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Sample records for 4-hydroxynonenal adduct accumulation

  1. PARP INHIBITION ALLEVIATES DIABETES-INDUCED SYSTEMIC OXIDATIVE STRESS AND NEURAL TISSUE 4-HYDROXYNONENAL ADDUCT ACCUMULATION: CORRELATION WITH PERIPHERAL NERVE FUNCTION

    PubMed Central

    Lupachyk, Sergey; Shevalye, Hanna; Maksimchyk, Yury; Drel, Viktor R.; Obrosova, Irina G.

    2011-01-01

    This study evaluated the role of poly(ADP-ribose) polymerase in systemic oxidative stress and 4-hydoxynonenal adduct accumulation in diabetic peripheral neuropathy. Control and streptozotocin-diabetic rats were maintained with or without treatment with the PARP inhibitor, 1,5-isoquinolinediol, 3 mg kg−1d−1, for 10 weeks after initial 2 weeks. Treatment efficacy was evaluated by poly(ADP-ribosyl)ated protein content in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons and non-neuronal cells (fluorescent immunohistochemistry), as well as by indices of peripheral nerve function. Diabetic rats displayed increased urinary isoprostane and 8-hydroxy-2'-deoxyguanosine excretion (ELISA), 4-hydroxynonenal adduct accumulation in endothelial and Schwann cells of the peripheral nerve, neurons, astrocytes, and oligodendrocytes of the spinal cord, and neurons and glial cells of the dorsal root ganglia (double-label fluorescent immunohistochemistry) as well as motor and sensory nerve conduction velocity deficits, thermal hypoalgesia, and tactile allodynia. PARP inhibition counteracted diabetes-induced systemic oxidative stress and 4-hydroxynonenal adduct accumulation in peripheral nerve and spinal cord (Western blot analysis) and dorsal root ganglion neurons (perikarya, fluorescent immunohistochemistry) which correlated with improvement of large and small nerve fiber function. The findings reveal the important role of PARP activation in systemic oxidative stress and 4-hydroxynonenal adduct accumulation in diabetic peripheral neuropathy. PMID:21300148

  2. Increased accumulation of 4-hydroxynonenal adducts in male GSTA4/PPAR alpha double knockout mice enhances injury during early stages of alcoholic liver disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hepatic lipid peroxidation and accumulation of aldehyde-adducted proteins occur early in alcohol-mediated injury and are postulated to mediate the subsequent pro-inflammatory and fibrotic responses observed in alcoholic liver disease. To test the significance of lipid peroxidation formation in the ...

  3. Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal.

    PubMed

    Shireman, Laura M; Kripps, Kimberly A; Balogh, Larissa M; Conner, Kip P; Whittington, Dale; Atkins, William M

    2010-12-15

    4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it. PMID:20836986

  4. S-adenosyl-L-methionine protection of acetaminophen mediated oxidative stress and identification of hepatic 4-hydroxynonenal protein adducts by mass spectrometry

    SciTech Connect

    Brown, James Mike; Kuhlman, Christopher; Terneus, Marcus V.; Labenski, Matthew T.; Lamyaithong, Andre Benja; Ball, John G.; Lau, Serrine S.; Valentovic, Monica A.

    2014-12-01

    Acetaminophen (APAP) hepatotoxicity is protected by S-adenosyl-L-methionine (SAMe) treatment 1 hour (h) after APAP in C57/Bl6 mice. This study examined protein carbonylation as well as mitochondrial and cytosolic protein adduction by 4-hydroxynonenal (4-HNE) using mass spectrometry (MS) analysis. Additional studies investigated the leakage of mitochondrial proteins and 4-HNE adduction of these proteins. Male C57/Bl6 mice (n = 5/group) were divided into the following groups and treated as indicated: Veh (15 ml/kg water, ip), SAMe (1.25 mmol/kg, ip), APAP (250 mg/kg), and SAMe given 1 h after APAP (S + A). APAP toxicity was confirmed by an increase (p < 0.05) in plasma ALT (U/l) and liver weight/10 g body weight relative to the Veh, SAMe and S + A groups 4 h following APAP treatment. SAMe administered 1 h post-APAP partially corrected APAP hepatotoxicity as ALT and liver weight/10 g body weights were lower in the S + A group compared the APAP group. APAP induced leakage of the mitochondrial protein, carbamoyl phosphate synthase-1 (CPS-1) into the cytosol and which was reduced in the S + A group. SAMe further reduced the extent of APAP mediated 4-HNE adduction of CPS-1. MS analysis of hepatic and mitochondrial subcellular fractions identified proteins from APAP treated mice. Site specific 4-HNE adducts were identified on mitochondrial proteins sarcosine dehydrogenase and carbamoyl phosphate synthase-1 (CPS-1). In summary, APAP is associated with 4-HNE adduction of proteins as identified by MS analysis and that CPS-1 leakage was greater in APAP treated mice. SAMe reduced the extent of 4-HNE adduction of proteins as well as leakage of CPS-1. - Highlights: • Acetaminophen (APAP) toxicity protected by S-adenosylmethionine (SAMe) • 4-Hydroxynonenal adducted to sarcosine dehydrogenase • 4-Hydroxynonenal adducted to carbamoyl phosphate synthetase-1 • SAMe reduced APAP mediated CPS-1 mitochondrial leakage.

  5. 4-hydroxynonenal, a lipid peroxidation product, rapidly accumulates following traumatic spinal cord injury and inhibits glutamate uptake.

    PubMed

    Springer, J E; Azbill, R D; Mark, R J; Begley, J G; Waeg, G; Mattson, M P

    1997-06-01

    Traumatic injury to the spinal cord initiates a host of pathophysiological events that are secondary to the initial insult. One such event is the accumulation of free radicals that damage lipids, proteins, and nucleic acids. A major reactive product formed following lipid peroxidation is the aldehyde, 4-hydroxynonenal (HNE), which cross-links to side chain amino acids and inhibits the function of several key metabolic enzymes. In the present study, we used immunocytochemical and immunoblotting techniques to examine the accumulation of protein-bound HNE, and synaptosomal preparations to study the effects of spinal cord injury and HNE formation on glutamate uptake. Protein-bound HNE increased in content in the damaged spinal cord at early times following injury (1-24 h) and was found to accumulate in myelinated fibers distant to the site of injury. Immunoblots revealed that protein-bound HNE levels increased dramatically over the same postinjury interval. Glutamate uptake in synaptosomal preparations from injured spinal cords was decreased by 65% at 24 h following injury. Treatment of control spinal cord synaptosomes with HNE was found to decrease significantly, in a dose-dependent fashion, glutamate uptake, an effect that was mimicked by inducers of lipid peroxidation. Taken together, these findings demonstrate that the lipid peroxidation product HNE rapidly accumulates in the spinal cord following injury and that a major consequence of HNE accumulation is a decrease in glutamate uptake, which may potentiate neuronal cell dysfunction and death through excitotoxic mechanisms. PMID:9166741

  6. S-adenosyl-l-methionine protection of acetaminophen mediated oxidative stress and identification of hepatic 4-hydroxynonenal protein adducts by mass spectrometry.

    PubMed

    Brown, James Mike; Kuhlman, Christopher; Terneus, Marcus V; Labenski, Matthew T; Lamyaithong, Andre Benja; Ball, John G; Lau, Serrine S; Valentovic, Monica A

    2014-12-01

    Acetaminophen (APAP) hepatotoxicity is protected by S-adenosyl-l-methionine (SAMe) treatment 1hour (h) after APAP in C57/Bl6 mice. This study examined protein carbonylation as well as mitochondrial and cytosolic protein adduction by 4-hydroxynonenal (4-HNE) using mass spectrometry (MS) analysis. Additional studies investigated the leakage of mitochondrial proteins and 4-HNE adduction of these proteins. Male C57/Bl6 mice (n=5/group) were divided into the following groups and treated as indicated: Veh (15ml/kg water, ip), SAMe (1.25mmol/kg, ip), APAP (250mg/kg), and SAMe given 1h after APAP (S+A). APAP toxicity was confirmed by an increase (p<0.05) in plasma ALT (U/l) and liver weight/10g body weight relative to the Veh, SAMe and S+A groups 4h following APAP treatment. SAMe administered 1h post-APAP partially corrected APAP hepatotoxicity as ALT and liver weight/10g body weights were lower in the S+A group compared the APAP group. APAP induced leakage of the mitochondrial protein, carbamoyl phosphate synthase-1 (CPS-1) into the cytosol and which was reduced in the S+A group. SAMe further reduced the extent of APAP mediated 4-HNE adduction of CPS-1. MS analysis of hepatic and mitochondrial subcellular fractions identified proteins from APAP treated mice. Site specific 4-HNE adducts were identified on mitochondrial proteins sarcosine dehydrogenase and carbamoyl phosphate synthase-1 (CPS-1). In summary, APAP is associated with 4-HNE adduction of proteins as identified by MS analysis and that CPS-1 leakage was greater in APAP treated mice. SAMe reduced the extent of 4-HNE adduction of proteins as well as leakage of CPS-1. PMID:25246065

  7. S-Adenosyl-L-Methionine Protection of Acetaminophen Mediated Oxidative Stress and Identification of Hepatic 4-Hydroxynonenal Protein Adducts by Mass Spectrometry

    PubMed Central

    Brown, James Mike; Kuhlman, Christopher; Terneus, Marcus V.; Labenski, Matthew T.; Lamyaithong, Andre Benja; Ball, John G.; Lau, Serrine S.; Valentovic, Monica A.

    2015-01-01

    Acetaminophen (APAP) hepatotoxicity is protected by S-adenosyl-L-methionine (SAMe) treatment 1 hour (h) after APAP in C57/Bl6 mice. This study examined protein carbonylation as well as mitochondrial and cytosolic protein adduction by 4-hydroxynonenal (4-HNE) using mass spectrometry (MS) analysis. Additional studies investigated the leakage of mitochondrial proteins and 4-HNE adduction of these proteins. Male C57/Bl6 mice (n=5/group) were divided into the following groups and treated as indicated: Veh (15 ml/kg water, ip), SAMe (1.25 mmol/kg, ip), APAP (250 mg/kg), and SAMe given 1 h after APAP (S+A). APAP toxicity was confirmed by an increase (p<0.05) in plasma ALT (U/L) and liver weight/10 g body weight relative to the Veh, SAMe and S+A groups 4 h following APAP treatment. SAMe administered 1 h post APAP partially corrected APAP hepatotoxicity as ALT and liver weight/10 g body weights were lower in the S+A group compared the APAP group. APAP induced leakage of the mitochondrial protein, carbamoyl phosphate synthase-1 (CPS-1) into the cytosol and which was reduced in the S+A group. SAMe further reduced the extent of APAP mediated 4-HNE adduction of CPS-1. MS analysis of hepatic and mitochondrial subcellular fractions identified proteins from APAP treated mice. Site specific 4-HNE adducts were identified on mitochondrial proteins sarcosine dehydrogenase and carbamoyl phosphate synthase-1 (CPS-1). In summary, APAP is associated with 4-HNE adduction of proteins as identified by MS analysis and that CPS-1 leakage was greater in APAP treated mice. SAMe reduced the extent of 4-HNE adduction of proteins as well as leakage of CPS-1. PMID:25246065

  8. Rapamycin improves motor function, reduces 4-hydroxynonenal adducted protein in brain, and attenuates synaptic injury in a mouse model of synucleinopathy

    PubMed Central

    Bai, Xiang; Wey, Margaret Chia-Ying; Fernandez, Elizabeth; Hart, Matthew J.; Gelfond, Jonathan; Bokov, Alex F.; Rani, Sheela; Strong, Randy

    2015-01-01

    Background Synucleinopathy is any of a group of age-related neurodegenerative disorders including Parkinson's disease, multiple system atrophy, and dementia with Lewy Bodies, which is characterized by α-synuclein inclusions and parkinsonian motor deficits affecting millions of patients worldwide. But there is no cure at present for synucleinopathy. Rapamycin has been shown to be neuroprotective in several in vitro and in vivo synucleinopathy models. However, there are no reports on the long-term effects of RAPA on motor function or measures of neurodegeneration in models of synucleinopathy. Methods We determined whether long-term feeding a rapamycin diet (14 ppm in diet; 2.25 mg/kg body weight/day) improves motor function in neuronal A53T α-synuclein transgenic mice (TG) and explored underlying mechanisms using a variety of behavioral and biochemical approaches. Results After 24 weeks of treatment, rapamycin improved performance on the forepaw stepping adjustment test, accelerating rotarod and pole test. Rapamycin did not alter A53T α-synuclein content. There was no effect of rapamycin treatment on midbrain or striatal monoamines or their metabolites. Proteins adducted to the lipid peroxidation product 4-hydroxynonenal were decreased in brain regions of both wild-type and TG mice treated with rapamycin. Reduced levels of the presynaptic marker synaptophysin were found in several brain regions of TG mice. Rapamycin attenuated the loss of synaptophysin protein in the affected brain regions. Rapamycin also attenuated the loss of synaptophysin protein and prevented the decrease of neurite length in SH-SY5Y cells treated with 4-hydroxynonenal. Conclusion Taken together, these data suggest that rapamycin, an FDA approved drug, may prove useful in the treatment of synucleinopathy. PMID:26306821

  9. Conformational Interconversion of the trans-4-Hydroxynonenal-Derived (6S,8R,11S) 1,N2-Deoxyguanosine Adduct When Mismatched with Deoxyadenosine in DNA

    PubMed Central

    Huang, Hai; Wang, Hao; Lloyd, R. Stephen; Rizzo, Carmelo J.; Stone, Michael P.

    2009-01-01

    The (6S,8R,11S) 1,N2-HNE-dG adduct of trans-4-hydroxynonenal (HNE) was incorporated into the duplex 5′-d(GCTAGCXAGTCC)-3′•5′-d(GGACTAGCTAGC)-3′ [X=(6S,8R,11S) HNE-dG], in which the lesion was mismatched opposite dA. The (6S,8R,11S) adduct maintained the ring-closed 1,N2-HNE-dG structure. This was in contrast to when this adduct was correctly paired with dC, conditions under which it underwent ring opening and re-arrangement to diastereomeric minor groove hemiacetals [Huang, H., Wang, H., Qi, N., Lloyd, R.S., Harris, T.M., Rizzo, C.J., & Stone, M.P. (2008) J. Am. Chem. Soc. 130, 10898–10906]. The (6S,8R,11S) adduct exhibited a syn/anti conformational equilibrium about the glycosyl bond. The syn conformation was predominant in acidic solution. Structural analysis of the syn conformation revealed that X7 formed a distorted base pair with the complementary protonated A18. The HNE moiety was located in the major groove. Structural perturbations were observed at the neighbor C6•G19 and A8•T17 base pairs. At basic pH, the anti conformation of X7 was the major species. At X7 the 1,N2-HNE-dG intercalated and displaced the complementary A18 in the 5′-direction, resulting in a bulge at the X7•A18 base pair. The HNE aliphatic chain was oriented towards the minor groove. The Watson-Crick hydrogen bonding of the neighboring A8•T17 base pair was also disrupted. PMID:19053179

  10. Chemistry and Biology of DNA Containing 1,N2-Deoxyguanosine Adducts of the α,β-Unsaturated Aldehydes Acrolein, Crotonaldehyde, and 4-Hydroxynonenal

    PubMed Central

    2009-01-01

    The α,β-unsaturated aldehydes (enals) acrolein, crotonaldehyde, and trans-4-hydroxynonenal (4-HNE) are products of endogenous lipid peroxidation, arising as a consequence of oxidative stress. The addition of enals to dG involves Michael addition of the N2-amine to give N2-(3-oxopropyl)-dG adducts, followed by reversible cyclization of N1 with the aldehyde, yielding 1,N2-dG exocyclic products. The 1,N2-dG exocyclic adducts from acrolein, crotonaldehyde, and 4-HNE exist in human and rodent DNA. The enal-induced 1,N2-dG lesions are repaired by the nucleotide excision repair pathway in both Escherichia coli and mammalian cells. Oligodeoxynucleotides containing structurally defined 1,N2-dG adducts of acrolein, crotonaldehyde, and 4-HNE were synthesized via a postsynthetic modification strategy. Site-specific mutagenesis of enal adducts has been carried out in E. coli and various mammalian cells. In all cases, the predominant mutations observed are G→T transversions, but these adducts are not strongly miscoding. When placed into duplex DNA opposite dC, the 1,N2-dG exocyclic lesions undergo ring opening to the corresponding N2-(3-oxopropyl)-dG derivatives. Significantly, this places a reactive aldehyde in the minor groove of DNA, and the adducted base possesses a modestly perturbed Watson−Crick face. Replication bypass studies in vitro indicate that DNA synthesis past the ring-opened lesions can be catalyzed by pol η, pol ι, and pol κ. It also can be accomplished by a combination of Rev1 and pol ζ acting sequentially. However, efficient nucleotide insertion opposite the 1,N2-dG ring-closed adducts can be carried out only by pol ι and Rev1, two DNA polymerases that do not rely on the Watson−Crick pairing to recognize the template base. The N2-(3-oxopropyl)-dG adducts can undergo further chemistry, forming interstrand DNA cross-links in the 5′-CpG-3′ sequence, intrastrand DNA cross-links, or DNA−protein conjugates. NMR and mass spectrometric analyses

  11. Circulating levels of linoleic acid and HDL-cholesterol are major determinants of 4-hydroxynonenal protein adducts in patients with heart failure☆

    PubMed Central

    Asselin, Caroline; Ducharme, Anique; Ntimbane, Thierry; Ruiz, Matthieu; Fortier, Annik; Guertin, Marie-Claude; Lavoie, Joël; Diaz, Ariel; Levy, Émile; Tardif, Jean-Claude; Des Rosiers, Christine

    2013-01-01

    Objective Measurements of oxidative stress biomarkers in patients with heart failure (HF) have yielded controversial results. This study aimed at testing the hypothesis that circulating levels of the lipid peroxidation product 4-hydroxynonenal bound to thiol proteins (4HNE-P) are strongly associated with those of its potential precursors, namely n-6 polyunsaturated fatty acids (PUFA). Methods and results Circulating levels of 4HNE-P were evaluated by gas chromatography-mass spectrometry in 71 control subjects and 61 ambulatory symptomatic HF patients along with various other clinically- and biochemically-relevant parameters, including other oxidative stress markers, and total levels of fatty acids from all classes, which reflect both free and bound to cholesterol, phospholipids and triglycerides. All HF patients had severe systolic functional impairment despite receiving optimal evidence-based therapies. Compared to controls, HF patients displayed markedly lower circulating levels of HDL- and LDL-cholesterol, which are major PUFA carriers, as well as of PUFA of the n-6 series, specifically linoleic acid (LA; P=0.001). Circulating 4HNE-P in HF patients was similar to controls, albeit multiple regression analysis revealed that LA was the only factor that was significantly associated with circulating 4HNE-P in the entire population (R2=0.086; P=0.02). In HF patients only, 4HNE-P was even more strongly associated with LA (P=0.003) and HDL-cholesterol (p<0.0002). Our results demonstrate that 4HNE-P levels, expressed relative to HDL-cholesterol, increase as HDL-cholesterol plasma levels decrease in the HF group only. Conclusion Results from this study emphasize the importance of considering changes in lipids and lipoproteins in the interpretation of measurements of lipid peroxidation products. Further studies appear warranted to explore the possibility that HDL-cholesterol particles may be a carrier of 4HNE adducts. PMID:24494189

  12. Replication Bypass of the trans-4-Hydroxynonenal-Derived (6S,8R,11S)-1,N[superscript 2]-Deoxyguanosine DNA Adduct by the Sulfolobus solfataricus DNA Polymerase IV

    SciTech Connect

    Banerjee, Surajit; Christov, Plamen P.; Kozekova, Albena; Rizzo, Carmelo J.; Egli, Martin; Stone, Michael P.

    2014-10-02

    trans-4-Hydroxynonenal (HNE) is the major peroxidation product of {omega}-6 polyunsaturated fatty acids in vivo. Michael addition of the N{sub 2}-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N{sub 2}-dGuo (1,N{sub 2}-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPs to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua {yields} Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct, the (6S,8R,11S)-1,N{sub 2}-dGuo lesion remained in the ring

  13. MALONDIALDEHYDE AND 4-HYDROXYNONENAL ADDUCTS ARE NOT FORMED ON CARDIAC RYANODINE RECEPTOR (RyR2) AND SARCO(ENDO)PLASMIC RETICULUM Ca2+ ATPase (SERCA2) IN DIABETES

    PubMed Central

    Moore, Caronda J.; Shao, Chun Hong; Nagai, Ryoji; Kutty, Shelby; Singh, Jaipaul; Bidasee, Keshore R.

    2013-01-01

    Recently, we reported an elevated level of glucose-derived carbonyl adducts on cardiac ryanodine receptor (RyR2) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2) in hearts of streptozotocin(STZ)-induced diabetic rats. We also showed these adduct impaired RyR2 and SERCA2 activities, and altered evoked Ca2+ transients. What is less clear is if lipid-derived malondialdehyde (MDA) and 4-hydroxy-2-nonenal (4-HNE) also chemically react with and impair RyR2 and SERCA2 activities in diabetes. This study used Western blot assays with adduct-specific antibodies and confocal microscopy to assess levels of MDA, 4-HNE, Nε-carboxy(methyl)lysine (CML), pentosidine and pyrraline adducts on RyR2 and SERCA2 and evoked intracellular transient Ca2+ kinetics in myocytes from control, diabetic and treated-diabetic rats. MDA and 4-HNE adducts were not detected on RyR2 and SERCA2 from control or 8 weeks diabetic rats with altered evoked Ca2+ transients. However, CML, pentosidine, and pyrraline adducts were elevated 3–5 fold (p<0.05). Treating diabetic rats with pyridoxamine (a scavenger of reactive carbonyl species, RCS) or aminoguanidine (a mixed reactive oxygen species-reactive carbonyl species scavenger) reduced CML, pentosidine and pyrraline adducts on RyR2 and SERCA2 and blunted SR Ca2+ cycling changes. Treating diabetic rats with the superoxide dismutase mimetic tempol had no impact on MDA and 4-HNE adducts on RyR2 and SERCA2, and on SR Ca2+ cycling. From these data we conclude that lipid-derived MDA and 4-HNE adducts are not formed on RyR2 and SERCA2 in this model of diabetes, and are therefore unlikely to be directly contributing to the SR Ca2+ dysregulation. PMID:23354458

  14. Differential metabolism of 4-hydroxynonenal in liver, lung and brain of mice and rats

    SciTech Connect

    Zheng, Ruijin; Dragomir, Ana-Cristina; Mishin, Vladimir; Richardson, Jason R.; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-08-15

    The lipid peroxidation end-product 4-hydroxynonenal (4-HNE) is generated in tissues during oxidative stress. As a reactive aldehyde, it forms Michael adducts with nucleophiles, a process that disrupts cellular functioning. Liver, lung and brain are highly sensitive to xenobiotic-induced oxidative stress and readily generate 4-HNE. In the present studies, we compared 4-HNE metabolism in these tissues, a process that protects against tissue injury. 4-HNE was degraded slowly in total homogenates and S9 fractions of mouse liver, lung and brain. In liver, but not lung or brain, NAD(P)+ and NAD(P)H markedly stimulated 4-HNE metabolism. Similar results were observed in rat S9 fractions from these tissues. In liver, lung and brain S9 fractions, 4-HNE formed protein adducts. When NADH was used to stimulate 4-HNE metabolism, the formation of protein adducts was suppressed in liver, but not lung or brain. In both mouse and rat tissues, 4-HNE was also metabolized by glutathione S-transferases. The greatest activity was noted in livers of mice and in lungs of rats; relatively low glutathione S-transferase activity was detected in brain. In mouse hepatocytes, 4-HNE was rapidly taken up and metabolized. Simultaneously, 4-HNE-protein adducts were formed, suggesting that 4-HNE metabolism in intact cells does not prevent protein modifications. These data demonstrate that, in contrast to liver, lung and brain have a limited capacity to metabolize 4-HNE. The persistence of 4-HNE in these tissues may increase the likelihood of tissue injury during oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a highly reactive aldehyde. • Rodent liver, but not lung or brain, is efficient in degrading 4-hydroxynonenal. • 4-hydroxynonenal persists in tissues with low metabolism, causing tissue damage.

  15. Increased accumulation of 4-hydroxynonenal adducts in female GSTA4/PPAR alpha double knockout mice enhance steatosis and inflammation in a model of pediatric nonalcoholic fatty liver disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hepatocellular injury resulting from increased lipid peroxidation products and oxidative stress is considered a potential mechanism driving the progression of nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitsis (NASH). To test the significance of lipid peroxidation and protein...

  16. Retinal proteins modified by 4-hydroxynonenal: identification of molecular targets.

    PubMed

    Kapphahn, Rebecca J; Giwa, Babatomiwa M; Berg, Kristin M; Roehrich, Heidi; Feng, Xiao; Olsen, Timothy W; Ferrington, Deborah A

    2006-07-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is a product of lipid peroxidation that can covalently modify and inactivate proteins. Previously, we reported increased HNE modification of select retinal proteins resolved by one-dimensional gel electrophoresis in aged Fisher 344 x Brown Norway rats (Louie, J.L., Kapphahn, R.J., Ferrington, D.A., 2002. Proteasome function and protein oxidation in the aged retina. Exp. Eye Res. 75, 271-284). In the current study, quantitative assessment of HNE molar content using slot blot immunoassays showed HNE content is increased 30% in aged rat retina. In contrast, there was no age-related difference in HNE content in individual spots resolved by 2D gel electrophoresis suggesting the increased modification is likely on membrane proteins that are missing on 2D gels. The HNE-immunoreactive proteins resolved by 2D gel electrophoresis were identified by MALDI-TOF mass spectrometry. These proteins are involved in metabolism, chaperone function, and fatty acid transport. Proteins that were frequently modified and had the highest molar content of HNE included triosephosphate isomerase, alpha enolase, heat shock cognate 70 and betaB2 crystallin. Immunochemical detection of HNE adducts on retinal sections showed greater immune reaction in ganglion cells, photoreceptor inner segment, and the inner plexiform layer. Identification of HNE modified proteins in two alternative model systems, human retinal pigment epithelial cells in culture (ARPE19) and human donor eyes, indicated that triosephosphate isomerase and alpha enolase are generally modified. These results identify a common subset of proteins that contain HNE adducts and suggest that select retinal proteins are molecular targets for HNE modification. PMID:16530755

  17. Carcinine Has 4-Hydroxynonenal Scavenging Property and Neuroprotective Effect in Mouse Retina

    PubMed Central

    Marchette, Lea D.; Wang, Huaiwen; Li, Feng; Babizhayev, Mark A.; Kasus-Jacobi, Anne

    2012-01-01

    Purpose. Oxidative stress induces retinal damage and contributes to vision loss in progressive retinopathies. Carcinine (β-alanyl-histamine) is a natural imidazole-containing peptide derivative with antioxidant activity. It is predicted to scavenge 4-hydroxynonenal (4-HNE), a toxic product of lipid oxidation. The aim of this study was to confirm the 4-HNE scavenging effect and evaluate the neuroprotective effect of carcinine in mouse retina subjected to oxidative stress. Methods. HPLC coupled with mass spectrometry was used to analyze carcinine and 4-HNE-carcinine adduct. Protection of retinal proteins from modification by 4-HNE was tested by incubating carcinine with retinal protein extract and 4-HNE. Modified retinal proteins were quantified by dot-blot analysis. Mice were treated with carcinine (intravitreal injection and gavage) and exposed to bright light to induce oxidative damage in the retina. Photoreceptor degeneration was measured by histology and electroretinography. Retinal levels of retinol dehydrogenase 12 (RDH12) were measured by immunoblot analysis, after exposure to bright light and in retinal explants after exposure to 4-HNE. Results. The ability of carcinine to form an adduct with 4-HNE, as well as to prevent and even reverse the adduction of retinal proteins by the toxic aldehyde was demonstrated in vitro. Carcinine, administered by intravitreal injection or gavage, strongly protected mouse retina against light-induced photoreceptor degeneration and had a protective effect on RHD12, a protein found specifically in photoreceptor cells. Conclusions. This study suggests that carcinine can be administered noninvasively to efficiently protect photoreceptor cells from oxidative damage. Carcinine could be administered daily to prevent vision loss in progressive retinopathies. PMID:22577078

  18. Reduced cellular redox status induces 4-hydroxynonenal-mediated caspase 3 activation leading to erythrocyte death during chronic arsenic exposure in rats

    SciTech Connect

    Biswas, Debabrata; Sen, Gargi; Biswas, Tuli

    2010-05-01

    Chronic exposure to arsenic in rats led to gradual accumulation of the toxicant in erythrocytes causing oxidative stress in these cells. 4-Hydroxynonenal (4-HNE), a major aldehyde product of lipid peroxidation, contributed significantly to the cytopathological events observed during oxidative stress in the erythrocytes of exposed rats. 4-HNE triggered death signal cascade that was initiated with the formation of HNE-protein adducts in cytosol. HNE-protein adduct formation resulted in depletion of cytosolic antioxidants followed by increased generation of ROS. Results showed accumulation of hydrogen peroxide (H{sub 2}O{sub 2}) from the early stages of arsenic exposure, while superoxide (O{sub 2}{sup c}entre dot{sup -}) and hydroxyl radical ({sup c}entre dotOH) also contributed to the oxidative stress during longer period of exposure. Suppression of antioxidant system coupled with increased generation of ROS eventually led to activation of caspase 3 during arsenic exposure. Attenuation of HNE-mediated activation of caspase 3 in presence of N-acetylcysteine (NAC) indicated the involvement of GSH in the process. Prevention of HNE-mediated degradation of membrane proteins in presence of Z-DEVD-FMK identified caspase 3 as the principal mediator of HNE-induced cellular damage during arsenic exposure. Degradation of band 3 followed by its aggregation on the red cell surface promoted immunologic recognition of redistributed band 3 by autologous IgG with subsequent attachment of C3b. Finally, the formation of C3b-IgG-band 3 immune complex accelerated the elimination of affected cells from circulation and led to the decline of erythrocyte life span during chronic arsenic toxicity.

  19. 4-hydroxynonenal regulates mitochondrial function in human small airway epithelial cells.

    PubMed

    Galam, Lakshmi; Failla, Athena; Soundararajan, Ramani; Lockey, Richard F; Kolliputi, Narasaiah

    2015-12-01

    Prolonged exposure to oxidative stress causes Acute Lung Injury (ALI) and significantly impairs pulmonary function. Previously we have demonstrated that mitochondrial dysfunction is a key pathological factor in hyperoxic ALI. While it is known that hyperoxia induces the production of stable, but toxic 4-hydroxynonenal (4-HNE) molecule, it is unknown how the reactive aldehyde disrupts mitochondrial function. Our previous in vivo study indicated that exposure to hyperoxia significantly increases 4-HNE-Protein adducts, as well as levels of MDA in total lung homogenates. Based on the in vivo studies, we explored the effects of 4-HNE in human small airway epithelial cells (SAECs). Human SAECs treated with 25 μM of 4-HNE showed a significant decrease in cellular viability and increased caspase-3 activity. Moreover, 4-HNE treated SAECs showed impaired mitochondrial function and energy production indicated by reduced ATP levels, mitochondrial membrane potential, and aconitase activity. This was followed by a significant decrease in mitochondrial oxygen consumption and depletion of the reserve capacity. The direct effect of 4-HNE on the mitochondrial respiratory chain was confirmed using Rotenone. Furthermore, SAECs treated with 25 μM 4-HNE showed a time-dependent depletion of total Thioredoxin (Trx) proteins and Trx activity. Taken together, our results indicate that 4-HNE induces cellular and mitochondrial dysfunction in human SAECs, leading to an impaired endogenous antioxidant response. PMID:26484418

  20. Stereoselective Effects of 4-Hydroxynonenal in Cultured Mouse Hepatocytes

    PubMed Central

    Dabrowski, Michael J.; Zolnerciks, Joseph K.; Balogh, Larissa M.; Greene, Robert J.; Kavanagh, Terrance J.; Atkins, William M.

    2010-01-01

    4-Hydroxynonenal (HNE) is produced from arachidonic acid or linoleic acid during oxidative stress. Although HNE is formed in tissues as a racemate, enantiospecific HNE effects have not been widely documented, nor considered. Therefore, a panel of cellular responses was compared after treatment with (R)-HNE, (S)-HNE, or racemic HNE. The phosphorylation status of Jun kinase (JNK) or Akt increased 28-fold or 2-3-fold, respectively, after treatment with 100 μM (S)-HNE and racemic HNE compared to (R)-HNE. In contrast, the increase in phosphorylation of MAPK was greatest for (R)-HNE. caspase-3-dependent cleavage of glutamate cysteine ligase (GCL) catalytic subunit and focal adhesion kinase (FAK) were greater in cells treated with (S)-HNE at 48 hrs. (S)-HNE also caused a greater number of subG1 nuclei, a hallmark of apoptosis, at 30 hours after treatment. Together, the results demonstrate different dose- and time-dependent responses to (R)-HNE and (S)-HNE. The results further suggest that HNE enantiomers could differentially contribute to the progression of different diseases or contribute by different mechanisms. PMID:20873854

  1. 4-Hydroxynonenal induces p53-mediated apoptosis in retinal pigment epithelial cells

    PubMed Central

    Sharma, Abha; Sharma, Rajendra; Chaudhary, Pankaj; Vatsyayan, Rit; Pearce, Virginia; Jeyabal, Prince V.S.; Zimniak, Piotr; Awasthi, Sanjay; Awasthi, Yogesh C.

    2009-01-01

    4-Hydroxynonenal (4-HNE) has been suggested to be involved in stress-induced signaling for apoptosis. In present studies, we have examined the effects of 4-HNE on the intrinsic apoptotic pathway associated with p53 in human retinal pigment epithelial (RPE and ARPE-19) cells. Our results show that 4-HNE causes induction, phosphorylation, and nuclear accumulation of p53 which is accompanied with down regulation of MDM2, activation of the pro-apoptotic p53 target genes viz. p21 and Bax, JNK, caspase3, and onset of apoptosis in treated RPE cells. Reduced expression of p53 by an efficient silencing of the p53 gene resulted in a significant resistance of these cells to 4-HNE-induced cell death. The effects of 4-HNE on the expression and functions of p53 are blocked in GSTA4-4 over expressing cells indicating that 4-HNE-induced, p53-mediated signaling for apoptosis is regulated by GSTs. Our results also show that the induction of p53 in tissues of mGsta4 (-/-) mice correlate with elevated levels of 4-HNE due to its impaired metabolism. Together, these studies suggest that 4-HNE is involved in p53-mediated signaling in in vitro cell cultures as well as in vivo that can be regulated by GSTs. PMID:18930016

  2. Role of the lipid peroxidation product, 4-hydroxynonenal, in the development of nitrate tolerance.

    PubMed

    D'Souza, Yohan; Kawamoto, Toshihiro; Bennett, Brian M

    2014-04-21

    Tolerance to nitrates such as nitroglycerin (GTN) is associated with oxidative stress, inactivation of aldehyde dehydrogenase 2 (ALDH2), and decreased GTN-induced cGMP accumulation and vasodilation. We hypothesized that GTN-induced inactivation of ALDH2 results in increased 4-hydroxy-2-nonenal (HNE) adduct formation of key proteins involved in GTN bioactivation, and, consequently, an attenuated vasodilator response to GTN (i.e., tolerance). We used an in vivo GTN tolerance model, a cell culture model of nitrate action, and Aldh2(-/-) mice to assess whether GTN exposure resulted in HNE adduct formation, and whether exogenous HNE affected GTN-induced relaxation and cGMP accumulation. Immunoblot analysis indicated a marked increase in HNE adduct formation in GTN-tolerant porcine kidney epithelial cells (PK1) and in aortae from GTN-tolerant rats and untreated Aldh2(-/-) mice. Preincubation of PK1 cells with HNE resulted in a dose-dependent decrease in GTN-induced cGMP accumulation, and pretreatment of isolated rat aorta with HNE resulted in dose-dependent decreases in the vasodilator response to GTN, thus mimicking GTN-tolerance. Pretreatment of aortae from Aldh2(-/-) mice with 10 μM HNE resulted in a desensitized vasodilator response to GTN. In the in vivo rat tolerance model, changes in HNE adduct formation correlated well with the onset of GTN tolerance and tolerance reversal. Furthermore, coadministration of an HNE scavenger during the tolerance induction protocol completely prevented HNE adduct formation and GTN tolerance but did not prevent the inactivation of ALDH2. The data are consistent with a novel mechanism of GTN tolerance suggesting a primary role of HNE adduct formation in the development of GTN tolerance. PMID:24555687

  3. 4-Hydroxynonenal, an aldehydic product of membrane lipid peroxidation, impairs glutamate transport and mitochondrial function in synaptosomes.

    PubMed

    Keller, J N; Mark, R J; Bruce, A J; Blanc, E; Rothstein, J D; Uchida, K; Waeg, G; Mattson, M P

    1997-10-01

    Removal of extracellular glutamate at synapses, by specific high-affinity glutamate transporters, is critical to prevent excitotoxic injury to neurons. Oxidative stress has been implicated in the pathogenesis of an array of prominent neurodegenerative conditions that involve degeneration of synapses and neurons in glutamatergic pathways including stroke, and Alzheimer's, Parkinson's and Huntington's diseases. Although cell culture data indicate that oxidative insults can impair key membrane regulatory systems including ion-motive ATPases and amino acid transport systems, the effects of oxidative stress on synapses, and the mechanisms that mediate such effects, are largely unknown. This study provides evidence that 4-hydroxynonenal, an aldehydic product of lipid peroxidation, mediates oxidation-induced impairment of glutamate transport and mitochondrial function in synapses. Exposure of rat cortical synaptosomes to 4-hydroxynonenal resulted in concentration- and time-dependent decreases in [3H]glutamate uptake, and mitochondrial function [assessed with the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Other related aldehydes including malondialdehyde and hexanal had little or no effect on glutamate uptake or mitochondrial function. Exposure of synaptosomes to insults known to induce lipid peroxidation (FeSO4 and amyloid beta-peptide) also impaired glutamate uptake and mitochondrial function. The antioxidants propyl gallate and glutathione prevented impairment of glutamate uptake and MTT reduction induced by FeSO4 and amyloid beta-peptide, but not that induced by 4-hydroxynonenal. Western blot analyses using an antibody to 4-hydroxynonenal-conjugated proteins showed that 4-hydroxynonenal bound to multiple cell proteins including GLT-1, a glial glutamate transporter present at high levels in synaptosomes. 4-Hydroxynonenal itself induced lipid peroxidation suggesting that, in addition to binding directly to membrane regulatory proteins, 4

  4. Proteinuria in passive Heymann nephritis is associated with lipid peroxidation and formation of adducts on type IV collagen.

    PubMed Central

    Neale, T J; Ojha, P P; Exner, M; Poczewski, H; Rüger, B; Witztum, J L; Davis, P; Kerjaschki, D

    1994-01-01

    Passive Heymann nephritis (PHN) is a model of human membranous nephropathy that is characterized by formation of granular subepithelial immune deposits in the glomerular capillary wall which results in complement activation. This is causally related to damage of the filtration barrier and subsequent proteinuria. The local accumulation of injurious reactive oxygen species (ROS) is a major effector mechanism in PHN. ROS may induce tissue damage by initiating lipid peroxidation (LPO). In turn, this leads to adduct formation between breakdown products of LPO with structural proteins, such as formation of malondialdehyde (MDA) or 4-hydroxynonenal-lysine adducts. To examine the role of LPO in the development of proteinuria we have localized MDA and 4-hydroxynonenal-lysine adducts in glomeruli of PHN rats by immunofluorescence microscopy, using specific monoclonal antibodies. By immunogold electron microscopy, MDA adducts were localized to cytoplasmic vesicles and cell membranes of glomerular epithelial cells, to the glomerular basement membrane (GBM), and also to immune deposits. Type IV collagen was specifically identified as being modified by MDA adducts, using a variety of techniques. Collagenase pretreatment of GBM extracts indicated that the NC-1 domain of type IV collagen was a site of adduct formation. When LPO was inhibited by pretreatment of PHN rats with the antioxidant probucol, proteinuria was reduced by approximately 85%, and glomerular immunostaining for dialdehyde adducts was markedly reduced, even though the formation of immune deposits was not affected. By contrast, lowering of the serum cholesterol levels had no influence on the development of proteinuria. These findings are consistent with the premise that ROS-induced glomerular injury in PHN involves LPO and that this results not only in damage of cell membranes but in modification of type IV collagen in the GBM as well. The close temporal correlation of the occurrence of LPO with proteinuria and the

  5. Routes to 4-Hydroxynonenal: Fundamental Issues in the Mechanisms of Lipid Peroxidation*S⃞

    PubMed Central

    Schneider, Claus; Porter, Ned A.; Brash, Alan R.

    2008-01-01

    Although investigation of the toxicological and physiological actions of α/β-unsaturated 4-hydroxyalkenals has made great progress over the last 2 decades, understanding of the chemical mechanism of formation of 4-hydroxynonenal and related aldehydes has advanced much less. The aim of this review is to discuss mechanistic evidence for these non-enzymatic routes, especially of the underappreciated intermolecular pathways that involve dimerized and oligomerized fatty acid derivatives as key intermediates. These cross-molecular reactions of fatty acid peroxyls have also important implications for understanding of the basic initiation and propagation steps during lipid peroxidation and the nature of the products that arise. PMID:18285327

  6. Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

    SciTech Connect

    Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir; Black, Adrienne T.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-03-01

    4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86–98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2 −/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. - Highlights: • Lipid peroxidation generates 4-hydroxynonenal, a reactive aldehyde. • 4-HNE induces antioxidant proteins in mouse keratinocytes. • Induction of

  7. Olive leaf extracts protect cardiomyocytes against 4-hydroxynonenal-induced toxicity in vitro: comparison with oleuropein, hydroxytyrosol, and quercetin.

    PubMed

    Bali, Elif Burcu; Ergin, Volkan; Rackova, Lucia; Bayraktar, Oğuz; Küçükboyaci, Nurgün; Karasu, Çimen

    2014-08-01

    Olive (Olea europaea) leaf, an important traditional herbal medicine, displays cardioprotection that may be related to the cellular redox modulating effects of its polyphenolic constituents. This study was undertaken to investigate the protective effect of the ethanolic and methanolic extracts of olive leaves compared to the effects of oleuropein, hydroxytyrosol, and quercetin as a positive standard in a carbonyl compound (4-hydroxynonenal)-induced model of oxidative damage to rat cardiomyocytes (H9c2). Cell viability was detected by the MTT assay; reactive oxygen species production was assessed by the 2',7'-dichlorodihydrofluorescein diacetate method, and the mitochondrial membrane potential was determined using a JC-1 dye kit. Phospho-Hsp27 (Ser82), phospho-MAPKAPK-2 (Thr334), phospho-c-Jun (Ser73), cleaved-caspase-3 (cl-CASP3) (Asp175), and phospho-SAPK/JNK (Thr183/Tyr185) were measured by Western blotting. The ethanolic and methanolic extracts of olive leaves inhibited 4-hydroxynonenal-induced apoptosis, characterized by increased reactive oxygen species production, impaired viability (LD50: 25 µM), mitochondrial dysfunction, and activation of pro-apoptotic cl-CASP3. The ethanolic and methanolic extracts of olive leaves also inhibited 4-hydroxynonenal-induced phosphorylation of stress-activated transcription factors, and the effects of extracts on p-SAPK/JNK, p-Hsp27, and p-MAPKAPK-2 were found to be concentration-dependent and comparable with oleuropein, hydroxytyrosol, and quercetin. While the methanolic extract downregulated 4-hydroxynonenal-induced p-MAPKAPK-2 and p-c-Jun more than the ethanolic extract, it exerted a less inhibitory effect than the ethanolic extract on 4-hydroxynonenal-induced p-SAPK/JNK and p-Hsp27. cl-CASP3 and p-Hsp27 were attenuated, especially by quercetin. Experiments showed a predominant reactive oxygen species inhibitory and mitochondrial protecting ability at a concentration of 1-10 µg/mL of each extract, oleuropein

  8. The lack of 4-hydroxynonenal in otosclerotic bone tissue in Ethiopian population.

    PubMed

    Rudic, Milan; Wagner, Richard; Willkinson, Eric; Danese, Giovanni; Kiros, Nega; Zarkovic, Kamelija; Zarkovic, Neven

    2015-10-01

    In Ethiopians, like in other Africans, the incidence of otosclerosis is lower than in Western and Asian populations. Unfortunately, due to the lack of available otorhinolaryngology specialists many patients are not treated and suffer the progression of the disease and severe hearing loss. This program of the Global ENT Outreach Organization (GEO) together with the Ethiopian partners was done to help some of these patients and in parallel to evaluate the presence of the oxidative stress bioactive marker 4-hydroxynonenal (HNE), which is known as major lipid peroxidation product and the second messenger of free radicals, in the otosclerotic bone specimens. Namely, we described recently that as HNE acts as a bone growth regulator associated with pathogenesis of otosclerosis. The prospective study conducted at the ENT Department of the Migbare Senay General Hospital, Addis Ababa, Ethiopia in June 2012, under the auspices of the Global ENT Outreach Organization, USA. Altogether 36 patients (male = 12, female = 24) underwent surgery due to the previous otosclerosis diagnosis based on the clinical and audiometric findings. The bone samples were harvested from patients with intraoperatively confirmed otosclerosis diagnosis. Immunohistochemistry for HNE-modified proteins was carried out on formalin-fixed paraffin-embedded specimens. The presence of HNE was found in almost all bone samples analyzed, without particular difference in the HNE distribution pattern between the otosclerotic and respective control bone specimens. Although there was no significant association between the HNE appearance and otosclerotic bone outgrowth observed, several cases have shown tendency of higher HNE expression in patients with more severe hearing loss. The results of the present study are in contrast with our previous findings obtained on European patients most likely due to the differences between studied population groups. PMID:25218196

  9. Modulation of keratinocyte expression of antioxidants by 4-hydroxynonenal, a lipid peroxidation end product

    PubMed Central

    Zheng, Ruijin; Heck, Diane E.; Mishin, Vladimir; Black, Adrienne T.; Shakarjian, Michael P.; Kong, Ah-Ng Tony; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-01-01

    4-Hydroxynonenal (4-HNE) is a lipid peroxidation end product generated in response to oxidative stress in the skin. Keratinocytes contain an array of antioxidant enzymes which protect against oxidative stress. In these studies, we characterized 4-HNE-induced changes in antioxidant expression in mouse keratinocytes. Treatment of primary mouse keratinocytes and PAM 212 keratinocytes with 4-HNE increased mRNA expression for heme oxygenase-1 (HO-1), catalase, NADPH:quinone oxidoreductase (NQO1) and glutathione S-transferase (GST) A1-2, GSTA3 and GSTA4. In both cell types, HO-1 was the most sensitive, increasing 86-98 fold within 6 h. Further characterization of the effects of 4-HNE on HO-1 demonstrated concentration- and time-dependent increases in mRNA and protein expression which were maximum after 6 h with 30 μM. 4-HNE stimulated keratinocyte Erk1/2, JNK and p38 MAP kinases, as well as PI3 kinase. Inhibition of these enzymes suppressed 4-HNE-induced HO-1 mRNA and protein expression. 4-HNE also activated Nrf2 by inducing its translocation to the nucleus. 4-HNE was markedly less effective in inducing HO-1 mRNA and protein in keratinocytes from Nrf2−/− mice, when compared to wild type mice, indicating that Nrf2 also regulates 4-HNE-induced signaling. Western blot analysis of caveolar membrane fractions isolated by sucrose density centrifugation demonstrated that 4-HNE-induced HO-1 is localized in keratinocyte caveolae. Treatment of the cells with methyl-β-cyclodextrin, which disrupts caveolar structure, suppressed 4-HNE-induced HO-1. These findings indicate that 4-HNE modulates expression of antioxidant enzymes in keratinocytes, and that this can occur by different mechanisms. Changes in expression of keratinocyte antioxidants may be important in protecting the skin from oxidative stress. PMID:24423726

  10. Alcohol, Aldehydes, Adducts and Airways.

    PubMed

    Sapkota, Muna; Wyatt, Todd A

    2015-01-01

    Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease. PMID:26556381

  11. Alcohol, Aldehydes, Adducts and Airways

    PubMed Central

    Sapkota, Muna; Wyatt, Todd A.

    2015-01-01

    Drinking alcohol and smoking cigarettes results in the formation of reactive aldehydes in the lung, which are capable of forming adducts with several proteins and DNA. Acetaldehyde and malondialdehyde are the major aldehydes generated in high levels in the lung of subjects with alcohol use disorder who smoke cigarettes. In addition to the above aldehydes, several other aldehydes like 4-hydroxynonenal, formaldehyde and acrolein are also detected in the lung due to exposure to toxic gases, vapors and chemicals. These aldehydes react with nucleophilic targets in cells such as DNA, lipids and proteins to form both stable and unstable adducts. This adduction may disturb cellular functions as well as damage proteins, nucleic acids and lipids. Among several adducts formed in the lung, malondialdehyde DNA (MDA-DNA) adduct and hybrid malondialdehyde-acetaldehyde (MAA) protein adducts have been shown to initiate several pathological conditions in the lung. MDA-DNA adducts are pre-mutagenic in mammalian cells and induce frame shift and base-pair substitution mutations, whereas MAA protein adducts have been shown to induce inflammation and inhibit wound healing. This review provides an insight into different reactive aldehyde adducts and their role in the pathogenesis of lung disease. PMID:26556381

  12. “Twin peaks”: Searching for 4-hydroxynonenal urinary metabolites after oral administration in rats

    PubMed Central

    Keller, Julia; Baradat, Maryse; Jouanin, Isabelle; Debrauwer, Laurent; Guéraud, Françoise

    2014-01-01

    4-Hydroxynonenal (HNE) is a cytotoxic and genotoxic lipid oxidation secondary product which is formed endogenously upon peroxidation of cellular n-6 fatty acids. However, it can also be formed in food or during digestion, upon peroxidation of dietary lipids. Several studies have evidenced that we are exposed through food to significant concentrations of HNE that could pose a toxicological concern. It is then of importance to known how HNE is metabolized after oral administration. Although its metabolism has been studied after intravenous administration in order to mimick endogenous formation, its in vivo fate after oral administration had never been studied. In order to identify and quantify urinary HNE metabolites after oral administration in rats, radioactive and stable isotopes of HNE were used and urine was analyzed by radio-chromatography (radio-HPLC) and chromatography coupled with High Resolution Mass Spectrometry (HPLC–HRMS). Radioactivity distribution revealed that 48% of the administered radioactivity was excreted into urine and 15% into feces after 24 h, while 3% were measured in intestinal contents and 2% in major organs, mostly in the liver. Urinary radio-HPLC profiles revealed 22 major peaks accounting for 88% of the urinary radioactivity. For identification purpose, HNE and its stable isotope [1,2-13C]-HNE were given at equimolar dose to be able to univocally identify HNE metabolites by tracking twin peaks on HPLC–HRMS spectra. The major peak was identified as 9-hydroxy-nonenoic acid (27% of the urinary radioactivity) followed by classical HNE mercapturic acid derivatives (the mercapturic acid conjugate of di-hydroxynonane (DHN-MA), the mercapturic acid conjugate of 4-hydroxynonenoic acid (HNA-MA) in its opened and lactone form) and by metabolites that are oxidized in the terminal position. New urinary metabolites as thiomethyl and glucuronide conjugates were also evidenced. Some analyses were also performed on feces and gastro

  13. A role for 4-hydroxynonenal, an aldehydic product of lipid peroxidation, in disruption of ion homeostasis and neuronal death induced by amyloid beta-peptide.

    PubMed

    Mark, R J; Lovell, M A; Markesbery, W R; Uchida, K; Mattson, M P

    1997-01-01

    Peroxidation of membrane lipids results in release of the aldehyde 4-hydroxynonenal (HNE), which is known to conjugate to specific amino acids of proteins and may alter their function. Because accumulating data indicate that free radicals mediate injury and death of neurons in Alzheimer's disease (AD) and because amyloid beta-peptide (A beta) can promote free radical production, we tested the hypothesis that HNE mediates A beta 25-35-induced disruption of neuronal ion homeostasis and cell death. A beta induced large increases in levels of free and protein-bound HNE in cultured hippocampal cells. HNE was neurotoxic in a time- and concentration-dependent manner, and this toxicity was specific in that other aldehydic lipid peroxidation products were not neurotoxic. HNE impaired Na+, K(+)-ATPase activity and induced an increase of neuronal intracellular free Ca2+ concentration. HNE increased neuronal vulnerability to glutamate toxicity, and HNE toxicity was partially attenuated by NMDA receptor antagonists, suggesting an excitotoxic component to HNE neurotoxicity. Glutathione, which was previously shown to play a key role in HNE metabolism in nonneuronal cells, attenuated the neurotoxicities of both A beta and HNE. The antioxidant propyl gallate protected neurons against A beta toxicity but was less effective in protecting against HNE toxicity. Collectively, the data suggest that HNE mediates A beta-induced oxidative damage to neuronal membrane proteins, which, in turn, leads to disruption of ion homeostasis and cell degeneration. PMID:8978733

  14. Relation between TLR4/NF-κB signaling pathway activation by 27-hydroxycholesterol and 4-hydroxynonenal, and atherosclerotic plaque instability

    PubMed Central

    Gargiulo, Simona; Gamba, Paola; Testa, Gabriella; Rossin, Daniela; Biasi, Fiorella; Poli, Giuseppe; Leonarduzzi, Gabriella

    2015-01-01

    It is now thought that atherosclerosis, although due to increased plasma lipids, is mainly the consequence of a complicated inflammatory process, with immune responses at the different stages of plaque development. Increasing evidence points to a significant role of Toll-like receptor 4 (TLR4), a key player in innate immunity, in the pathogenesis of atherosclerosis. This study aimed to determine the effects on TLR4 activation of two reactive oxidized lipids carried by oxidized low-density lipoproteins, the oxysterol 27-hydroxycholesterol (27-OH) and the aldehyde 4-hydroxynonenal (HNE), both of which accumulate in atherosclerotic plaques and play a key role in the pathogenesis of atherosclerosis. Secondarily, it examined their potential involvement in mediating inflammation and extracellular matrix degradation, the hallmarks of high-risk atherosclerotic unstable plaques. In human promonocytic U937 cells, both 27-OH and HNE were found to enhance cell release of IL-8, IL-1β, and TNF-α and to upregulate matrix metalloproteinase-9 (MMP-9) via TLR4/NF-κB-dependent pathway; these actions may sustain the inflammatory response and matrix degradation that lead to atherosclerotic plaque instability and to their rupture. Using specific antibodies, it was also demonstrated that these inflammatory cytokines increase MMP-9 upregulation, thus enhancing the release of this matrix-degrading enzyme by macrophage cells and contributing to plaque instability. These innovative results suggest that, by accumulating in atherosclerotic plaques, the two oxidized lipids may contribute to plaque instability and rupture. They appear to do so by sustaining the release of inflammatory molecules and MMP-9 by inflammatory and immune cells, for example, macrophages, through activation of TLR4 and its NF-κB downstream signaling. PMID:25757594

  15. Inhibition of glycolysis attenuates 4-hydroxynonenal-dependent autophagy and exacerbates apoptosis in differentiated SH-SY5Y neuroblastoma cells

    PubMed Central

    Dodson, Matthew; Liang, Qiuli; Johnson, Michelle S; Redmann, Matthew; Fineberg, Naomi; Darley-Usmar, Victor M; Zhang, Jianhua

    2013-01-01

    How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE. PMID:24145463

  16. Cell death and diseases related to oxidative stress:4-hydroxynonenal (HNE) in the balance

    PubMed Central

    Dalleau, S; Baradat, M; Guéraud, F; Huc, L

    2013-01-01

    During the last three decades, 4-hydroxy-2-nonenal (HNE), a major α,β-unsaturated aldehyde product of n-6 fatty acid oxidation, has been shown to be involved in a great number of pathologies such as metabolic diseases, neurodegenerative diseases and cancers. These multiple pathologies can be explained by the fact that HNE is a potent modulator of numerous cell processes such as oxidative stress signaling, cell proliferation, transformation or cell death. The main objective of this review is to focus on the different aspects of HNE-induced cell death, with a particular emphasis on apoptosis. HNE is a special apoptotic inducer because of its abilities to form protein adducts and to propagate oxidative stress. It can stimulate intrinsic and extrinsic apoptotic pathways and interact with typical actors such as tumor protein 53, JNK, Fas or mitochondrial regulators. At the same time, due to its oxidant status, it can also induce some cellular defense mechanisms against oxidative stress, thus being involved in its own detoxification. These processes in turn limit the apoptotic potential of HNE. These dualities can imbalance cell fate, either toward cell death or toward survival, depending on the cell type, the metabolic state and the ability to detoxify. PMID:24096871

  17. Modification of histidine residues in proteins by reaction with 4-hydroxynonenal.

    PubMed Central

    Uchida, K; Stadtman, E R

    1992-01-01

    We find that histidine residues in proteins are major targets for reaction with the lipid peroxidation product 4-hydroxynon-2-enal (HNE). Reaction of insulin (which contains no sulfhydryl groups) with HNE leads to the generation of HNE-protein adducts, which are converted to radioactive derivatives upon subsequent treatment with NaB[3H]H4. Amino acid analysis of the modified protein showed that the HNE treatment leads to the selective loss of histidine residues and the stiochiometric formation of 3H-labeled amino acid derivatives. The same labeled products were detected in acid hydrolysates of polyhistidine and N-acetylhistidine after their reactions with HNE and NaB[3H]H4. The reaction of N-acetylhistidine with HNE led to the production of two compounds. Upon acid hydrolysis, both derivatives yielded stoichiometric amounts of histidine. However, after reduction with NaBH4, acid hydrolysis led to a mixture of amino acid derivatives [presumably, isomeric forms of N pi (N tau)-1,4-dihydroxynonanylhistidine] that were indistinguishable from those obtained from insulin and polyhistidine after similar treatment. Although other possibilities are not excluded, it is suggested that the modification of histidine residues in proteins by HNE involves a Michael-type addition of the imidazole nitrogen atom of histidine to the alpha, beta-unsaturated bond of HNE, followed by secondary reaction involving the aldehyde group with the C-4 hydroxyl group of HNE. The reaction of histidine residues with HNE provides the basis for methods by which the contributions of HNE in the modification of proteins can be determined. PMID:1584790

  18. Role of the Lipoperoxidation Product 4-Hydroxynonenal in the Pathogenesis of Severe Malaria Anemia and Malaria Immunodepression

    PubMed Central

    Schwarzer, Evelin; Arese, Paolo; Skorokhod, Oleksii A.

    2015-01-01

    Oxidative stress plays an important role in the pathogenesis of falciparum malaria, a disease still claiming close to 1 million deaths and 200 million new cases per year. Most frequent complications are severe anemia, cerebral malaria, and immunodepression, the latter being constantly present in all forms of malaria. Complications are associated with oxidative stress and lipoperoxidation. Its final product 4-hydroxynonenal (4-HNE), a stable yet very reactive and diffusible molecule, forms covalent conjugates with proteins, DNA, and phospholipids and modulates important cell functions at very low concentrations. Since oxidative stress plays important roles in the pathogenesis of severe malaria, it appears important to explore the role of 4-HNE in two important malaria complications such as malaria anemia and malaria immunodepression where oxidative stress is considered to be involved. In this review we will summarize data about 4-HNE chemistry, its biologically relevant chemical properties, and its role as regulator of physiologic processes and as pathogenic factor. We will review studies documenting the role of 4-HNE in severe malaria with emphasis on malaria anemia and immunodepression. Data from other diseases qualify 4-HNE both as oxidative stress marker and as pathomechanistically important molecule. Further studies are needed to establish 4-HNE as accepted pathogenic factor in severe malaria. PMID:25969702

  19. Formation of Malondialdehyde, 4-Hydroxynonenal, and 4-Hydroxyhexenal during in Vitro Digestion of Cooked Beef, Pork, Chicken, and Salmon.

    PubMed

    Steppeler, Christina; Haugen, John-Erik; Rødbotten, Rune; Kirkhus, Bente

    2016-01-20

    Red meat high in heme iron may promote the formation of potentially genotoxic aldehydes during lipid peroxidation in the gastrointestinal tract. In this study, the formation of malondialdehyde (MDA) equivalents measured by the thiobarbituric acid reactive substances (TBARS) method was determined during in vitro digestion of cooked red meat (beef and pork), as well as white meat (chicken) and fish (salmon), whereas analysis of 4-hydroxyhexenal (HHE) and 4-hydroxynonenal (HNE) was performed during in vitro digestion of cooked beef and salmon. Comparing products with similar fat contents indicated that the amount of unsaturated fat and not total iron content was the dominating factor influencing the formation of aldehydes. It was also shown that increasing fat content in beef products caused increasing concentrations of MDA equivalents. The highest levels, however, were found in minced beef with added fish oil high in unsaturated fat. This study indicates that when ingested alone, red meat products low in unsaturated fat and low in total fat content contribute to relatively low levels of potentially genotoxic aldehydes in the gastrointestinal tract. PMID:26654171

  20. Substrate Specificity Combined with Stereopromiscuity in Glutathione Transferase A4-4-dependent Metabolism of 4-Hydroxynonenal

    PubMed Central

    Balogh, Larissa M.; Le Trong, Isolde; Kripps, Kimberly A.; Shireman, Laura M.; Stenkamp, Ronald E.; Zhang, Wei; Mannervik, Bengt; Atkins, William M.

    2010-01-01

    Conjugation to glutathione (GSH) by glutathione transferase A4-4 (GSTA4-4) is a major route of elimination for the lipid peroxidation product 4-hydroxynonenal (HNE), a toxic compound that contributes to numerous diseases. Both enantiomers of HNE are presumed to be toxic, and GSTA4-4 has negligible stereoselectivity towards them, despite its high catalytic chemospecificity for alkenals. In contrast to the highly flexible, and substrate promiscuous, GSTA1-1 isoform that has poor catalytic efficiency with HNE, GSTA4-4 has been postulated to be a rigid template that is pre-organized for HNE metabolism. However, the combination of high substrate chemoselectivity and low substrate stereoselectivity is intriguing. The mechanism by which GSTA4-4 achieves this combination is important, because it must metabolize both enantiomers of HNE to efficiently detoxify the biologically formed mixture. The crystal structures of GSTA4-4 and an engineered variant of GSTA1-1 with high catalytic efficiency toward HNE, co-crystallized with a GSH-HNE conjugate analog, demonstrate that GSTA4-4 undergoes no enantiospecific induced fit; instead the active site residue Arg15 is ideally located to interact with the 4-hydroxyl group of either HNE enantiomer. The results reveal an evolutionary strategy for achieving biologically useful stereopromiscuity towards a toxic racemate, concomitant with high catalytic efficiency and substrate specificity towards an endogenously formed toxin. PMID:20085333

  1. Substrate specificity combined with stereopromiscuity in glutathione transferase A4-4-dependent metabolism of 4-hydroxynonenal.

    PubMed

    Balogh, Larissa M; Le Trong, Isolde; Kripps, Kimberly A; Shireman, Laura M; Stenkamp, Ronald E; Zhang, Wei; Mannervik, Bengt; Atkins, William M

    2010-02-23

    Conjugation to glutathione (GSH) by glutathione transferase A4-4 (GSTA4-4) is a major route of elimination for the lipid peroxidation product 4-hydroxynonenal (HNE), a toxic compound that contributes to numerous diseases. Both enantiomers of HNE are presumed to be toxic, and GSTA4-4 has negligible stereoselectivity toward them, despite its high catalytic chemospecificity for alkenals. In contrast to the highly flexible, and substrate promiscuous, GSTA1-1 isoform that has poor catalytic efficiency with HNE, GSTA4-4 has been postulated to be a rigid template that is preorganized for HNE metabolism. However, the combination of high substrate chemoselectivity and low substrate stereoselectivity is intriguing. The mechanism by which GSTA4-4 achieves this combination is important, because it must metabolize both enantiomers of HNE to efficiently detoxify the biologically formed mixture. The crystal structures of GSTA4-4 and an engineered variant of GSTA1-1 with high catalytic efficiency toward HNE, cocrystallized with a GSH-HNE conjugate analogue, demonstrate that GSTA4-4 undergoes no enantiospecific induced fit; instead, the active site residue Arg15 is ideally located to interact with the 4-hydroxyl group of either HNE enantiomer. The results reveal an evolutionary strategy for achieving biologically useful stereopromiscuity toward a toxic racemate, concomitant with high catalytic efficiency and substrate specificity toward an endogenously formed toxin. PMID:20085333

  2. Impact of 4-hydroxynonenal on matrix metalloproteinase-9 regulation in lipopolysaccharide-stimulated RAW 264.7 cells.

    PubMed

    Schrimpe-Rutledge, Alexandra C; Fong, Kim Y; Wright, David W

    2015-03-01

    Tissue degradation and leukocyte extravasation suggest proteolytic destruction of the extracellular matrix (ECM) during severe malaria. Matrix metalloproteinases (MMPs) play an established role in ECM turnover, and increased MMP-9 protein abundance is correlated with malarial infection. The malaria pigment hemozoin (Hz) is a heme detoxification biomineral that is produced during infection and associated with biologically active lipid peroxidation products such as 4-hydroxynonenal (HNE) adsorbed to its surface. Hz has innate immunomodulatory activity, and many of its effects can be reproduced by exogenously added HNE. Hz phagocytosis enhances MMP-9 expression in monocytes; thus, this study was designed to examine the ability of HNE to alter MMP-9 regulation in activated cells of macrophage lineage. Data show that treatment of lipopolysaccharide-stimulated RAW 264.7 cells with HNE increased MMP-9 secretion and activity. HNE treatment abolished the cognate tissue inhibitor of metalloproteinase-1 protein levels, further decreasing MMP-9 regulation. Phosphorylation of both p38 mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase was induced by HNE, but only p38 MAPK inhibition lessened MMP-9 secretion. These results demonstrate the in vitro ability of HNE to cause MMP-9 dysregulation in an activated cell model. The findings may extend to myriad pathologies associated with lipid peroxidation and elevated MMP-9 levels leading to tissue damage. PMID:25663587

  3. Gsta4 Null Mouse Embryonic Fibroblasts Exhibit Enhanced Sensitivity to Oxidants: Role of 4-Hydroxynonenal in Oxidant Toxicity*

    PubMed Central

    McElhanon, Kevin E.; Bose, Chhanda; Sharma, Rajendra; Wu, Liping; Awasthi, Yogesh C.; Singh, Sharda P.

    2013-01-01

    The alpha class glutathione s-transferase (GST) isozyme GSTA4–4 (EC2.5.1.18) exhibits high catalytic efficiency to-wards 4-hydroxynon-2-enal (4-HNE), a major end product of oxidative stress induced lipid peroxidation. Exposure of cells and tissues to heat, radiation, and chemicals has been shown to induce oxidative stress resulting in elevated concentrations of 4-HNE that can be detrimental to cell survival. Alternatively, at physiological levels 4-HNE acts as a signaling molecule conveying the occurrence of oxidative events initiating the activation of adaptive pathways. To examine the impact of oxidative/electrophilic stress in a model with impaired 4-HNE metabolizing capability, we disrupted the Gsta4 gene that encodes GSTA4–4 in mice. The effect of electrophile and oxidants on embryonic fibroblasts (MEF) isolated from wild type (WT) and Gsta4 null mice were examined. Results indicate that in the absence of GSTA4–4, oxidant-induced toxicity is potentiated and correlates with elevated accumulation of 4-HNE adducts and DNA damage. Treatment of Gsta4 null MEF with 1,1,4-tris(acetyloxy)-2(E)-nonene [4-HNE(Ac)3], a pro-drug form of 4-HNE, resulted in the activation and phosphorylation of the c-jun-N-terminal kinase (JNK), extracellular-signal-regulated kinases (ERK 1/2) and p38 mitogen activated protein kinases (p38 MAPK) accompanied by enhanced cleavage of caspase-3. Interestingly, when recombinant mammalian or invertebrate GSTs were delivered to Gsta4 null MEF, activation of stress-related kinases in 4-HNE(Ac)3 treated Gsta4 null MEF were inversely correlated with the catalytic efficiency of delivered GSTs towards 4-HNE. Our data suggest that GSTA4–4 plays a major role in protecting cells from the toxic effects of oxidant chemicals by attenuating the accumulation of 4-HNE. PMID:24353929

  4. Roles of the Lipid Peroxidation Product 4-Hydroxynonenal in Obesity, the Metabolic Syndrome, and Associated Vascular and Neurodegenerative Disorders

    PubMed Central

    Mattson, Mark P.

    2009-01-01

    A rising tide of obesity and type 2 diabetes has resulted from the development of technologies that have made inexpensive high calorie foods readily available and exercise unnecessary for many people. Obesity and the metabolic syndrome (insulin resistance, visceral adiposity and dyslipidemia) wreak havoc on cells throughout the body thereby promoting cardiovascular and kidney disease, and degenerative diseases of the brain and body. Obesity and insulin resistance promote disease by increasing oxidative damage to proteins, lipids and DNA as the result of a combination of increased free radical production and an impaired ability of cells to detoxify the radicals and repair damaged molecules. By covalently modifying membrane-associated proteins, the membrane lipid peroxidation product 4-hydroxynonenal (HNE) may play particularly sinister roles in the metabolic syndrome and associated disease processes. HNE can damage pancreatic β cells and can impair the ability of muscle and liver cells to respond to insulin. HNE may promote atherosclerosis by modifying lipoproteins and can cause cardiac cell damage by impairing metabolic enzymes. An adverse role for HNE in the brain in obesity and the metabolic syndrome is suggested by studies showing that HNE levels are increased in brain cells with aging and Alzheimer’s disease. HNE can cause the dysfunction and degeneration of neurons by modifying membrane-associated glucose and glutamate transporters, ion-motive ATPases, enzymes involved in amyloid metabolism, and cytoskeletal proteins. Exercise and dietary energy restriction reduce HNE production and may also increase cellular systems for HNE detoxification including glutathione and oxidoreductases. The recent development of low molecular weight molecules that scavenge HNE suggests that HNE can be targeted in the design of drugs for the treatment of obesity, the metabolic syndrome, and associated disorders. PMID:19622391

  5. ACCUMULATION OF M1DG DNA ADDUCTS AFTER CHRONIC EXPOSURE TO PCBS, BUT NOT FROM ACUTE EXPOSURE TO DIOXIN-LIKE COMPOUNDS

    EPA Science Inventory

    ABSTRACT: Oxidative DNA damage is one of the key events leading to mutation and cancer. The present study examined the accumulation of M1dG DNA adducts, 3-(2’-deoxy-β-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one, after single or yearly exposur...

  6. Preferential binding of 4-hydroxynonenal to lysine residues in specific parasite proteins in plakortin-treated Plasmodium falciparum-parasitized red blood cells

    PubMed Central

    Schwarzer, Evelin; Gallo, Valentina; Valente, Elena; Ulliers, Daniela; Taglialatela-Scafati, Orazio; Arese, Paolo; Skorokhod, Oleksii A.

    2015-01-01

    The data show the frequencies by which the amino acid residues lysine, histidine and cysteine of six proteins of the malaria parasite Plasmodium falciparum are post-translationally modified by the lipoperoxydation endproduct 4-hydroxynonenal after challenging the parasitized red blood cell with plakortin. Plakortin is an antimalarial endoperoxide whose molecular anti-parasitic effect is described in Skorokhod et al. (2015) [1]. Plakortin did not elicit hemoglobin leakage from host red blood cells and did not oxidize reduced glutathione. PMID:26702418

  7. Increased 4-hydroxynonenal protein adducts in male GSTA4–4/PPAR-alpha double knockout mice enhance injury during early stages of alcoholic liver disease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male wild type 129/SvJ mice, and glutathione S-transferase A4-4 null (GSTA4-/-) mice for 40 d. GSTA4-/- mice were also crossed with peroxisome proliferator-activated ...

  8. 4-Hydroxynonenal Regulates TNF-α Gene Transcription Indirectly via ETS1 and microRNA-29b in Human Adipocytes Induced From Adipose Tissue-Derived Stromal Cells.

    PubMed

    Zhang, Xi-Mei; Guo, Lin; Huang, Xiang; Li, Qiu-Ming; Chi, Mei-Hua

    2016-08-01

    Obesity is characterized by an accumulation of excessive body fat and can be diagnosed by a variety of measures, such as BMI. However, in some obese individuals, oxidative stress is also thought to be an important pathogenic mechanism of obesity-associated metabolic syndrome. Oxidative stress increases the lipid peroxidation product, 4-hydroxynonenal (4-HNE), which is one of the most abundant and active lipid peroxides. Within the adipose tissue, adipocytes are derived from adipose tissue-derived stromal cells (ADSCs), which play a key role in the generation and metabolism of adipose tissue. Additionally, obesity is associated with low-grade inflammation. Specific microRNAs (miRNAs) that regulate obesity-associated inflammation are largely dysregulated in metabolic syndrome (MS). In this study, we aim to confirm whether 4-HNE and miRNAs play a role in the regulation of TNF-α gene transcription. We enrolled six obese individuals who were referred to Harbin Medical University (Heilongjiang, China) and six nonobese control participants. Plasma 4-HNE levels of the 12 subjects were determined by ELISA. Using qRT-PCR, we measured ETS1, miR-29b, SP1, and TNF-α levels in subcutaneous white adipose tissue (WAT). Furthermore, we examined the relationship between ETS1 and TNF-α using a luciferase reporter assay and a ChIP assay. Our results suggest that ETS1 promotes TNF-α gene transcription in adipocytes. In addition, we demonstrated that 4-HNE promotes TNF-α gene transcription through the inhibition of the miR-29b → SP1 → TNF-α pathway and promotion of the ETS1 → TNF-α pathway. Anat Rec, 299:1145-1152, 2016. © 2016 Wiley Periodicals, Inc. PMID:27164408

  9. The generation of 4-hydroxynonenal, an electrophilic lipid peroxidation end product, in rabbit cornea organ cultures treated with UVB light and nitrogen mustard

    SciTech Connect

    Zheng, Ruijin; Po, Iris; Mishin, Vladimir; Black, Adrienne T.; Heck, Diane E.; Laskin, Debra L.; Sinko, Patrick J.; Gerecke, Donald R.; Gordon, Marion K.; Laskin, Jeffrey D.

    2013-10-15

    The cornea is highly sensitive to oxidative stress, a process that can lead to lipid peroxidation. Ultraviolet light B (UVB) and nitrogen mustard (mechlorethamine) are corneal toxicants known to induce oxidative stress. Using a rabbit air-lifted corneal organ culture model, the oxidative stress responses to these toxicants in the corneal epithelium was characterized. Treatment of the cornea with UVB (0.5 J/cm{sup 2}) or nitrogen mustard (100 nmol) resulted in the generation of 4-hydroxynonenal (4-HNE), a reactive lipid peroxidation end product. This was associated with increased expression of the antioxidant, heme oxygenase-1 (HO-1). In human corneal epithelial cells in culture, addition of 4-HNE or 9-nitrooleic acid, a reactive nitrolipid formed during nitrosative stress, caused a time-dependent induction of HO-1 mRNA and protein; maximal responses were evident after 10 h with 30 μM 4-HNE or 6 h with 10 μM 9-nitrooleic acid. 4-HNE and 9-nitrooleic acid were also found to activate Erk1/2, JNK and p38 MAP kinases, as well as phosphoinositide-3-kinase (PI3)/Akt. Inhibition of p38 blocked 4-HNE- and 9-nitrooleic acid-induced HO-1 expression. Inhibition of Erk1/2, and to a lesser extent, JNK and PI3K/Akt, suppressed only 4-HNE-induced HO-1, while inhibition of JNK and PI3K/Akt, but not Erk1/2, partly reduced 9-nitrooleic acid-induced HO-1. These data indicate that the actions of 4-HNE and 9-nitrooleic acid on corneal epithelial cells are distinct. The sensitivity of corneal epithelial cells to oxidative stress may be an important mechanism mediating tissue injury induced by UVB or nitrogen mustard. - Highlights: • UVB or nitrogen mustard causes rabbit corneal epithelial injury. • 4-Hydroxynonenal (4-HNE) was formed and heme oxygenase-1 (HO-1) was increased. • 4-HNE induced HO-1 mRNA and protein expression in human corneal epithelial cells. • The induction of HO-1 by 4-HNE was through MAP kinase activation.

  10. Both wheat (Triticum aestivum) bran arabinoxylans and gut flora-mediated fermentation products protect human colon cells from genotoxic activities of 4-hydroxynonenal and hydrogen peroxide.

    PubMed

    Glei, Michael; Hofmann, Thomas; Küster, Katrin; Hollmann, Jürgen; Lindhauer, Meinolf G; Pool-Zobel, Beatrice L

    2006-03-22

    Dietary fibers are fermented by the gut flora to yield short chain fatty acids (SCFAs), which inhibit the growth of tumor cells, induce glutathione S-transferases (GSTs), and protect cells from the genotoxic activity of 4-hydroxynonenal (HNE). Here, we investigated effects of wheat bran-derived arabinoxylans and fermentation products on these parameters of chemoprevention. Newly isolated water extractable (WeAx) and alkali extractable arabinoxylans (AeAx) were fermented under anaerobic conditions with human feces. Resulting fermentation supernatants (FSs) were analyzed for SCFAs and used to treat HT29 colon cancer cells. Cell growth, cytotoxicity, antigenotoxicity against hydrogen peroxide (H2O2) or HNE, and GST activity were determined. Nonfermented WeAx decreased H2O2-induced DNA damage by 64%, thus demonstrating chemoprotective properties by this nonfermented wheat bran fiber. The fermentation of WeAx and AeAx resulted in 3-fold increases of SCFA, but all FSs (including the control without arabinoxylans) inhibited the growth of the HT29 cells, reduced the genotoxicity of HNE, and enhanced the activity of GSTs (FS WeAx, 2-fold; FS AeAx, 1.7-fold; and control FS, 1.4-fold), which detoxify HNE. Thus, increases in SCFAs were not reflected by enhanced functional effects. The conclusion is that fermentation mixtures contain modulatory compounds that arise from the feces and might add to the effectiveness of SCFAs. PMID:16536580

  11. Alterations in mitochondrial respiratory functions, redox metabolism and apoptosis by oxidant 4-hydroxynonenal and antioxidants curcumin and melatonin in PC12 cells

    SciTech Connect

    Raza, Haider John, Annie; Brown, Eric M.; Benedict, Sheela; Kambal, Amr

    2008-01-15

    Cellular oxidative stress and alterations in redox metabolisms have been implicated in the etiology and pathology of many diseases including cancer. Antioxidant treatments have been proven beneficial in controlling these diseases. We have recently shown that 4-hydroxynonenal (4-HNE), a by-product of lipid peroxidation, induces oxidative stress in PC12 cells by compromising the mitochondrial redox metabolism. In this study, we have further investigated the deleterious effects of 4-HNE on mitochondrial respiratory functions and apoptosis using the same cell line. In addition, we have also compared the effects of two antioxidants, curcumin and melatonin, used as chemopreventive agents, on mitochondrial redox metabolism and respiratory functions in these cells. 4-HNE treatment has been shown to cause a reduction in glutathione (GSH) pool, an increase in reactive oxygen species (ROS), protein carbonylation and apoptosis. A marked inhibition in the activities of the mitochondrial respiratory enzymes, cytochrome c oxidase and aconitase was observed after 4-HNE treatment. Increased nuclear translocation of NF-kB/p65 protein was also observed after 4-HNE treatment. Curcumin and melatonin treatments, on the other hand, maintained the mitochondrial redox and respiratory functions without a marked effect on ROS production and cell viability. These results suggest that 4-HNE-induced cytotoxicity may be associated, at least in part, with the altered mitochondrial redox and respiratory functions. The alterations in mitochondrial energy metabolism and redox functions may therefore be critical in determining the difference between cell death and survival.

  12. Rapid liquid chromatography-tandem mass spectrometry analysis of 4-hydroxynonenal for the assessment of oxidative degradation and safety of vegetable oils.

    PubMed

    Gabbanini, Simone; Matera, Riccardo; Valvassori, Alice; Valgimigli, Luca

    2015-04-15

    A novel method for the UHPLC-MS/MS analysis of (E)-4-hydroxynonenal (4-HNE) is described. The method is based on derivatization of 4-HNE with pentafluorophenylhydrazine (1) or 4-trifluoromethylphenylhydrazine (2) in acetonitrile in the presence of trifluoroacetic acid as catalyst at room temperature and allows complete analysis of one sample of vegetable oil in only 21 min, including sample preparation and chromatography. The method involving hydrazine 1, implemented in an ion trap instrument with analysis of the transition m/z 337→154 showed LOD=10.9 nM, average accuracy of 101% and precision ranging 2.5-4.0% RSD intra-day (2.7-4.1% RSD inter-day), with 4-HNE standard solutions. Average recovery from lipid matrices was 96.3% from vaseline oil, 91.3% from sweet almond oil and 105.3% from olive oil. The method was tested on the assessment of safety and oxidative degradation of seven samples of dietary oil (soybean, mixed seeds, corn, peanut, sunflower, olive) and six cosmetic-grade oils (avocado, blackcurrant, apricot kernel, echium, sesame, wheat germ) and effectively detected increased 4-HNE levels in response to chemical (Fenton reaction), photochemical, or thermal stress and aging, aimed at mimicking typical oxidation associated with storage or industrial processing. The method is a convenient, cost-effective and reliable tool to assess quality and safety of vegetable oils. PMID:25818139

  13. 4-Hydroxynonenal, an aldehydic product of lipid peroxidation, impairs signal transduction associated with muscarinic acetylcholine and metabotropic glutamate receptors: possible action on G alpha(q/11).

    PubMed

    Blanc, E M; Kelly, J F; Mark, R J; Waeg, G; Mattson, M P

    1997-08-01

    Considerable data indicate that oxidative stress and membrane lipid peroxidation contribute to neuronal degeneration in an array of age-related neurodegenerative disorders. In contrast, the impact of subtoxic levels of membrane lipid peroxidation on neuronal function is largely unknown. We now report that 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, disrupts coupling of muscarinic cholinergic receptors and metabotropic glutamate receptors to phospholipase C-linked GTP-binding proteins in cultured rat cerebrocortical neurons. At subtoxic concentrations, HNE markedly inhibited GTPase activity, inositol phosphate release, and elevation of intracellular calcium levels induced by carbachol (muscarinic agonist) and (RS)-3,5-dihydroxyphenyl glycine (metabotropic glutamate receptor agonist). Maximal impairment of agonist-induced responses occurred within 30 min of exposure to HNE. Other aldehydes, including malondialdehyde, had little effect on agonist-induced responses. Antioxidants that suppress lipid peroxidation did not prevent impairment of agonist-induced responses by HNE, whereas glutathione, which is known to bind and detoxify HNE, did prevent impairment of agonist-induced responses. HNE itself did not induce oxidative stress. Immunoprecipitation-western blot analysis using an antibody to HNE-protein conjugates showed that HNE can bind to G alpha(q/11). HNE also significantly suppressed inositol phosphate release induced by aluminum fluoride. Collectively, our data suggest that HNE plays a role in altering receptor-G protein coupling in neurons under conditions of oxidative stress that may occur both normally, and before cell degeneration and death in pathological settings. PMID:9231714

  14. The generation of 4-hydroxynonenal, an electrophilic lipid peroxidation end product, in rabbit cornea organ cultures treated with UVB light and nitrogen mustard

    PubMed Central

    Zheng, Ruijin; Po, Iris; Mishin, Vladimir; Black, Adrienne T.; Heck, Diane E.; Laskin, Debra L.; Sinko, Patrick J.; Gerecke, Donald R.; Gordon, Marion K.; Laskin, Jeffrey D.

    2013-01-01

    The cornea is highly sensitive to oxidative stress, a process that can lead to lipid peroxidation. Ultraviolet light B (UVB) and nitrogen mustard (mechlorethamine) are corneal toxicants known to induce oxidative stress. Using a rabbit air-lifted corneal organ culture model, the oxidative stress responses to these toxicants in the corneal epithelium was characterized. Treatment of the cornea with UVB (0.5 J/cm2) or nitrogen mustard (100 nmol) resulted in the generation of 4-hydroxynonenal (4-HNE), a reactive lipid peroxidation end product. This was associated with increased expression of the antioxidant, heme oxygenase-1 (HO-1). In human corneal epithelial cells in culture, addition of 4-HNE or 9-nitrooleic acid, a reactive nitrolipid formed during nitrosative stress, caused a time-dependent induction of HO-1 mRNA and protein; maximal responses were evident after 10 hr with 30 μM 4-HNE or 6 hr with 10 μM 9-nitrooleic acid. 4-HNE and 9-nitrooleic acid were also found to activate Erk1/2, JNK and p38 MAP kinases, as well as phosphoinositide-3-kinase (PI3)/Akt. Inhibition of p38 blocked 4-HNE- and 9-nitrooleic acid-induced HO-1 expression. Inhibition of Erk1/2, and to a lesser extent, JNK and PI3K/Akt, suppressed only 4-HNE-induced HO-1, while inhibition of JNK and PI3K/Akt, but not Erk1/2, partly reduced 9-nitrooleic acid-induced HO-1. These data indicate that the actions of 4-HNE and 9-nitrooleic acid on corneal epithelial cells are distinct. The sensitivity of corneal epithelial cells to oxidative stress may be an important mechanism mediating tissue injury induced by UVB or nitrogen mustard. PMID:23845594

  15. 4-Hydroxynonenal enhances MMP-9 production in murine macrophages via 5-lipoxygenase-mediated activation of ERK and p38 MAPK

    SciTech Connect

    Lee, Seung J.; Kim, Chae E.; Yun, Mi R.; Seo, Kyo W.; Park, Hye M.; Yun, Jung W.; Shin, Hwa K.; Bae, Sun S.; Kim, Chi D.

    2010-01-15

    Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B{sub 4} (LTB{sub 4}) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT{sub 1} (cysLT{sub 1}) receptor antagonist, REV-5901 as well as a BLT{sub 1} receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB{sub 4} and cysLT (LTC{sub 4} and LTD{sub 4}) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB{sub 4} and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.

  16. Interactive effects of ultraviolet-B radiation and pesticide exposure on DNA photo-adduct accumulation and expression of DNA damage and repair genes in Xenopus laevis embryos.

    PubMed

    Yu, Shuangying; Tang, Song; Mayer, Gregory D; Cobb, George P; Maul, Jonathan D

    2015-02-01

    though expression of XPC and CSA were induced by exposure to UVB or pesticides, XPA was the limiting factor in the NER pathway. Our results suggest that pesticides may increase the accumulation of UVB-induced DNA photo-adducts and one likely mechanism is the alteration of critical NER gene expression. The present study provides important implications for evaluating the combined risks of pesticide usage and potentially increasing UVB radiation in aquatic ecosystems. PMID:25569846

  17. Effects of Toxicologically Relevant Xenobiotics and the Lipid-Derived Electrophile 4-Hydroxynonenal on Macrophage Cholesterol Efflux: Silencing Carboxylesterase 1 Has Paradoxical Effects on Cholesterol Uptake and Efflux

    PubMed Central

    2015-01-01

    Cholesterol cycles between free cholesterol (unesterified) found predominantly in membranes and cholesteryl esters (CEs) stored in cytoplasmic lipid droplets. Only free cholesterol is effluxed from macrophages via ATP-binding cassette (ABC) transporters to extracellular acceptors. Carboxylesterase 1 (CES1), proposed to hydrolyze CEs, is inactivated by oxon metabolites of organophosphorus pesticides and by the lipid electrophile 4-hydroxynonenal (HNE). We assessed the ability of these compounds to reduce cholesterol efflux from foam cells. Human THP-1 macrophages were loaded with [3H]-cholesterol/acetylated LDL and then allowed to equilibrate to enable [3H]-cholesterol to distribute into its various cellular pools. The cholesterol-engorged cells were then treated with toxicants in the absence of cholesterol acceptors for 24 h, followed by a 24 h efflux period in the presence of toxicant. A concentration-dependent reduction in [3H]-cholesterol efflux via ABCA1 (up to 50%) was found for paraoxon (0.1–10 μM), whereas treatment with HNE had no effect. A modest reduction in [3H]-cholesterol efflux via ABCG1 (25%) was found after treatment with either paraoxon or chlorpyrifos oxon (10 μM each) but not HNE. No difference in efflux rates was found after treatments with either paraoxon or HNE when the universal cholesterol acceptor 10% (v/v) fetal bovine serum was used. When the re-esterification arm of the CE cycle was disabled in foam cells, paraoxon treatment increased CE levels, suggesting the neutral CE hydrolysis arm of the cycle had been inhibited by the toxicant. However, paraoxon also partially inhibited lysosomal acid lipase, which generates cholesterol for efflux, and reduced the expression of ABCA1 protein. Paradoxically, silencing CES1 expression in macrophages did not affect the percent of [3H]-cholesterol efflux. However, CES1 mRNA knockdown markedly reduced cholesterol uptake by macrophages, with SR-A and CD36 mRNA reduced 3- and 4-fold, respectively

  18. Detection of carcinogenic etheno-DNA adducts in children and adolescents with non-alcoholic steatohepatitis (NASH)

    PubMed Central

    Teufel, Ulrike; Peccerella, Teresa; Engelmann, Guido; Bruckner, Thomas; Flechtenmacher, Christa; Millonig, Gunda; Stickel, Felix; Hoffmann, Georg F.; Schirmacher, Peter; Mueller, Sebastian; Bartsch, Helmut

    2015-01-01

    Background Carcinogenic exocyclic-DNA adducts like 1,N6-etheno-2'-deoxyadenosine (εdA) are formed through reactive intermediates of 4-hydroxynonenal (4-HNE) or other lipid peroxidation (LPO) products with the DNA bases A, C, methyl-C and G. High levels of hepatic etheno-DNA adducts have been detected in cancer prone liver diseases including alcoholic liver disease (ALD). In ALD εdA levels correlated significantly with cytochrome P-450 2E1 (CYP2E1) expression which is also induced in non-alcoholic steatohepatitis (NASH). We investigated the occurrence of εdA adducts in children with NASH as a DNA damage marker. Methods Liver biopsies from 21 children/adolescents with histologically proven NASH were analysed for hepatic fat content, inflammation, and fibrosis. εdA levels in DNA, CYP2E1-expression and protein bound 4-hydroxynonenal (HNE) were semi-quantitatively evaluated by immunohistochemistry. Results Among 21 NASH children, εdA levels in the liver were high in 3, moderate in 5, weak in 9 and not elevated in 4 patients. There was a positive correlation between CYP2E1 and protein-bound 4-HNE (r=0.60; P=0.008) and a trend for a positive relationship for CYP2E1 vs. staining intensity of εdA (r=0.45; P=0.06). Inflammatory activity and fibrosis correlated significantly (r=0.49, P=0.023). Conclusions Our results demonstrate for the first time the presence of elevated carcinogenic etheno-DNA lesions (εdA) in the majority (17/21) of liver biopsies from young NASH patients. Our data suggest that LPO-derived etheno-adducts are implicated in NASH. Whether these adducts may serve as predictive risk markers in NASH children to develop hepatocellular cancer later in life remains to be investigated. PMID:26734629

  19. 4-HNE Adduct Stability Characterized by Collision-Induced Dissociation and Electron Transfer Dissociation Mass Spectrometry

    PubMed Central

    Fritz, Kristofer S.; Kellersberger, Katherine A.; Gomez, Jose D.; Petersen, Dennis R.

    2012-01-01

    4-hydroxynonenal (4-HNE) alters numerous proteomic and genomic processes. Understanding chemical mechanisms of 4-HNE interactions with biomolecules and their respective stabilities may lead to new discoveries in biomarkers for numerous diseases of oxidative stress. Collision-induced dissociation (CID) and electron transfer dissociation (ETD) MS/MS were utilized to examine the stability of a 4-HNE-Cys Michael adduct. CID conditions resulted in the neutral loss of 4-HNE, also known as a retro-Michael addition reaction (RMA). Consequently, performing ETD fragmentation on this same adduct did not result in RMA. Interestingly, 4-HNE adduct reduction via sodium borohydride (NaBH4) treatment stabilized against the CID induced RMA. In a direct comparison of three forms of 4-HNE adducts, computational modeling revealed sizeable shifts in the shape and orientation of the lowest unoccupied molecular orbital (LUMO) density around the 4-HNE-Cys moiety. These findings demonstrate that ETD MS/MS analysis can be used to improve the detection of 4-HNE-protein modifications by preventing RMA reactions from occurring. PMID:22404378

  20. Involvement of peroxisome proliferator-activated receptor β/δ (PPAR β/δ) in BDNF signaling during aging and in Alzheimer disease: possible role of 4-hydroxynonenal (4-HNE).

    PubMed

    Benedetti, Elisabetta; D'Angelo, Barbara; Cristiano, Loredana; Di Giacomo, Erica; Fanelli, Francesca; Moreno, Sandra; Cecconi, Francesco; Fidoamore, Alessia; Antonosante, Andrea; Falcone, Roberta; Ippoliti, Rodolfo; Giordano, Antonio; Cimini, Annamaria

    2014-01-01

    Aging and many neurological disorders, such as AD, are linked to oxidative stress, which is considered the common effector of the cascade of degenerative events. In this phenomenon, reactive oxygen species play a fundamental role in the oxidative decomposition of polyunsaturated fatty acids, resulting in the formation of a complex mixture of aldehydic end products, such as malondialdehyde, 4-hydroxynonenal, and other alkenals. Interestingly, 4-HNE has been indicated as an intracellular agonist of peroxisome proliferator-activated receptor β/δ. In this study, we examined, at early and advanced AD stages (3, 9, and 18 months), the pattern of 4-HNE and its catabolic enzyme glutathione S-transferase P1 in relation to the expression of PPARβ/δ, BDNF signaling, as mRNA and protein, as well as on their pathological forms (i.e., precursors or truncated forms). The data obtained indicate a novel detrimental age-dependent role of PPAR β/δ in AD by increasing pro-BDNF and decreasing BDNF/TrkB survival pathways, thus pointing toward the possibility that a specific PPARβ/δ antagonist may be used to counteract the disease progression. PMID:24621497

  1. 4-Hydroxynonenal induces mitochondrial oxidative stress, apoptosis and expression of glutathione S-transferase A4-4 and cytochrome P450 2E1 in PC12 cells

    SciTech Connect

    Raza, Haider . E-mail: h.raza@uaeu.ac.ae; John, Annie

    2006-10-15

    An excessive and sustained increase in reactive oxygen species (ROS) production and oxidative stress have been implicated in the pathogenesis of many diseases. In the present study, we have demonstrated that 4-hydroxynonenal (4-HNE), a product of lipid peroxidation, alters glutathione (GSH) pools and induces oxidative stress in PC12 cells in culture. This increase was accompanied by alterations in subcellular ROS and glutathione (GSH) metabolisms. The GSH homeostasis was affected as both mitochondrial and extramitochondrial GSH levels, GSH peroxidase and glutathione reductase activities were inhibited and glutathione S-transferase (GST) activity was increased after 4-HNE treatment. A concentration- and time-dependent increase in cytochrome P450 2E1 (CYP 2E1) activity in the mitochondria and postmitochondrial supernatant was also observed. 4-HNE-induced oxidative stress also caused an increase in the expression of GSTA4-4, CYP2E1 and Hsp70 proteins in the mitochondria. Increased oxidative stress in PC12 cells initiated apoptosis as indicated by the release of mitochondrial cytochrome c, activation of poly-(ADP-ribose) polymerase (PARP), DNA fragmentation and decreased expression of antiapoptotic Bcl-2 proteins. Mitochondrial respiratory and redox functions also appeared to be affected markedly by 4-HNE treatment. These results suggest that HNE-induced oxidative stress and apoptosis might be associated with altered mitochondrial functions and a compromised GSH metabolism and ROS clearance.

  2. Antiatherogenic and antitumoral properties of Opuntia cladodes: inhibition of low density lipoprotein oxidation by vascular cells, and protection against the cytotoxicity of lipid oxidation product 4-hydroxynonenal in a colorectal cancer cellular model.

    PubMed

    Keller, Julia; Camaré, Caroline; Bernis, Corinne; Astello-García, Marizel; de la Rosa, Ana-Paulina Barba; Rossignol, Michel; del Socorro Santos Díaz, María; Salvayre, Robert; Negre-Salvayre, Anne; Guéraud, Françoise

    2015-09-01

    Opuntia species have been used for thousands of years as a folk medicine in the treatment of diseases. However, the components and protective mechanisms are still unclear. We make the hypothesis that Opuntia species may protect the development of oxidative stress-associated diseases, such as atherosclerosis or colon cancer, via their antioxidant properties. We investigated the protective effect of Opuntia cladode powder against the oxidation of low-density lipoprotein (LDL) evoked by vascular endothelial cells, an important risk factor for atherosclerosis development, and the toxicity of 4-hydroxynonenal (a major lipid peroxidation product) on normal (Apc +/+) and preneoplastic (Apc min/+) immortalized epithelial colon cells. Various Opuntia species classified according to their degree of domestication, from the wildest (Opuntia streptacantha, Opuntia hyptiacantha, Opuntia megacantha), medium (Opuntia albicarpa), to the most domesticated (Opuntia ficus-indica) were tested. Cladode powders prepared from these Opuntia species significantly inhibited LDL oxidation induced by incubation with murine endothelial cells and the subsequent foam cell formation of RAW 264.7 murine macrophages and cytotoxicity on murine endothelial cells. Moreover, Opuntia cladode powder blocked the promotion of colon cancer development on an in vitro model of colonocytes. It may be noted that the phenolic acid and flavonoids content, the antioxidant capacity, and the protective effect were relatively similar in all the cladode powders from wild (O. streptacantha) and domesticated Opuntia. Altogether, these data confirm the therapeutic potential of Opuntia cladodes in diseases associated with oxidative stress. PMID:25840808

  3. DNA adducts in biomonitoring.

    PubMed

    Hemminki, K

    1995-05-01

    The types of occupational groups studied by postlabelling include foundry, coke oven and aluminium workers, roofers, garage and terminal workers, car mechanics and chimney sweeps. There does not seem to be a direct relationship between the exposure and adduct levels. However, the postlabelling assay is sensitive enough to show adducts in apparently unexposed individuals. The origin of such adducts is unknown; in the case of aromatic adducts, the origin is likely to be environmental and/or dietary. PMID:7618142

  4. Screening for DNA adducts by data-dependent constant neutral loss-triple stage mass spectrometry with a linear quadrupole ion trap mass spectrometer.

    PubMed

    Bessette, Erin E; Goodenough, Angela K; Langouët, Sophie; Yasa, Isil; Kozekov, Ivan D; Spivack, Simon D; Turesky, Robert J

    2009-01-15

    A two-dimensional linear quadrupole ion trap mass spectrometer (LIT/MS) was employed to simultaneously screen for DNA adducts of environmental, dietary, and endogenous genotoxicants, by data-dependent constant neutral loss scanning followed by triple-stage mass spectrometry (CNL-MS3). The loss of the deoxyribose (dR) from the protonated DNA adducts ([M + H - 116]+) in the MS/MS scan mode triggered the acquisition of MS3 product ion spectra of the aglycone adducts [BH2]+. Five DNA adducts of the tobacco carcinogen 4-aminobiphenyl (4-ABP) were detected in human hepatocytes treated with 4-ABP, and three DNA adducts of the cooked-meat carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) were identified in the livers of rats exposed to MeIQx, by the CNL-MS3 scan mode. Buccal cell DNA from tobacco smokers was screened for DNA adducts of various classes of carcinogens in tobacco smoke including 4-ABP, 2-amino-9H-pyrido[2,3-b]indole (AalphaC), and benzo[a]pyrene (BaP); the cooked-meat carcinogens MeIQx, AalphaC, and 2-amino-1-methyl-6-phenylmidazo[4,5-b]pyridine (PhIP); and the lipid peroxidation products acrolein (AC) and trans-4-hydroxynonenal (HNE). The CNL-MS3 scanning technique can be used to simultaneously screen for multiple DNA adducts derived from different classes of carcinogens, at levels of adduct modification approaching 1 adduct per 108 unmodified DNA bases, when 10 microg of DNA is employed for the assay. PMID:19086795

  5. Copper accumulation and lipid oxidation precede inflammation and myelin lesions in N,N-diethyldithiocarbamate peripheral myelinopathy

    SciTech Connect

    Viquez, Olga M.; Valentine, Holly L.; Amarnath, Kalyani; Milatovic, Dejan; Valentine, William M.

    2008-05-15

    Dithiocarbamates have a wide spectrum of applications in industry, agriculture and medicine with new applications being actively investigated. One adverse effect of dithiocarbamates is the neurotoxicity observed in humans and experimental animals. Results from previous studies have suggested that dithiocarbamates elevate copper and promote lipid oxidation within myelin membranes. In the current study, copper levels, lipid oxidation, protein oxidative damage and markers of inflammation were monitored as a function of N,N-diethyldithiocarbamate (DEDC) exposure duration in an established model for DEDC-mediated myelinopathy in the rat. Intra-abdominal administration of DEDC was performed using osmotic pumps for periods of 2, 4, and 8 weeks. Metals in brain, liver and tibial nerve were measured using ICP-MS and lipid oxidation assessed through HPLC measurement of malondialdehyde in tibial nerve, and GC/MS measurement of F{sub 2} isoprostanes in sciatic nerve. Protein oxidative injury of sciatic nerve proteins was evaluated through quantification of 4-hydroxynonenal protein adducts using immunoassay, and inflammation monitored by quantifying levels of IgGs and activated macrophages using immunoassay and immunohistochemistry methods, respectively. Changes in these parameters were then correlated to the onset of structural lesions, determined by light and electron microscopy, to delineate the temporal relationship of copper accumulation and oxidative stress in peripheral nerve to the onset of myelin lesions. The data provide evidence that DEDC mediates lipid oxidation and elevation of total copper in peripheral nerve well before myelin lesions or activated macrophages are evident. This relationship is consistent with copper-mediated oxidative stress contributing to the myelinopathy.

  6. The expression and function of vascular endothelial growth factor in retinal pigment epithelial (RPE) cells is regulated by 4-hydroxynonenal (HNE) and glutathione S-transferaseA4-4

    SciTech Connect

    Vatsyayan, Rit; Lelsani, Poorna Chandra Rao; Chaudhary, Pankaj; Kumar, Sushil; Awasthi, Sanjay; Awasthi, Yogesh C.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Low concentration of HNE (0.1-1.0 {mu}M) induced secretion of VEGF in RPE cells. Black-Right-Pointing-Pointer VEGF secreted medium of RPE cells promoted proliferation of endothelial cells. Black-Right-Pointing-Pointer VEGFR2 expression was attenuated with increasing concentrations of HNE. Black-Right-Pointing-Pointer These effects of HNE could be blocked by the over expression of GSTA4-4 in cells. -- Abstract: It is well established that 4-hydroxynonenal (HNE) plays a major role in oxidative stress-induced signaling and the toxicity of oxidants. Surprisingly our recent studies also demonstrate that low levels of HNE generated during oxidative stress promote cell survival mechanisms and proliferation. Since the expression and secretion of VEGF is known to be affected by Oxidative stress, during present studies, we have examined dose dependent effect of HNE on VEGF expression and secretion in a model of retinal pigment epithelial (RPE) cells in culture. Results of these studies showed that while inclusion of 0.1 {mu}M HNE in the medium caused increased secretion of VEGF, its secretion and expression was significantly suppressed in the presence of >5 {mu}M HNE in the media. These concentration dependent hormetic effects of HNE on VEGF secretion could be blocked by the over expression of GSTA4-4 indicating that these effects were specifically attributed to HNE and regulated by GSTA4-4. VEGF secreted into the media showed angiogenic properties as indicated by increased migration and tube formation of HUVEC in matrigel when grown in media from RPE cells treated with 1 {mu}M HNE. The corresponding media from GSTA4-4 over expressing RPE cells had no effect on migration and tube formation of HUVEC in matrigel. These results are consistent with earlier studies showing that at low concentrations, HNE promotes proliferative mechanisms and suggest that HNE induces VEGF secretion from RPE cells that acts in a paracrine fashion to induce

  7. Polychlorinated Biphenyls Induce Oxidative DNA Adducts in Female Sprague-Dawley Rats.

    PubMed

    Mutlu, Esra; Gao, Lina; Collins, Leonard B; Walker, Nigel J; Hartwell, Hadley J; Olson, James R; Sun, Wei; Gold, Avram; Ball, Louise M; Swenberg, James A

    2016-08-15

    Polychlorinated biphenyls (PCBs) are organic chemicals that were traditionally produced and widely used in industry as mixtures and are presently formed as byproducts of pigment and dye manufacturing. They are known to persist and bioaccumulate in the environment. Some have been shown to induce liver cancer in rodents. Although the mechanism of the toxicity of PCBs is unknown, it has been shown that they increase oxidative stress, including lipid peroxidation. We hypothesized that oxidative stress-induced DNA damage could be a contributor for PCB carcinogenesis and analyzed several DNA adducts in female Sprague-Dawley rats exposed to 3,3',4,4',5-pentachlorobiphenyl (PCB 126), 2,2',4,4',5,5'-hexachlorobiphenyl (PCB 153), and a binary mixture (PCB 126 + 153) for 14, 31, and 53 wks. Eight adducts were measured to profile oxidative DNA lesions, including 8-oxo-deoxyguanosine (8-oxo-dG), 1,N(6)-ethenodeoxyadenosine (1,N(6)-εdA), N(2),3-ethenoguanine (N(2),3-εG), 1,N(2)-ethenodeoxyguanosine (1,N(2)-εdG), as well as malondialdehyde (M1dG), acrolein (AcrdG), crotonaldehyde (CrdG), and 4-hydroxynonenal-derived dG adducts (HNEdG) by LC-MS/MS analysis. Statistically significant increases were observed for 8-oxo-dG and 1,N(6)-εdA concentrations in hepatic DNA of female rats exposed to the binary mixture (1000 ng/kg/day + 1000 μg/kg/day) but not in rats exposed to PCB 126 (1000 ng/kg/day) or PCB 153 (1000 μg/kg/day) for 14 and 31 wks. However, exposure to PCB 126 (1000 ng/kg/day) for 53 wks significantly increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, and M1dG. Exposure to PCB 153 (1000 μg/kg/day) for 53 wks increased 8-oxo-dG, and 1,N(6)-εdA. Exposure to the binary mixture for 53 wks increased 8-oxo-dG, 1,N(6)-εdA, AcrdG, 1,N(2)-εdG, and N(2),3-εG significantly above control groups. Increased hepatic oxidative DNA adducts following exposure to PCB 126, PCB 153, or the binary mixture shows that an increase in DNA damage may play an important role in hepatic toxicity and

  8. Isolevuglandin Adducts in Disease

    PubMed Central

    Bi, Wenzhao

    2015-01-01

    Abstract Significance: A diverse family of lipid-derived levulinaldehydes, isolevuglandins (isoLGs), is produced by rearrangement of endoperoxide intermediates generated through both cyclooxygenase (COX) and free radical-induced cyclooxygenation of polyunsaturated fatty acids and their phospholipid esters. The formation and reactions of isoLGs with other biomolecules has been linked to alcoholic liver disease, Alzheimer's disease, age-related macular degeneration, atherosclerosis, cardiac arythmias, cancer, end-stage renal disease, glaucoma, inflammation of allergies and infection, mitochondrial dysfunction, multiple sclerosis, and thrombosis. This review chronicles progress in understanding the chemistry of isoLGs, detecting their production in vivo and understanding their biological consequences. Critical Issues: IsoLGs have never been isolated from biological sources, because they form adducts with primary amino groups of other biomolecules within seconds. Chemical synthesis enabled investigation of isoLG chemistry and detection of isoLG adducts present in vivo. Recent Advances: The first peptide mapping and sequencing of an isoLG-modified protein present in human retina identified the modification of a specific lysyl residue of the sterol C27-hydroxylase Cyp27A1. This residue is preferentially modified by iso[4]LGE2 in vitro, causing loss of function. Adduction of less than one equivalent of isoLG can induce COX-associated oligomerization of the amyloid peptide Aβ1-42. Adduction of isoLGE2 to phosphatidylethanolamines causes gain of function, converting them into proinflammatory isoLGE2-PE agonists that foster monocyte adhesion to endothelial cells. Future Directions: Among the remaining questions on the biochemistry of isoLGs are the dependence of biological activity on isoLG isomer structure, the structures and mechanism of isoLG-derived protein–protein and DNA–protein cross-link formation, and its biological consequences. Antioxid. Redox Signal. 22

  9. Structural Elucidation of a Carnosine-Acrolein Adduct and its Quantification in Human Urine Samples

    PubMed Central

    Bispo, Vanderson S.; de Arruda Campos, Ivan P.; Di Mascio, Paolo; Medeiros, Marisa H. G.

    2016-01-01

    Aldehydes accumulate in inflammation, during myocardial infarction and have been associated with pain symptoms. One pathway of aldehyde detoxification is the conjugation with carnosine. A 3-methylpyridinium carnosine adduct from the reaction of carnosine and acrolein was characterized using extensive spectroscopic measurements. The adduct with urinary concentrations of 1.82 ± 0.68 nmol/mg of creatinine is one of the most abundant acrolein metabolites in urine and opens promising therapeutic strategies for carnosine. PMID:26783107

  10. Structural Elucidation of a Carnosine-Acrolein Adduct and its Quantification in Human Urine Samples.

    PubMed

    Bispo, Vanderson S; de Arruda Campos, Ivan P; Di Mascio, Paolo; Medeiros, Marisa H G

    2016-01-01

    Aldehydes accumulate in inflammation, during myocardial infarction and have been associated with pain symptoms. One pathway of aldehyde detoxification is the conjugation with carnosine. A 3-methylpyridinium carnosine adduct from the reaction of carnosine and acrolein was characterized using extensive spectroscopic measurements. The adduct with urinary concentrations of 1.82 ± 0.68 nmol/mg of creatinine is one of the most abundant acrolein metabolites in urine and opens promising therapeutic strategies for carnosine. PMID:26783107

  11. DNA adducts-chemical addons.

    PubMed

    Rajalakshmi, T R; AravindhaBabu, N; Shanmugam, K T; Masthan, K M K

    2015-04-01

    DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde). This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers. PMID:26015708

  12. DNA adducts-chemical addons

    PubMed Central

    Rajalakshmi, T. R.; AravindhaBabu, N.; Shanmugam, K. T.; Masthan, K. M. K.

    2015-01-01

    DNA adduct is a piece of DNA covalently bond to a chemical (safrole, benzopyrenediol epoxide, acetaldehyde). This process could be the start of a cancerous cell. When a chemical binds to DNA, it gets damaged resulting in abnormal replication. This could be the start of a mutation and without proper DNA repair, this can lead to cancer. It is this chemical that binds with the DNA is our prime area of concern. Instead of performing the whole body analysis for diagnosing cancer, this test could be carried out for early detection of cancer. When scanning tunneling microscope is used, the DNA results can be obtained earlier. DNA adducts in scientific experiments are used as biomarkers. PMID:26015708

  13. The boron trifluoride nitromethane adduct

    NASA Astrophysics Data System (ADS)

    Ownby, P. Darrell

    2004-02-01

    The separation of the boron isotopes using boron trifluoride·organic-donor, Lewis acid·base adducts is an essential first step in preparing 10B enriched and depleted crystalline solids so vital to nuclear studies and reactor applications such as enriched MgB 2, boron carbide, ZrB 2, HfB 2, aluminum boron alloys, and depleted silicon circuits for radiation hardening and neutron diffraction crystal structure studies. The appearance of this new adduct with such superior properties demands attention in the continuing search for more effective and efficient means of separation. An evaluation of the boron trifluoride nitromethane adduct, its thermodynamic and physical properties related to large-scale isotopic separation is presented. Its remarkably high separation factor was confirmed to be higher than the expected theoretical value. However, the reportedly high acid/donor ratio was proven to be an order of magnitude lower. On-going research is determining the crystal structure of deuterated and 11B enriched 11BF 3·CD 3NO 2 by X-ray and neutron diffraction.

  14. DNA adduct measurements in zebra mussels, Dreissena polymorpha, Pallas. Potential use for genotoxicant biomonitoring of fresh water ecosystems.

    PubMed

    Le Goff, J; Gallois, J; Pelhuet, L; Devier, M H; Budzinski, H; Pottier, D; André, V; Cachot, J

    2006-08-12

    The purpose of this study was to examine PAH accumulation and bulky DNA adduct formation in the digestive gland of zebra mussels exposed in their habitat or in controlled laboratory conditions to complex mixture of PAH. DNA adducts were measured using a 32P-postlabelling protocol with nuclease P1 enrichment adapted from Reddy and Randerath [Reddy, M.V., Randerath, K., 1986. Nuclease P1-mediated enhancement of sensitivity of 32P-postlabelling test for structurally diverse DNA adducts. Carcinogenesis 7, 1543-1551]. Specimens collected in the upper part of the Seine estuary were shown to accumulate higher levels of PAH (up to 1.6 microg g(-1) dry weight) in comparison to individuals from the reference site (0.053 microg g(-1) dry weight). The former exhibited elevated levels of DNA adducts (up to 4.0/10(8) nucleotides) and higher diversity of individual adducts with five distinct spots being specifically detected in individuals originating from the Seine estuary. Zebra mussels exposed for 5 days to 0.01% (v/v) of organic extract of sediment from the Seine estuary were shown to accumulate high amounts of PAH (up to 138 microg g(-1) dry weight) but exhibited relatively low levels of DNA adducts. Exposure to benzo[a]pyrene led to a dose-dependent accumulation of B[a]P (up to 7063 microg g(-1) dry weight) and a clear induction of DNA adduct formation in the digestive gland of mussels (up to 1.13/10(8) nucleotides). Comparisons with other bivalves exposed to the same model PAH, revealed similar levels of adducts and comparable adduct profiles with a main adduct spot and a second faint one. This study clearly demonstrated that zebra mussels are able to biotransform B[a]P and probably other PAH into reactive metabolites with DNA-binding activity. This work also demonstrated the applicability of the nuclease P1 enhanced 32P-postlabelling method for bulky adduct detection in the digestive gland of zebra mussels. DNA adduct measurement in zebra mussels could be a suitable

  15. DNA ADDUCTS OF THE ANTITUMOR AGENT DIAZIQUONE

    EPA Science Inventory

    We have studied adduct formation of the antineoplastic agent diaziquone with DNA and nucleotides in vitro. he aziridine moieties of AZQ can be expected to interact covalently with DNA which in turn presumably elicit the antitumor activity. e analyzed AZQ-DNA adducts by a modified...

  16. Recognition of cisplatin adducts by cellular proteins.

    PubMed

    Kartalou, M; Essigmann, J M

    2001-07-01

    Cisplatin is a widely used chemotherapeutic agent. It reacts with nucleophilic bases in DNA and forms 1,2-d(ApG), 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand crosslinks, interstrand crosslinks and monofunctional adducts. The presence of these adducts in DNA is through to be responsible for the therapeutic efficacy of cisplatin. The exact signal transduction pathway that leads to cell cycle arrest and cell death following treatment with the drug is not known but cell death is believed to be mediated by the recognition of the adducts by cellular proteins. Here we describe the structural information available for cisplatin and related platinum adducts, the interactions of the adducts with cellular proteins and the implications of these interactions for cell survival. PMID:11406166

  17. [DNA adducts in human female genital organs].

    PubMed

    Postawski, Krzysztof; Przadka-Rabaniuk, Dorota; Monist, Marta; Baranowski, Włodzimierz

    2007-12-01

    DNA adducts, one of genetic damages markers, precede and finally can lead to oncogenic mutations. They appear in genome as a result of DNA bases damages caused by various and numerous environmental factors eg. ultraviolet light, ionic radiation, toxins and also endogenic substances, for example estrogens. It is believed that the creation of DNA adducts is a necessary but insufficient process for the neoplastic transformation of the cell. The following review presents concise knowledge about the DNA adducts creation and their sequels served in healthy and cancerous tissues of the female genital organs, on the base of the available data. PMID:18411923

  18. Detection of cellular DNA adducts in human fibroblasts treated with A-ring saturated and A-NCR-DMBA

    SciTech Connect

    Kumari, H.L.; Abood, N.; Goswami, S.P.; Bhat, H.B.; Milo, G.E.; Vitiak, D.T.

    1986-05-01

    Previous reports from these laboratories revealed that 1,2,3,4-tetrahydro 7,12-dimethylbenz(a)anthracene (TH-DMBA) and 6,11-dimethylcyclopentano(a)anthracene (CP-DMA) transform human fibroblasts to an abnormal phenotype. The parent PAH 7,12-DMBA does not transform these cells. Since 7,12-DMBA but not TH-DMBA or CP-DMA is anticipated to form active bay region diol epoxides, they investigated whether the A-ring analogues form adducts with cellular DNA, when the cells are treated in S phase of the cell cycle. Treatment for 10 hr was carried out when the cells exhibited S phase entry i.e. 10 hr after Gl release from a nutritionally deficient block. Adducts were isolated using a modification of the /sup 32/P-post-labeling technique. Preliminary results indicate that TH-DMBA formed two different nucleotide adducts where as there were three different adducts identified with CP-DMA. These results suggest the level of modification to be circa 1-2 adducts/10/sup 7/ total nucleotides for TH-DMBA and 2-4 adducts/10/sup 7/ total nucleotides for CP-DMA. The present evidence accumulated to date strongly suggests that alternate mechanisms exist to oxygenate the FAH to reactive intermediates that subsequently form specific DNA adducts.

  19. Accumulate repeat accumulate codes

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative channel coding scheme called 'Accumulate Repeat Accumulate codes' (ARA). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes, thus belief propagation can be used for iterative decoding of ARA codes on a graph. The structure of encoder for this class can be viewed as precoded Repeat Accumulate (RA) code or as precoded Irregular Repeat Accumulate (IRA) code, where simply an accumulator is chosen as a precoder. Thus ARA codes have simple, and very fast encoder structure when they representing LDPC codes. Based on density evolution for LDPC codes through some examples for ARA codes, we show that for maximum variable node degree 5 a minimum bit SNR as low as 0.08 dB from channel capacity for rate 1/2 can be achieved as the block size goes to infinity. Thus based on fixed low maximum variable node degree, its threshold outperforms not only the RA and IRA codes but also the best known LDPC codes with the dame maximum node degree. Furthermore by puncturing the accumulators any desired high rate codes close to code rate 1 can be obtained with thresholds that stay close to the channel capacity thresholds uniformly. Iterative decoding simulation results are provided. The ARA codes also have projected graph or protograph representation that allows for high speed decoder implementation.

  20. New fluorescence methodology for detecting DNA adducts

    SciTech Connect

    Giese, R.W.

    1993-05-21

    A new reagent, BO-IMI, has been developed that achieves, single step, phosphate specific fluorescence labeling under aqueous conditions. Both 3 in. and 5 in. mononucleotides, including representative DNA adducts can be labeled. Included in this technique is a convenient procedure for postlabeling sample cleanup, leading to a practical detection of the products by capillary electrophoresis with laser fluorescencedetection. We consider that this new method will have a significant impact on the measurement of DNA adducts in human samples. This work was largely accomplished in the second half of our project. In the first half, we set up a new way to isolate DNA nucleotides from blood, worked with an initial, less specific technique for labeling DNA adducts, compared ionizing radiation vs oxidative damage to fluorescein labeled deoxyadenylic acid, and set up a capillary electrophoresis laser fluorescence detection system.

  1. Sperm DNA oxidative damage and DNA adducts.

    PubMed

    Jeng, Hueiwang Anna; Pan, Chih-Hong; Chao, Mu-Rong; Lin, Wen-Yi

    2015-12-01

    The objective of this study was to investigate DNA damage and adducts in sperm from coke oven workers who have been exposed to polycyclic aromatic hydrocarbons. A longitudinal study was conducted with repeated measurements during spermatogenesis. Coke-oven workers (n=112) from a coke-oven plant served the PAH-exposed group, while administrators and security personnel (n=67) served the control. Routine semen parameters (concentration, motility, vitality, and morphology) were analyzed simultaneously; the assessment of sperm DNA integrity endpoints included DNA fragmentation, bulky DNA adducts, and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo). The degree of sperm DNA fragmentation was measured using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and sperm chromatin structure assay (SCSA). The PAH-exposed group had a significant increase in bulky DNA adducts and 8-oxo-dGuo compared to the control subjects (Ps=0.002 and 0.045, respectively). Coke oven workers' percentages of DNA fragmentation and denaturation from the PAH-exposed group were not significantly different from those of the control subjects (Ps=0.232 and 0.245, respectively). Routine semen parameters and DNA integrity endpoints were not correlated. Concentrations of 8-oxo-dGuo were positively correlated with percentages of DNA fragmentation measured by both TUNEL and SCSA (Ps=0.045 and 0.034, respectively). However, the concentrations of 8-oxo-dGuo and percentages of DNA fragmentation did not correlate with concentrations of bulky DNA adducts. In summary, coke oven workers with chronic exposure to PAHs experienced decreased sperm DNA integrity. Oxidative stress could contribute to the degree of DNA fragmentation. Bulky DNA adducts may be independent of the formation of DNA fragmentation and oxidative adducts in sperm. Monitoring sperm DNA integrity is recommended as a part of the process of assessing the impact of occupational and environmental toxins on sperm

  2. Reduced variational space analysis of methane adducts

    SciTech Connect

    Cundari, T.R.; Klinckman, T.R.

    1998-10-05

    Methane is the major component of natural gas, and hence its catalytic conversion to functionalized products (e.g., methanol) is of great interest. A variety of transition metal complexes have been investigated experimentally for the selective activation of methane. Recent experiments and computations suggest that weakly bound methane adducts play a pivotal role in metal-mediated methane activation. Calculation of the intrinsic reaction coordinates for methane activation by d{sup 0} imidos indicates that the adduct lies along the pathway for methane activation. Isolation of a stable methane adduct, suitable for experimental characterization, would be aided by a greater understanding of their chemistry. Given the short-lived nature of these adducts and the limited direct experimental information, computational chemistry is a useful tool for understanding the bonding and structure of these catalytic intermediates. This research investigated the bonding forces in methane adducts of transition metal (TM) complexes. The calculations reported here employed effective core potential (ECP) methods within the Hartree-Fock approximation using the GAMESS quantum chemistry program. The reduced variational space self-consistent field (RVS-SCF) method developed by Stevens and Fink was employed. This technique was used to analyze the Coulomb and exchange energy (CEX), polarization energy (POL), and charge transfer energy (CT) contributions to the binding energy ({Delta}E{sub add}) of methane to a TM complex. Adducts of high-valent (d{sup 0}) transition metal complexes were studied. The role of metal, ligand, and charge on the different contributions to the binding energy were analyzed.

  3. Vitamin A-aldehyde adducts: AMD risk and targeted therapeutics.

    PubMed

    Sparrow, Janet R

    2016-04-26

    Although currently available treatment options for age-related macular degeneration (AMD) are limited, particularly for atrophic AMD, the identification of predisposing genetic variations has informed clinical studies addressing therapeutic options such as complement inhibitors and anti-inflammatory agents. To lower risk of early AMD, recommended lifestyle interventions such as the avoidance of smoking and the intake of low glycemic antioxidant-rich diets have largely followed from the identification of nongenetic modifiable factors. On the other hand, the challenge of understanding the complex relationship between aging and cumulative damage leading to AMD has fueled investigations of the visual cycle adducts that accumulate in retinal pigment epithelial (RPE) cells and are a hallmark of aging retina. These studies have revealed properties of these compounds that provide insights into processes that may compromise RPE and could contribute to disease mechanisms in AMD. This work has also led to the design of targeted therapeutics that are currently under investigation. PMID:27071115

  4. DETERMINATION OF HEMOGLOBIN ADDUCTS FOLLOWING ACRYLAMIDE EXPOSURE

    EPA Science Inventory

    The present project was undertaken to develop new methodologies for biological monitoring of exposure to the toxicant acrylamide in laboratory animals as well as humans. ethods were developed to measure the adducts of acrylamide and its epoxide metabolite glycinamide to cysteine ...

  5. 32P-postlabelling methods for cyclic DNA adducts.

    PubMed

    Watson, W P; Crane, A E; Steiner, S

    1993-01-01

    32P-Postlabelling procedures coupled with HPLC have been developed to detect and measure a range of cyclic DNA adducts formed by bifunctional genotoxic agents. The methods are based on reverse-phase HPLC, particularly column-switching HPLC, to enrich adduct 3'-monophosphates before labelling. Following 3'-dephosphorylation of the 3'5'-[5'-32P]bisphosphates with nuclease P1, the resulting 5'-[32P]monophosphate adducts are resolved, identified and characterized by co-chromatography with synthetic reference standards. The procedures have been applied to a number of cyclic adducts including those formed by chloroacetaldehyde, glycidaldehyde and malonaldehyde. In general, labelling efficiencies measured as chromatographed 5'-[32P]monophosphates were in the range 30-40%. However, the values for the malonaldehyde deoxyguanosine adduct were much lower. The techniques have been applied to studies on the formation of DNA adducts in the skin of male C3H mice treated cutaneously with glycidaldehyde. The HPLC-32P-postlabelling analysis of epidermal DNA hydrolysates indicated that a single major cyclic adduct was formed by reaction with deoxyadenosine residues in mouse skin DNA. The adduct was identified as a hydroxymethyl ethenodeoxyadenosine adduct by comparison with a synthetic standard. This adduct was highly fluorescent and it was possible to make quantitative comparisons of the amounts of adduct determined by either HPLC-32P-postlabelling or HPLC-fluorescence detection. PMID:8225493

  6. Accumulate-Repeat-Accumulate-Accumulate-Codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Dolinar, Sam; Thorpe, Jeremy

    2004-01-01

    Inspired by recently proposed Accumulate-Repeat-Accumulate (ARA) codes [15], in this paper we propose a channel coding scheme called Accumulate-Repeat-Accumulate-Accumulate (ARAA) codes. These codes can be seen as serial turbo-like codes or as a subclass of Low Density Parity Check (LDPC) codes, and they have a projected graph or protograph representation; this allows for a high-speed iterative decoder implementation using belief propagation. An ARAA code can be viewed as a precoded Repeat-and-Accumulate (RA) code with puncturing in concatenation with another accumulator, where simply an accumulator is chosen as the precoder; thus ARAA codes have a very fast encoder structure. Using density evolution on their associated protographs, we find examples of rate-lJ2 ARAA codes with maximum variable node degree 4 for which a minimum bit-SNR as low as 0.21 dB from the channel capacity limit can be achieved as the block size goes to infinity. Such a low threshold cannot be achieved by RA or Irregular RA (IRA) or unstructured irregular LDPC codes with the same constraint on the maximum variable node degree. Furthermore by puncturing the accumulators we can construct families of higher rate ARAA codes with thresholds that stay close to their respective channel capacity thresholds uniformly. Iterative decoding simulation results show comparable performance with the best-known LDPC codes but with very low error floor even at moderate block sizes.

  7. Functional importance of ICAM-1 in the mechanism of neutrophil-induced liver injury in bile duct-ligated mice.

    PubMed

    Gujral, Jaspreet S; Liu, Jie; Farhood, Anwar; Hinson, Jack A; Jaeschke, Hartmut

    2004-03-01

    Cholestasis-induced liver injury during bile duct obstruction causes an acute inflammatory response. To further characterize the mechanisms underlying the neutrophil-induced cell damage in the bile duct ligation (BDL) model, we performed experiments using wild-type (WT) and ICAM-1-deficient mice. After BDL for 3 days, increased ICAM-1 expression was observed along sinusoids, along portal veins, and on hepatocytes in livers of WT animals. Neutrophils accumulated in sinusoids [358 +/- 44 neutrophils/20 high-power fields (HPF)] and >50% extravasated into the parenchymal tissue. Plasma alanine transaminase (ALT) levels increased by 23-fold, and severe liver cell necrosis (47 +/- 11% of total cells) was observed. Chlorotyrosine-protein adducts (a marker for neutrophil-derived hypochlorous acid) and 4-hydroxynonenal adducts (a lipid peroxidation product) were detected in these livers. Neutrophils also accumulated in the portal venules and extravasated into the portal tracts. However, no evidence for chlorotyrosine or 4-hydroxynonenal protein adducts was detected in portal tracts. ICAM-1-deficient mice showed 67% reduction in plasma ALT levels and 83% reduction in necrosis after BDL compared with WT animals. The total number of neutrophils in the liver was reduced (126 +/- 25/20 HPF), and 85% of these leukocytes remained in sinusoids. Moreover, these livers showed minimal staining for chlorotyrosine and 4-hydroxynonenal adducts, indicating a substantially reduced oxidant stress and a diminished cytokine response. Thus neutrophils relevant for the aggravation of acute cholestatic liver injury in BDL mice accumulate in hepatic sinusoids, extravasate into the tissue dependent on ICAM-1, and cause cell damage involving reactive oxygen formation. PMID:14563671

  8. Inhibition by resistant starch of red meat-induced promutagenic adducts in mouse colon.

    PubMed

    Winter, Jean; Nyskohus, Laura; Young, Graeme P; Hu, Ying; Conlon, Michael A; Bird, Anthony R; Topping, David L; Le Leu, Richard K

    2011-11-01

    Population studies have shown that high red meat intake may increase colorectal cancer risk. Our aim was to examine the effect of different amounts and sources of dietary protein on induction of the promutagenic adduct O(6)-methyl-2-deoxyguanosine (O(6)MeG) in colonocytes, to relate these to markers of large bowel protein fermentation and ascertain whether increasing colonic carbohydrate fermentation modified these effects. Mice (n = 72) were fed 15% or 30% protein as casein or red meat or 30% protein with 10% high amylose maize starch as the source of resistant starch. Genetic damage in distal colonocytes was detected by immunohistochemical staining for O(6)MeG and apoptosis. Feces were collected for measurement of pH, ammonia, phenols, p-cresol, and short-chain fatty acids (SCFA). O(6)MeG and fecal p-cresol concentrations were significantly higher with red meat than with casein (P < 0.018), with adducts accumulating in cells at the crypt apex. DNA adducts (P < 0.01) and apoptosis (P < 0.001) were lower and protein fermentation products (fecal ammonia, P < 0.05; phenol, P < 0.0001) higher in mice fed resistant starch. Fecal SCFA levels were also higher in mice fed resistant starch (P < 0.0001). This is the first demonstration that high protein diets increase promutagenic adducts (O(6)MeG) in the colon and dietary protein type seems to be the critical factor. The delivery of fermentable carbohydrate to the colon (as resistant starch) seems to switch from fermentation of protein to that of carbohydrate and a reduction in adduct formation, supporting previous observations that dietary resistant starch opposes the mutagenic effects of dietary red meat. PMID:21885815

  9. Human DNA adduct measurements: state of the art.

    PubMed Central

    Poirier, M C; Weston, A

    1996-01-01

    Human DNA adduct formation (covalent modification of DNA with chemical carcinogens) is a promising biomarker for elucidating the molecular epidemiology of cancer. Classes of compounds for which human DNA adducts have been observed include polycyclic aromatic hydrocarbons (PAHs), nitrosamines, mycotoxins, aromatic amines, heterocyclic amines, ultraviolet light, and alkylating cancer chemotherapeutic agents. Most human DNA adduct exposure monitoring has been performed with either 32P-postlabeling or immunoassays, neither of which is able to chemically characterize specific DNA adducts. Recently developed combinations of methods with chemical and physical end points have allowed identification of specific adducts in human tissues. Studies are presented that demonstrate that high ambient levels of benzo[a]pyrene are associated with high levels of DNA adducts in human blood cell DNA and that the same DNA adduct levels drop when the ambient PAH levels decrease significantly. DNA adduct dosimetry, which has been achieved with some dietary carcinogens and cancer chemotherapeutic agents, is described, as well as studies correlating DNA adducts with other biomarkers. It is likely that some toxic, noncarcinogenic compounds may have genotoxic effects, including oxidative damage, and that adverse health outcomes other than cancer may be correlated with DNA adduct formation. The studies presented here may serve as useful prototypes for exploration of other toxicological end points. PMID:8933030

  10. Polymorphic acetylation of arylamines and DNA-adduct formation.

    PubMed

    Weber, W W; Levy, G N; Martell, K J

    1990-01-01

    Inbred mouse strains congenic for rapid and slow N-acetyltransferase (NAT) (A.B6, rapid and B6.A, slow) were used to separate the effect of the NAT polymorphism from the influence of other genetically polymorphic enzymes on DNA adduct formation induced by exposure to arylamine carcinogens. Adduct formation was measured by HPLC analysis of 32P-postlabeled nucleotides from DNA of the urinary bladder and liver. Acetylator phenotype was a significant determinant of DNA damage in females as slow acetylators had higher levels of bladder DNA adducts than rapids. This correlation was the reverse of that seen with liver DNA. Older mice (20-23 weeks) formed much higher bladder DNA adduct levels than young mice (7 week). The increase in bladder adduct formation with age was seen in both sexes of all mouse strains. The older male B6 mice showed a 26-fold increase in bladder adducts and the older females showed no more than a 2-fold increase. In addition, the older male B6 mice produced significant amounts of an unidentified, early eluting adduct peak. Biochemical studies of liver NAT and O-acetyltransferase (OAT) activities showed a direct correlation between the levels of liver 2-aminofluorene (AF) NAT activity and levels of liver DNA-adduct formation, but the role of OAT activity in adduct formation in the mouse remains unclear. These results indicate that the NAT phenotype, age and sex are all important determinants of arylamine-DNA adduct formation in mice. PMID:2134671

  11. Accumulate-Repeat-Accumulate-Accumulate Codes

    NASA Technical Reports Server (NTRS)

    Divsalar, Dariush; Dolinar, Samuel; Thorpe, Jeremy

    2007-01-01

    Accumulate-repeat-accumulate-accumulate (ARAA) codes have been proposed, inspired by the recently proposed accumulate-repeat-accumulate (ARA) codes. These are error-correcting codes suitable for use in a variety of wireless data-communication systems that include noisy channels. ARAA codes can be regarded as serial turbolike codes or as a subclass of low-density parity-check (LDPC) codes, and, like ARA codes they have projected graph or protograph representations; these characteristics make it possible to design high-speed iterative decoders that utilize belief-propagation algorithms. The objective in proposing ARAA codes as a subclass of ARA codes was to enhance the error-floor performance of ARA codes while maintaining simple encoding structures and low maximum variable node degree.

  12. Development of an immunoassay to detect benzene adducts in hemoglobin

    SciTech Connect

    Grassman, J.A.

    1993-01-01

    The purpose of this project was to develop an immunoassay to detect the adducts formed in hemoglobin after exposure to benzene, which is known to cause bone marrow degeneration and acute myelogenous leukemia. The use of benzene-adduct detection as a biological monitoring method would permit measurement of low exposures and exposures sustained weeks earlier. The reactivity of hydroquinone, an important benzene metabolite, with blood proteins and amino acids was investigated in order to decide which antigens and analytes were likely to be suitable for immunoassay development. The second section determined the combination of benzene-metabolite and antigen need to produce an immunoassay with the requisite low detection limit and specificity. The immunoassays with the best performance were tested on hemoglobin from benzene-exposed mice. In vitro studies showed that hydroquinone efficiently formed adducts with erythrocyte membranes and hemoglobin but not with albumin. Adduction efficiency was greater in incubations using purified hemoglobin than whole blood. Cysteine accounted for 15 to 27% of the adducts formed by hydroquinone. The site of the other adducts were not identified although there was evidence that the hemoglobin heme was adducted. Adducts were found on only 1 of the 2 globin chains. Tryptic digestion of the globin failed to associate the adducts with a specific peptide. Antigens made from hydroquinone-adducted hemoglobin but not hydroquinone-adducted cysteines coupled to carrier proteins effectively elicited adduct-specific antibodies. Interference due to reactivity to hemoglobin was controlled by using uniform quantities of hemoglobin in all wells. The mid-range of the best assays were approximately 12 pmoles HQ per well. Antibodies directed toward hemoglobin adducted with the benzene metabolites phenol, catechol and 1,2,4-trihydroxybenzene were also made. The performance of the anti-1,2,4-trihydroxybenzene were suitable for quantitative immunoassays.

  13. Covalent adduction of nitrogen mustards to model protein nucleophiles.

    PubMed

    Thompson, Vanessa R; DeCaprio, Anthony P

    2013-08-19

    Protein adducts have the potential to serve as unique biomarkers of exposure to compounds of interest. Many xenobiotics (or their metabolites) are electrophilic and therefore reactive with nucleophilic amino acid residues on proteins. Nitrogen mustards are reactive xenobiotics with potential use as chemical warfare agents (CWA) or agents of terrorist attack, in addition to being employed as chemotherapeutic agents. The present study utilized cysteine-, lysine-, and histidine-containing model peptides to characterize in vitro adduction of the nitrogen mustards mechloroethamine (HN-2) and tris-(2-chlorethyl)amine (HN-3) to these nucleophilic amino acid residues by means of liquid chromatography-tandem mass spectrometry. The study assessed the structure of adducts formed, the time course of adduct formation, concentration-response relationships, and temporal stability of adducts. Adduction was hypothesized to occur on all three model peptides via initial formation of a reactive aziridinium intermediate for both mechloroethamine and tris-(2-chlorethyl)amine, followed by covalent adduction to nucleophilic residues. While adduction was found to occur most readily with cysteine, it was also observed at lysine and histidine, demonstrating that adduction by mechloroethamine and tris-(2-chlorethyl)amine is possible at multiple nucleophilic sites. Following solid phase extraction cleanup, adducts formed with mechloroethamine were stable for up to three weeks. Adducts formed with tris-(2-chlorethyl)amine were less stable; however, hydrolyzed secondary adducts were observed throughout the three week period. This study demonstrates that the nitrogen mustards mechloroethamine and tris-(2-chlorethyl)amine form stable adducts with reactive protein nucleophiles other than cysteine. PMID:23859065

  14. Protein modification by acrolein: Formation and stability of cysteine adducts

    PubMed Central

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2010-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intra-molecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein-acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues. PMID:19231900

  15. Mutagenesis by site-specific arylamine adducts in plasmid DNA: Enhancing replication of the adducted strand alters mutation frequency

    SciTech Connect

    Reid, T.M.; Lee, Meisie; King, C.M. )

    1990-07-03

    Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by >7-fold to 2.9% and of the AAF adduct by >12-fold to 0.75%. The AF adduct produced primarily single-base deletions in the absence of uracil but only base substitutions in the uracil-containing constructs. The AAF adduct produced mutations only in the uracil-containing DNA, which included both frame shifts and base substitutions. Mutations produced by both adducts were SOS dependent.

  16. Cytochrome c adducts with PCB quinoid metabolites.

    PubMed

    Li, Miao; Teesch, Lynn M; Murry, Daryl J; Pope, R Marshal; Li, Yalan; Robertson, Larry W; Ludewig, Gabriele

    2016-02-01

    Polychlorinated biphenyls (PCBs) are a group of 209 individual congeners widely used as industrial chemicals. PCBs are found as by-products in dye and paint manufacture and are legacy, ubiquitous, and persistent as human and environmental contaminants. PCBs with fewer chlorine atoms may be metabolized to hydroxy- and dihydroxy-metabolites and further oxidized to quinoid metabolites both in vitro and in vivo. Specifically, quinoid metabolites may form adducts on nucleophilic sites within cells. We hypothesized that the PCB-quinones covalently bind to cytochrome c and, thereby, cause defects in the function of cytochrome c. In this study, synthetic PCB quinones, 2-(4'-chlorophenyl)-1,4-benzoquinone (PCB3-pQ), 4-4'-chlorophenyl)-1,2-benzoquinone (PCB3-oQ), 2-(3', 5'-dichlorophenyl)-1,4-benzoquinone, 2-(3',4', 5'-trichlorophenyl)-1,4-benzoquinone, and 2-(4'-chlorophenyl)-3,6-dichloro-1,4-benzoquinone, were incubated with cytochrome c, and adducts were detected by liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI TOF). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was employed to separate the adducted proteins, while trypsin digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were applied to identify the amino acid binding sites on cytochrome c. Conformation change of cytochrome c after binding with PCB3-pQ was investigated by SYBYL-X simulation and cytochrome c function was examined. We found that more than one molecule of PCB-quinone may bind to one molecule of cytochrome c. Lysine and glutamic acid were identified as the predominant binding sites. Software simulation showed conformation changes of adducted cytochrome c. Additionally, cross-linking of cytochrome c was observed on the SDS-PAGE gel. Cytochrome c was found to lose its function as electron acceptor after incubation with PCB quinones. These data provide evidence that the covalent

  17. Cigarette side-stream smoke lung and bladder carcinogenesis: inducing mutagenic acrolein-DNA adducts, inhibiting DNA repair and enhancing anchorage-independent-growth cell transformation

    PubMed Central

    Chin, Chiu; Huang, William; Lepor, Herbert; Wu, Xue-Ru; Rom, William N.; Chen, Lung-Chi; Tang, Moon-shong

    2015-01-01

    Second-hand smoke (SHS) is associated with 20–30% of cigarette-smoke related diseases, including cancer. Majority of SHS (>80%) originates from side-stream smoke (SSS). Compared to mainstream smoke, SSS contains more tumorigenic polycyclic aromatic hydrocarbons and acrolein (Acr). We assessed SSS-induced benzo(a)pyrene diol epoxide (BPDE)- and cyclic propano-deoxyguanosine (PdG) adducts in bronchoalveolar lavage (BAL), lung, heart, liver, and bladder-mucosa from mice exposed to SSS for 16 weeks. In SSS exposed mice, Acr-dG adducts were the major type of PdG adducts formed in BAL (p < 0.001), lung (p < 0.05), and bladder mucosa (p < 0.001), with no significant accumulation of Acr-dG adducts in heart or liver. SSS exposure did not enhance BPDE-DNA adduct formation in any of these tissues. SSS exposure reduced nucleotide excision repair (p < 0.01) and base excision repair (p < 0.001) in lung tissue. The levels of DNA repair proteins, XPC and hOGG1, in lung tissues of exposed mice were significantly (p < 0.001 and p < 0.05) lower than the levels in lung tissues of control mice. We found that Acr can transform human bronchial epithelial and urothelial cells in vitro. We propose that induction of mutagenic Acr-DNA adducts, inhibition of DNA repair, and induction of cell transformation are three mechanisms by which SHS induces lung and bladder cancers. PMID:26431382

  18. 40 CFR 721.4590 - Mannich-based adduct.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Mannich-based adduct. 721.4590 Section 721.4590 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.4590 Mannich-based adduct....

  19. PURIFICATION AND RECOVERY OF BULKY HYDROPHOBIC DNA ADDUCTS

    EPA Science Inventory

    For many years 32P postlabeling has detected DNA adducts at very low levels and yet has not been able to identify unknown adducts. Mass spectrometry offers substantially improved identification powers, albeit at some loss in detection limits. With this ultimate utilization of ma...

  20. Intramolecular Tetrylene Lewis Adducts: Synthesis and Reactivity.

    PubMed

    Schneider, Julia; Krebs, Kilian M; Freitag, Sarah; Eichele, Klaus; Schubert, Hartmut; Wesemann, Lars

    2016-07-01

    A series of benzyl(diphenylphosphino) and o-phenyl(diphenlyphosphino) substituted germylenes and plumbylenes were synthesized by nucleophilic substitution between the respective lithium reagent and tetrylene halide. The Lewis pairs were characterized by X-ray crystallography and NMR spectroscopy. The reactivity of the tetrylenes was investigated with respect to azide addition. In the germylene case, the germaniumimide was formed as the kinetically controlled product, which rearranges upon heating to give the phosphinimide. The stannylene and plumbylene derivatives react with adamantylazide to give the azide adducts. 1-Pentene reacts diastereoselectively with the phosphagermirane to give a cyclic addition product. Trimethysilylacetylene shows an addition with the benzylphosphino-substituted germylene and plumbylene to give the cycloheteropentene molecules. The addition product between phenylacetylene and the four membered Ge-P adduct shows after addition at room temperature a 1,4-phenylmigration to give a cyclic phosphine. Alkylnitrene insertion into a Ge-C bond of the alkyne addition product of the phosphagermirane was found in reaction with adamantylazide. PMID:27273819

  1. Role of pyridine in Wyodak-pyridine adducts

    SciTech Connect

    David L. Wertz; Amanda Winters; Tara Craft; Jami Holloway

    2006-02-01

    When pyridine (PYR) is added to powdered Wyodak subbituminous coal (WYO), the sample is converted to a paste, and the molecular-level adduct which is formed is stable for months. After the excess pyridine has evaporated from the WYO-PYR sample, the stoichiometry of the adduct is ca. two pyridine molecules per bilayer of WYO polycyclic units; this adduct exists even after mild vacuum treatment of the sample. The pyridine molecules in this adduct appear to be located between the bilayer lamellae and to be H-bonded to either H-O or H-N moieties attached to the poly-cyclic aromatic units of WYO. An H-bonded N- - -H-X distance of 2.6 {angstrom} has been calculated from a structural model of the WYO-PYR adduct. 37 refs., 12 figs., 4 tabs.

  2. Diet-related DNA adduct formation in relation to carcinogenesis.

    PubMed

    Hemeryck, Lieselot Y; Vanhaecke, Lynn

    2016-08-01

    The human diet contributes significantly to the initiation and promotion of carcinogenesis. It has become clear that the human diet contains several groups of natural foodborne chemicals that are at least in part responsible for the genotoxic, mutagenic, and carcinogenic potential of certain foodstuffs. Electrophilic chemicals are prone to attack nucleophilic sites in DNA, resulting in the formation of altered nucleobases, also known as DNA adducts. Since DNA adduct formation is believed to signal the onset of chemically induced carcinogenesis, the DNA adduct-inducing potential of certain foodstuffs has been investigated to gain more insight into diet-related pathways of carcinogenesis. Many studies have investigated diet-related DNA adduct formation. This review summarizes work on known or suspected dietary carcinogens and the role of DNA adduct formation in hypothesized carcinogenesis pathways. PMID:27330144

  3. General method for quantifying base adducts in specific mammalian genes

    SciTech Connect

    Thomas, D.C.; Morton, A.G.; Bohr, V.A.; Sancar, A.

    1988-06-01

    A general method has been developed to measure the formation and removal of DNA adducts in defined sequences of mammalian genomes. Adducted genomic DNA is digested with an appropriate restriction enzyme, treated with Escherichia coli UvrABC excision nuclease (ABC excinuclease), subjected to alkaline gel electrophoresis, and probed for specific sequences by Southern hybridization. The ABC excinuclease incises DNA containing bulky adducts and thus reduces the intensity of the full-length fragments in Southern hybridization in proportion to the number of adducts present in the probed sequence. This method is similar to that developed by Bohr et al. for quantifying pyrimidine dimers by using T4 endonuclease V. Because of the wide substrate range of ABC exinuclease, however, our method can be used to quantify a large variety of DNA adducts in specific genomic sequences.

  4. 32P-POSTLABELING DNA ADDUCT ASSAY: CIGARETTE SMOKE-INDUCED DNA ADDUCTS IN THE RESPIRATORY AND NONRESPIRATORY RAT TISSUES

    EPA Science Inventory

    An analysis of the tissue DNA adducts in rats by the sensitive 32P-postlabeling assay showed one to eight detectable DNA adducts in lung, trachea, larynx, heart and bladder of the sham controls. hronic exposure of animals to mainstream cigarette smoke showed a remarkable enhancem...

  5. Biocatalytic Reductions of Baylis - Hillman Adducts

    SciTech Connect

    A Walton; W Conerly; Y Pompeu; B Sullivan; J Stewart

    2011-12-31

    Baylis-Hillman adducts are highly useful synthetic intermediates; to enhance their value further, we sought enantiocomplementary alkene reductases to introduce chirality. Two solutions emerged: (1) a wild-type protein from Pichia stipitis (OYE 2.6), whose performance significantly outstrips that of the standard enzyme (Saccharomyces pastorianus OYE1), and (2) a series of OYE1 mutants at position 116 (Trp in the wild-type enzyme). To understand how mutations could lead to inverted enantioselectivity, we solved the X-ray crystal structure of the Trp116Ile OYE1 variant complexed with a cyclopentenone substrate. This revealed key protein-ligand interactions that control the orientation of substrate binding above the FMN cofactor.

  6. Glottal Adduction and Subglottal Pressure in Singing.

    PubMed

    Herbst, Christian T; Hess, Markus; Müller, Frank; Švec, Jan G; Sundberg, Johan

    2015-07-01

    Previous research suggests that independent variation of vocal loudness and glottal configuration (type and degree of vocal fold adduction) does not occur in untrained speech production. This study investigated whether these factors can be varied independently in trained singing and how subglottal pressure is related to average glottal airflow, voice source properties, and sound level under these conditions. A classically trained baritone produced sustained phonations on the endoscopic vowel [i:] at pitch D4 (approximately 294 Hz), exclusively varying either (a) vocal register; (b) phonation type (from "breathy" to "pressed" via cartilaginous adduction); or (c) vocal loudness, while keeping the others constant. Phonation was documented by simultaneous recording of videokymographic, electroglottographic, airflow and voice source data, and by percutaneous measurement of relative subglottal pressure. Register shifts were clearly marked in the electroglottographic wavegram display. Compared with chest register, falsetto was produced with greater pulse amplitude of the glottal flow, H1-H2, mean airflow, and with lower maximum flow declination rate (MFDR), subglottal pressure, and sound pressure. Shifts of phonation type (breathy/flow/neutral/pressed) induced comparable systematic changes. Increase of vocal loudness resulted in increased subglottal pressure, average flow, sound pressure, MFDR, glottal flow pulse amplitude, and H1-H2. When changing either vocal register or phonation type, subglottal pressure and mean airflow showed an inverse relationship, that is, variation of glottal flow resistance. The direct relation between subglottal pressure and airflow when varying only vocal loudness demonstrated independent control of vocal loudness and glottal configuration. Achieving such independent control of phonatory control parameters would be an important target in vocal pedagogy and in voice therapy. PMID:25944295

  7. DNA adduct formation by alachlor metabolites

    SciTech Connect

    Brown, M.A.; Kimmel, E.C.; Casida, J.E.

    1988-01-01

    The extent of DNA adduct formation by alachlor (ArN(CH/sub 2/OCH/sub 3/)C(O)CH/sub 2/Cl wherein Ar is 2,6-diethylphenyl) and its metabolites is used as a guide to deduce the causal agent(s) in the carcinogenicity of this major herbicide. (/sup 14/C-phenyl)Alachlor is compared to its two metabolic cleavage products, (/sup 14/C-phenyl) 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) (ArNHC(O)CH/sub 2/Cl) and (/sup 14/C-phenyl)2,6-diethylaniline (DEA) (ArNH/sub 2/), and to (/sup 14/C-methoxy)alachlor in various in vitro and in vivo systems. Horseradish peroxidase and hydrogen peroxide activate DEA, but not CEDPA or alachlor, for formation of adducts with calf thymus DNA, which probably involves 2,6-diethylnitrosobenzene (ArNO) as an intermediate. Mouse liver microsomes and NADPH are both required to enhance the binding from each labeled preparation to calf thymus DNA; 4-fold higher labeling is observed from (/sup 14/C-methoxy)- than from (/sup 14/C-phenyl)alachlor. This 4-fold preferential DNA labeling from the /sup 14/C-methoxy compound is likewise found in the liver of mice treated intraperitoneally. Mouse liver protein and hemoglobin are also labeled, in vivo, with (/sup 14/C-phenyl)alachlor, -CDEPA and -DEA, and, as with the DNA, the labeling of these proteins is 1.5- to 2-fold higher with (/sup 14/C-methoxy)alachlor.

  8. Immunodetection of Serum Albumin Adducts as Biomarkers for Organophosphorus Exposure

    PubMed Central

    Chen, Sigeng; Zhang, Jun; Lumley, Lucille

    2013-01-01

    A major challenge in organophosphate (OP) research has been the identification and utilization of reliable biomarkers for the rapid, sensitive, and efficient detection of OP exposure. Although Tyr 411 OP adducts to human serum albumin (HSA) have been suggested to be one of the most robust biomarkers in the detection of OP exposure, the analysis of HSA-OP adduct detection has been limited to techniques using mass spectrometry. Herein, we describe the procurement of two monoclonal antibodies (mAb-HSA-GD and mAb-HSA-VX) that recognized the HSA Tyr 411 adduct of soman (GD) or S-[2-(diisopropylamino)ethyl]-O-ethyl methylphosphonothioate (VX), respectively, but did not recognize nonphosphonylated HSA. We showed that mAb-HSA-GD was able to detect the HSA Tyr 411 OP adduct at a low level (i.e., human blood plasma treated with 180 nM GD) that could not be detected by mass spectrometry. mAb-HSA-GD and mAb-HSA-VX showed an extremely low-level detection of GD adducted to HSA (on the order of picograms). mAb-HSA-GD could also detect serum albumin OP adducts in blood plasma samples from different animals administered GD, including rats, guinea pigs, and monkeys. The ability of the two antibodies to selectively recognize nerve agents adducted to serum albumin suggests that these antibodies could be used to identify biomarkers of OP exposure and provide a new biologic approach to detect OP exposure in animals. PMID:23192655

  9. Derivatization of isothiocyanates and their reactive adducts for chromatographic analysis.

    PubMed

    Agerbirk, Niels; De Nicola, Gina Rosalinda; Olsen, Carl Erik; Müller, Caroline; Iori, Renato

    2015-10-01

    Isothiocyanates form adducts with a multitude of biomolecules, and these adducts need analytical methods. Likewise, analytical methods for hydrophilic isothiocyanates are needed. We considered reaction with ammonia to form thiourea derivatives. The hydrophilic, glycosylated isothiocyanate moringin, 4-(α-L-rhamnopyranosyloxy)benzyl isothiocyanate, was efficiently derivatized to the thiourea derivative by incubation with ammonia. The hydrophobic benzyl isothiocyanate was also efficiently derivatized to the thiourea derivative. The thiourea group provided a UV absorbing chromophore, and the derivatives showed expectable sodium and hydrogen adducts in ion trap mass spectrometry and were suitable for liquid chromatography analysis. Reactive dithiocarbamate adducts constitute the major type of reactive ITC adduct expected in biological matrices. Incubation of a model dithiocarbamate with ammonia likewise resulted in conversion to the corresponding thiourea derivative, suggesting that a variety of matrix-bound reactive isothiocyanate adducts can be determined using this strategy. As an example of the application of the method, recovery of moringin and benzyl isothiocyanate applied to cabbage leaf discs was studied in simulated insect feeding assays. The majority of moringin was recovered as native isothiocyanate, but a major part of benzyl isothiocyanate was converted to reactive adducts. PMID:26342619

  10. Quantitation of DNA adducts by stable isotope dilution mass spectrometry

    PubMed Central

    Tretyakova, Natalia; Goggin, Melissa; Janis, Gregory

    2012-01-01

    Exposure to endogenous and exogenous chemicals can lead to the formation of structurally modified DNA bases (DNA adducts). If not repaired, these nucleobase lesions can cause polymerase errors during DNA replication, leading to heritable mutations potentially contributing to the development of cancer. Due to their critical role in cancer initiation, DNA adducts represent mechanism-based biomarkers of carcinogen exposure, and their quantitation is particularly useful for cancer risk assessment. DNA adducts are also valuable in mechanistic studies linking tumorigenic effects of environmental and industrial carcinogens to specific electrophilic species generated from their metabolism. While multiple experimental methodologies have been developed for DNA adduct analysis in biological samples – including immunoassay, HPLC, and 32P-postlabeling – isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS) generally has superior selectivity, sensitivity, accuracy, and reproducibility. As typical DNA adducts concentrations in biological samples are between 0.01 – 10 adducts per 108 normal nucleotides, ultrasensitive HPLC-ESI-MS/MS methodologies are required for their analysis. Recent developments in analytical separations and biological mass spectrometry – especially nanoflow HPLC, nanospray ionization MS, chip-MS, and high resolution MS – have pushed the limits of analytical HPLC-ESI-MS/MS methodologies for DNA adducts, allowing researchers to accurately measure their concentrations in biological samples from patients treated with DNA alkylating drugs and in populations exposed to carcinogens from urban air, drinking water, cooked food, alcohol, and cigarette smoke. PMID:22827593

  11. Protein adduct formation as a molecular mechanism in neurotoxicity.

    PubMed

    Lopachin, Richard M; Decaprio, Anthony P

    2005-08-01

    Chemicals that cause nerve injury and neurological deficits are a structurally diverse group. For the majority, the corresponding molecular mechanisms of neurotoxicity are poorly understood. Many toxicants (e.g., hepatotoxicants) of other organ systems and/or their oxidative metabolites have been identified as electrophiles and will react with cellular proteins by covalently binding nucleophilic amino acid residues. Cellular toxicity occurs when adduct formation disrupts protein structure and/or function, which secondarily causes damage to submembrane organelles, metabolic pathways, or cytological processes. Since many neurotoxicants are also electrophiles, the corresponding pathophysiological mechanism might involve protein adduction. In this review, we will summarize the principles of covalent bond formation that govern reactions between xenobiotic electrophiles and biological nucleophiles. Because a neurotoxicant can form adducts with multiple nucleophilic residues on proteins, the challenge is to identify the mechanistically important adduct. In this regard, it is now recognized that despite widespread chemical adduction of tissue proteins, neurotoxicity can be mediated through binding of specific target nucleophiles in key neuronal proteins. Acrylamide and 2,5-hexanedione are prototypical neurotoxicants that presumably act through the formation of protein adducts. To illustrate both the promise and the difficulty of adduct research, these electrophilic chemicals will be discussed with respect to covalent bond formation, suspected protein sites of adduction, and proposed mechanisms of neurotoxicity. The goals of future investigations are to identify and quantify specific protein adducts that play a causal role in the generation of neurotoxicity induced by electrophilic neurotoxicants. This is a challenging but critical objective that will be facilitated by recent advances in proteomic methodologies. PMID:15901921

  12. Foam cell-derived 4-hydroxynonenal induces endothelial cell senescence in a TXNIP-dependent manner.

    PubMed

    Riahi, Yael; Kaiser, Nurit; Cohen, Guy; Abd-Elrahman, Ihab; Blum, Galia; Shapira, Oz M; Koler, Tomer; Simionescu, Maya; Sima, Anca V; Zarkovic, Neven; Zarkovic, Kamelija; Orioli, Marica; Aldini, Giancarlo; Cerasi, Erol; Leibowitz, Gil; Sasson, Shlomo

    2015-08-01

    Vascular endothelial cell (VEC) senescence is considered an early event in the development of atherosclerotic lesions. Stressful stimuli, in particular oxidative stress, have been linked to premature senescence in the vasculature. Foam cells are a major source of reactive oxygen species and may play a role in the induction of VEC senescence; hence, we investigated their involvement in the induction of VEC senescence in a co-culture transwell system. Primary bovine aortic endothelial cells, exposed to the secretome of THP-1 monocyte-derived foam cells, were analysed for the induction of senescence. Senescence associated β-galactosidase activity and the expression of p16 and p21 were increased, whereas phosphorylated retinoblastoma protein was reduced. This senescent phenotype was mediated by 4-hydroxnonenal (4-HNE), a lipid peroxidation product secreted from foam cells; scavenging of 4-HNE in the co-culture medium blunted this effect. Furthermore, both foam cells and 4-HNE increased the expression of the pro-oxidant thioredoxin-interacting protein (TXNIP). Molecular manipulation of TXNIP expression confirmed its involvement in foam cell-induced senescence. Previous studies showed that peroxisome proliferator-activated receptor (PPAR)δ was activated by 4-hydroalkenals, such as 4-HNE. Pharmacological interventions supported the involvement of the 4-HNE-PPARδ axis in the induction of TXNIP and VEC senescence. The association of TXNIP with VEC senescence was further supported by immunofluorescent staining of human carotid plaques in which the expression of both TXNIP and p21 was augmented in endothelial cells. Collectively, these findings suggest that foam cell-released 4-HNE activates PPARδ in VEC, leading to increased TXNIP expression and consequently to senescence. PMID:25754218

  13. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity

    SciTech Connect

    Jiang, Shuai; Pan, Amy W.; Lin, Tzu-yin; Zhang, Hongyong; Malfatti, Michael; Turteltaub, Kenneth; Henderson, Paul T.; Pan, Chong-xian

    2015-11-06

    This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 108 nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 108 nucleotides per hour in carboplatin alone (p = 0.021). In conclusion, this rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.

  14. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity

    DOE PAGESBeta

    Jiang, Shuai; Pan, Amy W.; Lin, Tzu-yin; Zhang, Hongyong; Malfatti, Michael; Turteltaub, Kenneth; Henderson, Paul T.; Pan, Chong-xian

    2015-11-06

    This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 108 nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 108 nucleotides per hour in carboplatin alone (p = 0.021). In conclusion, this rapid report provides themore » first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.« less

  15. Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity.

    PubMed

    Jiang, Shuai; Pan, Amy W; Lin, Tzu-yin; Zhang, Hongyong; Malfatti, Michael; Turteltaub, Kenneth; Henderson, Paul T; Pan, Chong-xian

    2015-12-21

    This rapid report focuses on the pharmacodynamic mechanism of the carboplatin/paclitaxel combination and correlates it with its cytotoxicity. Consistent with the synergistic to additive antitumor activity (the combination index ranging from 0.53 to 0.94), cells exposed to this combination had significantly increased carboplatin-DNA adduct formation when compared to that of carboplatin alone (450 ± 30 versus 320 ± 120 adducts per 10(8) nucleotides at 2 h, p = 0.004). Removal of paclitaxel increased the repair of carboplatin-DNA adducts: 39.4 versus 33.1 adducts per 10(8) nucleotides per hour in carboplatin alone (p = 0.021). This rapid report provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic. PMID:26544157

  16. DNA adducts as a dosimeter for risk estimation

    SciTech Connect

    Belinsky, S.A.; White, C.M.; Devereux, T.R.; Anderson, M.W.

    1987-12-01

    The dose response for O/sup 6/-methylguanine (O/sup 6/MG) formation and cytotoxicity was determined in lung and nasal mucosa from Fischer 344 rats during multiple dose administration of the tobacco-specific nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). O/sup 6/MG accumulated in the lung following treatment for 12 days with doses of NNK from 0.3 to 100 mgkgday. The dose response for NNK was nonlinear; the O/sup 6/MG-to-dose ratio, an index of alkylation efficiency, increased dramatically as the dose of carcinogen decreased. These data suggest that low- and high-K/sub m/ pathways may exist for activation to NNK to a methylating agent. Marked differences in O/sup 6/MG concentration were observed in specific lung cell populations. The presence of a high-affinity pathway in the Clara cell for activation of NNK may contribute to the potent carcinogenicity observed following low-dose exposure to this tobacco-specific carcinogen. The dose response for O/sup 6/MG formation differed considerably between the respiratory and olfactory mucosa from the nasal passages of the rat. These studies suggest that a low K/sub m/ pathway for NNK activation is also present in the nose and that this pathway is localized predominantly in the respiratory region. These data suggest that both the formation of promutagenic adducts and cell proliferation secondary to toxicity are required for the induction of neoplasia by NNK within the nose.

  17. Accumulate Repeat Accumulate Coded Modulation

    NASA Technical Reports Server (NTRS)

    Abbasfar, Aliazam; Divsalar, Dariush; Yao, Kung

    2004-01-01

    In this paper we propose an innovative coded modulation scheme called 'Accumulate Repeat Accumulate Coded Modulation' (ARA coded modulation). This class of codes can be viewed as serial turbo-like codes, or as a subclass of Low Density Parity Check (LDPC) codes that are combined with high level modulation. Thus at the decoder belief propagation can be used for iterative decoding of ARA coded modulation on a graph, provided a demapper transforms the received in-phase and quadrature samples to reliability of the bits.

  18. Relating protein adduction to gene expression changes: a systems approach

    PubMed Central

    Zhang, Bing; Shi, Zhiao; Duncan, Dexter T; Prodduturi, Naresh; Marnett, Lawrence J; Liebler, Daniel C

    2013-01-01

    Modification of proteins by reactive electrophiles such as the 4-hydroxy-2-nonenal (HNE) plays a critical role in oxidant-associated human diseases. However, little is known about protein adduction and the mechanism by which protein damage elicits adaptive effects and toxicity. We developed a systems approach for relating protein adduction to gene expression changes through the integration of protein adduction, gene expression, protein-DNA interaction, and protein-protein interaction data. Using a random walk strategy, we expanded a list of responsive transcription factors inferred from gene expression studies to upstream signaling networks, which in turn allowed overlaying protein adduction data on the network for the prediction of stress sensors and their associated regulatory mechanisms. We demonstrated the general applicability of transcription factor-based signaling network inference using 103 known pathways. Applying our workflow on gene expression and protein adduction data from HNE-treatment not only rediscovered known mechanisms of electrophile stress but also generated novel hypotheses regarding protein damage sensors. Although developed for analyzing protein adduction data, the framework can be easily adapted for phosphoproteomics and other types of protein modification data. PMID:21594272

  19. Covalent thiol adducts arising from reactive intermediates of cocaine biotransformation.

    PubMed

    Schneider, Kevin J; DeCaprio, Anthony P

    2013-11-18

    Exposure to cocaine results in the depletion of hepatocellular glutathione and macromolecular protein binding in humans. Such cocaine-induced responses have generally been attributed to oxidative stress and reactive metabolites resulting from oxidative activation of the cocaine tropane nitrogen. However, little conclusive data exists on the mechanistic pathways leading to protein modification or the structure and specificity of cocaine-derived adduction products. We now report a previously uncharacterized route of cocaine bioactivation leading to the covalent adduction of biological thiols, including cysteine and glutathione. Incubation of cocaine with biological nucleophiles in an in vitro biotransformation system containing human liver microsomes identified a monooxygenase-mediated event leading to the oxidation of, and subsequent sulfhydryl addition to, the cocaine aryl moiety. Adduct structures were confirmed using ultra-high performance liquid chromatography coupled to high resolution, high mass accuracy mass spectrometry. Examination of assays containing transgenic bactosomes expressing single human cytochrome P450 isoforms determined the role of P450s 1A2, 2C19, and 2D6 in the oxidation process resulting in adduct formation. P450-catalyzed aryl epoxide formation and subsequent attack by free nucleophilic moieties is consistent with the resulting adduct structures, mechanisms of formation, and the empirical observation of multiple structural and stereo isomers. Analogous adduction mechanisms were maintained across all sulfhydryl-containing nucleophile models examined; N-acetylcysteine, glutathione, and a synthetic cysteine-containing hexapeptide. Predictive in silico calculations of molecular reactivity and electrophilicity/nucleophilicity were compared to the results of in vitro assay incubations in order to better understand the adduction process using the principles of hard and soft acid and base (HSAB) theory. This study elucidated a novel metabolic

  20. Characterization of hemoglobin adduct formation in mice and rats after administration of ( sup 14 C)butadiene or ( sup 14 C)isoprene

    SciTech Connect

    Sun, J.D.; Dahl, A.R.; Bond, J.A.; Birnbaum, L.S.; Henderson, R.F. )

    1989-08-01

    Occupational exposures to 1,3-butadiene or isoprene occur through their use in the manufacture of rubber and other related polymer products. The purpose of this study was to determine if butadiene or isoprene administration would result in the formation of adducts with blood hemoglobin (Hb), and if such adducts can be used as a measure of previous exposure(s). Male B6C3F1 mice and male Sprague-Dawley rats were injected intraperitoneally with 1, 10, 100, or 1000 mumol (14C)butadiene or 0.3, 3.0, 300, 1000, or 3000 mumol (14C)isoprene per kilogram body weight. Animals were killed 24 hr later. Globin was isolated from blood samples and was analyzed for 14C by liquid scintillation spectroscopy. Hb adduct formation was linearly related to administered doses up to 100 mumol (14C)butadiene or 500 mumol (14C)isoprene per kilogram body weight for mice and rats, respectively. For (14C)butadiene, the efficiency of Hb adduct formation in mice and rats within the linear response range was 0.177 +/- 0.003 and 0.407 +/- 0.019 (pmol of 14C-adducts/mg globin)/(mumol of retained (14C)butadiene/kg body wt), respectively (mean +/- SE; n = 18). For (14C)isoprene, these values for mice and rats were 0.158 +/- 0.035 and 0.079 +/- 0.016 (pmol of 14C-adducts/mg globin)/(mumol of retained (14C)isoprene/kg body wt), respectively (mean +/- SE; n = 12). Hb adducts also accumulated linearly after repeated daily administration of 100 mumol (14C)butadiene or 500 mumol (14C)isoprene per kilogram body wt to mice and rats, respectively, for 3 days. (14C)Butadiene-derived Hb adducts in blood showed lifetimes of approximately 24 and approximately 65 days for mice and rats, respectively, which correlate with the reported lifetimes for red blood cells in these rodent species. Thus, levels of butadiene- or isoprene-derived adducts on Hb in circulating blood may be a useful measure of prior repeated exposures to these compounds.

  1. PROTEIN ADDUCTS AS BIOMAKERS OF EXPOSURE TO ORGANOPHOSPHORUS COMPOUNDS

    PubMed Central

    Marsillach, Judit; Costa, Lucio G.; Furlong, Clement E.

    2013-01-01

    Exposure to organophosphorus (OP) compounds can lead to serious neurological damage or death. Following bioactivation by the liver cytochromes P450, the OP metabolites produced are potent inhibitors of serine active-site enzymes including esterases, proteases and lipases. OPs may form adducts on other cellular proteins. Blood cholinesterases (ChEs) have long served as biomarkers of OP exposure in humans. However, the enzymatic assays used for biomonitoring OP exposures have several drawbacks. A more useful approach will focus on multiple biomarkers and avoid problems with the enzymatic activity assays. OP inhibitory effects result from a covalent bond with the active-site serine of the target enzymes. The serine OP adducts become irreversible following a process referred to as aging where one alkyl group dissociates over variable lengths of time depending on the OP adduct. The OP-adducted enzyme then remains in circulation until it is degraded, allowing for a longer window of detection compared with direct analysis of OPs or their metabolites. Mass spectrometry (MS) provides a very sensitive method for identification of post-translational protein modifications. MS analyses of the percentage adduction of the active-site serine of biomarker proteins such as ChEs will eliminate the need for basal activity levels of the individual and will provide for a more accurate determination of OP exposure. MS analysis of biomarker proteins also provides information about the OP that has caused inhibition. Other useful biomarker proteins include other serine hydrolases, albumin, tubulin and transferrin. PMID:23261756

  2. Characterization of deoxyguanosine adducts from hydroquinone/benzoquinone

    SciTech Connect

    Jowa, J.; Winkel, S.; Witz, G.; Snyder, R.

    1986-03-01

    Occupational exposure to benzene has long been associated with the development of pancytopenia and leukemia. This toxicity has been attributed to the action of benzene metabolites. The authors have chosen to investigate the reaction of hydroquinone (HQ)/benzoquinone(BQ) with deoxyguanosine(dG) and DNA. (/sup 14/C)HQ was incubated with (/sup 3/H)dG in potassium phosphate buffer pH7.2 for 24 hours. Two dual labeled products were found by HPLC and presumed to be adducts. The same result was obtained when BQ was substituted in the reaction for HQ. Both adducts were found in isolated DNA from Clostridium perfringens, Micrococcus lysodeikticus, human placenta and calf thymus reacted with HO under similar conditions. One of the dG adducts was proposed to be (/sup 3/'OH) benzetheno(1,N-2)deoxyguanosine based on NMR and mass spectral results. The other adduct was characterized by a molecular weight of 339. The latter adduct was found in greater amounts than the former when HQ was reacted with denatured DNA.

  3. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases

    NASA Astrophysics Data System (ADS)

    Karmakar, Saswata; Harcourt, Emily M.; Hewings, David S.; Lovejoy, Alexander F.; Kurtz, David M.; Ehrenschwender, Thomas; Barandun, Luzi J.; Roost, Caroline; Alizadeh, Ash A.; Kool, Eric T.

    2015-09-01

    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens.

  4. Organocatalytic removal of formaldehyde adducts from RNA and DNA bases.

    PubMed

    Karmakar, Saswata; Harcourt, Emily M; Hewings, David S; Scherer, Florian; Lovejoy, Alexander F; Kurtz, David M; Ehrenschwender, Thomas; Barandun, Luzi J; Roost, Caroline; Alizadeh, Ash A; Kool, Eric T

    2015-09-01

    Formaldehyde is universally used to fix tissue specimens, where it forms hemiaminal and aminal adducts with biomolecules, hindering the ability to retrieve molecular information. Common methods for removing these adducts involve extended heating, which can cause extensive degradation of nucleic acids, particularly RNA. Here, we show that water-soluble bifunctional catalysts (anthranilates and phosphanilates) speed the reversal of formaldehyde adducts of mononucleotides over standard buffers. Studies with formaldehyde-treated RNA oligonucleotides show that the catalysts enhance adduct removal, restoring unmodified RNA at 37 °C even when extensively modified, while avoiding the high temperatures that promote RNA degradation. Experiments with formalin-fixed, paraffin-embedded cell samples show that the catalysis is compatible with common RNA extraction protocols, with detectable RNA yields increased by 1.5-2.4-fold using a catalyst under optimized conditions and by 7-25-fold compared with a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens. PMID:26291948

  5. Isolation, identification, and assay of [3H]-porfiromycin adducts of EMT6 mouse mammary tumor cell DNA: effects of hypoxia and dicumarol on adduct patterns.

    PubMed

    Tomasz, M; Hughes, C S; Chowdary, D; Keyes, S R; Lipman, R; Sartorelli, A C; Rockwell, S

    1991-07-01

    [3H]-(N-la-methyl) Porfiromycin (POR) was employed to detect and identify the radiolabeled mono- and bis-adducts formed in living EMT6 mouse mammary tumor cells under different conditions. To provide authentic standard adducts, calf-thymus DNA was treated with POR under reductive activation, then digested to nucleosides and POR-nucleoside adducts. The three major adducts formed were isolated by HPLC and authenticated. Two were mono-adducts, composed of deoxyguanosine linked at its N2-position to C-1 of POR and of 10-decarbamoyl POR. The third was a bis-adduct, in which POR was crosslinked to two deoxyguanosines at their N2-positions. DNA from [3H]-POR treated EMT6 cells was digested an analyzed by HPLC. DNA-associated label was located in thymidine and in two mono-adducts and one bis-adduct identical to those described above. Label in thymidine resulted from N-demethylation of POR and reincorporation of label into new thymidylate residues. Adducts were formed more abundantly in hypoxia than in air. In addition, the mono-adduct to crosslink ratios were different, approximately 1:1 and 2:1 for hypoxic and aerobic cells, respectively. The different patterns of alkylation in air and hypoxia may be related to the greater toxicity of POR in hypoxia. When cells were treated simultaneously with POR and dicumarol, adduct levels were lower, and a new, unknown adduct was observed primarily under hypoxia; these changes may be related to the altered toxicity of POR in the presence of dicumarol. The HPLC assay detected simultaneously the full array of stable mono- and bis-adducts in DNA with good sensitivity (greater than or equal to 2 x 10(6) adducts/nucleotide) and excellent reproducibility. This assay should be generally applicable to all cells and tissues when MC or POR with high specific radioactivity can be employed. PMID:1714285

  6. Removal of aflatoxin B1-DNA adducts and in vitro transformation in mouse embryo fibroblasts C3H/10T1 1/2

    SciTech Connect

    Amstad, P.A.; Wang, T.V.; Cerutti, P.A.

    1983-01-01

    The mechanism of in vitro transformation of the mouse embryo fibroblast C3H/10T 1/2 clone 8 by aflatoxin B1 (AFB1) was studied in confluent holding (CH) experiments. Confluent cultures of C3H/10T 1/2 cells were treated with AFB1 for 16 hours, and the DNA adduct composition and concentration were determined by chromatographic procedures after 0, 8, 16, and 40 hours of CH when the cells were replated at low density for the expression of their colony-forming ability and the formation of transformed foci. Total adduct concentration and the concentration of the major primary adduct 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AFB1-N7-Gua) decreased continuously during CH due to spontaneous decomposition and probably also due to enzymatic repair processes. In contrast, the more chemically stable secondary product 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'-oxo-N5-pyrimidyl)-3-hydroxyaflatoxin B1 (AFB1-triamino-Py) accumulated in the DNA and reached its maximum concentration after 16 hours of CH. While the loss of total AFB1-DNA adducts during CH was reflected in recovery of viability, the potential to form transformed foci reached a maximum after 16 hours of CH and then decreased with continued CH below the initial value. Therefore, no simple relationship exists between the concentration of the total adducts AFB1-N7-Gua and AFB1-triamino-Py at the time of release from CH and the potential to form transformed foci. However, DNA lesions or abnormal DNA configurations formed during CH as a consequence of the cellular processing of AFB1-DNA adducts may play a role in the transformation process.

  7. QUANTITATIVE AND TEMPORAL RELATIONSHIPS BETWEEN DNA ADDUCT FORMATION IN TARGET AND SURROGATE TISSUES: IMPLICATIONS FOR BIOMONITORING

    EPA Science Inventory

    DNA-carcinogen adducts offer a potential dosimeter for environmental genotoxicants reaching the exposed individual. ecause the target tissues for many chemical carcinogens are not readily accessible for monitoring adducts in humans, peripheral blood lymphocytes (PBLS) have served...

  8. Quantitation of DNA Adducts Induced by 1,3-Butadiene

    NASA Astrophysics Data System (ADS)

    Sangaraju, Dewakar; Villalta, Peter W.; Wickramaratne, Susith; Swenberg, James; Tretyakova, Natalia

    2014-07-01

    Human exposure to 1,3-butadiene (BD) present in automobile exhaust, cigarette smoke, and forest fires is of great concern because of its potent carcinogenicity. The adverse health effects of BD are mediated by its epoxide metabolites such as 3,4-epoxy-1-butene (EB), which covalently modify genomic DNA to form promutagenic nucleobase adducts. Because of their direct role in cancer, BD-DNA adducts can be used as mechanism-based biomarkers of BD exposure. In the present work, a mass spectrometry-based methodology was developed for accurate, sensitive, and precise quantification of EB-induced N-7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) DNA adducts in vivo. In our approach, EB-GII adducts are selectively released from DNA backbone by neutral thermal hydrolysis, followed by ultrafiltration, offline HPLC purification, and isotope dilution nanoLC/ESI+-HRMS3 analysis on an Orbitrap Velos mass spectrometer. Following method validation, EB-GII lesions were quantified in human fibrosarcoma (HT1080) cells treated with micromolar concentrations of EB and in liver tissues of rats exposed to sub-ppm concentrations of BD (0.5-1.5 ppm). EB-GII concentrations increased linearly from 1.15 ± 0.23 to 10.11 ± 0.45 adducts per 106 nucleotides in HT1080 cells treated with 0.5-10 μM DEB. EB-GII concentrations in DNA of laboratory rats exposed to 0.5, 1.0, and 1.5 ppm BD were 0.17 ± 0.05, 0.33 ± 0.08, and 0.50 ± 0.04 adducts per 106 nucleotides, respectively. We also used the new method to determine the in vivo half-life of EB-GII adducts in rat liver DNA (2.20 ± 0.12 d) and to detect EB-GII in human blood DNA. To our knowledge, this is the first application of nanoLC/ESI+-HRMS3 Orbitrap methodology to quantitative analysis of DNA adducts in vivo.

  9. Chemistry and Biology of Aflatoxin-DNA Adducts

    SciTech Connect

    Stone, Michael P.; Banerjee, Surajit; Brown, Kyle L.; Egli, Martin

    2012-03-27

    Aspergillus flavus is a fungal contaminant of stored rice, wheat, corn, and other grainstuffs, and peanuts. This is of concern to human health because it produces the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}), which is genotoxic and is implicated in the etiology of liver cancer. AFB{sub 1} is oxidized in vivo by cytochrome P450 to form aflatoxin B{sub 1} epoxide, which forms an N7-dG adduct (AFB{sub 1}-N7-dG) in DNA. The latter rearranges to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative that equilibrates between {alpha} and {beta} anomers of the deoxyribose. In DNA, both the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts intercalate above the 5'-face of the damaged guanine. Each produces G {yields} T transversions in Escherichia coli, but the AFB{sub 1}-{beta}-FAPY adduct is more mutagenic. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) provides a model for understanding error-prone bypass of the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts. It bypasses the AFB{sub 1}-N7-dG adduct, but it conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including mis-insertion of dATP, consistent with the G {yields} T mutations characteristic of AFB{sub 1} mutagenesis in E. coli. Crystallographic analyses of a series of binary and ternary complexes with the Dpo4 polymerase revealed differing orientations of the N7-C8 bond of the AFB{sub 1}-N7-dG adduct as compared to the N{sup 5}-C8 bond in the AFB{sub 1}-{beta}-FAPY adduct, and differential accommodation of the intercalated AFB{sub 1} moieties within the active site. These may modulate AFB{sub 1} lesion bypass by this polymerase.

  10. Strategy for identifying unknown hemoglobin adducts using adductome LC-MS/MS data: Identification of adducts corresponding to acrylic acid, glyoxal, methylglyoxal, and 1-octen-3-one.

    PubMed

    Carlsson, Henrik; Törnqvist, Margareta

    2016-06-01

    Electrophilic compounds have the ability to form adducts with nucleophilic sites in proteins and DNA in tissues, and thereby constitute risks for toxic effects. Adductomic approaches are developed for systematic screening of adducts to DNA and blood proteins, with the aim to detect unknown internal exposures to electrophiles. In a previous adductomic screening of adducts to N-terminals in hemoglobin, using LC-MS/MS, 19 unknown adducts were detected in addition to seven previously identified adducts. The present paper describes the identification of four of these unknown adducts, as well as the strategy used to identify them. Using LC-MS data from the screening, hypotheses about adduct identities were formulated: probable precursor electrophiles with matching molecular weights were suggested based on the molecular weights of the modifications and the retention times of the analytes, in combination with comparisons of theoretical Log P calculations and databases. Reference adducts were generated by incubation of blood samples with the hypothesized precursor electrophiles. The four identified precursor electrophiles, corresponding to the observed unknown adducts, were glyoxal, methylglyoxal, acrylic acid and 1-octen-3-one. Possible origins/exposure sources and toxicological information concerning the electrophilic precursors are discussed. The identified adducts could be explored as possible biomarkers for exposure. PMID:27046699

  11. UNUSUALLY STABLE ADDUCT BETWEEN METHANOLYZED AMOXICILLIN OR AMPICILLIN AND THEIR DIKETOPIPERAZINE DERIVATIVES.

    PubMed

    Kosińska, Katarzyna; Frański, Rafał; Frańska, Magdalena

    2016-01-01

    Amoxicillin and ampicillin were subjected to methanolysis. As expected, the methanolysis products were observed by HPLC-ESI-MS. Besides these products, diketopiperazine derivatives were also detected. Additionally, unusually stable adduct formed between the products of methanolysis and diketopiperazine derivatives was also identified. Analogical adducts were detected when ethanolysis was performed instead of methanolysis. HPLC-ESI-MS analysis of the separated adducts confirmed that the adducts were composed of methanolysis products and diketopiperazine derivatives. PMID:27180422

  12. IMPROVED THIN-LAYER CHROMATOGRAPHIC SEPARATION OF 32P-POSTLABELING DNA ADDUCTS

    EPA Science Inventory

    DNA adducts represent the putative initiating event in the chemical process. 2P-Postlabeling is one of several assayswhich have been developed for the sensitive detection of DNA adducts. n integral part of the 32p-postlabeling assay is the separation of adducted nucleotides by mu...

  13. CANCER BIOMARKERS IN HUMAN ATHEROSCLEROTIC LESIONS: DETECTION OF DNA ADDUCTS

    EPA Science Inventory

    Since somatic mutations are suspected to contribute to the pathogenesis not only of cancer but also of atherosclerotic plaques, we measured DNA adducts in the smooth muscle layer of atherosclerotic lesions in abnormal aorta specimens taken at surgery from seven patients. NA adduc...

  14. DETERMINATION OF HEMOGLOBIN ADDUCTS IN HUMANS OCCUPATIONALLY EXPOSED TO ACRYLAMIDE

    EPA Science Inventory

    Hemoglobin (Hb) adduct determinations were used to monitor occupational exposure to acrylamide (AA) and acrylonitrile (AN). orth-one workers in a factory in the People's Republic of China who were involved in the synthesis of a AA by catalytic hydration of AN and the manufacturin...

  15. 40 CFR 721.4590 - Mannich-based adduct.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... reporting. (1) The chemical substance generically identified as a Mannich-based adduct (PMN P-93-66) is.... Requirements as specified in § 721.80(h). (ii) Release to water. Requirements as specified in § 721.90 (a)(4... 721.4590 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC...

  16. 40 CFR 721.4590 - Mannich-based adduct.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... reporting. (1) The chemical substance generically identified as a Mannich-based adduct (PMN P-93-66) is.... Requirements as specified in § 721.80(h). (ii) Release to water. Requirements as specified in § 721.90 (a)(4... 721.4590 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC...

  17. 40 CFR 721.4590 - Mannich-based adduct.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... reporting. (1) The chemical substance generically identified as a Mannich-based adduct (PMN P-93-66) is.... Requirements as specified in § 721.80(h). (ii) Release to water. Requirements as specified in § 721.90 (a)(4... 721.4590 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC...

  18. 40 CFR 721.4590 - Mannich-based adduct.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... reporting. (1) The chemical substance generically identified as a Mannich-based adduct (PMN P-93-66) is.... Requirements as specified in § 721.80(h). (ii) Release to water. Requirements as specified in § 721.90 (a)(4... 721.4590 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) TOXIC...

  19. Distortions induced in DNA by cis-platinum interstrand adducts

    SciTech Connect

    Sip, M.; Schwartz, A.; Vovelle, F.; Ptak, M.; Leng, M. )

    1992-03-10

    A 22 base pair double-stranded oligonucleotide containing a unique interstrand adduct resulting from chelation of the two guanine residues within the central sequence d(TGCT/AGCA) by a cis-platinum residue has been studied by means of gel electrophoresis, chemical probes, and molecular mechanics. The anomalously slow electrophoretic mobility of the multimers of the platinated and ligated oligomers suggests that the platinated oligonucleotide is bent. The two cytosine residues (complementary to the platinated guanines) are hyperreactive to hydroxylamine, indicating a large exposure of the two bases to the solvent. The adduct does not induce a local denaturation within the flanking sequences since the adenine residues are not reactive with diethyl pyrocarbonate. This is confirmed by the nonreactivity of the complementary T residues with osmium tetraoxide. These results and the molecular mechanics modeling suggest that the interstrand adduct bends the double helix by approximately 55{degree} toward the major groove, that the double helix conserves its average twist angle, and that the distortion induced by the adduct is localized at the platinated sequence d(GC/CG).

  20. Mass Spectrometric Analyses of Organophosphate Insecticide Oxon Protein Adducts

    PubMed Central

    Thompson, Charles M.; Prins, John M.; George, Kathleen M.

    2010-01-01

    Objective Organophosphate (OP) insecticides continue to be used to control insect pests. Acute and chronic exposures to OP insecticides have been documented to cause adverse health effects, but few OP-adducted proteins have been correlated with these illnesses at the molecular level. Our aim was to review the literature covering the current state of the art in mass spectrometry (MS) used to identify OP protein biomarkers. Data sources and extraction We identified general and specific research reports related to OP insecticides, OP toxicity, OP structure, and protein MS by searching PubMed and Chemical Abstracts for articles published before December 2008. Data synthesis A number of OP-based insecticides share common structural elements that result in predictable OP–protein adducts. The resultant OP–protein adducts show an increase in molecular mass that can be identified by MS and correlated with the OP agent. Customized OP-containing probes have also been used to tag and identify protein targets that can be identified by MS. Conclusions MS is a useful and emerging tool for the identification of proteins that are modified by activated organophosphate insecticides. MS can characterize the structure of the OP adduct and also the specific amino acid residue that forms the key bond with the OP. Each protein that is modified in a unique way by an OP represents a unique molecular biomarker that with further research can lead to new correlations with exposure. PMID:20056576

  1. CARCINOGEN-DNA ADDUCTS: INTRODUCTION, LITERATURE SUMMARY, AND RECOMMENDATIONS

    EPA Science Inventory

    The report summarizes the literature concerning adducts formed by xenobiotics with DNA and/or protein and discusses their feasibility as a monitoring tool for use in exposure and risk assessment. The report is divided into three segments. The first segment provides an introductio...

  2. NMR at the Picomole Level of a DNA Adduct

    PubMed Central

    Kautz, Roger; Wang, Poguang; Giese, Roger W.

    2014-01-01

    We investigate the limit of detection for obtaining NMR data of a DNA adduct using modern microscale NMR instrumentation, once the adduct has been isolated at the pmol level. Eighty nanograms (130 pmol) of a DNA adduct standard, N-(2′-deoxyguanosin-8-yl)-2-acetylaminofluorene 5′-monophosphate (AAF-dGMP), in 1.5 μL of D2O with 10% methanol-d4, in a vial, was completely picked up as a droplet suspended in a fluorocarbon liquid, and loaded efficiently into a microcoil probe. This work demonstrates a practical manual method of droplet microfluidic sample loading, previously demonstrated using automated equipment, which provides a several-fold advantage over conventional flow injection. Eliminating dilution during injection and confining the sample into the observed volume realizes the full theoretical mass sensitivity of a microcoil, comparable to a micro-cryo probe. With 80 ng, an NMR spectrum acquired over 40 hr showed all of the resonances seen in a standard spectrum of AAF-dGMP, with a S/N of at least 10, despite broadening due to previously-noted effects of conformational exchange. Also a 2D TOCSY spectrum (total correlation spectroscopy) was acquired on 1.6 μg in 18 hr. This work helps to define the utility of NMR in combination with other analytical methods for the structural characterization of a small amount of a DNA adduct. PMID:24028148

  3. Probing myo-inositol 1-phosphate synthase with multisubstrate adducts

    PubMed Central

    Deranieh, Rania M.; Greenberg, Miriam L.; Le Calvez, Pierre-B.; Mooney, Maura C.; Migaud, Marie E.

    2015-01-01

    The synthesis of a series of carbohydrate-nucleotide hybrids, designed to be multisubstrate adducts mimicking myo-inositol 1-phosphate synthase first oxidative transition state, is reported. Their ability to inhibit the synthase has been assessed and results have been rationalised computationally to estimate their likely binding mode. PMID:23132282

  4. Ion-molecule adduct formation in tandem mass spectrometry.

    PubMed

    Alechaga, Élida; Moyano, Encarnación; Galceran, Maria Teresa

    2016-02-01

    Nowadays most LC-MS methods rely on tandem mass spectrometry not only for quantitation and confirmation of compounds by multiple reaction monitoring (MRM), but also for the identification of unknowns from their product ion spectra. However, gas-phase reactions between charged and neutral species inside the mass analyzer can occur, yielding product ions at m/z values higher than that of the precursor ion, or at m/z values difficult to explain by logical losses, which complicate mass spectral interpretation. In this work, the formation of adduct ions in the mass analyzer was studied using several mass spectrometers with different mass analyzers (ion trap, triple quadrupole, and quadrupole-Orbitrap). Heterocyclic amines (AαC, MeAαC, Trp-P-1, and Trp-P-2), photo-initiators (BP and THBP), and pharmaceuticals (phenacetin and levamisole) were selected as model compounds and infused in LCQ Classic, TSQ Quantum Ultra AM, and Q-Exactive Orbitrap (ThermoFisher Scientific) mass spectrometers using electrospray as ionization method. The generation of ion-molecule adducts depended on the compound and also on the instrument employed. Adducts with neutral organic solvents (methanol and acetonitrile) were only observed in the ion trap instrument (LCQ Classic), because of the ionization source on-axis configuration and the lack of gas-phase barriers, which allowed inertial entrance of the neutrals into the analyzer. Adduct formation (only with water) in the triple quadrupole instruments was less abundant than in the ion trap and quadrupole-Orbitrap mass spectrometers, because of the lower residence time of the reactive product ions in the mass analyzer. The moisture level of the CID and/or damper gas had a great effect in beam-like mass analyzers such as triple quadrupole, but not in trap-like mass analyzers, probably because of the long residence time that allowed adduct formation even with very low concentrations of water inside the mass spectrometer. PMID:26700446

  5. Detection of protein adduction derived from dauricine by alkaline permethylation.

    PubMed

    Xie, Honglei; Liu, Yuyang; Peng, Ying; Zhao, Dongmei; Zheng, Jiang

    2016-06-01

    Dauricine is a bisbenzylisoquinoline alkaloid derivative and has shown multiple pharmacological properties. Despite this, our previous study demonstrated that dauricine induced severe lung toxicity in experimental animals. Metabolic activation of dauricine to the corresponding quinone methide intermediate is suggested to play an important role in dauricine-induced cytotoxicity. Protein adduction derived from the reactive intermediate is considered to initiate the process of the toxicity. In the present study, we developed an alkaline permethylation- and mass spectrometry-based approach to detect dauricine-derived protein adduction. Protein samples were permethylated in the presence of NaOH and CH3I at 80 °C, followed by LC-MS/MS analysis. A thioether product was produced in the reaction. Not only does this technique quantify dauricine-derived protein adduction but also it tells the nature of the interaction between the target proteins and the reactive intermediate of dauricine. The recovery, precision, limit of detection, limit of quantity, and method detection limit were found to be 102.8 %±1.7 %, 1.89 %, 1.32 fmol/mL, 4.93 fmol/mL and 3.37 fmol/mL respectively. The surrogate recovery and surrogate RSD values were 81.5-103.0 % and 2.59 %, respectively. This analytical method has proven sensitive, selective, reliable, and feasible to assess total protein adduction derived from dauricine, and will facilitate the mechanistic investigation of dauricine and other bisbenzylisoquinoline toxicities. Graphical Abstract Alkaline permethylation of dauricine derived protein adduct. PMID:27071763

  6. Quantification of Carnosine-Aldehyde Adducts in Human Urine.

    PubMed

    da Silva Bispo, Vanderson; Di Mascio, Paolo; Medeiros, Marisa

    2014-10-01

    Lipid peroxidation generates several reactive carbonyl species, including 4-hydroxy-2-nonenal (HNE), acrolein (ACR), 4-hydroxy-2-hexenal (HHE) and malondialdehyde. One major pathwayof aldehydes detoxification is through conjugation with glutathione catalyzed by glutathione-S-transferases or, alternatively, by conjugation with endogenous histidine containing dipeptides, such as carnosine (CAR). In this study, on-line reverse-phase high-performance liquid chromatography (HPLC) separation with tandem mass spectrometry detection was utilized for the accurate quantification of CAR- ACR, CAR-HHE and CAR-HNE adducts in human urinary samples from non-smokers young adults. Standard adducts were prepared and isolated by HPLC. The results showed the presence of a new product from the reaction of CAR with ACR. This new adduct was completely characterized by HPLC/MS-MSn, 1H RMN, COSY and HSQC. The new HPLC/MS/MS methodology employing stable isotope-labeled internal standards (CAR-HHEd5 and CAR-HNEd11) was developed for adducts quantification. This methodology permits quantification of 10pmol CAR-HHE and 1pmol of CAR-ACR and CAR-HNE. Accurate determinations in human urine sample were performed and showed 4.65±1.71 to CAR-ACR, 5.13±1.76 to CAR-HHE and 5.99±3.19nmol/mg creatinine to CAR-HNE. Our results indicate that carnosine pathways can be an important detoxification route of a, ß -unsaturated aldehydes. Moreover, carnosine adducts may be useful as redox stress indicator. PMID:26461323

  7. 32P-postlabeling DNA adduct assay: cigarette smoke-induced dna adducts in the respiratory and nonrespiratory rat tissues. Book chapter

    SciTech Connect

    Gupta, R.C.; Gairola, C.G.

    1990-01-01

    An analysis of the tissue DNA adducts in rats by the sensitive (32)p-postlabeling assay showed one to eight detectable DNA adducts in lung, trachea, larynx, heart and bladder of the sham controls. Chronic exposure of animals to mainstream cigarette smoke showed a remarkable enhancement of most adducts in the lung and heart DNA. Since cigarette smoke contains several thousand chemicals and a few dozen of them are known or potential carcinogens, the difference between the DNA adducts of nasal and the other tissues may reflect the diversity of reactive constituents and their differential absorption in different tissues. In comparison to the lung DNA adducts, the adducts in nasal DNA were less hydrophobic. Identity of the predominant adducts was further investigated by comparison with several reference DNA adducts from 10 PAH and aromatic amines. Since some of these chemicals are present in cigarette smoke, the results suggest that these constituents of cigarette smoke may not be directly responsible for formation of DNA adducts in the lung and heart of the smoke-exposed animals.

  8. Detection of benzo[a]pyrene diol epoxide-DNA adducts in peripheral blood lymphocytes and antibodies to the adducts in serum from coke oven workers.

    PubMed Central

    Harris, C C; Vahakangas, K; Newman, M J; Trivers, G E; Shamsuddin, A; Sinopoli, N; Mann, D L; Wright, W E

    1985-01-01

    Coke oven workers are exposed to high levels of carcinogenic polycyclic aromatic hydrocarbons, including benzo[a]pyrene (B[a]P), and are at increased risk of lung cancer. Since B[a]P is enzymatically activated to 7 beta,8 alpha-dihydroxy(9 alpha, 10 alpha)epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE) that forms adducts with DNA, the presence of these adducts was measured in DNA from peripheral blood lymphocytes by synchronous fluorescence spectrophotometry and enzyme radioimmunoassay. Approximately two-thirds of the workers had detectable levels of B[a]PDE-DNA adducts. Antibodies to the DNA adducts were also found in the serum of 27% of the workers. B[a]PDE-DNA adducts were not detectable in lymphocytes and antibodies to the adducts were not detected in sera from a control group of nonsmoking laboratory workers. DNA adducts and/or antibodies to the adducts indicate exposure to B[a]P and its metabolic activation to the carcinogenic metabolite that covalently binds to and damages DNA. Detection of adducts and antibodies to them may also be useful as internal dosimeters of the pathobiological effective doses of chemical carcinogens. PMID:2413443

  9. Chloroethyinitrosourea-derived ethano cytosine and adenine adducts are substrates for escherichia coli glycosylases excising analogous etheno adducts

    SciTech Connect

    Guliaev, Anton B.; Singer, B.; Hang, Bo

    2004-05-05

    Exocyclic ethano DNA adducts are saturated etheno ring derivatives formed mainly by therapeutic chloroethylnitrosoureas (CNUs), which are also mutagenic and carcinogenic. In this work, we report that two of the ethano adducts, 3,N{sup 4}-ethanocytosine (EC) and 1,N{sup 6}-ethanoadenine (EA), are novel substrates for the Escherichia coli mismatch-specific uracil-DNA glycosylase (Mug) and 3-methyladenine DNA glycosylase II (AlkA), respectively. It has been shown previously that Mug excises 3,N{sup 4}-ethenocytosine ({var_epsilon}C) and AlkA releases 1,N{sup 6}-ethenoadenine ({var_epsilon}A). Using synthetic oligonucleotides containing a single ethano or etheno adduct, we found that both glycosylases had a {approx}20-fold lower excision activity toward EC or EA than that toward their structurally analogous {var_epsilon}C or {var_epsilon}A adduct. Both enzymes were capable of excising the ethano base paired with any of the four natural bases, but with varying efficiencies. The Mug activity toward EC could be stimulated by E. coli endonuclease IV and, more efficiently, by exonuclease III. Molecular dynamics (MD) simulations showed similar structural features of the etheno and ethano derivatives when present in DNA duplexes. However, also as shown by MD, the stacking interaction between the EC base and Phe 30 in the Mug active site is reduced as compared to the {var_epsilon}C base, which could account for the lower EC activity observed in this study.

  10. Photochemistry of psoralen-DNA adducts, biological effects of psoralen-DNA adducts, applications of psoralen-DNA photochemistry

    SciTech Connect

    Shi, Yun-bo

    1988-03-01

    This thesis consists of three main parts and totally eight chapters. In Part I, The author will present studies on the photochemistry of psoralen-DNA adducts, specifically, the wavelength dependencies for the photoreversals of thymidine-HMT (4'-hydroxymethyl-4, 5', 8-trimenthylpsoralen) monoadducts and diadduct and the same adducts incorporated in DNA helices and the wavelength dependecies for the photocrossslinking of thymidine-HMT monoadducts in double-stranded helices. In Part II, The author will report some biological effects of psoralen-DNA adducts, i.e., the effects on double-stranded DNA stability, DNA structure, and transcription by E. coli and T7 RNA polymerases. Finally, The author will focus on the applications of psoralen-DNA photochemistry to investigation of protein-DNA interaction during transcription, which includes the interaction of E. coli and T7 RNA polymerases with DNA in elongation complexes arrested at specific psoralen-DNA adduct sites as revealed by DNase I footprinting experiments. 123 refs., 52 figs., 12 tabs.

  11. Oxidative DNA damage accumulation in gastric carcinogenesis

    PubMed Central

    Farinati, F; Cardin, R; Degan, P; Rugge, M; Di, M; Bonvicini, P; Naccarato, R

    1998-01-01

    Background—Gastric carcinogenesis is a multifactorial, multistep process, in which chronic inflammation plays a major role. 
Aims—In order to ascertain whether free radical mediated oxidative DNA damage is involved in such a process, concentrations of 8-hydroxydeoxyguanosine (8OHdG), a mutagenic/carcinogenic adduct, and thiobarbituric acid reactive substances (TBARS), as an indirect measure of free radical mediated damage, were determined in biopsy specimens from patients undergoing endoscopy. 
Patients—Eighty eight patients were divided into histological subgroups as follows: 27 with chronic non-atrophic gastritis, 41 with atrophic gastritis, six with gastric cancer, and 14 unaffected controls. 
Methods—Intestinal metaplasia, Helicobacter pylori infection, and disease activity were semiquantitatively scored. 8OHdG concentrations were assessed by HPLC with electrochemical detection, and TBARS concentrations were fluorimetrically assayed. 
Results—8OHdG concentrations (mean number of adducts/105 dG residues) were significantly higher in chronic atrophic gastritis (p=0.0009). Significantly higher concentrations were also detected in the presence of severe disease activity (p=0.02), intestinal metaplasia (p=0.035), and H pylori infection (p=0.001). TBARS concentrations were also higher in atrophic gastritis, though not significantly so. In a multiple logistic regression analysis, 8OHdG concentrations correlated best with the presence and severity of H pylori infection (r=0.53, p=0.002). 
Conclusions—Chronic gastritis is characterised by the accumulation of oxidative DNA damage with mutagenic and carcinogenic potential. H pylori infection is the major determinant for DNA adduct formation. 

 Keywords: free radicals; oxidative DNA damage; gastric carcinogenesis; precancerous changes; peroxidative damage PMID:9577340

  12. Human Biomonitoring of DNA Adducts by Ion Trap Multistage Mass Spectrometry.

    PubMed

    Guo, Jingshu; Turesky, Robert J

    2016-01-01

    Humans are continuously exposed to hazardous chemicals in the environment. These chemicals or their electrophilic metabolites can form adducts with genomic DNA, which can lead to mutations and the initiation of cancer. The identification of DNA adducts is required for understanding exposure and the etiological role of a genotoxic chemical in cancer risk. The analytical chemist is confronted with a great challenge because the levels of DNA adducts generally occur at <1 adduct per 10(7) nucleotides, and the amount of tissue available for measurement is limited. Ion trap mass spectrometry has emerged as an important technique to screen for DNA adducts because of the high level sensitivity and selectivity, particularly when employing multi-stage scanning (MS(n) ). The product ion spectra provide rich structural information and corroborate the adduct identities even at trace levels in human tissues. Ion trap technology represents a significant advance in measuring DNA adducts in humans. © 2016 by John Wiley & Sons, Inc. PMID:27584705

  13. UVR Exposure Sensitizes Keratinocytes to DNA Adduct Formation

    PubMed Central

    Nair, Sudhir; Kekatpure, Vikram D.; Judson, Benjamin L.; Rifkind, Arleen B.; Granstein, Richard D.; Boyle, Jay O.; Subbaramaiah, Kotha; Guttenplan, Joseph B.; Dannenberg, Andrew J.

    2009-01-01

    Ultraviolet radiation (UVR) and exposure to tobacco smoke, a source of polycyclic aromatic hydrocarbons (PAH), have been linked to skin carcinogenesis. UVR-mediated activation of the aryl hydrocarbon receptor (AhR) stimulates the transcription of CYP1A1 and CYP1B1, which encode proteins that convert PAH to genotoxic metabolites. We determined whether UVR exposure sensitized human keratinocytes to PAH-induced DNA adduct formation. UVR exposure induced CYP1A1 and CYP1B1 in HaCaT cells, an effect that was mimicked by photooxidized tryptophan (aTRP) and FICZ, a component of aTRP. UVR exposure or pretreatment with aTRP or FICZ also sensitized cells to benzo[a]pyrene (B[a]P) induced DNA adduct formation. α-Naphthoflavone (αNF), an AhR antagonist, suppressed UVR-, aTRP- and FICZ-mediated induction of CYP1A1 and CYP1B1 and inhibited B[a]P induced DNA adduct formation. Treatment with 17-AAG, a Hsp90 inhibitor, caused a marked decrease in levels of AhR, inhibited UVR-, aTRP- and FICZ-mediated induction of CYP1A1 and CYP1B1 and blocked the sensitization of HaCaT cells to B[a]P induced DNA adduct formation. FICZ has been suggested to be a physiological ligand of the AhR that may have systemic effects. Hence, studies of FICZ were also carried out in MSK-Leuk1 cells, a model of oral leukoplakia. Pretreatment with αNF or 17-AAG blocked FICZ-mediated induction of CYP1A1 and CYP1B1, and suppressed the increased B[a]P-induced DNA adduct formation. Collectively, these results suggest that sunlight may activate AhR signaling and thereby sensitize cells to PAH-mediated DNA adduct formation. Antagonists of AhR signaling may have a role in the chemoprevention of photocarcinogenesis. PMID:19789301

  14. Proteomic analysis of adducted butyrylcholinesterase for biomonitoring organophosphorus exposures

    PubMed Central

    Marsillach, Judit; Hsieh, Edward J.; Richter, Rebecca J.; MacCoss, Michael J.; Furlong, Clement E.

    2014-01-01

    Organophosphorus (OP) compounds include a broad group of toxic chemicals such as insecticides, chemical warfare agents and antiwear agents. The liver cytochromes P450 bioactivate many OPs to potent inhibitors of serine hydrolases. Cholinesterases were the first OP targets discovered and are the most studied. They are used to monitor human exposures to OP compounds. However, the assay that is currently used has limitations. The mechanism of action of OP compounds is the inhibition of serine hydrolases by covalently modifying their active-site serine. After structural rearrangement, the complex OP inhibitor-enzyme is irreversible and will remain in circulation until the modified enzyme is degraded. Mass spectrometry is a sensitive technology for analyzing protein modifications, such as OP-adducted enzymes. These analyses also provide some information about the nature of the OP adduct. Our aim is to develop high-throughput protocols for monitoring OP exposures using mass spectrometry. PMID:23123252

  15. Smoking related carcinogen-DNA adducts in biopsy samples of human urinary bladder: Identification of N-(deoxyguanosin-8-yl)-4-aminobiphenyl as a major adduct

    SciTech Connect

    Talaska, G. Univ. of Cincinnati, OH ); Al-Juburi, A.Z.S.S. ); Kadlubar, F.F. )

    1991-06-15

    The prevalence of covalent modifications to DNA (carcinogen-DNA adducts) in 42 human urinary bladder biopsy samples was investigated by {sup 32}P-postlabeling methods, with enhancement by both nuclease P1 treatment and 1-butanol extraction. Total mean carcinogen-DNA adduct levels and the mean levels of several specific adducts were significantly elevated in DNA samples of 13 current smokers, as opposed to 9 never smokers or 20 ex-smokers (5 years abstinence). There was no significant difference between the latter two groups. Several DNA adducts enhanced by nuclease P1 treatment were chromatographically similar to putative hydrocarbon DNA adducts reported earlier for placenta and lung DNA samples obtained from cigarette smokers. Putative aromatic amine adducts were detected by 1-butanol extraction that were not present when the samples were treated with nuclease P1. One of these displayed chromatographic behavior identical to the predominant adduct induced by the human urinary bladder carcinogen, 4-aminobiphenyl, which is present in cigarette smoke. This adduct comigrated in several thin-layer chromatographic systems with a synthetic N-(deoxyguanosin-8-yl)-4-amino(2,2{prime}-{sup 3}H)biphenyl-3{prime},5{prime}-bisphosphate marker. These data reinforce an association between cigarette smoking and DNA damage and suggest a molecular basis for the initiation of human urinary bladder cancer by cigarette smoke.

  16. Dispersant additives derived from lactone modified amido-amine adducts

    SciTech Connect

    Gutierrez, A.; Lundberg, R.D.

    1990-10-16

    This patent describes a lactone modified dispersant additive. It comprises one adduct of a polyolefin of 300 to 10,000 number average molecular weight substituted with at least 0.8 (e.g., from about 1 to 4) dicarboxylic acid producing moieties (preferably acid or anhydride moieties) per polyolefin molecule, an amido-amine or thioamido-amine characterized by being a reaction product of at least a polyamine and an alpha, beta-unsaturated compound.

  17. Ion Pairs or Neutral Molecule Adducts? Cooperativity in Hydrogen Bonding

    ERIC Educational Resources Information Center

    DeKock, Roger L.; Schipper, Laura A.; Dykhouse, Stephanie C.; Heeringa, Lee P.; Brandsen, Benjamin M.

    2009-01-01

    We performed theoretical studies on the systems NH[subscript 3] times HF times mH[subscript 2]O, NH[subscript 3] times HCl times mH[subscript 2]O, with m = 0, 1, 2, and 6. The molecules with m = 0 form hydrogen-bonded adducts with little tendency to form an ion-pair structure. The molecule NH[subscript 3] times HCl times H[subscript 2]O cannot be…

  18. 2' and 3' Carboranyl uridines and their diethyl ether adducts

    DOEpatents

    Soloway, Albert H.; Barth, Rolf F.; Anisuzzaman, Abul K.; Alam, Fazlul; Tjarks, Werner

    1992-01-01

    There is disclosed a process for preparing carboranyl uridine nucleoside compounds and their diethyl ether adducts, which exhibit a tenfold increase in boron content over prior art boron containing nucleoside compounds. Said carboranyl uridine nucleoside compounds exhibit enhanced lipophilicity and hydrophilic properties adequate to enable solvation in aqueous media for subsequent incorporation of said compounds in methods for boron neutron capture therapy in mammalian tumor cells.

  19. Protein tyrosine adduct in humans self-poisoned by chlorpyrifos

    SciTech Connect

    Li, Bin; Eyer, Peter; Eddleston, Michael; Jiang, Wei; Schopfer, Lawrence M.; Lockridge, Oksana

    2013-06-15

    Studies of human cases of self-inflicted poisoning suggest that chlorpyrifos oxon reacts not only with acetylcholinesterase and butyrylcholinesterase but also with other blood proteins. A favored candidate is albumin because in vitro and animal studies have identified tyrosine 411 of albumin as a site covalently modified by organophosphorus poisons. Our goal was to test this proposal in humans by determining whether plasma from humans poisoned by chlorpyrifos has adducts on tyrosine. Plasma samples from 5 self-poisoned humans were drawn at various time intervals after ingestion of chlorpyrifos for a total of 34 samples. All 34 samples were analyzed for plasma levels of chlorpyrifos and chlorpyrifos oxon (CPO) as a function of time post-ingestion. Eleven samples were analyzed for the presence of diethoxyphosphorylated tyrosine by mass spectrometry. Six samples yielded diethoxyphosphorylated tyrosine in pronase digests. Blood collected as late as 5 days after chlorpyrifos ingestion was positive for CPO-tyrosine, consistent with the 20-day half-life of albumin. High plasma CPO levels did not predict detectable levels of CPO-tyrosine. CPO-tyrosine was identified in pralidoxime treated patients as well as in patients not treated with pralidoxime, indicating that pralidoxime does not reverse CPO binding to tyrosine in humans. Plasma butyrylcholinesterase was a more sensitive biomarker of exposure than adducts on tyrosine. In conclusion, chlorpyrifos oxon makes a stable covalent adduct on the tyrosine residue of blood proteins in humans who ingested chlorpyrifos. - Highlights: • Chlorpyrifos-poisoned patients have adducts on protein tyrosine. • Diethoxyphosphate-tyrosine does not lose an alkyl group. • Proteins in addition to AChE and BChE are modified by organophosphates.

  20. A structurally-characterized NbCl5-NHC adduct.

    PubMed

    Bortoluzzi, Marco; Ferretti, Eleonora; Marchetti, Fabio; Pampaloni, Guido; Zacchini, Stefano

    2014-05-01

    The selective reactions of niobium pentachloride with two bulky NHC carbenes afforded NbCl5(NHC) complexes, bearing the highest oxidation state ever found for a metal centre in a transition metal halide-NHC adduct. The X-ray structure of 2a is the first one reported for a monodentate NHC-niobium species, and exhibits an abnormally long Nb-C bond. PMID:24658260

  1. 32P-postlabelling analysis of small aromatic and of bulky non-aromatic DNA adducts.

    PubMed

    Reddy, M V

    1993-01-01

    The 32P-postlabelling methodology for analysis of DNA adducts derived from carcinogens containing one aromatic ring (e.g., safrole, styrene oxide, benzene metabolites, 1-nitrosoindole-3-acetonitrile) or a bulky non-aromatic moiety (e.g., mitomycin C, diaziquone) is reviewed. Six steps are involved: digestion of DNA to 3'-nucleotides, enrichment of adducts, 32P-labelling of adducts, separation of labelled adducts by TLC, detection, and quantitation. The first step, DNA digestion with micrococcal nuclease and spleen phosphodiesterase, is applicable to DNA modified with most carcinogens independent of their size and structure. Of the two commonly used procedures for enrichment of aromatic adducts in DNA digests, the nuclease P1 treatment is substantially more effective than butanol extraction for small aromatic and bulky non-aromatic adducts. For initial purification of these adducts from unadducted material after 32P-labelling, multi-directional polyethyleneimine (PEI)-cellulose TLC using 1 M sodium phosphate, pH 6.0, as the D1 solvent is not suitable, because they are not retained on PEI-cellulose under these conditions. A higher concentration of sodium phosphate (e.g., 2.3 M) or development with D1 and D3 solvents in the same direction helps to retain adducts of safrole and of benzene metabolites. Also, transfer of adducts from multiple cut-outs above the origin after D1 chromatography, as adopted for analysis of I-compounds, is potentially applicable. However, initial purification by reverse-phase TLC, followed by in situ transfer and resolution by PEI-cellulose TLC has been found to be most effective for these adducts. Reverse-phase TLC at 4 degrees C or in a stronger salt solution further improves retention of some adducts (e.g., mitomycin C and diaziquone adducts). For adduct separation by PEI-cellulose TLC, salt solutions with or without urea are used. PMID:8225492

  2. Effect of phytochemical intervention on dibenzo[a,l]pyrene-induced DNA adduct formation.

    PubMed

    Russell, Gilandra K; Gupta, Ramesh C; Vadhanam, Manicka V

    2015-04-01

    Dibenzo[a,l]pyrene (DBP) has been found to be the most potent carcinogen of the polycyclic aromatic hydrocarbons (PAHs). Primary sources for DBP in the environment are combustion of wood and coal burning, gasoline and diesel exhaust, and tires. Given the likelihood of environmental exposure to DBP and strong experimental evidence of its potency, it is likely to contribute to lung cancer development. Intervention with compounds of natural origin ("phytochemicals") is considered an effective means to prevent cancer development and favorably modulate the underlying mechanisms, including DNA adduct formation. In this study, several agents have been identified that inhibit environmental carcinogen-induced DNA adduct formation using a cell-free microsomal system. Of the ten agents tested, resveratrol (648 ± 26 adducts/10(9) nucleotides), oltipraz (1007 ± 348 adducts/10(9) nucleotides), delphinidin (1252 ± 142 adducts/10(9) nucleotides), tanshinone I (1981 ± 213 adducts/10(9) nucleotides), tanshinone IIA (2606 ± 478 adducts/10(9) nucleotides) and diindoylmethane (3643 ± 469 adducts/10(9) nucleotides) were the most effective compared to vehicle treatment (14,062 ± 1097 adducts/10(9) nucleotides). DBP is metabolized by phase I metabolizing enzymes CYP1A1, CYP1A2, and CYP1B1. DBP-induced DNA adducts can be inhibited by several mechanisms. We found that all the test agents inhibited DNA adducts by inhibiting one or more of these enzymes. Oltipraz inhibited DNA adducts entirely by inhibiting the CYP450s, while resveratrol and delphinidin inhibited DNA adducts by also interacting directly with the carcinogenic metabolite, anti-dibenzo(a,l)pyrene-11,12-dihydrodiol-13,14-epoxide. PMID:25794985

  3. Effect of phytochemical intervention on dibenzo[a,l]pyrene-induced DNA adduct formation

    PubMed Central

    Russell, Gilandra K.; Gupta, Ramesh C.; Vadhanam, Manicka V.

    2015-01-01

    Dibenzo[a,l]pyrene (DBP) has been found to be the most potent carcinogen of the polycyclic aromatic hydrocarbons (PAHs). Primary sources for DBP in the environment are combustion of wood and coal burning, gasoline and diesel exhaust, and tires. Given the likelihood of environmental exposure to DBP and strong experimental evidence of its potency, it is likely to contribute to lung cancer development. Intervention with compounds of natural origin (“phytochemicals”) is considered an effective means to prevent cancer development and favorably modulate the underlying mechanisms, including DNA adduct formation. In this study, several agents have been identified that inhibit environmental carcinogen-induced DNA adduct formation using a cell-free microsomal system. Of the ten agents tested, resveratrol (648 ± 26 adducts/109 nucleotides), oltipraz (1007 ± 348 adducts/109 nucleotides), delphinidin (1252 ± 142 adducts/109 nucleotides), tanshinone I (1981 ± 213 adducts/109 nucleotides), tanshinone IIA (2606 ± 478 adducts/109 nucleotides) and diindoylmethane (3643 ± 469 adducts/109 nucleotides) were the most effective compared to vehicle treatment (14,062 ± 1097 adducts/109 nucleotides). DBP is metabolized by phase I metabolizing enzymes CYP1A1, CYP1A2, and CYP1B1. DBP-induced DNA adducts can be inhibited by several mechanisms. We found that all the test agents inhibited DNA adducts by inhibiting one or more of these enzymes. Oltipraz inhibited DNA adducts entirely by inhibiting the CYP450s, while resveratrol and delphinidin inhibited DNA adducts by also interacting directly with the carcinogenic metabolite, anti-dibenzo(a,l)pyrene-11,12-dihydrodiol-13,14-epoxide. PMID:25794985

  4. TRPA1-mediated accumulation of aminoglycosides in mouse cochlear outer hair cells.

    PubMed

    Stepanyan, Ruben S; Indzhykulian, Artur A; Vélez-Ortega, A Catalina; Boger, Erich T; Steyger, Peter S; Friedman, Thomas B; Frolenkov, Gregory I

    2011-12-01

    Aminoglycoside ototoxicity involves the accumulation of antibiotic molecules in the inner ear hair cells and the subsequent degeneration of these cells. The exact route of entry of aminoglycosides into the hair cells in vivo is still unknown. Similar to other small organic cations, aminoglycosides could be brought into the cell by endocytosis or permeate through large non-selective cation channels, such as mechanotransduction channels or ATP-gated P2X channels. Here, we show that the aminoglycoside antibiotic gentamicin can enter mouse outer hair cells (OHCs) via TRPA1, non-selective cation channels activated by certain pungent compounds and by endogenous products of lipid peroxidation. Using conventional and perforated whole-cell patch clamp recordings, we found that application of TRPA1 agonists initiates inward current responses in wild-type OHCs, but not in OHCs of homozygous Trpa1 knockout mice. Similar responses consistent with the activation of non-selective cation channels were observed in heterologous cells transfected with mouse Trpa1. Upon brief activation with TRPA1 agonists, Trpa1-transfected cells become loaded with fluorescent gentamicin-Texas Red conjugate (GTTR). This uptake was not observed in mock-transfected or non-transfected cells. In mouse organ of Corti explants, TRPA1 activation resulted in the rapid entry of GTTR and another small cationic dye, FM1-43, in OHCs and some supporting cells, even when hair cell mechanotransduction was disrupted by pre-incubation in calcium-free solution. This TRPA1-mediated entry of GTTR and FM1-43 into OHCs was observed in wild-type but not in Trpa1 knockout mice and was not blocked by PPADS, a non-selective blocker of P2X channels. Notably, TRPA1 channels in mouse OHCs were activated by 4-hydroxynonenal, an endogenous molecule that is known to be generated during episodes of oxidative stress and accumulate in the cochlea after noise exposure. We concluded that TRPA1 channels may provide a novel pathway for

  5. Thermal stability of DNA adducts induced by cyanomorpholinoadriamycin in vitro.

    PubMed Central

    Cullinane, C; Phillips, D R

    1993-01-01

    The Adriamycin derivative, cyanomorpholinoadriamycin (CMA) was reacted with DNA in vitro to form apparent interstrand crosslinks. The extent of interstrand crosslink formation was monitored by a gel electrophoresis assay and maximal crosslinking of DNA was observed within 1 hr with 5 microM of drug. The interstrand crosslinks were heat labile, with a midpoint melting temperature of 70 degrees C (10 min exposure to heat) in 45% formamide. When CMA-induced adducts were detected as blockages of lambda-exonuclease, 12 blockage sites were observed with 8 being prior to 5'-GG sequences, one prior to 5'-CC, one prior to 5'-GC and 2 at unresolved combinations of these sequences. These exonuclease-detected blockages reveal the same sites of CMA-induced crosslinking as detected by in vitro transcription footprinting and primer-extension blockages on single strand DNA, where the blockages at 5'-GG and 5'-CC were identified as sites of intrastrand crosslinking and the 5'-GC blockage as a probable site of interstrand crosslinking. The thermal stability of both types of crosslink (10 min exposure to heat) ranged from 63-70 degrees C at individual sites. High levels of adduct were detected with poly (dG-dC) but not with poly (dI-dC). These results suggest adduct formation involving an aminal linkage between the 3 position of the morpholino moiety and N2 of guanine. Images PMID:8493102

  6. Structural Characterization of Hydroxyl Radical Adducts in Aqueous Media

    NASA Astrophysics Data System (ADS)

    Janik, Ireneusz; Tripathi, G. N. R.

    2015-06-01

    The oxidation by the hydroxyl (OH) radical is one of the most widely studied reactions because of its central role in chemistry, biology, organic synthesis, and photocatalysis in aqueous environments, wastewater treatment, and numerous other chemical processes. Although the redox potential of OH is very high, direct electron transfer (ET) is rarely observed. If it happens, it mostly proceeds through the formation of elusive OH adduct intermediate which facilitates ET and formation of hydroxide anion. Using time resolved resonance Raman technique we structurally characterized variety of OH adducts to sulfur containing organic compounds, halide ions as well as some metal cations. The bond between oxygen of OH radical and the atom of oxidized molecule differs depending on the nature of solute that OH radical reacts with. For most of sulfur containing organics, as well as halide and pseudo-halide ions, our observation suggested that this bond has two-center three-electron character. For several metal aqua ions studied, the nature of the bond depends on type of the cation being oxidized. Discussion on spectral parameters of all studied hydroxyl radical adducts as well as the role solvent plays in their stabilization will be presented.

  7. DNA adducts and carcinogenicity of nitro-polycyclic aromatic hydrocarbons

    SciTech Connect

    Fu, P.P.; Herreno-Saenz, D.; Von Tungeln, L.S.

    1994-10-01

    We have been interested in the structure-activity relationships of nitro-polycyclic aromatic hydrocarbons (nitro-PAHs), and have focused on the correlation of structural and electronic features with biological activities, including mutagenicity and tumorigenicity. In our studies, we have emphasized 1-, 2-, 3-, and 6-nitrobenzo[a]pyrenes (nitro-B[a]Ps) and related compounds, all of which are derived from the potent carcinogen benzo[a]pyrene. While 1-, 2-, and 3-nitro-B[a]P are potent mutagens in Salmonella, 6-nitro-B[a]P is a weak mutagen. In vitro metabolism of 1- and 3-nitro-B[a]P has been found to generate multiple pathways for mutagenic activation. The formation of the corresponding trans-7,8-dihydrodiols and 7,8,9,10-tetrahydrotetrols suggests that 1- and 3-nitro-B[a]P trans-7,8-diol-anti-9, 10--epoxides are ultimate metabolites of the parent nitro-B[a]Ps. We have isolated a DNA adduct from the reaction between 3-nitro-B[a]P trans-7,8-diol-anti-9, 10-epoxide and calf thymus DNA, and identified it as 10-(deoxyguanosin-N{sup 2}-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3-nitro-B[a]P. The same adduct was identified from in vitro metabolism of [{sup 3}H]3-nitro-B[a]P by rat liver microsomes in the presence of calf thymus DNA. A DNA adduct of 3-nitro-B[a]P formed from reaction of N-hydroxy-3-amino-B[a]P, prepared in situ with calf thymus DNA was also isolated. This adduct was identified as 6-(deoxyguanosin-N{sup 2}-yl)-3-amino-B[a]P. The same adduct was obtained from incubating DNA with 3-nitro-B[a]P in the presence of the mammalian nitroreductase, xanthine oxidase, and hypoxanthine. 48 refs., 6 figs., 1 tab.

  8. DNA adducts of ethylene dibromide: Aspects of formation and mutagenicity

    SciTech Connect

    Cmarik, J.L.

    1991-01-01

    1,2-Dibromoethane (ethylene dibromide, EDB), a potential human carcinogen, undergoes bioactivation by the pathway of glutathione (GSH) conjugation, which generates a reactive intermediate capable of alkylating DNA. The major DNA adduct formed is S-[2-(N[sup 7]-guanyl)ethyl]GSH. This dissertation examined the bioactivation of EDB and the formation of DNA adducts. The selectivity of purified rat and human GSH S-transferases for EDB was examined in vitro. An assay was developed to measure the formation of S,S[prime]-ethylene-bis(GSH). The [alpha] class of the GSH S-transferases was responsible for the majority of EDB-GSH conjugation with both the rat and human enzymes. Human tissue samples for a victim of EDB poisoning were analyzed for S-[2-(N[sup 7]-guanyl)ethyl]GSH utilizing electrochemical detection. No adducts were detected in samples of brain, heart, or kidney. The pattern of alkylation of guanines in fragments of plasmid pBR322 DNA by S-(2-chloroethyl)GSH and related compounds was determined. Alkylation varied approximately ten-fold in intensity and was strongest in runs of guanines. Few differences were observed in the alkylation patterns generated by the different compounds tested. The spectrum of mutations caused by S-(2-chloroethyl)GSH was determined using an M13 bacteriophage forward mutation assay. The majority of mutations (70%) were G:C to A:T transitions. Participation of the N[sup 7]-guanyl adduct in the mutagenic process is strongly implicated. The sequence selectivity of alkylation in the region of M13 sequenced in the mutation assay was determined. Comparison of the sequence selectivity with the mutation spectrum revealed no obligate relationship between the extent of adduct formation and the number of mutations which resulted at different sites. Sequence context appears to exert a strong influence on the processing of lesions. These studies strongly implicate S-[2-(N[sup 7]-guanyl)-ethyl]GSH as a mutagenic lesion formed by EDB.

  9. Low response in white blood cell DNA adducts among workers in a highly polluted cokery environment.

    PubMed

    Kuljukka, T; Savela, K; Vaaranrinta, R; Mutanen, P; Veidebaum, T; Sorsa, M; Peltonen, K

    1998-06-01

    Coke oven workers are often heavily exposed to polynuclear aromatic hydrocarbons (PAHs); this exposure has been associated with higher cancer rates among these workers. We assessed the exposure of cokery workers in an oil shale processing plant. Personal hygienic monitoring, measurement of urinary 1-hydroxypyrene (1-OHP), and analysis of PAH-DNA adducts in white blood cells (WBCs) were performed. The 32P-postlabeling method was used for adduct measurement. The mean adduct value, 1.6 adducts per 10(8) nucleotides, did not differ significantly from the control value (P = 0.098). Smokers had significantly higher adduct levels than non-smoking workers (P = 0.002). 1-OHP levels measured in post-shift samples correlated with DNA adducts found in white blood cells (WBCs). We conclude that hygienic monitoring and measurement of urinary metabolites are essential background exposure data when the biologically effective dose of chemical carcinogens is assessed. PMID:9636933

  10. Zinc acetylacetonate hydrate adducted with nitrogen donor ligands: Synthesis, spectroscopic characterization, and thermal analysis

    NASA Astrophysics Data System (ADS)

    Brahma, Sanjaya; Shivashankar, S. A.

    2015-12-01

    We report synthesis, spectroscopic characterization, and thermal analysis of zinc acetylacetonate complex adducted by nitrogen donor ligands, such as pyridine, bipyridine, and phenanthroline. The pyridine adducted complex crystallizes to monoclinic crystal structure, whereas other two adducted complexes have orthorhombic structure. Addition of nitrogen donor ligands enhances the thermal property of these complexes as that with parent metal-organic complex. Zinc acetylacetonate adducted with pyridine shows much higher volatility (106 °C), decomposition temperature (202 °C) as that with zinc acetylacetonate (136 °C, 220 °C), and other adducted complexes. All the adducted complexes are thermally stable, highly volatile and are considered to be suitable precursors for metal organic chemical vapor deposition. The formation of these complexes is confirmed by powder X-ray diffraction, Fourier transform infrared spectroscopy, mass spectroscopy, and elemental analysis. The complexes are widely used as starting precursor materials for the synthesis of ZnO nanostructures by microwave irradiation assisted coating process.

  11. Structural aspects of adducts of N-phthaloylglycine and its derivatives

    NASA Astrophysics Data System (ADS)

    Barooah, Nilotpal; Sarma, Rupam J.; Batsanov, Andrei S.; Baruah, Jubaraj B.

    2006-06-01

    N-phthaloylglycine forms 2:1 adduct with 1,3-dihydroxybenzene and 1:2 adduct with 2-aminopyrimidine. Whereas N-phthaloylglycine form salts with 2,6-diaminopyridine and with 8-hydroxyquinoline. The 1:1 adduct of N, N'-bis(glycinyl)pyromellitic diimide with dimethylsulphoxide, 2-aminopyrimidine and 4,4'-dihydroxybiphenyl are prepared and characterised. The reaction of N, N'-bis(glycinyl)pyromellitic diimide with 2,6-diaminopyridine gives corresponding salt.

  12. Structure of adducts of isoindolo[2,1-a]benzimidazole derivatives with maleimides

    NASA Astrophysics Data System (ADS)

    Korolev, Oleksandr; Yegorova, Tatyana; Levkov, Igor; Malytskyy, Volodymyr; Shishkin, Oleg; Zubatyuk, Roman; Palamarchuk, Genadiy; Vedrenne, Marc; Baltas, Michel; Voitenko, Zoia

    2015-03-01

    The selectivity of formation and some mechanistic insights during the synthesis of substituted isoindolo[2,1-a]benzimidazoles are discussed. Furthermore, the reactions of the obtained products with maleimides were carried out. Two types rearrangement adducts together with intermediate Michael type adducts were isolated. The influence of the reaction conditions and reagents ratio is discussed. Specific spectral criteria for the identification of the Michael type adducts are indicated.

  13. Comparison of DNA adducts from exposure to complex mixtures in various human tissues and experimental systems

    PubMed Central

    Lewtas, Joellen; Mumford, Judy; Everson, Richard B.; Hulka, Barbara; Wilcosky, Tim; Kozumbo, Walter; Thompson, Claudia; George, Michael; Dobiáš, Lubomir; Šrám, Radim; Li, Xueming; Gallagher, Jane

    1993-01-01

    DNA adducts derived from complex mixtures of polycyclic aromatic compounds emitted from tobacco smoke are compared to industrial pollution sources (e.g., coke ovens and aluminum smelters), smoky coal burning, and urban air pollution. Exposures to coke oven emissions and smoky coal, both potent rodent skin tumor initiators and lung carcinogens in humans, result in high levels of DNA adducts compared to tobacco smoke in the in vitro calf thymus DNA model system, in cultured lymphocytes, and in the mouse skin assay. Using tobacco smoke as a model in human studies, we have compared relative DNA adduct levels detected in blood lymphocytes, placental tissue, bronchoalveolar lung lavage cells, sperm, and autopsy tissues of smokers and nonsmokers. Adduct levels in DNA isolated from smokers were highest in human heart and lung tissue with smaller but detectable differences in placental tissue and lung lavage cells. Comparison of the DNA adduct levels resulting from human exposure to different complex mixtures shows that emissions from coke ovens, aluminum smelters, and smoky coal result in higher DNA adduct levels than tobacco smoke exposure. These studies suggest that humans exposed to complex combustion mixtures will have higher DNA adduct levels in target cells (e.g., lung) as compared to nontarget cells (e.g., lymphocytes) and that the adduct levels will be dependent on the genotoxic and DNA adduct-forming potency of the mixture. ImagesFIGURE 1.FIGURE 1.FIGURE 2.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 3.FIGURE 4. PMID:8319665

  14. Detection and comparison of DNA adducts after in vitro and in vivo diesel emission exposures

    SciTech Connect

    Gallagher, J.; George, M.; Kohan, M.; Thompson, C.; Shank, T.

    1993-01-01

    Development of methodologies to evaluate certain classes of polycyclic aromatic compounds (PAC) detected in complex mixtures to which humans are exposed would greatly improve the diagnostic potential of (32)P-postlabeling analysis. Identification of DNA adduct patterns of specific exposure-related marker adducts would strengthen associations between observed DNA adducts and exposures to different environmental pollutants (e.g., kerosene, cigarette smoke, coke oven, and diesel). Diesel-modified DNA adduct patterns were compared in various in vitro and in vivo rodent model systems and then compared to DNA reactive oxidative and reductive metabolites of 1-nitropyrene. The formation of nitrated-polycyclic aromatic hydrocarbon (nitrated-PAH) DNA adducts, derived from the metabolism of diesel extract constituents, was enhanced relative to other PAH-derived DNA adducts via xanthine oxidase-catalyzed nitroreduction. These adducts were detectable only by the butanol extraction version of the postlabeling analysis. Marker adducts detected in the various test systems presented here will assist in characterizing nuclease-P1-sensitive nitrated PAH adducts in humans.

  15. Lifetimes and stabilities of familiar explosives molecular adduct complexes during ion mobility measurements

    PubMed Central

    McKenzie, Alan; DeBord, John Daniel; Ridgeway, Mark; Park, Melvin; Eiceman, Gary; Fernandez-Lima, Francisco

    2015-01-01

    Trapped ion mobility spectrometry coupled to mass spectrometry (TIMS-MS) was utilized for the separation and identification of familiar explosives in complex mixtures. For the first time, molecular adduct complex lifetimes, relative stability, binding energies and candidate structures are reported for familiar explosives. Experimental and theoretical results showed that the adduct size and reactivity, complex binding energy and the explosive structure tailors the stability of the molecular adduct complex. TIMS flexibility to adapt the mobility separation as a function of the molecular adduct complex stability (i.e., short or long IMS experiments / low or high IMS resolution) permits targeted measurements of explosives in complex mixtures with higher confidence levels. PMID:26153567

  16. Glutathione transferase A4-4 resists adduction by 4-hydroxynonenal☆

    PubMed Central

    Shireman, Laura M.; Kripps, Kimberly A.; Balogh, Larissa M.; Conner, Kip P.; Whittington, Dale; Atkins, William M.

    2010-01-01

    4-Hydroxy-2-trans-nonenal (HNE) is a lipid peroxidation product that contributes to the pathophysiology of several diseases with components of oxidative stress. The electrophilic nature of HNE results in covalent adduct formation with proteins, fatty acids and DNA. However, it remains unclear whether enzymes that metabolize HNE avoid inactivation by it. Glutathione transferase A4-4 (GST A4-4) plays a significant role in the elimination of HNE by conjugating it with glutathione (GSH), with catalytic activity toward HNE that is dramatically higher than the homologous GST A1-1 or distantly related GSTs. To determine whether enzymes that metabolize HNE resist its covalent adduction, the rates of adduction of these GST isoforms were compared and the functional effects of adduction on catalytic properties were determined. Although GST A4-4 and GST A1-1 have striking structural similarity, GST A4-4 was insensitive to adduction by HNE under conditions that yield modest adduction of GST A1-1 and extensive adduction of GST P1-1. Furthermore, adduction of GST P1-1 by HNE eliminated its activity toward the substrates 1-chloro- 2,4-dinitrobenzene (CDNB) and toward HNE itself. HNE effects on GST A4-4 and A1-1 were less significant. The results indicate that enzymes that metabolize HNE may have evolved structurally to resist covalent adduction by it. PMID:20836986

  17. Chromatographic and fluorescence spectroscopic studies of individual 7,12-dimethylbenz(a)anthracene--deoxyribonucleoside adducts

    SciTech Connect

    Moschel, R.C.; Pigott, M.A.; Costantino, N.; Dipple, A.

    1983-09-01

    Compared with standard Sephadex LH-20 column chromatography, a newly developed high pressure liquid chromatographic separation of hydrocarbon deoxyribonucleoside adducts derived from the DNA of mouse embryo cell cultures exposed to 7,12-dimethylbenz(a)anthracene (DMBA) provides markedly superior resolution. Once resolved, the fluorescence spectroscopic properties of the three major DMBA--DNA adducts indicate that the fluorescence exhibited by adducts derived from a bay region syn dihydrodiol epoxide of DMBA differs subtly from that exhibited by adducts derived from the isomeric anti dihydrodiol epoxide.

  18. Temporal and spatial features of the formation of DNA adducts in sulfur mustard-exposed skin

    SciTech Connect

    Batal, Mohamed; Boudry, Isabelle; Mouret, Stéphane; Wartelle, Julien; Emorine, Sandy; Bertoni, Marine; Bérard, Izabel; and others

    2013-12-15

    Sulfur mustard (SM) is a chemical warfare agent that targets skin where it induces large blisters. DNA alkylation is a critical step to explain SM-induced cutaneous symptoms. We determined the kinetics of formation of main SM–DNA adducts and compare it with the development of the SM-induced pathogenesis in skin. SKH-1 mice were exposed to 2, 6 and 60 mg/kg of SM and treated skin was biopsied between 6 h and 21 days. Formation of SM DNA adducts was dose-dependent with a maximum immediately after exposure. However, adducts were persistent and still detectable 21 days post-exposure. The time-dependent formation of DNA adducts was also found to be correlated with the appearance of apoptotic cells. This temporal correlation suggests that these two early events are responsible for the severity of the damage to the skin. Besides, SM–DNA adducts were also detected in areas located next to contaminated zone, thus suggesting that SM diffuses in skin. Altogether, this work provides for the first time a clear picture of SM-induced genotoxicity using DNA adducts as a marker. - Highlights: • Sulfur mustard adducts are formed in DNA after skin exposure. • DNA damage formation is an early event in the pathological process of skin burn. • The amount of SM–DNA adducts is maximal at the earliest time point investigated. • Adducts are still detected 3 weeks after exposure. • Sulfur mustard diffuses in skin especially when large doses are applied.

  19. Detection of mitomycin C-DNA adducts in vivo by 32P-postlabeling: time course for formation and removal of adducts and biochemical modulation.

    PubMed

    Warren, A J; Maccubbin, A E; Hamilton, J W

    1998-02-01

    Mitomycin C (MMC) is a DNA cross-linking agent that has been used in cancer chemotherapy for over 20 years, yet little is known either qualitatively or quantitatively about MMC-induced DNA adduct formation and repair in vivo. As an initial means of investigating this, we used a recently developed 32P-postlabeling assay to examine the formation and loss of MMC-DNA adducts in the tissues of a simple in vivo model test system, the chick embryo, following treatment with a chemotherapeutic dose of MMC. As early as 15 min after MMC treatment, four adducts could be detected in the liver which were tentatively identified as the (CpG) N2G-MMC-N2G interstrand cross-link, the bifunctionally activated MMC-N2G monoadduct, and two isomers (alpha and beta) of the monofunctionally activated MMC-N2G monoadduct. The (GpG) N2G-MMC-N2G intrastrand cross-link appears to be a poor substrate for nuclease P1 and/or T4 kinase and was not evaluable by this assay. Levels of all four detectable adducts increased substantially within the first 2 h after MMC treatment, reached maximal levels by 6 h, and decreased progressively thereafter through 24 h, although low levels of certain adducts persisted beyond 24 h. Lung and kidney had comparable levels of total MMC adducts, which were approximately 60% those of the liver, and there were no significant differences in the proportion of specific adducts among the three tissues. The interstrand cross-link represented approximately 13-14% of the total MMC adducts, which is approximately 5-fold greater than the proportion of CpG sites in the genome. In addition, the interstrand cross-link was selectively decreased after 16 h relative to the three monoadducts, suggesting preferential repair. The effect of modulating different components of the Phase I and Phase II drug metabolism on MMC adduct formation, using either glutethimide, 3,4,3',4'-tetrachlorobiphenyl, dexamethasone, buthionine sulfoximine, ethacrynic acid, or N-acetylcysteine pretreatments, was

  20. Metabolites and DNA adduct formation from flavoenzyme-activated porfiromycin.

    PubMed

    Pan, S S; Iracki, T

    1988-08-01

    Porfiromycin was reductively metabolized by NADPH cytochrome P-450 reductase and xanthine oxidase under anaerobic conditions. The production of metabolites varied with the pH and the contents of the reaction buffer. In Tris buffer, two major metabolites were produced at pH 7.5 and above, whereas one major metabolite was produced at pH 6.5. The three major metabolites were separated and isolated by HPLC. Identification by californium-252 plasma desorption mass spectrometry showed that the two major metabolites from pH 7.5 were (trans) and (cis)-forms of 7-amino-1-hydroxyl-2-methylaminomitosene and the major metabolite from pH 6.5 was 7-amino-2-methylaminomitosene. All three major metabolites showed substitutions at the C-1 position. DNA was alkylated readily by enzyme-activated porfiromycin. Digestion of porfiromycin-alkylated DNA by DNase, snake venom phosphodiesterase, and alkaline phosphatase resulted in an insoluble nuclease-resistant fraction and a soluble fraction. The nuclease-resistant fraction reflected a high content of cross-linked adducts. Upon HPLC analysis, the solubilized fraction contained two monofunctionally linked porfiromycin adducts and a possibly cross-linked dinucleotide. The major adduct was isolated by HPLC and identified by NMR, as N2-(2'-deoxyguanosyl)-7-amino-2-methylaminomitosene. The N2 position of deoxyguanosine appeared as the major monofunctional alkylating site for DNA alkylation by porfiromycin. Thus, mitomycin C and porfiromycin (which differs from mitomycin C only by the addition of a methyl group to the aziridine nitrogen) share the same enzymatic activating mechanism that leads to the formation of the same types of metabolites and the same specificity of DNA alkylation. PMID:3412325

  1. Group 13 Superacid Adducts of [PCl2N]3.

    PubMed

    Tun, Zin-Min; Heston, Amy J; Panzner, Matthew J; Scionti, Vincenzo; Medvetz, Doug A; Wright, Brian D; Johnson, Nicholas A; Li, Linlin; Wesdemiotis, Chrys; Rinaldi, Peter L; Youngs, Wiley J; Tessier, Claire A

    2016-04-01

    Irrespective of the order of the addition of reagents, the reactions of [PCl2N]3 with MX3 (MX3 = AlCl3, AlBr3, GaCl3) in the presence of water or gaseous HX give the air- and light-sensitive superacid adducts [PCl2N]3·HMX4. The reactions are quantitative when HX is used. These reactions illustrate a Lewis acid/Brønsted acid dichotomy in which Lewis acid chemistry can become Brønsted acid chemistry in the presence of adventitious water or HX. The crystal structures of all three [PCl2N]3·HMX4 adducts show that protonation weakens the two P-N bonds that flank the protonated nitrogen atom. Variable-temperature NMR studies indicate that exchange in solution occurs in [PCl2N]3·HMX4, even at lower temperatures than those for [PCl2N]3·MX3. The fragility of [PCl2N]3·HMX4 at or near room temperature and in the presence of light suggests that such adducts are not involved directly as intermediates in the high-temperature ring-opening polymerization (ROP) of [PCl2N]3 to give [PCl2N]n. Attempts to catalyze or initiate the ROP of [PCl2N]3 with the addition of [PCl2N]3·HMX4 at room temperature or at 70 °C were not successful. PMID:26974866

  2. Age-dependent increase of etheno-DNA-adducts in liver and brain of ROS overproducing OXYS rats

    SciTech Connect

    Nair, Jagadeesan; Sinitsina, Olga; Vasunina, Elena A.; Nevinsky, Georgy A.; Laval, Jacques; Bartsch, Helmut . E-mail: h.bartsch@dkfz.de

    2005-10-21

    Reactive oxygen species (ROS) and lipid peroxidation (LPO) play a role in aging and degenerative diseases. To correlate oxidative stress and LPO-derived DNA damage, we determined etheno-DNA-adducts in liver and brain from ROS overproducing OXYS rats in comparison with age-matched Wistar rats. Liver DNA samples from 3- and 15-month-old OXYS and Wistar rats were analyzed for 1,N {sup 6}-ethenodeoxyadenosine ({epsilon}dA) and 3,N {sup 4}-ethenodeoxycytidine ({epsilon}dC) by immunoaffinity/{sup 32}P-postlabelling. While {epsilon}dA and {epsilon}dC levels were not different in young rats, adduct levels were significantly higher in old OXYS rats when compared to old Wistar or young OXYS rats. Frozen rat brain sections were analyzed for {epsilon}dA by immunostaining of nuclei. Brains from old OXYS rats accumulated {epsilon}dA more frequently than age-matched Wistar rats. Our results demonstrate increased LPO-induced DNA damage in organs of OXYS rats which correlates with their known shorter life-span and elevated frequency of chronic degenerative diseases.

  3. Reduction of cisplatin-induced nephrotoxicity in vivo by selenomethionine: the effect on cisplatin-DNA adducts.

    PubMed

    García Sar, Daniel; Montes-Bayón, Maria; Blanco González, Elisa; Sierra Zapico, Luisa M; Sanz-Medel, Alfredo

    2011-06-20

    Cisplatin is one of the most effective chemotherapeutic agents, although its clinical use is limited by severe renal toxicity. This toxicity seems to be related to the accumulation of the drug in kidney tissues, leading to renal failure. For this reason, several compounds have been evaluated to ameliorate the nephrotoxicity induced by cisplatin. In the present investigation, we report the effect of the oral administration of selenomethionine before intraperitoneal cisplatin treatment. The preadministration of this Se species has been shown to have an important effect in reducing renal damage induced by cisplatin by increasing the excreted urea and improving creatinine clearance. Quantification of the level of DNA--cisplatin adducts in kidney and liver tissues was carried out by postcolumn isotope dilution analysis using liquid chromatography-inductively coupled plasma (LC-ICP-MS) as speciation set up. The level of DNA--cisplatin adducts in rats given Se-methionine in the drinking water before cisplatin administration was considerably lower in kidney tissues with respect to the animals drinking only water. Such effects were not observed in liver tissue. Initial speciation studies of Pt and Se conducted in kidney tissues of exposed animals by HPLC-ICP-MS have revealed the presence of cisplatin as part of a complex with Se-methionine, which can be eventually excreted into urine. This Pt--Se complex could explain the observed reduction of the kidney damage in Se-methionine-treated animals. PMID:21491944

  4. First Crystal Structure for a Gold Carbene-Protein Adduct.

    PubMed

    Ferraro, Giarita; Gabbiani, Chiara; Merlino, Antonello

    2016-07-20

    The X-ray structure of the adduct formed in the reaction between the gold N-heterocyclic carbene compound Au(NHC)Cl (with NHC = 1-butyl-3-methyl-imidazole-2-ylidene) and the model protein thaumatin is reported here. The structure reveals binding of Au(NHC)(+) fragments to distinct protein sites. Notably, binding of the gold compound occurs at lysine side chains and at the N-terminal tail; the metal binds the protein after releasing Cl(-) ligand, but retaining NHC fragment. PMID:27364343

  5. Unraveling the Photoswitching Mechanism in Donor-Acceptor Stenhouse Adducts.

    PubMed

    Lerch, Michael M; Wezenberg, Sander J; Szymanski, Wiktor; Feringa, Ben L

    2016-05-25

    Molecular photoswitches have opened up a myriad of opportunities in applications ranging from responsive materials and control of biological function to molecular logics. Here, we show that the photoswitching mechanism of donor-acceptor Stenhouse adducts (DASA), a recently reported class of photoswitches, proceeds by photoinduced Z-E isomerization, followed by a thermal, conrotatory 4π-electrocyclization. The photogenerated intermediate is manifested by a bathochromically shifted band in the visible absorption spectrum of the DASA. The identification of the role of this intermediate reveals a key step in the photoswitching mechanism that is essential to the rational design of switching properties via structural modification. PMID:27152878

  6. Lack of Involvement of CEP Adducts in TLR Activation and in Angiogenesis

    PubMed Central

    Gounarides, John; Cobb, Jennifer S.; Zhou, Jing; Cook, Frank; Yang, Xuemei; Yin, Hong; Meredith, Erik; Rao, Chang; Huang, Qian; Xu, YongYao; Anderson, Karen; De Erkenez, Andrea; Liao, Sha-Mei; Crowley, Maura; Buchanan, Natasha; Poor, Stephen; Qiu, Yubin; Fassbender, Elizabeth; Shen, Siyuan; Woolfenden, Amber; Jensen, Amy; Cepeda, Rosemarie; Etemad-Gilbertson, Bijan; Giza, Shelby; Mogi, Muneto; Jaffee, Bruce; Azarian, Sassan

    2014-01-01

    Proteins that are post-translationally adducted with 2-(ω-carboxyethyl)pyrrole (CEP) have been proposed to play a pathogenic role in age-related macular degeneration, by inducing angiogenesis in a Toll Like Receptor 2 (TLR2)-dependent manner. We have investigated the involvement of CEP adducts in angiogenesis and TLR activation, to assess the therapeutic potential of inhibiting CEP adducts and TLR2 for ocular angiogenesis. As tool reagents, several CEP-adducted proteins and peptides were synthetically generated by published methodology and adduction was confirmed by NMR and LC-MS/MS analyses. Structural studies showed significant changes in secondary structure in CEP-adducted proteins but not the untreated proteins. Similar structural changes were also observed in the treated unadducted proteins, which were treated by the same adduction method except for one critical step required to form the CEP group. Thus some structural changes were unrelated to CEP groups and were artificially induced by the synthesis method. In biological studies, the CEP-adducted proteins and peptides failed to activate TLR2 in cell-based assays and in an in vivo TLR2-mediated retinal leukocyte infiltration model. Neither CEP adducts nor TLR agonists were able to induce angiogenesis in a tube formation assay. In vivo, treatment of animals with CEP-adducted protein had no effect on laser-induced choroidal neovascularization. Furthermore, in vivo inactivation of TLR2 by deficiency in Myeloid Differentiation factor 88 (Myd88) had no effect on abrasion-induced corneal neovascularization. Thus the CEP-TLR2 axis, which is implicated in other wound angiogenesis models, does not appear to play a pathological role in a corneal wound angiogenesis model. Collectively, our data do not support the mechanism of action of CEP adducts in TLR2-mediated angiogenesis proposed by others. PMID:25343517

  7. Covalent adducts arising from the decomposition products of lipid hydroperoxides in the presence of cytochrome C

    PubMed Central

    Williams, Michelle V.; Wishnok, John S.; Tannenbaum, Steven R.

    2008-01-01

    Polyunsaturated fatty acids can be converted to lipid hydroperoxides through non-enzymatic and enzymatic pathways. The prototypic ω-6 lipid hydroperoxide 13-hydroperoxy-octadecadienoic acid (13-HPODE) decomposes homolytically to form highly reactiveα,β-unsaturated aldehydes, such as 9,12-dioxo-10(E)-dodecenoic acid (DODE), 4-oxo-2(E)-nonenal (ONE), 4,5-epoxy-2(E)-decenal (EDE), and 4-hydroxy-2(E)-nonenal (HNE), that can form covalent adducts with DNA. Both 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal can also modify proteins to form products that can potentially serve as biomarkers of lipid hydroperoxide-mediated macromolecule damage. In this study cytochrome C was used to identify and characterize the modification sites individually for each of these aldehydes and also to determine the most abundant adduct formed following decomposition of 13-HPODE. The adducts were characterized by ESI-TOF/MS analysis of the intact proteins and by a combination of ESI-ion-trap/MSn and quadrupole-TOF/MS/MS analysis of the tryptic and chymotryptic peptides. The major adducts included an HNE-His Michael adduct on H33, EDE-Lys adducts on K7 and K8, ONE-Lys ketoamide adducts on K5, K7, and K8, an apparent ONE-Lys Michael adduct on K5, and DODE-Lys carboxyl ketoamide adducts on K86 and K87. DODE was the most reactive aldehyde toward cytochrome C. The major adduct from this reaction was analogous to the most abundant adduct resulting from the decomposition of 13-HPODE in the presence of cytochrome C. PMID:17407328

  8. Dynamic and static control of the human knee joint in abduction-adduction.

    PubMed

    Zhang, L Q; Wang, G

    2001-09-01

    It is unclear whether humans can voluntarily control dynamic and static properties in knee abduction-adduction, which may be important in performing functional tasks and preventing injuries, whether the main load is about the abduction axis or not. A joint-driving device was used to perturb the knee in abduction-adduction at full knee extension under both passive (muscle relaxed) and active (muscle contracted in abduction or adduction) conditions. Dynamic control properties in knee abduction-adduction were characterized by joint stiffness, viscosity, and limb inertia, and quasi-static knee torque-angle relationship was characterized by knee abduction-adduction laxity and quasi-static stiffness (at a 20Nm moment). It was found that the subjects were capable of generating net abduction and adduction moment through differential co-contraction of muscles crossing the medial and lateral sides of the knee, which helped to reduce the abduction-adduction joint laxity (p< or =0.01) and increase stiffness (p<0.027) and viscous damping. Knee abduction laxity was significantly lower than adduction laxity (p=0.043) and the quasi-static abduction stiffness was significantly higher than adduction stiffness (p<0.001). The knee joint showed significantly higher stiffness and viscosity in abduction-adduction than their counterparts in knee flexion-extension at comparable levels of joint torque (p<0.05). Similar to dynamic flexion-extension properties, the system damping ratio remained constant over different levels of contraction, indicating simplified control tasks for the central nervous system; while the natural undamped frequency increased considerably with abduction-adduction muscle contraction, presumably making the knee a quicker system during strenuous tasks involving strong muscle contraction. PMID:11506781

  9. Ultrasensitive isolation, identification and quantification of DNA-protein adducts by ELISA-based RADAR assay.

    PubMed

    Kiianitsa, Kostantin; Maizels, Nancy

    2014-07-01

    Enzymes that form transient DNA-protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA-protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine. PMID:24914050

  10. MULTIPLE DNA ADDUCTS IN LYMPHOCYTES OF SMOKERS AND NONSMOKERS DETERMINED BY 32P-POSTLABELING ANALYSIS

    EPA Science Inventory

    Identification of DNA adducts in peripheral lymphocytes could serve as a means of monitoring human exposure to potential genotoxic agents. n this study, DNA from peripheral lymphocytes of smokers and nonsmokers was examined for adducts by the P1 nuclease 32P-postlabeling techniqu...

  11. CIGARETTE SMOKE-INDUCED DNA ADDUCTS IN THE RESPIRATORY AND NONRESPIRATORY TISSUE OF RATS

    EPA Science Inventory

    Formation of DNA adducts is regarded a- an essential initial step in the process of chemical carcinogenesis. To determine how chronic exposure to cigarette smoke affects the distribution of DNA adducts In selected respiratory and nonrespiratory tissues, we exposed male Sprague-Da...

  12. FORMATION OF HEMOGLOBIN ADDUCTS OF ACRYLAMIDE AND ITS EPOXIDE METABOLITE GLYCIDAMIDE IN THE RAT

    EPA Science Inventory

    A method was developed for the determination of hemoglobin (Hb) adducts form by the neurotoxic agent acrylamide and its mutagenic epoxide metabolite glycidamide. he method was based on simultaneous measurements of the cysteine adducts formed by these two agents by means of gas ch...

  13. Cyclooctyne [60]fullerene hexakis adducts: a globular scaffold for copper-free click chemistry.

    PubMed

    Ramos-Soriano, Javier; Reina, José J; Pérez-Sánchez, Alfonso; Illescas, Beatriz M; Rojo, Javier; Martín, Nazario

    2016-08-18

    The synthesis of a new highly symmetric hexakis adduct of C60 appended with 12 cyclooctyne moieties has been carried out. This compound has been used for the copper-free strain-promoted cycloaddition reaction to a series of azides with excellent yields. This strategy for the obtention of clicked adducts of [60]fullerene is of special interest for biological applications. PMID:27492263

  14. Significance of DNA adduct studies in animal models for cancer molecular dosimetry and risk assessment.

    PubMed Central

    Beland, F A; Poirier, M C

    1993-01-01

    To elucidate the relationship between DNA adduct formation and tumorigenesis, a number of experiments have been conducted to measure DNA adducts in target tissues from experimental animals during continuous exposure to carcinogens. With aflatoxins, aromatic amines, and polycyclic aromatic hydrocarbons, tumor induction appears to be associated with the major DNA adduct detected, whereas with N-nitrosamines the response is normally correlated with minor forms of DNA damage. During continuous carcinogen administration, steady-state adduct concentrations are generally obtained in the target tissues, and there is often a linear correlation between the carcinogen concentration and the steady-state DNA adduct level. Exceptions exist when the mechanism of activation changes or with the onset of significant toxicity. Steady-state DNA adduct levels are often linearly related to the tumorigenic response. Carcinogen-induced cell proliferation occurs when significant deviations from linearity are observed. Because DNA adducts detected in humans are chemically identical to those found in experimental animals, DNA adduct data in animals may contribute to our understanding of human cancer risk. PMID:8319658

  15. 40 CFR 721.3680 - Ethylene oxide adduct of fatty acid ester with pentaerythritol.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... ester with pentaerythritol. 721.3680 Section 721.3680 Protection of Environment ENVIRONMENTAL PROTECTION... New Uses for Specific Chemical Substances § 721.3680 Ethylene oxide adduct of fatty acid ester with... identified generically as ethylene oxide adduct of fatty acid ester with pentaerythritol (PMN P-91-442)...

  16. 40 CFR 721.3680 - Ethylene oxide adduct of fatty acid ester with pentaerythritol.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... ester with pentaerythritol. 721.3680 Section 721.3680 Protection of Environment ENVIRONMENTAL PROTECTION... New Uses for Specific Chemical Substances § 721.3680 Ethylene oxide adduct of fatty acid ester with... identified generically as ethylene oxide adduct of fatty acid ester with pentaerythritol (PMN P-91-442)...

  17. Turned head--adducted hip--truncal curvature syndrome.

    PubMed Central

    Hamanishi, C; Tanaka, S

    1994-01-01

    One hundred and eight neonates and infants who showed the clinical triad of a head turned to one side, adduction contracture of the hip joint on the occipital side of the turned head, and truncal curvature, which we named TAC syndrome, were studied. These cases included seven with congenital and five with late infantile dislocations of the hip joint and 14 who developed muscular torticollis. Forty one were among 7103 neonates examined by one of the authors. An epidemiological analysis confirmed the aetiology of the syndrome to be environmental. The side to which the head was turned and that of the adducted hip contracture showed a high correlation with the side of the maternal spine on which the fetus had been lying. TAC syndrome is an important asymmetrical deformity that should be kept in mind during neonatal examination, and may be aetiologically related to the unilateral dislocation of the hip joint, torticollis, and infantile scoliosis which develop after a vertex presentation. Images PMID:8048823

  18. Kinetics, mechanism and thermodynamics of bisulfite-aldehyde adduct formation

    SciTech Connect

    Olson, T.M.; Boyce, S.D.; Hoffmann, M.R.

    1986-04-01

    The kinetics and mechanism of bisulfite addition to benzaldehyde were studied at low pH in order to assess the importance of this reaction in stabilizing S(IV) in fog-, cloud-, and rainwater. Previously, the authors established that appreciable concentrations of the formaldehyde-bisulfite adduct (HMSA) are often present in fogwater. Measured HMSA concentrations in fogwater often do not fully account for observed excess S(IV) concentrations, however, so that other S(IV)-aldehyde adducts may be present. Reaction rates were determined by monitoring the disappearance of benzaldehyde by U.V. spectrophotometry under pseudo-first order conditions, (S(IV))/sub T/ >>(phi-CHO)/sub T/, in the pH range 0 - 4.4 at 25/sup 0/C. The equilibrium constant was determined by dissolving the sodium salt of the addition compound in a solution adjusted to pH 3.9, and measuring the absorbance of the equilibrated solution at 250 nm. A literature value of the extinction coefficient for benzaldehyde was used to calculate the concentration of free benzaldehyde. All solutions were prepared under an N/sub 2/ atmosphere using deoxygenated, deionized water and ionic strength was maintained at 1.0 M with sodium chloride.

  19. Spontaneous dehydrocoupling in peri-substituted phosphine-borane adducts.

    PubMed

    Taylor, Laurence J; Surgenor, Brian A; Wawrzyniak, Piotr; Ray, Matthew J; Cordes, David B; Slawin, Alexandra M Z; Kilian, Petr

    2016-02-01

    Bis(borane) adducts Acenap(PiPr2·BH3)(PRH·BH3) (Acenap = acenaphthene-5,6-diyl; 4a, R = Ph; 4b, R = ferrocenyl, Fc; 4c, R = H) were synthesised by the reaction of excess H3B·SMe2 with either phosphino-phosphonium salts [Acenap(PiPr2)(PR)](+)Cl(-) (1a, R = Ph; 1b, R = Fc), or bis(phosphine) Acenap(PiPr2)(PH2) (3). Bis(borane) adducts 4a-c were found to undergo dihydrogen elimination at room temperature, this spontaneous catalyst-free phosphine-borane dehydrocoupling yields BH2 bridged species Acenap(PiPr2)(μ-BH2)(PR·BH3) (5a, R = Ph; 5b, R = Fc; 5c, R = H). Thermolysis of 5c results in loss of the terminal borane moiety to afford Acenap(PiPr2)(μ-BH2)(PH) (14). Single crystal X-ray structures of 3, 4b and 5a-c are reported. PMID:26314761

  20. Melatonin affects conjugation of 4-hydroxynonenal with glutathione in liver of pacu, a hypoxia-tolerant fish.

    PubMed

    Bastos, F F; Tobar, S A L; Dantas, R F; Silva, E S; Nogueira, N P A; Paes, M C; Righi, B D P; Bastos, J Cunha; Bastos, V L F Cunha

    2013-10-01

    In cytosol from liver of pacu, Piaractus mesopotamicus, a hypoxia-tolerant fish that dwells in Pantanal, we found an enzyme activity capable of modulating the alkenal 4-hydroxy-2-nonenal (HNE) by conjugating it with glutathione (GST-HNE activity). HNE is a downstream metabolite from the oxidation of polyunsaturated fatty acids by reactive oxygen species arisen from mitochondria of animal cells. HNE production may increase more intensively under oxidative stress. Harmful effects to cell survival may occur when HNE increases over 10(-4) M. Pacus submitted to hypoxia in July (cold season in Pantanal) showed 40% less of this GST-HNE conjugating activity in their liver cytosol. Injecting pacus subjected to hypoxia during the cold season with a summer physiological dose of melatonin caused their liver cytosolic GST-HNE activity to increase up to the levels found in the warm season. From October to March (warm season in Pantanal), pacus are prone to oxidative stress particularly during potamodromous active oxygen-demanding swimming, when they migrate up rivers to spawn. Thus, our findings point out that the higher levels of melatonin in circulation during the summer are important to avoid the increase of 4-HNE inside liver cells of this fish species. PMID:23440384

  1. Quantitation of 4,4′-methylene diphenyl diisocyanate human serum albumin adducts

    PubMed Central

    Luna, Leah G.; Green, Brett J.; Zhang, Fagen; Arnold, Scott M.; Siegel, Paul D.; Bartels, Michael J.

    2016-01-01

    4,4′-Methylene diphenyl diisocyanate (herein 4,4′-MDI) is used in the production of polyurethane foams, elastomers, coatings, adhesives and the like for a wide range of commercial products. Occupational exposure to MDI levels above current airborne exposure limits can elicit immune mediated hypersensitivity reactions such as occupational asthma in sensitive individuals. To accurately determine exposure, there has been increasing interest in developing analytical methods to measure internal biomarkers of exposure to MDI. Previous investigators have reported methodologies for measuring MDI diamine metabolites and MDI-Lysine (4,4′-MDI-Lys) adducts. The purpose of this study was to develop and validate an ultra performance liquid chromatography isotope dilution tandem mass spectrometry (UPLC-ID/MS/MS) quantitation method via a signature peptide approach to enable biomonitoring of 4,4′-MDI adducted to human serum albumin (HSA) in plasma. A murine, anti-4,4′-MDI monoclonal IgM antibody was bound to magnetic beads and utilized for enrichment of the MDI adducted HSA. Following enrichment, trypsin digestion was performed to generate the expected 414 site (primary site of adduction) 4,4′-MDI-adducted HSA signature peptide that was quantified by UPLC-ID/MS/MS. An Agilent 6530 UPLC/quadrupole time of flight MS (QTOF) system was utilized for intact adducted protein analysis and an Agilent 6490 UPLC/MS/MS system operated in multiple reaction monitoring (MRM) mode was utilized for quantification of the adducted signature peptide biomarker both for in chemico and worker serum samples. Worker serum samples were initially screened utilizing the previously developed 4,4′-MDI-Lys amino acid method and results showed that 12 samples were identified as quantifiable for 4,4′-MDI-Lys adducts. The signature peptide adduct approach was applied to the 12 worker samples identified as quantifiable for 4,4′-MDI-Lys adducts. Results indicated no positive results were obtained

  2. Liquid chromatography-thermospray mass spectrometry of DNA adducts formed with mitomycin C, porfiromycin and thiotepa.

    PubMed

    Musser, S M; Pan, S S; Callery, P S

    1989-07-14

    High-performance liquid chromatography (HPLC) and thermospray mass spectrometry were combined for the analysis of DNA adducts formed from the interaction of the anticancer drugs mitomycin C, porfiromycin and thiotepa with calf thymus DNA. The adducts formed from reaction of mitomycin C and porfiromycin with DNA were separated from unmodified nucleosides by HPLC on a C18 column and identified by thermospray mass spectrometry. Thiotepa DNA adducts readily depurinated from DNA and were chromatographed and identified by thermospray liquid chromatography-mass spectrometry as the modified bases without the ribose moiety attached. The utility of thermospray mass spectrometry for the identification of microgram quantities of nucleoside adducts and depurinated base adducts of these anticancer drugs was demonstrated. PMID:2504760

  3. MRN, CtIP, and BRCA1 mediate repair of topoisomerase II–DNA adducts

    PubMed Central

    Aparicio, Tomas; Baer, Richard; Gottesman, Max

    2016-01-01

    Repair of DNA double-strand breaks (DSBs) with complex ends poses a special challenge, as additional processing is required before DNA ligation. For example, protein–DNA adducts must be removed to allow repair by either nonhomologous end joining or homology-directed repair. Here, we investigated the processing of topoisomerase II (Top2)–DNA adducts induced by treatment with the chemotherapeutic agent etoposide. Through biochemical analysis in Xenopus laevis egg extracts, we establish that the MRN (Mre11, Rad50, and Nbs1) complex, CtIP, and BRCA1 are required for both the removal of Top2–DNA adducts and the subsequent resection of Top2-adducted DSB ends. Moreover, the interaction between CtIP and BRCA1, although dispensable for resection of endonuclease-generated DSB ends, is required for resection of Top2-adducted DSBs, as well as for cellular resistance to etoposide during genomic DNA replication. PMID:26880199

  4. DNA adducts in marine mussel and fresh water fishes living in polluted and unpolluted environments

    SciTech Connect

    Kurelec, B.; Checko, M.; Krca, S.; Garg, A.; Gupta, R.C. Baylor College of Medicine, Houston, TX )

    1988-09-01

    {sup 32}P-postlabeling analysis of DNA adducts in the digestive gland of marine mussel Mytilus galloprovincialis from polluted and unpolluted sites near Rovinj, Northern Adriatic, revealed that majority of adducts are caused by natural environmental factors rather than by man-made chemicals. The only pollutant-specific adducts were observed in a mussel exposed to seawater experimentally polluted with aminofluorene, and in a population of mussel living at a site heavily polluted with a waste waters of an oil refinery. Fresh water fish species Leuciscus cephalus, Barbus barbus, Abramis brama and Rutilus pigus virgo living in a polluted Sava River, Yugoslavia, or in its unpolluted tributary Korana River, have induced in their livers qualitatively identical and quantitatively similar DNA adducts. These DNA adducts had a species-specific patterns and their appearance was seasonally-dependent.

  5. DNA adducts as a measure of lung cancer risk in humans exposed to polycyclic aromatic hydrocarbons.

    PubMed Central

    Kriek, E; Van Schooten, F J; Hillebrand, M J; Van Leeuwen, F E; Den Engelse, L; De Looff, A J; Dijkmans, A P

    1993-01-01

    Workers in the coking, foundry, and aluminum industry can be exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and are at increased risk for lung cancer, as are cigarette smokers. In recent years several studies on workers in the foundry and coking industries have been reported. In these studies, white blood cell(WBC) DNA was used for analysis of PAH-DNA adducts. Theoretically, DNA adduct formation is a more relevant biological parameter for assessing exposure risk than PAH in the work atmosphere, or the amount of a metabolite in the urine, because adduct levels reflect that part of the dose that escapes detoxification and binds to DNA. We analyzed WBC DNA from coke-oven workers and from workers in an aluminum production plant and demonstrated the presence of PAH-DNA adducts. Forty-seven percent of the coke-oven workers had detectable levels of PAH-DNA adducts in their WBC compared with 27% of the controls (p < 0.05), measured with ELISA. In both groups, smokers had significantly higher levels of PAH-DNA adducts than did nonsmokers. In the aluminum workers, no PAH-DNA adducts were detected by ELISA, although the benzo[a]pyrene concentrations in the work atmosphere were comparable to those of the coke-oven workers. The more sensitive 32P-postlabeling assay showed the presence of PAH-DNA adducts in 91% of the aluminum workers. There was no correlation of WBC adduct levels with the concentration of PAH in the work atmosphere. Recently we showed that total PAH-DNA adduct levels in WBC from lung cancer patients were much higher than those generally found in healthy smokers.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8319662

  6. Depurinating estrogen-DNA adducts, generators of cancer initiation: their minimization leads to cancer prevention.

    PubMed

    Cavalieri, Ercole L; Rogan, Eleanor G

    2016-03-01

    Estrogens can initiate cancer by reacting with DNA. Specific metabolites of endogenous estrogens, the catechol estrogen-3,4-quinones, react with DNA to form depurinating estrogen-DNA adducts. Loss of these adducts leaves apurinic sites in the DNA, generating mutations that can lead to the initiation of cancer. A variety of endogenous and exogenous factors can disrupt estrogen homeostasis, which is the normal balance between estrogen activating and protective enzymes. In fact, if estrogen metabolism becomes unbalanced and generates excessive catechol estrogen 3,4-quinones, formation of depurinating estrogen-DNA adducts increases and the risk of initiating cancer is greater. The levels of depurinating estrogen-DNA adducts are high in women diagnosed with breast cancer and those at high risk for the disease. High levels of depurinating estrogen-DNA adducts before the presence of breast cancer indicates that adduct formation is a critical factor in breast cancer initiation. Women with thyroid or ovarian cancer also have high levels of estrogen-DNA adducts, as do men with prostate cancer or non-Hodgkin lymphoma. Depurinating estrogen-DNA adducts are initiators of many prevalent types of human cancer. These findings and other discoveries led to the recognition that reducing the levels of estrogen-DNA adducts could prevent the initiation of human cancer. The dietary supplements N-acetylcysteine and resveratrol inhibit formation of estrogen-DNA adducts in cultured human breast cells and in women. These results suggest that the two supplements offer an approach to reducing the risk of developing various prevalent types of human cancer. Graphical abstract Major metabolic pathway in cancer initiation by estrogens. PMID:26979321

  7. /sup 32/P-postlabeling analysis of aromatic DNA adducts in fish from polluted areas

    SciTech Connect

    Dunn, B.P.; Black, J.J.; Maccubbin, A.

    1987-12-15

    Brown bullheads (Ictalurus nebulosus) were sampled from sites in the Buffalo and Detroit Rivers where fish are exposed to high levels of sediment bound polycyclic aromatic hydrocarbons, and suffer from an elevated frequency of liver cancer. DNA was isolated from the livers of these wild fish and from control specimens which were raised in clean aquariums. DNA was enzymatically digested to normal and adducted nucleotides, and hydrophobic/bulky adducts were enriched in the digests either by preparative reverse-phase high-pressure liquid chromatography, or selective nuclease P1 dephosphorylation of normal nucleotides. Aromatic DNA-carcinogen adducts were then quantitated using /sup 32/P-postlabeling analysis. Using both adduct enrichment procedures, chromatograms derived from DNA of fish from polluted areas showed a diffuse diagonal radioactive zone not present in DNA from aquarium raised fish. The diagonal zone appeared to consist at least in part of multiple overlapping discrete adduct spots which could be partially separated by gradient high-pressure liquid chromatography prior to /sup 32/P-postlabeling analysis, and most of which were more strongly retained on a reverse-phase column than the major benzo(a)pyrene-DNA adduct. The behavior of the adducts in the diagonal radioactive zone and of their unlabeled precursors is consistent with their identification as nucleotide adducts of a variety of bulky hydrophobic aromatic environmental compounds. Total pollution-related adduct levels as analyzed by HPLC adduct enrichment and /sup 32/P-postlabeling were 70.1 +/- 29 (SD) nmol/mol normal nucleotide in fish from the Buffalo River, and 52 and 56 nmol/mol for two specimens from the Detroit River.

  8. Line narrowing spectroscopic studies of DNA-carcinogen adducts and DNA-dye complexes

    SciTech Connect

    Suh, Myungkoo

    1995-12-06

    Laser-induced fluorescence line narrowing and non-line narrowing spectroscopic methods were applied to conformational studies of stable DNA adducts of the 7{beta}, 8{alpha}-dihydoxy-9{alpha}, l0{alpha}-epoxy-7,8,9, 10-tetrahydrobenzo[{alpha}]pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, ({minus})-trans-, (+)-cis- and ({minus})-cis adducts of anti-BPDE bound to exocyclic amino group of the central guanine in an 11-mer oligonucleotide, exist in a mixture of conformations in frozen aqueous buffer matrices. The (+)-trans adduct adopts primarily an external conformation with a smaller fraction ( {approximately} 25 %) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant {pi}-{pi} stacking interactions between the pyrenyl residues and the bases. Conformations of the trans-adduct of (+)-anti -BPDE in 11-mer oligonucleotides were studied as a function of flanking bases. In single stranded form the adduct at G{sub 2} or G{sub 3} (5 ft-flanking, base guanine) adopts a conformation with strong, interaction with the bases. In contrast, the adduct with a 5ft-flanking, thymine exists in a primarily helixexternal conformation. Similar differences were observed in the double stranded oligonucleotides. The nature of the 3ft-flanking base has little influence on the conformational equilibrium of the (+)-trans-anti BPDE-dG adduct. The formation and repair of BPDE-N{sup 2}-dG in DNA isolated from the skin of mice treated topically with benzo[{alpha}]pyrene (BP) was studied. Low-temperature fluorescence spectroscopy of the intact DNA identified the major adduct as (+)-trans-anti-BPDE-N-dG, and the minor adduct fraction consisted mainly of (+)-cis-anti-BPDE-N{sup 2}-dG.

  9. Absence of formation of benzo[a]pyrene/DNA adducts in the cuttlefish (Sepia officinalis, Mollusca: Cephalopoda)

    SciTech Connect

    Lee, P.G.; Lu, L.J.W.; Salazar, J.J.; Holoubek, V. )

    1994-01-01

    Benzo[a]pyrene (B[a]P) injected intramuscularly into the base of the arms of cuttlefish was released continuously from the injection site and removed from the organism. Only a portion of the compound accumulated in the body. Twenty-four hr after its injection, 75% of B[a]P applied in olive oil was removed from the cuttlefish, and 1.2% was found in the body outside the head, in site of injection. If the carcinogen was dissolved in dimethylformamide, the removal of B[a]P was slower, so that only 18% of the injected B[a]P was removed from the organism and 0.36% accumulated in the body outside the head 24 hr after injection. The high level of B[a]P in gills and hemolymph 4 hr after injection and the kinetics of the decrease of its concentration with time indicate that these two organs could be involved in the excretion of B[a]P from the body. The B[a]P/DNA adducts characteristic for vertebrates could not be demonstrated in gills, skin, brain, hepatopancreas, and lymphocytes of the cuttlefish 24 hr after injection. The dose of the carcinogene injected into the cuttlefish was 2-4 times higher than the dose resulting in the formation of a high level of B[a]P/DNA adducts in vertebrates. A different metabolism of B[a]P in the tissue of cephalopods, compared to vertebrates, could be less favorable to the process leading to malignant transformation and could explain the absence from the literature of reports of tumors in cephalopods. 15 refs., 1 fig., 2 tabs.

  10. (32)P-POSTLABELING ANALYSIS OF DNA ADDUCTS OF TWO NITRATED POLYCYCLIC AROMATIC HYDROCARBONS IN RABBIT TRACHEAL EPITHELIAL CELLS

    EPA Science Inventory

    The 1-nitropyrene (1-NPP and 3-nitrofluoranthene (3-NF) adducts have been analyzed by (32)P-postlabeling and with 1-NP have been compared to the total number of adducts estimated from (14)C binding in rabbit trachael epithelial (RTE) DNA samples. One adduct spot, by (32)P-postlab...

  11. ON BENZO[A]PYRENE DERIVED DNA ADDUCTS FORMED IN LUNG TISSUE OF MICE

    EPA Science Inventory

    On Benzo [a] pyrene Derived DNA Adducts Formed in Lung Tissue of Mice
    The previously identified major DNA adducts of benzo[a]pyrene (BP) in vitro and in vivo are the stable and unstable adducts formed by reaction of the bay-region diol epoxide of BP (BPDE) and BP radical catio...

  12. CYCLOPENTA-FUSED POLYCYCLIC AROMATIC HYDROCARBONS IN STRAIN A/J MOUSE LUNG: DNA ADDUCTS, ONCOGENE MUTATIONS, & TUMORIGENESIS

    EPA Science Inventory

    Cyclopenta-fused Polycyclic Aromatic Hydrocarbons in Strain AJJ Mouse Lung: DNA Adducts, Oncogene Mutations, and Tumorigenesis.

    We have examined the relationships between DNA adducts, Ki-ras oncogene mutations, DNA adducts, and adenoma induction in the lungs of strain A/J...

  13. IR spectra and structure of 4-hydroxybenzylidenemalononitrile, its oxyanion, cyanide adduct and adduct-oxyanion: experimental and ab initio studies

    NASA Astrophysics Data System (ADS)

    Velcheva, Evelina A.; Binev, Yuri I.; Petrova, Milena J.

    1999-01-01

    The structures of 4-hydroxybenzylidenemalononitrile (HO-C 6H 4-CHC(CN) 2, I), its oxyanion ( -O-C 6H 4-CHC(CN) 2, II), cyanide adduct (HO-C 6H 4-CH(CN)-C¯(CN) 2, III) and adduct-oxyanion ( -O-C 6H 4-CH(CN)-C¯(CN) 2, IV) have been studied by means of both quantitative IR spectra and ab initio force field calculations. The conversion of ( I) into the anionic species causes strong changes in the IR spectra: decreases in the ν CN frequency down to 110 cm -1, up to 7-fold increases in the ACN intensity, up to 58 cm -1 ν CN splitting, etc. The charge analysis shows that the intramolecular charge transfer between the electronegative [C(CN) 2] and electropositive fragments of ( I) is 0.34 e -. Nearly 0.6 e - of the oxyanionic charge of ( II) remains within the oxyphenylene fragment and nearly 0.5 e - of the carbanionic charge of ( III) delocalizes within the dicyanomethide fragment. The two charges in ( IV) are spread over the whole species.

  14. Accumulation of oxidative DNA damage in brain mitochondria in mouse model of hereditary ferritinopathy.

    PubMed

    Deng, Xiaoling; Vidal, Ruben; Englander, Ella W

    2010-07-19

    Tissue iron content is strictly regulated to concomitantly satisfy specialized metabolic requirements and avoid toxicity. Ferritin, a multi-subunit iron storage protein, is central to maintenance of iron homeostasis in the brain. Mutations in the ferritin light chain (FTL)-encoding gene underlie the autosomal dominant, neurodegenerative disease, neuroferritinopathy/hereditary ferritinopathy (HF). HF is characterized by progressive accumulation of ferritin and iron. To gain insight into mechanisms by which FTL mutations promote neurodegeneration, a transgenic mouse, expressing human mutant form of FTL, was recently generated. The FTL mouse exhibits buildup of iron in the brain and presents manifestations of oxidative stress reminiscent of the human disease. Here, we asked whether oxidative DNA damage accumulates in the FTL mouse brain. Long-range PCR (L-PCR) amplification-mediated DNA damage detection assays revealed that the integrity of mitochondrial DNA (mtDNA) in the brain was significantly compromised in the 12- but not 6-month-old FTL mice. Furthermore, L-PCR employed in conjunction with DNA modifying enzymes, which target specific DNA adducts, revealed the types of oxidative adducts accumulating in mtDNA in the FTL brain. Consistently with DNA damage predicted to form under conditions of excessive oxidative stress, detected adducts include, oxidized guanines, abasic sites and strand breaks. Elevated mtDNA damage may impair mitochondrial function and brain energetics and in the long term contribute to neuronal loss and exacerbate neurodegeneration in HF. PMID:20478358

  15. DNA adducts in human placenta as related to air pollution and to GSTM1 genotype.

    PubMed

    Topinka, J; Binková, B; Mracková, G; Stávková, Z; Benes, I; Dejmek, J; Lenícek, J; Srám, R J

    1997-04-24

    DNA adducts in human placenta have been studied in relation to metabolic genotype for glutathione S-transferase M1 (GSTM1) in 98 mothers living in two regions with a different annual average air pollution levels: Northern Bohemia-the district of Teplice as polluted industrial area (mines, brown coal power plants) and Southern Bohemia-the district of Prachatice as agricultural area without heavy industry. Forty-nine placenta samples (25 from the Teplice district and 24 from the Prachatice district) from non-smoking mothers with the date of delivery in the summer period and 49 placenta samples (25 from the Teplice district and 24 from Prachatice district) from mothers with the date of delivery in the winter period were analysed. The total DNA adduct levels were calculated as the sum of adducts in the diagnoal radioactive zone (DRZ) and one distinct spot outside of the DRZ (termed X), which was detected in almost all placenta samples. We found total DNA adduct levels of 1.40 +/- 0.87 (0.04-3.65) and 1.04 +/- 0.63 (0.11-3.08) adducts per 10(8) nucleotides for the Teplice and Prachatice districts, respectively. The significant difference between both districts in placental DNA adduct levels was found for the winter sampling period only (1.49 vs. 0.96 adducts per 10(8) nucleotides; p = 0.023). No seasonal variation was observed for DNA adduct levels in the overall population studied. A positive GSTM1 genotype was detected in 51 subjects, while GSTM1-null genotype was found in 47 subjects. Higher DNA adduct levels were detected in a group with GSTM1-null genotype (p = 0.009). This finding seems more significant for subjects in the Teplice district (p = 0.047) than for those in the Prachatice district (p = 0.092). Significant district and seasonal differences were found in subgroups carrying the GSTM1-null genotype. DNA adduct levels in placentas of mothers with GSTM1-null genotype living in the polluted district of Teplice were higher than those in Prachatice (p = 0

  16. Preparation and Characterization of Cysteine Adducts of Deoxynivalenol.

    PubMed

    Stanic, Ana; Uhlig, Silvio; Solhaug, Anita; Rise, Frode; Wilkins, Alistair L; Miles, Christopher O

    2016-06-15

    Conjugation with the biologically relevant thiol glutathione is one of the metabolic pathways for the mycotoxin deoxynivalenol (DON) in wheat. The occurrence of putative DON-cysteine conjugates has also been shown in wheat, likely in part as a result of degradation of the glutathione conjugates. It was reported that thiols react in vitro with DON at two positions: reversibly at C-10 of the α,β-unsaturated ketone and irreversibly at C-13 of the epoxy group. We synthesized pure DON-cysteine adducts and made analytical standards using quantitative NMR experiments. Compounds were characterized using NMR and LC-HRMS/MS and tested in vitro for toxicity. Cysteine conjugates were much less toxic than DON at the same concentration, and LC-HRMS analysis demonstrated that there was no detectable metabolism of the conjugates in human monocytes or human macrophages. PMID:27229448

  17. Non Covalent Interactions and Internal Dynamics in Adducts of Freons

    NASA Astrophysics Data System (ADS)

    Caminati, Walther; Gou, Qian; Evangelisti, Luca; Feng, Gang; Spada, Lorenzo; Vallejo-López, Montserrat; Lesarri, Alberto; Cocinero, Emilio J.

    2014-06-01

    The complexation of chlorofluorocarbons (CFCs) with atmospheric water and pollutants of the atmosphere affects their reactivity and it seems to accelerate, for example, the decomposition rate of freons in the atmosphere [1]. For this reason we characterized shapes, stabilities, nature of the non-covalent interactions, structures and internal dynamics of a number of complexes of CFCs with water and of their dimers or oligomers by rotational spectroscopy. It has been found that hydrogenated CFCs form adducts with other molecules through weak hydrogen bonds (WHBs). Their C-H groups can act as proton donors, enhanced by the electron withdrawing of the halogen atoms, interacting with the electron rich regions of the partner molecules [2]. Also in adducts or oligomers of hydrogenated CFCs the monomer units are held together by nets of WHBs [3]. When CFCs are perhalogenated, the positive electrostatic region ("σ-hole") can interact electrostatically with negative sites of another, or of the same molecular entity, giving rise, according to IUPAC, to the so called halogen bond (HaB). However, it has been observed that when the perhalogenated CFCs has a Π electron system, a lone pair•••Π interaction (Bürgi-Dunitz) is favoured [4]. We describe here the HaBs that CF4 and CF3Cl form with a variety of partner molecules such as water, ammonia, dimethyl ether, etc. Important spectroscopic features outline strong dynamics effects taking place in this kind of complex. References [1] V. Vaida, H. G. Kjaergaard, K. J. Feierabend, Int. Rev. Phys. Chem. 22 (2003) 203. [2] See, for example: W. Caminati, S. Melandri, A. Maris, P. Ottaviani, Angew. Chem. Int. Ed. 45 (2006) 2438. [3] G. Feng, L. Evangelisti, I. Cacelli, L. Carbonaro, G. Prampolini, W. Caminati, Chem. Commun. 50 (2014) 171. [4] Q. Gou, G. Feng, L. Evangelisti, W. Caminati, Angew. Chem. Int. Ed. 52 (2013) 52 11888.

  18. Synthesis and mutagenesis of the butadiene-derived N3 2'-deoxyuridine adducts.

    PubMed

    Fernandes, Priscilla H; Hackfeld, Linda C; Kozekov, Ivan D; Hodge, Richard P; Lloyd, R Stephen

    2006-07-01

    1,3-Butadiene is a known carcinogen and mutagen that acts through a variety of metabolic intermediates that react with DNA, forming stable and unstable lesions on dG, dA, dC, and dT. The N3 2'-deoxyuridine adducts are a highly stable, stereoisomeric mixture of adducts derived from the reaction of cytosine with the monoepoxide metabolite of butadiene, followed by spontaneous deamination. In this study, the phosphoramidites and subsequent oligodeoxynucleotides containing the N3 2'-deoxyuridine adducts have been constructed and characterized. Using a single-stranded shuttle vector DNA, the mutagenic potential of these adducts has been tested following replication in mammalian cells. Replication past the N3 2'-deoxyuridine adducts was found to be highly mutagenic with an overall mutation yield of approximately 97%. The major mutations that were observed were C to T transitions and C to A transversions. In vitro, these adducts posed a complete block to both the Klenow fragment of Escherichia coli polymerase I and polymerase epsilon, while these lesions significantly blocked polymerase delta. These data suggested a possible involvement of bypass polymerases in the in vivo replication of these lesions. Overall, these findings indicate that the N3 2'-deoxyuridine adducts are highly mutagenic lesions that may contribute to butadiene-mediated carcinogenesis. PMID:16841966

  19. Conformational Properties of Equilenin-DNA Adducts: Stereoisomer and Base Effects

    PubMed Central

    Ding, Shuang; Shapiro, Robert; Cai, Yuqin; Geacintov, Nicholas E.; Broyde, Suse

    2008-01-01

    Equilin and equilenin, components of the hormone replacement therapy drug Premarin, can be metabolized to the catechol 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dC, dA and dG to form unusual stable cyclic adducts, which have been found in human breast tumor tissue. Four stereoisomeric adducts have been identified for each base. These twelve Premarin-derived adducts provide a unique opportunity for analyzing effects of stereochemistry and base damage on DNA structure, and consequently its function. Our computational studies have shown that these adducts, with obstructed Watson-Crick hydrogen bond edges and near-perpendicular ring systems, have limited conformational flexibility, and near-mirror image conformations in stereoisomer pairs. The dC and dA adducts can adopt major and minor groove positions in the double helix, but the dG adducts are positioned only in the major groove. In all cases, opposite orientations of the equilenin rings with respect to the 5'→3' direction of the damaged strand are found in stereoisomer pairs derived from the same base, and no Watson-Crick pairing is possible. However, detailed structural properties in DNA duplexes are distinct for each stereoisomer of each damaged base. These differences may underlie observed differential stereoisomer and base-dependent mutagenicities and repair susceptibilities of these adducts. PMID:18416538

  20. A mathematical model for intracellular effects of toxins on DNA adduction and repair

    SciTech Connect

    Gaver, D.P.; Jacobs, P.A.; Carpenter, R.L.; Burkhart, J.G.

    1997-01-01

    The processes by which certain classes of toxic compounds or their metabolites may react with DNA to alter the genetic information contained in subsequent generations of cells or organisms are a major component of hazard associated with exposure to chemicals in the environment. Many classes of chemicals may form DNA adducts and there may or may not be a defined mechanism to remove a particular adduct from DNA independent of replication. Many compounds and metabolites that bind DNA also readily bind existing proteins; some classes of toxins and DNA adducts have the capacity to inactive a repair enzyme and divert the repair process competitively. This paper formulates an intracellular dynamic model for one aspect of the action of toxins that form DNA adducts, recognizing a capacity for removal of those adducts by a repair enzyme combined with reaction of the toxin and/or the DNA adduct to inactive the repair enzyme. This particular model illustrates the possible saturation of repair enzyme capacity by the toxin dosage and shows that bistable behavior can occur, with the potential to induce abrupt shifts away from steady-state equilibria. The model suggests that bistable behavior, dose and variation between individuals or tissues may combine under certain conditions to amplify the biological effect of dose observed as DNA adduction and its consequences as mutation. A model recognizing stochastic phenomena also indicates that variation in within-cell toxin concentration may promote jumps between stable equilibria.

  1. Polycyclic aromatic hydrocarbon-DNA adducts and the CYP1A1 restriction fragment length polymorphism

    SciTech Connect

    Shields, P.G.; Bowman, E.D.; Weston, A.; Harris, C.C.; Sugimura, H.; Caporaso, N.E.; Petruzzelli, S.F. ); Trump, B.F. )

    1992-11-01

    Human cancer risk assessment at a genetic level involves the investigation of carcinogen metabolism and DNA adduct formation. Wide interindividual differences in metabolism result in different DNA adduct levels. For this and other reasons, many laboratories have considered DNA adducts to be a measure of the biologically effective dose of a carcinogen. Techniques for studying DNA adducts using chemically specific assays are becoming available. A modification of the [sup 32]P-postlabeling assay for polycyclic aromatic hydrocarbon DNA adducts described here provides potential improvements in quantification. DNA adducts, however, reflect only recent exposure to carcinogens; in contrast, genetic testing for metabolic capacity indicates the extent to which carcinogens can be activated and exert genotoxic effects. Such studies may reflect both separate and integrated risk factors together with DNA adduct levels. A recently described restriction fragment length polymorphism for the CYP1A1, which codes for the cytochrome P450 enzyme primarily responsible for the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons, has been found to be associated with lung cancer risk in a Japanese population. In a subset of individuals enrolled in a US lung cancer case-control study, no association with lung cancer was found. 17 refs., 3 figs.

  2. 32P analysis of DNA adducts in tissues of benzene-treated rats.

    PubMed

    Reddy, M V; Blackburn, G R; Schreiner, C A; Mehlman, M A; Mackerer, C R

    1989-07-01

    Solid tumors have been reported in the Zymbal gland, oral and nasal cavities, liver, and mammary gland of Sprague-Dawley rats following chronic, high-dose administration of benzene. The carcinogenic activity of benzene is thought to be caused by activation to toxic metabolites that can interact with DNA, forming covalent adducts. A nuclease P1-enhanced 32P-postlabeling assay, having a sensitivity limit of 1 adduct in 10(9-10) DNA nucleotides, was found suitable for measuring aromatic DNA adducts derived in vitro from catechol, benzenetriol (BT), phenol, hydroquinone (HQ), and benzoquinone (BQ), potential metabolites of benzene. When DNA specimens isolated from tissues of female Sprague-Dawley rats at 24 hr after an oral gavage dose of 200 to 500 mg/kg, 5 days/week, in olive oil (3 mL/kg) for 1 day, 1 week, 5 weeks, and 10 weeks were analyzed by the 32P-postlabeling procedure, no aromatic adducts were detected unequivocally with DNA samples of liver, kidney, bone marrow, and mammary gland. With Zymbal gland DNA, three weak spots at levels totaling four lesions per 10(9) DNA nucleotides were seen only after 10 weeks of treatment, and these adducts did not correspond chromatographically to major adducts in vitro from the above specified compounds. Consequently, this finding requires confirmatory experiments. This distinct adduct pattern may relate to tumor induction in this organ following benzene administration. Our results also indicate that DNA adducts derived from catechol, BT, phenol, HQ, and BQ are either not formed in vivo with benzene or formed at levels below the detection limit of 1 adduct per 10(9-10) DNA nucleotides. PMID:2792046

  3. 32P analysis of DNA adducts in tissues of benzene-treated rats.

    PubMed Central

    Reddy, M V; Blackburn, G R; Schreiner, C A; Mehlman, M A; Mackerer, C R

    1989-01-01

    Solid tumors have been reported in the Zymbal gland, oral and nasal cavities, liver, and mammary gland of Sprague-Dawley rats following chronic, high-dose administration of benzene. The carcinogenic activity of benzene is thought to be caused by activation to toxic metabolites that can interact with DNA, forming covalent adducts. A nuclease P1-enhanced 32P-postlabeling assay, having a sensitivity limit of 1 adduct in 10(9-10) DNA nucleotides, was found suitable for measuring aromatic DNA adducts derived in vitro from catechol, benzenetriol (BT), phenol, hydroquinone (HQ), and benzoquinone (BQ), potential metabolites of benzene. When DNA specimens isolated from tissues of female Sprague-Dawley rats at 24 hr after an oral gavage dose of 200 to 500 mg/kg, 5 days/week, in olive oil (3 mL/kg) for 1 day, 1 week, 5 weeks, and 10 weeks were analyzed by the 32P-postlabeling procedure, no aromatic adducts were detected unequivocally with DNA samples of liver, kidney, bone marrow, and mammary gland. With Zymbal gland DNA, three weak spots at levels totaling four lesions per 10(9) DNA nucleotides were seen only after 10 weeks of treatment, and these adducts did not correspond chromatographically to major adducts in vitro from the above specified compounds. Consequently, this finding requires confirmatory experiments. This distinct adduct pattern may relate to tumor induction in this organ following benzene administration. Our results also indicate that DNA adducts derived from catechol, BT, phenol, HQ, and BQ are either not formed in vivo with benzene or formed at levels below the detection limit of 1 adduct per 10(9-10) DNA nucleotides. Images FIGURE 1. FIGURE 2. FIGURE 3. PMID:2792046

  4. Inhaled cigarette smoke induces the formation of DNA adducts in lungs of rats

    SciTech Connect

    Bond, J.A.; Chen, B.T.; Griffith, W.C.; Mauderly, J.L.

    1989-06-01

    Cigarette smoking causes a variety of adverse human health effects, including lung cancer. The molecular events associated with smoke-induced carcinogenesis are thought to be related in part to the genotoxic activities of the chemicals associated with smoke. The purpose of this investigation was to determine the molecular dosimetry of compounds in cigarette smoke in lungs of rats exposed by inhalation. These studies investigated the effects of exposure mode, sex, and time (adduct persistence) on the level of DNA adducts. Male and female F344/N rats were exposed 6 hr/day, 5 days/week for 22 days to cigarette smoke by nose-only intermittent (NOI), nose-only continuous (NOC), or whole-body continuous (WBC) exposures. Separate groups of rats were sham-exposed nose-only (NOS) or whole-body (WBS) to filtered air. All smoke exposure modes yielded daily smoke exposure concentration X time products of 600 mg particulate.hr/m3 for the first week and 1200 mg particulate.hour/m3 thereafter. Groups of rats were killed at 18 hr and 3 weeks after the 22-day exposure period and DNA adducts in lung tissues were quantified by the /sup 32/P-postlabeling method. There were significant (p less than 0.05) increases in levels of clearly resolved lung DNA adducts in male and female rats exposed to smoke compared to sham-exposed rats. There were no significant effects of exposure mode or sex on lung DNA adducts. Mean levels (+/- SE) of clearly resolved lung DNA adducts for both sexes combined in NOI, NOC, WBC, NOS, and WBS groups were 50 +/- 4, 52 +/- 6, 52 +/- 7, 21 +/- 6, and 22 +/- 4 adducts per 10(9) bases, respectively. Levels of clearly resolved DNA adducts were significantly less in lungs of rats killed 3 weeks after exposure and had declined to near control levels, suggesting that smoke-induced adducts are repaired by lung DNA repair enzymes.

  5. Polycyclic aromatic hydrocarbon-DNA adducts and survival among women with breast cancer

    SciTech Connect

    Sagiv, Sharon K. Gaudet, Mia M.; Eng, Sybil M.; Abrahamson, Page E.; Shantakumar, Sumitra; Teitelbaum, Susan L.; Bell, Paula; Thomas, Joyce A.; Neugut, Alfred I.; Santella, Regina M.; Gammon, Marilie D.

    2009-04-15

    Polycyclic aromatic hydrocarbons (PAH) are mammary carcinogens in animal studies, and a few epidemiologic studies have suggested a link between elevated levels of PAH-DNA adducts and breast cancer incidence. An association between PAH-DNA adducts and survival among breast cancer cases has not been previously reported. We conducted a survival analysis among women with newly diagnosed invasive breast cancer between 1996 and 1997, enrolled in the Long Island Breast Cancer Study Project. DNA was isolated from blood samples that were obtained from cases shortly after diagnosis and assayed for PAH-DNA adducts using ELISA. Among the 722 cases with PAH-DNA adduct measurements, 97 deaths (13.4%) from all causes and 54 deaths (7.5%) due to breast cancer were reported to National Death Index (NDI) by December 31, 2002. Using Cox proportional hazards models and controlling for age at diagnosis, we did not find evidence that all-cause mortality (hazard ratio (HR)=0.88; 95% confidence interval (CI): 0.57-1.37), or breast cancer mortality (HR=1.20; 95% CI: 0.63-2.28) was strongly associated with detectable PAH-DNA adduct levels compared with non-detectable adducts; additionally, no dose-response association was observed. Among a subgroup with treatment data (n=520), adducts were associated with over a two-fold higher mortality among those receiving radiation, but mortality for adducts was reduced among hormone therapy users. Results from this large population-based study do not provide strong support for an association between detectable PAH-DNA adducts and survival among women with breast cancer, except perhaps among those receiving radiation treatment.

  6. Determinants of 4-aminobiphenyl-DNA adducts in bladder cancer biopsies.

    PubMed

    Airoldi, Luisa; Orsi, Federica; Magagnotti, Cinzia; Coda, Renato; Randone, Donato; Casetta, Giovanni; Peluso, Marco; Hautefeuille, Agnes; Malaveille, Christian; Vineis, Paolo

    2002-05-01

    Exposure to 4-aminobiphenyl (4-ABP) is an important determinant of urinary bladder cancer in humans. We have analyzed by gas chromatography-mass spectrometry the DNA adducts of 4-ABP in 75 bladder cancer biopsies. The purpose was to understand whether smoking, N-acetyltransferase 2 (NAT2) polymorphism, diet or tumor grade were determinants of 4-ABP-DNA levels. 4-ABP-DNA adducts were above the detection limit of 0.1 fmol/microg DNA for 37/75 patients. Overall the level of adducts was 2.7 +/- 0.7 (mean +/- SE) fmol/microg DNA (86 +/- 22 adducts/10(8) normal nucleotides, mean +/- SE). A strong association with grade was observed. In the group of patients with detectable 4-ABP-DNA adducts the odds ratio for having a tumor grade of 2 or 3 was respectively 4.3 (95% CI 0.8-21.9) and 6 (1.3-27.5), compared with grade 1. A non-statistically significant association was found between adduct levels and the deduced slow acetylator phenotype in grades 2 and 3. The intake of fruit and vegetables produced a lower frequency of detectable adducts, though the association was not statistically significant. Detectable 4-ABP-DNA adducts were clearly associated with current smoking in higher tumor grades (grade 3 versus grades 1 + 2, odds ratios 10.4; 95% CI 1.7-63.1). Overall, our findings indicate that higher levels of DNA adducts characterize more invasive tumors (higher tumor grades). This seems to be facilitated by smoking and contrasted by the intake of fruit and vegetables. PMID:12016161

  7. 7-Alkylguanine adduct levels in urine, lungs and liver of mice exposed to styrene by inhalation

    SciTech Connect

    Vodicka, Pavel Erik . E-mail: pvodicka@biomed.cas.cz; Linhart, Igor; Novak, Jan; Koskinen, Mikko; Vodickova, Ludmila; Hemminki, Kari

    2006-01-15

    This study describes urinary excretion of two nucleobase adducts derived from styrene 7,8-oxide (SO), i.e., 7-(2-hydroxy-1-phenylethyl)guanine (N7{alpha}G) and 7-(2-hydroxy-2-phenylethyl)guanine (N7{beta}G), as well as a formation of N7-SO-guanine adducts in lungs and liver of two month old male NMRI mice exposed to styrene by inhalation in a 3-week subacute study. Strikingly higher excretion of both isomeric nucleobase adducts in the first day of exposure was recorded, while the daily excretion of nucleobase adducts in following time intervals reached the steady-state level at 4.32 + 1.14 and 6.91 + 1.17 pmol/animal for lower and higher styrene exposure, respectively. {beta}-SO-guanine DNA adducts in lungs increased with exposure in a linear way (F = 13.7 for linearity and 0.17 for non-linearity, respectively), reaching at the 21st day the level of 23.0 adducts/10{sup 8} normal nucleotides, i.e., 0.74 fmol/{mu}g DNA of 7-alkylguanine DNA adducts for the concentration of 1500 mg/m{sup 3}, while no 7-SO-guanine DNA adducts were detected in the liver after 21 days of inhalation exposure to both of styrene concentrations. A comparison of 7-alkylguanines excreted in urine with 7-SO-guanines in lungs (after correction for depurination and for missing {alpha}-isomers) revealed that persisting 7-SO-guanine DNA adducts in lungs account for about 0.5% of the total alkylation at N7 of guanine. The total styrene-specific 7-guanine alkylation accounts for about 1.0 x 10{sup -5}% of the total styrene uptake, while N1-adenine alkylation contributes to this percentage only negligibly.

  8. Urinary biomarkers suggest that estrogen-DNA adducts may play a role in the aetiology of non-Hodgkin lymphoma

    PubMed Central

    Gaikwad, Nilesh W.; Yang, Li; Weisenburger, Dennis D.; Vose, Julie; Beseler, Cheryl; Rogan, Eleanor G.; Cavalieri, Ercole L.

    2015-01-01

    A variety of evidence suggests that estrogens may induce non-Hodgkin lymphoma (NHL). The reaction of catechol estrogen quinones with DNA to form depurinating estrogen-DNA adducts is hypothesized to initiate this process. These adducts are released from DNA, shed from cells into the bloodstream and excreted in urine. The aim of this study was to determine whether or not the depurinating estrogen-DNA adducts might be involved in the aetiology of human NHL. Estrogen metabolites, conjugates and depurinating DNA adducts were identified and quantified in spot urine samples from 15 men with NHL and 30 healthy control men by using ultraperformance liquid chromatography/tandem mass spectrometry. The levels of estrogen-DNA adducts were significantly higher in the men with NHL than in the healthy control men. Thus, formation of estrogen-DNA adducts may play a critical role in the aetiology of NHL, and these adducts could be potential biomarkers of NHL risk. PMID:19863189

  9. Base-Displaced Intercalated Structure of the N-(2'-Deoxyguanosin-8-yl)-3-aminobenzanthrone DNA Adduct.

    PubMed

    Politica, Dustin A; Malik, Chanchal K; Basu, Ashis K; Stone, Michael P

    2015-12-21

    3-Nitrobenzanthrone (3-NBA), an environmental mutagen found in diesel exhaust and a suspected carcinogen, undergoes metabolic reduction followed by reaction with DNA to form aminobenzanthrone (ABA) adducts, with the major alkylation product being N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (C8-dG-ABA). Site-specific synthesis of the C8-dG-ABA adduct in the oligodeoxynucleotide 5'-d(GTGCXTGTTTGT)-3':5'-d(ACAAACACGCAC)-3'; X = C8-dG-ABA adduct, including codons 272-275 of the p53 gene, has allowed for investigation into the structural and thermodynamic properties of this adduct. The conformation of the C8-dG-ABA adduct was determined using NMR spectroscopy and was refined using molecular dynamics (MD) calculations restrained by experimentally determined interproton distance restraints obtained from NOE experiments. The refined structure revealed that the C8-dG-ABA adduct formed a base-displaced intercalated conformation. The adducted guanine was shifted into the syn conformation about the glycosidic bond. The 5'- and 3'-neighboring base pairs remained intact. While this facilitated π-stacking interactions between the ABA moiety and neighboring bases, the thermal melting temperature (Tm) of the adduct-containing duplex showed a decrease of 11 °C as compared to the corresponding unmodified oligodeoxynucleotide duplex. Overall, in this sequence, the base-displaced intercalated conformation of the C8-dG-ABA lesion bears similarity to structures of other arylamine C8-dG adducts. However, in this sequence, the base-displaced intercalated conformation for the C8-dG-ABA adduct differs from the conformation of the N(2)-dG-ABA adduct reported by de los Santos and co-workers, in which it is oriented in the minor groove toward the 5' end of the duplex, with the modified guanine remaining in the anti conformation about the glyosidic torsion angle, and the complementary base remaining within the duplex. The results are discussed in relationship to differences between the C8-d

  10. The role of polycyclic aromatic hydrocarbon-DNA adducts in inducing mutations in mouse skin

    PubMed Central

    Chakravarti, Dhrubajyoti; Venugopal, Divya; Mailander, Paula C.; Meza, Jane L.; Higginbotham, Sheila; Cavalieri, Ercole L.; Rogan, Eleanor G.

    2008-01-01

    Polycyclic aromatic hydrocarbons (PAH) form stable and depurinating DNA adducts in mouse skin to induce preneoplastic mutations. Some mutations transform cells, which then clonally expand to establish tumors. Strong clues about the mutagenic mechanism can be obtained if the PAH-DNA adducts can be correlated with both preneoplastic and tumor mutations. To this end, we studied mutagenesis in PAH-treated early preneoplastic skin (1 day after exposure) and in the induced papillomas in SENCAR mice. Papillomas were studied by PCR amplification of the H-ras gene and sequencing. For benzo[a]pyrene (BP), BP-7,8-dihydrodiol (BPDHD), 7,12-dimethylbenz[a]anthracene (DMBA) and dibenzo[a,l]pyrene (DB[a,l]P), the codon 13 (GGC to GTC) and codon 61 (CAA to CTA) mutations in papillomas corresponded to the relative levels of Gua and Ade-depurinating adducts, despite BP and BPDHD forming significant amounts of stable DNA adducts. Such a relationship was expected for DMBA and DB[a,l]P, as they formed primarily depurinating adducts. These results suggest that depurinating adducts play a major role in forming the tumorigenic mutations. To validate this correlation, preneoplastic skin mutations were studied by cloning H-ras PCR products and sequencing individual clones. DMBA- and DB[a,l]P-treated skin showed primarily A.T to G.C mutations, which correlated with the high ratio of the Ade/Gua-depurinating adducts. Incubation of skin DNA with T.G-DNA glycosylase eliminated most of these A.T to G.C mutations, indicating that they existed as G.T heteroduplexes, as would be expected if they were formed by errors in the repair of abasic sites generated by the depurinating adducts. BP and its metabolites induced mainly G.C to T.A mutations in preneoplastic skin. However, PCR over unrepaired anti-BPDE-N2dG adducts can generate similar mutations as artifacts of the study protocol, making it difficult to establish an adduct-mutation correlation for determining which BP-DNA adducts induce the early

  11. DNA Adduct Formation of 4-Aminobiphenyl and Heterocyclic Aromatic Amines in Human Hepatocytes

    PubMed Central

    Nauwelaers, Gwendoline; Bessette, Erin E.; Gu, Dan; Tang, Yijin; Rageul, Julie; Fessard, Valérie; Yuan, Jian-Min; Yu, Mimi C.; Langouët, Sophie; Turesky, Robert J.

    2011-01-01

    DNA adduct formation of the aromatic amine, 4-aminobiphenyl (4-ABP), a known human carcinogen present in tobacco smoke, and the heterocyclic aromatic amines (HAAs), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), potential human carcinogens, which are also present in tobacco smoke or formed during the high-temperature cooking of meats, was investigated in freshly cultured human hepatocytes. The carcinogens (10 μM) were incubated with hepatocytes derived from eight different donors for time periods up to 24 h. The DNA adducts were quantified by liquid chromatography-electrospray ionization mass spectrometry with a linear quadrupole ion trap mass spectrometer. The principal DNA adducts formed for all of the carcinogens were N-(deoxyguanosin-8-yl) (dG-C8) adducts. The levels of adducts ranged from 3.4 to 140 adducts per 107 DNA bases. The highest level of adduct formation occurred with AαC, followed by 4-ABP, then by PhIP, MeIQx, and IQ. Human hepatocytes formed dG-C8-HAA-adducts at levels that were up to 100-fold greater than the amounts of adducts produced in rat hepatocytes. In contrast to HAA adducts, the levels of dG-C8-4-ABP adduct formation were similar in human and rat hepatocytes. These DNA binding data demonstrate that the rat, an animal model that is used for carcinogenesis bioassays, significantly underestimates the potential hepatic genotoxicity of HAAs in humans. The high level of DNA adducts formed by AαC, a carcinogen produced in tobacco smoke at levels that are up to 100-fold higher than the amounts of 4-ABP, is noteworthy. The possible causal role of AαC in tobacco-associated cancers warrants investigation. PMID:21456541

  12. Preferential Formation of Benzo[a]pyrene Adducts at Lung Cancer Mutational Hotspots in P53

    NASA Astrophysics Data System (ADS)

    Denissenko, Mikhail F.; Pao, Annie; Tang, Moon-Shong; Pfeifer, Gerd P.

    1996-10-01

    Cigarette smoke carcinogens such as benzo[a]pyrene are implicated in the development of lung cancer. The distribution of benzo[a]pyrene diol epoxide (BPDE) adducts along exons of the P53 gene in BPDE-treated HeLa cells and bronchial epithelial cells was mapped at nucleotide resolution. Strong and selective adduct formation occurred at guanine positions in codons 157, 248, and 273. These same positions are the major mutational hotspots in human lung cancers. Thus, targeted adduct formation rather than phenotypic selection appears to shape the P53 mutational spectrum in lung cancer. These results provide a direct etiological link between a defined chemical carcinogen and human cancer.

  13. Adenine-DNA adducts derived from the highly tumorigenic dibenzo[a,l]pyrene are resistant to nucleotide excision repair while guanine adducts are not

    PubMed Central

    Kropachev, Konstantin; Kolbanovskiy, Marina; Liu, Zhi; Cai, Yuqin; Zhang, Lu; Schwaid, Adam G.; Kolbanovskiy, Alexander; Ding, Shuang; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

    2013-01-01

    The structural origins of differences in susceptibilities of various DNA lesions to nucleotide excision repair (NER) are poorly understood. Here we compared, in the same sequence context, the relative NER dual incision efficiencies elicited by two stereochemically distinct pairs of guanine (N2-dG) and adenine (N6-dA) DNA lesions, derived from enantiomeric genotoxic diol epoxides of the highly tumorigenic fjord region polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P). Remarkably, in cell-free HeLa cell extracts, the guanine adduct with R absolute chemistry at the N2-dG linkage site is ~ 35 times more susceptible to NER dual incisions than the stereochemically identical N6-dA adduct. For the guanine and adenine adducts with S stereochemistry, a similar, but somewhat smaller effect (factor of ~15) is observed. The striking resistance of the bulky N6-dA in contrast to the modest to good susceptibilities of the N2-dG adducts to NER are interpreted in terms of the balance between lesion-induced DNA-distorting and DNA-stabilizing van der Waals interactions in their structures, that are partly reflected in the overall thermal stabilities of the modified duplexes. Our results are consistent with the hypothesis that the high genotoxic activity of DB[a,l]P is related to the formation of NER-resistant and persistent DB[a,l]P-derived adenine adducts in cellular DNA. PMID:23570232

  14. Integrating S-phase Checkpoint Signaling with Trans-Lesion Synthesis of Bulky DNA Adducts

    PubMed Central

    Barkley, Laura R.; Ohmori, Haruo; Vaziri, Cyrus

    2011-01-01

    Bulky adducts are DNA lesions generated in response to environmental agents including benzo[a]pyrene (a combustion product) and solar ultraviolet radiation. Error-prone replication of adducted DNA can cause mutations, which may result in cancer. To minimize the detrimental effects of bulky adducts and other DNA lesions, S-phase checkpoint mechanisms sense DNA damage and integrate DNA repair with ongoing DNA replication. The essential protein kinase Chk1 mediates the S-phase checkpoint, inhibiting initiation of new DNA synthesis and promoting stabilization and recovery of stalled replication forks. Here we review the mechanisms by which Chk1 is activated in response to bulky adducts and potential mechanisms by which Chk1 signaling inhibits the initiation stage of DNA synthesis. Additionally, we discuss mechanisms by which Chk1 signaling facilitates bypass of bulky lesions by specialized Y-family DNA polymerases, thereby attenuating checkpoint signaling and allowing resumption of normal cell cycle progression. PMID:17652783

  15. 4-Aminobiphenyl-DNA adducts and p53 mutations in bladder cancer.

    PubMed

    Martone, T; Airoldi, L; Magagnotti, C; Coda, R; Randone, D; Malaveille, C; Avanzi, G; Merletti, F; Hautefeuille, A; Vineis, P

    1998-02-01

    Epidemiologic studies have suggested that smokers of air-cured tobacco (rich in arylamines) are at higher risk of bladder cancer than smokers of flue-cured tobacco. The risk has been shown to be modulated by the N-acetyltransferase genotype. We analyzed the biopsies of 45 patients with bladder cancer. p53 mutations were sought by direct sequencing, and 4-aminobiphenyl-DNA adducts were measured by negative ion gas chromatography-mass spectrometry. 4-Aminobiphenyl-DNA adducts were higher in smokers of air-cured tobacco and in current smokers, but no relationship with the number of cigarettes smoked was found. Adducts were higher in more advanced histologic grades of tumors. No pattern was evident for p53 mutations. Seven of 9 mutations occurred in grade 3 tumors. No association was found between 4-ABP adducts and GSTM1 or NAT2 genetic polymorphisms. PMID:9466649

  16. Metabolism of the Antibacterial Triclocarban by Human Epidermal Keratinocytes to Yield Protein Adducts

    PubMed Central

    Schebb, Nils Helge; Buchholz, Bruce A.; Hammock, Bruce D.; Rice, Robert H.

    2012-01-01

    Previous studies of triclocarban suggest that its biotransformation could yield reactive metabolites that form protein adducts. Since the skin is the major route of triclocarban exposure, present work examined this possibility in cultured human keratinocytes. The results provide evidence for considerable biotransformation and protein adduct formation when cytochrome P450 activity is induced in the cells by TCDD, a model Ah receptor ligand. Since detecting low adduct levels in cells and tissues is difficult, we utilized the novel approach of accelerator mass spectrometry for this purpose. Exploiting the sensitivity of the method, we demonstrated that a substantial portion of triclocarban forms adducts with keratinocyte protein under the P450 inducing conditions employed. PMID:22711420

  17. Methods for synthesizing alane without the formation of adducts and free of halides

    DOEpatents

    Zidan, Ragaiy; Knight, Douglas A; Dinh, Long V

    2013-02-19

    A process is provided to synthesize an alane without the formation of alane adducts as a precursor. The resulting product is a crystallized .alpha.-alane and is a highly stable product and is free of halides.

  18. Syndromes presenting adducted thumb with/without clubfoot and Dundar syndrome.

    PubMed

    Uzak, A Subasioglu; Fryns, J P; Dundar, M

    2014-01-01

    Congenital adducted thumb has been called variously as congenital clasped thumb, thumb in palm deformity or flexion adduction deformity of the thumb. This condition can be an isolated anomaly or associated with several genetic disorders. The syndromes that include adducted thumb as a cardinal feature such as Dundar Syndrome are few in the literature. This syndrome is an autosomal-recessive very rare disorder characterized by typical facial appearance with dysmorphic features that includes wasted build, hyperextensible, thin and translucent skin with atrophic scarring, severe congenital contractures of fingers and thumbs, club feet, severe kyphoscoliosis, joint instability, muscular hypotonia, and ocular involvement. Heart, kidney, and/or intestinal defects can also be observed. Up to date the syndrome is described in few families in the literature. Here we discuss the syndromes that include adducted thumb as a cardinal feature and also the differential diagnosis of the Dundar Syndrome according to the literature. PMID:25059014

  19. Fast carry accumulator design

    NASA Technical Reports Server (NTRS)

    Mastin, W. C.

    1971-01-01

    Simple iterative accumulator combined with gated-carry, carry-completion detection, and skip-carry circuits produces three accumulators with decreased carry propagation times. Devices are used in machine control, measurement equipment, and computer applications to increase speed of binary addition. NAND gates are used in combining network.

  20. DNA adducts in human carcinogenesis: etiological relevance and structure-activity relationship.

    PubMed

    Bartsch, H

    1996-06-01

    Sensitive methods for quantifying DNA adducts from (i) benzo[a]pyrene (BP), (ii) alkylation exposure, and (iii) etheno(epsilon)-DNA adduct-forming chemicals were developed and applied to humans and animal models. The aims were to identify hitherto unknown sources and mechanisms of exogenous and endogenous DNA damage, to examine the effect of drug polymorphism on BP adduct levels, and to develop QSAR between tumorigenic potency, heritable genetic damage and structural elements of alkylating carcinogens (Vogel and Nivard (1994) Mutation Res., 395, 13-32). (i) BP-DNA adducts: An HPLC/fluorimetry assay suitable for measuring (+)-anti-BP-diol-epoxide (BPDE) adducts in human tissues and white blood cells (WBC) was developed (Alexandrov et al. (1992) Cancer Res., 52, 6248-6253). In smokers, a positive correlation was found between pulmonary CYP1A1-related catalytic activity (AHH) and the level of lung BPDE-DNA adducts. In coke oven workers, an enhancing effect of smoking on BPDE-adduct levels in WBC was demonstrated (Rojas et al. (1995) Carcinogenesis, 16, 1373-1376). (ii) 3-Alkyladenines (3-alkAde): Alkylating carcinogens form 3-alkAde adducts in DNA which depurinate to yield 3-alkAde in urine, for which a detection method was developed (Friesen et al. (1991) Chem. Res. Toxicol., 4, 102-106; Prevost et al. (1990) Carcinogenesis, 11, 1747-1751), using immunoaffinity purification and GC-MS analysis. The usefulness of 3-alkAde analysis for the determination of the whole-body dose of alkylating agents derived from exogenous and endogenous sources was demonstrated. (iii) Etheno-DNA adduct-forming agents: Etheno(epsilon)-DNA base adducts (epsilon A, epsilon dC, epsilon dG) are promutagenic DNA lesions that are formed by occupational (vinyl halides) and environmental (urethane) carcinogens. An ultrasensitive detection method was developed (Nair et al. (1995) Carcinogenesis, 16, 613-617), based on immunoaffinity purification and 32P-postlabelling of epsilon-nucleoside 3

  1. Translesion synthesis past acrolein-derived DNA adducts by human mitochondrial DNA polymerase γ.

    PubMed

    Kasiviswanathan, Rajesh; Minko, Irina G; Lloyd, R Stephen; Copeland, William C

    2013-05-17

    Acrolein, a mutagenic aldehyde, is produced endogenously by lipid peroxidation and exogenously by combustion of organic materials, including tobacco products. Acrolein reacts with DNA bases forming exocyclic DNA adducts, such as γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (γ-HOPdG) and γ-hydroxy-1,N(6)-propano-2'-deoxyadenosine (γ-HOPdA). The bulky γ-HOPdG adduct blocks DNA synthesis by replicative polymerases but can be bypassed by translesion synthesis polymerases in the nucleus. Although acrolein-induced adducts are likely to be formed and persist in mitochondrial DNA, animal cell mitochondria lack specialized translesion DNA synthesis polymerases to tolerate these lesions. Thus, it is important to understand how pol γ, the sole mitochondrial DNA polymerase in human cells, acts on acrolein-adducted DNA. To address this question, we investigated the ability of pol γ to bypass the minor groove γ-HOPdG and major groove γ-HOPdA adducts using single nucleotide incorporation and primer extension analyses. The efficiency of pol γ-catalyzed bypass of γ-HOPdG was low, and surprisingly, pol γ preferred to incorporate purine nucleotides opposite the adduct. Pol γ also exhibited ∼2-fold lower rates of excision of the misincorporated purine nucleotides opposite γ-HOPdG compared with the corresponding nucleotides opposite dG. Extension of primers from the termini opposite γ-HOPdG was accomplished only following error-prone purine nucleotide incorporation. However, pol γ preferentially incorporated dT opposite the γ-HOPdA adduct and efficiently extended primers from the correctly paired terminus, indicating that γ-HOPdA is probably nonmutagenic. In summary, our data suggest that acrolein-induced exocyclic DNA lesions can be bypassed by mitochondrial DNA polymerase but, in the case of the minor groove γ-HOPdG adduct, at the cost of unprecedented high mutation rates. PMID:23543747

  2. In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies.

    PubMed

    Iwamoto, Taka-aki; Kobayashi, Nobuhiko; Imoto, Kyoko; Yamamoto, Aya; Nakamura, Yu; Yamauchi, Yukika; Okumura, Hiromi; Tanaka, Akiko; Hanaoka, Fumio; Shibutani, Shinya; Miyagawa, Sachiko; Mori, Toshio

    2004-11-01

    The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells. PMID:15380103

  3. Immunocytochemical analysis of cisplatin-induced platinum-DNA adducts with double-fluorescence video microscopy.

    PubMed Central

    Meijer, C.; de Vries, E. G.; Dam, W. A.; Wilkinson, M. H.; Hollema, H.; Hoekstra, H. J.; Mulder, N. H.

    1997-01-01

    To detect low-level DNA platination, a sensitive immunocyto- and histochemical technique was developed using a polyclonal antibody. The antibody GPt, derived after immunization of rabbits with highly platinated DNA and purified with affinity chromatography, detected the main platinum (Pt)-containing intrastrand and interstrand adducts. Double-fluorescence microscopy image analysis was used to quantify Pt-DNA adducts with Hoechst 33258 fluorescence to locate the nuclei and with fluorescein isothiocyanate fluorescence to measure the immunosignal. A two- to five-fold dose-dependent difference in the level of cisplatin (CDDP)-induced Pt-DNA adducts between a CDDP-sensitive and -resistant human tumour cell line was detected. Large differences in Pt-DNA adduct levels after in vitro CDDP incubation between human buccal cells, lymphocytes and biopsies of different tumour types were observed. Pt-DNA adduct levels were fivefold higher in human testicular tumours than in colon tumours, representing CDDP-sensitive and -resistant tumours, respectively, in the clinic. These data suggest the possibility of predictive testing by measuring Pt-DNA adduct levels. Pt-DNA adducts in patients after treatment with CDDP were shown in normal buccal cells and in imprints of fresh tumour biopsies as well as in paraffin-embedded tumour cells. The analysis of Pt-DNA adducts at a single-cell level in small samples of normal and tumour cells during and/or after treatment is feasible with GPt and will hopefully enable more selective treatment of patients. Images Figure 1 Figure 2 Figure 4 Figure 5 PMID:9252194

  4. Formation, Accumulation, and Hydrolysis of Endogenous and Exogenous Formaldehyde-Induced DNA Damage.

    PubMed

    Yu, Rui; Lai, Yongquan; Hartwell, Hadley J; Moeller, Benjamin C; Doyle-Eisele, Melanie; Kracko, Dean; Bodnar, Wanda M; Starr, Thomas B; Swenberg, James A

    2015-07-01

    Formaldehyde is not only a widely used chemical with well-known carcinogenicity but is also a normal metabolite of living cells. It thus poses unique challenges for understanding risks associated with exposure. N(2-)hydroxymethyl-dG (N(2)-HOMe-dG) is the main formaldehyde-induced DNA mono-adduct, which together with DNA-protein crosslinks (DPCs) and toxicity-induced cell proliferation, play important roles in a mutagenic mode of action for cancer. In this study, N(2)-HOMe-dG was shown to be an excellent biomarker for direct adduction of formaldehyde to DNA and the hydrolysis of DPCs. The use of inhaled [(13)CD2]-formaldehyde exposures of rats and primates coupled with ultrasensitive nano ultra performance liquid chromatography-tandem mass spectrometry permitted accurate determinations of endogenous and exogenous formaldehyde DNA damage. The results show that inhaled formaldehyde only reached rat and monkey noses, but not tissues distant to the site of initial contact. The amounts of exogenous adducts were remarkably lower than those of endogenous adducts in exposed nasal epithelium. Moreover, exogenous adducts accumulated in rat nasal epithelium over the 28-days exposure to reach steady-state concentrations, followed by elimination with a half-life (t1/2) of 7.1 days. Additionally, we examined artifact formation during DNA preparation to ensure the accuracy of nonlabeled N(2)-HOMe-dG measurements. These novel findings provide critical new data for understanding major issues identified by the National Research Council Review of the 2010 Environmental Protection Agency's Draft Integrated Risk Information System Formaldehyde Risk Assessment. They support a data-driven need for reflection on whether risks have been overestimated for inhaled formaldehyde, whereas underappreciating endogenous formaldehyde as the primary source of exposure that results in bone marrow toxicity and leukemia in susceptible humans and rodents deficient in DNA repair. PMID:25904104

  5. Identification of Rosmarinic Acid-Adducted Sites in Meat Proteins in a Gel Model under Oxidative Stress by Triple TOF MS/MS.

    PubMed

    Tang, Chang-Bo; Zhang, Wan-Gang; Wang, Yao-Song; Xing, Lu-Juan; Xu, Xing-Lian; Zhou, Guang-Hong

    2016-08-24

    Triple TOF MS/MS was used to identify adducts between rosmarinic acid (RosA)-derived quinones and meat proteins in a gel model under oxidative stress. Seventy-five RosA-modified peptides responded to 67 proteins with adduction of RosA. RosA conjugated with different amino acids in proteins, and His, Arg, and Lys adducts with RosA were identified for the first time in meat. A total of 8 peptides containing Cys, 14 peptides containing His, 48 peptides containing Arg, 64 peptides containing Lys, and 5 peptides containing N-termini that which participated in adduction reaction with RosA were identified, respectively. Seventy-seven adduction sites were subdivided into all adducted proteins including 2 N-terminal adduction sites, 3 Cys adduction sites, 4 His adduction sites, 29 Arg adduction sites, and 39 Lys adduction sites. Site occupancy analyses showed that approximately 80.597% of the proteins carried a single RosA-modified site, 14.925% retained two sites, 1.492% contained three sites, and the rest 2.985% had four or more sites. Large-scale triple TOF MS/MS mapping of RosA-adducted sites reveals the adduction regulations of quinone and different amino acids as well as the adduction ratios, which clarify phenol-protein adductions and pave the way for industrial meat processing and preservation. PMID:27486909

  6. Correlation of mutagenic potencies of various petroleum oils and oil coal tar mixtures with DNA adduct levels in vitro.

    PubMed

    Reddy, M V; Blackburn, G R; Schreiner, C A; Mackerer, C R

    1997-08-01

    An in vitro system was utilized to measure DNA adduct-forming ability of petroleum oils and oil coal tar mixtures to define correlations between DNA adduct levels and their mutagenic potencies. The system consisted of reaction of dimethyl sulfoxide extracts of oils with calf thymus DNA in the presence of Aroclor-induced hamster liver microsomes for 30 min. Following DNA extraction, DNA adducts were measured by the nuclease P1-enhanced postlabeling assay coupled with two-dimensional polyethyleneimine (PEI)-cellulose TLC. Thin layer plates showed putative aromatic DNA adducts, with levels ranging from 60 to 1400 adducts per 10(9) DNA nucleotides. TLC mobilities suggested adducts to be aromatic compounds containing 4 or more rings. A good correlation (coefficient of correlation = 0.91) was observed between DNA adduct levels and Salmonella mutagenicity for 19 oils. All 19 samples tested produced DNA adducts. To expedite the TLC procedure, adducts were resolved by one-dimensional TLC and the radioactivity measured using a mechanical scanner. Results were comparable to those obtained by two-dimensional TLC and quantification after scraping. Our data show that the in vitro incubation system coupled with the postlabeling adduct assay is a useful screening method to identify mutagenic and potentially carcinogenic oils. PMID:9288888

  7. The use of an artificial nucleotide for polymerase-based recognition of carcinogenic O6-alkylguanine DNA adducts.

    PubMed

    Wyss, Laura A; Nilforoushan, Arman; Williams, David M; Marx, Andreas; Sturla, Shana J

    2016-08-19

    Enzymatic approaches for locating alkylation adducts at single-base resolution in DNA could enable new technologies for understanding carcinogenesis and supporting personalized chemotherapy. Artificial nucleotides that specifically pair with alkylated bases offer a possible strategy for recognition and amplification of adducted DNA, and adduct-templated incorporation of an artificial nucleotide has been demonstrated for a model DNA adduct O(6)-benzylguanine by a DNA polymerase. In this study, DNA adducts of biological relevance, O(6)-methylguanine (O(6)-MeG) and O(6)-carboxymethylguanine (O(6)-CMG), were characterized to be effective templates for the incorporation of benzimidazole-derived 2'-deoxynucleoside-5'-O-triphosphates ( BENZI: TP and BIM: TP) by an engineered KlenTaq DNA polymerase. The enzyme catalyzed specific incorporation of the artificial nucleotide BENZI: opposite adducts, with up to 150-fold higher catalytic efficiency for O(6)-MeG over guanine in the template. Furthermore, addition of artificial nucleotide BENZI: was required for full-length DNA synthesis during bypass of O(6)-CMG. Selective incorporation of the artificial nucleotide opposite an O(6)-alkylguanine DNA adduct was verified using a novel 2',3'-dideoxy derivative of BENZI: TP. The strategy was used to recognize adducts in the presence of excess unmodified DNA. The specific processing of BENZI: TP opposite biologically relevant O(6)-alkylguanine adducts is characterized herein as a basis for potential future DNA adduct sequencing technologies. PMID:27378785

  8. Human serum albumin-benzo[a]pyrene anti-diol epoxide adduct structure elucidation by fluorescence line narrowing spectroscopy.

    PubMed

    Day, B W; Doxtader, M M; Rich, R H; Skipper, P L; Singh, K; Dasari, R R; Tannenbaum, S R

    1992-01-01

    Cryogenic (4-10 K) laser-induced vibrationless ground state and vibronic excited state fluorescence emission spectra of the adducts resulting from reaction in vitro of human serum albumin and the carcinogen (+-)-r-7,t-8-dihydroxy-c-9,c-10-epoxy-7,8,9,10- tetrahydrobenzo[a]-pyrene were recorded in order to determine the structures formed. Comparison of these fluorescence line-narrowed (FLN) spectra to those obtained from BaP-7,8,9,10- tetrahydrotetrols, synthetic N-t-BOC-alaninate ester, and N tau- and N pi-histidine amine anti-BaPDE adducts revealed that a mixture of adduct types are formed with the protein. Extensive dialysis of the adducted protein simplified the FLN spectrum, causing it to become nearly identical to the FLN spectrum obtained from the stable peptide adduct. Comparison of the FLN spectra of the synthetic histidine adducts to those obtained from peptide adducts isolated from enzymic digestion of the adducted protein indicated that only one of the imidazole nitrogens is the nucleophile which forms a stable adduct with anti-BaPDE. The FLN studies confirm that N tau-histidine adducts are formed between human serum albumin and the C-10 position of anti-BaPDE. PMID:1581540

  9. Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts

    DOE PAGESBeta

    Hang, Bo

    2010-01-01

    DNA adducts play a central role in chemical carcinogenesis. The analysis of formation and repair of smoking-related DNA adducts remains particularly challenging as both smokers and nonsmokers exposed to smoke are repetitively under attack from complex mixtures of carcinogens such as polycyclic aromatic hydrocarbons and N -nitrosamines. The bulky DNA adducts, which usually have complex structure, are particularly important because of their biological relevance. Several known cellular DNA repair pathways have been known to operate in human cells on specific types of bulky DNA adducts, for example, nucleotide excision repair, base excision repair, and direct reversal involving O 6more » -alkylguanine DNA alkyltransferase or AlkB homologs. Understanding the mechanisms of adduct formation and repair processes is critical for the assessment of cancer risk resulting from exposure to cigarette smoke, and ultimately for developing strategies of cancer prevention. This paper highlights the recent progress made in the areas concerning formation and repair of bulky DNA adducts in the context of tobacco carcinogen-associated genotoxic and carcinogenic effects.« less

  10. Abacavir forms novel cross-linking abacavir protein adducts in patients.

    PubMed

    Meng, Xiaoli; Lawrenson, Alexandre S; Berry, Neil G; Maggs, James L; French, Neil S; Back, David J; Khoo, Saye H; Naisbitt, Dean J; Park, B Kevin

    2014-04-21

    Abacavir (ABC), a nucleoside-analogue reverse transcriptase inhibitor, is associated with severe hypersensitivity reactions that are thought to involve the activation of CD8+ T cells in a HLA-B*57:01-restricted manner. Recent studies have claimed that noncovalent interactions of ABC with HLA-B*57:01 are responsible for the immunological reactions associated with ABC. However, the formation of hemoglobin-ABC aldehyde (ABCA) adducts in patients exposed to ABC suggests that protein conjugation might represent a pathway for antigen formation. To further characterize protein conjugation reactions, we used mass spectrometric methods to define ABCA modifications in patients receiving ABC therapy. ABCA formed a novel intramolecular cross-linking adduct on human serum albumin (HSA) in patients and in vitro via Michael addition, followed by nucleophilic adduction of the aldehyde with a neighboring protein nucleophile. Adducts were detected on Lys159, Lys190, His146, and Cys34 residues in the subdomain IB of HSA. Only a cysteine adduct and a putative cross-linking adduct were detected on glutathione S-transferase Pi (GSTP). These findings reveal that ABC forms novel types of antigens in all patients taking the drug. It is therefore vital that the immunological consequences of such pathways of haptenation are explored in the in vitro models that have been used by various groups to define new mechanisms of drug hypersensitivity exemplified by ABC. PMID:24571427

  11. Capturing Labile Sulfenamide and Sulfinamide Serum Albumin Adducts of Carcinogenic Arylamines by Chemical Oxidation

    PubMed Central

    Peng, Lijuan; Turesky, Robert J.

    2013-01-01

    Aromatic amines and heterocyclic aromatic amines (HAAs) are a class of structurally related carcinogens that are formed during the combustion of tobacco or during the high temperature cooking of meats. These procarcinogens undergo metabolic activation by N-oxidation of the exocyclic amine group to produce N-hydroxylated metabolites, which are critical intermediates implicated in toxicity and DNA damage. The arylhydroxylamines and their oxidized arylnitroso derivatives can also react with cysteine (Cys) residues of glutathione or proteins to form, respectively, sulfenamide and sulfinamide adducts. However, sulfur-nitrogen linked adducted proteins are often difficult to detect because they are unstable and undergo hydrolysis during proteolytic digestion. Synthetic N-oxidized intermediates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a carcinogenic HAA produced in cooked meats, and 4-aminobiphenyl, a carcinogenic aromatic amine present in tobacco smoke were reacted with human serum albumin (SA) and formed labile sulfenamide or sulfinamide adducts at the Cys34 residue. Oxidation of the carcinogen-modified SA with m-chloroperoxybenzoic acid (m-CPBA) produced the arylsulfonamide adducts, which were stable to heat and the chemical reduction conditions employed to denature SA. The sulfonamide adducts of PhIP and 4-ABP were identified, by liquid chromatography/mass spectrometry, in proteolytic digests of denatured SA. Thus, selective oxidation of arylamine-modified SA produces stable arylsulfonamide-SA adducts, which may serve as biomarkers of these tobacco and dietary carcinogens. PMID:23240913

  12. Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts

    PubMed Central

    Hang, Bo

    2010-01-01

    DNA adducts play a central role in chemical carcinogenesis. The analysis of formation and repair of smoking-related DNA adducts remains particularly challenging as both smokers and nonsmokers exposed to smoke are repetitively under attack from complex mixtures of carcinogens such as polycyclic aromatic hydrocarbons and N-nitrosamines. The bulky DNA adducts, which usually have complex structure, are particularly important because of their biological relevance. Several known cellular DNA repair pathways have been known to operate in human cells on specific types of bulky DNA adducts, for example, nucleotide excision repair, base excision repair, and direct reversal involving O6-alkylguanine DNA alkyltransferase or AlkB homologs. Understanding the mechanisms of adduct formation and repair processes is critical for the assessment of cancer risk resulting from exposure to cigarette smoke, and ultimately for developing strategies of cancer prevention. This paper highlights the recent progress made in the areas concerning formation and repair of bulky DNA adducts in the context of tobacco carcinogen-associated genotoxic and carcinogenic effects. PMID:21234336

  13. Malondialdehyde–Deoxyguanosine Adducts among Workers of a Thai Industrial Estate and Nearby Residents

    PubMed Central

    Peluso, Marco; Srivatanakul, Petcharin; Munnia, Armelle; Jedpiyawongse, Adisorn; Ceppi, Marcello; Sangrajrang, Suleeporn; Piro, Sara; Boffetta, Paolo

    2010-01-01

    Background Humans living near industrial point emissions can experience high levels of exposures to air pollutants. Map Ta Phut Industrial Estate in Thailand is the location of the largest steel, oil refinery, and petrochemical factory complexes in Southeast Asia. Air pollution is an important source of oxidative stress and reactive oxygen species, which interact with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively. Objective We measured the levels of malondialdehyde–deoxyguanosine (dG) adducts, a biomarker of oxidative stress and lipid peroxidation, in petrochemical workers, nearby residents, and subjects living in a control district without proximity to industrial sources. Design We conducted a cross-sectional study to compare the prevalence of malondialdehyde-dG adducts in groups of subjects experiencing various degrees of air pollution. Results The multivariate regression analysis shows that the adduct levels were associated with occupational and environmental exposures to air pollution. The highest adduct level was observed in the steel factory workers. In addition, the formation of DNA damage tended to be associated with tobacco smoking, but without reaching statistical significance. A nonsignificant increase in DNA adducts was observed after 4–6 years of employment among the petrochemical complexes. Conclusions Air pollution emitted from the Map Ta Phut Industrial Estate complexes was associated with increased adduct levels in petrochemical workers and nearby residents. Considering the mutagenic potential of DNA lesions in the carcinogenic process, we recommend measures aimed at reducing the levels of air pollution. PMID:20056580

  14. Cigarette smoke-induced DNA adducts in the respiratory and nonrespiratory tissues of rats

    SciTech Connect

    Gairola, C.G.; Gupta, R.C. )

    1991-01-01

    Formation of DNA adducts is regarded as an essential initial step in the process of chemical carcinogenesis. To determine how chronic exposure to cigarette smoke affects the distribution of DNA adducts in selected respiratory and nonrespiratory tissues. The authors exposed male Sprague-Dawley rats daily to fresh mainstream smoke from the Univ. of Kentucky reference cigarettes (2R1) in a nose-only exposure system for 32 weeks. Blood carboxyhemoglobin, total particulate matter (TPM) intake, and pulmonary aryl hydrocarbon hydroxylase values indicated effective exposure of animals to cigarette smoke. DNA was extracted from three respiratory (larynx, trachea, and lung) and three nonrespiratory (liver, heart, and bladder) tissues and analyzed for DNA adducts by the {sup 32}P-postlabeling assay under conditions capable of detecting low levels of diverse aromatic/hydrophobic adducts. Data showed that the total DNA adducts in the lung, heart, and trachea, and larynx were increased by 10- to 20-fold in the smoke-exposed group. These data suggest selective formation of DNA adducts in the tissues.

  15. Assay of Protein and Peptide Adducts of Cholesterol Ozonolysis Products by Hydrophobic and Click Enrichment Methods

    PubMed Central

    2015-01-01

    Cholesterol undergoes ozonolysis to afford a variety of oxysterol products, including cholesterol-5,6-epoxide (CholEp) and the isomeric aldehydes secosterol A (seco A) and secosterol B (seco B). These oxysterols display numerous important biological activities, including protein adduction; however, much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols. Here, we synthesized alkynyl derivatives of cholesterol-derived oxysterols and employed a straightforward detection method to establish secosterols A and B as the most protein-reactive of the oxysterols tested. Model adduction studies with an amino acid, peptides, and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction. Hydrophobic separation methods—Folch extraction and solid phase extraction (SPE)—were successfully applied to enrich oxysterol-adducted peptide species, and LC-MS/MS analysis of a model peptide–seco adduct revealed a unique fragmentation pattern (neutral loss of 390 Da) for that species. Coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol-modified peptides and proteins in an adducted protein. More broadly, these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases, including atherosclerosis, Alzheimer’s disease, Parkinson’s disease, and asthma. PMID:25185119

  16. Benzo(a)pyrene-albumin adducts in humans exposed to polycyclic aromatic hydrocarbons in an industrial area of Poland.

    PubMed Central

    Kure, E H; Andreassen, A; Ovrebø, S; Grzybowska, E; Fiala, Z; Strózyk, M; Chorazy, M; Haugen, A

    1997-01-01

    OBJECTIVES: The interaction of benzo(a)pyrene with serum albumin was measured in an attempt to identify the actual exposure and to evaluate albumin adduct measurements as biomarkers for exposure monitoring. METHODS: Benzo(a)pyrene-diol-epoxide (BPDE)-albumin adducts were measured by competitive enzyme linked immunosorbent assay (ELISA) in plasma of coke oven plant workers from three plants and from people living in a highly industrialised area of Silesia in Poland. Due to the high air concentrations of polycyclic aromatic hydrocarbons (PAHs) in this area, a control group was selected from a rural non-industrialised area in Poland. Breathing zone air measurements of PAHs were collected from some of the participants. RESULTS: Coke oven plant workers and non-occupationally exposed people had similar concentrations of albumin adducts whereas the rural controls were significantly lower (2.74 fmol adducts/microgram albumin (SEM 0.124)). The mean concentration of BPDE-albumin adduct in plasma of both the occupational and the environmental groups were significantly higher in the summer samples (4.34 fmol adducts/microgram albumin (SEM 0.335) and 4.55 fmol adducts/microgram albumin (SEM 0.296), respectively) than in the winter samples (3.06 fmol adducts/microgram albumin (SEM 0.187) and 3.04 fmol adducts/microgram albumin (SEM 0.184), respectively) even though the air measurements showed higher concentrations of PAHs in the winter. The statistical analysis did not show any effects of air exposures on concentrations of BPDE-albumin adduct. CONCLUSIONS: A multiple regression analysis of the measured concentrations of BPDE-albumin adducts for all the groups, during both seasons, indicates that occupational exposures do not contribute significantly to the formation of adducts. In general, the concentrations of albumin adducts found vary within relatively small limits for the two seasons and between the various groups of participants. No extreme differences were found. PMID

  17. Novel pyrano and vinylphenol adducts of deoxyanthocyanidins in sorghum sourdough.

    PubMed

    Bai, Yunpeng; Findlay, Brandon; Maldonado, Alma Fernanda Sanchez; Schieber, Andreas; Vederas, John C; Gänzle, Michael G

    2014-11-26

    This study determined the fate of deoxyanthocyanidins in sorghum sourdoughs. Sourdoughs prepared from the red sorghum variety Town were fermented with the caffeic acid-decarboxylating strains Lactobacillus plantarum FUA3171 and the decarboxylase negative L. casei FUA3166. Deoxyanthocyanidins were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Apigeninidin and methoxyapigeninidin were the major deoxyanthocyanidins prior to fermentation. During fermentation, novel deoxyanthocyanidins were formed. Purification by preparative LC, followed by NMR analysis and high-resolution MS identified two of the compounds as 6-deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin. To identify pathways for their formation, sorghum was fermented with single strains, L. plantarum or L. casei. 6-Deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin were formed only during fermentation with L. plantarum FUA3171, indicating a role of vinylphenol in their formation. Chemical synthesis confirmed that 6-deoxyanthocyanidin-vinylphenol and pyrano-3-deoxyanthocyanidin are formed from apigeninidin with vinylphenol but not with p-coumaric acid as reactants. In conclusion, the products of microbial decarboxylation of hydroxycinnamic acids convert apigeninidin and methoxyapigeninidin to pyrano-3-deoxyanthocyanidins and vinylphenol adducts. PMID:25370078

  18. Investigating the Role of Adducts in Protein Supercharging with Sulfolane

    NASA Astrophysics Data System (ADS)

    Douglass, Kevin Aart; Venter, Andre R.

    2012-03-01

    The supercharging effect of sulfolane on cytochrome c (cyt c) during electrospray ionization mass spectrometry (ESI-MS) in the absence of conformational effects was investigated. The addition of sulfolane on the order of 1 mM or greater to denaturing solutions of cyt c results in supercharging independent of protein concentration over the range of 0.1 to 10 μM. While supercharging was observed in the positive mode, no change in the charge state distribution was observed in the negative mode, ruling out polarity-independent factors such as conformational changes or surface tension effects. A series of sulfolane adducts observed with increasing intensity concurrent with increasing charge state suggests that a direct interaction between sulfolane and the charged sites of cyt c plays an important role in supercharging. We propose that charge delocalization occurring through large-scale dipole reordering of the highly polar supercharging reagent reduces the electrostatic barrier for proximal charging along the cyt c amino acid chain. Supporting this claim, supercharging was shown to increase with increasing dipole moment for several supercharging reagents structurally related to sulfolane.

  19. Cellulose based hybrid hydroxylated adducts for polyurethane foams

    NASA Astrophysics Data System (ADS)

    De Pisapia, Laura; Verdolotti, Letizia; Di Mauro, Eduardo; Di Maio, Ernesto; Lavorgna, Marino; Iannace, Salvatore

    2012-07-01

    Hybrid flexible polyurethane foams (HPU) were synthesized by using a hybrid hydroxilated adduct (HHA) based on renewable resources. In particular the HHA was obtained by dispersing cellulose wastes in colloidal silica at room temperature, pressure and humidity. The colloidal silica was selected for its ability of modifying the cellulose structure, by inducing a certain "destructurization" of the crystalline phase, in order to allow cellulose to react with di-isocyanate for the final synthesis of the polyurethane foam. In fact, cellulose-polysilicate complexes are engaged in the reaction with the isocyanate groups. This study provides evidence of the effects of the colloidal silica on the cellulose structure, namely, a reduction of the microfiber cellulose diameter and the formation of hydrogen bonds between the polysilicate functional groups and the hydroxyl groups of the cellulose, as assessed by IR spectroscopy and solid state NMR. The HHA was added to a conventional polyol in different percentages (between 5 and 20%) to synthesize HPU in presence of catalysts, silicone surfactant and diphenylmethane diisocyanate (MDI). The mixture was expanded in a mold and cured for two hours at room temperature. Thermal analysis, optical microscopy and mechanical tests were performed on the foams. The results highlighted an improvement of thermal stability and a decrease of the cell size with respect neat polyurethane foam. Mechanical tests showed an improvement of the elastic modulus and of the damping properties with increasing HHA amount.

  20. C18 thin-layer chromatographic enhancement of the 32P-postlabeling assay for aromatic or bulky carcinogen-DNA adducts: evaluation of adduct recoveries in comparison with nuclease P1 and butanol methods.

    PubMed

    Reddy, M V

    1993-05-01

    The suitability of C18 reversed-phase thin-layer chromatography (TLC) for enrichment of adducts in the 32P-postlabeling assay was investigated for structurally diverse classes of DNA adducts derived from benzo[a]pyrene, 2-acetylaminofluorene, benzoquinone, safrole, and mitomycin C. The TLC enrichment involved retention of adducts to the C18 phase followed by elution with organic solvent-water. Adduct patterns obtained by the C18 purification were qualitatively similar to those obtained by the nuclease P1 and butanol procedures, the two commonly used enrichment methods. Adduct recoveries by the C18 method varied for different adducts and were significantly lower than those obtained by the other two techniques. PMID:8314936

  1. Determining efficacy of cancer chemopreventive agents using a cell-free system concomitant with DNA adduction.

    PubMed

    Smith, W A; Gupta, R C

    1999-03-10

    The large (>2000) and expanding number of natural and synthetic agents with potential cancer chemopreventive properties renders it economically and physically impossible to test each of these agents for their efficacy in the widely accepted 2-year animal bioassay and clinical trials. Therefore, there is a growing need for relevant short-term screening tests to study these compounds such that only the most efficacious ones undergo extensive long-term studies. We have previously reported in a pilot study that the use of a microsome-mediated test system concomitant with DNA adduction is a pertinent and relevant model for rapidly studying the efficacy and mechanisms of cancer chemopreventive agents. We have extended this study to investigate 26 additional agents for their potential chemopreventive abilities by studying their effects on microsome-mediated benzo[a]pyrene (BP)-DNA adduction. These agents had differential effects on the two major adducts of BP-DNA, i.e., BP-7,8-diol-9,10-epoxide (BPDE)-deoxyguanosine (dG) and 9-OH-BP-dG-derived adducts. These agents were therefore categorized into five classes. Three test agents (ellagic acid, genistein and oltipraz) were strong inhibitors of both adducts. These agents diminished BP-DNA adduction by 65-95% and were categorized as Class I agents. Six other agents (benzyl isocyanate, R(+)-1-phenylethyl isocyanate, linoleic acid ethyl ester, (+)-biotin, indole-3-carboxylic acid and beta-carotene) moderately inhibited both BP-DNA adducts (25-64%); these compounds were identified as Class II agents. Six additional test agents inhibited only one adduct selectively and nine others were ineffective; these agents were categorized as Class III and Class IV, respectively. Interestingly, seven test agents enhanced BPDE-dG or 9-OH-BP-dG or both adducts and were categorized as Class V agents. Four of these Class V agents concomitantly inhibited BPDE-dG while enhancing 9-OH-BP-dG. This emphasizes the importance of studying individual DNA

  2. Simultaneous Detection of Multiple DNA Adducts in Human Lung Samples by Isotope-Dilution UPLC-MS/MS

    PubMed Central

    2015-01-01

    Recent studies have demonstrated that various DNA adducts can be detected in human tissues and fluids using liquid chromatography connected to tandem mass spectrometry (LC-MS/MS). However, the utility of a single DNA adduct as a biomarker in risk assessment is debatable because humans are exposed to many genotoxicants. We established a method to measure DNA adducts derived from 16 ubiquitous genotoxicants and developed an analytical technique for their simultaneous quantification by ultra performance liquid chromatography (UPLC)-MS/MS. Methods for the enrichment of the analytes from DNA hydrolysates and chromatographic separation preceding mass spectrometric analysis were optimized, and the resultant technique was used for the simultaneous analysis of the 16 DNA adducts in human lung biopsy specimens. Eleven adducts (formed by benzo[a]pyrene, 1-methylpyrene, 4-aminobiphenyl, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 1-methoxy-3-indolylmethylglucosinolate, 5-hydroxymethylfurfural, and malondialdehyde) were not detected in any tissue sample (limits of detection: 0.02–7.1 adducts/108 nucleosides). 3,N4-etheno-2′-deoxycytidine and 1,N6-etheno-2′-deoxyadenosine, formed from 2,3-epoxyaldehydes of endogenous lipid peroxidation products, were present in all subjects (16.9–115.3 and 27.2–179/108 nucleosides, respectively). The same was true for N2-(trans-methylisoeugenol-3′-yl)-2′-deoxyguanosine, the major adduct of methyleugenol (1.7–23.7/108 nucleosides). A minor adduct of methyleugenol and two adducts of furfuryl alcohol were detected in several pulmonary specimens. Taken together, we developed a targeted approach for the simultaneous mass spectrometric analyses of 16 DNA adducts, which can be easily extended by adducts formed from other mutagens. The method allowed one to detect adducts of furfuryl alcohol and methyleugenol in samples of human lung. PMID:25423194

  3. Simultaneous detection of multiple DNA adducts in human lung samples by isotope-dilution UPLC-MS/MS.

    PubMed

    Monien, Bernhard H; Schumacher, Fabian; Herrmann, Kristin; Glatt, Hansruedi; Turesky, Robert J; Chesné, Christophe

    2015-01-01

    Recent studies have demonstrated that various DNA adducts can be detected in human tissues and fluids using liquid chromatography connected to tandem mass spectrometry (LC-MS/MS). However, the utility of a single DNA adduct as a biomarker in risk assessment is debatable because humans are exposed to many genotoxicants. We established a method to measure DNA adducts derived from 16 ubiquitous genotoxicants and developed an analytical technique for their simultaneous quantification by ultra performance liquid chromatography (UPLC)-MS/MS. Methods for the enrichment of the analytes from DNA hydrolysates and chromatographic separation preceding mass spectrometric analysis were optimized, and the resultant technique was used for the simultaneous analysis of the 16 DNA adducts in human lung biopsy specimens. Eleven adducts (formed by benzo[a]pyrene, 1-methylpyrene, 4-aminobiphenyl, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 1-methoxy-3-indolylmethylglucosinolate, 5-hydroxymethylfurfural, and malondialdehyde) were not detected in any tissue sample (limits of detection: 0.02-7.1 adducts/10(8) nucleosides). 3,N(4)-etheno-2'-deoxycytidine and 1,N(6)-etheno-2'-deoxyadenosine, formed from 2,3-epoxyaldehydes of endogenous lipid peroxidation products, were present in all subjects (16.9-115.3 and 27.2-179/10(8) nucleosides, respectively). The same was true for N(2)-(trans-methylisoeugenol-3'-yl)-2'-deoxyguanosine, the major adduct of methyleugenol (1.7-23.7/10(8) nucleosides). A minor adduct of methyleugenol and two adducts of furfuryl alcohol were detected in several pulmonary specimens. Taken together, we developed a targeted approach for the simultaneous mass spectrometric analyses of 16 DNA adducts, which can be easily extended by adducts formed from other mutagens. The method allowed one to detect adducts of furfuryl alcohol and methyleugenol in samples of human lung. PMID:25423194

  4. The Frequency of 1,4-Benzoquinone-Lysine Adducts in Cytochrome c Correlate with Defects in Apoptosome Activation

    PubMed Central

    Fisher, Ashley A.; Labenski, Matthew T.; Chapman, John D.; Bratton, Shawn B.; Monks, Terrence J.; Lau, Serrine S.

    2011-01-01

    Electrophile-mediated post-translational modifications (PTMs) are known to cause tissue toxicities and disease progression. These effects are mediated via site-specific modifications and structural disruptions associated with such modifications. 1,4-Benzoquinone (BQ) and its quinone-thioether metabolites are electrophiles that elicit their toxicity via protein arylation and the generation of reactive oxygen species. Site-specific BQ-lysine adducts are found on residues in cytochrome c that are necessary for protein-protein interactions, and these adducts contribute to interferences in its ability to facilitate apoptosome formation. To further characterize the structural and functional impact of these BQ-mediated PTMs, the original mixture of BQ-adducted cytochrome c was fractionated by liquid isoelectric focusing to provide various fractions of BQ-adducted cytochrome c species devoid of the native protein. The fractionation process separates samples based on their isoelectric point (pI), and because BQ adducts form predominantly on lysine residues, increased numbers of BQ adducts on cytochrome c correlate with a lower protein pI. Each fraction was analyzed for structural changes, and each was also assayed for the ability to support apoptosome-mediated activation of caspase-3. Circular dichroism revealed that several of the BQ-adducted cytochrome c species maintained a slightly more rigid structure in comparison to native cytochrome c. BQ-adducted cytochrome c also failed to activate caspase-3, with increasing numbers of BQ-lysine adducts corresponding to a greater inability to activate the apoptosome. In summary, the specific site of the BQ-lysine adducts, and the nature of the adduct, are important determinants of the subsequent structural changes to cytochrome c. In particular, adducts at sites necessary for protein-protein interactions interfere with the proapoptotic function of cytochrome c. PMID:21527774

  5. Oligomerization and Membrane-binding Properties of Covalent Adducts Formed by the Interaction of α-Synuclein with the Toxic Dopamine Metabolite 3,4-Dihydroxyphenylacetaldehyde (DOPAL).

    PubMed

    Follmer, Cristian; Coelho-Cerqueira, Eduardo; Yatabe-Franco, Danilo Y; Araujo, Gabriel D T; Pinheiro, Anderson S; Domont, Gilberto B; Eliezer, David

    2015-11-13

    Oxidative deamination of dopamine produces the highly toxic aldehyde 3,4-dihydroxyphenylacetaldehyde (DOPAL), enhanced production of which is found in post-mortem brains of Parkinson disease patients. When injected into the substantia nigra of rat brains, DOPAL causes the loss of dopaminergic neurons accompanied by the accumulation of potentially toxic oligomers of the presynaptic protein α-synuclein (aS), potentially explaining the synergistic toxicity described for dopamine metabolism and aS aggregation. In this work, we demonstrate that DOPAL interacts with aS via formation of Schiff-base and Michael-addition adducts with Lys residues, in addition to causing oxidation of Met residues to Met-sulfoxide. DOPAL modification leads to the formation of small aS oligomers that may be cross-linked by DOPAL. Both monomeric and oligomeric DOPAL adducts potently inhibit the formation of mature amyloid fibrils by unmodified aS. The binding of aS to either lipid vesicles or detergent micelles, which results in a gain of α-helix structure in its N-terminal lipid-binding domain, protects the protein against DOPAL adduct formation and, consequently, inhibits DOPAL-induced aS oligomerization. Functionally, aS-DOPAL monomer exhibits a reduced affinity for small unilamellar vesicles with lipid composition similar to synaptic vesicles, in addition to diminished membrane-induced α-helical content in comparison with the unmodified protein. These results suggest that DOPAL could compromise the functionality of aS, even in the absence of protein oligomerization, by affecting the interaction of aS with lipid membranes and hence its role in the regulation of synaptic vesicle traffic in neurons. PMID:26381411

  6. Silver adducts of four-branched histidine rich peptides exhibit synergistic antifungal activity.

    PubMed

    Leng, Qixin; Woodle, Martin C; Liu, Yijia; Mixson, A James

    2016-09-01

    Previously, a four branched histidine-lysine rich peptide, H3K4b, was shown to demonstrate selective antifungal activity with minimal antibacterial activity. Due to the potential breakdown from proteases, H3K4b was further evaluated in the current study by varying the D- and l-amino acid content in its branches. Whereas analogues of H3K4b that selectively replaced l-amino acids (H3k4b, h3K4b) had improved antifungal activity, the all d-amino acid analogue, h3k4b, had reduced activity, suggesting that partial breakdown of the peptide may be necessary. Moreover, because histidines form coordination bonds with the silver ion, we examined whether silver adducts can be formed with these branched histidine-lysine peptides, which may improve antifungal activity. For Candida albicans, the silver adduct of h3K4b or H3k4b reduced the MIC compared to peptide and silver ions alone by 4- and 5-fold, respectively. For Aspergillus fumigatus, the silver adducts showed even greater enhancement of activity. Although the silver adducts of H3k4b or h3K4b showed synergistic activity, the silver adduct with the all l-amino acid H3K4b surprisingly showed the greatest synergistic and growth inhibition of A. fumigatus: the silver adduct of H3K4b reduced the MIC compared to the peptide and silver ions alone by 30- and 26-fold, respectively. Consistent with these antifungal efficacy results, marked increases in free oxygen radicals were produced with the H3K4b and silver combination. These studies suggest that there is a balance between stability and breakdown for optimal antifungal activity of the peptide alone and for the peptide-silver adduct. PMID:27387239

  7. Transplatin-conjugated triplex-forming oligonucleotides form adducts with both strands of DNA.

    PubMed

    Campbell, Meghan A; Miller, Paul S

    2009-12-01

    Triplex-forming oligonucleotides (TFOs) can bind to polypurine x polypyrimidine tracts in DNA and, as a consequence, perturb the normal functioning of a targeted gene. The effectiveness of such antigene TFOs can potentially be enhanced by covalent attachment of the TFO to its DNA target. Here, we report that attachment of N-7-platinated guanine nucleosides to the 3'- and/or 5'-ends of oligopyrimidine TFOs enables these TFOs to form highly stable adducts with target DNA deoxyguanosines or deoxyadenosines that are adjacent to the TFO binding site. Such adduct formation stably anchors the TFO to its target. Depending on the sequences adjacent to the TFO binding site, adduct formation can occur on either strand of the DNA. Adduct formation by 3',5'-bis-platinated TFOs can result in the formation of an interstrand cross-link between both strands of the DNA duplex. Formation of the adducts, which could be reversed by treatment with sodium cyanide, was dependent upon the ability of the TFO to bind to DNA and appeared to occur at a rate slower than that at which the TFO bound to the DNA duplex. The extent of adduct formation at 37 degrees C by platinated deoxyribo-TFOs diminished as the pH was increased from 6.5 to 7.4. In contrast, high levels (approximately 86%) of adduct formation by platinated 2'-O-methylribo-TFOs were observed at both pH 6.5 and pH 7.4. Platinated 2'-O-methylribo-TFOs were also shown to bind to plasmid DNA and inhibit transcription in vitro, and to inhibit plasmid replication in E. coli cells. These results suggest that platinum-conjugated TFOs may be good candidates for use as antigene agents. PMID:19950917

  8. Neutrophils amplify the formation of DNA adducts by benzo[a]pyrene in lung target cells.

    PubMed

    Borm, P J; Knaapen, A M; Schins, R P; Godschalk, R W; Schooten, F J

    1997-09-01

    Inflammatory cells and their reactive oxygen metabolites can cause mutagenic effects in lung cells. The purpose of this study was to investigate the ability of activated neutrophils to modulate DNA binding of benzo[a]pyrene (B[a]P), a known carcinogen, in lung target cells. Equivalent numbers of rat lung epithelial cells (RLE-6TN cell line) and freshly isolated human blood neutrophils (PMN) were coincubated in vitro for 2 hr after addition of benzo[a]pyrene (0.5 microM) or two of its trans-diol metabolites, with or without stimulation with phorbol myristate acetate (PMA). DNA adducts of B[a]P-metabolites were determined in target cells using 32P-postlabeling; oxidative DNA damage (7-hydro-8-oxo-2'-deoxyguanosine [8-oxodG]) was evaluated by high performance liquid chromatography with electrochemical detection. Increased DNA adducts were observed in lung cells coincubated with polymorphonuclear leukocytes (PMN). Activation of PMN with PMA, or addition of more activated PMN in relation to the number of lung cells, further increased the number of adducts, the latter in a dose-response manner. Incubation with B[a]P-4,5-diol did not result in any adduct formation, while B[a]P-7,8-diol led to a significant number of adducts. Moreover, PMA-activated PMN strongly enhanced adduct formation by B[a]P-7,8-diol, but not 8-oxodG, in lung cells. The addition of antioxidants to the coincubations significantly reduced the number of adducts. Results suggest that an inflammatory response in the lung may increase the biologically effective dose of polycyclic aromatic hydrocarbons (PAHs), and may be relevant to data interpretation and risk assessment of PAH-containing particulates. PMID:9400705

  9. Synthesis of an oligodeoxyribonucleotide adduct of mitomycin C by the postoligomerization method via a triamino mitosene.

    PubMed

    Champeil, Elise; Paz, Manuel M; Ladwa, Sweta; Clement, Cristina C; Zatorski, Andrzej; Tomasz, Maria

    2008-07-23

    The cancer chemotherapeutic agent mitomycin C (MC) alkylates and cross-links DNA monofunctionally and bifunctionally in vivo and in vitro, forming six major MC-deoxyguanosine adducts of known structures. The synthesis of one of the monoadducts (8) by the postoligomerization method was accomplished both on the nucleoside and oligonucleotide levels, the latter resulting in the site-specific placement of 8 in a 12-mer oligodeoxyribonucleotide 26. This is the first application of this method to the synthesis of a DNA adduct of a complex natural product. Preparation of the requisite selectively protected triaminomitosenes 14 and 24 commenced with removal of the 10-carbamoyl group from MC, followed by reductive conversion to 10-decarbamoyl-2,7-diaminomitosene 10. This substance was transformed to 14 or 24 in several steps. Both were successfully coupled to the 2-fluoro-O(6)-(2-trimethylsilylethyl)deoxyinosine residue of the 12-mer oligonucleotide. The N(2)-phenylacetyl protecting group of 14 after its coupling to the 12-mer oligonucleotide could not be removed by penicillinamidase as expected. Nevertheless, the Teoc protecting group of 24 after coupling to the 12-mer oligonucleotide was removed by treatment with ZnBr2 to give the adducted oligonucleotide 26. However, phenylacetyl group removal was successful on the nucleoside-level synthesis of adduct 8. Proof of the structure of the synthetic nucleoside adduct included HPLC coelution and identical spectral properties with a natural sample, and (1)H NMR. Structure proof of the adducted oligonucleotide 26 was provided by enzymatic digestion to nucleosides and authentic adduct 8, as well as MS and MS/MS analysis. PMID:18588303

  10. A new model for multiply charged adduct formation between peptides and anions in electrospray mass spectrometry.

    PubMed

    Liu, Xiaohua; Cole, Richard B

    2011-12-01

    A new model has been developed to account for adduct formation on multiply charged peptides observed in negative ion electrospray mass spectrometry. To obtain a stable adduct, the model necessitates an approximate matching of apparent gas-phase basicity (GB(app)) of a given proton bearing site on the peptide with the gas-phase basicity (GB) of the anion attaching at that site. Evidence supporting the model is derived from the fact that for [Glu] Fibrinopeptide B, higher GB anions dominated in adducts observed at higher negative charge states, whereas lower GB anions appeared predominately in lower charge state adducts. Singly charged adducts were only observed for lower GB anions: HSO(4)(-), I(-), CF(3)COO(-). Ions that have medium GBs (NO(3) (-), Br(-), H(2)PO(4)(-)) only form adducts having -2 charge states, whereas Cl(-) (higher GB) can form adducts having -3 charge states. The model portends that (1) carboxylate groups are much more basic than available amino groups; (2) apparent GBs of the various carboxylate groups on peptides do not vary substantially from one another; and (3) apparent GBs of the individual carboxylate and amino sites do not behave independently. This model was developed for negative ion attachment but an analogous mechanism is also proposed for the positive ion mode wherein (1) binding of a neutral at an amino site polarizes this amino group, but hardly affects apparent GBs of other sites; (2) proton addition (charge state augmentation) at one site can decrease the instrinsic GBs of other potential protonation sites and lower their apparent GBs. PMID:21997579

  11. A New Model for Multiply Charged Adduct Formation Between Peptides and Anions in Electrospray Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Liu, Xiaohua; Cole, Richard B.

    2011-12-01

    A new model has been developed to account for adduct formation on multiply charged peptides observed in negative ion electrospray mass spectrometry. To obtain a stable adduct, the model necessitates an approximate matching of apparent gas-phase basicity (GBapp) of a given proton bearing site on the peptide with the gas-phase basicity (GB) of the anion attaching at that site. Evidence supporting the model is derived from the fact that for [Glu] Fibrinopeptide B, higher GB anions dominated in adducts observed at higher negative charge states, whereas lower GB anions appeared predominately in lower charge state adducts. Singly charged adducts were only observed for lower GB anions: HSO{4/-}, I-, CF3COO-. Ions that have medium GBs (NO{3/-}, Br-, H2PO{4/-}) only form adducts having -2 charge states, whereas Cl- (higher GB) can form adducts having -3 charge states. The model portends that (1) carboxylate groups are much more basic than available amino groups; (2) apparent GBs of the various carboxylate groups on peptides do not vary substantially from one another; and (3) apparent GBs of the individual carboxylate and amino sites do not behave independently. This model was developed for negative ion attachment but an analogous mechanism is also proposed for the positive ion mode wherein (1) binding of a neutral at an amino site polarizes this amino group, but hardly affects apparent GBs of other sites; (2) proton addition (charge state augmentation) at one site can decrease the instrinsic GBs of other potential protonation sites and lower their apparent GBs.

  12. The analysis of DNA adducts: the transition from (32)P-postlabeling to mass spectrometry.

    PubMed

    Klaene, Joshua J; Sharma, Vaneet K; Glick, James; Vouros, Paul

    2013-06-28

    The technique of (32)P-postlabeling, which was introduced in 1982 for the analysis of DNA adducts, has long been the method of choice for in vivo studies because of its high sensitivity as it requires only <10μg DNA to achieve the detection of 1 adduct in 10(10) normal bases. (32)P-postlabeling has therefore been utilized in numerous human and animal studies of DNA adduct formation. Like all techniques (32)P-postlabeling does have several disadvantages including the use of radioactive phosphorus, lack of internal standards, and perhaps most significantly does not provide any structural information for positive identification of unknown adducts, a shortcoming that could significantly hamper progress in the field. Structural methods have since been developed to allow for positive identification of DNA adducts, but to this day, the same level of sensitivity and low sample requirements provided by (32)P-postlabeling have not been matched. In this mini review we will discuss the (32)P-postlabeling method and chronicle the transition to mass spectrometry via the hyphenation of gas chromatography, capillary electrophoresis, and ultimately liquid chromatography which, some 30years later, is only just starting to approach the sensitivity and low sample requirements of (32)P-postlabeling. This paper focuses on the detection of bulky carcinogen-DNA adducts, with no mention of oxidative damage or small alkylating agents. This is because the (32)P-postlabeling assay is most compatible with bulky DNA adducts. This will also allow a more comprehensive focus on a subject that has been our particular interest since 1990. PMID:22960573

  13. Formation of metal-ion adducts and evidence for surface-catalyzed ionization in electrospray analysis of pharmaceuticals and pesticides

    USGS Publications Warehouse

    Thurman, E.M.; Ferrer, I.

    2002-01-01

    The formation of metal ion adducts in liquid chromatography/mass spectrometry positive-ion electrospray analysis of pharmaceuticals and pesticides was investigated. The evidence of surface-catalyzed ionization in the electrospray analysis was also studied. Both positive and negative ion mass spectrometry were used for the analysis of the products. It was found that the sodium adducts formed in the analysis included single, double, and triple sodium adducts. Adduction was found to occur by attachment of the metal ion to carboxyl, carbonyl and aromatic pi electrons of the molecule.

  14. Formation of 1,4-dioxo-2-butene-derived adducts of 2'-deoxyadenosine and 2'-deoxycytidine in oxidized DNA.

    PubMed

    Chen, Bingzi; Vu, Choua C; Byrns, Michael C; Dedon, Peter C; Peterson, Lisa A

    2006-08-01

    Oxidation of deoxyribose in DNA produces a variety of electrophilic residues that are capable of reacting with nucleobases to form adducts such as M(1)dG, the pyrimidopurinone adduct of dG. We now report that deoxyribose oxidation in DNA leads to the formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA. We previously demonstrated that these adducts arise in reactions of nucleosides and DNA with trans-1,4-dioxo-2-butene, the beta-elimination product of the 2-phosphoryl-1,4-dioxobutane residue arising from 5'-oxidation of deoxyribose in DNA, and with cis-1,4-dioxo-2-butene, a metabolite of furan. Treatment of DNA with enediyne antibiotics capable of oxidizing the 5'-position of deoxyribose (calicheamicin and neocarzinostatin) led to a concentration-dependent formation of oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA, while the antibiotic bleomycin, which is capable of performing only 4-oxidation of deoxyribose, did not give rise to the adducts. The nonspecific DNA oxidant, gamma-radiation, also produced the adducts that represented approximately 0.1% of the 2-phosphoryl-1,4-dioxobutane residues formed during the irradiation. These results suggest that the oxadiazabicyclo(3.3.0)octaimine adducts of dC and dA could represent endogenous DNA lesions arising from oxidative stresses that also give rise to other DNA adducts. PMID:16918236

  15. Metabolism and hemoglobin adduct formation of acrylamide in humans.

    PubMed

    Fennell, Timothy R; Sumner, Susan C J; Snyder, Rodney W; Burgess, Jason; Spicer, Rebecca; Bridson, William E; Friedman, Marvin A

    2005-05-01

    Acrylamide (AM), used in the manufacture of polyacrylamide and grouting agents, is produced during the cooking of foods. Workplace exposure to AM can occur through the dermal and inhalation routes. The objectives of this study were to evaluate the metabolism of AM in humans following oral administration, to compare hemoglobin adduct formation on oral and dermal administration, and to measure hormone levels. The health of the people exposed under controlled conditions was continually monitored. Prior to conducting exposures in humans, a low-dose study was conducted in rats administered 3 mg/kg (1,2,3-13C3) AM by gavage. The study protocol was reviewed and approved by Institute Review Boards both at RTI, which performed the sample analysis, and the clinical research center conducting the study. (1,2,3-13C3) AM was administered in an aqueous solution orally (single dose of 0.5, 1.0, or 3.0 mg/kg) or dermally (three daily doses of 3.0 mg/kg) to sterile male volunteers. Urine samples (3 mg/kg oral dose) were analyzed for AM metabolites using 13C NMR spectroscopy. Approximately 86% of the urinary metabolites were derived from GSH conjugation and excreted as N-acetyl-S-(3-amino-3-oxopropyl)cysteine and its S-oxide. Glycidamide, glyceramide, and low levels of N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine were detected in urine. On oral administration, a linear dose response was observed for N-(2-carbamoylethyl)valine (AAVal) and N-(2-carbamoyl-2-hydroxyethyl)valine (GAVal) in hemoglobin. Dermal administration resulted in lower levels of AAVal and GAVal. This study indicated that humans metabolize AM via glycidamide to a lesser extent than rodents, and dermal uptake was approximately 6.6% of that observed with oral uptake. PMID:15625188

  16. Theoretical characterization of dihydrogen adducts with halide anions

    NASA Astrophysics Data System (ADS)

    Vitillo, Jenny G.; Damin, Alessandro; Zecchina, Adriano; Ricchiardi, Gabriele

    2006-06-01

    The interaction between a hydrogen molecule and the halide anions F-, Cl-, Br-, and I- has been studied at different levels of theory and with different basis sets. The most stable configurations of the complexes have a linear geometry, while the t-shaped complexes are saddle points on the potential energy surface, opposite to what is observed for alkali cations. An electrostatic analysis conducted on the resulting adducts has highlighted the predominance of the electrostatic term in the complexation energy and, in particular, of the quadrupole- and dipole-polarizability dependent contributions. Another striking difference with respect to the positive ions, is the fact that although the binding energies have similar values (ranging between 25 and 3kJ/mol for F- and I-, respectively), the vibrational shift of the ν˜H-H and in general the perturbation of the hydrogen molecule in complexes are much greater in the complexes with anions (Δν˜H-H ranges between -720 and -65cm-1). Another difference with respect to the interaction with cations is a larger charge transfer from the anion to the hydrogen molecule. The Δν˜ is the result of the cooperative role of the electrostatics and of the charge transfer in the interaction. The correlation between binding energies and vibrational shift is far from linear, contrary to what is observed for cation complexes, in accordance with the higher polarizability and dynamic polarizability of the molecule along the molecular axis. The observed correlation may be valuable in the interpretation of spectra and thermodynamic properties of adsorbed H2 in storage materials.

  17. Black tea polyphenols modulate xenobiotic-metabolizing enzymes, oxidative stress and adduct formation in a rat hepatocarcinogenesis model.

    PubMed

    Murugan, Ramalingam Senthil; Uchida, Koji; Hara, Yukihiko; Nagini, Siddavaram

    2008-10-01

    The present study was designed to investigate the modulatory effects of black tea polyphenols (Polyphenon-B) on phase I and phase II xenobiotic-metabolizing enzymes and oxidative stress in a rat model of hepatocellular carcinoma (HCC). Liver tumours induced in male Sprague-Dawley rats by dietary administration of rho-dimethylaminoazobenzene (DAB) increased cytochrome P450 (total and CYP1A1, 1A2 and 2B isoforms), cytochrome b(5), cytochrome b(5) reductase, glutathione S-transferase (GST total and GST-P isoform) and gamma-glutamyltranspeptidase (GGT) with decrease in quinone reductase (QR). This was accompanied by enhanced lipid and protein oxidation and compromised antioxidant defences associated with increased expression of the oxidative stress markers 4-hydroxynonenal (4-HNE), anti-hexanoyl lysine (HEL), dibromotyrosine (DiBrY) and 8-hydroxy 2-deoxyguanosine (8-OHdG). Dietary administration of Polyphenon-B effectively suppressed DAB-induced hepatocarcinogenesis, as evidenced by reduced preneoplastic and neoplastic lesions, modulation of xenobiotic-metabolizing enzymes and amelioration of oxidative stress. Thus, it can be concluded that Polyphenon-B acts as an effective chemopreventive agent by modulating xenobiotic-metabolizing enzymes and mitigating oxidative stress in an in vivo model of hepatocarcinogenesis. PMID:18985486

  18. Factors that influence the mutagenic patterns of DNA adducts from chemical carcinogens.

    PubMed

    Seo, K Y; Jelinsky, S A; Loechler, E L

    2000-10-01

    Carcinogens are generally mutagens, which is understandable given that tumor cells grow uncontrollably because they have mutations in critical genes involved in growth control. Carcinogens often induce a complex pattern of mutations (e.g., GC-->TA, GC-->AT, etc.). These mutations are thought to be initiated when a DNA polymerase encounters a carcinogen-DNA adduct during replication. In principle, mutational complexity could be due to either a collection of different adducts each inducing a single kind of mutation (Hypothesis 1a), or a single adduct inducing different kinds of mutations (Hypothesis 1b). Examples of each are discussed. Regarding Hypothesis 1b, structural factors (e.g., DNA sequence context) and biological factors (e.g., differing DNA polymerases) that can affect the pattern of adduct mutagenesis are discussed. This raises the question: how do structural and biological factors influence the pattern of adduct mutagenesis. For structural factors, three possibilities are considered: (Hypothesis 2a) a single conformation of an adduct giving rise to multiple mutations -- dNTP insertion by DNA polymerase being influenced by (e.g.) the surrounding DNA sequence context; (Hypothesis 2b) a variation on this ("dislocation mutagenesis"); or (Hypothesis 2c) a single adduct adopting multiple conformations, each capable of giving a different pattern of mutations. Hypotheses 2a, 2b and 2c can each in principle rationalize many mutational results, including how the pattern of adduct mutagenesis might be influenced by factors, such as DNA sequence context. Five lines of evidence are discussed suggesting that Hypothesis 2c can be correct for base substitution mutagenesis. For example, previous work from our laboratory was interpreted to indicate that [+ta]-B[a]P-N(2)-dG in a 5'-CGG sequence context (G115) could be trapped in a conformation giving predominantly G-->T mutations, but heating caused the adduct to equilibrate to its thermodynamic mixture of conformations

  19. Protein adducts as dosimeters of human exposure to styrene, styrene-7,8-oxide, and benzene.

    PubMed

    Rappaport, S M; Yeowell-O'Connell, K

    1999-09-01

    Cysteinyl adducts of hemoglobin (Hb) and albumin (Alb) formed via reactions with reactive species were measured in 48 subjects exposed to styrene (0.24-55.2 ppm) and to styrene-7,8-oxide (SO) (2.65-107 ppb) in a factory producing boats in the USA. Hb and Alb adducts were also investigated among 88 workers exposed to benzene (0-138 ppm) in several Chinese factories. The particular adducts were S-(2-hydroxy-1-phenylethyl) cysteine, from reactions of SO with Alb (designated SO-Alb), and S-phenylcysteine, from reactions of the CYP450 benzene metabolite, benzene oxide (BO), with Hb and Alb (designated BO-Hb and BO-Alb, respectively). The relationships between adduct levels and exposures were investigated in both studies. The estimated slopes varied considerably among the particular combinations of adduct and agent to which the workers were exposed, ranging from 0.815 pmol BO-Hb/g Hb per ppm benzene to 24400 pmol SO-Alb/g Alb per ppm SO. We used these estimated slopes, along with kinetic constants, to predict the systemic doses of SO and BO in humans per mg of styrene, SO or benzene per kg body weight, under certain assumptions. Using RX to signify the particular electrophile (SO or BO) the doses of RX to the blood per unit of dose varied between 2.21 and 4110 nM RX-h/mg agent per kg b.w. The dose of RX to the blood arising from inhalation of SO was almost 2000 times that of styrene (i.e. 4110 vs. 2.21 nM RX/mg agent per kg b.w.) and 430-781 times that of benzene (i.e. 4110 vs. 5.26-9.55 nM RX/mg agent per kg b.w.), depending upon the study. Comparable estimates of the blood dose of BO were obtained from adducts of Hb and Alb and two independent studies of BO-Alb yielded similar dose estimates. These results point to the utility of protein adducts as dosimeters of reactive electrophilic species in occupational studies. Finally, significant levels of background adducts of SO and BO with Hb and Alb were observed among workers, among control subjects and in commercial human

  20. Cisplatin intrastrand adducts sensitize DNA to base damage by hydrated electrons.

    PubMed

    Behmand, B; Wagner, J R; Sanche, L; Hunting, D J

    2014-05-01

    The oligonucleotide TTTTTGTGTTT with or without a cisplatin adduct was reacted with hydrated electrons generated by ionizing radiation. Hydroxyl radicals were quenched with ethylenediaminetetraacetic acid (EDTA), and the solutions were bubbled with wet nitrogen to eliminate oxygen, a scavenger of hydrated electrons. Prior to irradiation, the structure of the initial cisplatin adduct was identified by mass spectrometry as G-cisplatin-G. Radiation damage to DNA bases was quantified by high-performance liquid chromatography (HPLC), after enzymatic digestion of the TTTTTGTGTTT-cisplatin complex to deoxyribonucleosides. The masses of the platinum adducts following digestion and separation by HPLC were measured by mass spectrometry. Our results demonstrate that hydrated electrons induce damage to thymines as well as detachment of the cisplatin moiety from both guanines in the oligonucleotide. This detachment regenerates both unmodified guanine and damaged guanine, in equimolar amounts. At 1000 Gy, a net average of 2.5 thymines and 1 guanine are damaged for each platinum lost from the oligonucleotide. Given the extensive base damage that occurs for each cisplatin adduct lost, it is clear that, prior to undergoing detachment, these adducts must catalyze several cycles of reactions of hydrated electrons with DNA bases. It is likely that a single reaction leads to the loss of the cisplatin adduct and the damage observed on the guanine base; however, the damage to the thymine bases must require the continued presence of the cisplatin adduct, acting as a catalyst. To our knowledge, this is the first time that platinum-DNA adducts have been shown to have catalytic activity. We propose two pathways for the interaction of hydrated electrons with TTTTTGTGTTT-cisplatin: (1) the hydrated electron is initially captured by a thymine base and transferred by base to base electron hopping to the guanine site, where the cisplatin moiety detaches from the oligonucleotide via dissociative

  1. Brevetoxin Forms Covalent DNA Adducts in Rat Lung Following Intratracheal Exposure

    PubMed Central

    Radwan, Faisal F.Y.; Ramsdell, John S.

    2008-01-01

    Background Human exposure to brevetoxins produced by the red tide organism, Karenia brevis, is an increasing public health concern. Using in vitro exposure of rat liver cells to brevetoxin B (PbTx-2), the primary toxin product of K. brevis, we previously showed that it formed C27,28-epoxy brevetoxin metabolites capable of covalently binding to nucleic acids, a common initiation step for carcinogenesis. Objective This study was undertaken to evaluate nucleic acid adduction in lung following in vitro and in vivo brevetoxin exposures. Methods To clarify reactions of brevetoxin epoxide with DNA, we analyzed reaction products of PbTx-6 (a C27,28 epoxide metabolite of brevetoxin B) with nucleosides. We also analyzed adducts from nucleic acid hydrolysates of isolated rat lung cells treated with PbTx-2 or PbTx-6 in vitro and lung tissue from rats after intratracheal exposure to PbTx-2 or PbTx-6 at 45 μg toxin/kg body weight. Results Our results indicate that PbTx-2 forms DNA adducts with cytidine after treatment of isolated lung cells, and forms DNA adducts with adenosine and guanosine after intratracheal exposure. Conclusions These results are consistent with metabolic activation of highly reactive brevetoxin intermediates that bind to nucleic acid. These findings provide a basis for monitoring exposure and assessing the hazard associated with depurination of brevetoxin–nucleotide adducts in lung tissue. PMID:18629316

  2. DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

    PubMed

    Dingley, Karen H; Ubick, Esther A; Vogel, John S; Ognibene, Ted J; Malfatti, Michael A; Kulp, Kristen; Haack, Kurt W

    2014-01-01

    Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels. PMID:24623226

  3. Insights into the Mechanism of Protein Electrospray Ionization From Salt Adduction Measurements

    NASA Astrophysics Data System (ADS)

    Yue, Xuanfeng; Vahidi, Siavash; Konermann, Lars

    2014-08-01

    The mechanisms whereby protein ions are liberated from charged droplets during electrospray ionization (ESI) remain under investigation. Compact conformers electrosprayed from aqueous solution in positive ion mode likely follow the charged residue model (CRM), which envisions analyte release after solvent evaporation to dryness. The concentration of nonvolatile salts such as NaCl increases sharply within vanishing CRM droplets, promoting nonspecific pairing of Cl- and Na+ with charged groups on the protein surface. For unfolded proteins, it has been proposed that ion formation occurs via the chain ejection model (CEM). During the CEM proteins are expelled from the droplet long before complete solvent evaporation has taken place. Here we examine whether salt adduction levels support the view that folded and unfolded proteins follow different ESI mechanisms. Solvent evaporation during the CEM is expected to be less extensive and, hence, the salt concentration at the point of protein release should be substantially lower than for the CRM. CEM ions should therefore exhibit lower adduction levels than CRM species. We explore the adduction behavior of several proteins that were chosen to allow comparative studies on folded and unfolded structures in the same solution. In-source activation eliminates chloride adducts via HCl release, generating protein ions that are heterogeneously charged because of sodiation and protonation. Sodiation levels measured under such conditions provide estimates of the salt adduction behavior experienced by the "nascent" analyte ions. Sodiation levels are significantly reduced for unfolded proteins, supporting the view that these species are indeed formed via the CEM.

  4. Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts.

    PubMed

    Ngo, Phong D; Mansoorabadi, Steven O; Frey, Perry A

    2016-08-01

    Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties. PMID:27387593

  5. Formation of DNA Adducts by Ellipticine and Its Micellar Form in Rats — A Comparative Study

    PubMed Central

    Stiborova, Marie; Manhartova, Zuzana; Hodek, Petr; Adam, Vojtech; Kizek, Rene; Frei, Eva

    2014-01-01

    The requirements for early diagnostics as well as effective treatment of cancer diseases have increased the pressure on development of efficient methods for targeted drug delivery as well as imaging of the treatment success. One of the most recent approaches covering the drug delivery aspects is benefitting from the unique properties of nanomaterials. Ellipticine and its derivatives are efficient anticancer compounds that function through multiple mechanisms. Formation of covalent DNA adducts after ellipticine enzymatic activation is one of the most important mechanisms of its pharmacological action. In this study, we investigated whether ellipticine might be released from its micellar (encapsulated) form to generate covalent adducts analogous to those formed by free ellipticine. The 32P-postlabeling technique was used as a useful imaging method to detect and quantify covalent ellipticine-derived DNA adducts. We compared the efficiencies of free ellipticine and its micellar form (the poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PAGE-PEO) block copolymer, P 119 nanoparticles) to form ellipticine-DNA adducts in rats in vivo. Here, we demonstrate for the first time that treatment of rats with ellipticine in micelles resulted in formation of ellipticine-derived DNA adducts in vivo and suggest that a gradual release of ellipticine from its micellar form might produce the enhanced permeation and retention effect of this ellipticine-micellar delivery system. PMID:25479328

  6. Nitroreduction and formation of hemoglobin adducts in rats with a human intestinal microflora

    SciTech Connect

    Scheepers, P.T.J.; Straetemans, M.M.E.; Koopman, J.P.; Bos, R.P.

    1994-10-01

    In the covalent binding of nitroarenes to macromolecules, nitroreduction is an important step. The intestinal microflora represents an enormous potential of bacterial nitroreductase activity. As a consequence, the in vivo nitroreduction of orally administerednitroarenes is primarily located in the intestine. In this study, we have investigated the nitroreduction of 2-nitrofluorene (2-NF) by a human microflora in female Wistar rats. Germ-free (FG) rats were equipped with a bacterial flora derived from human feces. Nontreated GF rats and GF animals equipped with a conventional rat flora were used as controls. The composition of the human and the conventional microflora isolated from the rats were consistent with the microflora of the administered feces. In the rats receiving only sunflower seed oil, no adducts were detected. The animals equipped with a human or rat microflora that received 2-aminofluorene (2-AF) formed 2-AF hemoglobin (Hb)-adducts at average levels mean {+-} 0.003 and 0.043 {+-} 0.010 {mu}mole/g Hb, respectively. In the FG rats, an adduct level of 0.57 {+-} 0.09 was determined after 2-AF administration and non adducts were detected after 2-NF administration. The results show that nitroreduction by an acquired human intestinal microflora and subsequent adduct formation can be studied in the rate in vivo. 21 refs., 3 tabs.

  7. Mass spectrometric characterization of human hemoglobin adducts formed in vitro by hexahydrophthalic anhydride.

    PubMed

    Kristiansson, Monica H; Jönsson, Bo A G; Lindh, Christian H

    2002-04-01

    Primary structural information of anhydride binding to endogenous proteins is of interest in order to determine the mechanism causing the type-I allergy seen in many anhydride-exposed workers. In addition, studies on specific protein adducts may generate new methods for biological monitoring. In this study, the binding of hexahydrophthalic anhydride (HHPA) to human hemoglobin (Hb) in vitro was investigated. The in vitro synthesized conjugates were analyzed using a hybrid quadrupole-time-of-flight mass spectrometer (Q-TOF) with electrospray ionization (ESI) to determine the number of HHPA adducts per Hb molecule. Structural information on the locations of the adducts was obtained through nanospray Q-TOF, liquid chromatography-ESI mass spectrometric analysis, and gas chromatography/mass spectrometric analysis of Pronase E and tryptic digests. Up to six adducts were found on the alpha-chain and five on the beta-chain. The HHPA-adducts were localized to the N-terminal valine of the alpha- and beta-chains of Hb and to lysine residues at positions 7, 11, 16, and 40 of the alpha-chain and 8, 17, 59, 66, and 144 of the beta-chain. These results will constitute a basis for studies on structure-activity relationships as well as for development of methods for biological monitoring of acid anhydrides. PMID:11952343

  8. Metabolic stability of superoxide adducts derived from newly developed cyclic nitrone spin traps.

    PubMed

    Bézière, Nicolas; Hardy, Micael; Poulhès, Florent; Karoui, Hakim; Tordo, Paul; Ouari, Olivier; Frapart, Yves-Michel; Rockenbauer, Antal; Boucher, Jean-Luc; Mansuy, Daniel; Peyrot, Fabienne

    2014-02-01

    Reactive oxygen species are by-products of aerobic metabolism involved in the onset and evolution of various pathological conditions. Among them, the superoxide radical is of special interest as the origin of several damaging species such as H2O2, hydroxyl radical, or peroxynitrite (ONOO(-)). Spin trapping coupled with ESR is a method of choice to characterize these species in chemical and biological systems and the metabolic stability of the spin adducts derived from reaction of superoxide and hydroxyl radicals with nitrones is the main limit to the in vivo application of the method. Recently, new cyclic nitrones bearing a triphenylphosphonium or permethylated β-cyclodextrin moiety have been synthesized and their spin adducts demonstrated increased stability in buffer. In this article, we studied the stability of the superoxide adducts of four new cyclic nitrones in the presence of liver subcellular fractions and biologically relevant reductants using an original setup combining a stopped-flow device and an ESR spectrometer. The kinetics of disappearance of the spin adducts were analyzed using an appropriate simulation program. Our results highlight the interest of the new spin trapping agents CD-DEPMPO and CD-DIPPMPO for specific detection of superoxide with high stability of the superoxide adducts in the presence of liver microsomes. PMID:24161442

  9. Neurodegeneration with Brain Iron Accumulation

    MedlinePlus

    ... Diversity Find People About NINDS NINDS Neurodegeneration with Brain Iron Accumulation Information Page Synonym(s): Hallervorden-Spatz Disease, ... done? Clinical Trials Organizations What is Neurodegeneration with Brain Iron Accumulation? Neurodegeneration with brain iron accumulation (NBIA) ...

  10. Plastids and Carotenoid Accumulation.

    PubMed

    Li, Li; Yuan, Hui; Zeng, Yunliu; Xu, Qiang

    2016-01-01

    Plastids are ubiquitously present in plants and are the organelles for carotenoid biosynthesis and storage. Based on their morphology and function, plastids are classified into various types, i.e. proplastids, etioplasts, chloroplasts, amyloplasts, and chromoplasts. All plastids, except proplastids, can synthesize carotenoids. However, plastid types have a profound effect on carotenoid accumulation and stability. In this chapter, we discuss carotenoid biosynthesis and regulation in various plastids with a focus on carotenoids in chromoplasts. Plastid transition related to carotenoid biosynthesis and the different capacity of various plastids to sequester carotenoids and the associated effect on carotenoid stability are described in light of carotenoid accumulation in plants. PMID:27485226

  11. Formation of DNA adducts in rat lung following chronic inhalation of diesel emissions, carbon black and titanium dioxide particles.

    PubMed

    Gallagher, J; Heinrich, U; George, M; Hendee, L; Phillips, D H; Lewtas, J

    1994-07-01

    Exposure of rats to diesel emissions results in the development of lung tumors. The objective of this study was to determine whether the polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs or other polycyclic organic matter adsorbed to diesel particles induces the formation of DNA adducts in the lung when compared to particles with little or no adsorbed organic matter. Rats were exposed to diesel emissions containing particles with over 30% solvent-extractable adsorbed organic matter and to particles with < 0.1% adsorbed organic matter (carbon black particles and TiO2). Wistar rats were exposed to diesel emissions (7.5 mg/m3) for 2 months, 6 months and 2 years and for 2 years to carbon black (11.3 mg/m3) and TiO2 particles (10.4 mg/m3) to compare tumorigenic response and DNA adduct formation in the lung. Two versions of the 32P-postlabeling assay for the detection of DNA adducts were used to tentatively identify nitrated-amine or arylamine adducts formed relative to other nitro PAH based on the demonstrated sensitivity of these adducts to nuclease P1 treatment. Total adduct levels were determined for peripheral lung tissue DNA as detected in a diagonal radioactive zone. One major adduct which migrated outside this region (adduct 1) and a nuclease P1-sensitive adduct (adduct 2) were quantitated separately. Adduct 1 increased significantly over time in the filtered air exposed animals but decreased markedly at the 2 year time points regardless of particle type, presumably as a result of adduct dilution through de novo cell synthesis or cell proliferation invoked in response to particle loading and/or effect on the endogenous synthesis or degradation of DNA reactive moieties. The nuclease sensitive adduct (adduct 2), possibly resulting from exposure to nitro-PAHs, was detected in diesel-exposed rats but was not detected in the rats exposed to TiO2 and carbon black. No significant elevation in PAH-derived adducts, relative to the filtered air controls, was observed in

  12. Nuclease S1-mediated enhancement of the 32P-postlabeling assay for aromatic carcinogen-DNA adducts.

    PubMed

    Reddy, M V

    1991-09-01

    Treatment of DNA digests with nuclease P1 prior to 32P-labeling of adducts has previously been shown to enhance the sensitivity of the 32P-postlabeling assay for the detection of aromatic carcinogen-DNA adducts. The enhancement was based on the ability of nuclease P1 to remove the 3'-phosphate from normal nucleotides but not the corresponding phosphate from most aromatic adducted nucleotides. We investigated the utility of another 3'-dephosphorylating enzyme, nuclease S1, for this purpose, and found it to be as effective as nuclease P1. The recovery of DNA adducts derived from benzo[a]-pyrene (B[a]P), benzoquinone (BQ) and 2-acetylaminofluorene (AAF) was comparable after enhancement with either enzyme. Some differences were, however, observed. Recovery of a minor B[a]P adduct was 1.5 times higher by the S1 procedure. Among minor adducts of BQ, two showed higher values (2.8- and 6.1-fold) by the S1 procedure and one by the P1 procedure (2.4-fold). The major AAF adduct, deoxyguanosine-C8-AF, exhibited poorer recovery (1-11%) by either procedure, while the minor adducts, deoxyguanosine-N2-AAF and deoxyguanosine-C8-AAF, showed better recovery (2-3 times) than by the enhancement procedure involving extraction of adducts into butanol. Our results show that the nuclease S1 assay can complement the nuclease P1 assay, with improved recoveries for some adducts. Considering the complexity of the postlabeling assay, this additional variant may prove useful in unequivocal detection of DNA adducts. PMID:1893535

  13. Tyrosine-Lipid Peroxide Adducts from Radical Termination: Para-Coupling and Intramolecular Diels-Alder Cyclization

    PubMed Central

    Shchepin, Roman; Möller, Matias N.; Kim, Hye-young H.; Hatch, Duane M.; Bartesaghi, Silvina; Kalyanaraman, Balaraman; Radi, Rafael

    2013-01-01

    Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogs of tyrosine gave rise to lipid peroxide-tyrosine (phenol) adducts in both aqueous micellar and organic solutions. The novel adducts were isolated and characterized by 1D and 2D NMR as well as by mass spectrometry. The spectral data suggest that the polyunsaturated lipid peroxyl radicals give stable peroxide coupling products exclusively at the para position of the tyrosyl (phenoxy) radicals. These adducts have characteristic 13C chemical shifts at 185 ppm due to the cross-conjugated carbonyl of the phenol-derived cyclohexadienone. The primary peroxide adducts subsequently undergo intramolecular Diels-Alder (IMDA) cyclization, affording a number of diastereomeric tricyclic adducts that have characteristic carbonyl 13C chemical shifts at ~198 ppm. All NMR HMBC and HSQC correlations support the structure assignment of the primary and Diels-Alder adducts, as does MS collision induced dissociation. Kinetic rate constants and activation parameters for the IMDA reaction were determined and the primary adducts were reduced with cuprous ion giving a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found either in the primary or the cuprous reduction product mixtures. These studies provide a framework for understanding the nature of lipid-protein adducts formed by peroxyl-tyrosyl radical-radical termination processes. Coupling of lipid peroxyl radicals with tyrosyl radicals leads to cyclohexenone and cyclohexadienone adducts which are of interest in and of themselves since, as electrophiles, they are likely targets for protein nucleophiles. One consequence of lipid peroxyl reactions with tyrosyls may therefore be protein-protein crosslinks via interprotein Michael adducts. PMID:21090613

  14. Identification of DNA--cisplatin adducts in a blind trial of in situ scanning tunneling microscopy.

    PubMed

    Jeffrey, A M; Jing, T W; DeRose, J A; Vaught, A; Rekesh, D; Lu, F X; Lindsay, S M

    1993-12-25

    Scanning tunneling microscopy (STM) reveals nanometer scale details of hydrated DNA but the interpretation of the images is controversial because of substrate artifacts and the lack of a theory for image contrast. We demonstrate that we have overcome these problems by identifying five DNA samples by their STM images alone in a blinded trial. The samples were single-stranded and double-stranded DNA with and without covalent modification by the anti-tumor drug cisplatin. The cisplatin adducts were distinguished by substantial kinking at the drug binding site. The oligomers were 20 bases in length, which was too short to permit the kinking angle to be determined with precision. However, models with a 45 degree kink gave a better fit to the images of the duplex adducts than models with a 90 degrees kink. A variety of structures was observed for the single-stranded adducts. PMID:8290350

  15. The influence of antagonist muscle electrical stimulation on maximal hip adduction force

    PubMed Central

    Nakano, Sota; Wada, Chikamune

    2016-01-01

    [Purpose] The aim of this study was to determine whether electrical stimulation of the tensor fascia lata muscle decreases voluntary maximum resistance to passive abduction motion in participants without disease of the central nervous system. [Subjects] The participants were 16 healthy men. [Methods] The hip joint was moved from 10° adduction to 0° adduction with an angular velocity of 7°/s. During the passive leg motion, the subject was asked to resist the motion with maximum force. Two experimental conditions were prepared: (1) electrical stimulation provided to the tensor fascia lata muscle during the passive motion; and (2) no electrical stimulation provided. [Results] The force was 10.2 ± 3.5 kgf with electrical stimulation and 12.2 ± 3.8 kgf without electrical stimulation. [Conclusion] The results suggested that the maximum hip adduction force decreased in participants because of electrical stimulation of the tensor fascia lata muscle. PMID:26957742

  16. Isomerism and adduct formation in the hector's base series: A MNDO study of model compounds

    NASA Astrophysics Data System (ADS)

    Cuthbertson, Alastair F.; Glidewell, Christopher

    MNDO calculations on a series of a model compounds show that the observed structures for Hector's base, Dost's base and Dost's keto compound are the thermodynamically most stable tautomers and that the bond-switched structure observed for the 1:1 adduct of Hector's base with carbon disulphide and the non-bond-switched structure observed for the corresponding adducts with isocyanates and isothiocyanates are both the thermodynamically most favoured isomers, so that the occurrence or otherwise of a bond switch in these compounds is determined by thermodynamic rather than by mechanistic factors. Proposed mechanisms for the formation of the carbon disulphide adduct of Hector's base, and for its desulphurisation are supported by MNDO calculations.

  17. Atomic-resolution structure of an N5 flavin adduct in D-arginine dehydrogenase.

    PubMed

    Fu, Guoxing; Yuan, Hongling; Wang, Siming; Gadda, Giovanni; Weber, Irene T

    2011-07-26

    D-Arginine dehydrogenase (DADH) catalyzes the flavin-dependent oxidative deamination of D-arginine and other D-amino acids to the corresponding imino acids. The 1.07 Å atomic-resolution structure of DADH crystallized with D-leucine unexpectedly revealed a covalent N(5) flavin adduct, instead of the expected iminoleucine product in the active site. This acyl adduct has been successfully reproduced by photoreduction of DADH in the presence of 4-methyl-2-oxopentanoic acid (ketoleucine). The iminoleucine may be released readily because of weak interactions in the binding site, in contrast to iminoarginine, converted to ketoleucine, which reacts with activated FAD to form the covalently linked acyl adduct. PMID:21707047

  18. New fluorescence methodology for detecting DNA adducts. Final progress report, May 1, 1991--November 30, 1994

    SciTech Connect

    Giese, R.W.

    1994-12-19

    A new reagent, {open_quotes}BO-IMI{close_quotes}, has been developed that achieves, for the first time, single step, phosphate specific fluorescence labeling under aqueous conditions. Both 3{prime} and 5{prime} mononucleotides, including representative DNA adducts can be labeled. Included in this technique is a convenient procedure for postlabeling sample cleanup, leading to a practical detection of the products by capillary electrophoresis with laser fluorescence detection (CE-LIF). We consider that this new method will have a significant impact on the measurement of DNA adducts in human samples. This work was largely accomplished in the second half of our project. In the first half, we set up a new way to isolate DNA nucleotides from blood, worked with an initial, less specific technique for labeling DNA adducts, compared ionizing radiation vs oxidative damage to fluorescein labeled deoxyadenylic acid, and set up a capillary electrophoresis laser fluorescence detection system.

  19. New fluorescence methodology for detecting DNA adducts. Progress report, May 1, 1991--May 21, 1993

    SciTech Connect

    Giese, R.W.

    1993-05-21

    A new reagent, BO-IMI, has been developed that achieves, single step, phosphate specific fluorescence labeling under aqueous conditions. Both 3 in. and 5 in. mononucleotides, including representative DNA adducts can be labeled. Included in this technique is a convenient procedure for postlabeling sample cleanup, leading to a practical detection of the products by capillary electrophoresis with laser fluorescencedetection. We consider that this new method will have a significant impact on the measurement of DNA adducts in human samples. This work was largely accomplished in the second half of our project. In the first half, we set up a new way to isolate DNA nucleotides from blood, worked with an initial, less specific technique for labeling DNA adducts, compared ionizing radiation vs oxidative damage to fluorescein labeled deoxyadenylic acid, and set up a capillary electrophoresis laser fluorescence detection system.

  20. Characterization and Reactivity of a Terminal Nickel(III)-Oxygen Adduct

    PubMed Central

    Pirovano, Paolo; Farquhar, Erik R.; Swart, Marcel; Fitzpatrick, Anthony J.; Morgan, Grace G.; McDonald, Aidan R.

    2015-01-01

    High-valent terminal metal-oxygen adducts are hypothesized to be the potent oxidising reactants in late transition metal oxidation catalysis. In particular, examples of high-valent terminal nickel-oxygen adducts are sparse, meaning there is a dearth in the understanding of such oxidants. In this study, a monoanionic NiII-bicarbonate complex was found to react in a 1:1 ratio with the one-electron oxidant tris(4-bromophenyl)ammoniumyl hexachloroantimonate, yielding a thermally unstable intermediate in high yield (~95%). Electronic absorption, electronic paramagnetic resonance and X-ray absorption spectroscopies and density functional theory calculations confirm its description as a low-spin (S = ½), square planar NiIII-oxygen adduct. This rare example of a high-valent terminal nickel-oxygen complex performs oxidations of organic substrates, including 2,6-ditertbutylphenol and triphenylphosphine, which are indicative of hydrogen atom abstraction and oxygen atom transfer reactivity, respectively. PMID:25612563

  1. Selection of adduct-forming chemicals for human-monitoring studies

    SciTech Connect

    Not Available

    1991-07-01

    The U.S. EPA, through its Environmental Monitoring Systems Laboratory-Las Vegas (EMSL-LV) and its Health Effects Research Laboratory-Research Triangle Park (HERL-RTP) has been exploring the feasibility of using biological markers to monitor exposure to environmental chemicals. The participants began by compiling a list of chemicals of known or suspected health hazards and for which the potential for human exposure exists. The chemicals on the master list were then systematically evaluated for: (1) the potential for adduct-formation in vivo, (2) the availability of supportive adduct research data, (3) the identifiability of exposed population(s), and (4) the level of genetic activity. After considering all the relevant data, the participants selected and prioritized for further study a small group of chemicals considered to have the greatest potential for use in pilot, adduct-based, biological monitoring studies in human populations.

  2. Reactivity of adducts relevant to the deposition of hexagonal BN from first-principles calculations

    NASA Astrophysics Data System (ADS)

    Freitas, R. R. Q.; Gueorguiev, G. K.; de Brito Mota, F.; de Castilho, C. M. C.; Stafström, S.; Kakanakova-Georgieva, A.

    2013-09-01

    First-principles calculations, which also implement the nudged elastic band (NEB) code, are performed to investigate (i) the stability of the (C2H5)3B:NH3 adduct formed by the initial precursor molecules triethylborane (C2H5)3B and ammonia NH3 in the metal-chemical-vapor-deposition (MOCVD) of hexagonal BN, and (ii) the energy barrier to the first ethane elimination through consistent unimolecular, ammonia-assisted, and adduct-assisted reaction pathways. Comparison is done with the reference case of the (CH3)3Al:NH3 adduct, notoriously known for its high degree of stability and reactivity, which determines an overall severe parasitic gas-phase chemical reaction mechanism in the deposition of AlN.

  3. Hemoglobin adducts in workers exposed to 1,6-hexamethylene diisocyanate

    PubMed Central

    Flack, Sheila L.; Fent, Kenneth W.; Gaines, Linda G. T.; Thomasen, Jennifer M.; Whittaker, Stephen G.; Ball, Louise M.; Nylander-French, Leena A.

    2014-01-01

    We investigated the utility of 1,6-hexamethylene diamine (HDA) hemoglobin adducts as biomarkers of exposure to 1,6-hexamethylene diisocyanate (HDI) monomer. Blood samples from 15 spray painters applying HDI-containing paint were analyzed for hemoglobin HDA (HDA-Hb) and N-acetyl-1,6-hexamethylene diamine (monoacetyl-HDA-Hb) by GC-MS. HDA-Hb was detected in the majority of workers (≤1.2–37 ng/g Hb), whereas monoacetyl-HDA-Hb was detected in one worker (0.06 ng/g Hb). The stronger, positive association between HDA-Hb and cumulative HDI exposure (r2 = 0.3, p < 0.06) than same day exposure (p ≥ 0.13) indicates long-term elimination kinetics for HDA-Hb adducts. This association demonstrates the suitability of HDA-Hb adducts for further validation as a biomarker of HDI exposure. PMID:21506697

  4. Chimpanzee accumulative stone throwing.

    PubMed

    Kühl, Hjalmar S; Kalan, Ammie K; Arandjelovic, Mimi; Aubert, Floris; D'Auvergne, Lucy; Goedmakers, Annemarie; Jones, Sorrel; Kehoe, Laura; Regnaut, Sebastien; Tickle, Alexander; Ton, Els; van Schijndel, Joost; Abwe, Ekwoge E; Angedakin, Samuel; Agbor, Anthony; Ayimisin, Emmanuel Ayuk; Bailey, Emma; Bessone, Mattia; Bonnet, Matthieu; Brazolla, Gregory; Buh, Valentine Ebua; Chancellor, Rebecca; Cipoletta, Chloe; Cohen, Heather; Corogenes, Katherine; Coupland, Charlotte; Curran, Bryan; Deschner, Tobias; Dierks, Karsten; Dieguez, Paula; Dilambaka, Emmanuel; Diotoh, Orume; Dowd, Dervla; Dunn, Andrew; Eshuis, Henk; Fernandez, Rumen; Ginath, Yisa; Hart, John; Hedwig, Daniela; Ter Heegde, Martijn; Hicks, Thurston Cleveland; Imong, Inaoyom; Jeffery, Kathryn J; Junker, Jessica; Kadam, Parag; Kambi, Mohamed; Kienast, Ivonne; Kujirakwinja, Deo; Langergraber, Kevin; Lapeyre, Vincent; Lapuente, Juan; Lee, Kevin; Leinert, Vera; Meier, Amelia; Maretti, Giovanna; Marrocoli, Sergio; Mbi, Tanyi Julius; Mihindou, Vianet; Moebius, Yasmin; Morgan, David; Morgan, Bethan; Mulindahabi, Felix; Murai, Mizuki; Niyigabae, Protais; Normand, Emma; Ntare, Nicolas; Ormsby, Lucy Jayne; Piel, Alex; Pruetz, Jill; Rundus, Aaron; Sanz, Crickette; Sommer, Volker; Stewart, Fiona; Tagg, Nikki; Vanleeuwe, Hilde; Vergnes, Virginie; Willie, Jacob; Wittig, Roman M; Zuberbuehler, Klaus; Boesch, Christophe

    2016-01-01

    The study of the archaeological remains of fossil hominins must rely on reconstructions to elucidate the behaviour that may have resulted in particular stone tools and their accumulation. Comparatively, stone tool use among living primates has illuminated behaviours that are also amenable to archaeological examination, permitting direct observations of the behaviour leading to artefacts and their assemblages to be incorporated. Here, we describe newly discovered stone tool-use behaviour and stone accumulation sites in wild chimpanzees reminiscent of human cairns. In addition to data from 17 mid- to long-term chimpanzee research sites, we sampled a further 34 Pan troglodytes communities. We found four populations in West Africa where chimpanzees habitually bang and throw rocks against trees, or toss them into tree cavities, resulting in conspicuous stone accumulations at these sites. This represents the first record of repeated observations of individual chimpanzees exhibiting stone tool use for a purpose other than extractive foraging at what appear to be targeted trees. The ritualized behavioural display and collection of artefacts at particular locations observed in chimpanzee accumulative stone throwing may have implications for the inferences that can be drawn from archaeological stone assemblages and the origins of ritual sites. PMID:26923684

  5. Chimpanzee accumulative stone throwing

    PubMed Central

    Kühl, Hjalmar S.; Kalan, Ammie K.; Arandjelovic, Mimi; Aubert, Floris; D’Auvergne, Lucy; Goedmakers, Annemarie; Jones, Sorrel; Kehoe, Laura; Regnaut, Sebastien; Tickle, Alexander; Ton, Els; van Schijndel, Joost; Abwe, Ekwoge E.; Angedakin, Samuel; Agbor, Anthony; Ayimisin, Emmanuel Ayuk; Bailey, Emma; Bessone, Mattia; Bonnet, Matthieu; Brazolla, Gregory; Buh, Valentine Ebua; Chancellor, Rebecca; Cipoletta, Chloe; Cohen, Heather; Corogenes, Katherine; Coupland, Charlotte; Curran, Bryan; Deschner, Tobias; Dierks, Karsten; Dieguez, Paula; Dilambaka, Emmanuel; Diotoh, Orume; Dowd, Dervla; Dunn, Andrew; Eshuis, Henk; Fernandez, Rumen; Ginath, Yisa; Hart, John; Hedwig, Daniela; Ter Heegde, Martijn; Hicks, Thurston Cleveland; Imong, Inaoyom; Jeffery, Kathryn J.; Junker, Jessica; Kadam, Parag; Kambi, Mohamed; Kienast, Ivonne; Kujirakwinja, Deo; Langergraber, Kevin; Lapeyre, Vincent; Lapuente, Juan; Lee, Kevin; Leinert, Vera; Meier, Amelia; Maretti, Giovanna; Marrocoli, Sergio; Mbi, Tanyi Julius; Mihindou, Vianet; Moebius, Yasmin; Morgan, David; Morgan, Bethan; Mulindahabi, Felix; Murai, Mizuki; Niyigabae, Protais; Normand, Emma; Ntare, Nicolas; Ormsby, Lucy Jayne; Piel, Alex; Pruetz, Jill; Rundus, Aaron; Sanz, Crickette; Sommer, Volker; Stewart, Fiona; Tagg, Nikki; Vanleeuwe, Hilde; Vergnes, Virginie; Willie, Jacob; Wittig, Roman M.; Zuberbuehler, Klaus; Boesch, Christophe

    2016-01-01

    The study of the archaeological remains of fossil hominins must rely on reconstructions to elucidate the behaviour that may have resulted in particular stone tools and their accumulation. Comparatively, stone tool use among living primates has illuminated behaviours that are also amenable to archaeological examination, permitting direct observations of the behaviour leading to artefacts and their assemblages to be incorporated. Here, we describe newly discovered stone tool-use behaviour and stone accumulation sites in wild chimpanzees reminiscent of human cairns. In addition to data from 17 mid- to long-term chimpanzee research sites, we sampled a further 34 Pan troglodytes communities. We found four populations in West Africa where chimpanzees habitually bang and throw rocks against trees, or toss them into tree cavities, resulting in conspicuous stone accumulations at these sites. This represents the first record of repeated observations of individual chimpanzees exhibiting stone tool use for a purpose other than extractive foraging at what appear to be targeted trees. The ritualized behavioural display and collection of artefacts at particular locations observed in chimpanzee accumulative stone throwing may have implications for the inferences that can be drawn from archaeological stone assemblages and the origins of ritual sites. PMID:26923684

  6. Accumulation of the planets

    NASA Technical Reports Server (NTRS)

    Wetherill, G. W.

    1987-01-01

    In modeling the accumulation of planetesimals into planets, it is appropriate to distinguish between two stages: an early stage, during which approximately 10 km diameter planetesimals accumulate locally to form bodies approximate 10 to the 25th g in mass; and a later stage in which the approximately 10 to the 25th g planetesimals accumulate into the final planets. In the terrestrial planet region, an initial planetesimal swarm corresponding to the critical mass of dust layer gravitational instabilities is considered. In order to better understand the accumulation history of Mercury-sized bodies, 19 Monte-Carlo simulations of terrestrial planet growth were calculated. A Monte Carlo technique was used to investigate the orbital evolution of asteroidal collision debris produced interior to 2.6 AU. It was found that there are two regions primarily responsible for production of Earth-crossing meteoritic material and Apollo objects. The same techniques were extended to include the origin of Earth-approaching asteroidal bodies. It is found that these same two resonant mechanisms predict a steady-state number of Apollo-Amor about 1/2 that estimated based on astronomical observations.

  7. Formation of melamium adducts by pyrolysis of thiourea or melamine/NH4 Cl mixtures.

    PubMed

    Braml, Nicole E; Sattler, Andreas; Schnick, Wolfgang

    2012-02-01

    Pyrolysis of prominent precursor compounds for the synthesis of carbon nitride type materials (e.g., melamine, thiourea) have been studied in detail. Molecular adducts containing monoprotonated melamium C(6)N(11)H(10)(+) and melaminium HC(3)N(3)(NH(2))(3)(+) ions, respectively, have been identified as intermediates. The adduct C(6)N(11)H(10)Cl·0.5NH(4)Cl was obtained by the reaction of melamine C(3)N(3)(NH(2))(3) with NH(4)Cl at 450 °C. During the pyrolysis of thiourea, guanidinium thiocyanate was initially formed and subsequently the melamium thiocyanate melamine adduct C(6)N(11)H(10)SCN·2C(3)N(3)(NH(2))(3) was isolated at 300 °C. A second melaminium thiocyanate melamine adduct with the formula HC(3)N(3)(NH(2))(3)SCN·2C(3)N(3)(NH(2))(3) represents an intermediary reaction product that is best accessible at low pressures. The crystal structures of the compounds were solved by single-crystal XRD. Unequivocal proton localization at the C(6)N(11)H(10)(+) ion was established. A typical intramolecular and interannular hydrogen bridge and other characteristic hydrogen-bonding motifs were identified. Additionally, the adducts were investigated by solid-state NMR spectroscopy. Our study provides detailed insight into the thermal condensation of thiourea by identifying and characterizing key intermediates involved in the condensation process leading to carbon nitride type materials. Furthermore, factors promoting the formation of melamium adduct phases over melem are discussed. PMID:22223531

  8. Detection of DNA methylation adducts in Hodgkin's disease patients treated with procarbazine.

    PubMed

    Bianchini, F; Weiderpass, E; Kyrtopoulos, S; Souliotis, V L; Henry-Amar, M; Wild, C P; Boffetta, P

    1996-01-01

    Abstract The aim of the present study was to assess the relationship between dose of the methylating agent procarbazine (PCZ), DNA methylation adduct formation andresponse to chemotherapy treatment in 23 Hodgkin's disease patients receiving MOPP/ABV combination therapy. The DNA adducts, 7-methyldeoxyguanosine (7-medG) and0(6)-methyldeoxyguanosine (0(6)-medG), were measured in leucocytes at the end of the first cycle of PCZ treatment (77-100 mg m(Z) per day). 7-medG was detected in only two patients prior to treatment and0(6)-medG was below the detection limit (0.08 pole per mole dG) in all subjects prior to treatment. The mean levels after PCZ treatment were 12.55 pmole 7-medG per mole dG and0.254 μmole 0(6)-medG per mole dG with a 2-3 fold variation between individuals. No correlation was observed between the levels of the two adducts suggesting inter-individual differences in formation andremoval of the two adducts. Failure of treatment was observed in five patients andthis was not correlated with higher or lower levels of 7-medG or 0(6)-medG. Other adducts formed as a consequence of treatment with PCZ or other MOPP/ABV components could have more relevance in this respect. The ability to measure DNA methylation adducts at the individual level following exposure to PCZ or other methylating chemotherapeutic drugs (e.g. dacarbazine) could be useful in prospective studies of secondary cancer in Hodgkin's disease patients. PMID:23888989

  9. Xeroderma Pigmentosum Group A Suppresses Mutagenesis Caused by Clustered Oxidative DNA Adducts in the Human Genome

    PubMed Central

    Sassa, Akira; Kamoshita, Nagisa; Kanemaru, Yuki; Honma, Masamitsu; Yasui, Manabu

    2015-01-01

    Clustered DNA damage is defined as multiple sites of DNA damage within one or two helical turns of the duplex DNA. This complex damage is often formed by exposure of the genome to ionizing radiation and is difficult to repair. The mutagenic potential and repair mechanisms of clustered DNA damage in human cells remain to be elucidated. In this study, we investigated the involvement of nucleotide excision repair (NER) in clustered oxidative DNA adducts. To identify the in vivo protective roles of NER, we established a human cell line lacking the NER gene xeroderma pigmentosum group A (XPA). XPA knockout (KO) cells were generated from TSCER122 cells derived from the human lymphoblastoid TK6 cell line. To analyze the mutagenic events in DNA adducts in vivo, we previously employed a system of tracing DNA adducts in the targeted mutagenesis (TATAM), in which DNA adducts were site-specifically introduced into intron 4 of thymidine kinase genes. Using the TATAM system, one or two tandem 7,8-dihydro-8-oxoguanine (8-oxoG) adducts were introduced into the genomes of TSCER122 or XPA KO cells. In XPA KO cells, the proportion of mutants induced by a single 8-oxoG (7.6%) was comparable with that in TSCER122 cells (8.1%). In contrast, the lack of XPA significantly enhanced the mutant proportion of tandem 8-oxoG in the transcribed strand (12%) compared with that in TSCER122 cells (7.4%) but not in the non-transcribed strand (12% and 11% in XPA KO and TSCER122 cells, respectively). By sequencing the tandem 8-oxoG-integrated loci in the transcribed strand, we found that the proportion of tandem mutations was markedly increased in XPA KO cells. These results indicate that NER is involved in repairing clustered DNA adducts in the transcribed strand in vivo. PMID:26559182

  10. Environmental toxins and breast cancer on Long Island. I. Polycyclic aromatic hydrocarbon DNA adducts.

    PubMed

    Gammon, Marilie D; Santella, Regina M; Neugut, Alfred I; Eng, Sybil M; Teitelbaum, Susan L; Paykin, Andrea; Levin, Bruce; Terry, Mary Beth; Young, Tie Lan; Wang, Lian Wen; Wang, Qiao; Britton, Julie A; Wolff, Mary S; Stellman, Steven D; Hatch, Maureen; Kabat, Geoffrey C; Senie, Ruby; Garbowski, Gail; Maffeo, Carla; Montalvan, Pat; Berkowitz, Gertrud; Kemeny, Margaret; Citron, Marc; Schnabel, Freya; Schuss, Allan; Hajdu, Steven; Vinceguerra, Vincent

    2002-08-01

    Polycyclic aromatic hydrocarbons (PAH) are potent mammary carcinogens in rodents, but their effect on breast cancer development in women is not clear. To examine whether currently measurable PAH damage to DNA increases breast cancer risk, a population-based case-control study was undertaken on Long Island, NY. Cases were women newly diagnosed with in situ and invasive breast cancer; controls were randomly selected women frequency matched to the age distribution of cases. Blood samples were donated by 1102 (73.0%) and 1141 (73.3%) of case and control respondents, respectively. Samples from 576 cases and 427 controls were assayed for PAH-DNA adducts using an ELISA. The geometric mean (and geometric SD) of the log-transformed levels of PAH-DNA adducts on a natural scale was slightly, but nonsignificantly, higher among cases [7.36 (7.29)] than among controls [6.21 (4.17); P = 0.51]. The age-adjusted odds ratio (OR) for breast cancer in relation to the highest quintile of adduct levels compared with the lowest was 1.51 [95% confidence interval (CI), 1.04-2.20], with little or no evidence of substantial confounding (corresponding multivariate-adjusted OR, 1.49; 95% CI, 1.00-2.21). There was no consistent elevation in risk with increasing adduct levels, nor was there a consistent association between adduct levels and two of the main sources of PAH, active or passive cigarette smoking or consumption of grilled and smoked foods. These data indicate that PAH-DNA adduct formation may influence breast cancer development, although the association does not appear to be dose dependent and may have a threshold effect. PMID:12163319

  11. Diet, metabolic polymorphisms and dna adducts: the EPIC-Italy cross-sectional study.

    PubMed

    Palli, D; Vineis, P; Russo, A; Berrino, F; Krogh, V; Masala, G; Munnia, A; Panico, S; Taioli, E; Tumino, R; Garte, S; Peluso, M

    2000-08-01

    DNA adducts in peripheral leukocytes are considered a reliable indicator of internal dose exposure to genotoxic agents and, possibly, of cancer risk. We investigated their association with diet and other individual characteristics in healthy adults. The prospective study EPIC-Italy, a section of a larger European project, enrolled 47,749 men and women, aged 35-64 years, in 5 centres: all provided individual information about dietary and life-style habits and a blood sample. In a cross-sectional study, approximately 100 volunteers were randomly selected from each of the three main geographical study areas (Northern, Central and Southern Italy). DNA adducts and four polymorphic metabolic genotypes were determined in peripheral leukocytes by using (32)P-postlabelling technique and PCR methods. Among 309 subjects (153 men), 72.8% had detectable levels of DNA adducts (mean: 8.1 +/- 0.6 per 10(9) nucleotides). Strong negative associations emerged with the reported frequency of consumption of fresh fruit and vegetables, olive oil, and the intake of antioxidants. DNA adducts were higher in subjects with GSTT1 null genotype (p = 0.05). Significant differences between study centres emerged in multivariate analyses (mean levels: 11.0, 10.0, 7.2, 6.5 and 5.2 for Florence, Naples, Turin, Varese and Ragusa, respectively). A possible opposite seasonal variation was found according to latitude: adduct levels tended to be lower in winter in Florence and the southern centres, and during summer in the two northern centres. Frequent consumption of fresh fruit and vegetables is associated with reduced levels of DNA adducts, possibly contributing to the role of diet in modulating cancer risk. PMID:10897053

  12. Synthesis of Mitomycin C and Decarbamoylmitomycin C N(2) deoxyguanosine-adducts.

    PubMed

    Champeil, Elise; Cheng, Shu-Yuan; Huang, Bik Tzu; Conchero-Guisan, Marta; Martinez, Thibaut; Paz, Manuel M; Sapse, Anne-Marie

    2016-04-01

    Mitomycin C (MC) and Decarbamoylmitomycin C (DMC) - a derivative of MC lacking the carbamate on C10 - are DNA alkylating agents. Their cytotoxicity is attributed to their ability to generate DNA monoadducts as well as intrastrand and interstrand cross-links (ICLs). The major monoadducts generated by MC and DMC in tumor cells have opposite stereochemistry at carbon one of the guanine-mitosene bond: trans (or alpha) for MC and cis (or beta) for DMC. We hypothesize that local disruptions of DNA structure from trans or cis adducts are responsible for the different biochemical responses produced by MC and DMC. Access to DNA substrates bearing cis and trans MC/DMC lesions is essential to verify this hypothesis. Synthetic oligonucleotides bearing trans lesions can be obtained by bio-mimetic methods. However, this approach does not yield cis adducts. This report presents the first chemical synthesis of a cis mitosene DNA adduct. We also examined the stereopreference exhibited by the two drugs at the mononucleotide level by analyzing the formation of cis and trans adducts in the reaction of deoxyguanosine with MC or DMC using a variety of activation conditions. In addition, we performed Density Functional Theory calculations to evaluate the energies of these reactions. Direct alkylation under autocatalytic or bifunctional conditions yielded preferentially alpha adducts with both MC and DMC. DFT calculations showed that under bifunctional activation, the thermodynamically favored adducts are alpha, trans, for MC and beta, cis, for DMC. This suggests that the duplex DNA structure may stabilize/oriente the activated pro-drugs so that, with DMC, formation of the thermodynamically favored beta products are possible in a cellular environment. PMID:26894558

  13. MTHFR Polymorphisms, Folate Intake, and Carcinogen DNA Adducts in the Lung

    PubMed Central

    Lee, Mi-Sun; Asomaning, Kofi; Su, Li; Wain, John C.; Mark, Eugene J.; Christiani, David C.

    2011-01-01

    The methylenetetrahydrofolate reductase (MTHFR) genes and folate in one-carbon metabolism are essential for DNA methylation and synthesis. However, their role in carcinogen DNA damage in target lung tissue, a dosimeter for cancer risk, is not known. Our study aimed to investigate the association between genetic and nutritional one-carbon metabolism factors and DNA adducts in target lung. Data on 135 lung cancer cases from the Massachusetts General Hospital were studied. Genotyping was completed for MTHFR C677T (rs1801133) and A1298C (rs1801131). Information on dietary intake for one-carbon related micronutrients, folate and other B vitamin, was derived from a validated food frequency questionnaire. DNA adducts in lung were measured by 32P-postlabeling. After adjusting for potential confounders, DNA adduct levels in lung significantly increased by 69.2% [95% confidence interval (CI), 5.5% to 171.5%] for the MTHFR 1298AC+CC genotype. The high risk group, combining the A1298C (AC+CC) plus C677T (CT+TT) genotypes, had significantly enhanced levels of lung adducts by 210.7% (95% CI, 21.4% to 695.2%) in contrast to the A1298C (AA) plus C677T (CC) genotypes. Elevation of DNA adduct was pronounced - 111.3% (95% CI, −3.0 to 360.5%) among 1298AC+CC patients who consumed the lowest level of folate intake as compared with 1298AA individuals with highest tertile of intake. These results indicate that DNA adducts levels are influenced by MTHFR polymorphisms and low folate consumption, suggesting an important role of genetic and nutritional factors in protecting DNA damage from lung carcinogen in at-risk populations. PMID:22052259

  14. INVESTIGATION OF THE RADICAL-MEDIATED PRODUCTION OF BENZENE OXIDE PROTEIN ADDUCTS IN VITRO AND IN VIVO

    EPA Science Inventory

    High background levels of benzene oxide (BO) adducts with hemoglobin and albumin (BO-Hb and BO-Alb) have been measured in unexposed humans and animals. To test the influence of radical-mediated pathways on production of these BO-protein adducts, we employed Fenton chemistry to...

  15. DNA adducts in marine mussel Mytilus galloprovincialis living in polluted and unpolluted environments. Chapter 12. Book chapter

    SciTech Connect

    Kurelec, B.; Garg, A.; Krca, S.; Gupta, R.C.

    1990-01-01

    A generally applicable (32)P-postlabeling assay was used to examine the presence of DNA adducts in mussels experimentally exposed to known carcinogens and in mussels collected from sites impacted by wastewaters. Mussels exposed to seawater artificially polluted with 2-aminofluorene showed exclusively one adduct which was identified to be dG-C8-2-aminofluorene. Under the same experimental conditions, Diesel-2 oil did not induce any detectable adducts. When mussel digestive gland DNA was collected and analyzed from one unpolluted site, two moderately impacted sites, and one site heavily impacted by cannery wastewaters, mussel DNA from the unpolluted and only one moderately polluted site showed the presence of 6 to 10 adducts. This indicates they were not related to the pollution. This was further supported by the absence of dose-related adducts. Clear evidence for the presence of pollution-related DNA adducts was, however, found in juvenile mussels collected from an oil refinery site. One major and three minor adducts were detected in these mussels with no adducts detected in juvenile mussels from an unpolluted site.

  16. BINDING OF CARCINOGENS TO DNA AND COVALENT ADDUCTS DNA DAMAGE - PAH, AROMATIC AMINES, NITRO-AROMATIC COMPOUNDS, AND HALOGENATED COMPOUNDS

    EPA Science Inventory

    DNA adducts are the covalent addition products resulting from binding of reactive chemical species to DNA bases. The cancer initiating role of DNA adducts is well-established, and is clearly reflected in the high cancer incidence observed in individuals with deficiencies in any o...

  17. Synthesis and structural studies of flavin and alloxazine adducts with O-nucleophiles

    NASA Astrophysics Data System (ADS)

    Ménová, Petra; Eigner, Václav; Čejka, Jan; Dvořáková, Hana; Šanda, Miloslav; Cibulka, Radek

    2011-10-01

    Five flavin (isoalloxazine) and alloxazine adducts with O-nucleophiles, 5-ethyl-4a-hydroxy-3,7,8,10-tetramethyl-4a,5-dihydroisoalloxazine ( 1a-OH), 5-ethyl-4a-hydroxy-3,10-dimethyl-4a,5-dihydroisoalloxazine ( 1b-OH), 5-ethyl-4a-methoxy-3,10-dimethyl-4a,5-dihydroisoalloxazine ( 1b-OMe), 5-ethyl-4a-hydroxy-1,3-dimethyl-4a,5-dihydroalloxazine ( 2a-OH) and 5-ethyl-4a-methoxy-1,3-dimethyl-4a,5-dihydroalloxazine ( 2a-OMe) were prepared from the corresponding salts, 5-ethyl-3,7,8,10-tetramethylisoalloxazinium ( 1a), 5-ethyl-3,10-dimethylisoalloxazinium ( 1b) and 5-ethyl-1,3-dimethylalloxazinium ( 2a) perchlorates by the addition of a nucleophile (water or methanol) and triethylamine as a base. The prepared adducts represent artificial analogs of flavin cofactor derivatives which are essential for the functioning of flavoenzymes. They were characterized by 1H and 13C NMR, HR-MS and UV-VIS spectra. In the cases of 1a-OH, 1b-OH, and 2a-OMe, the crystal structures were determined by X-ray diffraction. Flavinium and alloxazinium salts are in rapid equilibria with their adducts in water or methanolic solutions without the presence of a base. It was found that the equilibrium constants for flavin adduct formation is higher by six orders of magnitude than those for alloxazine derivatives. The presence of the sp 3 hybridized C4a atom in the molecule of the adducts causes deviation from planarity. The interplanar angles between benzene and the pyrimidine ring were found to be 31.5°, 23.64° and 15.62° for 1a-OH, 1b-OH and 2a-OMe, respectively, which are much higher than those of previously published adducts of C-nucleophiles. In isoalloxazine adducts, delocalization of π electrons between the N10-C10a and C10a-N1 bonds was detected while the length of the N10-C10a and C10a-N1 bonds in the alloxazine adducts corresponds to a double and single bond, respectively.

  18. White blood cell DNA adducts and fruit and vegetable consumption in bladder cancer.

    PubMed

    Peluso, M; Airoldi, L; Magagnotti, C; Fiorini, L; Munnia, A; Hautefeuille, A; Malaveille, C; Vineis, P

    2000-02-01

    The 'Mediterranean diet', a diet rich in cereals, fruit and vegetables, has been associated with lowering the risk of a variety of cancers of the digestive tract and the bladder. In a previous study, we showed that the high phenolic content these dietary components produce in the urine could be associated with higher antimutagenic properties of the urine and lower arylamine-DNA adducts in exfoliated bladder cells. We have conducted a case-control study on 162 bladder cancer patients and 104 hospital controls. Total aromatic DNA adducts were measured in white blood cells (WBC) of all subjects by (32)P-post-labelling. Genetically based metabolic polymorphisms were analysed by PCR-RFLP (NAT2, GSTM1, GSTT1, GSTP1, COMT and NQO1). All subjects were interviewed about their tobacco use, dietary habits and other risk factors. The odds ratio (OR) for the risk of bladder cancer according to the presence/absence of WBC DNA adducts (detection limit 0.1 RALx10(8)) was 3.7 [95% confidence interval (CI) 2.2-6.3] and a dose-response relationship with levels of adducts was apparent. The association between case/control status and the presence of WBC DNA adducts was significantly stronger in the subjects who consumed fewer portions of fruit or vegetables per day (OR 7.80, 95% CI 3.0-20.30 for 0-1 portions of vegetables) than in the heavy consumers (OR 4.98 for consumers of 2 portions daily, OR 1.97 for consumers of > or =3 portions; similar but lower estimates were found for the intake of fruit). No association was noticed between tobacco smoking and WBC DNA adducts. Only NAT-2, among the several genotypes considered, was associated in a statistically significant way with the risk of bladder cancer (OR 1.72, 95% CI 1.03-2.87) and with the levels of WBC DNA adducts. Our report suggests that fruit and vegetables could protect against bladder cancer by inhibiting the formation of DNA adducts. PMID:10657956

  19. IR spectra and structure of benzylidenemalononitrile and its cyanide, methoxide and heptylamine adducts: experimental and ab initio studies

    NASA Astrophysics Data System (ADS)

    Binev, Ivan G.; Binev, Yuri I.; Stamboliyska, Bistra A.; Juchnovski, Ivan N.

    1997-12-01

    The potassium cyanide, alkali-metal methoxide and heptylamine adducts of benzylidenemalononitrile were prepared as dimethyl sulphoxide (DMSO) and DMSO- d6 solutions; their structures were studied by IR spectroscopy and ab initio force field calculations. The cyanide and methoxide adducts have a carbanionic structure, whereas heptylamine forms a zwitterion. The IR spectra of the adducts studied are characterized by very intense, low-frequency νCN bands with a strong νCNs- νCNas splitting. The changes in the structure and force field of benzylidenemalononitrile accompanying its conversion into the adducts studied are essential and are spread over the whole molecule. The anionic charge is localized mainly within the dicyanomethide groups of the adducts.

  20. Cigarette Smoking, BPDE-DNA Adducts, and Aberrant Promoter Methylations of Tumor Suppressor Genes (TSGs) in NSCLC from Chinese Population.

    PubMed

    Jin, Yongtang; Xu, Peiwei; Liu, Xinneng; Zhang, Chunye; Tan, Cong; Chen, Chunmei; Sun, Xiaoyu; Xu, Yingchun

    2016-01-01

    Non-small cell lung cancer (NSCLC) is related to the genetic and epigenetic factors. The goal of this study was to determine association of cigarette smoking and BPDE-DNA adducts with promoter methylations of several genes in NSCLC. Methylation of the promoters of p16, RARβ, DAPK, MGMT, and TIMP-3 genes of tumor tissues from 199 lung cancer patients was analyzed with methylation-specific PCR (MSP), and BPDE-DNA adduct level in lung cancer tissue was obtained by ELISA. Level of BPDE-DNA adduct increased significantly in males, aged people (over 60 years), and smokers; however, no significant difference was found while comparing the BPDE-DNA adduct levels among different tumor types, locations, and stages. Cigarette smoking was also associated with increased BPDE-DNA adducts level (OR = 2.43, p > .05) and increased methylation level in at least 1 gene (OR = 5.22, p < .01), both in dose-response manner. Similarly, cigarette smoking also significantly increase the risk of p16 or DAPK methylation (OR = 3.02, p < .05 for p16, and 3.66, p < .05 for DAPK). The highest risk of BPDE-DNA adducts was detected among individuals with cigarette smoking for more than 40 pack-years (OR = 4.21, p < .01). Furthermore, the present study did not show that BPDE-DNA adducts are significantly associated with abnormal TSGs methylations in NSCLC, including SCC and AdO, respectively. Conclusively, cigarette smoking is significantly associated with the increase of BPDE-DNA adduct level, promoter hypermethylation of p16 and DAPK genes, while BPDE-DNA adduct was not significantly related to abnormal promoter hypermethylation in TSGs, suggesting that BPDE-DNA adducts and TSGs methylations play independent roles in NSCLC. PMID:27042875

  1. Stereochemical Configuration of 4-Hydroxy-2-nonenal-Cysteine Adducts and Their Stereoselective Formation in a Redox-regulated Protein*

    PubMed Central

    Wakita, Chika; Maeshima, Takuya; Yamazaki, Atsushi; Shibata, Takahiro; Ito, Sohei; Akagawa, Mitsugu; Ojika, Makoto; Yodoi, Junji; Uchida, Koji

    2009-01-01

    4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, preferentially reacts with cysteine residues to form a stable HNE-cysteine Michael addition adduct possessing three chiral centers. Here, to gain more insight into sulfhydryl modification by HNE, we characterized the stereochemical configuration of the HNE-cysteine adducts and investigated their stereoselective formation in redox-regulated proteins. To characterize the HNE-cysteine adducts by NMR, the authentic (R)-HNE- and (S)-HNE-cysteine adducts were prepared by incubating N-acetylcysteine with each HNE enantiomer, both of which provided two peaks in reversed-phase high performance liquid chromatography (HPLC). The NMR analysis revealed that each peak was a mixture of anomeric isomers. In addition, mutarotation at the anomeric center was also observed in the analysis of the nuclear Overhauser effect. To analyze these adducts in proteins, we adapted a pyridylamination-based approach, using 2-aminopyridine in the presence of sodium cyanoborohydride, which enabled analyzing the individual (R)-HNE- and (S)-HNE-cysteine adducts by reversed-phase HPLC following acid hydrolysis. Using the pyridylamination method along with mass spectrometry, we characterized the stereoselective formation of the HNE-cysteine adducts in human thioredoxin and found that HNE preferentially modifies Cys73 and, to the lesser extent, the active site Cys32. More interestingly, the (R)-HNE- and (S)-HNE-cysteine adducts were almost equally formed at Cys73, whereas Cys32 exhibited a remarkable preference for the adduct formation with (R)-HNE. Finally, the utility of the method for the determination of the HNE-cysteine adducts was confirmed by an in vitro study using HeLa cells. The present results not only offer structural insight into sulfhydryl modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein S-associated aldehydes in vitro and in vivo. PMID:19692331

  2. Benzo(a)pyrenediolepoxide-hemoglobin adducts and 3-hydroxy-benzo(a)pyrene urinary excretion profiles in rats subchronically exposed to benzo(a)pyrene.

    PubMed

    Bouchard, M; Viau, C

    1995-01-01

    The time profiles of benzo(a)pyrenediolepoxide (BaPDE)-hemoglobin (Hb) adduct formation and 3-hydroxybenzo(a)pyrene (3-OHBaP) urinary excretion were studied in male Sprague-Dawley rats exposed to daily benzo(a)pyrene (BaP) intraperitoneal doses of 1.25, 6.25, and 31.25 mumol/kg administered Tuesday to Friday for 4 consecutive weeks. Blood was withdrawn weekly, on Tuesdays, prior to dosing. Twenty-four-hour urine samples were collected on Mondays (following 72 h without treatment) and Thursdays. Analytes were quantified by high performance liquid chromatography (HPLC)/fluorescence. Exposure to BaP resulted in the accumulation of BaPDE-Hb adducts, reaching an average of 1.2 +/- 0.3, 8.3 +/- 1.9, and 38.2 +/- 6.1 pmol/g Hb for the 1.25, 6.25, and 31.25 mumol/kg per day doses after 4 weeks of treatment. The expected saw tooth excretion profile of 3-OHBaP was observed, with peaks on Thursdays and troughs on Mondays, and showed a progressive rise on both Mondays and Thursdays. Increase in Monday values with time suggested a possible increase in BaP body burden during exposure. To verify this aspect further, the urinary excretion kinetic of 3-OHBaP following acute intraperitoneal dosing (31.25 mumol/kg) was determined. Urine samples were collected at frequent timed intervals for up to 164 h post-dosing. Two-step elimination was observed, the second step having a half-life of 25 h, presumably linked to the slow release of BaP accumulated in fatty tissues upon repeated treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8534197

  3. COMPARISON OF DNA ADDUCT LEVELS IN HUMAN PLACENTA FROM POLYCHLORINATED BIPHENYL EXPOSED WOMEN AND SMOKERS IN WHICH CYP 1A1 LEVELS ARE SIMILARLY ELEVATED

    EPA Science Inventory

    Cigarette smoking is associated with high elevations in levels of both cytochrome P450 1A1 (CYP 1A1) and DNA adducts in human placenta. he identity of the smoking related adducts is not known. he DNA adducts identified in placenta of smokers could result from chemicals resent in ...

  4. 32 P-POSTLABELING AND HPLC SEPARATION OF DNA ADDUCTS FORMED BYDIESEL EXHAUST EXTRACTS IN VITRO AND IN MOUSE SKIN AND LUNG AFTERTOPICAL TREATMENT

    EPA Science Inventory

    Diesel exhaust extracts contain many carcinogenic compounds which have been shown to form PAH- and nitrated-PAH-DNA adducts in rodent skin and lung. he aim of this study was to characterize by "P-postlabeling, TLC and HPLC the primary postlabeled PAH-DNA adduct(s) formed in vitro...

  5. An Adenine-DNA Adduct Derived from Nitroreduction of 6-Nitrochrysene is more Resistant to Nucleotide Excision Repair than Guanine-DNA Adducts

    PubMed Central

    Krzeminski, Jacek; Kropachev, Konstantin; Reeves, Dara; Kolbanovskiy, Aleksandr; Kolbanovskiy, Marina; Chen, Kun-Ming; Sharma, Arun K.; Geacintov, Nicholas; Amin, Shantu; El-Bayoumy, Karam

    2013-01-01

    Previous studies in rats, mice and in vitro systems showed that 6-NC can be metabolically activated by two major pathways: 1) the formation of N-hydroxy-6-aminochrysene by nitroreduction to yield three major adducts: N-(dG-8-yl)-6-AC, 5-(dG-N2-yl)-6-AC and N-(dA-8-yl)-6-AC, and 2) the formation of trans-1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C) by a combination of nitroreduction and ring oxidation pathways to yield: N-(dG-8-yl)-1,2-DHD-6-AC, 5-(dG-N2-yl)-1,2-DHD-6-AC and N-(dA-8-yl)-1,2-DHD-6-AC. These DNA lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. Here we compared for the first time, in HeLa cell extracts in vitro, the relative nucleotide excision repair (NER) efficiencies of DNA lesions derived from simple nitroreduction and from a combination of nitroreduction and ring oxidation pathways. We show that the N-(dG-8-yl)-1,2-DHD-6-AC adduct is more resistant to NER than the N-(dG-8-yl)-6-AC adduct by a factor of ~2. Furthermore, the N-(dA-8-yl)-6-AC is much more resistant to repair since its NER efficiency is ~ 8-fold lower than that of the N-(dG-8-yl)-6-AC adduct. On the basis of our previous study and the present investigation, lesions derived from 6-NC and benzo[a]pyrene can be ranked from the most to the least resistant lesion as follows: N-(dA-8-yl)-6-AC > N-(dG-8-yl)-1,2-DHD-6-AC > 5-(dG-N2-yl)-6-AC ~ N-(dG-8-yl)-6-AC ~ (+)-7R,8S,9S,10S-benzo[a]pyrene diol epoxide-derived trans-anti-benzo[a]pyrene-N2-dG adduct. The slow repair of the various lesions derived from 6-NC and thus their potential persistence in mammalian tissue, could in part account for the powerful carcinogenicity of 6-NC as compared to B[a]P in the rat mammary gland. PMID:24112095

  6. Conformational, IR spectroscopic and electronic properties of conium alkaloids and their adducts with C60 fullerene

    NASA Astrophysics Data System (ADS)

    Zabolotnyi, M. A.; Prylutskyy, Yu I.; Poluyan, N. A.; Evstigneev, M. P.; Dovbeshko, G. I.

    2016-08-01

    Conformational, IR spectroscopic and electronic properties of the components of Conium alkaloids (Conium maculatum) in aqueous environment were determined by model calculations and experiment. With the help of FT-IR spectroscopy the possibility of formation of an adduct between γ-coniceine alkaloid and C60 fullerene was demonstrated, which is important for further application of conium analogues in biomedical purposes.

  7. Adduct supported analysis of γ-hydroxybutyrate in human serum with LC-MS/MS.

    PubMed

    Dziadosz, Marek; Weller, Jens-Peter; Klintschar, Michael; Teske, Jörg

    2013-08-01

    To avoid the detection of small fragmentation products of γ-hydroxybutyrate (GHB), a liquid chromatography-tandem mass spectrometry GHB quantification method in human serum supported by adduct formation was developed and validated. The continuous infusion of GHB/GHB-D6 made the identification of two adducts possible and GHB/GHB-D6 sodium acetate adduct fragmentation was used as target mass transition. A Luna 5 μm C18 (2) 100 A, 150 mm × 2 mm analytical column and elution with a programmed flow of the mobile phase consisting of 10% A (H2O/methanol = 95/5, v/v) and 90% B (H2O/methanol = 3/97, v/v), both with 10 mM ammonium acetate and 0.1% acetic acid (pH = 3.2), were used. Protein precipitation with 1 mL of the mobile phase B was used as the sample preparation. The calculated limit of detection/quantification was 1 μg/mL. The presented study shows that the fragmentation of GHB sodium acetate adducts is an effective way of quantification of this small molecule and is an interesting alternative to other methods based on the detection of ions smaller than 85 Da. This fact together with the short analysis time of 3 min and the fast sample preparation make this method very attractive for forensic/clinical application. PMID:23712650

  8. PROTEIN ADDUCT FORMING CHEMICALS FOR EXPOSURE MONITORING: CHEMICALS SELECTED FOR FURTHER STUDY

    EPA Science Inventory

    The present report is an in-depth characterization of twenty-two chemicals and the protein (primarily hemoglobin) adducts that are formed following exposure to these compounds. These chemicals were recommended for further study in a recent internal report which described the stat...

  9. Spin-trap-radical chromatography of spin adducts produced from L-valine by. gamma. -irradiation

    SciTech Connect

    Makiino, K.; Suzuki, N.; Moriya, F.; Rokushika, S.; Hatano, H.

    1980-01-01

    Diastereomeric spin adducts produced by reaction of 2-methyl-2-nitrosopropane with the short-lived radicals from L-valine by ..gamma..-irradiation could be separated and identified by means of high performance liquid chromatography and ESR spectroscopy. 6 figures.

  10. DNA adducts induced by in vitro activation of extracts of diesel and biodiesel exhaust particles

    EPA Science Inventory

    AbstractContext: Biodiesel and biodiesel-blend fuels offer a renewable alternative to petroleum diesel, but few data are available concerning the carcinogenic potential of biodiesel exhausts. Objectives: We compared the formation of covalent DNA adducts by the in vitro metabol...

  11. IN VITRO METABOLISM AND DNA ADDUCT FORMATION FROM THE MUTAGENIC ENVIRONMENTAL CONTAMINANT 2-NITROFLURORANTHENE

    EPA Science Inventory

    The metabolism and DNA adduct formation by the mutagenic environmental contaminant 2-nitrofluoranthene (2-NFA) was studied. ncubation under aerobic conditions with liver microsomes of rats pretreated with 3-methylcholanthrene yielded 2-NFA tran-7.8-dihydrodiol, 2-NFA tran-9,10-di...

  12. Noni juice reduces lipid peroxidation-derived DNA adducts in heavy smokers.

    PubMed

    Wang, Mian-Ying; Peng, Lin; Jensen, Claude J; Deng, Shixin; West, Brett J

    2013-03-01

    Food plants provide important phytochemicals which help improve or maintain health through various biological activities, including antioxidant effects. Cigarette smoke-induced oxidative stress leads to the formation of lipid hydroperoxides (LOOHs) and their decomposition product malondialdehyde (MDA), both of which cause oxidative damage to DNA. Two hundred forty-five heavy cigarette smokers completed a randomized, double-blind, placebo-controlled clinical trial designed to investigate the effect of noni juice on LOOH- and MDA-DNA adducts in peripheral blood lymphocytes (PBLs). Volunteers drank noni juice or a fruit juice placebo every day for 1 month. DNA adducts were measured by (32)P postlabeling analysis. Drinking 29.5-118 mL of noni juice significantly reduced adducts by 44.6-57.4%. The placebo, which was devoid of iridoid glycosides, did not significantly influence LOOH- and MDA-DNA adduct levels in current smokers. Noni juice was able to mitigate oxidative damage of DNA in current heavy smokers, an activity associated with the presence of iridoids. PMID:24804023

  13. DNA ADDUCTS IN MARINE MUSSEL MYTILUS GALLOPROVINCIALIS LIVING IN POLLUTED AND UNPOLLUTED ENVIRONMENTS

    EPA Science Inventory

    We have used a generally applicable 32P-postlabeling assay to examine for the presence of DNA adducts in mussels experimentally exposed to known carcinogens and in mussels collected from sites impacted by wastewaters. Mussels exposed to seawater artificially polluted with 2-amino...

  14. Analysis of regiospecific triacylglycerols by electrospray ionization-mass spectrometry 3 of lithiated adducts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some regiospecific triacylglycerol standards containing normal fatty acids, e.g., 1,3-dioleoyl-2-palmitoyl-glycerol (OPO) and 1,2-dioleoyl-3-palmitoyl-rac-glycerol (OOP), were analyzed by the electrospray ionization MS3 of their lithiated adducts. The fragment ions of the MS3 from the loss of alpha,...

  15. Noni juice reduces lipid peroxidation–derived DNA adducts in heavy smokers

    PubMed Central

    Wang, Mian-Ying; Peng, Lin; Jensen, Claude J; Deng, Shixin; West, Brett J

    2013-01-01

    Food plants provide important phytochemicals which help improve or maintain health through various biological activities, including antioxidant effects. Cigarette smoke–induced oxidative stress leads to the formation of lipid hydroperoxides (LOOHs) and their decomposition product malondialdehyde (MDA), both of which cause oxidative damage to DNA. Two hundred forty-five heavy cigarette smokers completed a randomized, double-blind, placebo-controlled clinical trial designed to investigate the effect of noni juice on LOOH- and MDA-DNA adducts in peripheral blood lymphocytes (PBLs). Volunteers drank noni juice or a fruit juice placebo every day for 1 month. DNA adducts were measured by 32P postlabeling analysis. Drinking 29.5–118 mL of noni juice significantly reduced adducts by 44.6–57.4%. The placebo, which was devoid of iridoid glycosides, did not significantly influence LOOH- and MDA-DNA adduct levels in current smokers. Noni juice was able to mitigate oxidative damage of DNA in current heavy smokers, an activity associated with the presence of iridoids. PMID:24804023

  16. COMPARISON OF DNA ADDUCTS FROM COMPLEX MIXTURE EXPOSURES IN VARIOUS HUMAN TISSUES AND EXPERIMENTAL SYSTEMS

    EPA Science Inventory

    DNA adducts derived from complex mixtures of polycyclic aromatic compounds emitted from tobacco smoke are compared to industrial pollution sources (e.g., coke ovens and aluminum, smelters), smoky coal burning, and urban air pollution. xposures to coke oven emissions and smoky coa...

  17. Dynamic vocal fold parameters with changing adduction in ex-vivo hemilarynx experiments.

    PubMed

    Döllinger, Michael; Berry, David A; Kniesburges, Stefan

    2016-05-01

    Ex-vivo hemilarynx experiments allow the visualization and quantification of three-dimensional dynamics of the medial vocal fold surface. For three excised human male larynges, the vibrational output, the glottal flow resistance, and the sound pressure during sustained phonation were analyzed as a function of vocal fold adduction for varying subglottal pressure. Empirical eigenfunctions, displacements, and velocities were investigated along the vocal fold surface. For two larynges, an increase of adduction level resulted in an increase of the glottal flow resistance at equal subglottal pressures. This caused an increase of lateral and vertical oscillation amplitudes and velocity indicating an improved energy transfer from the airflow to the vocal folds. In contrast, the third larynx exhibited an amplitude decrease for rising adduction accompanying reduction of the flow resistance. By evaluating the empirical eigenfunctions, this reduced flow resistance was assigned to an unbalanced oscillation pattern with predominantly lateral amplitudes. The results suggest that adduction facilitates the phonatory process by increasing the glottal flow resistance and enhancing the vibrational amplitudes. However, this interrelation only holds for a maintained balanced ratio between vertical and lateral displacements. Indeed, a balanced vertical-lateral oscillation pattern may be more beneficial to phonation than strong periodicity with predominantly lateral vibrations. PMID:27250133

  18. New adduct of abietane-type diterpene from Salvia leriifolia Benth.

    PubMed

    Hussain, Amjad; Adhikari, Achyut; Iqbal Choudhary, M; Ayatollahi, Syed Abdulmajid; Atta-Ur-Rahman

    2016-07-01

    A new adduct of abietane-type diterpene, salvialeriicone (1), was isolated from Salvia leriifolia Benth., along with a new chemical entity nor-abietane diterpene, 2-isopropyl-8,8-dimethyl-7,8-dihydrophenanthrene-1,4,5(6H)-trione (2). Their structures were determined using mass spectrometry, and 1D- and 2D-NMR spectroscopy. PMID:27266891

  19. Enzyme-DNA biocolloids for DNA adduct and reactive metabolite detection by chromatography-mass spectrometry.

    PubMed

    Bajrami, Besnik; Hvastkovs, Eli G; Jensen, Gary C; Schenkman, John B; Rusling, James F

    2008-02-15

    Silica microbead bioreactors (0.5 microm diameter) coated with DNA and enzymes were fabricated to measure reactive metabolite and DNA-adduct formation rates relevant to genotoxicity screening. Cytochrome (cyt) P450 2E1, cyt P450(cam), and myoglobin (Mb) were incorporated into thin films with DNA using the electrostatic layer-by-layer (LbL) method. The utility of these biocolloids was demonstrated by oxidation of guaiacol, styrene, and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Enzyme turnover rates for formation of reactive metabolites were monitored using gas chromatography/mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC-MS). Capillary LC-MS/MS was employed to determine DNA nucleobase adducts after catalyzing the reactive metabolite formation with DNA-enzyme biocolloids and then using neutral thermal hydrolysis on the biocolloids. Dramatic improvements in surface area to volume ratio over similar films on macroscopic surfaces opens new avenues for genotoxicity screening and enabled the first use of pure cyt P450 enzymes in enzyme-DNA films to produce DNA adducts. The method makes possible identification and formation rate measurements of major and minor DNA adducts as well as the metabolites themselves in <5 min of reaction time using relevant human liver enzymes. PMID:18217727

  20. DNA ADDUCTS AND PERSONAL AIR MONITORING OF CARCINOGENIC POLYCYCLIC AROMATIC HYDROCARBONS IN AN ENVIRONMENTALLY EXPOSED POPULATION

    EPA Science Inventory

    The effect of personal exposure to air pollution on DNA adducts in humans was analyzed in a group (N=30) of women working outdoors (up to 8 h/day) as postal workers or gardeners in the city of Teplice, CZ (Northern Bohemia) where winter. inversions may result in high levels of ai...

  1. Biomonitoring of nitropolynuclear aromatic hydrocarbons via protein and DNA adducts. Research report, August 1987-July 1991

    SciTech Connect

    El-Bayoumy, K.; Johnson, B.E.; Roy, A.K.; Upadhyaya, P.; Partian, S.J.

    1994-01-01

    The authors developed methods to detect and quantify adducts of 1-nitropyrene and 1,6-dinitropyrene with proteins and with DNA. ((3)H)-Nitropyrene given to male or female F344 or Sprague-Dawley rats by gavage at 5 dose levels (0.1 to 1000 ug/kg) led to stable hemoglobin adducts, which accounted for 0.08% + or - 0.005% of the dose. The radioactivity was associated with the heme moiety rather than the globin and was cleared with a half-life of 13.6 days ((3)H)1,6-dintropyrene also bound to rat hemoglobin at a level that is 0.04% of the dose given by gavage and was bound to the heme moiety. Comparing the rather complex DNA adduct pattern formed with 1-nitropyrene to the simple adduct pattern formed with 1,6-dinitropyrene in the rat suggests that N-(deoxyguanosin-8-yl)-1-amino-6-nitropyrene, derived from 1,6-dinitropyrene, may be a better dosimeter, even though 1,6-dinitropyrene is present in environment in far lower concentrations than 1-nitropyrene.

  2. The use of lithiated adducts for structural analysis of acylglycerols by MS-ESI

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Electrospray ionization mass spectrometry (ESI-MS) using lithium adducts is the method of choice for the analysis of acyglycerols. The method can be used for the identification of the structures of fatty acid constituents, including the number and location of double bonds and hydroxyl groups. The me...

  3. Separation and characterization of oxidized isomeric lipid-peptide adducts by ion mobility mass spectrometry.

    PubMed

    Milic, Ivana; Kipping, Marc; Hoffmann, Ralf; Fedorova, Maria

    2015-12-01

    Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid-protein adducts by liquid chromatography-MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid-peptide adducts generated by incubating model peptides corresponding to the amphipathic β1 sheet sequence of apolipoprotein B-100 with 1-palmitoyl-2-(oxo-nonanoyl)-sn-glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide-lipid adducts was separated by TWIMS, which was especially important for the identification of two mono-PONPC-peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide-lipid adduct possessing most likely different orientations of the hydrophobic sn-1 fatty acyl residue and head group of PONPC, relative to the peptide backbone. PMID:26634972

  4. Lipoxidation adducts with peptides and proteins: deleterious modifications or signaling mechanisms?

    PubMed

    Domingues, Rosário M; Domingues, Pedro; Melo, Tânia; Pérez-Sala, Dolores; Reis, Ana; Spickett, Corinne M

    2013-10-30

    Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,β-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. PMID:23770299

  5. Situational Restriction of Elevation in Adduction Relieved by Faden on the Medial Rectus

    PubMed Central

    Muralidhar, R.; Vijayalakshmi, P.; Sujatha, K.; Shetty, Shashikanth; Malay, K.; Rosenberg, Steve

    2016-01-01

    We describe a patient with situational restriction of elevation in adduction in his left eye. Clinical examination pointed to instability of the left medial rectus pulley. This was corrected by Faden on the medial rectus. The importance of this relatively new concept in identifying and treating orbital pulley instability is discussed. PMID:27162460

  6. Structural analysis and aggregation propensity of reduced and nonreduced glycated insulin adducts.

    PubMed

    Alavi, Parnian; Yousefi, Reza; Amirghofran, Sara; Karbalaei-Heidari, Hamid Reza; Moosavi-Movahedi, Ali Akbar

    2013-06-01

    The milieu within pancreatic β cells represents a favorable environment for glycation of insulin. Therefore, in this study, insulin samples were individually subjected to glycation under reducing and nonreducing conditions. As monitored by ortho-phthalaldehyde and fluorescamine assays, the reduced glycated insulin adduct demonstrates extensively higher level of glycation than the nonreduced glycated counterpart. Also, gel electrophoresis experiments suggest a significant impact of glycation under a reducing system on the level of insulin oligomerization. Furthermore, reduced and nonreduced glycated insulin adducts respectively exhibit full and partial resistance against dithiothreitol-induced aggregation. The results of thioflavin T and Congo red assays suggest the existence of a significant quantity of amyloid-like entities in the sample of reduced glycated insulin adduct. Both fluorescence and far-ultraviolet circular dichroism studies respectively suggest that the extents of unfolding and secondary structural alteration were closely correlated to the level of insulin glycation. Moreover, the surface tension of two glycated insulin adducts was inversely correlated to their glycation extents and to the quantity of exposed hydrophobic patches. Overall, the glucose-modified insulin molecules under reducing and nonreducing systems display different structural features having significant consequences on aggregation behaviors and surface tension properties. The particular structural constraints of glycated insulin may reduce the binding interaction of this hormone to its receptor which is important for both insulin function and clearance. PMID:23584594

  7. MUTATIONAL SPECTRUM AND RECOMBINOGENIC EFFECTS INDUCED BY AMINOFLUORENE ADDUCTS IN BACTERIOPHAGE M13 (JOURNAL VERSION)

    EPA Science Inventory

    Double stranded replicative form (RFI) DNA of bacteriophage M13mp10 has been modified in vitro to various extents with N-hydroxy-2-aminofluorene (N-OH-AF) and then transfected into E. coli cells. HPLC analysis of the modified DNA shows that only dG-C8-AF adducts are formed. Appro...

  8. Hemoglobin adducts of aromatic amines: associations with smoking status and type of tobacco.

    PubMed Central

    Bryant, M S; Vineis, P; Skipper, P L; Tannenbaum, S R

    1988-01-01

    Hemoglobin adducts of 15 aromatic amines were determined in nonsmokers and smokers of blond- or black-tobacco cigarettes living in Turin, Italy. The subjects were all males age 55 or less and were representative of the population previously examined in a case/control study of bladder cancer. 4-Aminobiphenyl adduct levels were found to be significantly different in the three groups, and the differences were approximately proportional to the relative risk of each group. Adjustment for age and cigarette consumption did not materially influence the differences. A significant correlation of adduct levels with cigarette consumption was also observed for all smokers as well as for smokers of blond tobacco. Other amines for which significant differences between smokers and nonsmokers were observed were 3-aminobiphenyl, 2-naphthylamine, o- and p-toluidine, 2,4-dimethylaniline, and 2-ethylaniline. Some of these amines are human bladder carcinogens, and their occurrence in blood as hemoglobin adducts is evidence for their metabolic activation. Thus, by a combination of traditional epidemiological methods and modern chemical analyses, we have provided evidence for a biochemical basis for the often observed association between cigarette smoking and bladder cancer. PMID:3200858

  9. 40 CFR 721.465 - Alkoxylated alkylpolyol acrylates, adduct with alkylamine (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.465 Alkoxylated alkylpolyol acrylates, adduct with alkylamine (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The...

  10. 40 CFR 721.465 - Alkoxylated alkylpolyol acrylates, adduct with alkylamine (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.465 Alkoxylated alkylpolyol acrylates, adduct with alkylamine (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The...

  11. FORMATION OF CIGARETTE SMOKE-INDUCED DNA ADDUCTS IN THE RAT LUNG AND NASAL MUCOSA

    EPA Science Inventory

    The formation of DNA adducts in the nasal, lung, and liver tissues of rats exposed daily to fresh smoke from a University of Kentucky refernece cigarette (2R1) for up to 40 weeks was examined. he amount of smoke total particulate matter (TPM) inhaled and the blood carboxyhemoglob...

  12. Creating Context for the Use of DNA Adduct Data in Risk Assessment

    EPA Science Inventory

    Assessments of human cancer risk require the integration of diverse types of data. Advancing technologies for quantitative measurements at the sub-cellular domain raise the critical issue of interpretation and use of DNA adduct data in context with current understanding of cancer...

  13. MALDI-TOF analysis of steroid/PAH-modified DNA adducts at the femtomole level

    SciTech Connect

    Gooden, J.K.; Gross, M.L.; Stack, D.

    1995-12-31

    Covalent binding of polycyclic aromatic hydrocarbons (PAH`s) and steroids to DNA to form adducts is one of the first events in the process of tumor initiation in carcinogenesis. Structure elucidation and characterization of these adducts provide important information that leads to further understanding of their biological metabolic pathways. In in vivo and in vitro steroid/PAH-DNA binding studies, the reaction products (adducts) are often of low amount (low picomole to femtomole). Previous results from this laboratory have shown that the sensitivity of MALDI-TOF can be improved by proper matrix selection. An increase in sensitivity can also be obtained with the use of d-fucose as a co-matrix. In this study 4-phenyl-{alpha}-cyanocinnamic acid, PCC, 4-benzyloxy-{alpha}-cyanocinnamic acid, BCC, ferulic acid, FA, {alpha}-cyano-4-hydroxycinnamic acid, 4HCCA, and 3-(2-naphthyl)-2-cyanoacrylic NCA, were used in the determination of the limit of detection for two different DNA adducts dibenzocarbazole-5-N7Ade, and 4-hydroxyestrone-N7Gua.

  14. 78 FR 46260 - Sorbitan Monooleate Ethylene Oxide Adduct; Exemption From the Requirement of a Tolerance

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-31

    ... monooleate ethylene oxide adduct is low; the LD 50 was >25,000 mg/kg in the rat and mouse. Also, no systemic... considered nonmutagenic. Evidence of carcinogenicity was not observed in mice. In rats, the incidence of... studies in rats and mice. Also, toxicity was only observed at doses (>10,000 mg/kg/day) well above...

  15. The Utility of Naphthyl-Keratin Adducts as Biomarkers for Jet-Fuel Exposure

    EPA Science Inventory

    We investigated the association between biomarkers of dermal exposure, naphthyl-keratin adducts (NKA), and urine naphthalene biomarker levels in 105 workers routinely exposed to jet-fuel. A moderate correlation was observed between NKA and urine naphthalene levels (p = 0.061). Th...

  16. 40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.3700 Fatty acid, ester with styrenated phenol... chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  17. 40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.3700 Fatty acid, ester with styrenated phenol... chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  18. 40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.3700 Fatty acid, ester with styrenated phenol... chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  19. 40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.3700 Fatty acid, ester with styrenated phenol... chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  20. 40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... phenol, ethylene oxide adduct. 721.3700 Section 721.3700 Protection of Environment ENVIRONMENTAL... Significant New Uses for Specific Chemical Substances § 721.3700 Fatty acid, ester with styrenated phenol... chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene...

  1. Crystal structure of ball-milled mixture of sodium chloride and magnesium chloride-ethanol adduct

    SciTech Connect

    Jiang Xue; Tian Xiuzhi; Fan Zhiqiang

    2008-02-05

    NaCl doped MgCl{sub 2}.nEtOH adducts were prepared by ball-milling MgCl{sub 2}.2.5EtOH with NaCl. Both the ball-milled MgCl{sub 2}.nEtOH/NaCl mixture and pure MgCl{sub 2}.2.5EtOH adducts were analyzed by X-ray diffraction (XRD), transmission electron microscope (TEM), thermogravimetry (TG) and differencial scanning calorimetry (DSC). A simple MgCl{sub 2}.nEtOH/NaCl mixture without ball-milling treatment was also studied for comparison. Two kinds of mixed crystals, Na{sub 2}MgCl{sub 4} and NaMgCl{sub 3}, were found to be formed in a ball-milled mixture that contained 16 mol.% NaCl. TG and DSC analysis of the samples also provided indirect evidences supporting the presence of the mixed crystals in the ball-milled mixture. Adding certain amounts of NaCl in MgCl{sub 2}.2.5EtOH adduct, either by co-milling or by simple mixing, greatly increased the thermal stability of the adduct, but thermal decomposition behaviour of the ball-milled mixture was still different from that of a simple mixture.

  2. (32)P-ADDUCT ASSAY: PRINCIPLE AND APPLICATIONS TO CARCINOGEN-EXPOSED ANIMAL AND HUMAN DNA

    EPA Science Inventory

    There is growing evidence that carcinogens initiate the malignant process via specific alterations in DNA structure, i.e., the covalent binding of carcinogens to DNA bases. Thus, carcinogen-DNA adducts represent as markers for tumor initiation. Several new techniques have been re...

  3. Identification of adducts formed by reaction of N-acetoxy-3,5-dimethylaniline with DNA

    PubMed Central

    Cui, Liang; Sun, Hsiao-Lan; Wishnok, John S.; Tannenbaum, Steven R.; Skipper, Paul L.

    2008-01-01

    Aromatic amines constitute one of the most extensively studied classes of chemical carcinogens. Although monocyclic aromatic amines are generally regarded as weak carcinogens, a recent epidemiologic study of bladder cancer found that the arylamine 3,5-dimethylaniline (3,5-DMA) may play a significant role in the etiology of this disease in man. Investigations using experimental animals also strongly suggested that DNA adducts—of indeterminate structure—formed by 3,5-DMA might account for its presumptive activity. The present study was undertaken to determine the structures of the major DNA adducts formed in vitro by the known, and possibly carcinogenic, N-hydroxylated metabolite. Calf thymus DNA (ct-DNA) was modified by reaction with N-acetoxy-3,5-dimethylaniline (N-AcO-3,5-DMA). After enzymatic hydrolysis of DNA to individual 2'-deoxyribonucleosides, adduct profiles were determined using HPLC/MS. 3,5-DMA formed four major DNA adducts, one to 2’-deoxyguanosine (dG), two to 2’-deoxyadenosine (dA), and one to 2’-deoxycytidine (dC). Reactions of N-AcO-3,5-DMA with dG, dA, and dC produced the same adducts as reaction with ct-DNA with very similar profiles. Adducts were isolated chromatographically and unambiguously characterized as N-(deoxyguanosin-8-yl)-3,5-dimethylaniline (dG-C8−3,5-DMA), 4-(deoxyadenosin-N6-yl)-3,5-dimethylaniline (dA-N6-3,5-DMA), N-(deoxyadenosin-8-yl)-3,5-dimethylaniline (dA-C8−3,5-DMA), and N-(deoxycytidin-5-yl)-3,5-dimethylaniline (dC-C5−3,5-DMA) by high-resolution mass spectra (HR-MS) and NMR spectroscopy including 1H-NMR, 13C-NMR, and two-dimensional NMR. This report includes the first detailed description of a dC adduct of an aromatic amine. The present results provide chemical support for a carcinogenic mechanism of action by 3,5-DMA based on N-hydroxylation and the intermediacy of a nitrenium ion in the formation of DNA adducts. PMID:18020398

  4. Role of CYP1B1 in PAH-DNA adduct formation and breast cancer risk

    SciTech Connect

    Goth-Goldstein, Regine; Russell, Marion L.; Muller, A.P.; Caleffi, M.; Eschiletti, J.; Graudenz, M.; Sohn, Michael D.

    2010-04-01

    This study investigated the hypothesis that increased exposure to polycyclic aromatic hydrocarbons (PAHs) increases breast cancer risk. PAHs are products of incomplete burning of organic matter and are present in cigarette smoke, ambient air, drinking water, and diet. PAHs require metabolic transformation to bind to DNA, causing DNA adducts, which can lead to mutations and are thought to be an important pre-cancer marker. In breast tissue, PAHs appear to be metabolized to their cancer-causing form primarily by the cytochrome P450 enzyme CYP1B1. Because the genotoxic impact of PAH depends on their metabolism, we hypothesized that high CYP1B1 enzyme levels result in increased formation of PAH-DNA adducts in breast tissue, leading to increased development of breast cancer. We have investigated molecular mechanisms of the relationship between PAH exposure, CYP1B1 expression and breast cancer risk in a clinic-based case-control study. We collected histologically normal breast tissue from 56 women (43 cases and 13 controls) undergoing breast surgery and analyzed these specimens for CYP1B1 genotype, PAH-DNA adducts and CYP1B1 gene expression. We did not detect any difference in aromatic DNA adduct levels of cases and controls, only between smokers and non-smokers. CYP1B1 transcript levels were slightly lower in controls than cases, but the difference was not statistically significant. We found no correlation between the levels of CYP1B1 expression and DNA adducts. If CYP1B1 has any role in breast cancer etiology it might be through its metabolism of estrogen rather than its metabolism of PAHs. However, due to the lack of statistical power these results should be interpreted with caution.

  5. Quantitative analysis of positional isomers of triacylglycerols via electrospray ionization tandem mass spectrometry of sodiated adducts.

    PubMed

    Herrera, Lisandra Cubero; Potvin, Michael A; Melanson, Jeremy E

    2010-09-01

    Herein we report a reversed-phase high-performance liquid chromatography tandem mass spectrometry (RP-HPLC/MS/MS) method for the analysis of positional isomers of triacylglycerols (TAGs) in vegetable oils. The fragmentation behavior of [M + X](+) ions (X = NH(4), Li, Na or Ag) was studied on a quadrupole-time-of-flight (Q-TOF) mass spectrometer under low-energy collision-induced dissociation (CID) conditions. Mass spectra that were dependent on the X(+) ion and the nature and position of the acyl substituents were observed for four pairs of 'AAB/ABA'-type TAGs, namely PPO/POP, OOP/OPO, LLO/LOL and OOL/OLO (where P is 16:0, palmitic acid; O is 18:1, oleic acid; and L is 18:2, linoleic acid). For the majority of [M + X](+) adducts, the loss of the fatty acid in the outer positions (sn-1 or sn-3) was favored over the loss in the central position (sn-2), which enabled the determination of the fractional abundance of the isomers. Ratios of the intensity of fragment ions at various AAB/ABA compositions produced linear calibration curves with positive slopes, comparable to those obtained traditionally by ESI-MS/MS of [M + NH(4)](+) adducts. The only exceptions were the [M + Ag](+) adducts of the PPO/POP system, which produced calibration curves with negative slopes. Sodium adducts provided the most consistent level of isomeric discrimination for the TAGs studied and also offered the most convenience in that they required no additive to the mobile phase. Therefore, calibration curve data derived from [M + Na](+) adducts were applied to the quantification of TAG regioisomers in sunflower and olive oils. The regiospecific analysis showed that palmitic acid was typically located at positions sn-1 or sn-3, whereas unsaturated fatty acids, oleic and linoleic acids were mostly found at the sn-2 position. PMID:20814981

  6. Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity

    PubMed Central

    James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

    2015-01-01

    Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury. PMID:26208104

  7. Comparison of Bile Acids and Acetaminophen Protein Adducts in Children and Adolescents with Acetaminophen Toxicity.

    PubMed

    James, Laura; Yan, Ke; Pence, Lisa; Simpson, Pippa; Bhattacharyya, Sudeepa; Gill, Pritmohinder; Letzig, Lynda; Kearns, Gregory; Beger, Richard

    2015-01-01

    Metabolomics approaches have enabled the study of new mechanisms of liver injury in experimental models of drug toxicity. Disruption of bile acid homeostasis is a known mechanism of drug induced liver injury. The relationship of individual bile acids to indicators of oxidative drug metabolism (acetaminophen protein adducts) and liver injury was examined in children with acetaminophen overdose, hospitalized children with low dose exposure to acetaminophen, and children with no recent exposure to acetaminophen. Nine bile acids were quantified through targeted metabolomic analysis in the serum samples of the three groups. Bile acids were compared to serum levels of acetaminophen protein adducts and alanine aminotransferase. Glycodeoxycholic acid, taurodeoxycholic acid, and glycochenodeoxycholic acid were significantly increased in children with acetaminophen overdose compared to healthy controls. Among patients with acetaminophen overdose, bile acids were higher in subjects with acetaminophen protein adduct values > 1.0 nmol/mL and modest correlations were noted for three bile acids and acetaminophen protein adducts as follows: taurodeoxycholic acid (R=0.604; p<0.001), glycodeoxycholic acid (R=0.581; p<0.001), and glycochenodeoxycholic acid (R=0.571; p<0.001). Variability in bile acids was greater among hospitalized children receiving low doses of acetaminophen than in healthy children with no recent acetaminophen exposure. Compared to bile acids, acetaminophen protein adducts more accurately discriminated among children with acetaminophen overdose, children with low dose exposure to acetaminophen, and healthy control subjects. In children with acetaminophen overdose, elevations of conjugated bile acids were associated with specific indicators of acetaminophen metabolism and non-specific indicators of liver injury. PMID:26208104

  8. PAH-DNA adducts in cord blood and fetal and child development in a Chinese cohort

    SciTech Connect

    Tang, D.L.; Li, T.Y.; Liu, J.J.; Chen, Y.H.; Qu, L.R.; Perera, F.

    2006-08-15

    Polycyclic aromatic hydrocarbons (PAHs) are an important class of toxic pollutants released by fossil fuel combustion. Other pollutants include metals and particulate matter. PAH-DNA adducts, or benzo(a)pyrene (BaP) adducts as their proxy, provide a chemical-specific measure of individual biologically effective doses that have been associated with increased risk of cancer and adverse birth outcomes. In the present study we examined the relationship between prenatal PAH exposure and fetal and child growth and development in Tongliang, China, where a seasonally operated coal-fired power plant was the major pollution source. In a cohort of 150 nonsmoking women and their newborns enrolled between 4 March 2002 and 19 June 2002, BaP-DNA adducts were measured in maternal and umbilical cord blood obtained at delivery. High PAH-DNA adduct levels (above the median of detectable adduct level) were associated with decreased birth head circumference (p = 0.057) and reduced children's weight at 18 months, 24 months, and 30 months of age (p {lt} 0.05), after controlling for potential confounders. In addition, in separate models, longer duration of prenatal exposure was associated with reduced birth length (p = 0.033) and reduced children's height at 18 (p = 0.001), 24 (p {lt} 0.001), and 30 months of age (p {lt} 0.001). The findings suggest that exposure to elevated levels of PAHS, with the Tongliang power plant being a significant source, is associated with reduced fetal and child growth in this population.

  9. 1,N(2)-propanodeoxyguanosine adduct formation in aortic DNA following inhalation of acrolein.

    PubMed Central

    Penn, A; Nath, R; Pan, J; Chen, L; Widmer, K; Henk, W; Chung, F L

    2001-01-01

    Recent reports indicate that many of the cytotoxic and health-threatening components of environmental tobacco smoke (ETS) reside in the vapor phase of the smoke. We have reported previously that inhalation of 1,3-butadiene, a prominent vapor phase component of ETS, accelerates arteriosclerotic plaque development in cockerels. In this study we asked whether inhaled acrolein, a reactive aldehyde that is also a prominent vapor-phase component of ETS, damages artery-wall DNA and accelerates plaque development. Cockerels inhaled 0, 1, or 10 ppm acrolein mixed with HEPA-filtered air for 6 hr. Half were killed immediately (day 1 group) for detection of the stable, premutagenic 1,N(2)-propanodeoxyguanosine acrolein adduct (AdG3) in aortic DNA via a (32)P-postlabeling/HPLC method, and half were killed after 10 days (day 10 group) for indirect assessment of adduct repair. In the day 1 group, acrolein-DNA adducts were 5 times higher in the 1 and 10 ppm groups than in HEPA-filtered air controls. However, in the day 10 group, adduct levels in the 1 and 10 ppm acrolein groups were reduced to the control adduct level. For the plaque studies, cockerels inhaled 1 ppm acrolein (6 hr/day, 8 weeks), mixed with the same HEPA-filtered air inhaled by controls. Plaque development was measured blind by computerized morphometry. Unlike butadiene inhalation, acrolein inhalation did not accelerate plaque development. Thus, even though repeated exposure to acrolein alone has no effect on plaque size under the exposure conditions described here, a single, brief inhalation exposure to acrolein elicits repairable DNA damage to the artery wall. These results suggest that frequent exposure to ETS may lead to persistent artery-wall DNA damage and thus provide sites on which other ETS plaque accelerants can act. PMID:11333181

  10. Covalent adduct formation between the plasmalogen-derived modification product 2-chlorohexadecanal and phloretin

    PubMed Central

    Üllen, Andreas; Nusshold, Christoph; Glasnov, Toma; Saf, Robert; Cantillo, David; Eibinger, Gerald; Reicher, Helga; Fauler, Günter; Bernhart, Eva; Hallstrom, Seth; Kogelnik, Nora; Zangger, Klaus; Oliver Kappe, C.; Malle, Ernst; Sattler, Wolfgang

    2015-01-01

    Hypochlorous acid added as reagent or generated by the myeloperoxidase (MPO)-H2O2-Cl− system oxidatively modifies brain ether-phospholipids (plasmalogens). This reaction generates a sn2-acyl-lysophospholipid and chlorinated fatty aldehydes. 2-Chlorohexadecanal (2-ClHDA), a prototypic member of chlorinated long-chain fatty aldehydes, has potent neurotoxic potential by inflicting blood–brain barrier (BBB) damage. During earlier studies we could show that the dihydrochalcone-type polyphenol phloretin attenuated 2-ClHDA-induced BBB dysfunction. To clarify the underlying mechanism(s) we now investigated the possibility of covalent adduct formation between 2-ClHDA and phloretin. Coincubation of 2-ClHDA and phloretin in phosphatidylcholine liposomes revealed a half-life of 2-ClHDA of approx. 120 min, decaying at a rate of 5.9 × 10−3 min−1. NMR studies and enthalpy calculations suggested that 2-ClHDA-phloretin adduct formation occurs via electrophilic aromatic substitution followed by hemiacetal formation on the A-ring of phloretin. Adduct characterization by high-resolution mass spectroscopy confirmed these results. In contrast to 2-ClHDA, the covalent 2-ClHDA-phloretin adduct was without adverse effects on MTT reduction (an indicator for metabolic activity), cellular adenine nucleotide content, and barrier function of brain microvascular endothelial cells (BMVEC). Of note, 2-ClHDA-phloretin adduct formation was also observed in BMVEC cultures. Intraperitoneal application and subsequent GC–MS analysis of brain lipid extracts revealed that phloretin is able to penetrate the BBB of C57BL/6J mice. Data of the present study indicate that phloretin scavenges 2-ClHDA, thereby attenuating 2-ClHDA-mediated brain endothelial cell dysfunction. We here identify a detoxification pathway for a prototypic chlorinated fatty aldehyde (generated via the MPO axis) that compromises BBB function in vitro and in vivo. PMID:25576489

  11. Correlations between Photodegradation of Bisretinoid Constituents of Retina and Dicarbonyl Adduct Deposition.

    PubMed

    Zhou, Jilin; Ueda, Keiko; Zhao, Jin; Sparrow, Janet R

    2015-11-01

    Non-enzymatic collagen cross-linking and carbonyl adduct deposition are features of Bruch's membrane aging in the eye, and disturbances in extracellular matrix turnover are considered to contribute to Bruch's membrane thickening. Because bisretinoid constituents of the lipofuscin of retinal pigment epithelial (RPE) cells are known to photodegrade to mixtures of aldehyde-bearing fragments and small dicarbonyls (glyoxal (GO) and methylglyoxal (MG)), we investigated RPE lipofuscin as a source of the reactive species that covalently modify protein side chains. Abca4(-/-) and Rdh8(-/-)/Abca4(-/-) mice that are models of accelerated bisretinoid formation were studied and pre-exposure of mice to 430 nm light enriched for dicarbonyl release by bisretinoid photodegradation. MG protein adducts were elevated in posterior eyecups of mutant mice, whereas carbonylation of an RPE-specific protein was observed in Abca4(-/-) but not in wild-type mice under the same conditions. Immunolabeling of cryostat-sectioned eyes harvested from Abca4(-/-) mice revealed that carbonyl adduct deposition in Bruch's membrane was accentuated. Cell-based assays corroborated these findings in mice. Moreover, the receptor for advanced glycation end products that recognizes MG and GO adducts and glyoxylase 1 that metabolizes MG and GO were up-regulated in Abca4(-/-) mice. Additionally, in acellular assays, peptides were cross-linked in the presence of A2E (adduct of two vitamin A aldehyde and ethanolamine) photodegradation products, and in a zymography assay, reaction of collagen IV with products of A2E photodegradation resulted in reduced cleavage by the matrix metalloproteinases MMP2 and MMP9. In conclusion, these mechanistic studies demonstrate a link between the photodegradation of RPE bisretinoid fluorophores and aging changes in underlying Bruch's membrane that can confer risk of age-related macular degeneration. PMID:26400086

  12. Formation of Hydroxymethyl DNA Adducts in Rats Orally Exposed to Stable Isotope Labeled Methanol

    PubMed Central

    Lu, Kun; Gul, Husamettin; Upton, Patricia B.; Moeller, Benjamin C.; Swenberg, James A.

    2012-01-01

    Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol’s well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [13CD4]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [13CD4]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account. PMID:22157354

  13. Oxidative DNA adducts after Cu(2+)-mediated activation of dihydroxy PCBs: role of reactive oxygen species.

    PubMed

    Spencer, Wendy A; Lehmler, Hans-Joachim; Robertson, Larry W; Gupta, Ramesh C

    2009-05-15

    Polychlorinated biphenyls (PCBs) are toxic industrial chemicals, complete carcinogens, and efficacious tumor promoters. However, the mechanism(s) of PCB-mediated carcinogenicity remains largely undefined. One likely pathway by which these agents may play a role in carcinogenesis is the generation of oxidative DNA damage by redox cycling of dihydroxylated PCB metabolites. We have now employed a new (32)P-postlabeling system to examine novel oxidative DNA lesions induced by Cu(2+)-mediated activation of PCB metabolites. (32)P postlabeling of DNA incubated with various PCB metabolites resulted in over a dozen novel polar oxidative DNA adducts that were chromatographically similar for all active agents. The most potent metabolites tested were the hydroquinones (hydroxyl groups arranged para to each other), yielding polar oxidative adduct levels ranging from 55 to 142 adducts/10(6) nucleotides. PCB catechols, or ortho-dihydroxy metabolites, were up to 40% less active than their corresponding hydroquinone congeners, whereas monohydroxylated and quinone metabolites did not produce detectable oxidative damage over that of vehicle. With the exception of 2,4,5-Cl-2',5'-dihydroxybiphenyl, this oxidative DNA damage seemed to be inversely related to chlorine content: no chlorine approximately mono->di->trichlorinated metabolites. Importantly, copper, but not iron, was essential for activation of the PCB metabolites to these polar oxidative DNA adducts, because in its absence or in the presence of the Cu(+)-specific scavenger bathocuproine, no adducts were detected. Intervention studies with known reactive oxygen species (ROS) modifiers suggested that H(2)O(2), singlet oxygen, hydroxyl radical, and superoxide may also be involved in this PCB-mediated oxidative DNA damage. These data indicate a mechanistic role for several ROS, in addition to copper, in PCB-induced DNA damage and provide further support for oxidative DNA damage in PCB-mediated carcinogenesis. PMID:19233261

  14. Direct quantitation of methyl phosphonate adducts to human serum butyrylcholinesterase by immunomagnetic-UHPLC-MS/MS.

    PubMed

    Carter, Melissa D; Crow, Brian S; Pantazides, Brooke G; Watson, Caroline M; Thomas, Jerry D; Blake, Thomas A; Johnson, Rudolph C

    2013-11-19

    Hydrolysis of G- and V-series organophosphorus nerve agents (OPNAs) containing a phosphorus-methyl bond yields a methylphosphonic acid (MeP) product when adducted to human butyrylcholinesterase (BChE). The MeP adduct is considered a sign of "aging" and results in loss of the o-alkyl identifier specific to each nerve agent. After aging has occurred, common therapeutics such as oximes cannot reactivate the cholinesterase enzyme and relieve cholinergic inhibition. Until now, a direct, quantitative method for determination of the MeP adduct to BChE was unavailable. Aged adducts in serum samples were processed by immunomagnetic separation of BChE by antibody conjugated bead, isotope-dilution, pepsin digestion, followed by UHPLC separation and detection by conventional electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Ions were detected in selected reaction monitoring (SRM) mode, and transition m/z 874.3 → 778.3 was used for quantitation. The analytical response ratio was linearly proportional to the serum concentration of MeP-adducted peptide (MeP-P) over the nominal concentration range of 2.0-250 ng/mL, with a coefficient of determination of R(2) ≥ 0.997. Intrarun accuracy, expressed as %Relative Error (%RE), was ≤13.5%, 16.3%, and 3.20% at 2.0, 16, and 250 ng/mL, respectively; the corresponding precision expressed as %RSD was ≤11.9%, 6.15%, and 3.39%. Interday %RSD was ≤7.13%, 5.69%, and 1.91%. Recovery of MeP-P from serum was ≥68% across the validated concentration range, and contributions from matrix effects were minimal. The method provides a direct, quantitative measurement of MeP-P found in clinical samples suspected of nerve agent exposure and subjected to such post-sampling stresses as elevated temperature and extended shipping. PMID:24205842

  15. Development of sandwich ELISAs for the detection of aromatic diisocyanate adducts.

    PubMed

    Lemons, Angela R; Bledsoe, Toni A; Siegel, Paul D; Beezhold, Donald H; Green, Brett J

    2013-11-29

    Diisocyanates (dNCOs) are highly reactive low molecular weight chemicals commonly used in the manufacturing industry. Occupational exposures to dNCOs have been shown to elicit allergic sensitization and occupational asthma. Among the most commonly used dNCOs in industry are the aromatic dNCOs, toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI). This study aimed to develop enzyme linked immunosorbent assays (ELISA) utilizing aromatic dNCO-specific monoclonal antibodies (mAbs) for the detection of aromatic dNCO adducts. Two sandwich ELISAs were developed. The first sandwich ELISA utilized mAb 60G2 along with an anti-human serum albumin (HSA) polyclonal antibody. This assay detected MDI-, 2,4- and 2,6-TDI-HSA adducts with limits of detection (LOD) of 2.67, <0.10, and 1.70 ng/mL, respectively. When spiked into human serum, the LOD of this ELISA increased to 34.37, 7.64 and 24.06 ng/mL, respectively. The second ELISA utilized mAbs 62G5 and 60G2 for capture and detection. This assay was capable of detecting 2,4- and 2,6-TDI-HSA adducts with LODs of <4.90 and 26.92 ng/mL, respectively, and when spiked in human serum, <4.90 and 95.93 ng/mL, respectively. This 62G5-60G2 sandwich assay was also able to detect dNCO adducted transferrin, hemoglobin, keratin and actin, but with less sensitivity than dNCO-HSA. The results of this study demonstrate potential application of these ELISAs in the identification and characterization of aromatic dNCO adducts as well as in biomonitoring occupational and environmental dNCO exposures. PMID:24012971

  16. {sup 32}P-postlabeling analysis of DNA adducts in wild perch (Perca fluviatilis) and northern pike (Esox lucius)

    SciTech Connect

    Ericson, G.; Liewenborg, B.; Balk, L.

    1995-12-31

    Several previous studies have demonstrated a correlation between high concentrations of sediment-associated contaminants and elevated levels of aromatic/hydrophobic DNA adduct levels in the liver of benthic fish species. In the present study DNA adducts was analyzed in coastal populations of perch (Perca fluviatilis) and northern pike (Esox lucius). Fish were sampled from four different sites in a gradient from a heavily industrialized area at the Swedish Baltic coast. For comparison, fish were also caught in a reference area with no main industries and comparatively low levels of contaminants of anthropogenic origin. DNA was extracted from liver and several extrahepatic tissues and DNA adducts were analyzed by the nuclease PI version of the {sup 32}P-postlabeling assay. The autoradiograms derived from DNA of fish from the contaminated sites showed several adduct spots not visible on the autoradiograms derived from fish from the reference area. Total adduct levels were significantly elevated in several tissues in fish from contaminated sites compared to the reference area. Species and tissue-specific differences in adduct levels and the use of {sup 32}P-postlabeling analysis of DNA adducts as a biomarker to monitor the presence and effects of genotoxic chemicals in the aquatic environment are discussed.

  17. The use of in vitro DNA adduct formation to estimate the genotoxicity of residues at contaminated sites.

    PubMed

    Shaw, G; Connell, D; Barron, W

    1995-05-01

    Genotoxic carcinogens such as polycyclic aromatic hydrocarbons (PAHs) covalently bind to the bases in DNA to form adducts. The formation of DNA adducts is significant with respect to chemical carcinogenesis. Many contaminated sites contain quantities of carcinogens such as PAHs, and the evaluation of the genotoxicity of these soils has important implications for human risk assessment. DNA adducts can be formed using an in vitro system incorporating extracts from contaminated soils. The 32P-postlabelling assay is a sensitive technique for the detection of DNA adducts from complex mixtures of environmental carcinogens. These techniques have been used to form and detect DNA adducts using soils from a number of coal gasworks sites. The results show that the extent of adduct formation depends partially on the petroleum hydrocarbon content of samples, but also on other undetermined factors related to composition. While environmental weathering has been shown to effect the PAH composition of samples, this is not an important factor in controlling the genotoxicity of samples as estimated by DNA adduct formation. PMID:7780722

  18. Charge separated states and singlet oxygen generation of mono and bis adducts of C60 and C70

    NASA Astrophysics Data System (ADS)

    Dallas, Panagiotis; Rogers, Gregory; Reid, Ben; Taylor, Robert A.; Shinohara, Hisanori; Briggs, G. Andrew D.; Porfyrakis, Kyriakos

    2016-02-01

    We present a series of fullerene derivatives and a study on their photoluminescence properties, complete with their efficiency as singlet oxygen generation photosensitizers. We demonstrate the intramolecular charge transfer between pyrene donor and fullerene acceptor. The opposite effect in decay lifetime measurements is observed for the mono and bis adducts of C60 and C70 for the first time, indicating an interplay between charge-separation and locally excited states. A monoexponential decay was observed for the mono adduct of C60 and the bis adduct of C70, while a biexponential decay was observed for the bis adduct of C60 and the mono adduct of C70. The effect of these molecules as sensitizers of the singlet oxygen radical was tested using detailed 3D excitation photoluminescence maps. A quenching of the singlet oxygen for the C60-mono and C70-bis adducts was observed while a strong photosensitizing effect was observed for the C60-bis and C70-mono adducts.

  19. Characterization of DNA adducts of the carcinogen N-methyl-4-aminoazobenzene in vitro and in vivo.

    PubMed

    Beland, F A; Tullis, D L; Kadlubar, F F; Straub, K M; Evans, F E

    1980-07-01

    Since the susceptibility of specific tissues to tumor formation has been correlated with the persistence of DNA-carcinogen adducts, the identity and persistence of DNA adducts formed from the hepatocarcinogen N-methyl-4-aminoazobenzene (MAB) has been determined. The synthetic ultimate carcinogen N-benzoyloxy-N-methyl-4-aminoazobenzene (N-BxO-MAB) was reacted in vitro with either calf thymus or rat liver DNA to yield approx. 1 bound residue per 1000 nucleotides. After enzymatic hydrolysis of the DNA and high pressure liquid chromatographic analysis, at least six MAB adducts were detected. Two of the products cochromatographed with MAB-DNA adducts formed in rat liver in vivo following oral administration of the precarcinogen MAB. These two adducts were identified by mass, UV and nuclear magnetic resonance (NMR) spectroscopy as N-(deoxyguanosin-8-yl)- and 3-(deoxyguanosin-N2-yl)-MAB. The former adduct was initially the predominant product in vivo, but it could not be detected 7 days following treatment. The latter adduct remained at a constant level for 14 days and therefore appears to be a persistent lesion. PMID:7389004

  20. Characterization of the lysyl adducts of prostaglandin H-synthases that are derived from oxygenation of arachidonic acid.

    PubMed

    Boutaud, O; Brame, C J; Chaurand, P; Li, J; Rowlinson, S W; Crews, B C; Ji, C; Marnett, L J; Caprioli, R M; Roberts, L J; Oates, J A

    2001-06-12

    These investigations characterize the covalent binding of reactive products of prostaglandin H-synthases (PGHSs) to the enzyme and to other molecules. The intermediate product of oxygenation of arachidonic acid by the PGHSs, prostaglandin (PG) H2, undergoes rearrangement to the highly reactive gamma-keto aldehydes, levuglandin (LG) E2 and D2. We previously have demonstrated that LGE2 reacts with the epsilon-amine of lysine to form both the lysyl-levuglandin Shiff base and the pyrrole-derived lysyl-levuglandin lactam adducts. We now demonstrate that these lysyl-levuglandin adducts are formed on the PGHSs following the oxygenation of arachidonic acid; after reduction of the putative Schiff base, proteolytic digestion of the enzyme, and isolation of the adducted amino acid residues, these adducts were identified by liquid chromatography-tandem mass spectrometry. The reactivity of the LGs is reflected by the finding that virtually all of the LG predicted to be formed from PGH2 can be accounted for as adducts of the PGH-synthase and that oxygenation of arachidonic acid by PGH-synthases also leads to the formation of adducts of other proteins present in the reaction solution. The reactivity of the PGH-synthase adducts themselves is demonstrated by the formation of intermolecular cross-links. PMID:11389610

  1. sup 14 C-sulfur mustard adducts of calf thymus DNA. Final report, Aug-Sep 90

    SciTech Connect

    Yaverbaum, S.

    1991-02-01

    A grant was awarded to TNO-PML to develop immunochemical monitoring systems for the detection of DNA-HD and Protein-HD adducts in humans following exposure to HD. TNO-PML has been using 35S-HD to prepare adducts for their assays, which have inherent shortcomings that limit detection sensitivity. An experimental batch of 14C-HD-DNA adducts was prepared in an attempt to increase the assay sensitivity. Double - and single-stranded purified calf thymus DNA preparations were reacted with 142, 14.2 and 1.42 uM of 14C-HD under aqueousfree conditions. The 14C-HD-DNA adducts were isolated at -20C in 75% ethanol solution and freed of HD agent and organic solvents (i.e., acetone and alcohol). The 14C-HD-DNA adducts in aqueous buffer were analyzed for specific activity and purity. The ds-DNA-HD adducts were uncontaminated, but the ss-DNA-HD adducts were initially slightly contaminated with alcohol.

  2. Highly persistent polycyclic aromatic hydrocarbon-DNA adducts in mouse skin: detection by 32P-postlabeling analysis.

    PubMed

    Randerath, E; Agrawal, H P; Reddy, M V; Randerath, K

    1983-08-01

    A 32P-postlabeling method for carcinogen-DNA adduct analysis recently developed in our laboratory was applied to skin DNA from mice treated topically with polycyclic aromatic hydrocarbons (PAHs). After application of 4 doses of 1.2 mumol each of benzo[alpha]pyrene (BP), 3-methylcholanthrene (MC) and 7,12-dimethylbenz[alpha]anthracene (DMBA), respectively, total covalent adduct binding in mouse skin DNA initially amounted to 1 adduct in 6.0 X 10(4) - 1.3 X 10(5) nucleotides. Four weeks after treatment, these levels had declined to 1 adduct in 1.4 X 10(6) - 2.7 X 10(6) nucleotides. Substantial removal of DNA adducts occurred during the first 2 weeks after carcinogen application while adducts remaining thereafter underwent little or no repair between 2 and 4 weeks after treatment. These results raise the possibility that the persistent adducts occupy specific genomic sites in quiescent cells where they may not be amenable to repair because of localized conformational alterations of DNA or shielding by associated proteins. PMID:6318965

  3. Heat exchanger-accumulator

    DOEpatents

    Ecker, Amir L.

    1980-01-01

    What is disclosed is a heat exchanger-accumulator for vaporizing a refrigerant or the like, characterized by an upright pressure vessel having a top, bottom and side walls; an inlet conduit eccentrically and sealingly penetrating through the top; a tubular overflow chamber disposed within the vessel and sealingly connected with the bottom so as to define an annular outer volumetric chamber for receiving refrigerant; a heat transfer coil disposed in the outer volumetric chamber for vaporizing the liquid refrigerant that accumulates there; the heat transfer coil defining a passageway for circulating an externally supplied heat exchange fluid; transferring heat efficiently from the fluid; and freely allowing vaporized refrigerant to escape upwardly from the liquid refrigerant; and a refrigerant discharge conduit penetrating sealingly through the top and traversing substantially the length of the pressurized vessel downwardly and upwardly such that its inlet is near the top of the pressurized vessel so as to provide a means for transporting refrigerant vapor from the vessel. The refrigerant discharge conduit has metering orifices, or passageways, penetrating laterally through its walls near the bottom, communicating respectively interiorly and exteriorly of the overflow chamber for controllably carrying small amounts of liquid refrigerant and oil to the effluent stream of refrigerant gas.

  4. Aminofluorene-DNA adduct formation in Salmonella typhimurium exposed to the carcinogen N-hydroxy-2-acetylaminofluorene.

    PubMed Central

    Beranek, D T; White, G L; Heflich, R H; Beland, F A

    1982-01-01

    The DNA adducts formed during incubation of the hepatocarcinogen N-hydroxy-2-acetylaminofluorene with Salmonella typhimurium tester strain TA1538 were investigated to determine if the covalently bound products were identical to those adducts found in rat liver DNA and to establish the biological significance of the adducts in a mutational assay. When bacteria were exposed to N-hydroxy-2-acetylaminofluorene in the presence of a 9,000 x g supernatant from a rat liver homogenate (S9), only one adduct was detected. This adduct had chromatographic, pH-dependent partitioning, and UV spectral characteristics identical to those of N-(deoxyguanosin-8-yl)-2-aminofluorene. In the absence of S9 activation the same product was detected, but at a 85-90% lower level, which indicates that S. typhimurium also may be capable of metabolizing N-hydroxy-2-acetylaminofluorene to a reactive electrophile. When incubations were conducted with N-hydroxy-2-aminofluorene in the absence of the activation system, N-(deoxyguanosin-8-yl)-2-aminofluorene again was the major adduct. At equimolar concentrations, the arylhydroxylamine was approximately 10 times more efficient than the arylhydroxamic acid in inducing reversions in the bacteria. Comparison of the mutation rate to the level of binding in bacterial DNA gave a linear relationship with a slope of 0.96 and a correlation coefficient of 0.92. These data support previous suggestions that N-hydroxy-2-acetylaminofluorene is deacetylated by rat liver S9 to the ultimate mutagen, N-hydroxy-2-aminofluorene, and also indicate that S. typhimurium can mediate this reaction. The correlation between mutagenicity and the extent of N-(deoxyguanosin-8-yl)-2-aminofluorene adduct formation, coupled with the observation that this adduct is the major DNA adduct found in rat liver in vivo, suggests that N-(deoxyguanosin-8-yl)-2-aminofluorene may be a critical lesion for the initiation of hepatic tumorigenesis. PMID:6752940

  5. Adduct formation of 7,12-dimethylbenz(a)anthracene in the embryo of the Japanese medaka (Oryzias latipes)

    SciTech Connect

    Liu, H.; Cooper, K.R.

    1995-12-31

    DNA adduct formation of 7,1 2-dimethylbenz(a)anthracene (DMBA) in vivo in the Japanese medaka embryo were investigated using {sup 32}P-postlabeling analysis. 1-compounds (endogenous adducts) were not observed in the Japanese medaka embryo on days 4 (prior to liver formation), 6 (liver/swim bladder) or 10 (prior to hatch) of development. The level of DMBA:DNA adducts were concentration-dependent over the range of 0.625 ppm (Total Adducts 0.05707 pmol/mg of DNA) to 2.50 ppm (0.43341 pmol/mg of DNA) and decreased at 5.00 ppm (0.25338 pmol/mg of DNA) after medaka embryos were exposed to DMBA for 6 days from the day of fertilization. The decrease in DMBA:DNA adducts at 5.00 ppm was probably due to embryo toxicity (78% death). The level of DMBA:DNA adducts formed from the embryos exposed to DMBA for 24 hr decreased as the stage of development increased: day 4 > day 6 > day 10; 0.0262, 0.0179, 0.0129 pmol/mg of DNA, respectively. The level of DMBA:DNA adducts increased as the length of exposure increased: 4 day < 6 day < 10 day; 0.0233, 0.0614, 0.1502, respectively. There was both a time and dose dependence to the number of adducts detected. The data presented demonstrated the development of DM BA-DNA adducts in the developing Japanese medaka (Oryzias latipes) and the lack of I-compounds.

  6. Differences in hemoglobin adduct levels of acrylamide in the general population with respect to dietary intake, smoking habits and gender.

    PubMed

    Hagmar, Lars; Wirfält, Elisabet; Paulsson, Birgit; Törnqvist, Margareta

    2005-02-01

    The variation in dietary exposure to acrylamide (AA) has been studied through measurement of hemoglobin adduct levels from AA, as a measurement of internal dose, in a sample from the blood bank of the Malmö Diet and Cancer Cohort (n=28,098). The blood donors are well characterised with regard to their food habits, and 142 individuals were selected to obtain highest possible variation in the adduct levels from AA (none, random or high intake of coffee, fried potato, crisp bread and snacks, food items estimated to have high levels of AA). Among 70 non-smokers the AA-adduct levels varied by a factor of 5, and ranged between 0.02 and 0.1 nmol/g, with considerable overlap in AA-adduct levels between the different dietary groups. There was a significant difference between men with high dietary exposure to AA compared to men with low dietary exposure (P=0.04). No such difference was found for women. As expected a higher level (range: 0.03-0.43 nmol/g) of the AA-adduct, due to AA in tobacco smoke, was found in smokers. Smoking women with high dietary exposure to AA had significantly higher AA-adduct levels compared to smoking women with low dietary exposure (P=0.01). No such significant difference was found in smoking men. The median hemoglobin (Hb) adduct level in the randomly selected group of non-smokers was compatible with earlier studies (0.031 nmol/g). The variation in the average internal dose, measured as Hb adducts, was somewhat smaller than estimated for daily intake by food consumption questionnaires in other studies. Thus, the observed relatively narrow inter-individual variation in AA-adduct levels means that estimates of individual dietary AA intake have to be very precise if they should be useful in future cancer epidemiology. PMID:15668117

  7. Solids Accumulation Scouting Studies

    SciTech Connect

    Duignan, M. R.; Steeper, T. J.; Steimke, J. L.

    2012-09-26

    The objective of Solids Accumulation activities was to perform scaled testing to understand the behavior of remaining solids in a Double Shell Tank (DST), specifically AW-105, at Hanford during multiple fill, mix, and transfer operations. It is important to know if fissionable materials can concentrate when waste is transferred from staging tanks prior to feeding waste treatment plants. Specifically, there is a concern that large, dense particles containing plutonium could accumulate in poorly mixed regions of a blend tank heel for tanks that employ mixing jet pumps. At the request of the DOE Hanford Tank Operations Contractor, Washington River Protection Solutions, the Engineering Development Laboratory of the Savannah River National Laboratory performed a scouting study in a 1/22-scale model of a waste staging tank to investigate this concern and to develop measurement techniques that could be applied in a more extensive study at a larger scale. Simulated waste tank solids: Gibbsite, Zirconia, Sand, and Stainless Steel, with stainless steel particles representing the heavier particles, e.g., plutonium, and supernatant were charged to the test tank and rotating liquid jets were used to mix most of the solids while the simulant was pumped out. Subsequently, the volume and shape of the mounds of residual solids and the spatial concentration profiles for the surrogate for heavier particles were measured. Several techniques were developed and equipment designed to accomplish the measurements needed and they included: 1. Magnetic particle separator to remove simulant stainless steel solids. A device was designed and built to capture these solids, which represent the heavier solids during a waste transfer from a staging tank. 2. Photographic equipment to determine the volume of the solids mounds. The mounds were photographed as they were exposed at different tank waste levels to develop a composite of topographical areas. 3. Laser rangefinders to determine the volume of

  8. Determinants of formation of aflatoxin-albumin adducts: a seven-township study in Taiwan

    PubMed Central

    Sun, C-A; Wu, D-M; Wang, L-Y; Chen, C-J; You, S-L; Santella, R M

    2002-01-01

    Dietary exposure to aflatoxins is one of the major risk factors for hepatocellular carcinoma. Individual susceptibility to aflatoxin-induced hepatocarcinogenesis may be modulated by both genetic and environmental factors affecting metabolism. A cross-sectional study was performed to evaluate determinants of the formation of aflatoxin covalently bound to albumin (AFB1-albumin adducts). A total of 474 subjects who were free of liver cancer and cirrhosis and were initially selected as controls for previous case–control studies of aflatoxin-induced hepatocarcinogenesis in Taiwan, were employed in this study. Aflatoxin-albumin adducts were determined by competitive enzyme-linked immunosorbent assay, hepatitis B surface antigen and antibodies to hepatitis C virus by enzyme immunoassay, as well as genotypes of glutathione S-transferase M1-1 and T1-1 by polymerase chain reaction. The detection rate of AFB1-albumin adducts was significantly higher in males (42.5%) than in females (21.6%) (multivariate-adjusted odds ratio=2.6, 95% confidence interval=1.4–5.0). The formation of detectable albumin adducts was moderately higher in hepatitis B surface antigen carriers (42.8%) than in non-carriers (36.6%) (multivariate-adjusted odds ratio=1.4, 95% confidence interval=1.0–2.1). In addition, the detection rate of AFB1-albumin adducts tended to increase with the increasing number of null genotypes of glutathione S-transferase M1-1 and glutathione S-transferase T1-1. In conclusion, this cross-sectional study has assessed the relative contributions of environmental exposure and host susceptibility factors in the formation of AFB1-albumin adducts in a well characterised Chinese adult population. This study further emphasises the necessity to reduce aflatoxin exposure in people living in an area endemic for chronic hepatitis B virus infection. British Journal of Cancer (2002) 87, 966–970. doi:10.1038/sj.bjc.6600584 www.bjcancer.com © 2002 Cancer Research UK PMID:12434285

  9. Nuclear Magnetic Resonance Studies of an N2-Guanine Adduct Derived from the Tumorigen Dibenzo[a,l]pyrene in DNA: Impact of Adduct Stereochemistry, Size, and Local DNA Sequence on Solution Conformations

    PubMed Central

    2015-01-01

    The dimensions and arrangements of aromatic rings (topology) in adducts derived from the reactions of polycyclic aromatic hydrocarbon (PAH) diol epoxide metabolites with DNA influence the distortions and stabilities of double-stranded DNA, and hence their recognition and processing by the human nucleotide excision repair (NER) system. Dibenzo[a,l]pyrene (DB[a,l]P) is a highly tumorigenic six-ring PAH, which contains a nonplanar and aromatic fjord region that is absent in the structurally related bay region five-ring PAH benzo[a]pyrene (B[a]P). The PAH diol epoxide–DNA adducts formed include the stereoisomeric 14S and 14Rtrans-anti-DB[a,l]P-N2-dG and the stereochemically analogous 10S- and 10R-B[a]P-N2-dG (B[a]P-dG) guanine adducts. However, nuclear magnetic resonance (NMR) solution studies of the 14S-DB[a,l]P-N2-dG adduct in DNA have not yet been presented. Here we have investigated the 14S-DB[a,l]P-N2-dG adduct in two different sequence contexts using NMR methods with distance-restrained molecular dynamics simulations. In duplexes with dC opposite the adduct deleted, a well-resolved base-displaced intercalative adduct conformation can be observed. In full duplexes, in contrast to the intercalated 14R stereoisomeric adduct, the bulky DB[a,l]P residue in the 14S adduct is positioned in a greatly widened and distorted minor groove, with significant disruptions and distortions of base pairing at the lesion site and two 5′-side adjacent base pairs. These unique structural features are significantly different from those of the stereochemically analogous but smaller B[a]P-dG adduct. The greater size and different topology of the DB[a,l]P aromatic ring system lead to greater structurally destabilizing DNA distortions that are partially compensated by stabilizing DB[a,l]P-DNA van der Waals interactions, whose combined effects impact the NER response to the adduct. These structural results broaden our understanding of the structure–function relationship in NER. PMID

  10. Water-soluble noncovalent adducts of the heterometallic copper subgroup complexes and human serum albumin with remarkable luminescent properties.

    PubMed

    Chelushkin, P S; Krupenya, D V; Tseng, Yu-Jui; Kuo, Ting-Yi; Chou, Pi-Tai; Koshevoy, I O; Burov, S V; Tunik, S P

    2014-01-25

    Novel water-soluble noncovalent adducts of the heterometallic copper subgroup complexes and human serum albumin (HSA) display strong phosphorescence, internalize into HeLa cells and can be used in time-resolved fluorescent imaging. PMID:24296768

  11. Adaptive Response Enzyme AlkB Preferentially Repairs 1-Methylguanine and 3-Methylthymine Adducts in Double-Stranded DNA.

    PubMed

    Chen, Fangyi; Tang, Qi; Bian, Ke; Humulock, Zachary T; Yang, Xuedong; Jost, Marco; Drennan, Catherine L; Essigmann, John M; Li, Deyu

    2016-04-18

    The AlkB protein is a repair enzyme that uses an α-ketoglutarate/Fe(II)-dependent mechanism to repair alkyl DNA adducts. AlkB has been reported to repair highly susceptible substrates, such as 1-methyladenine and 3-methylcytosine, more efficiently in ss-DNA than in ds-DNA. Here, we tested the repair of weaker AlkB substrates 1-methylguanine and 3-methylthymine and found that AlkB prefers to repair them in ds-DNA. We also discovered that AlkB and its human homologues, ABH2 and ABH3, are able to repair the aforementioned adducts when the adduct is present in a mismatched base pair. These observations demonstrate the strong adaptability of AlkB toward repairing various adducts in different environments. PMID:26919079

  12. Structural and energetic characterization of the major DNA adduct formed from the food mutagen ochratoxin A in the NarI hotspot sequence: influence of adduct ionization on the conformational preferences and implications for the NER propensity

    PubMed Central

    Sharma, Purshotam; Manderville, Richard A.; Wetmore, Stacey D.

    2014-01-01

    The nephrotoxic food mutagen ochratoxin A (OTA) produces DNA adducts in rat kidneys, the major lesion being the C8-linked-2′-deoxyguanosine adduct (OTB-dG). Although research on other adducts stresses the importance of understanding the structure of the associated adducted DNA, site-specific incorporation of OTB-dG into DNA has yet to be attempted. The present work uses a robust computational approach to determine the conformational preferences of OTB-dG in three ionization states at three guanine positions in the NarI recognition sequence opposite cytosine. Representative adducted DNA helices were derived from over 2160 ns of simulation and ranked via free energies. For the first time, a close energetic separation between three distinct conformations is highlighted, which indicates OTA-adducted DNA likely adopts a mixture of conformations regardless of the sequence context. Nevertheless, the preferred conformation depends on the flanking bases and ionization state due to deviations in discrete local interactions at the lesion site. The structural characteristics of the lesion thus discerned have profound implications regarding its repair propensity and mutagenic outcomes, and support recent experiments suggesting the induction of double-strand breaks and deletion mutations upon OTA exposure. This combined structural and energetic characterization of the OTB-dG lesion in DNA will encourage future biochemical experiments on this potentially genotoxic lesion. PMID:25217592

  13. Contribution of artifacts to N-methylated piperazine cyanide adduct formation in vitro from N-alkyl piperazine analogs.

    PubMed

    Zhang, Minli; Resuello, Christina M; Guo, Jian; Powell, Mark E; Elmore, Charles S; Hu, Jun; Vishwanathan, Karthick

    2013-05-01

    In the liver microsome cyanide (CN)-trapping assays, piperazine-containing compounds formed significant N-methyl piperazine CN adducts. Two pathways for the N-methyl piperazine CN adduct formation were proposed: 1) The α-carbon in the N-methyl piperazine is oxidized to form a reactive iminium ion that can react with cyanide ion; 2) N-dealkylation occurs followed by condensation with formaldehyde and dehydration to produce N-methylenepiperazine iminium ion, which then reacts with cyanide ion to form the N-methyl CN adduct. The CN adduct from the second pathway was believed to be an artifact or metabonate. In the present study, a group of 4'-N-alkyl piperazines and 4'-N-[¹³C]methyl-labeled piperazines were used to determine which pathway was predominant. Following microsomal incubations in the presence of cyanide ions, a significant percentage of 4'-N-[¹³C]methyl group in the CN adduct was replaced by an unlabeled natural methyl group, suggesting that the second pathway was predominant. For 4'-N-alkyl piperazine, the level of 4'-N-methyl piperazine CN adduct formation was limited by the extent of prior 4'-N-dealkylation. In a separate study, when 4'-NH-piperaziens were incubated with potassium cyanide and [¹³C]-labeled formaldehyde, 4'-N-[¹³C]methyl piperazine CN-adduct was formed without NADPH or liver microsome suggesting a direct Mannich reaction is involved. However, when [¹³C]-labeled methanol or potassium carbonate was used as the one-carbon donor, 4'-N-[¹³C]methyl piperazine CN adduct was not detected without liver microsome or NADPH present. The biologic and toxicological implications of bioactivation via the second pathway necessitate further investigation because these one-carbon donors for the formation of reactive iminium ions could be endogenous and readily available in vivo. PMID:23431111

  14. Iridium-catalysed dehydrocoupling of aryl phosphine-borane adducts: synthesis and characterisation of high molecular weight poly(phosphinoboranes).

    PubMed

    Paul, Ursula S D; Braunschweig, Holger; Radius, Udo

    2016-06-30

    The thermal dehydrogenative coupling of aryl phosphine-borane adducts with iridium complexes bearing a bis(phosphinite) pincer ligand is reported. This catalysis produces high molecular weight poly(phosphinoboranes) [ArPH-BH2]n (Ar = Ph, (p)Tol, Mes). Furthermore, we investigated the reactivity of these pincer complexes towards primary phosphines and their respective borane adducts on a stoichiometric scale. PMID:27320239

  15. Synthesis and reactivity of a CAAC-aminoborylene adduct: a hetero-allene or an organoboron isoelectronic with singlet carbenes.

    PubMed

    Dahcheh, Fatme; Martin, David; Stephan, Douglas W; Bertrand, Guy

    2014-11-24

    A one-electron reduction of a cyclic (alkyl)(amino)carbene (CAAC)-bis(trimethylsilyl)aminodichloroborane adduct leads to a stable aminoboryl radical. A second one-electron reduction gives rise to a CAAC-aminoborylene adduct, which features an allenic structure. However, in manner similar to that of stable electrophilic singlet carbenes, this compound activates small molecules, such as CO and H2. PMID:25267591

  16. Immunoperoxidase detection of polycyclic aromatic hydrocarbon-DNA adducts in mouth floor and buccal mucosa cells of smokers and nonsmokers.

    PubMed

    Besaratinia, A; Besarati Nia, A; Van Straaten, H W; Godschalk, R W; Van Zandwijk, N; Balm, A J; Kleinjans, J C; Van Schooten, F J

    2000-01-01

    Tobacco smoking is a major risk factor for oral cancer; mouth floor and buccal mucosa are among the most and least cancer-prone subsites, respectively, in the oral cavity. We investigated the applicability of immunohistochemistry of smoking-induced DNA adducts in oral cells for assessing the exposure to carcinogens, and estimating the risk for oral cancer. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were measured in mouth floor and buccal mucosa cells of smokers (n = 26) and nonsmokers (n = 22) by means of a semiquantitative immunoperoxidase assay. Smokers had elevated levels of PAH-DNA adducts compared to nonsmokers in their mouth floor cells (0.045 +/- 0.022 versus 0.022 +/- 0.016, P = 0.0008 arbitrary units of immunohistochemistry) as well as in their buccal mucosa cells (0.058 +/- 0.028 versus 0.028 +/- 0.012, P = 0.001). Also, there was a correlation between the levels of PAH-DNA adducts in mouth floor cells and those in buccal mucosa cells (r = 0.4, P = 0.01). Furthermore, PAH-DNA adduct levels in both mouth floor and buccal mucosa cells were significantly related to current smoking indices (amount of tar and number of cigarettes consumed per day). Expectedly, the levels of PAH-DNA adducts neither in mouth floor cells nor in buccal mucosa cells, both being short-lived cells, were related to smoking history index (pack years). The levels of PAH-DNA adducts, however, in mouth floor cells as the cancer prone cells were lower than those in buccal mucosa cells (0.037 +/- 0.023 versus 0.044 +/- 0.026, P = 0.04). We conclude that immunohistochemistry of PAH-DNA adducts in oral cells can be used for exposure assessment of tobacco-related carcinogens, however, it cannot be used for oral cancer risk estimation. PMID:11013411

  17. Monitoring of environmental cancer initiators through hemoglobin adducts by a modified Edman degradation method

    SciTech Connect

    Toernqvist, M.M.; Mowrer, J.; Jensen, S.; Ehrenberg, L.

    1986-04-01

    Tissue doses of cancer initiators/mutagens are suitably monitored through hemoglobin adducts formed in vivo, but the use of this method has been hampered by a lack of sufficiently simple and fast procedures. It was previously observed that when the N-terminal amino acid in hemoglobin, valine, is alkylated it is cleaved off by the Edman sequencing reagent, phenyl isothiocyanate, in the neutral-alkaline coupling medium, as opposed to the acidic medium required by normal amino acids. Based on this principle, conditions for a functioning procedure for gas chromatography/mass spectrometry (GC/MS) determination of N-terminal alkylvalines in hemoglobin were worked out. Derivatizing the protein in formamide solution with pentafluorophenyl isothiocyanate, using a /sup 2/H-alkylated protein as internal standard, and applying on-column injection during analysis, permit reproducible determination of hydroxyethylvaline and other adducts down into the dose range where cancer risks may be considered acceptably low.

  18. Raman spectroscopic evaluation of DNA adducts of a platinum containing anticancer drug

    NASA Astrophysics Data System (ADS)

    Jangir, Deepak K.; Mehrotra, Ranjana

    2014-09-01

    Mechanistic understanding of the interaction of drugs with their target molecules is important for better understanding of their mode of action and to improve their efficacy. Carboplatin is a platinum containing anticancer drug, used to treat different type of tumors. In the present work, we applied Raman spectroscopy to study the interaction of carboplatin with DNA at molecular level using different carboplatin-DNA molar ratios. These Raman spectroscopic results provide comprehensive understanding on the carboplatin-DNA interactions and indicate that DNA cross-linked adducts formed by carboplatin are similar to cisplatin adducts. The results indicate that guanine N7 and adenine N7 are the putative sites for carboplatin interaction. It is observed that carboplatin has some affinity toward cytosine in DNA. Phosphate sugar backbone of DNA showed conformation perturbation in DNA which were easily sensible at higher concentrations of carboplatin. Most importantly, carboplatin interaction induces intermediate A- and B-DNA conformations at the cross-linking sites.

  19. Methylthiodeoxynivalenol (MTD): insight into the chemistry, structure and toxicity of thia-Michael adducts of trichothecenes.

    PubMed

    Fruhmann, Philipp; Weigl-Pollack, Theresa; Mikula, Hannes; Wiesenberger, Gerlinde; Adam, Gerhard; Varga, Elisabeth; Berthiller, Franz; Krska, Rudolf; Hametner, Christian; Fröhlich, Johannes

    2014-07-28

    Methylthiodeoxynivalenol (MTD), a novel derivative of the trichothecene mycotoxin deoxynivalenol (DON), was prepared by applying a reliable procedure for the formal Michael addition of methanethiol to the conjugated double bond of DON. Structure elucidation revealed the preferred formation of the hemiketal form of MTD by intramolecular cyclisation between C8 and C15. Computational investigations showed a negative total reaction energy for the hemiketalisation step and its decrease in comparison with theoretical model compounds. Therefore, this structural behaviour seems to be a general characteristic of thia-Michael adducts of type B trichothecenes. MTD was shown to be less inhibitory for a reticulocyte lysate based in vitro translation system than the parent compound DON, which supports the hypothesis that trichothecenes are detoxified through thia-adduct formation during xenobiotic metabolism. PMID:24903010

  20. Adduct of magnesium iodide with n, n-diethylnicotinamide, its thermolysis, and its interaction with pyridine

    SciTech Connect

    Shlapnikov, D.S.; Kazantseva, E.P.; Lirova, B.I.

    1985-05-01

    This paper investigates the interaction of magnesium iodide and its diether complex with nitrogen- and oxygen-containing heterocyclic compounds. The activities of the latter in the displacement of ether from magnesium iodide diether complex or toward anhydrous MgI/sub 2/ can serve as criteria in the comparative assessment of their electron-donor properties. The IR spectra of N,N-diethylnicotinamide (I) and its adduct with MgI/sub 2/ are shown. It is shown that the adduct MgI/sub 2/.2 (I) is decomposed by water, is insoluble in ether, and is very soluble in pyridine. The complete displacement of the compound (I) by pyridine does not occur either in ether medium, or when a mixture of pyridine and MgI/sub 2/.2(I) is heated.

  1. Monitoring the apple polyphenol oxidase-modulated adduct formation of phenolic and amino compounds.

    PubMed

    Reinkensmeier, Annika; Steinbrenner, Katrin; Homann, Thomas; Bußler, Sara; Rohn, Sascha; Rawel, Hashadrai M

    2016-03-01

    Minimally processed fruit products such as smoothies are increasingly coming into demand. However, they are often combined with dairy ingredients. In this combination, phenolic compounds, polyphenoloxidases, and amino compounds could interact. In this work, a model approach is presented where apple serves as a source for a high polyphenoloxidase activity for modulating the reactions. The polyphenoloxidase activity ranged from 128 to 333nakt/mL in different apple varieties. From these, 'Braeburn' was found to provide the highest enzymatic activity. The formation and stability of resulting chromogenic conjugates was investigated. The results show that such adducts are not stable and possible degradation mechanisms leading to follow-up products formed are proposed. Finally, apple extracts were used to modify proteins and their functional properties characterized. There were retaining antioxidant properties inherent to phenolic compounds after adduct formation. Consequently, such interactions may also be utilized to improve the textural quality of food products. PMID:26471529

  2. Lewis Acid-Base Adduct Approach for High Efficiency Perovskite Solar Cells.

    PubMed

    Lee, Jin-Wook; Kim, Hui-Seon; Park, Nam-Gyu

    2016-02-16

    Since the first report on the long-term durable 9.7% solid-state perovskite solar cell employing methylammonium lead iodide (CH3NH3PbI3), mesoporous TiO2, and 2,2',7,7'-tetrakis[N,N-di(4-methoxyphenyl)amino]-9,9'-spirobifluorene (spiro-MeOTAD) in 2012, following the seed technologies on perovskite-sensitized liquid junction solar cells in 2009 and 2011, a surge of interest has been focused on perovskite solar cells due to superb photovoltaic performance and extremely facile fabrication processes. The power conversion efficiency (PCE) of perovskite solar cells reached 21% in a very short period of time. Such an unprecedentedly high photovoltaic performance is due to the intrinsic optoelectronic property of organolead iodide perovskite material. Moreover, a high dielectric constant, sub-millimeter scale carrier diffusion length, an underlying ferroelectric property, and ion migration behavior can make organolead halide perovskites suitable for multifunctionality. Thus, besides solar cell applications, perovskite material has recently been applied to a variety fields of materials science such as photodetectors, light emitting diodes, lasing, X-ray imaging, resistive memory, and water splitting. Regardless of application areas, the growth of a well-defined perovskite layer with high crystallinity is essential for effective utilization of its excellent physicochemical properties. Therefore, an effective methodology for preparation of high quality perovskite layers is required. In this Account, an effective methodology for production of high quality perovskite layers is described, which is the Lewis acid-base adduct approach. In the solution process to form the perovskite layer, the key chemicals of CH3NH3I (or HC(NH2)2I) and PbI2 are used by dissolving them in polar aprotic solvents. Since polar aprotic solvents bear oxygen, sulfur, or nitrogen, they can act as a Lewis base. In addition, the main group compound PbI2 is known to be a Lewis acid. Thus, PbI2 has a chance

  3. Lipid peroxidation and etheno DNA adducts in white blood cells of liver fluke-infected patients: protection by plasma alpha-tocopherol and praziquantel.

    PubMed

    Dechakhamphu, Somkid; Pinlaor, Somchai; Sitthithaworn, Paiboon; Nair, Jagadeesan; Bartsch, Helmut; Yongvanit, Puangrat

    2010-01-01

    Chronic infection by the liver fluke Opisthorchis viverrini is a strong risk factor for cholangiocarcinoma. To clarify the involvement of oxidative stress and lipid peroxidation-derived DNA damage, etheno (epsilon)-DNA adducts (epsilondA, epsilondC) in WBC and plasma alpha-tocopherol were measured in samples collected from O. viverrini-infected Thai patients (n = 50) and healthy noninfected volunteers (n = 20). epsilondA and epsilondC levels were three to five times higher (P < 0.001) in infected patients than in controls; O. viverrini infection also increased two to three times in the plasma inflammatory indicators, 8-isoprostane, malondialdehyde, and nitrate/nitrite. Mean plasma alpha-tocopherol levels were two times lower in patients than in healthy controls (P < 0.001). Two months after a single dose to infected patients of the antiparasitic drug praziquantel, epsilondA and epsilondC levels in WBC were decreased to control level (P < 0.03); plasma 8-isoprostane, malondialdehyde, nitrate/nitrite, and alkaline phosphatase (ALP) were concomitantly lowered. epsilondA and epsilondC levels in WBC were positively correlated with plasma 8-isoprostane, malondialdehyde, and nitrate/nitrite levels and ALP activity, whereas plasma alpha-tocopherol levels showed inverse correlations. We conclude that chronic O.viverrini infection induces an accumulation of lipid peroxidation-derived DNA damage through oxidative/nitrative stress, which is lowered by the plasma alpha-tocopherol and by antiparasitic drug therapy. Etheno adducts in WBC and urine should be explored as a risk marker for opisthorchiasis-related cholangiocarcinoma, and to assess the efficacy of preventive and therapeutic interventions. PMID:20056652

  4. ITER helium ash accumulation

    SciTech Connect

    Hogan, J.T.; Hillis, D.L.; Galambos, J.; Uckan, N.A. ); Dippel, K.H.; Finken, K.H. . Inst. fuer Plasmaphysik); Hulse, R.A.; Budny, R.V. . Plasma Physics Lab.)

    1990-01-01

    Many studies have shown the importance of the ratio {upsilon}{sub He}/{upsilon}{sub E} in determining the level of He ash accumulation in future reactor systems. Results of the first tokamak He removal experiments have been analysed, and a first estimate of the ratio {upsilon}{sub He}/{upsilon}{sub E} to be expected for future reactor systems has been made. The experiments were carried out for neutral beam heated plasmas in the TEXTOR tokamak, at KFA/Julich. Helium was injected both as a short puff and continuously, and subsequently extracted with the Advanced Limiter Test-II pump limiter. The rate at which the He density decays has been determined with absolutely calibrated charge exchange spectroscopy, and compared with theoretical models, using the Multiple Impurity Species Transport (MIST) code. An analysis of energy confinement has been made with PPPL TRANSP code, to distinguish beam from thermal confinement, especially for low density cases. The ALT-II pump limiter system is found to exhaust the He with maximum exhaust efficiency (8 pumps) of {approximately}8%. We find 1<{upsilon}{sub He}/{upsilon}{sub E}<3.3 for the database of cases analysed to date. Analysis with the ITER TETRA systems code shows that these values would be adequate to achieve the required He concentration with the present ITER divertor He extraction system.

  5. Bilateral Adduction Palsy in a Patient with Myotonic Dystrophy Type 1

    PubMed Central

    Kim, Hong-Jeon; Oh, Jung-Hwan

    2016-01-01

    Myotonic dystrophy type 1 (DM1) is caused by CTG repeat expansion in the DMPK gene in chromosome 19q13.3. External ophthalmoplegia is a rare manifestation in DM1. We report a DM1 patient confirmed by the presence of 650 CTG triplet expansions in the DMPK gene and had limitation of adduction gaze bilaterally. Brain MRI showed bilateral medial rectus muscles atrophy. Our patient provides additional evidence of ocular motor muscle involvement in DM1. PMID:27358583

  6. 2[prime] and 3[prime] Carboranyl uridines and their diethyl ether adducts

    DOEpatents

    Soloway, A.H.; Barth, R.F.; Anisuzzaman, A.K.; Alam, F.; Tjarks, W.

    1992-12-15

    A process is described for preparing carboranyl uridine nucleoside compounds and their diethyl ether adducts, which exhibit a tenfold increase in boron content over prior art boron containing nucleoside compounds. The carboranyl uridine nucleoside compounds exhibit enhanced lipophilicity and hydrophilic properties adequate to enable solvation in aqueous media for subsequent incorporation of the compounds in methods for boron neutron capture therapy in mammalian tumor cells. No Drawings

  7. Reactions of adducts of phosphorus pentachloride and oxa-containing nucleophiles with arsenic trifluoride

    SciTech Connect

    Fridland, S.V.; Miftakhov, M.N.; Arkhipov, V.P.

    1987-12-20

    Results are given on the synthesis of phosphonofluoridates by the reactions of arsenic trifluoride with adducts of phosphorus pentachloride with oxa-containing nucleophiles. The nucleophiles used were saturated ethers, dioxolanes, and vinyl ethers. Reaction products were identified by means of NMR spectroscopy using H 1, P 31, and C 13. A full analysis of chemical shift and spin-spin coupling constant behavior as well as the spectral structure is conducted.

  8. Aberrant methylation of hypermethylated-in-cancer-1 and exocyclic DNA adducts in tobacco smokers.

    PubMed

    Peluso, Marco E M; Munnia, Armelle; Bollati, Valentina; Srivatanakul, Petcharin; Jedpiyawongse, Adisorn; Sangrajrang, Suleeporn; Ceppi, Marcello; Giese, Roger W; Boffetta, Paolo; Baccarelli, Andrea A

    2014-01-01

    Tobacco smoke has been shown to produce both DNA damage and epigenetic alterations. However, the potential role of DNA damage in generating epigenetic changes is largely underinvestigated in human studies. We examined the effects of smoking on the levels of DNA methylation in genes for tumor protein p53, cyclin-dependent kinase inhibitor2A, hypermethylated-in-cancer-1 (HIC1), interleukin-6, Long Interspersed Nuclear Element type1, and Alu retrotransposons in blood of 177 residents in Thailand using bisulfite-PCR andpyrosequencing. Then, we analyzed the relationship of this methylation with the oxidative DNA adduct, M₁dG (a malondialdehyde adduct), measured by ³²P-postlabeling. Multivariate statistical analyses showed that HIC1 methylation levels were significantly increased in smokers compared with nonsmokers (p ≤ .05). A dose response was observed, with the highest HIC1 methylation levels in smokers of ≥ 10 cigarettes/day relative to nonsmokers and intermediate values in smokers of 1-9 cigarettes/day (p for trend ≤ .001). No additional relationships were observed. We also evaluated correlations between M₁dG and the methylation changes at each HIC1 CpG site individually. The levels of this adduct in smokers showed a significant linear correlation with methylation at one of the 3 CpGs evaluated in HIC1: hypermethylation at position 1904864340 was significantly correlated with the adduct M₁dG (covariate-adjusted regression coefficient (β) = .224 ± .101 [SE], p ≤ .05). No other correlations were detected. Our study extends prior work by others associating hypermethylation of HIC1 with smoking; shows that a very specific hypermethylation event can arise from smoking; and encourages future studies that explore a possible role for M₁dG in connecting smoking to this latter hypermethylation. PMID:24154486

  9. Desorption-induced recombination-cationization of metal-adsorbate adducts from sulfur precovered Ru(0001)

    NASA Astrophysics Data System (ADS)

    Cocco, R. A.; Tatarchuk, B. J.

    1990-03-01

    Temperature programmed static secondary ion mass spectrometry (TPSS) studies of pyrrole, furan and water on sulfur precovered Ru(0001) reveal significant increases in the coverage corrected adduct yield when these species desorb from the surface in an autocatalytic fashion. This effect has been attributed to an enhancement in the recombination-cationization rate which occurs when sputtered-ruthenium species contact desorbed organics just after leaving the surface. Adsorbates which do not undergo autocatalytic/explosive desorption do not exhibit this effect.

  10. Free flow electrophoresis separation and AMS quantitation of 14C-naphthalene-protein adducts

    NASA Astrophysics Data System (ADS)

    Buchholz, Bruce A.; Haack, Kurt W.; Sporty, Jennifer L.; Buckpitt, Alan R.; Morin, Dexter

    2010-04-01

    Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose-(concentration)dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 μCi) of 14C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 h post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with 14C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

  11. Phosphatase activity in commercial spleen exonuclease decreases the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling.

    PubMed

    Adams, S P; Laws, G M; Selden, J R; Nichols, W W

    1994-05-15

    Spleen exonuclease, which degrades nucleic acids into single 3'-nucleotides, is used in the detection of DNA adducts by 32P-postlabeling. Contamination of the exonuclease with phosphatase activity can reduce the recovery of benzo[a]pyrene and N-hydroxy-2-naphthylamine DNA adducts by 32P-postlabeling. Four preparations of spleen exonuclease containing varying levels of phosphatase activity (< 1-62% of the unmodified 3'-nucleotides being dephosphorylated) were used to hydrolyze the DNA. The exonuclease with the lowest phosphatase activity produced a recovery of up to 9.60 mumol of benzo[a]pyrene adducts per mole of DNA. Recovery of benzo[a]pyrene adducts was reduced to 0.56 mumol of adduct per mole of DNA using the exonuclease with the highest phosphatase activity. Phosphatase in the exonucleases also dephosphorylated N-hydroxy-2-naphthylamine DNA adducts. Surprisingly, recovery of these DNA adducts was nearly 10 times greater using nuclease P1 than when using 1-butanol extraction for adduct enrichment, since arylamine DNA adducts have previously been reported to be poorly detected by 32P-postlabeling after nuclease P1 treatment. Our data indicate that the hydrolysis of DNA by spleen exonuclease may be an important source of variability in both qualitative and quantitative analysis of adducts by 32P-postlabeling. PMID:8059938

  12. Flight restriction prevents associative learning deficits but not changes in brain protein-adduct formation during honeybee ageing.

    PubMed

    Tolfsen, Christina C; Baker, Nicholas; Kreibich, Claus; Amdam, Gro V

    2011-04-15

    Honeybees (Apis mellifera) senesce within 2 weeks after they discontinue nest tasks in favour of foraging. Foraging involves metabolically demanding flight, which in houseflies (Musca domestica) and fruit flies (Drosophila melanogaster) is associated with markers of ageing such as increased mortality and accumulation of oxidative damage. The role of flight in honeybee ageing is incompletely understood. We assessed relationships between honeybee flight activity and ageing by simulating rain that confined foragers to their colonies most of the day. After 15 days on average, flight-restricted foragers were compared with bees with normal (free) flight: one group that foraged for ∼15 days and two additional control groups, for flight duration and chronological age, that foraged for ∼5 days. Free flight over 15 days on average resulted in impaired associative learning ability. In contrast, flight-restricted foragers did as well in learning as bees that foraged for 5 days on average. This negative effect of flight activity was not influenced by chronological age or gustatory responsiveness, a measure of the bees' motivation to learn. Contrasting their intact learning ability, flight-restricted bees accrued the most oxidative brain damage as indicated by malondialdehyde protein adduct levels in crude cytosolic fractions. Concentrations of mono- and poly-ubiquitinated brain proteins were equal between the groups, whereas differences in total protein amounts suggested changes in brain protein metabolism connected to forager age, but not flight. We propose that intense flight is causal to brain deficits in aged bees, and that oxidative protein damage is unlikely to be the underlying mechanism. PMID:21430210

  13. Earthworms repair H2O2-induced oxidative DNA adducts without removing UV-induced pyrimidine dimers.

    PubMed

    Chang, Wen-Shin; Tsai, Chia-Wen; Lin, Cheng-Chieh; Lin, Chih-Hsueh; Shen, Wu-Chung; Lin, Song-Shei; Bau, Da-Tian

    2011-01-01

    Ultraviolet (UV) radiation is a natural insult to various organisms. Earthworms, although possessing similar biomolecules to those in mammalian skin, do not suffer from skin cancer nor any other types of cancer as humans do. However, little is known about the molecular mechanism of the earthworm's tolerance to UV. In this study, we evaluated the genotoxicity of UV and the capacity of earthworm cell to repair UV-induced damage. The T4 UV endonuclease UV-incorporated comet assay was used to examine the excision and rejoining steps of UV-induced pyrimidine dimer. Earthworm testis cells were treated with a combination of 5 mM hydroxyurea plus 50 μM cytosine-β-D-arabinofuranoside for 6 h to block DNA rejoining capacity and to investigate excision dynamics. Compared with H(2)O(2)-induced oxidative repair capacity, the excision step of repair of UV-induced lesions in earthworm testis cells was significantly lower. After 6-h treatment of 5 mM hydroxyurea plus 50 μM cytosine-β-D-arabinofuranoside, the medium was totally replaced with fresh medium and cells were allowed to rejoin the accumulated DNA strand breaks. We found that the capacity for rejoining UV-induced breaks was also significantly lower than that for the H(2)O(2)-induced breaks. Our results strongly suggest that earthworms seem to be efficient at repairing H(2)O(2)-induced oxidative DNA adducts, but not so capable of removing UV-induced pyrimidine dimers from their genome. PMID:22021692

  14. Detection of benzo[a]pyrene-guanine adducts in single-stranded DNA using the α-hemolysin nanopore

    NASA Astrophysics Data System (ADS)

    Perera, Rukshan T.; Fleming, Aaron M.; Johnson, Robert P.; Burrows, Cynthia J.; White, Henry S.

    2015-02-01

    The carcinogenic precursor benzo[a]pyrene (BP), a polycyclic aromatic hydrocarbon, is released into the environment through the incomplete combustion of hydrocarbons. Metabolism of BP in the human body yields a potent alkylating agent (benzo[a]pyrene diol epoxide, BPDE) that reacts with guanine (G) in DNA to form an adduct implicated in cancer initiation. We report that the α-hemolysin (αHL) nanopore platform can be used to detect a BPDE adduct to G in synthetic oligodeoxynucleotides. Translocation of a 41-mer poly-2‧-deoxycytidine strand with a centrally located BPDE adduct to G through αHL in 1 M KCl produces a unique multi-level current signature allowing the adduct to be detected. This readily distinguishable current modulation was observed when the BPDE-adducted DNA strand translocated from either the 5‧ or 3‧ directions. This study suggests that BPDE adducts and other large aromatic biomarkers can be detected with αHL, presenting opportunities for the monitoring, quantification, and sequencing of mutagenic compounds from cellular DNA samples.

  15. Extraction of rare earth oxides using supercritical carbon dioxide modified with Tri-n-butyl phosphate–nitric acid adducts

    DOE PAGESBeta

    Baek, Donna L.; Fox, Robert V.; Case, Mary E.; Sinclair, Laura K.; Schmidt, Alex B.; McIlwain, Patrick R.; Mincher, Bruce J.; Wai, Chien M.

    2016-06-14

    A new tri-n-butylphosphate–nitric acid (TBP–HNO3) adduct was prepared by combining TBP and fuming (90%) HNO3. The adduct was characterized, and its phase-equilibrium behavior in supercritical carbon dioxide is reported. Supercritical carbon dioxide (sc-CO2) was modified with this new adduct [TBP(HNO3)5.2(H2O)1.7], and the extraction efficacies of selected rare earth oxides (Y, Ce, Eu, Tb, and Dy) at 338 K and 34.5 MPa were compared with those obtained using an adduct formed from concentrated (70%) HNO3 and TBP [TBP(HNO3)1.7(H2O)0.6]. All rare earth oxides tested with both adduct species could be extracted with the exception of cerium oxide. Furthermore, the water and acidmore » concentrations in the different adducts were found to play a significant role in rare earth oxide extraction efficiency.« less

  16. Molecular characterization of the boron adducts of the proteasome inhibitor bortezomib with epigallocatechin-3-gallate and related polyphenols.

    PubMed

    Glynn, Stephen J; Gaffney, Kevin J; Sainz, Marcos A; Louie, Stan G; Petasis, Nicos A

    2015-04-01

    The green tea polyphenol epigallocatechin-3-gallate (EGCG) was reported to effectively antagonize the ability of Bortezomib (BZM) to induce apoptosis in cancer cells. This interaction was attributed to the formation of a covalent adduct between a phenolic moiety of EGCG with the boronic acid group of Bortezomib. However, the structural details of this boron adduct and the molecular factors that contribute to its formation and its ability to inhibit Bortezomib's activity remain unclear. This paper describes the use of NMR spectroscopy and cell assays to characterize the structures and properties of the boron adducts of EGCG and related polyphenols. The observed boron adducts included both boronate and borate derivatives, and their structural characteristics were correlated with cell-based evaluation of the ability of EGCG and other phenols to antagonize the anticancer activity of Bortezomib. The enhanced stability of the BZM/EGCG adduct was attributed to electronic and steric reasons, and a newly identified intramolecular interaction of the boron atom of BZM with the adjacent amide bond. The reported approach provides a useful method for determining the potential ability of polyphenols to form undesired adducts with boron-based drugs and interfere with their actions. PMID:25669488

  17. The formation of lipid hydroperoxide-derived amide-type lysine adducts on proteins: a review of current knowledge.

    PubMed

    Kato, Yoji

    2014-01-01

    Lipid peroxidation is an important biological reaction. In particular, polyunsaturated fatty acid (PUFA) can be oxidized easily. Peroxidized lipids often react with other amines accompanied by the formation of various covalent adducts. Novel amide-type lipid-lysine adducts have been identified from an in vitro reaction mixture of lipid hydroperoxide with a protein, biological tissues exposed to conditions of oxidative stress and human urine from a healthy person. In this chapter, the current knowledge of amide type adducts is reviewed with a focus on the evaluation of functional foods and diseases with a history of discovery of hexanoyl-lysine (HEL). Although there is extensive research on HEL and other amide-type adducts, the mechanism of generation of the amide bond remains unclear. We have found that the decomposed aldehyde plus peroxide combined with a lysine moiety does not fully explain the formation of the amide-type lipid-lysine adduct that is generated by lipid hydroperoxide. Singlet oxygen or an excited state of the ketone generated from the lipid hydroperoxide may also contribute to the formation of the amide linkage. The amide-adducts may prove useful not only for the detection of oxidative stress induced by disease but also for the estimation of damage caused by an excess intake of PUFA. PMID:24374915

  18. Molecular characterization of the boron adducts of the proteasome inhibitor Bortezomib with epigallocatechin-3-gallate and related polyphenols

    PubMed Central

    Glynn, Stephen J.; Gaffney, Kevin J.; Sainz, Marcos A.; Louie, Stan G.

    2015-01-01

    The green tea polyphenol epigallocatechin-3-gallate (EGCG) was reported to effectively antagonize the ability of Bortezomib to induce apoptosis in cancer cells. This interaction was attributed to the formation of a covalent adduct between a phenolic moiety of EGCG with the boronic acid group of Bortezomib. However, the structural details of this boron adduct and the molecular factors that contribute to its formation and its ability to inhibit Bortezomib's activity remain unclear. This paper describes the use of NMR spectroscopy and cell assays to characterize the structures and properties of the boron adducts of EGCG and related polyphenols. The observed boron adducts included both boronate and borate derivatives, and their structural characteristics were correlated with cell-based evaluation of the ability of EGCG and other phenols to antagonize the anticancer activity of Bortezomib. The enhanced stability of the BZM/EGCG adduct was attributed to electronic and steric reasons, and a newly identified intramolecular interaction of the boron atom of BZM with the adjacent amide bond. The reported approach provides a useful method for determining the potential ability of polyphenols to form undesired adducts with boron-based drugs and interfere with their actions. PMID:25669488

  19. Novel methods for synthesizing halide-free alane without the formation of adducts

    NASA Astrophysics Data System (ADS)

    Dinh, Long V.; Knight, Douglas A.; Paskevicius, Mark; Buckley, Craig E.; Zidan, Ragaiy

    2012-04-01

    Many of the current synthesis methods for aluminum hydride (alane—AlH3) involve reacting AlCl3 and LiAlH4 in solvents. The reaction requires the formation of an alane adduct such as AlH3ṡ[(C2H5)2O] prior to obtaining crystallized stable α-AlH3. This process requires several hours of pumping in a vacuum system to remove the ether and convert the alane etherate into stable α-alane. This crystallization process is both costly and hazardous because a large amount of highly flammable material (e.g. ether) is removed by vacuum pumps over several hours. Conversely, the work presented herein describes novel methods to synthesize adduct-free alane. It is demonstrated here that AlH3 can form by mixing AlCl3 and LiAlH4 in the solid state and heating to 75∘C; only α-AlH3 was obtained. The α-AlH3 product can be washed with minimal solvents leading to zero formation of alane adducts. In addition, the unwanted LiCl by-product is also removed during the solvent wash, resulting in halide-free α-alane. Although simply mixing and heating the reactants led to a 40% yield of alane, having the reactants compacted and mechanically pressed while heating increases the yield to 60% crystalline α-AlH3.

  20. Theoretical study of coupling p-aminothiophenol to hydroazo- and azo-adducts on Au(111).

    PubMed

    Lang, Xiufeng; Liang, Yanhong; Liu, Siyan; Zhao, Shanshan; Lau, Woon-Ming

    2016-09-01

    Aminothiophenol/Au(111) has been adopted as an exemplary model in plasmonics research, including surface-enhanced Raman spectroscopy, due to its high plasmonic-induced spectral-signal enhancement. The present work was aimed at clarifying whether aminothiophenol on Au(111) is chemically stable in the absence of any photo- and plasmonic-induced effects. Briefly, first-principles calculations were employed to track the detailed mechanism of oxidative coupling of p-aminothiophenol (PATP) to its azo-adduct with an N = N bond, i.e., p,p'-dimercaptoazobenzene (DMAB). Our results show the following: first, in the presence of adsorbed O2, PATP fractures its N-H bond and transfers the hydrogen to a nearby oxygen. This pathway is more favorable than the transfer of H to Au, but the activation barrier of 0.9 eV is still too high for the reaction to occur in the absence of thermal-, photo-, or plasmonic-activation. If this bar can be lifted, two such dehydrogenated PATP can couple themselves to form an adduct with a N-N bond, i.e., p,p'-dimercaptohydroazobenzene (DMHAB), and this reaction is exoergic with an energy barrier of 0.57 eV. Again, this step is slow in the absence of moderate thermal activation or photo-/plasmonic-activation. Finally, dehydrogenation of DMHAB gives the azo-adduct of DMAB, and this reaction is spontaneous, with no energy barrier. PMID:27488103

  1. Supramolecular Adducts of Cucurbit[7]uril and Amino Acids in the Gas Phase

    NASA Astrophysics Data System (ADS)

    Kovalenko, Ekaterina; Vilaseca, Marta; Díaz-Lobo, Mireia; Masliy, A. N.; Vicent, Cristian; Fedin, Vladimir P.

    2016-02-01

    The complexation of the macrocyclic cavitand cucurbit[7]uril (Q7) with a series of amino acids (AA) with different side chains (Asp, Asn, Gln, Ser, Ala, Val, and Ile) is investigated by ESI-MS techniques. The 1:1 [Q7 + AA + 2H]2+ adducts are observed as the base peak when equimolar Q7:AA solutions are electrosprayed, whereas the 1:2 [Q7 + 2AA + 2H]2+ dications are dominant when an excess of the amino acid is used. A combination of ion mobility mass spectrometry (IM-MS) and DFT calculations of the 1:1 [Q7 + AA + 2H]2+ (AA = Tyr, Val, and Ser) adducts is also reported and proven to be unsuccessful at discriminating between exclusion or inclusion-type conformations in the gas phase. Collision induced dissociation (CID) revealed that the preferred dissociation pathways of the 1:1 [Q7 + AA + 2H]2+ dications are strongly influenced by the identity of the amino acid side chain, whereas ion molecule reactions towards N-butylmethylamine displayed a common reactivity pattern comprising AA displacement. Special emphasis is given on the differences between the gas-phase behavior of the supramolecular adducts with amino acids (AA = Asp, Asn, Gln, Ser, Ala, Val, and Ile) and those featuring basic (Lys and Arg) and aromatic (Tyr and Phe) side chains.

  2. The effect of vocal fold adduction on the acoustic quality of phonation: ex vivo investigations

    PubMed Central

    Regner, Michael F.; Tao, Chao; Ying, Di; Olszewski, Aleksandra; Zhang, Yu; Jiang, Jack J.

    2011-01-01

    OBJECTIVES The purpose of this study was to investigate the effect of vocal fold adduction on voice quality in an ex vivo larynx model. STUDY DESIGN Prospective, repeated-measures experiments. METHODS Ten excised canine larynges were mounted on an excised larynx phonation system and measurements were recorded for three different vocal fold adduction levels. Acoustic perturbation measurements of jitter, shimmer, and signal-to-noise ratio (SNR) were calculated from recorded radiated sound histories. RESULTS Ex vivo experiments indicated that statistically significant increases in the means of jitter (p=0.005), shimmer (p=0.002), and SNR (p=0.011) measures decreased with respect to vocal fold adduction as the independent variable. Theoretical results showed that the DC and AC component of glottal area increased monotonically with prephonatory glottal area. CONCLUSIONS Acoustic perturbation increased with the degree of vocal fold abduction. Ex vivo larynx measurements suggested that a hyperadducted state may be acoustically best. This may be explained theoretically by an increase in DC/AC ratio as the prephonatory area is increased. PMID:22578437

  3. 32P-postlabelling analysis of DNA adducted with urinary mutagens from smokers of black tobacco.

    PubMed

    Peluso, M; Castegnaro, M; Malaveille, C; Talaska, G; Vineis, P; Kadlubar, F; Bartsch, H

    1990-08-01

    In order to characterize the tobacco-derived mutagens excreted in the urine of tobacco smokers, 32P-postlabelling techniques were used to examine DNA adducts formed from these mutagens with calf thymus DNA in the presence of a metabolic activation system (rat liver S9, Aroclor 1254-induced, with or without acetyl coenzyme A). Using either nuclease P1 or butanol extraction procedures, four-six and three spots, respectively, were reproducibly found on the autoradiograms in the case of the urine extract from two smokers of black tobacco. Using the urinary extract from a non-smoker, only three faint spots were detected after nuclease P1 enrichment. DNA adducts produced in smokers' urine were then compared with those formed by four N-hydroxyarylamines, N-hydroxy-2-amino-3,8-dimethyl-3H-imidazo[4,5-f]quinoxaline, N-hydroxy-2-amino-3-methyl-imidazo[4,5-f]quinoxaline, N-hydroxy-2-naphthylamine and N-hydroxy-4-aminobiphenyl. Visual inspection revealed that none of the reference aromatic amines contributed to the adduct pattern produced by the urinary mutagen(s). However, primary aromatic amines are mainly implicated as urinary mutagens because: (i) they produce frameshift mutations in Salmonella typhimurium strains, (ii) they are easily extractable with blue cotton and (iii) their mutagenicity is abolished by a nitrite treatment procedure for deamination. PMID:2387016

  4. A three-dimensional model of vocal fold abductionÕadduction

    PubMed Central

    Hunter, Eric J.; Titze, Ingo R.; Alipour, Fariborz

    2006-01-01

    A three-dimensional biomechanical model of tissue deformation was developed to simulate dynamic vocal fold abduction and adduction. The model was made of 1721 nearly incompressible finite elements. The cricoarytenoid joint was modeled as a rocking–sliding motion, similar to two concentric cylinders. The vocal ligament and the thyroarytenoid muscle’s fiber characteristics were implemented as a fiber–gel composite made of an isotropic ground substance imbedded with fibers. These fibers had contractile and/or passive nonlinear stress–strain characteristics. The verification of the model was made by comparing the range and speed of motion to published vocal fold kinematic data. The model simulated abduction to a maximum glottal angle of about 31°. Using the posterior-cricoarytenoid muscle, the model produced an angular abduction speed of 405° per second. The system mechanics seemed to favor abduction over adduction in both peak speed and response time, even when all intrinsic muscle properties were kept identical. The model also verified the notion that the vocalis and muscularis portions of the thyroarytenoid muscle play significantly different roles in posturing, with the muscularis portion having the larger effect on arytenoid movement. Other insights into the mechanisms of abduction/adduction were given. PMID:15101653

  5. Development of Enantioselective Polyclonal Antibodies to Detect Styrene Oxide Protein Adducts

    PubMed Central

    Shen, Shuijie; Zhang, Fan; Zeng, Su; Tian, Ye; Chai, Xiaojuan; Gee, Shirley; Hammock, Bruce D.; Zheng, Jiang

    2012-01-01

    Styrene has been reported to be pneumotoxic and hepatotoxic in humans and animals. Styrene oxide, a major reactive metabolite of styrene, has been found to form covalent binding with proteins, such as albumin and hemoglobin. Styrene oxide has two optical isomers and it was reported that the (R)-enantiomer was more toxic than the (S)-enantiomer. The purpose of this study was to develop polyclonal antibodies that can stereoselectively recognize proteins modified by styrene oxide enantiomers at cysteine residues. Immunogens were prepared by alkylation of thiolated keyhole limpet hemocyanin (KLH) with styrene oxide enantiomers. Polyclonal antibodies were raised by immunization of rabbits with the chiral immunogens. Titration tests showed all six rabbits generated high titers of antisera that recognize (R)- or (S)-coating antigens accordingly. No cross-reaction was observed toward the carrier protein (BSA). All three rabbits immunized with (R)-immunogen produced antibodies that show enantioselectivity to the corresponding antigen, while only one among the three rabbits immunized with (S)-immunogen generated antibodies with enantioselectivity of the recognition. The enantioselectivity was also observed in competitive ELISA and immunoblot analysis. Additionally, competitive ELISA tests showed that the immunorecognition required the hydroxyl group of the haptens. Immunoblot analysis demonstrated that the immunorecognition depended on the amount of protein adducts blotted and hapten loading in protein adducts. In summary, we successfully developed polyclonal antibodies to stereoselectively detect protein adducts modified by styrene oxide enantiomers. PMID:19245253

  6. Molecular structures of five adducts assembled from p-dimethylaminobenzaldehyde and organic acids

    NASA Astrophysics Data System (ADS)

    Jin, Shouwen; Wang, Lanqing; Liu, Hui; Liu, Li; Zhang, Huan; Wang, Daqi; Li, Minghui; Guo, Jianzhong; Guo, Ming

    2016-07-01

    Five adducts 1-5 derived from p-dimethylaminobenzaldehyde have been prepared and characterized by X-ray diffraction analysis, IR, mp, and elemental analysis. Of the five adducts two are organic salts (1, and 2) and the other three (3-5) are cocrystals. In salts 1, and 2, the L molecules are protonated. The supramolecular architectures of the adducts 1-5 involve extensive intermolecular N-H⋯O, O-H⋯O, O-H⋯S, and C-H⋯O hydrogen bonds as well as other non-covalent interactions. The role of weak and strong non-covalent interactions in the crystal packing is ascertained. The complexes displayed 2D/3D framework structure for the synergistic effect of the various non-covalent interactions. The results presented herein tell that the strength and directionality of the N-H⋯O, O-H⋯O, and O-H⋯S hydrogen bonds between organic acids and p-dimethylaminobenzaldehyde are sufficient to bring about the formation of binary cocrystals or organic salts.

  7. Characterization and reactivity of a terminal nickel(III)-oxygen adduct.

    PubMed

    Pirovano, Paolo; Farquhar, Erik R; Swart, Marcel; Fitzpatrick, Anthony J; Morgan, Grace G; McDonald, Aidan R

    2015-02-23

    High-valent terminal metal-oxygen adducts are hypothesized to be the potent oxidizing reactants in late transition metal oxidation catalysis. In particular, examples of high-valent terminal nickel-oxygen adducts are scarce, meaning there is a dearth in the understanding of such oxidants. A monoanionic Ni(II)-bicarbonate complex has been found to react in a 1:1 ratio with the one-electron oxidant tris(4-bromophenyl)ammoniumyl hexachloroantimonate, yielding a thermally unstable intermediate in high yield (ca. 95%). Electronic absorption, electronic paramagnetic resonance, and X-ray absorption spectroscopies and density functional theory calculations confirm its description as a low-spin (S = 1/2), square planar Ni(III)-oxygen adduct. This rare example of a high-valent terminal nickel-oxygen complex performs oxidations of organic substrates, including 2,6-di-tert-butylphenol and triphenylphosphine, which are indicative of hydrogen atom abstraction and oxygen atom transfer reactivity, respectively. PMID:25612563

  8. The reversibility of the glutathionyl-quercetin adduct spreads oxidized quercetin-induced toxicity

    SciTech Connect

    Boots, Agnes W. . E-mail: a.boots@farmaco.unimaas.nl; Balk, Jiska M.; Bast, Aalt; Haenen, Guido R.M.M.

    2005-12-16

    Quercetin is one of the most prominent dietary antioxidants. During its antioxidant activity, quercetin becomes oxidized into its o-quinone/quinone methide QQ. QQ is toxic since it instantaneously reacts with thiols of, e.g., proteins. In cells, QQ will initially form an adduct with glutathione (GSH), giving GSQ. We have found that GSQ is not stable; it dissociates continuously into GSH and QQ with a half life of 2 min. Surprisingly, GSQ incubated with 2-mercapto-ethanol (MSH), a far less reactive thiol, results in the conversion of GSQ into the MSH-adduct MSQ. A similar conversion of GSQ into relatively stable protein thiol-quercetin adducts is expected. With the dithiol dihydrolipoic acid (L(SH){sub 2}), quercetin is formed out of GSQ. These results indicate that GSQ acts as transport and storage of QQ. In that way, the initially highly focussed toxicity of QQ is dispersed by the formation of GSQ that finally spreads QQ-induced toxicity, probably even over cells.

  9. Adducts of nitrous oxide and N-heterocyclic carbenes: syntheses, structures, and reactivity.

    PubMed

    Tskhovrebov, Alexander G; Vuichoud, Basile; Solari, Euro; Scopelliti, Rosario; Severin, Kay

    2013-06-26

    N-Heterocyclic carbenes (NHCs) react at ambient conditions with nitrous oxide to give covalent adducts. In the crystal, all compounds show a bent N2O group connected via the N-atom to the former carbene carbon atom. Most adducts are stable at room temperature, but heating induces decomposition into the corresponding ureas. Kinetic experiments show that the thermal stability of the NHC-N2O adducts depends on steric as well as electronic effects. The coordination of N2O to NHCs weakens the N-N bond substantially, and facile N-N bond rupture was observed in reactions with acid or acetyl chloride. On the other hand, reaction with tritylium tetrafluoroborate resulted in a covalent modification of the terminal O-atom, and cleavage of the C-N2O bond was observed in a reaction with thionyl chloride. The coordination chemistry of IMes-N2O (IMes = 1,3-dimesitylimidazol-2-ylidene) was explored in reactions with the complexes CuOTf, Fe(OTf)2, PhSnCl3, CuCl2, and Zn(C6F5)2. Structural analyses show that IMes-N2O is able to act as a N-donor, as an O-donor, or as a chelating N,O-donor. The different coordination modes go along with pronounced electronic changes as evidenced by a bond length analysis. PMID:23758062

  10. Catalytic activities of Werner protein are affected by adduction with 4-hydroxy-2-nonenal

    PubMed Central

    Czerwińska, Jolanta; Poznański, Jarosław; Dębski, Janusz; Bukowy, Zuzanna; Bohr, Vilhelm A.; Tudek, Barbara; Speina, Elżbieta

    2014-01-01

    4-Hydroxy-2-nonenal (HNE) is a reactive α,β-unsaturated aldehyde generated during oxidative stress and subsequent peroxidation of polyunsaturated fatty acids. Here, Werner protein (WRN) was identified as a novel target for modification by HNE. Werner syndrome arises through mutations in the WRN gene that encodes the RecQ DNA helicase which is critical for maintaining genomic stability. This hereditary disease is associated with chromosomal instability, premature aging and cancer predisposition. WRN appears to participate in the cellular response to oxidative stress and cells devoid of WRN display elevated levels of oxidative DNA damage. We demonstrated that helicase/ATPase and exonuclease activities of HNE-modified WRN protein were inhibited both in vitro and in immunocomplexes purified from the cell extracts. Sites of HNE adduction in human WRN were identified at Lys577, Cys727, His1290, Cys1367, Lys1371 and Lys1389. We applied in silico modeling of the helicase and RQC domains of WRN protein with HNE adducted to Lys577 and Cys727 and provided a potential mechanism of the observed deregulation of the protein catalytic activities. In light of the obtained results, we postulate that HNE adduction to WRN is a post-translational modification, which may affect WRN conformational stability and function, contributing to features and diseases associated with premature senescence. PMID:25170083

  11. Signal transduction in light–oxygen–voltage receptors lacking the adduct-forming cysteine residue

    PubMed Central

    Yee, Estella F.; Diensthuber, Ralph P.; Vaidya, Anand T.; Borbat, Peter P.; Engelhard, Christopher; Freed, Jack H.; Bittl, Robert; Möglich, Andreas; Crane, Brian R.

    2015-01-01

    Light–oxygen–voltage (LOV) receptors sense blue light through the photochemical generation of a covalent adduct between a flavin-nucleotide chromophore and a strictly conserved cysteine residue. Here we show that, after cysteine removal, the circadian-clock LOV-protein Vivid still undergoes light-induced dimerization and signalling because of flavin photoreduction to the neutral semiquinone (NSQ). Similarly, photoreduction of the engineered LOV histidine kinase YF1 to the NSQ modulates activity and downstream effects on gene expression. Signal transduction in both proteins hence hinges on flavin protonation, which is common to both the cysteinyl adduct and the NSQ. This general mechanism is also conserved by natural cysteine-less, LOV-like regulators that respond to chemical or photoreduction of their flavin cofactors. As LOV proteins can react to light even when devoid of the adduct-forming cysteine, modern LOV photoreceptors may have arisen from ancestral redox-active flavoproteins. The ability to tune LOV reactivity through photoreduction may have important implications for LOV mechanism and optogenetic applications. PMID:26648256

  12. Chemical discrimination between dC and 5MedC via their hydroxylamine adducts

    PubMed Central

    Münzel, Martin; Lercher, Lukas; Müller, Markus; Carell, Thomas

    2010-01-01

    The presence of the methylated nucleobase 5MedC in CpG islands is a key factor that determines gene silencing. False methylation patterns are responsible for deteriorated cellular development and are a hallmark of many cancers. Today genes can be sequenced for the content of 5MedC only with the help of the bisulfite reagent, which is based exclusively on chemical reactivity differences established by the additional methyl group. Despite intensive optimization of the bisulfite protocol, the method still has specificity problems. Most importantly ∼95% of the DNA analyte is degraded during the analysis procedure. We discovered that the reagent O-allylhydroxylamine is able to discriminate between dC and 5MedC. The reagent, in contrast to bisulfite, does not exploit reactivity differences but gives directly different reaction products. The reagent forms a stable mutagenic adduct with dC, which can exist in two states (E versus Z). In case of dC the allylhydroxylamine adduct switches into the E-isomeric form, which generates dC to dT transition mutations that can easily be detected by established methods. Significantly, the 5MedC-adduct adopts exclusively the Z-isomeric form, which causes the polymerase to stop. O-allylhydroxylamine does allow differentiation between dC and 5MedC with high accuracy, leading towards a novel and mild chemistry for methylation analysis. PMID:20813757

  13. Characterization and Reactivity of a Terminal Nickel(III)-Oxygen Adduct

    DOE PAGESBeta

    Pirovano, Paolo; Farquhar, Erik R.; Swart, Marcel; Fitzpatrick, Anthony J.; Morgan, Grace G.; McDonald, Aidan R.

    2015-01-22

    Here, high-valent terminal metal–oxygen adducts are hypothesized to be the potent oxidizing reactants in late transition metal oxidation catalysis. In particular, examples of high-valent terminal nickel–oxygen adducts are scarce, meaning there is a dearth in the understanding of such oxidants. A monoanionic NiII-bicarbonate complex has been found to react in a 1:1 ratio with the one-electron oxidant tris(4-bromophenyl)ammoniumyl hexachloroantimonate, yielding a thermally unstable intermediate in high yield (ca. 95%). Electronic absorption, electronic paramagnetic resonance, and X-ray absorption spectroscopies and density functional theory calculations confirm its description as a low-spin (S=1/2), square planar NiIII–oxygen adduct. Moreover, this rare example of amore » high-valent terminal nickel–oxygen complex performs oxidations of organic substrates, including 2,6-di-tert-butylphenol and triphenylphosphine, which are indicative of hydrogen atom abstraction and oxygen atom transfer reactivity, respectively.« less

  14. Characterization and Reactivity of a Terminal Nickel(III)-Oxygen Adduct

    SciTech Connect

    Pirovano, Paolo; Farquhar, Erik R.; Swart, Marcel; Fitzpatrick, Anthony J.; Morgan, Grace G.; McDonald, Aidan R.

    2015-01-22

    Here, high-valent terminal metal–oxygen adducts are hypothesized to be the potent oxidizing reactants in late transition metal oxidation catalysis. In particular, examples of high-valent terminal nickel–oxygen adducts are scarce, meaning there is a dearth in the understanding of such oxidants. A monoanionic NiII-bicarbonate complex has been found to react in a 1:1 ratio with the one-electron oxidant tris(4-bromophenyl)ammoniumyl hexachloroantimonate, yielding a thermally unstable intermediate in high yield (ca. 95%). Electronic absorption, electronic paramagnetic resonance, and X-ray absorption spectroscopies and density functional theory calculations confirm its description as a low-spin (S=1/2), square planar NiIII–oxygen adduct. Moreover, this rare example of a high-valent terminal nickel–oxygen complex performs oxidations of organic substrates, including 2,6-di-tert-butylphenol and triphenylphosphine, which are indicative of hydrogen atom abstraction and oxygen atom transfer reactivity, respectively.

  15. Protein adducts of the prostate carcinogen PhIP in children

    SciTech Connect

    Lawrence Livermore National Laboratory

    2004-02-20

    Prostate cancer is the second leading cause of cancer death in men in the United States. few epidemiology studies have indicated that exposure to PhIP, a rodent prostate carcinogen formed in meat during cooking, may be an important risk factor for prostate cancer in humans. Therefore, a highly sensitive biomarker assay is urgently needed to clarify the role of PhIP in prostate cancer. The goal of this project is to develop an assay that can be used to more accurately quantify human exposure to PhIP and potential prostate cancer risk. Our hypothesis is that an Accelerator Mass Spectrometry-based method can be developed to measure protein adducts of PhIP in the blood of humans. This will provide a measure of the internal dose, as well as the capacity for carcinogen bioactivation to a form that can initiate the cancer process. Towards this goal, we have characterized an adduct formed by PhIP in vitro with the amino acid cysteine. This adduct should provide a biomarker of dietary PhIP exposure and potential prostate cancer risk that could be used to identify individuals for prevention and for monitoring the effect chemoprevention strategies.

  16. DNA adduction by phenol, hydroquinone, or benzoquinone in vitro but not in vivo: nuclease P1-enhanced 32P-postlabeling of adducts as labeled nucleoside bisphosphates, dinucleotides and nucleoside monophosphates.

    PubMed

    Reddy, M V; Bleicher, W T; Blackburn, G R; Mackerer, C R

    1990-08-01

    The carcinogenicity of benzene has been considered to be in part mediated by its chemically reactive metabolic product benzoquinone (BQ), which is formed from the intermediary metabolites phenol and hydroquinone (HQ). We have evaluated the DNA-binding capability of these chemicals in vitro and in vivo by postlabeling. Treatment of rat Zymbal glands in culture with phenol and HQ or direct reaction of BQ with DNA produced DNA adducts, which were detectable by the nuclease P1-enhanced 32P-postlabeling assay as 5'-32P-labeled 3',5'-bisphosphate products. The enhancement of sensitivity in this assay is based on the previous finding that nuclease P1 hydrolyzes the phosphate attached to the 3' side of normal nucleotides but not the corresponding phosphate of most aromatic/bulky adducted nucleotides. Also based on this hydrolytic property of nuclease P1, we developed an additional sensitive procedure that permitted the detection of DNA lesions as 5'-32P-labeled products of dinucleotides, pXpN, or of nucleoside monophosphates, pX, where X and N indicate an adducted nucleoside and a normal nucleoside respectively. In the latter assay, adducted DNA was first digested with nuclease P1 and acid phosphatase to yield XpN and N. The latter were then 32P-labeled to yield [5'-32P] pXpN or 32P-labeled and treated with venom phosphodiesterase to obtain [5'-32P]pX. After optimization of enzymatic conditions, the modified nuclease P1 assay yielded adduct recoveries similar to those obtained by the bisphosphate assay for in vitro phenol-, HQ- and BQ-DNA adducts. Neither of the nuclease P1-enhanced postlabeling procedures showed exposure-specific adducts in vivo in the bone marrow, Zymbal gland, liver and spleen of female Sprague-Dawley rats at 24 h after the last of four single, daily p.o. doses of 75 mg/kg phenol or 150 mg/kg phenol/HQ (1:1). Our results show that phenol, HQ and BQ produce adducts in vitro, but corresponding adducts are not detected in vivo with phenol and phenol

  17. Induction of ovarian cancer and DNA adducts by dibenzo[a,l]pyrene in the mouse

    PubMed Central

    Chen, Kun-Ming; Zhang, Shang-Min; Aliaga, Cesar; Sun, Yuan-Wan; Cooper, Timothy; Gowdahalli, Krishnegowda; Zhu, Junjia; Amin, Shantu; El-Bayoumy, Karam

    2011-01-01

    Tobacco smoking is an etiological factor of ovarian cacner; however, the mechanisms remain largely undefined. Therefore, as an initial investigation we examined the carcinogenicity and DNA adducts formation in the ovary of mice treated with DB[a,l]P, a tobacco smoke constituent and environmental pollutant. Ovarian tumors in B6C3F1 mice were induced by direct application of DB[a,l]P (24, 12, 6, and 3 nmol/mouse, 3 times a week for 38 weeks) into the oral cavity of mice. At 6 nmol, DB[a,l]P induced the highest total ovarian tumor incidence (79%), but the incidence of malignancy was only 15%. However, at the dose of 12 nmol, the total ovarian tumor incidence was 75%, and the incidence of malignancy was 65%. In addition to ovarian tumors, at the dose of 24 nmol, DB[a,l]P induced lesions in sites distal from the ovaries including the skin, mammary, lung, and oral tissues which were rare at doses lower than 24 nmol. Another bioassay was conducted to detect and quantify DNA-adducts induced by DB[a,l]P (24 nmol, 3 times a week for 5 weeks) in the ovary at 48 h, 1, 2 and 4 weeks after the last administration of DB[a,l]P. DNA was isolated, and the dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE)-DNA adducts were analyzed by a LC-MS/MS method. DB[a,l]P resulted in the formation of (−)-anti-cis-DB[a,l]PDE-dA and (−)-anti-trans-DB[a,l]PDE-dA adducts, which were 0.8 and 1.6 fmol/106 dA respectively in ovaries of mice within 48 h, and the level of adducts decreased over a week. Our results indicated that DB[a,l]P can be metabolized to form (−)-anti-DB[a,l]PDE; the latter may, in part, account for DB[a,l]P-induced ovarian cancer. This animal model should assist to better understand the mechanisms, account for the induction of ovarian cancer by tobacco carcinogens, and facilitate the development of chemopreventive agents against ovarian cancer. PMID:22107356

  18. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

    PubMed

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø; Rizzo, Carmelo J; Guengerich, F Peter; Tudek, Barbara

    2015-06-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  19. Characterization of glycidol-hemoglobin adducts as biomarkers of exposure and in vivo dose

    SciTech Connect

    Honda, Hiroshi; Törnqvist, Margareta; Nishiyama, Naohiro; Kasamatsu, Toshio

    2014-03-15

    Hemoglobin adducts have been used as biomarkers of exposure to reactive chemicals. Glycidol, an animal carcinogen, has been reported to form N-(2,3-dihydroxy-propyl)valine adducts to hemoglobin (diHOPrVal). To support the use of these adducts as markers of glycidol exposure, we investigated the kinetics of diHOPrVal formation and its elimination in vitro and in vivo. Five groups of rats were orally administered a single dose of glycidol ranging from 0 to 75 mg/kg bw, and diHOPrVal levels were measured 24 h after administration. A dose-dependent increase in diHOPrVal levels was observed with high linearity (R{sup 2} = 0.943). Blood sampling at different time points (1, 10, 20, or 40 days) from four groups administered glycidol at 12 mg/kg bw suggested a linear decrease in diHOPrVal levels compatible with the normal turnover of rat erythrocytes (life span, 61 days), with the calculated first-order elimination rate constant (k{sub el}) indicating that the diHOPrVal adduct was chemically stable. Then, we measured the second-order rate constant (k{sub val}) for the reaction of glycidol with N-terminal valine in rat and human hemoglobin in in vitro experiments with whole blood. The k{sub val} was 6.7 ± 1.1 and 5.6 ± 1.3 (pmol/g globin per μMh) in rat and human blood, respectively, indicating no species differences. In vivo doses estimated from k{sub val} and diHOPrVal levels were in agreement with the area under the (concentration–time) curve values determined in our earlier toxicokinetic study in rats. Our results indicate that diHOPrVal is a useful biomarker for quantification of glycidol exposure and for risk assessment. - Highlight: • Glycidol-hemoglobin adduct (diHOPrVal) was characterized for exposure evaluation. • We studied the kinetics of diHOPrVal formation and elimination in vitro and in vivo. • Dose dependent formation and chemical stability were confirmed in the rat study. • In vivo dose (AUC) of glycidol could be estimated from diHOPrVal levels

  20. S-arylcysteine-keratin adducts as biomarkers of human dermal exposure to aromatic hydrocarbons.

    PubMed

    Kang-Sickel, Juei-Chuan C; Fox, Donii D; Nam, Tae-Gyu; Jayaraj, Karupiah; Ball, Louise M; French, John E; Klapper, David G; Gold, Avram; Nylander-French, Leena A

    2008-04-01

    To measure biomarkers of skin exposure to ubiquitous industrial and environmental aromatic hydrocarbons, we sought to develop an ELISA to quantitate protein adducts of metabolites of benzene and naphthalene in the skin of exposed individuals. We hypothesized that electrophilic arene oxides formed by CYP isoforms expressed in the human skin react with nucleophilic sites on keratin, the most abundant protein in the stratum corneum that is synthesized de novo during keratinocyte maturation and differentiation. The sulfhydryl groups of cysteines in the head region of the keratin proteins 1 (K1) and 10 (K10) are likely targets. The following synthetic S-arylcysteines were incorporated into 10-mer head sequences of K1 [GGGRFSS( S-aryl-C)GG] and K10 [GGGG( S-aryl-C)GGGGG] to form the predicted immunogenic epitopes for antibody production for ELISA: S-phenylcysteine-K1 (SPK1), S-phenylcysteine-K10 (SPK10), S-(1-naphthyl)cysteine-K1 (1NK1), S-(1-naphthyl)cysteine-K10 (1NK10), S-(2-naphthyl)cysteine-K1 (2NK1), and S-(2-naphthyl)cysteine-K10 (2NK10). Analysis by ELISA was chosen based on its high throughput and sensitivity, and low cost. The synthetic modified oligopeptides, available in quantity, served both as immunogens and as chemical standards for quantitative ELISA. Polyclonal rabbit antibodies produced against the naphthyl-modified keratins reacted with their respective antigens with threshold sensitivities of 15-31 ng/mL and high specificity over a linear range up to 500 ng/mL. Anti- S-phenylcysteine antibodies were not sufficiently specific or sensitive toward the target antigens for use in ELISA under our experimental conditions. In dermal tape-strip samples collected from 13 individuals exposed to naphthalene-containing jet fuel, naphthyl-conjugated peptides were detected at levels from 0.343 +/- 0.274 to 2.34 +/- 1.61 pmol adduct/microg keratin but were undetectable in unexposed volunteers. This is the first report of adducts of naphthalene (or of any polycyclic

  1. Inhibition of DNA adduct formation of PhIP in female F344 rats by dietary conjugated linoleic acid.

    PubMed

    Josyula, S; He, Y H; Ruch, R J; Schut, H A

    1998-01-01

    The dietary mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mammary carcinogen in the female Fischer (F344) rat and a colon carcinogen in the male F344 rat. To exert its carcinogenicity, it is believed that PhIP needs to form adducts with DNA, a process requiring N-hydroxylation of PhIP by cytochromes P-450 1A1 and/or 1A2 (CYP 1A1 and/or 1A2), as well as further esterification of the hydroxylamine thus formed. Dietary conjugated linoleic acid (CLA) inhibits chemical carcinogenesis in various experimental models. We have examined the effect of dietary CLA on PhIP-DNA adduct formation in female F344 rats. Four-week-old animals were maintained on AIN-76A diet without or with CLA (1%, 0.5%, and 0.1% wt/wt) for 57 days. PhIP was added to the diets (0.04% wt/wt) from Days 14-42. Animals were killed (4/group) on Days 43, 50, and 57. DNA isolated from liver, mammary epithelial cells (MEC), colon, and white blood cells (WBC) was analyzed for PhIP-DNA adducts by 32P-postlabeling assays. On Day 43, CLA inhibited adduct formation in the liver (up to 58%) in a dose-dependent manner. CLA also inhibited hepatic adduct levels (29-39%) on Day 50 (at 1.0% and 0.5% CLA) and on Day 57 (53% at 0.5% CLA). CLA significantly reduced adduct levels in the WBC on Day 50 (63-70%). Adducts in MEC and the colon were not affected by dietary CLA. On Day 57, adduct levels in MEC, liver, colon, and WBC were 0-30.3%, 8.6-41.7%, 21.5-50.7%, and 7.5-11.8%, respectively, of those on Day 43. Northern blot analysis of liver RNA showed that dietary CLA did not affect steady-state levels of CYP 1A1 or 1A2 mRNA. It is concluded that dietary CLA inhibits PhIP-DNA adduct formation in liver and WBC but that those in MEC and the colon are unaffected when a low-level dietary regimen of carcinogen and inhibitor was used. In inhibiting PhIP-DNA adduct formation, CLA does not appear to act by inhibiting CYP 1A1 or 1A2 expression. PMID:10050262

  2. Identification of adducts derived from reactions of (1-chloroethenyl)oxirane with nucleosides and calf thymus DNA.

    PubMed

    Munter, Tony; Cottrell, Lisa; Hill, Stuart; Kronberg, Leif; Watson, William P; Golding, Bernard T

    2002-12-01

    (1-Chloroethenyl)oxirane is a major mutagenic metabolite of chloroprene, an important large-scale petrochemical used in the manufacture of synthetic rubbers. The reactions of (1-chloroethenyl)oxirane with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, thymidine, and calf thymus DNA have been studied in aqueous buffered solutions. The adducts from the nucleosides were isolated by reversed-phase HPLC, and characterized by their UV absorbance and (1)H and (13)C NMR spectroscopic and mass spectrometric features. The reaction with 2'-deoxyguanosine gave one major adduct, N7-(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGI), and eight minor adducts which were identified as diastereoisomeric pairs of N1-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyguanosine (dGII, dGIII), N3,N7-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGIV, dGV), N7,N9-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGVI, dGVII), and N1,N7-bis(3-chloro-2-hydroxy-3-buten-1-yl)-guanine (dGVIII, dGIX). The reaction of 2'-deoxyadenosine with (1-chloroethenyl)oxirane gave two adducts: N1-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (dAI) and N(6)-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyadenosine (dAII). The adduct dAII was shown to arise via a Dimroth rearrangement of adduct dAI. The HPLC analyses of the reaction mixtures of (1-chloroethenyl)oxirane with 2'-deoxycytidine and thymidine showed the formation of one major product in each reaction. The adduct from 2'-deoxycytidine was identified as N3-(3-chloro-2-hydroxy-3-buten-1-yl)-2'-deoxyuridine (dCI) derived by alkylation at N-3 followed by deamination. The adduct from thymidine was identified as N3-(3-chloro-2-hydroxy-3-buten-1-yl)-thymidine (TI). Reaction of (1-chloroethenyl)oxirane with calf thymus DNA gave all of the adducts observed from the individual nucleosides except dGII and dGIII. However, there was selectivity for the formation of dGI and dCI. The adduct levels in DNA were 9,630 (dGI), 240 (dCI), 83 (dAI), 6 (dAII), and 28 (TI

  3. DNA adducts of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) in fetal tissues of patas monkeys after transplacental exposure.

    PubMed

    Josyula, S; Lu, L J; Salazar, J J; Nerurkar, P V; Jones, A B; Grady, J J; Snyderwine, E G; Anderson, L M

    2000-08-01

    Transplacental genotoxicity of the heterocyclic amine food-derived mutagen/carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) has been investigated by (32)P-postlabeling assay for IQ-DNA adducts in maternal liver, placenta, and several fetal tissues of patas monkeys, after exposure to 15, 35, or 50 mg/kg IQ near the end of gestation or to the highest dose in the first or second trimester. Dose-dependent adduct formation occurred in all tissues, with the highest levels occurring in maternal liver. Adduct amounts were similar among fetal tissues and placenta, except for lower levels in fetal brain and slightly more adducts in fetal liver. Adducts in placenta, fetal liver, lung, kidney, skin, and adrenal gland, but not in maternal liver or fetal brain, increased significantly as gestation progressed. Pretreatment with phenobarbital, which induces CYP enzymes that detoxify IQ, decreased adducts in maternal liver and possibly placenta, but not in fetal tissues. The CYP inducer beta-naphthoflavone caused a significant increase in IQ-DNA adducts in fetal lungs. Regression analysis suggested that IQ activation in maternal and fetal liver and possibly placenta contributed to adduct formation in fetal tissues; adducts in placenta and/or fetal liver were strong predictors for those in most fetal tissues. The results indicate that exposure of pregnant primates to IQ results in DNA adduct formation in most fetal tissues, especially late in gestation; that upregulation of maternal detoxification does not provide fetal protection; and that adducts in placenta indicate adduct levels in fetal tissues. PMID:10906279

  4. High-resolution anion-exchange and partition thin-layer chromatography for complex mixtures of 32P-postlabeled DNA adducts.

    PubMed

    Spencer-Beach, G G; Beach, A C; Gupta, R C

    1996-03-01

    32P-Postlabeling has emerged as a major tool for detecting DNA adducts resulting from exposure to complex carcinogen mixtures. An integral component of this assay is multi-directional PEI-cellulose TLC in which lipophilic 32P-adducts are resolved in high-salt, high-urea solvents following removal of the bulk of non-adduct radioactivity. This TLC system is very effective for adducts formed following exposure to individual carcinogens; however, adducts resulting from exposure to complex mixtures (e.g. cigarette smoke) generally appear in the form of the so-called diagonal radioactive zones. By using mixtures of polycyclic aromatic hydrocarbon- and aromatic amine-DNA adducts as well as adducts in mouse skin treated with cigarette smoke condensate, we have demonstrated that a combination of 0.3-0.4 M NH4OH and isopropanol-4 M NH4OH (1-1.4:1) solvents can provide more sharply defined adduct spots than the commonly used urea solvents. The non-urea solvents also result in excellent resolution of many adducts which otherwise may remain buried in diagonal radioactive zones when using the urea solvents. In addition, the signal-to-noise ratio is increased 2- to 5-fold over the urea solvents enabling detection of discrete adducts at < or = 3 adducts per 10(10) nucleotides. These partition TLC solvents also involve fewer manipulations (e.g. no water washes to remove salt and urea), and are likely to be more informative with regards to the type of individual adducts detected in the biomonitoring of humans than has hitherto been possible. PMID:8704930

  5. Time-resolved fluorescence spectroscopy investigation of the effect of 4-hydroxynonenal on endogenous NAD(P)H in living cardiac myocytes

    NASA Astrophysics Data System (ADS)

    Chorvatova, Alzbeta; Aneba, Swida; Mateasik, Anton; Chorvat, Dusan; Comte, Blandine

    2013-06-01

    Lipid peroxidation is a major biochemical consequence of the oxidative deterioration of polyunsaturated lipids in cell membranes and causes damage to membrane integrity and loss of protein function. 4-hydroxy-2-nonenal (HNE), one of the most reactive products of n-6 polyunsaturated fatty acid peroxidation of membrane phospholipids, has been shown to be capable of affecting both nicotinamide adenine dinucleotide (phosphate) reduced [NAD(P)H] as well as NADH production. However, the understanding of its effects in living cardiac cells is still lacking. Our goal was to therefore investigate HNE effects on NAD(P)H noninvasively in living cardiomyocytes. Spectrally resolved lifetime detection of endogenous fluorescence, an innovative noninvasive technique, was employed. Individual fluorescence components were resolved by spectral linear unmixing approach. Gathered results revealed that HNE reduced the amplitude of both resolved NAD(P)H components in a concentration-dependent manner. In addition, HNE increased flavoprotein fluorescence and responsiveness of the NAD(P)H component ratio to glutathione reductase (GR) inhibitor. HNE also increased the percentage of oxidized nucleotides and decreased maximal NADH production. Presented data indicate that HNE provoked an important cell oxidation by acting on NAD(P)H regulating systems in cardiomyocytes. Understanding the precise role of oxidative processes and their products in living cells is crucial for finding new noninvasive tools for biomedical diagnostics of pathophysiological states.

  6. Noise Reduction by Signal Accumulation

    ERIC Educational Resources Information Center

    Kraftmakher, Yaakov

    2006-01-01

    The aim of this paper is to show how the noise reduction by signal accumulation can be accomplished with a data acquisition system. This topic can be used for student projects. In many cases, the noise reduction is an unavoidable part of experimentation. Several techniques are known for this purpose, and among them the signal accumulation is the…

  7. Strategies for synthesis of adducts of omicron-quinone metabolites of carcinogenic polycyclic aromatic hydrocarbons with 2'-deoxyribonucleosides.

    PubMed

    Ran, Chongzhao; Dai, Qing; Ruan, Qian; Penning, Trevor M; Blair, Ian A; Harvey, Ronald G

    2008-02-01

    Polycyclic aromatic hydrocarbons (PAHs) are major environmental carcinogens produced in the combustion of fossil fuels, tobacco, and other organic matter. Current evidence indicates that PAHs are transformed enzymatically to active metabolites that react with DNA to form adducts that result in mutations. Three activation pathways have been proposed: the diol epoxide path, the radical-cation path, and the quinone path. The latter involves aldo-keto reductase mediated oxidation of PAH dihydrodiol metabolites to catechols that enter into redox cycles with quinones. This results in generation of reactive oxygen species (ROS) that attack DNA, and the PAH quinones also react with DNA to form adducts. Several strategies for synthesis of the stable adducts formed by the o-quinone metabolites of carcinogenic PAHs with 2'-deoxyribonucleosides were investigated and compared. The PAH quinones studied were benz[a]anthracene-3,4-dione and its 7-methyl- and 7,12-dimethyl- derivatives. The parent PAHs represent a range of carcinogenicity from inactive to highly potent. Two synthetic methods were devised that differ in the catalyst employed, Pd(OAc)(2) or CuI. The Pd-mediated method involved coupling a protected amino-catechol PAH derivative with a halo-2'-deoxyribonucleoside. The copper-mediated method entailed reaction of a halo-PAH catechol derivative with a 2'-deoxyribonucleoside. Adducts of benz[a]anthracene-3,4-dione (and its 7-methyl- and 7,12-dimethyl- derivatives) with 2'-deoxyadenosine and 2'-deoxyguanosine were prepared by these methods. Availability of adducts of these types through synthesis makes possible for the first time biological studies to determine the role of these adducts in tumorigenesis. The copper-mediated method offers advantages of economy, adaptability to large-scale preparation, utility for synthesis of (13)C- or (15)N-labeled analogues, and nonformation of bis-adducts as secondary products. PMID:18181642

  8. 32P-postlabeling assay for carcinogen-DNA adducts: nuclease P1-mediated enhancement of its sensitivity and applications.

    PubMed Central

    Reddy, M V; Randerath, K

    1987-01-01

    Exceedingly sensitive assays are required for the detection of DNA adducts formed in humans exposed to low levels of environmental genotoxicants and therapeutic drugs. A 32P-postlabeling procedure for detection and quantitation of aromatic carcinogen-DNA lesions with a sensitivity limit of 1 adduct in 10(7) to 10(8) nucleotides has been described previously. In the standard procedure, DNA is enzymatically digested to 3'-phosphorylated normal and adducted mononucleotides, which are 32P-labeled at 5'-hydroxyl groups by T4 polynucleotide kinase-catalyzed [32P]phosphate transfer from [gamma-32P]ATP. 32P-labeled derivatives are resolved by TLC, detected by autoradiography, and quantitated by counting. This assay has been recently utilized for the determination and partial characterization of DNA adducts formed in somatic and reproductive tissues of rats given the clinically used anticancer drug, mitomycin C. The drug exhibits similar levels of covalent binding to DNA in most tissues. Further studies have revealed that adducted nucleotides are primarily guanine derivatives that are resistant to 3'-dephosphorylation by Penicillium citrinum nuclease P1. The latter observation has been utilized to enhance the 32P-assay's sensitivity to 1 adduct in 10(10) nucleotides for a 10-micrograms DNA sample by postincubation of DNA digests with nuclease P1 before 32P-labeling. The enzyme dephosphorylates the normal nucleotides but not most aromatic and bulky nonaromatic adducts, so that only the latter serve as substrates for the kinase-catalyzed labeling reaction. The new assay has also shown utility in the analysis of very low levels of age- and tissue-related DNA modifications, which might arise from dietary or endogenous compounds, in untreated rats and in humans. Images FIGURE 2. FIGURE 5. PMID:2834194

  9. Detection of human butyrylcholinesterase-nerve gas adducts by liquid chromatography-mass spectrometric analysis after in gel chymotryptic digestion.

    PubMed

    Tsuge, Kouichiro; Seto, Yasuo

    2006-06-21

    To verify the exposure to nerve gas, a method for detecting human butyrylcholinesterase (BuChE)-nerve gas adduct was developed using LC-electrospray mass spectrometry (ESI-MS). Purified human serum BuChE was incubated with sarin, soman or VX, and the adduct was purified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and digested in gel by treatment with chymotrypsin. The resulting peptide mixture was subjected to LC-ESI-MS. From the chymotryptic digest of untreated human BuChE, one peak corresponding to the peptide fragment containing the active center serine residue was detected on the extracted ion chromatogram at m/z 948.5, and the sequence was ascertained to be "GESAGAASVSL" by MS/MS analysis. From the chymotryptic digest of the human BuChE-sarin adduct, a singly charged peptide peak was detected on the extracted ion chromatogram at m/z 1,069.5, and the sequence was ascertained to be "GEXAGAASVSL" by MS/MS analysis (X denotes isopropylmethylphosphonylated serine). The difference in molecular weight (120.0 Da) between the active center peptide fragments corresponding to the untreated BuChE and BuChE-sarin adduct was assumed to be derived from the addition of an isopropyl methylphosphonyl moiety to the serine residue. The formation of human BuChE adducts with soman, VX and an aged soman adduct was confirmed by detecting the respective active center peptide fragments using LC-ESI-MS. To apply the established method to an actual biological sample, human serum was incubated with VX, and the adduct was purified by procainamide affinity chromatography followed by SDS-PAGE. After chymotryptic in gel digestion, the ethylphosphonylated active center peptide fragment could be detected, and the structure of the residue was ascertained by LC-ESI-MS analysis. PMID:16569519

  10. Prenatal Exposure to Polycyclic Aromatic Hydrocarbons, Benzo[a]pyrene–DNA Adducts, and Genomic DNA Methylation in Cord Blood

    PubMed Central

    Tang, Deliang; Zhu, Deguang; Qu, Lirong; Sjödin, Andreas; Li, Zheng; Camann, David; Perera, Frederica P.

    2012-01-01

    Background: Polycyclic aromatic hydrocarbons (PAHs) are carcinogenic environmental pollutants generated during incomplete combustion. After exposure and during metabolism, PAHs can form reactive epoxides that can covalently bind to DNA. These PAH–DNA adducts are established markers of cancer risk. PAH exposure has been associated with epigenetic alterations, including genomic cytosine methylation. Both global hypomethylation and hypermethylation of specific genes have been associated with cancer and other diseases in humans. Experimental evidence suggests that PAH–DNA adduct formation may preferentially target methylated genomic regions. Early embryonic development may be a particularly susceptible period for PAH exposure, resulting in both increased PAH–DNA adducts and altered DNA methylation. Objective: We explored whether prenatal exposure to PAHs is associated with genomic DNA methylation in cord blood and whether methylation levels are associated with the presence of detectable PAH–DNA adducts. Methods: In a longitudinal cohort study of nonsmoking women in New York City, we measured PAH exposure during pregnancy using personal air monitors, assessed PAH internal dose using prenatal urinary metabolites (in a subset), and quantified benzo[a]pyrene–DNA adducts and genomic DNA methylation in cord blood DNA among 164 participants. Results: Prenatal PAH exposure was associated with lower global methylation in umbilical cord white blood cells (p = 0.05), but global methylation levels were positively associated with the presence of detectable adducts in cord blood (p = 0.01). Conclusions: These observations suggest that PAH exposure was adequate to alter global methylation in our study population. Additional epidemiologic studies that can measure site-specific cytosine methylation and adduct formation will improve our ability to understand this complex molecular pathway in vivo. PMID:22256332

  11. Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

    SciTech Connect

    Ganesan, Shanthi Keating, Aileen F.

    2015-02-01

    Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response. - Highlights: • PM forms ovarian DNA adducts. • DNA damage marker γH2AX increased by PM exposure. • PM induces ovarian DNA double strand break repair.

  12. Formation of 7-hydroxymethyl-12-methylbenz(a)anthracene-DNA adducts from 7,12-dimethylbenz(a)anthracene in mouse epidermis

    SciTech Connect

    DiGiovanni, J.; Nebzydoski, A.P.; Decina, P.C.

    1983-09-01

    The formation of DNA adducts from (/sup 3/H)-7-hydroxymethyl-12-methylbenz(a)anthracene (7-OHM-12-MBA) and (/sup 3/H)-7,12-dimethylbenz(a)anthracene (DMBA) in the epidermis of Sencar mice was analyzed. Comparison of Sephadex LH-20 chromatographic profiles of DNA samples isolated from mice treated with DMBA or 7-OHM-12-MBA suggested that the DMBA-treated animals contained DNA adduct(s) derived from the further metabolism of 7-OHM-12-MBA. Further analysis of DNA samples from DMBA-treated mice by high-pressure liquid chromatography demonstrated the presence of 5 DNA adducts which were chromatographically indistinguishable from the DNA adducts formed in 7-OHM-12-MBA-treated mice. Epidermal homogenates were utilized to catalyze the covalent binding of (/sup 3/H)DMBA and (/sup 3/H)-7-OHM-12-MBA to calf thymus DNA in vitro. Under conditions of limiting concentrations of (/sup 3/H)DMBA, the majority of the DNA adducts formed chromatographed in regions where 7-OHM-12-MBA-DNA adducts eluted. A major DMBA-DNA adduct formed in this in vitro system eluted with the same retention time as did the major 7-OHM-12-MBA-DNA adduct formed in mouse skin in vivo. These results when coupled with the in vivo data suggest that 7-OHM-12-MBA is an intermediate for at least some of the binding of DMBA to epidermal DNA in Sencar mice.

  13. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of titanium oxide-enriched peptides for detection of aged organophosphorus adducts on human butyrylcholinesterase.

    PubMed

    Jiang, Wei; Murashko, Ekaterina A; Dubrovskii, Yaroslav A; Podolskaya, Ekaterina P; Babakov, Vladimir N; Mikler, John; Nachon, Florian; Masson, Patrick; Schopfer, Lawrence M; Lockridge, Oksana

    2013-08-15

    Exposure to nerve agents or organophosphorus (OP) pesticides can have life-threatening effects. Human plasma butyrylcholinesterase (BChE) inactivates these poisons by binding them to Ser198. After hours or days, these OP adducts acquire a negative charge by dealkylation in a process called aging. Our goal was to develop a method for enriching the aged adduct to facilitate detection of exposure. Human BChE inhibited by OP toxicants was incubated for 4 days to 6 years. Peptides produced by digestion with pepsin were enriched by binding to titanium oxide (TiO2) and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. It was found that with two exceptions, all aged OP adducts in peptide FGES198AGAAS were enriched by binding to Titansphere tips. Cresyl saligenin phosphate yielded two types of aged adduct, cresylphosphate and phosphate, but only the phosphate adduct bound to Titansphere. The nerve agent VR yielded no aged adduct, supporting crystal structure findings that the VR adduct on BChE does not age. The irreversible nature of aged OP adducts was demonstrated by the finding that after 6 years at room temperature in sterile pH 7.0 buffer, the adducts were still detectable. It was concluded that TiO2 microcolumns can be used to enrich aged OP-modified BChE peptide. PMID:23624322

  14. p53 controls global nucleotide excision repair of low levels of structurally diverse benzo(g)chrysene-DNA adducts in human fibroblasts.

    PubMed

    Lloyd, Daniel R; Hanawalt, Philip C

    2002-09-15

    Benzo(g)chrysene is a widespread environmental contaminant and potent carcinogen. We have measured the formation and nucleotide excision repair of covalent DNA adducts formed by the DNA-reactive metabolite of this compound in human fibroblasts, in which expression of the p53 tumor suppressor gene could be controlled by a tetracycline-inducible promoter. Cells were exposed for 1 h to 0.01, 0.1, or 1.2 microM (+/-)-anti-benzo(g)chrysene diol-epoxide, and DNA adducts were assessed at various post-treatment times by subjecting isolated DNA to (32)P-postlabeling analysis. Four major DNA adducts were detected, corresponding to the reaction of either the (+)- or (-)-anti-benzo(g)chrysene diol-epoxide stereoisomer with adenine or guanine. Treatment with 1.2 microM resulted in a level of 1100 total adducts/10(8) nucleotides for both p53-proficient and -deficient cells; removal of adducts was not observed in either case. In cells treated with 0.1 microM, the maximum level of total adducts at 24 h was 150/10(8) nucleotides in p53-proficient cells and 210 adducts/10(8) nucleotides in p53-deficient cells. A concentration of 0.01 microM resulted in a maximum of 20 adducts/10(8) nucleotides in p53-proficient cells at 4 h, but 40 adducts/10(8) nucleotides persisted in p53-deficient cells at 24 h. Whereas there were clear differences in the time course of adduct levels in p53-proficient compared with p53-deficient cells treated with 0.1 microM or 0.01 microM, these levels did not decrease extensively over 3 days. This is likely because of the stabilization of the diol-epoxide in cells, and consequent exposure and formation of adducts for many hours after the initial treatment. Furthermore, despite minor quantitative differences, all 4 of the adducts behaved similarly with respect to the effect of p53 expression on their removal. p53 appears to minimize the appearance of benzo(g)chrysene adducts in human cells by up-regulating global nucleotide excision repair and reducing the

  15. 1,3-butadiene: cancer, mutations, and adducts. Part V: Hemoglobin adducts as biomarkers of 1,3-butadiene exposure and metabolism.

    PubMed

    Swenberg, J A; Christova-Gueorguieva, N I; Upton, P B; Ranasinghe, A; Scheller, N; Wu, K Y; Yen, T Y; Hayes, R

    2000-03-01

    1,3-Butadiene (BD) is an important chemical used largely in the manufacture of synthetic rubber and thermoplastic resins. In addition, it has been identified in cigarette smoke, automobile exhaust, and gasoline vapor. The objective of this research was to develop highly sensitive and specific assays for the detection and quantitation of hemoglobin adducts of three BD metabolites: 1,2-epoxy-3-butene (BDO), 1,2,3,4-diepoxybutane (BDO2), and 1,2-dihydroxy-3,4-epoxybutane (BDO-diol). We have successfully developed an assay for both N-(2-hydroxy-3-butenyl)valine (HBVal) and N-(2,3,4-trihydroxybutyl)valine (THBVal) in hemoglobin. The six adducts measured were the two diastereomers (isomers I and II) of HBVal and the four diastereomers of THBVal (isomers I through IV, which were eluted as three peaks, 1, 2, and 3). HBVal and THBVal were measured in control and exposed B6C3F1 mice and Sprague-Dawley rats (1,000 ppm BD for 13 weeks at 6 hours/day, 5 days/week). In a second set of animal exposures, total THBVal was determined in B6C3F1 female mice (n = 5) exposed to 1,250 ppm BD for 1, 5, or 10 days (6 hours/day, 5 days/week). THBVal adducts were also monitored in occupationally exposed Chinese workers and nonoccupationally exposed U.S. laboratory workers. This study utilized the modified Edman degradation method of Törnqvist and colleagues (1986). Briefly, the samples were subjected to Edman degradation, Centricon-30 ultrafiltration, washing on C18 columns, and acetylation for isomers of THBVal only, followed by gas chromatography-mass spectrometry (GC-MS) quantitation. For the HBVal assay, an authentic internal standard globin alkylated with [2H6]BDO was used; for the THBVal assay, a synthesized external standard, THB[13C5]Val, was used after Edman degradation. The mean +/- SD amounts of total HBVal measured in exposed mice (in pmol/g globin) were 16,560 +/- 3,910 for female mice (n = 4) and 12,400 +/- 2,030 for male mice (n = 5). The corresponding values for rats were 8

  16. Elevated polycyclic aromatic hydrocarbon–DNA adducts in benign prostate and risk of prostate cancer in African Americans

    PubMed Central

    Rybicki, Benjamin A.

    2013-01-01

    Carcinogen–DNA adducts, a marker of DNA damage, are capable of inducing mutations in oncogenes and tumor suppressor genes, resulting in carcinogenesis. We have shown previously that polycyclic aromatic hydrocarbon (PAH)–DNA adduct levels in prostate cancer cases vary by cellular histology and that higher adduct levels are associated with biochemical recurrence. A nested case–control study was conducted in a historical cohort of 6692 men with histopathologically benign prostate specimens. PAH-DNA adduct levels were determined by immunohistochemistry in benign prostate specimens from 536 prostate cancer case-control pairs (59% White and 41% African American). We estimated the overall and race-stratified risk of subsequent prostate cancer associated with higher adduct levels. Prostate cancer risk for men with elevated adduct levels (defined as greater than control group median) was slightly increased [odds ratio (OR) = 1.28, 95% confidence interval (CI) = 0.98–1.67, P = 0.07]. After race stratification, elevated adduct levels were significantly associated with increased risk in African American men (OR = 1.56, CI = 1.00–2.44, *P = 0.05) but not White men (OR = 1.14, CI = 0.82–1.59, P = 0.45). Elevated PAH-DNA adduct levels were significantly associated with 60% increased risk of prostate cancer among cases diagnosed 1–4 years after cohort entry (OR = 1.60, CI = 1.07–2.41) with a greater risk observed in African Americans within the first 4 years of follow-up (OR = 4.71, CI = 1.97–11.26, ***P = 0.0005). Analyses stratified by age or tumor grade revealed no additional significant heterogeneity in risk. Increased prostate cancer risk associated with high PAH-DNA adduct levels in benign prostate was found only in African Americans; risk was greatest within 4 years of follow-up, possibly reflecting a carcinogenic process not yet histologically detectable. PMID:23066084

  17. Mass spectrometry-based quantification of myocardial protein adducts with acrolein in an in vivo model of oxidative stress

    PubMed Central

    Wu, Jianyong; Stevens, Jan F.; Maier, Claudia S.

    2012-01-01

    Acrolein exposure leads to the formation of protein-acrolein adducts. Protein modification by acrolein has been associated with various chronic diseases including cardiovascular and neurodegenerative diseases. Here we report an analytical strategy that enables the quantification of Michael-type protein adducts of acrolein in mitochondrial proteome samples using liquid chromatography in combination with tandem mass spectrometry and selected ion monitoring (LC-MS/MS SRM) analysis. Our approach combines site-specific identification and relative quantification at the peptide level of protein–acrolein adducts in relation to the unmodified protein thiol pool. Treatment of 3-month old rats with CCl4, an established in vivo model of acute oxidative stress, resulted in significant increases in the ratios of distinct acrolein-adducted peptides to the corresponding unmodified thiol-peptides obtained from proteins that were isolated from cardiac mitochondria. The mitochondrial proteins that were found adducted by acrolein were malate dehydrogenase, NADH dehydrogenase [ubiquinone] flavoprotein 1, cytochrome c oxidase subunit VIb isoform 1, ATP synthase d chain, and ADP/ATP translocase 1. The findings indicate that protein modification by acrolein has potential value as an index of mitochondrial oxidative stress. PMID:21809440

  18. Cytochrome P4501A induction and DNA adduct formation in glaucous gulls (Larus hyperboreus), fed with environmentally contaminated gull eggs.

    PubMed

    Østby, Lene; Gabrielsen, Geir Wing; Krøkje, Ase

    2005-11-01

    This study indicates that complex mixtures of pollutants found in the Arctic marine environment have genotoxic effects in glaucous gulls (Larus hyperboreus). DNA adducts were quantified, by the (32)P-postlabeling technique, in liver samples from gulls fed with hen eggs (controls) and from gulls fed with environmentally contaminated gull eggs (exposed). All birds were grown and fed under laboratory conditions. Hepatic homologues to mammalian cytochrome P4501A (CYP1A) proteins were also determined by Western blotting. DNA adducts were detected in all but one liver sample, but the exposed birds had a significantly increased level of DNA adducts relative to that of the controls. There was no clear significant correlation between the DNA adduct level and the level of organochlorine compounds (OCs) in blood. The level of CYP1A protein was significantly higher in the liver of exposed male gulls than in the liver of control males and positively correlated, with significance, to the level of OC compounds measured in blood. There was no significant correlation between the level of DNA adducts and the CYP1A protein content. PMID:16216630

  19. A nontargeted screening method for covalent DNA adducts and DNA modification selectivity using liquid chromatography-tandem mass spectrometry.

    PubMed

    Yao, Chunhe; Feng, Yong-Lai

    2016-10-01

    A method for nontargeted screening for covalent DNA adducts was developed using combination of neutral loss scan and product ion scan in a hybrid linear-ion-trap - triple quadrupole mass spectrometer system. DNA 2'-deoxynucleosides and adducts eluted from liquid chromatography were first analyzed in neutral loss mode to screen for the neutral loss of the deoxyribose moiety ([M+H-116](+)) from the protonated molecular ion ([M+H](+)). The product ion scan was subsequently used to elucidate the structures for the molecular ions observed from the peaks in the neutral loss scan chromatogram. The synthesized DNA adducts were used to evaluate the developed method by reaction of 20-mer DNA oligonucleotide with two direct agents respectively, specifically phenyl glycidyl ether and styrene-7,8-oxide. The modification selectivity of two compounds to the four nitrogenous bases on DNA sequence was also investigated in this study. The results showed that the two compounds had different modification selectivity to the four bases. Both compounds could modify all four nitrogenous bases (i.e. adenine, guanine, thymine, and cytosine) on DNA sequences to form various covalent DNA adducts. While phenyl glycidyl ether modified almost all of thymidine on DNA sequence, styrene-7,8-oxide, on the other hand, modified only a small portion of thymidine. The developed method proved possibly a potential tool for screening of unknown DNA adducts as exposure biomarkers of contaminants to human in the environment. PMID:27474284

  20. Relationship Between the Medial Elbow Adduction Moment During Pitching and Ulnar Collateral Ligament Appearance During Magnetic Resonance Imaging Evaluation

    PubMed Central

    Hurd, Wendy J.; Kaufman, Kenton R.; Murthy, Naveen S.

    2014-01-01

    Background Medial elbow distraction during pitching as the primary mechanism contributing to adaptations in ulnar collateral ligament (UCL) appearance during magnetic resonance imaging (MRI) evaluation has not been established. Hypothesis Uninjured high school–aged pitchers with unilateral adaptations of the UCL exhibit a higher peak internal elbow adduction moment than those without UCL adaptations. Study Design Cohort study (Prevalence); Level of evidence, 2. Methods Twenty uninjured, asymptomatic high school–aged pitchers underwent bilateral elbow MRI examinations. Three-dimensional motion analysis testing was performed to collect throwing arm biomechanics as participants pitched from an indoor mound. Nonparametric tests were performed to compare peak internal elbow adduction moment in uninjured participants with and without adaptations in UCL appearance and to determine the nature of the relationship between the peak internal adduction moment and UCL appearance. Results Uninjured participants with UCL thickening exhibited a higher peak internal elbow adduction moment of 53.3 ± 6.8 N·m compared with uninjured participants without adaptations in UCL appearance, 38.8 ± 10.9 N·m (P = .05), as higher moments were correlated with ligament thickening (correlation coefficient, 0.45) (P = .02). Conclusion This study establishes the association between medial elbow distraction captured by the internal adduction moment during pitching and UCL adaptations visible during MRI evaluation. PMID:21335342

  1. Anion Effects on Sodium Ion and Acid Molecule Adduction to Protein Ions in Electrospray Ionization Mass Spectrometry

    PubMed Central

    Flick, Tawnya G.; Merenbloom, Samuel I.; Williams, Evan R.

    2012-01-01

    Gaseous protein–metal ion and protein–molecule complexes can be readily formed by electrospray ionization (ESI) from aqueous solutions containing proteins and millimolar concentrations of sodium salts of various anions. The extent of sodium and acid molecule adduction to multiply charged protein ions is inversely related and depends strongly on the proton affinity (PA) of the anion, with extensive sodium adduction occurring for anions with PA values greater than ~300 kcal·mol−1 and extensive acid molecule adduction occurring for anions with PA values less than 315 kcal·mol−1. The role of the anion on the extent of sodium and acid molecule adduction does not directly follow the Hofmeister series, suggesting that direct protein–ion interactions may not play a significant role in the observed effect of anions on protein structure in solution. These results indicate that salts with anions that have low PA values may be useful solution-phase additives to minimize nonspecific metal ion adduction in ESI experiments designed to identify specific protein-metal ion interactions. PMID:21952761

  2. Identification of two N{sup 2}-deoxyguanosinyl DNA adducts upon nitroreduction of the environmental mutagen 1-nitropyrene

    SciTech Connect

    Herreno-Saenz, D.; Evans, F.E.; Beland, F.A.

    1995-03-01

    1-Nitropyrene, the most abundant nitro-polycyclic aromatic hydrocarbon in the environment, is a known mammalian and bacterial mutagen and a tumorigen in animals. Early studies on DNA adduct characterization for 1-nitropyrene identified N-(deoxyguanosin-8-yl)-1-aminopyrene as the major product from the modification of calf thymus DNA with N-hydroxy-1-aminopyrene, the activated metabolite from nitroreduction of 1-nitropyrene. In this paper, we report the identification of two N{sup 2}-deoxyguanosinyl adducts, in addition to N-(deoxyguanosin-8-yl)-1-aminopyrene, formed from the reaction of N-hydroxy-1-aminopyrene, prepared in situ, with calf thymus DNA. These DNA adducts were identified as 6-(deoxyguanosin-N{sup 2}-yl)-1-aminopyrene and 8-(deoxyguanosin-N{sup 2}-yl)-1-aminopyrene. The two N{sup 2}-deoxyguanosinyl adducts were also identified in an ascorbic acid-catalyzed activation of 1-nitropyrene and in the mammary gland of female Sprague-Dawley rats administered 1-nitropyrene. The DNA adducts were also formed when 1-nitropyrene was metabolized by xanthine oxidase in the presence of calf thymus DNA, and when 1-nitropyrene was activated by rat liver microsomes and cytosols, as well as from DNA isolated from Salmonella typhimurium suspension cultures incubated with 1-nitropyrene. 45 refs., 7 figs., 1 tab.

  3. Crystallographic, Spectroscopic, and Computational Analysis of a Flavin-C4a-Oxygen Adduct in Choline Oxidase

    SciTech Connect

    Orville, A.M.; Lountos, G. T.; Finnegan, S.; Gadda, G.; Prabhakar, R.

    2009-02-03

    Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 {angstrom} resolution X-ray crystal structure of choline oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to X-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and C4a-OO(H) adducts and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. We propose that the enzyme-bound FAD is reduced in the X-ray beam. The aerobic crystals then form either a C4a-OH or C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures.

  4. Crystallographic, Spectroscopic, and Computational Analysis of a Flavin C4a-Oxygen Adduct in Choline Oxidase

    SciTech Connect

    Orville, A.; Lountos, G; Finnegan, S; Gadda, G; Prabhakar, R

    2009-01-01

    Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 {angstrom} resolution X-ray crystal structure of choline oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to X-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and C4a-OO(H) adducts and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. We propose that the enzyme-bound FAD is reduced in the X-ray beam. The aerobic crystals then form either a C4a-OH or C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures.

  5. An abnormal N-heterocyclic carbene-carbon dioxide adduct from imidazolium acetate ionic liquids: the importance of basicity.

    PubMed

    Kelemen, Zsolt; Péter-Szabó, Barbara; Székely, Edit; Hollóczki, Oldamur; Firaha, Dzmitry S; Kirchner, Barbara; Nagy, József; Nyulászi, László

    2014-09-26

    In the reaction of 1-ethyl-3-methylimidazolium acetate [C2C1Im][OAc] ionic liquid with carbon dioxide at 125 °C and 10 MPa, not only the known N-heterocyclic carbene (NHC)-CO2 adduct I, but also isomeric aNHC-CO2 adducts II and III were obtained. The abnormal NHC-CO2 adducts are stabilized by the presence of the polarizing basic acetate anion, according to static DFT calculations and ab initio molecular dynamics studies. A further possible reaction pathway is facilitated by the high basicity of the system, deprotonating the initially formed NHC-CO2 adduct I, which can then be converted in the presence of the excess of CO2 to the more stable 2-deprotonated anionic abnormal NHC-CO2 adduct via the anionic imidazolium-2,4-dicarboxylate according to DFT calculations on model compounds. This suggests a generalizable pathway to abnormal NHC complex formation. PMID:25137312

  6. Identification of Chemical-Adducted Proteins in Urine by Multi-dimensional Protein Identification Technology (LC/LC–MS/MS)

    PubMed Central

    Labenski, Matthew T.; Fisher, Ashley A.; Monks, Terrence J.; Lau, Serrine S.

    2014-01-01

    Recent technological advancements in mass spectrometry facilitate the detection of chemical-induced posttranslational modifications (PTMs) that may alter cell signaling pathways or alter the structure and function of the modified proteins. To identify such protein adducts (Kleiner et al., Chem Res Toxicol 11:1283–1290, 1998), multi-dimensional protein identification technology (MuDPIT) has been utilized. MuDPIT was first described by Link et al. as a new technique useful for protein identification from a complex mixture of proteins (Link et al., Nat Biotechnol 17:676–682, 1999). MuDPIT utilizes two different HPLC columns to further enhance peptide separation, increasing the number of peptide hits and protein coverage. The technology is extremely useful for proteomes, such as the urine proteome, samples from immunoprecipitations, and 1D gel bands resolved from a tissue homogenate or lysate. In particular, MuDPIT has enhanced the field of adduct hunting for adducted peptides, since it is more capable of identifying lesser abundant peptides, such as those that are adducted, than the more standard LC–MS/MS. The site-specific identification of covalently adducted proteins is a prerequisite for understanding the biological significance of chemical-induced PTMs and the subsequent toxicological response they elicit. PMID:20972764

  7. Cysteine amide adduct formation from carboxylic acid drugs via UGT-mediated bioactivation in human liver microsomes.

    PubMed

    Harada, H; Toyoda, Y; Endo, T; Kobayashi, M

    2015-10-01

    Although chemical trapping has been widely used to evaluate cytochrome P450-mediated drug bioactivation, thus far, only a few in vitro-trapping studies have been performed on UDP-glucuronosyltransferase (UGT)-mediated drug bioactivation. In this study, we used cysteine (Cys) as trapping agent to gain new insights into the UGT-mediated bioactivation involving acyl glucuronides of carboxylic acid drugs. Diclofenac, ketoprofen and ibuprofen were incubated in human liver microsomes with UDPGA and Cys, followed by analysis using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). The N-acyl-Cys amide adduct of diclofenac was characterized by mass analysis and was detectable even in photodiode array analysis. Our data indicated that the formation of such adducts reflects the reactivity of the corresponding acyl glucuronides. In addition, it was suggested that the adduct formation requires an N-terminal Cys moiety with both a free amine and a free thiol group, from the results using various cysteine derivatives. We propose that the S-acyl-Cys thioester adduct can form via transacylation of an acyl glucuronide and can then form to an N-acyl-Cys amide adduct through intramolecular S- to N-acyl rearrangement. This series of the reactions has important implications as a possible bioactivation mechanism for covalent binding of carboxylic acid drugs to macromolecules. PMID:26601426

  8. Quantification of nerve agent adducts with albumin in rat plasma using liquid chromatography-isotope dilution tandem mass spectrometry.

    PubMed

    Bao, Yi; Liu, Qin; Chen, Jia; Lin, Ying; Wu, Bidong; Xie, Jianwei

    2012-03-16

    A sensitive method for the determination of the organophosphorus nerve agents sarin, soman and VX adducts with tyrosine residue of albumin in rat plasma has been developed and validated using liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS). O-(O-Alkyl methylphosphonyl) tyrosine adducts and their deuterated products that were used as the internal standards were synthesised to establish the quantitative isotope-dilution method. Protein purification and solid-phase extraction (SPE) were applied to improve the recovery efficiency, reduce interference and achieve high sensitivity. The method provided a detection limit of 0.01 ng/mL for sarin and soman adducts and 0.05 ng/mL for the VX adduct. The value of the intra-day relative standard deviation over the calibration range was less than 6.16% (n=6), and that of the inter-day was less than 12.7% (n=6). The recovery varied from 86% to 111%. This sensitive method was successfully applied to the analysis of adducts in rat plasma after nerve agent exposure, and the results demonstrated the dose-effect relationships. PMID:22305360

  9. Determination of DNA adducts by combining acid-catalyzed hydrolysis and chromatographic analysis of the carcinogen-modified nucleobases.

    PubMed

    Leung, Elvis M K; Deng, Kailin; Wong, Tin-Yan; Chan, Wan

    2016-01-01

    The commonly used method of analyzing carcinogen-induced DNA adducts involves the hydrolysis of carcinogen-modified DNA samples by using a mixture of enzymes, followed by (32)P-postlabeling or liquid chromatography (LC)-based analyses of carcinogen-modified mononucleotides/nucleosides. In the present study, we report the development and application of a new approach to DNA adduct analysis by combining the H(+)/heat-catalyzed release of carcinogen-modified nucleobases and the use of LC-based methods to analyze DNA adducts. Results showed that heating the carcinogen-modified DNA samples at 70 °C for an extended period of 4 to 6 h in the presence of 0.05% HCl can efficiently induce DNA depurination, releasing the intact carcinogen-modified nucleobases for LC analyses. After optimizing the hydrolysis conditions, DNA samples with C8- and N (2) -modified 2'-deoxyguanosine, as well as N (6) -modified 2'-deoxyadenosine, were synthesized by reacting DNA with 1-nitropyrene, acetaldehyde, and aristolochic acids, respectively. These samples were then hydrolyzed, and the released nucleobase adducts were analyzed using LC-based analytical methods. Analysis results demonstrated a dose-dependent release of target DNA adducts from carcinogen-modified DNA samples, indicating that the developed H(+)/heat-catalyzed hydrolysis method was quantitative. Comparative studies with enzymatic digestion method on carcinogen-modified DNA samples revealed that the two hydrolysis methods did not yield systematically different results. PMID:26581621

  10. Neighborhood Socioeconomic Status Modifies the Association Between Individual Smoking Status and PAH-DNA Adduct Levels in Prostate Tissue

    PubMed Central

    Rundle, Andrew; Richards, Catherine; Neslund-Dudas, Christine; Tang, Deliang; Rybicki, Benjamin A.

    2013-01-01

    Interactions between smoking and neighborhood-level socioeconomic status (SES) as risk factors for higher polycyclic aromatic hydrocarbon (PAH) DNA adduct levels in prostate tissue were investigated. PAH-DNA adducts were measured by immunohistochemistry with staining intensity measured in optical density units by semiquantitative absorbance image analysis in tumor adjacent tissue from 400 prostatectomy specimens from the Henry Ford Health System in Detroit. For each subject, their U.S. Census tract of residence was classified as being of higher or lower SES using the median value of the distribution of the proportion of tract residents with a high-school education. Generalized estimating equation models were used to assess interactions between neighborhood-level SES and smoking status, adjusting for race, age, education level, tumor volume, primary Gleason grade and prostate specific antigen (PSA) at diagnosis. There was a statistical interaction (P = 0.004) between tract-level SES and smoking status. In lower SES tracts smoking status was not associated with adduct staining, but in higher SES tracts adduct staining intensity was 13% (P = 0.01) higher in ever-smokers as compared to never-smokers. Among never-smokers, living in a lower SES tract was associated with a 25% higher mean staining intensity (P < 0.001). Neighborhood SES modifies the association between individual smoking status and PAH-DNA adduct levels in prostate tissue. PMID:22467358

  11. Determining protein adducts of fipexide: mass spectrometry based assay for confirming the involvement of its reactive metabolite in covalent binding.

    PubMed

    Sleno, Lekha; Varesio, Emmanuel; Hopfgartner, Gérard

    2007-01-01

    Fipexide is a nootropic drug, withdrawn from the market due to its idiosyncratic drug reactions causing adverse effects in man. Previous work on its metabolites has identified several potential reactive metabolites which could be implicated in protein binding. Here, we investigated the formation of these metabolites in rat and human hepatocytes. Based on these results, the o-quinone of fipexide (FIP), formed via the demethylenation reaction through a catechol intermediate, was chosen for further investigation. Studies were then pursued in order to relate this metabolite to protein binding, and thus better understand potential mechanisms for the toxicity of the parent compound. An assay was developed for determining the fipexide catechol-cysteine adduct in the microsomal protein fractions following in vitro incubations. This method digests the entire protein fraction into amino acids, followed by the detection of the Cys-metabolite adduct by liquid chromatography/mass spectrometry (LC/MS). We have designed a strategy where drug metabolism taking place in microsomal incubations and involved in protein binding can be assessed after the proteins have been digested, with the detection of the specific amino acid adduct. In this study, the structure of the fipexide adduct was hypothesized using knowledge previously gained in glutathione and N-acetylcysteine trapping experiments. Acetaminophen was used as a positive control for detecting a drug metabolite-cysteine adduct by LC/MS. This approach has the potential to be applicable as a protein-binding assay in early drug discovery without the need for radioactive compounds. PMID:18022964

  12. Topical metal chelation therapy ameliorates oxidation-induced toxicity in diabetic cataract.

    PubMed

    Zhang, Min; Shoeb, Mohammad; Liu, Ping; Xiao, Tianlin; Hogan, Dale; Wong, Ira G; Campbell, Gerald A; Ansari, Naseem H

    2011-01-01

    Oxidative stress plays a critical role in cataractogenesis, the leading cause of blindness worldwide. Since transition metals generate reactive oxygen species (ROS) formation, metal chelation therapy has been proposed for treatment of cataracts. However, the effectiveness of most chelators is limited by low tissue penetrability. This study is the first to demonstrate that the topically applied divalent metal chelator ethylenediamine tetraacetic acid (EDTA) combined with the carrier and permeability enhancer methyl sulfonyl methane (MSM) ameliorates both oxidation-induced lens opacification and the associated toxic accumulation of protein-4-hydroxynonenal (HNE) adducts. Both in vitro (rat lens culture) and in vivo (diabetic rats), EDTA-MSM (1) significantly reduced lens opacification by about 40-50%, (2) significantly diminished lens epithelial cell proliferation and fiber cell swelling in early stages of cataract formation in vivo, and (3) notably decreased the levels of protein-HNE adducts. These findings have important implications specifically for the treatment of cataract and generally for other diseases in which oxidative stress plays a key pathogenic role. PMID:21271438

  13. Gypsum accumulation on carbonate stone

    USGS Publications Warehouse

    McGee, E.S.; Mossotti, V.G.

    1992-01-01

    The accumulation of gypsum on carbonate stone has been investigated through exposure of fresh samples of limestone and marble at monitored sites, through examination of alteration crusts from old buildings and through laboratory experiments. Several factors contribute to gypsum accumulation on carbonate stone. Marble or limestone that is sheltered from direct washing by rain in an urban environment with elevated pollution levels is likely to accumulate a gypsum crust. Crust development may be enhanced if the stone is porous or has an irregular surface area. Gypsum crusts are a surficial alteration feature; gypsum crystals form at the pore opening-air interface, where evaporation is greatest.

  14. Characterization of quinone derived protein adducts and their selective identification using redox cycling based chemiluminescence assay.

    PubMed

    Elgawish, Mohamed Saleh; Kishikawa, Naoya; Ohyama, Kaname; Kuroda, Naotaka

    2015-07-17

    The cytotoxic mechanism of many quinones has been correlated to covalent modification of cellular proteins. However, the identification of relevant proteins targets is essential but challenging goals. To better understand the quinones cytotoxic mechanism, human serum albumin (HSA) was incubated in vitro with different concentration of menadione (MQ). In this respect, the initial nucleophilic addition of proteins to quinone converts the conjugates to redox-cycling quinoproteins with altered conformation and secondary structure and extended life span than the short lived, free quinones. The conjugation of MQ with nucleophilic sites likewise, free cysteine as well as ɛ-amino group of lysine residue of HSA has been found to be in concentration dependent manner. The conventional methods for modified proteins identification in complex mixtures are complicated and time consuming. Herein, we describe a highly selective, sensitive, simple, and fast strategy for quinoproteins identification. The suggested strategy exploited the unique redox-cycling capability of quinoproteins in presence of a reductant, dithiothreitol (DTT), to generate reactive oxygen species (ROS) that gave sufficient chemiluminescence (CL) when mixed with luminol. The CL approach is highly selective and sensitive to detect the quinoproteins in ten-fold molar excess of native proteins without adduct enrichment. The approach was also coupled with gel filtration chromatography (GFC) and used to identify adducts in complex mixture of proteins in vitro as well as in rat plasma after MQ administration. Albumin was identified as the main protein in human and rat plasma forming adduct with MQ. Overall, the identification of quinoproteins will encourage further studies of toxicological impact of quinones on human health. PMID:26044383

  15. Chiral Differentiation of DNA Adducts Formed by Enantiomeric Analogues of Antitumor Cisplatin Is Sequence-Dependent

    PubMed Central

    Delalande, Olivier; Malina, Jaroslav; Brabec, Viktor; Kozelka, Jiří

    2005-01-01

    1,2-GG intrastrand cross-links formed in DNA by the enantiomeric complexes [PtCl2(R,R-2,3-diaminobutane (DAB))] and [PtCl2(S,S-DAB)] were studied by biophysical methods. Molecular modeling revealed that structure of the cross-links formed at the TGGT sequence was affected by repulsion between the 5′-directed methyl group of the DAB ligand and the methyl group of the 5′-thymine of the TGGT fragment. Molecular dynamics simulations of the solvated platinated duplexes and our recent structural data indicated that the adduct of [PtCl2(R,R-DAB)] alleviated this repulsion by unwinding the TpG step, whereas the adduct of [PtCl2(S,S-DAB)] avoided the unfavorable methyl-methyl interaction by decreasing the kink angle. Electrophoretic retardation measurements on DNA duplexes containing 1,2-GG intrastrand cross-links of Pt(R,R-DAB)2+ or Pt(S,S-DAB)2+ at a CGGA site showed that in this sequence both enantiomers distorted the double helix to the identical extent similar to that found previously for the same sequence containing the cross-links of the parent antitumor \\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\setlength{\\oddsidemargin}{-69pt} \\begin{document} \\begin{equation*}cis-{\\mathrm{Pt}}({\\mathrm{NH}}_{3})_{2}^{2+}\\end{equation*}\\end{document} (cisplatin). In addition, the adducts showed similar affinities toward the high-mobility-group box 1 proteins. Hence, whereas the structural perturbation induced in DNA by 1,2-GG intrastrand cross-links of cisplatin does not depend largely on the bases flanking the cross-links, the perturbation related to GG cross-linking by bulkier platinum diamine derivatives does. PMID:15805172

  16. Tracking matrix effects in the analysis of DNA adducts of polycyclic aromatic hydrocarbons.

    PubMed

    Klaene, Joshua J; Flarakos, Caroline; Glick, James; Barret, Jennifer T; Zarbl, Helmut; Vouros, Paul

    2016-03-25

    LC-MS using electrospray ionization is currently the method of choice in bio-organic analysis covering a wide range of applications in a broad spectrum of biological media. The technique is noted for its high sensitivity but one major limitation that hinders achievement of its optimal sensitivity is the signal suppression due to matrix inferences introduced by the presence of co-extracted compounds during the sample preparation procedure. The analysis of DNA adducts of common environmental carcinogens is particularly sensitive to such matrix effects as sample preparation is a multistep process which involves "contamination" of the sample due to the addition of enzymes and other reagents for digestion of the DNA in order to isolate the analyte(s). This problem is further exacerbated by the need to reach low levels of quantitation (LOQ in the ppb level) while also working with limited (2-5μg) quantities of sample. We report here on the systematic investigation of ion signal suppression contributed by each individual step involved in the sample preparation associated with the analysis of DNA adducts of polycyclic aromatic hydrocarbon (PAH) using as model analyte BaP-dG, the deoxyguanosine (dG) adduct of benzo[a]pyrene (BaP). The individual matrix contribution of each one of these sources to analyte signal was systematically addressed as were any interactive effects. The information was used to develop a validated analytical protocol for the target biomarker at levels typically encountered in vivo using as little as 2μg of DNA and applied to a dose response study using a metabolically competent cell line. PMID:26607319

  17. DNA adduct formation and induction of detoxification mechanisms in Dreissena polymorpha exposed to nitro-PAHs.

    PubMed

    Châtel, Amélie; Faucet-Marquis, Virginie; Pfohl-Leszkowicz, Annie; Gourlay-Francé, Catherine; Vincent-Hubert, Françoise

    2014-11-01

    Derived polycyclic aromatic hydrocarbons (PAHs) such as nitro-PAHs are present in the environment and are known to be much more toxic than PAHs compounds. However, very few studies have analysed their effects on the aquatic environment and none have investigated the freshwater environment. In the present study, we determined whether 1-nitropyrene (1-NP), a model of nitro-PAHs, can induce DNA adducts in gills and digestive glands of the freshwater mussel Dreissena polymorpha. Two concentrations of 1-NP (50 and 500 μM) were tested. In addition, in order to understand the metabolic pathways involved in 1-NP genotoxicity, mRNA expression of genes implicated in biotransformation mechanisms was assessed by quantitative reverse transcription-PCR. Results showed the presence of DNA adducts in both gills and digestive glands, with highest levels obtained after 5 days of exposure to 500 μM. Metallothionein mRNA levels were enhanced in digestive glands exposed to 50 μM. Surprisingly, at the higher concentration (500 μM), aryl hydrocarbon receptor and HSP70 genes were only up-regulated in digestive glands while PgP mRNA levels were increased in both tissues. Results suggested a cytotoxic and genotoxic effect of 1-NP. Mussels seemed to be able to partially detoxify this compound, in view of the low amount of DNA adducts observed after 5 days exposure to 50 μM. For the first time, 1-NP biotransformation and detoxification systems have been characterised in D. polymorpha. PMID:25209124

  18. Mass spectrometric detection of CYP450 adducts following oxidative desulfuration of methyl parathion.

    PubMed

    Kyle, Patrick B; Smith, Stanley V; Baker, Rodney C; Kramer, Robert E

    2013-07-01

    Cytochrome P450 (CYP)-mediated desulfuration of methyl parathion results in mechanism-based inhibition of the enzyme. Although previous data suggest that reactive sulfur is released and binds to the apoprotein, the identities of neither the adduct(s) nor the affected amino acid(s) have been clearly determined. In this study, nanospray tandem mass spectroscopy was used to analyze peptide digests of CYP resolved by SDS-PAGE from liver microsomes of male rats following incubation in the absence or presence of methyl parathion. Oxidative desulfuration was confirmed by measurement of methyl paraoxon, and inhibition of specific CYP isozymes was determined by measurement of testosterone hydroxylation. Total CYP content was quantified spectrophotometrically. Incubation of microsomes with methyl parathion decreased CYP content by 58%. This effect was not associated with a comparable increase in absorbance at 420 nm, suggesting the displacement of heme from the apoprotein. Rates of testosterone 2β- and 6β-hydroxylation, respectively, were reduced to 8 and 2%, implicating CYP3A and CYP2C11 in the oxidative desulfuration of methyl parathion. Mass spectrometric analysis identified 96 amu adducts to cysteines 64 and 378 of CYP3A1. In addition, a peptide containing cysteine 433 that coordinates with heme was possibly modified as it was detected in control, but not methyl parathion samples. A comparison of rat CYP3A1 with human CYP3A4 suggests that cysteines 64 and 378 reside along the substrate channel, remote from the active site. Alteration of these residues might modulate substrate entry to the binding pocket of the enzyme. PMID:22271348

  19. Relationship between DNA adduct formation and unscheduled DNA synthesis (UDS) in cultured mouse epidermal keratinocytes

    SciTech Connect

    Gill, R.D.; Nettikumara, A.N.; DiGiovanni, J. ); Butterworth, B.E. )

    1991-01-01

    Primary cultures of mouse epidermal keratinocytes from SENCAR mice were treated with 7,12-dimethylbenz(a)anthracene (DMBA), benzo(a)pyrene (B(a)P), ({plus minus}) 7{beta}-8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (({plus minus}) anti-BPDE), and ({plus minus}) 7{beta},8{alpha}-dihydroxy-9{beta},10{beta}-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (({plus minus})syn-BPDE) to examine the relationship between DNA adduct formation and the induction of unscheduled DNA synthesis (UDS). DNA adducts were measured as pmol hydrocarbon bound per mg of DNA, and UDS was quantitated autoradiographically as net grains per nucleus. A good correlation was observed between the levels of UDS detected and the amount of DNA adducts present int he cell population when comparing similar compounds within the linear dose-response range of 0.005 {mu}g/ml-0.25 {mu}g/ml. These results suggest that the present UDS assay with MEKs is a useful assay for the rapid screening of potential genotoxic agents. However, the limits of sensitivity are such that the current assay may be unable to detect a low level of DNA damage induced by some weakly genotoxic (carcinogenic) agents. In addition, while the limits of sensitivity determined in these experiments apply to the polycyclic aromatic hydrocarbon class, other classes of genotoxic compounds such as alkylating agents or crosslinking agents may exhibit different thresholds of detection.

  20. N-Acetylcysteine blocks formation of cancer-initiating estrogen-DNA adducts in cells

    PubMed Central

    Zahid, Muhammad; Saeed, Muhammad; Ali, Mohammed F.; Rogan, Eleanor G.; Cavalieri, Ercole L.