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Sample records for 1a2 cyp1a2 activity

  1. Parkinson's disease and CYP1A2 activity

    PubMed Central

    Forsyth, J T; Grünewald, R A; Rostami-Hodjegan, A; Lennard, M S; Sagar, H J; Tucker, G T

    2000-01-01

    Aims MPTP, a neurotoxin which induces parkinsonism is partially metabolized by the enzyme CYP1A2. Smoking appears to protect against Parkinson's disease (PD) and cigarette smoke induces CYP1A2 activity. Thus, we investigated the hypothesis that idiopathic PD is associated with lower CYP1A2 activity using caffeine as a probe compound. Methods CYP1A2 activity was assessed using saliva paraxanthine (PX) to caffeine (CA) ratios. Caffeine half-life was also estimated from salivary concentrations of caffeine at 2 and 5 h post dose. 117 treated and 40 untreated patients with PD and 105 healthy control subjects were studied. Results PX/CA ratios were 0.57, 0.93 and 0.77 in treated patients, untreated patients and healthy control subjects, respectively, with no significant differences between study groups (95% CI: treated patients vs controls −0.24, 0.57; untreated patients vs controls −0.75, 0.35). However, patients with PD (treated or untreated) had caffeine half-lives shorter than that in controls (treated patients: 262 min, untreated patients: 244 min, controls: 345 min; 95% CI: controls vs treated patients 23, 143 (P = 0.003); controls vs untreated patients 19, 184 (P = 0.011)). Amongst the patients with PD, caffeine half-life was also inversely related to the age of onset of disease (P = 0.012); gender and concomitant drugs did not influence this significantly. Conclusions Based on PX/CA ratio, there was no evidence of decreased CYP1A2 activity in patients compared with control subjects. The observed decrease in the elimination half-life of caffeine in PD may be caused by increased CYP2E1 activity, an enzyme that also contributes to the metabolism of caffeine. The latter warrants further investigation. PMID:11012552

  2. PCB exposure and in vivo CYP1A2 activity among Native Americans.

    PubMed

    Fitzgerald, Edward F; Hwang, Syni-An; Lambert, George; Gomez, Marta; Tarbell, Alice

    2005-03-01

    Cytochrome P-450 1A2 (CYP1A2) is an enzyme involved in the metabolic activation of some carcinogens and is believed to be induced by xenobiotics. Very few studies, however, have investigated the association between environmental exposures and in vivo CYP1A2 activity in humans. To address this issue, a study was conducted of CYP1A2 activity among Native Americans exposed to polychlorinated biphenyls (PCBs) from the consumption of fish from the St. Lawrence River. At the Mohawk Nation at Akwesasne (in New York and in Ontario and Quebec, Canada), 103 adults were interviewed, and they donated blood for serum PCB analysis and underwent the caffeine breath test (CBT), a safe and noninvasive procedure that uses caffeine as a probe for CYP1A2 activity in vivo. The results supported the findings of other studies that CBT values are higher among smokers and men and lower among women who use oral contraceptives. Despite a relatively low average total PCB body burden in this population, the sum of serum levels for nine mono- or di-ortho-substituted PCB congeners showed positive associations with CBT values (p = 0.052 wet weight and p = 0.029 lipid adjusted), as did toxic equivalent quantities (TEQs; p = 0.091 for wet weight and 0.048 for lipid adjusted). Regarding individual congeners, serum levels of PCB-153, PCB-170, and PCB-180 were significantly correlated with CBT values. The results support the notion that CYP1A2 activity may be a marker of an early biological effect of exposure to PCBs in humans and that the CBT may be a useful tool to monitor such effects.

  3. PCB Exposure and in Vivo CYP1A2 Activity among Native Americans

    PubMed Central

    Fitzgerald, Edward F.; Hwang, Syni-An; Lambert, George; Gomez, Marta; Tarbell, Alice

    2005-01-01

    Cytochrome P-450 1A2 (CYP1A2) is an enzyme involved in the metabolic activation of some carcinogens and is believed to be induced by xenobiotics. Very few studies, however, have investigated the association between environmental exposures and in vivo CYP1A2 activity in humans. To address this issue, a study was conducted of CYP1A2 activity among Native Americans exposed to polychlorinated biphenyls (PCBs) from the consumption of fish from the St. Lawrence River. At the Mohawk Nation at Akwesasne (in New York and in Ontario and Quebec, Canada), 103 adults were interviewed, and they donated blood for serum PCB analysis and underwent the caffeine breath test (CBT), a safe and noninvasive procedure that uses caffeine as a probe for CYP1A2 activity in vivo. The results supported the findings of other studies that CBT values are higher among smokers and men and lower among women who use oral contraceptives. Despite a relatively low average total PCB body burden in this population, the sum of serum levels for nine mono- or di-ortho-substituted PCB congeners showed positive associations with CBT values (p = 0.052 wet weight and p = 0.029 lipid adjusted), as did toxic equivalent quantities (TEQs; p = 0.091 for wet weight and 0.048 for lipid adjusted). Regarding individual congeners, serum levels of PCB-153, PCB-170, and PCB-180 were significantly correlated with CBT values. The results support the notion that CYP1A2 activity may be a marker of an early biological effect of exposure to PCBs in humans and that the CBT may be a useful tool to monitor such effects. PMID:15743714

  4. PCB Exposure and in Vivo CYP1A2 Activity among Native Americans

    PubMed Central

    Fitzgerald, Edward F.; Hwang, Syni-An; Lambert, George; Gomez, Marta; Tarbell, Alice

    2005-01-01

    Cytochrome P-450 1A2 (CYP1A2) is an enzyme involved in the metabolic activation of some carcinogens and is believed to be induced by xenobiotics. Very few studies, however, have investigated the association between environmental exposures and in vivo CYP1A2 activity in humans. To address this issue, a study was conducted of CYP1A2 activity among Native Americans exposed to polychlorinated biphenyls (PCBs) from the consumption of fish from the St. Lawrence River. At the Mohawk Nation at Akwesasne (in New York and in Ontario and Quebec, Canada), 103 adults were interviewed, and they donated blood for serum PCB analysis and underwent the caffeine breath test (CBT), a safe and noninvasive procedure that uses caffeine as a probe for CYP1A2 activity in vivo. The results supported the findings of other studies that CBT values are higher among smokers and men and lower among women who use oral contraceptives. Despite a relatively low average total PCB body burden in this population, the sum of serum levels for nine mono- or di-ortho-substituted PCB congeners showed positive associations with CBT values (p = 0.052 wet weight and p = 0.029 lipid adjusted), as did toxic equivalent quantities (TEQs; p = 0.091 for wet weight and 0.048 for lipid adjusted). Regarding individual congeners, serum levels of PCB-153, PCB-170, and PCB-180 were significantly correlated with CBT values. The results support the notion that CYP1A2 activity may be a marker of an early biological effect of exposure to PCBs in humans and that the CBT may be a useful tool to monitor such effects. PMID:15743714

  5. Urinary mutagenicity, CYP1A2 and NAT2 activity in textile industry workers.

    PubMed

    Fanlo, Ana; Sinuès, Blanca; Mayayo, Esteban; Bernal, Luisa; Soriano, Antonia; Martínez-Jarreta, Begoña; Martínez-Ballarín, Enrique

    2004-11-01

    The two major causes of bladder cancer have been recognised to be cigarette smoke and occupational exposure to arylamines. These compounds are present both in tobacco smoke and in the dyes used in textile production. Aromatic amines suffer oxidative metabolism via P450 cytochrome CYP1A2, and detoxification by the polymorphic NAT2. The aim of the present work was to assess the association between occupational-derived exposure to mutagens and CYP1A2 or NAT2 activity. This cross-sectional study included 117 textile workers exposed to dyes and 117 healthy controls. The urinary mutagenicity was determined in 24 h urine using TA98 Salmonella typhimurium strain with microsomal activation S9 (MIS9) or incubation with beta-glucuronidase (MIbeta). Urinary caffeine metabolite ratios: AFMU+1X+1U/17U, and AFMU/AFMU+1X+1U were calculated to assess CYP1A2 and NAT2 activities, respectively. The results show that workers present a strikingly higher urine mutagenicity than controls (p<0.0001), despite the implementation of the new restrictive norms forbidding the industrial use of the most carcinogenic arylamines. Neither NAT2 nor CYP1A2 activity had any effect on the markers of internal exposure to mutagens, since no significant differences were observed when the urinary mutagenicity of slow and fast acetylators (p>0.05) was compared, and the urinary mutagenicity was not significantly associated with the CYP1A2 activity marker (r=0.04 and r=-0.01 for MIS9 and MIbeta, respectively). This study clearly indicates the need for further protective policies to minimise exposure to the lowest feasible limit in order to avoid unnecessary risks.

  6. Influence of environmental and genetic factors on CYP1A2 activity in individuals of South Asian and European ancestry.

    PubMed

    Perera, V; Gross, A S; McLachlan, A J

    2012-10-01

    The drug-metabolizing enzyme CYP1A2 contributes to the metabolism of a number of commonly used medicines and displays wide interindividual variability. The aim of this study was to investigate CYP1A2 activity in a population of South Asian ancestry and compare it with a population of European ancestry. CYP1A2 activity was determined using the 4 h paraxanthine/caffeine saliva concentration ratio following a 100-mg oral dose of caffeine in healthy individuals of South Asian (n = 166) and European (n = 166) ancestry. Participants were surveyed for extrinsic ethnic factors and genotyped for polymorphisms in CYP1A2 and related genes. Significantly lower CYP1A2 activity was observed in South Asian participants (median: 0.42; range: 0.10-1.06) as compared with European participants (0.54; 0.12-1.64) (P < 0.01). Multiple linear regression indicated that 41% of the variability in CYP1A2 activity could be explained by the diet, lifestyle, and genetic factors studied.

  7. Cytochrome P450 1A2 (CYP1A2) activity and risk factors for breast cancer: a cross-sectional study

    PubMed Central

    Hong, Chi-Chen; Tang, Bing-Kou; Hammond, Geoffrey L; Tritchler, David; Yaffe, Martin; Boyd, Norman F

    2004-01-01

    Introduction Breast cancer risk may be determined by various genetic, metabolic, and lifestyle factors that alter sex hormone metabolism. Cytochrome P450 1A2 (CYP1A2) is responsible for the metabolism of estrogens and many exogenous compounds, including caffeine. Methods In a cross-sectional study of 146 premenopausal and 149 postmenopausal women, we examined the relationships between CYP1A2 activity and known or suspected risk factors for breast cancer. Blood levels of sex hormones, lipids, and growth factors were measured. In vivo CYP1A2 activity was assessed by measuring caffeine metabolites in urine. Stepwise and maximum R regression analyses were used to identify covariates related to CYP1A2 activity after adjustment for ethnicity. Results In both menopausal groups CYP1A2 activity was positively related to smoking and levels of sex hormone binding globulin. In premenopausal women, CYP1A2 activity was also positively related to insulin levels, caffeine intake, age, and plasma triglyceride levels, and negatively related with total cholesterol levels and body mass index. In postmenopausal women CYP1A2 activity was positively associated with insulin-like growth factor-1, and negatively associated with plasma triglyceride, high-density lipoprotein cholesterol, and age at menarche. Conclusion These results suggest that CYP1A2 activity is correlated with hormones, blood lipids, and lifestyle factors associated with breast cancer risk, although some of the observed associations were contrary to hypothesized directions and suggest that increased CYP1A2 function may be associated with increased risk for breast cancer. PMID:15217502

  8. Effects of Ziziphus jujuba fruit extracts on cytochrome P450 (CYP1A2) activity in rats.

    PubMed

    Jing, Xin-Yue; Peng, Yun-Ru; Wang, Xin-Min; Duan, Jin-Ao

    2015-08-01

    Drug-drug interactions have become a serious problem in the clinic, since plant-based medicines are extensively used. The present study investigated the effects of Ziziphus jujuba fruit (ZJ) extract on the pharmacokinetics of phenacetin, a typical substrate of a cytochrome P450 enzyme CYP 1A2, in rats. The rats were pretreated with the water extract (1.0 g · kg(-1)) or the ethanolic extract (3.6 g · kg(-1)) of ZJ for 10 days, and the pharmacokinetics of phenacetin was investigated after intravenous administration. In an in vitro assay, acetaminophen formation in the hepatic microsomes of ZJ-treated rats was investigated to assess CYP1A2 activity. Our results demonstrated that the treatment with the water and ethanolic extracts of ZJ decreased the plasma concentration of phenacetin and increased the plasma concentration of acetaminophen, resulting in a 43.2% and 15.5% reduction in the AUC0-120 of phenacetin, respectively, and a 53.2% and 64.9% increase in the AUC0-120 of acetaminophen, respectively after intravenous administration. The water or ethanolic extract of ZJ significantly increased the clearance of phenacetin and acetaminophen formation in hepatic microsomes. In conclusion, ZJ extracts displayed effects on the pharmacokinetics of phenacetin and increased the CYP1A2 activity in rats. Therefore, precaution on drug-drug interactions should be taken when ZJ is co-administered with drugs metabolized by CYP1A2, which may result in decreased concentrations of these drugs.

  9. Evidence from a population pharmacokinetics analysis for a major effect of CYP1A2 activity on inter- and intraindividual variations of clozapine clearance.

    PubMed

    Dailly, Eric; Urien, Saik; Chanut, Evelyne; Claudel, Bertrand; Guerra, Nathalie; Femandez, Christine; Jolliet, Pascale; Bourin, Michel

    2002-05-01

    Interindividual variations of clozapine clearance could be related to individual CYP1A2 activity. A population approach was used to investigate clozapine pharmacokinetics of multiple doses of clozapine in patients. Clozapine plasma concentrations were obtained in 23 patients from therapeutic drug monitoring (83 samples). CYP1A2 activity was estimated by the norclozapine/clozapine plasma levels ratio and data were processed by a nonlinear mixed-effect modelling method. Different covariates (age, body weight, height, CYP1A2 activity, daily dose of clozapine) were tested but CYP1A2 activity was the single parameter that improved significantly the predictive model. The best fit was obtained by integration of a linear relationship between clozapine clearance and CYP1A2 activity. The findings suggest that (i) CYP1A2 activity is a major factor that determines clozapine clearance and (ii) the norclozapine/clozapine ratio could constitute a valuable measure of the CYP1A2 activity. This ratio can be simply determined in the context of therapeutic drug monitoring and could explain the inter- and intraindividual variation of clozapine plasma levels.

  10. Thiomethylstilbenes as inhibitors of CYP1A1, CYP1A2 and CYP1B1 activities.

    PubMed

    Mikstacka, Renata; Baer-Dubowska, Wanda; Wieczorek, Marcin; Sobiak, Stanislaw

    2008-06-01

    Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a natural stilbene derivative occurring in grapes, peanuts and red wine. Its chemopreventive action has been established in studies on animal models. Recently, numerous classes of compounds with stilbene backbone have been investigated for their biological activity concerning cancer prevention; e. g. resveratrol methyl ethers appeared to be specific and potent inhibitors of cytochromes P450 (CYP) family 1 involved in the activation of procarcinogens. Since the replacement of the 4'-hydroxyl with a thiomethyl group is supposed to reduce toxicity of stilbene derivatives, the purpose of this study was the synthesis and evaluation of a series of 4-thiomethyl-trans-stilbene derivatives differing in a number and position of additional methoxy groups. Their inhibitory potency toward human recombinant CYPs: CYP1A1, CYP1A2 and CYP1B1 have been studied and compared with the effect of resveratrol and its analogues. Among compounds tested, 2-methoxy-4'-thiomethyl-trans-stilbene and 3-methoxy-4'-thiomethyl-trans-stilbene demonstrated the most potent and selective inhibitory effect on CYP1A1 and CYP1B1 activities. The results of our study indicate that modification of stilbene derivatives with thiomethyl group may influence the selectivity and inhibitory potency of these compounds toward P450 isozymes. Thus, it should be considered in developing new chemopreventive agents based on their mechanism of action.

  11. Breast Cancer Association with CYP1A2 Activity and Gene Polymorphisms--a Preliminary Case-control Study in Tunisia.

    PubMed

    Imene, Ayari; Maurice, Arnaud J; Arij, Mani; Sofia, Pavanello; Saad, Saguem

    2015-01-01

    The aim of the present study was to evaluate the relative contribution of CYP1A2 isoforms (-3860 G/A, -2467T/delT and -163C/A) in control subjects and breast cancer patients to the metabolism of caffeine in human liver. Restriction fragment length polymorphism analysis of PCR-amplified Fragments (PCR-RFLP) was used for the genotyping of CYP1A2 SNPs and HPLC allowed the phenotyping through the measurement of CYP1A2 activity using the 17X + 13X + 37X/137X urinary metabolite ratio (CMR) and plasma caffeine half life (T1/2). The CYP1A2 -3860A genotype was associated with a decreased risk of breast cancer. In contrast, distributions of the CYP1A2 -2467T/delT or -2467delT/delT and -163A/C or A/A genotypes among breast cancer patients and controls were similar. When the genotype and phenotype relationship was measured by comparing the mean CMR ratios and caffeine half life within the genotype groups between subjects and breast cancer patients, there were no significant differences except for -3860 A, most of them being homozygous for the -3860 G/G SNP and had a significant higher mean CMR ratio and half life than those with -3860 G/A (P=0.02). The results of this preliminary study show a significant association between CP1A2 -3860 G variant and CYP1A2 phenotype which must be confirmed by further large-size case-control studies. PMID:25921178

  12. Echinacea purpurea up-regulates CYP1A2, CYP3A4 and MDR1 gene expression by activation of pregnane X receptor pathway

    PubMed Central

    Awortwe, Charles; Manda, Vamshi K.; Avonto, Cristina; Khan, Shabana I.; Khan, Ikhlas A.; Walker, Larry A.; Bouic, Patrick J.; Rosenkranz, Bernd

    2015-01-01

    This study investigated the mechanism underlying Echinacea-mediated induction of CYP1A2, CYP3A4 and MDR1 in terms of human pregnane X receptor (PXR) activation. Crude extracts and fractions of Echinacea purpurea were tested for PXR activation in HepG2 cells by a reporter gene assay. Quantitative real-time PCR was carried out to determine their effects on CYP1A2 and CYP3A4 mRNA expressions. Capsules and fractions were risk ranked as high, intermediate and remote risk of drug-metabolizing enzymes induction based on EC50 values determined for respective CYPs. Fractions F1, F2 and capsule (2660) strongly activated PXR with 5-, 4- and 3.5-fold increase in activity, respectively. Echinacea preparations potentiated up-regulation of CYP1A2, CYP3A4 and MDR1 via PXR activation. Thus E. purpurea preparations cause herb–drug interaction by up-regulating CYP1A2, CYP3A4 and P-gp via PXR activation. PMID:25377539

  13. The extent and determinants of changes in CYP2D6 and CYP1A2 activities with therapeutic doses of sertraline.

    PubMed

    Ozdemir, V; Naranjo, C A; Herrmann, N; Shulman, R W; Sellers, E M; Reed, K; Kalow, W

    1998-02-01

    The extent of changes in CYP2D6 and CYP1A2 activities with higher therapeutic dosages (>50 mg/day) of sertraline is not well established in vivo. This study assessed the extent and determinants of changes in CYP2D6 and CYP1A2 isozyme activities after treatment with clinically relevant doses of sertraline. Patients and healthy volunteers aged 19 to 85 years (N = 21) were treated with sertraline for 5 to 55 days. The dosage of sertraline ranged from 25 to 150 mg/day (93.5+/-26.4 mg/day; mean +/- SD). All subjects had an extensive metabolizer phenotype for CYP2D6 and received a single oral dose of dextromethorphan (30 mg) and caffeine (100 mg) before and after sertraline treatment. The log O-demethylation ratio (ODMR) of dextromethorphan and the caffeine metabolic ratio (CMR) in overnight urine were used as in vivo indices of the CYP2D6 and CYP1A2 isozyme activities, respectively. Concurrent medications and lifestyle habits (e.g., smoking and diet) were monitored during the study. Baseline log ODMR (-2.33+/-0.45) but not CMR (5.1+/-1.9) (mean +/- SD) significantly changed after sertraline treatment (-2.19+/-0.62; 4.5+/-1.6, respectively) (p: ODMR = 0.04, CMR = 0.10). There was no significant effect of age, dose, duration of treatment, gender, sertraline and/or desmethylsertraline plasma concentration, subject type (patient or volunteer), and weight on the extent of changes in log ODMR or CMR (p > 0.05). In conclusion, sertraline treatment at a mean daily dosage of 94.0 mg did not significantly change CYP1A2 activity and resulted in a modest inhibition of CYP2D6 activity.

  14. Coffee and tea consumption, genotype-based CYP1A2 and NAT2 activity and colorectal cancer risk-results from the EPIC cohort study.

    PubMed

    Dik, Vincent K; Bueno-de-Mesquita, H B As; Van Oijen, Martijn G H; Siersema, Peter D; Uiterwaal, Cuno S P M; Van Gils, Carla H; Van Duijnhoven, Fränzel J B; Cauchi, Stéphane; Yengo, Loic; Froguel, Philippe; Overvad, Kim; Bech, Bodil H; Tjønneland, Anne; Olsen, Anja; Boutron-Ruault, Marie-Christine; Racine, Antoine; Fagherazzi, Guy; Kühn, Tilman; Campa, Daniele; Boeing, Heiner; Aleksandrova, Krasimira; Trichopoulou, Antonia; Peppa, Eleni; Oikonomou, Eleni; Palli, Domenico; Grioni, Sara; Vineis, Paolo; Tumino, Rosaria; Panico, Salvatore; Peeters, Petra H M; Weiderpass, Elisabete; Engeset, Dagrun; Braaten, Tonje; Dorronsoro, Miren; Chirlaque, María-Dolores; Sánchez, María-José; Barricarte, Aurelio; Zamora-Ros, Raul; Argüelles, Marcial; Jirström, Karin; Wallström, Peter; Nilsson, Lena M; Ljuslinder, Ingrid; Travis, Ruth C; Khaw, Kay-Tee; Wareham, Nick; Freisling, Heinz; Licaj, Idlir; Jenab, Mazda; Gunter, Marc J; Murphy, Neil; Romaguera-Bosch, Dora; Riboli, Elio

    2014-07-15

    Coffee and tea contain numerous antimutagenic and antioxidant components and high levels of caffeine that may protect against colorectal cancer (CRC). We investigated the association between coffee and tea consumption and CRC risk and studied potential effect modification by CYP1A2 and NAT2 genotypes, enzymes involved in the metabolization of caffeine. Data from 477,071 participants (70.2% female) of the European Investigation into Cancer and Nutrition (EPIC) cohort study were analyzed. At baseline (1992-2000) habitual (total, caffeinated and decaffeinated) coffee and tea consumption was assessed with dietary questionnaires. Cox proportional hazards models were used to estimate adjusted hazard ratio's (HR) and 95% confidence intervals (95% CI). Potential effect modification by genotype-based CYP1A2 and NAT2 activity was studied in a nested case-control set of 1,252 cases and 2,175 controls. After a median follow-up of 11.6 years, 4,234 participants developed CRC (mean age 64.7 ± 8.3 years). Total coffee consumption (high vs. non/low) was not associated with CRC risk (HR 1.06, 95% CI 0.95-1.18) or subsite cancers, and no significant associations were found for caffeinated (HR 1.10, 95% CI 0.97-1.26) and decaffeinated coffee (HR 0.96, 95% CI 0.84-1.11) and tea (HR 0.97, 95% CI 0.86-1.09). High coffee and tea consuming subjects with slow CYP1A2 or NAT2 activity had a similar CRC risk compared to non/low coffee and tea consuming subjects with a fast CYP1A2 or NAT2 activity, which suggests that caffeine metabolism does not affect the link between coffee and tea consumption and CRC risk. This study shows that coffee and tea consumption is not likely to be associated with overall CRC. PMID:24318358

  15. INHIBITION OF HUMAN AND RAT CYP1A2 BY TCDD AND DIOXIN-LIKE CHEMICALS

    EPA Science Inventory

    Dioxins have been shown to bind and induce rodent CYP1A2, producing a dose-dependent hepatic sequestration in vivo. The induction of CYP1A2 activity has been used as a noninvasive biomarker for human exposure to dioxins; while there is a consistent relationship between exposure ...

  16. 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.

  17. Association between common CYP1A2 polymorphisms and theophylline metabolism in non-smoking healthy volunteers.

    PubMed

    Wang, Liqing; Hu, Zheyi; Deng, Xun; Wang, Yong; Zhang, Zhongyi; Cheng, Ze-Neng

    2013-04-01

    This study was designed to investigate the impact of cytochrome P450 (CYP) 1A2 polymorphisms on theophylline metabolism in a non-smoking healthy male Chinese population. Four polymorphisms CYP1A2 1C (G-3860A), G-3113A, CYP1A2 1F (C-163A) and CYP1A2 1B (C-5347T) were screened in 238 unrelated male volunteers. Then, a single oral 200-mg dose of theophylline was administered to 37 volunteers, who were selected from 238 volunteers based on the CYP1A2 genotype. CYP1A2 activities were evaluated by plasma 1,7-dimethylxanthine/caffeine ratios (17X/137X) after administration of 100-mg caffeine. The plasma concentrations of theophylline, 17X and 137X were determined by high-performance liquid chromatography. The activity of CYP1A2 was lower in volunteers with the -3113 AA genotype compared with those with the -3113 AG genotype (0.35 ± 0.04 versus 0.48 ± 0.07, p = 0.016) or the -3113 GG genotype (0.35 ± 0.04 versus 0.58 ± 0.22, p = 0.037). CYP1A2 1F polymorphisms were associated with increased CYP1A2 activity in volunteers with -3860G/-3113G/5347C homozygosity (0.66 ± 0.24 versus 0.46 ± 0.05, p = 0.034). However, theophylline metabolism showed no difference among volunteers carrying different haplotype pairs. CYP1A2 genetic polymorphisms influenced CYP1A2 enzyme activity as measured by caffeine, but CYP1A2 gene polymorphisms appeared to have limited influence on theophylline metabolism in our study.

  18. Effects of Panax notoginseng saponins on the activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in rats in vivo.

    PubMed

    Liu, Rui; Qin, Mengnan; Hang, Pengzhou; Liu, Yan; Zhang, Zhiren; Liu, Gaofeng

    2012-08-01

    The aim of this study was to assess the influence of the Panax notoginseng saponins (PNS) on the activities of the drug-metabolizing enzymes cytochrome P450 (CYP450) 1A2, 2 C9, 2D6 and 3A4 in rats. The activities of CYP1A2, 2 C9, 2D6 and 3A4 were measured using specific probe drugs. After pretreatment for 1 week with PNS or physiological saline (control group), probe drugs caffeine (10 mg/kg; CYP1A2 activity), tolbutamide (15 mg/kg; CYP2C9 activity), metoprolol (20 mg/kg; CYP2D6 activity) and dapsone (10 mg/kg; CYP3A4 activity) were administered to rats by intraperitoneal injection. The blood was then collected at different times for ultra performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) analysis. The data showed that PNS exhibited an induction effect on CYP1A2 by decreasing caffeine C(max) (36.3%, p < 0.01) and AUC(0-∞) (22.77%, p < 0.05) and increasing CL/F (27.03%, p < 0.05) compared with those of the control group. Western blot analysis was used to detect the effect of PNS on the protein level of CYP1A2, and the results showed that PNS could upregulate the protein expression of CYP1A2. However, no significant changes in CYP2C9, 2D6 or 3A4 activities were observed. In conclusion, the results indicate that PNS could induce CYP1A2, which may affect the disposition of medicines primarily dependent on the CYP1A2 pathway. Our work may be the basis of related herb-drug interactions in the clinic.

  19. 2,3,7, 8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)-MEDIATED OXIDATIVE STRESS IN FEMALE CYP1A-2 KNOCKOUT (CYP1A2-/-) MICE

    EPA Science Inventory

    2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD)-Mediated Oxidative Stress in Female CYP1A2 Knockout (CYP1A2-/-) Mice

    Deborah Burgin1, Janet Diliberto2, Linda Birnbaum2
    1UNC Toxicology; 2USEPA/ORD/NHEERL, RTP, NC

    Most of the effects due to TCDD exposure are mediated via...

  20. Caffeine intake and CYP1A2 variants associated with high caffeine intake protect non-smokers from hypertension.

    PubMed

    Guessous, Idris; Dobrinas, Maria; Kutalik, Zoltán; Pruijm, Menno; Ehret, Georg; Maillard, Marc; Bergmann, Sven; Beckmann, Jacques S; Cusi, Daniele; Rizzi, Federica; Cappuccio, Franco; Cornuz, Jacques; Paccaud, Fred; Mooser, Vincent; Gaspoz, Jean-Michel; Waeber, Gérard; Burnier, Michel; Vollenweider, Peter; Eap, Chin B; Bochud, Murielle

    2012-07-15

    The 15q24.1 locus, including CYP1A2, is associated with blood pressure (BP). The CYP1A2 rs762551 C allele is associated with lower CYP1A2 enzyme activity. CYP1A2 metabolizes caffeine and is induced by smoking. The association of caffeine consumption with hypertension remains controversial. We explored the effects of CYP1A2 variants and CYP1A2 enzyme activity on BP, focusing on caffeine as the potential mediator of CYP1A2 effects. Four observational (n = 16 719) and one quasi-experimental studies (n = 106) including European adults were conducted. Outcome measures were BP, caffeine intake, CYP1A2 activity and polymorphisms rs762551, rs1133323 and rs1378942. CYP1A2 variants were associated with hypertension in non-smokers, but not in smokers (CYP1A2-smoking interaction P = 0.01). Odds ratios (95% CIs) for hypertension for rs762551 CC, CA and AA genotypes were 1 (reference), 0.78 (0.59-1.02) and 0.66 (0.50-0.86), respectively, P = 0.004. Results were similar for the other variants. Higher CYP1A2 activity was linearly associated with lower BP after quitting smoking (P = 0.049 and P = 0.02 for systolic and diastolic BP, respectively), but not while smoking. In non-smokers, the CYP1A2 variants were associated with higher reported caffeine intake, which in turn was associated with lower odds of hypertension and lower BP (P = 0.01). In Mendelian randomization analyses using rs1133323 as instrument, each cup of caffeinated beverage was negatively associated with systolic BP [-9.57 (-16.22, -2.91) mmHg]. The associations of CYP1A2 variants with BP were modified by reported caffeine intake. These observational and quasi-experimental results strongly support a causal role of CYP1A2 in BP control via caffeine intake.

  1. Polymorphisms in the cytochrome P450 CYP1A2 gene (CYP1A2) in colorectal cancer patients and controls: allele frequencies, linkage disequilibrium and influence on caffeine metabolism

    PubMed Central

    Sachse, Christoph; Bhambra, Upinder; Smith, Gillian; Lightfoot, Tracy J; Barrett, Jennifer H; Scollay, Jenna; Garner, R Colin; Boobis, Alan R; Wolf, C Roland; Gooderham, Nigel J

    2003-01-01

    Aim Several single nucleotide polymorphisms (SNPs) of the cytochrome P450 enzyme 1A2 gene (CYP1A2) have been reported. Here, frequencies, linkage disequilibrium and phenotypic consequences of six SNPs are described. Methods From genomic DNA, 114 British Caucasians (49 colorectal cancer cases and 65 controls) were genotyped for the CYP1A2 polymorphisms −3858G→A (allele CYP1A2*1C), −2464T→delT (CYP1A2*1D), −740T→G (CYP1A2*1E and *1G), −164A→C (CYP1A2*1F), 63C→G (CYP1A2*2), and 1545T→C (alleles CYP1A2*1B, *1G, *1H and *3), using polymerase chain reaction–restriction fragment length polymorphism assays. All patients and controls were phenotyped for CYP1A2 by h.p.l.c. analysis of urinary caffeine metabolites. Results In 114 samples, the most frequent CYP1A2 SNPs were 1545T→C (38.2% of tested chromosomes), −164A→C (CYP1A2*1F, 33.3%) and −2464T→delT (CYP1A2*1D, 4.82%). The SNPs were in linkage disequilibrium: the most frequent constellations were found to be −3858G/−2464T/−740T/−164A/63C/1545T (61.8%), −3858G/−2464T/−740T/−164C/63C/1545C (33.3%), and −3858G/−2464delT/−740T/−164A/63C/1545C (3.51%), with no significant frequency differences between cases and controls. In the phenotype analysis, lower caffeine metabolic ratios were detected in cases than in controls. This was significant in smokers (n = 14, P = 0.020), and in a subgroup of 15 matched case-control pairs (P = 0.007), but it was not significant in nonsmokers (n = 100, P = 0.39). There was no detectable association between CYP1A2 genotype and caffeine phenotype. Conclusions (i) CYP1A2 polymorphisms are in linkage disequilibrium. Therefore, only −164A→C (CYP1A2*1F) and −2464T→delT (CYP1A2*1D) need to be analysed in the routine assessment of CYP1A2 genotype; (ii) in vivo CYP1A2 activity is lower in colorectal cancer patients than in controls, and (iii) CYP1A2 genotype had no effect on phenotype (based on the caffeine metabolite ratio). However, this

  2. Enoxacin is an inducer of CYP1A2 in rat liver.

    PubMed

    Schulz, T G; Stahlmann, R; Edwards, R J; Debri, K; Davies, D S; Neubert, D

    1995-10-26

    The induction of cytochrome P450 by enoxacin, ciprofloxacin, and ofloxacin was investigated in female Wistar rats. Animals were treated orally with daily doses ranging from 10 to 400 mg enoxacin per kg body wt, 400 mg ciprofloxacin, or 400 mg ofloxacin per kg body wt for up to 7 days. Activities of methoxyresorufin O-demethylase (MROD) and ethoxyresorufin O-deethylase (EROD) were determined fluorimetrically in hepatic microsomes. MROD activity was increased 2.6-fold after treatment with 100 mg enoxacin per kg body wt for 7 days. Lower doses of enoxacin did not induce MROD activity significantly. Antipeptide antibodies directed specifically against different rat cytochrome P450 enzymes demonstrated that CYP1A2, but not CYP1A1, was induced in rats treated with enoxacin. After ciprofloxacin or ofloxacin treatment, no induction of MROD or EROD activity was observed. Neither ciprofloxacin nor ofloxacin caused any change in CYP1A1 or CYP1A2 apoprotein levels. Further investigations with antipeptide antibodies showed that there was no induction of CYP2B1, CYP2B2, CYP2E1, CYP3A1, CYP3A2, CYP4A1, or CYP4A2 following treatment with enoxacin, ciprofloxacin, or ofloxacin. It is concluded that enoxacin, but not ciprofloxacin or ofloxacin, is an inducer of CYP1A2 in rat liver.

  3. Evidence for the involvement of CYP1A2 in the metabolism of bromodichloromethane in rat liver.

    PubMed

    Allis, John W; Anderson, Brian P; Zhao, Guangyu; Ross, Tracey M; Pegram, Rex A

    2002-07-01

    Bromodichloromethane (BDCM) is a drinking water disinfectant by-product that has been implicated in liver, kidney and intestinal cancers in rodents and in intestinal tumors and low birth weight effects in humans. BDCM is also hepatotoxic and requires metabolic activation for both toxicity and carcinogenicity. We have recently reported that CYP1A2 may participate in that metabolism and we now report experiments to support that implication. Induction of CYP1A2 in male F344 rats without inducing CYP2E1 or CYP2B1/2, using TCDD, increased the hepatotoxicity of BDCM when compared to earlier work conducted under similar protocols. Inhibition of CYP1A2, with isosafrole, reduced the metabolism and toxicity of BDCM in the previously induced rats. In addition, specific activities and Western blots for these CYP isoenzymes were measured 24 h after exposure. Activity data show that only CYP1A2 was inhibited by isosafrole; isosafrole forms a complex with CYP1A2 that persists for more than 24 h. Western blot results generally agree with the activity data except that isosafrole induced the protein for all isoenzymes measured. A physiologically based pharmacokinetic model, developed previously, estimated that BDCM metabolism was complete about 7 h after gavage dosing. It is noteworthy that the reduction in CYP1A2 activity was still measurable despite the production of additional CYP1A2 protein during the period of approximately 18 h after BDCM metabolism was complete. These results demonstrate that CYP1A2 does metabolize BDCM and does contribute to hepatotoxicity under certain conditions.

  4. CYP1A2 polymorphism and theophylline clearance in Korean non-smoking asthmatics

    PubMed Central

    Yim, Eun-Young; Kang, Hye-Ryun; Jung, Jae-Woo; Sohn, Seong-Wook

    2013-01-01

    Background Theophylline is mainly metabolized by cytochrome P450 (CYP) 1A2 and CYP2E1 which show inter-individual variations. However, the underlying mechanism remains unknown in humans. We investigated the relationship between differences in theophylline clearance and genetic polymorphisms in the CYP1A2 and CYP2E1 gene in 89 Korean asthmatic patients. Methods Polymerase chain reaction (PCR) was performed on the 5'-flanking region of those genes. PCR products were directly sequenced and confirmed using the SNaP shot method. We determined whether the detected SNPs affected gene transcription using electrophoretic mobility shift assay (EMSA). Theophylline clearance (mL/kg/h) was assessed by using a Bayesian approach. Results Genetic polymorphisms were identified at 7 sites in the CYP1A2 gene and at 10 sites in the CYP2E1. Among them, subjects with genotypes (GA+AA) of the -3860G>A polymorphism were found to show higher theophylline clearance than those with genotypes GG (29.11 ± 0.91 mL/kg/h vs. 26.12 ± 0.80 mL/kg/h, p = 0.014). This polymorphic site was revealed to be a protein binding site by conducting EMSA on nuclear hepatocyte extracts. Conclusion In conclusion, increased theophylline clearance was significantly related to the -3860G>A polymorphism, which could be associated with increased CYP1A2 inducibility in Korean non-smoking asthmatics. PMID:24260728

  5. COMPARING ENVIRONMENTALLY RELEVANT PCBS TO TCDD IN CYP1A2 NULL AND WILDTYPE MICE

    EPA Science Inventory


    The role of CYP1A2 on the interactions of TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin, dioxin), dioxin-like (DL) and non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) was compared in multiple responses of different laboratory-defined mixtures, based on mass ratios found in...

  6. Contribution of the activities of CYP3A, CYP2D6, CYP1A2 and other potential covariates to the disposition of methadone in patients undergoing methadone maintenance treatment

    PubMed Central

    Shiran, Mohammad-Reza; Lennard, Martin S; Iqbal, Mohammad-Zafar; Lagundoye, Oldwale; Seivewright, Nicholas; Tucker, Geoffrey T; Rostami-Hodjegan, Amin

    2009-01-01

    AIMS To investigate the influence of different cytochrome P450 (CYP) activities and other potential covariates on the disposition of methadone in patients on methadone maintenance therapy (MMT). METHODS Eighty-eight patients (58 male; 21–55 years; 84 White) on MMT were studied. CYP2D6 activity [3 h plasma metabolic ratio of dextromethorphan (DEX) to dextrorphan (DOR)] was determined in 44 patients (29 male; 24–55 years), CYP1A2 activity (salivary caffeine elimination half-life) in 44 patients (21 male; 24–55 years) and CYP3A activity (oral clearance of midazolam) in 49 patients (33 male; 23–55 years). Data on all three CYPs were obtained from 32 subjects. Total plasma concentrations of (RS)-methadone and total and unbound plasma concentrations of both enantiomers were measured by LC/MS. Population pharmacokinetics and subsequent multiple regression analysis were used to calculate methadone oral clearance and to identify its covariates. RESULTS Between 61 and 68% of the overall variation in total plasma trough concentrations of (RS)-, (R)- and (S)-methadone was explained by methadone dose, duration of addiction before starting MMT, CYP3A activity and illicit morphine use. CYP3A activity explained 22, 16, 15 and 23% of the variation in unbound (R)-, unbound (S)-, total (RS)- and total (S)-methadone clearances, respectively. Neither CYP2D6 nor CYP1A2 activity was related to methadone disposition. CONCLUSIONS CYP3A activity has a modest influence on methadone disposition. Inhibitors and inducers of this enzyme should be monitored in patients taking methadone. PMID:19133059

  7. Identification and characterization of reactive metabolites in myristicin-mediated mechanism-based inhibition of CYP1A2.

    PubMed

    Yang, Ai-Hong; He, Xin; Chen, Jun-Xiu; He, Li-Na; Jin, Chun-Huan; Wang, Li-Li; Zhang, Fang-Liang; An, Li-Jun

    2015-07-25

    Myristicin belongs to the methylenedioxyphenyl or allyl-benzene family of compounds, which are found widely in plants of the Umbelliferae family, such as parsley and carrot. Myristicin is also the major active component in the essential oils of mace and nutmeg. However, this compound can cause adverse reactions, particularly when taken inappropriately or in overdoses. One important source of toxicity of natural products arises from their metabolic biotransformations into reactive metabolites. Myristicin contains a methylenedioxyphenyl substructure, and this specific structural feature may allow compounds to cause a mechanism-based inhibition of cytochrome P450 enzymes and produce reactive metabolites. Therefore, the aim of this work was to identify whether the role of myristicin in CYP enzyme inhibition is mechanism-based inhibition and to gain further information regarding the structure of the resulting reactive metabolites. CYP cocktail assays showed that myristicin most significantly inhibits CYP1A2 among five CYP enzymes (CYP1A2, CYP2D6, CYP2E1, CYP3A4 and CYP2C19) from human liver microsomes. The 3.21-fold IC50 shift value of CYP1A2 indicates that myristicin may be a mechanism-based inhibitor of CYP1A2. Next, reduced glutathione was shown to block the inhibition of CYP1A2, indicating that myristicin utilized a mechanism-based inhibition. Phase I metabolism assays identified two metabolites, 5-allyl-1-methoxy-2,3-dihydroxybenzene (M1) and 1'-hydroxymyristicin or 2',3'-epoxy-myristicin (M2). Reduced glutathione capturing assays captured the glutathione-M1 adduct, and the reactive metabolites were identified using UPLC-MS(2) as a quinone and its tautomer. Thus, it was concluded that myristicin is a mechanism-based inhibitor of CYP1A2, and the reactive metabolites are quinone tautomers. Additionally, the cleavage process of the glutathione-M1 adduct was analyzed in further detail. This study provides additional information on the metabolic mechanism of myristicin

  8. Inhibition of heme oxygenase-1 partially reverses the arsenite-mediated decrease of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 catalytic activity in isolated rat hepatocytes.

    PubMed

    Anwar-Mohamed, Anwar; Klotz, Lars-Oliver; El-Kadi, Ayman O S

    2012-03-01

    Heme oxygenase (HO-1), the rate-limiting enzyme in the physiological breakdown of heme, is ubiquitous, and its expression can be increased by arsenite [As(III)], and similar other stimuli that induce cellular oxidative stress. Interestingly, it has been shown that the As(III)-induced HO-1 is inversely correlated with a decrease in cytochromes P450 (P450s) activity; however, the direct role for HO-1 in the inhibition of P450 enzymes remains unknown. Our results showed that As(III) at a concentration of 5 μM decreased the constitutive and inducible expression of CYP1A1, CYP1A2, CYP3A23, and CYP3A2 at the mRNA, protein, and catalytic activity levels. Moreover, As(III) decreased the nuclear accumulation of aryl hydrocarbon receptor (AhR) and pregnane X receptor without increasing their degradation. As(III) also increased the binding of cytosolic AhR to heat shock protein 90 and hepatitis B virus X-associated protein 2. In the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin as an inducer for CYP1A and rifampin as an inducer for CYP3A, As(III) decreased the enzymatic activity of the four P450s more than it decreased their mRNA or protein expression levels. It is noteworthy that treatment with the competitive HO-1 inhibitor, tin-mesoporphyrin, or supplementing external heme partially reversed the As(III)-mediated decrease in activities of the four P450s. In conclusion, the current study provides the first evidence that As(III) decreases CYP1A1, CYP1A2, CYP3A23, and CYP3A2 expression in freshly isolated rat primary hepatocytes. Furthermore, inhibiting the As(III)-mediated induction of HO-1 partially restores the enzymatic activity of these P450s that was initially decreased by As(III), confirming the direct role of HO-1 in the inhibition of P450s.

  9. Effects of mexiletine, a CYP1A2 inhibitor, on tizanidine pharmacokinetics and pharmacodynamics.

    PubMed

    Momo, Kenji; Homma, Masato; Osaka, Yoshiko; Inomata, Shin-ichi; Tanaka, Makoto; Kohda, Yukinao

    2010-03-01

    The aim of this study was to determine whether mexiletine, a CYP1A2 inhibitor, altered the pharmacokinetics and pharmacodynamics of tizanidine. The pharmacokinetics of tizanidine were examined in an open-label study in 12 healthy participants after a single dose of tizanidine (2 mg) with and without mexiletine coadministration (50 mg, 3 times as a pretreatment for a day and 2 times on the study day). Compared with tizanidine alone, mexiletine coadministration increased the peak plasma concentration (1.8 +/- 0.8 vs 5.3 +/- 1.8 ng/mL), area under the curve (4.5 +/- 2.2 vs 15.4 +/- 6.5 ng x h/mL), and the half-life (1.3 +/- 0.2 vs 1.8 +/- 0.7 h) of tizanidine, respectively (P < .05). Reduction in systolic blood pressure (-10 +/- 8 vs -24 +/- 7 mm Hg) and diastolic blood pressure (-10 +/- 7 vs -18 +/- 8 mm Hg) after tizanidine administration was also significantly enhanced by coadministration of mexiletine (P < .01). Of the 15 patients treated with tizanidine and mexiletine, 4 suffered tizanidine-induced adverse effects such as drowsiness and dry mouth in the retrospective survey. Present results suggested that coadministration of mexiletine increased blood tizanidine concentrations and enhanced tizanidine pharmacodynamics in terms of reduction in blood pressure and adverse symptoms.

  10. Measurement of human CYP1A2 induction by inhalation exposure to benzo(a)pyrene based on in vivo isotope breath method.

    PubMed

    Duan, Xiaoli; Shen, Guofeng; Yang, Hongbiao; Lambert, George; Wei, Fusheng; Zhang, Junfeng Jim

    2016-01-01

    Cytochrome P450 1A2 (CYP1A2) is an enzyme involved in the metabolic activation of certain carcinogens, and inducible by toxic substrates. To date, few studies have investigated in vivo CYP1A2 induction in humans and its relationship to polycylic aromatic hydrocarbons (PAHs) like benzo(a)pyrene (BaP). Non-smoking healthy male coke-oven workers (n = 30) were recruited as 'exposure' group, and non-smoking healthy office workers in the same city (n = 10) were selected as 'control' group, to test whether high inhalation exposure to PAHs can induce CYP1A2 activity in human livers. Significantly higher inhalation exposure of PAHs were found among the exposure group compared to the control. Inhalation BaP exposure concentration in the exposure group was more than 30 times higher than the control group (p < 0.001). However, the exposure group did not exhale significant higher levels of (13)CO2/(12)CO2 in breath samples (p = 0.81), and no significant relationship was found between the inhaled BaP concentration and the (13)CO2/(12)CO2 ratio (p = 0.91). A significant association was found between the (13)CO2/(12)CO2 exhalation and dietary BaP intake level. Hepatic CYP1A2 activity/induction level was not effected by inhaled BaP but was altered by ingestion of BaP.

  11. Measurement of human CYP1A2 induction by inhalation exposure to benzo(a)pyrene based on in vivo isotope breath method.

    PubMed

    Duan, Xiaoli; Shen, Guofeng; Yang, Hongbiao; Lambert, George; Wei, Fusheng; Zhang, Junfeng Jim

    2016-01-01

    Cytochrome P450 1A2 (CYP1A2) is an enzyme involved in the metabolic activation of certain carcinogens, and inducible by toxic substrates. To date, few studies have investigated in vivo CYP1A2 induction in humans and its relationship to polycylic aromatic hydrocarbons (PAHs) like benzo(a)pyrene (BaP). Non-smoking healthy male coke-oven workers (n = 30) were recruited as 'exposure' group, and non-smoking healthy office workers in the same city (n = 10) were selected as 'control' group, to test whether high inhalation exposure to PAHs can induce CYP1A2 activity in human livers. Significantly higher inhalation exposure of PAHs were found among the exposure group compared to the control. Inhalation BaP exposure concentration in the exposure group was more than 30 times higher than the control group (p < 0.001). However, the exposure group did not exhale significant higher levels of (13)CO2/(12)CO2 in breath samples (p = 0.81), and no significant relationship was found between the inhaled BaP concentration and the (13)CO2/(12)CO2 ratio (p = 0.91). A significant association was found between the (13)CO2/(12)CO2 exhalation and dietary BaP intake level. Hepatic CYP1A2 activity/induction level was not effected by inhaled BaP but was altered by ingestion of BaP. PMID:26552516

  12. Design synthesis and evaluation of the inhibitory selectivity of novel trans-resveratrol analogues on human recombinant CYP1A1 CYP1A2 and CYP1B1

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A series of trans-stilbene derivatives containing 4’-thiomethyl substituent were synthesized and evaluated for inhibitory activities on human recombinant cytochrome P450(s): CYP1A1, CYP1A2, and CYP1B1. CYP1A2-related metabolism of stilbene derivatives was estimated by using NADPH oxidation assay. A...

  13. Cytochrome P450 expression system for high-throughput real-time detection of genotoxicity: Application to the study of human CYP1A2 variants.

    PubMed

    Palma, Bernardo Brito; Moutinho, Daniela; Urban, Philippe; Rueff, José; Kranendonk, Michel

    2016-08-01

    Individual variations in cytochrome P450-mediated metabolism are believed to contribute to individual susceptibility to chemical carcinogenesis. CYP1A2 is one of the major forms of cytochrome P450 involved in drug metabolism and bioactivation of carcinogens. We have applied a recently developed high-throughput Salmonella typhimurium TA1535 system for detection of DNA damaging agents to the study of CYP1A2 polymorphisms. Non-synonymous variants T83M [CYP1A2*9], S212C [CYP1A2*12], S298R [part of CYP1A2*21], G299S [CYP1A2*13], I314V [no allele designation], I386F [CYP1A2*4], C406Y [CYP1A2*5] and R456H [CYP1A2*8] were examined. The cDNAs for each of these variants and the wild-type were co-expressed with human NADPH cytochrome P450 oxidoreductase in the TA1535-based system. The bioactivation capacity of these CYP1A2 variants was investigated using three CYP1A2-dependent pro-mutagens, 1-aminopyrene (1AP), 2-aminoanthracene (2AA), and 2-amino-3-methylimidazo(4,5-f)quinoline (IQ). All CYP1A2 variants except R456H, T83M, and I386F gave positive responses with all three compounds. Variant R456H generated no detectable holoenzyme and no detectable response for any of the compounds; I386F did not bioactivate IQ; T83M did not bioactivate 1AP. Multivariate analysis indicated variant T83M to be substantially altered in catalytic properties when compared with wild-type CYP1A2; variants G299S and I386F are slightly but significantly different. These results corroborate our previous studies, indicating the effectiveness of this new high-throughput system, not only for examining the effect of CYP1A2 polymorphisms on pro-mutagen bioactivation, but also for obtaining insights on CYP1A2 function at the mechanistic level. PMID:27476332

  14. A simple chromatographic method for determining norfloxacin and enoxacin in pharmacokinetic study assessing CYP1A2 inhibition.

    PubMed

    Kobayashi, Toshimi; Homma, Masato; Momo, Kenji; Kobayashi, Daisuke; Kohda, Yukinao

    2011-04-01

    We developed a simple assay method for the determination of serum and urine norfloxacin and enoxacin using reversed-phase high-performance liquid chromatography and perchloric acid precipitation for sample pre-treatment. Optimized conditions can permit detection of norfloxacin and enoxacin in the same chromatogram, so either compound can be used as an internal standard for another determinant. Supernatants of the precipitated samples were analyzed by the octadecylsilyl silica-gel column under ambient temperature and an ultraviolet wavelength of 272  nm. A mobile phase solvent consisting of 20 mm sodium dihydrogenphosphate (pH 3.0) and acetonitrile (85:15, v/v) was pumped at a flow rate of 1.0 mL/min. The calibration curves for norfloxacin and enoxacin at a concentration of 62.5-1000 ng/mL for serum and 250-4000 ng/mL for urine were linear (r > 0.9997). The recoveries of norfloxacin and enoxacin from serum and urine were >94% with the coefficient of variations (CV) <5%. The CVs for intra- and inter-day assay of norfloxacin and enoxacin were <4.2 and <5.5%, respectively. This method can be applied to the pharmacokinetic study of norfloxacin and enoxacin after repeated administration to assess changes in CYP1A2 activity in healthy subjects.

  15. The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains Is Governed by Their N-terminal and Internal Protein Regions.

    PubMed

    Park, Ji Won; Reed, James R; Backes, Wayne L

    2015-12-01

    In cellular membranes, different lipid species are heterogeneously distributed forming domains with different characteristics. Ordered domains are tightly packed with cholesterol, sphingomyelin, and saturated fatty acids, whereas disordered domains contain high levels of unsaturated fatty acids. Our laboratory has shown that membrane heterogeneity affects the organization of cytochrome P450s and their cognate redox partner, the cytochrome P450 reductase (CPR). Despite the high degree of sequence similarity, CYP1A1 was found to localize to disordered regions, whereas CYP1A2 resided in ordered domains. We hypothesized that regions of amino acid sequence variability may contain signal motifs that direct CYP1A proteins into ordered or disordered domains. Thus, chimeric constructs of CYP1A1 and CYP1A2 were created, and their localization was tested in HEK293T cells. CYP1A2, containing the N-terminal regions from CYP1A1, no longer localized in ordered domains, whereas the N terminus of CYP1A2 partially directed CYP1A1 into ordered regions. In addition, intact CYP1A2 containing a 206-302-residue peptide segment of CYP1A1 had less affinity to bind to ordered microdomains. After expression, the catalytic activity of CYP1A2 was higher than that of the CYP1A1-CYP1A2 chimera containing the N-terminal end of CYP1A1 with subsaturating CPR concentrations, but it was approximately equal with excess CPR suggesting that the localization of the CYP1A enzyme in ordered domains favored its interaction with CPR. These data demonstrate that both the N-terminal end and an internal region of CYP1A2 play roles in targeting CYP1A2 to ordered domains, and domain localization may influence P450 function under conditions that resemble those found in vivo. PMID:26468279

  16. The Localization of Cytochrome P450s CYP1A1 and CYP1A2 into Different Lipid Microdomains Is Governed by Their N-terminal and Internal Protein Regions.

    PubMed

    Park, Ji Won; Reed, James R; Backes, Wayne L

    2015-12-01

    In cellular membranes, different lipid species are heterogeneously distributed forming domains with different characteristics. Ordered domains are tightly packed with cholesterol, sphingomyelin, and saturated fatty acids, whereas disordered domains contain high levels of unsaturated fatty acids. Our laboratory has shown that membrane heterogeneity affects the organization of cytochrome P450s and their cognate redox partner, the cytochrome P450 reductase (CPR). Despite the high degree of sequence similarity, CYP1A1 was found to localize to disordered regions, whereas CYP1A2 resided in ordered domains. We hypothesized that regions of amino acid sequence variability may contain signal motifs that direct CYP1A proteins into ordered or disordered domains. Thus, chimeric constructs of CYP1A1 and CYP1A2 were created, and their localization was tested in HEK293T cells. CYP1A2, containing the N-terminal regions from CYP1A1, no longer localized in ordered domains, whereas the N terminus of CYP1A2 partially directed CYP1A1 into ordered regions. In addition, intact CYP1A2 containing a 206-302-residue peptide segment of CYP1A1 had less affinity to bind to ordered microdomains. After expression, the catalytic activity of CYP1A2 was higher than that of the CYP1A1-CYP1A2 chimera containing the N-terminal end of CYP1A1 with subsaturating CPR concentrations, but it was approximately equal with excess CPR suggesting that the localization of the CYP1A enzyme in ordered domains favored its interaction with CPR. These data demonstrate that both the N-terminal end and an internal region of CYP1A2 play roles in targeting CYP1A2 to ordered domains, and domain localization may influence P450 function under conditions that resemble those found in vivo.

  17. Inhibitory effect of grapefruit juice and its bitter principal, naringenin, on CYP1A2 dependent metabolism of caffeine in man.

    PubMed Central

    Fuhr, U; Klittich, K; Staib, A H

    1993-01-01

    1. The effects of grapefruit juice and naringenin on the activity of the human cytochrome P450 isoform CYP1A2 were evaluated using caffeine as a probe substrate. 2. In vitro naringin was a potent competitive inhibitor of caffeine 3-demethylation by human liver microsomes (Ki = 7-29 microM). 3. In vivo grapefruit juice (1.2 l day-1 containing 0.5 g l-1 naringin, the glycone form of naringenin) decreased the oral clearance of caffeine by 23% (95% CI: 7%-30%) and prolonged its half-life by 31% (95% CI: 20%-44%) (n = 12). 4. We conclude that grapefruit juice and naringenin inhibit CYP1A2 activity in man. However, the small effect on caffeine clearance in vivo suggests that in general the ingestion of grapefruit juice should not cause clinically significant inhibition of the metabolism of other drugs that are substrates of CYPIA2. PMID:8485024

  18. Development and validation of a reversed-phase HPLC method for CYP1A2 phenotyping by use of a caffeine metabolite ratio in saliva.

    PubMed

    Begas, Elias; Kouvaras, Evangelos; Tsakalof, Andreas K; Bounitsi, Maria; Asprodini, Eftihia Konstadinos

    2015-11-01

    CYP1A2 is important for metabolizing various clinically used drugs. Phenotyping of CYP1A2 may prove helpful for drug individualization therapy. Several HPLC methods have been developed for quantification of caffeine metabolites in plasma and urine. Aim of the present study was to develop a valid and simple HPLC method for evaluating CYP1A2 activity during exposure in xenobiotics by the use of human saliva. Caffeine and paraxanthine were isolated from saliva by liquid-liquid extraction (chlorophorm/isopropanol 85/15v/v). Extracts were analyzed by reversed-phase HPLC on a C18 column with mobile phase 0.1% acetic acid/methanol/acetonitrile (80/20/2 v/v) and detected at 273nm. Caffeine and paraxanthine elution times were <13min with no interferences from impurities or caffeine metabolites. Detector response was linear (0.10-8.00µg/ml, R(2) >0.99), recovery was >93% and bias <4.47%. Intra- and inter-day precision was <5.14% (n=6). The limit of quantitation was 0.10µg/ml and the limit of detection was 0.018±0.002µg/mL for paraxanthine and 0.032±0.002µg/ml for caffeine. Paraxanthine/caffeine ratio of 34 healthy volunteers was significantly higher in smokers (p<0.001). Saliva paraxanthine/caffeine ratios and urine metabolite ratios were highly correlated (r=0.85, p<0.001). The method can be used for the monitoring of CYP1A2 activity in clinical practice and in studies relevant to exposure to environmental and pharmacological xenobiotics.

  19. Development and validation of a reversed-phase HPLC method for CYP1A2 phenotyping by use of a caffeine metabolite ratio in saliva.

    PubMed

    Begas, Elias; Kouvaras, Evangelos; Tsakalof, Andreas K; Bounitsi, Maria; Asprodini, Eftihia Konstadinos

    2015-11-01

    CYP1A2 is important for metabolizing various clinically used drugs. Phenotyping of CYP1A2 may prove helpful for drug individualization therapy. Several HPLC methods have been developed for quantification of caffeine metabolites in plasma and urine. Aim of the present study was to develop a valid and simple HPLC method for evaluating CYP1A2 activity during exposure in xenobiotics by the use of human saliva. Caffeine and paraxanthine were isolated from saliva by liquid-liquid extraction (chlorophorm/isopropanol 85/15v/v). Extracts were analyzed by reversed-phase HPLC on a C18 column with mobile phase 0.1% acetic acid/methanol/acetonitrile (80/20/2 v/v) and detected at 273nm. Caffeine and paraxanthine elution times were <13min with no interferences from impurities or caffeine metabolites. Detector response was linear (0.10-8.00µg/ml, R(2) >0.99), recovery was >93% and bias <4.47%. Intra- and inter-day precision was <5.14% (n=6). The limit of quantitation was 0.10µg/ml and the limit of detection was 0.018±0.002µg/mL for paraxanthine and 0.032±0.002µg/ml for caffeine. Paraxanthine/caffeine ratio of 34 healthy volunteers was significantly higher in smokers (p<0.001). Saliva paraxanthine/caffeine ratios and urine metabolite ratios were highly correlated (r=0.85, p<0.001). The method can be used for the monitoring of CYP1A2 activity in clinical practice and in studies relevant to exposure to environmental and pharmacological xenobiotics. PMID:25891161

  20. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

    SciTech Connect

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-05-15

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1{sub C}YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+){sub s}evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  1. CYP1A1 and CYP1A2 expression: comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines.

    PubMed

    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W

    2009-05-15

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how "human-like" can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1_CYP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+)_severe-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs. PMID:19285097

  2. In Utero and Lactational Exposure to PCBs in Mice: Adult Offspring Show Altered Learning and Memory Depending on Cyp1a2 and Ahr Genotypes

    PubMed Central

    Curran, Christine P.; Genter, Mary Beth; Patel, Krishna V.; Schaefer, Tori L.; Skelton, Matthew R.; Williams, Michael T.; Vorhees, Charles V.

    2011-01-01

    Background: Both coplanar and noncoplanar polychlorinated biphenyls (PCBs) exhibit neurotoxic effects in animal studies, but individual congeners do not always produce the same effects as PCB mixtures. Humans genetically have > 60-fold differences in hepatic cytochrome P450 1A2 (CYP1A2)-uninduced basal levels and > 12-fold variability in aryl hydrocarbon receptor (AHR)affinity; because CYP1A2 is known to sequester coplanar PCBs and because AHR ligands include coplanar PCBs, both genotypes can affect PCB response. Objectives: We aimed to develop a mouse paradigm with extremes in Cyp1a2 and Ahr genotypes to explore genetic susceptibility to PCB-induced developmental neurotoxicity using an environmentally relevant mixture of PCBs. Methods: We developed a mixture of eight PCBs to simulate human exposures based on their reported concentrations in human tissue, breast milk, and food supply. We previously characterized specific differences in PCB congener pharmacokinetics and toxicity, comparing high-affinity–AHR Cyp1a2 wild-type [Ahrb1_Cyp1a2(+/+)], poor-affinity–AHR Cyp1a2 wild-type [Ahrd_Cyp1a2(+/+)], and high-affinity–AHR Cyp1a2 knockout [Ahrb1_Cyp1a2(–/–)] mouse lines [Curran CP, Vorhees CV, Williams MT, Genter MB, Miller ML, Nebert DW. 2011. In utero and lactational exposure to a complex mixture of polychlorinated biphenyls: toxicity in pups dependent on the Cyp1a2 and Ahr genotypes. Toxicol Sci 119:189–208]. Dams received a mixture of three coplanar and five noncoplanar PCBs on gestational day 10.5 and postnatal day (PND) 5. In the present study we conducted behavioral phenotyping of exposed offspring at PND60, examining multiple measures of learning, memory, and other behaviors. Results: We observed the most significant deficits in response to PCB treatment in Ahrb1_Cyp1a2(–/–) mice, including impaired novel object recognition and increased failure rate in the Morris water maze. However, all PCB-treated genotypes showed significant differences on

  3. Caffeine Impact on Metabolic Syndrome Components Is Modulated by a CYP1A2 Variant.

    PubMed

    Platt, Daniel E; Ghassibe-Sabbagh, Michella; Salameh, Pascale; Salloum, Angelique K; Haber, Marc; Mouzaya, Francis; Gauguier, Dominique; Al-Sarraj, Yasser; El-Shanti, Hatem; Zalloua, Pierre A; Abchee, Antoine B

    2016-01-01

    Cultural, dietary, and lifestyle factors are the main modulators of type 2 diabetes mellitus (T2DM) disease risk. Coffee is one of the most popular worldwide beverages, and recent epidemiological studies have showed that coffee consumption is associated with a lower risk of T2DM. This study investigates the impact of coffee intake on T2DM risk and assesses the effect of CYP variants with caffeine exposures on T2DM. Data from 7,607 study subjects were analyzed by logistic regression models, among whom 3,290 GWAS data were available for CYP variants association studies using Plink analysis. These data suggest a protective relationship for women, but not for men; however, the results were not statistically significant in this dataset and there is a significant interaction in favor of women regarding heavy coffee consumption. The interaction between male gender and heavy coffee consumption becomes significant, thereby tending to cancel the protective effect of coffee for males. CYP rs2470890 allele 'C' increases the odds of T2DM by a factor of around 1.2 but decreases the odds of caffeine boosting T2DM of 1.7 by a factor of 0.77. rs2470890 showed an association with T2DM only when the interaction with coffee was considered, thereby setting an example of genetic activation by dietary changes associating with metabolic syndrome. PMID:26588584

  4. Modulation of CYP1A1 and CYP1A2 hepatic enzymes after oral administration of Chios mastic gum to male Wistar rats.

    PubMed

    Katsanou, Efrosini S; Kyriakopoulou, Katerina; Emmanouil, Christina; Fokialakis, Nikolas; Skaltsounis, Alexios-Leandros; Machera, Kyriaki

    2014-01-01

    Chios mastic gum (CMG), a resin derived from Pistacia lentiscus var. chia, is known since ancient times for its pharmacological activities. CYP1A1 and CYP1A2 enzymes are among the most involved in the biotransformation of chemicals and the metabolic activation of pro-carcinogens. Previous studies referring to the modulation of these enzymes by CMG have revealed findings of unclear biological and toxicological significance. For this purpose, the modulation of CYP1A1 and CYP1A2 enzymes in the liver of male Wistar rats following oral administration of CMG extract (CMGE), at the levels of mRNA and CYP1A1 enzyme activity, was compared to respective enzyme modulation following oral administration of a well-known bioactive natural product, caffeine, as control compound known to involve hepatic enzymes in its metabolism. mRNA levels of Cyp1a1 and Cyp1a2 were measured by reverse transcription real-time polymerase chain reaction and their relative quantification was calculated. CYP1A1 enzyme induction was measured through the activity of ethoxyresorufin-O-deethylase (EROD). The results indicated that administration of CMGE at the recommended pharmaceutical dose does not induce significant transcriptional modulation of Cyp1a1/2 and subsequent enzyme activity induction of CYP1A1 while effects of the same order of magnitude were observed in the same test system following the administration of caffeine at the mean daily consumed levels. The outcome of this study further confirms the lack of any toxicological or biological significance of the specific findings on liver following the administration of CMGE. PMID:24950217

  5. In Utero and Lactational Exposure to a Complex Mixture of Polychlorinated Biphenyls: Toxicity in Pups Dependent on the Cyp1a2 and Ahr Genotypes

    PubMed Central

    Curran, Christine P.; Vorhees, Charles V.; Williams, Michael T.; Genter, Mary Beth; Miller, Marian L.; Nebert, Daniel W.

    2011-01-01

    Polychlorinated biphenyls (PCBs) are persistent toxic pollutants occurring as complex mixtures in the environment. Humans are known genetically to have > 60-fold differences in hepatic cytochrome P450 1A2 (CYP1A2) levels and > 12-fold differences in aryl hydrocarbon receptor (AHR) affinity, both of which could affect PCB pharmacokinetics. Thus, we compared Ahrb1_Cyp1a2(+/+) high-affinity AHR wild-type, Ahrd_Cyp1a2(+/+) poor affinity AHR wild-type, Ahrb1_Cyp1a2(−/−) knockout, and Ahrd_Cyp1a2(−/−) knockout mouse lines. We chose a mixture of three coplanar and five noncoplanar PCBs to reproduce that seen in human tissues, breast milk, and the food supply. The mixture was given by gavage to the mother on gestational day 10.5 (GD10.5) and postnatal day 5 (PND5); tissues were collected from pups and mothers at GD11.5, GD18.5, PND6, PND13, and PND28. Ahrb1_Cyp1a2(−/−) pups showed lower weight at birth and slower rate of growth postnatally. Absence of CYP1A2 resulted in significant splenic atrophy at PND13 and PND28. Presence of high-affinity AHR enhanced thymic atrophy and liver hypertrophy in the pups. Concentrations of each congener were analyzed at all time points: maximal noncoplanar congener levels in maternal tissues were observed from GD18 until PND6, whereas the highest levels in pups were found between PND6 and PND28. Coplanar PCB concentrations were generally higher in Ahrd-containing pup tissues; these findings are consistent with earlier studies demonstrating the crucial importance of AHR-mediated inducible CYP1 in the gastrointestinal tract as a means of detoxication of oral planar polycyclic aromatic hydrocarbons. PMID:20961953

  6. The effect of dose on 2,3,7,8-TCDD tissue distribution, metabolism and elimination in CYP1A2 (-/-) knockout and C57BL/6N parental strains of mice

    SciTech Connect

    Hakk, Heldur; Diliberto, Janet J.; Birnbaum, Linda S.

    2009-11-15

    Numerous metabolism studies have demonstrated that the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver-to-fat concentration ratios. This study was initiated to determine whether TCDD was inherently poorly metabolized or unavailable for metabolism because of sequestration to CYP1A2. [{sup 3}H]TCDD was administered as a single, oral dose (0.1 and 10 mug/kg) to 12 male C57BL/6N mice or 12 CYP1A2 (-/-) mice. At 96 h, less than 5% of the dose was eliminated in the urine of all groups, and TCDD detected in urine was bound to mouse major urinary protein (mMUP). Feces were the major elimination pathway (24-31% of dose), and fecal extracts and non-extractables were quantitated by HPLC for metabolites. No great differences in urinary or fecal elimination (% dose) were observed between the high and low dose treatments. TCDD concentrations were the highest in adipose tissue for CYP1A2 knockout mice but in liver for C57BL/6N mice supporting the role of hepatic CYP1A2 in the sequestration of TCDD. Overall metabolism between parental and knockout strains showed no statistical differences at either the high or low doses. The data suggested that metabolism of TCDD is inherently slow, due principally to CYP1A1, and that hepatic CYP1A2 is not an active participant in the metabolism of TCDD in male mice. Rather, CYP1A2 governs the pharmacokinetics of TCDD by making it unavailable for hepatic CYP1A1 through sequestration and attenuating extrahepatic tissue disposition.

  7. Modelling the metabolic action of human and rat CYP1A2 and its relationship with the carcinogenicity of heterocyclic amines

    NASA Astrophysics Data System (ADS)

    da Fonseca, Rute; Menziani, Maria Cristina; Melo, André; João Ramos, Maria

    Cytochrome P450 (CYP) is a family of enzymes responsible for organism detoxification. However, some of the members of the CYP1A subfamily also catalyse the activation of heterocyclic amines (HAs), present in cooked meat, to carcinogenic compounds which have been shown to increase the risk of breast, colorectal and lung cancer. In humans, CYP1A2 is the enzyme with the most significant action in HA metabolism but in rodents CYP1A1 is also important in this biotransformation. Understanding the metabolic action of these enzymes is essential to predict the factors that enable the formation of the carcinogenic products. We have built two models of CYP1A2, one for the human enzyme and one for the rat homologue. The templates chosen include the only X-ray structure published to date for a mammal CYP, a quimeric C2A5 from rabbit, as well as CYPs belonging to Bacillus megaterium (CYPBm-3), Pseudomonas putida (CYPcam), Pseudomonas sp. (CYPterp), and Saccharopolyspora erythraea (CYPeryf). Two HAs, MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) and MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline), known substrates of human and rat CYPIA2, were docked in the active site of the models, providing information regarding the different catalytic rates associated with the metabolisms in both enzymes. This is important for analysing the behaviour of animal models concerning the testing of anticancer drugs.

  8. Effect of myricetin on cytochrome P450 isoforms CYP1A2, CYP2C9 and CYP3A4 in rats.

    PubMed

    Guo, Yu-Jin; Zheng, Shuang-Li

    2014-04-01

    Myricetin is one of the main ingredients of Chinese bayberry, which is used as a traditional medicine. The purpose of this study was to find out whether myricetin influences the rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9 and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg) and midazolam (10 mg/kg), was orally administered to rats treated for 14 days with myricetin. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. Our study showed that treatment with multiple doses of myricetin had no effects on rat CYP1A2. However, CYP2C9 and CYP3A4 enzyme activities were inhibited after multiple doses of myricetin. Therefore, caution is needed when myricetin is co-administered with CYP2C9 or CYP3A4 substrates, which may result in herb-drug interactions.

  9. Quantification of caffeine in human saliva by Nuclear Magnetic Resonance as an alternative method for cytochrome CYP1A2 phenotyping.

    PubMed

    Schievano, Elisabetta; Finotello, Claudia; Navarini, Luciano; Mammi, Stefano

    2015-08-01

    The first step in caffeine metabolism is mediated for over 95% by the CYP1A2 isoform of cytochrome P450. Therefore, CYP1A2 activity is most conveniently measured through the determination of caffeine clearance. The HPLC quantification of caffeine is fully validated and is the most widely used method. It can be performed on saliva, which is gaining importance as a diagnostic biofluid and permits easy and low invasive sampling. Here, we present a quantitative (1)H nuclear magnetic resonance (NMR) method to determine caffeine in human saliva. The procedure is simple because it involves only an ultra-filtration step and a direct extraction in a deuterated solvent, yielding a matrix that is then analyzed. The reliability of this NMR method was demonstrated in terms of linearity, accuracy, recovery, and limits of detection (LoD). Good precision (relative standard deviation, RSD <4%), a recovery of >95% and LoD of 6.8·10(-7) mol L(-1) were obtained. The method was applied to samples collected from different volunteers over 24h following a single oral dose of about 100mg of caffeine administered with either coffee beverage or a capsule.

  10. Quantification of caffeine in human saliva by Nuclear Magnetic Resonance as an alternative method for cytochrome CYP1A2 phenotyping.

    PubMed

    Schievano, Elisabetta; Finotello, Claudia; Navarini, Luciano; Mammi, Stefano

    2015-08-01

    The first step in caffeine metabolism is mediated for over 95% by the CYP1A2 isoform of cytochrome P450. Therefore, CYP1A2 activity is most conveniently measured through the determination of caffeine clearance. The HPLC quantification of caffeine is fully validated and is the most widely used method. It can be performed on saliva, which is gaining importance as a diagnostic biofluid and permits easy and low invasive sampling. Here, we present a quantitative (1)H nuclear magnetic resonance (NMR) method to determine caffeine in human saliva. The procedure is simple because it involves only an ultra-filtration step and a direct extraction in a deuterated solvent, yielding a matrix that is then analyzed. The reliability of this NMR method was demonstrated in terms of linearity, accuracy, recovery, and limits of detection (LoD). Good precision (relative standard deviation, RSD <4%), a recovery of >95% and LoD of 6.8·10(-7) mol L(-1) were obtained. The method was applied to samples collected from different volunteers over 24h following a single oral dose of about 100mg of caffeine administered with either coffee beverage or a capsule. PMID:26048820

  11. SNP genetic polymorphisms of MDR-1, CYP1A2 and CYPB11 genes in four canine breeds upon toxicological evaluation

    PubMed Central

    Gagliardi, Rosa; Llambí, Silvia

    2015-01-01

    The fields of pharmacogenetics and pharmacogenomics have become increasingly promising regarding the clinical application of genetic data to aid in prevention of adverse reactions. Specific screening tests can predict which animals express modified proteins or genetic sequences responsible for adverse effects associated with a drug. Among the genetic variations that have been investigated in dogs, the multidrug resistance gene (MDR) is the best studied. However, other genes such as CYP1A2 and CYP2B11 control the protein syntheses involved in the metabolism of many drugs. In the present study, the MDR-1, CYP1A2 and CYP2B11 genes were examined to identify SNP polymorphisms associated with these genes in the following four canine breeds: Uruguayan Cimarron, Border Collie, Labrador Retriever and German Shepherd. The results revealed that several SNPs of the CYP1A2 and CYP2B11 genes are potential targets for drug sensitivity investigations. PMID:25797294

  12. [Association of polymorph variants of CYP1A2 and CYP1A1 genes with reproductive and thyroid diseases in female workers of petrochemical industry].

    PubMed

    Irmiakova, A R; Kochetova, O V; Gaĭnullina, M K; Sivochalova, O V; Viktorova, T V

    2012-01-01

    The article presents results obtained in study of relationship between polymorph variants of CYP1A1 and CYP1A2 genes with reproductive and thyroid diseases risk in female workers of petrochemical industry, when compared with reference group females. Variants TD and DD of CYP1A2 gene appeared to be associated with nodes formation in uterus and breast in female workers and reference group females. Following liability markers are obtained: homozygous in rare allele genotype CC of CYP1A1 gene for reproductive and thyroid diseaes (fibrous cystic mastopathy and nodular goitre), heterozygous genotype AG of CYP1A1 gene in uterine myoma and fibrous cystic mastopathy, homozygous in deleted T genotype of CYP1A2 gene in autoimmune thyroiditis. Occupational hazards and long length of service at hazardous industries increase effects of rare alleles of the genes studied.

  13. Differential expression of CYP1A1 and CYP1A2 genes in H4IIE rat hepatoma cells exposed to TCDD and PAHs.

    PubMed

    Kaisarevic, Sonja; Dakic, Vanja; Hrubik, Jelena; Glisic, Branka; Lübcke-von Varel, Urte; Pogrmic-Majkic, Kristina; Fa, Svetlana; Teodorovic, Ivana; Brack, Werner; Kovacevic, Radmila

    2015-01-01

    Rat hepatoma cells H4IIE were treated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs) (dibenz(a,h)anthracene, benzo(a)pyrene, benz(a)anthracene, chrysene), low-concentration mixtures of PAHs and TCDD, and environmental mixtures contaminated by PAHs and their derivatives. Expression of the gene battery comprising cytochrome P450 Cyp1a1, Cyp1a2, Cyp1b1, and glutathione-s-transferase Gsta2 and Gstp was investigated using quantitative real time polymerase chain reaction (qRT-PCR) analysis. The results revealed that TCDD induce Cyp1a1>Cyp1a2>Cyp1b1, while PAHs and PAH-containing environmental mixtures induce Cyp1a2>Cyp1a1>Cyp1b1 gene expression pattern. While low-concentration mixtures elicited a more pronounced response in comparison to single treatments, the typical gene expression patterns were not observed. In all samples, Gsta2 was predominantly expressed relative to Gstp. These findings indicate that differential Cyp1a1 and Cyp1a2 expression in the H4IIE cells might be used for detection of PAHs in highly contaminated environmental mixtures, but not in low-concentration mixtures of these compounds.

  14. CYP1A2 DOES NOT PLAY A CRITICAL ROLE IN 2, 3 7, 8-TETRACHLORODIBENZO-P-DIOXIN-INDUCED IMMUNOSUPRESSION

    EPA Science Inventory

    CYP1A2 IS NOT REQUIRED FOR 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN-INDUCED IMMUNOSUPPRESSION Smialowicz, Ralph J1; Burgin, Deborah E2; Williams, Wanda C1; Diliberto, Janet J1; Birnbaum, Linda S1
    1 Experimental Toxicology Division, US EPA, RTP, NC, USA; 2Curriculum in Toxicology, U...

  15. Enzymatic characterization of in vitro-expressed Baikal seal cytochrome P450 (CYP) 1A1, 1A2, and 1B1: implication of low metabolic potential of CYP1A2 uniquely evolved in aquatic mammals.

    PubMed

    Iwata, Hisato; Yamaguchi, Keisuke; Takeshita, Yoko; Kubota, Akira; Hirakawa, Shusaku; Isobe, Tomohiko; Hirano, Masashi; Kim, Eun-Young

    2015-05-01

    This study aimed to elucidate the catalytic function of cytochrome P450 (CYP) 1 enzymes in aquatic mammals. Alkoxyresorufin O-dealkylation (AROD) activities including methoxy- (MROD), ethoxy- (EROD), pentoxy- (PROD), and benzyloxyresorufin O-dealkylation (BROD), and 2- and 4-hydroxylation activities of 17β-estradiol (E2) were measured by using yeast-expressed Baikal seal (Pusa sibirica) CYP1A1, 1A2, and 1B1 proteins. Heterologous protein expression of the Baikal seal CYP1s (bsCYP1s) in yeast microsomes was confirmed by reduced CO-difference spectra and immunoblotting. Heterologously expressed human CYP1 enzyme (hCYP1) activities were simultaneously measured and compared with those of bsCYP1 isozymes. Recombinant bsCYP1A1 protein showed the highest Vmax of EROD, followed by MROD, PROD, and BROD, similar to that of hCYP1A1. Vmax/Km ratios of all AROD activities catalyzed by bsCYP1A1 were lower than those catalyzed by hCYP1A1, suggesting less potential for AROD by bsCYP1A1. Enzymatic assays for bsCYP1A2 showed no or minimal AROD activities, while hCYP1A2 displayed MROD and EROD activities. bsCYP1B1 showed an AROD profile (EROD>BROD>MROD>PROD) similar to that of hCYP1B1; however, Vmax/Km ratios of all AROD activities by bsCYP1B1 were higher. Yeast microsomes containing bsCYP1A1 and 1B1 and hCYP1A1, 1A2, and 1B1 metabolized E2 to 2-OHE2 and 4-OHE2, whereas bsCYP1A2 showed no such activity. Comparison of 4- and 2-hydroxylations of E2 by CYP1As suggests that bsCYP1A1, hCYP1A1, and 1A2 preferentially catalyze 2- rather than 4-hydroxylation. As for CYP1B1, the Vmax/Km ratios suggest that both Baikal seal and human CYPs catalyze 4- rather than 2-hydroxylation. Interspecies comparison showed that bsCYP1B1 has higher metabolic potencies for both E2 hydroxylations than does hCYP1B1, whereas the activity of bsCYP1A1 was lower than that of hCYP1A1. Messenger RNA expression levels of bsCYP1s in the liver of Baikal seals indicated that bsCYP1A1 and 1A2 enzymes contributed to 16

  16. Sequencing and characterization of mixed function monooxygenase genes CYP1A1 and CYP1A2 of Mink (Mustela vison) to facilitate study of dioxin-like compounds

    SciTech Connect

    Zhang Xiaowei; Moore, Jeremy N.; Newsted, John L.; Hecker, Markus Zwiernik, Matthew J.; Jones, Paul D.; Bursian, Steven J.

    2009-02-01

    As part of an ongoing effort to understand aryl hydrocarbon receptor (AhR) mediated toxicity in mink, cDNAs encoding for CYP1A1 and the CYP1A2 mixed function monooxygenases were cloned and characterized. In addition, the effects of selected dibenzofurans on the expression of these genes and the presence of their respective proteins (P4501A) were investigated, and then correlated with the catalytic activities of these proteins as measured by ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) activities. The predicted protein sequences for CYP1A1 and CYP1A2 comprise 517 and 512 amino acid residues, respectively. The phylogenetic analysis of the mink CYP1As with protein sequences of other mammals revealed high sequence homology with sea otter, seals and the dog, with amino acid identities ranging from 89 to 95% for CYP1A1 and 81 to 93% for CYP1A2. Since exposure to both 2,3,7,8-Tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) resulted in dose-dependent increases of CYP1A1 mRNA, CYP1A2 mRNA and CYP1A protein levels an underlying AhR-mediated mechanism is suggested. The up-regulation of CYP1A mRNA in liver was more consistent to the sum adipose TEQ concentration than to the liver TEQ concentration in minks treated with TCDF or PeCDF. The result suggested that the hepatic-sequestered fraction of PeCDF was biologically inactive to the induction of CYP1A1 and CYP1A2.

  17. Predicting drug metabolism by CYP1A1, CYP1A2, and CYP1B1: insights from MetaSite, molecular docking and quantum chemical calculations.

    PubMed

    Pragyan, Preeti; Kesharwani, Siddharth S; Nandekar, Prajwal P; Rathod, Vijay; Sangamwar, Abhay T

    2014-11-01

    Recently, CYP1 enzymes are documented for selective metabolism of anticancer leads in cancer prevention and/or progression. Elucidation of specificity of substrates/inhibitors of CYP1 isoforms plays a vital role in design of more selective and potent anticancer leads. However, an area of concern is the broad range of substrate specificities and planar nature of substrates with limited dataset which makes it difficult to predict their site of metabolism (SOM) accurately. In the present study, various models for prediction of site of metabolism in case of CYP1A1, CYP1A2, and CYP1B1 substrates were developed using MetaSite, molecular docking, and quantum chemical descriptors. The predictive accuracy of MetaSite, molecular docking, and quantum chemical descriptors in identifying experimental site of metabolism was analyzed at three levels; top rank, top three ranks, and top five ranks. Two quantum chemical descriptors, chemical hardness and local nucleophilicity are proposed for the prediction of CYP-mediated SOM for the first time. The predictive accuracy shown by chemical hardness at top three ranks was 83.3, 85.7, and 84.6 % for CYP1A1, CYP1A2 and CYP1B1, respectively, whereas local nucleophilicity gave poor predictions of 50, 42.8, and 46.2 %, respectively. The predictability of chemical hardness descriptor outperformed at all three levels of ranks for CYP1A1, CYP1A2, and CYP1B1. Hence, we propose chemical hardness as an useful quantum chemical descriptor for prediction of metabolically vulnerable prints in CYP1A1, CYP1A2, and CYP1B1 mediated metabolism and support the optimization efforts in drug discovery and development programs.

  18. COMPARISON OF OVERALL METABOLISM OF 1, 2, 7, 8-PECDD IN CYP1A2(-L-) KNOCKOUT AND C57BL/6N PARENTAL STRAINS OF MICE

    EPA Science Inventory

    COMPARISON OF OVERALL METABOLISM OF 1,2,3,7,8-PeCDD
    IN CYP1A2 (-/-) KNOCKOUT AND C57BL/6N PARENTAL
    STRAINS OF MICE

    Heldur Hakk1 and Janet J. Diliberto2

    1 USDA-ARS, Biosciences Research Laboratory, P.O. Box 5674, Fargo, ND, USA
    2 US EPA, ORD, National Heal...

  19. [In vivo evaluation of the metabolic ratio of CYP2C9 and CYP1A2 drug markers after administration of afobazole in comparison to standard inducers and inhibitors of cytochromes].

    PubMed

    Novitskaia, Ia G; Gribakina, O G; Kolyvanov, G B; Zherdev, V P; Smirnov, V V; Seredenin, S B

    2013-01-01

    The effect of subchronic peroral administration in effective doses of afobazole (5 mg/kg), and cytochrome P450 inductors (rifampicin, 13.4 mg/kg; phenytoin, 10.4 mg/kg) and inhibitors (fluconazole, 35.7 mg/kg; ciprofloxacin, 44.0 mg/kg) on the metabolic ratio (MR) of drugs-markers of CYP2C9 and CYP1A2 activity was studied in rats. Afobazole did not change the MR of compounds metabolized by the P450 isoforms studied. After peroral administration of standard P450 inductors and inhibitors, statistically significant bidirectional effects were identified, which demonstrated the expedience of administering a complex of selected compounds, markers, and CYP2C9 and CYP1A2 activity modificators for comparative evaluation of the effects of new drugs in rats. It is recommended to evaluate the activity of CYP1A2 by determining the MR for one of two caffeine metabolites, paraxanthine or theobromine, and the activity of CYP2C9 by determining the MR of metabolite Exp-3174 to losartan.

  20. Metabolism of the anthelmintic drug niclosamide by cytochrome P450 enzymes and UDP-glucuronosyltransferases: metabolite elucidation and main contributions from CYP1A2 and UGT1A1.

    PubMed

    Lu, Danyi; Ma, Zhiguo; Zhang, Tianpeng; Zhang, Xingwang; Wu, Baojian

    2016-01-01

    1. Niclosamide is an old anthelmintic drug that shows potential in fighting against cancers. Here, we characterized the metabolism of niclosamide by cytochrome P450 enzymes (CYPs) and UDP-glucuronosyltransferases (UGTs) using human liver microsomes (HLM) and expressed enzymes. 2. NADPH-supplemented HLM (and liver microsomes from various animal species) generated one hydroxylated metabolite (M1) from niclosamide; and UDPGA-supplemented liver microsomes generated one mono-O-glucuronide (M2). The chemical structures of M1 (3-hydroxy niclosamide) and M2 (niclosamide-2-O-glucuronide) were determined through LC-MS/MS and/or NMR analyses. 3. Reaction phenotyping revealed that CYP1A2 was the main enzyme responsible for M1 formation. The important role of CYP1A2 in niclosamide metabolism was further confirmed by activity correlation analyses as well as inhibition experiments using specific inhibitors. 4. Although seven UGT enzymes were able to catalyze glucuronidation of niclosamide, UGT1A1 and 1A3 were the enzymes showed the highest metabolic activities. Activity correlation analyses demonstrated that UGT1A1 played a predominant role in hepatic glucuronidation of niclosamide, whereas the role of UGT1A3 was negligible. 5. In conclusion, niclosamide was subjected to efficient metabolic reactions hydroxylation and glucuronidation, wherein CYP1A2 and UGT1A1 were the main contributing enzymes, respectively.

  1. Analysis of caffeine and paraxanthine in human saliva with ultra-high-performance liquid chromatography for CYP1A2 phenotyping.

    PubMed

    Jordan, Nan Yeun; Mimpen, Jolet Y; van den Bogaard, Willie J M; Flesch, Frits M; van de Meent, Michiel H M; Torano, Javier Sastre

    2015-07-15

    Cytochrome P450 1A2 (CYP1A2) plays an important role in drug metabolism. Caffeine (CAF) is converted into paraxanthine (PX) by this enzyme and is used as a xenobiotic substrate to determine the CYP1A2 phenotype in humans. A method for the quantification of CAF and PX in saliva was developed using liquid-liquid extraction with ethyl acetate and analysis with ultra-high-performance liquid chromatography. Peaks from CAF, PX and internal standard were resolved within 6min. The method was validated from 0.05 to 5μgmL(-1) CAF and 0.025-2.5μgmL(-1) PX. Inter- and intra-day accuracies ranged from 91.2 to 107.2% with precisions <13.5%. The limits of detection were 0.16 and 0.63 ngmL(-1) for PX and CAF, respectively. PX/CAF concentration ratios from volunteers were 0.26-1.09 with mean ratios of 0.78±0.26 and 0.38±0.10 for regular and light/non-coffee drinkers, respectively. PMID:26038236

  2. Analysis of caffeine and paraxanthine in human saliva with ultra-high-performance liquid chromatography for CYP1A2 phenotyping.

    PubMed

    Jordan, Nan Yeun; Mimpen, Jolet Y; van den Bogaard, Willie J M; Flesch, Frits M; van de Meent, Michiel H M; Torano, Javier Sastre

    2015-07-15

    Cytochrome P450 1A2 (CYP1A2) plays an important role in drug metabolism. Caffeine (CAF) is converted into paraxanthine (PX) by this enzyme and is used as a xenobiotic substrate to determine the CYP1A2 phenotype in humans. A method for the quantification of CAF and PX in saliva was developed using liquid-liquid extraction with ethyl acetate and analysis with ultra-high-performance liquid chromatography. Peaks from CAF, PX and internal standard were resolved within 6min. The method was validated from 0.05 to 5μgmL(-1) CAF and 0.025-2.5μgmL(-1) PX. Inter- and intra-day accuracies ranged from 91.2 to 107.2% with precisions <13.5%. The limits of detection were 0.16 and 0.63 ngmL(-1) for PX and CAF, respectively. PX/CAF concentration ratios from volunteers were 0.26-1.09 with mean ratios of 0.78±0.26 and 0.38±0.10 for regular and light/non-coffee drinkers, respectively.

  3. Effects of capsicine on rat cytochrome P450 isoforms CYP1A2, CYP2C19, and CYP3A4.

    PubMed

    Zhu, Hui-dan; Gu, Ni; Wang, Meng; Kong, Hong-ru; Zhou, Meng-tao

    2015-01-01

    Due to the frequent consumption of capsaicin (CAP) and its current therapeutic application, the correct assessment of this compound is important from a public health standpoint. The purpose of this study was to find out whether CAP affects rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (15 mg/kg), omeprazole (15 mg/kg), and midazolam (10 mg/kg), was given orally to rats treated for 7 d with oral administration of CAP. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS. The results showed that treatment with multiple doses of CAP had no significant effect on rat CYP1A2. However, CAP had a significant inhibitory effect on CYP2C19 and an inductive effect on CYP3A4. Therefore, caution is needed when CAP is co-administered with some CYP substrates clinically because of potential drug-CAP interactions.

  4. CYP1A2 and NAT2 phenotyping and 3-aminobiphenyl and 4-aminobiphenyl hemoglobin adduct levels in smokers and non-smokers

    SciTech Connect

    Sarkar, Mohamadi; Stabbert, Regina; Kinser, Robin D.; Oey, Jan; Rustemeier, Klaus; Holt, Klaus von; Schepers, Georg; Walk, Roger A.; Roethig, Hans J.

    2006-06-15

    Some aromatic amines are considered to be putative bladder carcinogens. Hemoglobin (Hb) adducts of 3-aminobiphenyl (3-ABP) and 4-aminobiphenyl (4-ABP) have been used as biomarkers of exposure to aromatic amines from cigarette smoke. One of the goals of this study was to determine intra- and inter-individual variability in 3-ABP and 4-ABP Hb adducts and to explore the predictability of ABP Hb adduct levels based on caffeine phenotyping. The study was conducted in adult smokers (S, n = 65) and non-smokers (NS, n 65). The subjects were phenotyped for CYP1A2 and NAT2 using urinary caffeine metabolites. Blood samples were collected twice within 6 weeks and adducts measured by GC/MS. The levels of 4-ABP Hb adducts were significantly (p < 0.0001) greater in S (34.5 {+-} 21.06 pg/g Hb) compared to NS (6.3 {+-} 3.02 pg/g Hb). The levels of 3-ABP Hb adducts were below the limit of quantification (BLOQ) in most (82%) of the NS and about 10-fold lower in S (3.6 {+-} 3.29 pg/g Hb) compared to 4-ABP Hb adducts. No differences were observed in the adduct levels between weeks 1 and 6 in the smokers, suggesting that a single sample would be adequate to monitor cigarette smoke exposure. The regression model developed with CYP1A2, NAT2 phenotype and number of cigarettes smoked (NCIG) accounted for 47% of the variability in 3-ABP adducts, whereas 32% variability in 4-ABP adducts was accounted by CYP1A2 and NCIG. The ratio of 4-ABP Hb adducts in adult S:NS was {approx} 5:1, whereas 3-ABP Hb adducts levels were BLOQ in some S, exhibited large interindividual variability ({approx} 91% compared to 57% for 4-ABP Hb) and poor dose response relationship. Therefore, 4-ABP Hb adduct levels may be a more useful biomarker of aminobiphenyl exposure from cigarette smoke.

  5. Coffee consumption and CYP1A2 genotype in relation to bone mineral density of the proximal femur in elderly men and women: a cohort study

    PubMed Central

    2010-01-01

    Background Drinking coffee has been linked to reduced calcium conservation, but it is less clear whether it leads to sustained bone mineral loss and if individual predisposition for caffeine metabolism might be important in this context. Therefore, the relation between consumption of coffee and bone mineral density (BMD) at the proximal femur in men and women was studied, taking into account, for the first time, genotypes for cytochrome P450 1A2 (CYP1A2) associated with metabolism of caffeine. Methods Dietary intakes of 359 men and 358 women (aged 72 years), participants of the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS), were assessed by a 7-day food diary. Two years later, BMD for total proximal femur, femoral neck and trochanteric regions of the proximal femur were measured by Dual-energy X-ray absorptiometry (DXA). Genotypes of CYP1A2 were determined. Adjusted means of BMD for each category of coffee consumption were calculated. Results Men consuming 4 cups of coffee or more per day had 4% lower BMD at the proximal femur (p = 0.04) compared with low or non-consumers of coffee. This difference was not observed in women. In high consumers of coffee, those with rapid metabolism of caffeine (C/C genotype) had lower BMD at the femoral neck (p = 0.01) and at the trochanter (p = 0.03) than slow metabolizers (T/T and C/T genotypes). Calcium intake did not modify the relation between coffee and BMD. Conclusion High consumption of coffee seems to contribute to a reduction in BMD of the proximal femur in elderly men, but not in women. BMD was lower in high consumers of coffee with rapid metabolism of caffeine, suggesting that rapid metabolizers of caffeine may constitute a risk group for bone loss induced by coffee. PMID:20175915

  6. Higher gene expression of CYP1A2, 2B1 and 2D2 in the brain of female compared with male rats.

    PubMed

    Nagai, K; Fukuno, S; Suzuki, H; Konishi, H

    2016-06-01

    Cytochrome P450 (CYP) in the brain plays an essential role in the local metabolism of various compounds, including clinically used drugs, toxins, and endogenous substances. In the present study, we compared the expression profiles of mRNAs for several CYP subtypes in the brain between male and female rats. The expression of CYP1A2, CYP2B1, and CYP2D2 in females was significantly higher than that in males. On the other hand, the expression level of the other CYP subtypes examined in the male brain was similar to that in the female brain. These results strongly suggest that marked gender differences exist in the expression profiles of some CYP subtypes in rat brain. PMID:27455552

  7. Higher gene expression of CYP1A2, 2B1 and 2D2 in the brain of female compared with male rats.

    PubMed

    Nagai, K; Fukuno, S; Suzuki, H; Konishi, H

    2016-06-01

    Cytochrome P450 (CYP) in the brain plays an essential role in the local metabolism of various compounds, including clinically used drugs, toxins, and endogenous substances. In the present study, we compared the expression profiles of mRNAs for several CYP subtypes in the brain between male and female rats. The expression of CYP1A2, CYP2B1, and CYP2D2 in females was significantly higher than that in males. On the other hand, the expression level of the other CYP subtypes examined in the male brain was similar to that in the female brain. These results strongly suggest that marked gender differences exist in the expression profiles of some CYP subtypes in rat brain.

  8. Genome-wide association analysis of coffee drinking suggests association with CYP1A1/CYP1A2 and NRCAM

    PubMed Central

    Amin, N; Byrne, E; Johnson, J; Chenevix-Trench, G; Walter, S; Nolte, I M; Vink, J M; Rawal, R; Mangino, M; Teumer, A; Keers, J C; Verwoert, G; Baumeister, S; Biffar, R; Petersmann, A; Dahmen, N; Doering, A; Isaacs, A; Broer, L; Wray, N R; Montgomery, G W; Levy, D; Psaty, B M; Gudnason, V; Chakravarti, A; Sulem, P; Gudbjartsson, D F; Kiemeney, L A; Thorsteinsdottir, U; Stefansson, K; van Rooij, F J A; Aulchenko, Y S; Hottenga, J J; Rivadeneira, F R; Hofman, A; Uitterlinden, A G; Hammond, C J; Shin, S-Y; Ikram, A; Witteman, J C M; Janssens, A C J W; Snieder, H; Tiemeier, H; Wolfenbuttel, B H R; Oostra, B A; Heath, A C; Wichmann, E; Spector, T D; Grabe, H J; Boomsma, D I; Martin, N G; van Duijn, C M

    2012-01-01

    Coffee consumption is a model for addictive behavior. We performed a meta-analysis of genome-wide association studies (GWASs) on coffee intake from 8 Caucasian cohorts (N=18 176) and sought replication of our top findings in a further 7929 individuals. We also performed a gene expression analysis treating different cell lines with caffeine. Genome-wide significant association was observed for two single-nucleotide polymorphisms (SNPs) in the 15q24 region. The two SNPs rs2470893 and rs2472297 (P-values=1.6 × 10−11 and 2.7 × 10−11), which were also in strong linkage disequilibrium (r2=0.7) with each other, lie in the 23-kb long commonly shared 5′ flanking region between CYP1A1 and CYP1A2 genes. CYP1A1 was found to be downregulated in lymphoblastoid cell lines treated with caffeine. CYP1A1 is known to metabolize polycyclic aromatic hydrocarbons, which are important constituents of coffee, whereas CYP1A2 is involved in the primary metabolism of caffeine. Significant evidence of association was also detected at rs382140 (P-value=3.9 × 10−09) near NRCAM—a gene implicated in vulnerability to addiction, and at another independent hit rs6495122 (P-value=7.1 × 10−09)—an SNP associated with blood pressure—in the 15q24 region near the gene ULK3, in the meta-analysis of discovery and replication cohorts. Our results from GWASs and expression analysis also strongly implicate CAB39L in coffee drinking. Pathway analysis of differentially expressed genes revealed significantly enriched ubiquitin proteasome (P-value=2.2 × 10−05) and Parkinson's disease pathways (P-value=3.6 × 10−05). PMID:21876539

  9. Human cytochrome P450-catalyzed conversion of the proestrogenic pesticide methoxychlor into an estrogen. Role of CYP2C19 and CYP1A2 in O-demethylation.

    PubMed

    Stresser, D M; Kupfer, D

    1998-09-01

    1,1,1-Trichloro-2,2-bis(4-methoxyphenyl)ethane (methoxychlor) is a widely used pesticide that is pro-estrogenic. We have elucidated the human cytochrome P450 enzymes responsible for conversion of methoxychlor into its major metabolite, the mono-O-demethylated derivative (mono-OH-M) that is estrogenic. Incubation of methoxychlor with microsomes from insect cells overexpressing either CYP1A2, CYP2C18, or CYP2C19 yielded mono-OH-M with turnover numbers of 14.9, 15.5, and 39.1 nmol/min/nmol of P450, respectively. CYP2B6 and CYP2C9 were much less active. Incubations with purified CYP2C19 and CYP2C18 resulted in formation of mono-OH-M, and also the bis-demethylated metabolite. Co-incubation of liver microsomes with methoxychlor and various P450 isoform-selective inhibitors suggested involvement of several P450s in mono-O-demethylation, including CYP1A2, CYP2A6, CYP2C9, and CYP2C19. A role for CYP2C19, CYP1A2, and CYP2A6 was also indicated by multivariate regression analysis of the mono-O-demethylase activity in a panel of human liver microsomes characterized for isoform-specific catalytic activities (R2 = 0.96). Based on the totality of the evidence, CYP2C19 appears to be the major catalyst of methoxychlor mono-O-demethylation. However, in individuals lacking functional CYP2C19 (e.g. the "poor metabolizer" phenotype), CYP1A2 may play the predominant role. CYP2A6, CYP2C9, and CYP2B6 probably contribute to a lesser extent. Although CYP2C18 is an efficient methoxychlor demethylase, its expression in liver is reportedly low or absent, suggesting a negligible role for this enzyme in methoxychlor metabolism. Lengthy incubations of liver microsomes with methoxychlor produced other secondary and tertiary metabolites. Efficient conversion of methoxychlor to estrogenic mono-OH-M by liver microsomes suggests that methoxychlor has the potential to be estrogenic in humans, as observed in several animal species.

  10. Increased exposure of vitamin A by Chrysanthemum morifolium Ramat extract in rat was not via induction of CYP1A1, CYP1A2, and CYP2B1.

    PubMed

    Wang, Ping; Pan, Xian; Chen, Guanming; Li, Jia; Liu, Li; Liu, Xiaodong; Jin, Shi; Xie, Lin; Wang, Guangji

    2012-06-01

    The aim of this study was to investigate the effect of Chrysanthemum morifolium Ramat (CM) extract on the pharmacokinetics of retinol and activities of cytochrome P450s (CYP450s) related to retinoid metabolism. Rats were treated with CM extract for 15 d. Plasma concentrations of retinol were measured following oral administration of retinol (45 mg/kg). Basal levels of retinol and retinoic acid in serum and liver were also measured. 7-Ethoxyresorufin-O-deethylase activity, phenacetin-O-deethylase activity, and 7-pentoxyresorufin-O-deethylase activities were used to assay the activities of CYP1A1, CYP1A2, and CYP2B1 in hepatic microsomes of rats, respectively. Protein expressions of the 3 CYP450s were measured by western blot. Our studies demonstrated that CM extract dose-dependently increased basal level of retinol in serum. In pharmacokinetic experiment, CM extract dose-dependently increased plasma concentrations of retinol after oral administration of retinol to rats treated with CM extract. But activities and expressions of CYP1A1, CYP1A2, and CYP2B1 in hepatic microsomes of rats were also induced by CM extract.

  11. In Silico Predictions of Drug - Drug Interactions Caused by CYP1A2, 2C9 and 3A4 Inhibition - a Comparative Study of Virtual Screening Performance.

    PubMed

    Kaserer, Teresa; Höferl, Martina; Müller, Klara; Elmer, Sebastian; Ganzera, Markus; Jäger, Walter; Schuster, Daniela

    2015-06-01

    The cytochrome P450 (CYP) superfamily represents the major enzyme class responsible for the metabolism of exogenous compounds. Investigation of clearance pathways is therefore an integral part in early drug development, as any alteration of metabolic enzymes may markedly influence the toxicological profile and efficacy of novel compounds. In silico methods are widely applied in drug development to complement experimental approaches. Several different tools are available for that purpose, however, for CYP enzymes they have only been applied retrospectively so far. Within this study, pharmacophore- and shape-based models and a docking protocol were generated for the prediction of CYP1A2, 2C9, and 3A4 inhibition. All theoretically validated models, the validated docking workflow, and additional external bioactivity profiling tools were applied independently and in parallel to predict the CYP inhibition of 29 compounds from synthetic and natural origin. After subsequent experimental assessment of the in silico predictions, we analyzed and compared the prospective performance of all methods, thereby defining the suitability of the applied techniques for CYP enzymes. We observed quite substantial differences in the performances of the applied tools, suggesting that the rational selection of that virtual screening method that proved to perform best can largely improve the success rates when it comes to CYP inhibition prediction. PMID:27490388

  12. Effects of CYP2B6 and CYP1A2 Genetic Variation on Nevirapine Plasma Concentration and Pharmacodynamics as Measured by CD4 Cell Count in Zimbabwean HIV-Infected Patients.

    PubMed

    Mhandire, Doreen; Lacerda, Miguel; Castel, Sandra; Mhandire, Kudakwashe; Zhou, Danai; Swart, Marelize; Shamu, Tinei; Smith, Peter; Musingwini, Tutsirai; Wiesner, Lubbe; Stray-Pedersen, Babill; Dandara, Collet

    2015-09-01

    The extremely high prevalence of HIV/AIDS in sub-Saharan Africa and limitations of current antiretroviral medicines demand new tools to optimize therapy such as pharmacogenomics for person-to-person variations. African populations exhibit greater genetic diversity than other world populations, thus making it difficult to extrapolate findings from one population to another. Nevirapine, an antiretroviral medicine, displays large plasma concentration variability which adversely impacts therapeutic virological response. This study, therefore, aimed to identify sources of variability in nevirapine pharmacokinetics and pharmacodynamics, focusing on genetic variation in CYP2B6 and CYP1A2. Using a cross-sectional study design, 118 HIV-infected adult Zimbabwean patients on nevirapine-containing highly active antiretroviral therapy (HAART) were characterized for three key functional single nucleotide polymorphisms (SNPs), CYP2B6 c.516G>T (rs3745274), CYP2B6 c.983T>C (rs28399499), and CYP1A2 g.-163C>A (rs762551). We investigated whether genotypes at these loci were associated with nevirapine plasma concentration, a therapeutic biomarker, and CD4 cell count, a biomarker of disease progression. CYP2B6 and CYP1A2 were chosen as the candidate genes based on reports in literature, as well as their prominence in the metabolism of efavirenz, a drug in the same class with nevirapine. Nevirapine plasma concentration was determined using LC-MS/MS. The mean nevirapine concentration for CYP2B6 c.516T/T genotype differed significantly from that of 516G/G (p < 0.001) and 516G/T (p < 0.01) genotypes, respectively. There were also significant differences in mean nevirapine concentration between CYP2B6 c.983T > C genotypes (p = 0.04). Importantly, the CYP1A2 g.-163C>A SNP was significantly associated with the pharmacodynamics endpoint, the CD4 cell count (p = 0.012). Variant allele frequencies for the three SNPs observed in this Zimbabwean group were similar to other

  13. Effects of CYP2B6 and CYP1A2 Genetic Variation on Nevirapine Plasma Concentration and Pharmacodynamics as Measured by CD4 Cell Count in Zimbabwean HIV-Infected Patients

    PubMed Central

    Mhandire, Doreen; Lacerda, Miguel; Castel, Sandra; Mhandire, Kudakwashe; Zhou, Danai; Swart, Marelize; Shamu, Tinei; Smith, Peter; Musingwini, Tutsirai; Wiesner, Lubbe; Stray-Pedersen, Babill

    2015-01-01

    Abstract The extremely high prevalence of HIV/AIDS in sub-Saharan Africa and limitations of current antiretroviral medicines demand new tools to optimize therapy such as pharmacogenomics for person-to-person variations. African populations exhibit greater genetic diversity than other world populations, thus making it difficult to extrapolate findings from one population to another. Nevirapine, an antiretroviral medicine, displays large plasma concentration variability which adversely impacts therapeutic virological response. This study, therefore, aimed to identify sources of variability in nevirapine pharmacokinetics and pharmacodynamics, focusing on genetic variation in CYP2B6 and CYP1A2. Using a cross-sectional study design, 118 HIV-infected adult Zimbabwean patients on nevirapine-containing highly active antiretroviral therapy (HAART) were characterized for three key functional single nucleotide polymorphisms (SNPs), CYP2B6 c.516G>T (rs3745274), CYP2B6 c.983T>C (rs28399499), and CYP1A2 g.-163C>A (rs762551). We investigated whether genotypes at these loci were associated with nevirapine plasma concentration, a therapeutic biomarker, and CD4 cell count, a biomarker of disease progression. CYP2B6 and CYP1A2 were chosen as the candidate genes based on reports in literature, as well as their prominence in the metabolism of efavirenz, a drug in the same class with nevirapine. Nevirapine plasma concentration was determined using LC-MS/MS. The mean nevirapine concentration for CYP2B6 c.516T/T genotype differed significantly from that of 516G/G (p < 0.001) and 516G/T (p < 0.01) genotypes, respectively. There were also significant differences in mean nevirapine concentration between CYP2B6 c.983T > C genotypes (p = 0.04). Importantly, the CYP1A2 g.-163C>A SNP was significantly associated with the pharmacodynamics endpoint, the CD4 cell count (p = 0.012). Variant allele frequencies for the three SNPs observed in this Zimbabwean group were similar to

  14. Pharmacokinetic assessment of a five-probe cocktail for CYPs 1A2, 2C9, 2C19, 2D6 and 3A

    PubMed Central

    Turpault, Sandrine; Brian, William; Van Horn, Robert; Santoni, Alix; Poitiers, Franck; Donazzolo, Yves; Boulenc, Xavier

    2009-01-01

    AIMS To assess the pharmacokinetics (PK) of selective substrates of CYP1A2 (caffeine), CYP2C9 (S-warfarin), CYP2C19 (omeprazole), CYP2D6 (metoprolol) and CYP3A (midazolam) when administered orally and concurrently as a cocktail relative to the drugs administered alone. METHODS This was an open-label, single-dose, randomized, six-treatment six-period six-sequence William's design study with a wash-out of 7 or 14 days. Thirty healthy male subjects received 100 mg caffeine, 100 mg metoprolol, 0.03 mg kg−1 midazolam, 20 mg omeprazole and 10 mg warfarin individually and in combination (cocktail). Poor metabolizers of CYP2C9, 2C19 and 2D6 were excluded. Plasma samples were obtained up to 48 h for caffeine, metoprolol and omeprazole, 12 h for midazolam, 312 h for warfarin and the cocktail. Three different validated liquid chromatography tandem mass spectrometry methods were used. Noncompartmental PK parameters were calculated. Log-transformed Cmax, AUClast and AUC for each analyte were analysed with a linear mixed effects model with fixed term for treatment, sequence and period, and random term for subject within sequence. Point estimates (90% CI) for treatment ratios (individual/cocktail) were computed for each analyte Cmax, AUClast and AUC. RESULTS There was no PK interaction between the probe drugs when administered in combination as a cocktail, relative to the probes administered alone, as the 90% CI of the PK parameters was within the prespecified bioequivalence limits of 0.80, 1.25. CONCLUSION The lack of interaction between probes indicates that this cocktail could be used to evaluate the potential for multiple drug–drug interactions in vivo. PMID:20002088

  15. The Effect of Dose on 2,3,7,8-TCDD Tissue Distribution, Metabolism and Elimination in CYP1A2 (-/-) Knockout and C57BL/6N Parental Strains of Mice

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Numerous metabolism studies have demonstrated that the highly toxic 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver to fat concentration ratios. This study was in...

  16. A COMPARISON OF THE METABOLISM OF METHOXYRESORUFIN, ACETANILIDE AND CAFFIENE IN RAT AND HUMAN CYP1A2 SUPERSOMES AND THEIR INHIBITION BY 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)

    EPA Science Inventory

    A COMPARISON OF THE METABOLISM OF METHOXYRESORUFIN, ACETANILIDE AND CAFFIENE IN RAT AND HUMAN CYP1A2 SUPERSOMES AND THEIR INHIBITION BY 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD). DF Staskal1, DG Ross2, LS Birnbaum2 and MJ DeVito2 1Curriculum In Toxicology, UNC-CH, Chapel Hill ...

  17. The effect of dose on 2,3,7,8-TCDD tissue distribution, metabolism and elimination in CYP1A2(-/_) knockout and C57BL/6N parental strains of mice

    EPA Science Inventory

    Numerous metabolism studies have demonstrated that the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is poorly metabolized. A hallmark feature of TCDD exposure is induction of hepatic CYP1A2 and subsequent sequestration leading to high liver-to-fat concentration ra...

  18. USE OF A PHYSIOLOGICALLY BASED PHARMACOKINETIC MODEL (PBPK) FOR RATS TO STUDY THE INFLUENCE OF BODY FAT MASS AND INDUCTION OF CYP1A2 ON THE PHARMACOKINETICS OF TCDD

    EPA Science Inventory

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly lipophilic chemical which distributes into adipose tissue, especially at low doses. However, at high doses TCDD sequesters in liver because it induces CYP1A2 that binds TCDD. A physiologically based pharmacokinetic (PBPK) mod...

  19. COMPARISON OF OVERALL METABOLISM OF 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIOXIN IN CYP1A2(-/-) KNOCKOUT AND C57BL/6N PARENTAL STRAINS OF MICE

    EPA Science Inventory

    Comparison of Overall Metabolism of 2,3,7,8-TCDD
    in CYP1A2 (-/-) Knockout and C57BL/6N Parental Strains of Mice

    Heldur Hakk* and Janet J. Diliberto**

    * USDA-ARS Biosciences Research Laboratory, P.O. Box 5674, Fargo, ND, USA
    ** US-EPA ORD, National Health Eff...

  20. Combination Analysis in Genetic Polymorphisms of Drug-Metabolizing Enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese Population

    PubMed Central

    Ota, Tomoko; Kamada, Yuka; Hayashida, Mariko; Iwao-Koizumi, Kyoko; Murata, Shigenori; Kinoshita, Kenji

    2015-01-01

    The Cytochrome P450 is the major enzyme involved in drug metabolism. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is one important factor that contributes to drug therapy failure. We have developed a new straightforward TaqMan PCR genotyping assay to investigate the prevalence of the most common allelic variants of polymorphic CYP enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A5 in the Japanese population. Moreover, we focused on the combination of each genotype for clinical treatment. The genotype analysis identified a total of 139 out of 483 genotype combinations of five genes in the 1,003 Japanese subjects. According to our results, most of subjects seemed to require dose modification during clinical treatment. In the near future, modifications should be considered based on the individual patient genotype of each treatment. PMID:25552922

  1. Induction of CYP1A1, CYP1A2, CYP1B1, increased oxidative stress and inflammation in the lung and liver tissues of rats exposed to incense smoke.

    PubMed

    Hussain, Tajamul; Al-Attas, Omar S; Al-Daghri, Nasser M; Mohammed, Arif A; De Rosas, Edgard; Ibrahim, Shebl; Vinodson, Benjamin; Ansari, Mohammed G; El-Din, Khaled I Alam

    2014-06-01

    Incense smoke is increasingly being recognized as a potential environmental contaminant and is linked to malignant and non-malignant respiratory diseases. The detoxification of environmental contaminants including polycyclic aromatic hydrocarbons (PAHs) involves the induction of cytochrome P-450 family enzymes (CYPs) by PAHs. However, the detoxification of PAHs also results in the generation of reactive and unstable intermediary metabolites which are implicated in the oxidative stress, DNA damage, and inflammation. It is unclear whether CYPs are similarly induced by incense smoke, which incidentally contains substantial amounts of PAHs. Here, we examined the impact of long-term incense smoke exposure on the induction of CYPs in male Wister Albino rats. Incense smoke exposure significantly induced the expression of CYP1A1, CYP1A2, and CYP1B1 mRNAs in both lung and liver tissues. The extent of CYP1A1 and CYP1B1 induction was significantly higher in the liver compared to that in the lung, while that of CYP1A2 was greater in the lung than in liver. Incense smoke exposure also increased malondialdehyde and reduced glutathione levels in lung and liver tissues, and the catalase activity in the liver tissues to significant levels. Furthermore incense smoke exposure led to a marked increase in TNF-α and IL-4 levels. The data demonstrate for the first time the capacity of incense smoke to induce CYP1 family enzymes in the target and non-target tissues. Induction of CYPs increased oxidative stress and inflammation appear to be intimately linked to promote the carcinogenesis and health complications in people chronically exposed to incense smoke.

  2. Polymorphisms in the cytochrome P450 genes CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1, CYP19A1 and colorectal cancer risk

    PubMed Central

    Bethke, Lara; Webb, Emily; Sellick, Gabrielle; Rudd, Matthew; Penegar, Stephen; Withey, Laura; Qureshi, Mobshra; Houlston, Richard

    2007-01-01

    Background Cytochrome P450 (CYP) enzymes have the potential to affect colorectal cancer (CRC) risk by determining the genotoxic impact of exogenous carcinogens and levels of sex hormones. Methods To investigate if common variants of CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1 and CYP19A1 influence CRC risk we genotyped 2,575 CRC cases and 2,707 controls for 20 single nucleotide polymorphisms (SNPs) that have not previously been shown to have functional consequence within these genes. Results There was a suggestion of increased risk, albeit insignificant after correction for multiple testing, of CRC for individuals homozygous for CYP1B1 rs162558 and heterozygous for CYP1A2 rs2069522 (odds ratio [OR] = 1.36, 95% confidence interval [CI]: 1.03–1.80 and OR = 1.34, 95% CI: 1.00–1.79 respectively). Conclusion This study provides some support for polymorphic variation in CYP1A2 and CYP1B1 playing a role in CRC susceptibility. PMID:17615053

  3. Hyalinosis and Ym1/Ym2 Gene Expression in the Stomach and Respiratory Tract of 129S4/SvJae and Wild-Type and CYP1A2-Null B6,129 Mice

    PubMed Central

    Ward, Jerrold M.; Yoon, Michung; Anver, Miriam R.; Haines, Diana C.; Kudo, Gen; Gonzalez, Frank J.; Kimura, Shioko

    2001-01-01

    The C57BL/6, 129, and B6,129 mouse strains or stocks have been commonly used to generate targeted mutant mice. The pathology of these mice is not well characterized. In studies of these aging mice, we found high incidences of hyalinosis (eosinophilic cytoplasmic change) in the glandular stomach, respiratory tract, bile duct, and gall bladder of B6,129 CYP1A2-null and wild-type mice as well as in both sexes of the background 129S4/SvJae strain. The gastric lesions of the glandular stomach were found in 95.7% of female CYP1A2-null mice as well as in 45.7% of female 129S4/SvJae animals. The eosinophilic protein isolated from characteristic hyaline gastric lesions was identified as Ym2, a member of the chitinase family. Immunohistochemistry, using rabbit polyclonal antibodies to oligopeptides derived from the Ym1 sequence, detected focal to diffuse reactivity within both normal and abnormal nasal olfactory and respiratory epithelium, pulmonary alveolar macrophages, bone marrow myeloid cells, and the squamous epithelium of the forestomach and epithelium of the glandular stomach. Alveolar macrophages in acidophilic pneumonia, a major cause of death of aging 129 mice, and in mice with the me mutation also were highly immunoreactive. The possible cause of this protein excess in gastric and other lesions and its possible functions are discussed. PMID:11141507

  4. Hepatic CYP1A, 2B, 2C, 2E and 3A regulation by methoxychlor in male and female rats.

    PubMed

    Oropeza-Hernández, Luis F; López-Romero, Ricardo; Albores, Arnulfo

    2003-09-15

    The effect on liver cytochrome P450 (CYP) by i.p. injections of methoxychlor (MXC) in corn oil at 0, 100, 150, 200 or 250 mg/kg twice daily for 3 days was investigated in adult male and female Wistar rats. The MXC injection (100 mg/kg b.w.) caused a similar increase of total CYP content in males and females as compared with controls who received the vehicle only. In males, this increase continued up to 250 mg/kg. As to the induction of specific CYP activities, the effect of MXC was found to be sex dependent with three different patterns. Males showed the greatest increases of ethoxy- and methoxyresorufin-O-dealkylase (EROD and MROD, respectively), two CYP1A1/1A2-related activities. On the contrary, females were more responsive than males for pentoxyresorufin-O-dealkylase (PROD) and benzyloxyresorufin-O-dearylase (BROD), two CYP2B-related activities. Finally, p-nitrophenol hydroxylase (PNPH), a CYP2E1-related activity, showed a similar small, although statistically significant, increase for both sexes. As to CYP apoprotein levels, CYP1A1 and CYP2B1/2B2 showed greater increases in females than in males; whereas, interestingly, CYP2E1 induction was higher in males than in females. These results indicate overall that gender modulates CYP expression after MXC injection both qualitatively and quantitatively, and, therefore, this pesticide is not a pure PB inducer. Moreover, the statistically significant increase of CYP3A2 apoprotein expression observed in females and also, to a lower extent, in males, and the decrease of CYP2C11 apoprotein found in males, two sex-related enzymes, may explain the reported endocrine disrupting effect of MXC. The relevance of the different patterns of rat liver CYP induction observed after MXC treatment, in relationship to the speculated endocrine disrupting potential of MXC in humans potentially exposed to this pesticide, needs further investigation.

  5. African Lettuce (Launaea taraxacifolia) Displays Possible Anticancer Effects and Herb-Drug Interaction Potential by CYP1A2, CYP2C9, and CYP2C19 Inhibition.

    PubMed

    Thomford, Nicholas E; Mkhize, Buyisile; Dzobo, Kevin; Mpye, Keleabetswe; Rowe, Arielle; Parker, M Iqbal; Wonkam, Ambroise; Skelton, Michelle; September, Alison V; Dandara, Collet

    2016-09-01

    Medicinal plants are part of the healthcare systems worldwide, especially in low- and middle-income countries. African lettuce (Launaea taraxacifolia) is cultivated extensively in Africa, from Senegal in the west to Ethiopia and Tanzania in the east, and in Southern Africa. Potential anticancer effects of L. taraxacifolia have been suggested, but little is known about putative molecular mechanisms or potential for herb-drug interactions through inhibition or induction of drug-metabolizing enzymes. We investigated the effects of crude aqueous extracts of L. taraxacifolia on growth kinetics and cell cycle progression of the WHC01 esophageal cancer cells. Antiproliferative and apoptotic effects were evaluated using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometry, while examining, in parallel, the genes regulating apoptosis and cell cycle in this cell culture model. In addition, we tested the inhibitory and enzyme kinetic effects of the aqueous L. taraxacifolia using recombinant human CYP450 isozyme model systems (CYP1A2, CYP2C9, and CYP2C19). L. taraxacifolia exhibited a significant growth inhibitory effect on the WHC01 cancer cells. Most cell cycle genes were downregulated. Cell cycle analysis showed a G0-G1 cell cycle arrest in WHC01 cells in the presence of L. taraxacifolia extract, accompanied by morphological changes. L. taraxacifolia extract treatment resulted in downregulation of expression levels of CYP1A2 (p < 0.0005) and CYP2C19 (p < 0.003) by 50-70%. L. taraxacifolia extract caused reversible and time-dependent inhibition of the recombinant CYP1A2, CYP2C9, and CYP2C19. This study provides new insights on possible anticancer effects of L. taraxacifolia, a widely used medicinal plant in parts of Africa and across the world especially by patients with cancer. Further mechanistic studies expanding on these observations would be timely and contribute to the field of global precision medicine that requires

  6. Effects of Radix Astragali and Radix Rehmanniae, the components of an anti-diabetic foot ulcer herbal formula, on metabolism of model CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4 probe substrates in pooled human liver microsomes and specific CYP isoforms.

    PubMed

    Or, Penelope M Y; Lam, Francis F Y; Kwan, Y W; Cho, C H; Lau, C P; Yu, H; Lin, G; Lau, Clara B S; Fung, K P; Leung, P C; Yeung, John H K

    2012-04-15

    The present study investigated the effects of Radix Astragali (RA) and Radix Rehmanniae (RR), the major components of an anti-diabetic foot ulcer herbal formula (NF3), on the metabolism of model probe substrates of human CYP isoforms, CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, which are important in the metabolism of a variety of xenobiotics. The effects of RA or RR on human CYP1A2 (phenacetin O-deethylase), CYP2C9 (tolbutamide 4-hydroxylase), CYP2D6 (dextromethorphan O-demethylase), CYP2E1 (chlorzoxazone 6-hydroxylase) and CYP3A4 (testosterone 6β-hydroxylase) activities were investigated using pooled human liver microsomes. NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.98mg/ml) and CYP3A4 (IC(50)=0.76mg/ml), with K(i) of 0.67 and 1.0mg/ml, respectively. With specific human CYP2C9 and CYP3A4 isoforms, NF3 competitively inhibited activities of CYP2C9 (IC(50)=0.86mg/ml) and CYP3A4 (IC(50)=0.88mg/ml), with K(i) of 0.57 and 1.6mg/ml, respectively. Studies on RA or RR individually showed that RR was more important in the metabolic interaction with the model CYP probe substrates. RR dose-dependently inhibited the testosterone 6β-hydroxylation (K(i)=0.33mg/ml) while RA showed only minimal metabolic interaction potential with the model CYP probe substrates studied. This study showed that RR and the NF3 formula are metabolized mainly by CYP2C9 and/or CYP3A4, but weakly by CYP1A2, CYP2D6 and CYP2E1. The relatively high K(i) values of NF3 (for CYP2C9 and CYP3A4 metabolism) and RR (for CYP3A4 metabolism) would suggest a low potential for NF3 to cause herb-drug interaction involving these CYP isoforms.

  7. Repeated dose toxicity and relative potency of 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for induction of CYP1A1, CYP1A2 and thymic atrophy in female Harlan Sprague-Dawley rats.

    PubMed

    Hooth, Michelle J; Nyska, Abraham; Fomby, Laurene M; Vasconcelos, Daphne Y; Vallant, Molly; DeVito, Michael J; Walker, Nigel J

    2012-11-15

    In this study we assessed the relative toxicity and potency of the chlorinated naphthalenes 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) and 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Chemicals were administered in corn oil:acetone (99:1) by gavage to female Harlan Sprague-Dawley rats at dosages of 0 (vehicle), 500, 1500, 5000, 50,000 and 500,000 ng/kg (PCN 66 and PCN 67) and 1, 3, 10, 100, and 300 ng/kg (TCDD) for 2 weeks. Histopathologic changes were observed in the thymus, liver and lung of TCDD treated animals and in the liver and thymus of PCN treated animals. Significant increases in CYP1A1 and CYP1A2 associated enzyme activity were observed in all animals exposed to TCDD, PCN 66 and PCN 67. Dose response modeling of CYP1A1, CYP1A2 and thymic atrophy gave ranges of estimated relative potencies, as compared to TCDD, of 0.0015-0.0072, for PCN 66 and 0.00029-0.00067 for PCN 67. Given that PCN 66 and PCN 67 exposure resulted in biochemical and histopathologic changes similar to that seen with TCDD, this suggests that they should be included in the WHO toxic equivalency factor (TEF) scheme, although the estimated relative potencies indicate that these hexachlorinated naphthalenes should not contribute greatly to the overall human body burden of dioxin-like activity.

  8. [Modeling of a three-dimensional structure of cytochrome P-450 1A2 and search for its new ligands].

    PubMed

    Belkina, N V; Skvortsov, V S; Ivanov, A S; Archakov, A I

    1998-01-01

    The substances inhibiting cytochrome P450 1A2 (CYP1A2) represent a perspective class of new drugs, which application in clinical practice can become the important part in preventive maintenance in oncology. The present work is devoted to computer modelling of 3-D structure of CYP1A2 and searching of new inhibitors by database mining. The modelling of CYP1A2 was done based on homology with 4 bacterial cytochromes P450 with known 3-D structure. For optimization of CYP1A2 active site structure the models of its complexes with characteristic substrates (caffeine and 7-ethoxyresorufin) were designed. These complexes were optimized by molecular dynamics simulation in water. The models of 24 complexes of CYP1A2 with known ligands with known Kd were designed by means of DockSearch and LeapFrog programs. 3D-QSAR model with good predictive force was created based on these complexes. On a final stage the search of knew CYP1A2 ligands in testing database (more than 23.000 substances from database Maybridge and 112 known CYP1A2 ligands from database Metabolite, MDL) was executed. 680 potential ligands of CYP1A2 with Kd values, comparable with known ones were obtained. This number has included 73 compounds from 112 known ligands, introduced in tested database as the internal control. PMID:9916262

  9. Cytochrome P450 1A2 Metabolizes 17β-Estradiol to Suppress Hepatocellular Carcinoma

    PubMed Central

    Ren, Jianwai; Chen, George G.; Liu, Yi; Su, Xianwei; Hu, Baoguang; Leung, Billy C. S.; Wang, Y.; Ho, Rocky L. K.; Yang, Shengli; Lu, Gang; Lee, C. G.; Lai, Paul B. S.

    2016-01-01

    Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy. PMID:27093553

  10. Cytochrome P450 1A2 Metabolizes 17β-Estradiol to Suppress Hepatocellular Carcinoma.

    PubMed

    Ren, Jianwai; Chen, George G; Liu, Yi; Su, Xianwei; Hu, Baoguang; Leung, Billy C S; Wang, Y; Ho, Rocky L K; Yang, Shengli; Lu, Gang; Lee, C G; Lai, Paul B S

    2016-01-01

    Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy. PMID:27093553

  11. 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

  12. Caffeine metabolites in umbilical cord blood, cytochrome P-450 1A2 activity, and intrauterine growth restriction.

    PubMed

    Grosso, Laura M; Triche, Elizabeth W; Belanger, Kathleen; Benowitz, Neal L; Holford, Theodore R; Bracken, Michael B

    2006-06-01

    Studies investigating antenatal caffeine consumption and reproductive outcomes show conflicting results, and most studies have used maternal self-reported caffeine consumption to estimate fetal exposure. This study (n=1,606) was specifically designed to test the association of caffeine and its primary metabolites in umbilical cord blood with intrauterine growth restriction (IUGR). Pregnant women were recruited from 56 obstetric practices and 15 clinics affiliated with six hospitals in Connecticut and Massachusetts between September 1996 and January 2000. In an adjusted model including caffeine only, levels in all quartiles were associated with reduced risk of IUGR. In adjusted analyses including paraxanthine and caffeine, serum paraxanthine levels in the highest quartile were associated with increased risk of IUGR (adjusted odds ratio=3.29, 95% confidence interval: 1.17, 9.22); caffeine remained protective. These conflicting findings suggest that cytochrome P-450 1A2 (CYP1A2) metabolic activity may be associated with IUGR, so the ratio of paraxanthine to caffeine was then modeled. The likelihood of IUGR increased 21% for every one standard deviation change in the ratio (adjusted odds ratio=1.21, 95% confidence interval: 1.07, 1.37), suggesting that CYP1A2 activity, and not the absolute levels of paraxanthine, influences fetal growth. No associations were observed between caffeine or any metabolites and preterm delivery.

  13. Augmented oxygen-mediated transcriptional activation of cytochrome P450 (CYP)1A expression and increased susceptibilities to hyperoxic lung injury in transgenic mice carrying the human CYP1A1 or mouse 1A2 promoter in vivo.

    PubMed

    Jiang, Weiwu; Couroucli, Xanthi I; Wang, Lihua; Barrios, Roberto; Moorthy, Bhagavatula

    2011-04-01

    Supplemental oxygen administration is frequently administered to pre-term and term infants having pulmonary insufficiency. However, hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Cytochrome P450 (CYP)A enzymes have been implicated in hyperoxic lung injury. In this study, we tested the hypothesis that hyperoxia induces CYP1A1 and 1A2 enzymes by transcriptional activation of the corresponding promoters in vivo, and transgenic mice expressing the human CYP1A1 or the mouse 1A2 promoter would be more susceptible to hyperoxic lung injury than wild type (WT) mice. Adult WT (CD-1) (12week-old) mice, transgenic mice carrying a 10kb human CYP1A1 promoter and the luciferase (luc) reporter gene (CYP1A1-luc), or mice expressing the mouse CYP1A2 promoter (CYP1A2-luc) were maintained in room air or exposed to hyperoxia for 24-72h. Hyperoxia exposure of CYP1A1-luc mice for 24 and 48h resulted in 2.5- and 1.25-fold increases, respectively, in signal intensities, compared to room air controls. By 72h, the induction had declined to control levels. CYP1A2-luc mice also showed enhanced luc expression after 24-48h, albeit to a lesser extent than those expressing the CYP1A1 promoter. Also, these mice showed decreased levels of endogenous CYP1A1 and 1A2 expression after prolonged hyperoxia, and were also more susceptible to lung injury than similarly exposed WT mice, with CYP1A2-luc mice showing the greatest injury. Our results support the hypothesis that hyperoxia induces CYP1A enzymes by transcriptional activation of its corresponding promoters, and that decreased endogenous expression of these enzymes contribute to the increased susceptibilities to hyperoxic lung injury in the transgenic animals. In summary, this is the first report providing direct evidence of hyperoxia-mediated induction of CYP1A1 and CYP1A2 expression in vivo by mechanisms entailing transcriptional activation of the corresponding promoters, a phenomenon that has

  14. Variability of cytochrome P450 1A2 activity over time in young and elderly healthy volunteers

    PubMed Central

    Simon, T; Becquemont, L; Hamon, B; Nouyrigat, E; Chodjania, Y; Poirier, J M; Funck-Brentano, C; Jaillon, P

    2001-01-01

    Aims To assess the age-associated changes over time of plasma paraxanthine/caffeine (PAX/CAF) ratios used as a probe for CYP1A2 activity. Methods Intraindividual and interindividual variabilities in PAX/CAF ratio were compared by phenotyping with caffeine, 16 young and 16 elderly healthy subjects on five occasions. Results PAX/CAF ratio variability was comparable regardless of age (intraindividual CV: 17.6 ± 6% and 16.2 ± 5.9%, interindividual CV: 48.1 ± 2.9% and 42.7 ± 3.6% in young and elderly, respectively). The PAX/CAF ratio was lower in elderly than in young subjects (95% CI for the difference: 0.004, 0.32) but the difference was not significant in nonsmokers compared separately. Conclusions The variability over time of the PAX/CAF ratio is not influenced by age. PMID:11736870

  15. Detection of a novel cytochrome P-450 1A2 polymorphism (F21L) in Chinese.

    PubMed

    Huang, J D; Guo, W C; Lai, M D; Guo, Y L; Lambert, G H

    1999-01-01

    Despite a wide interindividual variation of cytochrome P-450 1A2 (CYP1A2) activity, genetic polymorphism of CYP1A2 has not been reported. By amplification of exons of CYP1A2 by polymerase chain reaction in eight Chinese subjects, the polymerase chain reaction products were directly sequenced. One subject showed heterozygous C2866-->G (Phe21-->Leu) polymorphism. DNA from 157 Chinese subjects (104 polychlorinated biphenyl-exposed subjects and 53 control subjects) was screened for polymorphism by single-strand conformation polymorphism method and MboII endonuclease digestion. Only 1 of 157 samples showed another heterozygous C2866-->G mutation. The subject was previously exposed to polychlorinated biphenyl and showed a value of 3.5% in the caffeine breath test. The value is not significantly higher than the mean value of polychlorinated biphenyl-exposed subjects (3.12 +/- 0.29%, mean +/- S.E.M.). The incidence of the point mutation in these Chinese subjects is less than 1%. The prevalence of the F21L mutation in other ethnic groups and its effect on the metabolic activity of CYP1A2 remain to be further evaluated. PMID:9884316

  16. Inhibition of CYP3A4 and CYP1A2 b Aegle marmelos and its constituents

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aegle marmelos (bael) is a popular tree in India and other Southeast Asian countries. The fruit is usually consumed as dried, fresh or juice and is reported to have a high nutritional value and many perceived health benefits. Despite of the edible nature and therapeutic properties of A. marmelos, no...

  17. CYP1A2 IS NOT REQUIRED FOR 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIOXIN-INDUCED IMMUNOSUPPRESSION

    EPA Science Inventory

    ABSTRACT
    One of the most sensitive and reproducible immunotoxic endpoints of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) exposure is suppression of the antibody response to sheep red blood cells (SRBCs) in mice. Immunosuppression occurs in concert with hepatomegaly and associ...

  18. Inhibition of cytochrome P450 1A2-mediated metabolism and production of reactive oxygen species by heme oxygenase-1 in rat liver microsomes.

    PubMed

    Reed, James R; Cawley, George F; Backes, Wayne L

    2011-01-01

    Heme oxygenase-1 (HO-1) is induced in most cell types by many forms of environmental stress and is believed to play a protective role in cells exposed to oxidative stress. Metabolism by cytochromes P450 (P450) is highly inefficient as the oxidation of substrate is associated with the production of varying proportions of hydrogen peroxide and/or superoxide. This study tests the hypothesis that heme oxygenase-1 (HO-1) plays a protective role against oxidative stress by competing with P450 for binding to the common redox partner, the NADPH P450 reductase (CPR) and in the process, diminishing P450 metabolism and the associated production of reactive oxygen species (ROS). Liver microsomes were isolated from uninduced rats and rats that were treated with cadmium and/or β-napthoflavone (BNF) to induce HO-1 and/or CYP1A2. HO-1 induction was associated with slower rates of metabolism of the CYP1A2-specific substrate, 7-ethoxyresorufin. Furthermore, HO-1 induction also was associated with slower rates of hydrogen peroxide and hydroxyl radical production by microsomes from rats induced for CYP1A2. The inhibition associated with HO-1 induction was not dependent on the addition of heme to the microsomal incubations. The effects of HO-1 induction were less dramatic in the absence of substrate for CYP1A2, suggesting that the enzyme was more effective in inhibiting the CYP1A2-related activity than the CPR-related production of superoxide (that dismutates to form hydrogen peroxide).

  19. Differential inducing effect of benzo[a]pyrene on gene expression and enzyme activity of cytochromes P450 1A1 and 1A2 in Sprague-Dawley and Wistar rats.

    PubMed

    Floreani, Maura; Gabbia, Daniela; Barbierato, Massimo; DE Martin, Sara; Palatini, Pietro

    2012-01-01

    The objective of this study was to compare RT-PCR, Western blot and determination of enzyme activity in the assessment of the induction of cytochromes P450 (CYPs) 1A1 and 1A2 by benzo[a]pyrene (BaP) in Sprague-Dawley and Wistar rats. Inhibition studies and kinetic analyses confirmed literature data indicating that methoxyresorufin is a specific CYP1A2 substrate in both uninduced and BaP-treated rats, whereas ethoxyresorufin is a specific CYP1A1 substrate only in BaP-treated rats. BaP treatment increased mRNA and protein expressions of both CYP1A enzymes to a greater extent in Wistar than Sprague-Dawley rats. It consistently caused a higher increase in mRNA and protein expression of the aryl hydrocarbon receptor in the former rats. By contrast, CYP1A2 enzyme activity was much more markedly increased in Sprague-Dawley than Wistar rats and CYP1A1 activity was induced to similar levels. A BaP-induced increase in the turnover number of CYP1A enzymes in Sprague-Dawley rats, relative to Wistar rats, may provide a plausible explanation for the differential effect of BaP on gene expression and enzyme activity. These results have methodological implications, since they show that RT-PCR and Western blot may not provide a quantitative measure of induction of CYP1A activity, which is the actual measure of the change in CYP1A-mediated metabolism.

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

    PubMed

    Reed, James R; dela Cruz, Albert Leo N; Lomnicki, Slawo M; Backes, Wayne L

    2015-12-01

    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 reductase 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.

  1. EFFECT OF METALS ON POLYCYCLIC AROMATIC HYDROCARBON INDUCTION OF CYP1A1 AND CYP1A2 IN HUMAN HEPATOCYTES CULTURES. (R827180)

    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...

  2. EFFECT OF METALS ON POLYCYCLIC AROMATIC HYDROCARBON INDUCTION OF CYP1A1 AND CYP1A2 IN HUMAN HEPATOCYTE CULTURES. (R827180)

    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...

  3. In Silico Docking of Ligands to Drug Oxidation Enzymes Cytochrome P450 3A4 and Cytochrome P450 1A2.

    NASA Astrophysics Data System (ADS)

    Smith, David; Guglielmon, Jonathan; Glenn, Marsch; Peter, Guengerich F.

    2009-03-01

    Cytochrome P450 3A4 (CYP3A4) and Cytochrome P450 1A2 (CYP1A2) oxidize most drugs in humans. Protein modeling toolkits from OpenEye Scientific Software were used to examine the interaction of drug substrates with CYP3A4 and CYP1A2. Conformers and partial atomic charges were generated for each drug molecule. User-defined volumes were defined around CYP3A4 and CYP1A2 active sites. Ligands were docked assuming protein and substrates as rigid bodies. To assess rigid docking accuracy, x-ray diffraction coordinates of CYP3A4-erythromycin and CYP3A4-metyrapone complexes were obtained. Rigid re-docking of erythromycin and metyrapone into CYP3A4 yielded poses similar to the crystal structures. Rigid docking revealed two other energetically-favorable CYP3A4-metyrapone poses. The best poses were obtained by using all the Open Eye scoring functions. Optimization of protein-ligand interactions within 5-10 Angstroms of the docked ligand was then performed using the Merck Molecular Force Field in which the protein was assumed to be flexible and the ligand to be rigid. Nearby protein residues pulled slightly closer to the substrate, reducing the volume of the active site.

  4. Efficient synthesis of frutinone A and its derivatives through palladium-catalyzed C - H activation/carbonylation.

    PubMed

    Shin, Yongje; Yoo, Changho; Moon, Youngtaek; Lee, Yunho; Hong, Sungwoo

    2015-04-01

    Frutinone A, a biologically active ingredient of an antimicrobial herbal extract, demonstrates potent inhibitory activity towards the CYP1A2 enzyme. A three-step total synthesis of frutinone A with an overall yield of 44 % is presented. The construction of the chromone-annelated coumarin core was achieved through palladium-catalyzed CH carbonylation of 2-phenolchromones. The straightforward synthetic route allowed facile substitutions around the frutinone A core and thus rapid exploration of the structure-activity relationship (SAR) profile of the derivatives. The inhibitory activity of the synthesized frutinone A derivatives were determined for CYP1A2, and ten compounds exhibited one-to-two digit nanomolar inhibitory activity towards the CYP1A2 enzyme.

  5. Induction of cytochrome P450 1A2 by musk analogues and other inducing agents in rat liver.

    PubMed

    Iwata, N; Suzuki, K; Minegishi, K; Kawanishi, T; Hara, S; Endo, T; Takahashi, A

    1993-10-01

    We characterized the inducing effects of two musk analogues, musk xylene and musk ambrette, on phase I and phase II drug-metabolizing enzymes in rat liver and compared their effects with 3-methylcholanthrene, isosafrole and 2(3)-tertbutylhydroxyanisole (BHA) at 0.1 mmol/kg dose level. Musk xylene and isosafrole increased more efficiently the metabolic activation of 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1) to mutagen than that of benzo(a)pyrene. Musk ambrette increased both the activation of Glu-P-1 and benzo(a)pyrene to the same extent. Western blot analyses revealed that musk xylene, musk ambrette, isosafrole and BHA induced more strongly cytochrome P450 1A2 (CYP1A2) in microsomes than CYP1A1. 3-Methylcholanthrene induced CYP1A1 in preference to CYP1A2. On the other hand, all drugs except for 3-methylcholanthrene did not show remarkable increases in phase II enzyme activities, such as DT-diaphorase, glutathione S-transferase and UDP-glucuronyltransferase, at 0.1 mmol/kg dose level. These results show that musk xylene, musk ambrette, isosafrole and BHA at the dose level used in this study possess the potency to induce CYP1A2 without remarkable induction of CYP1A1 and phase II enzyme activities as observed for 3-methylcholanthrene, although they have been considered to induce both phase I and phase II drug-metabolizing enzymes at higher doses.

  6. Effect of vanillin and ethyl vanillin on cytochrome P450 activity in vitro and in vivo.

    PubMed

    Chen, Xiao-min; Wei, Min; Zhang, Hai-mou; Luo, Cheng-hao; Chen, Yi-kun; Chen, Yong

    2012-06-01

    Food safety is of extreme importance to human health. Vanillin and ethyl vanillin are the widely used food additives and spices in foods, beverages, cosmetics and drugs. The objective of the present work was to evaluate the impact of vanillin and ethyl vanillin on the activities of CYP2C9, CYP2E1, CYP3A4, CYP2B6 and CYP1A2 in human liver microsomes (HLM) in vitro, and impact on the activities of CYP1A2, CYP2C, CYP3A and CYP2E1 in rat liver microsomes (RLM) in vivo. The in vitro results demonstrated that vanillin and ethyl vanillin had no significant effect on the activity of five human CYP450 enzymes with concentration ranged from 8 to 128 μM. However, after rats were orally administered vanillin or ethyl vanillin once a day for seven consecutive days, CYP2E1 activity was increased and CYP1A2 activity was decreased in RLM. The in vivo results revealed that drug interaction between vanillin/ethyl vanillin and the CYP2E1/CYP1A2-metabolizing drugs might be possible, and also suggested that the application of the above additives in foods and drugs should not be unlimited so as to avoid the adverse interaction.

  7. Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1

    PubMed Central

    Milichovský, Jan; Bárta, František; Schmeiser, Heinz H.; Arlt, Volker M.; Frei, Eva; Stiborová, Marie; Martínek, Václav

    2016-01-01

    Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are NAD(P)H:quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using 32P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast

  8. INFLUENCE OF TYPE II DIABETES, OBESITY, AND EXPOSURE TO 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIOXIN (TCDD) EXPOSURE ON THE EXPRESSION OF HEPATIC CYP1A2 IN A MURIN MODEL OF TYPE II DIABETES

    EPA Science Inventory

    Influence of type II diabetes, obesity and exposure 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on the expression of hepatic CYPIA2 in a murine model of type II diabetes. SJ Godin', VM Richardson2, JJ Diliberto2, LS Birnbaum', MJ DeVito2; 'Curriculum In Toxicology, UNC-CH...

  9. COMPARISON OF OVERALL METABOLISM OF 1,2,3,7,8-PENTACHLORODIBENZO-P-DIOXIN (PECDD) IN CYP1A2(-L-)KNOCKOUT (KO) AND C57BL/6N PARENTAL STRAINS OF MICE

    EPA Science Inventory

    Assessment of immune responses to Penicillium chrysogenum and characterization of its allergens

    Yongjoo Chung1, Michael E Viana2, Lisa B Copeland3, and MaryJane K Selgrade3, Marsha D W Ward3. 1 UNC, SPH, Chapel Hill, NC, 2NCSU, CVM, Raleigh, NC, 3US EPA, ORD, NHEERL, RTP,...

  10. Rough Set Theory as an Interpretable Method for Predicting the Inhibition of Cytochrome P450 1A2 and 2D6.

    PubMed

    Burton, Julien; Petit, Joachim; Danloy, Emeric; Maggiora, Gerald M; Vercauteren, Daniel P

    2013-07-01

    Early prediction of ADME properties such as the cytochrome P450 (CYP) mediated drug-drug interactions is an important challenge in the drug discovery area. In this study, we propose to couple an original data mining approach based on Rough Set Theory (RST) to a structural description of molecules. The latter was achieved by using two types of structural keys: (1) the MACCS keys and (2) a set of five in-house fingerprints based on properties of the electron density distributions of chemical groups. The compounds considered are involved in the inhibition of CYP1A2 and CYP2D6. RST allowed the extraction of rules further used as classifiers to predict the inhibitory profile of an independent set of molecules. The results reached prediction accuracies of 90.6 and 88.2 % for CYP1A2 and CYP2D6, respectively. In addition, these classifiers were analyzed to determine which structural fragments were most used for building the rules, revealing relationships between the occurrence of particular molecular fragments and CYP inhibition. The results assessed RST as a suitable tool to build strongly predictive models and infer structure-activity rules associated with potency.

  11. [Clinical survey of tizanidine-induced adverse effects--impact of concomitant drugs providing cytochrome P450 1A2 modification--].

    PubMed

    Momo, Kenji; Homma, Masato; Matsumoto, Sayaka; Sasaki, Tadanori; Kohda, Yukinao

    2013-01-01

    The drug-drug interactions of tizanidine and cytochrome (CYP) P450 1A2 inhibitors, which potentially alter the hepatic metabolism of tizanidine, were investigated by retrospective survey of medical records with regard to prescription. One thousand five hundred sixty-three patients treated with tizanidine at University of Tsukuba Hospital were investigated. Of those, 713 patients (45.6%) were treated with coadministration of tizanidine and CYP1A2 inhibitors (37 drugs). The patients who received a combination of tizanidine and CYP1A2 inhibitors were characterized as elderly, having multiple diseases, and taking a large number of comedications (over 10 drugs) for a long period as compared with the patients who did not receive CYP1A2 inhibitors. Tizanidine-induced adverse effects were examined in 100 patients treated with coadministration of tizanidine and 8 CYP1A2 inhibitors. Adverse effects (e.g., drowsiness: 10 patients; low blood pressure: 9 patients; low heart rate: 9 patients) were observed in 23 patients (23%) 8±10 days after CYP1A2 inhibitors were coadministered. The patients with tizanidine-induced adverse effects were of older age (64.3±9.8 vs. 57.5±18.1 years, p<0.05) and received a higher daily dose of tizanidine (3.00±0.74 vs. 2.56±0.86 mg/day, p<0.05) than the patients without adverse effects. The present results suggest that coadministration of tizanidine and CYP1A2 inhibitors enhances tizanidine-induced adverse effects, especially in elderly patients treated with a higher dose of tizanidine.

  12. Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants.

    PubMed

    Azab, Ehab; Hegazy, Ahmad K; El-Sharnouby, Mohamed E; Abd Elsalam, Hassan E

    2016-01-01

    The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants. PMID:26771455

  13. Linear Interaction Energy Based Prediction of Cytochrome P450 1A2 Binding Affinities with Reliability Estimation

    PubMed Central

    Capoferri, Luigi; Verkade-Vreeker, Marlies C. A.; Buitenhuis, Danny; Commandeur, Jan N. M.; Pastor, Manuel; Vermeulen, Nico P. E.; Geerke, Daan P.

    2015-01-01

    Prediction of human Cytochrome P450 (CYP) binding affinities of small ligands, i.e., substrates and inhibitors, represents an important task for predicting drug-drug interactions. A quantitative assessment of the ligand binding affinity towards different CYPs can provide an estimate of inhibitory activity or an indication of isoforms prone to interact with the substrate of inhibitors. However, the accuracy of global quantitative models for CYP substrate binding or inhibition based on traditional molecular descriptors can be limited, because of the lack of information on the structure and flexibility of the catalytic site of CYPs. Here we describe the application of a method that combines protein-ligand docking, Molecular Dynamics (MD) simulations and Linear Interaction Energy (LIE) theory, to allow for quantitative CYP affinity prediction. Using this combined approach, a LIE model for human CYP 1A2 was developed and evaluated, based on a structurally diverse dataset for which the estimated experimental uncertainty was 3.3 kJ mol-1. For the computed CYP 1A2 binding affinities, the model showed a root mean square error (RMSE) of 4.1 kJ mol-1 and a standard error in prediction (SDEP) in cross-validation of 4.3 kJ mol-1. A novel approach that includes information on both structural ligand description and protein-ligand interaction was developed for estimating the reliability of predictions, and was able to identify compounds from an external test set with a SDEP for the predicted affinities of 4.6 kJ mol-1 (corresponding to 0.8 pKi units). PMID:26551865

  14. Carbon-Carbon Bond Cleavage in Activation of the Prodrug Nabumetone

    PubMed Central

    Varfaj, Fatbardha; Zulkifli, Siti N. A.; Park, Hyoung-Goo; Challinor, Victoria L.; De Voss, James J.

    2014-01-01

    Carbon-carbon bond cleavage reactions are catalyzed by, among others, lanosterol 14-demethylase (CYP51), cholesterol side-chain cleavage enzyme (CYP11), sterol 17β-lyase (CYP17), and aromatase (CYP19). Because of the high substrate specificities of these enzymes and the complex nature of their substrates, these reactions have been difficult to characterize. A CYP1A2-catalyzed carbon-carbon bond cleavage reaction is required for conversion of the prodrug nabumetone to its active form, 6-methoxy-2-naphthylacetic acid (6-MNA). Despite worldwide use of nabumetone as an anti-inflammatory agent, the mechanism of its carbon-carbon bond cleavage reaction remains obscure. With the help of authentic synthetic standards, we report here that the reaction involves 3-hydroxylation, carbon-carbon cleavage to the aldehyde, and oxidation of the aldehyde to the acid, all catalyzed by CYP1A2 or, less effectively, by other P450 enzymes. The data indicate that the carbon-carbon bond cleavage is mediated by the ferric peroxo anion rather than the ferryl species in the P450 catalytic cycle. CYP1A2 also catalyzes O-demethylation and alcohol to ketone transformations of nabumetone and its analogs. PMID:24584631

  15. Significant inhibitory impact of dibenzyl trisulfide and extracts of Petiveria alliacea on the activities of major drug-metabolizing enzymes in vitro: An assessment of the potential for medicinal plant-drug interactions.

    PubMed

    Murray, J; Picking, D; Lamm, A; McKenzie, J; Hartley, S; Watson, C; Williams, L; Lowe, H; Delgoda, R

    2016-06-01

    Dibenzyl trisulfide (DTS) is the major active ingredient expressed in Petiveria alliacea L., a shrub widely used for a range of conditions, such as, arthritis, asthma and cancer. Given its use alone and concomitantly with prescription medicines, we undertook to investigate its impact on the activities of important drug metabolizing enzymes, the cytochromes P450 (CYP), a key family of enzymes involved in many adverse drug reactions. DTS and seven standardized extracts from the plant were assessed for their impact on the activities of CYPs 1A2, 2C19, 2C9, 2D6 and 3A4 on a fluorometric assay. DTS revealed significant impact against the activities of CYPs 1A2, 2C19 and 3A4 with IC50 values of 1.9, 4.0 and 3.2μM, respectively, which are equivalent to known standard inhibitors of these enzymes (furafylline, and tranylcypromine), and the most potent interaction with CYP1A2 displayed irreversible enzyme kinetics. The root extract, drawn with 96% ethanol (containing 2.4% DTS), displayed IC50 values of 5.6, 3.9 and 4.2μg/mL respectively, against the same isoforms, CYPs 1A2, 2C19 and 3A4. These investigations identify DTS as a valuable CYP inhibitor and P. alliacea as a candidate plant worthy of clinical trials to confirm the conclusions that extracts yielding high DTS may lead to clinically relevant drug interactions, whilst extracts yielding low levels of DTS, such as aqueous extracts, are unlikely to cause adverse herb-drug interactions.

  16. Significant inhibitory impact of dibenzyl trisulfide and extracts of Petiveria alliacea on the activities of major drug-metabolizing enzymes in vitro: An assessment of the potential for medicinal plant-drug interactions.

    PubMed

    Murray, J; Picking, D; Lamm, A; McKenzie, J; Hartley, S; Watson, C; Williams, L; Lowe, H; Delgoda, R

    2016-06-01

    Dibenzyl trisulfide (DTS) is the major active ingredient expressed in Petiveria alliacea L., a shrub widely used for a range of conditions, such as, arthritis, asthma and cancer. Given its use alone and concomitantly with prescription medicines, we undertook to investigate its impact on the activities of important drug metabolizing enzymes, the cytochromes P450 (CYP), a key family of enzymes involved in many adverse drug reactions. DTS and seven standardized extracts from the plant were assessed for their impact on the activities of CYPs 1A2, 2C19, 2C9, 2D6 and 3A4 on a fluorometric assay. DTS revealed significant impact against the activities of CYPs 1A2, 2C19 and 3A4 with IC50 values of 1.9, 4.0 and 3.2μM, respectively, which are equivalent to known standard inhibitors of these enzymes (furafylline, and tranylcypromine), and the most potent interaction with CYP1A2 displayed irreversible enzyme kinetics. The root extract, drawn with 96% ethanol (containing 2.4% DTS), displayed IC50 values of 5.6, 3.9 and 4.2μg/mL respectively, against the same isoforms, CYPs 1A2, 2C19 and 3A4. These investigations identify DTS as a valuable CYP inhibitor and P. alliacea as a candidate plant worthy of clinical trials to confirm the conclusions that extracts yielding high DTS may lead to clinically relevant drug interactions, whilst extracts yielding low levels of DTS, such as aqueous extracts, are unlikely to cause adverse herb-drug interactions. PMID:27105957

  17. Modulatory effects of extracts of vinegar-baked Radix Bupleuri and saikosaponins on the activity of cytochrome P450 enzymes in vitro.

    PubMed

    Yu, Tongya; Chen, Xianzhi; Wang, Yinjie; Zhao, Ruizhi; Mao, Shirui

    2014-10-01

    1. In this article, the modulatory effects of extracts from vinegar-baked Radix Bupleuri (VBRB) and saikosaponins on the activity of CYP1A2, CYP2C9 and CYP3A4 were investigated in vitro. 2. Microsomal in vitro incubation method was utilized to simulate metabolic reaction under physiological environment by incubating the marker with liver microsomes in the absence or presence of VBRB and saikosaponins. The contents of 4-acetamidophenol, 6β-hydroxyltestosterone and 4-hydroxydiclofenac, the metabolites of phenacetin, testosterone and diclofenac, which were selected as specific probe drugs of CYP1A2, CYP2C9 and CYP3A4, respectively, were analyzed by high-performance liquid chromatography. 3. The production of the metabolites was incubation time dependent. The modulatory effects of different VBRB extracts and saikosaponins on CYP isoforms increased with concentration. Among all the extracts studied, BC1 has a strong inhibition effect compared to the three CYP isoforms tested, while the others have only significant inhibition on the activity of CYP2C9. 4. This in vitro study demonstrated that various extracts of VBRB tested in this study have negligible potential to interfere with CYP1A2- and CYP3A4-metabolized drugs; risk of herb-drug interaction might occur when VBRB is concurrently taken with CYP2C9 substrates.

  18. Induction of cytochromes P450 1A1 and 1A2 by tanshinones in human HepG2 hepatoma cell line

    SciTech Connect

    Zhang Rong; Sun Jianguo; Ma Liping; Wu Xiaolan; Pan Guoyu; Hao Haiping; Zhou Fang; Jiye, A; Liu Changhui; Ai Hua; Shang Lili; Gao Haiyan; Peng Ying; Wan Ping; Wu Hui; Wang Guangji

    2011-04-01

    Diterpenoid tanshinones including tanshinone IIA (TIIA), cryptotanshinone (CTS), tanshinone I (TI) and dihydrotanshinone I (DHTI) are the major bioactive components from Danshen. The major aim of our present study was to investigate the induction potential of these four main components of tanshinones (TIIA, CTS, TI, and DHTI) on the expression of CYP1A1 and CYP1A2 in HepG2 cells. Our results showed that all of these four tanshinones caused a significant time- and concentration-dependent increase in the amount of CYP1A1/2 expression in HepG2 cells. These induction effects were further characterized through transcriptional regulation: the induction of CYP1A1/2 mRNA level by tanshinones was completely blocked by the transcription inhibitor actinomycin D; the expression of CYP1A1/2 heterogeneous nuclear RNA was induced by tanshinone treatment; and CYP1A1 mRNA stability was not influenced by these tanshinones. Interestingly, tanshinones plus B[a]P produced additive/synergistic effect on CYP1A1/2 induction. In addition, the tanshinone-induced CYP1A1/2 expression was abolished by the aryl hydrocarbon receptor (AhR) antagonist resveratrol, suggesting an AhR dependent transcription mechanism. In the reporter gene assay, while TI and DHTI significantly induced AhR-dependent luciferase activity, TIIA and CTS failed to induce this activity. Collectively, the tanshinones could induce CYP1A1 and CYP1A2 expression through transcriptional activation mechanism and exert differential effects on activating AhR in HepG2 cells. Our findings suggest that rational administration of tanshinones should be considered with respect to their effect on AhR and CYP1A1/2 expression.

  19. Quantum Mechanics/Molecular Mechanics Modeling of Drug Metabolism: Mexiletine N-Hydroxylation by Cytochrome P450 1A2.

    PubMed

    Lonsdale, Richard; Fort, Rachel M; Rydberg, Patrik; Harvey, Jeremy N; Mulholland, Adrian J

    2016-06-20

    The mechanism of cytochrome P450(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (R)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM(B3LYP-D)/MM(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynamics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but this is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism.

  20. Quantum Mechanics/Molecular Mechanics Modeling of Drug Metabolism: Mexiletine N-Hydroxylation by Cytochrome P450 1A2.

    PubMed

    Lonsdale, Richard; Fort, Rachel M; Rydberg, Patrik; Harvey, Jeremy N; Mulholland, Adrian J

    2016-06-20

    The mechanism of cytochrome P450(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (R)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM(B3LYP-D)/MM(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynamics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but this is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism. PMID:27064685

  1. Fruits and vegetables protect against the genotoxicity of heterocyclic aromatic amines activated by human xenobiotic-metabolizing enzymes expressed in immortal mammalian cells.

    PubMed

    Platt, K L; Edenharder, R; Aderhold, S; Muckel, E; Glatt, H

    2010-12-21

    Heterocyclic aromatic amines (HAAs) can be formed during the cooking of meat and fish at elevated temperatures and are associated with an increased risk for cancer. On the other hand, epidemiological findings suggest that foods rich in fruits and vegetables can protect against cancer. In the present study three teas, two wines, and the juices of 15 fruits and 11 vegetables were investigated for their protective effect against the genotoxic effects of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). To closely mimic the enzymatic activation of these HAAs in humans, genetically engineered V79 Chinese hamster fibroblasts were employed that express human cytochrome P450-dependent monooxygenase (hCYP) 1A2 (responsible for the first step of enzymatic activation) and human N(O)-acetyltransferase (hNAT) 2*4 or human sulfotransferase (hSULT)1A1*1 (responsible for the second step of enzymatic activation): V79-hCYP1A2-hNAT2*4 for IQ activation and V79-hCYP1A2-hSULT1A1*1 for PhIP activation. HAA genotoxicity was determined by use of the comet assay. Black, green and rooibos tea moderately reduced the genotoxicity of IQ (IC(50)=0.8-0.9%), whereas red and white wine were less active. From the fruit juices, sweet cherry juice exhibited the highest inhibitory effect on IQ genotoxicity (IC(50)=0.17%), followed by juices from kiwi fruit, plum and blueberry (IC(50)=0.48-0.71%). The juices from watermelon, blackberry, strawberry, black currant, and Red delicious apple showed moderate suppression, whereas sour cherry, grapefruit, red currant, and pineapple juices were only weakly active. Granny Smith apple juice and orange juice proved inactive. Of the vegetable juices, strong inhibition of IQ genotoxicity was only seen with spinach and onion juices (IC(50)=0.42-0.54%). Broccoli, cauliflower, beetroot, sweet pepper, tomato, chard, and red-cabbage juices suppressed IQ genotoxicity only moderately, whereas cucumber juice was

  2. Role of Metabolic Enzymes P450 (CYP) on Activating Procarcinogen and their Polymorphisms on the Risk of Cancers.

    PubMed

    He, Xin; Feng, Shan

    2015-01-01

    Cytochrome P450 (CYP450) enzymes are the most important metabolizing enzyme family exists among all organs. Apart from their role in the deactivation of most endogenous compounds and xenobiotics, they also mediate most procarcinogens oxidation to ultimate carcinogens. There are several modes of CYP450s activation of procarcinogens. 1) Formation of epoxide and diol-epoxides intermediates, such as CYP1A1 and CYP1B1 mediates PAHs oxidation to epoxide intermediates; 2) Formation of diazonium ions, such as CYP2A6, CYP2A13 and CYP2E1 mediates activation of most nitrosamines to unstable metabolites, which can rearrange to give diazonium ions. 3) Formation of reactive semiquinones and quinines, such as CYP1A1 and CYP1B1 transformation of estradiol to catechol estrogens, subsequently formation semiquinones; 4) Formation of toxic O-esterification, such as CYP1A1 and CYP1A2 metabolizes PhIP to N(2)-acetoxy-PhIP and N(2)-sulfonyloxy-PhIP, which are carcinogenic metabolites. 5) Formation of free radical, such as CYP2E1 is involved in activation tetrachloromethane to free radicals. While for CYP2B6 and CYP2D6, only a minor role has been found in procarcinogens activation. In addition, as the gene polymorphisms reflected, the polymorphisms of CYP1A1 (-3801T/C and -4889A/G), CYP1A2 (- 163C/A and -2467T/delT), CYP1B1 (-48G/C, -119G/T and -432G/C), CYP2E1 (-1293G/C and -1053 C/T) have been associated with an increased risk of lung cancer. The polymorphisms CYP1A1 (-3801T/C and -4889A/G), and CYP2E1 (PstI/Rsa and 9-bp insertion) have an association with higher risk colon cancers, whereas CYP1A2 (-163C/A and -3860G/A) polymorphism is found to be among the protective factors. The polymorphisms CYP1A1 (-3801T/C and -4889A/G), CYP1B1 -432G/C, CYP2B6 (-516G/T and -785A/G) may increase the risk of breast cancer. In conclusion, CYP1A1, CYP1A2, CYP1B1, CYP2A6, and CYP2E1 are responsible for most of the procarcinogens activation, and their gene polymorphisms are associated with the risk of

  3. Effects of Seijo-bofu-to, a traditional Japanese herbal medicine containing furanocoumarin derivatives, on the drug-metabolizing enzyme activities in healthy male volunteers.

    PubMed

    Saruwatari, Junji; Takashima, Ayaka; Yoshida, Kousuke; Soraoka, Hiromi; Ding, Tong-Bin; Uchiyashiki, Yoshihiro; Tsuda, Yoshiyuki; Imamura, Motoki; Oniki, Kentaro; Miyata, Keishi; Nakagawa, Kazuko

    2014-10-01

    Seijo-bofu-to, a traditional medicine used to treat acne in Asian countries, contains twelve herbal components, including Angelica dahurica root, a source of furanocoumarin derivatives. In this study, we investigated potential herb-drug interactions of seijo-bofu-to in healthy male volunteers. Thirty-two young, healthy, non-smoking males were assessed for the baseline activity of cytochrome P450 (CYP) 1A2, CYP3A, CYP2D6, N-acetyltransferase 2 and xanthine oxidase according to the urinary metabolic indices of 8-hr urine samples collected after the administration of a 150-mg dose of caffeine and a 30-mg dose of dextromethorphan, and the ratio of urinary excretion of 6β-hydroxycortisol to cortisol. Thereafter, the volunteers received 3.75 g of seijo-bofu-to twice daily for 7 days and underwent the same tests on post-dose day 7. The geometric mean ratio of the CYP1A2 activity on day 7 to that observed at baseline was 0.66 (95% CI, 0.55-0.79, p = 0.001). The geometric mean phenotypic indices for CYP3A, CYP2D6, N-acetyltransferase 2 and xanthine oxidase on day 7 did not differ from the baseline values. The findings of the present study suggest that seijo-bofu-to may inhibit the activity of CYP1A2, whereas it is unlikely to participate in herb-drug interactions involving medications predominantly metabolized by CYP3A, CYP2D6, N-acetyltransferase 2 or xanthine oxidase.

  4. Inhibition of transforming growth factor-beta-induced liver fibrosis by a retinoic acid derivative via the suppression of Col 1A2 promoter activity.

    PubMed

    Yang, Kun-Lin; Chang, Wen-Teng; Hung, Kuo-Chen; Li, Eric I C; Chuang, Chia-Chang

    2008-08-22

    Transforming growth factor-beta1 (TGF-beta1) mediates expression of collagen 1A2 (Col 1A2) gene via a synergistic cooperation between Smad2/Smad3 and Sp1, both act on the Col 1A2 gene promoter. In our previous study, we reported that a retinoic acid derivative obtained from Phellinus linteus (designated PL) antagonizes TGF-beta-induced liver fibrosis through regulation of ROS and calcium influx. In this continuing study we seek further the effect of PL on the Smad signaling pathway. We used a Col 1A2 promoter-luciferase construct to study the action of PL on Smad through TGF-beta. We found that PL decreases the promoter activity of Col 1A2, hinders the translocalization of phosphorylated Smad2/3-Smad 4 complex from cytosol into nucleus and inhibits Sp1 binding activity. These results suggest that PL inhibits TGF-beta1-induced Col 1A2 promoter activity through blocking ROS and calcium influx as well as impeding Sp1 binding and translocalization of pSmad 2/3-Smad4 complex into nucleus.

  5. Species differences in intestinal metabolic activities of cytochrome P450 isoforms between cynomolgus monkeys and humans.

    PubMed

    Nishimuta, Haruka; Sato, Kimihiko; Mizuki, Yasuyuki; Yabuki, Masashi; Komuro, Setsuko

    2011-06-01

    The oral bioavailability of some drugs is markedly lower in cynomolgus monkeys than in humans. One of the reasons for the low bioavailability in cynomolgus monkeys may be the higher metabolic activity of intestinal CYP3A; however, the species differences in intestinal metabolic activities of other CYP isoforms between cynomolgus monkeys and humans are not well known. In the present study, we investigated the intrinsic clearance (CL(int)) values in pooled intestinal microsomes from cynomolgus monkeys and humans using 25 substrates of human CYP1A2, CYP2J2, CYP2C, and CYP2D6. As in humans, intestinal CL(int) values of human CYP1A2 and CYP2D6 substrates in cynomolgus monkeys were low. On the other hand, intestinal CL(int) values of human CYP2J2 and CYP2C substrates in cynomolgus monkeys were greatly higher than those in humans. Using immunoinhibitory antibodies and chemical inhibitors, we showed that the higher intestinal CL(int) values of the human CYP2J2 and CYP2C substrates in cynomolgus monkeys might be caused by monkey CYP4F and CYP2C subfamily members, respectively. In conclusion, there is a possibility that the greatly higher metabolic activity of CYP2C and CYP4F in cynomolgus monkey intestine is one of the causes of the species difference of intestinal first-pass metabolism between cynomolgus monkeys and humans. PMID:21383522

  6. Effect of Radix Sophorae Flavescentis on activity of CYP450 isoforms in rats

    PubMed Central

    Chen, Lianguo; Cai, Jinzhang; Wang, Shuanghu; Hu, Lufeng; Yang, Xuezhi

    2015-01-01

    Kushen (Radix Sophorae Flavescentis) is the dried roots of Sophora Flavescens Ait, alkaloids and flavonoids are the main active constituents of Radix Sophorae Flavescentis. The influence of Radix Sophorae Flavescentis on the activities of CYP450 isoforms CYP2B6, CYP2C19, CYP1A2, CYP2C9, CYP3A4 and CYP2D6 were evaluated by cocktail method. The rats were randomly divided into Radix Sophorae Flavescentis group and control group. The Radix Sophorae Flavescentis group rats were given 5 g/kg Radix Sophorae Flavescentis decoction by intragastric administration. The six probe drugs (bupropion, omeprazole, phenacetin, tolbutamide, midazolam and metroprolol) were given to rats through intragastric administration, and the plasma concentration were determined by UPLC-MS/MS. The result of Radix Sophorae Flavescentis group compared to control group, there were statistical pharmacokinetics difference for omeprazole, phenacetin, tolbutamide and metroprolol. It indicated that the Radix Sophorae Flavescentis may induce the activities of CYP2D6, and inhibit of CYP2C19, CYP1A2 and CYP2C9 of rats. As other drugs are always used after Radix Sophorae Flavescentis, interactions between other drugs and Radix Sophorae Flavescentis undertake the risk of either diminished efficacy or adverse effects. This may give advising for reasonable drug use after Radix Sophorae Flavescentis. PMID:26885078

  7. Effects of suberoylanilide hydroxamic acid on rat cytochrome P450 enzyme activities.

    PubMed

    Lin, Kezhi; Zhang, Qingwei; Liu, Zezheng; Yang, Suping; Lin, Yingying; Wen, Congcong; Zheng, Yuancai

    2015-01-01

    Vorinostat (suberoylanilide hydroxamic acid, SAHA) is the first approved histone deacetylase (HDAC) inhibitor for the treatment of cutaneous T-cell lymphoma after progressive disease following two systemic therapies. The rats were randomly divided into SAHA groups (low, medium and high dosage) and control group. The SAHA group rats were given 12.3, 24.5, and 49 mg/kg SAHA, respectively, by continuous intragastric administration for 7 days. The influence of SAHA on the activities of CYP450 isoforms CYP2B6, CYP1A2, CYP2C19, CYP2D6 and CYP2C9 were evaluated by cocktail method, they were responsed by the changes of pharmacokinetic parameters of bupropion, phenacetin, tolbutamide, metroprolol and omeprazole. The five probe drugs were given to rats through intragastric administration, and the plasma concentration were determined by UPLC-MS/MS. The result of SAHA group compared to control group, there were statistical pharmacokinetics difference for bupropion, phenacetin, tolbutamide and metroprolol. Continuous intragastric administration for 7 days may induce the activities of CYP2C19 of rats, inhibit CYP1A2 and slightly inhibit CYP2B6 and CYP2D6 of rats. This may give advising for reasonable drug use after co-used with SAHA. The results indicated that drug co-administrated with SAHA may need dose adjustment. Furthermore, continuous intragastric administration of SAHA for 7 days, liver cell damaged, causing liver cell edema, in liver metabolism process.

  8. 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…

  9. Simultaneous determination of cytochrome P450 1A, 2A and 3A activities in porcine liver microsomes.

    PubMed

    Johansson, Monika; Tomankova, Jana; Li, Shengjie; Zamaratskaia, Galia

    2012-09-01

    The aim of this study was to develop a robust method for the simultaneous determination of the activities of three porcine CYP450 enzymes in hepatic microsomes. A cocktail consisting of three selective CYP450 probe substrates, 7-ethoxyresorufin (CYP1A), coumarin (CYP2A) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC; CYP3A), was incubated with porcine liver microsomes. The presence of 7-ethoxyresorufin appears to significantly influence the kinetics of coumarin hydroxylation and BFC O-debenzylation. These results indicate that the use of 7-ethoxyresorufin in substrate cocktails together with coumarin and BFC should be avoided.

  10. Vavilosides A1/A2-B1/B2, new furostane glycosides from the bulbs of Allium vavilovii with cytotoxic activity.

    PubMed

    Zolfaghari, Behzad; Sadeghi, Masoud; Troiano, Raffaele; Lanzotti, Virginia

    2013-04-01

    A phytochemical analysis of the bulbs of Allium vavilovii M. Pop. & Vved. was attained for the first time extensively, affording to the isolation of four new furostanol saponins, named vavilosides A1/A2-B1/B2 (1a/b-2a/2b), as two couple of isomers in equilibrium, together with ascalonicoside A1/A2 (3a/3b) and 22-O-methyl ascalonicoside A1/A2 (4a/4b), previously isolated from shallot, Allium ascalonicum. High concentrations of kaempferol, kaempferide, and kaempferol 4(I)-glucoside were also isolated. The chemical structures of the new compounds, established through a combination of extensive nuclear magnetic resonance, mass spectrometry and chemical analyses, were identified as (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-galactopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside A1), (25R)-furost-5(6)-en-1β,3β,22β,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-galactopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside A2), (25R)-furost-5(6)-en-1β,3β,22α,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-xylopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside B1), (25R)-furost-5(6)-en-1β,3β,22β,26-tetraol 1-O-α-L-rhamnopyranosyl-(1→2)-O-β-d-xylopyranosyl 26-O-α-L-rhamnopyranoside (vaviloside B2). The isolated saponins showed cytotoxic activity on J-774, murine monocyte/macrophage, and WEHI-164, murine fibrosarcoma, cell lines with the following rank: vaviloside B1/B2>ascalonicoside A1/A2>vaviloside A1/A2. PMID:23415085

  11. Cytochrome P450 expression and activities in human tongue cells and their modulation by green tea extract

    SciTech Connect

    Yang, S.-P.; Raner, Gregory M. . E-mail: gmraner@uncg.edu

    2005-01-15

    The expression, inducibility, and activities of several cytochrome P450 (CYP) enzymes were investigated in a human tongue carcinoma cell model, CAL 27, and compared with the human liver model HepG2 cells. The modulation effects of green tea on various CYP isoforms in both cell lines were also examined. RT-PCR analysis of CAL 27 cells demonstrated constitutive expression of mRNA for CYPs 1A1, 1A2, 2C, 2E1, 2D6, and 4F3. The results were negative for CYP2A6, 2B6/7, 3A3/4, and 3A7. Both cell lines displayed identical expression and induction profiles for all of the isoforms examined in this study except 3A7 and 2B6/7, which were produced constitutively in HepG2 but not Cal-27 cells. CYP1A1 and 1A2 were both induced by treatment with {beta}-napthoflavone as indicated by RT-PCR and Western blotting, while CYP2C mRNA was upregulated by all-trans retinoic acid and farnesol. RT-PCR and Western blot analysis showed that the expressions of CYP1A1 and 1A2 were induced by green tea extract (GTE), which also caused an increase in mRNA for CYP2E1, CYP2D6, and CYP2C isoforms. The four tea catechins, EGC, EC, EGCG and ECG, applied to either HepG2 or Cal-27 cells at the concentration found in GTE failed to induce CYP1A1 or CYP1A2, as determined by RT-PCR. Of the isoforms that were apparently induced by GTE, only 7-ethoxycoumarin deethylase (ECOD) activity could be detected in CAL 27 or HepG2 cells. Interestingly, mRNA and protein for CYP1A1 and CYP1A2 were detected in both cell lines, and although protein and mRNA levels of CYP1A1 and CYP1A2 were increased by GTE, the observed ECOD activity in both cell lines was decreased.

  12. [Inhibitory effect of imperatorin and isoimperatorin on activity of cytochrome P450 enzyme in human and rat liver microsomes].

    PubMed

    Cao, Yan; Zhong, Yu-Huan; Yuan, Mei; Li, Hua; Zhao, Chun-Jie

    2013-04-01

    Imperatorin (IM) and isoimperatorin (ISOIM) are major active components of common herbal medicines from Umbelliferae plants, and widely used in clinic. This article studies the inhibitory effect of IM and ISOIM on the activity of cytochrome P450 (CYP) enzyme, and assesses their potential drug-drug interaction. IM and ISOIM were incubated separately with human or rat liver microsomes for 30 min, with phenacetin, bupropion, tolbutamide, S-mephenytoin, dextromethorphan and midazolam as probe substrates. Metabolites of the CYP probe substrates were determined by LC-MS/MS, and IC50 values were calculated to assess the inhibitory effect of the two drugs on human CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 enzymes, as well as on rat CYP1A2, 2B6, 2D2 and 3A1/2, and grade their inhibitory intensity. In human liver microsomes, IM and ISOIM showed different inhibitory effects on all of the six CYP isoenzymes. They were strong inhibitors for 1A2 and 2B6. The IC50 values were 0.05 and 0.20 micromol x L(-1) for 1A2, and 0.18 and 1.07 micromol x L(-1) for 2B6, respectively. They also showed moderate inhibitory effect on 2C19, and weak effect on 2C9, 2D6 and 3A4. In rat liver microsomes, IM and ISOIM were identified as moderate inhibitors for 1A2, with IC50 values of 1.95 and 2.98 micromol x L(-1). They were moderate and weak inhibitors for 2B6, with IC50 values of 6.22 and 21.71 micromol x L(-1), respectively. They also had weaker inhibitory effect on 2D2 and 3A1/2. The results indicated that IM and ISOIM had extensive inhibitory effects on human CYP enzymes. They are strong inhibitors of CYP1 A2 and 2B6 enzymes. However, it is worth noting the interaction arising from the inhibitory effect of CYP enzymes in clinic.

  13. Gene sequences for cytochromes p450 1A1 and 1A2: the need for biomarker development in sea otters (Enhydra lutris).

    PubMed

    Hook, Sharon E; Cobb, Michael E; Oris, James T; Anderson, Jack W

    2008-11-01

    There has been recent public concern regarding the impacts of environmental pollution on populations of otters. Population level impacts have been seen with otter (Lutra lutra) populations in Europe due to polychlorinated biphenyls, and with some segments of the Prince William Sound, AK, sea otter (Enhydra lutris) population following the Exxon Valdez oil spill. Despite public interest in these animals and their ecological significance, there are few tools that allow for the study of otter's response to contaminant exposure. Cytochrome p450 1A (CYP1A) performs the first step in metabolizing many xenobiotics, including many polychlorinated biphenyls and polycyclic aromatic hydrocarbons. CYP1A induction is a frequently used biomarker of exposure to these compounds. Despite the potential importance of this gene in ecological risk assessment, the complete coding sequence has not been published for any otter species. This study's objective was to isolate the gene for CYP1A1 and CYP1A2 in sea otters using a series of PCR-based approaches. The coding sequences from CYP1A1 and CYP1A2 from sea otters were identified and published in GenBank. Both CYP1A sequences are homologous to those obtained from marine mammals and other carnivores. These sequences will be useful as tools for researchers assessing contaminant exposure in mustelid populations. PMID:18761099

  14. AhR activation underlies the CYP1A autoinduction by A-998679 in rats

    PubMed Central

    Liguori, Michael J.; Lee, Chih-Hung; Liu, Hong; Ciurlionis, Rita; Ditewig, Amy C.; Doktor, Stella; Andracki, Mark E.; Gagne, Gerard D.; Waring, Jeffrey F.; Marsh, Kennan C.; Gopalakrishnan, Murali; Blomme, Eric A. G.; Yang, Yi

    2012-01-01

    Xenobiotic-mediated induction of cytochrome P450 (CYP) drug metabolizing enzymes (DMEs) is frequently encountered in drug discovery and can influence disposition, pharmacokinetic, and toxicity profiles. The CYP1A subfamily of DMEs plays a central role in the biotransformation of several drugs and environmental chemicals. Autoinduction of drugs through CYP3A enzymes is a common mechanism for their enhanced clearance. However, autoinduction via CYP1A is encountered less frequently. In this report, an experimental compound, A-998679 [3-(5-pyridin-3-yl-1,2,4-oxadiazol-3-yl) benzonitrile], was shown to enhance its own clearance via induction of Cyp1a1 and Cyp1a2. Rats were dosed for 5 days with 30, 100, and 200 mg/kg/day A-998679. During the dosing period, the compound's plasma AUC decreased at 30 mg/kg (95%) and 100 mg/kg (80%). Gene expression analysis and immunohistochemistry of the livers showed a large increase in the mRNA and protein levels of Cyp1a, which was involved in the biotransformation of A-998679. Induction of CYP1A was confirmed in primary rat, human, and dog hepatocytes. The compound also weakly inhibited CYP1A2 in human liver microsomes. A-998679 activated the aryl hydrocarbon receptor (AhR) in a luciferase gene reporter assay in HepG2 cells, upregulated expression of genes associated with AhR activation in rat liver and enhanced nuclear migration of AhR in HepG2 cells. Collectively these results demonstrate that A-998679 is an AhR activator that induces Cyp1a1 and Cyp1a2 expression, resulting in an autoinduction phenomenon. The unique properties of A-998679, along with its novel structure distinct from classical polycyclic aromatic hydrocarbons (PAHs), may warrant its further evaluation as a tool compound for use in studies involving AhR biology and CYP1A-related mechanisms of drug metabolism and toxicity. PMID:23112805

  15. Dual effects of phloretin on aflatoxin B1 metabolism: activation and detoxification of aflatoxin B1.

    PubMed

    Gao, Shang Shang; Chen, Xiao Yan; Zhu, Ri Zhe; Choi, Byung-Min; Kim, Sun Jun; Kim, Bok-Ryang

    2012-01-01

    Typically, chemopreventive agents involve either induction of phase II detoxifying enzymes and/or inhibition of cytochrome P450 enzymes (CYPs) that are required for the activation of procarcinogens. In this study, we investigated the protective effects of phloretin against aflatoxin B1 (AFB1) activation to the ultimate carcinogenic intermediate, AFB(1)-8, 9-epoxide (AFBO), and its subsequent detoxification. Phloretin markedly inhibited formation of the epoxide with human liver microsomes in a dose-dependent manner. Phloretin also inhibited the activities of nifedipine oxidation and ethoxyresorufin O-deethylase (EROD) in human liver microsomes. These data show that phloretin strongly inhibits CYP1A2 and CYP3A4 activities, which are involved in the activation of AFB1. Phloretin increased glutathione S-transferase (GST) activity of alpha mouse liver 12 (AML 12) cells in a dose-dependent manner. GST activity toward AFBO in cell lysates treated with 20 μM phloretin was 23-fold that of untreated control cell lysates. The expression of GSTA3, GSTA4, GSTM1, GSTP1 and GSTT1 was induced by phloretin in a dose-dependent manner in AML 12 cells. GSTP1, GSTM1, and GSTT1 were able to significantly increase the conjugation of AFBO with glutathione. Concurrently, induction of the GST isozyme genes was partially associated with the Nrf2/ARE pathway. Taken together, the results demonstrate that phloretin has a strong chemopreventive effect against AFB1 through its inhibitory effect on CYP1A2, CYP3A4, and its inductive effect on GST activity. PMID:22253071

  16. Inhibition of human cytochrome P450 1B1, 1A1 and 1A2 by antigenotoxic compounds, purpurin and alizarin.

    PubMed

    Takahashi, Eizo; Fujita, Ken-ichi; Kamataki, Tetsuya; Arimoto-Kobayashi, Sakae; Okamoto, Keinosuke; Negishi, Tomoe

    2002-10-31

    Recently we have shown that anthraquinone food pigments such as purpurin and alizarin suppress the genotoxic activities of several mutagens including heterocyclic amines and polycyclic aromatic hydrocarbons in the Drosophila DNA repair test and in the Ames test. To investigate the mechanism of this inhibition, we have now examined the effects of these anthraquinone pigments on enzymes that metabolize xenobiotics. The activities of eight human recombinant cytochrome P450 (CYP) isozymes were measured in the presence of purpurin, alizarin or carminic acid. Purpurin and alizarin strongly inhibited the activities of CYP1A1, CYP1A2 and CYP1B1, and weakly suppressed those of CYP2A6 and CYP2E1 in a dose-dependent manner, but did not inhibit those of CYP2C19, CYP3A4 and CYP3A5. Carminic acid did not affect the activities of any CYPs tested. CYP1B1 was the most strongly affected CYP molecule by purpurin and alizarin among CYPs examined in this study. From kinetic analysis, it was shown that the inhibition by purpurin on CYP1B1 was both competitive and non-competitive, and that by alizarin was competitive. The values of slopes obtained from Lineweaver-Burk plots are proportional to the square of purpurin concentration. This observation suggests that two molecules of purpurin are interacting with one molecule of CYP1B1. The K(m) value of CYP1B1 was 11 microM, and the K(i) value of purpurin and alizarin against CYP1B1 was 0.7 microM(2) and 0.5 microM, respectively. We also examined the effects of these pigments on the mutagenicities of MeIQx and B[a]P in the Ames test, using Salmonella typhimurium TA1538 co-expressing each form of human CYP and NADPH-cytochrome P450 reductase (OR). The mutagenicity of MeIQx in TA1538 1A2/OR or 1B1/OR was suppressed by purpurin and alizarin but not by carminic acid. Purpurin also reduced the mutagenicity of B[a]P in TA1538 1A1/OR or 1B1/OR. These results suggest that the antigenotoxic activities of purpurin and alizarin can be explained by

  17. 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.

  18. Evaluation of the assumptions of an ontogeny model of rat hepatic cytochrome P450 activity.

    PubMed

    Alcorn, Jane; Elbarbry, Fawzy A; Allouh, Mohammed Z; McNamara, Patrick J

    2007-12-01

    We previously reported an ontogeny model of hepatic cytochrome P450 (P450) activity that predicts in vivo P450 elimination from in vitro intrinsic clearance. The purpose of this study was to conduct investigations into key assumptions of the P450 ontogeny model using the developing rat model system. We used two developmentally dissimilar enzymes, CYP2E1 and CYP1A2, and male rats (n = 4) at age groups representing critical developmental stages. Total body and liver weights and hepatic microsomal protein contents were measured. Following high-performance liquid chromatography analysis, apparent K(M) and V(max) estimates were calculated using nonlinear regression analysis for CYP2E1- and CYP1A2-mediated chlorzoxazone 6-hydroxylation and methoxyresorufin O-dealkylation, and V(max) estimates for p-nitrophenol and phenacetin hydroxylations, respectively. Hepatic scaling factors and V(max) values provided estimates for infant scaling factors (ISF). The data show microsomal protein contents increased with postnatal age and reached adult values after postnatal day (PD) 7. Apparent K(M) values were similar at all developmental stages except at < or =PD7. Developmental increases in probe substrate V(max) values did not correlate with the biphasic increase in immunoquantifiable P450. The activity of two different probe substrates for each P450 covaried as a function of age. A plot of observed ISF values as a function of age reflected the developmental pattern of rat hepatic P450. In summation, these observations diverge from several of the model's assumptions. Further investigations are required to explain these inconsistencies and to investigate whether the developing rat may provide a predictive paradigm for pediatric risk assessment for P450-mediated elimination processes.

  19. Propiconazole-induced cytochrome P450 gene expression and enzymatic activities in rat and mouse liver.

    PubMed

    Sun, Guobin; Thai, Sheau-Fung; Tully, Douglas B; Lambert, Guy R; Goetz, Amber K; Wolf, Douglas C; Dix, David J; Nesnow, Stephen

    2005-02-15

    Propiconazole is a N-substituted triazole used as a fungicide on fruits, grains, seeds, hardwoods, and conifers. In the present study, propiconazole was examined for its effects on the expression of hepatic cytochrome P450 genes and on the activities of P450 enzymes in male Sprague-Dawley rats and male CD-1 mice. Rats and mice were administered propiconazole by gavage daily for 14 days at doses of 10, 75, and 150 mg/kg body weight/day. Quantitative real time RT-PCR assays of rat hepatic RNA samples from animals treated at the 150 mg/kg body weight/day dose revealed significant mRNA overexpression of the following genes compared to control: CYP1A2 (1.62-fold), CYP2B1 (10.8-fold), CYP3A1/CYP3A23 (2.78-fold), and CYP3A2 (1.84-fold). In mouse liver, propiconazole produced mRNA overexpression of Cyp2b10 (2.39-fold) and Cyp3a11 (5.19-fold). mRNA expression of CYP1A1 was not detected in liver tissues from treated or controls animals from either species. Propiconazole significantly induced both pentoxyresorufin O-dealkylation (PROD) and methoxyresorufin O-dealkylation (MROD) activities in both rat and mouse liver at the 150 mg/kg body weight/day and 75 mg/kg body weight/day doses. In summary, these results indicated that propiconazole induced CYP1A2 in rat liver and CYP2B and CYP3A families of isoforms in rat and mouse liver.

  20. Effects of long-term smoking on the activity and mRNA expression of CYP isozymes in rats

    PubMed Central

    He, Xiao-Meng; Zhou, Ying; Xu, Ming-Zhen; Li, Yang; Li, Hu-Qun

    2015-01-01

    Background To investigate the effect of long-term smoking on the activity and mRNA expression of cytochrome P450 (CYP) enzymes. Methods Sprague-Dawley rats were exposed to passive smoking 6 cigarettes per day for 180 days. A cocktail solution which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/kg) was given orally to rats. Blood samples were collected at pre-specified time points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by DAS 3.0. In addition, real-time RT-PCR was used to analyze the mRNA expression of CYP1A2, CYP2C11, CYP2E1 and CYP3A1 in rat liver. Results There were no significant influences of pharmacokinetic profiles of chlorzoxazone in long-term smoking pretreated rats. But many pharmacokinetic profiles of phenacetin, tolbutamide, and midazolam in long-term smoking pretreated rats were affected significantly (P<0.05). The results suggested that long-term smoking had significant inhibition effects on CYP2C11 and CYP3A1 while CYP1A2 enzyme activity was induced. Furthermore, Long-term smoking had no effects on rat CYP2E1. The mRNA expression results were consistent with the pharmacokinetic results. Conclusions Alterations of CYP450 enzyme activities may fasten or slow down excretion with corresponding influence on drug efficacy or toxicity in smokers compared to nonsmokers, which may lead to clinical failures of lung cancer therapy or toxicity in smokers. PMID:26623094

  1. Role of cytochrome p4501 family members in the metabolic activation of polycyclic aromatic hydrocarbons in mouse epidermis.

    PubMed

    Kleiner, Heather E; Vulimiri, Suryanarayana V; Hatten, William B; Reed, Melissa J; Nebert, Daniel W; Jefcoate, Colin R; DiGiovanni, John

    2004-12-01

    Polycyclic aromatic hydrocarbons (PAHs) are known to be activated by the cytochrome P450 (P450) 1 family. However, the precise role of individual P4501 family members in PAH bioactivation remains to be fully elucidated. We therefore investigated the formation of PAH-DNA adducts in the epidermis of Cyp1a2(-/-), Cyp1b1(-/-), and Ahr(-/-) knockout mice. A panel of different PAHs was used, ranging in carcinogenic potency. Mice were treated topically on the dorsal skin with the following tritium-labeled PAHs: dibenzo[a,l]pyre-ne (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (B[a]P), dibenzo[a,h]anthracene (DB[a,h]A), benzo[g]chrysene (B[g]C), and benzo[c]phenanthrene (B[c]P). At 24 h after treatment, mice (two male and two female mice per group) were sacrificed, and epidermal DNA was isolated and hydrolyzed with DNase I; subsequently, DNA adducts were quantitated by liquid scintillation counting. In the DB[a,l]P-treated mice, levels of DNA adducts were significantly lower in Cyp1a2(-/-) and Cyp1b1(-/-) mice by 57 and 46%, respectively, as compared to wild-type (WT) mice (C57BL/6 background). The levels of DB[a,l]P DNA adducts formed in Ahr(-/-) mice were 26% lower, but this was not statistically significant. The levels of DMBA-DNA adducts in Cyp1a2(-/-) mice were not different than the WT mice but were significantly lower in Cyp1b1(-/-) and Ahr(-/-) mice by 64 and 52%, respectively. DMBA-DNA adduct samples were further analyzed by HPLC following further digestion to deoxyribonucleosides. HPLC analysis of individual DMBA-DNA adducts revealed differences in the ratio of syn-DMBA-diol epoxide- to anti-DMBA-diol epoxide-derived adducts in the Ahr(-/-) and Cyp1b1(-/-) mice. The ratio of syn-/anti-derived adducts in WT mice was 0.49. This ratio was 0.23 in the Cyp1b1(-/-) mice and 0.87 in the Ahr(-/-) mice. In contrast to the results with DB[a,l]P and DMBA, the levels of B[a]P-, DB[a,h]A-, B[g]C-, and B[c]P-DNA adducts were significantly lower in Ahr

  2. The Effect of Yokukansan, a Traditional Herbal Preparation Used for the Behavioral and Psychological Symptoms of Dementia, on the Drug-Metabolizing Enzyme Activities in Healthy Male Volunteers.

    PubMed

    Soraoka, Hiromi; Oniki, Kentaro; Matsuda, Kazuki; Ono, Tatsumasa; Taharazako, Kosuke; Uchiyashiki, Yoshihiro; Kamihashi, Ryoko; Kita, Ayana; Takashima, Ayaka; Nakagawa, Kazuko; Yasui-Furukori, Norio; Kadowaki, Daisuke; Miyata, Keishi; Saruwatari, Junji

    2016-01-01

    The concomitant use of herb and prescription medications is increasing globally. Herb-drug interactions are therefore a clinically important problem. Yokukansan (YKS), a Japanese traditional herbal medicine, is one of the most frequently used herbal medicines. It is effective for treating the behavioral and psychological symptoms of dementia. We investigated the potential effects of YKS on drug-metabolizing enzyme activities in humans. An open-label repeat-dose study was conducted in 26 healthy Japanese male volunteers (age: 22.7±2.3 years) with no history of smoking. An 8-h urine sample was collected after a 150-mg dose of caffeine and a 30-mg dose of dextromethorphan before and after the administration of YKS (2.5 g, twice a day for 1 week). The activities of cytochrome P450 (CYP) 1A2, CYP2D6, CYP3A, xanthine oxidase (XO) and N-acetyltransferase 2 (NAT2) were assessed based on the urinary metabolic indices of caffeine and dextromethorphan, and the urinary excretion ratio of 6β-hydroxycortisol to cortisol. There were no statistically significant differences in the activities of the examined enzymes before or after the 7-d administration of YKS. Although further studies assessing the influence of YKS on the pharmacokinetics and pharmacodynamics of the substrates of the drug-metabolizing enzymes are needed to verify the present results, YKS is unlikely that a pharmacokinetic interaction will occur with concomitantly administered medications that are predominantly metabolized by the CYP1A2, CYP2D6, CYP3A, XO and NAT2. PMID:27582327

  3. The Effect of Yokukansan, a Traditional Herbal Preparation Used for the Behavioral and Psychological Symptoms of Dementia, on the Drug-Metabolizing Enzyme Activities in Healthy Male Volunteers.

    PubMed

    Soraoka, Hiromi; Oniki, Kentaro; Matsuda, Kazuki; Ono, Tatsumasa; Taharazako, Kosuke; Uchiyashiki, Yoshihiro; Kamihashi, Ryoko; Kita, Ayana; Takashima, Ayaka; Nakagawa, Kazuko; Yasui-Furukori, Norio; Kadowaki, Daisuke; Miyata, Keishi; Saruwatari, Junji

    2016-01-01

    The concomitant use of herb and prescription medications is increasing globally. Herb-drug interactions are therefore a clinically important problem. Yokukansan (YKS), a Japanese traditional herbal medicine, is one of the most frequently used herbal medicines. It is effective for treating the behavioral and psychological symptoms of dementia. We investigated the potential effects of YKS on drug-metabolizing enzyme activities in humans. An open-label repeat-dose study was conducted in 26 healthy Japanese male volunteers (age: 22.7±2.3 years) with no history of smoking. An 8-h urine sample was collected after a 150-mg dose of caffeine and a 30-mg dose of dextromethorphan before and after the administration of YKS (2.5 g, twice a day for 1 week). The activities of cytochrome P450 (CYP) 1A2, CYP2D6, CYP3A, xanthine oxidase (XO) and N-acetyltransferase 2 (NAT2) were assessed based on the urinary metabolic indices of caffeine and dextromethorphan, and the urinary excretion ratio of 6β-hydroxycortisol to cortisol. There were no statistically significant differences in the activities of the examined enzymes before or after the 7-d administration of YKS. Although further studies assessing the influence of YKS on the pharmacokinetics and pharmacodynamics of the substrates of the drug-metabolizing enzymes are needed to verify the present results, YKS is unlikely that a pharmacokinetic interaction will occur with concomitantly administered medications that are predominantly metabolized by the CYP1A2, CYP2D6, CYP3A, XO and NAT2.

  4. Non-coplanar 2,2',3,3',4,4',5,5',6,6'-decachlorobiphenyl (PCB 209) did not induce cytochrome P450 enzyme activities in primary cultured rat hepatocytes, was not genotoxic, and did not exhibit endocrine-modulating activities.

    PubMed

    Han, Xing; O'Connor, John C; Donner, E Maria; Nabb, Diane L; Mingoia, Robert T; Snajdr, Suzanne I; Clarke, Jane J; Kaplan, A Michael

    2009-01-31

    2,2',3,3',4,4',5,5',6,6'-Decachlorobiphenyl (PCB 209) is a fully chlorinated, non-coplanar biphenyl. To demonstrate that PCB 209 is not likely to exhibit human health hazards common to coplanar PCBs it was tested for cytochrome P450 (P450) enzyme induction potentials, genetic toxicity, and endocrine-modulating activity. PCB 209 (dose from 0.005 to 5000 ng/mL) did not significantly induce P450 CYP1A, 2A, 2B, 3A, or 4A enzyme activities in primary cultured rat hepatocytes. In contrast, Aroclor 1260, a PCB mixture that contains approximately 60% chlorine by weight, showed significant induction of P450 CYP1A, 2A, 2B, and 3A within the same dose range. PCB 209 (dose from 100 to 5000 microg/plate) was negative in the bacterial mutagenicity (Ames) test in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 or in Eschericia coli strain WP2uvrA. PCB 209 (dose from 25 to 150 microg/mL) was also negative for forward mutations at the thymidine kinase (TK+/-) locus of L5178Y mouse lymphoma cells. The Ames and the mouse lymphoma assays were both conducted in the absence and presence of rat liver S9 fraction. PCB 209 (dose from 500 to 2000 mg/kg by single dose oral gavage) did not induce an increase in the frequency of micronuclei in polychromatic erythrocytes in mouse bone marrow in vivo. PCB 209 did not induce estrogenic effects when administered by gavage to ovariectomized adult female rats at 500 and 1000 mg/kg for 4 days, nor did it produce alterations consistent with endocrine-modulating activity in adult intact male rats when administered by gavage at 500 and 1000 mg/kg for 15 consecutive days.

  5. Induction of cytochrome P4501A1 by aryl hydrocarbon receptor agonists in porcine aorta endothelial cells in culture and cytochrome P4501A1 activity in intact cells.

    PubMed

    Stegeman, J J; Hahn, M E; Weisbrod, R; Woodin, B R; Joy, J S; Najibi, S; Cohen, R A

    1995-02-01

    Endothelium is a single-cell layer lining blood vessels and constituting capillaries and could be a primary site of chemical effects in the cardiovasculature and systemically. Cytochrome P4501A1 (CYP1A1) is strongly inducible in vertebrate endothelium in vivo by aryl hydrocarbon receptor (AhR) agonists [Mol. Pharmacol. 36:723-729 (1989); Mol. Pharmacol. 41:1039-1046 (1992)]. We investigated CYP1A expression and activity in porcine aorta endothelial cells (PAEC) exposed in culture to the AhR agonists 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4'-tetrachlorobiphenyl (TCB), benzo[a]pyrene (BP), or beta-naphthoflavone (BNF). Immunoblotting with monoclonal anti-CYP1A1 and polyclonal anti-CYP1A1 and anti-CYP1A2 antibodies showed that CYP1A1 was induced in cultures exposed to TCDD, TCB, BP, or BNF but was not detectable in untreated or dimethylsulfoxide-exposed cultures. CYP1A1 was strongly induced at intermediate concentrations (0.1 microM or 1.0 microM) of TCB, BP, or BNF, but induction was suppressed by higher concentrations, a response not due to general toxicity; cell viability (trypan blue exclusion) was > 97% with BNF or TCB at up to 10 microM. CYP1A1 induction by TCDD was maximal at 0.3-1.0 nM. ED50 values for induction of CYP1A1 by TCDD, TCB, and BP were 0.016 nM, 3-10 nM, and 180 nM, respectively. Immunohistochemical analysis confirmed CYP1A1 induction in PAEC but also showed that only some cells in the cultures were induced. Subcellular fractionation, marker enzyme analysis, and immunoblot analysis showed that PAEC had a typical complement of microsomal electron-transport components. NADPH-cytochrome P450 reductase showed comparable rates (approximately 40 nmol/min/mg) in induced and control cultures. Cultures maximally induced by 0.1 microM TCB had microsomal CYP1A1 [ethoxyresorufin-O-deethylase (EROD)] activity averaging 25 pmol/min/mg. Addition of purified rat reductase to PAEC microsomes increased the EROD rates 3-fold. EROD rates measured in intact

  6. UPLC-MS-MS method for simultaneous determination of caffeine, tolbutamide, metoprolol, and dapsone in rat plasma and its application to cytochrome P450 activity study in rats.

    PubMed

    Liu, Yan; Li, Xiang; Yang, Chunjuan; Tai, Sheng; Zhang, Xiangning; Liu, Gaofeng

    2013-01-01

    A specific ultra-performance liquid chromatography tandem mass spectrometry method has been described for the simultaneous determination of caffeine, tolbutamide, metoprolol and dapsone in rat plasma, which are the four probe drugs of the four cytochrome P450 (CYP450) isoforms CYP1A2, CYP2C9, CYP2D6 and CYP3A4. The chromatographic separation was achieved using a Waters Acquity UPLC BEH HILIC C(18) column (2.1 × 50 mm, 1.7 µm). The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) (15:85, v/v). The triple quadrupole mass spectrometric detection was operated by positive electrospray ionization. Phenacetin was chosen as internal standard. Plasma samples were extracted with dichloromethane-butanol (10:1, v/v). The recoveries ranged from 67.5% to 98.5%. The calibration curves in plasma were linear in the range of 2.5-1,000 ng/mL for caffeine and dapsone, 5-5,000 ng/mL for tolbutamide and 2.5-250 ng/mLfor metoprolol, with correlation coefficient (r(2)) of 0.9936, 0.9966, 0.9990 and 0.9998, respectively. The method was successfully applied to pharmacokinetic studies of the four probe drugs of the four CYP450 isoforms and used to evaluate the effects of breviscapine on the activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in rats.

  7. Simultaneous determination of bupropion, metroprolol, midazolam, phenacetin, omeprazole and tolbutamide in rat plasma by UPLC-MS/MS and its application to cytochrome P450 activity study in rats.

    PubMed

    Ma, Jianshe; Wang, Shuanghu; Zhang, Meiling; Zhang, Qingwei; Zhou, Yunfang; Lin, Chongliang; Lin, Guanyang; Wang, Xianqin

    2015-08-01

    A specific ultra-performance liquid chromatography tandem mass spectrometry method is described for the simultaneous determination of bupropion, metroprolol, midazolam, phenacetin, omeprazole and tolbutamide in rat plasma with diazepam as internal standard, which are the six probe drugs of the six cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9. Plasma samples were protein precipitated with acetonitrile. The chromatographic separation was achieved using a UPLC® BEH C18 column (2.1 × 100 mm, 1.7 µm). The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) with gradient elution. The triple quadrupole mass spectrometric detection was operated by multiple reaction monitoring in positive electrospray ionization. The precisions were <13%, and the accuracy ranged from 93.3 to 110.4%. The extraction efficiency was >90.5%, and the matrix effects ranged from 84.3 to 114.2%. The calibration curves in plasma were linear in the range of 2-2000 ng/mL, with correlation coefficient (r(2) ) >0.995. The method was successfully applied to pharmacokinetic studies of the six probe drugs of the six CYP450 isoforms and used to evaluate the effects of erlotinib on the activities of CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19 and CYP2C9 in rats. Erlotinib may inhibit the activity of CYP2B6 and CYP3A4, and may induce CYP2C9 of rats.

  8. Cold-induced activities of cytochromes P450 1A1 and 1A2 in rat liver: putative role of endogenous compounds in induction mechanism.

    PubMed

    Perepechaeva, M L; Grishanova, A Yu

    2013-03-01

    Adaptation to cold includes adaptive changes at the organism and molecular levels. One of the interesting facts is induction of cytochromes P450 subfamily 1A (CYP1A) in the liver of rats, inducible enzymes participating in biotransformation of procarcinogenic xenobiotics, under the effect of moderate cold exposure. Cold activation of CYP1A can be mediated by adaptive changes and the resultant redistribution or intensification of the synthesis of mediator compounds. This hypothesis is verified in the present study. The role of bilirubin, tocopherol, and corticosterone as mediators of cold induction of CYP1A in the rat liver was evaluated. The results indicate that these compounds can be involved in cold induction of CYP1A, but none of them is the only mediator in this process.

  9. Cytochrome b(5) shifts oxidation of the anticancer drug ellipticine by cytochromes P450 1A1 and 1A2 from its detoxication to activation, thereby modulating its pharmacological efficacy.

    PubMed

    Kotrbová, Věra; Mrázová, Barbora; Moserová, Michaela; Martínek, Václav; Hodek, Petr; Hudeček, Jiří; Frei, Eva; Stiborová, Marie

    2011-09-15

    Ellipticine is a pro-drug, whose activation is dependent on its oxidation by cytochromes P450 (CYP) and peroxidases. Cytochrome b(5) alters the ratio of ellipticine metabolites formed by isolated reconstituted CYP1A1 and 1A2, favoring formation of 12-hydroxy- and 13-hydroxyellipticine metabolites implicated in ellipticine-DNA adduct formation, at the expense of 9-hydroxy- and 7-hydroxyellipticine that are detoxication products. Cytochrome b(5) enhances the production of 12-hydroxy and 13-hydroxyellipticine. The change in metabolite ratio results in an increased formation of covalent ellipticine-DNA adducts, one of the DNA-damaging mechanisms of ellipticine antitumor action. This finding explains previous apparent discrepancies found with isolated enzymes and in vivo, where CYP1A enzymatic activation correlated with ellipticine-DNA-adduct levels while isolated CYP1A1 or 1A2 in reconstituted systems were much less effective than CYP3A4. The effect of cytochrome b(5) might be even more pronounced in vivo, since, as we show here, ellipticine increases levels of cytochrome b(5) in rat liver. Our results demonstrate that both the native 3D structure of cytochrome b(5) and the presence of the heme as an electron transfer agent in this protein enable a shift in ellipticine metabolites formed by CYP1A1/2.

  10. Effect of high sucrose diet on liver enzyme content and activity and aflatoxin B1-induced mutagenesis.

    PubMed

    Peters, Leandra P; Teel, Robert W

    2003-01-01

    Aflatoxin B1 (AFB1) is a fungal toxin and contaminant that has been implicated in human liver carcinogenesis. In this study we evaluated the effect of a 65% of total calories from sucrose diet (HSD) for 90 days on hepatic cytochrome P450 (CYP450) and glutathione-S-transferase (GST) activity compared to rats maintained on standard lab chow (0% sucrose). There was a statistically significant increase in the number of S. typhimurium His+ revertants (p < 0.001) generated from the incubation of AFB1 with hepatic microsomes from rats fed a HSD. The HSD did not affect the total microsomal CYP450 content nor content of CYP450 1A2, 2B1, 2 isoforms which activate AFB1. Alkoxyresorufin O-dealkylase activity (MROD, PROD) of microsomes from animals fed HSD was decreased by 73% and 49%, respectively. MROD activity is linked to CYP 1A2 activity while PROD is linked to CYP 2B1,2 activity. Although the amount of CYP 3A was significantly decreased in rats fed a HSD, its activity, determined by the presence of the fluorometric metabolite 7-hydroxyquinidine, was unchanged. GST activity was significantly lower in the rats fed HSD.

  11. Activity of xenobiotic-metabolizing enzymes in the liver of rats with multi-vitamin deficiency.

    PubMed

    Tutelyan, Victor A; Kravchenko, Lidia V; Aksenov, Ilya V; Trusov, Nikita V; Guseva, Galina V; Kodentsova, Vera M; Vrzhesinskaya, Oksana A; Beketova, Nina A

    2013-01-01

    The purpose of the study was to determine how multi-vitamin deficiency affects xenobiotic-metabolizing enzyme (XME) activities in the rat liver. Vitamin levels and XME activities were studied in the livers of male Wistar rats who were fed for 4 weeks with semi-synthetic diets containing either adequate (100 % of recommended vitamin intake) levels of vitamins (control), or decreased vitamin levels (50 % or 20 % of recommended vitamin intake). The study results have shown that moderate vitamin deficiency (50 %) leads to a decrease of vitamin A levels only, and to a slight increase, as compared with the control, in the following enzyme activities: methoxyresorufin O-dealkylase (MROD) activity of CYP1 A2 - by 34 % (p < 0.05), UDP-glucuronosyl transferase - by 26 % (p < 0.05), and quinone reductase - by 55 % (p < 0.05). Profound vitamin deficiency (20 %) led to a decrease of vitamins A, E, B1, B2, and C, and enzyme activities in the liver: MROD - to 78 % of the control level (p < 0.05), 4-nitrophenol hydroxylase - to 74 % (p < 0.05), heme oxygenase-1 - to 83 % (p < 0.05), and quinone reductase - to 60 % (p < 0.05). At the same time, the UDP-glucuronosyl transferase activity and ethoxyresorufin O-dealkylase activity of CYP1A1, pentoxyresorufin O-dealkylase activity of CYP2B1/2 and 6β-testosterone hydroxylase, as well as the total activity of glutathione transferase did not differ from the control levels. The study has demonstrated that profound multi-vitamin deficiency is associated with a decrease in the expression of CYP1A2 and CYP3A1 mRNAs to 62 % and 79 %, respectively. These data indicated that a short-term but profound multi-vitamin deficiency in rats leads to a decrease in the activities and expression of the some XME that play an important role in detoxification of xenobiotics and metabolism of drugs and antioxidant protection. PMID:24220160

  12. Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 2. Expression of receptor mRNA in resting and lipopolysaccharide-activated mouse RAW 264.7 macrophages.

    PubMed

    Streitová, D; Hofer, M; Holá, J; Vacek, A; Pospísil, M

    2010-01-01

    Expression of mRNA for adenosine receptor subtypes A(1), A(2a), A(2b), and A(3) in normal and lipopolysaccharide (LPS)-activated murine RAW 264.7 macrophages has been investigated using the method of quantitative real-time polymerase chain reaction. The results have shown a very low, unquantifiable expression of adenosine A(1) receptor mRNA in both normal and LPS-activated macrophages. The other three adenosine receptor mRNAs have been found to be expressed at various but always quantifiable levels. Activation of the macrophages by LPS induced upregulation of the expression of adenosine receptor A(2a) and A(2b) mRNA, whereas the expression of adenosine receptor A(3) mRNA was downregulated. Unstimulated macrophages exhibited a high expression of the A(2b) adenosine receptor mRNA. The findings are discussed from the point of view of the antiinflammatory and hematopoiesis-stimulating roles of the adenosine receptor signaling.

  13. Inhibition of human cytochrome P450 enzymes by the natural hepatotoxin safrole.

    PubMed

    Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw

    2005-05-01

    The hepatotoxin, safrole is a methylenedioxy phenyl compound, found in sassafras oil and certain other essential oils. Recombinant cytochrome P450 (CYP, P450) and human liver microsomes were studied to investigate the selective inhibitory effects of safrole on human P450 enzymes and the mechanisms of action. Using Escherichia coli-expressed human P450, our results demonstrated that safrole was a non-selective inhibitor of CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 in the IC(50) order CYP2E1 < CYP1A2 < CYP2A6 < CYP3A4 < CYP2D6. Safrole strongly inhibited CYP1A2, CYP2A6, and CYP2E1 activities with IC(50) values less than 20 microM. Safrole caused competitive, non-competitive, and non-competitive inhibition of CYP1A2, CYP2A6 and CYP2E1 activities, respectively. The inhibitor constants were in the order CYP1A2 < CYP2E1 < CYP2A6. In human liver microsomes, 50 microM safrole strongly inhibited 7-ethoxyresorufin O-deethylation, coumarin hydroxylation, and chlorzoxazone hydroxylation activities. These results revealed that safrole was a potent inhibitor of human CYP1A2, CYP2A6, and CYP2E1. With relatively less potency, CYP2D6 and CYP3A4 were also inhibited.

  14. Age-Related Changes in Hepatic Activity and Expression of Detoxification Enzymes in Male Rats

    PubMed Central

    Vyskočilová, Erika; Szotáková, Barbora; Skálová, Lenka; Bártíková, Hana; Hlaváčová, Jitka

    2013-01-01

    Process of aging is accompanied by changes in the biotransformation of xenobiotics and impairment of normal cellular functions by free radicals. Therefore, this study was designed to determine age-related differences in the activities and/or expressions of selected drug-metabolizing and antioxidant enzymes in young and old rats. Specific activities of 8 drug-metabolizing enzymes and 4 antioxidant enzymes were assessed in hepatic subcellular fractions of 6-week-old and 21-month-old male Wistar rats. Protein expressions of carbonyl reductase 1 (CBR1) and glutathione S-transferase (GST) were determined using immunoblotting. Remarkable age-related decrease in specific activities of CYP2B, CYP3A, and UDP-glucuronosyl transferase was observed, whereas no changes in activities of CYP1A2, flavine monooxygenase, aldo-keto reductase 1C, and antioxidant enzymes with advancing age were found. On the other hand, specific activity of CBR1 and GST was 2.4 folds and 5.6 folds higher in the senescent rats compared with the young ones, respectively. Interindividual variability in CBR1 activity increased significantly with rising age. We suppose that elevated activities of GST and CBR1 may protect senescent rats against xenobiotic as well as eobiotic electrophiles and reactive carbonyls, but they may alter metabolism of drugs, which are CBR1 and especially GSTs substrates. PMID:23971034

  15. Effects of zedoary turmeric oil on P450 activities in rats with liver cirrhosis induced by thioacetamide.

    PubMed

    Cheng, Jing-Jing; Yang, Nai-Bin; Wu, Liang; Lin, Jia-Le; Dai, Ge-Xin; Zhu, Jia-Yin

    2014-01-01

    The aim of this study was to elucidate the effects of zedoary turmeric oil (ZTO) on P450 activities (CYP1A2, CYP2C9, CYP2C19, CYP2B6, CYP2D6 and CYP3A4) in rats with liver cirrhosis induced by thioacetamide (TAA). For the induction of liver cirrhosis, rats were given TAA in their drinking water at a concentration of 0.03% for consecutive 5 weeks and then 0.04% for the next consecutive 5 weeks throughout the establishment of cirrhosis. Then the cirrhotic rats were ip given saline, ZTO 100, 200 and 400 mg/kg, respectively, once daily for 2 weeks. When cirrhosis model was established at week 10, all rats of five groups were administered intragastrically with 15 mg/kg phenacetin, 0.6 mg/kg tolbutamide, 15 mg/kg omeprazole, 15 mg/kg bupropion, 15 mg/kg metoprolol, and 10 mg/kg midazolam. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The degree of liver cirrhosis was assessed by HE staining. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) from the model group increased by approximately 4-fold, and a decreased level of albumin (Alb) was also observed, as compared to the control group (P < 0.05). However, ZTO was found to reverse those changes of serum levels observed in the model group, and the 200 mg/kg ZTO treatment group showed the most obvious reverse tendency with significantly decreased ALT, AST and increased Alb levels (P < 0.05). The results indicated that ZTO with the dose of 100 mg/kg could inhibit the activities of CYP450 isoforms CYP2C9 and CYP2D6 in vivo in cirrhotic rats induced by TAA, while ZTO with the dose of 400 mg/kg could induce the activity of CYP2C19 in vivo in cirrhotic rats induced by TAA. However, ZTO showed no influence on cirrhotic rat hepatic CYP1A2, CYP2B6 and CYP3A4 activity in vivo. This has certain guiding significance to clinical treatment.

  16. Intact glucosinolates modulate hepatic cytochrome P450 and phase II conjugation activities and may contribute directly to the chemopreventive activity of cruciferous vegetables.

    PubMed

    Abdull Razis, Ahmad F; Bagatta, Manuela; De Nicola, Gina R; Iori, Renato; Ioannides, Costas

    2010-11-01

    The currently accepted view is that the chemopreventive activity of glucosinolates is exclusively mediated by their degradation products, such as isothiocyanates. In the present study, evidence is presented for the first time that intact glucosinolates can modulate carcinogen-metabolising enzyme systems. The glucosinolates glucoraphanin and glucoerucin were isolated from cruciferous vegetables and incubated with precision-cut rat liver slices. Both glucosinolates elevated the O-dealkylations of methoxy- and ethoxyresorufin, markers for CYP1 activity; supplementation of the incubation medium with myrosinase, the enzyme that converts glucosinolates to their corresponding isothiocyanates, abolished these effects. Moreover, both glucoerucin and glucoraphanin increased the apoprotein levels of microsomal CYP1A1, CYP1A2 and CYP1B1. At higher concentrations, both glucosinolates enhanced quinone reductase activity, whereas glucoraphanin also elevated glutathione S-transferase; in this instance, however, supplementation of the incubation medium with myrosinase exacerbated the inductive effect. Finally, both glucosinolates increased modestly cytosolic quinone reductase, GSTα and GSTμ protein levels, which became more pronounced when myrosinase was added to the incubations with the glucosinolate. It may be inferred that intact glucosinolates can modulate the activity of hepatic carcinogen-metabolising enzyme systems and this is likely to impact on the chemopreventive activity linked to cruciferous vegetable consumption.

  17. Structure-Activity Relationship and Substrate-Dependent Phenomena in Effects of Ginsenosides on Activities of Drug-Metabolizing P450 Enzymes

    PubMed Central

    Hao, Miao; Zhao, Yuqing; Chen, Peizhan; Huang, He; Liu, Hong; Jiang, Hualiang; Zhang, Ruiwen; Wang, Hui

    2008-01-01

    Ginseng, a traditional herbal medicine, may interact with several co-administered drugs in clinical settings, and ginsenosides, the major active components of ginseng, may be responsible for these ginseng-drug interactions (GDIs). Results from previous studies on ginsenosides' effects on human drug-metabolizing P450 enzymes are inconsistent and confusing. Herein, we first evaluated the inhibitory effects of fifteen ginsenosides and sapogenins on human CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 enzymes by using commercially available fluorescent probes. The structure-activity relationship of their effects on the P450s was also explored and a pharmacophore model was established for CYP3A4. Moreover, substrate-dependent phenomena were found in ginsenosides' effects on CYP3A4 when another fluorescent probe was used, and were further confirmed in tests with conventional drug probes and human liver microsomes. These substrate-dependent effects of the ginsenosides may provide an explanation for the inconsistent results obtained in previous GDI reports. PMID:18628990

  18. The relationship between DNA adduct formation by benzo[a]pyrene and expression of its activation enzyme cytochrome P450 1A1 in rat.

    PubMed

    Hodek, Petr; Koblihová, Jitka; Kizek, René; Frei, Eva; Arlt, Volker M; Stiborová, Marie

    2013-11-01

    Benzo[a]pyrene (BaP) is a human carcinogen requiring metabolic activation prior to reaction with DNA. Cytochrome P450 (CYP) 1A1 is the most important hepatic and intestinal enzyme in both BaP activation and detoxification. CYP1A2 is also capable of oxidizing BaP, but to a lesser extent. The induction of CYP1A1/2 by BaP and/or β-naphthoflavone in liver and small intestine of rats was investigated. Both BaP and β-naphthoflavone induced CYP1A expression and increased enzyme activities in both organs. Moreover, the induction of CYP1A enzyme activities resulted in an increase in formation of BaP-DNA adducts detected by (32)P-postlabeling in rat liver and in the distal part of small intestine in vivo. The increases in CYP1A enzyme activity were also associated with bioactivation of BaP and elevated BaP-DNA adduct levels in ex vivo incubations of microsomes of both organs with DNA and BaP. These findings indicate a stimulating effect of both compounds on BaP-induced carcinogenesis.

  19. Metabolic activation of clopidogrel: in vitro data provide conflicting evidence for the contributions of CYP2C19 and PON1.

    PubMed

    Polasek, Thomas M; Doogue, Matthew P; Miners, John O

    2011-12-01

    The recent report that clopidogrel efficacy may be more dependent on paraoxonase-1 (PON1) than on cytochrome P450 2C19 (CYP2C19) activity raises questions about the roles of these and other enzymes in clopidogrel activation. To provide insight into the emerging PON1 versus CYP2C19 debate, this commentary summarizes the clinical evidence on the pharmacokinetic determinants of clopidogrel efficacy. We then review the in vitro studies investigating the enzymes involved in clopidogrel activation, and comment on their strengths and limitations. There is agreement amongst in vitro studies regarding the involvement of CYP1A2 and CYP2B6 in the metabolism of clopidogrel to 2-oxo-clopidogrel. However, the evidence for other CYP enzymes in the first activation step (e.g. CYP2C19 and CYP3A4) is inconsistent and dependent on the in vitro test system and laboratory. All major drug metabolizing CYP enzymes are capable of converting 2-oxo-clopidogrel to sulfenic acid intermediates that subsequently form the active thiol metabolite. However, the extent of CYP involvement in this second step has been challenged, and new evidence suggests that CYP-independent hydrolytic cleavage of the thioester bond may be more important than oxidative metabolism.

  20. 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.

  1. 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

  2. Effect of standardized cranberry extract on the activity and expression of selected biotransformation enzymes in rat liver and intestine.

    PubMed

    Bártíková, Hana; Boušová, Iva; Jedličková, Pavla; Lněničková, Kateřina; Skálová, Lenka; Szotáková, Barbora

    2014-09-18

    The use of dietary supplements containing cranberry extract is a common way to prevent urinary tract infections. As consumption of these supplements containing a mixture of concentrated anthocyanins and proanthocyanidins has increased, interest in their possible interactions with drug-metabolizing enzymes has grown. In this in vivo study, rats were treated with a standardized cranberry extract (CystiCran®) obtained from Vaccinium macrocarpon in two dosage schemes (14 days, 0.5 mg of proanthocyanidins/kg/day; 1 day, 1.5 mg of proanthocyanidins/kg/day). The aim of this study was to evaluate the effect of anthocyanins and proanthocyanidins contained in this extract on the activity and expression of intestinal and hepatic biotransformation enzymes: cytochrome P450 (CYP1A1, CYP1A2, CYP2B and CYP3A), carbonyl reductase 1 (CBR1), glutathione-S-transferase (GST) and UDP-glucuronosyl transferase (UGT). Administration of cranberry extract led to moderate increases in the activities of hepatic CYP3A (by 34%), CYP1A1 (by 38%), UGT (by 40%), CBR1 (by 17%) and GST (by 13%), while activities of these enzymes in the small intestine were unchanged. No changes in the relative amounts of these proteins were found. Taken together, the interactions of cranberry extract with simultaneously administered drugs seem not to be serious.

  3. Simultaneous Screening of Activities of Five Cytochrome P450 and Four Uridine 5'-Diphospho-glucuronosyltransferase Enzymes in Human Liver Microsomes Using Cocktail Incubation and Liquid Chromatography-Tandem Mass Spectrometry.

    PubMed

    Lee, Boram; Ji, Hyeon-Kyeong; Lee, Taeho; Liu, Kwang-Hyeon

    2015-07-01

    Cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT) are major metabolizing enzymes in the biotransformation of most drugs. Altered P450 and UGT activities are a potential cause of adverse drug-drug interaction. A method for the simultaneous evaluation of the activities of five P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) and four UGTs (UGT1A1, UGT1A4, UGT1A9, and UGT2B7) was developed using in vitro cocktail incubation and tandem mass spectrometry. The nine probe substrates used in this assay were phenacetin (CYP1A2), diclofenac (CYP2C9), S-mephenytoin (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), 7-ethyl-10-hydroxy-camptothecin (SN-38) (UGT1A1), trifluoperazine (UGT1A4), mycophenolic acid (UGT1A9), and naloxone (UGT2B7). This new method involves incubation of two cocktail doses and single cassette analysis. The two cocktail doses and the concentration of each probe substrate in vitro were determined to minimize mutual drug interactions among substrates. Cocktail A comprised phenacetin, diclofenac, S-mephenytoin, dextromethorphan, and midazolam, whereas cocktail B comprised SN-38, trifluoperazine, mycophenolic acid, and naloxone. In the incubation study of these cocktails, the reaction mixtures were pooled and simultaneously analyzed using liquid chromatography-tandem mass spectrometry. The method was validated by comparing inhibition data obtained from the incubation of each probe substrate alone with data from the cocktail method. The IC50 values obtained in both cocktail and individual incubations were in agreement with values previously reported in the literature. This cocktail method offers a rapid and robust way to simultaneously evaluate phase I and II enzyme inhibition profiles of many new chemical entities. This new method will also be useful in the drug discovery process and for advancing the mechanistic understanding of drug interactions. PMID:25904760

  4. Effects of capsaicin and dihydrocapsaicin on human and rat liver microsomal CYP450 enzyme activities in vitro and in vivo.

    PubMed

    Zhang, Qing-Hao; Hu, Jin-Ping; Wang, Bao-Lian; Li, Yan

    2012-01-01

    Capsaicin and dihydrocapsaicin, the two most abundant members of capsaicinoids in chili peppers, are widely used as food additives and for other purposes. In this study, we examined the inhibitory potentials of capsaicin and dihydrocapsaicin against CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 activities in human liver microsomes. The effects of these two capsaicinoids on CYP450 enzymes were also evaluated in vivo in rats. The results demonstrated that capsaicin and dihydrocapsaicin moderately inhibited five isozymes (IC₅₀) values ranging from 4.4 to 61.8 μM), with the exception of CYP2E1 (IC₅₀ > 200 μM). Both capsaicinoids exhibited competitive, mixed, and noncompetitive inhibition on these isozymes (K (i) = 3.1 ± 0.5 - 78.6 ± 8.4 μM). Time-dependent inhibition of CYP3A4/5 by capsaicin was found. After multiple administrations of capsaicin and dihydrocapsaicin (1, 4, and 10 mg/kg) to rats, chlorzoxazone 6-hydroxylase activity and the expression of CYP2E1 were increased in liver microsomes. Our findings indicated that the possibility of food-drug interactions mediated by capsaicin and dihydrocapsaicin could not be excluded, and provided the useful information for evaluating the anticarcinogenic potentials of these two capsaicinoids. PMID:22375877

  5. Comparative 1-substituted imidazole inhibition of cytochrome p450 isozyme-selective activities in human and mouse hepatic microsomes.

    PubMed

    Franklin, Michael R; Constance, Jonathan E

    2007-01-01

    Inhibition of cytochrome P450(CYP)-selective reactions in a single human and a single mouse hepatic microsome preparation by fourteen 1-substituted imidazoles provides a simultaneous ranking of reaction susceptibility to a specific imidazole and the relative inhibitory potency of the imidazoles for a given reaction. CYP3A4/5 activity was inhibited (IC(50) <5 microM) by the greatest number of imidazoles, followed closely by CYP2C9. Seven imidazoles exhibited IC(50) values for CYP3A4/5 <0.3 microM (none for CYP2C9) and were exclusively above 300 MW. Nafimidone (MW, 236) exhibited an IC(50) value <0.3 microM towards CYP2D6 and CYP1A2 reactions. CYP2E1 and CYP2A6 were exclusively inhibited (IC(50) <5 microM) by imidazoles with MWs below approximately 200. In general, mouse activities exhibited lower IC(50) values than in human microsomes. PMID:17786623

  6. Casein Kinase 2 Is a Novel Regulator of the Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) Trafficking.

    PubMed

    Chan, Ting; Cheung, Florence Shin Gee; Zheng, Jian; Lu, Xiaoxi; Zhu, Ling; Grewal, Thomas; Murray, Michael; Zhou, Fanfan

    2016-01-01

    Human organic anion transporting polypeptides (OATPs) mediate the influx of many important drugs into cells. Casein kinase 2 (CK2) is a critical protein kinase that phosphorylates >300 protein substrates and is dysregulated in a number of disease states. Among the CK2 substrates are several transporters, although whether this includes human OATPs has not been evaluated. The current study was undertaken to evaluate the regulation of human OATP1A2 by CK2. HEK-239T cells in which OATP1A2 was overexpressed were treated with CK2 specific inhibitors or transfected with CK2 specific siRNA, and the activity, expression, and subcellular trafficking of OATP1A2 was evaluated. CK2 inhibition decreased the uptake of the prototypic OATP1A2 substrate estrone-3-sulfate (E3S). Kinetic studies revealed that this was due to a decrease in the maximum velocity (Vmax) of E3S uptake, while the Michaelis constant was unchanged. The cell surface expression, but not the total cellular expression of OATP1A2, was impaired by CK2 inhibition and knockdown of the catalytic α-subunits of CK2. CK2 inhibition decreased the internalization of OATP1A2 via a clathrin-dependent pathway, decreased OATP1A2 recycling, and likely impaired OATP1A2 targeting to the cell surface. Consistent with these findings, CK2 inhibition also disrupted the colocalization of OATP1A2 and Rab GTPase (Rab)4-, Rab8-, and Rab9-positive endosomal and secretory vesicles. Taken together, CK2 has emerged as a novel regulator of the subcellular trafficking and stability of OATP1A2. Because OATP1A2 transports many molecules of physiological and pharmacological importance, the present data may inform drug selection in patients with diseases in which CK2 and OATP1A2 are dysregulated.

  7. Comparison of three different in vitro mutation assays used for the investigation of cytochrome P450-mediated mutagenicity of nitro-polycyclic aromatic hydrocarbons.

    PubMed

    Kappers, W A; van Och, F M; de Groene, E M; Horbach, G J

    2000-03-23

    Three different in vitro mutation assays were used to investigate the involvement of cytochrome P450 enzymes in the activation of the nitro-polycyclic aromatic hydrocarbons (nitroPAHs) 1-nitropyrene and 2-nitrofluorene and their reduced metabolites amino-polycyclic aromatic hydrocarbons (aminoPAHs) 1-aminopyrene and 2-aminofluorene. Mutagenicity was investigated at the HPRT locus in Chinese hamster V79 cells with (V79-NH) or without (V79-MZ) endogenous acetyltransferase activity, stably expressing human cytochrome P450 cDNAs; in NIH/3T3 control or stably expressing human CYP1A2 cells, in combination with a shuttle vector containing a reporter gene; and in Salmonella typhimurium TA98, by inhibition of cytochrome P450 enzymes in rat liver S9 mix. Both the HPRT assay and the Ames test did not show any involvement of CYP3A in the activation of 1-nitropyrene to a mutagenic metabolite. In addition, a clear involvement of CYP1A2 in the activation of the nitroPAH 1-nitropyrene was demonstrated in both mutation assays using eukaryotic cells. However, no activation of 1-nitropyrene was seen in the eukaryotic cell lines when expressing only CYP1A2 (V79-MZ1A2) or acetyltransferase (V79-NH, 3T3-LNCX). The reduced metabolite of 1-nitropyrene, 1-aminopyrene, was also shown to be activated to a mutagenic metabolite by CYP1A2, using 3T3-1A2 cells in combination with a shuttle vector, and the Amestest in combination with the specific CYP1A2 inhibitor furafylline. No clear involvement of cytochrome P450 could be demonstrated for activation of 2-nitrofluorene to a mutagenic metabolite, whereas a role for CYP1A2 in the bioactivation of 2-aminofluorene is suggested. In the present study, we have demonstrated the complementary value of the three in vitro mutation assays in the examination of promutagen activation pathways. PMID:10727902

  8. Isoxanthohumol--Biologically active hop flavonoid.

    PubMed

    Żołnierczyk, Anna Katarzyna; Mączka, Wanda Krystyna; Grabarczyk, Małgorzata; Wińska, Katarzyna; Woźniak, Edyta; Anioł, Mirosław

    2015-06-01

    Isoxanthohumol (IXN), apart from xanthohumol (XN) and 8-prenylnaringenin (8PN), is one of the most important prenylflavonoids found in hops. Another natural source of this compound is a shrub Sophora flavescens, used in traditional Chinese medicine. Main dietary source of IXN is beer, and the compound is produced from XN during wort boiling. In the human body, the compound is O-demethylated to 8PN, the strongest known phytoestrogen. This process takes place in the liver and in the intestine, where it is mediated by local microflora. It has been reported in some studies that even though beer contains small amounts of hops and its preparations, these compounds may affect the functioning of the human body. IXN exhibits an antiproliferative activity against human cell lines typical for breast cancer (MCF-7), ovarian cancer (A-2780), prostate cancer (DU145 and PC-3), and colon cancer (HT-29 and SW620) cells. It strongly inhibits the activation of the following carcinogens: 2-amino-3-methylimidazol-[4,5-f]quinoline and aflatoxin B1 (AFB1) via human cytochrome P450 (CYP1A2). It also inhibits the production of prostate specific antigen (PSA). IXN significantly reduces the expression of transforming growth factor-β (TGF-β) in the case of invasive breast cancer MDA-MB-231. It interferes with JAK/STAT signaling pathway and inhibits the expression of pro1inflammatory genes in the monoblastic leukemia cell line (MonoMac6). It activates apoptosis in human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMCs). In addition, IXN shows an antiviral activity towards herpes viruses (HSV1 and HSV2) and bovine viral diarrhea virus (BVDV). PMID:25771121

  9. Protein-protein interactions between rat hepatic cytochromes P450 (P450s) and UDP-glucuronosyltransferases (UGTs): evidence for the functionally active UGT in P450-UGT complex.

    PubMed

    Ishii, Yuji; Iwanaga, Megumi; Nishimura, Yoshio; Takeda, Shuso; Ikushiro, Shin-Ichi; Nagata, Kiyoshi; Yamazoe, Yasushi; Mackenzie, Peter I; Yamada, Hideyuki

    2007-10-01

    The interaction between cytochrome P450s (CYP, P450) and UDP-glucuronosyltransferases (UGTs) was studied by co-immunoprecipitation. P450 isoform-selective antibody was used as a probe to co-precipitate UGTs with the P450s from solubilized rat liver microsomes. Antibodies toward CYP3A2, CYP2B2, CYP2C11/13 and CYP1A2 co-precipitated UGTs with corresponding P450s. However, calnexin, a type-I membrane protein, in the endoplasmic reticulum was not co-precipitated by anti-P450 antibodies. UGT activity toward 4-methylumbelliferone was detected in all co-precipitates, suggesting that UGT in the complex with P450s is functionally active. Repeated washing of co-immunoprecipitates revealed differences among P450 isoforms with regard to the affinity for UGT. Larger amounts of UGT1A1 and UGT1A6, compared with UGT2B1, were washed out from UGTs-CYP2C11/13 co-precipitates, whereas UGT-CYP3A2 and UGT-CYP2Bs complexes were resistant to thorough washing. Thus, CYP2C11/13 could associate with UGTs, but the affinity is assumed to be weaker than that of CYP2B/3As. These results suggest that there is isoform specificity in the interaction between P450s and UGTs.

  10. Aryl hydrocarbon receptor activation in lactotropes and gonadotropes interferes with estradiol-dependent and -independent preprolactin, glycoprotein alpha and luteinizing hormone beta gene expression.

    PubMed

    Cao, Jinyan; Patisaul, Heather B; Petersen, Sandra L

    2011-02-20

    Arylhydrocarbon receptor (Ahr) activation by 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) interferes with female reproductive functions, but there is little information on the specific targets of TCDD in the hypothalamic-pituitary-gonadal (HPG) axis. In these studies, we found that TCDD upregulated known AhR target genes, cytochrome p450 1a1 (Cyp1a1), Cyp1a2 and Cyp1b1 in the rat pituitary gland. Moreover, 75% of pituitary lactotropes and 45% of gonadotropes contained Ahr mRNA, and most Ahr-containing cells were estrogen receptor 1 (Esr1)-positive. TCDD abrogated estradiol (E(2))-induced prolactin (Prl) expression in vivo and in vitro; conversely, E(2) blocked TCDD upregulation of luteinizing hormone beta (Lhb) and glycoprotein hormone alpha polypeptide (Cga) expression. TCDD had no effect on levels of Ahr mRNA, but upregulated Esr1 mRNA. E(2) independently repressed Ahr and Esr1 expression and blocked TCDD upregulation of Esr1. Thus, complex interactions between Ahr and Esr alter Prl and luteinizing hormone (LH) synthesis by direct actions in lactotropes and gonadotropes. These findings provide important insights into how TCDD disrupts female reproductive functions.

  11. Aryl Hydrocarbon Receptor Activation in Lactotropes and Gonadotropes Interferes with Estradiol-Dependent and -Independent Preprolactin, Glycoprotein Alpha and Luteinizing Hormone Beta Gene Expression

    PubMed Central

    Cao, JinYan; Patisaul, Heather B.; Petersen, Sandra L.

    2011-01-01

    Arylhydrocarbon receptor (Ahr) activation by 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) interferes with female reproductive functions, but there is little information on the specific targets of TCDD in the hypothalamic-pituitary-gonadal (HPG) axis. In these studies, we found that TCDD upregulated known AhR target genes, cytochrome p450 1a1 (Cyp1a1), Cyp1a2 and Cyp1b1 in the rat pituitary gland. Moreover, 75% of pituitary lactotropes and 45% of gonadotropes contained Ahr mRNA, and most Ahr-containing cells were estrogen receptor 1 (Esr1)-positive. TCDD abrogated estradiol (E2)-induced prolactin (Prl) expression in vivo and in vitro; conversely, E2 blocked TCDD upregulation of luteinizing hormone beta (Lhb) and glycoprotein hormone alpha polypeptide (Cga) expression. TCDD had no effect on levels of Ahr mRNA, but upregulated Esr1 mRNA. E2 independently repressed Ahr and Esr1 expression and blocked TCDD upregulation of Esr1. Thus, complex interactions between Ahr and Esr alter Prl and luteinizing hormone (LH) synthesis by direct actions in lactotropes and gonadotropes. These findings provide important insights into how TCDD disrupts female reproductive functions. PMID:21187122

  12. Inhibition of Cytochrome P450 by Propolis in Human Liver Microsomes

    PubMed Central

    Ryu, Chang Seon; Oh, Soo Jin; Oh, Jung Min; Lee, Ji-Yoon; Lee, Sang Yoon; Chae, Jung-woo; Kwon, Kwang-il; Kim, Sang Kyum

    2016-01-01

    Although propolis is one of the most popular functional foods for human health, there have been no comprehensive studies of herb-drug interactions through cytochrome P450 (CYP) inhibition. The purpose of this study was to determine the inhibitory effects of propolis on the activities of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 using pooled human liver microsomes (HLMs). Propolis inhibited CYP1A2, CYP2E1 and CYP2C19 with an IC50 value of 6.9, 16.8, and 43.1 μg/mL, respectively, whereas CYP2A6, 2B6, 2C9, 2D6, and 3A4 were unaffected. Based on half-maximal inhibitory concentration shifts between microsomes incubated with and without nicotinamide adenine dinucleotide phosphate, propolis-induced CYP1A2, CYP2C19, and CYP2E1 inhibition was metabolism-independent. To evaluate the interaction potential between propolis and therapeutic drugs, the effects of propolis on metabolism of duloxetine, a serotonin-norepinephrine reuptake inhibitor, were determined in HLMs. CYP1A2 and CYP2D6 are involved in hydroxylation of duloxetine to 4-hydroxy duloxetine, the major metabolite, which was decreased following propolis addition in HLMs. These results raise the possibility of interactions between propolis and therapeutic drugs metabolized by CYP1A2. PMID:27437087

  13. Identification of cytochrome P450 isoform involved in the metabolism of YM992, a novel selective serotonin re-uptake inhibitor, in human liver microsomes.

    PubMed

    Noguchi, K; Mera, A; Watanabe, T; Higuchi, S; Chiba, K

    2000-05-01

    1. In vitro studies were conducted to identify the hepatic cytochrome P450 isoform involved in the metabolism of YM992, ((S)-2-[[(fluoro-4-indanyl)oxy]methyl]morpholine monohydrochloride), a novel serotonin re-uptake inhibitor, in human liver microsomes. 2. Microsomes prepared from yeast expressing CYP1A1, CYP1A2 and CYP2D6 effectively metabolized YM992. A significant correlation was observed between the rate of YM992 metabolism and 7-ethoxyresorufin O-deethylation, CYP1A1/2 specific activity, in liver microsomes from 16 individual donors (r2 = 0.628, p < 0.001). Alpha-naphtoflavone and isosafrole, CYP1A1/2 inhibitors, suppressed the metabolism of YM992 in human liver microsomes in a concentration-dependent manner. 3. The metabolism of YM992 in human liver microsomes was inhibited by approximately 95% by antibodies which recognize both CYP1A1 and CYP1A2 whereas antibodies specific for CYP1A1 did not show inhibitory effects. 4. The same major metabolites, M6 and M7, were generated from YM992 after incubation with human liver microsomes and recombinant human CYP1A2. 5. These results suggest that the metabolism of YM992 in human liver microsomes is mainly catalysed by CYP1A2, and that YM992 might increase plasma concentration of concomitant drugs metabolized by CYP1A2 due to competitive inhibition.

  14. Genetic Variation in Melatonin Pathway Enzymes in Children with Autism Spectrum Disorder and Comorbid Sleep Onset Delay

    PubMed Central

    Pendergast, Julie S.; Allen, Melissa J.; Leu, Roberta M.; Johnson, Carl Hirschie; Elsea, Sarah H.; Malow, Beth A.

    2014-01-01

    Sleep disruption is common in individuals with autism spectrum disorder (ASD). Genes whose products regulate endogenous melatonin modify sleep patterns and have been implicated in ASD. Genetic factors likely contribute to comorbid expression of sleep disorders in ASD. We studied a clinically unique ASD subgroup, consisting solely of children with comorbid expression of sleep onset delay. We evaluated variation in two melatonin pathway genes, acetylserotonin O-methyltransferase (ASMT) and cytochrome P450 1A2 (CYP1A2). We observed higher frequencies than currently reported (p < 0.04) for variants evidenced to decrease ASMT expression and related to decreased CYP1A2 enzyme activity (p ≤ 0.0007). We detected a relationship between genotypes in ASMT and CYP1A2 (r2 = 0.63). Our results indicate that expression of sleep onset delay relates to melatonin pathway genes. PMID:25059483

  15. Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies.

    PubMed

    Hoffmann, Stefan A; Müller-Vieira, Ursula; Biemel, Klaus; Knobeloch, Daniel; Heydel, Sandra; Lübberstedt, Marc; Nüssler, Andreas K; Andersson, Tommy B; Gerlach, Jörg C; Zeilinger, Katrin

    2012-12-01

    Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/10(6) cells followed by a period of stable conversion of about 100 pmol/h/10(6) cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver.

  16. Mechanism-based inhibitory and peroxisome proliferator-activated receptor α-dependent modulating effects of silybin on principal hepatic drug-metabolizing enzymes.

    PubMed

    Wang, Hong; Yan, Tingting; Xie, Yuan; Zhao, Min; Che, Yuan; Zhang, Jun; Liu, Huiying; Cao, Lijuan; Cheng, Xuefang; Xie, Yang; Li, Feiyan; Qi, Qu; Wang, Guangji; Hao, Haiping

    2015-04-01

    Silybin, a major pharmacologically active compound in silymarin, has been widely used in combination with other prescriptions in the clinic to treat hepatitis and a host of other diseases. Previous studies suggested that silybin is a potential inhibitor of multiple drug-metabolizing enzymes (DMEs); however, the in vitro to in vivo translation and the mechanisms involved remain established. The aim of this study was to provide a mechanistic understanding of the regulatory effects of silybin on principal DMEs. Silybin (50 or 150 mg/kg/d) was administered to mice for a consecutive 14 days. The plasma and hepatic exposure of silybin were detected; the mRNA, protein levels, and enzyme activities of principal DMEs were determined. The results demonstrated that the enzyme activities of CYP1A2, CYP2C, CYP3A11, and UGT1A1 were significantly repressed, whereas little alteration of the mRNA and protein levels was observed. Silybin inhibits these DMEs in a mechanism-based and/or substrate-competitive manner. More importantly, silybin was found to be a weak agonist of peroxisome proliferator-activated receptor (PPAR)α, as evidenced from the molecular docking, reporter gene assay, and the targeting gene expression analysis. However, silybin could significantly compromise the activation of PPARα by fenofibrate, characterized with significantly repressed expression of PPARα targeting genes, including L-FABP, ACOX1, and UGT1A6. This study suggests that silybin, despite its low bioavailability, may inhibit enzyme activities of multiple DMEs in a mechanism-based mode, and more importantly, may confer significant drug-drug interaction with PPARα agonists via the repression of PPARα activation in a competitive mode. PMID:25587127

  17. Metabolism of methiocarb and carbaryl by rat and human livers and plasma, and effect on their PXR, CAR and PPARα activities.

    PubMed

    Fujino, Chieri; Tamura, Yuki; Tange, Satoko; Nakajima, Hiroyuki; Sanoh, Seigo; Watanabe, Yoko; Uramaru, Naoto; Kojima, Hiroyuki; Yoshinari, Kouichi; Ohta, Shigeru; Kitamura, Shigeyuki

    2016-01-01

    The oxidative, reductive, and hydrolytic metabolism of methiocarb and the hydrolytic metabolism of carbaryl by liver microsomes and plasma of rats or humans were examined. The effects of the metabolism of methiocarb and carbaryl on their nuclear receptor activities were also examined. When methiocarb was incubated with rat liver microsomes in the presence of NADPH, methiocarb sulfoxide, and a novel metabolite, methiocarb sulfone were detected. Methiocarb sulfoxide was oxidized to the sulfone by liver microsomes and reduced back to methiocarb by liver cytosol. Thus, the interconversion between methiocarb and the sulfoxide was found to be a new metabolic pathway for methiocarb by liver microsomes. The product of methiocarb hydrolysis, which is methylthio-3,5-xylenol (MX), was also oxidized to sulfoxide form by rat liver microsomes. The oxidations were catalyzed by human flavin-containing monooxygenase isoform (FMO1). CYP2C19, which is a human cytochrome P450 (CYP) isoform, catalyzed the sulfoxidations of methiocarb and MX, while CYP1A2 also exhibited oxidase activity toward MX. Methiocarb and carbaryl were not enzymatically hydrolyzed by the liver microsomes, but they were mainly hydrolyzed by plasma and albumin to MX and 1-naphthol, respectively. Both methiocarb and carbaryl exhibited PXR and PPARα agonistic activities; however, methiocarb sulfoxide and sulfone showed markedly reduced activities. In fact, when methiocarb was incubated with liver microsomes, the receptor activities were decreased. In contrast, MX and 1-naphthol showed nuclear receptor activities equivalent to those of their parent carbamates. Thus, the hydrolysis of methiocarb and carbaryl and the oxidation of methiocarb markedly modified their nuclear receptor activities. PMID:27665777

  18. 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

  19. Genotoxic activity and induction of biotransformation enzymes in two human cell lines after treatment by Erika fuel extract.

    PubMed

    Amat-Bronnert, Agnès; Castegnaro, Marcel; Pfohl-Leszkowicz, Annie

    2007-01-01

    On 12 December 1999, the tanker Erika broke in two parts at about 60km from the Brittany French coasts (Point of Penmarc'h, Sud Finistère, France). About 10,000tonnes of heavy oil fuel were released in the sea. DNA adduct have been detected in fish liver and mussels digestive gland exposed to the Erika oil spill. In order to investigate the mechanism by which Erika fuel extract exhibits genotoxic effects the induction of DNA adducts by an Erika fuel extract have been analysed on two cell lines, human epithelial bronchial cells (WI) and human hepatoma cells. DNA adducts, reflected by a diagonal radioactive zone and individual adducts are detected only in hepatoma cells indicating biotransformation via CYP 1A2 and CYP 1B1. In addition, Erika fuel extract induces some metabolizing enzymes such CYP 1A2, COX2 and 5-LOX, the two later are involved in cancer processes. Formation of leucotrienes B4 (LTB(4)), a mediator playing a role in inflammation, is induced in epithelial bronchial cells. Since inhalation is one of the ways of contamination for human, the above results are important for human health and prevention. PMID:21783741

  20. Inhibitory Effects of Aschantin on Cytochrome P450 and Uridine 5'-diphospho-glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

    PubMed

    Kwon, Soon-Sang; Kim, Ju-Hyun; Jeong, Hyeon-Uk; Cho, Yong Yeon; Oh, Sei-Ryang; Lee, Hye Suk

    2016-01-01

    Aschantin is a bioactive neolignan found in Magnolia flos with antiplasmodial, Ca(2+)-antagonistic, platelet activating factor-antagonistic, and chemopreventive activities. We investigated its inhibitory effects on the activities of eight major human cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes of human liver microsomes to determine if mechanistic aschantin-enzyme interactions were evident. Aschantin potently inhibited CYP2C8-mediated amodiaquine N-de-ethylation, CYP2C9-mediated diclofenac 4'-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4'-hydroxylation, and CYP3A4-mediated midazolam 1'-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. Aschantin at 100 µM negligibly inhibited CYP1A2-mediated phenacetin O-de-ethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, and CYP2D6-mediated bufuralol 1'-hydroxylation. At 200 µM, it weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively, but did not show inhibition against UGT1A3, UGT1A4, or UGT2B7 up to 200 µM. These in vitro results indicate that aschantin should be examined in terms of potential interactions with pharmacokinetic drugs in vivo. It exhibited potent mechanism-based inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4. PMID:27128896

  1. Evaluation of the effect of TM208 on the activity of five cytochrome P450 enzymes using on-line solid-phase extraction HPLC-DAD: a cocktail approach.

    PubMed

    Lin, Wensi; Zhang, Jianmei; Ling, Xiaomei; Yu, Ning; Li, Jing; Yang, Haisong; Li, Runtao; Cui, Jingrong

    2013-04-01

    A rapid, simple, and sensitive on-line solid-phase extraction HPLC-DAD method for simultaneous evaluation of the activity of five CYP450 isoforms (CYP1A2, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) in vivo has been developed and validated. The five specific probe substrates include caffeine (1A2), metoprolol (2D6), dapsone (3A4), omeprazole (2C19) and chlorzoxazone (2E1). Automated pre-purification of plasma and enrichment of analytes were performed using a C18 on-line solid-phase extraction cartridge. After being eluted from the cartridge, the analytes and the internal standard antipyrine were separated on a C18 RP analytical column and analyzed by DAD. The method was validated to quantify the concentration ranges of 0.05-50.0 μg/ml for dapsone and omeprazole, 0.1-50.0 μg/ml for caffeine and 0.2-50.0 μg/ml for metoprolol and chlorzoxazone. The linearity (R(2)) for all analytes tested was exceeded 0.99. The intra-day precision ranged from 0.29 to 13% and the inter-day precision ranged from 5.0 to 15%, respectively. The intra-day and inter-day accuracy were between 86.7% and 113.6%. The extraction recoveries were in the range 82.8-109.9% for all the analytes and internal standard antipyrine. This method was successfully applied to evaluate the effects of TM208 on rat five CYP450 isoforms.

  2. Mixed-ligand copper(II) complexes activate aryl hydrocarbon receptor AhR and induce CYP1A genes expression in human hepatocytes and human cell lines.

    PubMed

    Kubešová, Kateřina; Dořičáková, Aneta; Trávníček, Zdeněk; Dvořák, Zdeněk

    2016-07-25

    The effects of four copper(II) mixed-ligand complexes [Cu(qui1)(L)]NO3·H2O (1-3) and [Cu(qui2)(phen)]NO3 (4), where qui1=2-phenyl-3-hydroxy-4(1H)-quinolinone, Hqui2=2-(4-amino-3,5-dichlorophenyl)-N-propyl-3-hydroxy-4(1H)-quinolinone-7-carboxamide, L=1,10-phenanthroline (phen) (1), 5-methyl-1,10-phenanthroline (mphen) (2), bathophenanthroline (bphen) (3), on transcriptional activities of steroid receptors, nuclear receptors and xenoreceptors have been studied. The complexes (1-4) did not influence basal or ligand-inducible activities of glucocorticoid receptor, androgen receptor, thyroid receptor, pregnane X receptor and vitamin D receptor, as revealed by gene reporter assays. The complexes 1 and 2 dose-dependently induced luciferase activity in stable gene reporter AZ-AhR cell line, and this induction was reverted by resveratrol, indicating involvement of aryl hydrocarbon receptor (AhR) in the process. The complexes 1, 2 and 3 induced CYP1A1 mRNA in LS180 cells and CYP1A1/CYP1A2 in human hepatocytes through AhR. Electrophoretic mobility shift assay EMSA showed that the complexes 1 and 2 transformed AhR in its DNA-binding form. Collectively, we demonstrate that the complexes 1 and 2 activate AhR and induce AhR-dependent genes in human hepatocytes and cancer cell lines. In conclusion, the data presented here might be of toxicological importance, regarding the multiple roles of AhR in human physiology and pathophysiology. PMID:27180721

  3. Transcription coactivator peroxisome proliferator-activated receptor-binding protein/mediator 1 deficiency abrogates acetaminophen hepatotoxicity

    PubMed Central

    Jia, Yuzhi; Guo, Grace L.; Surapureddi, Sailesh; Sarkar, Joy; Qi, Chao; Guo, Dongsheng; Xia, Jun; Kashireddi, Papreddy; Yu, Songtao; Cho, Young-Wook; Rao, M. Sambasiva; Kemper, Byron; Ge, Kai; Gonzalez, Frank J.; Reddy, Janardan K.

    2005-01-01

    Peroxisome proliferator-activated receptor-binding protein (PBP), also known as thyroid hormone receptor-associated protein 220/vitamin D receptor-interacting protein 205/mediator 1, an anchor for multisubunit mediator transcription complex, functions as a transcription coactivator for nuclear receptors. Disruption of the PBP gene results in embryonic lethality around embryonic day 11.5 by affecting placental and multiorgan development. Here, we report that targeted deletion of PBP in liver parenchymal cells (PBPLiv-/-) results in the abrogation of hypertrophic and hyperplastic influences in liver mediated by constitutive androstane receptor (CAR) ligands phenobarbital (PB) and 1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene, and of acetaminophen-induced hepatotoxicity. CAR interacts with the two nuclear receptor-interacting LXXLL (L, leucine; X, any amino acid) motifs in PBP in a ligand-dependent manner. We also show that PBP interacts with the C-terminal portion of CAR, suggesting that PBP is involved in the regulation of CAR function. Although the full-length PBP only minimally increased CAR transcriptional activity, a truncated form of PBP (amino acids 487-735) functioned as a dominant negative repressor, establishing that PBP functions as a coactivator for CAR. A reduction in CAR mRNA and protein level observed in PBPLiv-/- mouse liver suggests that PBP may regulate hepatic CAR expression. PBP-deficient hepatocytes in liver failed to reveal PB-dependent translocation of CAR to the nucleus. Adenoviral reconstitution of PBP in PBPLiv-/- mouse livers restored PB-mediated nuclear translocation of CAR as well as inducibility of CYP1A2, CYP2B10, CYP3A11, and CYP7A1 expression. We conclude that transcription coactivator PBP/TRAP220/MED1 is involved in the regulation of hepatic CAR function and that PBP deficiency in liver abrogates acetaminophen hepatotoxicity. PMID:16109766

  4. 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...

  5. Tritium analyses of COBRA-1A2 beryllium pebbles

    SciTech Connect

    Baldwin, D.L.

    1998-03-01

    Selected tritium measurements have been completed for the COBRA-1A2 experiment C03 and D03 beryllium pebbles. The completed results, shown in Tables 1, 2, and 3, include the tritium assay results for the 1-mm and 3-mm C03 pebbles, and the 1-mm D03 pebbles, stepped anneal test results for both types of 1-mm pebbles, and the residual analyses for the stepped-anneal specimens. All results have been reported with date-of-count and are not corrected for decay. Stepped-anneal tritium release response is provided in addenda.

  6. In vivo prediction of CYP-mediated metabolic interaction potential of formononetin and biochanin A using in vitro human and rat CYP450 inhibition data.

    PubMed

    Arora, Sumit; Taneja, Isha; Challagundla, Muralikrishna; Raju, Kanumuri Siva Rama; Singh, Sheelendra Pratap; Wahajuddin, Muhammad

    2015-11-19

    Formononetin (FMN) and Biochanin A (BCA) are the principal isoflavones present in commercially available extracts of red clover that are widely been consumed for various health benefits. We investigated the in vitro effects of FMN and BCA on catalytic activity of human/rat cytochrome P450 enzymes to assess the drug interaction potential of red clover. IC50 and Ki values of FMN and BCA for CYPs were determined in human/rat liver microsomes. FMN and BCA showed concentration-dependent inhibition of CYP1A2 activity with IC50 values of 13.42 and 24.98μM in human liver microsomes and 38.57 and 11.86μM in rat liver microsomes, respectively. The mode of inhibition of human CYP1A2 by FMN was found to be competitive with apparent Ki value of 10.13±1.96μM. FMN also inhibited human CYP2D6. BCA exerted moderately inhibitory effects on human CYP2C9. The predicted in vivo inhibition for CYP1A2 was insignificant (R value <1.1) at hepatic level while at intestinal level, it was significant (R value >11). The inhibitory effects on other CYPs were found to be minimal. Red clover may be considered safe to be consumed along with co-prescribed medications; however, precaution must be taken while co-administering it with CYP1A2 substrates.

  7. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)-induced hepatocarcinogenesis is not enhanced by CYP1A inducers, alpha- and beta-naphthoflavone: relationship to intralobular distribution of CYP1A expression.

    PubMed

    Suzuki, Shugo; Takeshita, Kentaro; Doi, Yuko; Asamoto, Makoto; Takahashi, Satoru; Naiki-Ito, Aya; Shirai, Tomoyuki

    2010-06-01

    Interaction of more than two chemicals from foods is a very important factor for carcinogenic risk assessment and management. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant carcinogenic heterocyclic amines in cooked foods, is speculated to be a human liver carcinogen. MeIQx is metabolically activated by CYP1A2 and then N-acetyltransferase (NAT), findings that suggest that its carcinogenic potential might be enhanced by simultaneous exposure to chemical(s) inducing CYP1A2. Therefore, we here investigated the effects of alpha- and beta-naphthoflavone as CYP1A2 inducers on MeIQx-induced rat hepatocarcinogenesis in a medium-term rat liver bioassay. Unexpectedly, no modifying influence of naphthoflavones on MeIQx-induced hepatocarcinogenesis was demonstrated with reference to glutathione S-transferase placental form (GST-P) positive foci in the liver, although up-regulation of CYP1A2 was detected on Western blot analysis. Activity of NAT was not affected. In MeIQx-treated rats, CYP1A expression was mainly detected in zone 3 of the liver where GST-P positive foci were preferentially located, while naphthoflavones alone or combinations of naphthoflavones and MeIQx induced CYP1A expression in zone 1. This difference in intralobular distribution of CYP1A might be related to the fact that MeIQx hepatocarcinogenesis was not modified by the two CYP1A inducers.

  8. Identification of human liver cytochrome P450 enzymes involved in the metabolism of SCH 530348 (Vorapaxar), a potent oral thrombin protease-activated receptor 1 antagonist.

    PubMed

    Ghosal, Anima; Lu, Xiaowen; Penner, Natalia; Gao, Lan; Ramanathan, Ragu; Chowdhury, Swapan K; Kishnani, Narendra S; Alton, Kevin B

    2011-01-01

    Vorapaxar (SCH 530348), a potent oral thrombin protease-activated receptor 1 antagonist, is being developed as an antiplatelet agent for patients with established vascular disease. The objective of this study was to identify the human liver cytochrome P450 (P450) enzyme(s) responsible for the metabolism of SCH 530348. Human liver microsomes metabolized SCH 530348 to M19, an amine metabolite formed via carbamate cleavage, and M20 (monohydroxy-SCH 530348). Recombinant human CYP3A4 exhibited the most activity (11.5% profiled radioactivity) for the formation of M19, followed by markedly less substrate conversion with CYP1A1 and CYP2C19. Trace levels of M19, a major excreted human metabolite, were detected with CYP1A2, CYP3A5, and CYP4F3A. Formation of M19 by human liver microsomes was inhibited 89% by ketoconazole (IC(50), 0.73 μM), 34% by tranylcypromine, and 89% by anti-CYP3A4 monoclonal antibody. There was a significant correlation between the rate of M19 formation and midazolam 1'-hydroxylation (r = 0.75) or M19 formation and testosterone 6β-hydroxylation (r = 0.92). The results of screening, inhibition, and correlation studies confirmed that CYP3A4 is the major P450 enzyme responsible for M19 formation from SCH 530348. In contrast, formation of M20, a major circulating human metabolite at steady state, was primarily catalyzed by CYP3A4 and CYP2J2. M20 is pharmacologically equipotent to SCH 530348, whereas M19 is an inactive metabolite. Formation of M20 by human liver microsomes was inhibited 89% by ketoconazole, 75% by astemizole (a CYP2J2 inhibitor), and 43% by CYP3A4 monoclonal antibody. These results suggest that CYP3A4 and CYP2J2 are both involved in the formation of M20 metabolite. PMID:20926621

  9. In vitro evaluation of cytochrome P450 induction and the inhibition potential of mitragynine, a stimulant alkaloid.

    PubMed

    Lim, Ee Lin; Seah, Tiong Chai; Koe, Xue Fen; Wahab, Habibah Abdul; Adenan, Mohd Ilham; Jamil, Mohd Fadzly Amar; Majid, Mohamed Isa Abdul; Tan, Mei Lan

    2013-03-01

    CYP450 enzymes are key determinants in drug toxicities, reduced pharmacological effect and adverse drug reactions. Mitragynine, an euphoric compound was evaluated for its effects on the expression of mRNAs encoding CYP1A2, CYP2D6 and CYP3A4 and protein expression and resultant enzymatic activity. The mRNA and protein expression of CYP450 isoforms were carried out using an optimized multiplex qRT-PCR assay and Western blot analysis. CYP1A2 and CYP3A4 enzyme activities were evaluated using P450-Glo™ assays. The effects of mitragynine on human CYP3A4 protein expression were determined using an optimized hCYP3A4-HepG2 cell-based assay. An in silico computational method to predict the binding conformation of mitragynine to the active site of the CYP3A4 enzyme was performed and further validated using in vitro CYP3A4 inhibition assays. Mitragynine was found to induce mRNA and protein expression of CYP1A2. For the highest concentration of 25 μM, induction of mRNA was approximately 70% that of the positive control and was consistent with the increased CYP1A2 enzymatic activity. Thus, mitragynine is a significant in vitro CYP1A2 inducer. However, it appeared to be a weak CYP3A4 inducer at the transcriptional level and a weak CYP3A4 enzyme inhibitor. It is therefore, unlikely to have any significant clinical effects on CYP3A4 activity. PMID:23274770

  10. Liquid chromatography/tandem mass spectrometry method for simultaneous evaluation of activities of five cytochrome P450s using a five-drug cocktail and application to cytochrome P450 phenotyping studies in rats.

    PubMed

    Zhang, Shaoyu; Song, Naining; Li, Quansheng; Fan, Huirong; Liu, Changxiao

    2008-08-01

    A reliable liquid chromatography/tandem mass spectrometry has been developed for simultaneous evaluation of the activities of five cytochrome P450s (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A) in rat plasma and urine. The five-specific probe substrates/metabolites include phenacetin/paracetamol (CYP1A2), tolbutamide/4-hydroxytolbutamide and carboxytolbutamide (CYP2C9), mephenytoin/4'-hydroxymephenytoin (CYP2C19), dextromethorphan/dextrorphan (CYP2D6), and midazolam/1'-hydroxymidazolam (CYP3A). Internal standards were brodimoprim (for phenacetin, paracetamol, midazolam and 1'-hydroxymidazolam), ofloxacin (for 4'-hydroxymephenytoin, dextromethorphan and dextrorphan) and meloxicam (for tolbutamide, 4-hydroxytolbutamide and carboxytolbutamide). Sample preparation was conducted with solid-phase extraction using Oasis HLB cartridges. The chromatography was performed using a C(18) column with mobile phase consisting of methanol/0.1% formic acid in 20 mM ammonium formate (75:25). The triple-quadrupole mass spectrometric detection was operated in both positive mode (for phenacetin, paracetamol, midazolam, 1'-hydroxymidazolam, brodimoprim, 4'-hydroxymephenytoin, dextromethorphan, dextrorphan and ofloxacin) and negative mode (for tolbutamide, 4-hydroxytolbutamide, carboxytolbutamide and meloxicam). Multiple reaction monitoring mode was used for data acquisition. Calibration ranges in plasma were 2.5-2500 ng/mL for phenacetin, 2.5-2500 ng/mL for paracetamol, 5-500 ng/mL for midazolam, and 0.5-500 ng/mL for 1'-hydroxymidazolam. In urine calibration ranges were 5-1000 ng/mL for dextromethorphan, 0.05-10 microg/mL for dextrorphan and 4'-hydroxymephenytoin, 5-2000 ng/mL for tolbutamide, 0.05-20 microg/mL for 4-hydroxytolbutamide and 0.025-10 microg/mL for carboxytolbutamide. The intra- and inter-day precision were 4.3-12.4% and 1.5-14.8%, respectively for all of the above analytes. The intra- and inter-day accuracy ranged from -9.1 to 8.3% and -10 to 9.2%, respectively for all of

  11. Erectile Dysfunction Drugs Changed the Protein Expressions and Activities of Drug-Metabolising Enzymes in the Liver of Male Rats

    PubMed Central

    Hassan, Mostafa

    2016-01-01

    Erectile dysfunction (ED) is a major health problem and is mainly associated with the persistent inability of men to maintain sufficient erection for satisfactory sexual performance. Millions of men are using sildenafil, vardenafil, and/or tadalafil for ED treatment. Cytochrome P450s (CYPs) play a central role in the metabolism of a wide range of xenobiotics as well as endogenous compounds. Susceptibility of individuals to the adverse effects of different drugs is mainly dependent on the expression of CYPs proteins. Therefore, changes in activities of phase I drug-metabolising enzymes [arylhydrocarbon hydroxylase (AHH), dimethylnitrosamine N-demethylase (DMN-dI), 7-ethoxycoumarin-O-deethylase (ECOD), and ethoxyresorufin-O-deethylase ((EROD)] and the protein expression of different CYPs isozymes (CYP1A2, CYP2E1, CYP2B1/2, CYP3A4, CYP2C23, and CYP2C6) were determined after treatment of male rats with either low or high doses of sildenafil (Viagra), tadalafil (Cialis), and/or vardenafil (Levitra) for 3 weeks. The present study showed that low doses of tadalafil and vardenafil increased DMN-dI activity by 32 and 23%, respectively. On the other hand, high doses of tadalafil, vardenafil, and sildenafil decreased such activity by 50, 56, and 52%, respectively. In addition, low doses of tadalafil and vardenafil induced the protein expression of CYP2E1. On the other hand, high doses of either tadalafil or sildenafil were more potent inhibitors to CYP2E1 expression than vardenafil. Moreover, low doses of both vardenafil and sildenafil markedly increased AHH activity by 162 and 247%, respectively, whereas high doses of tadalafil, vardenafil, and sildenafil inhibited such activity by 36, 49, and 57% and inhibited the EROD activity by 39, 49, and 33%, respectively. Low and high doses of tadalafil, vardenafil, and sildenafil inhibited the activity of NADPH-cytochrome c reductase as well as its protein expression. In addition, such drugs inhibited the expression of CYP B1/2 along

  12. Translation Elongation Factor eEF1A2 is a Novel Anticancer Target for the Marine Natural Product Plitidepsin

    PubMed Central

    Losada, Alejandro; Muñoz-Alonso, María José; García, Carolina; Sánchez-Murcia, Pedro A.; Martínez-Leal, Juan Fernando; Domínguez, Juan Manuel; Lillo, M. Pilar; Gago, Federico; Galmarini, Carlos M.

    2016-01-01

    eEF1A2 is one of the isoforms of the alpha subunit of the eukaryotic Elongation Factor 1. It is overexpressed in human tumors and is endowed with oncogenic properties, favoring tumor cell proliferation while inhibiting apoptosis. We demonstrate that plitidepsin, an antitumor agent of marine origin that has successfully completed a phase-III clinical trial for multiple myeloma, exerts its antitumor activity by targeting eEF1A2. The drug interacts with eEF1A2 with a KD of 80 nM and a target residence time of circa 9 min. This protein was also identified as capable of binding [14C]-plitidepsin in a cell lysate from K-562 tumor cells. A molecular modelling approach was used to identify a favorable binding site for plitidepsin at the interface between domains 1 and 2 of eEF1A2 in the GTP conformation. Three tumor cell lines selected for at least 100-fold more resistance to plitidepsin than their respective parental cells showed reduced levels of eEF1A2 protein. Ectopic expression of eEF1A2 in resistant cells restored the sensitivity to plitidepsin. FLIM-phasor FRET experiments demonstrated that plitidepsin localizes in tumor cells sufficiently close to eEF1A2 as to suggest the formation of drug-protein complexes in living cells. Altogether, our results strongly suggest that eEF1A2 is the primary target of plitidepsin. PMID:27713531

  13. Overexpression of cerebral and hepatic cytochrome P450s alters behavioral activity of rat offspring following prenatal exposure to lindane

    SciTech Connect

    Johri, Ashu; Yadav, Sanjay; Dhawan, Alok; Parmar, Devendra

    2007-12-15

    Oral administration of different doses (0.0625, 0.125 or 0.25 mg/kg corresponding to 1/1400th, 1/700th or 1/350th of LD{sub 50}) of lindane to the pregnant Wistar rats from gestation days 5 to 21 were found to produce a dose-dependent increase in the activity of cytochrome P450 (CYP)-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of CYP monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have suggested that increase in CYP activity may possibly lead to the formation of metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic CYPs was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.125 and 0.25 mg/kg) of lindane and up to 9 weeks at the highest dose (0.25 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6-week of age, have further indicated that due to the reduced activity of the CYPs during the ontogeny, lindane and its metabolites may not be effectively cleared from the brain. The data suggest that low dose prenatal exposure to the pesticide has the potential to produce overexpression of xenobiotic metabolizing CYPs in brain and liver of the offspring which may account for the behavioral changes observed in the offspring.

  14. PDZK1 and NHERF1 regulate the function of human organic anion transporting polypeptide 1A2 (OATP1A2) by modulating its subcellular trafficking and stability.

    PubMed

    Zheng, Jian; Chan, Ting; Cheung, Florence Shin Gee; Zhu, Ling; Murray, Michael; Zhou, Fanfan

    2014-01-01

    The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (V(max): 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)⁻¹ in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability.

  15. PDZK1 and NHERF1 Regulate the Function of Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) by Modulating Its Subcellular Trafficking and Stability

    PubMed Central

    Zheng, Jian; Chan, Ting; Cheung, Florence Shin Gee; Zhu, Ling; Murray, Michael; Zhou, Fanfan

    2014-01-01

    The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (Vmax: 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)−1 in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability. PMID:24728453

  16. In vitro evaluation of hepatotoxic drugs in human hepatocytes from multiple donors: Identification of P450 activity as a potential risk factor for drug-induced liver injuries.

    PubMed

    Utkarsh, Doshi; Loretz, Carol; Li, Albert P

    2016-08-01

    A possible risk factor for drug-induced hepatotoxicity is drug metabolizing enzyme activity, which is known to vary among individuals due to genetic (genetic polymorphism) and environmental factors (environmental pollutants, foods, and medications that are inhibitors or inducers of drug metabolizing enzymes). We hypothesize that hepatic cytochrome P450-dependent monooxygenase (CYP) activity is one of the key risk factors for drug induced liver injuries (DILI) in the human population, especially for drugs that are metabolically activated to cytotoxic/reactive metabolites. Human hepatocytes from 19 donors were evaluated for the activities of 8 major P450 isoforms: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Extensive individual variations were observed, consistent with what is known to be in the human population. As CYP3A4 is known to be one of the most important P450 isoforms for drug metabolism, studies were performed to evaluate the relationship between the in vitro cytotoxicity of hepatotoxic drugs and CYP3A4 activity. In a proof of concept study, hepatocytes from six donors (lots) representing the observed range of CYP3A4 activities were chosen for the evaluation of in vitro hepatotoxicity of four drugs known to be associated with acute liver failure: acetaminophen, cyclophosphamide, ketoconazole, and tamoxifen. The hepatocytes were cultured in collagen-coated plates and treated with the hepatotoxicants for approximately 24 h, followed by viability determination based on cellular adenosine triphosphate (ATP) contents. HH1023, the lot of hepatocytes with the highest CYP3A4 activity, was found to be the most sensitive to the cytotoxicity of all 4 hepatotoxic drugs, thereby suggesting that high CYP3A4 activity may be a risk factor. To further validate the relationship, a second study was performed with hepatocytes from 16 donors. In this study, the hepatocytes were quantified for CYP3A4 activity at the time of treatment. Results of the

  17. [Interactions of 1A2 insulator with promoter of hsp 70 gene in Drosophila melanogaster].

    PubMed

    Chetverina, D A; Elizar'ev, P V; Georgiev, P G; Erokhin, M M

    2013-04-01

    Insulators are regulatory DNA elements that participate in the modulation of the interactions between enhancers and promoters. Depending on the situation, insulators can either stabilize or destroy the contacts between enhancers and promoters. A possible explanation for the activity of insulators is their ability to directly interact with gene promoters. In the present study, it was demonstrated that, in model systems, a 1A2 insulator could interact with the core sequence of an hsp70 promoter. In this case, the insulator protein CP190 is found on the hsp70 promoter, which depends on the presence of an insulator in the transgene. The data obtained are consistent with the model, which implies that direct contacts between insulators and promoters make a considerable contribution to the modulation of the interactions between insulators and promoters. PMID:23866619

  18. Establishing population distribution of drug-metabolizing enzyme activities for the use of salivary caffeine as a dynamic liver function marker in a Singaporean Chinese population.

    PubMed

    Chia, Hazel Yiting; Yau, Wai-Ping; Ho, Han Kiat

    2016-04-01

    The salivary paraxanthine/caffeine molar ratio has been proposed as a novel dynamic liver function test to guide dose adjustments of drugs hepatically cleared by CYP1A2. Its usability requires an established population norm as well as the factors influencing the ratio and actual concentrations. To address this knowledge gap, salivary caffeine and paraxanthine concentrations were measured at 4 h post caffeine dose in healthy Chinese individuals who had undergone 24 h of caffeine abstinence. The metabolic ratio was calculated and statistical analysis was performed. From the 52 participants (26 males; 30 regular caffeine consumers) recruited, the salivary paraxanthine/caffeine molar ratio was normally distributed with a mean and SD of 0.5 ± 0.2. No statistically significant factors (BMI, body weight, gender and regularity of caffeine intake) affecting the metabolic ratio were found. The caffeine concentration and total caffeine plus paraxanthine concentrations were lower in males than in females, and lower in regular caffeine consumers than in non-regular caffeine consumers. The 4 h salivary metabolic ratio (mean: 0.5) was generally not significantly different from the literature reported salivary, serum and plasma ratios measured at 4-9 h in healthy individuals (mean range 0.4-0.7) but was significantly higher than the literature reported 6 h plasma ratio and salivary ratios measured at 1-6 h in patients with liver disease or mild abnormal liver function tests (mean range 0.03-0.2). Overall, the population norm of the salivary metabolic ratio in a Singaporean Chinese population established in this study is distinct from individuals with liver disease or mild abnormal liver function tests and provides the benchmark for dosage adjustments of drugs metabolized by CYP1A2. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26862045

  19. Sprague-Dawley rats display metabolism-mediated sex differences in the acute toxicity of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy)

    SciTech Connect

    Fonsart, Julien ||; Menet, Marie-Claude |; Decleves, Xavier ||; Galons, Herve |; Crete, Dominique; Debray, Marcel; Scherrmann, Jean-Michel ||; Noble, Florence ||

    2008-07-01

    The use of the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has been associated with unexplained deaths. Male humans and rodents are more sensitive to acute toxicity than are females, including a potentially lethal hyperthermia. MDMA is highly metabolized to five main metabolites, by the enzymes CYP1A2 and CYP2D. The major metabolite in rats, 3,4-methylenedioxyamphetamine (MDA), also causes hyperthermia. We postulated that the reported sex difference in rats is due to a sexual dimorphism(s). We therefore determined (1) the LD50 of MDMA and MDA, (2) their hyperthermic effects, (3) the activities of liver CYP1A2 and CYP2D, (4) the liver microsomal metabolism of MDMA and MDA, (5) and the plasma concentrations of MDMA and its metabolites 3 h after giving male and female Sprague-Dawley (SD) rats MDMA (5 mg.kg{sup -1} sc). The LD50 of MDMA was 2.4-times lower in males than in females. MDMA induced greater hyperthermia (0.9 deg. C) in males. The plasma MDA concentration was 1.3-fold higher in males, as were CYP1A2 activity (twice) and N-demethylation to MDA (3.3-fold), but the plasma MDMA concentration (1.4-fold) and CYP2D activity (1.3-fold) were higher in females. These results suggest that male SD rats are more sensitive to MDMA acute toxicity than are females, probably because their CYP1A2 is more active, leading to higher N-demethylation and plasma MDA concentration. This metabolic pathway could be responsible for the lethality of MDMA, as the LD50 of MDA is the same in both sexes. These data strongly suggest that the toxicity of amphetamine-related drugs largely depends on metabolic differences.

  20. Caffeine test in predicting flutamide-induced hepatic injury in patients with prostate cancer.

    PubMed

    Ozono, S; Yamaguchi, A; Mochizuki, H; Kawakami, T; Fujimoto, K; Otani, T; Yoshida, K; Ichinei, M; Yamashita, T; Hirao, Y

    2002-01-01

    The caffeine test measures the activity of cytochrome p450 (CYP1A2) which is a major enzyme involved in the activation of flutamide. The usefulness of this test in predicting flutamide-induced hepatic injury in patients with prostate cancer was examined. The subjects were: (1). five patients whose aspartate aminotransferase (AST) or alanine aminotransferase (ALT) level rose to 100 IU/l or higher following the start of flutamide (moderately injured group); (2). four patients whose AST and ALT levels were higher than normal but less than 100 IU/l (mildly injured group); and (3). two patients whose hepatic function remained normal (normal group). The subjects were each given canned coffee to drink. Urinary caffeine (137X), paraxanthine (17X) and 1, 7-dimethyluric acid (17U) levels were measured 4-5 h later. The metabolite ratio, (17U+17X)/137X, was calculated to serve as an indicator of CYP1A2 activity. The metabolite ratio for the moderately injured group (3.98+/-1.56) and the mildly injured group (5.55+/-1.42) were lower than that for the normal group (9.56). The results suggest that a decrease in CYP1A2 activity is involved in the onset of flutamide-induced hepatic injury, and that the caffeine test seems to provide a useful means of its prediction. PMID:12497002

  1. Inhibitory and inductive effects of Phikud Navakot extract on human cytochrome P450.

    PubMed

    Chiangsom, Abhiruj; Lawanprasert, Somsong; Oda, Shingo; Kulthong, Kornphimol; Luechapudiporn, Rataya; Yokoi, Tsuyoshi; Maniratanachote, Rawiwan

    2016-06-01

    Effects of the hydroethanolic extract of Phikud Navakot (PN), a Thai traditional remedy, on human cytochrome P450s (CYPs) were investigated in vitro. Selective substrates of CYPs were used to investigate the effects and kinetics of PN on CYP inhibition using human liver microsomes. Primary human hepatocytes were used to assess the inductive effects of PN on CYP enzyme activities and protein expressions. The results showed that PN inhibited the activities of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 with half maximal inhibitory concentration (IC50) values of 13, 62, 67, and 88 μg/mL, respectively. Meanwhile, it had no effect on the activities of CYP2C19 and CYP2E1 (IC50 > 1 mg/mL). PN exhibited competitive inhibition of CYP1A2 (Ki = 34 μg/mL), mixed type inhibition of CYP2C9 and CYP2D6 (Ki = 80 and 12 μg/mL, respectively), and uncompetitive inhibition of CYP3A4 (Ki = 150 μg/mL). PN did not have an inductive effect on CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in primary human hepatocytes, which is an advantageous characteristic of the extract. However the extract may cause herb-drug interactions via inhibition of CYP1A2, CYP2C9, CYP2D6 and CYP3A4, and precautions should be taken when PN is coadministered with drugs that are metabolized by these CYP enzymes. PMID:27212065

  2. Inhibition of human cytochrome P450 enzymes by licochalcone A, a naturally occurring constituent of licorice.

    PubMed

    He, Wei; Wu, Jing-Jing; Ning, Jing; Hou, Jie; Xin, Hong; He, Yu-Qi; Ge, Guang-Bo; Xu, Wei

    2015-10-01

    Licochalcone A (LCA) is a major bioactive compound in traditional Chinese herbal liquorice that possesses multiple pharmacological activities. However, the effects of the potential herb-drug interactions (HDIs) between LCA and therapeutic drugs on the inhibition of human cytochrome P450 (CYP) enzymes remain unclear. In the present study, the inhibitory effects of LCA on seven major human CYP isoforms, including CYP1A2, 2D6, 2E1, 2C19, 2C8, 2C9 and 3A4, were investigated in human liver microsomes (HLMs). The results demonstrated that LCA significantly inhibited the activities of CYP1A2, 2C19, 2C8, 2C9 and 3A4 and exhibited weak inhibitory effects on CYP2E1 and CYP2D6. Dixon and Lineweaver-Burk plots revealed that the inhibition types of LCA against CYP1A2, 2C9, 2C19 and 2C8 were best fit as mixed-type inhibitions, while LCA was a competitive inhibitor towards CYP3A4. The inhibition kinetic parameters (K(i)) were calculated to be 1.02 μM, 0.17 μM, 3.89 μM 0.89 μM, and 2.29 μM, for CYP1A2, 2C9, 2C19, 2C8, and 3A4, respectively. Furthermore, the areas under the plasma concentration-time curves (AUCs) of several drugs that are primarily metabolized by CYPs were estimated to increase by 2-398% in the presence of LCA, which suggested that LCA exhibited high HDI potentials via CYP inhibition. These data are significant for the clinical applications of LCA-containing herbs.

  3. Rapid induction of colon carcinogenesis in CYP1A-humanized mice by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and dextran sodium sulfate.

    PubMed

    Cheung, Connie; Loy, Shea; Li, Guang Xun; Liu, Anna B; Yang, Chung S

    2011-02-01

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant heterocyclic amine produced during the cooking of meats and fish, is suspected to be a human carcinogen. Metabolic activation of PhIP is primarily mediated by the enzyme cytochrome P450 (CYP) 1A2. Metabolism of PhIP by CYP1A2 differs considerably between humans and rodents, with more N(2)-hydroxylation (activation) and less 4'-hydroxylation (detoxication) in humans. Transgenic CYP1A-humanized mice (hCYP1A-mice), which have the human CYP1A1 and CYP1A2 genes but lack the murine orthologs Cyp1a1 and Cyp1a2, provide an excellent opportunity to develop a relevant model to study dietary-induced colon carcinogenesis. The treatment with 200 mg/kg PhIP by oral gavage, followed by 1.5% dextran sodium sulfate (DSS) in the drinking water for 7 days, was found to be an effective combination to induce colon carcinogenesis in hCYP1A-mice. Tumor multiplicity at week 6 was calculated to be 3.75 ± 0.70 and for week 10 was 3.90 ± 0.61 with 80-95% of the tumors being adenocarcinomas. No tumors were found in the similarly treated wild-type mice. Western blots revealed overexpression of β-catenin, c-Myc, cyclin D1, inducible nitric oxide synthase and cyclooxygenase-2 in colon tumor samples. Strong nuclear localization of β-catenin was observed in tumors. These results illustrate that PhIP and DSS combination produces rapid colon carcinogenesis in hCYP1A-mice and this is an effective model to mimic human colon carcinogenesis.

  4. Effect of Ginkgo biloba extract on procarcinogen-bioactivating human CYP1 enzymes: Identification of isorhamnetin, kaempferol, and quercetin as potent inhibitors of CYP1B1

    SciTech Connect

    Chang, Thomas K.H. . E-mail: tchang@interchange.ubc.ca; Chen Jie; Yeung, Eugene Y.H.

    2006-05-15

    In the present study, we investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the catalytic activity of human cytochrome P450 enzymes CYP1B1, CYP1A1, and CYP1A2. G. biloba extract of known abundance of terpene trilactones and flavonol glycosides inhibited 7-ethoxyresorufin O-dealkylation catalyzed by human recombinant CYP1B1, CYP1A1, and CYP1A2, and human liver microsomes, with apparent K {sub i} values of 2 {+-} 0.3, 5 {+-} 0.5, 16 {+-} 1.4, and 39 {+-} 1.2 {mu}g/ml (mean {+-} SE), respectively. In each case, the mode of inhibition was of the mixed type. Bilobalide, ginkgolides A, B, C, and J, quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, and isorhamentin 3-O-rutinoside were not responsible for the inhibition of CYP1 enzymes by G. biloba extract, as determined by experiments with these individual chemicals at the levels present in the extract. In contrast, the aglycones of quercetin, kaempferol, and isorhamentin inhibited CYP1B1, CYP1A1, and CYP1A2. Among the three flavonol aglycones, isorhamentin was the most potent in inhibiting CYP1B1 (apparent K {sub i} = 3 {+-} 0.1 nM), whereas quercetin was the least potent in inhibiting CYP1A2 (apparent K {sub i} 418 {+-} 50 nM). The mode of inhibition was competitive, noncompetitive, or mixed, depending on the enzyme and the flavonol. G. biloba extract also reduced benzo[a]pyrene hydroxylation, and the effect was greater with CYP1B1 than with CYP1A1 as the catalyst. Overall, our novel findings indicate that G. biloba extract and the flavonol aglycones isorhamnetin, kaempferol, and quercetin preferentially inhibit the in vitro catalytic activity of human CYP1B1.

  5. Chlorogenic acid prevents acetaminophen-induced liver injury: the involvement of CYP450 metabolic enzymes and some antioxidant signals.

    PubMed

    Pang, Chun; Sheng, Yu-chen; Jiang, Ping; Wei, Hai; Ji, Li-li

    2015-07-01

    Chlorogenic acid (CGA), a polyphenolic compound, is abundant in fruits, dietary vegetables, and some medicinal herbs. This study investigated the prevention of CGA against acetaminophen (AP)-induced hepatotoxicity and its engaged mechanisms. CGA reversed the decreased cell viability induced by AP in L-02 cells in vitro. In addition, CGA reduced the AP-induced increased serum levels of alanine/aspartate aminotransferase (ALT/AST) in vivo. The effect of CGA on cytochrome P450 (CYP) enzymatic (CYP2E1, CYP1A2, and CYP3A4) activities showed that CGA caused very little inhibition on CYP2E1 and CYP1A2 enzymatic activities, but not CYP3A4. The measurement of liver malondialdehyde (MDA), reactive oxygen species (ROS), and glutathione (GSH) levels showed that CGA prevented AP-induced liver oxidative stress injury. Further, CGA increased the AP-induced decreased mRNA expression of peroxiredoxin (Prx) 1, 2, 3, 5, 6, epoxide hydrolase (Ephx) 2, and polymerase (RNA) II (DNA directed) polypeptide K (Polr2k), and nuclear factor erythroid-2-related factor 2 (Nrf2). In summary, CGA ameliorates the AP-induced liver injury probably by slightly inhibiting CYP2E1 and CYP1A2 enzymatic properties. In addition, cellular important antioxidant signals such as Prx1, 2, 3, 5, 6, Ephx2, Polr2k, and Nrf2 also contributed to the protection of CGA against AP-induced oxidative stress injury. PMID:26160718

  6. Dose validation of PhIP hair level as a biomarker of heterocyclic aromatic amines exposure: a feeding study.

    PubMed

    Le Marchand, Loïc; Yonemori, Kim; White, Kami K; Franke, Adrian A; Wilkens, Lynne R; Turesky, Robert J

    2016-07-01

    Hair measurement of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a promising biomarker of exposure to this carcinogen formed in cooked meats. However, the dose relationship between normal range intake and hair levels and the modulating effects of CYP1A2 metabolism and hair melanin need to be evaluated. We conducted a randomized, cross-over feeding study among 41 non-smokers using ground beef cooked to two different levels of doneness, 5 days a week for 1 month. PhIP was measured by liquid chromatography/mass spectrometry in food (mean low dose = 0.72 µg/serving; mean high dose = 2.99 µg/serving), and change in PhIP hair level was evaluated. CYP1A2 activity was assessed in urine with the caffeine challenge test and head hair melanin was estimated by UV spectrophotometry. We observed a strong dose-dependent increase in hair PhIP levels. This increase was highly correlated with dose received (ρ = 0.68, P < 0.0001). CYP1A2 activity and normalizing for hair melanin did not modify the response to the intervention. Consumption of PhIP at doses similar to those in the American diet results in a marked dose-dependent accumulation of PhIP in hair. Hair PhIP levels may be used as a biomarker of dietary exposure in studies investigating disease risk. PMID:27207666

  7. Effects of icaritin on cytochrome P450 enzymes in rats.

    PubMed

    Liang, Dong-Lou; Zheng, Shuang-Li

    2014-04-01

    The purpose of this study was to find out whether icaritin influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (20 mg/kg), tolbutamide (5 mg/kg), chlorzoxazone (20 mg/kg) and midazolam (10 mg/kg), was orally administered to rats treated with multiple doses of icaritin. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. Treatment with multiple doses of icaritin had inhibitive effects on rat CYP1A2, CYP2C9 and CYP3A4 enzyme activities. However, icaritin has no inductive or inhibitory effect on the activity of CYP2E1. Therefore, caution is needed when icaritin is co-administered with some CYP1A2, CYP2C9 or CYP3A4 substrates, which may result in treatment failure and herb-drug interactions.

  8. Chlorogenic acid prevents acetaminophen-induced liver injury: the involvement of CYP450 metabolic enzymes and some antioxidant signals*

    PubMed Central

    Pang, Chun; Sheng, Yu-chen; Jiang, Ping; Wei, Hai; Ji, Li-li

    2015-01-01

    Chlorogenic acid (CGA), a polyphenolic compound, is abundant in fruits, dietary vegetables, and some medicinal herbs. This study investigated the prevention of CGA against acetaminophen (AP)-induced hepatotoxicity and its engaged mechanisms. CGA reversed the decreased cell viability induced by AP in L-02 cells in vitro. In addition, CGA reduced the AP-induced increased serum levels of alanine/aspartate aminotransferase (ALT/AST) in vivo. The effect of CGA on cytochrome P450 (CYP) enzymatic (CYP2E1, CYP1A2, and CYP3A4) activities showed that CGA caused very little inhibition on CYP2E1 and CYP1A2 enzymatic activities, but not CYP3A4. The measurement of liver malondialdehyde (MDA), reactive oxygen species (ROS), and glutathione (GSH) levels showed that CGA prevented AP-induced liver oxidative stress injury. Further, CGA increased the AP-induced decreased mRNA expression of peroxiredoxin (Prx) 1, 2, 3, 5, 6, epoxide hydrolase (Ephx) 2, and polymerase (RNA) II (DNA directed) polypeptide K (Polr2k), and nuclear factor erythroid-2-related factor 2 (Nrf2). In summary, CGA ameliorates the AP-induced liver injury probably by slightly inhibiting CYP2E1 and CYP1A2 enzymatic properties. In addition, cellular important antioxidant signals such as Prx1, 2, 3, 5, 6, Ephx2, Polr2k, and Nrf2 also contributed to the protection of CGA against AP-induced oxidative stress injury. PMID:26160718

  9. Evaluation of the effects of Mitragyna speciosa alkaloid extract on cytochrome P450 enzymes using a high throughput assay.

    PubMed

    Kong, Wai Mun; Chik, Zamri; Ramachandra, Murali; Subramaniam, Umarani; Aziddin, Raja Elina Raja; Mohamed, Zahurin

    2011-01-01

    The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC(50)) values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC(50) of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC(50) of CYP2C19 could not be determined, however, because inhibition was <50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6), ketoconazole (CYP3A4), tranylcypromine (CYP2C19) and furafylline (CYP1A2) were ACCESSused as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2. PMID:21876481

  10. Characterization of the cytochrome P450 enzymes involved in the metabolism of a new cardioprotective agent KR-33028.

    PubMed

    Kim, Hyojin; Yoon, Yune-Jung; Kim, Hyunmi; Kang, Suil; Cheon, Hyae Gyeong; Yoo, Sung-Eun; Shin, Jae-Gook; Liu, Kwang-Hyeon

    2006-10-10

    KR-33028 (N-[4-cyano-benzo[b]thiophene-2-carbonyl]guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. This study was performed to characterize the cytochrome P450 (CYP) enzymes that are involved in the metabolism of KR-33028. Hydroxylation (5-hydroxy- and 7-hydroxy-KR-33028) is major pathways for the metabolism of KR-33028 in human liver microsomes. Among the nine c-DNA expressed CYP isoforms tested, KR-33028 was 5-hydroxylated by CYP3A4 and 7-hydroxylated by CYP1A2, CYP3A4, and CYP2C19. These findings were supported by the combination of chemical inhibition studies in human liver microsomes and correlation analysis. Furafylline and ketoconazole potently inhibited hydroxylation of KR-33028 in human liver microsomes. Correlation analysis between the known CYP enzyme activities and the rates of the formation of 5-hydroxy- and 7-hydroxy-KR-33028 in the 16 human liver microsomes has showed significant correlations with CYP3A4-mediated midazolam 1'-hydroxylation and CYP1A2-mediated phenacetin O-deethylation, respectively. A 7-hydroxy-KR-33028 formation is also weakly correlated with CYP3A4-mediated midazolam 1'-hydroxylation. The kinetics of the major biotransformation of KR-33028 were studied: CYP3A4 mediated the formation of 5-hydroxy-KR-33028 from KR-33028 with Cl(int)=0.22microl/min/pmol CYP. The intrinsic clearance for 7-hydroxy-KR-33028 formation by CYP1A2, CYP2C19, and CYP3A4 were 0.26, 0.19, and 0.03microl/min/pmol CYP, respectively. Taken together, these results provide evidence that CYP3A4 and CYP1A2 are the major isoforms responsible for the hydroxy metabolites formation from KR-33028.

  11. Tanshinone I protects mice from aristolochic acid I-induced kidney injury by induction of CYP1A.

    PubMed

    Feng, Chenchen; Xie, Xiaofeng; Wu, Mengjun; Li, Chunzhu; Gao, Man; Liu, Mingxia; Qi, Xinming; Ren, Jin

    2013-11-01

    Hepatic CYP1A especially CYP1A2 plays an important role in the reduction of aristolochic acid I (AAI) nephrotoxicity. In this study, we investigated the effects of tanshinone I, a strong inducer of Cyp1a, on the nephrotoxicity induced by AAI. Histopathology and blood biochemistry assays showed that tanshinone I could reduce AAI-induced acute kidney injury. Pharmacokinetics analysis revealed that tanshinone I markedly decreased AUC of AAI in plasma and the content of AAI in both liver and kidney, indicating the enhancement of AAI metabolism. Real-time PCR and Western blot analysis confirmed that tanshinone I effectively increased the mRNA and protein levels of hepatic CYP1A1 and CYP1A2 in vivo. Luciferase assay showed that tanshinone I strongly increased the transcriptional activity of CYP1A1 and CYP1A2 in the similar extent. In summary, our data suggested that tanshinone I facilitated the metabolism of AAI and prevented AAI-induced kidney injury by induction of hepatic CYP1A 1/2 in vivo. PMID:23981375

  12. Thermal ramp tritium release in COBRA-1A2 C03 beryllium pebbles

    SciTech Connect

    Baldwin, D.L.

    1998-03-01

    Tritium release kinetics, using the method of thermal ramp heating at three linear ramp rates, were measured on the COBRA-1A2 C03 1-mm beryllium pebbles. This report includes a brief discussion of the test, and the test data in graph format.

  13. 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...

  14. 29 CFR 1917.28 - Hazard communication (See also § 1917.1(a)(2)(vi)).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 7 2010-07-01 2010-07-01 false Hazard communication (See also § 1917.1(a)(2)(vi)). 1917.28 Section 1917.28 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND HEALTH ADMINISTRATION, DEPARTMENT OF LABOR (CONTINUED) MARINE TERMINALS Marine Terminal Operations § 1917.28...

  15. Familial Hemiplegic Migraine with Severe Attacks: A New Report with ATP1A2 Mutation

    PubMed Central

    Martínez, E.; Moreno, R.; López-Mesonero, L.; Vidriales, I.; Ruiz, M.; Tellería, J. J.

    2016-01-01

    Introduction. Familial hemiplegic migraine (FHM) is a rare disorder characterized by migraine attacks with motor weakness during the aura phase. Mutations in CACNA1A, ATP1A2, SCN1A, and PRRT2 genes have been described. Methods. To describe a mutation in ATP1A2 gene in a FHM case with especially severe and prolonged symptomatology. Results. 22-year-old woman was admitted due to migraine-type headache and sudden onset of right-sided weakness and aphasia; she had similar episodes in her childhood. Her mother was diagnosed with hemiplegic migraine without genetic confirmation. She presented with fever, decreased consciousness, left gaze preference, mixed aphasia, right facial palsy, right hemiplegia, and left crural paresis. Computed tomography (CT) showed no lesion and CT perfusion study evidenced oligohemia in left hemisphere. A normal brain magnetic resonance (MR) was obtained. Impaired consciousness and dysphasia began to improve three days after admission and mild dysphasia and right hemiparesis lasted for 10 days. No recurrences were reported during a follow-up of two years. We identified a variant in heterozygous state in ATP1A2 gene (p.Thr364Met), pathogenic according to different prediction algorithms (SIFT, PolyPhen2, MutationTaster, and Condel). Conclusion. Prolonged and severe attacks with diffuse hypoperfusion in a FHM seemed to be specially related to ATP1A2 mutations, and p.T364M should be considered.

  16. SLC1A2 Variant Is Associated with Essential Tremor in Taiwanese Population

    PubMed Central

    Yu, Shao-wen; Chen, Chiung-Mei; Chen, Yi-Chun; Chang, Chia Wen; Chang, Hong-Shiu; Lyu, Rong-Kuo; Ro, Long-Sun; Wu, Yih-Ru

    2013-01-01

    Essential tremor (ET), which is one of the most common movement disorders, may lead to severe interference in quality of life. The first genome-wide association study (GWAS) has identified an association of the LINGO1 variant (rs9652490) with ET in Americans and Europeans. Recently, a second GWAS that was performed in a European population has discovered a new variant (rs3794087) of the main glial glutamate transporter (SLC1A2) that increases the risk of ET with an odds ratio of about 1.4. SLC1A2 encodes for the major glial high-affinity glutamate reuptake transporter in the brain and is a potential ET susceptibility gene. Because replication in a different ethnic population is important for validating a finding, we conducted a case-control study to investigate the SLC1A2 variant in an Asian cohort with ET in Taiwan. A total of 542 subjects (273 ET patients and 269 controls) were included. The results showed that rs3794087 was associated with ET among the Taiwanese. The odds ratio was 1.37. Our results were similar to those of the second GWAS of ET in Europeans, and this confirms that SLC1A2 may be a good functional candidate gene for ET. A replication study in another independent population is of importance to validate this association. PMID:23951268

  17. Inhibition of aryl hydrocarbon receptor transactivation and DNA adduct formation by CYP1 isoform-selective metabolic deactivation of benzo[a]pyrene

    SciTech Connect

    Endo, Kaori; Uno, Shigeyuki; Seki, Taiichiro; Ariga, Toyohiko; Kusumi, Yoshiaki; Mitsumata, Masako; Yamada, Sachiko; Makishima, Makoto

    2008-07-15

    Benzo[a]pyrene (BaP), a polyaromatic hydrocarbon produced by the combustion of cigarettes and coke ovens, is a known procarcinogen. BaP activates the aryl hydrocarbon receptor (AhR) and induces the expression of a battery of genes, including CYP1A1, which metabolize BaP to toxic compounds. The possible role of CYP1 enzymes in mediating BaP detoxification or metabolic activation remains to be elucidated. In this study, we assessed the effects of CYP1 enzymes (CYP1A1, CYP1A2 and CYP1B1) on BaP-induced AhR transactivation and DNA adduct formation in HEK293 cells and HepG2 cells. Transfection of CYP1A1 and CYP1B1, but not CYP1A2, suppressed BaP-induced activation of AhR. Expression of CYP1A1 and CYP1A2, but not CYP1B1, inhibited DNA adduct formation in BaP-treated HepG2 cells. These results indicate that CYP1A1 and CYP1B1 play a role in deactivation of BaP on AhR and that CYP1A1 and CYP1A2 are involved in BaP detoxification by suppressing DNA adduct formation. BaP treatment did not induce DNA adduct formation in HEK293 cells, even after transfection of CYP1 enzymes, suggesting that expression of CYP1 enzymes is not sufficient for DNA adduct formation. Lower expression of epoxide hydrolase and higher expression of glutathione S-transferase P1 (GSTP1) and GSTM1/M2 were observed in HEK293 cells compared with HepG2 cells. Dynamic expression of CYP1A1, CYP1A2 and CYP1B1 along with expression of other enzymes such as epoxide hydrolase and phase II enzymes may determine the detoxification or metabolic activation of BaP.

  18. Increased urinary excretion of 8-oxo-2'-deoxyguanosine, a biomarker of oxidative DNA damage, in urban bus drivers.

    PubMed

    Loft, S; Poulsen, H E; Vistisen, K; Knudsen, L E

    1999-04-26

    Oxidative damage to DNA could be involved in the increased risk of cancer associated with exposure to polluted urban air, which contains a number of oxidants. CYP1A2 is induced by and metabolizes polyaromatic hydrocarbons (PAH) and aromatic amines and could modify effects of exposure to ambient air pollution. Similarly, DNA repair may be influenced by occupational and other exposures as well as modify the effect of DNA damaging agents. As part of a large investigation of the genotoxic burden to diesel exposed workers in transport sectors we studied oxidative DNA damage in 57 non-smoking bus drivers from the greater Copenhagen area. The drivers were studied on a workday and on a day off work. Comparisons were made between drivers from the central (n=30) and rural/suburban (n=27) areas of Copenhagen. The rate of oxidative DNA damage was estimated from 24 h urinary excretion of 8-oxo-2'-deoxyguanosine (8-oxodG), a repair product of the highly mutagenic oxidation of guanine in DNA or the cellular pool of GTP. CYP1A2 activity was estimated from the urinary excretion of metabolites of dietary caffeine. The DNA repair was estimated by unscheduled DNA synthesis (UDS) in mononuclear cells isolated on the workday. Repeated measures ANOVA and multifactorial ANCOVA with CYP1A2 activity, age and UDS as covariates were used for statistical evaluation. On the workday, the 8-oxodG excretion was 190+/-108 and 146+/-89 pmol/kg 24 h in the bus drivers from central and the suburban/rural areas Copenhagen, respectively (p<0.05). The 8-oxodG excretion was not significantly different between the workday and the day off. CYP1A2 activity was not affected by driving area but was correlated with the 8-oxodG excretion on the workday (r=0.53; p<0.05). UDS was not significantly affected by driving area or correlated with the 8-oxodG excretion. The increased excretion of 8-oxodG in bus drivers from central Copenhagen as compared with drivers from rural/suburban greater Copenhagen suggests that

  19. Stabile Expression von Sulfotransferasen - allein oder in Kombination mit Cytochrom P450 - in Zelllinien für Mutagenitätsuntersuchungen

    NASA Astrophysics Data System (ADS)

    Pabel, Ulrike

    2003-10-01

    amino group, usually catalysed by cytochrome P450 1A2 (CYP1A2) and subsequent O-conjugation by phase-II enzymes e.g. sulfotransferases (SULT) or N-acetyltransferases. The bioactivation constitutes a critical parameter for the transfer of results from animal models on man. Recombinant in vitro systems expressing xenobiotic metabolizing enzymes of different species allow the comparative study of the bioactivation in humans and animal models. The aim of this project was to elucidate the bioactivation of aAA by human xenobiotic enzymes. The investigation focused on the role of SULT in this process. SULT-cDNAs were cloned into the mammalian expression vector pMPSV and transfected in V79 Chinese Hamster cells, which represent standard indicator cells for mutagenicity tests. Selected SULT-cDNAs were also co-expressed with human CYP1A2. These cells were able to catalyse internally both enzymatic reactions that are necessary for the bioactivation of aAA. The expression level of CYP1A2 and SULT in the co-expressing cell clones was characterised by immunoblot analysis and radiometric SULT-activity measurement. The mutagenicity of four aAA model compounds, 2-aminoanthracene, 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene and 2,4-diaminotoluene, at the hprt locus of the recombinant cell lines was investigated. These aAA were not or only marginally mutagenic in wild type cells or in recombinant cells expressing CYP1A2 alone. If CYP1A2 was co-expressed with SULT forms of the 1A subfamily clear mutagenic effects occured in low concentrations of the aAA (0,3 µM for 2-acetylaminofluorene and 3′-methyl-4-dimethylaminoazobenzene; 0,1 µM for 2-aminoanthracene; 10 µM for 2,4-diaminotoluene). The strongest activation of 2-acetylaminofluorene and 3'-methyl-4-dimethylaminoazobenzene was mediated by SULTA2 and of 2-aminoanthracene and 2,4-diaminotoluene by SULT1A1. SULT1A1 and SULT1A2 are expressed polymorphically in humans. Differences in the activation potency of distinct

  20. Dioxin activation of CYP1A5 promoter/enhancer regions from two avian species, common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus): Association with aryl hydrocarbon receptor 1 and 2 isoforms

    SciTech Connect

    Lee, Jin-Seon; Kim, Eun-Young Iwata, Hisato

    2009-01-01

    The present study focuses on the molecular mechanism and interspecies differences in susceptibility of avian aryl hydrocarbon receptor (AHR)-cytochrome P4501A (CYP1A) signaling pathway. By the cloning of 5'-flanking regions of CYP1A5 gene from common cormorant (Phalacrocorax carbo) and chicken (Gallus gallus), seven putative xenobiotic response elements (XREs) were identified within 2.7 kb upstream region of common cormorant CYP1A5 (ccCYP1A5), and six XREs were found within 0.9 kb of chicken CYP1A5 (ckCYP1A5). Analysis of sequential deletion and mutagenesis of the binding sites in avian CYP1A5 genes by in vitro reporter gene assays revealed that two XREs at -613 bp and -1585 bp in ccCYP1A5, and one XRE at -262 bp in ckCYP1A5 conferred TCDD-responsiveness. The binding of AHR1 with AHR nuclear translocator 1 (ARNT1) to the functional XRE in a TCDD-dependent manner was verified with gel shift assays, suggesting that avian CYP1A5 is induced by TCDD through AHR1/ARNT1 signaling pathway as well as mammalian CYP1A1 but through a distinct pathway from mammalian CYP1A2, an ortholog of the CYP1A5. TCDD-EC{sub 50} for the transcriptional activity in both cormorant AHR1- and AHR2-ccCYP1A5 reporter construct was 10-fold higher than that in chicken AHR1-ckCYP1A5 reporter construct. In contrast, chicken AHR2 showed no TCDD-dependent response. The TCDD-EC{sub 50} for CYP1A5 transactivation was altered by switching AHR1 between the two avian species, irrespective of the species from which the regulatory region of CYP1A5 gene originates. Therefore, the structural difference in AHR, not the CYP1A5 regulatory region may be a major factor to account for the dioxin susceptibility in avian species.

  1. Production of {sup 4}He and tritium from Be in the COBRA-1A2 irradiation

    SciTech Connect

    Greenwood, L.R.

    1998-03-01

    The production of {sup 4}He and tritium has been calculated for beryllium irradiated in the COBRA-1A2 experiment in the Experimental Breeder Reactor II. Reaction rates were based on adjusted neutron spectra determined from reactor dosimetry measurements at three different elevations in the region of the beryllium capsules. Equations are given so that gas production can be calculated for any specific capsule elevation.

  2. Novel action of FOXL2 as mediator of Col1a2 gene autoregulation.

    PubMed

    Marongiu, Mara; Deiana, Manila; Marcia, Loredana; Sbardellati, Andrea; Asunis, Isadora; Meloni, Alessandra; Angius, Andrea; Cusano, Roberto; Loi, Angela; Crobu, Francesca; Fotia, Giorgio; Cucca, Francesco; Schlessinger, David; Crisponi, Laura

    2016-08-01

    FOXL2 belongs to the evolutionarily conserved forkhead box (FOX) superfamily and is a master transcription factor in a spectrum of developmental pathways, including ovarian and eyelid development and bone, cartilage and uterine maturation. To analyse its action, we searched for proteins that interact with FOXL2. We found that FOXL2 interacts with specific C-terminal propeptides of several fibrillary collagens. Because these propeptides can participate in feedback regulation of collagen biosynthesis, we inferred that FOXL2 could thereby affect the transcription of the cognate collagen genes. Focusing on COL1A2, we found that FOXL2 indeed affects collagen synthesis, by binding to a DNA response element located about 65Kb upstream of this gene. According to our hypothesis we found that in Foxl2(-/-) mouse ovaries, Col1a2 was elevated from birth to adulthood. The extracellular matrix (ECM) compartmentalizes the ovary during folliculogenesis, (with type I, type III and type IV collagens as primary components), and ECM composition changes during the reproductive lifespan. In Foxl2(-/-) mouse ovaries, in addition to up-regulation of Col1a2, Col3a1, Col4a1 and fibronectin were also upregulated, while laminin expression was reduced. Thus, by regulating levels of extracellular matrix components, FOXL2 may contribute to both ovarian histogenesis and the fibrosis attendant on depletion of the follicle reserve during reproductive aging and menopause. PMID:27212026

  3. Genetic variation in the CYP1A1 gene is related to circulating PCB118 levels in a population-based sample

    SciTech Connect

    Lind, Lars; Penell, Johanna; Syvänen, Anne-Christine; Axelsson, Tomas; Ingelsson, Erik; Morris, Andrew P.; Lindgren, Cecilia; Salihovic, Samira; Bavel, Bert van; Lind, P. Monica

    2014-08-15

    Several of the polychlorinated biphenyls (PCBs), i.e. the dioxin-like PCBs, are known to induce the P450 enzymes CYP1A1, CYP1A2 and CYP1B1 by activating the aryl hydrocarbon receptor (Ah)-receptor. We evaluated if circulating levels of PCBs in a population sample were related to genetic variation in the genes encoding these CYPs. In the population-based Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS) study (1016 subjects all aged 70), 21 SNPs in the CYP1A1, CYP1A2 and CYP1B1 genes were genotyped. Sixteen PCB congeners were analysed by high-resolution chromatography coupled to high-resolution mass spectrometry (HRGC/ HRMS). Of the investigated relationships between SNPs in the CYP1A1, CYP1A2 and CYP1B1 and six PCBs (congeners 118, 126, 156, 169, 170 and 206) that captures >80% of the variation of all PCBs measured, only the relationship between CYP1A1 rs2470893 was significantly related to PCB118 levels following strict adjustment for multiple testing (p=0.00011). However, there were several additional SNPs in the CYP1A2 and CYP1B1 that showed nominally significant associations with PCB118 levels (p-values in the 0.003–0.05 range). Further, several SNPs in the CYP1B1 gene were related to both PCB156 and PCB206 with p-values in the 0.005–0.05 range. Very few associations with p<0.05 were seen for PCB126, PCB169 or PCB170. Genetic variation in the CYP1A1 was related to circulating PCB118 levels in the general elderly population. Genetic variation in CYP1A2 and CYP1B1 might also be associated with other PCBs. - Highlights: • We studied the relationship between PCBs and the genetic variation in the CYP genes. • Cross sectional data from a cohort of elderly were analysed. • The PCB levels were evaluated versus 21 SNPs in three CYP genes. • PCB 118 was related to variation in the CYP1A1 gene.

  4. ATP1A2 Mutations in Migraine: Seeing through the Facets of an Ion Pump onto the Neurobiology of Disease

    PubMed Central

    Friedrich, Thomas; Tavraz, Neslihan N.; Junghans, Cornelia

    2016-01-01

    Mutations in four genes have been identified in familial hemiplegic migraine (FHM), from which CACNA1A (FHM type 1) and SCN1A (FHM type 3) code for neuronal voltage-gated calcium or sodium channels, respectively, while ATP1A2 (FHM type 2) encodes the α2 isoform of the Na+,K+-ATPase's catalytic subunit, thus classifying FHM primarily as an ion channel/ion transporter pathology. FHM type 4 is attributed to mutations in the PRRT2 gene, which encodes a proline-rich transmembrane protein of as yet unknown function. The Na+,K+-ATPase maintains the physiological gradients for Na+ and K+ ions and is, therefore, critical for the activity of ion channels and transporters involved neuronal excitability, neurotransmitter uptake or Ca2+ signaling. Strikingly diverse functional abnormalities have been identified for disease-linked ATP1A2 mutations which frequently lead to changes in the enzyme's voltage-dependent properties, kinetics, or apparent cation affinities, but some mutations are truly deleterious for enzyme function and thus cause full haploinsufficiency. Here, we summarize structural and functional data about the Na+,K+-ATPase available to date and an overview is provided about the particular properties of the α2 isoform that explain its physiological relevance in electrically excitable tissues. In addition, current concepts about the neurobiology of migraine, the correlations between primary brain dysfunction and mechanisms of headache pain generation are described, together with insights gained recently from modeling approaches in computational neuroscience. Then, a survey is given about ATP1A2 mutations implicated in migraine cases as documented in the literature with focus on mutations that were described to completely destroy enzyme function, or lead to misfolded or mistargeted protein in particular model cell lines. We also discuss whether or not there are correlations between these most severe mutational effects and clinical phenotypes. Finally, perspectives

  5. ATP1A2 Mutations in Migraine: Seeing through the Facets of an Ion Pump onto the Neurobiology of Disease.

    PubMed

    Friedrich, Thomas; Tavraz, Neslihan N; Junghans, Cornelia

    2016-01-01

    Mutations in four genes have been identified in familial hemiplegic migraine (FHM), from which CACNA1A (FHM type 1) and SCN1A (FHM type 3) code for neuronal voltage-gated calcium or sodium channels, respectively, while ATP1A2 (FHM type 2) encodes the α2 isoform of the Na(+),K(+)-ATPase's catalytic subunit, thus classifying FHM primarily as an ion channel/ion transporter pathology. FHM type 4 is attributed to mutations in the PRRT2 gene, which encodes a proline-rich transmembrane protein of as yet unknown function. The Na(+),K(+)-ATPase maintains the physiological gradients for Na(+) and K(+) ions and is, therefore, critical for the activity of ion channels and transporters involved neuronal excitability, neurotransmitter uptake or Ca(2+) signaling. Strikingly diverse functional abnormalities have been identified for disease-linked ATP1A2 mutations which frequently lead to changes in the enzyme's voltage-dependent properties, kinetics, or apparent cation affinities, but some mutations are truly deleterious for enzyme function and thus cause full haploinsufficiency. Here, we summarize structural and functional data about the Na(+),K(+)-ATPase available to date and an overview is provided about the particular properties of the α2 isoform that explain its physiological relevance in electrically excitable tissues. In addition, current concepts about the neurobiology of migraine, the correlations between primary brain dysfunction and mechanisms of headache pain generation are described, together with insights gained recently from modeling approaches in computational neuroscience. Then, a survey is given about ATP1A2 mutations implicated in migraine cases as documented in the literature with focus on mutations that were described to completely destroy enzyme function, or lead to misfolded or mistargeted protein in particular model cell lines. We also discuss whether or not there are correlations between these most severe mutational effects and clinical phenotypes

  6. ALDH1A2 (RALDH2) genetic variation in human congenital heart disease

    PubMed Central

    2009-01-01

    Background Signaling by the vitamin A-derived morphogen retinoic acid (RA) is required at multiple steps of cardiac development. Since conversion of retinaldehyde to RA by retinaldehyde dehydrogenase type II (ALDH1A2, a.k.a RALDH2) is critical for cardiac development, we screened patients with congenital heart disease (CHDs) for genetic variation at the ALDH1A2 locus. Methods One-hundred and thirty-three CHD patients were screened for genetic variation at the ALDH1A2 locus through bi-directional sequencing. In addition, six SNPs (rs2704188, rs1441815, rs3784259, rs1530293, rs1899430) at the same locus were studied using a TDT-based association approach in 101 CHD trios. Observed mutations were modeled through molecular mechanics (MM) simulations using the AMBER 9 package, Sander and Pmemd programs. Sequence conservation of observed mutations was evaluated through phylogenetic tree construction from ungapped alignments containing ALDH8 s, ALDH1Ls, ALDH1 s and ALDH2 s. Trees were generated by the Neighbor Joining method. Variations potentially affecting splicing mechanisms were cloned and functional assays were designed to test splicing alterations using the pSPL3 splicing assay. Results We describe in Tetralogy of Fallot (TOF) the mutations Ala151Ser and Ile157Thr that change non-polar to polar residues at exon 4. Exon 4 encodes part of the highly-conserved tetramerization domain, a structural motif required for ALDH oligomerization. Molecular mechanics simulation studies of the two mutations indicate that they hinder tetramerization. We determined that the SNP rs16939660, previously associated with spina bifida and observed in patients with TOF, does not affect splicing. Moreover, association studies performed with classical models and with the transmission disequilibrium test (TDT) design using single marker genotype, or haplotype information do not show differences between cases and controls. Conclusion In summary, our screen indicates that ALDH1A2 genetic

  7. Overexpression of MMP-7 increases collagen 1A2 in the aging kidney

    PubMed Central

    Ślusarz, Anna; Nichols, LaNita A; Grunz-Borgmann, Elizabeth A; Chen, Gang; Akintola, Adebayo D; Catania, Jeffery M; Burghardt, Robert C; Trzeciakowski, Jerome P; Parrish, Alan R

    2013-01-01

    The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis that leads to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however, it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, with over a 500-fold upregulation in 2-year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of protein kinase A (PKA), src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 upregulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src, and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD. PMID:24273653

  8. Overexpression of MMP-7 Increases Collagen 1A2 in the Aging Kidney.

    PubMed

    Oelusarz, Anna; Nichols, Lanita A; Grunz-Borgmann, Elizabeth A; Chen, Gang; Akintola, Adebayo D; Catania, Jeffery M; Burghardt, Robert C; Trzeciakowski, Jerome P; Parrish, Alan R

    2013-10-01

    The percentage of the U.S. population over 65 is rapidly increasing, as is the incidence of chronic kidney disease (CKD). The kidney is susceptible to age-dependent alterations in structure, specifically tubulointerstitial fibrosis, that lead to CKD. Matrix metalloproteinases (MMPs) were initially characterized as extracellular matrix (ECM) proteinases; however it is clear that their biological role is much larger. We have observed increased gene expression of several MMPs in the aging kidney, including MMP-7. MMP-7 overexpression was observed starting at 16 months, and over a 500 fold up-regulation in 2 year-old animals. Overexpression of MMP-7 is not observed in age-matched, calorically restricted controls that do not develop fibrosis and renal dysfunction, suggesting a role in the pathogenesis. In order to delineate the contributions of MMP-7 to renal dysfunction, we overexpressed MMP-7 in NRK-52E cells. High-throughput sequencing of the cells revealed that two collagen genes, Col1a2 and Col3a1, were elevated in the MMP-7 overexpressing cells. These two collagen genes were also elevated in aging rat kidneys and temporally correlated with increased MMP-7 expression. Addition of exogenous MMP-7, or conditioned media from MMP-7 overexpressing cells also increased Col1A2 expression. Inhibition of PKA, src, and MAPK signaling at p38 and ERK was able to attenuate the MMP-7 up-regulation of Col1a2. Consistent with this finding, increased phosphorylation of PKA, src and ERK was seen in MMP-7 overexpressing cells and upon exogenous MMP-7 treatment of NRK-52E cells. These data suggest a novel mechanism by which MMP-7 contributes to the development of fibrosis leading to CKD. PMID:24273653

  9. The structures of the human calcium channel {alpha}{sub 1} subunit (CACNL1A2) and {beta} subunit (CACNLB3) genes

    SciTech Connect

    Yamada, Yuichiro; Masuda, Kazuhiro; Li, Qing

    1995-05-20

    Calcium influx in pancreatic {beta}-cells is regulated mainly by L-type voltage-dependent calcium channels (VDCCs) and triggers insulin secretion. The {alpha}{sub 1} subunit (CACN4) and the {beta} subunit ({beta}{sub 3}) of VDCCs, both of which are expressed in pancreatic islets, are major components for the VDCC activity, and so they may play a critical role in the regulation of insulin secretion. The authors have determined the structures of the human CACN4 (CACNL1A2) and the human {beta}{sub 3} (CACNLB3) genes. The CACNL1A2 gene spans more than 155 kb and has 49 exons. Most of the positions interrupted by introns are well conserved between the CACNL1A2 gene and the previously reported L-type VDCC {alpha}{sub 1} subunit, CACNL1A1, gene. On the other hand, the CACNLB3 gene distributes in {approximately} 8 kb and comprises 13 exons, most of which are located together within {approximately} 5 kb. Comparisons of the genomic sequences of CACNL1A2 with the previously reported cDNA sequences indicate that there are a number of polymorphisms in the human CACNL1A2 gene. In addition, the PCR-SSCP procedure of exon 1 of CACNL1A2 revealed a change from 7 to 8 ATG trinucleotide repeats in a patient with noninsulin-dependent diabetes mellitus (NIDDM), resulting in an addition of methionine at the amino-terminus of CACN4. The determination of the structures of the human CACNL1A2 and CACNLB3 genes should facilitate study of the role of these genes in the development of NIDDM and also other genetic diseases such as long QT syndrome. 39 refs., 3 figs., 3 tabs.

  10. Drug interactions of diclofenac and its oxidative metabolite with human liver microsomal cytochrome P450 1A2-dependent drug oxidation.

    PubMed

    Ohyama, Katsuhiro; Murayama, Norie; Shimizu, Makiko; Yamazaki, Hiroshi

    2014-01-01

    1.  The purpose of this study was to investigate the inhibitory effects of diclofenac on human cytochrome P450 1A2-, 2C19- and 3A4-mediated drug oxidations and to evaluate the drug interaction potential of diclofenac and 4'-hydroxydiclofenac. 2.  Diclofenac was converted to 4'-hydroxydiclofenac by recombinantly expressed human P450 1A2 with Km and Vmax values of 33 µM and 0.20 min(-1), respectively. Diclofenac and 4'-hydroxydiclofenac suppressed flurbiprofen 4'-hydroxylation by P450 2C9 strongly and moderately, respectively; however, they did not affect P450 2C19-dependent S-mephenytoin hydroxylation or P450 3A4-dependent midazolam hydroxylation. 3.  Although the caffeine 3-N-demethylation activity of liver microsomal P450 1A2 was inhibited by simultaneous incubation with diclofenac, the riluzole N-hydroxylation activities of recombinant P450 1A2 and human liver microsomes were inhibited after preincubation with diclofenac or 4'-hydroxydiclofenac for 20 min in the presence of NADPH. Using the inhibition constant (37 µM) of diclofenac on caffeine 3-N-demethylation and the reported 95th percentiles of maximum plasma concentration (10.5 µM) after an oral dose of diclofenac, the in vivo estimated increase in area under the plasma concentration-time curve was 29%. 4.  These results suggest that diclofenac could inhibit drug clearance to a clinically important degree that depends on P450 1A2. Clinically relevant drug interactions in vivo with diclofenac are likely to be invoked via human P450 1A2 function in addition to those caused by the effect of diclofenac on P450 2C9.

  11. The Caffeine Cytochrome P450 1A2 Metabolic Phenotype Does Not Predict the Metabolism of Heterocyclic Aromatic Amines in Humans

    PubMed Central

    Turesky, Robert J.; White, Kami K.; Wilkens, Lynne R.; Marchand, Loïc Le

    2015-01-01

    2-Amino-1-methylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) are carcinogenic heterocyclic aromatic amines (HAAs) formed in well-done cooked meats. Chemicals that induce cytochrome P450 (P450) 1A2, a major enzyme involved in the bioactivation of HAAs, also form in cooked meat. Therefore, well-done cooked meat may pose an increase in cancer risk because it contains both inducers of P450 1A2 and procarcinogenic HAAs. We examined the influence of components in meat to modulate P450 1A2 activity and the metabolism of PhIP and MeIQx in volunteers during a 4 week feeding study of well-done cooked beef. The mean P450 1A2 activity, assessed by caffeine metabolic phenotyping, ranged from 6.3 to 7.1 before the feeding study commenced and from 9.6 to 10.4 during the meat feeding period: the difference in means was significant (P < 0.001). Unaltered PhIP, MeIQx, and their P450 1A2 metabolites, N2-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HON-PhIP-N2-Gl); N3-(β-1-glucosiduronyl-2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (HON-PhIP-N3-Gl); 2-amino-3-methylimidazo-[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH); and 2-amino-8-(hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH2OH-IQx) were measured in urine during days 2, 14, and 28 days of the meat diet. Significant correlations were observed on these days between the levels of the unaltered HAAs and their oxidized metabolites, when expressed as percent of dose ingested or as metabolic ratios. However, there was no statistically significant correlation between the caffeine P450 1A2 phenotype and any urinary HAA biomarker. Although the P450 1A2 activity varied by greater than 20-fold among the subjects, there was a large intra-individual variation of the P450 1A2 phenotype and inconsistent responses to inducers of P450 1A2. The coefficient of variation of the P450 1A2 phenotype within-individual ranged between 1 to 112% (median=40

  12. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3....

  13. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3....

  14. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1091 Ship radio equipment—Sea areas A1, A2, and A3....

  15. Up-regulation of eEF1A2 promotes proliferation and inhibits apoptosis in prostate cancer

    SciTech Connect

    Sun, Yue; Du, Chengli; Wang, Bo; Zhang, Yanling; Liu, Xiaoyan; Ren, Guoping

    2014-07-18

    Highlights: • The expression of eEF1A2 is up-regulated in prostate cancer tissues. • Suppression of eEF1A2 inhibits the proliferation and promotes apoptosis. • Inhibition of eEF1A2 enhances the expression of apoptotic relevant proteins. • The expressions of eEF1A2 and cleavage-caspase3 are inversely correlated. - Abstract: Background: eEF1A2 is a protein translation factor involved in protein synthesis, which possesses important function roles in cancer development. This study aims at investigating the expression pattern of eEF1A2 in prostate cancer and its potential role in prostate cancer development. Methods: We examined the expression level of eEF1A2 in 30 pairs of prostate cancer tissues by using RT-PCR and immunohistochemical staining (IHC). Then we applied siRNA specifically targeting eEF1A2 to down-regulate its expression in DU-145 and PC-3 cells. Flow cytometer was used to explore apoptosis and Western-blot was used to detect the pathway proteins of apoptosis. Results: Our results showed that the expression level of eEF1A2 in prostate cancer tissues was significantly higher compared to their corresponding normal tissues. Reduction of eEF1A2 expression in DU-145 and PC-3 cells led to a dramatic inhibition of proliferation accompanied with enhanced apoptosis rate. Western blot revealed that apoptosis pathway proteins (caspase3, BAD, BAX, PUMA) were significantly up-regulated after suppression of eEF1A2. More importantly, the levels of eEF1A2 and caspase3 were inversely correlated in prostate cancer tissues. Conclusion: Our data suggests that eEF1A2 plays an important role in prostate cancer development, especially in inhibiting apoptosis. So eEF1A2 might serve as a potential therapeutic target in prostate cancer.

  16. Organophosphorothionate pesticides inhibit the bioactivation of imipramine by human hepatic cytochrome P450s

    SciTech Connect

    Di Consiglio, Emma; Meneguz, Annarita; Testai, Emanuela . E-mail: testai@iss.it

    2005-06-15

    The drug-toxicant interaction between the antidepressant imipramine (IMI) and three organophosphorothionate pesticides (OPTs), to which humans may be chronically and simultaneously exposed, has been investigated in vitro. Concentrations of IMI (2-400 {mu}M) and OPTs ({<=}10 {mu}M) representative of actual human exposure have been tested with recombinant human CYPs and human liver microsomes (HLM). The different CYPs involved in IMI demethylation to the pharmacologically active metabolite desipramine (DES) were CYP2C19 > CYP1A2 > CYP3A4. The OPTs significantly inhibited (up to >80%) IMI bioactivation catalyzed by the recombinant CYPs tested, except CYP2D6, and by HLM; the inhibition was dose-dependent and started at low pesticide concentrations (0.25-2.5 {mu}M). The OPTs, having lower K {sub m} values, efficiently competed with IMI for the enzyme active site, as in the case of CYP2C19. However, with CYP1A2 and CYP3A4, a time- and NADPH-dependent mechanism-based inactivation also occurred, consistently with irreversible inhibition due to the release of the sulfur atom, binding to the active CYP during OPT desulfuration. At low IMI and OPT concentrations, lower IC50 values have been obtained with recombinant CYP1A2 (0.7-1.1 {mu}M) or with HLM rich in 1A2-related activity (2-10.8 {mu}M). The K {sub i} values (2-14 {mu}M), independent on substrate concentrations, were quite low and similar for the three pesticides. Exposure to OPTs during IMI therapeutic treatments may lead to decreased DES formation, resulting in high plasma levels of the parent drug, eventual impairment of its pharmacological action and possible onset of adverse drug reactions (ADRs)

  17. Utility of B-13 Progenitor-Derived Hepatocytes in Hepatotoxicity and Genotoxicity Studies

    PubMed Central

    Probert, Philip M. E.

    2014-01-01

    AR42J-B-13 (B-13) cells form hepatocyte-like (B-13/H) cells in response to glucocorticoid treatment. To establish its utility in toxicity and genotoxicity screening, cytochrome P450 (CYP) induction, susceptibility to toxins, and transporter gene expression were examined. Conversion to B-13/H cells resulted in expression of male-specific CYP2C11 and sensitivity to methapyrilene. B-13/H cells constitutively expressed CYP1A, induced expression in response to an aryl hydrocarbon receptor agonist, and activated benzo[α]pyrene to a DNA-damaging species. Functional CYP1A2 was not expressed due to deletions in the Cyp1a2 gene. A B-13 cell line stably expressing the human CYP1A2 was therefore engineered (B-13−TR/h1A2) and the derived B-13/H cells expressed metabolically functional CYP1A2. Treatment with the cooked food mutagen 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine resulted in a dose-dependent increase in DNA damage. B-13/H cells expressed constitutive androstane receptor (CAR) and induced CYP2B1 mRNA levels in response to classical CAR activators. However, translation to functional CYP2B1 protein was low and increased minimally by CAR activator treatment. B-13/H cells expressed high levels of pregnane X-receptor (PXR) and induced CYP3A1 in response to classical PXR activators. CYP3A genes were inducible, functional, and activated aflatoxin B1 to a DNA-damaging species. All 23 major hepatic transporters were induced when B-13 cells were converted to B-13/H cells, although in many cases, levels remained below those present in adult rat liver. However, bile salt export pump, Abcb1b, multidrug resistance-associated protein, and breast cancer resistance protein transporters were functional in B-13/H cells. These data demonstrate that the B-13 cell generates hepatocyte-like cells with functional drug metabolism and transporter activities, which can alone—or in a humanized form—be used to screen for hepatotoxic and genotoxic endpoints in vitro. PMID:24235770

  18. Involvement of oxidative stress in hepatocellular tumor-promoting activity of oxfendazole in rats.

    PubMed

    Dewa, Yasuaki; Nishimura, Jihei; Muguruma, Masako; Jin, Meilan; Kawai, Masaomi; Saegusa, Yukie; Okamura, Toshiya; Umemura, Takashi; Mitsumori, Kunitoshi

    2009-05-01

    The tumor-promoting effects of oxfendazole (OX), a benzimidazole anthelmintic, were investigated using a medium-term rat hepatocarcinogenesis model. Six-week-old male F344 rats received an intraperitoneal injection of N-diethylnitrosamine (DEN) and were given a powdered diet containing 0 or 500 ppm OX for 6 weeks from 2 weeks after DEN treatment. All animals were subjected to two-thirds partial hepatectomy 1 week after OX treatment. The numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci were significantly increased in the livers of rats treated with OX, with concomitantly increased cell proliferation, compared with those in the livers of the DEN alone group. Quantitative real-time RT-PCR analysis revealed that OX induced not only mRNA expression of phase I enzymes Cyp1a1, Cyp1a2, but also Nrf2-regulated phase II enzymes such as Gpx2, Nqo1, Yc2, Akr7a3 and Gstm1, presumably due to an adaptive response against OX-induced oxidative stress. Reactive oxygen species production increased in microsomes isolated from the livers of OX-treated rats. Furthermore, OX enhanced oxidative DNA damage (as assessed by 8-hydroxydeoxyguanosine; 8-OHdG) and lipid peroxidation (as assessed by thiobarbituric acid-reactive substances; TBARS). These results suggest that administration of OX at a high dose and for a long term enhances oxidative stress responses, which may contribute to its tumor-promoting potential in rats.

  19. In vitro oxidative metabolism of cajaninstilbene Acid by human liver microsomes and hepatocytes: involvement of cytochrome p450 reaction phenotyping, inhibition, and induction studies.

    PubMed

    Hua, Xin; Peng, Xiao; Tan, Shengnan; Li, Chunying; Wang, Wei; Luo, Meng; Fu, Yujie; Zu, Yuangang; Smyth, Hugh

    2014-10-29

    Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid), an active constituent of pigeonpea leaves, an important tropical crop, is known for its clinical effects in the treatment of diabetes, hepatitis, and measles and its potential antitumor effect. In this study, the effect of the cytochrome P450 isozymes on the activity of CSA was investigated. Two hydroxylation metabolites were identified in the study. The reaction phenotype study showed that CYP3A4, CYP2C9, and CYP1A2 were the major cytochrome P450 isozymes in the metabolism of CSA. The metabolic food-drug interaction potential was also evaluated in vitro. The effect of CSA inhibition/induction of enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro was estimated by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques. CSA showed different inhibitory effects on different isozymes. CSA reversibly inhibited CYP3A4 and CYP2C9 activities in human liver microsomes with IC50 values of 28.3 and 31.3 μM, respectively, but exhibited no inhibition activities to CYP1A2, CYP2A6, CYP2C19, CYP2D6, and CYP2E1. CSA showed a weak effect on CYP450 enzymes in a time-dependent manner. CSA did not substantially induce CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 at concentrations up to 30 μM in primary human hepatocytes. The results of our experiments may be helpful to predict clinically significant food-drug interactions when other drugs are administered in combination with CSA. PMID:25272989

  20. [Effect of Fuzheng Huayu recipe on CYP450 isozymes in normal and liver fibrosis rats].

    PubMed

    Zheng, Tian-hui; Liu, Wei; Li, Shu-ping; Yang, Tao; Wang, Chang-hong; Liu, Cheng-hai

    2015-03-01

    To study the effect of Fuzheng Huayu recipe (FZHY) on five types of isozymes of cytochrome P450 (CYP450) of normal and liver fibrosis rats by using the cocktail probe method. Dimethylnitrosamine ( DMN) was injected to induce the liver fibrosis model. After the tail vein injection with Cocktail probe solutions prepared with five CYP450s probe substrates (phenacetin-CYP1A2, omeprazole-CYP2C9, tolbutamide-CYP2C19, dextromethorphan-CYP2D6, midazolam-CYP3A4), the plasma concentrations of the five probe substrates were determined by LC-MS/MS, and the pharmacokinetic parameters were calculated by PK solutions 2. After the oral administration with FZHY, normal rats given phenacetin, omeprazole, tolbutamide and dextromethorphan showed increase in AUC(0-t) and decrease in CL to varying degrees, indicating that FZHY obviously inhibited the activities of CYP1A2, CYP2C9, CYP2C19 and CYP2D6 in normal rats, but with no obvious effect on the activity of CYP3A4. After the oral administration with FZHY, liver fibrosis rats treated with CYP2C9 showed the significant increase in AUC(0-t) and significant decrease in Vd, hut with no obvious changes in the pharmacokinetic parameters of other four types of prove substances, suggesting that FZHY could significantly inhibit the activity of CYP2C9 in rats but had no effect on the activities of CYP1A2, CYP2C19, CYP2D6 and CYP3A4. The changes in the activity of CYP450 isozymes in liver fibrosis rats may be the reason for FZHY's different effects on CYP450 isozymes in normal and liver fibrosis rats. PMID:26226765

  1. Differential inhibition of CYP1-catalyzed regioselective hydroxylation of estradiol by berberine and its oxidative metabolites.

    PubMed

    Chang, Yu-Ping; Huang, Chiung-Chiao; Shen, Chien-Chang; Tsai, Keng-Chang; Ueng, Yune-Fang

    2015-10-01

    Berberine is a pharmacologically active alkaloid present in widely used medicinal plants, such as Coptis chinensis (Huang-Lian). The hormone estradiol is oxidized by cytochrome P450 (CYP) 1B1 to primarily form the genotoxic metabolite 4-hydroxyestradiol, whereas CYP1A1 and CYP1A2 predominantly generate 2-hydroxyestradiol. To illustrate the effect of berberine on the regioselective oxidation of estradiol, effects of berberine and its metabolites on CYP1 activities were studied. Among CYP1s, CYP1B1.1, 1.3 (L432V), and 1.4 (N453S)-catalyzed 4-hydroxylation were preferentially inhibited by berberine. Differing from the competitive inhibition of CYP1B1.1 and 1.3, N453S substitution in CYP1B1 allowed a non-competitive or mixed-type pattern. An N228T in CYP1B1 highly decreased its activity and preference to 4-hydroxylation. A reverse mutation of T223N in CYP1A2 retained its 2-hydroxylation preference, but enhanced its inhibition susceptibility to berberine. Compared with berberine, metabolites demethyleneberberine and thalifendine caused weaker inhibition of CYP1A1 and CYP1B1 activities. Unexpectedly, thalifendine was more potent than berberine in the inhibition of CYP1A2, in which case an enhanced interaction through polar hydrogen-π bond was predicted from the docking analysis. These results demonstrate that berberine preferentially inhibits the estradiol 4-hydroxylation activity of CYP1B1 variants, suggesting that 4-hydroxyestradiol-mediated toxicity might be reduced by berberine, especially in tissues/tumors highly expressing CYP1B1.

  2. De novo EEF1A2 mutations in patients with characteristic facial features, intellectual disability, autistic behaviors and epilepsy.

    PubMed

    Nakajima, J; Okamoto, N; Tohyama, J; Kato, M; Arai, H; Funahashi, O; Tsurusaki, Y; Nakashima, M; Kawashima, H; Saitsu, H; Matsumoto, N; Miyake, N

    2015-04-01

    Eukaryotic elongation factor 1, alpha-2 (eEF1A2) protein is involved in protein synthesis, suppression of apoptosis, and regulation of actin function and cytoskeletal structure. EEF1A2 gene is highly expressed in the central nervous system and Eef1a2 knockout mice show the neuronal degeneration. Until now, only one missense mutation (c.208G > A, p.Gly70Ser) in EEF1A2 has been reported in two independent patients with neurological disease. In this report, we described two patients with de novo mutations (c.754G > C, p.Asp252His and c.364G > A, p.Glu122Lys) in EEF1A2 found by whole-exome sequencing. Common clinical features are shared by all four individuals: severe intellectual disability, autistic behavior, absent speech, neonatal hypotonia, epilepsy and progressive microcephaly. Furthermore, the two patients share the similar characteristic facial features including a depressed nasal bridge, tented upper lip, everted lower lip and downturned corners of the mouth. These data strongly indicate that a new recognizable disorder is caused by EEF1A2 mutations.

  3. SIRT1 deacetylates RFX5 and antagonizes repression of collagen type I (COL1A2) transcription in smooth muscle cells

    SciTech Connect

    Xia, Jun; Wu, Xiaoyan; Yang, Yuyu; Zhao, Yuhao; Fang, Mingming; Xie, Weiping; Wang, Hong; Xu, Yong

    2012-11-16

    Highlights: Black-Right-Pointing-Pointer SIRT1 interacts with and deacetylates RFX5. Black-Right-Pointing-Pointer SIRT1 activation attenuates whereas SIRT1 inhibition enhances collagen repression by RFX5 in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 promotes cytoplasmic localization and proteasomal degradation of RFX5 and cripples promoter recruitment of RFX5. Black-Right-Pointing-Pointer IFN-{gamma} represses SIRT1 expression in vascular smooth muscle cells. Black-Right-Pointing-Pointer SIRT1 agonist alleviates collagen repression by IFN-{gamma} in vascular smooth muscle cells. -- Abstract: Decreased expression of collagen by vascular smooth muscle cells (SMCs) within the atherosclerotic plaque contributes to the thinning of the fibrous cap and poses a great threat to plaque rupture. Elucidation of the mechanism underlying repressed collagen type I (COL1A2) gene would potentially provide novel solutions that can prevent rupture-induced complications. We have previously shown that regulatory factor for X-box (RFX5) binds to the COL1A2 transcription start site and represses its transcription. Here we report that SIRT1, an NAD-dependent, class III deacetylase, forms a complex with RFX5. Over-expression of SIRT1 or NAMPT, which synthesizes NAD+ to activate SIRT1, or treatment with the SIRT1 agonist resveratrol decreases RFX5 acetylation and disrupts repression of the COL1A2 promoter activity by RFX5. On the contrary, knockdown of SIRT1 or treatment with SIRT1 inhibitors induces RFX5 acetylation and enhances the repression of collagen transcription. SIRT1 antagonizes RFX5 activity by promoting its nuclear expulsion and proteasomal degradation hence dampening its binding to the COL1A2 promoter. The pro-inflammatory cytokine IFN-{gamma} represses COL1A2 transcription by down-regulating SIRT1 expression in SMCs. Therefore, our data have identified as novel pathway whereby SIRT1 maintains collagen synthesis in SMCs by modulating RFX5 activity.

  4. Structure Reassignment and Synthesis of Jenamidines A1/A2, Synthesis of (+)-NP25302, and Formal Synthesis of SB-311009 Analogues

    PubMed Central

    Duvall, Jeremy R.; Wu, Fanghui; Snider, Barry B.

    2008-01-01

    The proposed structures of jenamidines A, B, and C (1−3) were revised to jenamidines A1/A2, B1/B2, and C (8-10). Jenamidines A1/A2 (8) were synthesized from activated proline derivative 43 by conversion to 26 in two steps and 50% overall yield. Acylation of 26 with acid chloride 38d gave 39d, which was deprotected with TFA and then mild base to give 8 in 45% yield from 26. (−)-trans-2,5-Dimethylproline ethyl ester (49) was prepared by the enantioselective Michael reaction of ethyl 2-nitropropionate (51) and methyl vinyl ketone (50) using modified dihydroquinine 60 as the catalyst. Further elaboration converted 49 to natural (+)-NP25302 (12). A Wittig reaction of proline NCA (76) with ylide 79 gave 72 as a 9/1 E/Z mixture in 27% yield completing a one step formal synthesis of SB-311009 analogues. PMID:17064037

  5. In vitro inhibition and induction of human liver cytochrome P450 enzymes by gentiopicroside: potent effect on CYP2A6.

    PubMed

    Deng, Yating; Wang, Lu; Yang, Yong; Sun, Wenji; Xie, Renming; Liu, Xueying; Wang, Qingwei

    2013-01-01

    Gentiopicroside (GE), a naturally occurring iridoid glycoside, has been developed into a Novel Traditional Chinese Drug named gentiopicroside injection, and it was approved for the treatment of acute jaundice and chronic active hepatitis by SFDA. However, the inhibitory and inducible effects of GE on the activity of cytochrome P450 (CYP450) are unclear. The purpose of this study was to evaluate the ability of GE to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, GE inhibited CYP2A6 and CYP2E1 in a concentration-dependent manner, with IC₅₀ values of 21.8 µg/ml and 594 µg/ml, respectively, and the IC₅₀ of CYP2A6 was close to the C(max) value observed clinically. GE was a non-competitive inhibitor of CYP2A6 at lower concentrations and a competitive inhibitor at higher concentrations. GE did not produce inhibition of CYP2C9, CYP2D6, CYP1A2 or CYP3A4 activities. However, a significant increase of CYP1A2 and CYP3A4 activity was observed at high concentrations. In cultured human hepatocytes no significant induction of CYP1A2, CYP3A4 or CYP2B6 was observed. Given these results, the in vivo potential inhibition of GE on CYP2A6 deserves further investigation, and it seems that the hepatoprotective effect of GE is irrelevant to its effect on P450s.

  6. In vitro metabolism of nobiletin, a polymethoxy-flavonoid, by human liver microsomes and cytochrome P450.

    PubMed

    Koga, Nobuyuki; Ohta, Chiho; Kato, Yoshihisa; Haraguchi, Koichi; Endo, Tetsuya; Ogawa, Kazunori; Ohta, Hideaki; Yano, Masamichi

    2011-11-01

    Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4'-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4'-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4'-OH-NBL. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

  7. Enantioselective metabolism of the endocrine disruptor pesticide methoxychlor by human cytochromes P450 (P450s): major differences in selective enantiomer formation by various P450 isoforms.

    PubMed

    Hu, Yiding; Kupfer, David

    2002-12-01

    Methoxychlor, a currently used pesticide that in mammals elicits proestrogenic/estrogenic activity and reproductive toxicity, has been classified as a prototype endocrine disruptor. Methoxychlor is prochiral, and its metabolites 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-M); 1,1,1-trichloro- 2-(4-methoxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (catechol-M); and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(3, 4-dihydroxyphenyl)ethane (tris-OH-M) are chiral; whereas 1,1,1-trichloro-2, 2-bis(4-hydroxyphenyl)ethane (bis-OH-M) is achiral. These metabolites are formed during methoxychlor incubation with liver microsomes or recombinant cytochrome p450s (rp450s). Since methoxychlor-metabolite enantiomers may have different estrogenic/antiestrogenic/antiandrogenic activities than corresponding racemates, the possibility that p450s preferentially generate or use R or S enantiomers, was examined. Indeed, rCYP1A2 and r2A6 mono-demethylated methoxychlor primarily into (R)-mono-OH-M at 91 and 75%, respectively, whereas rCYP1A1, 2B6, 2C8, 2C9, 2C19, and 2D6 formed the (S)-enantiomer at 69, 66, 75, 95, 96, and 80%, respectively. However, rCYP3A4, 3A5, and 2B1(rat) weakly demethylated methoxychlor without enantioselectivity. Human liver microsomes generated (S)-mono-OH-M (77-87%), suggesting that CYP1A2 and 2A6 display only minor catalytic contribution. P450 inhibitors demonstrated that CYP2C9 and possibly 2C19 are major hepatic catalysts forming (S)-mono-OH-M, and CYP1A2 is primarily involved in forming the (R)-mono-OH-M. Demethylation rate of (S)-mono-OH-M versus (R)-mono-OH-M forming achiral bis-OH-M by rCYP1A2 was 97/3, compared with 15/85 and 17/83 for rCYP2C9 and 2C19, respectively, indicating opposite substrate enantioselectivity of rCYP1A2 versus 2C9 and 2C19. Also, rCYP1A2 preferentially O-demethylated (R)-catechol-M into (R)-tris-OH-M (at 80%), contrasting r2C9 and r2C19 that yielded (S)-tris-OH-M at 80 and 77%, respectively. Ortho-hydroxylation of

  8. Assignment of human elongation factor 1{alpha} genes: EEF1A maps to chromosome 6q14 and EEF1A2 to 20q13.3

    SciTech Connect

    Lund, A.; Clark, B.; Knudsen, S.M.

    1996-09-01

    The human elongation factor 1 {alpha} gene family consists of at least 2 actively transcribed genes, EEF1A and EEF1A2, and more than 18 homologous loci. EEF1A2 is expressed ubiquitously, and both of them can function in translation. An EEF1A cDNA probe has previously been shown to cross-hybridize with several human chromosomes, but the location of the functional gene has not been established. We have mapped the functional EEF1A gene to 6q14 by combined fluorescence in situ hybridization (FISH) and PCR analysis of a somatic cell hybrid panel and mapped EEF1A2 to 20q13.3 by FISH. In addition, the 11 strongest cross-hybridizing loci (EEF1AL2-EEF1AL13) were mapped by FISH to 12p12, 9q34, 7p15-p21, 19q13, 3q26-q27, 7q33-q35, 1p13-p22, 2q12-q14, 5p12-q11, 1q31-q32, and Xq21. 17 refs., 1 fig., 1 tab.

  9. CYP2C subfamily, primarily CYP2C9, catalyses the enantioselective demethylation of the endocrine disruptor pesticide methoxychlor in human liver microsomes: use of inhibitory monoclonal antibodies in P450 identification.

    PubMed

    Hu, Y; Krausz, K; Gelboin, H V; Kupfer, D

    2004-02-01

    1. The endocrine disruptor pesticide methoxychlor undergoes O-demethylation by mammalian liver microsomes forming chiral mono-phenolic (1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane, i.e. mono-OH-M) and achiral bis-phenolic oestrogenic metabolites. Human liver microsomes (HLM) generated primarily the S-mono-OH-M. 2. Inhibitory monoclonal antibodies (MAb) identified those P450s catalysing the enantioselective O-demethylation of methoxychlor. In HLM, O-demethylation was inhibited by MAb anti-2C9 (30-40%), diminishing the per cent of S-mono-OH-M from about 80 to 55-60%. MAb anti-CYP1A2, 2A6, 2B6, 2C8, 2C19, 2D6 and 3A4 did not affect the demethylation rate in HLM. Nevertheless, MAb anti-CYP1A2 decreased the formation of R-mono-OH-M from 21-23 to 10-17%, indicating that CYP1A2 exhibits a role in generating the R-enantiomer. 3. Among cDNA-expressed human P450s (supersomes), CYP2C19 was the most active in demethylation, but in HLM, CYP2C19 appeared inactive (no inhibition by MAb anti-CYP2C19). There was a substantial difference in the per cent inhibition of demethylation by MAb anti-CYP2C9 and anti-rat CYP2C (MAb inhibiting all human CYP2C forms) and in altering the enantioselectivity, suggesting that demethylation by combined CYP2C8, 2C18 and 2C19 was significant (20-30%). 4. Polymorphism of methoxychlor demethylation was examined with supersomes and HLM-expressing CYP2C9 allelic variants. CYP2C9*1 and 2C9*2 were highly active; however, CYP2C9*3 appeared inactive.

  10. In vitro inhibitory effects of scutellarin on six human/rat cytochrome P450 enzymes and P-glycoprotein.

    PubMed

    Han, Yong-Long; Li, Dan; Yang, Quan-Jun; Zhou, Zhi-Yong; Liu, Li-Ya; Li, Bin; Lu, Jin; Guo, Cheng

    2014-05-05

    Inhibition of cytochrome P450 (CYP) and P-glycoprotein (P-gp) are regarded as the most frequent and clinically important pharmacokinetic causes among the various possible factors for drug-drug interactions. Scutellarin is a flavonoid which is widely used for the treatment of cardiovascular diseases. In this study, the in vitro inhibitory effects of scutellarin on six major human CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and six rat CYPs (CYP1A2, CYP2C7, CYP2C11, CYP2C79, CYP2D4, and CYP3A2) activities were examined by using liquid chromatography-tandem mass spectrometry. Meanwhile, the inhibitory effects of scutellarin on P-gp activity were examined on a human metastatic malignant melanoma cell line WM-266-4 by calcein-AM fluorometry screening assay. Results demonstrated that scutellarin showed negligible inhibitory effects on the six major CYP isoenzymes in human/rat liver microsomes with almost all of the IC50 values exceeding 100 μM, whereas it showed values of 63.8 μM for CYP2C19 in human liver microsomes, and 63.1 and 85.6 μM for CYP2C7 and CYP2C79 in rat liver microsomes, respectively. Scutellarin also showed weak inhibitory effect on P-gp. In conclusion, this study demonstrates that scutellarin is unlikely to cause any clinically significant herb-drug interactions in humans when co-administered with substrates of the six CYPs (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) and P-gp.

  11. Transfer of PCBs via lactation simultaneously induces the expression of P450 isoenzymes and the protooncogenes c-Ha-ras and c-raf in neonates.

    PubMed

    Borlak, J T; Scott, A; Henderson, C J; Jenke, H J; Wolf, C R

    1996-02-23

    At the first day of lactation, maternal rats were injected with a single i.p. dose of 100 or 250 mg/kg body weight of a mixture of polychlorinated biphenyls (Aroclor 1254). This treatment caused significant increases in both material and neonatal hepatic cytochrome P-450, cytochrome b5, and cytochrome-c-(P-450) reductase. Transfer of PCBs via lactation resulted in significant increases in hepatic enzyme activities catalysed by neonatal CYP1A1, CYP1A2, CYP2B1, CYP3A1, and CYP2E1 using a variety of substrates. In contrast, the metabolism of dimethylnitrosamine and aminopyrine was only marginally (up to 2-fold) increased in maternal animals four days post treatment. Further measurements showed significant increases in maternal and neonatal epoxide hydrolase, glutathione-S-transferase, and UDP-glucuronyl transferase activities, thus suggesting a coordinated response for an induction of CYP1A1, CYP1A2, CYP2A1, CYP2B1, CYP2E1, CYP3A1, and CYP4A1 in both maternal and neonatal CYP2C6, and at the higher dose the expression of neonatal CYP2E1 was significantly reduced. Northern blot analysis provided further evidence for significant increases in maternal and neonatal hepatic CYP1A1, CYP1A2, CYP2B1, and CYP2E1 mRNA, but reduced amounts of CYP2C7 and CYP4A1 mRNA. Additional Northern blot hybridization experiments may suggest an increased expression of the protooncogenes c-Ha-ras and c-raf in the mother and the neonate upon treatment of maternal rats with Aroclor 1254. Lactation itself may result in an increased expression of the latter protooncogenes, but the mRNA of the protooncogenes c-erb A and c-erb B was not detected in any of the tissues examined.

  12. Evaluation of the inhibition potential of plumbagin against cytochrome P450 using LC-MS/MS and cocktail approach

    PubMed Central

    Chen, Ang; Zhou, Xiaojing; Tang, Shuowen; Liu, Mingyao; Wang, Xin

    2016-01-01

    Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a natural naphthoquinone compound isolated from roots of Plumbago zeylanica L., has drawn a lot of attention for its plenty of pharmacological properties including antidiabetes and anti-cancer. The aim of this study was to investigate the effects of plumbagin on CYP1A2, CYP2B1/6, CYP2C9/11, CYP2D1/6, CYP2E1 and CYP3A2/4 activities in human and rat liver and evaluate the potential herb-drug interactions using the cocktail approach. All CYP substrates and their metabolites were analyzed using high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS). Plumbagin presented non-time-dependent inhibition of CYP activities in both human and rat liver. In humans, plumbagin was not only a mixed inhibitor of CYP2B6, CYP2C9, CYP2D6, CYP2E1 and CYP3A4, but also a non-competitive inhibitor of CYP1A2, with Ki values no more than 2.16 μM. In rats, the mixed inhibition of CYP1A2 and CYP2D1, and competitive inhibition for CYP2B1, CYP2C11 and CYP2E1 with Ki values less than 9.93 μM were observed. In general, the relatively low Ki values of plumbagin in humans would have a high potential to cause the toxicity and drug interactions involving CYP enzymes. PMID:27329697

  13. Regioselective differences in C(8)- and N-oxidation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by human and rat liver microsomes and cytochromes P450 1A2.

    PubMed

    Turesky, R J; Parisod, V; Huynh-Ba, T; Langouët, S; Guengerich, F P

    2001-07-01

    The metabolism of the mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was investigated with human and rat liver microsomes, recombinant human cytochrome P450 1A2 (P450 1A2) expressed in Escherichia coli cells, and rat P450 1A2. Human liver microsomes and human P450 1A2 catalyzed the oxidation of the exocyclic amine group of MeIQx to form the genotoxic product 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline (HONH-MeIQx). Human P450 1A2 also catalyzed the oxidation of C(8)-methyl group of MeIQx to form 2-amino-(8-hydroxymethyl)-3-methylimidazo[4,5-f]quinoxaline (8-CH(2)OH-IQx), 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carbaldehyde (IQx-8-CHO), and 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH). Thus, chemically stable C(8)-oxidation products of MeIQx may be useful biomarkers of P450 1A2 activity in humans. Rat liver microsomes were 10-15-fold less active than the human counterpart at both N-oxidation and C(8)-oxidation of MeIQx when expressed as nanomoles of product formed per minute per nanomoles of P450 1A2. Differences in regioselective oxidation of MeIQx were also observed with human and rat liver microsomes and the respective P450 1A2 orthologs. In contrast to human liver microsomes and P450 1A2, rat liver microsomes and purified rat P4501A2 were unable to catalyze the oxidation of MeIQx to the carboxylic derivative IQx-8-COOH, an important detoxication product formed in humans. However, rat liver microsomes and rat P4501A2, but not human liver microsomes or human P450 1A2, extensively catalyzed ring oxidation at the C-5 position of MeIQx to form the detoxication product 2-amino-3,8-dimethyl-5-hydroxyimidazo[4,5-f]quinoxaline (5-HO-MeIQx). There are important differences between human and rat P450 1A2, both in catalytic activities and oxidation pathways of MeIQx, that may affect the biological activity of this carcinogen and must be considered when assessing human health risk.

  14. 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

  15. In vitro inhibition and induction of human cytochrome P450 enzymes by mirabegron, a potent and selective β3-adrenoceptor agonist.

    PubMed

    Takusagawa, Shin; Miyashita, Aiji; Iwatsubo, Takafumi; Usui, Takashi

    2012-12-01

    The potential for mirabegron, a β(3)-adrenoceptor agonist for the treatment of overactive bladder, to cause drug-drug interactions via inhibition or induction of cytochrome P450 (CYP) enzymes was investigated in vitro. Mirabegron was shown to be a time-dependent inhibitor of CYP2D6 in the presence of NADPH as the IC(50) value in human liver microsomes decreased from 13 to 4.3 μM after 30-min pre-incubation. Further evaluation indicated that mirabegron may act partly as an irreversible or quasi-irreversible metabolism-dependent inhibitor of CYP2D6. Therefore, the potential of mirabegron to inhibit the metabolism of CYP2D6 substrates in vivo cannot be excluded. Mirabegron was predicted not to cause clinically significant metabolic drug-drug interactions via inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2E1, or CYP3A4/5 because the IC(50) values for these enzymes both with and without pre-incubation were >100 μM (370 times maximum human plasma concentration [C(max)]). Whereas positive controls (100 µM omeprazole and 10 µM rifampin) caused the anticipated CYP induction, the highest concentration of mirabegron (10 µM; 37 times plasma C(max)) had minimal effect on CYP1A2 and CYP3A4/5 activity, and CYP1A2 and CYP3A4 mRNA levels in freshly isolated human hepatocytes, suggesting that mirabegron is not an inducer of these enzymes.

  16. Cellular responses to oxidative stress: the [Ah] gene battery as a paradigm.

    PubMed Central

    Nebert, D W; Petersen, D D; Fornace, A J

    1990-01-01

    A major source of oxidative stress in animals is plant stress metabolites, also termed phytoalexins. The aromatic hydrocarbon-responsive [Ah] gene battery is considered here as a model system in which we can study metabolically coordinated enzymes that respond to phytoalexin-induced oxidative stress. In the mouse, the [Ah] battery comprises at least six genes: two Phase I genes, CYP1A1 and CYP1A2; and four Phase II genes, Nmo-1, Aldh-1, Ugt-1, and Gt-1. All six genes appear to be regulated positively by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other ligands of the Ah receptor. In the absence of foreign inducer, the control of Nmo-1 gene expression is independent of the control of CYP1A1 and CYP1A2 gene expression. The radiation deletion homozygote c14CoS/c14CoS mouse is lacking about 1.1 centiMorgans of chromosome 7. Although having no detectable CYP1A1 or CYP1A2 activation, the untreated c14CoS/c14CoS mouse exhibits markedly elevated transcripts of the Nmo-1 gene and three growth arrest- and DNA damage-inducible (gadd) genes. These data suggest that the missing region on chromosome 7 in the c14CoS/c14CoS mouse contains a gene(s), which we propose to call Nmo-1n, encoding a trans-acting factor(s) that is a negative effector of the Nmo-1 and gadd genes. The three other [Ah] battery Phase II genes behave similarly to Nmo-1 in the c14CoS/c14CoS mouse. This coordinated response to oxidative stress and DNA damage, by way of the release of a mammalian battery of genes from negative control, bears an interesting resemblance to the SOS response in bacteria. PMID:2272308

  17. Characterization of in vitro oxidative and conjugative metabolic pathways for brevetoxin (PbTx-2).

    PubMed

    Radwan, Faisal F Y; Ramsdell, John S

    2006-01-01

    Brevetoxins are potent marine toxins produced by the dinoflagellate Karenia brevis, the causative organism of Florida red tides. An in vitro metabolism of PbTx-2 was performed using purified cDNA-expressed rat liver cytochrome P-450 (CYP) enzymes and freshly isolated rat hepatocytes. The metabolic activities of six CYP enzymes, CYP1A2, CYP2A2, CYP2C11, CYP2D1, CYP2E1, and CYP3A1, were examined by incubation with PbTx-2 for up to 4 h in the presence of a NADPH-generating system. Further identification of the metabolites produced by CYP1A2 and CYP3A1 was preformed using high performance liquid chromatography-mass spectrometry (LC/MS). Both CYP1A2 and CYP3A1 metabolized PbTx-2 to PbTx-3 (MH+: m/z 897), PbTx-9 (MH+: m/z 899), and a newly recorded diol brevetoxin-2 metabolite (MH+: m/z 929). CYP3A1 also produced a considerably higher amount of BTX-B5 (MH+: m/z 911). Subsequent incubation of PbTx-2 with rat hepatocytes produced additional phase 1 metabolites of MH+: m/z 911, 913, 915, 917, and 931, indicating a CYP-catalyzed epoxidation at H-ring (C27,C28-double bond) and a subsequent A-ring hydrolysis of PbTx-2 metabolic products. A conjugation metabolism was identified by the production of a glutathione-brevetoxin conjugate (MH+: m/z 1222) and a cysteine-brevetoxin conjugate (MH+: m/z 1018). Structures of the new metabolites are postulated, and a likely CYP-catalyzed metabolism pathway of PbTx-2 metabolism are discussed. PMID:16221966

  18. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 47 Telecommunication 5 2010-10-01 2010-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1,...

  19. Screening of CACNA1A and ATP1A2 genes in hemiplegic migraine: clinical, genetic, and functional studies

    PubMed Central

    Carreño, Oriel; Corominas, Roser; Serra, Selma Angèlica; Sintas, Cèlia; Fernández-Castillo, Noèlia; Vila-Pueyo, Marta; Toma, Claudio; Gené, Gemma G; Pons, Roser; Llaneza, Miguel; Sobrido, María-Jesús; Grinberg, Daniel; Valverde, Miguel Ángel; Fernández-Fernández, José Manuel; Macaya, Alfons; Cormand, Bru

    2013-01-01

    Hemiplegic migraine (HM) is a rare and severe subtype of autosomal dominant migraine, characterized by a complex aura including some degree of motor weakness. Mutations in four genes (CACNA1A, ATP1A2, SCN1A and PRRT2) have been detected in familial and in sporadic cases. This genetically and clinically heterogeneous disorder is often accompanied by permanent ataxia, epileptic seizures, mental retardation, and chronic progressive cerebellar atrophy. Here we report a mutation screening in the CACNA1A and ATP1A2 genes in 18 patients with HM. Furthermore, intragenic copy number variant (CNV) analysis was performed in CACNA1A using quantitative approaches. We identified four previously described missense CACNA1A mutations (p.Ser218Leu, p.Thr501Met, p.Arg583Gln, and p.Thr666Met) and two missense changes in the ATP1A2 gene, the previously described p.Ala606Thr and the novel variant p.Glu825Lys. No structural variants were found. This genetic screening allowed the identification of more than 30% of the disease alleles, all present in a heterozygous state. Functional consequences of the CACNA1A-p.Thr501Met mutation, previously described only in association with episodic ataxia, and ATP1A2-p.Glu825Lys, were investigated by means of electrophysiological studies, cell viability assays or Western blot analysis. Our data suggest that both these variants are disease-causing. PMID:24498617

  20. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 47 Telecommunication 5 2014-10-01 2014-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1,...

  1. 47 CFR 80.1091 - Ship radio equipment-Sea areas A1, A2, and A3.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... accordance with 5 U.S.C. 552(a) and 1 CFR part 51. Copies of these standards can be inspected at the Federal... 47 Telecommunication 5 2011-10-01 2011-10-01 false Ship radio equipment-Sea areas A1, A2, and A3... SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System...

  2. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 47 Telecommunication 5 2011-10-01 2011-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1,...

  3. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 47 Telecommunication 5 2013-10-01 2013-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1,...

  4. 47 CFR 80.1093 - Ship radio equipment-Sea areas A1, A2, A3, and A4.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 47 Telecommunication 5 2012-10-01 2012-10-01 false Ship radio equipment-Sea areas A1, A2, A3, and... AND SPECIAL RADIO SERVICES STATIONS IN THE MARITIME SERVICES Global Maritime Distress and Safety System (GMDSS) Equipment Requirements for Ship Stations § 80.1093 Ship radio equipment—Sea areas A1,...

  5. Relationship between Genotypes Sult1a2 and Cyp2d6 and Tamoxifen Metabolism in Breast Cancer Patients

    PubMed Central

    Fernández-Santander, Ana; Gaibar, María; Novillo, Apolonia; Romero-Lorca, Alicia; Rubio, Margarita; Chicharro, Luis Miguel; Tejerina, Armando; Bandrés, Fernando

    2013-01-01

    Tamoxifen is a pro-drug widely used in breast cancer patients to prevent tumor recurrence. Prior work has revealed a role of cytochrome and sulfotransferase enzymes in tamoxifen metabolism. In this descriptive study, correlations were examined between concentrations of tamoxifen metabolites and genotypes for CYP2D6, CYP3A4, CYP3A5, SULT1A1, SULT1A2 and SULT1E1 in 135 patients with estrogen receptor-positive breast cancer. Patients were genotyped using the Roche-AmpliChip® CYP450 Test, and Real-Time and conventional PCR-RFLP. Plasma tamoxifen, 4-hydroxy-tamoxifen, N-desmethyl-tamoxifen, endoxifen and tamoxifen-N-oxide were isolated and quantified using a high-pressure liquid chromatography-tandem mass spectrometry system. Significantly higher endoxifen levels were detected in patients with the wt/wt CYP2D6 compared to the v/v CYP2D6 genotype (p<0.001). No differences were detected in the remaining tamoxifen metabolites among CYP2D6 genotypes. Patients featuring the SULT1A2*2 and SULT1A2*3 alleles showed significantly higher plasma levels of 4-hydroxy-tamoxifen and endoxifen (p = 0.025 and p = 0.006, respectively), as likely substrates of the SULT1A2 enzyme. Our observations indicate that besides the CYP2D6 genotype leading to tamoxifen conversion to potent hydroxylated metabolites in a manner consistent with a gene-dose effect, SULT1A2 also seems to play a role in maintaining optimal levels of both 4-hydroxy-tamoxifen and endoxifen. PMID:23922954

  6. Captan impairs CYP-catalyzed drug metabolism in the mouse.

    PubMed

    Paolini, M; Barillari, J; Trespidi, S; Valgimigli, L; Pedulli, G F; Cantelli-Forti, G

    1999-11-30

    To investigate whether the fungicide captan impairs CYP-catalyzed drug metabolism in murine liver, kidney and lung, the modulation of the regio- and stereo-selective hydroxylation of testosterone, including 6beta-(CYP3A), 6alpha-(CYP2A1 and CYP2B1) and 16alpha-(CYP2B9) oxidations was studied. Specific substrates as probes for different CYP isoforms such as p-nitrophenol (CYP2E1), pentoxyresorufin (CYP2B1), ethoxyresorufin (CYP1A1), aminopyrine (CYP3A), phenacetin and methoxyresorufin (CYP1A2), and ethoxycoumarin (mixed) were also considered. Daily doses of captan (7.5 or 15 mg/kg b.w., i.p.) were administered to different groups of Swiss Albino CD1 mice of both sexes for 1 or 3 consecutive days. While a single dose of this fungicide did not affect CYP-machinery, repeated treatment significantly impaired the microsomal metabolism; in the liver, for example, a general inactivating effect was observed, with the sole exception of testosterone 2alpha-hydroxylase activity which was induced up to 8.6-fold in males. In vitro studies showed that the mechanism-based inhibition was related to captan metabolites rather than the parental compound. In the kidney, both CYP3A- and CYP1A2-linked monooxygenases were significantly induced (2-fold) by this pesticide. Accelerated phenacetin and methoxyresorufin metabolism (CYP1A2) was also observed in the lung. Data on CYP3A (kidney) and CYP1A2 (kidney and lung) induction were corroborated by Western immunoblotting using rabbit polyclonal anti-CYP3A1/2 and CYP1A1/2 antibodies. By means of electron spin resonance (EPR) spectrometry coupled to a spin-trapping technique, it was found that the recorded induction generates a large amounts of the anion radical superoxide (O*2-) either in kidney or lung microsomes. These findings suggest that alterations in CYP-associated activities by captan exposure may result in impaired (endogenous) metabolism as well as of coadministered drugs with significant implications for their disposition. The

  7. Cytochrome P450 inhibition potential of new psychoactive substances of the tryptamine class.

    PubMed

    Dinger, Julia; Woods, Campbell; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

    2016-01-22

    New psychoactive substances (NPS) are not tested for their cytochrome P450 (CYP) inhibition potential before consumption. Therefore, this potential was explored for tryptamine-derived NPS (TDNPS) including alpha-methyl tryptamines (AMTs), dimethyl tryptamines (DMTs), diallyl tryptamines (DALTs), and diisopropyl tryptamines (DiPTs) using test substrates preferred by the Food and Drug Administration in a cocktail assay. All tested TDNPS with the exception of DMT inhibited CYP2D6 activity with IC50 values below 100μM. DALTs inhibited CYP2D6 activity similar to paroxetine and quinidine and CYP1A2 activity comparable to fluvoxamine. 5-Methoxy-N,N-diallyltryptamine reduced in vivo the caffeine metabolism in rats consistent with in vitro results. Five of the AMTs also inhibited CYP1A2 activity comparable to amiodarone. AMT and 6-F-AMT inhibited CYP2A6 activity in the range of the test inhibitor tranylcypromine. CYP2B6 activity was inhibited by 19 tryptamines, but weakly compared to efavirenz. CYP2C8 activity was inhibited by five of the tested TDNPS and three showed values comparable to trimethoprim and gemfibrozil. Six tryptamines inhibited CYP2C9 and seven CYP2C19 activities comparable to fluconazole and chloramphenicol, respectively. Nineteen compounds showed inhibition of CYP2E1 and 18 of CYP3A activity, respectively. These results showed that the CYP inhibition by TDNPS might be clinically relevant, but clinical studies are needed to explore this further. PMID:26599973

  8. Autoantibodies against CYP2D6 and other drug-metabolizing enzymes in autoimmune hepatitis type 2.

    PubMed

    Mizutani, Takaharu; Shinoda, Masakazu; Tanaka, Yuta; Kuno, Takuya; Hattori, Asuka; Usui, Toru; Kuno, Nayumi; Osaka, Takashi

    2005-01-01

    Autoimmune hepatitis (AIH) is a disease of unknown etiology, characterized by liver-related autoantibodies. Autoimmune hepatitis is subdivided into two major types: AIH type 1 is characterized by the detection of ANA, SMA, ANCA, anti-ASGP-R, and anti-SLA/LP. Autoimmune hepatitis type 2 is characterized to be mainly related with drug-metabolizing enzymes as autoantigens, such as anti-LKM (liver-kidney microsomal antigen)-1 against CYP2D6, anti-LKM-2 against CYP2C9-tienilic acid, anti-LKM-3 against UGT1A, and anti-LC1 (liver cytosol antigen)-1 and anti-APS (autoimmune polyglandular syndrome type-1) against CYP1A2, CYP2A6, and others. Anti-LKM-1 sera inhibited CYP2D6 activity in vitro but did not inhibit cellular drug metabolism in vivo. CYP2D6 is the major target autoantigen of LKM-1 and expressed on plasma membrane (PM) of hepatocytes, suggesting a pathogenic role for anti-LKM-1 in liver injury as a trigger. Anti-CYP1A2 was observed in dihydralazine-induced hepatitis, and radiolabeled CYP1A2 disappeared from the PM with a half-life of less than 30 min, whereas microsomal CYP1A2 was stably radiolabeled for several hours. Main antigenic epitopes on CYP2D6 are aa 193-212, aa 257-269, and aa 321-351; and D263 is essential. The third epitope is located on the surface of the protein CYP2D6 and displays a hydrophobic patch that is situated between an aromatic residue (W316) and histidine (H326). Some drugs such as anticonvulsants (phenobarbital, phenytoin, and carbamazepine) and halothane are suggested to induce hepatitis with anti-CYP3A and anti-CYP2E1, respectively. Autoantibodies against CYP11A1, CYP17, and/or CYP21 involved in the synthesis of steroid hormones are also detected in patients with adrenal failure, gonadal failure, and/or Addison disease.

  9. In vivo study of the effect of antiviral acyclic nucleotide phosphonate (R)-9-[2-(phosphonomethoxy)propyl]adenine (PMPA, tenofovir) and its prodrug tenofovir disoproxil fumarate on rat microsomal cytochrome P450.

    PubMed

    Anzenbacherová, E; Anzenbacher, P; Zídek, Z; Buchar, E; Kmonícková, E; Potmesil, P; Nekvindová, J; Veinlichová, A; Holý, A

    2008-01-01

    The total content of rat liver microsomal cytochrome P450 (CYP) significantly decreased after repeated i.p. administration of the antiviral agent tenofovir ((R)-9-[2-(phosphonomethoxy)propyl] adenine) and tenofovir disoproxil at a daily dose 25 mg/kg, although the content of liver microsomal protein did not change. The decrease of the CYP content was accompanied by concomitant increase of the amount of inactive CYP form, cytochrome P420. This effect was confirmed by a parallel study of the activities of selected CYP forms, CYP2E1 (p-nitrophenol hydroxylation) and CYP1A2 (7-ethoxyresorufin deethylation). The activity (expressed relatively to the protein content) of both CYP forms decreased significantly following the decrease of the total CYP. On the other hand, the CYP2E1 activity expressed relatively to the decreasing total CYP content remained unchanged. However, CYP1A2 activity also decreased when calculated relatively to the total native CYP content indicating lower stability of this form. Semiquantitative RT-PCR showed no significant changes in expression of major rat liver microsomal CYP forms after tenofovir treatment. In conclusion, repeated administration of tenofovir in higher doses led to significant decrease of the relative proportion of active liver microsomal CYPs accompanied by a conversion of these enzymes to the inactive form (CYP420) maintaining the sum of CYP proteins unchanged.

  10. 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

  11. The anticancer drug ellipticine is a potent inducer of rat cytochromes P450 1A1 and 1A2, thereby modulating its own metabolism.

    PubMed

    Aimová, Dagmar; Svobodová, Lucie; Kotrbová, Vera; Mrázová, Barbora; Hodek, Petr; Hudecek, Jirí; Václavíková, Radka; Frei, Eva; Stiborová, Marie

    2007-10-01

    Ellipticine is an antineoplastic agent whose mode of action is based mainly on DNA intercalation, inhibition of topoisomerase II, and formation of covalent DNA adducts mediated by cytochromes P450 (P450s) and peroxidases. Here, this drug was found to induce CYP1A1 and/or 1A2 enzymes and their enzymatic activities in livers, lungs, and kidneys of rats treated (i.p.) with ellipticine. The induction is transient. In the absence of repeated administration of ellipticine, the levels and activities of the induced CYP1A decreased almost to the basal level 2 weeks after treatment. The ellipticine-mediated CYP1A induction increases the DNA adduct formation by the compound. When microsomal fractions from livers, kidneys, and lungs of rats treated with ellipticine were incubated with ellipticine, DNA adduct formation, measured by (32)P-postlabeling analysis, was up to 3.8-fold higher in incubations with microsomes from pretreated rats than with controls. The observed stimulation of DNA adduct formation by ellipticine was attributed to induction of CYP1A1 and/or 1A2-mediated increase in ellipticine oxidative activation to 13-hydroxy- and 12-hydroxyellipticine, the metabolites generating two major DNA adducts in human and rat livers. In addition to these metabolites, increased formation of the excretion products 9-hydroxy- and 7-hydroxyellipticine was also observed in microsomes of rats treated with ellipticine. Taken together, these results demonstrate for the first time that by inducing CYP1A1/2, ellipticine increases its own metabolism, leading both to an activation of this drug to reactive species-forming DNA adducts and to detoxication metabolites, thereby modulating to some extent its pharmacological and/or genotoxic potential.

  12. Two Rare Mutations in the COL1A2 Gene Associate With Low Bone Mineral Density and Fractures in Iceland.

    PubMed

    Styrkarsdottir, Unnur; Thorleifsson, Gudmar; Eiriksdottir, Berglind; Gudjonsson, Sigurjon A; Ingvarsson, Thorvaldur; Center, Jacqueline R; Nguyen, Tuan V; Eisman, John A; Christiansen, Claus; Thorsteinsdottir, Unnur; Sigurdsson, Gunnar; Stefansson, Kari

    2016-01-01

    We conducted a genome-wide association study of low bone mineral density (BMD) at the hip and spine utilizing sequence variants found through whole-genome sequencing of 2636 Icelanders. We found two rare missense mutations, p.Gly496Ala and p.Gly703Ser, in the COL1A2 gene that associate with measures of osteoporosis in Icelanders. Mutations in COL1A2 are known to cause the autosomal dominant disorder osteogenesis imperfecta. Both variants associate with low BMD and with osteoporotic fractures. p.Gly496Ala (frequency of 0.105%) shows the strongest association with low BMD at the spine (p = 1.8 × 10(-7) , odds ratio [OR] = 4.61 [95% confidence interval (CI) 2.59, 8.18]), whereas p.Gly703Ser (frequency of 0.050%) is most strongly associated with low BMD at the hip (p = 1.9 × 10(-8) , OR = 9.34 [95% CI 4.28, 20.3]). Association with fractures was p = 2.2 × 10(-5) , OR = 3.75 (95% CI 2.03, 6.93) and p = 0.0023, OR = 4.32 (95% CI 1.69, 11.1), respectively. The carriers of these variants do not have signs of osteogenesis imperfecta other than low BMD, demonstrating that similar mutations in COL1A2 can affect skeletal phenotypes in more than one way.

  13. Distribution of A1A2BO and Rho (D) blood groups in tribal populations of Andhra Pradesh, South India.

    PubMed

    Goud, J D; Rao, P R

    1979-06-01

    The paper reports the distribution of A1A2BO and Rho (D) blood groups among five tribal populations, Koya Dora, Raj Gond, Naikpod, Pardhan and Lambadi from three districts of Andhra Pradesh, South India. Blood samples from a total of 1090 unrelated individuals were tested. Koya Doras were, however, sampled from five distant localities to find out intratribal variation, if any. In A1A2BO blood group system the combined frequencies of "P1" and "P2" among the five Koya Groups always exceeded the frequency of "q", a characteristic feature of many tribal populations of Andhra Pradesh. However, among Raj Gond, Naikpod, Pardhan and Lambadi tribes the frequency of "q" is higher than "p" with the maximum in Pardhans. The frequency of "r" is always higher than the combined frequencies of "p1" and "p2" except in Raj Gonds. The higher frequency of "q" over "p" among Naikpod, Pardhan and Lambadi tribes is indicative of a tendency towards the distribution pattern found in North India. A few Rh negative persons were detected only in Koya Dora, Raj Gond and Lambadis indicating that the allele r (cde) is present in these populations, although in a low frequency.

  14. Structural determinants of odorant recognition by the human olfactory receptors OR1A1 and OR1A2.

    PubMed

    Schmiedeberg, Kristin; Shirokova, Elena; Weber, Hans-Peter; Schilling, Boris; Meyerhof, Wolfgang; Krautwurst, Dietmar

    2007-09-01

    An interaction of odorants with olfactory receptors is thought to be the initial step in odorant detection. However, ligands have been reported for only 6 out of 380 human olfactory receptors, with their structural determinants of odorant recognition just beginning to emerge. Guided by the notion that amino acid positions that interact with specific odorants would be conserved in orthologs, but variable in paralogs, and based on the prediction of a set of 22 of such amino acid positions, we have combined site-directed mutagenesis, rhodopsin-based homology modelling, and functional expression in HeLa/Olf cells of receptors OR1A1 and OR1A2. We found that (i) their odorant profiles are centred around citronellic terpenoid structures, (ii) two evolutionary conserved amino acid residues in transmembrane domain 3 are necessary for the responsiveness of OR1A1 and the mouse ortholog Olfr43 to (S)-(-)-citronellol, (iii) changes at these two positions are sufficient to account for the differential (S)-(-)-citronellol responsiveness of the paralogs OR1A1 and OR1A2, and (iv) the interaction sites for (S)-(-)-citronellal and (S)-(-)-citronellol differ in both human receptors. Our results show that the orientation of odorants within a homology modelling-derived binding pocket of olfactory receptor orthologs is defined by evolutionary conserved amino acid positions.

  15. Inhibitory effect of Aristolochia fruit on Cytochrome P450 isozymes in vitro and in vivo.

    PubMed

    Zhu, Shuzhen; Gao, Jingwen; Shi, Zhan; QLi, George; Chen, Zuanguang; Yao, Meicun

    2015-05-01

    The mature fruits of Aristolochia debilis, known in China by the name, "Madouling" has been popularly prescribed in Asia, particularly in China, to treat a range of conditions including gynaecological problems, arthritis and wound healing. This study was aimed to evaluate the potential effect of Madouling on the cytochrome P450 (CYP) isozymes in vitro in microsomal fractions and in vivo in rats. The influence of Madouling on CYPs activity was first explored by an in vitro method of estimating levels of four respective metabolites in rat liver microsomes. The results were re-examined in vivo in rats by using a cocktail approach involving the probe drugs theophylline, tolbutamide, chlorzoxazone and dapsone. Pharmacokinetics of the four substrates was used to analyze the activities of the targeting isozymes. In vitro study revealed that Madouling decreased the activity of CYP1A2, 3A1 and 2E1. However, no significant influence on CYP2C6 was found. These results coincided with those of in vivo study to a great degree except that in vivo estimation the herb didn't inhibit CYP1A2 significantly. From the data obtained, Madouling is suggested as a candidate for clinically significant CYP interactions. Drug co-administrated with Madouling may need dose adjustment.

  16. In Vitro Evaluation of the Effects of Eurycoma longifolia Extract on CYP-Mediated Drug Metabolism

    PubMed Central

    Han, Young Min; Kim, In Sook; Rehman, Shaheed Ur; Choe, Kevin; Yoo, Hye Hyun

    2015-01-01

    Eurycoma longifolia (Simaroubaceae) is a popular folk medicine that has traditionally been used in Southeast Asia as an antimalarial, aphrodisiac, antidiabetic, and antimicrobial and in antipyretic remedies. This study evaluates the effects of Eurycoma longifolia extract on cytochrome P450 (CYP) enzyme-mediated drug metabolism to predict the potential for herb-drug interactions. Methanolic extract of E. longifolia root was tested at concentrations of 1, 3, 10, 30, 100, 300, and 1000 µg/mL in human liver microsomes or individual recombinant CYP isozymes. The CYP inhibitory activity was measured using the cocktail probe assay based on liquid chromatography-tandem mass spectrometry. E. longifolia showed weak, concentration-dependent inhibition of CYP1A2, CYP2A6, and CYP2C19. The inhibitory effects on these CYP isozymes were further tested using individual recombinant CYP isozymes, showing IC50 values of 324.9, 797.1, and 562.9 μg/mL, respectively. In conclusion, E. longifolia slightly inhibited the metabolic activities of CYP1A2, CYP2A6, and CYP2C19 but this issue requires careful attention in taking herbal medicines or dietary supplements containing E. longifolia extracts. PMID:26240600

  17. Polymorphisms in metabolic genes related to tobacco smoke and the risk of gastric cancer in the European prospective investigation into cancer and nutrition.

    PubMed

    Agudo, Antonio; Sala, Núria; Pera, Guillem; Capellá, Gabriel; Berenguer, Antonio; García, Nadia; Palli, Domenico; Boeing, Heiner; Del Giudice, Giuseppe; Saieva, Calogero; Carneiro, Fatima; Berrino, Franco; Sacerdote, Carlotta; Tumino, Rosario; Panico, Salvatore; Berglund, Göran; Simán, Henrik; Stenling, Roger; Hallmans, Göran; Martínez, Carmen; Bilbao, Roberto; Barricarte, Aurelio; Navarro, Carmen; Quirós, José R; Allen, Naomi; Key, Tim; Bingham, Sheila; Khaw, Kay-Tee; Linseisen, Jakob; Nagel, Gabriele; Overvad, Kim; Tjonneland, Anne; Olsen, Anja; Bueno-de-Mesquita, H Bas; Boshuizen, Hendriek C; Peeters, Petra H; Numans, Mattijs E; Clavel-Chapelon, Françoise; Boutron-Ruault, Marie-Christine; Trichopoulou, Antonia; Lund, Eiliv; Offerhaus, Johan; Jenab, Mazda; Ferrari, Pietro; Norat, Teresa; Riboli, Elio; González, Carlos A

    2006-12-01

    Metabolizing enzymes, which often display genetic polymorphisms, are involved in the activation of compounds present in tobacco smoke that may be relevant to gastric carcinogenesis. We report the results of a study looking at the association between risk of gastric adenocarcinoma and polymorphisms in genes CYP1A1, CYP1A2, EPHX1, and GSTT1. A nested case-control study was carried out within the European Prospective Investigation into Cancer and Nutrition, developed in 10 European countries. The study includes 243 newly diagnosed cases of histologically confirmed gastric adenocarcinoma and 946 controls matched by center, age, sex, and date of blood collection. Genotypes were determined in nuclear DNA from WBCs. We found an increased risk of gastric cancer for homozygotes for C (histidine) variant in Y113H of EPHX1 (odds ratio, 1.91; 95% confidence interval, 1.19-3.07) compared with subjects with TC/TT. There was also a significant increased risk for smokers carrying at least one variant allele A in Ex7+129C>A (m4) of CYP1A1 and never smokers with null GSTT1 and allele A in the locus -3859G>A of CYP1A2. Most of these genes are involved in the activation and detoxification of polycyclic aromatic hydrocarbons, suggesting a potential role of these compounds in gastric carcinogenesis. PMID:17164366

  18. Variable Bone Fragility Associated With an Amish COL1A2 Variant and a Knock-in Mouse Model

    PubMed Central

    Daley, Ethan; Streeten, Elizabeth A; Sorkin, John D; Kuznetsova, Natalia; Shapses, Sue A; Carleton, Stephanie M; Shuldiner, Alan R; Marini, Joan C; Phillips, Charlotte L; Goldstein, Steven A; Leikin, Sergey; McBride, Daniel J

    2010-01-01

    Osteogenesis imperfecta (OI) is a heritable form of bone fragility typically associated with a dominant COL1A1 or COL1A2 mutation. Variable phenotype for OI patients with identical collagen mutations is well established, but phenotype variability is described using the qualitative Sillence classification. Patterning a new OI mouse model on a specific collagen mutation therefore has been hindered by the absence of an appropriate kindred with extensive quantitative phenotype data. We benefited from the large sibships of the Old Order Amish (OOA) to define a wide range of OI phenotypes in 64 individuals with the identical COL1A2 mutation. Stratification of carrier spine (L1–4) areal bone mineral density (aBMD) Z-scores demonstrated that 73% had moderate to severe disease (less than −2), 23% had mild disease (−1 to −2), and 4% were in the unaffected range (greater than −1). A line of knock-in mice was patterned on the OOA mutation. Bone phenotype was evaluated in four F1 lines of knock-in mice that each shared approximately 50% of their genetic background. Consistent with the human pedigree, these mice had reduced body mass, aBMD, and bone strength. Whole-bone fracture susceptibility was influenced by individual genomic factors that were reflected in size, shape, and possibly bone metabolic regulation. The results indicate that the G610C OI (Amish) knock-in mouse is a novel translational model to identify modifying genes that influence phenotype and for testing potential therapies for OI. © 2010 American Society for Bone and Mineral Research PMID:19594296

  19. The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas

    SciTech Connect

    Sun, Yu; Wong, Nicholas; Guan, Yinghui; Salamanca, Clara M.; Cheng, Jung Chien; Lee, Jonathan M.; Gray, Joe W.; Auersperg, Nelly

    2008-04-25

    Ovarian epithelial carcinomas (OEC) frequently exhibit amplifications at the 20q13 locus which is the site of several oncogenes, including the eukaryotic elongation factor EEF1A2 and the transcription factor ZNF217. We reported previously that overexpressed ZNF217 induces neoplastic characteristics in precursor cells of OEC. Unexpectedly, ZNF217, which is a transcriptional repressor, enhanced expression of eEF1A2. In this study, array comparative genomic hybridization, single nucleotide polymorphism and Affymetrix analysis of ZNF217-overexpressing cell lines confirmed consistently increased expression of eEF1A2 but not of other oncogenes, and revealed early changes in EEF1A2 gene copy numbers and increased expression at crisis during immortalization. We defined the influence of eEF1A2 overexpression on immortalized ovarian surface epithelial cells, and investigated interrelationships between effects of ZNF217 and eEF1A2 on cellular phenotypes. Lentivirally induced eEF1A2 overexpression caused delayed crisis, apoptosis resistance and increases in serum-independence, saturation densities, and anchorage independence. siRNA to eEF1A2 reversed apoptosis resistance and reduced anchorage independence in eEF1A2-overexpressing lines. Remarkably, siRNA to eEF1A2 was equally efficient in inhibiting both anchorage independence and resistance to apoptosis conferred by ZNF217 overexpression. Our data define neoplastic properties that are caused by eEF1A2 in nontumorigenic ovarian cancer precursor cells, and suggest that eEF1A2 plays a role in mediating ZNF217-induced neoplastic progression.

  20. Membrane-Anchored Cytochrome P450 1A2-Cytochrome b5 Complex Features an X-Shaped Contact between Antiparallel Transmembrane Helices.

    PubMed

    Jeřábek, Petr; Florián, Jan; Martínek, Václav

    2016-04-18

    Eukaryotic cytochromes P450 (P450) are membrane-bound enzymes oxidizing a broad spectrum of hydrophobic substrates, including xenobiotics. Protein-protein interactions play a critical role in this process. In particular, the formation of transient complexes of P450 with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates catalytic activities of several P450s. To lay a structural foundation for the investigation of these effects, we constructed a model of the membrane-bound full-length human P450 1A2-cyt b5 complex. The model was assembled from several parts using a multiscale modeling approach covering all-atom and coarse-grained molecular dynamics (MD). For soluble P450 1A2-cyt b5 complexes, these simulations yielded three stable binding modes (sAI, sAII, and sB). The membrane-spanning transmembrane domains were reconstituted with the phospholipid bilayer using self-assembly MD. The predicted full-length membrane-bound complexes (mAI and mB) featured a spontaneously formed X-shaped contact between antiparallel transmembrane domains, whereas the mAII mode was found to be unstable in the membrane environment. The mutual position of soluble domains in binding mode mAI was analogous to the sAI complex. Featuring the largest contact area, the least structural flexibility, the shortest electron transfer distance, and the highest number of interprotein salt bridges, mode mAI is the best candidate for the catalytically relevant full-length complex. PMID:26918755

  1. Fennel and raspberry leaf as possible inhibitors of acetaminophen oxidation.

    PubMed

    Langhammer, Astrid Jordet; Nilsen, Odd Georg

    2014-10-01

    In addition to CYP2E1, several CYP isoenzymes, notably CYP1A2, 2D6, and 3A4, are suggested to contribute in acetaminophen oxidation and formation of the hepatotoxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). The in vitro CYP2E1 inhibitory potentials of fennel and raspberry leaf, herbs previously found to inhibit CYP1A2, 2D6, and 3A4 activities in vitro, were investigated. Extracts from commercially available herbal products were incubated with recombinant cDNA-expressed human CYP2E1. A validated LC/MS/MS methodology was applied for determination of 6-hydroxychlorzoxazone formation with disulfiram used as a positive inhibitory control. CYP2E1 IC50 inhibition constants were found to be 23 ± 4 and 27 ± 5 µg/ml for fennel and raspberry leaf, respectively, constants significantly lower than those presented in the literature for other herbal extracts. Together with previous findings, the presented in vitro data for CYP2E1 inhibition suggest that fennel and raspberry leaf have a significant potential of inhibiting all the major metabolic pathways for acetaminophen oxidation and NAPQI formation. Both herbs should be further investigated for their in vivo ability of inhibiting acetaminophen oxidation and NAPQI formation.

  2. Stereoselective metabolism of tetrahydropalmatine enantiomers in rat liver microsomes.

    PubMed

    Zhao, Ming; Li, Li-Ping; Sun, Dong-Li; Sun, Si-Yuan; Huang, Shan-Ding; Zeng, Su; Jiang, Hui-Di

    2012-05-01

    Tetrahydropalmatine (THP), with one chiral center, is an active alkaloid ingredient in Rhizoma Corydalis. The aim of the present paper is to study whether THP enantiomers are metabolized stereoselectively in rat, mouse, dog, and monkey liver microsomes, and then, to elucidate which Cytochrome P450 (CYP) isoforms are predominately responsible for the stereoselective metabolism of THP enantiomers in rat liver microsomes (RLM). The results demonstrated that (+)-THP was preferentially metabolized by liver microsomes from rats, mice, dogs, and monkeys, and the intrinsic clearance (Cl(int)) ratios of (+)-THP to (-)-THP were 2.66, 2.85, 4.24, and 1.67, respectively. Compared with the metabolism in untreated RLM, the metabolism of (-)-THP and (+)-THP was significantly increased in dexamethasone (Dex)-induced and β-naphthoflavone (β-NF)-induced RLM; meanwhile, the Cl(int) ratios of (+)-THP to (-)-THP in Dex-induced and β-NF-induced RLM were 5.74 and 0.81, respectively. Ketoconazole had stronger inhibitory effect on (+)-THP than (-)-THP, whereas fluvoxamine had stronger effect on (-)-THP in untreated and Dex-induced or β-NF-induced RLM. The results suggested that THP enantiomers were predominately metabolized by CYP3A1/2 and CYP1A2 in RLM, and CYP3A1/2 preferred to metabolize (+)-THP, whereas CYP1A2 preferred (-)-THP.

  3. Effect of steady-state enoxacin on single-dose pharmacokinetics of roflumilast and roflumilast N-oxide.

    PubMed

    Lahu, Gezim; Nassr, Nassr; Herzog, Rolf; Elmlinger, Martin; Ruth, Peter; Hinder, Markus; Huennemeyer, Andreas

    2011-04-01

    Roflumilast is an oral phosphodiesterase 4 (PDE4) inhibitor for the treatment of chronic obstructive pulmonary disease (COPD). It is metabolized by CYP1A2 and CYP3A4 to its primary metabolite, roflumilast N-oxide, through which >90% total PDE4 inhibitory activity (tPDE4i) is mediated. Fluoroquinolones, of which enoxacin is the most potent CYP1A2 inhibitor, are used to treat COPD exacerbations. This phase I, open, nonrandomized, fixed-sequence, 2-period study evaluated the effects of steady-state enoxacin on the single-dose pharmacokinetics of roflumilast and roflumilast N-oxide. Twenty healthy participants received roflumilast, 500 µg once daily, on days 1 and 12, and enoxacin, 400 mg twice daily, on days 7 to 18. Pharmacokinetic profiles were obtained for days 1 to 6 and 12 to 19. The safety and tolerability of all treatments were also assessed. In 19 evaluable participants, coadministration led to 56% higher mean systemic exposure, 20% higher mean peak concentrations, and 36% lower mean apparent oral clearance compared with roflumilast alone. For roflumilast N-oxide, 23% higher mean systemic exposure and 14% lower mean peak concentrations were seen after coadministration. Roflumilast was well tolerated both alone and in combination with enoxacin. A weak interaction was shown between roflumilast and enoxacin, as mean tPDE4i increased by 25%, but is unlikely to have clinical relevance.

  4. Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

    SciTech Connect

    Jaruchotikamol, Atika; Jarukamjorn, Kanokwan Sirisangtrakul, Wanna; Sakuma, Tsutomu; Kawasaki, Yuki; Nemoto, Nobuo

    2007-10-15

    The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[a]anthracene, {beta}-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression.

  5. Baicalin Protects Mice from Aristolochic Acid I-Induced Kidney Injury by Induction of CYP1A through the Aromatic Hydrocarbon Receptor

    PubMed Central

    Wang, Ke; Feng, Chenchen; Li, Chenggang; Yao, Jun; Xie, Xiaofeng; Gong, Likun; Luan, Yang; Xing, Guozhen; Zhu, Xue; Qi, Xinming; Ren, Jin

    2015-01-01

    Exposure to aristolochic acid I (AAI) can lead to aristolochic acid nephropathy (AAN), Balkan endemic nephropathy (BEN) and urothelial cancer. The induction of hepatic CYP1A, especially CYP1A2, was considered to detoxify AAI so as to reduce its nephrotoxicity. We previously found that baicalin had the strong ability to induce CYP1A2 expression; therefore in this study, we examined the effects of baicalin on AAI toxicity, metabolism and disposition, as well as investigated the underlying mechanisms. Our toxicological studies showed that baicalin reduced the levels of blood urea nitrogen (BUN) and creatinine (CRE) in AAI-treated mice and attenuated renal injury induced by AAI. Pharmacokinetic analysis demonstrated that baicalin markedly decreased AUC of AAI in plasma and the content of AAI in liver and kidney. CYP1A induction assays showed that baicalin exposure significantly increased the hepatic expression of CYP1A1/2, which was completely abolished by inhibitors of the Aromatic hydrocarbon receptor (AhR), 3ʹ,4ʹ-dimethoxyflavone and resveratrol, in vitro and in vivo, respectively. Moreover, the luciferase assays revealed that baicalin significantly increased the luciferase activity of the reporter gene incorporated with the Xenobiotic response elements recognized by AhR. In summary, baicalin significantly reduced the disposition of AAI and ameliorated AAI-induced kidney toxicity through AhR-dependent CYP1A1/2 induction in the liver. PMID:26204831

  6. CYP450 Enzyme-Mediated Metabolism of TCAS and Its Inhibitory and Induced Effects on Metabolized Enzymes in Vitro.

    PubMed

    Shen, Guolin; Wang, Cheng; Zhou, Lili; Li, Lei; Chen, Huiming; Yu, Wenlian; Li, Haishan

    2015-09-02

    In this study, we investigated the enzymes catalyzing the phase I metabolism of thiacalixarene (TCAS) based on in vitro system including cDNA-expressed P450 enzymes, human liver microsomes plus inhibitors and monoclonal antibodies. In addition, the inhibitory potential of TCAS on major CYP450 drug metabolizing enzymes (CYP1A2, CYP2C9, CYP2B6, CYP2D6 and CYP3A4) was assessed. The results showed that CYP1A2 and CYP2C9 mediated TCAS hydroxylation. IC50 values for TCAS in rat and human liver microsomes were greater than 50 µM, and it demonstrated a weak inhibition of rat and human CYP450 enzymes. Finally, sandwiched hepatocytes were used to evaluate the induction of CYP1A and CYP3A to define the function of TCAS in vivo. The results showed that incubation of TCAS at different concentrations for 72 h failed to induce CYP1A and CYP3A. However, incubation of the cells with 50 and 100 µM TCAS caused a profound decrease in the activities of CYP1A and CYP3A, which was probably due to cytotoxic effects, suggesting that exposure to TCAS might be a health concern.

  7. CYP450 Enzyme-Mediated Metabolism of TCAS and Its Inhibitory and Induced Effects on Metabolized Enzymes in Vitro

    PubMed Central

    Shen, Guolin; Wang, Cheng; Zhou, Lili; Li, Lei; Chen, Huiming; Yu, Wenlian; Li, Haishan

    2015-01-01

    In this study, we investigated the enzymes catalyzing the phaseⅠmetabolism of thiacalixarene (TCAS) based on in vitro system including cDNA-expressed P450 enzymes, human liver microsomes plus inhibitors and monoclonal antibodies. In addition, the inhibitory potential of TCAS on major CYP450 drug metabolizing enzymes (CYP1A2, CYP2C9, CYP2B6, CYP2D6 and CYP3A4) was assessed. The results showed that CYP1A2 and CYP2C9 mediated TCAS hydroxylation. IC50 values for TCAS in rat and human liver microsomes were greater than 50 µM, and it demonstrated a weak inhibition of rat and human CYP450 enzymes. Finally, sandwiched hepatocytes were used to evaluate the induction of CYP1A and CYP3A to define the function of TCAS in vivo. The results showed that incubation of TCAS at different concentrations for 72 h failed to induce CYP1A and CYP3A. However, incubation of the cells with 50 and 100 µM TCAS caused a profound decrease in the activities of CYP1A and CYP3A, which was probably due to cytotoxic effects, suggesting that exposure to TCAS might be a health concern. PMID:26404338

  8. Induction of cytochromes P450 1A1 and 1A2 suppresses formation of DNA adducts by carcinogenic aristolochic acid I in rats in vivo

    PubMed Central

    Dračínská, Helena; Bárta, František; Levová, Kateřina; Hudecová, Alena; Moserová, Michaela; Schmeiser, Heinz H.; Kopka, Klaus; Frei, Eva; Arlt, Volker M.; Stiborová, Marie

    2016-01-01

    Aristolochic acid I (AAI) is a natural plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. One of the most efficient enzymes reductively activating AAI to species forming AAI-DNA adducts is cytosolic NAD(P)H:quinone oxidoreductase 1. AAI is also either reductively activated or oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by microsomal cytochrome P450 (CYP) 1A1 and 1A2. Here, we investigated which of these two opposing CYP1A1/2-catalyzed reactions prevails in AAI metabolism in vivo. The formation of AAI-DNA adducts was analyzed in liver, kidney and lung of rats treated with AAI, Sudan I, a potent inducer of CYP1A1/2, or AAI after pretreatment with Sudan I. Compared to rats treated with AAI alone, levels of AAI-DNA adducts determined by the 32P-postlabeling method were lower in liver, kidney and lung of rats treated with AAI after Sudan I. The induction of CYP1A1/2 by Sudan I increased AAI detoxification to its O-demethylated metabolite AAIa, thereby reducing the actual amount of AAI available for reductive activation. This subsequently resulted in lower AAI-DNA adduct levels in the rat in vivo. Our results demonstrate that CYP1A1/2-mediated oxidative detoxification of AAI is the predominant role of these enzymes in rats in vivo, thereby suppressing levels of AAI-DNA adducts. PMID:26845733

  9. The first Japanese case of the arthrochalasia type of Ehlers-Danlos syndrome with COL1A2 gene mutation.

    PubMed

    Hatamochi, Atsushi; Hamada, Takahiro; Yoshino, Makoto; Hashimoto, Takashi

    2014-03-15

    This is the first report for a Japanese case of arthrochalasia type of Ehlers-Danlos syndrome (EDS). A 46-year-old woman consulted us for joint hypermobility and skin hyperextensibility that had been present soon after birth. There was no family history of a similar disease. She was diagnosed as having bilateral congenital hip dislocation and bilateral habitual shoulder dislocation at her childhood. Her skin was velvety, doughy and hyperextensible. She showed hypermobility of the joints of the hands and feet and generalized joint laxity, with no evidence of scoliosis. Electrophoretic analysis of collagenous proteins revealed the presence of an additional band in the position of pNα2(I) in the sample from culture medium of the patient fibroblasts. Analysis of the α2 chains of type I collagen gene, COL1A2, showed a heterozygous G to T transition at the +1 position of the exon 6 donor splice site (c.279+1G>T). This mutation resulted in skipping of exon 6, which leads to deficient processing of the amino-terminal end of proα2(I) chains of type I collagen. Based on these findings, we made a diagnosis of the arthrochalasia type of EDS, which corresponds to EDS type VIIB in the former classification.

  10. The effect of enzyme inhibition on the metabolism and activation of tacrine by human liver microsomes.

    PubMed Central

    Spaldin, V; Madden, S; Pool, W F; Woolf, T F; Park, B K

    1994-01-01

    1. Tacrine (1,2,3,4-tetrahydro-9-aminoacridine-hydrochloride: THA) underwent metabolism in vitro by a panel (n = 12) of human liver microsomes genotyped for CYP2D6, in the presence of NADPH, to both protein-reactive and stable metabolites. 2. There was considerable variation in the extent of THA metabolism amongst human livers. Protein-reactive metabolite formation showed a 10-fold variation (0.6 +/- 0.1%-5.2 +/- 0.8% of incubated radioactivity mg-1 protein) whilst stable metabolites showed a 3-fold variation (24.3 +/- 1.7%-78.6 +/- 2.6% of incubated radioactivity). 3. Using cytochrome P450 isoform specific inhibitors CYP1A2 was identified as the major enzyme involved in all routes of THA metabolism. 4. There was a high correlation between aromatic and alicyclic hydroxylation (r = 0.92, P < 0.0001) consistent with these biotransformations being catalysed by the same enzymes. 5. Enoxacin (ENOX), cimetidine (CIM) and chloroquine (CQ) inhibited THA metabolism by a preferential decrease in the bioactivation to protein-reactive, and hence potentially toxic, species. The inhibitory potency of ENOX and CIM was increased significantly upon pre-incubation with microsomes and NADPH. 6. Covalent binding correlated with 7-OH-THA formation before (r = 0.792, P < 0.0001) and after (r = 0.73, P < 0.0001) inhibition by CIM, consistent with a two-step mechanism in the formation of protein-reactive metabolite(s) via a 7-OH intermediate. 7. The use of enzyme inhibitors may provide a useful tool for examining the relationship between the metabolism and toxicity of THA in vivo. PMID:7946932

  11. Evaluation of inhibitory effects of caffeic acid and quercetin on human liver cytochrome p450 activities.

    PubMed

    Rastogi, Himanshu; Jana, Snehasis

    2014-12-01

    When herbal drugs and conventional allopathic drugs are used together, they can interact in our body which can lead to the potential for herb-drug interactions. This work was conducted to evaluate the herb-drug interaction potential of caffeic acid and quercetin mediated by cytochrome P450 (CYP) inhibition. Human liver microsomes (HLMs) were added to each selective probe substrates of cytochrome P450 enzymes with or without of caffeic acid and quercetin. IC50 , Ki values, and the types of inhibition were determined. Both caffeic acid and quercetin were potent competitive inhibitors of CYP1A2 (Ki = 1.16 and 0.93 μM, respectively) and CYP2C9 (Ki = 0.95 and 1.67 μM, respectively). Caffeic acid was a potent competitive inhibitor of CYP2D6 (Ki = 1.10 μM) and a weak inhibitor of CYP2C19 and CYP3A4 (IC50  > 100 μM). Quercetin was a potent competitive inhibitor of CYP 2C19 and CYP3A4 (Ki = 1.74 and 4.12 μM, respectively) and a moderate competitive inhibitor of CYP2D6 (Ki = 18.72 μM). These findings might be helpful for safe and effective use of polyphenols in clinical practice. Our data indicated that it is necessary to study the in vivo interactions between drugs and pharmaceuticals with dietary polyphenols. PMID:25196644

  12. De Novo Mutations in SLC1A2 and CACNA1A Are Important Causes of Epileptic Encephalopathies.

    PubMed

    2016-08-01

    Epileptic encephalopathies (EEs) are the most clinically important group of severe early-onset epilepsies. Next-generation sequencing has highlighted the crucial contribution of de novo mutations to the genetic architecture of EEs as well as to their underlying genetic heterogeneity. Our previous whole-exome sequencing study of 264 parent-child trios revealed more than 290 candidate genes in which only a single individual had a de novo variant. We sought to identify additional pathogenic variants in a subset (n = 27) of these genes via targeted sequencing in an unsolved cohort of 531 individuals with a diverse range of EEs. We report 17 individuals with pathogenic variants in seven of the 27 genes, defining a genetic etiology in 3.2% of this unsolved cohort. Our results provide definitive evidence that de novo mutations in SLC1A2 and CACNA1A cause specific EEs and expand the compendium of clinically relevant genotypes for GABRB3. We also identified EEs caused by genetic variants in ALG13, DNM1, and GNAO1 and report a mutation in IQSEC2. Notably, recurrent mutations accounted for 7/17 of the pathogenic variants identified. As a result of high-depth coverage, parental mosaicism was identified in two out of 14 cases tested with mutant allelic fractions of 5%-6% in the unaffected parents, carrying significant reproductive counseling implications. These results confirm that dysregulation in diverse cellular neuronal pathways causes EEs, and they will inform the diagnosis and management of individuals with these devastating disorders. PMID:27476654

  13. Severe osteogenesis imperfecta caused by double glycine substitutions near the amino-terminal triple helical region in COL1A2.

    PubMed

    Takagi, Masaki; Shinohara, Hiroyuki; Narumi, Satoshi; Nishimura, Gen; Hasegawa, Yukihiro; Hasegawa, Tomonobu

    2015-07-01

    Most cases of osteogenesis imperfecta (OI) are caused by heterozygous mutations in COL1A1 or COL1A2, the genes encoding the two type I procollagen alpha chains, proα1 (I) and proα2 (I). We report on a unique case of severe OI, a long term survivor of lethal type II OI, rather than progressively deforming type III, due to double substitutions of glycine residues in COL1A2 (p.Gly208Glu and p.Gly235Asp), located on the same allele. To the best of our knowledge, this is the first example of a patient with double COL1A2 glycine substitution mutations on the same allele. We show for the first time that double COL1A2 glycine substitution mutations located near the amino-terminal triple helical region, which individually are likely to result in mild OI, cause severe OI in combination. PMID:25858481

  14. NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I

    SciTech Connect

    Levova, Katerina; Moserova, Michaela; Nebert, Daniel W.; Phillips, David H.; Frei, Eva; Schmeiser, Heinz H.; Arlt, Volker M.; Stiborova, Marie

    2012-12-15

    Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)—the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1(−/−), Cyp1a2(−/−) and Cyp1a1/1a2(−/−) knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential. Highlights: ► NAD(P)H:quinone oxidoreductase expression in Cyp1a knockout and humanized CYP1A mice ► Reductive activation of the nephrotoxic and carcinogenic aristolochic acid I (AAI) ► NAD(P)H:quinone oxidoreductase is induced in mice treated with AAI. ► Induced hepatic enzyme activity resulted in elevated AAI-DNA adduct levels.

  15. Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus).

    PubMed

    Feng, Ruijuan; Fang, Lingling; Cheng, Yunying; He, Xue; Jiang, Wentao; Dong, Ranran; Shi, Hongjuan; Jiang, Dongneng; Sun, Lina; Wang, Deshou

    2015-01-01

    Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts. PMID:25976364

  16. Retinoic acid homeostasis through aldh1a2 and cyp26a1 mediates meiotic entry in Nile tilapia (Oreochromis niloticus)

    PubMed Central

    Feng, Ruijuan; Fang, Lingling; Cheng, Yunying; He, Xue; Jiang, Wentao; Dong, Ranran; Shi, Hongjuan; Jiang, Dongneng; Sun, Lina; Wang, Deshou

    2015-01-01

    Meiosis is a process unique to the differentiation of germ cells. Retinoic acid (RA) is the key factor controlling the sex-specific timing of meiotic initiation in tetrapods; however, the role of RA in meiotic initiation in teleosts has remained unclear. In this study, the genes encoding RA synthase aldh1a2, and catabolic enzyme cyp26a1 were isolated from Nile tilapia (Oreochromis niloticus), a species without stra8. The expression of aldh1a2 was up-regulated and expression of cyp26a1 was down-regulated before the meiotic initiation in ovaries and in testes. Treatment with RA synthase inhibitor or disruption of Aldh1a2 by CRISPR/Cas9 resulted in delayed meiotic initiation, with simultaneous down-regulation of cyp26a1 and up-regulation of sycp3. By contrast, treatment with an inhibitor of RA catabolic enzyme and disruption of cyp26a1 resulted in earlier meiotic initiation, with increased expression of aldh1a2 and sycp3. Additionally, treatment of XY fish with estrogen (E2) and XX fish with fadrozole led to sex reversal and reversion of meiotic initiation. These results indicate that RA is indispensable for meiotic initiation in teleosts via a stra8 independent signaling pathway where both aldh1a2 and cyp26a1 are critical. In contrast to mammals, E2 is a major regulator of sex determination and meiotic initiation in teleosts. PMID:25976364

  17. Inhibition of CYP1 by berberine, palmatine, and jatrorrhizine: Selectivity, kinetic characterization, and molecular modeling

    SciTech Connect

    Lo, Sheng-Nan; Chang, Yu-Ping; Tsai, Keng-Chang; Chang, Chia-Yu; Wu, Tian-Shung; Ueng, Yune-Fang

    2013-11-01

    Cytochrome P450 (P450, CYP) 1 family plays a primary role in the detoxification and bioactivation of polycyclic aromatic hydrocarbons. Human CYP1A1, CYP1A2, and CYP1B1 exhibit differential substrate specificity and tissue distribution. Berberine, palmatine, and jatrorrhizine are protoberberine alkaloids present in several medicinal herbs, such as Coptis chinensis (Huang-Lian) and goldenseal. These protoberberines inhibited CYP1A1.1- and CYP1B1.1-catalyzed 7-ethoxyresorufin O-deethylation (EROD) activities, whereas CYP1A2.1 activity was barely affected. Kinetic analysis revealed that berberine noncompetitively inhibited EROD activities of CYP1A1.1 and CYP1B1.1, whereas palmatine and jatrorrhizine caused either competitive or mixed type of inhibition. Among protoberberines, berberine caused the most potent and selective inhibitory effect on CYP1B1.1 with the least K{sub i} value of 44 ± 16 nM. Berberine also potently inhibited CYP1B1.1 activities toward 7-ethoxycoumarin and 7-methoxyresorufin, whereas the inhibition of benzo(a)pyrene hydroxylation activity was less pronounced. Berberine inhibited the polymorphic variants, CYP1B1.3 (V432L) and CYP1B1.4 (N453S), with IC{sub 50} values comparable to that for CYP1B1.1 inhibition. Berberine-mediated inhibition was abolished by a mutation of Asn228 to Thr in CYP1B1.1, whereas the inhibition was enhanced by a reversal mutation of Thr223 to Asn in CYP1A2.1. This result in conjugation with the molecular modeling revealed the crucial role of hydrogen-bonding interaction of Asn228 on CYP1B1.1 with the methoxy moiety of berberine. These findings demonstrate that berberine causes a selective CYP1B1-inhibition, in which Asn228 appears to be crucial. The inhibitory effects of berberine on CYP1B1 activities toward structurally diverse substrates can be different. - Highlights: • Berberine preferentially inhibited CYP1B1 activity. • Berberine caused similar inhibitory effects on CYP1B1.1, CYP1B1.3 and CYP1B1.4. • Asn228 in CYP

  18. Spatiotemporal brain dynamics of emotional face processing modulations induced by the serotonin 1A/2A receptor agonist psilocybin.

    PubMed

    Bernasconi, Fosco; Schmidt, André; Pokorny, Thomas; Kometer, Michael; Seifritz, Erich; Vollenweider, Franz X

    2014-12-01

    Emotional face processing is critically modulated by the serotonergic system. For instance, emotional face processing is impaired by acute psilocybin administration, a serotonin (5-HT) 1A and 2A receptor agonist. However, the spatiotemporal brain mechanisms underlying these modulations are poorly understood. Here, we investigated the spatiotemporal brain dynamics underlying psilocybin-induced modulations during emotional face processing. Electrical neuroimaging analyses were applied to visual evoked potentials in response to emotional faces, following psilocybin and placebo administration. Our results indicate a first time period of strength (i.e., Global Field Power) modulation over the 168-189 ms poststimulus interval, induced by psilocybin. A second time period of strength modulation was identified over the 211-242 ms poststimulus interval. Source estimations over these 2 time periods further revealed decreased activity in response to both neutral and fearful faces within limbic areas, including amygdala and parahippocampal gyrus, and the right temporal cortex over the 168-189 ms interval, and reduced activity in response to happy faces within limbic and right temporo-occipital brain areas over the 211-242 ms interval. Our results indicate a selective and temporally dissociable effect of psilocybin on the neuronal correlates of emotional face processing, consistent with a modulation of the top-down control. PMID:23861318

  19. Spatiotemporal brain dynamics of emotional face processing modulations induced by the serotonin 1A/2A receptor agonist psilocybin.

    PubMed

    Bernasconi, Fosco; Schmidt, André; Pokorny, Thomas; Kometer, Michael; Seifritz, Erich; Vollenweider, Franz X

    2014-12-01

    Emotional face processing is critically modulated by the serotonergic system. For instance, emotional face processing is impaired by acute psilocybin administration, a serotonin (5-HT) 1A and 2A receptor agonist. However, the spatiotemporal brain mechanisms underlying these modulations are poorly understood. Here, we investigated the spatiotemporal brain dynamics underlying psilocybin-induced modulations during emotional face processing. Electrical neuroimaging analyses were applied to visual evoked potentials in response to emotional faces, following psilocybin and placebo administration. Our results indicate a first time period of strength (i.e., Global Field Power) modulation over the 168-189 ms poststimulus interval, induced by psilocybin. A second time period of strength modulation was identified over the 211-242 ms poststimulus interval. Source estimations over these 2 time periods further revealed decreased activity in response to both neutral and fearful faces within limbic areas, including amygdala and parahippocampal gyrus, and the right temporal cortex over the 168-189 ms interval, and reduced activity in response to happy faces within limbic and right temporo-occipital brain areas over the 211-242 ms interval. Our results indicate a selective and temporally dissociable effect of psilocybin on the neuronal correlates of emotional face processing, consistent with a modulation of the top-down control.

  20. Modulation of aryl hydrocarbon receptor-regulated enzymes by trimethylarsine oxide in C57BL/6 mice: In vivo and in vitro studies.

    PubMed

    Elshenawy, Osama H; El-Kadi, Ayman O S

    2015-10-01

    Arsenic is a worldwide environmental pollutant that is associated with skin and several types of internal cancers. Recent reports revealed that arsenic biomethylation could activate the toxic and carcinogenic potential of arsenic. Therefore, we investigated the effect of trimethylarsine oxide (TMAO) on the activation of AhR-regulated genes in vivo and in vitro. In vivo, C57BL/6 mice received TMAO (13mg/kg i.p.) with or without the prototypical AhR ligand, TCDD (15μg/kg), then the livers were harvested at 6 and 24h post-treatment. In vitro, isolated hepatocytes from C57BL/6 mice were treated with TMAO (5μM) in the absence and presence of TCDD (1nM) for 6 and 24h. Our in vivo results demonstrated that, TMAO alone increased Cyp1a1, Cyp1a2, Cyp1b1, Nqo1, Gsta1, and Ho-1 at mRNA level. Upon co-exposure to TMAO and TCDD, TMAO potentiated the TCDD-mediated induction of Cyp1a1, Cyp1b1, and Nqo1 mRNA levels. Western blotting revealed that, TMAO alone increased Cyp1a1, Cyp1a2, Nqo1, Gsta1/2, and Ho-1 protein levels, and potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 protein level. In addition, TMAO alone significantly increased Cyp1a1, Cyp1a2, Nqo1, Gst, and Ho-1 activities and significantly potentiated the TCDD-mediated induction of Cyp1a1 activity. At the in vitro level, TMAO induced Cyp1a1 and potentiated the TCDD-mediated induction of Cyp1a1 at mRNA, protein and activity levels. In addition, TMAO increased the nuclear localization of AhR and AhR-dependent XRE-driven luciferase activity. Our results demonstrate that the TMAO, modulates AhR-regulated genes which could potentially participate, at least in part, in arsenic induced toxicity and carcinogenicity.

  1. Modulation of aryl hydrocarbon receptor-regulated enzymes by trimethylarsine oxide in C57BL/6 mice: In vivo and in vitro studies.

    PubMed

    Elshenawy, Osama H; El-Kadi, Ayman O S

    2015-10-01

    Arsenic is a worldwide environmental pollutant that is associated with skin and several types of internal cancers. Recent reports revealed that arsenic biomethylation could activate the toxic and carcinogenic potential of arsenic. Therefore, we investigated the effect of trimethylarsine oxide (TMAO) on the activation of AhR-regulated genes in vivo and in vitro. In vivo, C57BL/6 mice received TMAO (13mg/kg i.p.) with or without the prototypical AhR ligand, TCDD (15μg/kg), then the livers were harvested at 6 and 24h post-treatment. In vitro, isolated hepatocytes from C57BL/6 mice were treated with TMAO (5μM) in the absence and presence of TCDD (1nM) for 6 and 24h. Our in vivo results demonstrated that, TMAO alone increased Cyp1a1, Cyp1a2, Cyp1b1, Nqo1, Gsta1, and Ho-1 at mRNA level. Upon co-exposure to TMAO and TCDD, TMAO potentiated the TCDD-mediated induction of Cyp1a1, Cyp1b1, and Nqo1 mRNA levels. Western blotting revealed that, TMAO alone increased Cyp1a1, Cyp1a2, Nqo1, Gsta1/2, and Ho-1 protein levels, and potentiated the TCDD-mediated induction of Cyp1a1 and Cyp1b1 protein level. In addition, TMAO alone significantly increased Cyp1a1, Cyp1a2, Nqo1, Gst, and Ho-1 activities and significantly potentiated the TCDD-mediated induction of Cyp1a1 activity. At the in vitro level, TMAO induced Cyp1a1 and potentiated the TCDD-mediated induction of Cyp1a1 at mRNA, protein and activity levels. In addition, TMAO increased the nuclear localization of AhR and AhR-dependent XRE-driven luciferase activity. Our results demonstrate that the TMAO, modulates AhR-regulated genes which could potentially participate, at least in part, in arsenic induced toxicity and carcinogenicity. PMID:26144063

  2. Effects of vitexin on the pharmacokinetics and mRNA expression of CYP isozymes in rats.

    PubMed

    Wang, Xin-shuai; Hu, Xiao-chen; Chen, Gui-ling; Yuan, Xiang; Yang, Rui-na; Liang, Shuo; Ren, Jing; Sun, Jia-chun; Kong, Guo-qiang; Gao, She-gan; Feng, Xiao-shan

    2015-03-01

    In traditional therapy with Chinese medicine, vitexin has several pharmacological properties, including antinociceptive, antispasmodic, antioxidant, antimyeloperoxidase, and α-glucosidase inhibitory activities. Recently, vitexin was shown to protect the heart against ischemia/reperfusion injury in an in vitro model by inhibiting apoptosis. The purpose of this study was to find out whether vitexin influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C11, and CYP3A1) by using cocktail probe drugs in vivo; the influence on the levels of CYP mRNA was also studied. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (10 mg/kg), tolbutamide (1 mg/kg), and midazolam (5 mg/kg), was given as oral administration to rats treated with short or long period of intravenous vitexin via the caudal vein. Blood samples were collected at a series of time points, and the concentrations of probe drugs in plasma were determined by HPLC-mass spectrometry (MS)/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0. In addition, real-time reverse transcription-polymerase chain reaction was performed to determine the effects of vitexin on the mRNA expression of CYP1A2, CYP2C11, and CYP3A1 in rat liver. Treatment with short or long period of vitexin had no effects on rat CYP1A2. However, CYP3A1 enzyme activity was inhibited by vitexin in a concentration-dependent and time-dependent manner. Furthermore, CYP2C11 enzyme activity was induced after short period treatment but inhibited after long period of vitexin treatment. The mRNA expression results were in accordance with the pharmacokinetic results. In conclusion, vitexin can either inhibit or induce activities of CYP2C11 and CYP3A1. Therefore, caution is needed when vitexin is co-administered with some CYP2C11 or CYP3A1 substrates in clinic, which may result in treatment failure and herb-drug interactions.

  3. 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

  4. A missense variant of the ATP1A2 gene is associated with a novel phenotype of progressive sensorineural hearing loss associated with migraine.

    PubMed

    Oh, Se-Kyung; Baek, Jeong-In; Weigand, Karl M; Venselaar, Hanka; Swarts, Herman G P; Park, Seong-Hyun; Hashim Raza, Muhammad; Jung, Da Jung; Choi, Soo-Young; Lee, Sang-Heun; Friedrich, Thomas; Vriend, Gert; Koenderink, Jan B; Kim, Un-Kyung; Lee, Kyu-Yup

    2015-05-01

    Hereditary sensorineural hearing loss is an extremely clinical and genetic heterogeneous disorder in humans. Especially, syndromic hearing loss is subdivided by combinations of various phenotypes, and each subtype is related to different genes. We present a new form of progressive hearing loss with migraine found to be associated with a variant in the ATP1A2 gene. The ATP1A2 gene has been reported as the major genetic cause of familial migraine by several previous studies. A Korean family presenting progressive hearing loss with migraine was ascertained. The affected members did not show any aura or other neurologic symptoms during migraine attacks, indicating on a novel phenotype of syndromic hearing loss. To identify the causative gene, linkage analysis and whole-exome sequencing were performed. A novel missense variant, c.571G>A (p.(Val191Met)), was identified in the ATP1A2 gene that showed co-segregation with the phenotype in the family. In silico studies suggest that this variant causes a change in hydrophobic interactions and thereby slightly destabilize the A-domain of Na(+)/K(+)-ATPase. However, functional studies failed to show any effect of the p.(Val191Met) substitution on the catalytic rate of this enzyme. We describe a new phenotype of progressive hearing loss with migraine associated with a variant in the ATP1A2 gene. This study suggests that a variant in Na(+)/K(+)-ATPase can be involved in both migraine and hearing loss.

  5. Inhibition selectivity of grapefruit juice components on human cytochromes P450.

    PubMed

    Tassaneeyakul, W; Guo, L Q; Fukuda, K; Ohta, T; Yamazoe, Y

    2000-06-15

    Five compounds including furanocoumarin monomers (bergamottin, 6', 7'-dihydroxybergamottin (DHB)), furanocoumarin dimers (4-¿¿6-hydroxy-71-¿(1-hydroxy-1-methyl)ethyl-4-methyl-6-(7-oxo-7H- furo¿3,2-g1benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl- 2-octenyl]oxy]-7H-furo[3,2-g]¿1benzopyran-7-one (GF-I-1) and 4-¿¿6-hydroxy-7¿¿4-methyl-1-(1-methylethenyl)-6-(7-oxo-7H-furo¿3, 2-g1benzopyran-4-yl)-4-hexenylŏxy-3, 7-dimethyl-2-octenylŏxy-7H-furo¿3,2-g1benzopyran-7-one (GF-I-4)), and a sesquiterpene nootkatone have been isolated from grapefruit juice and screened for their inhibitory effects toward human cytochrome P450 (P450) forms using selective substrate probes. Addition of ethyl acetate extract of grapefruit juice into an incubation mixture resulted in decreased activities of CYP3A4, CYP1A2, CYP2C9, and CYP2D6. All four furanocoumarins clearly inhibited CYP3A4-catalyzed nifedipine oxidation in concentration- and time-dependent manners, suggesting that these compounds are mechanism-based inhibitors of CYP3A4. Of the furanocoumarins investigated, furanocoumarin dimers, GF-I-1 and GF-I-4, were the most potent inhibitors of CYP3A4. Inhibitor concentration required for half-maximal rate of inactivation (K(I)) values for bergamottin, DHB, GF-I-1, and GF-I-4 were calculated, respectively, as 40.00, 5. 56, 0.31, and 0.13 microM, whereas similar values were observed on their inactivation rate constant at infinite concentration of inhibitor (k(inact), 0.05-0.08 min(-1)). Apparent selectivity toward CYP3A4 does occur with the furanocoumarin dimers. In contrast, bergamottin showed rather stronger inhibitory effect on CYP1A2, CYP2C9, CYP2C19, and CYP2D6 than on CYP3A4. DHB inhibited CYP3A4 and CYP1A2 activities at nearly equivalent potencies. Among P450 forms investigated, CYP2E1 was the least sensitive to the inhibitory effect of furanocoumarin components. A sesquiterpene nootkatone has no significant effect on P450 activities investigated except for CYP2A6 and CYP2C19

  6. MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2.

    PubMed

    Zhu, Qiuzhen; Zhang, Yuefan; Liu, Yulan; Cheng, Hao; Wang, Jing; Zhang, Yue; Rui, Yaocheng; Li, Tiejun

    2016-01-01

    Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury. PMID:26918757

  7. MLIF Alleviates SH-SY5Y Neuroblastoma Injury Induced by Oxygen-Glucose Deprivation by Targeting Eukaryotic Translation Elongation Factor 1A2

    PubMed Central

    Liu, Yulan; Cheng, Hao; Wang, Jing; Zhang, Yue; Rui, Yaocheng; Li, Tiejun

    2016-01-01

    Monocyte locomotion inhibitory factor (MLIF), a heat-stable pentapeptide, has been shown to exert potent anti-inflammatory effects in ischemic brain injury. In this study, we investigated the neuroprotective action of MLIF against oxygen-glucose deprivation (OGD)-induced injury in human neuroblastoma SH-SY5Y cells. MTT assay was used to assess cell viability, and flow cytometry assay and Hoechst staining were used to evaluate apoptosis. LDH assay was used to exam necrosis. The release of inflammatory cytokines was detected by ELISA. Levels of the apoptosis associated proteins were measured by western blot analysis. To identify the protein target of MLIF, pull-down assay and mass spectrometry were performed. We observed that MLIF enhanced cell survival and inhibited apoptosis and necrosis by inhibiting p-JNK, p53, c-caspase9 and c-caspase3 expression. In the microglia, OGD-induced secretion of inflammatory cytokines was markedly reduced in the presence of MLIF. Furthermore, we found that eukaryotic translation elongation factor 1A2 (eEF1A2) is a downstream target of MLIF. Knockdown eEF1A2 using short interfering RNA (siRNA) almost completely abrogated the anti-apoptotic effect of MLIF in SH-SY5Y cells subjected to OGD, with an associated decrease in cell survival and an increase in expression of p-JNK and p53. These results indicate that MLIF ameliorates OGD-induced SH-SY5Y neuroblastoma injury by inhibiting the p-JNK/p53 apoptotic signaling pathway via eEF1A2. Our findings suggest that eEF1A2 may be a new therapeutic target for ischemic brain injury. PMID:26918757

  8. Consequences of POR mutations and polymorphisms

    PubMed Central

    Miller, Walter L.; Agrawal, Vishal; Sandee, Duanpen; Tee, Meng Kian; Huang, Ningwu; Choi, Ji Ha; Morrissey, Kari; Giacomini, Kathleen M.

    2015-01-01

    P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ~40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ~60%, is found on ~28% of human alleles. A promoter polymorphism (~8% of Asians and ~13% of Caucasians) at −152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ~30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically-relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism. PMID:21070833

  9. Formation of DNA adducts in wild-type and transgenic mice expressing human sulfotransferases 1A1 and 1A2 after oral exposure to furfuryl alcohol

    PubMed Central

    Høie, Anja Hortemo; Monien, Bernhard Hans; Sakhi, Amrit Kaur; Glatt, Hansruedi; Hjertholm, Hege; Husøy, Trine

    2015-01-01

    Furfuryl alcohol (FFA) is present in many heat-treated foods as a result of its formation via dehydration of pentoses. It is also used legally as a flavouring agent. In an inhalation study conducted in the National Toxicology Program, FFA showed some evidence of carcinogenic activity in rats and mice. FFA was generally negative in conventional genotoxicity assays, which suggests that it may be a non-genotoxic carcinogen. However, it was recently found that FFA is mutagenic in Salmonella strains expressing appropriate sulfotransferases (SULTs), such as human or mouse SULT1A1. The same DNA adducts that were formed by FFA in these strains, mainly N 2-((furan-2-yl)methyl)-2′-deoxyguanosine (N 2-MF-dG), were also detected in tissues of FFA-exposed mice and even in human lung specimens. In the present study, a single oral dose of FFA (250mg/kg body weight) or saline was administered to FVB/N mice and transgenic mice expressing human SULT1A1/1A2 on the FVB/N background. The transgenic mice were used, since human and mouse SULT1A1 substantially differ in substrate specificity and tissue distribution. DNA adducts were studied in liver, kidney, proximal and distal small intestine as well as colon, using isotope-dilution ultra performance liquid chromatography (UPLC–MS/MS). Surprisingly, low levels of adducts that may represent N 2-MF-dG were detected even in tissues of untreated mice. FFA exposure enhanced the adduct levels in colon and liver, but not in the remaining investigated tissues of wild-type (wt) mice. The situation was similar in transgenic mice, except that N 2-MF-dG levels were also strongly enhanced in the proximal small intestine. These different results between wt and transgenic mice may be attributed to the fact that human SULT1A1, but not the orthologous mouse enzyme, is strongly expressed in the small intestine. PMID:25904584

  10. Effect of herbal teas on hepatic drug metabolizing enzymes in rats.

    PubMed

    Maliakal, P P; Wanwimolruk, S

    2001-10-01

    We have investigated the effect of herbal teas (peppermint, chamomile and dandelion) on the activity of hepatic phase I and phase II metabolizing enzymes using rat liver microsomes. Female Wistar rats were divided into six groups (n = 5 each). Three groups had free access to a tea solution (2%) while the control group had water. Two groups received either green tea extract (0.1%) or aqueous caffeine solution (0.0625%). After four weeks of pretreatment, different cytochrome P450 (CYP) isoforms and phase II enzyme activities were determined by incubation of liver microsomes or cytosol with appropriate substrates. Activity of CYP1A2 in the liver microsomes of rats receiving dandelion, peppermint or chamomile tea was significantly decreased (P < 0.05) to 15%, 24% and 39% of the control value, respectively. CYP1A2 activity was significantly increased by pretreatment with caffeine solution. No alterations were observed in the activities of CYP2D and CYP3A in any group of the pretreated rats. Activity of CYP2E in rats receiving dandelion or peppermint tea was significantly lower than in the control group, 48% and 60% of the control, respectively. There was a dramatic increase (244% of control) in the activity of phase II detoxifying enzyme UDP-glucuronosyl transferase in the dandelion tea-pretreated group. There was no change in the activity of glutathione-S-transferase. The results suggested that, like green and black teas, certain herbal teas can cause modulation of phase I and phase II drug metabolizing enzymes.

  11. Effects of musk xylene and musk ketone on rat hepatic cytochrome P450 enzymes.

    PubMed

    Lehman-McKeeman, L D; Caudill, D; Vassallo, J D; Pearce, R E; Madan, A; Parkinson, A

    1999-12-20

    The purpose of the present work was to characterize the effect of musk xylene (MX) and musk ketone (MK) treatment on rat hepatic cytochrome P450 enzymes. Male F344 rats were dosed orally with MX (10, 50 or 200 mg/kg) or MK (20, 100 or 200 mg/kg) for 7 days, after which CYP1A, 2B and 3A enzyme activities and protein levels were determined. MX treatment resulted in a two- to four-fold increase in the activity of CYP1A, 2B and 3A enzymes. For CYP1A and 3A, these changes were consistent with small increases in immunoreactive proteins. However, for CYP2B, despite only a three-fold increase in enzyme activity, protein levels were increased nearly 50-fold relative to control. This induction occurred by transcriptional activation of the CYP2B1 gene as evidenced by increased steady state CYP2B1 mRNA levels. In contrast to MX, MK treatment increased CYP2B activity, protein and mRNA levels. However MK treatment also increased CYP1A enzyme activity nearly 30-fold higher than control rats, a profile that was markedly different from MX, and very different from its effects in mice (Stuard, S.B., Caudill, D., Lehman-Mc-Keeman, L.D., 1997. Characterization of the effects of musk ketone on mouse cytochrome P450 enzymes. Fund. Appl. Toxicol. 40, 264-271). These results indicate that in rats, MX is an inducer of CYP2B enzymes, but these enzymes are not functionally active. In contrast, MK also induces CYP2B enzymes, with no concurrent inactivation. MK also exhibits a unique pattern of cytochrome P450 induction by increasing both CYP1A and CYP2B in rats.

  12. Discovery of Western European R1b1a2 Y chromosome variants in 1000 genomes project data: an online community approach.

    PubMed

    Rocca, Richard A; Magoon, Gregory; Reynolds, David F; Krahn, Thomas; Tilroe, Vincent O; Op den Velde Boots, Peter M; Grierson, Andrew J

    2012-01-01

    The authors have used an online community approach, and tools that were readily available via the Internet, to discover genealogically and therefore phylogenetically relevant Y-chromosome polymorphisms within core haplogroup R1b1a2-L11/S127 (rs9786076). Presented here is the analysis of 135 unrelated L11 derived samples from the 1000 Genomes Project. We were able to discover new variants and build a much more complex phylogenetic relationship for L11 sub-clades. Many of the variants were further validated using PCR amplification and Sanger sequencing. The identification of these new variants will help further the understanding of population history including patrilineal migrations in Western and Central Europe where R1b1a2 is the most frequent haplogroup. The fine-grained phylogenetic tree we present here will also help to refine historical genetic dating studies. Our findings demonstrate the power of citizen science for analysis of whole genome sequence data. PMID:22911832

  13. Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes Modulated by ToxCast Chemicals

    EPA Science Inventory

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  14. Modulation of Xenobiotic Metabolizing Enzyme and Transporter Gene Expression in Primary Cultures of Human Hepatocytes by ToxCast Chemicals

    EPA Science Inventory

    ToxCast chemicals were assessed for induction or suppression of xenobiotic metabolizing enzyme and transporter gene expression using primary human hepatocytes. The mRNA levels of 14 target and 2 control genes were measured: ABCB1, ABCB11, ABCG2, SLCO1B1, CYP1A1, CYP1A2, CYP2B6, C...

  15. Cytochrome P450 induction properties of food and herbal-derived compounds using a novel multiplex RT-qPCR in vitro assay, a drug-food interaction prediction tool.

    PubMed

    Koe, Xue Fen; Tengku Muhammad, Tengku Sifzizul; Chong, Alexander Shu-Chien; Wahab, Habibah Abdul; Tan, Mei Lan

    2014-09-01

    A multiplex RT-qPCR was developed to examine CYP1A2, CYP2D6, and CYP3A4 induction properties of compounds from food and herbal sources. The induction of drug metabolizing enzymes is an important pharmacokinetic interaction with unique features in comparison with inhibition of metabolizing enzymes. Cytochrome induction can lead to serious drug-drug or drug-food interactions, especially if the coadministered drug plasma level is critical as it can reduce therapeutic effects and cause complications. Using this optimized multiplex RT-qPCR, cytochrome induction properties of andrographolide, curcumin, lycopene, bergamottin, and resveratrol were determined. Andrographolide, curcumin, and lycopene produced no significant induction effects on CYP1A2, CYP2D6, and CYP3A4. However, bergamottin appeared to be a significant in vitro CYP1A2 inducer starting from 5 to 50 μmol/L with induction ranging from 60 to 100-fold changes. On the other hand, resveratrol is a weak in vitro CYP1A2 inducer. Examining the cytochrome induction properties of food and herbal compounds help complement CYP inhibition studies and provide labeling and safety caution for such products. PMID:25473508

  16. Enzyme kinetic study of a new cardioprotective agent, KR-32570 using human liver microsomes and recombinant CYP isoforms.

    PubMed

    Kim, Hyojin; Seo, Kyung-Ah; Kim, Hyunmi; Lee, Hye Suk; Lee, Choong-Hwan; Shin, Jae-Gook; Liu, Kwang-Hyeon

    2007-04-01

    KR-32570 (5-(2-Methoxy-5-chlorophenyl)furan-2-ylcarbonyl)guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. Human liver microsomal incubation of KR-32570 in the presence of NADPH resulted in the formation of two metabolites, hydroxy-KR-32570 and O-desmethyl-KR-32570. In this study, a kinetic analysis of the metabolism of two metabolites from KR-32570 was performed in human liver microsomes, and recombinant CYP1A2, and CYP3A4. The metabolism for hydroxy- and O-desmethyl-KR-32570 formation from KR-32570 by human liver microsomes was best described by a Michaelis-Menten equation and a Hill equation, respectively. The Cl(int) values of hydroxy- and O-desmethyl-KR-32570 formation were similar to each other (0.03 vs 0.04 microL/min/pmol CYP, respectively). CYP3A4 mediated the formation of hydroxy-KR-32570 from KR-32570 with Cl(int) = 0.24 microL/min/pmol CYP3A4. The intrinsic clearance for O-desmethyl-KR-32570 formation by CYP1A2 was 0.83 AL/min/pmol CYP1A2. These findings suggest that CYP3A4 and CYP1A2 enzymes are major enzymes contributing to the metabolism of KR-32570.

  17. Ethanol and 4-methylpyrazole increase DNA adduct formation of furfuryl alcohol in FVB/N wild-type mice and in mice expressing human sulfotransferases 1A1/1A2.

    PubMed

    Sachse, Benjamin; Meinl, Walter; Glatt, Hansruedi; Monien, Bernhard H

    2016-03-01

    Furfuryl alcohol (FFA) is a carcinogenic food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. The activation by sulfotransferases (SULTs) yields a DNA reactive and mutagenic sulfate ester. The most prominent DNA adduct, N(2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N(2)-MF-dG), was detected in FFA-treated mice and also in human tissue samples. The dominant pathway of FFA detoxification is the oxidation via alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). The activity of these enzymes may be greatly altered in the presence of inhibitors or competitive substrates. Here, we investigated the impact of ethanol and the ADH inhibitor 4-methylpyrazole (4MP) on the DNA adduct formation by FFA in wild-type and in humanized mice that were transgenic for human SULT1A1/1A2 and deficient in the mouse (m) Sult1a1 and Sult1d1 genes (h1A1/1A2/1a1(-)/1d1(-)). The administration of FFA alone led to hepatic adduct levels of 4.5 N(2)-MF-dG/10(8) nucleosides and 33.6 N(2)-MF-dG/10(8) nucleosides in male and female wild-type mice, respectively, and of 19.6 N(2)-MF-dG/10(8) nucleosides and 95.4 N(2)-MF-dG/10(8) nucleosides in male and female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 1.6g ethanol/kg body weight increased N(2)-MF-dG levels by 2.3-fold in male and by 1.7-fold in female wild-type mice and by 2.5-fold in male and by 1.5-fold in female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 100mg 4MP/kg body weight had a similar effect on the adduct levels. These findings indicate that modulators of the oxidative metabolism, e.g. the drug 4MP or consumption of alcoholic beverages, may increase the genotoxic effects of FFA also in humans.

  18. Ethanol and 4-methylpyrazole increase DNA adduct formation of furfuryl alcohol in FVB/N wild-type mice and in mice expressing human sulfotransferases 1A1/1A2.

    PubMed

    Sachse, Benjamin; Meinl, Walter; Glatt, Hansruedi; Monien, Bernhard H

    2016-03-01

    Furfuryl alcohol (FFA) is a carcinogenic food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. The activation by sulfotransferases (SULTs) yields a DNA reactive and mutagenic sulfate ester. The most prominent DNA adduct, N(2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N(2)-MF-dG), was detected in FFA-treated mice and also in human tissue samples. The dominant pathway of FFA detoxification is the oxidation via alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). The activity of these enzymes may be greatly altered in the presence of inhibitors or competitive substrates. Here, we investigated the impact of ethanol and the ADH inhibitor 4-methylpyrazole (4MP) on the DNA adduct formation by FFA in wild-type and in humanized mice that were transgenic for human SULT1A1/1A2 and deficient in the mouse (m) Sult1a1 and Sult1d1 genes (h1A1/1A2/1a1(-)/1d1(-)). The administration of FFA alone led to hepatic adduct levels of 4.5 N(2)-MF-dG/10(8) nucleosides and 33.6 N(2)-MF-dG/10(8) nucleosides in male and female wild-type mice, respectively, and of 19.6 N(2)-MF-dG/10(8) nucleosides and 95.4 N(2)-MF-dG/10(8) nucleosides in male and female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 1.6g ethanol/kg body weight increased N(2)-MF-dG levels by 2.3-fold in male and by 1.7-fold in female wild-type mice and by 2.5-fold in male and by 1.5-fold in female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 100mg 4MP/kg body weight had a similar effect on the adduct levels. These findings indicate that modulators of the oxidative metabolism, e.g. the drug 4MP or consumption of alcoholic beverages, may increase the genotoxic effects of FFA also in humans. PMID:26775039

  19. First and second line imatinib treatment in chronic myelogenous leukemia patients expressing rare e1a2 or e19a2 BCR-ABL transcripts.

    PubMed

    Andrikovics, Hajnalka; Nahajevszky, Sarolta; Szilvási, Anikó; Bors, András; Adám, Emma; Kozma, András; Kajtár, Béla; Barta, Anikó; Poros, Anna; Tordai, Attila

    2007-09-01

    During the formation of the Philadelphia (Ph) chromosome, in the majority of chronic myelogenous leukemia (CML) patients, the chromosome 22 breakpoint is located in the major breakpoint cluster region of the BCR gene (M-bcr). Minor and micro breakpoint cluster regions (m-bcr with e1a2 transcript and micro-bcr with e19a2 transcript) are rarely affected and have been suggested to be associated with peculiar CML phenotypes. Despite the different clinical characteristics, it is currently not established, whether different therapeutic options are preferably recommended for the treatment of e1a2 or e19a2 CML. Here we report two patients with e1a2 and one patient with e19a2 translocations, treated with different approaches including imatinib. First and second line imatinib treatments induced haematologic response in all of the three patients, and major cytogenetic response in one patient with e1a2, as well as in the patient with e19a2 CML. However, relapse occurred in the patient with e19a2 CML, possibly caused by the presence of additional chromosomal abnormalities such as an extra Ph chromosome, and loss of chromosome Y. Stem cell transplantation (SCT) therapy caused complete haematologic response with molecular remission; however, the patient died of infectious complication. We conclude that in patients with rare BCR-ABL variants, the effectiveness of imatininb treatment may be influenced by the CML stage besides the actual molecular type of the rare transcript. However, this conclusion cannot be generalized to larger patient groups with rare BCR-ABL variants for which further studies may be needed.

  20. Consistent linkage of dominantly inherited osteogenesis imperfecta to the type I collagen loci: COL1A1 and COL1A2.

    PubMed

    Sykes, B; Ogilvie, D; Wordsworth, P; Wallis, G; Mathew, C; Beighton, P; Nicholls, A; Pope, F M; Thompson, E; Tsipouras, P

    1990-02-01

    The segregation of COL1A1 and COL1A2, the two genes which encode the chains of type I collagen, was analyzed in 38 dominant osteogenesis imperfecta (OI) pedigrees by using polymorphic markers within or close to the genes. This was done in order to estimate the consistency of linkage of OI genes to these two loci. None of the 38 pedigrees showed evidence of recombination between the OI gene and both collagen loci, suggesting that the frequency of unlinked loci in the population must be low. From these results, approximate 95% confidence limits for the proportion of families linked to the type I collagen genes can be set between .91 and 1.00. This is high enough to base prenatal diagnosis of dominantly inherited OI on linkage to these genes even in families which are too small for the linkage to be independently confirmed to high levels of significance. When phenotypic features were compared with the concordant collagen locus, all eight pedigrees with Sillence OI type IV segregated with COL1A2. On the other hand, Sillence OI type I segregated with both COL1A1 (17 pedigrees) and COL1A2 (7 pedigrees). The concordant locus was uncertain in the remaining six OI type I pedigrees. Of several other features, the presence or absence of presenile hearing loss was the best predictor of the mutant locus in OI type I families, with 13 of the 17 COL1A1 segregants and none of the 7 COL1A2 segregants showing this feature.

  1. Dopamine D2-Receptor Antagonists Down-Regulate CYP1A1/2 and CYP1B1 in the Rat Liver.

    PubMed

    Harkitis, P; Daskalopoulos, E P; Malliou, F; Lang, M A; Marselos, M; Fotopoulos, A; Albucharali, G; Konstandi, M

    2015-01-01

    Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens. PMID:26466350

  2. 2,3,7,8-Tetrachlorodibenzo-p-dioxin treatment alters eicosanoid levels in several organs of the mouse in an aryl hydrocarbon receptor-dependent fashion

    SciTech Connect

    Bui, Peter; Solaimani, Parrisa; Wu, Xiaomeng; Hankinson, Oliver

    2012-03-01

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) adversely affects many mammalian organs and tissues. These effects are mediated by the aryl hydrocarbon receptor (AHR). CYP1A1, CYP1A2 and CYP1B1 are upregulated by the liganded AHR. These (and other) cytochromes P450 can metabolize arachidonic acid into a variety of bioactive eicosanoids. Towards investigating a potential role of eicosanoids in TCDD toxicity, arachidonic acid, two other unsaturated long-chain fatty acids, and up to twenty-five eicosanoids were measured in five organs/tissues of male and female wild-type and Ahr null mice treated or untreated with TCDD. TCDD generally increased the levels of the four dihydroxyeicosatrienoic acids (DHETs) and (where measured) 5,6-epoxyeicosatrienoic acid and 18-, 19- and 20-hydroxyeicosatrienoic acids (HETEs) in the serum, liver, spleen and lungs, but not the heart, of both sexes, and increased the levels in the serum, liver and spleen of several metabolites that are usually considered products of lipoxygenase activity, but which may also be generated by cytochromes P450. TCDD also increased the levels of the esterified forms of these eicosanoids in the liver in parallel with the corresponding free forms. The levels of prostanoids were generally not affected by TCDD. The above changes did not occur in Ahr null mice, and are therefore mediated by the AHR. TCDD increased the mRNA levels of Cyp1a1, Cyp1a2, Cyp1b1 and the Pla2g12a form of phospholipase A{sub 2} to varying degrees in the different organs, and these increases correlated with some but not all the changes in eicosanoids levels in the organs, suggesting that other enzymes may also be involved. -- Highlights: ► TCDD treatment increases the levels of many eicosanoids in several mouse organs. ► Products of both the cytochrome P450 and classical lipoxygenase pathways are increased. ► These increases are dependent on the aryl hydrocarbon receptor. ► Cyp1a1, Cyp1a2 and Cyp1b1 appear to be responsible for much but

  3. Dopamine D2-Receptor Antagonists Down-Regulate CYP1A1/2 and CYP1B1 in the Rat Liver.

    PubMed

    Harkitis, P; Daskalopoulos, E P; Malliou, F; Lang, M A; Marselos, M; Fotopoulos, A; Albucharali, G; Konstandi, M

    2015-01-01

    Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens.

  4. Dopamine D2-Receptor Antagonists Down-Regulate CYP1A1/2 and CYP1B1 in the Rat Liver

    PubMed Central

    Harkitis, P.; Lang, M. A.; Marselos, M.; Fotopoulos, A.; Albucharali, G.; Konstandi, M.

    2015-01-01

    Dopaminergic systems regulate the release of several hormones including growth hormone (GH), thyroid hormones, insulin, glucocorticoids and prolactin (PRL) that play significant roles in the regulation of various Cytochrome P450 (CYP) enzymes. The present study investigated the role of dopamine D2-receptor-linked pathways in the regulation of CYP1A1, CYP1A2 and CYP1B1 that belong to a battery of genes controlled by the Aryl Hydrocarbon Receptor (AhR) and play a crucial role in the metabolism and toxicity of numerous environmental toxicants. Inhibition of dopamine D2-receptors with sulpiride (SULP) significantly repressed the constitutive and benzo[a]pyrene (B[a]P)-induced CYP1A1, CYP1A2 and CYP1B expression in the rat liver. The expression of AhR, heat shock protein 90 (HSP90) and AhR nuclear translocator (ARNT) was suppressed by SULP in B[a]P-treated livers, whereas the AhRR expression was increased by the drug suggesting that the SULP-mediated repression of the CYP1 inducibility is due to inactivation of the AhR regulatory system. At signal transduction level, the D2-mediated down-regulation of constitutive CYP1A1/2 and CYP1B1 expression appears to be mediated by activation of the insulin/PI3K/AKT pathway. PRL-linked pathways exerting a negative control on various CYPs, and inactivation of the glucocorticoid-linked pathways that positively control the AhR-regulated CYP1 genes, may also participate in the SULP-mediated repression of both, the constitutive and induced CYP1 expression. The present findings indicate that drugs acting as D2-dopamine receptor antagonists can modify several hormone systems that regulate the expression of CYP1A1, CYP1A2 and CYP1B1, and may affect the toxicity and carcinogenicity outcome of numerous toxicants and pre-carcinogenic substances. Therefore, these drugs could be considered as a part of the strategy to reduce the risk of exposure to environmental pollutants and pre-carcinogens. PMID:26466350

  5. Functional and pharmacological characterization of two different ASIC1a/2a heteromers reveals their sensitivity to the spider toxin PcTx1

    PubMed Central

    Joeres, Niko; Augustinowski, Katrin; Neuhof, Andreas; Assmann, Marc; Gründer, Stefan

    2016-01-01

    Acid Sensing Ion Channels (ASICs) detect extracellular proton signals and are involved in synaptic transmission and pain sensation. ASIC subunits assemble into homo- and heteromeric channels composed of three subunits. Single molecule imaging revealed that heteromers composed of ASIC1a and ASIC2a, which are widely expressed in the central nervous system, have a flexible 2:1/1:2 stoichiometry. It was hitherto not possible, however, to functionally differentiate these two heteromers. To have a homogenous population of ASIC1a/2a heteromers with either 2:1 or 1:2 stoichiometry, we covalently linked subunits in the desired configuration and characterized their functional properties in Xenopus oocytes. We show that the two heteromers have slightly different proton affinity, with an additional ASIC1a subunit increasing apparent affinity. Moreover, we found that zinc, which potentiates ASIC2a-containing ASICs but not homomeric ASIC1a, potentiates both heteromers. Finally, we show that PcTx1, which binds at subunit-subunit interfaces of homomeric ASIC1a, inhibits both heteromers suggesting that ASIC2a can also contribute to a PcTx1 binding site. Using this functional fingerprint, we show that rat cortical neurons predominantly express the ASIC1a/2a heteromer with a 2:1 stoichiometry. Collectively, our results reveal the contribution of individual subunits to the functional properties of ASIC1a/2a heteromers. PMID:27277303

  6. Functional and pharmacological characterization of two different ASIC1a/2a heteromers reveals their sensitivity to the spider toxin PcTx1.

    PubMed

    Joeres, Niko; Augustinowski, Katrin; Neuhof, Andreas; Assmann, Marc; Gründer, Stefan

    2016-01-01

    Acid Sensing Ion Channels (ASICs) detect extracellular proton signals and are involved in synaptic transmission and pain sensation. ASIC subunits assemble into homo- and heteromeric channels composed of three subunits. Single molecule imaging revealed that heteromers composed of ASIC1a and ASIC2a, which are widely expressed in the central nervous system, have a flexible 2:1/1:2 stoichiometry. It was hitherto not possible, however, to functionally differentiate these two heteromers. To have a homogenous population of ASIC1a/2a heteromers with either 2:1 or 1:2 stoichiometry, we covalently linked subunits in the desired configuration and characterized their functional properties in Xenopus oocytes. We show that the two heteromers have slightly different proton affinity, with an additional ASIC1a subunit increasing apparent affinity. Moreover, we found that zinc, which potentiates ASIC2a-containing ASICs but not homomeric ASIC1a, potentiates both heteromers. Finally, we show that PcTx1, which binds at subunit-subunit interfaces of homomeric ASIC1a, inhibits both heteromers suggesting that ASIC2a can also contribute to a PcTx1 binding site. Using this functional fingerprint, we show that rat cortical neurons predominantly express the ASIC1a/2a heteromer with a 2:1 stoichiometry. Collectively, our results reveal the contribution of individual subunits to the functional properties of ASIC1a/2a heteromers. PMID:27277303

  7. Differential toxicity of heterocyclic aromatic amines and their mixture in metabolically competent HepaRG cells

    SciTech Connect

    Dumont, Julie; Josse, Rozenn; Lambert, Carine; Antherieu, Sebastien; Le Hegarat, Ludovic; Aninat, Caroline; Robin, Marie-Anne; Guguen-Guillouzo, Christiane

    2010-06-01

    Human exposure to heterocyclic aromatic amines (HAA) usually occurs through mixtures rather than individual compounds. However, the toxic effects and related mechanisms of co-exposure to HAA in humans remain unknown. We compared the effects of two of the most common HAA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), individually or in combination, in the metabolically competent human hepatoma HepaRG cells. Various endpoints were measured including cytotoxicity, apoptosis, oxidative stress and DNA damage by the comet assay. Moreover, the effects of PhIP and/or MeIQx on mRNA expression and activities of enzymes involved in their activation and detoxification pathways were evaluated. After a 24 h treatment, PhIP and MeIQx, individually and in combination, exerted differential effects on apoptosis, oxidative stress, DNA damage and cytochrome P450 (CYP) activities. Only PhIP induced DNA damage. It was also a stronger inducer of CYP1A1 and CYP1B1 expression and activity than MeIQx. In contrast, only MeIQx exposure resulted in a significant induction of CYP1A2 activity. The combination of PhIP with MeIQx induced an oxidative stress and showed synergistic effects on apoptosis. However, PhIP-induced genotoxicity was abolished by a co-exposure with MeIQx. Such an inhibitory effect could be explained by a significant decrease in CYP1A2 activity which is responsible for PhIP genotoxicity. Our findings highlight the need to investigate interactions between HAA when assessing risks for human health and provide new insights in the mechanisms of interaction between PhIP and MeIQx.

  8. Skatole metabolism in the pigs with reduced testicular oestrogen synthesis.

    PubMed

    Zamaratskaia, G; Berger, T

    2014-04-01

    The objectives of the study were to investigate the involvement of oestrogens in the regulation of skatole levels in pigs. In total, 44 intact male pigs, siblings from 10 litters, were included in the study. Pigs were orally treated weekly with either 0.1 mg letrozole/kg body weight to reduce endogenous oestrogens or the canola oil vehicle. Fat and liver samples were collected at slaughter at 16, 20 and 40 weeks of age. Skatole and androstenone levels in fat and activities of hepatic cytochrome P4501A1, CYP1A2, CYP2A19 and CYP2E1 were analysed. Letrozole treatment did not significantly change either the levels of skatole or activities of skatole-metabolising enzymes, suggesting that oestrogens are not responsible for gender-related differences in skatole concentrations in porcine tissues. PMID:24460981

  9. Investigation of selective inhibitory effects of glycyrol on human CYP 1A1 and 2C9.

    PubMed

    Kim, Sun Joo; Kim, Su Jin; Hong, Miri; Choi, Hyun Gyu; Kim, Jeong Ah; Lee, Sangkyu

    2016-10-01

    1. Glycyrol is a coumarin derivative isolated from the roots of Glycyrrhiza uralensis called Gamcho in Korea and commonly used as a sweetener in oriental medicine. Glycyrol shows several biological activities, including anti-oxidative, anti-inflammatory, antibacterial, anti-angiogenic, and anti-allergenic properties. Although there have been studies on the biological effects of glycyrol, the inhibitory effects of glycyrol on cytochrome P450 (CYP) activities have not been investigated. 2. We investigated the inhibitory effects of glycyrol on the activities of CYP isoforms using a cocktail of probe substrates in pooled human liver microsome (HLM) and human recombinant cDNA-expressed CYPs. Glycyrol strongly inhibited CYP1A-mediated phenacetin O-deethylation and CYP2C9-mediated diclofenac 4'-hydroxylation in HLMs, which were the result of competitive inhibition as revealed by a Dixon plot. In addition, glycyrol showed selective inhibition of CYP1A1- and CYP1A2-catalyzed phenacetin O-deethylase activity with a half-maximal inhibitory concentration of (IC50) 1.3 and 16.1 μM in human recombinant cDNA-expressed CYP1A1 and CYP1A2, respectively. 3. Glycyrol decreased CYP2C9-catalyzed diclofenac 4'-hydroxylation activity with IC50 values of 0.67 μM in human recombinant cDNA-expressed CYP2C9. This is the first investigation of competitive inhibitory effects on CYP1A1 and CYP2C9 in HLMs. PMID:26750984

  10. Investigation of selective inhibitory effects of glycyrol on human CYP 1A1 and 2C9.

    PubMed

    Kim, Sun Joo; Kim, Su Jin; Hong, Miri; Choi, Hyun Gyu; Kim, Jeong Ah; Lee, Sangkyu

    2016-10-01

    1. Glycyrol is a coumarin derivative isolated from the roots of Glycyrrhiza uralensis called Gamcho in Korea and commonly used as a sweetener in oriental medicine. Glycyrol shows several biological activities, including anti-oxidative, anti-inflammatory, antibacterial, anti-angiogenic, and anti-allergenic properties. Although there have been studies on the biological effects of glycyrol, the inhibitory effects of glycyrol on cytochrome P450 (CYP) activities have not been investigated. 2. We investigated the inhibitory effects of glycyrol on the activities of CYP isoforms using a cocktail of probe substrates in pooled human liver microsome (HLM) and human recombinant cDNA-expressed CYPs. Glycyrol strongly inhibited CYP1A-mediated phenacetin O-deethylation and CYP2C9-mediated diclofenac 4'-hydroxylation in HLMs, which were the result of competitive inhibition as revealed by a Dixon plot. In addition, glycyrol showed selective inhibition of CYP1A1- and CYP1A2-catalyzed phenacetin O-deethylase activity with a half-maximal inhibitory concentration of (IC50) 1.3 and 16.1 μM in human recombinant cDNA-expressed CYP1A1 and CYP1A2, respectively. 3. Glycyrol decreased CYP2C9-catalyzed diclofenac 4'-hydroxylation activity with IC50 values of 0.67 μM in human recombinant cDNA-expressed CYP2C9. This is the first investigation of competitive inhibitory effects on CYP1A1 and CYP2C9 in HLMs.

  11. Severe liver cirrhosis markedly reduces AhR-mediated induction of cytochrome P450 in rats by decreasing the transcription of target genes.

    PubMed

    Floreani, Maura; De Martin, Sara; Gabbia, Daniela; Barbierato, Massimo; Nassi, Alberto; Mescoli, Claudia; Orlando, Rocco; Bova, Sergio; Angeli, Paolo; Gola, Elisabetta; Sticca, Antonietta; Palatini, Pietro

    2013-01-01

    Although the induction of cytochrome P450 (CYP) has long been investigated in patients with cirrhosis, the question whether liver dysfunction impairs the response to CYP inducers still remains unresolved. Moreover, the mechanism underlying the possible effect of cirrhosis on induction has not been investigated. Since ethical constraints do not permit methodologically rigorous studies in humans, this question was addressed by investigating the effect of the prototypical inducer benzo[a]pyrene (BP) on CYP1A1 and CYP1A2 in cirrhotic rats stratified according to the severity of liver dysfunction. We simultaneously assessed mRNA level, protein expression and enzymatic activity of the CYP1A enzymes, as well as mRNA and protein expressions of the aryl hydrocarbon receptor (AhR), which mediates the BP effect. Basal mRNA and protein expressions of CYP1A1 were virtually absent in both healthy and cirrhotic rats. On the contrary, CYP1A2 mRNA, protein and enzyme activity were constitutively present in healthy rats and decreased significantly as liver function worsened. BP treatment markedly increased the concentrations of mRNA and immunodetectable protein, and the enzymatic activities of both CYP1A enzymes to similar levels in healthy and non-ascitic cirrhotic rats. Induced mRNA levels, protein expressions and enzymatic activities of both CYPs were much lower in ascitic rats and were proportionally reduced. Both constitutive and induced protein expressions of AhR were significantly lower in ascitic than in healthy rats. These results indicate that the inducibility of CYP1A enzymes is well preserved in compensated cirrhosis, whereas it is markedly reduced when liver dysfunction becomes severe. Induction appears to be impaired at the transcriptional level, due to the reduced expression of AhR, which controls the transcription of CYP1A genes.

  12. Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes

    SciTech Connect

    Richert, L. Tuschl, G.; Pekthong, D.; Mantion, G.; Weber, J.-C.; Mueller, S.O.

    2009-02-15

    It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman{sup TM} Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.

  13. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes.

    PubMed

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5-50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1'-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans.

  14. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes

    PubMed Central

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5–50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1′-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans. PMID:25395831

  15. Effect of honokiol on the induction of drug-metabolizing enzymes in human hepatocytes.

    PubMed

    Cho, Yong-Yeon; Jeong, Hyeon-Uk; Kim, Jeong-Han; Lee, Hye Suk

    2014-01-01

    Honokiol, 2-(4-hydroxy-3-prop-2-enyl-phenyl)-4-prop-2-enyl-phenol, an active component of Magnolia officinalis and Magnolia grandiflora, exerts various pharmacological activities such as antitumorigenic, antioxidative, anti-inflammatory, neurotrophic, and antithrombotic effects. To investigate whether honokiol acts as a perpetrator in drug interactions, messenger ribonucleic acid (mRNA) levels of phase I and II drug-metabolizing enzymes, including cytochrome P450 (CYP), UDP-glucuronosyltransferase (UGT), and sulfotransferase 2A1 (SULT2A1), were analyzed by real-time reverse transcription polymerase chain reaction following 48-hour honokiol exposure in three independent cryopreserved human hepatocyte cultures. Honokiol treatment at the highest concentration tested (50 μM) increased the CYP2B6 mRNA level and CYP2B6-catalyzed bupropion hydroxylase activity more than two-fold in three different hepatocyte cultures, indicating that honokiol induces CYP2B6 at higher concentrations. However, honokiol treatment (0.5-50 μM) did not significantly alter the mRNA levels of phase I enzymes (CYP1A2, CYP3A4, CYP2C8, CYP2C9, and CYP2C19) or phase II enzymes (UGT1A1, UGT1A4, UGT1A9, UGT2B7, and SULT2A1) in cryopreserved human hepatocyte cultures. CYP1A2-catalyzed phenacetin O-deethylase and CYP3A4-catalyzed midazolam 1'-hydroxylase activities were not affected by 48-hour honokiol treatment in cryopreserved human hepatocytes. These results indicate that honokiol is a weak CYP2B6 inducer and is unlikely to increase the metabolism of concomitant CYP2B6 substrates and cause pharmacokinetic-based drug interactions in humans. PMID:25395831

  16. HTR-PROTEUS Pebble Bed Experimental Program Cores 1, 1A, 2, and 3: Hexagonal Close Packing with a 1:2 Moderator-to-Fuel Pebble Ratio

    SciTech Connect

    John D. Bess; Barbara H. Dolphin; James W. Sterbentz; Luka Snoj; Igor Lengar; Oliver Köberl

    2013-03-01

    In its deployment as a pebble bed reactor (PBR) critical facility from 1992 to 1996, the PROTEUS facility was designated as HTR-PROTEUS. This experimental program was performed as part of an International Atomic Energy Agency (IAEA) Coordinated Research Project (CRP) on the Validation of Safety Related Physics Calculations for Low Enriched HTGRs. Within this project, critical experiments were conducted for graphite moderated LEU systems to determine core reactivity, flux and power profiles, reaction-rate ratios, the worth of control rods, both in-core and reflector based, the worth of burnable poisons, kinetic parameters, and the effects of moisture ingress on these parameters. Four benchmark experiments were evaluated in this report: Cores 1, 1A, 2, and 3. These core configurations represent the hexagonal close packing (HCP) configurations of the HTR-PROTEUS experiment with a moderator-to-fuel pebble ratio of 1:2. Core 1 represents the only configuration utilizing ZEBRA control rods. Cores 1A, 2, and 3 use withdrawable, hollow, stainless steel control rods. Cores 1 and 1A are similar except for the use of different control rods; Core 1A also has one less layer of pebbles (21 layers instead of 22). Core 2 retains the first 16 layers of pebbles from Cores 1 and 1A and has 16 layers of moderator pebbles stacked above the fueled layers. Core 3 retains the first 17 layers of pebbles but has polyethylene rods inserted between pebbles to simulate water ingress. The additional partial pebble layer (layer 18) for Core 3 was not included as it was used for core operations and not the reported critical configuration. Cores 1, 1A, 2, and 3 were determined to be acceptable benchmark experiments.

  17. HTR-PROTEUS Pebble Bed Experimental Program Cores 1, 1A, 2, and 3: Hexagonal Close Packing with a 1:2 Moderator-to-Fuel Pebble Ratio

    SciTech Connect

    John D. Bess; Barbara H. Dolphin; James W. Sterbentz; Luka Snoj; Igor Lengar; Oliver Köberl

    2012-03-01

    In its deployment as a pebble bed reactor (PBR) critical facility from 1992 to 1996, the PROTEUS facility was designated as HTR-PROTEUS. This experimental program was performed as part of an International Atomic Energy Agency (IAEA) Coordinated Research Project (CRP) on the Validation of Safety Related Physics Calculations for Low Enriched HTGRs. Within this project, critical experiments were conducted for graphite moderated LEU systems to determine core reactivity, flux and power profiles, reaction-rate ratios, the worth of control rods, both in-core and reflector based, the worth of burnable poisons, kinetic parameters, and the effects of moisture ingress on these parameters. Four benchmark experiments were evaluated in this report: Cores 1, 1A, 2, and 3. These core configurations represent the hexagonal close packing (HCP) configurations of the HTR-PROTEUS experiment with a moderator-to-fuel pebble ratio of 1:2. Core 1 represents the only configuration utilizing ZEBRA control rods. Cores 1A, 2, and 3 use withdrawable, hollow, stainless steel control rods. Cores 1 and 1A are similar except for the use of different control rods; Core 1A also has one less layer of pebbles (21 layers instead of 22). Core 2 retains the first 16 layers of pebbles from Cores 1 and 1A and has 16 layers of moderator pebbles stacked above the fueled layers. Core 3 retains the first 17 layers of pebbles but has polyethylene rods inserted between pebbles to simulate water ingress. The additional partial pebble layer (layer 18) for Core 3 was not included as it was used for core operations and not the reported critical configuration. Cores 1, 1A, 2, and 3 were determined to be acceptable benchmark experiments.

  18. Arginine vasotocin V1a2 receptor and GnRH-I co-localize in preoptic neurons of the sex changing grouper, Epinephelus adscensionis.

    PubMed

    Kline, Richard J; Holt, G Joan; Khan, Izhar A

    2016-01-01

    The arginine vasotocin/vasopressin (AVT/AVP) and gonadotropin releasing hormone (GnRH) systems are known to control sexual behaviors and reproduction, respectively, in different vertebrate groups. However, a direct functional connection between these two neuroendocrine systems has not been demonstrated for any vertebrate species. Therefore, the objective of this research was to test the hypothesis that AVT acts on the GnRH system via an AVT V1a receptor in a sex changing grouper species, the rock hind, Epinephelus adscensionis. AVT V1a2 receptors were co-localized with GnRH-I on neurons in the preoptic anterior hypothalamus identifying a structural linkage between the AVT system and GnRH-I. Transcripts for avt, gnrh-I, and two AVT receptor subtypes (v1a1 and v1a2) were isolated and characterized for E. adscensionis and their expression was measured in males and females by q-RT-PCR. Translation of V1a-type cDNA sequences revealed two distinct forms of the AVT V1a receptor in E. adscensionis brain similar to those reported for other species. The observation of significantly higher gnrh-I mRNA in the POA+H of rock hind males as compared to females suggests differential regulation of the gnrh-I transcripts in the two sexes of this protogynous species. In male E. adscensionis, but not in females, a negative relationship was seen between plasma 11-ketotestosterone (11-KT) and the v1a1 receptor mRNA levels in the POA+H, while a positive trend was observed between 11-KT and v1a2 receptor mRNA levels, indicating that these receptor forms may be differentially regulated. PMID:26361870

  19. Hypervelocity Impact (HVI). Volume 2; WLE Small-Scale Fiberglass Panel Flat Multi-Layer Targets A-1, A-2, and B-1

    NASA Technical Reports Server (NTRS)

    Gorman, Michael R.; Ziola, Steven M.

    2007-01-01

    During 2003 and 2004, the Johnson Space Center's White Sands Testing Facility in Las Cruces, New Mexico conducted hypervelocity impact tests on the space shuttle wing leading edge. Hypervelocity impact tests were conducted to determine if Micro-Meteoroid/Orbital Debris impacts could be reliably detected and located using simple passive ultrasonic methods. The objective of Targets A-1, A-2, and B-2 was to study hypervelocity impacts through multi-layered panels simulating Whipple shields on spacecraft. Impact damage was detected using lightweight, low power instrumentation capable of being used in flight.

  20. Genetic polymorphism of estrogen metabolizing enzymes in Siberian women with breast cancer.

    PubMed

    Khvostova, Ekaterina P; Pustylnyak, Vladimir O; Gulyaeva, Lyudmila F

    2012-03-01

    Allelic variants of cytochrome P450: CYP1A1, CYP1A2, CYP19 (aromatase), and the II-phase enzyme sulfotransferase 1A1 (SULT1A1) genes are associated with a high risk of hormone-dependent cancers. We estimated the frequency of these allelic variants in the female population of the Novosibirsk district and their association with the elevated risk of breast cancer. A DNA bank of patients with gynecologic oncology, patients with breast cancer (n = 335), and healthy women (n = 530) was created, and the following single-nucleotide polymorphisms were examined: CYP1A1 M1 polymorphism, that is, T264-C transition in the 30-noncoding region; CYP1A2*1F polymorphism, that is, C734-A transversion in the CYP1A2 gene; C-T transition (Arg264Cys) in exon 7 of CYP19; and SULT1A1*2 polymorphism, that is, G638-A transition (Arg213His) in the SULT1A1 gene. The results of our study indicate that women with mutant allele C and genotypes C/T, C/C of the CYP1A1 gene, wild-allele C, and genotype C/C of the CYP1A2 gene, mutant allele A and genotypes A/G, A/A of the SULT1A1 gene have an increased risk of development of breast cancer. Women with body mass index ≥ 30 and the heterozygous genotype C/T of the CYP19 gene have an increased risk of breast cancer. The CYP1A2 heterozygous variant genotype C/A is associated with an increased risk of an estrogen receptor (ER(+)) tumor.

  1. Semiphysiologically Based Pharmacokinetic Model of Leflunomide Disposition in Rheumatoid Arthritis Patients.

    PubMed

    Hopkins, A M; Wiese, M D; Proudman, S M; O'Doherty, C E; Foster, Djr; Upton, R N

    2015-06-01

    A semiphysiologically based pharmacokinetic (semi-PBPK) population model was used to evaluate the influence of enterohepatic recycling and protein binding, as well as the effect of genetic variability in CYP1A2, CYP2C19, and ABCG2, on the large interindividual variability of teriflunomide (active metabolite) concentrations following leflunomide administration in rheumatoid arthritis (RA) patients. The model was developed with total and free teriflunomide concentrations determined in RA patients taking leflunomide, as well as mean teriflunomide concentrations following the administration of leflunomide or teriflunomide extracted from the literature. Once developed, the 15-compartment model was able to predict total and free teriflunomide concentrations and was used to screen demographic and genotypic covariates, of which only fat-free mass and liver function (ALT) improved prediction. This approach effectively evaluated the effects of multiple covariates on both total and free teriflunomide concentrations, which have only been explored previously through simplistic one-compartment models for total teriflunomide. PMID:26225264

  2. The detection of small organic molecules based on novel functionalized surface plasmon resonance sensors

    NASA Astrophysics Data System (ADS)

    Zheng, Rui; Cameron, Brent D.

    2010-02-01

    The objective of this study was to develop rapid, inexpensive, and easily applied in vivo phenotyping strategies for characterizing drug-metabolizing phenotypes with reference to the cytochrome P450 (CYP) enzymes in biological fluids. Therefore, the accurate detection of low concentration of theophylline, which can be used as a probe for cytochrome P450 (CYP450) enzymes (e.g. CYP1A2) activity, could benefit drug-metabolizing studies. In this study, a portable, specific, and sensitive functionalized surface plasmon resonance (SPR) sensor using polyacrylamide molecularly imprinted polymers (MIPs) as the highly specific selector is developed for the detection of low concentration theophylline in the presence of other confounding components, such as, caffeine which has a very similar chemical structure.

  3. The effect of ZnO nanoparticles on liver function in rats.

    PubMed

    Tang, Hua-Qiao; Xu, Min; Rong, Qian; Jin, Ru-Wen; Liu, Qi-Ji; Li, Ying-Lun

    2016-01-01

    Zinc oxide (ZnO) is widely incorporated as a food additive in animal diets. In order to optimize the beneficial effects of ZnO and minimize any resultant environmental pollution, ZnO nanoparticles are often used for delivery of the zinc. However, the possible toxic effects of ZnO nanoparticles, including effects on cytochrome P450 (CYP450) enzymes, have not been evaluated. In this study, we investigated the effect of ZnO nanoparticles, in doses used in animal feeds, on CYP450 enzymes, liver and intestinal enzymes, liver and kidney histopathology, and hematologic indices in rats. We found that liver and kidney injury occurred when the concentrations of ZnO nanoparticles in feed were 300-600 mg/kg. Also, liver mRNA expression for constitutive androstane receptor was suppressed and mRNA expression for pregnane X receptor was induced when feed containing ZnO nanoparticles was given at a concentration of 600 mg/kg. Although the expression of mRNA for CYP 2C11 and 3A2 enzymes was induced by ZnO nanoparticles, the activities of CYP 2C11 and 3A2 were suppressed. While liver CYP 1A2 mRNA expression was suppressed, CYP 1A2 activity remained unchanged at all ZnO nanoparticle doses. Therefore, it has been concluded that ZnO nanoparticles, in the doses customarily added to animal feed, changed the indices of hematology and blood chemistry, altered the expression and activity of hepatic CYP enzymes, and induced pathological changes in liver and kidney tissues of rats. These findings suggest that greater attention needs to be paid to the toxic effects of ZnO nanoparticles in animal feed, with the possibility that the doses of ZnO should be reduced. PMID:27621621

  4. The effect of ZnO nanoparticles on liver function in rats

    PubMed Central

    Tang, Hua-Qiao; Xu, Min; Rong, Qian; Jin, Ru-Wen; Liu, Qi-Ji; Li, Ying-Lun

    2016-01-01

    Zinc oxide (ZnO) is widely incorporated as a food additive in animal diets. In order to optimize the beneficial effects of ZnO and minimize any resultant environmental pollution, ZnO nanoparticles are often used for delivery of the zinc. However, the possible toxic effects of ZnO nanoparticles, including effects on cytochrome P450 (CYP450) enzymes, have not been evaluated. In this study, we investigated the effect of ZnO nanoparticles, in doses used in animal feeds, on CYP450 enzymes, liver and intestinal enzymes, liver and kidney histopathology, and hematologic indices in rats. We found that liver and kidney injury occurred when the concentrations of ZnO nanoparticles in feed were 300–600 mg/kg. Also, liver mRNA expression for constitutive androstane receptor was suppressed and mRNA expression for pregnane X receptor was induced when feed containing ZnO nanoparticles was given at a concentration of 600 mg/kg. Although the expression of mRNA for CYP 2C11 and 3A2 enzymes was induced by ZnO nanoparticles, the activities of CYP 2C11 and 3A2 were suppressed. While liver CYP 1A2 mRNA expression was suppressed, CYP 1A2 activity remained unchanged at all ZnO nanoparticle doses. Therefore, it has been concluded that ZnO nanoparticles, in the doses customarily added to animal feed, changed the indices of hematology and blood chemistry, altered the expression and activity of hepatic CYP enzymes, and induced pathological changes in liver and kidney tissues of rats. These findings suggest that greater attention needs to be paid to the toxic effects of ZnO nanoparticles in animal feed, with the possibility that the doses of ZnO should be reduced.

  5. The effect of ZnO nanoparticles on liver function in rats

    PubMed Central

    Tang, Hua-Qiao; Xu, Min; Rong, Qian; Jin, Ru-Wen; Liu, Qi-Ji; Li, Ying-Lun

    2016-01-01

    Zinc oxide (ZnO) is widely incorporated as a food additive in animal diets. In order to optimize the beneficial effects of ZnO and minimize any resultant environmental pollution, ZnO nanoparticles are often used for delivery of the zinc. However, the possible toxic effects of ZnO nanoparticles, including effects on cytochrome P450 (CYP450) enzymes, have not been evaluated. In this study, we investigated the effect of ZnO nanoparticles, in doses used in animal feeds, on CYP450 enzymes, liver and intestinal enzymes, liver and kidney histopathology, and hematologic indices in rats. We found that liver and kidney injury occurred when the concentrations of ZnO nanoparticles in feed were 300–600 mg/kg. Also, liver mRNA expression for constitutive androstane receptor was suppressed and mRNA expression for pregnane X receptor was induced when feed containing ZnO nanoparticles was given at a concentration of 600 mg/kg. Although the expression of mRNA for CYP 2C11 and 3A2 enzymes was induced by ZnO nanoparticles, the activities of CYP 2C11 and 3A2 were suppressed. While liver CYP 1A2 mRNA expression was suppressed, CYP 1A2 activity remained unchanged at all ZnO nanoparticle doses. Therefore, it has been concluded that ZnO nanoparticles, in the doses customarily added to animal feed, changed the indices of hematology and blood chemistry, altered the expression and activity of hepatic CYP enzymes, and induced pathological changes in liver and kidney tissues of rats. These findings suggest that greater attention needs to be paid to the toxic effects of ZnO nanoparticles in animal feed, with the possibility that the doses of ZnO should be reduced. PMID:27621621

  6. Comparison of various urine collection intervals for caffeine and dextromethorphan phenotyping in children.

    PubMed

    Kennedy, Mary Jayne; Abdel-Rahman, Susan M; Kashuba, Angela D M; Leeder, J Steven

    2004-07-01

    Caffeine and dextromethorphan have been used successfully both alone and in combination to assess phenotype and enzyme activity in children of various ages. Previous pediatric phenotyping studies with these agents have used varying durations of urine collection. However, the minimum duration required for accurate phenotypic assessment with these compounds in children remains unknown. We calculated the cumulative metabolite recoveries and molar ratios in urine collected from children for 2, 4, 6, and 8 hours after caffeine and dextromethorphan administration to determine when respective urinary molar ratios stabilize and thus likely accurately reflect enzyme activity. Subjects (n = 24, ages 3-8 years) were given 4 oz of Coca-Cola(R) ( approximately 11.5 mg caffeine) and a single oral dose of dextromethorphan (0.5 mg/kg). Urine was collected at discrete intervals (0-2, 2-4, 4-6, and 6-8 h) during an 8-hour period, and the cumulative metabolite recoveries and urinary molar ratios were calculated. CYP2D6 genotyping was also performed in 21 of 24 subjects. In CYP2D6 extensive metabolizers, the extent of recovery for relevant metabolites was equivalent by 4 hours and represented 45% to 60% of the total amount recovered in the 8-hour period. The 2-hour CYP1A2 ratio was significantly different from those of longer collection intervals. Metabolite ratios for all other enzymes (i.e., NAT-2, XO, and CYP2D6) were independent of the duration of urine collection. These data suggest that a 4-hour urine collection is adequate for the concurrent assessment of hepatic CYP1A2, NAT-2, XO, and CYP2D6 activity in children ages 3 to 8 years who are CYP2D6 extensive metabolizers, using standard caffeine and dextromethorphan phenotyping methods. Longer collection periods may be required, however, in younger children or CYP2D6 poor metabolizers. PMID:15199075

  7. Polybrominated diphenyl ethers alter hepatic phosphoenolpyruvate carboxykinase enzyme kinetics in male Wistar rats: implications for lipid and glucose metabolism.

    PubMed

    Nash, Jessica T; Szabo, David T; Carey, Gale B

    2013-01-01

    Xenobiotics such as phenobarbital, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and Aroclor 1254 significantly suppress the activity of a key gluconeogenic and glyceroneogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), suggesting that xenobiotics disrupt hepatic glucose and fat metabolism. The effects of polybrominated diphenyl ethers (PBDE), a family of synthetic flame-retardant chemicals, on PEPCK activity is unknown. This study investigated the effect of DE-71, a commercial PBDE mixture, on PEPCK enzyme kinetics. Forty-eight 1-mo-old male Wistar rats were gavaged daily with either corn oil or corn oil containing 14 mg/kg DE-71 for 3, 14, or 28 d (n = 8/group). At each time point, fasting plasma glucose, insulin, and C-peptide were measured and hepatic PEPCK activity, lipid content, and three cytochrome P-450 enzymes (CYP1A, -2B, and -3A) were assayed. PBDE treatment for 28 d significantly decreased PEPCK Vmax ( μ mol/min/g liver weight) by 43% and increased liver lipid by 20%, compared to control. CYP1A, -2B, and -3A Vmax values were enhanced by 5-, 6-, and 39-fold, respectively, at both 14 and 28 d in treated rats compared to control. There was a significant inverse and temporal correlation between CYP3A and PEPCK Vmax for the treatment group. Fasting plasma glucose, insulin, and C-peptide levels were not markedly affected by treatment, but the glucose:insulin ratio was significantly higher in treated compared to control rats. Data suggest that in vivo PBDE treatment compromises liver glucose and lipid metabolism, and may influence whole-body insulin sensitivity.

  8. Assessment of a dry extract from milk thistle (Silybum marianum) for interference with human liver cytochrome-P450 activities.

    PubMed

    Doehmer, Johannes; Weiss, Gabriele; McGregor, Gerard P; Appel, Kurt

    2011-02-01

    The effect of a standardised dry extract from Silybum marianum (HEPAR-PASC®) on the enzyme kinetics of cytochrome-P450 isoenzymes (CYP) was investigated with primary human hepatocytes and human liver microsomes in order to assess the potential for drug-drug interactions. A cytotoxic effect on hepatocytes was observed at concentrations at and above 50 μg/ml. The EC(50) value was calculated to be 72.0 μg/ml. Therefore, the chosen test concentrations for CYP induction on human hepatocytes were 50, 10, and 1.5 μg/ml, which allowed for interpretation of the clinical significance of the data with a range of 50-1-fold c(max) at maximal recommended doses. No induction was observed at the lowest concentration of 1.5 μg/ml, which is close to c(max). The extract did not induce CYP 3A4 at any of the tested concentrations. A low or marginal induction of 1A2, 2B6, and 2E1 at the maximum concentration of 50 μg/ml was observed. CYP inhibition on human microsomes was tested at concentrations of 150, 15, and 1.5 μg/ml. No or minor CYP inhibition was observed for all CYPs tested at the lowest concentration of 1.5 μg/ml, i.e. CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. At concentrations of 15 and 150 μg/ml the extract significantly inhibited CYP 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4. In these cases, K(i) values were determined. All K(i) values exceeded c(max) by at least a factor of 10-fold. According to FDA regulations 1>c(max)/K(i)>0.1 indicates, that drug-drug interactions are possible for CYPs 2C8, and 2C9, but not likely, and are remote for CYPs 2C19, 2D6, and 3A4.

  9. Technical note: use of PCR-single-strand conformation polymorphism analysis for detection of bovine beta-casein variants A1, A2, A3, and B.

    PubMed

    Barroso, A; Dunner, S; Cañón, J

    1999-10-01

    We have optimized the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) technique to screen the most frequent variants (A1, A2, A3, and B) of the bovine beta-casein gene. Five partly overlapping PCR products (233, 234, 265, 466, and 498 bp) of Exon VII of the beta-casein gene that encompass the target point mutations were heat-denatured, separated on nondenaturing polyacrylamide gels, and silver-stained. Simultaneous detection of all variants in reference samples of known genotypes (A1A2, A2A2, A1A3, A1B, and A2B) was best achieved on 17% polyacrylamide (100:1 acrylamide:bis-acrylamide ratio) gels with the PCR product of 234 bp. These results were confirmed by sequencing the allele-specific SSCP bands directly excised from polyacrylamide gels. A population of 65 anonymous samples belonging to various breeds was then analyzed twice, without discrepancies in a blind trial. Routine beta-casein genotyping using PCR-SSCP is proposed as a cost-effective, fast, and sensitive technique.

  10. Mutation in a gene for type I procollagen (COL1A2) in a woman with postmenopausal osteoporosis: evidence for phenotypic and genotypic overlap with mild osteogenesis imperfecta.

    PubMed Central

    Spotila, L D; Constantinou, C D; Sereda, L; Ganguly, A; Riggs, B L; Prockop, D J

    1991-01-01

    Mutations in the two genes for type I collagen (COL1A1 or COL1A2) cause osteogenesis imperfecta (OI), a heritable disease characterized by moderate to extreme brittleness of bone early in life. Here we show that a 52-year-old postmenopausal woman with severe osteopenia and a compression fracture of a thoracic vertebra had a mutation in the gene for the alpha 2(I) chain of type I collagen (COL1A2) similar to mutations that cause OI. cDNA was prepared from the woman's skin fibroblast RNA and assayed for the presence of a mutation by treating DNA heteroduplexes with carbodiimide. The results indicated a sequence variation in the region encoding amino acid residues 660-667 of the alpha 2(I) chain. Further analysis demonstrated a single-base mutation that caused a serine-for-glycine substitution at position 661 of the alpha 2(I) triple-helical domain. The substitution produced posttranslational overmodification of the collagen triple helix, as is seen with most glycine substitutions that cause OI. The patient had a history of five previous fractures, slightly blue sclerae, and slight hearing loss. Therefore, the results suggest that there may be phenotypic and genotypic overlap between mild osteogenesis imperfecta and postmenopausal osteoporosis, and that a subset of women with postmenopausal osteoporosis may have mutations in the genes for type I procollagen. Images PMID:2052622

  11. Mutation in a gene for type I procollagen (COL1A2) in a woman with postmenopausal osteoporosis: Evidence for phenotypic and genotypic overlap with mild osteogenesis imperfecta

    SciTech Connect

    Spotila, L.D.; Constantinou, C.D.; Sereda, L.; Ganguly, A.; Prockop, D.J. ); Riggs, B.L. )

    1991-06-15

    Mutations in the two genes for type I collagen (COL1A1 or COL1A2) cause osteogenesis imperfecta (OI), a heritable disease characterized by moderate to extreme brittleness of bone early in life. Here, the authors show that a 52-year-old post menopausal woman with severe osteopenia and a compression fracture of a thoracic vertebra had a mutation in the gene for the {alpha}2(I) chain of type I collagen (COL1A2) similar to mutations that cause OI. cDNA was prepared from the woman's skin fibroblast RNA and assayed for the presence of a mutation by treating DNA heteroduplexes with carbodiimide. The results indicated a sequence variation in the region encoding amino acid residues 660-667 of the {alpha}2(I) chain. Further analysis demonstrated a single-base mutation that caused a serine-for-glycine substitution at position 661 of the {alpha}2(I) triple-helical domain. The substitution produced posttranslational overmodification of the collagen triple helix, as is seen with most glycine substitutions that cause OI. The patient had a history of five previous fractures, slightly blue sclerae, and slight hearing loss. Therefore, the results suggest that there may be phenotypic and genotypic overlap between mild osteogenesis imperfecta and postmenopausal osteoporosis, and that a subset of women with postmenopausal osteoporosis may have mutations in the genes for type I procollagen.

  12. Exacerbation of Acetaminophen Hepatotoxicity by the Anthelmentic Drug Fenbendazole

    PubMed Central

    Gardner, Carol R.; Mishin, Vladimir; Laskin, Jeffrey D.; Laskin, Debra L.

    2012-01-01

    Fenbendazole is a broad-spectrum anthelmintic drug widely used to prevent or treat nematode infections in laboratory rodent colonies. Potential interactions between fenbendazole and hepatotoxicants such as acetaminophen are unknown, and this was investigated in this study. Mice were fed a control diet or a diet containing fenbendazole (8–12 mg/kg/day) for 7 days prior to treatment with acetaminophen (300 mg/kg) or phosphate buffered saline. In mice fed a control diet, acetaminophen administration resulted in centrilobular hepatic necrosis and increases in serum transaminases, which were evident within 12 h. Acetaminophen-induced hepatotoxicity was markedly increased in mice fed the fenbendazole-containing diet, as measured histologically and by significant increases in serum transaminase levels. Moreover, in mice fed the fenbendazole-containing diet, but not the control diet, 63% mortality was observed within 24 h of acetaminophen administration. Fenbendazole by itself had no effect on liver histology or serum transaminases. To determine if exaggerated hepatotoxicity was due to alterations in acetaminophen metabolism, we analyzed sera for the presence of free acetaminophen and acetaminophen-glucuronide. We found that there were no differences in acetaminophen turnover. We also measured cytochrome P450 (cyp) 2e1, cyp3a, and cyp1a2 activity. Whereas fenbendazole had no effect on the activity of cyp2e1 or cyp3a, cyp1a2 was suppressed. A prolonged suppression of hepatic glutathione (GSH) was also observed in acetaminophen-treated mice fed the fenbendazole-containing diet when compared with the control diet. These data demonstrate that fenbendazole exacerbates the hepatotoxicity of acetaminophen, an effect that is related to persistent GSH depletion. These findings are novel and suggest a potential drug-drug interaction that should be considered in experimental protocols evaluating mechanisms of hepatotoxicity in rodent colonies treated with fenbendazole. PMID

  13. Estimating pediatric doses of drugs metabolized by cytochrome P450 (CYP) isozymes, based on physiological liver development and serum protein levels.

    PubMed

    Suzuki, Shinya; Murayama, Yuka; Sugiyama, Erika; Hirunpanich, Vilasinee; Saito, Kiyomi; Sekiyama, Masao; Sato, Hitoshi

    2010-04-01

    We established a method for estimating pediatric doses of drugs metabolized by cytochrome P450 (CYP) isozymes, using the free fraction of drug in plasma (fu), serum protein level (P), liver volume (LV), and CYP activity (Vmax/Km) as indices of physiological and biochemical development in children up to 15 years old. This method allows the child/adult dose ratio (D(C)/D(A))=child/adult oral clearance ratio (CL((PO)(C))/CL((PO)(A))) of drugs mainly metabolized in the liver to be estimated by the following equation: [formula: see text]. Major metabolism of drugs was ascribed to CYP1A2 for theophylline and caffeine, and CYP1A2 and CYP2D6 for propranolol and mexiletine. For theophylline and caffeine, CL((PO)(C))/CL((PO)(A)) calculated from the child/adult body surface area ratio (BSA ratio) and the value calculated by our method were compared, using CL((PO)(C))/CL((PO)(A)) calculated from the clearance ratio based on population pharmacokinetics (PPK ratio) as a reference. For all drugs, pediatric doses calculated from the Crawford equation and our equation were compared, with predetermined doses as the reference. For theophylline and caffeine, the relative accuracy of our method was significantly higher than that of BSA-based estimation when the PPK ratio was used for reference. For theophylline, caffeine, and propranolol, the relative accuracy of our method was significantly higher than that of BSA-based estimation when predetermined doses were used for reference. These findings indicate the validity of our method which considers the physiological and biochemical development (i.e., fu, P, LV, and CYP activity) for pediatric dose estimation. PMID:20372009

  14. Gene Silencing and Haploinsufficiency of Csk Increase Blood Pressure

    PubMed Central

    Kim, Sung-Moon; Ji, Su-Min; Park, So-Yon; Kim, Marina E.; Jigden, Baigalmaa; Lim, Ji Eun; Hwang, Sue-Yun; Lee, Young-Ho; Oh, Bermseok

    2016-01-01

    Objective Recent genome-wide association studies have identified 33 human genetic loci that influence blood pressure. The 15q24 locus is one such locus that has been confirmed in Asians and Europeans. There are 21 genes in the locus within a 1-Mb boundary, but a functional link of these genes to blood pressure has not been reported. We aimed to identify a causative gene for blood pressure change in the 15q24 locus. Methods and Results CSK and ULK3 were selected as candidate genes based on eQTL analysis studies that showed the association between gene transcript levels and the lead SNP (rs1378942). Injection of siRNAs for mouse homologs Csk, Ulk3, and Cyp1a2 (negative control) showed reduced target gene mRNA levels in vivo. However, Csk siRNA only increased blood pressure while Ulk3 and Cyp1a2 siRNA did not change it. Further, blood pressure in Csk+/- heterozygotes was higher than in wild-type, consistent with what we observed in Csk siRNA-injected mice. We confirmed that haploinsufficiency of Csk increased the active form of Src in Csk+/- mice aorta. We also showed that inhibition of Src by PP2, a Src inhibitor decreased high blood pressure in Csk+/- mice and the active Src in Csk+/- mice aorta and in Csk knock-down vascular smooth muscle cells, suggesting blood pressure regulation by Csk through Src. Conclusions Our study demonstrates that Csk is a causative gene in the 15q24 locus and regulates blood pressure through Src, and these findings provide a novel therapeutic target for the treatment of hypertension. PMID:26751575

  15. Metabolism of (-)-cis- and (-)-trans-rose oxide by cytochrome P450 enzymes in human liver microsomes.

    PubMed

    Nakahashi, Hiroshi; Yamamura, Yuuki; Usami, Atsushi; Rangsunvigit, Pramoch; Malakul, Pomthong; Miyazawa, Mitsuo

    2015-12-01

    The in vitro metabolism of (-)-cis- and (-)-trans-rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9-oxidized metabolite were determined using gas chromatography-mass spectrometry (GC-MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (-)-cis- and (-)-trans-rose oxide. CYP1A2 also efficiently oxidized (-)-cis-rose oxide at the 9-position but not (-)-trans-rose oxide. α-Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti-CYP2B6 antibody inhibited (-)-cis-rose oxide 9-hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (-)-trans-rose oxide was suppressed by thioTEPA and anti-CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations correlated with (S)-mephenytoin N-demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (-)-cis- and (-)-trans-rose oxide in human liver microsomes.

  16. Inhibition of human cytochrome P450 enzymes by Bacopa monnieri standardized extract and constituents.

    PubMed

    Ramasamy, Seetha; Kiew, Lik Voon; Chung, Lip Yong

    2014-02-24

    Bacopa monnieri and the constituents of this plant, especially bacosides, possess various neuropharmacological properties. Like drugs, some herbal extracts and the constituents of their extracts alter cytochrome P450 (CYP) enzymes, causing potential herb-drug interactions. The effects of Bacopa monnieri standardized extract and the bacosides from the extract on five major CYP isoforms in vitro were analyzed using a luminescent CYP recombinant human enzyme assay. B. monnieri extract exhibited non-competitive inhibition of CYP2C19 (IC50/Ki = 23.67/9.5 µg/mL), CYP2C9 (36.49/12.5 µg/mL), CYP1A2 (52.20/25.1 µg/mL); competitive inhibition of CYP3A4 (83.95/14.5 µg/mL) and weak inhibition of CYP2D6 (IC50 = 2061.50 µg/mL). However, the bacosides showed negligible inhibition of the same isoforms. B. monnieri, which is orally administered, has a higher concentration in the gut than the liver; therefore, this herb could exhibit stronger inhibition of intestinal CYPs than hepatic CYPs. At an estimated gut concentration of 600 µg/mL (based on a daily dosage of 300 mg/day), B. monnieri reduced the catalytic activities of CYP3A4, CYP2C9 and CYP2C19 to less than 10% compared to the total activity (without inhibitor = 100%). These findings suggest that B. monnieri extract could contribute to herb-drug interactions when orally co-administered with drugs metabolized by CYP1A2, CYP3A4, CYP2C9 and CYP2C19.

  17. Evaluation of the impact of Flos Daturae on rat hepatic cytochrome P450 enzymes by cocktail probe drugs.

    PubMed

    Geng, Peiwu; Wang, Shuanghu; Wang, Chunjie; Chen, Jianmiao; Zhang, Lijing; Yang, Suping; Wen, Congcong; Zhou, Yunfang; Zhang, Meiling

    2015-01-01

    Flos Daturae, known as "baimantuoluo" or "yangjinhua" in China, has been used for centuries in Traditional Chinese Medicine for the treatment of asthma, convulsions, pain, and rheumatism. To investigate the influences of Flos Daturae on the activities of rat CYP450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2B6, CYP2D6 and CYP3A4) using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained phenacetin (10 mg/kg), tolbutamide (1 mg/kg), omeprazole (10 mg/kg), bupropion (10 mg/kg), metoprolol (10 mg/kg) and testosterone (10 mg/kg), was intragastric administered to rats treated with a single low or high dose of Flos Daturae decotion for 7days. Blood samples collected at a series of time-points in plasma were determined by UPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 3.0. The results from the present in vivo study showed that Flos Daturae induce the activity of CYP2D6 enzyme with the decreased Cmax, AUC(0-∞) (P < 0.05) and the increased CL (P < 0.05). However, there were no significant differences of other probe drugs in plasma concentration and pharmacokinetic parameters. There were no significant effects on rat CYP1A2, CYP3A4, CYP2B6, CYP2C9 and CYP2C19 by Flos Daturae. Therefore, the resulting data suggested that caution was needed when Flos Daturae was co-administered with CYP2D6 substrates, which may result in treatment failure and herb-drug interactions. PMID:26885208

  18. [Importance of drug interactions with smoking in modern drug research].

    PubMed

    Laki, Szilvia; Kalapos-Kovács, Bernadett; Antal, István; Klebovich, Imre

    2013-01-01

    Drug interaction is a process during which a drug's fate in the body or its pharmacological properties are altered by an influencing factor. The extent of the drug interaction's effect can vary. The interaction could result from the modulation by another drug, food, alcohol, caffeine, narcotics, a drug influencing absorption or smoking. Moreover, transporter interactions with smoking could also have a major impact on many drug's efficacy. Clinically relevant drug interactions with smoking were classified in terms of their effect: pharmacokinetic, pharmacodynamic and transporter interactions. Policyclic aromatic carbohydrates, found in cigarette smoke, have enzyme inducing properties. The interaction affects mainly the hepatic isoenzyme CYP1A2. Interactions caused by smoking have an effect on all drugs being substrates of and therefore metabolised by CYP1A2. Pharmacokinetic alteration can also occur during the absorption, distribution and elimination process. The pharmacodynamic interactions are mainly caused by the effects of nicotine, a cigarette smoke component. Through interactions, smoking could also modify the activity of transporter proteins, altering this way the ADME properties of many drugs. Since smoking is one of the deadliest artefact in the history of human civilisation, identifying drug interactions with smoking is the physician's and pharmacist's major responsibility and task. Moreover, it is necessary to identify the patient's smoking habits during a medical treatment. This review aims to investigate the main types of drug interactions (PK/PD), identify factors influencing the activity of CYP enzymes and transporters, and also summarize the mechanisms of the most important drug interactions with smoking and their clinically relevant consequences (Table II-VI.). Drugs, with effects somehow altered by smoking-interactions, have been studied. PMID:24575657

  19. Exacerbation of acetaminophen hepatotoxicity by the anthelmentic drug fenbendazole.

    PubMed

    Gardner, Carol R; Mishin, Vladimir; Laskin, Jeffrey D; Laskin, Debra L

    2012-02-01

    Fenbendazole is a broad-spectrum anthelmintic drug widely used to prevent or treat nematode infections in laboratory rodent colonies. Potential interactions between fenbendazole and hepatotoxicants such as acetaminophen are unknown, and this was investigated in this study. Mice were fed a control diet or a diet containing fenbendazole (8-12 mg/kg/day) for 7 days prior to treatment with acetaminophen (300 mg/kg) or phosphate buffered saline. In mice fed a control diet, acetaminophen administration resulted in centrilobular hepatic necrosis and increases in serum transaminases, which were evident within 12 h. Acetaminophen-induced hepatotoxicity was markedly increased in mice fed the fenbendazole-containing diet, as measured histologically and by significant increases in serum transaminase levels. Moreover, in mice fed the fenbendazole-containing diet, but not the control diet, 63% mortality was observed within 24 h of acetaminophen administration. Fenbendazole by itself had no effect on liver histology or serum transaminases. To determine if exaggerated hepatotoxicity was due to alterations in acetaminophen metabolism, we analyzed sera for the presence of free acetaminophen and acetaminophen-glucuronide. We found that there were no differences in acetaminophen turnover. We also measured cytochrome P450 (cyp) 2e1, cyp3a, and cyp1a2 activity. Whereas fenbendazole had no effect on the activity of cyp2e1 or cyp3a, cyp1a2 was suppressed. A prolonged suppression of hepatic glutathione (GSH) was also observed in acetaminophen-treated mice fed the fenbendazole-containing diet when compared with the control diet. These data demonstrate that fenbendazole exacerbates the hepatotoxicity of acetaminophen, an effect that is related to persistent GSH depletion. These findings are novel and suggest a potential drug-drug interaction that should be considered in experimental protocols evaluating mechanisms of hepatotoxicity in rodent colonies treated with fenbendazole.

  20. Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes.

    PubMed

    Price, R J; Scott, M P; Giddings, A M; Walters, D G; Stierum, R H; Meredith, C; Lake, B G

    2008-06-01

    1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200 microM TB produced concentration-dependent increases in CYP1A2, CYP2B6 and CYP3A4 mRNA levels, whereas treatment with BHT increased CYP2B6 and CYP3A4 mRNA levels. CYP1A2, CYP2B6 and CYP3A4 mRNA levels were induced around 48-, 21- and 9-fold, respectively, by 200 microM TB, with CYP2B6 and CYP 3A4 mRNA levels being induced around 12- and 7-fold, respectively, by 200 microM BHT. 3. In contrast, the treatment of human hepatocytes for 72 h with PG and CC had little or no effect on CYP mRNA levels. 4. The treatment of human hepatocytes with TB also induced CYP1A-dependent 7-ethoxyresorufin O-deethylase activity, whereas BHT induced CYP3A-dependent testosterone 6beta-hydroxylase activity. 5. In summary, the results demonstrate that TB is a mixed inducer of CYP forms in human hepatocytes inducing CYP1A, CYP2B and CYP3A forms, whereas BHT is an inducer of CYP2B and CYP3A forms.

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

    PubMed Central

    Sun, Hongmao; Veith, Henrike; Xia, Menghang; Austin, Christopher P.; Tice, Raymond R.; Huang, Ruili

    2012-01-01

    The human cytochrome P450 (CYP) enzyme family is involved in the biotransformation of many xenobiotics. As part of the U.S. Tox21 Phase I effort, we profiled the CYP activity of approximately three thousand compounds, primarily those of environmental concern, against human CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 isoforms in a quantitative high throughput screening (qHTS) format. In order to evaluate the extent to which computational models built from a drug-like library screened in these five CYP assays under the same conditions can accurately predict the outcome of an environmental compound library, five support vector machines (SVM) models built from over 17,000 drug-like compounds were challenged to predict the CYP activities of the Tox21 compound collection. Although a large fraction of the test compounds fall outside of the applicability domain (AD) of the models, as measured by k-nearest neighbor (k-NN) similarities, the predictions were largely accurate for CYP1A2, CYP2C9, and CYP3A4 ioszymes with area under the receiver operator characteristic curves (AUC-ROC) ranging between 0.82 and 0.84. The lower predictive power of the CYP2C19 model (AUC-ROC = 0.76) is caused by experimental errors and that of the CYP2D6 model (AUC-ROC = 0.76) can be rescued by rebalancing the training data. Our results demonstrate that decomposing molecules into atom types enhanced the coverage of the AD and that computational models built from drug-like molecules can be used to predict the ability of non-drug like compounds to interact with these CYPs. PMID:23459712

  2. High-quality permanent draft genome sequence of Bradyrhizobium sp. Ai1a-2; a microsymbiont of Andira inermis discovered in Costa Rica

    SciTech Connect

    Tian, Rui; Parker, Matthew; Seshadri, Rekha; Reddy, T. B. K.; Markowitz, Victor; Ivanova, Natalia; Pati, Amrita; Woyke, Tanja; Baeshen, Mohammed; Baeshen, Nabih; Kyrpides, Nikos; Reeve, Wayne

    2015-06-14

    Bradyrhizobium sp. Ai1a-2 is is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen fixing root nodule of Andira inermis collected from Tres Piedras in Costa Rica. In this report we describe, for the first time, the genome sequence information and annotation of this legume microsymbiont. The 9,029,266 bp genome has a GC content of 62.56% with 247 contigs arranged into 246 scaffolds. The assembled genome contains 8,482 protein-coding genes and 102 RNA-only encoding genes. Lastly, this rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Root Nodule Bacteria (GEBA-RNB) project proposal.

  3. High-quality permanent draft genome sequence of Bradyrhizobium sp. Ai1a-2; a microsymbiont of Andira inermis discovered in Costa Rica

    DOE PAGESBeta

    Tian, Rui; Parker, Matthew; Seshadri, Rekha; Reddy, T. B. K.; Markowitz, Victor; Ivanova, Natalia; Pati, Amrita; Woyke, Tanja; Baeshen, Mohammed; Baeshen, Nabih; et al

    2015-06-14

    Bradyrhizobium sp. Ai1a-2 is is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an effective nitrogen fixing root nodule of Andira inermis collected from Tres Piedras in Costa Rica. In this report we describe, for the first time, the genome sequence information and annotation of this legume microsymbiont. The 9,029,266 bp genome has a GC content of 62.56% with 247 contigs arranged into 246 scaffolds. The assembled genome contains 8,482 protein-coding genes and 102 RNA-only encoding genes. Lastly, this rhizobial genome was sequenced as part of the DOE Joint Genome Institute 2010 Genomic Encyclopedia for Bacteria and Archaea-Rootmore » Nodule Bacteria (GEBA-RNB) project proposal.« less

  4. High-throughput pharmacogenetics identifies SLCO1A2 polymorphisms as candidates to elucidate the risk of febrile neutropenia in the breast cancer RAPP-01 trial.

    PubMed

    Callens, Céline; Debled, Marc; Delord, Marc; Turbiez-Stalain, Isabelle; Veyret, Corinne; Bièche, Ivan; Brain, Etienne

    2015-09-01

    The RAPP-01 clinical trial compared two adjuvant chemotherapies, doxorubicin plus docetaxel (arm A) versus doxorubicin plus cyclophosphamide (arm B), in 627 women with breast cancer. It stopped prematurely when three severe adverse events occurred among patients with febrile neutropenia (FN), all in the arm A. FN occurred in 40.8% (126/311) in arm A versus 7.1% (22/316) in arm B. We investigated Single Nucleotide Polymorphisms (SNPs) in drug transporter and metabolism genes potentially incriminated in this excess of FN. Using a dedicated DNA chip, we tested association of SNPs belonging to 97 transporter and 68 metabolizing genes with FN occurrence in 155 patients enrolled in the RAPP-01 trial, 85 in arm A and 70 in arm B. Association study in the 85 patients receiving docetaxel identified two SNPs, rs4762699 and rs2857468, both located in the SLCO1A2 gene. Haplotype T-T was associated with a high risk of FN: 83.3% of patients with at least one copy of T-T versus 32.8% in patients with other haplotypes (odds ratio = 10.25, P = 1.4e-4). In a multivariate logistic model adjusted for treatment arm, effect of haplotype T-T remained significant (odds ratio = 6.84, P = 1.15e-4). FN in patients receiving docetaxel in the RAPP-01 trial is significantly associated with the haplotype T-T in rs4762699 and rs2857468 in the SLCO1A2 transporter gene. This result should be validated in an independent cohort.

  5. Evaluation of cytochrome P450 inductions by anti-epileptic drug oxcarbazepine, 10-hydroxyoxcarbazepine, and carbamazepine using human hepatocytes and HepaRG cells.

    PubMed

    Sugiyama, Ikuo; Murayama, Norie; Kuroki, Ayaka; Kota, Jagannath; Iwano, Shunsuke; Yamazaki, Hiroshi; Hirota, Takashi

    2016-09-01

    Anti-epileptic drug oxcarbazepine is structurally related to carbamazepine, but has reportedly different metabolic pathway. Auto-induction potentials of oxcarbazepine, its pharmacologically active metabolite 10-hydroxyoxcarbazepine and carbamazepine were evaluated by cytochrome P450 (CYP) 1A2, CYP2B6 and CYP3A4 mRNA levels and primary metabolic rates using human hepatocytes and HepaRG cells. For the CYP1A2 the induction potential determined as the fold change in mRNA levels was 7.2 (range: 2.3-11.5) and 10.0 (6.2-13.7) for oxcarbazepine and carbamazepine, respectively, while 10-hydroxyoxcarbazepine did not induce. The fold change in mRNA levels for CYP2B6 was 11.5 (3.2-19.3), 7.0 (2.5-10.8) and 14.8 (3.1-29.1) for oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine, respectively. The fold change for CYP3A4 induction level by oxcarbazepine, 10-hydroxyoxcarbazepine and carbamazepine was 3.5 (1.2-7.4), 2.7 (0.8-5.7) and 8.3 (3.5-14.5), respectively. The data suggest lower induction potential of oxcarbazepine and 10-hydroxyoxcarbazepine relative to carbamazepine. The results in HepaRG cells showed similar trend as the human hepatocytes. After incubation for 72 h in hepatocytes and HepaRG cells, auto-induction was evident for only carbamazepine metabolism. The 10-keto group instead of double bond at C10 position is evidently a determinant factor for limited auto-induction of P450 enzymes by oxcarbazepine. PMID:26711482

  6. In vitro inhibitory effects of asiaticoside and madecassoside on human cytochrome P450.

    PubMed

    Winitthana, T; Niwattisaiwong, N; Patarapanich, C; Tantisira, M H; Lawanprasert, S

    2011-06-01

    The inhibitory effects and types of inhibition of asiaticoside and madecassoside on human CYPs were studied in vitro using recombinant human CYPs. The median inhibitory concentrations (IC50) of asiaticoside and madecassoside were determined for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. Asiaticoside inhibited CYP2C19 (IC50 = 412.68 ± 15.44 μM) and CYP3A4 (IC50=343.35 ± 29.35 μM). Madecassoside also inhibited CYP2C19 (IC50 = 539.04 ± 14.18 μM) and CYP3A4 (IC50 = 453.32 ± 39.33 μM). Asiaticoside and madecassoside had no effect on the activities of CYP1A2, CYP2C9 and CYP2D6 and CYP2E1. Assessment of mechanism-based inhibition and the type of inhibition were performed for asiaticoside and madecassoside with CYP2C19 and CYP3A4. These results suggested that madecassoside is a mechanism-based inhibitor of CYP2C19 and CYP3A4. Assessment of mechanism-based inhibition by asiaticoside was limited by its low solubility. Asiaticoside exhibited non-competitive inhibition of CYP2C19 (Ki=385.24 ± 8.75 μM) and CYP3A4 (Ki = 535.93 ± 18.99 μM). Madecassoside also showed non-competitive inhibition of CYP2C19 (Ki = 109.62 ± 6.14 μM) and CYP3A4 (Ki = 456.84 ± 16.43 μM). These results suggest that asiaticoside and madecassoside could cause drug-drug interactions via inhibition of CYP2C19 and CYP3A4. An in vivo study is needed to examine this further.

  7. Fluvoxamine inhibition and carbamazepine induction of the metabolism of clozapine: evidence from a therapeutic drug monitoring service.

    PubMed

    Jerling, M; Lindström, L; Bondesson, U; Bertilsson, L

    1994-08-01

    Therapeutic drug monitoring data for clozapine were used to study interactions with other drugs. The distribution of the ratio concentration/dose (C/D) of clozapine was compared in four matched groups--patients simultaneously treated with benzodiazepines, patients on drugs that inhibit the cytochrome P450 enzyme CYP2D6, patients taking carbamazepine, and those not taking any of these drugs. No difference was seen among the monotherapy, CYP2D6, and benzodiazepine groups. Patients on carbamazepine had a mean 50% lower C/D than the monotherapy group (p < 0.001), indicating that carbamazepine is an inducer of the metabolism of clozapine. The C/D was inversely correlated to the daily dose of carbamazepine. Intraindividual comparisons in eight patients, with analyses both on and off carbamazepine, confirmed a substantial decrease of the clozapine concentration when carbamazepine was introduced. Four patients treated with clozapine were concomitantly given the antidepressant fluvoxamine. Three of them exhibited a much higher C/D ratio when on fluvoxamine compared with the monotherapy group. Two had their clozapine levels analyzed when on and off fluvoxamine. The dose-normalized clozapine concentration increased by a factor of 5-10 when fluvoxamine was added. We conclude that carbamazepine causes decreased clozapine plasma levels, while fluvoxamine increases the levels. The pathways are not known with certainty, but CYP1A2 may be of major importance for the metabolism of clozapine, since fluvoxamine is a potent inhibitor of this enzyme. A recent panel study suggests that determination of CYP1A2 activity with the caffeine test may be very useful for the dosing of clozapine. The induction of clozapine metabolism by carbamazepine might be partly mediated by CYP3A4.

  8. Cellular responses to oxidative stress: The (Ah) gene battery as a paradigm

    SciTech Connect

    Nebert, D.W.; Petersen, D.D.; Fornace, A.J. Jr. )

    1990-08-01

    A major source of oxidative stress in animals is plant stress metabolites, also termed phytoalexins. The aromatic hydrocarbon-responsive (Ah) gene battery is considered here as a model system in which the authors can study metabolically coordinated enzymes that respond to phytoalexin-induced oxidative stress. In the mouse, the (Ah) battery comprises at least six genes: two Phase I genes, CYP1A1 and CYP1A2; and four Phase II genes, Nmo-1, Aldh-1, Ugt-1, and Gt-1. All six genes appear to be regulated positively by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other ligands of the Ah receptor. The radiation deletion homozygote c{sup 14CoS}/c{sup 14CoS} mouse is lacking about 1.1 centiMorgans of chromosome 7. Although having no detectable CYP1A1 or CYP1A2 activation, the untreated c{sup 14CoS}/c{sup 14CoS} mouse exhibits markedly elevated transcripts of the Nmo-1 gene and three growth arrest- and DNA damage-inducible (gadd) genes. These data suggest that the missing region on chromosome 7 in the c{sup 14Cos}/c{sup 14CoS} mouse contains a gene(s), which they propose to call Nmo-1n, encoding a trans-acting factor(s) that is a negative effector of the Nmo-1 and gadd genes. The three other (Ah) battery Phase II genes behave similarly to Nmo-1 in the c{sup 14CoS}/c{sup 14CoS} mouse. This coordinated response to oxidative stress and DNA damage, by way of the release of a mammalian battery of genes from negative control, bears an interesting resemblance to the SOS response in bacteria.

  9. Strain differences in hepatic cytochrome P450 1A and 3A expression between Sprague-Dawley and Wistar rats.

    PubMed

    Kishida, Tomoyuki; Muto, Shin-ichi; Hayashi, Morimichi; Tsutsui, Masaru; Tanaka, Satoru; Murakami, Makoto; Kuroda, Junji

    2008-10-01

    Expression of hepatic cytochrome P450 (CYP) isoforms was compared in Sprague-Dawley (SD) and Wistar (WI) rats, which are commonly used strains in preclinical studies. Basal CYP1A1, CYP1A2, and CYP3A2 mRNA levels were higher in WI rats than in SD rats (by 8-, 3- and 2-fold, respectively). Treatment with phenobarbital, a potent CYP inducer, increased the predominance of expression of these three mRNAs in WI rats (by 26-, 4-, and 2-fold, respectively) along with the predominance of increased microsomal total P450 contents and smooth-surface endoplasmic reticulum in the centrilobular hepatocytes. CYP1A enzymatic activity was also higher in WI rats than in SD rats. No strain differences were observed in phenobarbital induction of CYP2B1/2, CYP2C6, or CYP3A1. CYP3A2 mRNA was more strongly induced by dexamethasone, a typical inducer of CYP3A, together with CYP3A1 mRNA, in WI rats than in SD rats (by 2-fold), whereas the CYP1A1 and CYP1A2 mRNA expression induced by beta-naphtoflavone, a typical inducer of CYP1A, did not differ between the two strains. Furthermore, WI rats exhibited predominantly arylhydrocarbon receptor, pregnane X receptor, and constitutive androstane receptor mRNAs, responsible for CYP1A or CYP3A induction, with phenobarbital or dexamethasone induction. In conclusion, significant, predominant expression of hepatic CYP1A and CYP3A mRNAs in WI rats was observed, possibly related to nuclear receptor-mediated induction. Considering the pharmacokinetic and toxicological importance of CYP1A and CYP3A, different outcomes might arise depending on the rat strains used in preclinical studies of drugs metabolized typically or mainly by both isoforms.

  10. Urinary Metabolites of the Dietary Carcinogen PhIP are Predictive of Colon DNA Adducts After a Low Dose Exposure in Humans

    SciTech Connect

    Malfatti, M; Dingley, K; Nowell, S; Ubick, E; Mulakken, N; Nelson, D; Lang, N; Felton, J; Turteltaub, K

    2006-04-28

    Epidemiologic evidence indicates that exposure to heterocyclic amines (HAs) in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of HAs which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of HA bioactivation in humans, the most mass abundant HA, 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers were administered a dietary relevant dose of [{sup 14}C]PhIP 48-72 h prior to surgery to remove colon tumors. Urine was collected for 24 h after dosing for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All ten subjects were phenotyped for CYP1A2, NAT2, and SULT1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N{sup 2}-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N{sup 2}-glucuronide, had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N{sup 2}-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.

  11. Differential expression of cytochrome P450 enzymes in normal and tumor tissues from childhood rhabdomyosarcoma.

    PubMed

    Molina-Ortiz, Dora; Camacho-Carranza, Rafael; González-Zamora, José Francisco; Shalkow-Kalincovstein, Jaime; Cárdenas-Cardós, Rocío; Ností-Palacios, Rosario; Vences-Mejía, Araceli

    2014-01-01

    Intratumoral expression of genes encoding Cytochrome P450 enzymes (CYP) might play a critical role not only in cancer development but also in the metabolism of anticancer drugs. The purpose of this study was to compare the mRNA expression patterns of seven representative CYPs in paired tumor and normal tissue of child patients with rabdomyosarcoma (RMS). Using real time quantitative RT-PCR, the gene expression pattern of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, and CYP3A5 were analyzed in tumor and adjacent non-tumor tissues from 13 child RMS patients. Protein concentration of CYPs was determined using Western blot. The expression levels were tested for correlation with the clinical and pathological data of the patients. Our data showed that the expression levels of CYP1A1 and CYP1A2 were negligible. Elevated expression of CYP1B1 mRNA and protein was detected in most RMS tumors and adjacent normal tissues. Most cancerous samples exhibit higher levels of both CYP3A4 and CYP3A5 compared with normal tissue samples. Expression of CYP2E1 mRNA was found to be significantly higher in tumor tissue, however no relation was found with protein levels. CYP2W1 mRNA and/or protein are mainly expressed in tumors. In conclusion, we defined the CYP gene expression profile in tumor and paired normal tissue of child patients with RMS. The overexpression of CYP2W1, CYP3A4 and CYP3A5 in tumor tissues suggests that they may be involved in RMS chemoresistance; furthermore, they may be exploited for the localized activation of anticancer prodrugs.

  12. Subacute nicotine co-exposure has no effect on 2,2',3,5',6- pentachlorobiphenyl disposition but alters hepatic cytochrome P450 expression in the male rat.

    PubMed

    Stamou, Marianna; Uwimana, Eric; Flannery, Brenna M; Kania-Korwel, Izabela; Lehmler, Hans-Joachim; Lein, Pamela J

    2015-12-01

    Polychlorinated biphenyls (PCBs) are metabolized by cytochrome P450 2B enzymes (CYP2B) and nicotine is reported to alter CYP2B activity in the brain and liver. To test the hypothesis that nicotine influences PCB disposition, 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and its metabolites were quantified in tissues of adult male Wistar rats exposed to PCB 95 (6mg/kg/d, p.o.) in the absence or presence of nicotine (1.0mg/kg/d of the tartrate salt, s.c.) for 7 consecutive days. PCB 95 was enantioselectively metabolized to hydroxylated (OH-) PCB metabolites, resulting in a pronounced enrichment of E1-PCB 95 in all tissues investigated. OH-PCBs were detected in blood and liver tissue, but were below the detection limit in adipose, brain and muscle tissues. Co-exposure to nicotine did not change PCB 95 disposition. CYP2B1 mRNA and CYP2B protein were not detected in brain tissues but were detected in liver. Co-exposure to nicotine and PCB 95 increased hepatic CYP2B1 mRNA but did not change CYP2B protein levels relative to vehicle control animals. However, hepatic CYP2B protein in animals co-exposed to PCB 95 and nicotine were reduced compared to animals that received only nicotine. Quantification of CYP2B3, CYP3A2 and CYP1A2 mRNA identified significant effects of nicotine and PCB 95 co-exposure on hepatic CYP3A2 and hippocampal CYP1A2 transcripts. Our findings suggest that nicotine co-exposure does not significantly influence PCB 95 disposition in the rat. However, these studies suggest a novel influence of PCB 95 and nicotine co-exposure on hepatic cytochrome P450 (P450) expression that may warrant further attention due to the increasing use of e-cigarettes and related products.

  13. Pharmacogenetics of drug oxidation via cytochrome P450 (CYP) in the populations of Denmark, Faroe Islands and Greenland.

    PubMed

    Brosen, Kim

    2015-09-01

    Denmark, the Faroe Islands and Greenland are three population-wise small countries on the northern part of the Northern Hemisphere, and studies carried out here on the genetic control over drug metabolism via cytochrome P450 have led to several important discoveries. Thus, CYP2D6 catalyzes the 2-hydroxylation, and CYP2C19 in part catalyzes the N-demethylation of imipramine. The phenomenon of phenocopy with regard to CYP2D6 was first described when Danish patients changed phenotype from extensive to poor metabolizers during treatment with quinidine. It was a Danish extensive metabolizer patient that became a poor metabolizer during paroxetine treatment, and this was due to the potent inhibition of CYP2D6 by paroxetine, which is also is metabolized by this enzyme. Fluoxetine and norfluoxetine are also potent inhibitors of CYP2D6, and fluvoxamine is a potent inhibitor of both CYP1A2 and CYP2C19. The bioactivation of proguanil to cycloguanil is impaired in CYP2C19 poor metabolizers. The O-demethylation of codeine and tramadol to their respective my-opioid active metabolites, morphine and (+)-O-desmethyltramadol was markedly impaired in CYP2D6 poor metabolizers compared to extensive metabolizers, and this impairs the hypoalgesic effect of the two drugs in the poor metabolizers. The frequency of CYP2D6 poor metabolizers is 2%-3% in Greenlanders and nearly 15% in the Faroese population. The frequency of CYP2C19 poor metabolizers in East Greenlanders is approximately 10%. A study in Danish mono and dizygotic twins showed that the non-polymorphic 3-N-demethylation of caffeine catalyzed by CYP1A2 is subject to approximately 70% genetic control.

  14. Differential expression of cytochrome P450 enzymes in normal and tumor tissues from childhood rhabdomyosarcoma.

    PubMed

    Molina-Ortiz, Dora; Camacho-Carranza, Rafael; González-Zamora, José Francisco; Shalkow-Kalincovstein, Jaime; Cárdenas-Cardós, Rocío; Ností-Palacios, Rosario; Vences-Mejía, Araceli

    2014-01-01

    Intratumoral expression of genes encoding Cytochrome P450 enzymes (CYP) might play a critical role not only in cancer development but also in the metabolism of anticancer drugs. The purpose of this study was to compare the mRNA expression patterns of seven representative CYPs in paired tumor and normal tissue of child patients with rabdomyosarcoma (RMS). Using real time quantitative RT-PCR, the gene expression pattern of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, and CYP3A5 were analyzed in tumor and adjacent non-tumor tissues from 13 child RMS patients. Protein concentration of CYPs was determined using Western blot. The expression levels were tested for correlation with the clinical and pathological data of the patients. Our data showed that the expression levels of CYP1A1 and CYP1A2 were negligible. Elevated expression of CYP1B1 mRNA and protein was detected in most RMS tumors and adjacent normal tissues. Most cancerous samples exhibit higher levels of both CYP3A4 and CYP3A5 compared with normal tissue samples. Expression of CYP2E1 mRNA was found to be significantly higher in tumor tissue, however no relation was found with protein levels. CYP2W1 mRNA and/or protein are mainly expressed in tumors. In conclusion, we defined the CYP gene expression profile in tumor and paired normal tissue of child patients with RMS. The overexpression of CYP2W1, CYP3A4 and CYP3A5 in tumor tissues suggests that they may be involved in RMS chemoresistance; furthermore, they may be exploited for the localized activation of anticancer prodrugs. PMID:24699256

  15. Transcriptome analysis provides new insights into liver changes induced in the rat upon dietary administration of the food additives butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole.

    PubMed

    Stierum, Rob; Conesa, Ana; Heijne, Wilbert; Ommen, Ben van; Junker, Karin; Scott, Mary P; Price, Roger J; Meredith, Clive; Lake, Brian G; Groten, John

    2008-08-01

    Transcriptomics was performed to gain insight into mechanisms of food additives butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG), and thiabendazole (TB), additives for which interactions in the liver can not be excluded. Additives were administered in diets for 28 days to Sprague-Dawley rats and cDNA microarray experiments were performed on hepatic RNA. BHT induced changes in the expression of 10 genes, including phase I (CYP2B1/2; CYP3A9; CYP2C6) and phase II metabolism (GST mu2). The CYP2B1/2 and GST expression findings were confirmed by real time RT-PCR, western blotting, and increased GST activity towards DCNB. CC altered the expression of 12 genes. Three out of these were related to peroxisomes (phytanoyl-CoA dioxygenase, enoyl-CoA hydratase; CYP4A3). Increased cyanide insensitive palmitoyl-CoA oxidation was observed, suggesting that CC is a weak peroxisome proliferator. TB changed the expression of 12 genes, including CYP1A2. In line, CYP1A2 protein expression was increased. The expression level of five genes, associated with p53 was found to change upon TB treatment, including p53 itself, GADD45alpha, DN-7, protein kinase C beta and serum albumin. These array experiments led to the novel finding that TB is capable of inducing p53 at the protein level, at least at the highest dose levels employed above the current NOAEL. The expression of eight genes changed upon PG administration. This study shows the value of gene expression profiling in food toxicology in terms of generating novel hypotheses on the mechanisms of action of food additives in relation to pathology.

  16. In vitro inhibition of the cytochrome P450 (CYP450) system by the antiplatelet drug ticlopidine: potent effect on CYP2C19 and CYP2D6

    PubMed Central

    Ko, Jae Wook; Desta, Zeruesenay; Soukhova, Nadia V; Tracy, Timothy; Flockhart, David A

    2000-01-01

    Aims To examine the potency of ticlopidine (TCL) as an inhibitor of cytochrome P450s (CYP450s) in vitro using human liver microsomes (HLMs) and recombinant human CYP450s. Methods Isoform-specific substrate probes of CYP1A2, 2C19, 2C9, 2D6, 2E1 and 3A4 were incubated in HLMs or recombinant CYPs with or without TCL. Preliminary data were generated to simulate an appropriate range of substrate and inhibitor concentrations to construct Dixon plots. In order to estimate accurately inhibition constants (Ki values) of TCL and determine the type of inhibition, data from experiments with three different HLMs for each isoform were fitted to relevant nonlinear regression enzyme inhibition models by WinNonlin. Results TCL was a potent, competitive inhibitor of CYP2C19 (Ki = 1.2 ± 0.5 µm) and of CYP2D6 (Ki = 3.4 ± 0.3 µm). These Ki values fell within the therapeutic steady-state plasma concentrations of TCL (1–3 µm). TCL was also a moderate inhibitor of CYP1A2 (Ki = 49 ± 19 µm) and a weak inhibitor of CYP2C9 (Ki > 75 µm), but its effect on the activities of CYP2E1 (Ki = 584 ± 48 µm) and CYP3A (> 1000 µm) was marginal. Conclusions TCL appears to be a broad-spectrum inhibitor of the CYP isoforms, but clinically significant adverse drug interactions are most likely with drugs that are substrates of CYP2C19 or CYP2D6. PMID:10759690

  17. The Pseudomonas aeruginosa PA14 ABC Transporter NppA1A2BCD Is Required for Uptake of Peptidyl Nucleoside Antibiotics

    PubMed Central

    Braun, Yvonne; Dubiley, Svetlana; Lafon, Corinne; Köhler, Thilo; Page, Malcolm G. P.; Mourez, Michael; Severinov, Konstantin

    2015-01-01

    ABSTRACT Analysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14. IMPORTANCE One of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the

  18. Adenosine A(1), A(2a), A(2b), and A(3) receptors in hematopoiesis. 1. Expression of receptor mRNA in four mouse hematopoietic precursor cells.

    PubMed

    Streitová, D; Sefc, L; Savvulidi, F; Pospísil, M; Holá, J; Hofer, M

    2010-01-01

    Four mouse bone marrow or thymus cell populations, namely granulopoietic/monocytopoietic, erythropoietic, B-lymphopoietic, and T-lymphopoietic precursor cells have been assayed by RT-PCR technique for the presence and relative amounts of adenosine A(1), A(2a), A(2b), and A(3) receptor mRNA. It has been found that (i) all four populations studied express all four adenosine receptor subtypes, (ii) the A(1), receptor is the least expressed in all populations studied, (iii) the A(3) receptor is markedly expressed in the populations of granulopoietic/monocytopoietic and erythropoietic cells, (iv) the A(2a) receptor is markedly expressed in the populations of B-lymphopoietic and T-lymphopoietic cells, and v) the A(2b) receptor does not predominate in any of the precursor cells studied. Our data offer a new possibility for the assessment of the readiness of these cells to respond, by receptor-mediated mechanisms, to adenosine or its analogs present in the tissues as a result of endogenous processes and/or following their administration.

  19. Genetic studies on Vysyas of Andhra Pradesh, S. India: A1A2BO, Rh (O) D, transferrin, group specific component, haptoglobin and pseudocholinesterase types.

    PubMed

    Gopalam, K B; Rao, P R

    1981-01-01

    Vysya population, an endogamous Hindu caste group, was sampled from five distant localities of Andhra Pradesh, S. India and examined for A1A2BO, Rh blood groups, Tf, Hp, Gc, cholinesterase E1 and E2 loci, albumin and ceruloplasmin types. The blood group A has shown an exceptionally low value in these groups and consequently there is a rise in O group frequencies (0.7429 to 0.8144). The incidence of Rh negative individuals is very low (0.1115-0.1571), being absent from one of the groups. Tf DChi is found with a frequency ranging from 0.0043 to 0.0333 with a single fast moving Tf B variant in one of the sub-populations. Hp1 gene frequencies ranged from 0.1271 to 0.2130 and Gc2 from 0.1504 to 0.2773. Silent variants at E1 locus of pseudocholinesterase were present in very high frequencies (0.0115 to 0.1925), the overall frequency being 0.1040. Only a single C+5 was found and dibucaine as well as fluoride resistant variants were rare. No variants were found at the loci of albumin and ceruloplasmin. Differentiation in the distribution of these variants in the five sub-populations of Vysyas reported is evident from these studies.

  20. DNA adducts induced by food mutagen PhIP in a mouse model expressing human sulfotransferases 1A1 and 1A2.

    PubMed

    Høie, Anja Hortemo; Monien, Bernhard Hans; Glatt, Hansruedi; Hjertholm, Hege; Husøy, Trine

    2016-04-25

    Food processing contaminant 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) has previously been shown to induce formation of DNA adducts in vivo. In a previous study the adduct levels were found to increase in a mouse model expressing human (h) sulfotransferases (SULTs) 1A1 and 1A2 after PhIP exposure, detected by (32)P-postlabelling. Isotope dilution ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) is emerging as the method of choice for selective and reproducible detection of known DNA adducts. In the present study we investigated the level and distribution of PhIP induced DNA adducts in male FVB mice 9-11 weeks of age with hSULT mice or wild-type mice (wt) using UPLC-MS/MS. Mice received a single administration of 75 mg/kg bw PhIP by oral gavage, and DNA was analysed 3h after exposure. C8-(2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine- N(2)-yl)-2'-deoxyguanosine (C8-PhIP-dG) adduct levels are significantly higher in PhIP exposed hSULT mice compared with PhIP exposed wt mice. The liver was the least affected organ in wild-type mice, whereas it was the most affected organ in hSULT mice with a 14-fold higher adduct level. PMID:26940682

  1. Partial isodisomy for maternal chromosome 7 and short stature in an individual with a mutation at the COL1A2 locus

    SciTech Connect

    Spotila, L.D.; Sereda, L.; Prockop, D.J. )

    1992-12-01

    Uniparental disomy for chromosome 7 has been described previously in two individuals with cystic fibrosis. Here, the authors describe a third case that was discovered because the proband was homozygous for a mutation in the COL1A2 gene for type I procollagen, although his mother was heterozygous and his father did not have the mutation. Phenotypically, the proband was similar to the two previously reported cases with uniparental disomy for chromosome 7, in that he was short in stature and growth retarded. Paternity was assessed with five polymorphic markers. Chromosome 7 inheritance in the proband was analyzed using 12 polymorphic markers distributed along the entire chromosome. Similar analysis of the proband's two brothers established the phase of the alleles at the various loci, assuming minimal recombination. The proband inherited only maternal alleles at five loci and was homozygous at all loci examined, except one. He was heterozygous for an RFLP at the IGBP-1 locus at 7p13-p12. The results suggest that the isodisomy was not complete because of a recombination event involving the proximal short arms of two maternal chromosomes. In addition, the phenotype of proportional dwarfism in the proband suggests imprinting of one or more growth-related genes on chromosome 7. 42 refs., 5 figs., 3 tabs.

  2. Mammalian translation elongation factor eEF1A2: X-ray structure and new features of GDP/GTP exchange mechanism in higher eukaryotes.

    PubMed

    Crepin, Thibaut; Shalak, Vyacheslav F; Yaremchuk, Anna D; Vlasenko, Dmytro O; McCarthy, Andrew; Negrutskii, Boris S; Tukalo, Michail A; El'skaya, Anna V

    2014-11-10

    Eukaryotic elongation factor eEF1A transits between the GTP- and GDP-bound conformations during the ribosomal polypeptide chain elongation. eEF1A*GTP establishes a complex with the aminoacyl-tRNA in the A site of the 80S ribosome. Correct codon-anticodon recognition triggers GTP hydrolysis, with subsequent dissociation of eEF1A*GDP from the ribosome. The structures of both the 'GTP'- and 'GDP'-bound conformations of eEF1A are unknown. Thus, the eEF1A-related ribosomal mechanisms were anticipated only by analogy with the bacterial homolog EF-Tu. Here, we report the first crystal structure of the mammalian eEF1A2*GDP complex which indicates major differences in the organization of the nucleotide-binding domain and intramolecular movements of eEF1A compared to EF-Tu. Our results explain the nucleotide exchange mechanism in the mammalian eEF1A and suggest that the first step of eEF1A*GDP dissociation from the 80S ribosome is the rotation of the nucleotide-binding domain observed after GTP hydrolysis.

  3. Modulation of carcinogen metabolizing cytochromes P450 in rat liver and kidney by cabbage and sauerkraut juices: comparison with the effects of indole-3-carbinol and phenethyl isothiocyanate.

    PubMed

    Szaefer, Hanna; Krajka-Kuźniak, Violetta; Bartoszek, Agnieszka; Baer-Dubowska, Wanda

    2012-08-01

    This study investigated the effect of raw cabbage and sauerkraut juices on the activity and expression of CYP1A1, 1A2, 1B1 and 2B in Wistar rat liver and kidney. The results were compared with those of two commercially available products of glucosinolates degradation: indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC). Significant differences in the effect of the cabbage juices as well as I3C and PEITC between the liver and kidney were found. In the liver, both juices decreased the activities of enzymatic markers of CYP1A1 and CYP1A2 after 10 days of the experiment, while in the kidney an enhancement of the activity of these enzymes was observed on days 4 and 10. The increased activity of these enzymes and CYP1A1/1A2 protein level in the liver was found after 30 days of treatment with sauerkraut juice. A similar effect was observed as a result of PEITC treatment. I3C increased the expression and activity of hepatic CYPs at all time points investigated. In conclusion, the present study demonstrates that raw cabbage and sauerkraut juices may affect CYPs involved in the activation of carcinogens/xenobiotics and in this way exert anticarcinogenic activity. The final effects, however, depend on the time-frame of exposure and the type of tissue.

  4. Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses.

    PubMed

    Gottwein, Judith M; Jensen, Sanne B; Li, Yi-Ping; Ghanem, Lubna; Scheel, Troels K H; Serre, Stéphanie B N; Mikkelsen, Lotte; Bukh, Jens

    2013-03-01

    With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi-FL) recombinants relying only on the JFH1 NS3 helicase, NS5B, and the 3' untranslated region. With identified adaptive mutations, semi-FL recombinants of genotypes(isolates) 1a(TN) and 3a(S52) produced supernatant infectivity titers of ~4 log(10) focus-forming units/ml in Huh7.5 cells. Genotype 1a(TN) adaptive mutations allowed generation of 1a(H77) semi-FL virus. Concentration-response profiles revealed the higher efficacy of the NS3 protease inhibitor asunaprevir (BMS-650032) and the NS5A inhibitor daclatasvir (BMS-790052) against 1a(TN and H77) than 3a(S52) viruses. Asunaprevir had intermediate efficacy against previously developed 2a recombinants J6/JFH1 and J6cc. Daclatasvir had intermediate efficacy against J6/JFH1, while low sensitivity was confirmed against J6cc. Using a cross-titration scheme, infected cultures were treated until viral escape or on-treatment virologic suppression occurred. Compared to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations. Inhibitors showed synergism at drug concentrations reported in vivo. In summary, semi-FL HCV recombinants, including the most advanced reported genotype 3a infectious culture system, permitted genotype-specific analysis of combination treatment in the context of the complete viral life cycle. Despite differential sensitivity to lead compound NS3 protease and NS5A inhibitors, genotype 1a, 2a, and 3a viruses were suppressed by combination treatment with relatively low

  5. In vitro digestion of purified β-casein variants A(1), A(2), B, and I: effects on antioxidant and angiotensin-converting enzyme inhibitory capacity.

    PubMed

    Petrat-Melin, B; Andersen, P; Rasmussen, J T; Poulsen, N A; Larsen, L B; Young, J F

    2015-01-01

    Genetic polymorphisms of bovine milk proteins affect the protein profile of the milk and, hence, certain technological properties, such as casein (CN) number and cheese yield. However, reports show that such polymorphisms may also affect the health-related properties of milk. Therefore, to gain insight into their digestion pattern and bioactive potential, β-CN was purified from bovine milk originating from cows homozygous for the variants A(1), A(2), B, and I by a combination of cold storage, ultracentrifugation, and acid precipitation. The purity of the isolated β-CN was determined by HPLC, variants were verified by mass spectrometry, and molar extinction coefficients at λ=280nm were determined. β-Casein from each of the variants was subjected to in vitro digestion using pepsin and pancreatic enzymes. Antioxidant and angiotensin-converting enzyme (ACE) inhibitory capacities of the hydrolysates were assessed at 3 stages of digestion and related to that of the undigested samples. Neither molar extinction coefficients nor overall digestibility varied significantly between these 4 variants; however, clear differences in digestion pattern were indicated by gel electrophoresis. In particular, after 60min of pepsin followed by 5min of pancreatic enzyme digestion, one ≈4kDa peptide with the N-terminal sequence (106)H-K-E-M-P-F-P-K- was absent from β-CN variant B. This is likely a result of the (122)Ser to (122)Arg substitution in variant B introducing a novel trypsin cleavage site, leading to the changed digestion pattern. All investigated β-CN variants exhibited a significant increase in antioxidant capacity upon digestion, as measured by the Trolox-equivalent antioxidant capacity assay. After 60min of pepsin + 120min of pancreatic enzyme digestion, the accumulated increase in antioxidant capacity was ≈1.7-fold for the 4 β-CN variants. The ACE inhibitory capacity was also significantly increased by digestion, with the B variant reaching the highest inhibitory

  6. Modulatory effect of the 5-HT1A agonist buspirone and the mixed non-hallucinogenic 5-HT1A/2A agonist ergotamine on psilocybin-induced psychedelic experience.

    PubMed

    Pokorny, Thomas; Preller, Katrin H; Kraehenmann, Rainer; Vollenweider, Franz X

    2016-04-01

    The mixed serotonin (5-HT) 1A/2A/2B/2C/6/7 receptor agonist psilocybin dose-dependently induces an altered state of consciousness (ASC) that is characterized by changes in sensory perception, mood, thought, and the sense of self. The psychological effects of psilocybin are primarily mediated by 5-HT2A receptor activation. However, accumulating evidence suggests that 5-HT1A or an interaction between 5-HT1A and 5-HT2A receptors may contribute to the overall effects of psilocybin. Therefore, we used a double-blind, counterbalanced, within-subject design to investigate the modulatory effects of the partial 5-HT1A agonist buspirone (20mg p.o.) and the non-hallucinogenic 5-HT2A/1A agonist ergotamine (3mg p.o.) on psilocybin-induced (170 µg/kg p.o.) psychological effects in two groups (n=19, n=17) of healthy human subjects. Psychological effects were assessed using the Altered State of Consciousness (5D-ASC) rating scale. Buspirone significantly reduced the 5D-ASC main scale score for Visionary Restructuralization (VR) (p<0.001), which was mostly driven by a reduction of the VR item cluster scores for elementary and complex visual hallucinations. Further, buspirone also reduced the main scale score for Oceanic Boundlessness (OB) including derealisation and depersonalisation phenomena at a trend level (p=0.062), whereas ergotamine did not show any effects on the psilocybin-induced 5D-ASC main scale scores. The present finding demonstrates that buspirone exerts inhibitory effects on psilocybin-induced effects, presumably via 5-HT1A receptor activation, an interaction between 5-HT1A and 5-HT2A receptors, or both. The data suggest that the modulation of 5-HT1A receptor activity may be a useful target in the treatment of visual hallucinations in different psychiatric and neurological diseases. PMID:26875114

  7. Modulatory effect of the 5-HT1A agonist buspirone and the mixed non-hallucinogenic 5-HT1A/2A agonist ergotamine on psilocybin-induced psychedelic experience.

    PubMed

    Pokorny, Thomas; Preller, Katrin H; Kraehenmann, Rainer; Vollenweider, Franz X

    2016-04-01

    The mixed serotonin (5-HT) 1A/2A/2B/2C/6/7 receptor agonist psilocybin dose-dependently induces an altered state of consciousness (ASC) that is characterized by changes in sensory perception, mood, thought, and the sense of self. The psychological effects of psilocybin are primarily mediated by 5-HT2A receptor activation. However, accumulating evidence suggests that 5-HT1A or an interaction between 5-HT1A and 5-HT2A receptors may contribute to the overall effects of psilocybin. Therefore, we used a double-blind, counterbalanced, within-subject design to investigate the modulatory effects of the partial 5-HT1A agonist buspirone (20mg p.o.) and the non-hallucinogenic 5-HT2A/1A agonist ergotamine (3mg p.o.) on psilocybin-induced (170 µg/kg p.o.) psychological effects in two groups (n=19, n=17) of healthy human subjects. Psychological effects were assessed using the Altered State of Consciousness (5D-ASC) rating scale. Buspirone significantly reduced the 5D-ASC main scale score for Visionary Restructuralization (VR) (p<0.001), which was mostly driven by a reduction of the VR item cluster scores for elementary and complex visual hallucinations. Further, buspirone also reduced the main scale score for Oceanic Boundlessness (OB) including derealisation and depersonalisation phenomena at a trend level (p=0.062), whereas ergotamine did not show any effects on the psilocybin-induced 5D-ASC main scale scores. The present finding demonstrates that buspirone exerts inhibitory effects on psilocybin-induced effects, presumably via 5-HT1A receptor activation, an interaction between 5-HT1A and 5-HT2A receptors, or both. The data suggest that the modulation of 5-HT1A receptor activity may be a useful target in the treatment of visual hallucinations in different psychiatric and neurological diseases.

  8. Differential cellular expression of organic anion transporting peptides OATP1A2 and OATP2B1 in the human retina and brain: implications for carrier-mediated transport of neuropeptides and neurosteriods in the CNS.

    PubMed

    Gao, Bo; Vavricka, Stephan R; Meier, Peter J; Stieger, Bruno

    2015-07-01

    Organic anion transporting polypeptides (OATPs) are polyspecific organic anion transporters, which are expressed in the blood-brain barrier, the choroid plexus, and other organs. The physiologic function of OATPs in extrahepatic tissues remains ambiguous. In rat retina, members of the OATP family are expressed. We therefore investigated the human retina for the expression of OATP1A2 and OATP2B1 and extended the study to human brain. Furthermore, we searched for peptide neurotransmitters as novel OATP substrates. OATP1A2 displayed a broad expression pattern in human retina as assessed by immunofluorescence localization. It is expressed in photoreceptor bodies and somas of amacrine cells. OATP1B2 expression is restricted to the inner nuclear layer and to the inner plexiform layer. Using paraffin sections from human cortex, cerebellum, and hippocampus, OATP1A2 was localized to neurons and neuronal processes, while OATP2B1 is expressed in endothelial cells of brain capillaries. Substance P and vasoactive intestinal peptide were identified as substrates for OATP1A2 and OATP2B1. Double-labeling immunofluorescence of human retina demonstrated the presence of substance P and of vasoactive intestinal peptides in neurons expressing OATP1A2 and OATP2B1, respectively. The expression of OATP1A2 and OATP2B1 in retinal neurons implies a role of these transporters in the reuptake of peptide neurotransmitters released from retinal neurons. The abundant expression of OATP1A2 in brain neurons points to the possibility that OATP1A2 could be involved in the homeostasis of neurosteroids. The high expression of OATP2B1 in brain capillaries supports an important function of OATPs in substance penetration across the blood-brain barrier.

  9. A complete X-ray sample of the high latitude sky from HEAO-1 A-2: log N lo S and luminosity functions

    NASA Technical Reports Server (NTRS)

    Piccinotti, G.; Mushotzky, R. F.; Boldt, E. A.; Holt, S. S.; Marshall, F. E.; Serlemitsos, P. J.; Shafer, R. A.

    1981-01-01

    An experiment was performed in which a complete X-ray survey of the 8.2 steradians of the sky at galactic latitudes where the absolute value of b is 20 deg down to a limiting sensitivity of 3.1 x ten to the minus 11th power ergs/sq cm sec in the 2-10 keV band. Of the 85 detected sources 17 were identified with galactic objects, 61 were identified with extragalactic objects, and 7 remain unidentified. The log N - log S relation for the non-galactic objects is well fit by the Euclidean relationship. The X-ray spectra of these objects were used to construct log N - log S in physical units. The complete sample of identified sources was used to construct X-ray luminosity functions, using the absolute maximum likelihood method, for clusters galaxies and active galactic nuclei.

  10. Isolation and characterization of new onionins A2 and A3 from Allium cepa, and of onionins A1, A2, and A3 from Allium fistulosum.

    PubMed

    Nohara, Toshihiro; Fujiwara, Yukio; Kudo, Rino; Yamaguchi, Koki; Ikeda, Tsuyoshi; Murakami, Kotaro; Ono, Masateru; Kajimoto, Tetsuya; Takeya, Motohiro

    2014-01-01

    In this study, the new stable sulfur-containing compounds onionins A2 (1) and A3 (2) were isolated from the acetone extracts of the bulbs of Allium cepa L. and identified as the stereoisomers of onionin A1 discovered in our previous study. Their chemical structures, 3,4-dimethyl-5-(1E-propenyl)-tetrahydrothiophene-2-sulfenic acid-S-oxides, were characterized using various spectroscopic techniques. In addition, 1 and 2 together with onionin A1 were successfully isolated from the leaves of the Welsh onion, Allium fistulosum L. The onion-extracted fractions showed good potential to inhibit the polarization of M2 activated macrophages, indicating their possible ability to inhibit tumor cell proliferation. PMID:25366317

  11. Toxicity studies with 5-hydroxymethylfurfural and its metabolite 5-sulphooxymethylfurfural in wild-type mice and transgenic mice expressing human sulphotransferases 1A1 and 1A2.

    PubMed

    Bauer-Marinovic, Morana; Taugner, Felicitas; Florian, Simone; Glatt, Hansruedi

    2012-05-01

    5-Sulphooxymethylfurfural (SMF), an electrophilic metabolite of the abundant Maillard product 5-hydroxymethylfurfural (HMF), was intraperitoneally administered to FVB/N mice. At a dosage of 250 mg/kg, most animals died after 5-11 days due to massive damage to proximal tubules. At lower dosages, administered repeatedly, tubules also were the major target of toxicity, with regeneration and atypical hyperplasia occurring at later periods. Additionally, hepatotoxic effects and serositis of peritoneal tissues were observed. SMF is a minor metabolite of HMF in conventional mice, but HMF is an excellent substrate for a major sulphotransferase (hSULT1A1) in humans. Parental FVB/N mice and FVB/N-hSULT1A1/2 mice, carrying multiple copies of the hSULT1A1/2 gene cluster, were exposed to HMF in drinking water (0, 134 and 536 mg/kg body mass/day) for 12 weeks. Nephrotoxic effects and enhanced proliferation of hepatocytes were only detected at the high dosage. They were mild and, surprisingly, unaffected by hSULT1A1/2 expression. Thus, SMF was a potent nephrotoxicant when administered as a bolus, but did not reach levels sufficient to produce serious toxicity when generated from HMF administered continuously via drinking water. This was even the case in transgenic mice expressing clearly higher HMF sulphation activity in liver and kidney than humans.

  12. Cytochrome P450 system proteins reside in different regions of the endoplasmic reticulum.

    PubMed

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

    2014-12-01

    Cytochrome P450 (P450) function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of the present study was to determine whether the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4 and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450 in specific membrane regions may provide a novel mechanism for modulating P450 function. PMID:25236845

  13. Continuous monitoring of Naproxen by a cytochrome P450-based electrochemical sensor.

    PubMed

    Baj-Rossi, C; Rezzonico Jost, T; Cavallini, A; Grassi, F; De Micheli, G; Carrara, S

    2014-03-15

    This paper reports the characterization of an electrochemical biosensor for the continuous monitoring of Naproxen based on cytochrome P450. The electrochemical biosensor is based on the drop-casting of multi-walled carbon-nanotubes (MWCNTs) and microsomal cytochrome P4501A2 (msCYP1A2) on a graphite screen-printed electrode (SPE). The proposed biosensor was employed to monitor Naproxen (NAP), a well-known anti-inflammatory compound, through cyclic voltammetry. The dynamic linear range for the amperometric detection of NAP had an upper limit of 300 µM with a corresponding limit of detection (LOD) of 16 ± 1 µM (S/N=3), which is included in NAP physiological range (9-300 µM). The MWCNT/msCYP1A2-SPE sensor was also calibrated for NAP detection in mouse serum that was previously extracted from mice, showing a slightly higher LOD (33 ± 18 µM). The stability of the msCYP1A2-based biosensor was assessed by longtime continuous cyclic voltammetric measurements. The ability of the sensor to monitor drug delivery was investigated by using a commercial micro-osmotic pump. Results show that the MWCNT/msCYP1A2-SPE sensor is capable of precisely monitoring the real-time delivery of NAP for 16 h. This work proves that the proposed electrochemical sensor might represent an innovative point-of-care solution for the personalization of drug therapies, as well as for pharmacokinetic studies in both animals and humans.

  14. Identification and phylogenetic analysis of novel cytochrome P450 1A genes from ungulate species.

    PubMed

    Darwish, Wageh Sobhy; Kawai, Yusuke; Ikenaka, Yoshinori; Yamamoto, Hideaki; Muroya, Tarou; Ishizuka, Mayumi

    2010-09-01

    As part of an ongoing effort to understand the biological response of wild and domestic ungulates to different environmental pollutants such as dioxin-like compounds, cDNAs encoding for CYP1A1 and CYP1A2 were cloned and characterized. Four novel CYP1A cDNA fragments from the livers of four wild ungulates (elephant, hippopotamus, tapir and deer) were identified. Three fragments from hippopotamus, tapir and deer were classified as CYP1A2, and the other fragment from elephant was designated as CYP1A1/2. The deduced amino acid sequences of these fragment CYP1As showed identities ranging from 76 to 97% with other animal CYP1As. The phylogenetic analysis of these fragments showed that both elephant and hippopotamus CYP1As made separate branches, while tapir and deer CYP1As were located beside that of horse and cattle respectively in the phylogenetic tree. Analysis of dN/dS ratio among the identified CYP1As indicated that odd toed ungulate CYP1A2s were exposed to different selection pressure.

  15. Identification and phylogenetic analysis of novel cytochrome P450 1A genes from ungulate species.

    PubMed

    Darwish, Wageh Sobhy; Kawai, Yusuke; Ikenaka, Yoshinori; Yamamoto, Hideaki; Muroya, Tarou; Ishizuka, Mayumi

    2010-09-01

    As part of an ongoing effort to understand the biological response of wild and domestic ungulates to different environmental pollutants such as dioxin-like compounds, cDNAs encoding for CYP1A1 and CYP1A2 were cloned and characterized. Four novel CYP1A cDNA fragments from the livers of four wild ungulates (elephant, hippopotamus, tapir and deer) were identified. Three fragments from hippopotamus, tapir and deer were classified as CYP1A2, and the other fragment from elephant was designated as CYP1A1/2. The deduced amino acid sequences of these fragment CYP1As showed identities ranging from 76 to 97% with other animal CYP1As. The phylogenetic analysis of these fragments showed that both elephant and hippopotamus CYP1As made separate branches, while tapir and deer CYP1As were located beside that of horse and cattle respectively in the phylogenetic tree. Analysis of dN/dS ratio among the identified CYP1As indicated that odd toed ungulate CYP1A2s were exposed to different selection pressure. PMID:20448415

  16. Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection.

    PubMed

    Kawakami, Hirotaka; Ohtsuki, Sumio; Kamiie, Junichi; Suzuki, Takashi; Abe, Takaaki; Terasaki, Tetsuya

    2011-01-01

    Cytochrome P450 (CYP) proteins are involved in the biological oxidation and reduction of xenobiotics, affecting the pharmacological efficiency of drugs. This study aimed to establish a method to simultaneously quantify 11 CYP isoforms by multiplexed-multiple reaction monitoring analysis with liquid chromatography tandem mass spectrometry and in silico peptide selection to clarify CYP isoform expression profiles in human liver tissue. CYP1A2, 2A6, and 2D6 target peptides were identified by shot-gun proteomic analysis, and those of other isoforms were selected by in silico peptide selection criteria. The established quantification method detected target peptides at 10  fmol, and the dynamic range of calibration curves was at least 500-fold. The quantification value of CYP1A2 in Supersomes was not significantly different between the established method and quantitative immunoblot analysis. The absolute protein expression levels of 11 CYP isoforms were determined from one pooled and 10 individual human liver microsomes. In the individual microsomes, CYP2C9 showed the highest protein expression level, and CYP1A2, 2A6, 2C19, and 3A4 protein expression exhibited more than a 20-fold difference among individuals. This highly sensitive and selective quantification method is a useful tool for the analysis of highly homologous CYP isoforms and the contribution made by each CYP isoform to drug metabolism. PMID:20564338

  17. Estimation of the binding modes with important human cytochrome P450 enzymes, drug interaction potential, pharmacokinetics, and hepatotoxicity of ginger components using molecular docking, computational, and pharmacokinetic modeling studies

    PubMed Central

    Qiu, Jia-Xuan; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Zhou, Shu-Feng; Zhu, Shengrong

    2015-01-01

    Ginger is one of the most commonly used herbal medicines for the treatment of numerous ailments and improvement of body functions. It may be used in combination with prescribed drugs. The coadministration of ginger with therapeutic drugs raises a concern of potential deleterious drug interactions via the modulation of the expression and/or activity of drug-metabolizing enzymes and drug transporters, resulting in unfavorable therapeutic outcomes. This study aimed to determine the molecular interactions between 12 main active ginger components (6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, ar-curcumene, β-bisabolene, β-sesquiphelandrene, 6-gingerdione, (−)-zingiberene, and methyl-6-isogingerol) and human cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 and to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the 12 ginger components using computational approaches and comprehensive literature search. Docking studies showed that ginger components interacted with a panel of amino acids in the active sites of CYP1A2, 2C9, 2C19, 2D6, and 3A4 mainly through hydrogen bond formation, to a lesser extent, via π–π stacking. The pharmacokinetic simulation studies showed that the [I]/[Ki] value for CYP2C9, 2C19, and 3A4 ranged from 0.0002 to 19.6 and the R value ranged from 1.0002 to 20.6 and that ginger might exhibit a high risk of drug interaction via inhibition of the activity of human CYP2C9 and CYP3A4, but a low risk of drug interaction toward CYP2C19-mediated drug metabolism. Furthermore, it has been evaluated that the 12 ginger components possessed a favorable ADMET profiles with regard to the solubility, absorption, permeability across the blood–brain barrier, interactions with CYP2D6, hepatotoxicity, and plasma protein binding. The validation results showed that there was no remarkable effect of ginger on the metabolism of warfarin in humans, whereas concurrent use of ginger and nifedipine exhibited

  18. Estimation of the binding modes with important human cytochrome P450 enzymes, drug interaction potential, pharmacokinetics, and hepatotoxicity of ginger components using molecular docking, computational, and pharmacokinetic modeling studies.

    PubMed

    Qiu, Jia-Xuan; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Zhou, Shu-Feng; Zhu, Shengrong

    2015-01-01

    Ginger is one of the most commonly used herbal medicines for the treatment of numerous ailments and improvement of body functions. It may be used in combination with prescribed drugs. The coadministration of ginger with therapeutic drugs raises a concern of potential deleterious drug interactions via the modulation of the expression and/or activity of drug-metabolizing enzymes and drug transporters, resulting in unfavorable therapeutic outcomes. This study aimed to determine the molecular interactions between 12 main active ginger components (6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, ar-curcumene, β-bisabolene, β-sesquiphelandrene, 6-gingerdione, (-)-zingiberene, and methyl-6-isogingerol) and human cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 and to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the 12 ginger components using computational approaches and comprehensive literature search. Docking studies showed that ginger components interacted with a panel of amino acids in the active sites of CYP1A2, 2C9, 2C19, 2D6, and 3A4 mainly through hydrogen bond formation, to a lesser extent, via π-π stacking. The pharmacokinetic simulation studies showed that the [I]/[Ki ] value for CYP2C9, 2C19, and 3A4 ranged from 0.0002 to 19.6 and the R value ranged from 1.0002 to 20.6 and that ginger might exhibit a high risk of drug interaction via inhibition of the activity of human CYP2C9 and CYP3A4, but a low risk of drug interaction toward CYP2C19-mediated drug metabolism. Furthermore, it has been evaluated that the 12 ginger components possessed a favorable ADMET profiles with regard to the solubility, absorption, permeability across the blood-brain barrier, interactions with CYP2D6, hepatotoxicity, and plasma protein binding. The validation results showed that there was no remarkable effect of ginger on the metabolism of warfarin in humans, whereas concurrent use of ginger and nifedipine exhibited a

  19. Activities.

    ERIC Educational Resources Information Center

    Kincaid, Charlene; And Others

    1993-01-01

    Presents an activity in which students collect and organize data from a real-world simulation of the scientific concept of half life. Students collect data using a marble sifter, analyze the data using a graphing calculator, and determine an appropriate mathematical model. Includes reproducible worksheets. (MDH)

  20. Activities.

    ERIC Educational Resources Information Center

    Mathematics Teacher, 1982

    1982-01-01

    The material presented is designed to help students explore geometric patterns involving Fibonnaci numbers and the golden ratio, and to aid in review of basic geometry skills. Worksheet masters intended for duplication are provided. Suggestions are made of possible classroom extensions to the initial activities. (MP)

  1. Dietary modulation of the biotransformation and genotoxicity of aflatoxin B(1).

    PubMed

    Gross-Steinmeyer, Kerstin; Eaton, David L

    2012-09-28

    chemoprotective dietary components may also arise through a decrease in the rate of activation of AFB to AFBO. Dietary consumption of apiaceous vegetables inhibits CYP1A2 activity in humans, and it has been demonstrated that some compounds in those vegetables act as potent inhibitors of human CYP1A2 and cause reduced hCYP1A2-mediated mutagenicity of AFB. Other dietary compounds of different origin (e.g., constituents of brassica vegetables and hops) have been shown to modify expression of human hepatic enzymes involved in the oxidation of AFB. SFN has been shown to protect animals from AFB-induced tumors, to reduce AFB biomarkers in humans in vivo and to reduce efficiently AFB adduct formation in human hepatocytes, although it appears that this protective effect is the result of repression of human hepatic CYP3A4 expression, rather than induction of protective GSTs, at least in human hepatocytes. If this mechanism were to occur in vivo in humans, it would raise safety concerns for the use of SFN as a chemoprotective agent as it may have important implications for drug-drug interactions in humans. A dietary chemoprevention pathway that is independent of AFB biotransformation is represented by the potential for dietary components, such as chlorophyllin, to tightly bind to and reduce the bioavailability of aflatoxins. Chlorophyllin has been shown to significantly reduce genotoxic AFB biomarkers in humans, and it therefore holds promise as a practical means of reducing the incidence of AFB-induced liver cancer. Recent reports have demonstrated that DNA repair mechanisms are inducible in mammalian systems and some diet-derived compounds elevated significantly the gene expression of enzymes potentially involved in nucleotide excision repair of AFB-DNA adducts. However, these are initial observations and more research is needed to determine if dietary modulation of DNA repair is a safe and effective approach to chemoprevention of AFB-induced liver cancer.

  2. Dietary modulation of the biotransformation and genotoxicity of aflatoxin B(1).

    PubMed

    Gross-Steinmeyer, Kerstin; Eaton, David L

    2012-09-28

    chemoprotective dietary components may also arise through a decrease in the rate of activation of AFB to AFBO. Dietary consumption of apiaceous vegetables inhibits CYP1A2 activity in humans, and it has been demonstrated that some compounds in those vegetables act as potent inhibitors of human CYP1A2 and cause reduced hCYP1A2-mediated mutagenicity of AFB. Other dietary compounds of different origin (e.g., constituents of brassica vegetables and hops) have been shown to modify expression of human hepatic enzymes involved in the oxidation of AFB. SFN has been shown to protect animals from AFB-induced tumors, to reduce AFB biomarkers in humans in vivo and to reduce efficiently AFB adduct formation in human hepatocytes, although it appears that this protective effect is the result of repression of human hepatic CYP3A4 expression, rather than induction of protective GSTs, at least in human hepatocytes. If this mechanism were to occur in vivo in humans, it would raise safety concerns for the use of SFN as a chemoprotective agent as it may have important implications for drug-drug interactions in humans. A dietary chemoprevention pathway that is independent of AFB biotransformation is represented by the potential for dietary components, such as chlorophyllin, to tightly bind to and reduce the bioavailability of aflatoxins. Chlorophyllin has been shown to significantly reduce genotoxic AFB biomarkers in humans, and it therefore holds promise as a practical means of reducing the incidence of AFB-induced liver cancer. Recent reports have demonstrated that DNA repair mechanisms are inducible in mammalian systems and some diet-derived compounds elevated significantly the gene expression of enzymes potentially involved in nucleotide excision repair of AFB-DNA adducts. However, these are initial observations and more research is needed to determine if dietary modulation of DNA repair is a safe and effective approach to chemoprevention of AFB-induced liver cancer. PMID:22640941

  3. Fasting-Induced Changes in Hepatic P450 Mediated Drug Metabolism Are Largely Independent of the Constitutive Androstane Receptor CAR

    PubMed Central

    de Vries, E. M.; Lammers, L. A.; Achterbergh, R.; Klümpen, H-J; Mathot, R. A. A.; Boelen, A.; Romijn, J. A.

    2016-01-01

    Introduction Hepatic drug metabolism by cytochrome P450 enzymes is altered by the nutritional status of patients. The expression of P450 enzymes is partly regulated by the constitutive androstane receptor (CAR). Fasting regulates the expression of both P450 enzymes and CAR and affects hepatic drug clearance. We hypothesized that the fasting-induced alterations in P450 mediated drug clearance are mediated by CAR. Methods To investigate this we used a drug cocktail validated in humans consisting of five widely prescribed drugs as probes for specific P450 enzymes: caffeine (CYP1A2), metoprolol (CYP2D6), omeprazole (CYP2C19), midazolam (CYP3A4) and s-warfarin (CYP2C9). This cocktail was administered to wild type (WT, C57Bl/6) mice or mice deficient for CAR (CAR-/-) that were either fed ad libitum or fasted for 24 hours. Blood was sampled at predefined intervals and drug concentrations were measured as well as hepatic mRNA expression of homologous/orthologous P450 enzymes (Cyp1a2, Cyp2d22, Cyp3a11, Cyp2c37, Cyp2c38 and Cyp2c65). Results Fasting decreased Cyp1a2 and Cyp2d22 expression and increased Cyp3a11 and Cyp2c38 expression in both WT and CAR-/- mice. The decrease in Cyp1a2 was diminished in CAR-/- in comparison with WT mice. Basal Cyp2c37 expression was lower in CAR-/- compared to WT mice. Fasting decreased the clearance of all drugs tested in both WT and CAR-/- mice. The absence of CAR was associated with an decrease in the clearance of omeprazole, metoprolol and midazolam in fed mice. The fasting-induced reduction in clearance of s-warfarin was greater in WT than in CAR-/-. The changes in drug clearance correlated with the expression pattern of the specific P450 enzymes in case of Cyp1a2-caffeine and Cyp2c37-omeprazole. Conclusion We conclude that CAR is important for hepatic clearance of several widely prescribed drugs metabolized by P450 enzymes. However the fasting-induced alterations in P450 mediated drug clearance are largely independent of CAR. PMID

  4. Cytochrome P450 enzyme mediated herbal drug interactions (Part 2)

    PubMed Central

    Wanwimolruk, Sompon; Phopin, Kamonrat; Prachayasittikul, Virapong

    2014-01-01

    To date, a number of significant herbal drug interactions have their origins in the alteration of cytochrome P450 (CYP) activity by various phytochemicals. Among the most noteworthy are those involving St. John's wort and drugs metabolized by human CYP3A4 enzyme. This review article is the continued work from our previous article (Part 1) published in this journal (Wanwimolruk and Prachayasittikul, 2014[ref:133]). This article extends the scope of the review to six more herbs and updates information on herbal drug interactions. These include black cohosh, ginseng, grape seed extract, green tea, kava, saw palmetto and some important Chinese medicines are also presented. Even though there have been many studies to determine the effects of herbs and herbal medicines on the activity of CYP, most of them were in vitro and in animal studies. Therefore, the studies are limited in predicting the clinical relevance of herbal drug interactions. It appeared that the majority of the herbal medicines have no clear effects on most of the CYPs examined. For example, the existing clinical trial data imply that black cohosh, ginseng and saw palmetto are unlikely to affect the pharmacokinetics of conventional drugs metabolized by human CYPs. For grape seed extract and green tea, adverse herbal drug interactions are unlikely when they are concomitantly taken with prescription drugs that are CYP substrates. Although there were few clinical studies on potential CYP-mediated interactions produced by kava, present data suggest that kava supplements have the ability to inhibit CYP1A2 and CYP2E1 significantly. Therefore, caution should be taken when patients take kava with CYP1A2 or CYP2E1 substrate drugs as it may enhance their therapeutic and adverse effects. Despite the long use of traditional Chinese herbal medicines, little is known about the potential drug interactions with these herbs. Many popularly used Chinese medicines have been shown in vitro to significantly change the

  5. Genotoxicity of tamoxifen, tamoxifen epoxide and toremifene in human lymphoblastoid cells containing human cytochrome P450s.

    PubMed

    Styles, J A; Davies, A; Lim, C K; De Matteis, F; Stanley, L A; White, I N; Yuan, Z X; Smith, L L

    1994-01-01

    The clastogenicity of tamoxifen and toremifene was tested in six human lymphoblastoid cell lines each expressing increased monooxygenase activity associated with a specific transfected human cytochrome P450 cDNA (CYP1A1, CYP1A2, CYP2D6, CYP2E1 or CYP3A4). The chemicals were also tested in a cell line (MCL-5) expressing elevated native CYP1A1 and containing transfected CYP1A2, CYP2A6, CYP2E1 and CYP3A4 and epoxide hydrolase, and in a cell line containing only the viral vector (Ho1). Dose-related increases in micronuclei were observed when cells expressing 2E1, 3A4, 2D6 or MCL-5 cells were exposed to tamoxifen. The positive responses in the cell lines were in the order MCL-5 > 2E1 > 3A4 > 2D6. Toremifene also gave positive results with 2E1, 3A4 and MCL-5 cells, although the responses were less marked and the positive effects required higher doses than with tamoxifen. A synthesized epoxide of tamoxifen was also tested in these cell lines and produced similar increases in the incidences of micronucleated cells. The increases in the responses observed with the epoxide were greater than with tamoxifen or toremifene. The P450 isoenzyme activities in these cells were in a range similar to those of human tumour-derived cell lines. Microsomes (1A1, 2A2, 2A6, 2B6, 2E1, 3A4 and 2D6) from these cells all metabolized tamoxifen. The major metabolite detected by HPLC was N-desmethyltamoxifen, and 4-hydroxytamoxifen was also detected in cells with cytochrome P450 2E1 and 2D6. These results are consistent with the following conclusions. (1) Tamoxifen requires metabolic activation to DNA-reactive species by specific CYP monooxygenases in order to exert its genotoxic effects. (2) The positive clastogenic effects elicited in lymphoblastoid cells by tamoxifen epoxide suggest that the genotoxic (and possibly the carcinogenic) effects of tamoxifen may be due to one or more epoxide metabolites that are generated intracellularly, probably in close proximity to the nucleus. (3) Tamoxifen is

  6. Small-scale fluctuations and angular correlations of the X-ray background in the HEAO 1 A-2 energy band - Constraints on clustering of X-ray sources

    NASA Technical Reports Server (NTRS)

    Martin-Mirones, J. M.; De Zotti, G.; Franceschini, A.; Boldt, E. A.; Marshall, F. E.; Danese, L.; Persic, M.

    1991-01-01

    HEAO 1 A-2 all-sky survey data have been used to determine the amplitude of intensity fluctuations of the extragalactic 2-10 keV X-ray background (XRB) over an effective solid angle of 1.84 sq deg and their angular correlation function on angular scales of less than 3 deg. A good empirical fit to the data is obtained assuming that the integral counts in the A-2 band have a slope of 1.65 + 0.06 or - 0.07. Alternatively, the data may imply a significant clustering of extragalactic X-ray sources.

  7. Metabolism of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in human hepatocytes: 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid is a major detoxification pathway catalyzed by cytochrome P450 1A2.

    PubMed

    Langouët, S; Welti, D H; Kerriguy, N; Fay, L B; Huynh-Ba, T; Markovic, J; Guengerich, F P; Guillouzo, A; Turesky, R J

    2001-02-01

    Metabolic pathways of the mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) remain incompletely characterized in humans. In this study, the metabolism of MeIQx was investigated in primary human hepatocytes. Six metabolites were characterized by UV and mass spectroscopy. Novel metabolites were additionally characterized by 1H NMR spectroscopy. The carcinogenic metabolite, 2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline, which is formed by cytochrome P450 1A2 (P450 1A2), was found to be transformed into the N(2)-glucuronide conjugate, N(2)-(beta-1-glucosiduronyl)-2-(hydroxyamino)-3,8-dimethylimidazo[4,5-f]quinoxaline. The phase II conjugates N(2)-(3,8-dimethylimidazo[4,5-f]quinoxalin-2-yl)sulfamic acid and N(2)-(beta-1-glucosiduronyl)-2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, as well as the 7-oxo derivatives of MeIQx and N-desmethyl-MeIQx, 2-amino-3,8-dimethyl-6-hydro-7H-imidazo[4,5-f]quinoxalin-7-one (7-oxo-MeIQx), and 2-amino-6-hydro-8-methyl-7H-imidazo[4,5-f]quinoxalin-7-one (N-desmethyl-7-oxo-MeIQx), thought to be formed exclusively by the intestinal flora, were also identified. A novel metabolite was characterized as 2-amino-3-methylimidazo[4,5-f]quinoxaline-8-carboxylic acid (IQx-8-COOH), and it was the predominant metabolite formed in hepatocytes exposed to MeIQx at levels approaching human exposure. IQx-8-COOH formation is catalyzed by P450 1A2. This metabolite is a detoxication product and does not induce umuC gene expression in Salmonella typhimurium strain NM2009. IQx-8-COOH is also the principal oxidation product of MeIQx excreted in human urine [Turesky, R., et al. (1998) Chem. Res. Toxicol. 11, 217-225]. Thus, P450 1A2 is involved in both the metabolic activation and detoxication of this procarcinogen in humans. Analogous metabolism experiments were conducted with hepatocytes of untreated rats and rats pretreated with the P450 inducer 3-methylcholanthrene. Unlike human hepatocytes, the rat cell preparations did not produce IQx-8

  8. CYP 450 enzyme induction by chronic oral musk xylene in adult and developing rats.

    PubMed

    Suter-Eichenberger, R; Boelsterli, U A; Conscience-Egli, M; Lichtensteiger, W; Schlumpf, M

    2000-04-10

    Developmental and adult toxicity of musk xylene was studied in Long Evans (LE) rats fed with chow containing musk xylene (MX) in food pellets in concentrations of 1 mg, 10 mg, 33 mg, 100 mg and 1000 mg MX per 1 kg chow corresponding to a daily intake of 0.07-0.08 mg MX/kg up to 70-80 mg MX/kg body weight. Adult male and female rats were MX exposed for a minimum of 10 weeks before mating. Exposure continued throughout pregnancy, birth and lactation. The effects of MX on CYP1A1/1A2 were studied in liver microsomes by EROD (7-ethoxyresorufin-rosomes deethylase) for CYP1A1 and by MROD (methoxyresorufin-o-demethylase) for CYP1A2 activity and by Western blotting. MX induced these enzymes dose dependently in adult and developing rats at PN (postnatal day) 1 and 14. The lowest effective maternal dose was 2-3 mg MX/kg/day. Western blot data of CYP2B and CYP3A indicated the induction of both P450 enzyme proteins in developing rats at PN 14 at the higher dose of 70-80 mg MX/kg/day. In contrast, upon high MX exposure CYP2B but not CYP3A was found to be induced in adult first generation male and female rats, indicating differential sensitivity to MX in development.

  9. CYP 450 enzyme induction by chronic oral musk xylene in adult and developing rats.

    PubMed

    Suter-Eichenberger, R; Boelsterli, U A; Conscience-Egli, M; Lichtensteiger, W; Schlumpf, M

    1999-12-20

    Developmental and adult toxicity of musk xylene was studied in Long Evans (LE) rats fed with chow containing musk xylene (MX) in food pellets in concentrations of 1 mg, 10 mg, 33 mg, 100 mg and 1000 mg MX per 1 kg chow corresponding to a daily intake of 0.07-0.08 mg MX/kg up to 70-80 mg MX/kg body weight. Adult male and female rats were MX exposed for a minimum of 10 weeks before mating. Exposure continued throughout pregnancy, birth and lactation. The effects of MX on CYP1A1/1A2 were studied in liver microsomes by EROD (7-ethoxyresorufin-o-deethylase) for CYP1A1 and by MROD (methoxyresorufin-o-demethylase) for CYP1A2 activity and by Western blotting. MX induced these enzymes dose dependently in adult and developing rats at PN (postnatal day) 1 and 14. The lowest effective maternal dose was 2-3 mg MX/kg/day. Western blot data of CYP2B and CYP3A indicated the induction of both P450 enzyme proteins in developing rats at PN 14 at the higher dose of 70-80 mg MX/kg/day. In contrast, upon high MX exposure CYP2B but not CYP3A was found to be induced in adult first generation male and female rats, indicating differential sensitivity to MX in development.

  10. CYP450 phenotyping and metabolite identification of quinine by accurate mass UPLC-MS analysis: a possible metabolic link to blackwater fever

    PubMed Central

    2013-01-01

    Background The naturally occurring alkaloid drug, quinine is commonly used for the treatment of severe malaria. Despite centuries of use, its metabolism is still not fully understood, and may play a role in the haemolytic disorders associated with the drug. Methods Incubations of quinine with CYPs 1A2, 2C9, 2C19, 2D6, and 3A4 were conducted, and the metabolites were characterized by accurate mass UPLC-MSE analysis. Reactive oxygen species generation was also measured in human erythrocytes incubated in the presence of quinine with and without microsomes. Results The metabolites 3-hydroxyquinine, 2’-oxoquininone, and O-desmethylquinine were observed after incubation with CYPs 3A4 (3-hydroxyquinine and 2’-oxoquininone) and 2D6 (O-desmethylquinine). In addition, multiple hydroxylations were observed both on the quinoline core and the quinuclidine ring system. Of the five primary abundance CYPs tested, 3A4, 2D6, 2C9, and 2C19 all demonstrated activity toward quinine, while 1A2 did not. Further, quinine produced robust dose-dependent oxidative stress in human erythrocytes in the presence of microsomes. Conclusions Taken in context, these data suggest a CYP-mediated link between quinine metabolism and the poorly understood haemolytic condition known as blackwater fever, often associated with quinine ingestion. PMID:23800033

  11. Inhibitory effects of kale ingestion on metabolism by cytochrome P450 enzymes in rats.

    PubMed

    Yamasaki, Izumi; Yamada, Masayoshi; Uotsu, Nobuo; Teramoto, Sachiyuki; Takayanagi, Risa; Yamada, Yasuhiko

    2012-01-01

    Kale (Brassica oleracea L. var acephala DC) is a leafy green vegetable belonging to the cabbage family (Brassicaceae) that contains a large amount of health-promoting phytochemicals. There are any reports about the effects of kale ingestion on the chemoprevention function and mechanism, but the interactions between kale and drugs have not been researched. We investigated the effects of kale intake on cytochrome P450 (CYP) metabolism by using cocktail probe drugs, including midazolam (for CYP3A4), caffeine (for CYP1A2), dextromethorphan (for CYP2D6), tolbutamide (for CYP2C9), omeprazole (for CYP2C19), and chlorzoxazone (for CYP2E1). Cocktail drugs were administered into rats treated with kale and cabbage (2000 mg/kg) for a week. The results showed that kale intake induced a significant increase in plasma levels and the AUC of midazolam, caffeine, and dextromethorphan. In addition, the plasma concentration and AUC of omeprazole tended to increase. Additionally, no almost differences in the mRNA expression levels of CYP enzymes in the liver were observed. In conclusion, kale ingestion was considered to have an inhibitory effect on the activities of CYP3A4, 1A2, 2D6, and 2C19 for a reason competitive inhibition than inhibitory changes in the mRNA expressions.

  12. Metabolism of 7-benzyloxy-4-trifluoromethyl-coumarin by human hepatic cytochrome P450 isoforms.

    PubMed

    Renwick, A B; Surry, D; Price, R J; Lake, B G; Evans, D C

    2000-10-01

    1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the