To examine the transcriptional activity of the CYP3A4 gene, DNA fragments in the 5'-flanking region of the gene were inserted in front of the simian virus 40 promoter and the chloramphenicol acetyltransferase structural gene, and the constructs were transfected in HepG2 cells. The analysis of the chloramphenicol acetyltransferase activity indicated that (a) specific element(s) which could bind with a factor(s) in livers was present in the 5'-flanking region of the CYP3A4 gene to show the transcriptional activity.

  16. Requirement of histidine 217 for ubiquinone reductase activity (Qi site) in the cytochrome bc1 complex.

    PubMed

    Gray, K A; Dutton, P L; Daldal, F

    1994-01-25

    Folding models suggest that the highly conserved histidine 217 of the cytochrome b subunit from the cytochrome bc1 complex is close to the quinone reductase (Qi) site. This histidine (bH217) in the cytochrome b polypeptide of the photosynthetic bacterium Rhodobacter capsulatus has been replaced with three other residues, aspartate (D), arginine (R), and leucine (L). bH217D and bH217R are able to grow photoheterotrophically and contain active cytochrome bc1 complexes (60% of wild-type activity), whereas the bH217L mutant is photosynthetically incompetent and contains a cytochrome bc1 complex that has only 10% of the wild-type activity. Single-turnover flash-activated electron transfer experiments show that cytochrome bH is reduced via the Qo site with near native rates in the mutant strains but that electron transfer between cytochrome bH and quinone bound at the Qi site is greatly slowed. These results are consistent with redox midpoint potential (Em) measurements of the cytochrome b subunit hemes and the Qi site quinone. The Em values of cyt bL and bH are approximately the same in the mutants and wild type, although the mutant strains have a larger relative concentration of what may be the high-potential form of cytochrome bH, called cytochrome b150. However, the redox properties of the semiquinone at the Qi site are altered significantly. The Qi site semiquinone stability constant of bH217R is 10 times higher than in the wild type, while in the other two strains (bH217D and bH217L) the stability constant is much lower than in the wild type. Thus H217 appears to have major effects on the redox properties of the quinone bound at the Qi site. These data are incorporated into a suggestion that H217 forms part of the binding pocket of the Qi site in a manner reminiscent of the interaction between quinone bound at the Qb site and H190 of the L subunit of the bacterial photosynthetic reaction center.

  17. Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.

    2013-10-01

    Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes,more » intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. - Highlights: • Piperine induces PXR-mediated CYP3A4 and MDR1 expression. • Piperine activates PXR by binding to PXR and recruiting coactivator SRC-1. • Piperine induces PXR activation in vivo. • Caution should be taken in piperine consumption during drug treatment.« less

  18. The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase.

    PubMed

    Gilmour, R; Goodhew, C F; Pettigrew, G W; Prazeres, S; Moura, J J; Moura, I

    1994-06-15

    In work that is complementary to our investigation of the spectroscopic features of the cytochrome c peroxidase from Paracoccus denitrificans [Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Biochem. J. 294, 745-752], we have studied the kinetics of oxidation of cytochrome c by this enzyme. The enzyme, as isolated, is in the fully oxidized form and is relatively inactive. Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzyme. Full activation is achieved by addition of 1 mM CaCl2. Enzyme activation is associated with formation of a high-spin state at the oxidized low-potential haem. EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA of the reduced, partially activated, form abolishes the activity. We conclude that the active enzyme is a mixed-valence form with the low-potential haem in a high-spin state that is stabilized by Ca2+. Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution. Most of the activity lost upon dilution can be recovered after reconcentration. The M(r) of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of M(r) obtained are intermediate between those of a monomer (39,565) and a dimer. We propose that the active form of the enzyme is a dimer which dissociates at high dilution to give inactive monomers. From the activity of the enzyme at different dilutions, a KD of 0.8 microM can be calculated for the monomerdimer equilibrium. The cytochrome c peroxidase oxidizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations. The maximal catalytic-centre activity ('turnover number') under the assay conditions used is 62,000 min-1, with a half-saturating ferrocytochrome c concentration of 3.3 microM. The corresponding values for the

  19. Assessing the clinical significance of botanical supplementation on human cytochrome P450 3A activity: comparison of a milk thistle and black cohosh product to rifampin and clarithromycin.

    PubMed

    Gurley, Bill; Hubbard, Martha A; Williams, D Keith; Thaden, John; Tong, Yudong; Gentry, W Brooks; Breen, Philip; Carrier, Danielle J; Cheboyina, Shreekar

    2006-02-01

    Phytochemical-mediated modulation of cytochrome P450 enzymes (CYPs) may underlie many herb-drug interactions. This study's purpose was to assess the effects of milk thistle and black cohosh supplementation on CYP3A activity and compare them to a clinically recognized inducer, rifampin, and inhibitor, clarithromycin. Healthy volunteers were randomly assigned to receive a standardized milk thistle (900 mg) or black cohosh (80 mg) supplement for 14 days. Subjects also received rifampin (600 mg) and clarithromycin (1000 mg) for 7 days as positive controls for CYP3A induction and inhibition, respectively. Midazolam was administered orally before and after each supplementation and control period. The effects of milk thistle, black cohosh, rifampin, and clarithromycin on midazolam pharmacokinetics were determined using noncompartmental techniques. Unlike those observed for rifampin and clarithromycin, midazolam pharmacokinetics was unaffected by milk thistle or black cohosh. Milk thistle and black cohosh appear to have no clinically relevant effect on CYP3A activity in vivo.

  20. Assessing the clinical significance of botanical supplementation on human cytochrome P450 3A activity: Comparison of a milk thistle and black cohosh product to rifampin and clarithromycin

    PubMed Central

    Gurley, Bill; Hubbard, Martha A.; Williams, D. Keith; Thaden, John; Tong, Yudong; Gentry, W. Brooks; Breen, Philip; Carrier, Danielle J.; Cheboyina, Shreekar

    2007-01-01

    Phytochemical-mediated modulation of cytochrome P450 enzymes (CYPs) may underlie many herb-drug interactions. This study’s purpose was to assess the effects of milk thistle and black cohosh supplementation on CYP3A activity and compare them to a clinically recognized inducer, rifampin, and inhibitor, clarithromycin. Healthy volunteers were randomly assigned to receive a standardized milk thistle (900 mg) or black cohosh (80 mg) supplement for 14 days. Subjects also received rifampin (600 mg) and clarithromycin (1000 mg) for 7 days as positive controls for CYP3A induction and inhibition, respectively. Midazolam was administered orally before and after each supplementation and control period. The effects of milk thistle, black cohosh, rifampin, and clarithromycin on midazolam pharmacokinetics were determined using noncompartmental techniques. Unlike those observed for rifampin and clarithromycin, midazolam pharmacokinetics were unaffected by milk thistle or black cohosh. Milk thistle and black cohosh appear to have no clinically relevant effect on CYP3A activity in vivo. PMID:16432272

  1. Renal cytochrome P450 omega-hydroxylase and epoxygenase activity are differentially modified by nitric oxide and sodium chloride.

    PubMed

    Oyekan, A O; Youseff, T; Fulton, D; Quilley, J; McGiff, J C

    1999-10-01

    Renal function is perturbed by inhibition of nitric oxide synthase (NOS). To probe the basis of this effect, we characterized the effects of nitric oxide (NO), a known suppressor of cytochrome P450 (CYP) enzymes, on metabolism of arachidonic acid (AA), the expression of omega-hydroxylase, and the efflux of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated kidney. The capacity to convert [(14)C]AA to HETEs and epoxides (EETs) was greater in cortical microsomes than in medullary microsomes. Sodium nitroprusside (10-100 microM), an NO donor, inhibited renal microsomal conversion of [(14)C]AA to HETEs and EETs in a dose-dependent manner. 8-bromo cGMP (100 microM), the cell-permeable analogue of cGMP, did not affect conversion of [(14)C]AA. Inhibition of NOS with N(omega)-nitro-L-arginine-methyl ester (L-NAME) significantly increased conversion of [(14)C]AA to HETE and greatly increased the expression of omega-hydroxylase protein, but this treatment had only a modest effect on epoxygenase activity. L-NAME induced a 4-fold increase in renal efflux of 20-HETE, as did L-nitroarginine. Oral treatment with 2% sodium chloride (NaCl) for 7 days increased renal epoxygenase activity, both in the cortex and the medulla. In contrast, cortical omega-hydroxylase activity was reduced by treatment with 2% NaCl. Coadministration of L-NAME and 2% NaCl decreased conversion of [(14)C]AA to HETEs without affecting epoxygenase activity. Thus, inhibition of NOS increased omega-hydroxylase activity, CYP4A expression, and renal efflux of 20-HETE, whereas 2% NaCl stimulated epoxygenase activity.

  2. Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Grinkova, Yelena V.; Denisov, Ilia G.; McLean, Mark A.

    Highlights: ► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs. -- Abstract: The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involvesmore » release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP

  3. Differential effect of Amyloid Beta on the Cytochrome P450 epoxygenase activity in rat brain

    PubMed Central

    Sarkar, Pallabi; Narayanan, Jayashree; Harder, David R.

    2011-01-01

    One of the prominent features of Alzheimer's disease is the excessive accumulation of the protein amyloid beta (Aβ) in certain areas of the brain leading to neurodegeneration. Aβ is cytotoxic and disrupts several cytoprotective pathways. Recent literature has demonstrated that certain cytochrome P450 (CYP) products are neuroprotective, including epoxide metabolites of arachidonic acid (AA), epoxyeicosatrienoic acids (EETs). The action of Aβ with respect to regionally produced EETs in the brain has yet to be defined. Epoxygenases metabolize AA into 4 regioisomers of EETs (14,15 -, 11,12-, 8,9- and 5,6-EET). EETs are rapidly degraded into dihydroxyeicosatrienoic acids (DiHETEs) by soluble epoxide hydrolase (sEH). To determine the effect of Aβ on the epoxygenase activity in different regions of the brain, microsomes were prepared from the cerebrum and cerebellum of adult Sprague-Dawley rats and incubated with 1 and 10 μM Aβ for 30 minutes after which epoxygenase activity assay was performed. Mass spectrometry indicated that incubation with Aβ reduced 14,15-EET production by 30% as compared to vehicle in the cerebrum, but not in the cerebellum. When we separated the cerebrum into cortex and hippocampus, significant decrease in the production of total EETs and DiHETEs were seen in presence of Aβ (81% and 74%) in the cortex. Moreover, 11, 12-EET production was decreased to ∼70% of vehicle in both cortex and hippocampus. Epoxygenase activity in the cultured astrocytes and neurons also showed reduction in total EET and DiHETE production (to 80% and ∼70% of vehicle respectively) in presence of Aβ. Altogether, our data suggest that Aβ reduces epoxygenase activity differentially in a region-specific and cell-specific manner. The reduction of cytoprotective EETs by Aβ in the cerebrum may make it more prone to degeneration than the cerebellum. Further understanding of these interactions will improve our ability to protect against the pathology of Alzheimer

  4. Adaptation of cytochrome-b5 reductase activity and methaemoglobinaemia in areas with a high nitrate concentration in drinking-water.

    PubMed Central

    Gupta, S. K.; Gupta, R. C.; Seth, A. K.; Gupta, A. B.; Bassin, J. K.; Gupta, A.

    1999-01-01

    An epidemiological investigation was undertaken in India to assess the prevalence of methaemoglobinaemia in areas with high nitrate concentration in drinking-water and the possible association with an adaptation of cytochrome-b5 reductase. Five areas were selected, with average nitrate ion concentrations in drinking-water of 26, 45, 95, 222 and 459 mg/l. These areas were visited and house schedules were prepared in accordance with a statistically designed protocol. A sample of 10% of the total population was selected in each of the areas, matched for age and weight, giving a total of 178 persons in five age groups. For each subject, a detailed history was documented, a medical examination was conducted and blood samples were taken to determine methaemoglobin level and cytochrome-b5 reductase activity. Collected data were subjected to statistical analysis to test for a possible relationship between nitrate concentration, cytochrome-b5 reductase activity and methaemoglobinaemia. High nitrate concentrations caused methaemoglobinaemia in infants and adults. The reserve of cytochrome-b5 reductase activity (i.e. the enzyme activity not currently being used, but which is available when needed; for example, under conditions of increased nitrate ingestion) and its adaptation with increasing water nitrate concentration to reduce methaemoglobin were more pronounced in children and adolescents. PMID:10534899

  5. The Chemically Inducible Plant Cytochrome P450 CYP76B1 Actively Metabolizes Phenylureas and Other Xenobiotics1

    PubMed Central

    Robineau, Tiburce; Batard, Yannick; Nedelkina, Svetlana; Cabello-Hurtado, Francisco; LeRet, Monique; Sorokine, Odile; Didierjean, Luc; Werck-Reichhart, Danièle

    1998-01-01

    Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants. On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin. In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas. CYP76B1 catalyzes the double N-dealkylation of phenylureas with turnover rates comparable to those reported for physiological substrates and produces nonphytotoxic compounds. Potential uses for CYP76B1 thus include control of herbicide tolerance and selectivity, as well as soil and groundwater bioremediation. PMID:9808750

  6. Effects of atrazine on cytochrome P450 enzymes of zebrafish (Danio rerio).

    PubMed

    Dong, Xiaoli; Zhu, Lusheng; Wang, Jinhua; Wang, Jun; Xie, Hui; Hou, Xinxin; Jia, Wentao

    2009-10-01

    In this study, the effects of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in males and females of adult zebrafish (Danio rerio) were studied. The liver microsomal cytochrome P450 content, NADPH-P450 reductase, aminopyrine N-demethylase (APND), and erythromycin N-demethylase (ERND) activity were measured. Zebrafish were exposed to control and 3 treatments (0.01, 0.1, and 1 mg L(-1)) of atrazine for 5, 10, 15, 20, and 25 days. The results indicated that, within the range of test atrazine concentrations, either P450 content or P450 isozyme activities could be induced by atrazine. Compared to controls, P450 content was significantly increased at all atrazine concentrations at days 10, 15, and 20; thereafter, at day 25, all concentrations decreased to approximately the control levels, both in males and females. In addition, the strongest induction of P450 content was observed on day 15 in males and day 10 in females at treatment concentrations of 1 mg L(-1). NADPH-P450 reductase activities showed mild increase in males; however, the females exhibited significant induction on days 15, 20, and 25; especially, at concentrations of 0.01 mg L(-1), the induction level was consistently increased during the experiment. The inducements of APND and ERND in males were mainly observed on the days 5, 10, and 15, which showed less distinct induction, while significant induction was observed in cases of treatments during all days in females. In conclusion, atrazine induces P450 enzymes in zebrafish, and the effects may function as significant toxicity mechanisms in zebrafish. Additionally, it also confirms the importance of using a combined multi-time and multi-index diagnostic method to enhance the sensitivity and effectiveness of the indices adopted.

  7. Does Compound I Vary Significantly between Isoforms of Cytochrome P450?

    PubMed Central

    2011-01-01

    The cytochrome P450 (CYP) enzymes are important in many areas, including pharmaceutical development. Subtle changes in the electronic structure of the active species, Compound I, have been postulated previously to account partly for the experimentally observed differences in reactivity between isoforms. Current predictive models of CYP metabolism typically assume an identical Compound I in all isoforms. Here we present a method to calculate the electronic structure and to estimate the Fe–O bond enthalpy of Compound I, and apply it to several human and bacterial CYP isoforms. Conformational flexibility is accounted for by sampling large numbers of structures from molecular dynamics simulations, which are subsequently optimized with density functional theory (B3LYP) based quantum mechanics/molecular mechanics. The observed differences in Compound I between human isoforms are small: They are generally smaller than the spread of values obtained for the same isoform starting from different initial structures. Hence, it is unlikely that the variation in activity between human isoforms is due to differences in the electronic structure of Compound I. A larger difference in electronic structure is observed between the human isoforms and P450cam and may be explained by the slightly different hydrogen-bonding environment surrounding the cysteinyl sulfur. The presence of substrate in the active site of all isoforms studied appears to cause a slight decrease in the Fe–O bond enthalpy, apparently due to displacement of water out of the active site, suggesting that Compound I is less stable in the presence of substrate. PMID:21863858

  8. Metabolism of styrene to styrene oxide and vinylphenols in cytochrome P450 2F2- and P450 2E1-knockout mouse liver and lung microsomes.

    PubMed

    Shen, Shuijie; Li, Lei; Ding, Xinxin; Zheng, Jiang

    2014-01-21

    Pulmonary toxicity of styrene is initiated by cytochromes P450-dependent metabolic activation. P450 2E1 and P450 2F2 are considered to be two main cytochrome P450 enzymes responsible for styrene metabolism in mice. The objective of the current study was to determine the correlation between the formation of styrene metabolites (i.e., styrene oxide and 4-vinylphenol) and pulmonary toxicity of styrene, using Cyp2e1- and Cyp2f2-null mouse models. A dramatic decrease in the formation of styrene glycol and 4-vinylphenol was found in Cyp2f2-null mouse lung microsomes relative to that in the wild-type mouse lung microsomes; however, no significant difference in the production of the styrene metabolites was observed between lung microsomes obtained from Cyp2e1-null and the wild-type mice. The knockout and wild-type mice were treated with styrene (6.0 mmol/kg, ip), and cell counts and LDH activity in bronchoalveolar lavage fluids were monitored to evaluate the pulmonary toxicity induced by styrene. Cyp2e1-null mice displayed a susceptibility to lung toxicity of styrene similar to that of the wild-type animals; however, Cyp2f2-null mice were resistant to styrene-induced pulmonary toxicity. In conclusion, both P450 2E1 and P450 2F2 are responsible for the metabolic activation of styrene. The latter enzyme plays an important role in styrene-induced pulmonary toxicity. Both styrene oxide and 4-vinylphenol are suggested to participate in the development of lung injury induced by styrene.

  9. Role of Cytochrome P450 Hydroxylase in the Decreased Accumulation of Vitamin E in Muscle from Turkeys Compared to that from Chickens

    PubMed Central

    Perez, Dale M.; Richards, Mark P.; Parker, Robert S.; Berres, Mark E.; Wright, Aaron T.; Sifri, Mamduh; Sadler, Natalie C; Tatiyaborworntham, Nantawat; Li, Na

    2016-01-01

    Turkeys and chickens reared to 5 weeks of age and fed diets with feedstuffs low in endogenous tocopherols were examined. Treatments included feed supplemented with RRR (natural source vitamin E) alpha tocopheryl acetate (AcT, 35 mg/kg feed) and all-racemic (synthetic vitamin E) AcT (10 and 58 mg/kg feed). Alpha tocopherol hydroxylase activity was greater in liver microsomes prepared from turkeys compared to that from chickens (p < 0.01). Alpha and gamma tocopherol metabolites were higher in turkey bile than in chicken when assessing the RRR AcT diet and the all-racemic AcT diet at 58 mg/kg feed (p < 0.01). Turkey cytochrome P450 2C29 was increased relative to its chicken ortholog on the basis of RNA-Seq transcript abundance (p < 0.001) and activity-based protein profiling (p < 0.01) of liver tissue. Alpha tocopherol concentrations in plasma, liver, and muscle from turkey were lower than the respective tissues from chicken (p < 0.05). Lipid oxidation was greater in turkey thigh than in chicken (p < 0.05). These results suggest that elevated tocopherol metabolism by cytochrome P450 hydroxylase(s) in turkeys contributes to the decreased accumulation of alpha tocopherol in turkey tissues compared to that of chickens. PMID:26653675

  10. The Influence of Cytochrome P450 Pharmacogenetics on Disposition of Common Antidepressant and Antipsychotic Medications

    PubMed Central

    van der Weide, Jan; Hinrichs, John WJ

    2006-01-01

    Since the identification of all the major drug-metabolising cytochrome P450 (CYP) enzymes and their major gene variants, pharmacogenetics has had a major impact on psychotherapeutic drug therapy. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is an important reason for drug therapy failure. Variability in CYP activity may be caused by various factors, including endogenous factors such as age, gender and morbidity as well as exogenous factors such as co-medication, food components and smoking habit. However, polymorphisms, present in most CYP genes, are responsible for a substantial part of this variability. Although CYP genotyping has been shown to predict the majority of aberrant phenotypes, it is currently rarely performed in clinical practice. PMID:16886044

  11. Deletion of P399{sub E}401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Flueck, Christa E., E-mail: christa.flueck@dkf.unibe.ch; Mallet, Delphine; Hofer, Gaby

    2011-09-09

    Highlights: {yields} Mutations in human POR cause congenital adrenal hyperplasia. {yields} We are reporting a novel 3 amino acid deletion mutation in POR P399{sub E}401del. {yields} POR mutation P399{sub E}401del decreased P450 activities by 60-85%. {yields} Impairment of steroid metabolism may be caused by multiple hits. {yields} Severity of aromatase inhibition is related to degree of in utero virilization. -- Abstract: P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399{sub E}401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant PORmore » proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399{sub E}401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17{alpha}-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399{sub E}401 revealed reduced stability and flexibility of the mutant. In conclusion, P399{sub E}401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399{sub E}401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.« less

  12. INTERINDIVIDUAL VARIANCE OF CYTOCHROME P450 FORMS IN HUMAN HEPATIC MICROSOMES: CORRELATION OF INDIVIDUAL FORMS WITH XENOBIOTIC METABOLISM AND IMPLICATIONS IN RISK ASSESSMENT

    EPA Science Inventory

    Differences in biotransformation activities may alter the bioavailability or efficacy of drugs, provide protection from certain xenobiotic and environmental agents, or increase toxicity of others. Cytochrome P450 (CYP450) enzymes are responsible for the majority of oxidation reac...

  13. Buprofezin Is Metabolized by CYP353D1v2, a Cytochrome P450 Associated with Imidacloprid Resistance in Laodelphax striatellus.

    PubMed

    Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Han, Zhaojun

    2017-11-29

    CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus . This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. The results showed the nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of buprofezin (eluting at 8.7 min) and parallel formation of an unknown metabolite (eluting 9.5 min). However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. The recombinant CYP353D1v2 protein efficiently catalyzed the model substrate p -nitroanisole with a maximum velocity of 9.24 nmol/min/mg of protein and a Michaelis constant of Km = 6.21 µM. In addition, imidacloprid was metabolized in vitro by the recombinant CYP353D1v2 microsomes (catalytic constant Kcat) 0.064 pmol/min/pmol P450, Km = 6.41 µM. The mass spectrum of UPLC-MS analysis shows that the metabolite was a product of buprofezin, which was buprofezin sulfone. This result provided direct evidence that L. striatellus cytochrome P450 CYP353D1v2 is capable of metabolizing imidacloprid and buprofezin.

  14. Buprofezin Is Metabolized by CYP353D1v2, a Cytochrome P450 Associated with Imidacloprid Resistance in Laodelphax striatellus

    PubMed Central

    Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Han, Zhaojun

    2017-01-01

    CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus. This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. The results showed the nicotinamide–adenine dinucleotide phosphate (NADPH)-dependent depletion of buprofezin (eluting at 8.7 min) and parallel formation of an unknown metabolite (eluting 9.5 min). However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. The recombinant CYP353D1v2 protein efficiently catalyzed the model substrate p-nitroanisole with a maximum velocity of 9.24 nmol/min/mg of protein and a Michaelis constant of Km = 6.21 µM. In addition, imidacloprid was metabolized in vitro by the recombinant CYP353D1v2 microsomes (catalytic constant Kcat) 0.064 pmol/min/pmol P450, Km = 6.41 µM. The mass spectrum of UPLC-MS analysis shows that the metabolite was a product of buprofezin, which was buprofezin sulfone. This result provided direct evidence that L. striatellus cytochrome P450 CYP353D1v2 is capable of metabolizing imidacloprid and buprofezin. PMID:29186030

  15. Metabolism of bilirubin by human cytochrome P450 2A6

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Abu-Bakar, A'edah, E-mail: a.abubakar@uq.edu.au; Arthur, Dionne M.; Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Adelaide

    2012-05-15

    The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K{sub i} of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdinmore » and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme. -- Highlights: ► Human CYP2A6 interacts with bilirubin with a high affinity. ► Bilirubin docking to the CYP2A6 active site is more stable than biliverdin docking. ► Recombinant CYP2A6 microsomes metabolised bilirubin to biliverdin. ► Bilirubin increased the

  16. Short-term fasting alters cytochrome P450-mediated drug metabolism in humans.

    PubMed

    Lammers, Laureen A; Achterbergh, Roos; de Vries, Emmely M; van Nierop, F Samuel; Klümpen, Heinz-Josef; Soeters, Maarten R; Boelen, Anita; Romijn, Johannes A; Mathôt, Ron A A

    2015-06-01

    Experimental studies indicate that short-term fasting alters drug metabolism. However, the effects of short-term fasting on drug metabolism in humans need further investigation. Therefore, the aim of this study was to evaluate the effects of short-term fasting (36 h) on P450-mediated drug metabolism. In a randomized crossover study design, nine healthy subjects ingested a cocktail consisting of five P450-specific probe drugs [caffeine (CYP1A2), S-warfarin (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6), and midazolam (CYP3A4)] on two occasions (control study after an overnight fast and after 36 h of fasting). Blood samples were drawn for pharmacokinetic analysis using nonlinear mixed effects modeling. In addition, we studied in Wistar rats the effects of short-term fasting on hepatic mRNA expression of P450 isoforms corresponding with the five studied P450 enzymes in humans. In the healthy subjects, short-term fasting increased oral caffeine clearance by 20% (P = 0.03) and decreased oral S-warfarin clearance by 25% (P < 0.001). In rats, short-term fasting increased mRNA expression of the orthologs of human CYP1A2, CYP2C19, CYP2D6, and CYP3A4 (P < 0.05), and decreased the mRNA expression of the ortholog of CYP2C9 (P < 0.001) compared with the postabsorptive state. These results demonstrate that short-term fasting alters cytochrome P450-mediated drug metabolism in a nonuniform pattern. Therefore, short-term fasting is another factor affecting cytochrome P450-mediated drug metabolism in humans. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  17. Pyrethroid Activity-Based Probes for Profiling Cytochrome P450 Activities Associated with Insecticide Interactions

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ismail, Hanafy M.; O'Neill, Paul M.; Hong, David

    2014-01-18

    Pyrethroid insecticides are used to control a diverse spectrum of diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid metabolizing and non-metabolizing mosquito P450s, as well as rodent microsomes to measure labeling specificity, plus CPR and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using a deltamethrin mimetic PyABP we were able to profile active enzymes in rat liver microsomes and identify pyrethroid metabolizing enzymes in the targetmore » tissue. The most reactive enzyme was a P450, CYP2C11, which is known to metabolize deltamethrin. Furthermore, several other pyrethroid metabolizers were identified (CYPs 2C6, 3A4, 2C13 and 2D1) along with related detoxification enzymes, notably UDP-g’s 2B1 - 5, suggesting a network of associated pyrethroid metabolizing enzymes, or ‘pyrethrome’. Considering the central role that P450s play in metabolizing insecticides, we anticipate that PyABPs will aid the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of new tools for disease control.« less

  18. Cytochrome P4501A biomarker indication of the timeline of chronic exposure of Barrow's goldeneyes to residual Exxon Valdez oil.

    PubMed

    Esler, Daniel; Ballachey, Brenda E; Trust, Kimberly A; Iverson, Samuel A; Reed, John A; Miles, A Keith; Henderson, John D; Woodin, Bruce R; Stegeman, John J; McAdie, Malcolm; Mulcahy, Daniel M; Wilson, Barry W

    2011-03-01

    We examined hepatic EROD activity, as an indicator of CYP1A induction, in Barrow's goldeneyes captured in areas oiled during the 1989 Exxon Valdez spill and those from nearby unoiled areas. We found that average EROD activity differed between areas during 2005, although the magnitude of the difference was reduced relative to a previous study from 1996/1997, and we found that areas did not differ by 2009. Similarly, we found that the proportion of individuals captured from oiled areas with elevated EROD activity (≥ 2 times unoiled average) declined from 41% in winter 1996/1997 to 10% in 2005 and 15% in 2009. This work adds to a body of literature describing the timelines over which vertebrates were exposed to residual Exxon Valdez oil and indicates that, for Barrow's goldeneyes in Prince William Sound, exposure persisted for many years with evidence of substantially reduced exposure by 2 decades after the spill. Copyright © 2010 Elsevier Ltd. All rights reserved.

  19. Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c.

    PubMed

    González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A

    2015-08-11

    Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin's transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ's histone chaperone activity.

  20. Quantitative Assessment of the Influence of Cytochrome P450 1A2 Gene Polymorphism and Colorectal Cancer Risk

    PubMed Central

    Rewuti, Abudouaini; Ma, Yu-Shui; Wang, Xiao-Feng; Xia, Qing; Fu, Da; Han, Yu-Song

    2013-01-01

    Cytochrome P450 1A2 (CYP1A2) encodes a member of the cytochrome P450 superfamily of enzymes, which play a central role in activating and detoxifying many carcinogens and endogenous compounds thought to be involved in the development of colorectal cancer (CRC). The CYP1A2*C (rs2069514) and CYP1A2*F (rs762551) polymorphism are two of the most commonly studied polymorphisms of the gene for their association with risk of CRC, but the results are conflicting. To derive a more precise estimation of the relationship between CYP1A2 and genetic risk of CRC, we performed a comprehensive meta-analysis which included 7088 cases and 7568 controls from 12 published case-control studies. In a combined analysis, the summary per-allele odds ratio for CRC was 0.91 (95% CI: 0.83–1.00, P = 0.04), and 0.91 (95% CI: 0.68–1.22, P = 0.53), for CYP1A2 *F and *C allele, respectively. In the subgroup analysis by ethnicity, significant associations were found in Asians for CYP1A2*F and CYP1A2*C, while no significant associations were detected among Caucasian populations. Similar results were also observed using dominant genetic model. Potential sources of heterogeneity were explored by subgroup analysis and meta-regression. No significant heterogeneity was detected in most of comparisons. This meta-analysis suggests that the CYP1A2 *F and *C polymorphism is a protective factor against CRC among Asians. PMID:23951174

  1. Differential Consequences of Tramadol in Overdosing: Dilemma of a Polymorphic Cytochrome P450 2D6-Mediated Substrate.

    PubMed

    Srinivas, Nuggehally R

    2015-09-01

    Tramadol is a centrally acting opioid analgesic that is prone to polymorphic metabolism via cytochrome P450 (CYP) 2D6. The generation of the active metabolite, O-desmethyltramadol, which occurs through the CYP 2D6 pathway, significantly contributes to the drug's activity. However, dosage adjustments of tramadol are typically not practiced in the clinic when treating patients who are homozygous extensive metabolizers, heterozygous extensive metabolizers, or poor metabolizers. In the event of a tramadol overdose, the consequences may be influenced importantly by the genotype or phenotype status of the subject. Depending on the individual subject's CYP 2D6 status, one may see excessive miotic-related toxicity driven by the excessive availability of O-desmethyltramadol or one may manifest mydriatic-related toxicity driven by the excessive availability of tramadol. This report provides pharmacokinetic perspectives in situations of tramadol overdosing.

  2. 7,12-Dimethylbenzanthracene induces apoptosis in RL95-2 human endometrial cancer cells: Ligand-selective activation of cytochrome P450 1B1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kim, Ji Young; Medical Research Science Center, Dong-A University, Busan 602-714; Lee, Seung Gee

    2012-04-15

    7,12-Dimethylbenzanthracene (DMBA), a polycyclic aromatic hydrocarbon, exhibits mutagenic, carcinogenic, immunosuppressive, and apoptogenic properties in various cell types. To achieve these functions effectively, DMBA is modified to its active form by cytochrome P450 1 (CYP1). Exposure to DMBA causes cytotoxicity-mediated apoptosis in bone marrow B cells and ovarian cells. Although uterine endometrium constitutively expresses CYP1A1 and CYP1B1, their apoptotic role after exposure to DMBA remains to be elucidated. Therefore, we chose RL95-2 endometrial cancer cells as a model system for studying DMBA-induced cytotoxicity and cell death and hypothesized that exposure to DMBA causes apoptosis in this cell type following CYP1A1 and/ormore » CYP1B1 activation. We showed that DMBA-induced apoptosis in RL95-2 cells is associated with activation of caspases. In addition, mitochondrial changes, including decrease in mitochondrial potential and release of mitochondrial cytochrome c into the cytosol, support the hypothesis that a mitochondrial pathway is involved in DMBA-induced apoptosis. Exposure to DMBA upregulated the expression of AhR, Arnt, CYP1A1, and CYP1B1 significantly; this may be necessary for the conversion of DMBA to DMBA-3,4-diol-1,2-epoxide (DMBA-DE). Although both CYP1A1 and CYP1B1 were significantly upregulated by DMBA, only CYP1B1 exhibited activity. Moreover, knockdown of CYP1B1 abolished DMBA-induced apoptosis in RL95-2 cells. Our data show that RL95-2 cells are susceptible to apoptosis by exposure to DMBA and that CYP1B1 plays a pivotal role in DMBA-induced apoptosis in this system. -- Highlights: ► Cytotoxicity-mediated apoptogenic action of DMBA in human endometrial cancer cells. ► Mitochondrial pathway in DMBA-induced apoptosis of RL95-2 endometrial cancer cells. ► Requirement of ligand-selective activation of CYP1B1 in DMBA-induced apoptosis.« less

  3. Biomonitoring the cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in hair: impact of exposure, hair pigmentation, and cytochrome P450 1A2 phenotype.

    PubMed

    Turesky, Robert J; Liu, Lin; Gu, Dan; Yonemori, Kim M; White, Kami K; Wilkens, Lynne R; Le Marchand, Loïc

    2013-03-01

    Hair is a promising tissue to assess exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen formed in cooked meats. However, an understanding of how dietary exposure to PhIP, cytochrome P450 1A2 activity-a key enzyme involved in PhIP metabolism, and hair pigmentation affect the level of PhIP accrued in hair is required to determine the reliability of the PhIP hair level as a biomarker of exposure to this carcinogen. We examined the impact of PhIP exposure, cytochrome P450 1A2 activity, and hair pigmentation on the levels of PhIP accumulated in the hair of volunteers on a 4-week semicontrolled diet of cooked meat containing known quantities of PhIP. The amount of PhIP in hair increased, on average, 15-fold in light- and dark-haired individuals during consumption of cooked meat. PhIP levels in hair were correlated to PhIP intake (ρ = 0.53; P < 0.001), and the relationship was strengthened when PhIP levels were normalized for the melanin content of hair (ρ = 0.71; P < 0.001). However, PhIP accrual in hair was not correlated to cytochrome P450 1A2 activity, as assessed by the caffeine test, or to the levels of unmetabolized PhIP in urine or to the metabolic ratio of the major urinary metabolite N(2)-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine to unmetabolized PhIP. The use of the PhIP hair biomarker should take hair pigmentation into account for accurate exposure assessment of PhIP. PhIP hair levels can serve as a biomarker in epidemiologic studies investigating the association of heterocyclic aromatic amine (HAA), cooked meat, and cancer risk.

  4. Identification of three cytochrome P450 genes in the Chagas' disease vector Triatoma infestans: Expression analysis in deltamethrin susceptible and resistant populations.

    PubMed

    Grosso, Carla G; Blariza, María J; Mougabure-Cueto, Gastón; Picollo, María I; García, Beatriz A

    2016-10-01

    Cytochrome P450 monooxygenases play a predominant role in the metabolism of insecticides. Many insect P450 genes have frequently been associated with detoxification processes allowing the insect to become tolerant or resistant to insecticides. The increases of expression of P450 genes at transcriptional level are often consider responsible for increasing the metabolism of insecticides and seems to be a common phenomenon in the evolution of resistance development in insects. As pyrethroid resistance has been detected in Triatoma infestans, it was of interest to analyze genes associated with resistance to insecticides such as those encoding for cytochromes P450. With this purpose, the cDNA sequences of three cytochrome P450 genes (CYP4EM7, CYP3085B1, and CYP3092A6) were identified in this species. Primers and specific Taqman probes were designed from these sequences to determine their expression by quantitative PCR. The mRNA levels of the cytochrome P450 genes identified were determined from total RNA extracted from pools of fat body collected from individuals of different resistant and susceptible strains of T. infestans, and at different interval times after the topical application of the lethal doses 50% (LD50) of deltamethrin on the ventral abdomen of insects belonging to the different populations analyzed. It was detected overexpression of the CYP4EM7 gene in the most resistant strain of T. infestans and the expression of the three cytochrome P450 genes isolated was induced by deltamethrin in the susceptible and resistant populations included in this study. These results suggest that these genes would be involved in the detoxification of deltamethrin and support the hypothesis that considers to the cytochrome P450 genes of importance in the development of pyrethroid resistance. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Contaminants in eggs of colonial waterbirds and hepatic cytochrome P450 enzyme levels in pipped tern embryos, Washington State.

    PubMed

    Blus, L J; Melancon, M J; Hoffman, D J; Henny, C J

    1998-10-01

    Eggs of Forster's terns (Sterna forsteri) collected in 1991 from nesting colonies on Crescent Island (Columbia River) and the Potholes Reservoir in south central Washington generally contained low residues of organochlorine pesticides and metabolites, 2,3,7, 8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and polychlorinated biphenyls (PCBs). Hepatic cytochrome P450 enzyme activity in pipped embryos of Forster's terns from the two colonies seemed unaffected by contaminants. At Crescent Island, examination of 23 Forster's tern eggs with large embryos (19 viable [10 pipped] and four dead [two pipped]) revealed developmental abnormalities in two viable pipped embryos (missing maxilla and deformed pelvic girdle) and a viable prepipping embryo (shortened beak). Our limited sample sizes and number of compounds analyzed preclude us from determining whether or not the abnormalities are related to contaminants. No abnormalities were noted in 10 pipped eggs (nine viable and one dead at collection) of Forster's terns collected from the Potholes Reservoir colony. Eggs of Caspian terns (Sterna caspia) collected from Crescent Island in 1991 also contained generally low residues of contaminants, only one developmental abnormality was noted, and limited data indicated that cytochrome P450 enzyme activity apparently was unaffected by contaminants. Organochlorine contaminants were generally low in addled eggs of American white pelicans (Pelecanus erythrorhynchos) collected from Crescent Island in 1994.

  6. Contaminants in eggs of colonial waterbirds and hepatic cytochrome P450 enzyme levels in pipped tern embryos, Washington State

    USGS Publications Warehouse

    Blus, L.J.; Melancon, M.J.; Hoffman, D.J.; Henny, C.J.

    1998-01-01

    Eggs of Forster's terns (Sterna forsteri) collected in 1991 from nesting colonies on Crescent Island (Columbia River) and the Potholes Reservoir in south central Washington generally contained low residues of organochlorine pesticides and metabolites, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzofuran, and polychlorinated biphenyls (PCBs). Hepatic cytochrome P450 enzyme activity in pipped embryos of Forster's terns from the two colonies seemed unaffected by contaminants. At Crescent Island, examination of 23 Forster's tern eggs with large embryos (19 viable [10 pipped] and four dead [two pipped]) revealed developmental abnormalities in two viable pipped embryos (missing maxilla and deformed pelvic girdle) and a viable prepipping embryo (shortened beak). Our limited sample sizes and number of compounds analyzed preclude us from determining whether or not the abnormalities are related to contaminants. No abnormalities were noted in 10 pipped eggs (nine viable and one dead at collection) of Forster's terns collected from the Potholes Reservoir colony. Eggs of Caspian terns (Sterna caspia) collected from Crescent Island in 1991 also contained generally low residues of contaminants, only one developmental abnormality was noted, and limited data indicated that cytochrome P450 enzyme activity apparently was unaffected by contaminants. Organochlorine contaminants were generally low in addled eggs of American white pelicans (Pelecanus erythrorhynchos) collected from Crescent Island in 1994.

  7. Cytochromes P450 Catalyze the Reduction of α,β-Unsaturated Aldehydes

    PubMed Central

    Amunom, Immaculate; Dieter, Laura J.; Tamasi, Viola; Cai, Jan; Conklin, Daniel J.; Srivastava, Sanjay; Martin, Martha V.; Guengerich, F. Peter; Prough, Russell A.

    2011-01-01

    The metabolism of α,β-unsaturated aldehydes, e.g. 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O2, and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ≅ P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 & rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions, but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and α-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of reduction of α,β-unsaturated aldehydes in liver. PMID:21766881

  8. QUANTITATIVE EVALUATION OF BROMODICHLOROMETHANE METABOLISM BY RECOMBINANT RAT AND HUMAN CYTOCHROME P450S

    EPA Science Inventory

    ABSTRACT
    We report quantitative estimates of the parameters for metabolism of bromodichloromethane (BDCM) by recombinant preparations of hepatic cytochrome P450s (CYPs) from rat and human. BDCM is a drinking water disinfectant byproduct that has been implicated in liver, kidn...

  9. Human Cytochrome P450 Enzyme Modulation by Gymnema sylvestre: A Predictive Safety Evaluation by LC-MS/MS

    PubMed Central

    Rammohan, Bera; Samit, Karmakar; Chinmoy, Das; Arup, Saha; Amit, Kundu; Ratul, Sarkar; Sanmoy, Karmakar; Dipan, Adhikari; Tuhinadri, Sen

    2016-01-01

    Background: Traditionally GS is used to treat diabetes mellitus. Drug-herb interaction of GS via cytochrome P450 enzyme system by substrate cocktail method using HLM has not been reported. Objective: To evaluate the in-vitro modulatory effects of GS extracts (aqueous, methanol, ethyl acetate, chloroform and n-hexane) and deacylgymnemic acid (DGA) on human CYP1A2, 2C8, 2C9, 2D6 and 3A4 activities in HLM. Material and Methods: Probe substrate-based LCMS/MS method was established for all CYPs. The metabolite formations were examined after incubation of probe substrates with HLM in the presence or absence of extracts and DGA. The inhibitory effects of GS extracts and DGA were characterized with kinetic parameters IC50 and Ki values. Results: GS extracts showed differential effect on CYP activities in the following order of inhibitory potency: ethyl acetate > Chloroform > methanol > n-hexane > aqueous > DGA. This differential effect was observed against CYP1A2, 2C9 and less on CYP3A4 and 2C8 but all CYPs were unaffected by aqueous extract and DGA. The ethyl acetate and chloroform extract exhibited moderate inhibition towards CYP1A2 and 3A4. The aqueous extract and DGA however showed negligible inhibition towards all five major human CYPs with very high IC50 values (>90μg/ml). Conclusion: The results of our study revealed that phytoconstituents contained in GS, particularly in ethyl acetate and chloroform extracts, were able to inhibit CYP1A2, 3A4 and 2C9. The presence of relatively small, lipophillic yet slightly polar compounds within the GS extracts may be attributed for inhibition activities. These suggest that the herb or its extracts should be examined for potential pharmacokinetic drug interactions in vivo. Abbreviations used: GS: Gymnema sylvestre, GSE: Gymnema sylvestre extract, DGA: deacyl gymnemic acid, CYP: cytochrome P450, DMSO: dimethylsulphoxide, HLM: human liver microsomes, LC-MS/MS: liquid chromatography tandem mass spectroscopy, NADPH: reduced

  10. Human Cytochrome P450 Enzyme Modulation by Gymnema sylvestre: A Predictive Safety Evaluation by LC-MS/MS.

    PubMed

    Rammohan, Bera; Samit, Karmakar; Chinmoy, Das; Arup, Saha; Amit, Kundu; Ratul, Sarkar; Sanmoy, Karmakar; Dipan, Adhikari; Tuhinadri, Sen

    2016-07-01

    Traditionally GS is used to treat diabetes mellitus. Drug-herb interaction of GS via cytochrome P450 enzyme system by substrate cocktail method using HLM has not been reported. To evaluate the in-vitro modulatory effects of GS extracts (aqueous, methanol, ethyl acetate, chloroform and n -hexane) and deacylgymnemic acid (DGA) on human CYP1A2, 2C8, 2C9, 2D6 and 3A4 activities in HLM. Probe substrate-based LCMS/MS method was established for all CYPs. The metabolite formations were examined after incubation of probe substrates with HLM in the presence or absence of extracts and DGA. The inhibitory effects of GS extracts and DGA were characterized with kinetic parameters IC50 and Ki values. GS extracts showed differential effect on CYP activities in the following order of inhibitory potency: ethyl acetate > Chloroform > methanol > n -hexane > aqueous > DGA. This differential effect was observed against CYP1A2, 2C9 and less on CYP3A4 and 2C8 but all CYPs were unaffected by aqueous extract and DGA. The ethyl acetate and chloroform extract exhibited moderate inhibition towards CYP1A2 and 3A4. The aqueous extract and DGA however showed negligible inhibition towards all five major human CYPs with very high IC50 values (>90μg/ml). The results of our study revealed that phytoconstituents contained in GS, particularly in ethyl acetate and chloroform extracts, were able to inhibit CYP1A2, 3A4 and 2C9. The presence of relatively small, lipophillic yet slightly polar compounds within the GS extracts may be attributed for inhibition activities. These suggest that the herb or its extracts should be examined for potential pharmacokinetic drug interactions in vivo . Abbreviations used: GS: Gymnema sylvestre , GSE: Gymnema sylvestre extract, DGA: deacyl gymnemic acid, CYP: cytochrome P450, DMSO: dimethylsulphoxide, HLM: human liver microsomes, LC-MS/MS: liquid chromatography tandem mass spectroscopy, NADPH: reduced nicotinamide adeninedinucleotide phosphate, NRS: nicotinamide

  11. Differential transcription of cytochrome P450s and glutathione S transferases in DDT-susceptible and resistant Drosophila melanogaster strains in response to DDT and oxidative stress

    USDA-ARS?s Scientific Manuscript database

    Metabolic DDT resistance in Drosophila melanogaster has previously been associated with constitutive over-transcription of cytochrome P450s. Increased P450 activity has also been associated with increased oxidative stress. In contrast, over-transcription of glutathione S transferases (GSTs) has been...

  12. The Effect of Vinpocetine on Human Cytochrome P450 Isoenzymes by Using a Cocktail Method.

    PubMed

    Kong, Lingti; Song, Chunli; Ye, Linhu; Guo, Daohua; Yu, Meiling; Xing, Rong

    2016-01-01

    Vinpocetine is a derivative of the alkaloid vincamine, which had been prescribed for chronic cerebral vascular ischemia and acute ischemic stroke or used as a dietary supplement for its several different mechanisms of biological activities. However, information on the cytochrome P450 (CYP) enzyme-mediated drug metabolism has not been previously studied. The present study was performed to investigate the effects of vinpocetine on CYPs activity, and cocktail method was used, respectively. To evaluate the effects of vinpocetine on the activity of human CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, human liver microsomes were utilized to incubate with the mixed CYPs probe substrates and the target components. The results indicate that vinpocetine exhibited weak inhibitory effect on the CYP2C9, where the IC50 value is 68.96 μM, whereas the IC50 values for CYP3A4, CYP2C19, CYP2D6, and CYP2E1 were all over range of 100 μM, which showed that vinpocetine had no apparent inhibitory effects on these CYPs. In conclusion, the results indicated that drugs metabolized by CYP2C9 coadministrated with vinpocetine may require attention or dose adjustment.

  13. The Effect of Vinpocetine on Human Cytochrome P450 Isoenzymes by Using a Cocktail Method

    PubMed Central

    Kong, Lingti; Song, Chunli; Ye, Linhu; Guo, Daohua; Yu, Meiling; Xing, Rong

    2016-01-01

    Vinpocetine is a derivative of the alkaloid vincamine, which had been prescribed for chronic cerebral vascular ischemia and acute ischemic stroke or used as a dietary supplement for its several different mechanisms of biological activities. However, information on the cytochrome P450 (CYP) enzyme-mediated drug metabolism has not been previously studied. The present study was performed to investigate the effects of vinpocetine on CYPs activity, and cocktail method was used, respectively. To evaluate the effects of vinpocetine on the activity of human CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, human liver microsomes were utilized to incubate with the mixed CYPs probe substrates and the target components. The results indicate that vinpocetine exhibited weak inhibitory effect on the CYP2C9, where the IC50 value is 68.96 μM, whereas the IC50 values for CYP3A4, CYP2C19, CYP2D6, and CYP2E1 were all over range of 100 μM, which showed that vinpocetine had no apparent inhibitory effects on these CYPs. In conclusion, the results indicated that drugs metabolized by CYP2C9 coadministrated with vinpocetine may require attention or dose adjustment. PMID:27006677

  14. Structural and Functional Characterization of a Cytochrome P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen Bond

    PubMed Central

    2015-01-01

    The structural basis of the regulation of microsomal cytochrome P450 (P450) activity was investigated by mutating the highly conserved heme binding motif residue, Phe429, on the proximal side of cytochrome P450 2B4 to a histidine. Spectroscopic, pre-steady-state and steady-state kinetic, thermodynamic, theoretical, and structural studies of the mutant demonstrate that formation of an H-bond between His429 and the unbonded electron pair of the Cys436 axial thiolate significantly alters the properties of the enzyme. The mutant lost >90% of its activity; its redox potential was increased by 87 mV, and the half-life of the oxyferrous mutant was increased ∼37-fold. Single-crystal electronic absorption and resonance Raman spectroscopy demonstrated that the mutant was reduced by a small dose of X-ray photons. The structure revealed that the δN atom of His429 forms an H-bond with the axial Cys436 thiolate whereas the εN atom forms an H-bond with the solvent and the side chain of Gln357. The amide of Gly438 forms the only other H-bond to the tetrahedral thiolate. Theoretical quantification of the histidine–thiolate interaction demonstrates a significant electron withdrawing effect on the heme iron. Comparisons of structures of class I–IV P450s demonstrate that either a phenylalanine or tryptophan is often found at the location corresponding to Phe429. Depending on the structure of the distal pocket heme, the residue at this location may or may not regulate the thermodynamic properties of the P450. Regardless, this residue appears to protect the thiolate from solvent, oxidation, protonations, and other deleterious reactions. PMID:25029089

  15. Interaction of rocuronium with human liver cytochromes P450.

    PubMed

    Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

    2015-02-01

    Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  16. Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b₅ gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

    PubMed

    Chen, Xi'en; Zhang, Yalin

    2015-03-10

    NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.

    PubMed

    Ewen, Kerstin M; Schiffler, Burkhard; Uhlmann-Schiffler, Heike; Bernhardt, Rita; Hannemann, Frank

    2008-05-01

    Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.

  18. Electrochemistry of Cytochrome P450 BM3 in Sodium Dodecyl Sulfate Films

    PubMed Central

    Udit, Andrew K.; Hill, Michael G.; Gray, Harry B.

    2008-01-01

    Direct electrochemistry of the cytochrome P450 BM3 heme domain (BM3) was achieved by confining the protein within sodium dodecyl sulfate (SDS) films on the surface of basal-plane graphite (BPG) electrodes. Cyclic voltammetry revealed the heme FeIII/II redox couple at −330 mV (vs. Ag/AgCl, pH 7.4). Up to 10 V/s, the peak current was linear with scan rate, allowing us to treat the system as surface-confined within this regime. The standard heterogeneous rate constant determined at 10 V/s was estimated to be 10 s−1. Voltammograms obtained for the BM3-SDS-BPG system in the presence of dioxygen exhibited catalytic waves at the onset of FeIII reduction. The altered heme reduction potential of the BM3-SDS-graphite system indicates that SDS is likely bound in the enzyme active-site region. Compared to other P450-surfactant systems, we find redox potentials and electron transfer rates that differ by ~ 100 mV and > 10-fold, respectively, indicating that the nature of the surfactant environment has a significant effect on the observed heme redox properties. PMID:17129070

  19. Investigation of cytochrome P450 inhibitory properties of maslinic acid, a bioactive compound from Olea europaea L., and its structure-activity relationship.

    PubMed

    Sun, Min; Tang, Yu; Ding, Tonggui; Liu, Mingyao; Wang, Xin

    2015-01-15

    Maslinic acid (MA), the main pentacyclic triterpene of Olea europaea L. fruit, possesses a variety of pharmacological actions, including hypoglycemic, antioxidant, cardioprotective and antitumoral activities. Despite its importance, little is known about its effects on the cytochrome P450 (CYP) activity in both humans and animals. Therefore, the aim of this study was to investigate the effects of MA on the CYP 1A2, 2C9/11, 2D1/6, 2E1 and 3A2/4 activities by human and rat liver microsomes and specific CYP isoforms. In humans, MA only weakly inhibited CYP3A4 activity in human liver microsomes and specific CYP3A4 isoform with IC50 value at 46.1 and 62.3µM, respectively. In rats, MA also exhibited weak inhibition on CYP2C11, CYP2E1 and CYP3A2 activities with IC50 values more than 100µM. Enzyme kinetic studies showed that the MA was not only a competitive inhibitor of CYP3A4 in humans, but also a competitive inhibitor of CYP2C11 and 3A2 in rats, with Ki of 18.4, 98.7 and 66.3µM, respectively. Moreover, the presence of hydroxyl group at C-2 position of triterpenic acid in MA compared with oleanolic acid could magnify its competitive inhibition on human CYP3A4 activity. The relatively high Ki values of MA would have a low potential to cause the possible toxicity and drug interactions involving CYP enzymes, thus suggesting a sufficient safety for its putative use as a nutraceutical taken together with drugs. Copyright © 2014 Elsevier GmbH. All rights reserved.

  20. Effect of calcium(ion) uptake by rat adrenal mitochondria on pregnenolone formation and spectral properties of cytochrome P-450.

    PubMed

    Simpson, E R; Williams-Smith, D L

    1975-10-09

    The effect of calcium on pregnenolone formation from endogenous precursors has been studied in mitochondria from rat decapsulated and capsular adrenal glands. In the presence of succinate, addition of calcium chloride in the concentration range 20-150 muM caused an inhibition of pregnenolone formation in both decapsulated and capsular adrenal mitochondria. 11beta-hydroxylation of added deoxycosticosterone in decapsulated adrenal mitochondria was also inhibited. Under these conditions, calcium inhibited the reduction of adrenodoxin, a component of the cytochrome P-450 reductase system, presumably because uptake of calcium by the mitochondria competes with energy-linked transhydrogenase for high-energy intermediates. For this reason, incubations were carried out in the presence of succinate plus isocitrate plus NADP+. Under these conditions, calcium chloride in the concentration range 120-875 muM caused a 2-4-fold stimulation of pregnenolone formation, but had no effect on corticosterone formation from added deoxycorticosterone. The effect of calcium on the optical spectra of cytochrome P-450 has also been examined in mitochondria from decapsulated and capsular rat adrenals. In the presence of succinate, calcium induced a spectral change resembling a type I difference spectrum of cytochrome P-450. Thus it appears that uptake of calcium by adrenal mitochondria can stimulate pregnenolone formation by increasing the interaction of mitochondrial cytochrome P-450 with endogenous substrate.

  1. Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.

    PubMed

    Jones, Robert T; Bakker, Saskia E; Stone, Deborah; Shuttleworth, Sally N; Boundy, Sam; McCart, Caroline; Daborn, Phillip J; ffrench-Constant, Richard H; van den Elsen, Jean M H

    2010-10-01

    Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450-substrate interactions. Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X-ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also 'V'-shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT-CYP6G1 complex and a non-resistant CYP6A2 homology model implies that tight-fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. The CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities.

  2. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography*

    PubMed Central

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

    2015-01-01

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. PMID:25670859

  3. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

    DOE PAGES

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; ...

    2015-02-10

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

  4. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

  5. Effects of sex, weight, diet and hCG administration on levels of skatole and indole in the liver and hepatic activities of cytochromes P4502E1 and P4502A6 in pigs.

    PubMed

    Zamaratskaia, G; Chen, G; Lundström, K

    2006-02-01

    Cytochromes P4502E1 (CYP2E1) and P4502A6 (CYP2A6) catalyse metabolic reactions of skatole and indole metabolism. The objectives of this study were as follows: to evaluate whether activities of CYP2E1 and CYP2A6 in pigs of two live weights (LW) differ between males and females; to investigate whether activities of CYP2E1 and CYP2A6 are affected by hCG stimulation; and to investigate whether the levels of skatole and indole in the liver and the activities of CYP2E1 and CYP2A6 are affected by raw potato starch (RPS). Female pigs expressed higher CYP2A6 activity at 90kg LW, and higher CYP2E1 activity at 115kg LW compared to male pigs. Skatole levels in the liver were higher in male pigs than in female pigs at both LW, whereas indole levels were higher in males only at 115 kg LW. Neither levels of indolic compounds in the liver nor enzyme activities were affected by hCG stimulation. The inclusion of RPS in the diet reduced skatole levels in the liver in both sexes and increased CYP2A6 activity in female pigs. It was concluded that the incidence of boar taint may depend on both skatole amount, which reach the liver, and the activities of enzymes involved in skatole metabolism, which may vary depending on sex, live weight, and diet.

  6. Significantly shorter Fe–S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase

    DOE PAGES

    Krest, Courtney M.; Silakov, Alexey; Rittle, Jonathan; ...

    2015-08-03

    Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate-ligated haem proteins that catalyse the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C–H bonds. In this paper, to provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I using variable-temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe–S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe–S bond lengths can be understood in terms ofmore » variations in the hydrogen-bonding patterns within the ‘cys-pocket’ (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe–S bond, which enables greater electron donation from the axial thiolate ligand. Finally, this observation may in part explain P450's greater propensity for C–H bond activation.« less

  7. Pharmacokinetics and Differential Regulation of Cytochrome P450 Enzymes in Type 1 Allergic Mice.

    PubMed

    Tanino, Tadatoshi; Komada, Akira; Ueda, Koji; Bando, Toru; Nojiri, Yukie; Ueda, Yukari; Sakurai, Eiichi

    2016-12-01

    Type 1 allergic diseases are characterized by elevated production of specific immunoglobulin E (IgE) for each antigen and have become a significant health problem worldwide. This study investigated the effect of IgE-mediated allergy on drug pharmacokinetics. To further understand differential suppression of hepatic cytochrome P450 (P450) activity, we examined the inhibitory effect of nitric oxide (NO), a marker of allergic conditions. Seven days after primary sensitization (PS7) or secondary sensitization (SS7), hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities were decreased to 45%-75% of the corresponding control; however, CYP2D activity was not downregulated. PS7 and SS7 did not change the expression levels of five P450 proteins. Disappearance of CYP1A2 and CYP2D substrates from the plasma was not significantly different between allergic mice and control mice. In contrast, the area under the curve of a CYP1A2-mediated metabolite in PS7 and SS7 mice was reduced by 50% of control values. Total clearances of a CYP2E1 substrate in PS7 and SS7 mice were significantly decreased to 70% and 50% respectively, of the control without altering plasma protein binding. Hepatic amounts of CYP1A2 and CYP2E1 substrates were enhanced by allergic induction, being responsible for each downregulated activity. NO scavenger treatment completely improved the downregulated P450 activities. Therefore, our data suggest that the onset of IgE-mediated allergy alters the pharmacokinetics of major P450-metabolic capacity-limited drugs except for CYP2D drugs. NO is highly expected to participate in regulatory mechanisms of the four P450 isoforms. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  8. n-Alkane and clofibrate, a peroxisome proliferator, activate transcription of ALK2 gene encoding cytochrome P450alk2 through distinct cis-acting promoter elements in Candida maltosa

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kogure, Takahisa; Faculty of Applied Life Sciences, Niigata University of Pharmacy and Applied Life Sciences, Higashijima 265-1, Niitsu, Niigata 956-8603; Takagi, Masamichi

    2005-04-01

    The ALK2 gene, encoding one of the n-alkane-hydroxylating cytochromes P450 in Candida maltosa, is induced by n-alkanes and a peroxisome proliferator, clofibrate. Deletion analysis of this gene's promoter revealed two cis-acting elements-an n-alkane-responsive element (ARE2) and a clofibrate-responsive element (CRE2)-that partly overlap in sequence but have distinct functions. ARE2-mediated activation responded to n-alkanes but not to clofibrate and was repressed by glucose. CRE2-mediated activation responded to polyunsaturated fatty acids and steroid hormones as well as to peroxisome proliferators but not to n-alkanes, and it was not repressed by glucose. Both elements mediated activation by oleic acid. Mutational analysis demonstrated thatmore » three CCG sequences in CRE2 were critical to the activation by clofibrate as well as to the in vitro binding of a specific protein to this element. These findings suggest that ALK2 is induced by peroxisome proliferators and steroid hormones through a specific CRE2-mediated regulatory mechanism.« less

  9. Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks

    NASA Astrophysics Data System (ADS)

    Bayburt, Timothy H.; Sligar, Stephen G.

    2002-05-01

    The architecture of membrane proteins in their native environment of the phospholipid bilayer is critical for understanding physiological function, but has been difficult to realize experimentally. In this communication we describe the incorporation of a membrane-anchored protein into a supported phospholipid bilayer. Cytochrome P450 2B4 solubilized and purified from the hepatic endoplasmic reticulum was incorporated into phospholipid bilayer nanostructures and oriented on a surface for visualization by atomic force microscopy. Individual P450 molecules were observed protruding from the bilayer surface. Problems associated with deformation of the protein by the atomic force microscopy probe were avoided by analyzing force-dependent height measurements to quantitate the height of the protein above the bilayer surface. Measurements of the atomic force microscopy cantilever deflection as a function of probe-sample separation reveal that the top of the P450 opposite the N-terminal membrane anchor region sits 3.5 nanometers above the phospholipid-water boundary. Models of the orientation of the enzyme are presented and discussed in relation to membrane interactions and interaction with cytochrome P450 reductase.

  10. Metabolism of styrene to styrene oxide and vinylphenols in cytochrome P450 2F2- and P450 2E1-knockout mouse liver and lung microsomes

    PubMed Central

    Shen, Shuijie; Li, Lei; Ding, Xinxin; Zheng, Jiang

    2014-01-01

    Pulmonary toxicity of styrene is initiated by cytochromes P450-dependent metabolic activation. P450 2E1 and P450 2F2 are considered to be two main cytochrome P450 (CYP) enzymes responsible for styrene metabolism in mice. The objective of the current study was to determine the correlation between the formation of styrene metabolites (i.e. styrene oxide and 4-vinylphenol) and pulmonary toxicity of styrene, using Cyp2e1- and Cyp2f2-null mouse models. Dramatic decrease in the formation of styrene glycol and 4-vinylphenol was found in Cyp2f2-null mouse lung microsomes, relative to that in the wild-type mouse lung microsomes. However, no significant difference in the production of the styrene metabolites was observed between lung microsomes obtained from Cyp2e1-null and the wild-type mice. The knock–out and wild-type mice were treated with styrene (6.0 mmol/kg, ip), and cell counts and LDH activity in bronchoalveolar lavage fluids were monitored to evaluate the pulmonary toxicity induced by styrene. Cyp2e1-null mice displayed similar susceptibility to lung toxicity of styrene as the wild-type animals. However, Cyp2f2-null mice were resistant to styrene-induced pulmonary toxicity. In conclusion, both P450 2E1 and P450 2F2 are responsible for the metabolic activation of styrene. The latter enzyme plays an important role in styrene-induced pulmonary toxicity. Both styrene oxide and 4-vinylphenol are suggested to participate in the development of lung injury induced by styrene. PMID:24320693

  11. Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias*

    PubMed Central

    Butler, Christopher F.; Peet, Caroline; Mason, Amy E.; Voice, Michael W.; Leys, David; Munro, Andrew W.

    2013-01-01

    Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such “gatekeeper” mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme. PMID:23828198

  12. Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

    PubMed Central

    Kelly, Steven L.; Kelly, Diane E.

    2013-01-01

    The first eukaryote genome revealed three yeast cytochromes P450 (CYPs), hence the subsequent realization that some microbial fungal genomes encode these proteins in 1 per cent or more of all genes (greater than 100) has been surprising. They are unique biocatalysts undertaking a wide array of stereo- and regio-specific reactions and so hold promise in many applications. Based on ancestral activities that included 14α-demethylation during sterol biosynthesis, it is now seen that CYPs are part of the genes and metabolism of most eukaryotes. In contrast, Archaea and Eubacteria often do not contain CYPs, while those that do are frequently interesting as producers of natural products undertaking their oxidative tailoring. Apart from roles in primary and secondary metabolism, microbial CYPs are actual/potential targets of drugs/agrochemicals and CYP51 in sterol biosynthesis is exhibiting evolution to resistance in the clinic and the field. Other CYP applications include the first industrial biotransformation for corticosteroid production in the 1950s, the diversion into penicillin synthesis in early mutations in fungal strain improvement and bioremediation using bacteria and fungi. The vast untapped resource of orphan CYPs in numerous genomes is being probed and new methods for discovering function and for discovering desired activities are being investigated. PMID:23297358

  13. Structural basis for inhibition of the histone chaperone activity of SET/TAF-Iβ by cytochrome c

    PubMed Central

    González-Arzola, Katiuska; Díaz-Moreno, Irene; Cano-González, Ana; Díaz-Quintana, Antonio; Velázquez-Campoy, Adrián; Moreno-Beltrán, Blas; López-Rivas, Abelardo; De la Rosa, Miguel A.

    2015-01-01

    Chromatin is pivotal for regulation of the DNA damage process insofar as it influences access to DNA and serves as a DNA repair docking site. Recent works identify histone chaperones as key regulators of damaged chromatin’s transcriptional activity. However, understanding how chaperones are modulated during DNA damage response is still challenging. This study reveals that the histone chaperone SET/TAF-Iβ interacts with cytochrome c following DNA damage. Specifically, cytochrome c is shown to be translocated into cell nuclei upon induction of DNA damage, but not upon stimulation of the death receptor or stress-induced pathways. Cytochrome c was found to competitively hinder binding of SET/TAF-Iβ to core histones, thereby locking its histone-binding domains and inhibiting its nucleosome assembly activity. In addition, we have used NMR spectroscopy, calorimetry, mutagenesis, and molecular docking to provide an insight into the structural features of the formation of the complex between cytochrome c and SET/TAF-Iβ. Overall, these findings establish a framework for understanding the molecular basis of cytochrome c-mediated blocking of SET/TAF-Iβ, which subsequently may facilitate the development of new drugs to silence the oncogenic effect of SET/TAF-Iβ’s histone chaperone activity. PMID:26216969

  14. Cytochrome c oxidase loses catalytic activity and structural integrity during the aging process in Drosophila melanogaster

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ren, Jian-Ching; Rebrin, Igor; Klichko, Vladimir

    2010-10-08

    Research highlights: {yields} Cytochrome c oxidase loses catalytic activity during the aging process. {yields} Abundance of seven nuclear-encoded subunits of cytochrome c oxidase decreased with age in Drosophila. {yields} Cytochrome c oxidase is specific intra-mitochondrial site of age-related deterioration. -- Abstract: The hypothesis, that structural deterioration of cytochrome c oxidase (CcO) is a causal factor in the age-related decline in mitochondrial respiratory activity and an increase in H{sub 2}O{sub 2} generation, was tested in Drosophila melanogaster. CcO activity and the levels of seven different nuclear DNA-encoded CcO subunits were determined at three different stages of adult life, namely, young-, middle-,more » and old-age. CcO activity declined progressively with age by 33%. Western blot analysis, using antibodies specific to Drosophila CcO subunits IV, Va, Vb, VIb, VIc, VIIc, and VIII, indicated that the abundance these polypeptides decreased, ranging from 11% to 40%, during aging. These and previous results suggest that CcO is a specific intra-mitochondrial site of age-related deterioration, which may have a broad impact on mitochondrial physiology.« less

  15. Identification of in vitro cytochrome P450 modulators to detect induction by prototype inducers in the mallard duckling (Anas platyrhynchos

    USGS Publications Warehouse

    Renauld, A.E.; Melancon, M.J.; Sordillo, L.M.

    1999-01-01

    Seven modulators of mammalian monooxygenase activity were screened for their ability to selectively stimulate or inhibit in vitro monooxygenase activities of hepatic microsomes from mallard ducklings treated with phenobarbital, β-naphthoflavone, 3,3′,4,4′,5-pentachlorobiphenyl or vehicle. Microsomes were assayed fluorometrically for four monooxygenases: benzyloxy-, ethoxy-, methoxy-, and pentoxyresorufin-O-dealkylase, in combination with each of the seven modulators. Four combinations: α-naphthoflavone and 2-methylbenzimidazole with benzyloxyresorufin, and Proadifen with methoxy- and ethoxyresorufin, respectively, were evaluated further. β-Naphthoflavone-treated groups were clearly distinguished from the corn oil vehicle control group by all of the assays and by the effects of the modulators in three of the four assay/modulator combinations. Enzyme activities of the phenobarbital and saline groups were statistically similar (P≥0.05) when assayed without modulator added, but each assay/modulator combination distinguished between these groups. The PCB-treated group was distinguished from the corn oil vehicle control group only for BROD activity, with or without the presence of modulator. Graphing of per cent modulation of BROD activity versus initial BROD activity provided the clearest distinction between all of the study groups. Identification of these selective in vitro modulators may improve detection and measurement of low level cytochrome P450 induction in avian species. Also, both the monooxygenase activities induced and the impacts of the modulators indicated differences between mammalian and avian cytochromes P450.

  16. Biomonitoring the Cooked Meat Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in Hair: Impact of Exposure, Pigmentation and Cytochrome P450 1A2 Phenotype

    PubMed Central

    Turesky, Robert J.; Liu, Lin; Gu, Dan; Yonemori, Kim M.; White, Kami K.; Wilkens, Lynne R.; Marchand, Loic Le

    2013-01-01

    Background Hair is a promising tissue to assess exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogen formed in cooked meats. However, an understanding of how dietary exposure to PhIP, cytochrome P450 1A2 activity - a key enzyme involved in PhIP metabolism, and hair pigmentation affect the level of PhIP accrued in hair is required in order to determine the reliability of the PhIP hair level as a biomarker of exposure to this carcinogen. Methods We examined the impact of PhIP exposure, cytochrome P450 1A2 activity, and hair pigmentation on the levels of PhIP accumulated in the hair of volunteers on a 4-week semi-controlled diet of cooked meat containing known quantities of PhIP. Results The amount of PhIP in hair increased, on average, 15-fold in light- and dark-haired individuals during consumption of cooked meat. PhIP levels in hair were correlated to PhIP intake (ρ = 0.53; p < 0.001), and the relationship was strengthened when PhIP levels were normalized for the melanin content of hair (ρ = 0.71; p < 0.001). However, PhIP accrual in hair was not correlated to cytochrome P450 1A2 activity, as assessed by the caffeine test, or to the levels of unmetabolized PhIP in urine, or to the metabolic ratio of the major urinary metabolite N2-(ß-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine to unmetabolized PhIP. Conclusions The employment of the PhIP hair biomarker should take hair pigmentation into account for accurate exposure assessment. Impact PhIP hair levels can serve as a biomarker in epidemiological studies investigating the association of HAAs, cooked meat and cancer risk. PMID:23329727

  17. Insights into Hydrocarbon Assimilation by Eurotialean and Hypocrealean Fungi: Roles for CYP52 and CYP53 Clans of Cytochrome P450 Genes.

    PubMed

    Huarte-Bonnet, Carla; Kumar, Suresh; Saparrat, Mario C N; Girotti, Juan R; Santana, Marianela; Hallsworth, John E; Pedrini, Nicolás

    2018-03-01

    Several filamentous fungi are able to concomitantly assimilate both aliphatic and polycyclic aromatic hydrocarbons that are the biogenic by-products of some industrial processes. Cytochrome P450 monooxygenases catalyze the first oxidation reaction for both types of substrate. Among the cytochrome P450 (CYP) genes, the family CYP52 is implicated in the first hydroxylation step in alkane-assimilation processes, while genes belonging to the family CYP53 have been linked with oxidation of aromatic hydrocarbons. Here, we perform a comparative analysis of CYP genes belonging to clans CYP52 and CYP53 in Aspergillus niger, Beauveria bassiana, Metarhizium robertsii (formerly M. anisopliae var. anisopliae), and Penicillium chrysogenum. These species were able to assimilate n-hexadecane, n-octacosane, and phenanthrene, exhibiting a species-dependent modification in pH of the nutrient medium during this process. Modeling of the molecular docking of the hydrocarbons to the cytochrome P450 active site revealed that both phenanthrene and n-octacosane are energetically favored as substrates for the enzymes codified by genes belonging to both CYP52 and CYP53 clans, and thus appear to be involved in this oxidation step. Analyses of gene expression revealed that CYP53 members were significantly induced by phenanthrene in all species studied, but only CYP52X1 and CYP53A11 from B. bassiana were highly induced with n-alkanes. These findings suggest that the set of P450 enzymes involved in hydrocarbon assimilation by fungi is dependent on phylogeny and reveal distinct substrate and expression specificities.

  18. Solution structural ensembles of substrate-free cytochrome P450(cam).

    PubMed

    Asciutto, Eliana K; Young, Matthew J; Madura, Jeffry; Pochapsky, Susan Sondej; Pochapsky, Thomas C

    2012-04-24

    Removal of substrate (+)-camphor from the active site of cytochrome P450(cam) (CYP101A1) results in nuclear magnetic resonance-detected perturbations in multiple regions of the enzyme. The (1)H-(15)N correlation map of substrate-free diamagnetic Fe(II) CO-bound CYP101A permits these perturbations to be mapped onto the solution structure of the enzyme. Residual dipolar couplings (RDCs) were measured for (15)N-(1)H amide pairs in two independent alignment media for the substrate-free enzyme and used as restraints in solvated molecular dynamics (MD) simulations to generate an ensemble of best-fit structures of the substrate-free enzyme in solution. Nuclear magnetic resonance-detected chemical shift perturbations reflect changes in the electronic environment of the NH pairs, such as hydrogen bonding and ring current shifts, and are observed for residues in the active site as well as in hinge regions between secondary structural features. RDCs provide information about relative orientations of secondary structures, and RDC-restrained MD simulations indicate that portions of a β-rich region adjacent to the active site shift so as to partially occupy the vacancy left by removal of the substrate. The accessible volume of the active site is reduced in the substrate-free enzyme relative to the substrate-bound structure calculated using the same methods. Both symmetric and asymmetric broadening of multiple resonances observed upon substrate removal as well as localized increased errors in RDC fits suggest that an ensemble of enzyme conformations are present in the substrate-free form.

  19. Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor.

    PubMed

    Su, Fei; Takaya, Naoki; Shoun, Hirofumi

    2004-02-01

    Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.

  20. Inhibition of cytochrome P450 2B4 by environmentally persistent free radical-containing particulate matter

    PubMed Central

    Reed, James R.; dela Cruz, Albert Leo N.; Lomnicki, Slawo M.; Backes, Wayne L.

    2015-01-01

    Combustion processes generate particulate matter (PM) that can affect human health. The presence of redox-active metals and aromatic hydrocarbons in the post-combustion regions results in the formation of air-stable, environmentally persistent free radicals (EPFRs) on entrained particles. Exposure to EPFRs has been shown to negatively influence pulmonary and cardiovascular functions. Cytochromes P450 (P450/CYP) are endoplasmic reticulum resident proteins that are responsible for the metabolism of foreign compounds. Previously, it was shown that model EPFRs, generated by exposure of silica containing 5% copper oxide (CuO-Si) to either dicholorobenzene (DCB230) or 2-monochlorophenol (MCP230) at ≥ 230°C, inhibited six forms of P450 in rat liver microsomes (Toxicol. Appl. Pharmacol. (2014) 277:200-209). In this study, the inhibition of P450 by MCP230 was examined in more detail by measuring its effect on the rate of metabolism of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) and 7-benzyloxyresorufin (7BRF) by the purified, reconstituted CYP2B4 system. MCP230 inhibited the CYP2B4-mediated metabolism of 7EFC at least 10-fold more potently than non-EPFR controls (CuO-Si, silica, and silica generated from heating silica and MCP at 50°C, so that EPFRs were not formed (MCP50)). The inhibition by EPFRs was specific for the P450 and did not affect the ability of the redox partner, P450 reductase (CPR) from reducing cytochrome c. All of the PM inhibited CYP2B4-mediated metabolism noncompetitively with respect to substrate. When CYP2B4-mediated metabolism of 7EFC was measured as a function of the CPR concentration, the mechanism of inhibition was competitive. EPFRs likely inhibit CYP2B4-mediated substrate metabolism by physically disrupting the CPR•P450 complex. PMID:25817938

  1. Cytochrome P450 1A induction in gudgeon Gobio gobio : Laboratory and Field Studies.

    PubMed

    Flammarion, P

    1999-01-01

    The induction of cytochrome P450 1A was studied in gudgeon (Gobio gobio), a common European cyprinid, using both farm-raised and field-caught fish. The effects of sex, reproductive status and past exposure to xenobiotics were assessed. When exposed to beta-naphthoflavone (bNF), reared gudgeon showed a dose-dependent increase of EROD activity with a plateau observed at doses from 20 mg kg-1 (females) and 5 mg kg-1 (males). The sexual difference in EROD activity was related to the gonadosomatic index (GSI) of the female whatever the level of induction. Dose and sex effects were confirmed by the immunodetection of CYP1A protein. More than 1 month was necessary for EROD activity to decrease to baseline levels. A second bNF injection after 32 days gave similar levels of induction, suggesting that EROD induction by bNF was not impaired by a pretreatment. Wild fish were brought from two sites in the Rhone river basin: a low contaminated site (Ain) and a highly contaminated site (Rhone). Wild gudgeon were highly induced by bNF in laboratory conditions, except males from the Rhone site which exhibited EROD levels as high as the EROD plateau found in laboratory conditions. A 2- month depuration period in clean water was necessary for EROD activity in wild gudgeon to decrease to baseline levels. These results provide better knowledge of the main factors of modulation of the induction in gudgeon as well as on the influence of the history of exposure to inducers.

  2. Identification of a novel cytochrome P450 gene, CYP321E1 from the diamondback moth, Plutella xylostella (L.) and RNA interference to evaluate its role in chlorantraniliprole resistance.

    PubMed

    Hu, Z; Lin, Q; Chen, H; Li, Z; Yin, F; Feng, X

    2014-12-01

    Insect cytochrome P450 monooxygenases (P450s) play an important role in catalysis of many reactions leading to insecticides resistance. Our previous studies on transcriptome analysis of chlorantraniliprole-resistant development in the diamondback moth, Plutella xylostella revealed that up-regulation of cytochrome P450s are one of the main factors leading to the development of chlorantraniliprole resistance. Here, we report for the first time a novel cytochrome P450 gene CYP321E1, which belongs to the cytochrome P450 gene family CYP321. Real-time quantitative PCR (RT-qPCR) analyses indicated that CYP321E1 was expressed at all developmental stages of P. xylostella but was highest in the fourth-instar larvae; furthermore, the relatively high expression was observed in the midgut of the fourth-instar larvae, followed by fat bodies and epidermis. The expression of CYP321E1 in P. xylostella was differentially affected by three representative insecticides, including alphamethrin, abamectin and chlorantraniliprole. Among them, the exposure to chlorantraniliprole resulted in the largest transcript level of this cytochrome P450 gene. The findings suggested potential involvement of CYP321E1 in chlorantraniliprole resistance of P. xylostella. To assess the functional link of CYP321E1 to chlorantraniliprole resistance, RNA interference (RNAi)-mediated gene silencing by double stranded RNA (dsRNA) injecting was used. Results revealed that injection delivery of dsRNA can greatly reduce gene expression after 24 h. As a consequence of RNAi, a significant increment in mortality of larvae injected CYP321E1 dsRNA was observed after 24 h of exposure to chlorantraniliprole. These results strongly support our notion that this novel cytochrome P450 gene plays an important role in chlorantraniliprole detoxification in the diamondback moth and is partly responsible for its resistance.

  3. Camptothecin Attenuates Cytochrome P450 3A4 Induction by Blocking the Activation of Human Pregnane X ReceptorS⃞

    PubMed Central

    Chen, Yakun; Tang, Yong; Robbins, Gregory T.

    2010-01-01

    Differential regulation of drug-metabolizing enzymes (DMEs) is a common cause of adverse drug effects in cancer therapy. Due to the extremely important role of cytochrome P450 3A4 (CYP3A4) in drug metabolism and the dominant regulation of human pregnane X receptor (hPXR) on CYP3A4, finding inhibitors for hPXR could provide a unique tool to control drug efficacies in cancer therapy. Camptothecin (CPT) was demonstrated as a novel and potent inhibitor (IC50 = 0.58 μM) of an hPXR-mediated transcriptional regulation on CYP3A4 in this study. In contrast, one of its analogs, irinotecan (CPT-11), was found to be an hPXR agonist in the same tests. CPT disrupted the interaction of hPXR with steroid receptor coactivator-1 but had effects on neither the competition of ligand binding nor the formation of the hPXR and retinoid X receptor α heterodimer, nor the interaction between the regulatory complex and DNA-responsive elements. CPT treatment resulted in delayed metabolism of nifedipine in human hepatocytes treated with rifampicin, suggesting a potential prevention of drug-drug interactions between CYP3A4 inducers and CYP3A4-metabolized drugs. Because CPT is the leading compound of topoisomerase I inhibitors, which comprise a quickly developing class of anticancer agents, the findings indicate the potential of a new class of compounds to modify hPXR activity as agonists/inhibitors and are important in the development of CPT analogs. PMID:20504912

  4. Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission.

    PubMed

    Caranto, Jonathan D; Vilbert, Avery C; Lancaster, Kyle M

    2016-12-20

    Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N 2 O). It has been proposed that N 2 O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH 2 OH) quantitatively to N 2 O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH 2 OH to nitrite ([Formula: see text]) under aerobic conditions. Although we observe [Formula: see text] formation under aerobic conditions, its concentration is not stoichiometric with the NH 2 OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH 2 OH to N 2 O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an Fe III -NH 2 OH adduct of cyt P460 is oxidized to an {FeNO} 6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH 2 OH, forming the N-N bond of N 2 O during a bimolecular, rate-determining step. We propose that [Formula: see text] results when nitric oxide (NO) dissociates from the {FeNO} 6 intermediate and reacts with dioxygen. Thus, [Formula: see text] is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH 2 OH contributes to NO and N 2 O emissions from nitrifying microorganisms.

  5. Synthesis and room temperature photo-induced electron transfer in biologically active bis(terpyridine)ruthenium(II)-cytochrome c bioconjugates and the effect of solvents on the bioconjugation of cytochrome c.

    PubMed

    Peterson, Joshua R; Smith, Trevor A; Thordarson, Pall

    2010-01-07

    Photo-active bis(terpyridine)ruthenium(ii) chromophores were synthesised and attached to the redox enzyme iso-1 cytochrome c in a mixed solvent system to form photo-induced bioconjugates in greater than 40% yield after purification. The effects of up to 20% (v/v) of acetonitrile, tetrahydrofuran, dimethylformamide, or dimethyl sulfoxide at 4, 25 and 35 degrees C on the stability and biological activity of cytochrome c and its reactivity towards the model compound 4,4'-dithiodipyridine (DTDP) was measured. The second-order rate constant for the DTDP reaction was found to range between k = 2.5-4.3 M(-1) s(-1) for reactions with 5% organic solvent added compared to k = 5.6 M(-1) s(-1) in pure water at 25 degrees C. Use of 20% solvent generally results in significant protein oxidation, and 20% acetonitrile and tetrahydrofuran in particular result in significant protein dimerization, which competes with the bioconjugation reaction. Cyclic voltammetry studies indicated that the rate of electron transfer to the heme in solution was reduced in the bis(terpyridine)ruthenium(ii) cytochrome c bioconjugates compared to unmodified cytochrome c. Steady-state fluorescence studies on these bioconjugates showed that energy or electron transfer is taking place between the bis(terpyridine)ruthenium(ii) chromophores and cytochrome c. The bis(terpyridine)ruthenium(ii) cytochrome c bioconjugates demonstrate room temperature photo-activated electron transfer from the bis(terpyridine)ruthenium(ii) donor to the protein acceptor. Two sacrificial donors were used; in 50% glycerol, the bioconjugates were reduced in about 15 min while in 20 mM EDTA the bioconjugates were fully reduced in less than 5 min upon irradiation with a xenon lamp source. Under these conditions, the reduction of the non-covalent mixture of cytochrome c and bis(terpyridine)ruthenium(ii) mixtures took over 30 min. Control experiments showed that the photo-induced reduction of cytochrome c only occurs in the absence of

  6. Optical biosensing of nitrite ions using cytochrome cd1 nitrite reductase encapsulated in a sol-gel matrix.

    PubMed

    Ferretti, S; Lee, S K; MacCraith, B D; Oliva, A G; Richardson, D J; Russell, D A; Sapsford, K E; Vidal, M

    2000-11-01

    Nitrite is an important human health and environmental analyte. As such, the European Union (EU) has imposed a limit for nitrite in potable water of 0.1 mg l-1 (2.18 microM). In order to develop an optical biosensing system for the determination of nitrite ions in environmental waters, cytochrome cd1 nitrite reductase has been extracted and purified from the bacterium Paracoccus pantotrophus. The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cytochrome cd1 enzyme, i.e., Km, Vmax and kcat have been determined. The influence of pH on the activity of the cytochrome cd1 has been investigated and the results suggest that this enzyme can be used for the determination of nitrite in the pH range 6-9. Biosensing experiments with the cytochrome cd1 in solution suggested that the decrease in intensity of the absorption band associated with the d1 haem (which is the nitrite binding site), at 460 nm, with increasing nitrite concentrations would enable the measurement of this analyte with the optimum limit of detection. The cytochrome cd1 has been encapsulated in a bulk sol-gel monolith with no structural changes observed and retention of enzymatic activity. The detection of nitrite ions in the range 0.075-1.250 microM was achieved, with a limit of detection of 0.075 microM. In order to increase the speed of response, a sol-gel sandwich thin film structure was formulated with the cytochrome cd1. This structure enabled the determination of nitrite concentrations within ca. 5 min. The sol-gel sandwich entrapped cytochrome cd1 enzyme was found to be stable for several months when the films were stored at 4 degrees C.

  7. Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Swinney, D.C.; Ryan, D.E.; Thomas, P.E.

    1988-07-26

    Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6..beta..-hydroxyprogesterone and 16..cap alpha..-hydroxyprogesterone), two secondary metabolites (6..beta..,16..cap alpha..-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16..cap alpha..-hydroxyprogesterone). The K/sub m,app/ for the formation of these products from progesterone was determined to be approximately 0.5 ..mu..M, while the K/sub m,app/ for metabolism of 6..beta..- and 16..cap alpha..-hydroxyprogesterone was found to be 5-10 ..mu..M. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 ..mu..M, and a lag in formation of secondary metabolites was not observed inmore » 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6..beta..,16..cap alpha..-dihydroxyprogesterone and 6-keto-16..cap alpha..-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6..beta..-hydroxyprogesterone or 16..cap alpha..-hydroxyprogesterone. Incubation of 6..beta..-hydroxyprogesterone with a reconstituted system in an atmosphere of /sup 18/I/sub 2/ resulted in > 90% incorporation of /sup 18/O in the 16..cap alpha..-position of 6..beta..,16..cap alpha..-dihydroxyprogesterone but no incorporation of /sup 18/O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O/sub 2/, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored.« less

  8. Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley

    PubMed Central

    Koopmann, Edda; Hahlbrock, Klaus

    1997-01-01

    Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H. PMID:9405720

  9. Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline.

    PubMed

    Utoh, Masahiro; Murayama, Norie; Uno, Yasuhiro; Onose, Yui; Hosaka, Shinya; Fujino, Hideki; Shimizu, Makiko; Iwasaki, Kazuhide; Yamazaki, Hiroshi

    2013-12-01

    Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

  10. Role of Cytochrome c in Apoptosis: Increased Sensitivity to Tumor Necrosis Factor Alpha Is Associated with Respiratory Defects but Not with Lack of Cytochrome c Release▿

    PubMed Central

    Vempati, Uma D.; Diaz, Francisca; Barrientos, Antoni; Narisawa, Sonoko; Mian, Abdul M.; Millán, José Luis; Boise, Lawrence H.; Moraes, Carlos T.

    2007-01-01

    Although the role of cytochrome c in apoptosis is well established, details of its participation in signaling pathways in vivo are not completely understood. The knockout for the somatic isoform of cytochrome c caused embryonic lethality in mice, but derived embryonic fibroblasts were shown to be resistant to apoptosis induced by agents known to trigger the intrinsic apoptotic pathway. In contrast, these cells were reported to be hypersensitive to tumor necrosis factor alpha (TNF-α)-induced apoptosis, which signals through the extrinsic pathway. Surprisingly, we found that this cell line (CRL 2613) respired at close to normal levels because of an aberrant activation of a testis isoform of cytochrome c, which, albeit expressed at low levels, was able to replace the somatic isoform for respiration and apoptosis. To produce a bona fide cytochrome c knockout, we developed a mouse knockout for both the testis and somatic isoforms of cytochrome c. The mouse was made viable by the introduction of a ubiquitously expressed cytochrome c transgene flanked by loxP sites. Lung fibroblasts in which the transgene was deleted showed no cytochrome c expression, no respiration, and resistance to agents that activate the intrinsic and to a lesser but significant extent also the extrinsic pathways. Comparison of these cells with lines with a defective oxidative phosphorylation system showed that cells with defective respiration have increased sensitivity to TNF-α-induced apoptosis, but this process was still amplified by cytochrome c. These studies underscore the importance of oxidative phosphorylation and apoptosome function to both the intrinsic and extrinsic apoptotic pathways. PMID:17210651

  11. Ligand-independent activation of the arylhydrocarbon receptor by ETK (Bmx) tyrosine kinase helps MCF10AT1 breast cancer cells to survive in an apoptosis-inducing environment.

    PubMed

    Fujisawa, Yasuko; Li, Wen; Wu, Dalei; Wong, Patrick; Vogel, Christoph; Dong, Bin; Kung, Hsing-Jien; Matsumura, Fumio

    2011-10-01

    It has been reported that the arylhydrocarbon receptor (AHR) is overexpressed in certain types of breast tumors. However, so far no concrete evidence has been provided yet as to why and how the overexpressed AHR in those cancer cells is functionally activated without exogenous ligands. Here we show that the AHR was functionally activated when estrogen receptor-negative, AHR overexpressing MCF10AT1 human breast cancer cells (designated P20E) were subjected to serum starvation. Transfection of cells with ETK-KQ, a plasmid for kinase-dead epithelial and endothelial tyrosine kinase (ETK), attenuated this AHR activation. Artificial over-expression of ETK in P20E cells through transfection with wild-type ETK plasmid (ETK-wt) caused up-regulation of cytochrome P4501a1 (CYP1A1; a marker of functional activation of AHR). Furthermore, ablation of ETK expression by a specific antisense oligonucleotide or AG879, a specific inhibitor of ETK kinase suppressed activation of AHR induced by omeprazole, a strong ligand-independent activator of AHR. Activation of ETK in those cells conferred them resistance to UVB- as well as doxorubicin-induced apoptosis, both of which were reversed by ETK-KQ. Together, these findings support our conclusion that ETK is the tyrosine kinase responsible for the functional activation of the AHR in these mammary epithelial cells.

  12. Biosynthesis of Costunolide, Dihydrocostunolide, and Leucodin. Demonstration of Cytochrome P450-Catalyzed Formation of the Lactone Ring Present in Sesquiterpene Lactones of Chicory

    PubMed Central

    de Kraker, Jan-Willem; Franssen, Maurice C.R.; Joerink, Maaike; de Groot, Aede; Bouwmeester, Harro J.

    2002-01-01

    Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-costunolide. This (+)-costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of 18O2 resulted in the incorporation of one atom of 18O into (+)-costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C6-hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C12-atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-costunolide into 11(S),13-dihydrocostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme. PMID:12011356

  13. Biosynthesis of costunolide, dihydrocostunolide, and leucodin. Demonstration of cytochrome p450-catalyzed formation of the lactone ring present in sesquiterpene lactones of chicory.

    PubMed

    de Kraker, Jan-Willem; Franssen, Maurice C R; Joerink, Maaike; de Groot, Aede; Bouwmeester, Harro J

    2002-05-01

    Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-costunolide. This (+)-costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of 18O2 resulted in the incorporation of one atom of 18O into (+)-costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C6-hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C12-atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-costunolide into 11(S),13-dihydrocostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme.

  14. Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

    PubMed

    Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

    1989-03-15

    Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

  15. The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons.

    PubMed

    Finta, C; Zaphiropoulos, P G

    2000-12-30

    Using a bacterial artificial chromosome (BAC) clone, we have mapped the human cytochrome P450 3A (CYP3A) locus containing the genes encoding for CYP3A4, CYP3A5 and CYP3A7. The genes lie in a head-to-tail orientation in the order of 3A4, 3A7 and 3A5. In both intergenic regions (3A4-3A7 and 3A7-3A5), we have detected several additional cytochrome P450 3A exons, forming two CYP3A pseudogenes. These pseudogenes have the same orientation as the CYP3A genes. To our surprise, a 3A7 mRNA species has been detected in which the exons 2 and 13 of one of the pseudogenes (the one that is downstream of 3A7) are spliced after the 3A7 terminal exon. This results in an mRNA molecule that consists of the 13 3A7 exons and two additional exons at the 3' end. The additional two exons originating from the pseudogene are in an altered reading frame and consequently have the capability to code a completely different amino acid sequence than the canonical CYP3A exons 2 and 13. These findings may represent a generalized evolutionary process with genes having the potential to capture neighboring sequences and use them as functional exons.

  16. Comparison of mibefradil and derivative NNC 55-0396 effects on behavior, cytochrome P450 activity, and tremor in mouse models of essential tremor

    PubMed Central

    Quesada, Arnulfo; Bui, Peter H.; Homanics, Gregg E.; Hankinson, Oliver; Handforth, Adrian

    2014-01-01

    NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2, 3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride], is a mibefradil derivative that retains potent in vitro T-type calcium channel antagonist efficacy. We compared the two compounds for behavioral toxicity, effects on cytochrome P450 activity, and efficacy against tremor in the γ-aminobutyric acid type A (GABAA) receptor subunit α1-null mouse, and the harmaline tremor model of essential tremor in wild-type mice. NNC 55-0396 was better tolerated than mibefradil in the horizontal wire test of sedation/motor function, with 3/6 failing at 300 and 30 mg/kg respectively. To assess for a potential interaction with harmaline, mice were given the drugs, followed by harmaline or vehicle, and tested 30 min later in the inverted wire grid test. Mibefradil exacerbated, whereas NNC 55-0396 ameliorated harmaline-induced test deficits. In mouse liver microsomes, NNC 55-0396 was a less potent inhibitor of harmaline O-demethylation than mibefradil (Ki: 0.95 and 0.29 µM respectively), and also less potent at inhibiting testosterone 6-β-hydroxylation (Ki: 0.71 and 0.12 µM respectively). In the GABAA α1-null model, NNC 55-0396 but not mibefradil, (each at 20 mg/kg), suppressed tremor while NNC 55-0396 at 12.5 mg/kg suppressed harmaline-induced tremor by half by 20–100 min, whereas mibefradil at the same dose did not significantly affect tremor. In contrast to mibefradil, NNC 55-0396 is well tolerated and suppresses tremor, and exerts less cytochrome P450 inhibition. These results suggest potential clinical utility for NNC 55-0396 or similar derivatives as a T-type calcium antagonist. PMID:21256842

  17. Cytochrome p450 architecture and cysteine nucleophile placement impact raloxifene-mediated mechanism-based inactivation.

    PubMed

    VandenBrink, Brooke M; Davis, John A; Pearson, Josh T; Foti, Robert S; Wienkers, Larry C; Rock, Dan A

    2012-11-01

    The propensity for cytochrome P450 (P450) enzymes to bioactivate xenobiotics is governed by the inherent chemistry of the xenobiotic itself and the active site architecture of the P450 enzyme(s). Accessible nucleophiles in the active site or egress channels of the P450 enzyme have the potential of sequestering reactive metabolites through covalent modification, thereby limiting their exposure to other proteins. Raloxifene, a drug known to undergo CYP3A-mediated reactive metabolite formation and time-dependent inhibition in vitro, was used to explore the potential for bioactivation and enzyme inactivation of additional P450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A5). Every P450 tested except CYP2E1 was capable of raloxifene bioactivation, based on glutathione adduct formation. However, raloxifene-mediated time-dependent inhibition only occurred in CYP2C8 and CYP3A4. Comparable inactivation kinetics were achieved with K(I) and k(inact) values of 0.26 μM and 0.10 min(-1) and 0.81 μM and 0.20 min(-1) for CYP2C8 and CYP3A4, respectively. Proteolytic digests of CYP2C8 and CYP3A4 Supersomes revealed adducts to Cys225 and Cys239 for CYP2C8 and CYP3A4, respectively. For each P450 enzyme, proposed substrate/metabolite access channels were mapped and active site cysteines were identified, which revealed that only CYP2C8 and CYP3A4 possess accessible cysteine residues near the active site cavities, a result consistent with the observed kinetics. The combined data suggest that the extent of bioactivation across P450 enzymes does not correlate with P450 inactivation. In addition, multiple factors contribute to the ability of reactive metabolites to form apo-adducts with P450 enzymes.

  18. Analysis of cytochrome P450 CYP119 ligand-dependent conformational dynamics by two-dimensional NMR and X-ray crystallography.

    PubMed

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N; Nishida, Clinton R; de Montellano, Paul R Ortiz

    2015-04-17

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional (1)H,(15)N HSQC chemical shift perturbation mapping of (15)N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Soybean meal fermented by Aspergillus awamori increases the cytochrome P-450 content of the liver microsomes of mice.

    PubMed

    Kishida, T; Ataki, H; Takebe, M; Ebihara, K

    2000-04-01

    The effect of soybean meal fermented by Aspergillus awamori on the acute lethality of acetaldehyde, pentobarbital sleeping time, and cytochrome P-450 content of the hepatic microsomes was studied in mice. Most of the daidzin and genistin in soybean meal (SBM) were converted into the respective aglycones, daidzein and genistein, by fermentation. In experiment 1, mice were fed isonitrogenic test diets with one of the following five protein sources for 28 d: casein, SBM, fermented and hot-air-dried SBM (FSBM-HD), fermented and freeze-dried SBM (FSBM-FD), or methanol-extracted FSBM-FD (FSMB-FD-R). The acute lethality of acetaldehyde in mice fed the FSBM-FD diet was significantly lower than that in mice fed the SBM, FSBM-HD, or FSBM-FD-R diet. In experiments 2 and 3, mice were fed isonitrogenic test diets with one of the following four protein sources for 28 d: casein, SBM, FSBM-FD, and FSBM-FD-R. The pentobarbital sleeping time was significantly shorter and the cytochrome P-450 content was significantly higher in the mice fed the FSBM-FD diet than the respective value in mice fed the other test diets. In experiment 4, mice were fed one of eight diets which contained different levels of aglycone obtained by varying the proportion of FSBM-FD and FSBM-FD-R, for 28 d. The cytochrome P-450 content in hepatic microsomes increased as the dietary level of isoflavonoid aglycones increased, but there was a saturation phenomenon. These results suggest that soy isoflavonoid aglycones are more potent inducers of cytochrome P-450 than isoflavonoid glycosides.

  20. Different structure of the complexes of two cytochrome P-450 isozymes with acetanilide by 1H-NMR relaxation and spectrophotometry.

    PubMed

    Woldman YaYu; Weiner, L M; Lyakhovich, V V

    1993-05-28

    The functional and spectral characteristics of the interaction of acetanilide with phenobarbital- and methylcholanthrene- induced rat liver microsomes, as well as with corresponding major isozymes (cytochromes P-450b and P-450c) have been compared. The magnitude of the reverse 1st type binding spectra proved to be negatively correlated with the acetanilide oxidation on isozymes under study. The data on paramagnetic relaxation of acetanilide protons in the presence of P-450 have shown the structure of the enzyme-substrate complex to be different for two isozymes, acetanilide molecule being closer to Fe ion in the active site in the case of P-450c, which is active towards acetanilide oxidation. For the P-450c-acetanilide complex the group oxidized (phenyl) is the closest to Fe ion.

  1. Cytochrome P450-mediated metabolic engineering: current progress and future challenges.

    PubMed

    Renault, Hugues; Bassard, Jean-Etienne; Hamberger, Björn; Werck-Reichhart, Danièle

    2014-06-01

    Cytochromes P450 catalyze a broad range of regiospecific, stereospecific and irreversible steps in the biosynthetic routes of plant natural metabolites with important applications in pharmaceutical, cosmetic, fragrance and flavour, or polymer industries. They are consequently essential drivers for the engineered bioproduction of such compounds. Two ground-breaking developments of commercial products driven by the engineering of P450s are the antimalarial drug precursor artemisinic acid and blue roses or carnations. Tedious optimizations were required to generate marketable products. Hurdles encountered in P450 engineering and their potential solutions are summarized here. Together with recent technical developments and novel approaches to metabolic engineering, the lessons from this pioneering work should considerably boost exploitation of the amazing P450 toolkit emerging from accelerated sequencing of plant genomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. Effects of aging and rifampicin pretreatment on the pharmacokinetics of human cytochrome P450 probes caffeine, warfarin, omeprazole, metoprolol and midazolam in common marmosets genotyped for cytochrome P450 2C19.

    PubMed

    Toda, Akiko; Uehara, Shotaro; Inoue, Takashi; Utoh, Masahiro; Kusama, Takashi; Shimizu, Makiko; Uno, Yasuhiro; Mogi, Masayuki; Sasaki, Erika; Yamazaki, Hiroshi

    2018-07-01

    1. The pharmacokinetics were investigated for human cytochrome P450 probes after single intravenous and oral administrations of 0.20 and 1.0 mg/kg, respectively, of caffeine, warfarin, omeprazole, metoprolol and midazolam to aged (10-14 years old, n = 4) or rifampicin-treated/young (3 years old, n = 3) male common marmosets all genotyped as heterozygous for a cytochrome P450 2C19 variant. 2. Slopes of the plasma concentration-time curves after intravenous administration of warfarin and midazolam were slightly, but significantly (two-way analysis of variance), decreased in aged marmosets compared with young marmosets. The mean hepatic clearances determined by in silico fitting for individual pharmacokinetic models of warfarin and midazolam in the aged group were, respectively, 23% and 56% smaller than those for the young group. 3. Significantly enhanced plasma clearances of caffeine, warfarin, omeprazole and midazolam were evident in young marmosets pretreated with rifampicin (25 mg/kg daily for 4 days). Two- to three-fold increases in hepatic intrinsic clearance values were observed in the individual pharmacokinetic models. 4. The in vivo dispositions of multiple simultaneously administered drugs in old, young and P450-enzyme-induced marmosets were elucidated. The results suggest that common marmosets could be experimental models for aged, induced or polymorphic P450 enzymes in P450-dependent drug metabolism studies.

  3. Identification of promoter polymorphisms in the cytochrome P450 CYP6AY1 linked with insecticide resistance in the brown planthopper, Nilaparvata lugens.

    PubMed

    Pang, R; Li, Y; Dong, Y; Liang, Z; Zhang, Y; Zhang, W

    2014-12-01

    Imidacloprid resistance in the brown planthopper, Nilaparvata lugens, is primarily the result of the over-expression of cytochrome P450 monooxygenases. Here, a field-collected strain of N. lugens was shown to be highly resistant to both imidacloprid and buprofezin. Insecticide exposure and quantitative real-time PCR revealed that its resistance was mainly associated with a cytochrome P450 gene, CYP6AY1. CYP6AY1 is known to metabolize imidacloprid but its effect on buprofezin is unclear. In the 5'-untranslated region of CYP6AY1, a novel alternative splicing was detected. After a 1990-bp promoter region was cloned, its basal luciferase activity was assessed. Furthermore, genotyping studies identified 12 variations in the promoter region that discriminated between the field-collected and control strain. Finally, survival bioassays revealed a single nucleotide polymorphism and an insertion-deletion polymorphism linked to buprofezin and imidacloprid resistance. Mutagenesis of these sites enhanced the promoter activity of CYP6AY1. These results suggest that promoter polymorphisms may affect P450-mediated multiple insecticide resistance of pests. © 2014 The Royal Entomological Society.

  4. Novel phacB-encoded cytochrome P450 monooxygenase from Aspergillus nidulans with 3-hydroxyphenylacetate 6-hydroxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities.

    PubMed

    Ferrer-Sevillano, Francisco; Fernández-Cañón, José M

    2007-03-01

    Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (delta phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).

  5. Susceptibility to endometrial cancer: influence of allelism at p53, glutathione S-transferase (GSTM1 and GSTT1) and cytochrome P-450 (CYP1A1) loci.

    PubMed Central

    Esteller, M.; García, A.; Martínez-Palones, J. M.; Xercavins, J.; Reventós, J.

    1997-01-01

    A case-control study was designed to identify associations between polymorphisms at p53, cytochrome P-450 (CYP1A1) and glutathione-S-transferases and endometrial cancer susceptibility. Among all polymorphisms analysed, an insertional variant in p53 (P53PIN3) and two polymorphisms in the 3'-end and exon 7 of CYP1A1 showed significant association with enhanced endometrial cancer risk. Images Figure 1 Figure 2 PMID:9155064

  6. Selecting of a cytochrome P450cam SeSaM library with 3-chloroindole and endosulfan - Identification of mutants that dehalogenate 3-chloroindole.

    PubMed

    Kammoonah, Shaima; Prasad, Brinda; Balaraman, Priyadarshini; Mundhada, Hemanshu; Schwaneberg, Ulrich; Plettner, Erika

    2018-01-01

    Cytochrome P450 cam (a camphor hydroxylase) from the soil bacterium Pseudomonas putida shows potential importance in environmental applications such as the degradation of chlorinated organic pollutants. Seven P450 cam mutants generated from Sequence Saturation Mutagenesis (SeSaM) and isolated by selection on minimal media with either 3-chloroindole or the insecticide endosulfan were studied for their ability to oxidize of 3-chloroindole to isatin. The wild-type enzyme did not accept 3-chloroindole as a substrate. Mutant (E156G/V247F/V253G/F256S) had the highest maximal velocity in the conversion of 3-chloroindole to isatin, whereas mutants (T56A/N116H/D297N) and (G60S/Y75H) had highest k cat /K M values. Six of the mutants had more than one mutation, and within this set, mutation of residues 297 and 179 was observed twice. Docking simulations were performed on models of the mutant enzymes; the wild-type did not accommodate 3-chloroindole in the active site, whereas all the mutants did. We propose two potential reaction pathways for dechlorination of 3-chloroindole. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. 1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro

    PubMed Central

    Xing, Fuqiang; Zhan, Qiuqiang; He, Yiduo; Cui, Jiesheng; He, Sailing; Wang, Guanyu

    2016-01-01

    Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR) at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS) generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant). Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor) and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis. PMID:27689798

  8. Kinetics of interprotein electron transfer between cytochrome c6 and the soluble CuA domain of cyanobacterial cytochrome c oxidase.

    PubMed

    Paumann, Martina; Feichtinger, Markus; Bernroitner, Margit; Goldfuhs, Judith; Jakopitsch, Christa; Furtmüller, Paul G; Regelsberger, Günther; Peschek, Günter A; Obinger, Christian

    2004-10-08

    Cytochrome c6 is a soluble metalloprotein located in the periplasmic space and the thylakoid lumen of many cyanobacteria and is known to carry electrons from cytochrome b6f to photosystem I. The CuA domain of cytochrome c oxidase, the terminal enzyme which catalyzes the four-electron reduction of molecular oxygen in the respiratory chains of mitochondria and many bacteria, also has a periplasmic location. In order to test whether cytochrome c6 could also function as a donor for cytochrome c oxidase, we investigated the kinetics of the electron transfer between recombinant cytochrome c6 (produced in high yield in Escherichia coli by coexpressing the maturation proteins encoded by the ccmA-H gene cluster) and the recombinant soluble CuA domain (i.e., the donor binding and electron entry site) of subunit II of cytochrome c oxidase from Synechocystis PCC 6803. The forward and the reverse electron transfer reactions were studied by the stopped-flow technique and yielded apparent bimolecular rate constants of (3.3 +/- 0.3) x 10(5) M(-1) s(-1) and (3.9 +/- 0.1) x 10(6) M(-1) s(-1), respectively, in 5 mM potassium phosphate buffer, pH 7, containing 20 mM potassium chloride and 25 degrees C. This corresponds to an equilibrium constant Keq of 0.085 in the physiological direction (DeltarG'0 = 6.1 kJ/mol). The reduction of the CuA fragment by cytochrome c6 is almost independent on ionic strength, which is in contrast to the reaction of the CuA domain with horse heart cytochrome c, which decreases with increasing ionic strength. The findings are discussed with respect to the potential role of cytochrome c6 as mobile electron carrier in both cyanobacterial electron transport pathways. Copyright 2004 Federation of European Biochemical Societies

  9. Interaction of tributyltin with 3,3',4,4',5-pentachlorobiphenyl-induced ethoxyresorufin O-deethylase activity in rat hepatoma cells.

    PubMed

    Kannan, K; Villeneuve, D L; Blankenship, A L; Giesy, J P

    1998-11-13

    Interaction of tributyltin (TBT) with 3,3',4,4',5-pentachlorobiphenyl (PCB-126)-induced ethoxyresorufin O-deethylase (EROD) activity was examined in vitro using H4IIE rat hepatoma cells. H4IIE cells were exposed to TBT and PCB-126, individually or in combination, at different concentrations. TBT was cytotoxic at concentrations greater than 98 nM. PCB-126 was not cytotoxic in the concentration range of 49 to 3140 pM. At concentrations greater than 49 nM, PCB-126 enhanced the cytotoxicity of TBT in the 24-98 nM range. In the absence of inducers of EROD activity, TBT significantly inhibited constitutive EROD activity in H4IIE cells in a concentration-dependent manner. EROD activity in H4IIE cells was significantly increased by exposure to PCB-126 alone. This effect was potentiated by coexposure to low, noncytotoxic concentrations of TBT. The induction of cytochrome P-4501A (CYP1A) activity in the presence of both an inducer (PCB-126) and low concentrations of an inhibitor (TBT) indicates that TBT does not interfere with the Ah receptor binding, but acts at the transcriptional level. Potentiation of EROD activity and cytotoxicity as a consequence of coexposure to PCB-126 and TBT is of considerable toxicological significance, given their coaccumulation in a variety of marine organisms.

  10. Human reductive halothane metabolism in vitro is catalyzed by cytochrome P450 2A6 and 3A4.

    PubMed

    Spracklin, D K; Thummel, K E; Kharasch, E D

    1996-09-01

    The anesthetic halothane undergoes extensive oxidative and reductive biotransformation, resulting in metabolites that cause hepatotoxicity. Halothane is reduced anaerobically by cytochrome P450 (P450) to the volatile metabolites 2-chloro-1,1-difluoroethene (CDE) and 2-chloro-1,1,1-trifluoroethane (CTE). The purpose of this investigation was to identify the human P450 isoform(s) responsible for reductive halothane metabolism. CDE and CTE formation from halothane metabolism by human liver microsomes was determined by GC/MS analysis. Halothane metabolism to CDE and CTE under reductive conditions was completely inhibited by carbon monoxide, which implicates exclusively P450 in this reaction. Eadie-Hofstee plots of both CDE and CTE formation were nonlinear, suggesting multiple P450 isoform involvement. Microsomal CDE and CTE formation were each inhibited 40-50% by P450 2A6-selective inhibitors (coumarin and 8-methoxypsoralen) and 55-60% by P450 3A4-selective inhibitors (ketoconazole and troleandomycin). P450 1A-, 2B6-, 2C9/10-, and 2D6-selective inhibitors (7,8-benzoflavone, furafylline, orphenadrine, sulfaphenazole, and quinidine) had no significant effect on reductive halothane metabolism. Measurement of product formation catalyzed by a panel of cDNA-expressed P450 isoforms revealed that maximal rates of CDE formation occurred with P450 2A6, followed by P450 3A4. P450 3A4 was the most effective catalyst of CTE formation. Among a panel of 11 different human livers, there were significant linear correlations between the rate of CDE formation and both 2A6 activity (r = 0.64, p < 0.04) and 3A4 activity (r = 0.64, p < 0.03). Similarly, there were significant linear correlations between CTE formation and both 2A6 activity (r = 0.55, p < 0.08) and 3A4 activity (r = 0.77, p < 0.005). The P450 2E1 inhibitors 4-methylpyrazole and diethyldithiocarbamate inhibited CDE and CTE formation by 20-45% and 40-50%, respectively; however, cDNA-expressed P450 2E1 did not catalyze

  11. Human cytochromes P450 in health and disease

    PubMed Central

    Nebert, Daniel W.; Wikvall, Kjell; Miller, Walter L.

    2013-01-01

    There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases—confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development. PMID:23297354

  12. Cytochrome P4501A biomarker indication of the timeline of chronic exposure of Barrow’s goldeneyes to residual Exxon Valdez oil

    USGS Publications Warehouse

    Esler, Daniel N.; Ballachey, Brenda E.; Trust, Kimberly A.; Iverson, Samuel A.; Reed, John A.; Miles, A. Keith; Henderson, John D.; Woodin, Bruce R.; Stegeman, John J.; McAdie, Malcolm; Mulcahy, Daniel M.; Wilson, Barry W.

    2011-01-01

    We examined hepatic EROD activity, as an indicator of CYP1A induction, in Barrow’s goldeneyes captured in areas oiled during the 1989 Exxon Valdez spill and those from nearby unoiled areas. We found that average EROD activity differed between areas during 2005, although the magnitude of the difference was reduced relative to a previous study from 1996/1997, and we found that areas did not differ by 2009. Similarly, we found that the proportion of individuals captured from oiled areas with elevated EROD activity (⩾2 times unoiled average) declined from 41% in winter 1996/1997 to 10% in 2005 and 15% in 2009. This work adds to a body of literature describing the timelines over which vertebrates were exposed to residual Exxon Valdez oil and indicates that, for Barrow’s goldeneyes in Prince William Sound, exposure persisted for many years with evidence of substantially reduced exposure by 2 decades after the spill.

  13. Effects of obesity on liver cytochromes P450 in various animal models.

    PubMed

    Tomankova, Veronika; Anzenbacher, Pavel; Anzenbacherova, Eva

    2017-06-01

    The prevalence of obesity and other obesity-related diseases is increasing worldwide. Obesity is a disease characterized by increased body weight, or a condition resulting from excessive accumulation of body fat. Due to increased body fat deposits, obesity has also been associated with increased mortality resulting from higher incidence rates of hypertension, diabetes, or various types of cancer, such as breast, colorectal, cervical and prostate cancer. Physiological changes associated with obesity are likely to result in altered drug biotransformation. The main enzymes enabling the oxidative biotransformation of most drugs are cytochromes P450 (CYPs). The review summarizes how pathophysiological factors, especially obesity, affect properties (e.g. enzyme activity, protein expression, gene expression) of CYP enzymes in various experimental models of human obesity. Results reported by various authors suggest that obesity is associated with a decrease of CYP activities (except for the CYP2C and CYP2E1 enzymes). The only exception is mouse obesity induced by monosodium glutamate (administered to newborn mice) as it usually leads to increased CYP expression. Selecting an animal model that is as close as possible to the properties of human obesity is of paramount importance.

  14. A novel role of Drosophila cytochrome P450-4e3 in permethrin insecticide tolerance.

    PubMed

    Terhzaz, Selim; Cabrero, Pablo; Brinzer, Robert A; Halberg, Kenneth A; Dow, Julian A T; Davies, Shireen-A

    2015-12-01

    The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  15. INDUCTION AND POST-TRANSCRIPTIONAL SUPPRESSION OF HEPATIC CYTOCHROME P450 1A1 BY 3,3',4,4'-TETRACHLOROBIPHENYL. (R827102)

    EPA Science Inventory

    Abstract

    <p>3,3',4,4'-Tetrachlorobiphenyl (TCB) can induce and inhibit cytochrome P450 1A1 (CYP1A1) in vertebrates. TCB may also suppress CYP1A1 protein levels, but the mechanism is unknown. This study examined transcriptional and translational aspects of hepatic ...

  16. Worldwide Distribution of Cytochrome P450 Alleles: A Meta-analysis of Population-scale Sequencing Projects.

    PubMed

    Zhou, Y; Ingelman-Sundberg, M; Lauschke, V M

    2017-10-01

    Genetic polymorphisms in cytochrome P450 (CYP) genes can result in altered metabolic activity toward a plethora of clinically important medications. Thus, single nucleotide variants and copy number variations in CYP genes are major determinants of drug pharmacokinetics and toxicity and constitute pharmacogenetic biomarkers for drug dosing, efficacy, and safety. Strikingly, the distribution of CYP alleles differs considerably between populations with important implications for personalized drug therapy and healthcare programs. To provide a global distribution map of CYP alleles with clinical importance, we integrated whole-genome and exome sequencing data from 56,945 unrelated individuals of five major human populations. By combining this dataset with population-specific linkage information, we derive the frequencies of 176 CYP haplotypes, providing an extensive resource for major genetic determinants of drug metabolism. Furthermore, we aggregated this dataset into spectra of predicted functional variability in the respective populations and discuss the implications for population-adjusted pharmacological treatment strategies. © 2017 The Authors Clinical Pharmacology & Therapeutics published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

  17. Characterization of human liver cytochrome P-450 enzymes involved in the O-demethylation of a new P-glycoprotein inhibitor HM-30181.

    PubMed

    Paek, In Bok; Kim, Sung Yeon; Kim, Maeng Sup; Kim, John; Lee, Gwansun; Lee, Hye Suk

    2007-08-01

    HM-30181, 4-oxo-4H-chromene-2-carboxylic acid [2-(2-{4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-ethyl]-phenyl}-2H-tetrazol-5-yl)-4,5-dimethoxy-phenyl]-amide, is a new P-glycoprotein inhibitor with the potential to increase the cytotoxic activity of orally coadministered paclitaxel. This study was performed to characterize human cytochrome P-450 (CYP) enzymes involved in the metabolism of HM-30181 to 4- or 5-O-desmethyl-HM-30181 (M2) and 6- or 7-O-desmethyl-HM-30181 (M3) and to investigate the inhibitory potential of HM-30181 on CYP enzymes in human liver microsomes. CYP3A4 was identified as the major isozyme responsible for the O-demethylation of HM-30181 to M2 and M3 based on the correlation analysis, chemical inhibition and immuno-inhibition study and metabolism in cDNA-expressed human CYP isozymes. HM-30181 itself had no inhibitory effects on CYPs 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, and 3A4 in human liver microsomes, suggesting the possibility that the pharmacokinetics of HM-30181 could be changed with coadministration of known CYP3A4 inducers or inhibitors.

  18. Functional Analysis of Cytochrome P450s Involved in Streptovaricin Biosynthesis and Generation of Anti-MRSA Analogues.

    PubMed

    Liu, Yuanzhen; Chen, Xu; Li, Zhengyuan; Xu, Wei; Tao, Weixin; Wu, Jie; Yang, Jian; Deng, Zixin; Sun, Yuhui

    2017-10-20

    The streptovaricins, chemically related to the rifamycins, are highly effective antibacterial agents, particularly against mycobacteria. Herein, a bioassay-guided investigation of Streptomyces spectabilis CCTCC M2017417 has led to the characterization of streptovaricins as potent compounds against methicillin-resistant Staphylococcus aureus (MRSA). We identified the streptovaricin biosynthetic gene cluster from S. spectabilis CCTCC M2017417 based on genomic sequencing and bioinformatic analysis. Targeted in-frame deletion of five cytochrome P450 genes (stvP1-P5) resulted in the identification of four new streptovaricin analogues and revealed the functions of these genes as follows: stvP1, stvP4, and stvP5 are responsible for the hydroxylation of C-20, Me-24, and C-28, respectively. stvP2 is possibly involved in formation of the methylenedioxy bridge, and stvP3, a conserved gene found in the biosynthetic cluster for naphthalenic ansamycins, might be related to the formation of a naphthalene ring. Biochemical verification of the hydroxylase activity of StvP1, StvP4, and StvP5 was performed, and StvP1 showed unexpected biocatalytic specificity and promiscuity. More importantly, anti-MRSA studies of streptovaricins and derivatives revealed significant structure-activity relationships (SARs): The hydroxyl group at C-28 plays a vital role in antibacterial activity. The hydroxyl group at C-20 substantially enhances activity in the absence of the methoxycarbonyl side chain at C-24, which can increase the activity regardless of the presence of a hydroxyl group at C-20. The inner lactone ring between C-21 and C-24 shows a positive effect on activity. This work provides meaningful information on the SARs of streptovaricins and demonstrates the utility of the engineering of streptovaricins to yield novel anti-MRSA molecules.

  19. A Conserved Cytochrome P450 Evolved in Seed Plants Regulates Flower Maturation.

    PubMed

    Liu, Zhenhua; Boachon, Benoît; Lugan, Raphaël; Tavares, Raquel; Erhardt, Mathieu; Mutterer, Jérôme; Demais, Valérie; Pateyron, Stéphanie; Brunaud, Véronique; Ohnishi, Toshiyuki; Pencik, Ales; Achard, Patrick; Gong, Fan; Hedden, Peter; Werck-Reichhart, Danièle; Renault, Hugues

    2015-12-07

    Global inspection of plant genomes identifies genes maintained in low copies across taxa and under strong purifying selection, which are likely to have essential functions. Based on this rationale, we investigated the function of the low-duplicated CYP715 cytochrome P450 gene family that appeared early in seed plants and evolved under strong negative selection. Arabidopsis CYP715A1 showed a restricted tissue-specific expression in the tapetum of flower buds and in the anther filaments upon anthesis. cyp715a1 insertion lines showed a strong defect in petal development, and transient alteration of pollen intine deposition. Comparative expression analysis revealed the downregulated expression of genes involved in pollen development, cell wall biogenesis, hormone homeostasis, and floral sesquiterpene biosynthesis, especially TPS21 and several key genes regulating floral development such as MYB21, MYB24, and MYC2. Accordingly, floral sesquiterpene emission was suppressed in the cyp715a1 mutants. Flower hormone profiling, in addition, indicated a modification of gibberellin homeostasis and a strong disturbance of the turnover of jasmonic acid derivatives. Petal growth was partially restored by the active gibberellin GA3 or the functional analog of jasmonoyl-isoleucine, coronatine. CYP715 appears to function as a key regulator of flower maturation, synchronizing petal expansion and volatile emission. It is thus expected to be an important determinant of flower-insect interaction. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  20. Transgenic Production of Epoxy Fatty Acids by Expression of a Cytochrome P450 Enzyme from Euphorbia lagascae Seed

    PubMed Central

    Cahoon, Edgar B.; Ripp, Kevin G.; Hall, Sarah E.; McGonigle, Brian

    2002-01-01

    Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Δ12-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Δ12-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developing E. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to the CYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Δ9,15) in cultures supplied with α-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Δ9,15. Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Δ12-epoxy group of vernolic acid. PMID:11842164

  1. Some mutations of exon-7 in cytochrome P450 gene 3A4 and their effect on 6beta-hydroxylation of cortisol.

    PubMed

    Shchepotina, E G; Vavilin, V A; Goreva, O B; Lyakhovich, V V

    2006-06-01

    Analysis of variants of exon 7 sequences in cytochrome P450 gene 3A4 in a sample of Caucasoid persons was carried out. The effect of these variants on activity of CYP3A was assessed by the level of cortisol 6beta-hydroxylation. Alleles CYP3A4*5 and *17 were not detected: probably, these mutations are rare and consequently they have little effect on the character of polymorphic distribution of CYP3A4 activity in this population. The incidence of CYP3A4*2 was 5.26%. The 6betaOH-cortisol/cortisol ratio in an individual with CYP3A4*2/*2 genotype was 7.408, which corresponded to "slow metabolizer" phenotype in this sample.

  2. Protective Effects of Diallyl Sulfide against Thioacetamide-Induced Toxicity: A Possible Role of Cytochrome P450 2E1.

    PubMed

    Kim, Nam Hee; Lee, Sangkyu; Kang, Mi Jeong; Jeong, Hye Gwang; Kang, Wonku; Jeong, Tae Cheon

    2014-02-01

    Effects of diallyl sulfide (DAS) on thioacetamide-induced hepatotoxicity and immunotoxicity were investigated. When male Sprague-Dawley rats were treated orally with 100, 200 and 400 mg/kg of DAS in corn oil for three consecutive days, the activity of cytochrome P450 (CYP) 2E1-selective p-nitrophenol hydroxylase was dose-dependently suppressed. In addition, the activities of CYP 2B-selective benzyloxyresorufin O-debenzylase and pentoxyresorufin O-depentylase were significantly induced by the treatment with DAS. Western immunoblotting analyses also indicated the suppression of CYP 2E1 protein and/or the induction of CYP 2B protein by DAS. To investigate a possible role of metabolic activation by CYP enzymes in thioacetamide-induced hepatotoxicity, rats were pre-treated with 400 mg/kg of DAS for 3 days, followed by a single intraperitoneal treatment with 100 and 200 mg/kg of thioacetamide in saline for 24 hr. The activities of serum alanine aminotransferase and aspartate aminotransferase significantly elevated by thioacetamide were protected in DAS-pretreated animals. Likewise, the suppressed antibody response to sheep erythrocytes by thioacetamide was protected by DAS pretreatment in female BALB/c mice. Taken together, our present results indicated that thioacetamide might be activated to its toxic metabolite(s) by CYP 2E1, not by CYP 2B, in rats and mice.

  3. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes.

    PubMed

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-07-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members' duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes.

  4. Synergistic Effects of Mutations in Cytochrome P450cam Designed to Mimic CYP101D1

    PubMed Central

    Batabyal, Dipanwita; Li, Huiying; Poulos, Thomas L.

    2013-01-01

    A close ortholog to the cytochrome P450cam (CYP101A1) that catalyzes the same hydroxylation of camphor to 5-exo hydroxycamphor is CYP101D1. There are potentially important differences in and around the active site that could contribute to subtle functional differences. Adjacent to the heme iron ligand, Cys357, is Leu358 in P450cam while this residue is Ala in CYP101D1. Leu358 plays a role in binding of the P450cam redox partner, putidaredoxin (Pdx). On the opposite side of the heme about 15 – 20 Å away Asp251 in P450cam plays a critical role in a proton relay network required for O2 activation but forms strong ion pairs with Arg186 and Lys178. In CYP101D1 a Gly replaces Lys178. Thus, the local electrostatic environment and ion pairing is substantially different in CYP101D1. These sites have been systematically mutated in P450cam to the corresponding residues in CYP101D1 and the mutants analyzed by crystallography, kinetics, and UV/Vis spectroscopy. Individually the mutants have little effect on activity or structure but in combination there is a major drop in enzyme activity. This loss in activity is due the mutants being locked in the low-spin state which prevents electron transfer from the P450cam redox partner, Pdx. These studies illustrate the strong synergistic effects on well separated parts of the structure in controlling the equilibrium between the open (low-spin) and closed (high-spin) conformational states. PMID:23865948

  5. Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification1[W][OPEN

    PubMed Central

    Sundin, Lisa; Vanholme, Ruben; Geerinck, Jan; Goeminne, Geert; Höfer, René; Kim, Hoon; Ralph, John; Boerjan, Wout

    2014-01-01

    ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production. PMID:25315601

  6. An assay of optimal cytochrome c oxidase activity in fish gills.

    PubMed

    Hu, Yau-Chung; Chung, Meng-Han; Lee, Tsung-Han

    2018-07-15

    Cytochrome c oxidase (COX) catalyzes the terminal oxidation reaction in the electron transport chain (ETC) of aerobic respiratory systems. COX activity is an important indicator for the evaluation of energy production by aerobic respiration in various tissues. On the basis of the respiratory characteristics of muscle, we established an optimal method for the measurement of maximal COX activity. To validate the measurement of cytochrome c absorbance, different ionic buffer concentrations and tissue homogenate protein concentrations were used to investigate COX activity. The results showed that optimal COX activity is achieved when using 50-100 μg fish gill homogenate in conjunction with 75-100 mM potassium phosphate buffer. Furthermore, we compared branchial COX activities among three species of euryhaline teleost (Chanos chanos, Oreochromis mossambicus, and Oryzias dancena) to investigate differences in aerobic respiration of osmoregulatory organs. COX activities in the gills of these three euryhaline species were compared with COX subunit 4 (COX4) protein levels. COX4 protein abundance and COX activity patterns in the three species occurring in environments with various salinities increased when fish encountered salinity challenges. This COX activity assay therefore provides an effective and accurate means of assessing aerobic metabolism in fish. Copyright © 2018 Elsevier Inc. All rights reserved.

  7. Bcs1p can rescue a large and productive cytochrome bc(1) complex assembly intermediate in the inner membrane of yeast mitochondria.

    PubMed

    Conte, Laura; Trumpower, Bernard L; Zara, Vincenzo

    2011-01-01

    The yeast cytochrome bc(1) complex, a component of the mitochondrial respiratory chain, is composed of ten distinct protein subunits. In the assembly of the bc(1) complex, some ancillary proteins, such as the chaperone Bcs1p, are actively involved. The deletion of the nuclear gene encoding this chaperone caused the arrest of the bc(1) assembly and the formation of a functionally inactive bc(1) core structure of about 500-kDa. This immature bc(1) core structure could represent, on the one hand, a true assembly intermediate or, on the other hand, a degradation product and/or an incorrect product of assembly. The experiments here reported show that the gradual expression of Bcs1p in the yeast strain lacking this protein was progressively able to rescue the bc(1) core structure leading to the formation of the functional homodimeric bc(1) complex. Following Bcs1p expression, the mature bc(1) complex was also progressively converted into two supercomplexes with the cytochrome c oxidase complex. The capability of restoring the bc(1) complex and the supercomplexes was also possessed by the mutated yeast R81C Bcsp1. Notably, in the human ortholog BCS1L, the corresponding point mutation (R45C) was instead the cause of a severe bc(1) complex deficiency. Differently from the yeast R81C Bcs1p, two other mutated Bcs1p's (K192P and F401I) were unable to recover the bc(1) core structure in yeast. This study identifies for the first time a productive assembly intermediate of the yeast bc(1) complex and gives new insights into the molecular mechanisms involved in the last steps of bc(1) assembly. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Cytochrome P450 and organochlorine contaminants in black-crowned night-herons from the Chesapeake Bay region, USA

    USGS Publications Warehouse

    Rattner, Barnett A.; Melancon, Mark J.; Rice, Clifford P.; Riley, Walter; Eisemann, John D.; Hines, Randy K.

    1997-01-01

    Black-crowned night-heron (Nycticorax nycticorax) offspring were collected from a relatively uncontaminated coastal reference site (next to Chincoteague National Wildlife Refuge, VA, USA) and two sites in the Chesapeake Bay watershed (Baltimore Harbor, MD and Rock Creek Park, Washington, DC, USA). Hepatic microsomal activities of benzyloxyresorufin-O-dealkylase and ethoxyresorufin-O-dealkylase were significantly elevated (up to sixfold and ninefold induction, respectively) in pipping embryos from the Baltimore Harbor colony compared to the reference site, whereas values in embryos from the Rock Creek Park colony were intermediate. Concentrations of organochlorine pesticides and metabolites in pipping embryos from both sites in the Chesapeake watershed were greater than at the reference site but below the known threshold for reproductive impairment. However, concentrations of 10 arylhydrocarbon receptor-active polychlorinated biphenyl (PCB) congeners and estimated toxic equivalents were up to 37-fold greater in embryos collected from these two sites in the Chesapeake Bay region, with values for toxic congeners 77 and 126 exceeding those observed in pipping heron embryos from the Great Lakes. Monooxygenase activity of pipping embryos was associated with concentrations of several organochlorine pesticides, total PCBs, arylhydrocarbon receptor-active PCB congeners, and toxic equivalents (r = 0.30–0.59), providing further evidence of the value of cytochrome P450 as a biomarker of organic contaminant exposure. Organochlorine contaminant levels were greater in 10-d-old nestlings from Baltimore Harbor than the reference site but had no apparent effect on monooxygenase activity or growth. These findings demonstrate induction of cytochrome P450 in pipping black-crowned night-heron embryos in the Chesapeake Bay region, probably by exposure to PCB congeners of local origin, and the accumulation of organochlorine pesticides and metabolites in nestling herons from Baltimore

  9. Efficacy of UV-C photolysis of bisphenol A on transcriptome alterations of genes in zebrafish embryos.

    PubMed

    Saeed, Asma; Hashmi, Imran; Zare, Ava; Mehrabani-Zeinabad, Mitra; Achari, Gopal; Habibi, Hamid R

    2016-09-18

    The purpose of this study was to investigate the efficacy of UV-C direct photolysis of bisphenol A (BPA) as a remediation method of BPA contamination. We used zebrafish embryos as a model organism to test the toxicity and residual biological activity by measuring cytochrome P4501A1 (CYP1A), aromatase B (Aro B) and heat shock proteins (HSP-70) transcript levels. The mRNA levels of CYP1A gene increased about two fold while exposure of zebrafish embryos at 72 hpf resulted in significant induction (P = 0.048) of Aro B at 100 µg/L of BPA. Exposure of zebrafish embryos at 72 hpf to increasing concentrations of BPA resulted in significant induction (P = 0.0031) of HSP-70 transcript level. UV treatment of BPA resulted in a significant reduction in toxicity by reducing mortality of zebrafish embryos. The results suggest that UV-C direct photolysis may be an effective method for remediation of BPA contamination. Further studies will be necessary for better understanding of the identity and relative activity of the UV degradation by-products.

  10. Induction of cytochrome P450 enzymes in rat liver by two conazoles, myclobutanil and triadimefon.

    PubMed

    Sun, G; Grindstaff, R D; Thai, S F; Lambert, G R; Tully, D B; Dix, D J; Nesnow, S

    2007-02-01

    This study was undertaken to examine the inductive effects of two triazole antifungal agents, myclobutanil and triadimefon, on the expression of hepatic cytochrome P450 (CYP) genes and on the activities of CYP enzymes in male Sprague Dawley rats. Rats were dosed with the conazoles at three dose levels by gavage for 14 days: myclobutanil (150, 75, and 10mgkg(-1) body weight day(-1); triadimefon (115, 50, and 10 mg kg(-1) body weight day-'), which included their maximum tolerated dose levels (MTD). Both myclobutanil and triadimefon significantly induced pentoxyresorufin O-depentylase activities at their MTD levels: myclobutanil, 8.1-fold at 150mgkg(-1) body weight day- ; and triadimefon, 18.5-fold at 115mgkg(-1) body weight day-'. Benzyloxyresorufin O-debenzylase activities were similarly increased: myclobutanil, 13.3-fold; triadimefon, 27.7-fold. Quantitative real-time reverse-transcription polymerase chain reaction assays were used to characterize the mRNA expression of specific CYP genes induced by these two conazoles. Myclobutanil and triadimefon treatment at their MTD levels significantly increased rat hepatic mRNA expression of CYP2B1 (14.3- and 54.6-fold), CYP3A23/3A1 (2.2- and 7.3-fold), and CYP3A2 (1.5- and 1.7-fold). Western immunoblots of rat hepatic microsomal proteins identified significantly increased levels of CYP isoforms after myclobutanil or triadimefon treatment at their MTD levels: CYP2BI/2 (4.8- and 5.3-fold), and CYP3A1 (2.2- and 2.9-fold). Triadimefon also increased CYP3A2 immunoreactive protein levels 1.8-fold. These results indicate that triadimefon and myclobutanil, like other triazole-containing conazoles, induced CYP2B and CYP3A families of cytochromes in rat liver.

  11. Building Structure Feature-based Models for Predicting Isoform-specific Human Cytochrome P-450 (hCYP 3A4, 2D6 and 2C9) Inhibition Assay Results in ToxCast

    EPA Science Inventory

    EPA’s ToxCast project is using high-throughput screening (HTS) to profile and prioritize chemicals for further testing. ToxCast Phase I evaluated 309 unique chemicals, the majority pesticide actives, in over 500 HTS assays. These included 3 human cytochrome P450 (hCYP3A4, hCYP2...

  12. Cytochrome P450 Genetic Variation Associated with Tamoxifen Biotransformation in American Indian and Alaska Native People

    PubMed Central

    Khan, Burhan A.; Robinson, Renee; Fohner, Alison E.; Muzquiz, LeeAnna I.; Schilling, Brian D.; Beans, Julie A.; Olnes, Matthew J.; Trawicki, Laura; Frydenlund, Holly; Laukes, Cindi; Beatty, Patrick; Phillips, Brian; Nickerson, Deborah; Howlett, Kevin; Dillard, Denise A.; Thornton, Timothy A.; Thummel, Kenneth E.

    2018-01-01

    Abstract Despite evidence that pharmacogenetics can improve tamoxifen pharmacotherapy, there are few studies with American Indian and Alaska Native (AIAN) people. We examined variation in cytochrome P450 (CYP) genes (CYP2D6, CYP3A4, CYP3A5, and CYP2C9) and tamoxifen biotransformation in AIAN patients with breast cancer (n = 42) from the Southcentral Foundation in Alaska and the Confederated Salish and Kootenai Tribes in Montana. We tested for associations between CYP diplotypes and plasma concentrations of tamoxifen and metabolites. Only the CYP2D6 variation was significantly associated with concentrations of endoxifen (P = 0.0008) and 4‐hydroxytamoxifen (P = 0.0074), tamoxifen's principal active metabolites, as well as key metabolic ratios. The CYP2D6 was also the most significant predictor of active metabolites and metabolic ratios in a multivariate regression model, including all four genes as predictors, with minor roles for other CYP genes. In AIAN populations, CYP2D6 is the largest contributor to tamoxifen bioactivation, illustrating the importance of validating pharmacogenetic testing for therapy optimization in an understudied population. PMID:29436156

  13. The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway

    PubMed Central

    Helliwell, Chris A.; Chandler, Peter M.; Poole, Andrew; Dennis, Elizabeth S.; Peacock, W. James

    2001-01-01

    We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA12. A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains. Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified. Arabidopsis thaliana has two CYP88A genes, both of which are expressed. Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA12. Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase. PMID:11172076

  14. Influence of different proton pump inhibitors on activity of cytochrome P450 assessed by [(13)C]-aminopyrine breath test.

    PubMed

    Kodaira, Chise; Uchida, Shinya; Yamade, Mihoko; Nishino, Masafumi; Ikuma, Mutsuhiro; Namiki, Noriyuki; Sugimoto, Mitsushige; Watanabe, Hiroshi; Hishida, Akira; Furuta, Takahisa

    2012-03-01

    Aminopyrine is metabolized by cytochrome P450 (CYP) in the liver. The investigators evaluated influences of different PPIs on CYP activity as assessed by the [(13)C]-aminopyrine breath test ([(13)C]-ABT). Subjects were 15 healthy volunteers with different CYP2C19 status (5 rapid metabolizers [RMs], 5 intermediate metabolizers [IMs], and 5 poor metabolizers [PMs]). Breath samples were collected before and every 15 to 30 minutes for 3 hours after oral ingestion of [(13)C]-aminopyrine 100 mg on day 8 of each of the following regimens: control; omeprazole 20 mg and 80 mg, lansoprazole 30 mg, and rabeprazole 20 mg. Changes in carbon isotope ratios in carbon dioxide ((13)CO(2)/(12)CO(2)) in breath samples were measured by infrared spectrometry and expressed as delta-over-baseline (DOB) ratios (‰). Mean areas under the curve of DOB from 0 to 3 h (AUC(0-3h) of DOB) were significantly decreased by omeprazole 20 mg and lansoprazole 30 mg but not by rabeprazole 20 mg. Conversely, higher PPI dose (ie, omeprazole 80 mg) seemed to further decrease AUC(0-3h) of DOB in RMs but increased it in PMs. Omeprazole and lansoprazole at the standard doses inhibit CYP activity but rabeprazole does not, whereas high-dose omeprazole seems to induce CYPs.

  15. Comparison of brain mitochondrial cytochrome c oxidase activity with cyanide LD(50) yields insight into the efficacy of prophylactics.

    PubMed

    Marziaz, Mandy L; Frazier, Kathryn; Guidry, Paul B; Ruiz, Robyn A; Petrikovics, Ilona; Haines, Donovan C

    2013-01-01

    Cyanide inhibits cytochrome c oxidase, the terminal oxidase of the mitochondrial respiratory pathway, therefore inhibiting the cell oxygen utilization and resulting in the condition of histotoxic anoxia. The enzyme rhodanese detoxifies cyanide by utilizing sulfur donors to convert cyanide to thiocyanate, and new and improved sulfur donors are actively sought as researchers seek to improve cyanide prophylactics. We have determined brain cytochrome c oxidase activity as a marker for cyanide exposure for mice pre-treated with various cyanide poisoning prophylactics, including sulfur donors thiosulfate (TS) and thiotaurine (TT3). Brain mitochondria were isolated by differential centrifugation, the outer mitochondrial membrane was disrupted by a maltoside detergent, and the decrease in absorbance at 550 nm as horse heart ferrocytochrome c (generated by the dithiothreitol reduction of ferricytochrome c) was oxidized was monitored. Overall, the TS control prophylactic treatment provided significant protection of the cytochrome c oxidase activity. The TT3-treated mice showed reduced cytochrome c oxidase activity even in the absence of cyanide. In both treatment series, addition of exogenous Rh did not significantly enhance the prevention of cytochrome c oxidase inhibition, but the addition of sodium nitrite did. These findings can lead to a better understanding of the protection mechanism by various cyanide antidotal systems. Copyright © 2011 John Wiley & Sons, Ltd.

  16. Gene engineering in yeast for biodegradation: Immunological cross-reactivity among cytochrome p-450 system proteins of saccharomyces cerevisiae and candida tropicalis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Loper, J.C.; Chen, C.; Dey, C.R.

    1993-01-01

    Yeasts are eukaryotic microorganisms whose cytochrome P-450 monooxygenase systems may be amenable to genetic engineering for the hydroxylation and detoxication of polychlorinated aromatic hydrocarbons. The molecular genetic properties of strains of bakers yeast, Saccharomyces cerevisiae, and an n-alkane utilizing yeast, Candida tropicalis ATCC750 are examined. Standard methods were used to purify cytochrome P-450 and NADPH-cytochrome c (P-450) reductase proteins from cells cultured by semi-anaerobic glucose fermentation (S. cerevisiae, C. tropicalis) and by growth on tetradecane (C. tropicalis). Polyvalent antisera prepared in rabbits to some of these proteins were used in tests of immunological relatedness among the purified proteins using sodiummore » dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose filter immunoblots. The results provide evidence for gene relationships which should prove useful in gene isolation and subsequent engineering of P-450 enzyme systems in yeast.« less

  17. Crystallization and Preliminary X-ray Analysis of Allene Oxide Synthase, Cytochrome P450 CYP74A2, from Parthenium argentatum

    USDA-ARS?s Scientific Manuscript database

    Oxylipins are oxygenated derivatives of fatty acids and pivotal signaling molecules in plants and animals. Allene oxide synthase (AOS) is a key cytochrome P450 CYP74 enzyme involved in the biosynthesis of plant oxylipin jasmonates to convert 13(S)-hydroperoxide to allene oxide. Guayule (Parthenium a...

  18. Effect of mosquito mats (pyrethroid-based) vapor inhalation on rat brain cytochrome P450s.

    PubMed

    Vences-Mejía, Araceli; Gómez-Garduño, Josefina; Caballero-Ortega, Heriberto; Dorado-González, Víctor; Nosti-Palacios, Rosario; Labra-Ruíz, Norma; Espinosa-Aguirre, J Javier

    2012-01-01

    The effect of transfluthrin (TF) or D-allethrin (DA) pyrethroid (PYR) vapors, often contained as main ingredients in two commercially available mosquito repellent mats, on cytochrome P450 (CYP) enzymes of rat brain and liver was assessed. Immunodetection of CYP2E1 and CYP3A2 proteins revealed their induction in cerebrum and cerebellum, but not in liver microsomes of rats exposed by inhalation to TF or DA. This overexpression of proteins correlated with an increase of their catalytic activities. The specifically increased expression of CYP isoenzymes, due to PYR exposure in the rat brain, could perturb the normal metabolism of endogenous and xenobiotic compounds and leads to increased risks of neurotoxicity by bioactivation, lipid peroxidation and DNA damage.

  19. Jumpstarting the cytochrome P450 catalytic cycle with a hydrated electron.

    PubMed

    Erdogan, Huriye; Vandemeulebroucke, An; Nauser, Thomas; Bounds, Patricia L; Koppenol, Willem H

    2017-12-29

    Cytochrome P450cam (CYP101Fe 3+ ) regioselectively hydroxylates camphor. Possible hydroxylating intermediates in the catalytic cycle of this well-characterized enzyme have been proposed on the basis of experiments carried out at very low temperatures and shunt reactions, but their presence has not yet been validated at temperatures above 0 °C during a normal catalytic cycle. Here, we demonstrate that it is possible to mimic the natural catalytic cycle of CYP101Fe 3+ by using pulse radiolysis to rapidly supply the second electron of the catalytic cycle to camphor-bound CYP101[FeO 2 ] 2+ Judging by the appearance of an absorbance maximum at 440 nm, we conclude that CYP101[FeOOH] 2+ (compound 0) accumulates within 5 μs and decays rapidly to CYP101Fe 3+ , with a k 440 nm of 9.6 × 10 4 s -1 All processes are complete within 40 μs at 4 °C. Importantly, no transient absorbance bands could be assigned to CYP101[FeO 2+ por •+ ] (compound 1) or CYP101[FeO 2+ ] (compound 2). However, indirect evidence for the involvement of compound 1 was obtained from the kinetics of formation and decay of a tyrosyl radical. 5-Hydroxycamphor was formed quantitatively, and the catalytic activity of the enzyme was not impaired by exposure to radiation during the pulse radiolysis experiment. The rapid decay of compound 0 enabled calculation of the limits for the Gibbs activation energies for the conversions of compound 0 → compound 1 → compound 2 → CYP101Fe 3+ , yielding a Δ G ‡ of 45, 39, and 39 kJ/mol, respectively. At 37 °C, the steps from compound 0 to the iron(III) state would take only 4 μs. Our kinetics studies at 4 °C complement the canonical mechanism by adding the dimension of time. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Contribution of cytochrome P450 1B1 to hypertension and associated pathophysiology: a novel target for antihypertensive agents.

    PubMed

    Malik, Kafait U; Jennings, Brett L; Yaghini, Fariborz A; Sahan-Firat, Seyhan; Song, Chi Young; Estes, Anne M; Fang, Xiao R

    2012-08-01

    The aim of this review is to discuss the contribution of cytochrome P450 (CYP) 1B1 in vascular smooth muscle cell growth, hypertension, and associated pathophysiology. CYP1B1 is expressed in cardiovascular and renal tissues, and mediates angiotensin II (Ang II)-induced activation of NADPH oxidase and generation of reactive oxygen species (ROS), and vascular smooth muscle cell migration, proliferation, and hypertrophy. Moreover, CYP1B1 contributes to the development and/or maintenance of hypertension produced by Ang II-, deoxycorticosterone (DOCA)-salt-, and N(ω)-nitro-L-arginine methyl ester-induced hypertension and in spontaneously hypertensive rats. The pathophysiological changes, including cardiovascular hypertrophy, increased vascular reactivity, endothelial and renal dysfunction, injury and inflammation associated with Ang II- and/or DOCA-salt induced hypertension in rats, and Ang II-induced hypertension in mice are minimized by inhibition of CYP1B1 activity with 2,4,3',5'-tetramethoxystilbene or by Cyp1b1 gene disruption in mice. These pathophysiological changes appear to be mediated by increased production of ROS via CYP1B1-dependent NADPH oxidase activity and extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src. Copyright © 2011 Elsevier Inc. All rights reserved.