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

Chemistry and Biology of Aflatoxin-DNA Adducts

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

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

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 abovemore » 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.« less

Sequence distribution of acetaldehyde-derived N2-ethyl-dG adducts along duplex DNA.

PubMed

Matter, Brock; Guza, Rebecca; Zhao, Jianwei; Li, Zhong-ze; Jones, Roger; Tretyakova, Natalia

2007-10-01

Acetaldehyde (AA) is the major metabolite of ethanol and may be responsible for an increased gastrointestinal cancer risk associated with alcohol beverage consumption. Furthermore, AA is one of the most abundant carcinogens in tobacco smoke and induces tumors of the respiratory tract in laboratory animals. AA binding to DNA induces Schiff base adducts at the exocyclic amino group of dG, N2-ethylidene-dG, which are reversible on the nucleoside level but can be stabilized by reduction to N2-ethyl-dG. Mutagenesis studies in the HPRT reporter gene and in the p53 tumor suppressor gene have revealed the ability of AA to induce G-->A transitions and A-->T transversions, as well as frameshift and splice mutations. AA-induced point mutations are most prominent at 5'-AGG-3' trinucleotides, possibly a result of sequence specific adduct formation, mispairing, and/or repair. However, DNA sequence preferences for the formation of acetaldehyde adducts have not been previously examined. In the present work, we employed a stable isotope labeling-HPLC-ESI+-MS/MS approach developed in our laboratory to analyze the distribution of acetaldehyde-derived N2-ethyl-dG adducts along double-stranded oligodeoxynucleotides representing two prominent lung cancer mutational "hotspots" and their surrounding DNA sequences. 1,7,NH 2-(15)N-2-(13)C-dG was placed at defined positions within DNA duplexes derived from the K-ras protooncogene and the p53 tumor suppressor gene, followed by AA treatment and NaBH 3CN reduction to convert N2-ethylidene-dG to N2-ethyl-dG. Capillary HPLC-ESI+-MS/MS was used to quantify N2-ethyl-dG adducts originating from the isotopically labeled and unlabeled guanine nucleobases and to map adduct formation along DNA duplexes. We found that the formation of N2-ethyl-dG adducts was only weakly affected by the local sequence context and was slightly increased in the presence of 5-methylcytosine within CG dinucleotides. These results are in contrast with sequence

Structures of exocyclic R,R- and S,S-N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine adducts induced by 1,2,3,4-diepoxybutane.

PubMed

Kowal, Ewa A; Seneviratne, Uthpala; Wickramaratne, Susith; Doherty, Kathleen E; Cao, Xiangkun; Tretyakova, Natalia; Stone, Michael P

2014-05-19

1,3-Butadiene (BD) is an industrial and environmental chemical present in urban air and cigarette smoke, and is classified as a human carcinogen. It is oxidized by cytochrome P450 to form 1,2,3,4-diepoxybutane (DEB); DEB bis-alkylates the N(6) position of adenine in DNA. Two enantiomers of bis-N(6)-dA adducts of DEB have been identified: R,R-N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (R,R-DHB-dA), and S,S-N(6),N(6)-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (S,S-DHB-dA) [ Seneviratne , U. , Antsypovich , S. , Dorr , D. Q. , Dissanayake , T. , Kotapati , S. , and Tretyakova , N. ( 2010 ) Chem. Res. Toxicol. 23 , 1556 -1567 ]. Herein, the R,R-DHB-dA and S,S-DHB-dA adducts have been incorporated into the 5'-d(C(1)G(2)G(3)A(4)C(5)X(6)A(7)G(8)A(9)A(10)G(11))-3':5'-d(C(12)T(13)T(14)C(15)T(16)T(17)G(18)T(19)C(20)C(21)G(22))-3' duplex [X(6) = R,R-DHB-dA (R(6)) or S,S-DHB-dA (S(6))]. The structures of the duplexes were determined by molecular dynamics calculations, which were restrained by experimental distances obtained from NMR data. Both the R,R- and S,S-DHB-dA adducts are positioned in the major groove of DNA. In both instances, the bulky 3,4-dihydroxypyrrolidine rings are accommodated by an out-of-plane rotation about the C6-N(6) bond of the bis-alkylated adenine. In both instances, the directionality of the dihydroxypyrrolidine ring is evidenced by the pattern of NOEs between the 3,4-dihydroxypyrrolidine protons and DNA. Also in both instances, the anti conformation of the glycosyl bond is maintained, which combined with the out-of-plane rotation about the C6-N(6) bond, allows the complementary thymine, T(17), to remain stacked within the duplex, and form one hydrogen bond with the modified base, between the imine nitrogen of the modified base and the T(17) N3H imino proton. The loss of the second Watson-Crick hydrogen bonding interaction at the lesion sites correlates with the lower thermal stabilities of the R,R- and S,S-DHB-dA duplexes, as

Biological exposure indices of pyrrole adducts in serum and urine for hazard assessment of n-hexane exposure.

PubMed

Yin, Hongyin; Zhang, Chunling; Guo, Ying; Shao, Xiaoying; Zeng, Tao; Zhao, Xiulan; Xie, Keqin

2014-01-01

Pyrrole adducts might be used as a biomarker for monitoring occupational exposure to n-hexane, but the Biological Exposure Indices of pyrrole adducts in serum and urine are still unknown. The current study was designed to investigate the biological exposure limit of pyrrole adducts for hazard assessment of n-hexane. Male Wistar rats were given daily dose of 500, 1000, 1500, 2000, 4000 mg/kg bw n-hexane by gavage for 24 weeks. The levels of pyrrole adducts in serum and urine were determined at 8, 24 hours postdose once a week. The Biological Exposure Indices was evaluated by neurological evaluation and the levels of pyrrole adducts. The difference in pyrrole adducts formation between humans and rats were estimated by using in vitro test. Dose-dependent effects were observed between the doses of n-hexane and pyrrole adducts in serum and urine, and the levels of pyrrole adduct in serum and urine approached a plateau at week 4. There was a significantly negative correlation between the time to paralysis and the level of pyrrole adducts in serum and urine, while a positive correlation between gait score and levels of pyrrole adducts in serum and urine was observed. In vitro, pyrrole adducts formed in human serum was about two times more than those in rat serum at the same level of 2,5-HD. It was concluded that the BEIs of pyrrole adducts in humans were 23.1 ± 5.91 nmol/ml in serum 8 h postdose, 11.7 ± 2.64 nmol/ml in serum 24 h postdose, 253.8 ± 36.3 nmol/ml in urine 8 h postdose and 54.6 ± 15.42 nmol/ml in urine 24 h postdose.

Biological Exposure Indices of Pyrrole Adducts in Serum and Urine for Hazard Assessment of n-Hexane Exposure

PubMed Central

Yin, Hongyin; Zhang, Chunling; Guo, Ying; Shao, Xiaoying; Zeng, Tao; Zhao, Xiulan; Xie, Keqin

2014-01-01

Background Pyrrole adducts might be used as a biomarker for monitoring occupational exposure to n-hexane, but the Biological Exposure Indices of pyrrole adducts in serum and urine are still unknown. The current study was designed to investigate the biological exposure limit of pyrrole adducts for hazard assessment of n-hexane. Methods Male Wistar rats were given daily dose of 500, 1000, 1500, 2000, 4000 mg/kg bw n-hexane by gavage for 24 weeks. The levels of pyrrole adducts in serum and urine were determined at 8, 24 hours postdose once a week. The Biological Exposure Indices was evaluated by neurological evaluation and the levels of pyrrole adducts. The difference in pyrrole adducts formation between humans and rats were estimated by using in vitro test. Results Dose-dependent effects were observed between the doses of n-hexane and pyrrole adducts in serum and urine, and the levels of pyrrole adduct in serum and urine approached a plateau at week 4. There was a significantly negative correlation between the time to paralysis and the level of pyrrole adducts in serum and urine, while a positive correlation between gait score and levels of pyrrole adducts in serum and urine was observed. In vitro, pyrrole adducts formed in human serum was about two times more than those in rat serum at the same level of 2,5-HD. Conclusion It was concluded that the BEIs of pyrrole adducts in humans were 23.1±5.91 nmol/ml in serum 8 h postdose, 11.7±2.64 nmol/ml in serum 24 h postdose, 253.8±36.3 nmol/ml in urine 8 h postdose and 54.6±15.42 nmol/ml in urine 24 h postdose. PMID:24465904

Ethanol and 4-methylpyrazole increase DNA adduct formation of furfuryl alcohol in FVB/N wild-type mice and in mice expressing human sulfotransferases 1A1/1A2.

PubMed

Sachse, Benjamin; Meinl, Walter; Glatt, Hansruedi; Monien, Bernhard H

2016-03-01

Furfuryl alcohol (FFA) is a carcinogenic food contaminant, which is formed by acid- and heat-catalyzed degradation of fructose and glucose. The activation by sulfotransferases (SULTs) yields a DNA reactive and mutagenic sulfate ester. The most prominent DNA adduct, N(2)-((furan-2-yl)methyl)-2'-deoxyguanosine (N(2)-MF-dG), was detected in FFA-treated mice and also in human tissue samples. The dominant pathway of FFA detoxification is the oxidation via alcohol dehydrogenases (ADHs) and aldehyde dehydrogenases (ALDHs). The activity of these enzymes may be greatly altered in the presence of inhibitors or competitive substrates. Here, we investigated the impact of ethanol and the ADH inhibitor 4-methylpyrazole (4MP) on the DNA adduct formation by FFA in wild-type and in humanized mice that were transgenic for human SULT1A1/1A2 and deficient in the mouse (m) Sult1a1 and Sult1d1 genes (h1A1/1A2/1a1(-)/1d1(-)). The administration of FFA alone led to hepatic adduct levels of 4.5 N(2)-MF-dG/10(8) nucleosides and 33.6 N(2)-MF-dG/10(8) nucleosides in male and female wild-type mice, respectively, and of 19.6 N(2)-MF-dG/10(8) nucleosides and 95.4 N(2)-MF-dG/10(8) nucleosides in male and female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 1.6g ethanol/kg body weight increased N(2)-MF-dG levels by 2.3-fold in male and by 1.7-fold in female wild-type mice and by 2.5-fold in male and by 1.5-fold in female h1A1/1A2/1a1(-)/1d1(-) mice. The coadministration of 100mg 4MP/kg body weight had a similar effect on the adduct levels. These findings indicate that modulators of the oxidative metabolism, e.g. the drug 4MP or consumption of alcoholic beverages, may increase the genotoxic effects of FFA also in humans. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Structures of Exocyclic R,R- and S,S-N6,N6-(2,3-Dihydroxybutan-1,4-diyl)-2′-Deoxyadenosine Adducts Induced by 1,2,3,4-Diepoxybutane

PubMed Central

2015-01-01

1,3-Butadiene (BD) is an industrial and environmental chemical present in urban air and cigarette smoke, and is classified as a human carcinogen. It is oxidized by cytochrome P450 to form 1,2,3,4-diepoxybutane (DEB); DEB bis-alkylates the N6 position of adenine in DNA. Two enantiomers of bis-N6-dA adducts of DEB have been identified: R,R-N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (R,R-DHB-dA), and S,S-N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (S,S-DHB-dA) [SeneviratneU., AntsypovichS., DorrD. Q., DissanayakeT., KotapatiS., and TretyakovaN. (2010) Chem. Res. Toxicol.23, 1556−156720873715]. Herein, the R,R-DHB-dA and S,S-DHB-dA adducts have been incorporated into the 5′-d(C1G2G3A4C5X6A7G8A9A10G11)-3′:5′-d(C12T13T14C15T16T17G18T19C20C21G22)-3′ duplex [X6 = R,R-DHB-dA (R6) or S,S-DHB-dA (S6)]. The structures of the duplexes were determined by molecular dynamics calculations, which were restrained by experimental distances obtained from NMR data. Both the R,R- and S,S-DHB-dA adducts are positioned in the major groove of DNA. In both instances, the bulky 3,4-dihydroxypyrrolidine rings are accommodated by an out-of-plane rotation about the C6-N6 bond of the bis-alkylated adenine. In both instances, the directionality of the dihydroxypyrrolidine ring is evidenced by the pattern of NOEs between the 3,4-dihydroxypyrrolidine protons and DNA. Also in both instances, the anti conformation of the glycosyl bond is maintained, which combined with the out-of-plane rotation about the C6-N6 bond, allows the complementary thymine, T17, to remain stacked within the duplex, and form one hydrogen bond with the modified base, between the imine nitrogen of the modified base and the T17 N3H imino proton. The loss of the second Watson–Crick hydrogen bonding interaction at the lesion sites correlates with the lower thermal stabilities of the R,R- and S,S-DHB-dA duplexes, as compared to the corresponding unmodified duplex. The reduced base stacking at the

Major Groove Orientation of the (2S)-N6-(2-Hydroxy-3-buten-1-yl)-2′-deoxyadenosine DNA Adduct Induced by 1,2-Epoxy-3-butene

PubMed Central

2015-01-01

1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. It is oxidized by cytochrome P450 monooxygenases to 1,2-epoxy-3-butene (EB), which alkylates DNA. BD exposures lead to large numbers of mutations at A:T base pairs even though alkylation of guanines is more prevalent, suggesting that one or more adenine adducts of BD play a role in BD-mediated genotoxicity. However, the etiology of BD-mediated genotoxicity at adenine remains poorly understood. EB alkylates the N6 exocyclic nitrogen of adenine to form N6-(hydroxy-3-buten-1-yl)-2′-dA ((2S)-N6-HB-dA) adducts (TretyakovaN., LinY., SangaiahR., UptonP. B., and SwenbergJ. A. (1997) Carcinogenesis18, 137−1479054600). The structure of the (2S)-N6-HB-dA adduct has been determined in the 5′-d(C1G2G3A4C5Y6A7G8A9A10G11)-3′:5′-d(C12T13T14C15T16T17G18T19 C20C21G22)-3′ duplex [Y = (2S)-N6-HB-dA] containing codon 61 (underlined) of the human N-ras protooncogene, from NMR spectroscopy. The (2S)-N6-HB-dA adduct was positioned in the major groove, such that the butadiene moiety was oriented in the 3′ direction. At the Cα carbon, the methylene protons of the modified nucleobase Y6 faced the 5′ direction, which placed the Cβ carbon in the 3′ direction. The Cβ hydroxyl group faced toward the solvent, as did carbons Cγ and Cδ. The Cβ hydroxyl group did not form hydrogen bonds with either T16O4 or T17O4. The (2S)-N6-HB-dA nucleoside maintained the anti conformation about the glycosyl bond, and the modified base retained Watson–Crick base pairing with the complementary base (T17). The adduct perturbed stacking interactions at base pairs C5:G18, Y6:T17, and A7:T16 such that the Y6 base did not stack with its 5′ neighbor C5, but it did with its 3′ neighbor A7. The complementary thymine T17 stacked well with both 5′ and 3′ neighbors T16 and G18. The presence of the (2S)-N6-HB-dA resulted in a 5 °C reduction in the Tm of the duplex, which is attributed to less

Atomic-Resolution Structure of an N(5) Flavin Adduct in D-Arginine Dehydrogenase

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

Fu, Guoxing; Yuan, Hongling; Wang, Siming

2011-09-06

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 {angstrom} 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.

N-acetylcysteine prevents the geldanamycin cytotoxicity by forming geldanamycin-N-acetylcysteine adduct.

PubMed

Mlejnek, Petr; Dolezel, Petr

2014-09-05

Geldanamycin (GDN) is a benzoquinone ansamycin antibiotic with anti-proliferative activity on tumor cells. GDN cytotoxicity has been attributed to the disruption of heat shock protein 90 (Hsp90) binding and stabilizing client proteins, and by the induction of oxidative stress with concomitant glutathione (GSH) depletion. The later mechanism of cytotoxicity can be abrogated by N-acetylcysteine (NAC). It was suggested that NAC prevents GDN cytotoxicity mainly by the restoring of glutathione (GSH) level (Clark et al., 2009). Here we argue that NAC does not protect cells from the GDN cytotoxicity by restoring the level of GSH. A detailed LC/MS/MS analysis of cell extracts indicated formation of GDN adducts with GSH. The amount of the GDN-GSH adduct is proportional to the GDN concentration and increases with incubation time. While nanomolar and low micromolar GDN concentrations induce cell death without an apparent GSH decrease, only much higher micromolar GDN concentrations cause a significant GSH decrease. Therefore, only high micromolar GDN concentrations can cause cell death which might be related to GSH depletion. Addition of NAC leads to the formation of adducts with GDN which diminish formation of GDN adducts with GSH. NAC also forms stable adducts with GDN extracellularly. Although NAC induces an increase in the GSH pool, this effect is not crucial for abrogation of GDN cytotoxicity. Indeed, the presence of NAC in the growth medium causes a rapid conversion of GDN into the GDN-NAC adduct, which is the real cause of the abrogated GDN cytotoxicity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Evaluation of the DNA damaging potential of cannabis cigarette smoke by the determination of acetaldehyde derived N2-ethyl-2'-deoxyguanosine adducts.

PubMed

Singh, Rajinder; Sandhu, Jatinderpal; Kaur, Balvinder; Juren, Tina; Steward, William P; Segerbäck, Dan; Farmer, Peter B

2009-06-01

Acetaldehyde is an ubiquitous genotoxic compound that has been classified as a possible carcinogen to humans. It can react with DNA to form primarily a Schiff base N(2)-ethylidene-2'-deoxyguanosine (N(2)-ethylidene-dG) adduct. An online column-switching valve liquid chromatography tandem mass spectrometry (LC-MS/MS) selected reaction monitoring (SRM) method was developed for the determination of N(2)-ethylidene-dG adducts in DNA following reduction with sodium cyanoborohydride (NaBH(3)CN) to the chemically stable N(2)-ethyl-2'-deoxyguanosine (N(2)-ethyl-dG) adduct. Accurate quantitation of the adduct was obtained by the addition of the [(15)N(5)]N(2)-ethyl-dG stable isotope-labeled internal standard prior to enzymatic hydrolysis of the DNA samples to 2'-deoxynucleosides with the incorporation of NaBH(3)CN in the DNA hydrolysis buffer. The method required 50 microg of hydrolyzed DNA on column for the analysis, and the limit of detection for N(2)-ethyl-dG was 2.0 fmol. The analysis of calf thymus DNA treated in vitro with acetaldehyde (ranging from 0.5 to 100 mM) or with the smoke generated from 1, 5, and 10 cannabis cigarettes showed linear dose-dependent increases in the level of N(2)-ethyl-dG adducts (r = 0.954 and r = 0.999, respectively). Similar levels (332.8 +/- 21.9 vs 348.4 +/- 19.1 adducts per 10(8) 2'-deoxynucleosides) of N(2)-ethyl-dG adducts were detected following the exposure of calf thymus DNA to 10 tobacco or 10 cannabis cigarettes. No significant difference was found in the levels of N(2)-ethyl-dG adducts in human lung DNA obtained from nonsmokers (n = 4) and smokers (n = 4) with the average level observed as 13.3 +/- 0.7 adducts per 10(8) 2'-deoxynucleosides. No N(2)-ethyl-dG adducts were detected in any of the DNA samples following analysis with the omission of NaBH(3)CN from the DNA hydrolysis buffer. In conclusion, these results provide evidence for the DNA damaging potential of cannabis smoke, implying that the consumption of cannabis

Nearest-neighbor thermodynamics of deoxyinosine pairs in DNA duplexes

PubMed Central

Watkins, Norman E.; SantaLucia, John

2005-01-01

Nearest-neighbor thermodynamic parameters of the ‘universal pairing base’ deoxyinosine were determined for the pairs I·C, I·A, I·T, I·G and I·I adjacent to G·C and A·T pairs. Ultraviolet absorbance melting curves were measured and non-linear regression performed on 84 oligonucleotide duplexes with 9 or 12 bp lengths. These data were combined with data for 13 inosine containing duplexes from the literature. Multiple linear regression was used to solve for the 32 nearest-neighbor unknowns. The parameters predict the Tm for all sequences within 1.2°C on average. The general trend in decreasing stability is I·C > I·A > I·T ≈ I· G > I·I. The stability trend for the base pair 5′ of the I·X pair is G·C > C·G > A·T > T·A. The stability trend for the base pair 3′ of I·X is the same. These trends indicate a complex interplay between H-bonding, nearest-neighbor stacking, and mismatch geometry. A survey of 14 tandem inosine pairs and 8 tandem self-complementary inosine pairs is also provided. These results may be used in the design of degenerate PCR primers and for degenerate microarray probes. PMID:16264087

Monitoring exposure to acrylonitrile using adducts with N-terminal valine in hemoglobin.

PubMed

Osterman-Golkar, S M; MacNeela, J P; Turner, M J; Walker, V E; Swenberg, J A; Sumner, S J; Youtsey, N; Fennell, T R

1994-12-01

Human exposure to acrylonitrile (ACN), a carcinogen in rats, may occur in industrial settings, through waste water and tobacco smoke. ACN is an electrophilic compound and binds covalently to nucleophilic sites in macromolecules. Measurements of adducts with hemoglobin could be utilized for improved exposure assessments. In this study, a method for quantification of N-(2-cyanoethyl)valine (CEVal), the product of reaction of ACN with N-terminal valine in hemoglobin has been developed. The method is based on the N-alkyl Edman procedure, which involves derivatization of the globin with pentafluorophenyl isothiocyanate and gas chromatographic-mass spectrometric analysis of the resulting thiohydantoin. An internal standard was prepared by reacting valylglycylglycine with [2H3]ACN, spiked with [14C]ACN to a known sp. act. Levels of CEVal were measured in globin from rats exposed to 3-300 p.p.m. ACN in drinking water for 105 days and from humans (four smokers and four non-smokers). CEVal was detected at all exposure levels in the drinking water study. The relationship between adduct level and water concentration was linear at concentrations of 10 p.p.m. (corresponding to an average daily uptake of c. 0.74 mg ACN/kg body wt during the 65 days prior to sacrifice) and below, with a slope of 37.7 pmol CEVal/g globin/p.p.m. At higher concentrations, adduct levels increased sublinearly, indicating saturation of a metabolic process for elimination of ACN. Comparison of adduct formation with the estimated dose (mg/kg/day) of ACN indicated that at low dose (0-10 p.p.m.) CEVal = 0.508 x ACN dose + 0.048 and at high dose (35-300 p.p.m.) CEVal = 1.142 x ACN dose - 1.098. Globin from the smokers (10-20 cigarettes/day) contained about 90 pmol CEVal/g, whereas the adduct levels in globin from non-smokers were below the detection limit. The analytical sensitivity should be sufficient to allow monitoring of occupationally exposed workers at levels well below the current Occupational Safety

Base-Displaced Intercalated Conformation of the 2-Amino-3-methylimidazo[4,5-f]quinoline N2-dG DNA Adduct Positioned at the Nonreiterated G1 in the NarI Restriction Site

PubMed Central

2016-01-01

The conformation of an N2-dG adduct arising from the heterocyclic amine 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), a potent food mutagen, was determined in 5′-d(C1T2C3X4G5C6G7C8C9A10T11C12)-3′:5′-d(G13A14T15G16G17C18G19C20C21G22A23G24)-3′; X = N2-dG-IQ, in which the modified nucleotide X4 corresponds to G1 in the 5′-d(G1G2CG3CC)-3′ NarI restriction endonuclease site. Circular dichroism (CD) revealed blue shifts relative to the unmodified duplex, consistent with adduct-induced twisting, and a hypochromic effect for the IQ absorbance in the near UV region. NMR revealed that the N2-dG-IQ adduct adopted a base-displaced intercalated conformation in which the modified guanine remained in the anti conformation about the glycosidic bond, the IQ moiety intercalated into the duplex, and the complementary base C21 was displaced into the major groove. The processing of the N2-dG-IQ lesion by hpol η is sequence-dependent; when placed at the reiterated G3 position, but not at the G1 position, this lesion exhibits a propensity for frameshift replication [Choi, J. Y., et al. (2006) J. Biol. Chem., 281, 25297–25306]. The structure of the N2-dG-IQ adduct at the nonreiterated G1 position was compared to that of the same adduct placed at the G3 position [Stavros, K. M., et al. (2014) Nucleic Acids Res., 42, 3450–3463]. CD indicted minimal spectral differences between the G1 vs G3N2-dG-IQ adducts. NMR indicated that the N2-dG-IQ adduct exhibited similar base-displaced intercalated conformations at both the G1 and G3 positions. This result differed as compared to the corresponding C8-dG-IQ adducts placed at the same positions. The C8-dG-IQ adduct adopted a minor groove conformation when placed at position G1 but a base-displaced intercalated conformation when placed at position G3 in the NarI sequence. The present studies suggest that differences in lesion bypass by hpol η may be mediated by differences in the 3′-flanking sequences, perhaps modulating the ability

Extraction of rare earth oxides using supercritical carbon dioxide modified with Tri- n-butyl phosphate–nitric acid adducts

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

Baek, Donna L.; Fox, Robert V.; Case, Mary E.

A new tri- n-butylphosphate–nitric acid (TBP–HNO 3) adduct was prepared by combining TBP and fuming (90%) HNO 3. The adduct was characterized, and its phase-equilibrium behavior in supercritical carbon dioxide is reported. Supercritical carbon dioxide (sc-CO 2) was modified with this new adduct [TBP(HNO 3) 5.2(H 2O) 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%) HNO 3 and TBP [TBP(HNO 3) 1.7(H 2O) 0.6]. All rare earth oxides tested with both adduct species couldmore » be extracted with the exception of cerium oxide. Furthermore, the water and acid concentrations in the different adducts were found to play a significant role in rare earth oxide extraction efficiency.« less

Extraction of rare earth oxides using supercritical carbon dioxide modified with Tri- n-butyl phosphate–nitric acid adducts

DOE PAGES

Baek, Donna L.; Fox, Robert V.; Case, Mary E.; ...

2016-06-14

A new tri- n-butylphosphate–nitric acid (TBP–HNO 3) adduct was prepared by combining TBP and fuming (90%) HNO 3. The adduct was characterized, and its phase-equilibrium behavior in supercritical carbon dioxide is reported. Supercritical carbon dioxide (sc-CO 2) was modified with this new adduct [TBP(HNO 3) 5.2(H 2O) 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%) HNO 3 and TBP [TBP(HNO 3) 1.7(H 2O) 0.6]. All rare earth oxides tested with both adduct species couldmore » be extracted with the exception of cerium oxide. Furthermore, the water and acid concentrations in the different adducts were found to play a significant role in rare earth oxide extraction efficiency.« less

Recognition of platinum-DNA adducts by HMGB1a.

PubMed

Ramachandran, Srinivas; Temple, Brenda; Alexandrova, Anastassia N; Chaney, Stephen G; Dokholyan, Nikolay V

2012-09-25

Cisplatin (CP) and oxaliplatin (OX), platinum-based drugs used widely in chemotherapy, form adducts on intrastrand guanines (5'GG) in genomic DNA. DNA damage recognition proteins, transcription factors, mismatch repair proteins, and DNA polymerases discriminate between CP- and OX-GG DNA adducts, which could partly account for differences in the efficacy, toxicity, and mutagenicity of CP and OX. In addition, differential recognition of CP- and OX-GG adducts is highly dependent on the sequence context of the Pt-GG adduct. In particular, DNA binding protein domain HMGB1a binds to CP-GG DNA adducts with up to 53-fold greater affinity than to OX-GG adducts in the TGGA sequence context but shows much smaller differences in binding in the AGGC or TGGT sequence contexts. Here, simulations of the HMGB1a-Pt-DNA complex in the three sequence contexts revealed a higher number of interface contacts for the CP-DNA complex in the TGGA sequence context than in the OX-DNA complex. However, the number of interface contacts was similar in the TGGT and AGGC sequence contexts. The higher number of interface contacts in the CP-TGGA sequence context corresponded to a larger roll of the Pt-GG base pair step. Furthermore, geometric analysis of stacking of phenylalanine 37 in HMGB1a (Phe37) with the platinated guanines revealed more favorable stacking modes correlated with a larger roll of the Pt-GG base pair step in the TGGA sequence context. These data are consistent with our previous molecular dynamics simulations showing that the CP-TGGA complex was able to sample larger roll angles than the OX-TGGA complex or either CP- or OX-DNA complexes in the AGGC or TGGT sequences. We infer that the high binding affinity of HMGB1a for CP-TGGA is due to the greater flexibility of CP-TGGA compared to OX-TGGA and other Pt-DNA adducts. This increased flexibility is reflected in the ability of CP-TGGA to sample larger roll angles, which allows for a higher number of interface contacts between the Pt

Influence of GSTM1 and NAT2 genotypes on placental DNA adducts in an environmentally exposed population.

PubMed

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

1997-01-01

The placenta bulky DNA adducts have been studied in relation to metabolic genotypes for glutathione S-transferase M1 (GSTM1) and N-acetyl transferase 2 (NAT2) in 158 mothers (113 nonsmokers and 45 smokers) living in two regions with different annual average air pollution levels of sulphur dioxide, nitrogen oxides, particulate matter < 10 microns, and polycyclic aromatic hydrocarbons. One region was the district of Teplice as the polluted industrial region with mines and brown coal power plants, and the other was the district of Prachatice, an agricultural region without heavy industry. DNA adduct levels were determined by using a butanol extraction enrichment procedure of 32P-postlabeling. GSTM1 and NAT2 genotypes were studied by using polymerase chain reaction. The total DNA adduct levels included a diagonal radioactive zone (DRZ) and one distinct spot outside DRZ (termed X), which was detected in almost all placenta samples and correlated with DRZ (r = .682; P < .001). We found the total DNA adduct levels 2.12 +/- 1.46 (0.04-7.70) and 1.48 +/- 1.09 (0.11-4.98) adducts per 10(8) nucleotides for Teplice and Prachatice districts, respectively, indicating significant differences between both regions studied (P = .004). Elevated DNA adduct levels were found in smoking mothers (10 or more cigarettes per day) by comparison with nonsmoking mothers (3.21 +/- 1.39 versus 1.32 +/- 0.88 adducts per 10(8) nucleotides; P < .001). Placental DNA adduct levels in smokers correlated with cotinine measured in plasma (r = .432; P = .003). This relation indicates that cigarette smoking could be predominantly responsible for DNA adduct formation in placentas of smoking mothers. DNA adduct levels were evaluated separately for non-smokers (1.50 +/- 1.00 vs. 1.09 +/- 0.66 adducts/10(8) nucleotides for the Teplice and Prachatice districts, respectively; P = .046) and smokers (3.35 +/- 1.47 vs. 2.91 +/- 1.20 adducts/10(8) nucleotides for Teplice and Prachatice districts, respectively; P

Correlation between levels of 2, 5-hexanedione and pyrrole adducts in tissues of rats exposure to n-hexane for 5-days.

PubMed

Yin, Hongyin; Guo, Ying; Zeng, Tao; Zhao, Xiulan; Xie, Keqin

2013-01-01

The formation of pyrrole adducts might be responsible for peripheral nerve injury caused by n-hexane. The internal dose of pyrrole adducts would supply more information for the neurotoxicity of n-hexane. The current study was designed to investigate the tissue distributions of 2, 5-hexanedione (2,5-HD) and pyrrole adducts in rats exposed to n-hexane, and analyze the correlation between pyrrole adducts and 2,5-HD in tissues. Male Wistar rats were given daily dose of 500,1000, 2000, 4000 mg/kg bw n-hexane by gavage for 5 days. The rats were sacrificed 24 hours after the last administration. The levels of 2, 5-hexanedione and pyrrole adducts in tissues were measured by gas chromatography and Ehrlich's reagent, respectively. The correlations between 2, 5-hexanedione and pyrrole adducts were analyzed by linear regression. Dose-dependent effects were observed between the dosage of n-hexane and 2, 5-hexanedione, and pyrrole adducts in tissues. The highest level of 2, 5-hexanedione was found in urine and the lowest in sciatic nerve, while the highest level of pyrrole adducts was seen in liver and the lowest in serum. There were significant correlations among the free 2, 5-hexanedione, total 2, 5-hexanedione and pyrrole adducts within the same tissues. Pyrrole adducts in serum showed the most significant correlation with free 2, 5-hexanedione or pyrrole adducts in tissues. The findings suggested that pyrrole adducts in serum might be a better indicator for the internal dose of free 2, 5-hexanedione and pyrrole adducts in tissues.

A Sandwich ELISA for Adducts of Polycyclic Aromatic Hydrocarbons with Human Serum Albumin1

PubMed Central

Chung, Ming Kei; Riby, Jacques; Li, He; Iavarone, Anthony T.; Williams, Evan R.; Zheng, Yuxin; Rappaport, Stephen M.

2010-01-01

Adducts of benzo[α]pyrene-diolepoxide (BPDE)2 with blood nucleophiles have been used as biomarkers of exposure to polycyclic aromatic hydrocarbons (PAHs). The most popular such assay is a competitive ELISA which employs monoclonal antibody 8E11 to detect benzo[α]pyrene tetrols following hydrolysis of BPDE adducts from lymphocyte DNA or human serum albumin (HSA). Here we use 8E11 as the capture antibody in a sandwich ELISA to detect BPDE-HSA adducts directly in 1 mg samples of HSA or 20 μL of serum/plasma. The assay employs an anti-HSA antibody for detection, which is amplified by an avidin/biotinylated horseradish peroxidase complex. The sandwich ELISA has advantages of specificity and simplicity and is about 10 times more sensitive than the competitive ELISA. To validate the assay, HSA samples were assayed from three populations with known high (coke-oven workers), medium (steel-factory control workers), and low (volunteer subjects) PAH exposures (n = 30). The respective geometric mean levels of BPDE-HSA adducts, i.e., 67.8, 14.7 and 1.93 ng/mg HSA (1,010, 220 and 28.9 fmol BPDE equivalents/mg HSA), were significantly different (p < 0.05). The sandwich ELISA will be useful for screening PAH exposures in large epidemiologic studies and can be extended to other adducts for which capture antibodies are available. PMID:20083082

The impact of glutathione s-transferase M1 and cytochrome P450 1A1 genotypes on white-blood-cell polycyclic aromatic hydrocarbon-DNA adduct levels in humans.

PubMed

Rothman, N; Shields, P G; Poirier, M C; Harrington, A M; Ford, D P; Strickland, P T

1995-09-01

Carcinogenic polycyclic aromatic hydrocarbons (PAHs) form DNA adducts via a complex metabolic activation pathway that includes cytochrome P450 (CYP) 1A1, whereas intermediate metabolites can be detoxified by conjugation through pathways including glutathione s-transferase M1 (GSTM1). PAH-DNA adducts can be measured in peripheral white blood cells (WBCs) and should reflect the net effect of competing activation and detoxification pathways and DNA repair as well as exposure. We have previously shown that WBC PAH-DNA adducts measured by an enzyme-linked immunosorbent assay (ELISA) were associated with recent, frequent consumption of charbroiled food among 47 nonsmoking wildland fire-fighters who provided two blood samples 8 wk apart. In the investigation reported here, which was performed in the same population, we measured the association between the GSTM1 null genotype, which results in loss of enzyme activity, and PAH-DNA adduct levels, hypothesizing that subjects with this genotype would have higher levels of DNA adducts because of their decreased ability to detoxify PAH metabolites. However, PAH-DNA adduct levels were nonsignificantly lower in subjects with the GSTM1 null genotype (n = 28) compared with other subjects (n = 19) (median 0.04 fmol/microgram DNA vs 0.07 fmol/microgram DNA, respectively, P = 0.45, Wilcoxon rank-sum test). Adduct levels were also lower in the nine subjects heterozygous or homozygous for the CYP1A1 exon 7 polymorphism (which codes for a valine rather than isoleucine and is thought to be associated with greater CYP1A1 activity) compared with the 38 wild-type subjects (P = 0.12). In the entire group, there was a positive association between consuming charbroiled food and PAH-DNA adduct formation (r = 0.24, P = 0.02, Spearman rank-order correlation). This association was weaker in the subgroup of subjects with the GSTM1 null genotype (r = 0.03, P = 0.84) and stronger among the remaining subjects (r = 0.57, P = 0.0002). These results

Formation of oligonucleotide adducts in pharmaceutical formulations.

PubMed

Krotz, Achim H; Gaus, Hans; Hardee, Gregory E

2005-01-01

During preformulation studies, we observed that oligonucleotide extracted from topical formulations contained considerable amounts of covalently modified oligonucleotide adducts. In this report, we describe the identification and characterization of reaction products that form when PS-oligodeoxyribonucleotide ISIS 2302 (1) is brought into contact with aqueous solutions of glycerol-derived excipients. Compatibility tests showed that the presence of certain glycerides in the formulation lead to adduct formation (1+58x amu, 1+72x amu, 1+58x+72y amu, x, and y are the number of modifications on one oligonucleotide strand). No adduct formation was observed in the presence of triglycerides or propylene glycol-derived excipients used in the study. Using nucleosides as model compounds, two modifications of deoxyguanosine were isolated by preparative reversed phase (RP)-high pressure liquid chromatography (HPLC) and characterized by nuclear magnetic resonance (NMR) and HPLC-mass spectrometry (MS). Modifications were identified as N2-(1-carboxymethyl)- and N2-(1-carboxyethyl) derivatives of 2'-deoxyguanosine. The mechanism of formation of these adducts may involve advanced glycation reactions possibly caused by excipient impurities or degradation products such as glyceraldehyde or glyceraldehyde derivatives.

Correlation between Levels of 2, 5-Hexanedione and Pyrrole Adducts in Tissues of Rats Exposure to n-Hexane for 5-Days

PubMed Central

Yin, Hongyin; Guo, Ying; Zeng, Tao; Zhao, Xiulan; Xie, Keqin

2013-01-01

Background The formation of pyrrole adducts might be responsible for peripheral nerve injury caused by n-hexane. The internal dose of pyrrole adducts would supply more information for the neurotoxicity of n-hexane. The current study was designed to investigate the tissue distributions of 2, 5-hexanedione (2,5-HD) and pyrrole adducts in rats exposed to n-hexane, and analyze the correlation between pyrrole adducts and 2,5-HD in tissues. Methods Male Wistar rats were given daily dose of 500,1000, 2000, 4000 mg/kg bw n-hexane by gavage for 5 days. The rats were sacrificed 24 hours after the last administration. The levels of 2, 5-hexanedione and pyrrole adducts in tissues were measured by gas chromatography and Ehrlich’s reagent, respectively. The correlations between 2, 5-hexanedione and pyrrole adducts were analyzed by linear regression Results Dose-dependent effects were observed between the dosage of n-hexane and 2, 5-hexanedione, and pyrrole adducts in tissues. The highest level of 2, 5-hexanedione was found in urine and the lowest in sciatic nerve, while the highest level of pyrrole adducts was seen in liver and the lowest in serum. There were significant correlations among the free 2, 5-hexanedione, total 2, 5-hexanedione and pyrrole adducts within the same tissues. Pyrrole adducts in serum showed the most significant correlation with free 2, 5-hexanedione or pyrrole adducts in tissues. Conclusion The findings suggested that pyrrole adducts in serum might be a better indicator for the internal dose of free 2, 5-hexanedione and pyrrole adducts in tissues. PMID:24098756

Identification of the major tamoxifen-DNA adducts in rat liver by mass spectroscopy.

PubMed

Rajaniemi, H; Rasanen, I; Koivisto, P; Peltonen, K; Hemminki, K

1999-02-01

We present here the first mass spectroscopic (MS) identification of the main tamoxifen-induced DNA adducts in rat liver. The two main adducts were isolated by high-performance liquid chromatography (HPLC) and identified by MS, MS-MS and ultraviolet spectroscopy. Adduct 1 was the N-desmethyltamoxifen-deoxyguanosine adduct in which the alpha-position of the metabolite N-desmethyltamoxifen is linked covalently to the amino group of deoxyguanosine. Adduct 2 was confirmed to be the trans isomer of alpha-(N2-deoxyguanosinyl)tamoxifen, as previously suggested by co-chromatography.

LC-MS/MS screening strategy for unknown adducts to N-terminal valine in hemoglobin applied to smokers and nonsmokers.

PubMed

Carlsson, Henrik; von Stedingk, Hans; Nilsson, Ulrika; Törnqvist, Margareta

2014-12-15

Electrophilically reactive compounds have the ability to form adducts with nucleophilic sites in DNA and proteins, constituting a risk for toxic effects. Mass spectrometric detection of adducts to N-terminal valine in hemoglobin (Hb) after detachment by modified Edman degradation procedures is one approach for in vivo monitoring of exposure to electrophilic compounds/metabolites. So far, applications have been limited to one or a few selected reactive species, such as acrylamide and its metabolite glycidamide. This article presents a novel screening strategy for unknown Hb adducts to be used as a basis for an adductomic approach. The method is based on a modified Edman procedure, FIRE, specifically developed for LC-MS/MS analysis of N-terminal valine adducts in Hb detached as fluorescein thiohydantoin (FTH) derivatives. The aim is to detect and identify a priori unknown Hb adducts in human blood samples. Screening of valine adducts was performed by stepwise scanning of precursor ions in small mass increments, monitoring four fragments common for the FTH derivative of valine with different N-substitutions in the multiple-reaction mode, covering a mass range of 135 Da (m/z 503-638). Samples from six smokers and six nonsmokers were analyzed. Control experiments were performed to compare these results with known adducts and to check for artifactual formation of adducts. In all samples of smokers and nonsmokers, seven adducts were identified, of which six have previously been studied. Nineteen unknown adducts were observed, and 14 of those exhibited fragmentation patterns similar to earlier studied FTH derivatives of adducts to valine. Identification of the unknown adducts will be the focus of future work. The presented methodology is a promising screening tool using Hb adducts to indicate exposure to potentially toxic electrophilic compounds and metabolites.

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

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

Sarkar, Mohamadi; Department of Pharmaceutics, Virginia Commonwealth University, Richmond, VA 23298; Stabbert, Regina

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

K-ras gene sequence effects on the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-DNA adducts.

PubMed

Ziegel, Rebecca; Shallop, Anthony; Jones, Roger; Tretyakova, Natalia

2003-04-01

The tobacco specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolically activated to electrophilic species that form methyl and pyridyloxobutyl adducts with genomic DNA, including O(6)-methylguanine, N7-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine. If not repaired, these lesions could lead to mutations and the initiation of cancer. Previous studies used ligation-mediated polymerase chain reaction (LMPCR) in combination with PAGE to examine the distribution of NNK-induced strand breaks and alkali labile lesions (e.g., N7-methylguanine) within gene sequences. However, LMPCR cannot be used to establish the distribution patterns of highly promutagenic O(6)-methylguanine and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts of NNK. We have developed methods based on stable isotope labeling HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS) that enable us to accurately quantify NNK-induced adducts at defined sites within DNA sequences. In the present study, the formation of N7-methylguanine, O(6)-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts at specific positions within a K-ras gene-derived double-stranded DNA sequence (5'-G(1)G(2)AG(3)CTG(4)G(5)TG(6)G(7)CG(8)TA G(9)G(10)C-3') was investigated following treatment with activated NNK metabolites. All three lesions preferentially formed at the second position of codon 12 (GGT), the major mutational hotspot for G-->A and G-->T base substitutions observed in smoking-induced lung tumors. Therefore, our data support the involvement of NNK and other tobacco specific nitrosamines in mutagenesis and carcinogenesis.

Formation of formaldehyde adducts in the reactions of DNA and deoxyribonucleosides with alpha-acetates of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and N-nitrosodimethylamine (NDMA).

PubMed

Cheng, Guang; Wang, Mingyao; Upadhyaya, Pramod; Villalta, Peter W; Hecht, Stephen S

2008-03-01

The cytochrome P450-mediated alpha-hydroxylation of the carcinogenic nitrosamines N-nitrosodimethylamine (NDMA, 1), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 6a), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, 6b) produces diazonium ions and formaldehyde. The DNA-binding properties of the diazonium ions have been thoroughly characterized, and there is no doubt that they are critical in cancer induction by these nitrosamines. However, the possibility of additional DNA damage via released formaldehyde has not been reported. In this study, we used acetoxymethylmethylnitrosamine (5), 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (10a), and 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (10b) as stable precursors to the alpha-hydroxymethylnitrosamines that would be formed in the metabolism of NDMA, NNK, and NNAL. These alpha-acetates were incubated with calf thymus DNA in the presence of esterase at pH 7.0 and 37 degrees C. The DNA was isolated and enzymatically hydrolyzed to deoxyribonucleosides, and the hydrolysates were analyzed by liquid chromatography-electrospray ionization-mass spectrometry-selected ion monitoring for formaldehyde DNA adducts. Convincing evidence for the formation of the formaldehyde adducts N6-hydroxymethyl-dAdo (11), N4-hydroxymethyl-dCyd (12), N2-hydroxymethyl-dGuo (13), and the cross-links di-(N6-deoxyadenosyl)methane (14), (N6-deoxyadenosyl- N2-deoxyguanosyl)methane (15), and di-(N2-deoxyguanosyl)methane (16) was obtained in these reactions. These results demonstrate that NDMA, NNK, and NNAL have the potential to be bident carcinogens, damaging DNA through the metabolic formation of both diazonium ions and formaldehyde.

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

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

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 thanmore » 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.« less

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

Identification of butyrylcholinesterase adducts after inhibition with isomalathion using mass spectrometry: difference in mechanism between (1R)- and (1S)-stereoisomers.

PubMed

Doorn, J A; Schall, M; Gage, D A; Talley, T T; Thompson, C M; Richardson, R J

2001-10-15

Previous kinetic studies found that butyrylcholinesterase (BChE) inhibited by (1R)-isomalathions readily reactivated, while enzyme inactivated by (1S)-isomers did not. This study tested the hypothesis that (1R)- and (1S)-isomers inhibit BChE by different mechanisms, yielding distinct adducts identifiable by peptide mass mapping with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Equine BChE (EBChE) was inhibited to <10% of control activity with each isomer of isomalathion and the reference compound isoparathion methyl. Control and treated enzyme was digested with trypsin, and peptides were fractionated with HPLC. Separated and unseparated peptides were analyzed with MALDI-TOF-MS. Identity of an organophosphorus peptide adduct was confirmed by fragmentation using postsource decay analysis. EBChE inhibited by (1R)-isomalathions or (S)-isoparathion methyl readily reactivated after oxime treatment with 30-40% activity recovered. Enzyme inactivated by (1S)-isomalathions or (R)-isoparathion methyl recovered <2% and <5% activity, respectively, after oxime treatment. MALDI-TOF-MS analysis revealed that inhibition of EBChE by (1R)-isomalathions and (R)- or (S)-isoparathion methyl yielded O,S-dimethyl phosphate adducts. Enzyme inactivated by (1S)-isomalathions produced only O-methyl phosphate adduct. EBChE modified by (1R)-isomalathions or either enantiomer of isoparathion methyl yielded an O-methyl phosphate adduct as well. The results indicate that EBChE inhibition by (1R)-isomalathions proceeds with loss of diethyl thiosuccinate, but inactivation by (1S)-isomers occurs with loss of thiomethyl as the primary leaving group followed by rapid expulsion of diethyl thiosuccinate to yield an aged enzyme. Furthermore, the data suggest that aging of the O,S-dimethyl phosphate adduct occurs via an S(N)2 process with loss of thiomethyl. Copyright 2001 Academic Press.

Loss of Dermatan-4-Sulfotransferase 1 Function Results in Adducted Thumb-Clubfoot Syndrome

PubMed Central

Dündar, Munis; Müller, Thomas; Zhang, Qi; Pan, Jing; Steinmann, Beat; Vodopiutz, Julia; Gruber, Robert; Sonoda, Tohru; Krabichler, Birgit; Utermann, Gerd; Baenziger, Jacques U.; Zhang, Lijuan; Janecke, Andreas R.

2009-01-01

Adducted thumb-clubfoot syndrome is an autosomal-recessive disorder characterized by typical facial appearance, wasted build, thin and translucent skin, congenital contractures of thumbs and feet, joint instability, facial clefting, and coagulopathy, as well as heart, kidney, or intestinal defects. We elucidated the molecular basis of the disease by using a SNP array-based genome-wide linkage approach that identified distinct homozygous nonsense and missense mutations in CHST14 in each of four consanguineous families with this disease. The CHST14 gene encodes N-acetylgalactosamine 4-O-sulfotransferase 1 (D4ST1), which catalyzes 4-O sulfation of N-acetylgalactosamine in the repeating iduronic acid-α1,3-N-acetylgalactosamine disaccharide sequence to form dermatan sulfate. Mass spectrometry of glycosaminoglycans from a patient's fibroblasts revealed absence of dermatan sulfate and excess of chondroitin sulfate, showing that 4-O sulfation by CHST14 is essential for dermatan sulfate formation in vivo. Our results indicate that adducted thumb-clubfoot syndrome is a disorder resulting from a defect specific to dermatan sulfate biosynthesis and emphasize roles for dermatan sulfate in human development and extracellular-matrix maintenance. PMID:20004762

Role of TP53 in repair of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human transitional cell carcinoma of the urinary bladder.

PubMed

Torino, J L; Burger, M S; Reznikoff, C A; Swaminathan, S

2001-01-01

The global genomic repair of DNA adducts was examined in human papillary transitional cell carcinoma (TCC) cell lines after exposure to N:-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), the proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). (32)P-post-labeling analysis of TCC cultures exposed to N-OH-AABP revealed a major adduct, identified as the 3',5'-bisphosphate derivative of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). The amount of adduct formation in TCC10 was dependent upon the dose and the duration of exposure and ranged between 1 and 5 adducts/10(7) nucleotides. To test if p53 regulates repair of the dG-C8-ABP adduct in genomic DNA, an isogeneic set of cell lines was obtained by infection of the TCC10 cultures with a retroviral construct expressing a trans-dominant mutant of p53, namely a Val-->Ala mutation at codon 143. The TDM143-TCC10 line expressing the mutant form of p53 was selected. The rate of repair of dG-C8-ABP was compared between TCC10 and TDM143-TCC10 cultures after treatment with 15 microM N-OH-AABP. The rate of disappearance of the adduct was monitored over a period of time after chemical treatment. (32)P-post-labeling analysis of dG-C8-ABP in parental TCC10 showed its rapid removal, the majority of adducts disappearing within 48 h. In contrast to TCC10, TDM143-TCC10 was relatively slower in removal of dG-C8-ABP. After 24 h DNA repair TDM143-TCC10 showed an approximately 3-fold greater amount of dG-C8-ABP compared with TCC10. These results imply that p53 plays a role in the repair of ABP adducts and that in p53 null cells the unrepaired DNA damage could cause accumulation of mutations, which might contribute to increased genomic instability and neoplastic progression.

Development of an ultra performance LC/MS method to quantify cisplatin 1,2 intrastrand guanine-guanine adducts

PubMed Central

Baskerville-Abraham, Irene M.; Boysen, Gunnar; Troutman, J. Mitchell; Mutlu, Esra; Collins, Leonard; deKrafft, Kathryn E.; Lin, Wenbin; King, Candice; Chaney, Stephen G.; Swenberg, James A.

2009-01-01

Platinum chemotherapeutic agents have been widely used in the treatment of cancer. Cisplatin was the first of the platinum based chemotherapeutic agents and therefore has been extensively studied as an anti-tumor agent since the late 1960s. Because this agent forms several DNA adducts, a highly sensitive and specific quantitative assay is needed to correlate the molecular dose of individual adducts with the effects of treatment. An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for quantification of 1,2 guanine-guanine intrastrand cisplatin adducts [CP-d(GpG)], using 15N10 CP-d(GpG) as an internal standard, was developed. The internal standard was characterized by MS/MS and its concentration was validated by ICP-MS. Samples containing CP-d(GpG) in DNA were purified by enzyme hydrolysis , centrifugal filtration and HPLC with fraction collection prior to quantification by UPLC-MS/MS in the selective reaction monitoring (SRM) mode (m/z 412.5→248.1 for CP-d(GpG); m/z 417.5→253.1 for [15N10] CP-d(GpG)). Recovery of standards was >90% and quantification was unaffected by increasing concentrations of calf thymus DNA. This method utilizes 25 μg of DNA per injection. The limit of quantification was 3 fmol or 3.7 adducts per 108 nucleotides, which approaches the sensitivity of the 32P postlabeling method for this adduct. These data suggested that this method is suitable for in vitro and in vivo assessment of CP-d(GpG) adducts formed by cisplatin and carboplatin. Subsequently the method was applied to studies using ovarian carcinoma cell lines and C57/BL6 mice to illustrate that this method is capable of quantifying CP-d(GpG) adducts using biologically relevant systems and doses. The development of biomarkers to determine tissue-specific molecular dosimetry during treatment will lead to a more complete understanding of both therapeutic and adverse effects of cisplatin and carboplatin. This will support the refinement of therapeutic

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.

Theoretical characterization of stable eta1-N2O-, eta2-N2O-, eta1-N2-, and eta2-N2-bound species: intermediates in the addition reactions of nitrogen hydrides with the pentacyanonitrosylferrate(II) ion.

PubMed

Olabe, José A; Estiú, Guillermina L

2003-08-11

The addition of nitrogen hydrides (hydrazine, hydroxylamine, ammonia, azide) to the pentacyanonitrosylferrate(II) ion has been analyzed by means of density functional calculations, focusing on the identification of stable intermediates along the reaction paths. Initial reversible adduct formation and further decomposition lead to the eta(1)- and eta(2)-linkage isomers of N(2)O and N(2), depending on the nucleophile. The intermediates (adducts and gas-releasing precursors) have been characterized at the B3LYP/6-31G level of theory through the calculation of their structural and spectroscopic properties, modeling the solvent by means of a continuous approach. The eta(2)-N(2)O isomer is formed at an initial stage of adduct decompositions with the hydrazine and azide adducts. Further conversion to the eta(1)-N(2)O isomer is followed by Fe-N(2)O dissociation. Only the eta(1)-N(2)O isomer is predicted for the reaction with hydroxylamine, revealing a kinetically controlled N(2)O formation. eta(1)-N(2) and eta(2)-N(2) isomers are also predicted as stable species.

Identification of 4-(3-Pyridyl)-4-oxobutyl-2'-deoxycytidine Adducts Formed in the Reaction of DNA with 4-(Acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone: A Chemically Activated Form of Tobacco-Specific Carcinogens.

PubMed

Michel, Anna K; Zarth, Adam T; Upadhyaya, Pramod; Hecht, Stephen S

2017-03-31

Metabolic activation of the carcinogenic tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1 ) and N '-nitrosonornicotine (NNN, 2 ) results in the formation of 4-(3-pyridyl)-4-oxobutyl (POB)-DNA adducts, several of which have been previously identified both in vitro and in tissues of laboratory animals treated with NNK or NNN. However, 2'-deoxycytidine adducts formed in this process have been incompletely examined in previous studies. Therefore, in this study we prepared characterized standards for the identification of previously unknown 2'-deoxycytidine and 2'-deoxyuridine adducts that could be produced in these reactions. The formation of these products in reactions of 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 3 ), a model 4-(3-pyridyl)-4-oxobutylating agent, with DNA was investigated. The major 2'-deoxycytidine adduct, identified as its stable cytosine analogue O 2 -[4-(3-pyridyl)-4-oxobut-1-yl]-cytosine ( 12 ), was O 2 -[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxycytidine ( 13 ), whereas lesser amounts of 3-[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxycytidine ( 14 ) and N 4 -[4-(3-pyridyl)-4-oxobut-1-yl]-2'-deoxycytidine ( 15 ) were also observed. The potential conversion of relatively unstable 2'-deoxycytidine adducts to stable 2'-deoxyuridine adducts by treatment of the adducted DNA with bisulfite was also investigated, but the harsh conditions associated with this approach prevented quantitation. The results of this study provide new validated standards for the study of 4-(3-pyridyl)-4-oxobutylation of DNA, a critical reaction in the carcinogenesis by 1 and 2 , and demonstrate the presence of previously unidentified 2'-deoxycytidine adducts in this DNA.

Formation of tamoxifen-DNA adducts in multiple organs of adult female cynomolgus monkeys dosed with tamoxifen for 30 days.

PubMed

Schild, Laura J; Divi, Rao L; Beland, Frederick A; Churchwell, Mona I; Doerge, Daniel R; Gamboa da Costa, Gonçalo; Marques, M Matilde; Poirier, Miriam C

2003-09-15

The use of the antiestrogen tamoxifen (TAM) is associated with an increase in endometrial cancer. TAM-induced endometrial carcinogenesis may proceed through a genotoxin-mediated pathway, although the detection of endometrial TAM-DNA adducts in exposed women is still controversial. In this study, a monkey model has been used to investigate the question of TAM-DNA adduct formation in primates. Two methods have been used to determine TAM-DNA adducts: a TAM-DNA chemiluminescence immunoassay (TAM-DNA CIA), using an antiserum that has specificity for (E)-alpha-(deoxyguanosin-N(2)-yl)-tamoxifen (dG-TAM) and (E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen (dG-desmethyl-TAM) and electrospray ionization tandem mass spectrometry (ES-MS/MS) coupled with on-line sample preparation and high-performance liquid chromatography (HPLC). Mature (19 year old) cynomolgus monkeys were given either vehicle control (n = 1) or TAM (n = 3) twice daily for a total dose of 2 mg of TAM/kg body weight (bw)/day for 30 days by naso-gastric intubation. Tissues were harvested, and DNA was isolated from uterus, ovary, liver, brain cortex, and kidney. By TAM-DNA CIA, values for uterine TAM-DNA adducts in two monkeys were 0.9 and 1.7 adducts/10(8) nucleotides, whereas values for ovarian TAM-DNA adducts in the same animals were 0.4 and 0.5 adducts/10(8) nucleotides. Liver, brain cortex, and kidney DNA samples from the three exposed monkeys had TAM-DNA levels of 2.1-4.2 adducts/10(8) nucleotides, 0.4-5.0 adducts/10(8) nucleotides, and 0.7-2.1 adducts/10(8) nucleotides, respectively. By HPLC-ES-MS/MS, the levels of TAM-DNA adducts detected in all tissues were comparable with those observed by TAM-DNA CIA. Thus, values for uterine TAM-DNA adducts ranged from 0.5 to 1.4 adducts/10(8) nucleotides, whereas values for ovarian TAM-DNA adducts, measurable in two monkeys, were 0.2 and 0.3 adducts/10(8) nucleotides. Liver DNA contained the highest TAM-DNA adduct levels (7.0-11.1 adducts/10(8) nucleotides

Role of CYP1B1 in PAH-DNA adduct formation and breast cancer risk

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

Goth-Goldstein, Regine; Russell, Marion L.; Muller, A.P.

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 resultmore » 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.« less

Hemoglobin adducts as biomarkers of 1,3-butadiene in occupationally low exposed Italian workers and a few diesel-exposed miners.

PubMed

Begemann, P; Upton, P B; Ranasinghe, A; Swenberg, J A; Soleo, L; Vimercati, L; Gelormini, A; Fustinoni, S; Zwirner-Baier, I; Neumann, H G

2001-06-01

Hemoglobin adducts were determined as biomarkers of 1,3-butadiene (BD) in 30 workers and 10 controls from an Italian BD plant and in 14 diesel-exposed miners. N-(2,3,4-trihydroxybutyl)valine (THBVal), an N-terminal valine globin adduct of reactive butadiene metabolites, was analyzed by gas chromatography/high resolution mass spectrometry after a modified Edman degradation and further acetylation. The BD exposure for the plant workers was 31 microg/m(3) (personal sampling). Whereas there was no detectable difference in hemoglobin adduct levels (range 17.7-61.4 pmol/g globin) between the total group of exposed and controls, slight but significant differences could be found between two subgroups of workers from different production units as well as one subgroup and controls (P<0.05), between smoking (n=13) and non-smoking exposed workers (n=17; P=0.066) as well as between smoking exposed workers and controls (P=0.055). Adduct levels of the miners (all non-smokers) were in the same range as those of the Italian BD-workers and controls. The internal exposure and strain measured by THBVal levels resulting from a very low occupational BD exposure was in the range of the contribution of moderate smoking.

Role of CYP1B1 in PAH-DNA Adduct Formation and Breast Cancer Risk

DTIC Science & Technology

2006-03-01

32 cases and 11 controls) undergoing surgery and analyzed these specimens for CYP1B1 gene expression, CYP1B1 genotype and PAH-DNA adducts. CYP1B1...quantitated and its purity determined by its 260/280 nm absorption. Samples were aliqoted for later measurements of CYP1B1 genotype and DNA adducts...19.78) 0.06 – 73.7 d. Perform CYP1B1 genotype analysis The CYP1B1 genotype at two polymorphic sites located in the catalytic side of the enzyme

The fate of H atom adducts to 3'-uridine monophosphate.

PubMed

Wang, Ran; Zhang, Ru Bo; Eriksson, Leif A

2010-07-29

The stabilities of the adducts deriving from H free radical addition to the O2, O4, and C5 positions of 3'-uridine monophosphate (3'UMP) are studied by the hybrid density functional B3LYP approach. Upon H atom addition at the O2 position, a concerted low-barrier proton-transfer process will initially occur, followed by the potential ruptures of the N-glycosidic or beta-phosphate bonds. The rupture barriers are strongly influenced by the rotational configuration of the phosphate group at the 3' terminal, and are influenced by bulk solvation effects. The O4-H adduct has the highest thermal stability, as the localization of the unpaired electron does not enable cleavage of either the C1'-N1 or the C3'-O(P) bonds. For the most stable adduct, with H atom added to the C5 position, the rate-controlled step is the H2'a abstraction by the C6 radical site, after which the subsequent strand rupture reactions proceed with low barriers. The main unpaired electron densities are presented for the transient species. Combined with previous results, it is concluded that the H atom adducts are more facile to drive the strand scission rather than N-glycosidic bond ruptures within the nucleic acid bases.

Stereospecific Formation of Interstrand Carbinolamine DNA Crosslinks by Crotonaldehyde- and Acetaldehyde-Derived α-CH3-γ-OH-1,N2-Propano-2’-deoxyguanosine Adducts in the 5′-CpG-3′ Sequence

PubMed Central

Cho, Young-Jin; Wang, Hao; Kozekov, Ivan D.; Kurtz, Andrew J.; Jacob, Jaison; Voehler, Markus; Smith, Jarrod; Harris, Thomas M.; Lloyd, R. Stephen; Rizzo, Carmelo J.; Stone, Michael P.

2008-01-01

The crotonaldehyde- and acetaldehyde-derived R- and S-α-CH3-γ-OH-1,N2-propanodeoxyguanosine adducts were monitored in single-stranded and duplex oligodeoxynucleotides using NMR spectroscopy. In both instances the cis and trans diastereomers of the α-CH3 and γ-OH groups underwent slow exchange, with the trans diastereomers being favored. In single-stranded oligodeoxynucleotides, the aldehyde intermediates were not detected spectroscopically, but their presence was revealed through the formation of N-terminal conjugates with the tetrapeptide KWKK. When annealed into 5′-d(GCTAGCXAGTCC)-3′•5′-d(GGACTCYCTAGC)-3′ containing the 5′-CpG-3′ sequence context (X=R- or S-α-CH3-γ-13C-OH-PdG; Y=15N2-dG), at pH 7, partial opening of the R- or S-α-CH3-γ-13C-OH-PdG adducts to the corresponding N2-(3-oxo-1-methyl-propyl)-dG aldehydes was observed at temperatures below the Tm of the duplexes. These aldehydes equilibrated with their geminal diol hydrates; higher temperatures favored the aldehydes. When annealed opposite to T, the S-α-CH3-γ-13C-OH-PdG adduct was stable. At 37 °C, an interstrand DNA crosslink was observed spectroscopically only for the R-α-CH3-γ-OH-PdG adduct. Molecular modeling predicted that the interstrand crosslink formed by the R-α-CH3-γ-OH-PdG adduct introduced less disruption into the duplex structure than did the crosslink arising from the S-α-CH3-γ-OH-PdG adduct, due to differing orientations of the R- and S-CH3 groups. Modeling also predicted that the α-methyl group of the aldehyde arising from the R-α-CH3-γ-OH-PdG adduct oriented in the 3′ direction in the minor groove, facilitating crosslinking. In contrast, the α-methyl group of the aldehyde arising from the S-α-CH3-γ-OH-PdG adduct oriented in the 5′ direction within the minor groove potentially hindering crosslinking. NMR revealed that for the R-α-CH3-γ-OH-PdG adduct, the carbinolamine form of the crosslink was favored in duplex DNA, in situ, with the imine or

Exposure to the chlorofluorocarbon substitute 2,2-dichloro-1,1,1- trifluoroethane and the anesthetic agent halothane is associated with transient protein adduct formation in the heart

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

Huwyler, J.; Gut, J.

1992-05-15

Hydrochlorofluorocarbons (HCFCs) that are structural analogues of the anesthetic agent halothane may follow a common pathway of bioactivation and formation of adducts to cellular targets of distinct tissues. Exposure of rats to a single dose of HCFC 123 (2,2-dichloro- 1,1,1-trifluoroethane) or its structural analogue halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in vivo resulted in the formation of one prominent trifluoroacetylated protein adduct (TFA-protein adduct) in the heart. In contrast, a variety of distinct TFA-protein adducts were formed in the liver and the kidney of the same animals. The TFA-protein adduct in the heart was processed rapidly; t1/2 of the intact TFA-protein adduct was lessmore » than 12 h.« less

Tamoxifen-DNA adduct formation in monkey and human reproductive organs.

PubMed

Hernandez-Ramon, Elena E; Sandoval, Nicole A; John, Kaarthik; Cline, J Mark; Wood, Charles E; Woodward, Ruth A; Poirier, Miriam C

2014-05-01

The estrogen analog tamoxifen (TAM), used for adjuvant therapy of breast cancer, induces endometrial and uterine tumors in breast cancer patients. Proliferation stimulus of the uterine endometrium is likely involved in tumor induction, but genotoxicity may also play a role. Formation of TAM-DNA adducts in human tissues has been reported but remains controversial. To address this issue, we examined TAM-DNA adducts in uteri from two species of monkeys, Erythrocebus patas (patas) and Macaca fascicularis (macaque), and in human endometrium and myometrium. Monkeys were given 3-4 months of chronic TAM dosing scaled to be equivalent to the daily human dose. In the uteri, livers and brains from the patas (n = 3), and endometrium from the macaques (n = 4), TAM-DNA adducts were measurable by TAM-DNA chemiluminescence immunoassay. Average TAM-DNA adduct values for the patas uteri (23 adducts/10(8) nucleotides) were similar to those found in endometrium of the macaques (19 adducts/10(8) nucleotides). Endometrium of macaques exposed to both TAM and low-dose estradiol (n = 5) averaged 34 adducts/10(8) nucleotides. To examine TAM-DNA persistence in the patas, females (n = 3) were exposed to TAM for 3 months and to no drug for an additional month, resulting in low or non-detectable TAM-DNA in livers and uteri. Human endometrial and myometrial samples from women receiving (n = 8) and not receiving (n = 8) TAM therapy were also evaluated. Women receiving TAM therapy averaged 10.3 TAM-DNA adducts/10(8) nucleotides, whereas unexposed women showed no detectable TAM-DNA. The data indicate that genotoxicity, in addition to estrogen agonist effects, may contribute to TAM-induced human endometrial cancer.

Adduct formation of ionic and nanoparticular silver with amino acids and glutathione

NASA Astrophysics Data System (ADS)

Blaske, Franziska; Stork, Lisa; Sperling, Michael; Karst, Uwe

2013-09-01

To investigate the interaction of ionic and nanoparticular silver with amino acids and small peptides, an electrospray ionization time-of-flight mass spectrometry method was developed. Monomeric and oligomeric silver adducts were formed with amino acids including cysteine (Cys), methionine, histidine, lysine, or the tripeptide glutathione (GSH). The obtained spectra for ionic silver show clusters in different ratios between Ag+ and the reaction partners (X) including [Ag n X m - ( n + 1)H]- ( n = 1-4, m = 1-3). Regarding Cys, adduct clusters up to n = 5 and m = 4 were observed as well. Considering silver-GSH interactions, even doubly charged oligomers occur generating [Ag( a + 1)GSH a - ( a + 3)H]2- ( a = 5-7) and [Ag b GSH b - ( b + 2)H]2- ( b = 4-8) ions. 1H NMR data of free GSH compared to that after treatment with Ag+ confirm sulfur-metal interactions due to changing chemical shifts for the protons located adjacent to the thiol group. Density functional theory calculations for silver-GSH clusters may explain the formation of experimentally recorded large clusters due to cooperative effects between silver and carboxylic acid side chains. Both sets of experiments indicate the presence of these adducts in the liquid phase. For silver nanoparticles, the respective data confirm the release of silver ions and the subsequent adduct formation.

Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences.

PubMed

Rajesh, Mathur; Wang, Gang; Jones, Roger; Tretyakova, Natalia

2005-02-15

The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene

2-Nitrosoamino-3-methylimidazo[4,5-f]quinoline activated by the inflammatory response forms nucleotide adducts.

PubMed

Lakshmi, Vijaya M; Schut, Herman A J; Zenser, Terry V

2005-11-01

Heterocyclic amines and inflammation have been implicated in the etiology of colon cancer. We have recently demonstrated that during autoxidation of the inflammatory mediator nitric oxide 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) undergoes nitrosation to form 2-nitrosoamino-3-methylimidazo[4,5-f]quinoline (N-NO-IQ). This study evaluates the genotoxicity of N-NO-IQ and compares the adducts it forms to those of 2-hydroxyamino-3-methylimidazo[4,5-f]quinoline (N-OH-IQ). N-NO-IQ was incubated with 2'-deoxyguanosine 3'-monophosphate (dGp) under a variety of inflammatory conditions. 32P-Postlabeling demonstrated the presence of multiple adducts. Incubation of N-OH-IQ with dGp at pH 7.4, 5.5, or 2.0 resulted in the formation of a single major adduct, N-(deoxyguanosin-8-yl)-IQ (dG-C8-IQ). Using a combination of 32P-postlabeling, HPLC, and nuclease P1 treatment, N-NO-IQ was shown to produce dG-C8-IQ under several different conditions. HOCl oxidation of N-NO-IQ increased dG-C8-IQ formation, and this was further increased as pH decreased from 7.4 to 5.5. Oxidation of N-NO-IQ formed a new adduct, adduct 2, while in the absence of oxidants adduct m was the major adduct. Adducts 2 and m were not formed by N-OH-IQ and not further identified. The results demonstrate that N-NO-IQ forms N-(deoxyguanosin-8-yl)-IQ, is genotoxic, is activated by conditions that mediate inflammatory responses, and is a possible cancer risk factor for individuals with colitis, inflammation of the colon.

Translesion Synthesis of the N(2)-2'-Deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells: Error-Free Replication by DNA Polymerase κ and Mutagenic Bypass by DNA Polymerases η, ζ, and Rev1.

PubMed

Bose, Arindam; Millsap, Amy D; DeLeon, Arnie; Rizzo, Carmelo J; Basu, Ashis K

2016-09-19

Translesion synthesis (TLS) of the N(2)-2'-deoxyguanosine (dG-N(2)-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5'-CG1G2CG3CC-3'). TLS efficiency was 38%, 29%, and 25% for the dG-N(2)-IQ located at G1, G2, and G3, respectively, which suggests that dG-N(2)-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8-35% in cells with knockdown of pol η, pol κ, pol ι, pol ζ, or Rev1. Up to 75% reduction in TLS occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N(2)-IQ bypass. Mutation frequencies (MFs) of dG-N(2)-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ( ( 2015 ) Nucleic Acids Res. 43 , 8340 - 8351 ). The major type of mutation induced by dG-N(2)-IQ was targeted G → T, as was reported for dG-C8-IQ. In each site, knockdown of pol κ resulted in an increase in MF, whereas MF was reduced when pol η, pol ι, pol ζ, or Rev1 was knocked down. The reduction in MF was most pronounced when pol η, pol ζ, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol κ predominantly performs error-free TLS of the dG-N(2)-IQ adduct, whereas pols η, pol ζ, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ζ and κ showed that the former was inefficient in full-length primer extension on dG-N(2)-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences

Accurate characterization of carcinogenic DNA adducts using MALDI tandem time-of-flight mass spectrometry

NASA Astrophysics Data System (ADS)

Barnes, Charles A.; Chiu, Norman H. L.

2009-01-01

Many chemical carcinogens and their in vivo activated metabolites react readily with genomic DNA, and form covalently bound carcinogen-DNA adducts. Clinically, carcinogen-DNA adducts have been linked to various cancer diseases. Among the current methods for DNA adduct analysis, mass spectroscopic method allows the direct measurement of unlabeled DNA adducts. The goal of this study is to explore the use of matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to determine the identity of carcinogen-DNA adducts. Two of the known carcinogenic DNA adducts, namely N-(2'-deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenyl-imidazo [4,5-b] pyridine (dG-C8-PhIP) and N-(2'-deoxyguanosin-8yl)-4-aminobiphenyl (dG-C8-ABP), were selected as our models. In MALDI-TOF MS measurements, the small matrix ion and its cluster ions did not interfere with the measurements of both selected dG adducts. To achieve a higher accuracy for the characterization of selected dG adducts, 1 keV collision energy in MALDI-TOF/TOF MS/MS was used to measure the adducts. In comparison to other MS/MS techniques with lower collision energies, more extensive precursor ion dissociations were observed. The detection of the corresponding fragment ions allowed the identities of guanine, PhIP or ABP, and the position of adduction to be confirmed. Some of the fragment ions of dG-C8-PhIP have not been reported by other MS/MS techniques.

Electron Capture Dissociation of Divalent Metal-adducted Sulfated N-Glycans Released from Bovine Thyroid Stimulating Hormone

NASA Astrophysics Data System (ADS)

Zhou, Wen; Håkansson, Kristina

2013-11-01

Sulfated N-glycans released from bovine thyroid stimulating hormone (bTSH) were ionized with the divalent metal cations Ca2+, Mg2+, and Co by electrospray ionization (ESI). These metal-adducted species were subjected to infrared multiphoton dissociation (IRMPD) and electron capture dissociation (ECD) and the corresponding fragmentation patterns were compared. IRMPD generated extensive glycosidic and cross-ring cleavages, but most product ions suffered from sulfonate loss. Internal fragments were also observed, which complicated the spectra. ECD provided complementary structural information compared with IRMPD, and all observed product ions retained the sulfonate group, allowing sulfonate localization. To our knowledge, this work represents the first application of ECD towards metal-adducted sulfated N-glycans released from a glycoprotein. Due to the ability of IRMPD and ECD to provide complementary structural information, the combination of the two strategies is a promising and valuable tool for glycan structural characterization. The influence of different metal ions was also examined. Calcium adducts appeared to be the most promising species because of high sensitivity and ability to provide extensive structural information.

Detection of serum AFB1-lysine adduct in Malaysia and its association with liver and kidney functions.

PubMed

Mohd Redzwan, S; Rosita, Jamaluddin; Mohd Sokhini, A M; Nurul 'Aqilah, A R; Wang, Jia-Sheng; Kang, Min-Su; Zuraini, Ahmad

2014-01-01

Aflatoxin is ubiquitously found in many foodstuffs and produced by Aspergillus species of fungi. Of many aflatoxin metabolites, AFB1 is classified by the International Agency for Research on Cancer (IARC) as group one carcinogen and linked to the development of hepatocellular carcinoma (HCC). The study on molecular biomarker of aflatoxin provides a better assessment on the extent of human exposure to aflatoxin. In Malaysia, the occurrences of aflatoxin-contaminated foods have been documented, but there is a lack of data on human exposure to aflatoxin. Hence, this study investigated the occurrence of AFB1-lysine adduct in serum samples and its association with liver and kidney functions. 5ml fasting blood samples were collected from seventy-one subjects (n=71) for the measurement of AFB1-lysine adduct, albumin, total bilirubin, AST (aspartate aminotransferase), ALT (alanine transaminase), ALP (alkaline phosphatase), GGT (gamma-glutamyl transpeptidase), creatinine and BUN (blood urea nitrogen). The AFB1-lysine adduct was detected in all serum samples (100% detection rate) with a mean of 6.85±3.20pg/mg albumin (range: 1.13-18.85pg/mg albumin). Male subjects (mean: 8.03±3.41pg/mg albumin) had significantly higher adduct levels than female subjects (mean: 5.64±2.46pg/mg albumin) (p<0.01). It was noteworthy that subjects with adduct levels greater than average (>6.85pg/mg albumin) had significantly elevated level of total bilirubin (p<0.01), GGT (p<0.05) and creatinine (p<0.01). Nevertheless, only the level of total bilirubin, (r=0.347, p-value=0.003) and creatinine (r=0.318, p-value=0.007) showed significant and positive correlation with the level of AFB1-lysine adduct. This study provides a valuable insight on human exposure to aflatoxin in Malaysia. Given that aflatoxin can pose serious problem to the health, intervention strategies should be implemented to limit/reduce human exposure to aflatoxin. Besides, a study with a big sample size should be warranted in

Exocyclic Deoxyadenosine Adducts of 1,2,3,4-Diepoxybutane: Synthesis, Structural Elucidation, and Mechanistic Studies

PubMed Central

Seneviratne, Uthpala; Antsypovich, Sergey; Goggin, Melissa; Dorr, Danae Quirk; Guza, Rebecca; Moser, Adam; Thompson, Carrie; York, Darrin M.; Tretyakova, Natalia

2009-01-01

1,2,3,4-Diepoxybutane (DEB)1 is considered the ultimate carcinogenic metabolite of 1,3-butadiene, an important industrial chemical and environmental pollutant present in urban air. Although it preferentially modifies guanine within DNA, DEB induces a large number of A → T transversions, suggesting that it forms strongly mispairing lesions at adenine nucleobases. We now report the discovery of three potentially mispairing exocyclic adenine lesions of DEB: N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (compound 2), 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (compound 3), and 1,N6-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxyadenosine (compound 4). The structures and stereochemistry of the novel DEB-dA adducts were determined by a combination of UV and NMR spectroscopy, tandem mass spectrometry, and independent synthesis. We found that synthetic N6-(2-hydroxy-3,4-epoxybut-1-yl)-2′-deoxyadenosine (compound 1) representing the product of N6-adenine alkylation by DEB spontaneously cyclizes to form 3 under aqueous conditions or 2 under anhydrous conditions in the presence of organic base. Compound 3 can be interconverted with 4 by a reversible unimolecular pericyclic reaction favoring 4 as a more thermodynamically stable product. Both 3 and 4 are present in double stranded DNA treated with DEB in vitro and in liver DNA of laboratory mice exposed to 1,3-butadiene by inhalation. We propose that in DNA under physiological conditions, DEB alkylates the N-1 position of adenine in DNA to form N1-(2-hydroxy-3,4-epoxybut-1-yl)-adenine adducts, which undergo an SN2-type intramolecular nucleophilic substitution and rearrangement to give 3 (minor) and 4 (major). Formation of exocyclic DEB-adenine lesions following exposure to 1,3-butadiene provides a possible mechanism of mutagenesis at the A:T base pairs. PMID:19883087

Translesion Synthesis of the N2-2′-Deoxyguanosine Adduct of the Dietary Mutagen IQ in Human Cells: Error-Free Replication by DNA Polymerase κ and Mutagenic Bypass by DNA Polymerases η, ζ, and Rev1

PubMed Central

2016-01-01

Translesion synthesis (TLS) of the N2-2′-deoxyguanosine (dG-N2-IQ) adduct of the carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) was investigated in human embryonic kidney 293T cells by replicating plasmid constructs in which the adduct was individually placed at each guanine (G1, G2, or G3) of the NarI sequence (5′-CG1G2CG3CC-3′). TLS efficiency was 38%, 29%, and 25% for the dG-N2-IQ located at G1, G2, and G3, respectively, which suggests that dG-N2-IQ is bypassed more efficiently by one or more DNA polymerases at G1 than at either G2 or G3. TLS efficiency was decreased 8–35% in cells with knockdown of pol η, pol κ, pol ι, pol ζ, or Rev1. Up to 75% reduction in TLS occurred when pol η, pol ζ, and Rev1 were simultaneously knocked down, suggesting that these three polymerases play important roles in dG-N2-IQ bypass. Mutation frequencies (MFs) of dG-N2-IQ at G1, G2, and G3 were 23%, 17%, and 11%, respectively, exhibiting a completely reverse trend of the previously reported MF of the C8-dG adduct of IQ (dG-C8-IQ), which is most mutagenic at G3 ((2015) Nucleic Acids Res.43, 8340−835126220181). The major type of mutation induced by dG-N2-IQ was targeted G → T, as was reported for dG-C8-IQ. In each site, knockdown of pol κ resulted in an increase in MF, whereas MF was reduced when pol η, pol ι, pol ζ, or Rev1 was knocked down. The reduction in MF was most pronounced when pol η, pol ζ, and Rev1 were simultaneously knocked down and especially when the adduct was located at G3, where MF was reduced by 90%. We conclude that pol κ predominantly performs error-free TLS of the dG-N2-IQ adduct, whereas pols η, pol ζ, and Rev1 cooperatively carry out the error-prone TLS. However, in vitro experiments using yeast pol ζ and κ showed that the former was inefficient in full-length primer extension on dG-N2-IQ templates, whereas the latter was efficient in both error-free and error-prone extensions. We believe that the observed differences between

Characterization of the Major Purine and Pyrimidine Adducts Formed after Incubations of 1-Chloro-3-buten-2-one with Single-/Double-Stranded DNA and Human Cells.

PubMed

Liu, Ling-Yan; Zheng, Jin; Kong, Cong; An, Jing; Yu, Ying-Xin; Zhang, Xin-Yu; Elfarra, Adnan A

2017-02-20

We have previously shown that 1-chloro-3-buten-2-one (CBO), a potential reactive metabolite of 1,3-butadiene (BD), exhibits potent cytotoxicity and genotoxicity that have been attributed in part to its reactivity toward DNA. In an effort to identify the DNA adducts of CBO, we characterized the CBO reactions with 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), and 2'-deoxyadenosine (dA) under in vitro physiological conditions (pH 7.4, 37 °C). In the present study, we investigated the CBO reaction with 2'-deoxythymidine (dT) and compared the rate constants of the reactions of CBO with dA, dC, dG, and dT at both individual- and mixed-nucleosides levels. We also investigated the reactions of CBO with single- and double-stranded DNA using HPLC with UV detection after adducts were released by either acid or enzymatic hydrolysis of DNA. Consistent with the results from the nucleoside reactions and the rate constant experiments, 1,N 6 -(1-hydroxy-1-chloromethylpropan-1,3-diyl)adenine (A-2D) was identified as the major DNA adduct detected after acid hydrolysis, followed by N7-(4-chloro-3-oxobutyl)guanine (CG-2H) and a small amount of 1,N 6 -(1-hydroxy-1-hydroxymethylpropan-1,3-diyl)adenine (A-1D). After enzymatic hydrolysis, 1,N 6 -(1-hydroxy-1-hydroxymethylpropan-1,3-diyl)-2'-dexoyadenosine (dA-1), 3,N 4 -(1-hydroxy-1-hydroxymethylpropan-1,3-diyl)-2'-deoxycytidine (dC-1/2), and 1,N 2 -(3-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2'-dexoyguanosine (CG-1) were detected, with dA-1 being the major product, followed by dC-1/2. When a nontoxic concentration of CBO (1 μM) was incubated with HepG2 cells, no adducts could be detected by LC-MS. However, pretreatment of cells with l-buthionine sulfoximine to deplete GSH levels allowed A-2D to be consistently detected in cellular DNA. These results may contribute to a better understanding of the role of the DNA adducts in CBO genotoxicity and mutagenicity. It also suggests that A-2D could be developed as a biomarker of CBO formation

Formation of Fused-Ring 2′-Deoxycytidine Adducts from 1-Chloro-3-buten-2-one, an in Vitro 1,3-Butadiene Metabolite, under in Vitro Physiological Conditions

PubMed Central

Sun, Liang; Pelah, Avishay; Zhang, Dong-Ping; Zhong, Yu-Fang; An, Jing; Yu, Ying-Xin; Zhang, Xin-Yu; Elfarra, Adnan A.

2013-01-01

1-Chloro-3-buten-2-one (CBO) is a potential metabolite of 1,3-butadiene (BD), a carcinogenic air pollutant. CBO is a bifunctional alkylating agent that readily reacts with glutathione (GSH) to form mono-GSH and di-GSH adducts. Recently, CBO and its precursor 1-chloro-2-hydroxy-3-butene (CHB) were found to be cytotoxic and genotoxic in human liver cells in culture with CBO being approximately 100-fold more potent than CHB. In the present study, CBO was shown to react readily with 2′-deoxycytidine (dC) under in vitro physiological conditions (pH 7.4, 37 °C) to form four dC adducts with the CBO moieties forming fused rings with the N3 and N4 atoms of dC. The four products were structurally characterized as 2-hydroxy-2-hydroxymethyl-7-(2-deoxy-β-D-erythro-pentofuranosyl)-1,2,3,4-tetrahy dro-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-1 and dC-2, a pair of diastereomers), 4-chloromethyl-4-hydroxy-7-(2-deoxy-β-D-erythro-pentofuranosyl)-1,2,3,4-tetrahydr o-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-3), and 2-chloromethyl-2-hydroxy-7-(2-deoxy-β-D-erythro-pentofuranosyl)-1,2,3,4-tetrahydr o-6-oxo-6H,7H-pyrimido[1,6-a]pyrimidin-5-ium (dC-4). Interestingly, dC-1 and dC-2 were stable under our experimental conditions (pH 7.4, 37 °C, 6 h) and existed in equilibrium as indicated by HPLC analysis, whereas dC-3 and dC-4 were labile with the half-lives being 3.0 ± 0.36 and 1.7 ± 0.06 h, respectively. Decomposition of dC-4 produced both dC-1 and dC-2, whereas acid hydrolysis of dC-1/dC-2 and dC-4 in 1 M HCl at 100 °C for 30 min yielded the deribosylated adducts dC-1H/dC-2H and dC-4H, respectively. Because fused-ring dC adducts of other chemicals are mutagenic, the characterized CBO-dC adducts could be mutagenic and play a role in the cytotoxicity and genotoxicity of CBO and its precursors, CHB and BD. The CBO-dC adducts may also be used as standards to characterize CBO-DNA adducts and to develop potential biomarkers for CBO formation in vivo. PMID:24020501

Activation of aminoimidazole carcinogens by nitrosation: mutagenicity and nucleotide adducts

PubMed Central

Zenser, Terry V.; Lakshmi, Vijaya M.; Schut, Herman A. J.; Zhou, Hui-jia; Josephy, P. David

2009-01-01

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) are heterocyclic amines (HCA) derived from high temperature cooking of meat and thought to cause colon cancer in humans. Reactive nitrogen oxygen species, which are mediators of the inflammatory response, can convert these amines to the corresponding N-nitrosamines, N-NO-IQ and N-NO-MeIQx. This study was designed to evaluate whether these N-nitrosamines are genotoxic and could be responsible, in part, for the high incidence of colon cancer in individuals with colitis. Such an association would counsel reduced intake of well-done red meat by colitis patients. Mutagenicity was evaluated by reversion of a lacZ frameshift allele in three different E. coli strains. Strains DJ701 and DJ702 express recombinant (S. typhimurium) aromatic amine N-acetyltransferase; DJ702 also expresses recombinant human cytochrome P450 1A2 and NADPH-P450 reductase; and DJ2002 served as an N-acetyltransferase-negative control. In strain DJ701, N-NO-IQ and N-NO-MeIQx elicited dose-dependent mutagenicity, which was not further increased in DJ702. Neither nitrosamine was mutagenic in strain DJ2002. While both N-nitrosamines are stable for >4 hours (pH 7.4, 37°C), they react with DNA or 2′-deoxyguanosine 3′-monophosphate at lower pH (5.5) to form adducts. HOCl, a component of the inflammatory response, increased adduct formation, as measured by 32P-postlabeling. Following treatment with nuclease P1 and separation by two-dimensional thin-layer chromatography and then HPLC, N-NO-IQ and N-NO-MeIQx were shown to form the same adducts as those formed by N-OH-MeIQx or N-OH-IQ, namely N-(deoxyguanosin-8-yl) adducts. In summary, these N-nitrosamines are genotoxic and might be alternatives to their hydroxylamine analogues as activated intermediates leading to initiation of colon cancer in individuals with colitis. PMID:19449459

Quantification of tamoxifen DNA adducts using on-line sample preparation and HPLC-electrospray ionization tandem mass spectrometry.

PubMed

Gamboa da Costa, Gonçalo; Marques, M Matilde; Beland, Frederick A; Freeman, James P; Churchwell, Mona I; Doerge, Daniel R

2003-03-01

The nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agent for the treatment of all stages of hormone-dependent breast cancer and more recently as a chemopreventive agent in women with elevated risk of developing the disease. While clearly beneficial for the treatment of breast cancer, tamoxifen has been reported to increase the risk of endometrial cancer in women. Furthermore, it has been shown to be hepatocarcinogenic in rats. Tamoxifen is clearly genotoxic in rat liver, as indicated by the formation of DNA adducts; the occurrence of tamoxifen DNA adducts in human endometrial tissue is more controversial. The detection and quantitation of tamoxifen DNA adducts have relied primarily upon (32)P-postlabeling, with other techniques, such as immunoassays and accelerator mass spectrometry, being used to a much lesser extent. To expand the range of available analytical methodologies for quantifying tamoxifen DNA adducts, we have developed an assay using on-line sample preparation, coupled with HPLC and electrospray ionization tandem mass spectrometry (ES-MS/MS). alpha-Acetoxytamoxifen was reacted with salmon testis DNA at ratios between 0.1 ng and 1 mg alpha-acetoxytamoxifen per mg DNA. After enzymatic hydrolysis to nucleosides, the most highly modified DNA samples were analyzed by HPLC-UV, which indicated the presence of two adduct peaks in approximately a 1:4 ratio. The major adduct was isolated, rigorously characterized as (E)-alpha-(deoxyguanosin-N(2)-yl)tamoxifen, and quantified on the basis of its molar extinction coefficient. A similar reaction was conducted with [N(CD(3))(2)]-alpha-acetoxytamoxifen to prepare a deuterated adduct that could serve as an internal standard for ES-MS/MS. The limit of detection for the HPLC-ES-MS/MS method was approximately 5 adducts/10(9) nucleotides, with an intra- and interassay precision of 3% relative standard deviation. The method was validated over the range of 8-1 520,000 adducts/10(8) nucleotides

Hepatic DNA adduct dosimetry in rats fed tamoxifen: a comparison of methods.

PubMed

Schild, Laura J; Phillips, David H; Osborne, Martin R; Hewer, Alan; Beland, Frederick A; Churchwell, Mona I; Brown, Karen; Gaskell, Margaret; Wright, Elizabeth; Poirier, Miriam C

2005-03-01

Liver homogenates from rats fed tamoxifen (TAM) in the diet were shared among four different laboratories. TAM-DNA adducts were assayed by high pressure liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES-MS/MS), TAM-DNA chemiluminescence immunoassay (TAM-DNA CIA), and (32)P-postlabeling with either thin layer ((32)P-P-TLC) or liquid chromatography ((32)P-P-HPLC) separation. In the first study, rats were fed a diet containing 500 p.p.m. TAM for 2 months, and the values for measurements of the (E)-alpha-(deoxyguanosin-N(2)-yl)-tamoxifen (dG-N(2)-TAM) adduct in replicate rat livers varied by 3.5-fold when quantified using 'in house' TAM-DNA standards, or other approaches where appropriate. In the second study, rats were fed 0, 50, 250 or 500 p.p.m. TAM for 2 months, and TAM-DNA values were quantified using both 'in house' approaches as well as a newly synthesized [N-methyl-(3)H]TAM-DNA standard that was shared among all the participating groups. In the second study, the total TAM-DNA adduct values varied by 2-fold, while values for the dG-N(2)-TAM varied by 2.5-fold. Ratios of dG-N(2)-TAM:(E)-alpha-(deoxyguanosin-N(2)-yl)-N-desmethyltamoxifen (dG-N(2)-N-desmethyl-TAM) in the second study were approximately 1:1 over the range of doses examined. The study demonstrated a remarkably good agreement for TAM-DNA adduct measurements among the diverse methods employed.

Influence of pH on EPR spectra of radical adducts with a new spin trap 1,2,2,5,5-pentamethyl-3-imidazoline 3-oxide

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

Skubnevskaya, G.I.; Dul'tseva, G.G.; Shchukin, G.I.

1987-08-10

1,2,2,5,5-Pentamethyl-3-imidazoline 3-oxide is an effective spin trap for short-lived free radicals, forming spin adducts with life time of > 10 min. Protonation of the amine N atom of imidazoline is manifested in the EPR spectra of the spin adducts, which makes it possible to measure pH in the range of 2.5 to 4.

Quantification of 3-nitrobenzanthrone-DNA adducts using online column-switching HPLC-electrospray tandem mass spectrometry.

PubMed

Gamboa da Costa, Gonçalo; Singh, Rajinder; Arlt, Volker M; Mirza, Amin; Richards, Meirion; Takamura-Enya, Takeji; Schmeiser, Heinz H; Farmer, Peter B; Phillips, David H

2009-11-01

The aromatic nitroketone 3-nitrobenzanthrone (3-nitro-7H-benz[de]anthracen-7-one; 3-NBA) is an extremely potent mutagen and a suspected human carcinogen detected in the exhaust of diesel engines and in airborne particulate matter. 3-NBA is metabolically activated via reduction of the nitro group to the hydroxylamine (N-OH-3-ABA) to form covalent DNA adducts. Thus far, the detection and quantification of covalent 3-NBA-DNA adducts has relied solely on (32)P-postlabeling methodologies. In order to expand the range of available techniques for the detection and improved quantification of 3-NBA-DNA adducts, we have developed a method based upon online column-switching HPLC coupled to electrospray tandem mass spectrometry, with isotopic dilution of (15)N-labeled internal standards. This methodology was applied to the determination of three 3-NBA-derived adducts: 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone (dG-N(2)-3-ABA), N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG-C8-N-3-ABA) and 2-(2'-deoxyguanosine-8-yl)-3-aminobenzanthrone (dG-C8-C2-3-ABA). Dose-dependent increases were observed for all three adducts when salmon testis DNA was reacted with N-acetoxy-3-aminobenzanthrone (N-AcO-3-ABA). dG-C8-C2-3-ABA was detected at much lower levels (overall 1%) than the other two adducts. DNA samples isolated from tissues of rats treated either intratracheally with 3-NBA or intraperitoneally with N-OH-3-ABA were analyzed by mass spectrometry, and the results compared to those obtained by (32)P-postlabeling. The method required 50 microg of hydrolyzed animal DNA on column and the limit of detection was 2.0 fmol for each adduct. dG-C8-C2-3-ABA was not observed in any of the samples providing confirmation that it is not formed in vivo. Linear regression analysis of the levels of dG-N(2)-3-ABA and dG-C8-N-3-ABA in the rat DNA showed a reasonable correlation between the two methods (R(2) = 0.88 and 0.93, respectively). In summary, the mass spectrometric method is a faster, more

Toxicokinetic study of pyrrole adducts and its potential application for biological monitoring of 2,5-hexanedione subacute exposure.

PubMed

Yin, Hong-Yin; Guo, Ying; Song, Fu-Yong; Zeng, Tao; Xie, Ke-Qin

2014-08-01

The formation of pyrrole adducts might be responsible for peripheral nerve injury caused by n-hexane, but there is not an effective biomarker for monitoring occupational exposure of n-hexane. The current study was designed to investigate the changes of pyrrole adducts in serum and urine of rats exposed to 2,5-hexanedione (2,5-HD) and analyze the correlation between pyrrole adducts and 2,5-HD. Two groups of male Wistar rats (n = 8) were administered a single dose of 200 and 400 mg/kg 2,5-HD (i.p.), and another two groups (n = 8) were given daily dose of 200 and 400 mg/kg 2,5-HD (i.p.) for 5 days. Pyrrole adducts and 2,5-HD in serum and urine were determined, at different time points after dosing, using Ehrlich’s reagent and gas chromatography, respectively. The levels of pyrrole adducts in serum accumulated in a time-dependant manner after repeated exposure to 2,5-HD, while pyrrole adducts in urine, and 2,5-HD in serum and urine were kept stable. The half-life times (t1/2) of 2,5-HD and pyrrole adducts in serum were 2.27 ± 0.28 and 25.3 ± 3.34 h, respectively. Furthermore, the levels of pyrrole adducts in urine were significantly correlated with the levels of 2,5-HD in serum (r = 0.736, P < 0.001) and urine (r = 0.730, P < 0.001), and the levels of pyrrole adducts in serum were correlated with the cumulative dosage of 2,5-HD (r = 0.965, P < 0.001). The results suggested that pyrrole adducts in serum and urine might be markers of chronic exposure to n-hexane or 2,5-HD.

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.

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts and breast cancer: modification by gene promoter methylation in a population-based study.

PubMed

White, Alexandra J; Chen, Jia; McCullough, Lauren E; Xu, Xinran; Cho, Yoon Hee; Teitelbaum, Susan L; Neugut, Alfred I; Terry, Mary Beth; Hibshoosh, Hanina; Santella, Regina M; Gammon, Marilie D

2015-12-01

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts have been associated with breast cancer incidence. Aberrant changes in DNA methylation may be an early event in carcinogenesis. However, possible relations between PAH-DNA adducts, methylation, and breast cancer are unknown. The objectives of this study were to (1) assess associations between PAH-DNA adducts, and breast cancer, stratified by DNA methylation markers and (2) examine interactions between adducts and DNA methylation in association with breast cancer and tumor subtype. In a population-based case-control study, promoter methylation of 13 breast cancer-related genes was measured in tumor tissue (n = 765-851 cases). Blood DNA from breast cancer cases (n = 873) and controls (n = 941) was used to assess PAH-DNA adducts and global methylation. Logistic regression was used to estimate adjusted odds ratios (ORs) and 95% confidence intervals (CI); and the ratio of the OR (ROR) was used to assess heterogeneity. Women with detectable PAH-DNA adducts and methylated RARβ (ROR 2.69, 95% CI 1.02-7.12; p for interaction = 0.03) or APC (ROR 1.76, 95% CI 0.87-3.58; p for interaction = 0.09) genes were more likely to have hormone receptor-positive tumors than other subtypes. Interactions with other methylation markers were not apparent (p ≥ 0.10). The association between adducts and breast cancer did not vary by methylation status of the tumor nor did adducts associate with global methylation in the controls. Gene-specific methylation of RARβ, and perhaps APC, may interact with PAH-DNA adducts to increase risk of hormone receptor-positive breast cancer. There was little evidence that adducts were associated with or interacted with other methylation markers of interest.

How does airway exposure of aflatoxin B1 affect serum albumin adduct concentrations? Evidence based on epidemiological study and animal experimentation.

PubMed

Mo, Xianwei; Lai, Hao; Yang, Yang; Xiao, Jun; He, Ke; Liu, Chao; Chen, Jiansi; Lin, Yuan

2014-08-01

Aflatoxin B1 (AFB1) airway inhalation represents an additional route of exposure to this toxin. However, the association between AFB1 inhalation and serum AFB1 albumin adducts remains unclear. The aim of this study was to explore the association between airway exposure to AFB1 and serum AFB1 albumin adduct concentrations via an epidemiological study, as well as in an AFB1 airway exposure animal model. Our epidemiological study was conducted in a sugar factory in the Guangxi Autonomous Region of China. In order to examine fungal contamination, air samples were obtained in the workshop and areas outside the workshop, such as the office and nearby store. Dust samples were also collected from the bagasse warehouse and presser workshop, and were analyzed using an indirect competitive enzyme-linked immunosorbent assay (ELISA). Additionally, blood samples were collected from a total of 121 workshop workers, and a control group (n = 80) was comprised of workers who undertook administrative tasks or other work outside the workshop. The animal experiment was conducted in the laboratory animal center of Guangxi Medical University, where a total of 60 adult male rabbits were involved in this study. By intubation, AFB1 was administered in three groups of rabbits daily, at dose rates of 0.075, 0.05 and 0.025 mg/kg/day for a period of 7 days. Blood samples were collected on day 1, day 3, day 7 and day 21, and the measurements of the AFB1 albumin adducts in the serum were performed by a double antibody sandwich ELISA. The epidemiological study showed that serum albumin adducts were detected in 67 workshop workers (55.37%), and the values ranged 6.4 pg/mg albumin to 212 pg/mg albumin (mean value: 51 ± 4.62 pg/mg albumin). In contrast, serum albumin adducts were detected in only 7 control group participants, with the values ranging from 9 pg AFB1/mg albumin to 59 pg/mg albumin (mean value: 20 ± 13.72 pg/mg albumin). The animal experiment revealed that the rabbits had detectable

Electronic structure and optical properties of Eu(III) tris-β-diketonate adducts with 1,10-phenanthroline

NASA Astrophysics Data System (ADS)

Shurygin, A. V.; Korochentsev, V. V.; Cherednichenko, A. I.; Mirochnik, A. G.; Kalinovskaya, I. V.; Vovna, V. I.

2018-03-01

Adducts of tris-β-diketonates of the rare earth metal Eu(III) with 1,10-phenanthroline are studied by photoelectron spectroscopy and quantum chemistry methods. The electronic structure, peculiarities of the nature of chemical bonds, and the geometric structure of the adducts are determined. The interpretation of UV photoelectron spectra of vapors and X-ray photoelectron spectra of solid is carried out with the chosen technique. DFT/TDDFT methods make it possible to study the 1,10-phenanthroline molecule influence on the adduct electronic structure and to analyze the electronic effects of substitution of methyl groups by trifluoromethyl groups in the ligands. At transition from the tris-β-diketonate complexes to the adducts, it is observed an increase of the absorption region and a decrease in the energy gap that contributes to the efficiency growth in electronic excitation energy transfer in the ligand-metal. Moreover, phenanthroline displaces water groups, that are luminescence quenchers, from the first coordination sphere, closes coordination in the adduct, and blocks their further attachment. Both factors contribute to an increase in the luminescence intensity.

Self-assembly in solvates of 2,4-diamino-6-(4-methyl- phenyl)-1,3,5-triazine and in its molecular adducts with some aliphatic dicarboxylic acids

NASA Astrophysics Data System (ADS)

Nandy, Purnendu; Nayak, Amrita; Biswas, Sharmita Nandy; Pedireddi, V. R.

2016-03-01

Solid state structures of 2,4-diamino-6-(4-methylphenyl)-1,3,5-triazine, 1, in the form of methanol and dimethylsulfoxide (DMSO) solvates, as well as supramolecular assemblies of 1 with various aliphatic dicarboxylic acids, oxalic (a), malonic (b), succinic (c), glutaric (d) and adipic (e) have been reported. Analysis of the assemblies has been carried out by single crystal X-ray diffraction and thermal methods. Triazine 1 yields anhydrous molecular adducts with acids a-d, upon co-crystallization either from CH3OH and DMSO solvents. However acid e gives anhydrous adduct from DMSO solvent, while it gives a methanol adduct from CH3OH. Structure determination reveals that molecular adducts 1a, 1d and 1e are in a 2:1 ratio of 1 and the corresponding acid. However the ratio is 1:1, in 1b, perhaps due to the involvement of one of the acid groups in the intramolecular hydrogen bonding and in adduct 1c the ratio observed is 3:2. Structural features in all these assemblies have been rationalised in terms of various recognition patterns formed between the acceptor and donor groups. A noteworthy feature is that -COOH groups in acid a establish interaction with 1 through amino groups, while such interactions are observed to be through hetero -N atoms in case of the acids b-e.

Increased levels of the acetaldehyde-derived DNA adduct N 2-ethyldeoxyguanosine in oral mucosa DNA from Rhesus monkeys exposed to alcohol

PubMed Central

Balbo, Silvia; Juanes, Rita Cervera; Khariwala, Samir; Baker, Erich J.; Daunais, James B.; Grant, Kathleen A.

2016-01-01

Alcohol is a human carcinogen. A causal link has been established between alcohol drinking and cancers of the upper aerodigestive tract, colon, liver and breast. Despite this established association, the underlying mechanisms of alcohol-induced carcinogenesis remain unclear. Various mechanisms may come into play depending on the type of cancer; however, convincing evidence supports the concept that ethanol’s major metabolite acetaldehyde may play a major role. Acetaldehyde can react with DNA forming adducts which can serve as biomarkers of carcinogen exposure and potentially of cancer risk. The major DNA adduct formed from this reaction is N 2-ethylidenedeoxyguanosine, which can be quantified as its reduced form N 2-ethyl-dG by LC-ESI-MS/MS. To investigate the potential use of N 2-ethyl-dG as a biomarker of alcohol-induced DNA damage, we quantified this adduct in DNA from the oral, oesophageal and mammary gland tissues from rhesus monkeys exposed to alcohol drinking over their lifetimes and compared it to controls. N 2-Ethyl-dG levels were significantly higher in the oral mucosa DNA of the exposed animals. Levels of the DNA adduct measured in the oesophageal mucosa of exposed animals were not significantly different from controls. A correlation between the levels measured in the oral and oesophageal DNA, however, was observed, suggesting a common source of formation of the DNA adducts. N 2-Ethyl-dG was measured in mammary gland DNA from a small cohort of female animals, but no difference was observed between exposed animals and controls. These results support the hypothesis that acetaldehyde induces DNA damage in the oral mucosa of alcohol-exposed animals and that it may play role in the alcohol-induced carcinogenic process. The decrease of N 2-ethyl-dG levels in exposed tissues further removed from the mouth also suggests a role of alcohol metabolism in the oral cavity, which may be considered separately from ethanol liver metabolism in the investigation of

Evaluation of polycyclic aromatic hydrocarbon-DNA adducts in exfoliated oral cells by an immunohistochemical assay.

PubMed

Romano, G; Sgambato, A; Boninsegna, A; Flamini, G; Curigliano, G; Yang, Q; La Gioia, V; Signorelli, C; Ferro, A; Capelli, G; Santella, R M; Cittadini, A

1999-01-01

Polycyclic aromatic hydrocarbon-DNA adducts were evaluated in oral cells from 98 healthy volunteers by an immunohistochemical method using a specific antiserum against benzo(a)pyrene-DNA adducts revealed by the immunoperoxidase reaction. Mean adduct content, determined as relative staining intensity by absorbance image analyzer, was significantly higher in the cells from tobacco smokers compared with nonsmokers (330 +/- 98, n = 33 versus 286 +/- 83, n = 64, respectively) with a P = 0.013 obtained by two-sample t test with equal variances. We found that in the smoker group, the PAH-DNA adduct content increases with the number of cigarettes. Thus, the relative staining intensity was 305 +/- 105 in the group smoking 1-10 cigarettes/day (n = 16), 347 +/- 77 in the 11-20 group (n = 14), and 386 +/- 112 in the group smoking more than 20 cigarettes/day (n = 3; P = 0.03 by nonparametric test for trend). No significant association was detected between PAH-DNA adducts in oral cells and variables such as residential area, oral infections, alcohol or vitamin intake, grilled food consumption, and professional activity. This work confirms and extends previous data suggesting that this immunohistochemical method might be used as a valuable dosimeter of genotoxic damage in a carcinogen-exposed population, although further studies are needed to verify the applicability of the test in high-risk populations other than smokers.

Detection of Adriamycin–DNA adducts by accelerator mass spectrometry at clinically relevant Adriamycin concentrations

PubMed Central

Coldwell, Kate E.; Cutts, Suzanne M.; Ognibene, Ted J.; Henderson, Paul T.; Phillips, Don R.

2008-01-01

Limited sensitivity of existing assays has prevented investigation of whether Adriamycin–DNA adducts are involved in the anti-tumour potential of Adriamycin. Previous detection has achieved a sensitivity of a few Adriamycin–DNA adducts/104 bp DNA, but has required the use of supra-clinical drug concentrations. This work sought to measure Adriamycin–DNA adducts at sub-micromolar doses using accelerator mass spectrometry (AMS), a technique with origins in geochemistry for radiocarbon dating. We have used conditions previously validated (by less sensitive decay counting) to extract [14C]Adriamycin–DNA adducts from cells and adapted the methodology to AMS detection. Here we show the first direct evidence of Adriamycin–DNA adducts at clinically-relevant Adriamycin concentrations. [14C]Adriamycin treatment (25 nM) resulted in 4.4 ± 1.0 adducts/107 bp (∼1300 adducts/cell) in MCF-7 breast cancer cells, representing the best sensitivity and precision reported to date for the covalent binding of Adriamycin to DNA. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin–DNA adduct detection and revealed adduct formation within an hour of drug treatment. This method has been shown to be highly reproducible for the measurement of Adriamycin–DNA adducts in tumour cells in culture and can now be applied to the detection of these adducts in human tissues. PMID:18632763

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

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

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 7β, 8α-dihydoxy-9α, l0α-epoxy-7,8,9, 10-tetrahydrobenzo[α]pyrene (anti-BPDE). Stereochemically distinct (+)-trans-, (-)-trans-, (+)-cis- and (-)-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 ( ~25 %) exists in a partially base-stacked conformation. Both cis adducts were found to be intercalated with significant π-π stacking interactions between the pyrenyl residues and the bases.more » 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 2 or G 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 2-dG in DNA isolated from the skin of mice treated topically with benzo[α]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 2-dG.« less

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

Adducts of nitrogenous ligands with rhodium(II) tetracarboxylates and tetraformamidinate: NMR spectroscopy and density functional theory calculations.

PubMed

Cmoch, Piotr; Głaszczka, Rafał; Jaźwiński, Jarosław; Kamieński, Bohdan; Senkara, Elżbieta

2014-03-01

Complexation of tetrakis(μ2-N,N'-diphenylformamidinato-N,N')-di-rhodium(II) with ligands containing nitrile, isonitrile, amine, hydroxyl, sulfhydryl, isocyanate, and isothiocyanate functional groups has been studied in liquid and solid phases using (1)H, (13)C and (15)N NMR, (13)C and (15)N cross polarisation-magic angle spinning NMR, and absorption spectroscopy in the visible range. The complexation was monitored using various NMR physicochemical parameters, such as chemical shifts, longitudinal relaxation times T1 , and NOE enhancements. Rhodium(II) tetraformamidinate selectively bonded only unbranched amine (propan-1-amine), pentanenitrile, and (1-isocyanoethyl)benzene. No complexation occurred in the case of ligands having hydroxyl, sulfhydryl, isocyanate, and isothiocyanate functional groups, and more expanded amine molecules such as butan-2-amine and 1-azabicyclo[2.2.2]octane. Such features were opposite to those observed in rhodium(II) tetracarboxylates, forming adducts with all kind of ligands. Special attention was focused on the analysis of Δδ parameters, defined as a chemical shift difference between signal in adduct and corresponding signal in free ligand. In the case of (1)H NMR, Δδ values were either negative in adducts of rhodium(II) tetraformamidinate or positive in adducts of rhodium(II) tetracarboxylates. Experimental findings were supported by density functional theory molecular modelling and gauge independent atomic orbitals chemical shift calculations. The calculation of chemical shifts combined with scaling procedure allowed to reproduce qualitatively Δδ parameters. Copyright © 2013 John Wiley & Sons, Ltd.

Sperm DNA oxidative damage and DNA adducts

PubMed Central

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

2015-01-01

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

DNA adducts: Mass spectrometry methods and future prospects

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

Farmer, P.B.; Brown, K.; Tompkins, E.

2005-09-01

Detection of DNA adducts is widely used for the monitoring of exposure to genotoxic carcinogens. Knowledge of the nature and amounts of DNA adducts formed in vivo also gives valuable information regarding the mutational effects that may result from particular exposures. The power of mass spectrometry (MS) to achieve qualitative and quantitative analyses of human DNA adducts has increased greatly in recent years with the development of improved chromatographic interfaces and ionisation sources. Adducts have been detected on nucleic acid bases, 2'-deoxynucleosides or 2'-deoxynucleotides, with LC-MS/MS being the favoured technique for many of these analyses. Our current applications of thismore » technique include the determination of N7-(2-carbamoyl-2-hydroxyethyl)-guanine, which was postulated to be found as a DNA repair product in urine following exposure to acrylamide, and of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydro-2'-deoxyadenosine, as markers of oxidative damage in human lymphocyte DNA. Higher sensitivity (with a detection limit of 1-10 adducts/10{sup 12} nucleotides) may be achieved by the use of accelerator mass spectrometry (AMS), although this requires the presence of certain isotopes, such as [{sup 14}C], in the material being analysed. In order to make this technique more amenable for studies of human exposure to environmental carcinogens, new postlabelling techniques, incorporating [{sup 14}C] into specific DNA adducts after formation, are being developed. It is expected that combining the use of advanced MS techniques with existing {sup 32}P-postlabelling and immunochemical methodologies will contribute greatly to the understanding of the burden of human exposure to environmental carcinogens.« less

Infrared spectra of some acetone—magnesium adducts

NASA Astrophysics Data System (ADS)

Hisatsune, I. C.

Co-deposition of atomic magnesium with excess acetone at liquid-nitrogen temperature produces an unstable charge-transfer complex with a characteristic carbonyl infrared band at 1595 cm -1 and stable acetone adducts in which the metal atom bridges the carbonyl bond (π-complex) or coordinates to the oxygen atom (σ-complex). Infrared spectra of these complexes with (CH 3) 2CO and (CD 3) 2CO have been obtained. Corroborations for these adducts were obtained from infrared studies of acetone matrices with atomic copper and acetaldehyde matrices with atomic magnesium and with atomic copper. Infrared spectra of an acetone adduct and a dimethyl ether adduct of the Grignard reagent CH 3MgI have also been obtained. Hydrolysis of a σ-adduct gives acetone but isopropyl alcohol is obtained from hydrolysis of the π-adduct.

Conformations of 1,3,3,5,7,7-Hexamethyl-1,5-diazacyclooctane and Its Bis-BH(3) Adduct. Mono- and Bis-BH(3) Adducts of Di-Tertiary Amines.

PubMed

Livant, P.; Majors, A. W.; Webb, T. R.

1996-05-03

A variable-temperature (1)H- and (13)C-NMR study revealed a conformational equilibrium for 1,3,3,5,7,7-hexamethyl-1,5-diazacyclooctane (4) having DeltaG() = 8.8 +/- 0.6 kcal/mol at 184 K. This activation barrier connects a major and a minor form of 4. Molecular mechanics calculations on 4 led to the conclusion that the major form is a set of twist-chair-chairs interconverting rapidly via the chair-chair and that the minor form is most likely a set of twist-boat-boats interconverting rapidly via the boat-boat. The proximity of the two nitrogen lone pairs in the major form of 4 made plausible the expectation that 4, as well as a related diamine with apposed nitrogens, 3,7-dimethyl-3,7-diazabicyclo[3.3.1]nonane (3), might bind a Lewis acid, namely BH(3), using both lone pairs simultaneously and equally. This proved not to be the case: for 3 only the bis-BH(3) adduct was found and for 4 the mono-BH(3) adduct utilized only one nitrogen lone pair. The structure of the bis-BH(3) adduct of 4 (12) was determined by X-ray crystallography to be a twist-boat-boat with BH(3)s cis. Molecular mechanics calculations on 12 were consistent with the solid state conformation found.

Immunohistochemical detection of a substituted 1,N(2)-ethenodeoxyguanosine adduct by omega-6 polyunsaturated fatty acid hydroperoxides in the liver of rats fed a choline-deficient, L-amino acid-defined diet.

PubMed

Kawai, Yoshichika; Kato, Yoji; Nakae, Dai; Kusuoka, Osamu; Konishi, Yoichi; Uchida, Koji; Osawa, Toshihiko

2002-03-01

Endogenous lipid peroxidation products react with DNA and form exocyclic DNA adducts. The purpose of this study was to investigate the in vivo formation of 7-(2-oxo-heptyl)-substituted 1,N(2)-etheno-2'-deoxyguanosine adduct (Oxo-heptyl-varepsilondG), one of the major products from the reaction of 13-hydroperoxyoctadecadienoic acid (13-HPODE) with dG. The monoclonal antibody specific to Oxo-heptyl-varepsilondG was prepared using a chemically synthesized conjugate of Oxo-heptyl-varepsilondG and carrier protein as immunogen. The characterization showed that the obtained antibody (mAb6A3) is specific to the Oxo-heptyl-varepsilondG moiety. Using the novel antibody, the presence of the Oxo-heptyl-varepsilondG adduct in vivo was immunohistochemically revealed in the liver of rats fed a choline-deficient, L-amino acid-defined diet, an endogenous carcinogenesis model, for 3 days. In addition, the Oxo-heptyl-varepsilondG formation was confirmed in DNAs treated with peroxidized linoleic acid, arachidonic acid and gamma-linolenic acid, respectively, suggesting that the hydroperoxides of omega-6 polyunsaturated fatty acids could be the potential sources of Oxo-heptyl-varepsilondG formation in vivo. Collectively, the results in this study suggest the first evidence that the formation of Oxo-heptyl-varepsilondG, the omega-6 lipid hydroperoxide-mediated DNA adduct, may be a potential biomarker for the early phase of carcinogenesis.

Courses of change in knee adduction moment and lateral thrust differ up to 1 year after TKA.

PubMed

Shimada, Noboru; Deie, Masataka; Hirata, Kazuhiko; Hiate, Yasuhiko; Orita, Naoya; Iwaki, Daisuke; Ito, Yoshihiro; Kimura, Hiroaki; Pappas, Evangelos; Ochi, Mitsuo

2016-08-01

In total knee arthroplasty (TKA), dynamic knee loading may loosen the artificial joint and bone or cause polyethylene wear after prolonged use. TKA decreases knee adduction moment at 6 months, but this effect is lost by 1 year post-operatively. However, lateral thrust after TKA has not been clarified. We hypothesized that like knee adduction moment, lateral thrust would return to baseline levels by 1 year post-operatively. Participants were 15 patients who underwent TKA for medial knee OA. Japanese Orthopaedic Association (JOA) score, numeric rating scale, and gait analysis (measurement of peak knee adduction moment, knee varus angle at peak knee adduction moment, lateral thrust, and gait speed) were performed preoperatively (baseline) and 3 weeks, 3 and 6 months, and 1 year post-operatively. JOA score improved from 55 ± 9.8 to 78 ± 12.1 at 1 year post-operatively, and pain decreased significantly from baseline at each follow-up (p < 0.001). Significant increases in gait speed were observed at 6 months and 1 year (p < 0.001). Peak knee adduction moment during stance phase was significantly lower at 3 weeks, 3 months, and 6 months compared to baseline (p < 0.05), but no significant changes were seen at 1 year. Knee varus at peak knee adduction moment did not differ significantly between any measurement points, while lateral thrust was decreased at 6 months and 1 year compared to baseline (p < 0.05). Temporal courses of changes up to 1 year after TKA differed between knee adduction moment and lateral thrust, so our hypothesis was rejected. IV.

DNA Sequence Modulates Geometrical Isomerism of the trans-8,9-Dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy Aflatoxin B1 Adduct

PubMed Central

2016-01-01

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus, is oxidized by cytochrome P450 enzymes to aflatoxin B1-8,9-epoxide, which alkylates DNA at N7-dG. Under basic conditions, this N7-dG adduct rearranges to yield the trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B1 (AFB1–FAPY) adduct. The AFB1–FAPY adduct exhibits geometrical isomerism involving the formamide moiety. NMR analyses of duplex oligodeoxynucleotides containing the 5′-XA-3′, 5′-XC-3′, 5′-XT-3′, and 5′-XY-3′ sequences (X = AFB1–FAPY; Y = 7-deaza-dG) demonstrate that the equilibrium between E and Z isomers is controlled by major groove hydrogen bonding interactions. Structural analysis of the adduct in the 5′-XA-3′ sequence indicates the preference of the E isomer of the formamide group, attributed to formation of a hydrogen bond between the formyl oxygen and the N6 exocyclic amino group of the 3′-neighbor adenine. While the 5′-XA-3′ sequence exhibits the E isomer, the 5′-XC-3′ sequence exhibits a 7:3 E:Z ratio at equilibrium at 283 K. The E isomer is favored by a hydrogen bond between the formyl oxygen and the N4-dC exocyclic amino group of the 3′-neighbor cytosine. The 5′-XT-3′ and 5′-XY-3′ sequences cannot form such a hydrogen bond between the formyl oxygen and the 3′-neighbor T or Y, respectively, and in these sequence contexts the Z isomer is favored. Additional equilibria between α and β anomers and the potential to exhibit atropisomers about the C5–N5 bond do not depend upon sequence. In each of the four DNA sequences, the AFB1–FAPY adduct maintains the β deoxyribose configuration. Each of these four sequences feature the atropisomer of the AFB1 moiety that is intercalated above the 5′-face of the damaged guanine. This enforces the Ra axial conformation for the C5–N5 bond. PMID:25587868

DNA Sequence Modulates Geometrical Isomerism of the trans-8,9- Dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)- 9-hydroxy Aflatoxin B1 Adduct.

PubMed

Li, Liang; Brown, Kyle L; Ma, Ruidan; Stone, Michael P

2015-02-16

Aflatoxin B(1) (AFB(1)), a mycotoxin produced by Aspergillus flavus, is oxidized by cytochrome P450 enzymes to aflatoxin B(1)-8,9-epoxide, which alkylates DNA at N7-dG. Under basic conditions, this N7-dG adduct rearranges to yield the trans-8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxy aflatoxin B(1) (AFB(1)−FAPY) adduct. The AFB(1)−FAPY adduct exhibits geometrical isomerism involving the formamide moiety. NMR analyses of duplex oligodeoxynucleotides containing the 5′-XA-3′, 5′-XC-3′, 5′-XT-3′, and 5′-XY-3′ sequences (X = AFB(1)−FAPY; Y = 7-deaza-dG)demonstrate that the equilibrium between E and Z isomers is controlled by major groove hydrogen bonding interactions.Structural analysis of the adduct in the 5′-XA-3′ sequence indicates the preference of the E isomer of the formamide group,attributed to formation of a hydrogen bond between the formyl oxygen and the N(6) exocyclic amino group of the 3′-neighboradenine. While the 5′-XA-3′ sequence exhibits the E isomer, the 5′-XC-3′ sequence exhibits a 7:3 E:Z ratio at equilibrium at 283K. The E isomer is favored by a hydrogen bond between the formyl oxygen and the N(4)-dC exocyclic amino group of the 3′-neighbor cytosine. The 5′-XT-3′ and 5′-XY-3′ sequences cannot form such a hydrogen bond between the formyl oxygen and the 3′-neighbor T or Y, respectively, and in these sequence contexts the Z isomer is favored. Additional equilibria between α and β anomers and the potential to exhibit atropisomers about the C5−N(5) bond do not depend upon sequence. In each of the four DNA sequences, the AFB(1)−FAPY adduct maintains the β deoxyribose configuration. Each of these four sequences feature the atropisomer of the AFB(1) moiety that is intercalated above the 5′-face of the damaged guanine. This enforces the Ra axialc onformation for the C5−N(5) bond.

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.

PARP1 impact on DNA repair of platinum adducts: preclinical and clinical read-outs.

PubMed

Olaussen, Ken A; Adam, Julien; Vanhecke, Elsa; Vielh, Philippe; Pirker, Robert; Friboulet, Luc; Popper, Helmut; Robin, Angélique; Commo, Fréderic; Thomale, Jürgen; Kayitalire, Louis; Filipits, Martin; Le Chevalier, Thierry; André, Fabrice; Brambilla, Elisabeth; Soria, Jean-Charles

2013-05-01

Evaluation of DNA repair proteins might provide meaningful information in relation to prognosis and chemotherapy efficacy in Non-Small Cell Lung Cancer (NSCLC) patients. The role of Poly(ADP-Ribose) Polymerase (PARP) in DNA repair of platinum adducts has not been firmly established. We used a DNA repair functional test based on antibody recognition of cisplatin intrastrand platinum adducts on DNA. We evaluated the effect of PARP inhibition on DNA repair functionality in a panel of cisplatin cell lines treated by the clinical-grade pharmacological inhibitor CEP8983 (a 4-methoxy-carbazole derivate) and the commercially available inhibitor PJ34 (phenanthridinone). We determined PARP1 protein expression in whole tumor sections from the International Adjuvant Lung cancer Trial (IALT)-bio study and tested a 3-marker PARP1/MSH2/ERCC1 algorithm combining PARP1 tumor status with previously published data. Chemosensitivity of cisplatin in NSCLC cell lines was correlated with the accumulation of cisplatin DNA adducts (P=0.0004). Further, the pharmacological inhibition of PARP induced a 1.7 to 2.3-fold increase in platinum adduct accumulation (24h) in A549 cell line suggesting a slow-down of platinum DNA-adduct repair capacity. In parallel, PARP1 inhibition increased the sensitivity to cisplatin treatment. In patient samples, PARP1 expression levels did not influence patient survival or the effect of platinum-based post-operative chemotherapy in the global IALT-bio population (interaction P=0.79). Among cases with high expression of all three markers (triple positive), untreated patients had prolonged survival with a median DFS of 7.8 years, (HR=0.34, 95%CI [0.19-0.61], adjusted P=0.0003) compared to triple negative patients (1.4 years). Remarkably, triple positive patients suffered from a detrimental effect (4.9-year reduction of median DFS) by post-operative cisplatin-based chemotherapy (HR=1.79, 95%CI [1.01-3.17], adjusted P=0.04, chemotherapy vs. control). Combinatorial

DNA adducts of ethylene dibromide: Aspects of formation and mutagenicity

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

Cmarik, J.L.

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 victimmore » 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.« less

Threefold interweaving of (4,4) nets built from R(10)10(58) rings inthe hydrogen-bonded adduct 1,4-diazabicyclo

PubMed

Burchell; Ferguson; Lough; Glidewell

2000-09-01

The 1:1 adduct of 1,4-diazabicyclo[2.2.2]octane and 5-hydroxyisophthalic acid is a salt, [H(C(6)H(12)N(2))](+). [HOC(6)H(3)(COOH)COO](-) or C(6)H(13)N(2)(+).C(8)H(5)O(5)(-). The ions are linked by three types of hydrogen bond, i.e. N-H.O, O-H.O and O-H.N, into continuous two-dimensional (4,4) nets built from a single type of R(10)(10)(58) ring. Six independent sheets of this type make up the structure and these are interwoven in sets of three.

Conformational preferences of DNA following damage by aristolochic acids: Structural and energetic insights into the different mutagenic potential of the ALI and ALII-N(6)-dA adducts.

PubMed

Kathuria, Preetleen; Sharma, Purshotam; Abendong, Minette N; Wetmore, Stacey D

2015-04-21

Aristolochic acids (AAI and AAII), produced by the Aristolochiaceae family of plants, are classified as group I (human) carcinogens by the International Agency for Research on Cancer. These acids are metabolized in cells to yield aristolactams (ALI and ALII, respectively), which further form bulky adducts with the purine nucleobases. Specifically, the adenine lesions are more persistent in cells and have been associated with chronic renal diseases and related carcinogenesis. To understand the structural basis of the nephrotoxicity induced by AAs, the ALI-N(6)-dA and ALII-N(6)-dA lesions are systematically studied using computational methods. Density functional theory calculations indicate that the aristolactam moiety intrinsically prefers a planar conformation with respect to adenine. Nucleoside and nucleotide models suggest that the anti and syn orientations about the glycosidic bond are isoenergetic for both adducts. Molecular dynamics simulations and free energy calculations reveal that the anti base-displaced intercalated conformation is the most stable conformer for both types of AL-N(6)-dA adducted DNA, which agrees with previous experimental work on the ALII-N(6)-dA adduct and thereby validates our approach. Interestingly, this conformer differs from the dominant conformations adopted by other N6-linked adenine lesions, including those derived from polycyclic aromatic hydrocarbons. Furthermore, the second most stable syn base-displaced intercalated conformation lies closer in energy to the anti base-displaced intercalated conformation for ALI-N(6)-dA compared to ALII-N(6)-dA. This indicates that a mixture of conformations may be detectable for ALI-N(6)-dA in DNA. If this enhanced conformational flexibility of double-stranded DNA persists when bound to a lesion-bypass polymerase, this provides a possible structural explanation for the previously observed greater nephrotoxic potential for the ALI versus ALII-N(6)-dA adduct. In addition, the structural

Increased N7-methyldeoxyguanosine DNA adducts after occupational exposure to pesticides and influence of genetic polymorphisms of paraoxonase-1 and glutathione S-transferase M1 and T1.

PubMed

Gómez-Martín, Antonio; Altakroni, Bashar; Lozano-Paniagua, David; Margison, Geoffrey P; de Vocht, Frank; Povey, Andrew C; Hernández, Antonio F

2015-06-01

There are concerns about genetic risks associated with long-term exposure to pesticides as these compounds may damage DNA, resulting in mutations that eventually lead to cancer, neurological, and reproductive adverse health effects. This study assessed DNA damage in intensive agricultural workers exposed to pesticides by determining the levels of N7-methyldeoxyguanosine (N7-MedG), an adduct known to be a robust biomarker of recent exposure to chemical methylating agents. A cohort of 39 plastic greenhouse workers was assessed for changes in lymphocyte DNA N7-MedG levels between low level and high level exposures during the course of a spraying season. The contributions of genetic polymorphisms of the pesticide-metabolizing enzymes paraoxonase-1 (PON1) and the glutathione S-transferases, GSTM1 and GSTT1, on N7-MedG levels and other potential confounders were also assessed. N7-MedG increased in the period of high pesticide exposure as compared to the low exposure period (0.23 and 0.18 µmol N7-MedG/mol dG for the unadjusted and adjusted linear mixed models, P = 0.02 and 0.08, respectively). Significant decreased levels of erythrocyte acetylcholinesterase and plasma cholinesterase were observed in the high versus low exposure period in both the unadjusted (2.85 U/g hemoglobin and 213.13 U/L, respectively) and adjusted linear mixed models (2.99 U/g hemoglobin and 230.77 U/L, respectively), indicating pesticide intake. In intensive agriculture workers, higher pesticide exposure increased DNA alkylation levels, further demonstrating the genotoxicity of pesticides in man. In addition, pesticide-exposed individuals with inherited susceptible metabolic genotypes (particularly, null genotype for GSTM1 and the PON1 192R allele) appear to have an increased risk of genotoxic DNA damage. Environ. Mol. Mutagen. 56:437-445, 2015. © 2014 Wiley Periodicals, Inc. © 2014 Wiley Periodicals, Inc.

Electron Detachment Dissociation of Underivatized Chloride-Adducted Oligosaccharides

NASA Astrophysics Data System (ADS)

Kornacki, James R.; Adamson, Julie T.; Håkansson, Kristina

2012-11-01

Chloride anion attachment has previously been shown to aid determination of saccharide anomeric configuration and generation of linkage information in negative ion post-source decay MALDI tandem mass spectrometry. Here, we employ electron detachment dissociation (EDD) and collision activated dissociation (CAD) for the structural characterization of underivatized oligosaccharides bearing a chloride ion adduct. Both neutral and sialylated oligosaccharides are examined, including maltoheptaose, an asialo biantennary glycan (NA2), disialylacto- N-tetraose (DSLNT), and two LS tetrasaccharides (LSTa and LSTb). Gas-phase chloride-adducted species are generated by negative ion mode electrospray ionization. EDD and CAD spectra of chloride-adducted oligosaccharides are compared to the corresponding spectra for doubly deprotonated species not containing a chloride anion to assess the role of chloride adduction in the stimulation of alternative fragmentation pathways and altered charge locations allowing detection of additional product ions. In all cases, EDD of singly chloridated and singly deprotonated species resulted in an increase in observed cross-ring cleavages, which are essential to providing saccharide linkage information. Glycosidic cleavages also increased in EDD of chloride-adducted oligosaccharides to reveal complementary structural information compared to traditional (non-chloride-assisted) EDD and CAD. Results indicate that chloride adduction is of interest in alternative anion activation methods such as EDD for oligosaccharide structural characterization.

Aflatoxin B1-induced DNA adduct formation and p53 mutations in CYP450-expressing human liver cell lines.

PubMed

Macé, K; Aguilar, F; Wang, J S; Vautravers, P; Gómez-Lechón, M; Gonzalez, F J; Groopman, J; Harris, C C; Pfeifer, A M

1997-07-01

Epidemiological evidence has been supporting a relationship between dietary aflatoxin B1 (AFB1) exposure, development of human primary hepatocellular carcinoma (HCC) and mutations in the p53 tumor suppressor gene. However, the correlation between the observed p53 mutations, the AFB1 DNA adducts and their activation pathways has not been elucidated. Development of relevant cellular in vitro models, taking into account species and tissue specificity, could significantly contribute to the knowledge of cytotoxicity and genotoxicity mechanisms of chemical procarcinogens, such as AFB1, in humans. For this purpose a non-tumorigenic SV40-immortalized human liver epithelial cell line (THLE cells) which retained most of the phase II enzymes, but had markedly reduced phase I activities was used for stable expression of the human CYP1A2, CYP2A6, CYP2B6 and CYP3A4 cDNA. The four genetically engineered cell lines (T5-1A2, T5-2A6, T5-2B6 and T5-3A4) produced high levels of the specific CYP450 proteins and showed comparable or higher catalytic activities related to the CYP450 expression when compared to human hepatocytes. The T5-1A2, T5-2A6, T5-2B6 and T5-3A4 cell lines exhibited a very high sensitivity to the cytotoxic effects of AFB1 and were approximately 125-, 2-, 2- and 15-fold, respectively, more sensitive than the control T5-neo cells, transfected with an expressing vector which does not contain CYP450 cDNA. In the CYP450-expressing cells, nanomolar doses of AFB1-induced DNA adduct formation including AFB1-N7-guanine, -pyrimidyl and -diol adducts. In addition, the T5-1A2 cells showed AFM1-DNA adducts. At similar levels of total DNA adducts, both the T5-1A2 and T5-3A4 cells showed, at codon 249 of the p53 gene, AGG to AGT transversions at a relative frequency of 15x10(-6). In contrast, only the T5-3A4 cells showed CCC to ACC transversion at codon 250 at a high frequency, whereas the second most frequent mutations found in the T5-1A2 cells were C to T transitions at the first

Simultaneous detection of five different 2-hydroxyethyl-DNA adducts formed by ethylene oxide exposure, using a high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry assay.

PubMed

Tompkins, Elaine M; Jones, Donald J L; Lamb, John H; Marsden, Debbie A; Farmer, Peter B; Brown, Karen

2008-01-01

A method has been developed for the simultaneous detection and quantitation of five different 2-hydroxyethyl-DNA (HE-DNA) adducts that could be formed as a result of exposure to ethylene oxide (EO). In addition to the major N7-HE-guanine (N7-HEG) adducts this assay can also measure the less prevalent but potentially more biologically significant N1-HE-2'-deoxyadenosine (N1-HEdA), O(6)-HE-2'-deoxyguanosine (O(6)-HEdG), N(6)-HE-2'-deoxyadenosine (N(6)-HEdA) and N3-HE-2'-deoxyuridine adducts (N3-HEdU). The method involves the isolation of HE adducts from the unmodified nucleosides by either neutral thermal hydrolysis or enzymatic digestion, followed by high-performance liquid chromatographic (HPLC) purification, before detection and quantification by liquid chromatography tandem mass spectrometry (LC/MS/MS) using selective reaction monitoring (SRM). The limits of detection were in the range 0.5-25 fmol for each individual adduct, making this one of the most sensitive assays available for the detection of N7-HEG. To illustrate the possible applications of the assay, it has been employed in the measurement of endogenous/background and EO-induced HE adducts in a variety of DNA samples.

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

Alcohol, Aldehydes, Adducts and Airways.

PubMed

Sapkota, Muna; Wyatt, Todd A

2015-11-05

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.

Human DNA adduct measurements: State of the art

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

Poirier, M.C.; Weston, A.

1996-10-01

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 {sup 32}P-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 presentedmore » 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. 156 refs., 1 fig., 3 tabs.« less

Formation and Repair of Tobacco Carcinogen-Derived Bulky DNA Adducts

DOE PAGES

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 6 -alkylguaninemore » 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

N-terminal valine adduct from the anti-HIV drug abacavir in rat haemoglobin as evidence for abacavir metabolism to a reactive aldehyde in vivo

PubMed Central

Charneira, C; Grilo, NM; Pereira, SA; Godinho, ALA; Monteiro, EC; Marques, MM; Antunes, AMM

2012-01-01

BACKGROUND AND PURPOSE The aim of this study was to obtain evidence for the activation of the nucleoside reverse transcriptase inhibitor abacavir to reactive aldehyde metabolites in vivo. Protein haptenation by these reactive metabolites may be a factor in abacavir-induced toxic events. EXPERIMENTAL APPROACH The formation of N-terminal valine adducts from the abacavir-derived aldehydes was investigated in the haemoglobin of Wistar rats treated with eight daily doses (120 mg·kg−1) of abacavir. The analyses were conducted by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry upon comparison with synthetic standards. KEY RESULTS An N-terminal valine haemoglobin adduct derived from an α,β-unsaturated aldehyde metabolite of abacavir was identified in vivo for the first time. CONCLUSIONS AND IMPLICATIONS This preliminary work on abacavir metabolism provides the first unequivocal evidence for the formation of an α,β-unsaturated aldehyde metabolite in vivo and of its ability to form haptens with proteins. The methodology described herein can be used to assess the formation of this metabolite in human samples and has the potential to become a valuable pharmacological tool for mechanistic studies of abacavir toxicity. In fact, the simplicity of the method suggests that the abacavir adduct with the N-terminal valine of haemoglobin could be used to investigate abacavir-induced toxicity for accurate risk/benefit estimations. PMID:22725138

N-terminal valine adduct from the anti-HIV drug abacavir in rat haemoglobin as evidence for abacavir metabolism to a reactive aldehyde in vivo.

PubMed

Charneira, C; Grilo, N M; Pereira, S A; Godinho, A L A; Monteiro, E C; Marques, M M; Antunes, A M M

2012-11-01

The aim of this study was to obtain evidence for the activation of the nucleoside reverse transcriptase inhibitor abacavir to reactive aldehyde metabolites in vivo. Protein haptenation by these reactive metabolites may be a factor in abacavir-induced toxic events. The formation of N-terminal valine adducts from the abacavir-derived aldehydes was investigated in the haemoglobin of Wistar rats treated with eight daily doses (120 mg·kg(-1)) of abacavir. The analyses were conducted by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry upon comparison with synthetic standards. An N-terminal valine haemoglobin adduct derived from an α,β-unsaturated aldehyde metabolite of abacavir was identified in vivo for the first time. This preliminary work on abacavir metabolism provides the first unequivocal evidence for the formation of an α,β-unsaturated aldehyde metabolite in vivo and of its ability to form haptens with proteins. The methodology described herein can be used to assess the formation of this metabolite in human samples and has the potential to become a valuable pharmacological tool for mechanistic studies of abacavir toxicity. In fact, the simplicity of the method suggests that the abacavir adduct with the N-terminal valine of haemoglobin could be used to investigate abacavir-induced toxicity for accurate risk/benefit estimations. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Data-Independent Mass Spectrometry Approach for Screening and Identification of DNA Adducts.

PubMed

Guo, Jingshu; Villalta, Peter W; Turesky, Robert J

2017-11-07

Long-term exposures to environmental toxicants and endogenous electrophiles are causative factors for human diseases including cancer. DNA adducts reflect the internal exposure to genotoxicants and can serve as biomarkers for risk assessment. Liquid chromatography-multistage mass spectrometry (LC-MS n ) is the most common method for biomonitoring DNA adducts, generally targeting single exposures and measuring up to several adducts. However, the data often provide limited evidence for a role of a chemical in the etiology of cancer. An "untargeted" method is required that captures global exposures to chemicals, by simultaneously detecting their DNA adducts in the genome; some of which may induce cancer-causing mutations. We established a wide selected ion monitoring tandem mass spectrometry (wide-SIM/MS 2 ) screening method utilizing ultraperformance-LC nanoelectrospray ionization Orbitrap MS n with online trapping to enrich bulky, nonpolar adducts. Wide-SIM scan events are followed by MS 2 scans to screen for modified nucleosides by coeluting peaks containing precursor and fragment ions differing by -116.0473 Da, attributed to the neutral loss of deoxyribose. Wide-SIM/MS 2 was shown to be superior in sensitivity, specificity, and breadth of adduct coverage to other tested adductomic methods with detection possible at adduct levels as low as 4 per 10 9 nucleotides. Wide-SIM/MS 2 data can be analyzed in a "targeted" fashion by generation of extracted ion chromatograms or in an "untargeted" fashion where a chromatographic peak-picking algorithm can be used to detect putative DNA adducts. Wide-SIM/MS 2 successfully detected DNA adducts, derived from chemicals in the diet and traditional medicines and from lipid peroxidation products, in human prostate and renal specimens.

Influence of GSTM1 null and low repair XPC PAT+ on anti-B[a]PDE-DNA adduct in mononuclear white blood cells of subjects low exposed to PAHs through smoking and diet.

PubMed

Pavanello, Sofia; Pulliero, Alessandra; Clonfero, Erminio

2008-02-01

The influence of low-activity NER genotypes (XPC PAT-/+, XPA-A23G, XPD Asp312Asn, XPD Lys751Gln) and GSTM1 (active or null) was evaluated on anti-benzo[a]pyrene diol epoxide-(B[a]PDE)-DNA adduct formed in the lymphocyte plus monocyte fraction (LMF). The sample population consisted of 291 healthy subjects with low exposure to polycyclic aromatic hydrocarbons (PAHs) (B[a]P) through their smoking (n=126 smokers) or dietary habits (n=165 non-smokers with high (>or=52 times/year) consumption of charcoaled meat or pizza). The bulky anti-B[a]PDE-DNA adduct levels were detected by HPLC/fluorescence analysis and genotypes by PCR. Anti-B[a]PDE-DNA was present (>or=0.5 adducts/10(8) nucleotides) in 163 (56%) subjects (median (range) 0.77 (0.125-32.0) adducts/10(8) nucleotides), with smokers showing a significantly higher adduct level than non-smokers with high consumption of PAH-rich meals (P<0.01). Our exposed-sample population with unfavourable XPC PAT+/- or +/+ and GSTM1 null genotypes has the significantly highest adduct level (P<0.01). Taking into account tobacco smoke and diet as sources of exposure to B[a]P, low-activity XPC PAT+ shows a major role in smokers (P<0.05) and GSTM1 null in non-smokers with frequent consumption of PAH-rich meals (P<0.01). The modulation of anti-B[a]PDE-DNA adduct in the LMF by GSTM1 null and low-activity XPC PAT+ polymorphisms may be considered as potential genetic susceptibility factors that can modify individual responses to low PAH (B[a]P) genotoxic exposure, with the consequent risk of cancer in the general population.

Linking the generation of DNA adducts to lung cancer.

PubMed

Ceppi, Marcello; Munnia, Armelle; Cellai, Filippo; Bruzzone, Marco; Peluso, Marco E M

2017-09-01

Worldwide, lung cancer is the leading cause of cancer death. DNA adducts are considered a reliable biomarker that reflects carcinogen exposure to tobacco smoke, but the central question is what is the relationship of DNA adducts and cancer? Therefore, we investigated this relationship by a meta-analysis of twenty-two studies with bronchial adducts for a total of 1091 subjects, 887 lung cancer cases and 204 apparently healthy individuals with no evidence of lung cancer. Our study shows that these adducts are significantly associated to increase lung cancer risk. The value of Mean Ratio lung-cancer (MR) of bronchial adducts resulting from the random effects model was 2.64, 95% C.I. 2.00-3.50, in overall lung cancer cases as compared to controls. The significant difference, with lung cancer patients having significant higher levels of bronchial adducts than controls, persisted after stratification for smoking habits. The MR lung-cancer value between lung cancer patients and controls for smokers was 2.03, 95% C.I. 1.42-2.91, for ex-smokers 3.27, 95% C.I. 1.49-7.18, and for non-smokers was 3.81, 95% C.I. 1.85-7.85. Next, we found that the generation of bronchial adducts is significantly related to inhalation exposure to tobacco smoke carcinogens confirming its association with volatile carcinogens. The MR smoking estimate of bronchial adducts resulting from meta-regression was 2.28, 95% Confidence Interval (C.I.) 1.10-4.73, in overall smokers in respect to non-smokers. The present work provides strengthening of the hypothesis that bronchial adducts are not simply relate to exposure, but are a cause of chemical-induced lung cancer. Copyright © 2017 Elsevier B.V. All rights reserved.

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

The sphingosine 1-phosphate breakdown product, (2E)-hexadecenal, forms protein adducts and glutathione conjugates in vitro.

PubMed

Schumacher, Fabian; Neuber, Corinna; Finke, Hannah; Nieschalke, Kai; Baesler, Jessica; Gulbins, Erich; Kleuser, Burkhard

2017-08-01

Sphingosine 1-phosphate (S1P), a bioactive lipid involved in various physiological processes such as cell proliferation and apoptosis, can be irreversibly cleaved by S1P lyase, yielding phosphoethanolamine and (2 E )-hexadecenal (2 E HD). The latter metabolite, an α,β-unsaturated fatty aldehyde, may be susceptible to nucleophilic attack by cellular biomolecules. Hence, we studied whether 2 E HD forms reaction products with GSH and proteins in vitro. Using LC-MS/MS and stable isotopically labeled reference material, we identified a total of nine novel reaction products of 2 E HD in a cell-free approach: two GSH conjugates and seven l-amino acid adducts. Both GSH conjugates were also found in HepG2 cell lysates incubated with 2 E HD. Likewise, we detected four out of seven amino acid adducts released from the model protein, BSA, and proteins extracted from HepG2 cells. On this occasion, the 2 E HD Michael adduct with l-histidine proved to be the most prominent adduct. Most interestingly, inhibition of the enzymatically driven oxidative degradation of 2 E HD resulted in increased levels of both GSH conjugates and protein adducts in HepG2 cell lysates. Hence, our data provide new insights into sphingolipid metabolism and will be useful to investigate certain disorders linked to an impaired fatty aldehyde metabolism in more detail. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

The Elusive Palladium-Diazo Adduct Captured: Synthesis, Isolation and Structural Characterization of [(ArNHC-PPh2 )Pd(η2 -N2 C(Ph)CO2 Et)].

PubMed

Rull, Silvia G; Álvarez, Eleuterio; Fructos, Manuel R; Belderrain, Tomás R; Pérez, Pedro J

2017-06-07

The first example of a diazo palladium adduct is reported. The complexes [(ArNHC-PPh 2 )M(η 2 -N 2 C(Ph)CO 2 Et)] (M=Ni, 3; M=Pd, 4; ArNHC-PPh 2 =3-(2,6-diisopropylphenyl)-1-[(diphenylphosphino)ethyl]imidazol-2-ylidene) were prepared by ligand exchange with styrene-coordinated precursors [(ArNHC-PPh 2 )M(styrene)] (M=Ni, 1; M=Pd, 2). Complex 4 was fully characterized, including X-ray analyses; this constitutes the first example of a diazo adduct compound with palladium, thereby closing the gap between Groups 8 and 10 regarding this type of compounds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determination of isocyanate specific albumin-adducts in workers exposed to toluene diisocyanates.

PubMed

Sabbioni, Gabriele; Gu, Qi; Vanimireddy, Lakshiminiranjan Reddy

2012-03-01

Toluene diisocyanates (2,4-TDI and 2,6-TDI) are important intermediates in the chemical industry. Among the main damages after low levels of TDI exposure are lung sensitization and asthma. It is therefore necessary to have sensitive and specific methods to monitor isocyanate exposure of workers. Urinary metabolites or protein adducts have been used as biomarkers in workers exposed to TDI. However, with these methods it was not possible to determine if the biomarkers result from exposure to TDI or to the corresponding toluene diamines (TDA). This work presents a new procedure for the determination of isocyanate-specific albumin adducts. Isotope dilution mass spectrometry was used to measure the adducts in albumin present in workers exposed to TDI. 2,4-TDI and 2,6-TDI formed adducts with lysine: N(ϵ)-[({3-amino-4-methylphenyl}amino)carbonyl]-lysine, N(ϵ)-[({5-amino-2-methylphenyl}amino)carbonyl]-lysine, and N(ϵ)- [({3-amino-2-methylphenyl}amino)carbonyl]-lysine. In future studies, this new method can be applied to measure TDI-exposures in workers.

Analysis of 4-aminobiphenyl-DNA adducts in human urinary bladder and lung by alkaline hydrolysis and negative ion gas chromatography-mass spectrometry.

PubMed Central

Lin, D; Lay, J O; Bryant, M S; Malaveille, C; Friesen, M; Bartsch, H; Lang, N P; Kadlubar, F F

1994-01-01

Analysis of carcinogen-DNA adducts has been regarded as a useful means of assessing human exposure to chemical carcinogens. We have established a method for quantitation of 4-aminobiphenyl (4-ABP)-DNA adducts by alkaline hydrolysis and gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS). Aliquots of DNA (typically 100 micrograms/ml) were spiked with an internal standard, d9-4-ABP, and were hydrolyzed in 0.05 N NaOH at 130 degrees C overnight. The liberated 4-ABP was extracted with hexane and derivatized using pentafluoropropionic anhydride in trimethylamine for 30 min at room temperature prior to GC-NICI-MS. With in vitro [3H]N-hydroxy-4-ABP modified DNA standards, we observed 59 +/- 7% (n = 9) recovery of the 4-ABP and a linear correlation between hydrolyzed 4-ABP and the adduct levels ranging from about 1 in 10(8) to 1 in 10(4) nucleotides (r = 0.999, n = 9). The method was further validated by comparison of the results with that obtained by the 32P-postlabeling method. There was excellent agreement (r = 0.994, p < 0.001) between the two methods for quantitation of the adduct in eight samples of Salmonella typhimurium DNA treated with 4-ABP and rat liver S9, although the 32P-postlabeling method gave slightly higher values. The DNA adducts in 11 human lung and 8 urinary bladder mucosa specimens were then determined by our GC-NICI-MS method. The adduct levels were found to be < 0.32 to 49.5 adducts per 10(8) nucleotides in the lungs and < 0.32 to 3.94 adducts per 10(8) nucleotides in the bladder samples.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 4. A Figure 4. B PMID:7889831

Oxidative transformation of tunichromes - Model studies with 1,2-dehydro-N-acetyldopamine and N-acetylcysteine.

PubMed

Kuang, Qun F; Abebe, Adal; Evans, Jason; Sugumaran, Manickam

2017-08-01

Tunichromes are 1,2-dehydrodopa containing bioactive peptidyl derivatives found in blood cells of several tunicates. They have been implicated in metal sequestering, tunic formation, wound healing and defense reaction. Earlier studies conducted on these compounds indicate their extreme liability, high reactivity and easy oxidative polymerization. Their reactions are also complicated by the presence of multiple dehydrodopyl units. Since they have been invoked in crosslinking and covalent binding, to understand the reactivities of these novel compounds, we have taken a simple model compound that possess the tunichrome reactive group viz., 1,2-dehydro-N-acetyldopamine (Dehydro NADA) and examined its reaction with N-acetylcysteine in presence of oxygen under both enzymatic and nonenzymatic conditions. Ultraviolet and visible spectral studies of reaction mixtures containing dehydro NADA and N-acetylcysteine in different molar ratios indicated the production of side chain and ring adducts of N-acetylcysteine to dehydro NADA. Liquid chromatography and mass spectral studies supported this contention and confirmed the production of several different products. Mass spectral analysis of these products show the potentials of dehydro NADA to form side chain adducts that can lead to polymeric products. This is the first report demonstrating the ability of dehydro dopyl units to form adducts and crosslinks with amino acid side chains. Copyright © 2017 Elsevier Inc. All rights reserved.

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein.

PubMed Central

Requena, J R; Fu, M X; Ahmed, M U; Jenkins, A J; Lyons, T J; Baynes, J W; Thorpe, S R

1997-01-01

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of < 1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo. PMID:9078279

Urinary Metabolites of the Dietary Carcinogen PhIP are Predictive of Colon DNA Adducts After a Low Dose Exposure in Humans

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

Malfatti, M; Dingley, K; Nowell, S

2006-04-28

Epidemiologic evidence indicates that exposure to heterocyclic amines (HAs) in the diet is an important risk factor for the development of colon cancer. Well-done cooked meats contain significant levels of HAs which have been shown to cause cancer in laboratory animals. To better understand the mechanisms of HA bioactivation in humans, the most mass abundant HA, 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), was used to assess the relationship between PhIP metabolism and DNA adduct formation. Ten human volunteers were administered a dietary relevant dose of [{sup 14}C]PhIP 48-72 h prior to surgery to remove colon tumors. Urine was collected for 24 h after dosingmore » for metabolite analysis, and DNA was extracted from colon tissue and analyzed by accelerator mass spectrometry for DNA adducts. All ten subjects were phenotyped for CYP1A2, NAT2, and SULT1A1 enzyme activity. Twelve PhIP metabolites were detected in the urine samples. The most abundant metabolite in all volunteers was N-hydroxy-PhIP-N{sup 2}-glucuronide. Metabolite levels varied significantly between the volunteers. Interindividual differences in colon DNA adducts levels were observed between each individual. The data showed that individuals with a rapid CYP1A2 phenotype and high levels of urinary N-hydroxy-PhIP-N{sup 2}-glucuronide, had the lowest level of colon PhIP-DNA adducts. This suggests that glucuronidation plays a significant role in detoxifying N-hydroxy-PhIP. The levels of urinary N-hydroxy-PhIP-N{sup 2}-glucuronide were negatively correlated to colon DNA adduct levels. Although it is difficult to make definite conclusions from a small data set, the results from this pilot study have encouraged further investigations using a much larger study group.« less

Micronuclei related to anti-B[a]PDE-DNA adduct in peripheral blood lymphocytes of heavily polycyclic aromatic hydrocarbon-exposed nonsmoking coke-oven workers and controls.

PubMed

Pavanello, Sofia; Kapka, Lucyna; Siwinska, Ewa; Mielzyñska, Danuta; Bolognesi, Claudia; Clonfero, Erminio

2008-10-01

Micronuclei (MN) frequency associated to biologically effective dose of polycyclic aromatic hydrocarbons [PAH; anti-benzo[a]pyrene diolepoxide (B[a]PDE)-DNA] within the same subjects' peripheral blood lymphocytes (PBL) was evaluated. Study subjects were nonsmoking male Polish coke-oven workers (n=49) and matched controls (n=45) verified for PAH exposure by urinary 1-pyrenol. We found that coke-oven workers, heavily exposed to PAHs (80% workers exceeded the urinary 1-pyrenol biological exposure index value), presented significantly higher MN frequency in PBLs than controls (P<0.01). Substantial difference was also found for adduct levels in PBLs (P<0.01). Increase in MN levels was significantly related to anti-B[a]PDE-DNA formation, key adduct of the ultimate carcinogenic metabolite of B[a]P (n=94; r=0.47; P<0.001). The dose-response relationship was improved when subjects with adduct levels above the 3rd tertile (>or=4.35 adducts/10(8) nucleotides) were excluded (n=61; r=0.69; P<0.001). Saturation of adduct/MN formation at high levels may disturb the underlying relationship. Linear multiple regression analysis, without subjects of 3rd tertile adduct level (n=61), revealed that adduct formation (t=4.61; P<0.001), but not 1-pyrenol, was the significant determinant in increasing MN. In conclusion, the increase in MN frequency is mainly related to the specific anti-B[a]PDE-DNA formation within PBLs of the same subject. Our results substantiate, with the use of an early indicator of biological effect as well, that workers are at higher cancer risk than controls.

Including the Copenhagen Adduction Exercise in the FIFA 11+ Provides Missing Eccentric Hip Adduction Strength Effect in Male Soccer Players: A Randomized Controlled Trial.

PubMed

Harøy, Joar; Thorborg, Kristian; Serner, Andreas; Bjørkheim, André; Rolstad, Linn E; Hölmich, Per; Bahr, Roald; Andersen, Thor Einar

2017-11-01

The FIFA 11+ was developed as a complete warm-up program to prevent injuries in soccer players. Although reduced hip adduction strength is associated with groin injuries, none of the exercises included in the FIFA 11+ seem to specifically target hip adduction strength. To investigate the effect on eccentric hip adduction strength of the FIFA 11+ warm-up program with or without the Copenhagen adduction exercise. Randomized controlled trial; Level of evidence, 1. We recruited 45 eligible players from 2 U19 elite male soccer teams. Players were randomized into 2 groups; 1 group carried out the standard FIFA 11+ program, while the other carried out the FIFA 11+ but replaced the Nordic hamstring exercise with the Copenhagen adduction exercise. Both groups performed the intervention 3 times weekly for 8 weeks. Players completed eccentric strength and sprint testing before and after the intervention. Per-protocol analyses were performed, and 12 players were excluded due to low compliance (<67% of sessions completed). The main outcome was eccentric hip adduction strength (N·m/kg). Between-group analyses revealed a significantly greater increase in eccentric hip adduction strength of 0.29 Nm/kg (8.9%; P = .01) in favor of the group performing the Copenhagen adduction exercise, whereas no within-group change was noted in the group that used the standard FIFA 11+ program (-0.02 N·m/kg [-0.7%]; P = .69). Including the Copenhagen adduction exercise in the FIFA 11+ program increases eccentric hip adduction strength, while the standard FIFA 11+ program does not. Registration: Registration: ISRCTN13731446 (International Standard Randomised Controlled Trial Number registry).

Isotope Dilution nanoLC/ESI+-HRMS3 Quantitation of Urinary N7-(1-Hydroxy-3-buten-2-yl) Guanine Adducts in Humans and Their Use as Biomarkers of Exposure to 1,3-Butadiene.

PubMed

Sangaraju, Dewakar; Boldry, Emily J; Patel, Yesha M; Walker, Vernon; Stepanov, Irina; Stram, Daniel; Hatsukami, Dorothy; Tretyakova, Natalia

2017-02-20

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a known human carcinogen. Occupational exposure to BD in the polymer and monomer industries is associated with an increased incidence of lymphoma. BD is present in automobile exhaust, cigarette smoke, and forest fires, raising concern about potential exposure of the general population to this carcinogen. Following inhalation exposure, BD is bioactivated to 3,4-epoxy-1-butene (EB). If not detoxified, EB is capable of modifying guanine and adenine bases of DNA to form nucleobase adducts, which interfere with accurate DNA replication and cause cancer-initiating mutations. We have developed a nanoLC/ESI + -HRMS 3 methodology for N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts in human urine (limit of detection: 0.25 fmol/mL urine; limit of quantitation: 1.0 fmol/mL urine). This new method was successfully used to quantify EB-GII in urine of F344 rats treated with 0-200 ppm of BD, occupationally exposed workers, and smokers belonging to two different ethnic groups. EB-GII amounts increased in a dose-dependent manner in urine of laboratory rats exposed to 0, 62.5, or 200 ppm of BD. Urinary EB-GII levels were significantly increased in workers occupationally exposed to 0.1-2.2 ppm of BD (1.25 ± 0.51 pg/mg of creatinine) as compared to administrative controls exposed to <0.01 ppm of BD (0.22 ± 0.08 and pg/mg of creatinine) (p = 0.0024), validating the use of EB-GII as a biomarker of human exposure to BD. EB-GII was also detected in smokers' urine with European American smokers excreting significantly higher amounts of EB-GII than African American smokers (0.48 ± 0.09 vs 0.12 ± 0.02 pg/mg of creatinine, p = 3.1 × 10 -7 ). Interestingly, small amounts of EB-GII were observed in animals and humans with no known exposure to BD, providing preliminary evidence for its endogenous formation. Urinary EB-GII adduct levels and urinary mercapturic acids of BD (MHBMA, DHBMA) were compared

Isolevuglandin adducts in disease.

PubMed

Salomon, Robert G; Bi, Wenzhao

2015-06-20

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. 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. 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. 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.

NMR solution structure of an N2-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: Intercalation from the minor groove with ruptured Watson-Crick base pairing

PubMed Central

Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H.; Cai, Yuqin; Rodriguez, Fabian A.; Sayer, Jane M.; Jerina, Donald M.; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E.

2012-01-01

The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the non-planar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely-studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14-position with the exocyclic amino group of guanine. Here, we present the first NMR solution structure of a DB[a,l]P-derived adduct, the 14R (+)-trans-anti-DB[a,l]P–N2-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N2-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3’-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3’-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE - DNA adduct conformation differs from: (1) the classical intercalation motif where Watson-Crick base-pairing is intact at the lesion site, and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix . The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed. PMID:23121427

Effects of alkyl chain length and substituent pattern of fullerene bis-adducts on film structures and photovoltaic properties of bulk heterojunction solar cells.

PubMed

Tao, Ran; Umeyama, Tomokazu; Kurotobi, Kei; Imahori, Hiroshi

2014-10-08

A series of alkoxycarbonyl-substituted dihydronaphthyl-based [60]fullerene bis-adduct derivatives (denoted as C2BA, C4BA, and C6BA with the alkyl chain of ethyl, n-butyl, and n-hexyl, respectively) have been synthesized to investigate the effects of alkyl chain length and substituent pattern of fullerene bis-adducts on the film structures and photovoltaic properties of bulk heterojunction polymer solar cells. The shorter alkyl chain length caused lower solubility of the fullerene bis-adducts (C6BA > C4BA > C2BA), thereby resulting in the increased separation difficulty of respective bis-adduct isomers. The device performance based on poly(3-hexylthiophene) (P3HT) and the fullerene bis-adduct regioisomer mixtures was enhanced by shortening the alkyl chain length. When using the regioisomerically separated fullerene bis-adducts, the devices based on trans-2 and a mixture of trans-4 and e of C4BA exhibited the highest power conversion efficiencies of ca. 2.4%, which are considerably higher than those of the C6BA counterparts (ca. 1.4%) and the C4BA regioisomer mixture (1.10%). The film morphologies as well as electron mobilities of the P3HT:bis-adduct blend films were found to affect the photovoltaic properties considerably. These results reveal that the alkyl chain length and substituent pattern of fullerene bis-adducts significantly influence the photovoltaic properties as well as the film structures of bulk heterojunction solar cells.

Analysis of 4-hydroxy-1-(-3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in human exfoliated oral mucosa cells by liquid chromatography-electrospray ionization-tandem mass spectrometry

PubMed Central

Stepanov, Irina; Muzic, John; Le, Chap T.; Sebero, Erin; Villalta, Peter; Ma, Bin; Jensen, Joni; Hatsukami, Dorothy; Hecht, Stephen S.

2013-01-01

Quantitation of DNA adducts could provide critical information on the relationship between exposure to tobacco smoke and cancer risk in smokers. In this study, we developed a robust and sensitive liquid chromatography-tandem mass spectrometry method for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB1)-releasing DNA adducts in human oral cells, a non-invasive source of DNA for biomarker studies. Isolated DNA undergoes acid hydrolysis, after which samples are purified by solid-phase extraction and analyzed by LC-ESI-MS/MS. The developed method was applied for analysis of samples obtained via collection with a commercial mouthwash from 30 smokers and 15 nonsmokers. In smokers, the levels of HPB-releasing DNA adducts averaged 12.0 pmol HPB/mg DNA (detected in 20 out of 28 samples with quantifiable DNA yield) and in nonsmokers, the levels of adducts averaged 0.23 pmol/mg DNA (detected in 3 out of 15 samples). For the 30 smoking subjects, matching buccal brushings were also analyzed and HPB-releasing DNA adducts were detected in 24 out of 27 samples with quantifiable DNA yield, averaging 44.7 pmol HPB/mg DNA. The levels of adducts in buccal brushings correlated with those in mouthwash samples of smokers (R = 0.73, p < 0.0001). Potentially the method can be applied in studies of individual susceptibility to tobacco-induced cancers in humans. PMID:23252610

Quantification of phase I / II metabolizing enzyme gene expression and polycyclic aromatic hydrocarbon-DNA adduct levels in human prostate

PubMed Central

John, Kaarthik; Ragavan, Narasimhan; Pratt, M. Margaret; Singh, Paras B.; Al-Buheissi, Salah; Matanhelia, Shyam S.; Phillips, David H.; Poirier, Miriam C.; Martin, Francis L.

2008-01-01

BACKGROUND Studies of migrant populations suggest that dietary and/or environmental factors play a crucial role in the aetiology of prostatic adenocarcinoma (CaP). The human prostate consists of the peripheral zone (PZ), transition zone (TZ) and central zone (CZ); CaP occurs most often in the PZ. METHODS To investigate the notion that an underlying differential expression of phase I/II genes, and/or the presence of polycyclic aromatic hydrocarbon (PAH)-DNA adducts might explain the elevated PZ susceptibility, we examined prostate tissues (matched tissue sets consisting of PZ and TZ) from men undergoing radical retropubic prostatectomy for CaP (n=26) or cystoprostatectomy (n=1). Quantitative gene expression analysis was employed for cytochrome P450 (CYP) isoforms CYP1A1, CYP1B1 and CYP1A2, as well as N-acetyltransferase 1 and 2 (NAT1 and NAT2) and catechol-O-methyl transferase (COMT). RESULTS CYP1B1, NAT1 and COMT were expressed in all tissue sets; levels of CYP1B1 and NAT1 were consistently higher in the PZ compared to TZ. Immunohistochemistry confirmed the presence of CYP1B1 (nuclear-associated and primarily in basal epithelial cells) and NAT1. Tissue sections from 23 of these aforementioned 27 matched tissue sets were analyzed for PAH-DNA adduct levels using antiserum elicited against DNA modified with r7, t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydro-benzo[a]pyrene (BPDE). PAH-DNA adduct levels were highest in glandular epithelial cells, but a comparison of PZ and TZ showed no significant differences. CONCLUSION Although expression of activating and/or detoxifying enzymes may be higher in the PZ, PAH-DNA adduct levels appear to be similar in both zones. Therefore, factors other than PAH-DNA adducts may be responsible for promotion of tumour formation in the human prostate. PMID:19143007

Condensed tannin-resorcinol adducts in laminating adhesives

Treesearch

Richard W. Hemingway; Roland E. Kreibich

1985-01-01

A condensed tannin-resorcinol adduct made by co-reaction of an extract from southern pine bark with resorcinol at a 2 to 1 weight ratio was used to prepare a laminating resin in which the entire amount of resorcinol normally used was replaced by this adduct. The resin was formulated into a room temperature setting adhesive that meets the basic criteria of product...

Nuclear magnetic resonance at the picomole level of a DNA adduct.

PubMed

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

2013-10-21

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 picomole 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 severalfold advantage over conventional flow injection. Eliminating dilution during injection and confining the sample to the observed volume produce the full theoretical mass sensitivity of a microcoil, comparable to that of a microcryo probe. With 80 ng, an NMR spectrum acquired over 40 h showed all of the resonances seen in a standard spectrum of AAF-dGMP, with a signal-to-noise ratio of at least 10, despite broadening due to previously noted effects of conformational exchange. Even with this broadening to 5 Hz, a two-dimensional total correlation spectroscopy spectrum was acquired on 1.6 μg in 18 h. 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.

Mutation and repair induced by the carcinogen 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) in the dihydrofolate reductase gene of Chinese hamster ovary cells and conformational modeling of the dG-C8-PhIP adduct in DNA.

PubMed

Carothers, A M; Yuan, W; Hingerty, B E; Broyde, S; Grunberger, D; Snyderwine, E G

1994-01-01

Three experiments using 20 microM 2-(hydroxyamino)-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP) were performed to induce mutations in the dihydrofolate reductase (DHFR) gene of a hemizygous Chinese hamster ovary (CHO) cell line (UA21). Metabolized forms of this chemical primarily bind at the C-8 position of guanine in DNA. In total, 21 independent induced mutants were isolated and 20 were characterized. DNA sequencing showed that the preferred mutation type found in 75% of the induced DHFR- clones was G.C-->T.A single and tandem double transversions. In addition to base substitutions, one mutant carried a-1 frameshift and another one had lost the entire locus by deletion. The induced changes affected purine targets on the nontranscribed strand of the gene in nearly all of the mutants sequenced (18/19). At the time that the first two experiments were performed, the initial adduct levels were quantitated in treated cells at the mutagenic dose by 32P-postlabeling. While the induced frequency of mutation was relatively low (approximately 5 x 10(-6), the adduct levels after a 1-h exposure of UA21 cells to 20 microM N-OH-PhIP were relatively high (13 adducts x 10(-6) nucleotides). This latter method was then employed to learn if the induced mutation frequency correlated with rapid overall genome repair of PhIP-DNA adducts. Total adduct levels, determined using DNA samples from treated cells collected after intervals of time, were reduced by about 50% after 6 h, and about 70% after 24 h. Since overall genome repair in CHO cells is relatively slow compared with preferential gene repair, the removal of dG-C8-PhIP adducts was apparently efficient. In order to better understand the mutational and repair results, we performed computational modeling to determine the lowest energy structure for the major dG-C8-PhIP adduct in a repetitively mutated duplex sequence opposite dA. Results of this analysis indicate that the PhIP-modified base resembles previous structural

Use of LC-MS/MS and Stable Isotopes to Differentiate Hydroxymethyl and Methyl DNA Adducts from Formaldehyde and Nitrosodimethylamine

PubMed Central

Lu, Kun; Craft, Sessaly; Nakamura, Jun; Moeller, Benjamin C.; Swenberg, James A.

2012-01-01

Formaldehyde is a known human and animal carcinogen that forms DNA adducts, and causes mutations. While there is widespread exposure to formaldehyde in the environment, formaldehyde is also an essential biochemical in all living cells. The presence of both endogenous and exogenous sources of formaldehyde makes it difficult to develop exposure-specific DNA biomarkers. Furthermore, chemicals such as nitrosodimethylamine form one mole of formaldehyde for every mole of methylating agent, raising questions about potential co-carcinogenesis. Formaldehyde-induced hydroxymethyl DNA adducts are not stable and need to be reduced to stable methyl adducts for detection, which adds another layer of complexity to identifying the origins of these adducts. In this study, highly sensitive mass spectrometry methods and isotope labeled compounds were used to differentiate between endogenous and exogenous hydroxymethyl and methyl DNA adducts. We demonstrate that N2-hydroxymethyl-dG is the primary DNA adduct formed in cells following formaldehyde exposure. In addition, we show that alkylating agents induce methyl adducts at N2-dG and N6-dA positions, which are identical to the reduced forms of hydroxymethyl adducts arising from formaldehyde. The use of highly sensitive LC-MS/MS and isotope labeled compounds for exposure solves these challenges and provides mechanistic insights on the formation and role of these DNA adducts. PMID:22148432

Novel LC-ESI/MS/MSn Method for the Characterization and Quantification of 2′-Deoxyguanosine Adducts of the Dietary Carcinogen 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine by 2-D Linear Quadrupole Ion Trap Mass Spectrometry

PubMed Central

Goodenough, Angela K.; Schut, Herman A. J.; Turesky, Robert J.

2008-01-01

An accurate and sensitive liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MSn) technique has been developed for the characterization and quantification of 2′-deoxyguanosine (dG) adducts of the dietary mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). PhIP is an animal and potential human carcinogen that occurs in grilled meats. Following enzymatic digestion and adduct enrichment by solid-phase extraction (SPE), PhIP—DNA adducts were analyzed by MS/MS and MSn scan modes on a 2-D linear quadrupole ion trap mass spectrometer (QIT/MS). The major DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-PhIP), was detected in calf thymus (CT) DNA modified in vitro with a bioactivated form of PhIP and in the colon and liver of rats given PhIP as part of the diet. The lower limit of detection (LOD) was 1 adduct per 108 DNA bases, and the limit of quantification (LOQ) was 3 adducts per 108 DNA bases in both MS/MS and MS3 scan modes, using 27 μg of DNA for analysis. Measurements were based on isotope dilution with the internal standard, N-(deoxyguanosin-8-yl)-2-amino-1-(trideutero)methyl-6-phenylimidazo[4,5-b]pyridine (dG-C8-[2H3C]-PhIP). The selected reaction monitoring (SRM) scan mode in MS/MS was employed to monitor the loss of deoxyribose (dR) from the protonated molecules of the adducts ([M + H - 116]+). The consecutive reaction monitoring (CRM) scan modes in MS3 and MS4 were used to measure and further characterize product ions of the aglycone ion (BH2+) (Guanyl-PhIP). The MS3 scan mode was effective in eliminating isobaric interferences observed in the MS/MS scan mode and resulted in an improved signal-to-noise (S/N) ratio. Moreover, the product ion spectra obtained by the MSn scan modes provided rich structural information about the adduct and were used to corroborate the identity of dG-C8-PhIP. In addition, an isomeric dG-PhIP adduct was detected in vivo. This LCESI

Impact of genetic modulation of SULT1A enzymes on DNA adduct formation by aristolochic acids and 3-nitrobenzanthrone.

PubMed

Arlt, Volker M; Meinl, Walter; Florian, Simone; Nagy, Eszter; Barta, Frantisek; Thomann, Marlies; Mrizova, Iveta; Krais, Annette M; Liu, Maggie; Richards, Meirion; Mirza, Amin; Kopka, Klaus; Phillips, David H; Glatt, Hansruedi; Stiborova, Marie; Schmeiser, Heinz H

2017-04-01

Exposure to aristolochic acid (AA) causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN). Conflicting results have been found for the role of human sulfotransferase 1A1 (SULT1A1) contributing to the metabolic activation of aristolochic acid I (AAI) in vitro. We evaluated the role of human SULT1A1 in AA bioactivation in vivo after treatment of transgenic mice carrying a functional human SULT1A1-SULT1A2 gene cluster (i.e. hSULT1A1/2 mice) and Sult1a1(-/-) mice with AAI and aristolochic acid II (AAII). Both compounds formed characteristic DNA adducts in the intact mouse and in cytosolic incubations in vitro. However, we did not find differences in AAI-/AAII-DNA adduct levels between hSULT1A1/2 and wild-type (WT) mice in all tissues analysed including kidney and liver despite strong enhancement of sulfotransferase activity in both kidney and liver of hSULT1A1/2 mice relative to WT, kidney and liver being major organs involved in AA metabolism. In contrast, DNA adduct formation was strongly increased in hSULT1A1/2 mice compared to WT after treatment with 3-nitrobenzanthrone (3-NBA), another carcinogenic aromatic nitro compound where human SULT1A1/2 is known to contribute to genotoxicity. We found no differences in AAI-/AAII-DNA adduct formation in Sult1a1(-/-) and WT mice in vivo. Using renal and hepatic cytosolic fractions of hSULT1A1/2, Sult1a1(-/-) and WT mice, we investigated AAI-DNA adduct formation in vitro but failed to find a contribution of human SULT1A1/2 or murine Sult1a1 to AAI bioactivation. Our results indicate that sulfo-conjugation catalysed by human SULT1A1 does not play a role in the activation pathways of AAI and AAII in vivo, but is important in 3-NBA bioactivation.

Paclitaxel Enhances Carboplatin-DNA Adduct Formation and Cytotoxicity

DOE PAGES

Jiang, Shuai; Pan, Amy W.; Lin, Tzu-yin; ...

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 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). In conclusion, this rapid reportmore » provides the first pharmacodynamics data to support the use of carboplatin/paclitaxel combination in the clinic.« less

Mass spectrometric analysis of sulfur mustard-induced biomolecular adducts: Are DNA adducts suitable biomarkers of exposure?

PubMed

Zubel, Tabea; Bürkle, Alexander; Mangerich, Aswin

2018-09-01

The bi-functional chemical warfare agent sulfur mustard (SM), whose release in asymmetric conflicts or terrorist attacks represents a realistic threat, induces several kinds of biomolecular adducts, including highly toxic DNA adducts. Isotope dilution liquid chromatographic tandem mass spectrometry (ID-LC-MS/MS) is considered the gold standard for highly accurate, precise, specific and sensitive quantification of DNA adducts in general. Recently, a number of LC-MS/MS approaches have been established to analyze SM-induced protein and DNA adducts in cell culture and rodent animal models. As DNA adducts are mechanism-based biomarkers for SM exposure, results from such studies provide a deeper understanding of the etiology of SM-induced pathologies, especially of long-term effects such as cancer formation. As a result, medical treatment of SM-exposed individuals might be improved. Yet, despite the progress that has been made during the last years, there is still a need for advanced methods of ID-LC-MS/MS for the detection and quantitation of SM adducts. Copyright © 2017 Elsevier B.V. All rights reserved.

Inert Reassessment Document for Poly(oxyethylene) adducts of mixed phytosterols

EPA Pesticide Factsheets

Poly(oxyethy1ene) adducts of mixed phytosterols is uncategorized as to list classification status. Based upon the reasonable certainty of no harm safety finding, the List 4B classification for poly(oxyethy1ene) adducts of mixed phytosterols is affirmed.

Solution structure of a DNA duplex containing a cis-diammineplatinum(II) 1,3-d(GTG) intrastrand cross-link, a major adduct in cells treated with the anticancer drug carboplatin.

PubMed

Teuben, J M; Bauer, C; Wang, A H; Reedijk, J

1999-09-21

The platinum 1,3-d(GXG) intrastrand cross-link is one of the adducts formed in the reaction of the antitumor drug cisplatin with DNA, and in fact the major adduct found in cells treated with the cisplatin analogue carboplatin. To determine the 3D structure of this adduct, the duplex d(CTCTGTGTCTC).d(GAGACACAGAG)], where GTG denotes a platinum 1,3-intrastrand cross-link, was prepared and studied with high-resolution (1)H NMR. The solution structure was determined using the SPEDREF protocol, which includes an iterative NOE-restrained refinement procedure. Calculated and recorded NOE spectra were found to be in good agreement (NMR R factor 22%). The studied duplex is more distorted from B-DNA than previously determined structures of the 1,2-d(GG) intrastrand adducts. The base pairing is lost for the 5'G-C and the central T-A base pair in the GTG lesion, and the central thymine is extruded from the minor groove. To accommodate this lesion, the minor groove is widened, and the 5'-guanine ribose adopts an N-type conformation. The helix is unwound locally and is significantly bent toward the major groove. Significant difference between the structural distortion of the 1, 3-d(GTG) cross-link and other Pt-DNA cross-links sheds new light on the observed differences in protein recognition of these lesions, and thus on the possible differences in mechanisms of action of the various Pt-DNA adducts formed in treatment with platinum anticancer complexes.

Mutagenic Spectra Arising from Replication Bypass of the 2,6-diamino-4-hydroxy-N5-methyl Formamidopyrimidine Adduct in Primate Cells

PubMed Central

Earley, Lauriel F.; Minko, Irina G.; Christov, Plamen P.; Rizzo, Carmelo J.; Lloyd, R. Stephen

2013-01-01

DNA exposures to electrophilic methylating agents that are commonly used during chemotherapeutic treatments cause diverse chemical modifications of nucleobases, with reaction at N7-dG being the most abundant. Although this base modification frequently results in destabilization of the glycosyl bond and spontaneous depurination, the adduct can react with hydroxide ion to yield a stable, ring-opened MeFapy-dG and this lesion has been reported to persist in animal tissues. Results from prior in vitro replication bypass investigations of the MeFapy-dG adduct had revealed complex spectra of replication errors that differed depending on the identity of DNA polymerase and the local sequence context. In this study, a series of nine site-specifically modified MeFapy-dG-containing oligodeoxynucleotides were engineered into a shuttle vector and subjected to replication in primate cells. In all nine sequence contexts examined, MeFapy-dG was shown to be associated with a strong mutator phenotype, predominantly causing base substitutions, with G to T transversions being most common. Single and dinucleotide deletions were also found in a subset of the sequence contexts. Interestingly, single-nucleotide deletions occurred not only at the adducted site, but also one nucleotide downstream of the adduct. Standard models for primer-template misalignment could account for some, but not all mutations observed. These data demonstrate that in addition to mutagenesis predicted from replication of DNAs containing O6-Me-dG and O4-Me-dT, the MeFapy-dG adduct likely contributes to mutagenic events following chemotherapeutic treatments. PMID:23763662

STABILITY OF HEMOGLOBIN AND ALBUMIN ADDUCTS OF BENZENE OXIDE AND 1,4-BENZOQUINONE AFTER ADMINISTRATION OF BENZENE TO F344 RATS

EPA Science Inventory

The stability of cysteinyl adducts of benzene oxide (BO) and mono-S-substituted cysteinyl adducts of 1,4-benzoquinone (1,4-BQ) was investigated in both hemoglobin (Hb) and albumin (Alb) following administration of a single oral dose of 400 mg [U-14C/13C6]benzene/kg body weight ...

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

Red Wine Consumption is inversely associated with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-DNA Adduct Levels in Prostate

PubMed Central

Rybicki, Benjamin A.; Neslund-Dudas, Christine; Bock, Cathryn H.; Nock, Nora L.; Rundle, Andrew; Jankowski, Michelle; Levin, Albert M.; Beebe-Dimmer, Jennifer; Savera, Adnan T.; Takahashi, Satoru; Shirai, Tomoyuki; Tang, Deliang

2011-01-01

In humans, genetic variation and dietary factors may alter the biologic effects of exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), one of the major heterocyclic amines generated from cooking meats at high temperatures that has carcinogenic potential through the formation of DNA adducts. Previously, we reported grilled red meat consumption associated with PhIP-DNA adduct levels in human prostate. In the present study, we expanded our investigation to estimate the associations between beverage consumption and PhIP-DNA adduct levels in prostate for 391 prostate cancer cases. Of the 15 beverages analyzed, red wine consumption had the strongest association with PhIP-DNA adduct levels showing an inverse correlation in both tumor (p=0.006) and non-tumor (p=0.002) prostate cells. Red wine consumption differed significantly between African-American and white cases, but PhIP-DNA adduct levels in prostate did not vary by race. In African Americans compared with whites, however, associations between red wine consumption and PhIP-DNA adduct levels were not as strong as associations with specific (e.g., SULT1A1 and UGT1A10 genotypes) and non-specific (e.g., African ancestry) genetic variation. In a multivariable model, the covariate for red wine consumption explained a comparable percentage (13-16%) of the variation in PhIP-DNA adduct levels in prostate across the two racial groups, but the aforementioned genetic factors explained 33% of the PhIP-DNA adduct variation in African-American cases, while only 19% of the PhIPDNA adduct variation in whites. We conclude that red wine consumption may counteract biologic effects of PhIP exposure in human prostate, but genetic factors may play an even larger role, particularly in African Americans. PMID:21846795

Reaction of glyoxal with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, cytidine, thymidine, and calf thymus DNA: identification of DNA adducts.

PubMed

Olsen, Raymond; Molander, Paal; Øvrebø, Steinar; Ellingsen, Dag G; Thorud, Syvert; Thomassen, Yngvar; Lundanes, Elsa; Greibrokk, Tyge; Backman, Josefin; Sjöholm, Rainer; Kronberg, Leif

2005-04-01

Glyoxal (ethanedial) is an increasingly used industrial chemical that has been found to be mutagenic in bacteria and mammalian cells. In this study, the reactions of glyoxal with 2'-deoxyguanosine, 2'-deoxyadenosine, 2'-deoxycytidine, cytidine, thymidine, and calf thymus DNA have been studied in aqueous buffered solutions. The nucleoside adducts were isolated by reversed-phase liquid chromatography and characterized by their UV absorbance and 1H and 13C NMR spectroscopic and mass spectrometric features. The reaction with 2'-deoxyguanosine gave one adduct, the previously known 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one adduct. The reaction of 2'-deoxyadenosine with glyoxal resulted in the formation of a previously not reported N6-(hydroxyacetyl)-2'-deoxyadenosine adduct. In the reaction of glyoxal with 2'-deoxycytidine and cytidine at neutral conditions and 37 degrees C, 5-hydroxyacetyl pyrimidine derivatives were obtained. When the cytidine reaction was performed at pH 4.5 and 50 degrees C, the 5-hydroxyacetyl derivative of uridine was formed through deamination of cytidine-glyoxal. Adducts in the thymidine reaction could not be detected. In the reaction of glyoxal with calf thymus DNA, the 2'-deoxyguanosine-glyoxal and 2'-deoxyadenosine-glyoxal adducts were obtained, the former being the major adduct.

Characterization of glycidol-hemoglobin adducts as biomarkers of exposure and in vivo dose

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

Honda, Hiroshi, E-mail: honda.hiroshi@kao.co.jp; Törnqvist, Margareta; Nishiyama, Naohiro

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 differentmore » 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 di

Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients.

PubMed

Zayas, Beatriz; Stillwell, Sara W; Wishnok, John S; Trudel, Laura J; Skipper, Paul; Yu, Mimi C; Tannenbaum, Steven R; Wogan, Gerald N

2007-02-01

We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10(9) bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW=443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444-->328. The method was validated by analysis of DNA (100 microg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 microg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10(9) bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.

Chemistry and Chemical Equilibrium Dynamics of BMAA and Its Carbamate Adducts

PubMed Central

Diaz-parga, Pedro

2018-01-01

Beta-N-methylamino-L-alanine (BMAA) has been demonstrated to contribute to the onset of the ALS/Parkinsonism-dementia complex (ALS/PDC) and is implicated in the progression of other neurodegenerative diseases. While the role of BMAA in these diseases is still debated, one of the suggested mechanisms involves the activation of excitatory glutamate receptors. In particular, the excitatory effects of BMAA are shown to be dependent on the presence of bicarbonate ions, which in turn forms carbamate adducts in physiological conditions. The formation of carbamate adducts from BMAA and bicarbonate is similar to the formation of carbamate adducts from non-proteinogenic amino acids. Structural, chemical, and biological information related to non-proteinogenic amino acids provide insight into the formation of and possible neurological action of BMAA. This article reviews the carbamate formation of BMAA in the presence of bicarbonate ions, with a particular focus on how the chemical equilibrium of BMAA carbamate adducts may affect the molecular mechanism of its function. Highlights of nuclear magnetic resonance (NMR)-based studies on the equilibrium process between free BMAA and its adducts are presented. The role of divalent metals on the equilibrium process is also explored. The formation and the equilibrium process of carbamate adducts of BMAA may answer questions on their neuroactive potency and provide strong motivation for further investigations into other toxic mechanisms. PMID:28921378

Chemistry and Chemical Equilibrium Dynamics of BMAA and Its Carbamate Adducts.

PubMed

Diaz-Parga, Pedro; Goto, Joy J; Krishnan, V V

2018-01-01

Beta-N-methylamino-L-alanine (BMAA) has been demonstrated to contribute to the onset of the ALS/Parkinsonism-dementia complex (ALS/PDC) and is implicated in the progression of other neurodegenerative diseases. While the role of BMAA in these diseases is still debated, one of the suggested mechanisms involves the activation of excitatory glutamate receptors. In particular, the excitatory effects of BMAA are shown to be dependent on the presence of bicarbonate ions, which in turn forms carbamate adducts in physiological conditions. The formation of carbamate adducts from BMAA and bicarbonate is similar to the formation of carbamate adducts from non-proteinogenic amino acids. Structural, chemical, and biological information related to non-proteinogenic amino acids provide insight into the formation of and possible neurological action of BMAA. This article reviews the carbamate formation of BMAA in the presence of bicarbonate ions, with a particular focus on how the chemical equilibrium of BMAA carbamate adducts may affect the molecular mechanism of its function. Highlights of nuclear magnetic resonance (NMR)-based studies on the equilibrium process between free BMAA and its adducts are presented. The role of divalent metals on the equilibrium process is also explored. The formation and the equilibrium process of carbamate adducts of BMAA may answer questions on their neuroactive potency and provide strong motivation for further investigations into other toxic mechanisms.

Nuclear magnetic resonance solution structure of an N(2)-guanine DNA adduct derived from the potent tumorigen dibenzo[a,l]pyrene: intercalation from the minor groove with ruptured Watson-Crick base pairing.

PubMed

Tang, Yijin; Liu, Zhi; Ding, Shuang; Lin, Chin H; Cai, Yuqin; Rodriguez, Fabian A; Sayer, Jane M; Jerina, Donald M; Amin, Shantu; Broyde, Suse; Geacintov, Nicholas E

2012-12-04

The most potent tumorigen identified among the polycyclic aromatic hydrocarbons (PAH) is the nonplanar fjord region dibenzo[a,l]pyrene (DB[a,l]P). It is metabolically activated in vivo through the widely studied diol epoxide (DE) pathway to form covalent adducts with DNA bases, predominantly guanine and adenine. The (+)-11S,12R,13R,14S DE enantiomer forms adducts via its C14 position with the exocyclic amino group of guanine. Here, we present the first nuclear magnetic resonance solution structure of a DB[a,l]P-derived adduct, the 14R-(+)-trans-anti-DB[a,l]P-N(2)-dG (DB[a,l]P-dG) lesion in double-stranded DNA. In contrast to the stereochemically identical benzo[a]pyrene-derived N(2)-dG adduct (B[a]P-dG) in which the B[a]P rings reside in the B-DNA minor groove on the 3'-side of the modifed deoxyguanosine, in the DB[a,l]P-derived adduct the DB[a,l]P rings intercalate into the duplex on the 3'-side of the modified base from the sterically crowded minor groove. Watson-Crick base pairing of the modified guanine with the partner cytosine is broken, but these bases retain some stacking with the bulky DB[a,l]P ring system. This new theme in PAH DE-DNA adduct conformation differs from (1) the classical intercalation motif in which Watson-Crick base pairing is intact at the lesion site and (2) the base-displaced intercalation motif in which the damaged base and its partner are extruded from the helix. The structural considerations that lead to the intercalated conformation of the DB[a,l]P-dG lesion in contrast to the minor groove alignment of the B[a]P-dG adduct, and the implications of the DB[a,l]P-dG conformational motif for the recognition of such DNA lesions by the human nucleotide excision repair apparatus, are discussed.

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

Formation of DNA adducts from oil-derived products analyzed by 32P-HPLC.

PubMed

Akkineni, L K; Zeisig, M; Baranczewski, P; Ekström, L G; Möller, L

2001-01-01

substances tested were, in increasing order, 9,10-dimethylanthracene, 7,12-dimethylbenz[a]anthracene and 9-vinylanthracene. Metabolic activation with human and rat liver extracts gave rise to the same DNA adduct clusters. When bioactivation with material from different human individuals was used, there was a significant correlation between the CYP 1A1 activity and the capacity to form DNA adducts. This pattern was also confirmed using the CYP 1A1 inhibitor ellipticine. The 32P-HPLC method was shown to be sensitive and reproducible, and it had the capacity to separate DNA adduct-forming substances when applied to a great variety of petroleum products.

Environmental, Dietary, Maternal, and Fetal Predictors of Bulky DNA Adducts in Cord Blood: A European Mother–Child Study (NewGeneris)

PubMed Central

Mendez, Michelle A.; Schoket, Bernadette; Godschalk, Roger W.; Espinosa, Ana; Landström, Anette; Villanueva, Cristina M.; Merlo, Domenico F.; Fthenou, Eleni; Gracia-Lavedan, Esther; van Schooten, Frederik-J.; Hoek, Gerard; Brunborg, Gunnar; Meltzer, Helle M.; Alexander, Jan; Nielsen, Jeanette K.; Sunyer, Jordi; Wright, John; Kovács, Katalin; de Hoogh, Kees; Gutzkow, Kristine B.; Hardie, Laura J.; Chatzi, Leda; Knudsen, Lisbeth E.; Anna, Lívia; Ketzel, Matthias; Haugen, Margaretha; Botsivali, Maria; Nieuwenhuijsen, Mark J.; Cirach, Marta; Toledano, Mireille B.; Smith, Rachel B.; Fleming, Sarah; Agramunt, Silvia; Kyrtopoulos, Soterios A.; Lukács, Viktória; Kleinjans, Jos C.; Segerbäck, Dan; Kogevinas, Manolis

2015-01-01

Background: Bulky DNA adducts reflect genotoxic exposures, have been associated with lower birth weight, and may predict cancer risk. Objective: We selected factors known or hypothesized to affect in utero adduct formation and repair and examined their associations with adduct levels in neonates. Methods: Pregnant women from Greece, Spain, England, Denmark, and Norway were recruited in 2006–2010. Cord blood bulky DNA adduct levels were measured by the 32P-postlabeling technique (n = 511). Diet and maternal characteristics were assessed via questionnaires. Modeled exposures to air pollutants and drinking-water disinfection by-products, mainly trihalomethanes (THMs), were available for a large proportion of the study population. Results: Greek and Spanish neonates had higher adduct levels than the northern European neonates [median, 12.1 (n = 179) vs. 6.8 (n = 332) adducts per 108 nucleotides, p < 0.001]. Residence in southern European countries, higher maternal body mass index, delivery by cesarean section, male infant sex, low maternal intake of fruits rich in vitamin C, high intake of dairy products, and low adherence to healthy diet score were statistically significantly associated with higher adduct levels in adjusted models. Exposure to fine particulate matter and nitrogen dioxide was associated with significantly higher adducts in the Danish subsample only. Overall, the pooled results for THMs in water show no evidence of association with adduct levels; however, there are country-specific differences in results with a suggestion of an association in England. Conclusion: These findings suggest that a combination of factors, including unknown country-specific factors, influence the bulky DNA adduct levels in neonates. Citation: Pedersen M, Mendez MA, Schoket B, Godschalk RW, Espinosa A, Landström A, Villanueva CM, Merlo DF, Fthenou E, Gracia-Lavedan E, van Schooten FJ, Hoek G, Brunborg G, Meltzer HM, Alexander J, Nielsen JK, Sunyer J, Wright J, Kovács K, de

Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of bis-(2-Chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

PubMed Central

Gruppi, Francesca; Hejazi, Leila; Christov, Plamen P.; Krishnamachari, Sesha; Turesky, Robert J.; Rizzo, Carmelo J.

2015-01-01

A robust, quantitative ultraperformance liquid chromatography ion trap multistage scanning mass spectrometric (UPLC/MS3) method was established to characterize and measure five deoxyguanosine (dG) adducts formed by reaction of the chemotherapeutic nitrogen mustard (NM) bis-(2-chloroethyl)ethylamine with calf thymus (CT) DNA. In addition to the known N7-guanine (NM-G) adduct and its crosslink (G-NM-G), the ring-opened formamidopyrimidine (FapyG) mono-adduct (NM-FapyG) and cross-links in which one (FapyG-NM-G) or both (FapyG-NM-FapyG) guanines underwent ring-opening to FapyG units were identified. Authentic standards of all adducts were synthesized and characterized by NMR and mass spectrometry. These adducts were quantified in CT DNA treated with NM (1 μM) as their deglycosylated bases. A two-stage neutral thermal hydrolysis was developed to mitigate the artifactual formation of ring-opened FapyG adducts involving hydrolysis of the cationic adduct at 37 °C, followed by hydrolysis of the FapyG adducts at 95 °C. The limit of quantification values ranged between 0.3 and 1.6 adducts per 107 DNA bases, when the equivalent of 5 μg DNA hydrolysate was assayed on column. The principal adduct formed was the G-NM-G cross-link, followed by the NM-G mono-adduct; the FapyG-NM-FapyG adduct was at the limit of detection. The NM-FapyG adducts formed in CT DNA at a level of ~20% that of the NM-G adduct. NM-FapyG has not been previously quanitified and the FapyG-NM-G and FapyG-NM-FapyG adducts have not be previously characterized. Our validated analytical method was then applied to measure DNA adduct formation in the MDA-MB-231 mammary tumor cell line exposed to NM (100 μM) for 24 h. The major adduct formed was NM-G (970 adducts per 107 bases), followed by G-NM-G (240 adducts per 107 bases) and NM-FapyG (180 adducts per 107 bases), and lastly the FapyG-NM-G cross-link adduct (6.0 adducts per 107 bases). These lesions are expected to contribute to the NM-mediated toxicity and

Malondialdehyde-acetaldehyde (MAA) adducted surfactant protein induced lung inflammation is mediated through scavenger receptor a (SR-A1).

PubMed

Sapkota, Muna; DeVasure, Jane M; Kharbanda, Kusum K; Wyatt, Todd A

2017-02-13

Co-exposure to cigarette smoke and alcohol leads to the generation of high concentrations of acetaldehyde and malondialdehyde in the lung. These aldehydes being highly electrophilic in nature react with biologically relevant proteins such as surfactant protein D (SPD) through a Schiff base reaction to generate SPD adducted malondialdehyde-acetaldehyde adduct (SPD-MAA) in mouse lung. SPD-MAA results in an increase in lung pro-inflammatory chemokine, keratinocyte chemoattractant (KC), and the recruitment of lung lavage neutrophils. Previous in vitro studies in bronchial epithelial cells and macrophages show that scavenger receptor A (SR-A1/CD204) is a major receptor for SPD-MAA. No studies have yet examined the in vivo role of SR-A1 in MAA-mediated lung inflammation. Therefore, we hypothesize that in the absence of SR-A1, MAA-induced inflammation in the lung is reduced or diminished. To test this hypothesis, C57BL/6 WT and SR-A1 KO mice were nasally instilled with 50 μg/mL of SPD-MAA for 3 weeks (wks). After 3 weeks, bronchoalveolar lavage (BAL) fluid was collected and assayed for a total cell count, a differential cell count and CXCL1 (KC) chemokine. Lung tissue sections were stained with hematoxylin and eosin (H&E) and antibodies to MAA adduct. Results showed that BAL cellularity and influx of neutrophils were decreased in SR-A1 KO mice as compared to WT following repetitive SPD-MAA exposure. MAA adduct staining in the lung epithelium was decreased in SR-A1 KO mice. In comparison to WT, no increase in CXCL1 was observed in BAL fluid from SR-A1 KO mice over time. Overall, the data demonstrate that SR-A1/CD204 plays an important role in SPD-MAA induced inflammation in lung.

Methemoglobin Formation and Characterization of Hemoglobin Adducts of Carcinogenic Aromatic Amines and Heterocyclic Aromatic Amines.

PubMed

Pathak, Khyatiben V; Chiu, Ting-Lan; Amin, Elizabeth Ambrose; Turesky, Robert J

2016-03-21

Arylamines (AAs) and heterocyclic aromatic amines (HAAs) are structurally related carcinogens formed during the combustion of tobacco or cooking of meat. They undergo cytochrome P450 mediated N-hydroxylation to form metabolites which bind to DNA and lead to mutations. The N-hydroxylated metabolites of many AAs also can undergo a co-oxidation reaction with oxy-hemolgobin (HbO2) to form methemoglobin (met-Hb) and the arylnitroso intermediates, which react with the β-Cys(93) chain of Hb to form Hb-arylsulfinamide adducts. The biochemistry of arylamine metabolism has been exploited to biomonitor certain AAs through their Hb arylsulfinamide adducts in humans. We examined the reactivity of HbO2 with the N-hydroxylated metabolites of 4-aminobiphenyl (ABP, HONH-ABP), aniline (ANL, HONH-ANL), and the HAAs 2-amino-9H-pyrido[2,3-b]indole (AαC, HONH-AαC), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, HONH-PhIP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx, HONH-MeIQx). HONH-ABP, HO-ANL, and HONH-AαC induced methemoglobinemia and formed Hb sulfinamide adducts. However, HONH-MeIQx and HONH-PhIP did not react with the oxy-heme complex, and met-Hb formation and chemical modification of the β-Cys(93) residue were negligible. Molecular modeling studies showed that the distances between the H-ON-AA or H-ON-HAA substrates and the oxy-heme complex of HbO2 were too far away to induce methemoglobinemia. Different conformational changes in flexible helical and loop regions around the heme pocket induced by the H-ON-AA or H-ON-HAAs may explain the different proclivities of these chemicals to induce methemoglobinemia. Hb-Cys(93β) sulfinamide and sulfonamide adducts of ABP, ANL, and AαC were identified, by Orbitrap MS, following the proteolysis of Hb with trypsin, Glu-C, or Lys-C. Hb sulfinamide and sulfonamide adducts of ABP were identified in the blood of mice exposed to ABP, by Orbitrap MS. This is the first report of the identification of intact Hb

DNA adduct profiling of in vitro colonic meat digests to map red vs. white meat genotoxicity.

PubMed

Hemeryck, Lieselot Y; Rombouts, Caroline; De Paepe, Ellen; Vanhaecke, Lynn

2018-05-01

The consumption of red meat has been linked to an increased colorectal cancer (CRC) risk. One of the major hypotheses states that heme iron (present in red meat) stimulates the formation of genotoxic N-nitroso compounds (NOCs) and lipid peroxidation products (LPOs). By means of DNA adductomics, chemically induced DNA adduct formation can be mapped in relation to e.g. dietary exposures. In this study, this state-of-the-art methodology was used to investigate alkylation and (lipid per)oxidation induced DNA adduct formation in in vitro red vs. white meat digests. In doing so, 90 alkylation and (lipid per)oxidation induced DNA adduct types could be (tentatively) identified. Overall, 12 NOC- and/or LPO-related DNA adduct types, i.e. dimethyl-T (or ethyl-T), hydroxymethyl-T, tetramethyl-T, methylguanine (MeG), guanidinohydantoin, hydroxybutyl-C, hydroxymethylhydantoin, malondialdehyde-x3-C, O 6 -carboxymethylguanine, hydroxyethyl-T, carboxyethyl-T and 3,N 4 -etheno-C were singled out as potential heme-rich meat digestion markers. The retrieval of these DNA adduct markers is in support of the heme, NOC and LPO hypotheses, suggesting that DNA adduct formation may indeed contribute to red meat related CRC risk. Copyright © 2018 Elsevier Ltd. All rights reserved.

New diorganotin(IV) derivatives of 7-hydroxycoumarin (umbelliferone) and their adducts with 1,10-phenanthroline

NASA Astrophysics Data System (ADS)

Nath, Mala; Jairath, Ruchi; Eng, George; Song, Xueqing; Kumar, Ashok

2005-10-01

New diorganotin(IV) derivatives of the general formula R 2Sn(Umb) 2 (where R = n-Bu, n-Oct and Ph; Umb = umbelliferone anion) have been synthesized either by the reaction of R 2SnO with umbelliferone under azeotropic removel of water or by the reaction of R 2SnCl 2 with sodium salt of umbelliferone. Further, the adducts of the general formula R 2Sn(Umb) 2·phen (where R = n-Bu and n-Oct; phen = 1,10-phenanthroline) have also been synthesized by the interaction of R 2Sn(Umb) 2 with 1,10-phenanthroline. The bonding and coordination behavior in these derivatives are discussed on the basis of IR and 119Sn Mössbauer spectroscopic studies in solid state. Their coordination behavior in solution is discussed by the multinuclear ( 1H, 13C and 119Sn) NMR spectral studies. The Mössbauer and IR studies indicate that umbelliferone acts as a monoanionic bidentate ligand in R 2Sn(Umb) 2 coordinating through O(7) and O(1). A distorted octahedral geometry around tin has been proposed for R 2Sn(Umb) 2 as well as for R 2Sn(Umb) 2·phen in solid state. The newly synthesized derivatives have been tested for their anti-inflammatory and cardiovascular activities. The average LD 50 value >1000 mg kg -1 of these compounds indicates their safety margin.

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

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

Endo, Kaori; Uno, Shigeyuki; Seki, Taiichiro

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

Site-Specific Protein Adducts of 4-Hydroxy-2(E)-Nonenal in Human THP-1 Monocytic Cells: Protein Carbonylation Is Diminished by Ascorbic Acid

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

Chavez, Juan; Chung, Woon-Gye; Miranda, Cristobal L.

2010-01-18

The protein targets and sites of modification by 4-hydroxy-2(E)-nonenal (HNE) in human monocytic THP-1 cells after exogenous exposure to HNE were examined using a multipronged proteomic approach involving electrophoretic, immunoblotting, and mass spectrometric methods. Immunoblot analysis using monoclonal anti-HNE antibodies showed several proteins as targets of HNE adduction. Pretreatment of THP-1 cells with ascorbic acid resulted in reduced levels of HNE-protein adducts. Biotinylation of Michael-type HNE adducts using an aldehyde-reactive hydroxylamine-functionalized probe (aldehyde-reactive probe, ARP) and subsequent enrichment facilitated the identification and site-specific assignment of the modifications by LC-MS/MS analysis. Sixteen proteins were unequivocally identified as targets of HNE adduction,more » and eighteen sites of HNE modification at Cys and His residues were assigned. HNE exposure of THP-1 cells resulted in the modification of proteins involved in cytoskeleton organization and regulation, proteins associated with stress responses, and enzymes of the glycolytic and other metabolic pathways. Finally, this study yielded the first evidence of site-specific adduction of HNE to Cys-295 in tubulin α-1B chain, Cys-351 and Cys-499 in α-actinin-4, Cys-328 in vimentin, Cys-369 in d-3-phosphoglycerate dehydrogenase, and His-246 in aldolase A.« less

Enantioselective syntheses of carbanucleosides from the Pauson-Khand adduct of trimethylsilylacetylene and norbornadiene.

PubMed

Vázquez-Romero, Ana; Rodríguez, Julia; Lledó, Agustí; Verdaguer, Xavier; Riera, Antoni

2008-10-16

A new enantioselective approach to carbanucleosides from Pauson-Khand (PK) adduct 1 is disclosed. The chiral cyclopentenone 1 is readily accessible in enantiomerically pure form via PK reaction of trimethylsilylacetylene and norbornadiene using N-benzyl-N-diphenylphosphino-tert-butyl-sulfinamide as a chiral P,S ligand. (-)-Carbavir and (-)-Abacavir were enantioselectively synthesized starting from (-)-1. The key steps of the sequence are a photochemical conjugate addition of a hydroxymethyl radical, a retro-Diels-Alder reaction, and a palladium catalyzed allylic substitution to introduce the nucleobase.

A fluorescent-based HPLC assay for quantification of cysteine and cysteamine adducts in Escherichia coli-derived proteins.

PubMed

Soriano, Brian D; Tam, Lei-Ting T; Lu, Hsieng S; Valladares, Violeta G

2012-01-01

Recombinant proteins expressed in Escherichia coli are often produced as unfolded, inactive forms accumulated in inclusion bodies. Redox-coupled thiols are typically employed in the refolding process in order to catalyze the formation of correct disulfide bonds at maximal folding efficiency. These thiols and the recombinant proteins can form mixed disulfide bonds to generate thiol-protein adducts. In this work, we apply a fluorescent-based assay for the quantification of cysteine and cysteamine adducts as observed in E. coli-derived proteins. The thiols are released by reduction of the adducted protein, collected and labeled with a fluorescent reagent, 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. The derivatized thiols are separated by reversed-phase HPLC and can be accurately quantified after method optimization. The estimated thiol content represents total amount of adducted forms present in the analyzed samples. The limit of quantification (LOQ) was established; specifically, the lowest amount of quantifiable cysteine adduction is 30 picograms and the lowest amount of quantifiable cysteamine adduction is 60 picograms. The assay is useful for quantification of adducts in final purified products as well as in-process samples from various purification steps. The assay indicates that the purification process accomplishes a decrease in cysteine adduction from 0.19 nmol adduct/nmol protein to 0.03 nmol adduct/nmol protein as well as a decrease in cysteamine adduction from 0.24 nmol adduct/nmol protein to 0.14 nmol adduct/nmol protein. Copyright © 2011. Published by Elsevier B.V.

Base-displaced intercalation of the 2-amino-3-methylimidazo[4,5-f]quinolone N2-dG adduct in the NarI DNA recognition sequence

PubMed Central

Stavros, Kallie M.; Hawkins, Edward K.; Rizzo, Carmelo J.; Stone, Michael P.

2014-01-01

2-Amino-3-methylimidazo[4,5-f]quinolone (IQ), a heterocyclic amine found in cooked meats, undergoes bioactivation to a nitrenium ion, which alkylates guanines at both the C8-dG and N2-dG positions. The conformation of a site-specific N2-dG-IQ adduct in an oligodeoxynucleotide duplex containing the iterated CG repeat restriction site of the NarI endonuclease has been determined. The IQ moiety intercalates, with the IQ H4a and CH3 protons facing the minor groove, and the IQ H7a, H8a and H9a protons facing the major groove. The adducted dG maintains the anti-conformation about the glycosyl bond. The complementary dC is extruded into the major groove. The duplex maintains its thermal stability, which is attributed to stacking between the IQ moiety and the 5′- and 3′-neighboring base pairs. This conformation is compared to that of the C8-dG-IQ adduct in the same sequence, which also formed a ‘base-displaced intercalated’ conformation. However, the C8-dG-IQ adopted the syn conformation placing the Watson−Crick edge of the modified dG into the major groove. In addition, the C8-dG-IQ adduct was oriented with the IQ CH3 group and H4a and H5a facing the major groove. These differences may lead to differential processing during DNA repair and replication. PMID:24366876

Organocatalytic Removal of Formaldehyde Adducts from RNA and DNA Bases

PubMed Central

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.

2015-01-01

Formaldehyde is universally employed 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, and avoiding 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 to a commercial kit. Such catalytic strategies show promise for general use in reversing formaldehyde adducts in clinical specimens. PMID:26291948

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.

Electrospray ionization-tandem mass spectrometry and 32P-postlabeling analyses of tamoxifen-DNA adducts in humans.

PubMed

Beland, Frederick A; Churchwell, Mona I; Doerge, Daniel R; Parkin, Daniel R; Malejka-Giganti, Danuta; Hewer, Alan; Phillips, David H; Carmichael, Paul L; Gamboa da Costa, Gonçalo; Marques, M Matilde

2004-07-21

Although the nonsteroidal antiestrogen tamoxifen is used as an adjuvant chemotherapeutic agent to treat hormone-dependent breast cancer and as a chemopreventive agent in women with elevated risk of breast cancer, it has also been reported to increase the risk of endometrial cancer. Reports of low levels of tamoxifen-DNA adducts in human endometrial tissue have suggested that tamoxifen induces endometrial cancer by a genotoxic mechanism. However, these findings have been controversial. We used electrospray ionization-tandem mass spectrometry (ES-MS/MS) and 32P-postlabeling analyses to investigate the presence of tamoxifen-DNA adducts in human endometrial tissue. Endometrial DNA from eight tamoxifen-treated women and eight untreated women was hydrolyzed to nucleosides and assayed for (E)-alpha-(deoxyguanosin-N2-yl)-tamoxifen (dG-Tam) and (E)-alpha-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-desMeTam), the two major tamoxifen-DNA adducts that have been reported to be present in humans and/or experimental animals treated with tamoxifen, using on-line sample preparation coupled with high-performance liquid chromatography (HPLC) and ES-MS/MS. The same DNA samples were assayed for the presence of dG-Tam and dG-desMeTam by (32)P-postlabeling methodology, using two different DNA digestion and labeling protocols, followed by both thin-layer chromatography and HPLC. We did not detect either tamoxifen-DNA adduct by HPLC-ES-MS/MS analyses (limits of detection for dG-Tam and dG-desMeTam were two adducts per 10(9) nucleotides and two adducts per 10(8) nucleotides, respectively) or by 32P-postlabeling analyses (limit of detection for both adducts was one adduct per 10(9) nucleotides) in any of the endometrial DNA samples. The initiation of endometrial cancer by tamoxifen is probably not due to a genotoxic mechanism involving the formation of dG-Tam or dG-desMeTam.

Biomonitoring of Aristolactam-DNA Adducts in Human Tissues using Ultra-Performance Liquid Chromatography/Ion-Trap Mass Spectrometry

PubMed Central

Yun, Byeong Hwa; Rosenquist, Thomas; Sidorenko, Viktoriya; Iden, Charles; Chung-Hsin, Chen; Pu, Yeong-Shiau; Bonala, Radha; Johnson, Francis; Dickman, Kathleen G.; Grollman, Arthur P.; Turesky, Robert J.

2012-01-01

Aristolochic acids (AAs) are a structurally-related family of nephrotoxic and carcinogenic nitrophenanthrene compounds found in Aristolochia herbaceous plants, many of which have been used worldwide for medicinal purposes. AAs have been implicated in the etiology of so-called Chinese herbs nephropathy and of Balkan endemic nephropathy. Both of these disease syndromes are associated with carcinomas of the upper urinary tract (UUC). 8-Methoxy-6-nitrophenanthro-[3,4-d]-1,3-dioxolo-5-carboxylic acid (AA-I) is a principal component of Aristolochia herbs. Following metabolic activation, AA-I reacts with DNA to form aristolactam (AL-I)-DNA adducts. We have developed a sensitive analytical method, using ultra-performance liquid chromatography-electrospray ionization/multistage mass spectrometry (UPLC-ESI/MSn) with a linear quadrupole ion-trap mass spectrometer, to measure 7-(deoxyadenosin-N6-yl) aristolactam I (dA-AL-I) and 7-(deoxyguanosin-N2-yl) aristolactam I (dG-AL-I) adducts. Using 10 μg of DNA for measurements, the lower limits of quantitation of dA-AL-I and dG-AL-I are, respectively, 0.3 and 1.0 adducts per 108 DNA bases. We have used UPLC-ESI/MSn to quantify AL-DNA adducts in tissues of rodents exposed to AA, and in the renal cortex of patients with UUC who reside in Taiwan, where the incidence of this uncommon cancer is the highest reported for any country in the world. In human tissues, dA-AL-I was detected at levels ranging from 9 to 338 adducts per 108 DNA bases, whereas dG-AL-I was not found. We conclude that UPLC-ESI/MSn is a highly sensitive, specific and robust analytical method, positioned to supplant 32P-postlabeling techniques currently used for biomonitoring of DNA adducts in human tissues. Importantly, UPLC-ESI/MSn could be used to document exposure to AA, the toxicant responsible for AA nephropathy and its associated UUC. PMID:22515372

40 CFR 721.3700 - Fatty acid, ester with styrenated phenol, ethylene oxide adduct.

Code of Federal Regulations, 2011 CFR

2011-07-01

.... Requirements as specified in § 721.90 (a)(4), (b)(4), and (c)(4) (where N = 400 ppb). (b) Specific requirements.... (1) Recordkeeping requirements. Requirements as specified in § 721.125 (a), (b), (c), (f), (g), (h... adduct (P-90-364) is subject to reporting under this section for the significant new uses described in...

1 : 2 Adducts of copper(I) halides with 1,2-bis(di-2-pyridylphosphino)ethane: solid state and solution structural studies and antitumour activity.

PubMed

Bowen, Richard J; Navarro, Maribel; Shearwood, Anne-Marie J; Healy, Peter C; Skelton, Brian W; Filipovska, Aleksandra; Berners-Price, Susan J

2009-12-28

The 1 : 2 adducts of copper(I) halides with 1,2-bis(2-pyridylphosphino)ethane (d2pype) have been synthesized and solution properties characterized by variable temperature (1)H, (31)P and (65)Cu NMR spectroscopy. Single-crystal structure determinations for the chloride, bromide and iodide complexes show these to crystallize from acetonitrile in the triclinic space group P1 as isostructural centrosymmetric dimers [(d2pype)Cu(mu-d2pype)(2)Cu(d2pype)]X(2).(solvent) with a approximately 12.6, b approximately 12.7, c approximately 15.3 A, alpha approximately 84, beta approximately 67, gamma approximately 84 degrees. In contrast to the analogous AuCl:2(d2pype) and AgNO(3):2(d2pype) adducts, in solution these CuX:2(d2pype) adducts (where X = Cl, Br and I) exist almost exclusively as bis-chelated monomeric [Cu(d2pype)(2)]X; evidence for an equilibrium between monomeric and dimeric forms is detected only for the CuCl adduct in methanol. Cytotoxicity studies in two human breast cancer lines and two matched liver progenitor cell lines indicate that [Cu(d2pype)(2)]Cl is non selectively toxic to both non-tumourigenic and tumourigenic cells. However, the analogous Au(I) compound [Au(d2pype)(2)]Cl, is toxic to highly tumourigenic cells and more selective in its toxicity to tumourigenic cells compared to non-tumourigenic cells. The significance of these results to the further development of selective, mitochondria-targeted, Au(I) antitumour complexes is discussed.

Differences in micronucleus frequency and acrylamide adduct levels with hemoglobin between vegetarians and non-vegetarians.

PubMed

Kotova, Natalia; Frostne, Cecilia; Abramsson-Zetterberg, Lilianne; Tareke, Eden; Bergman, Rolf; Haghdoost, Siamak; Paulsson, Birgit; Törnqvist, Margareta; Segerbäck, Dan; Jenssen, Dag; Grawé, Jan

2015-10-01

Nutrients and food constituents can prevent or contribute to genotoxicity. In this study, the possible influence of a vegetarian/non-vegetarian diet on genotoxic effects was investigated in 58 non-smoking healthy vegetarians (V) and non-vegetarians (NV), age 21-37 years from the Stockholm area in Sweden. Physical activity and dietary habits were similar in both groups, with the exception of the intake of meat and fish. Using flow cytometry, we determined the formation of micronuclei (MN) in transferrin-positive immature peripheral blood reticulocytes (Trf-Ret) (Total: n = 53; V: n = 27; NV: n = 26). Dietary exposure to acrylamide was measured through hemoglobin (Hb) adducts in peripheral erythrocytes (Total: n = 53; V: n = 29; NV: n = 24). Hb adducts of both acrylamide and its genotoxic metabolite glycidamide were monitored as a measure of the corresponding in vivo doses. Our data demonstrated that compared with the non-vegetarians, the vegetarians exhibited lower frequencies of MN (fMN) in the Trf-Ret (p < 0.01, Student's t test). A multivariate analysis demonstrated that there was no association between the fMN and factors such as age, sex, intake of vitamins/minerals, serum folic acid and vitamin B12 levels, physical activity, and body mass index. The mean Hb adduct levels of acrylamide and glycidamide showed no significant differences between vegetarians and non-vegetarians. Furthermore, there were no significant relationships between the adduct levels and fMN in the individuals. The ratio of the Hb adduct levels from glycidamide and acrylamide, however, showed a significant difference (p < 0.04) between the two groups. These data suggest that the vegetarian diet might be beneficial in lowering genomic instability in healthy individuals. The measured Hb adduct levels indicate that the total intake of acrylamide does not differ between the two studied groups and does not contribute to the observed difference in fMN, although an influence of the diet on the

Basis of Miscoding of the DNA Adduct N2,3-Ethenoguanine by Human Y-family DNA Polymerases*

PubMed Central

Zhao, Linlin; Pence, Matthew G.; Christov, Plamen P.; Wawrzak, Zdzislaw; Choi, Jeong-Yun; Rizzo, Carmelo J.; Egli, Martin; Guengerich, F. Peter

2012-01-01

N2,3-Ethenoguanine (N2,3-ϵG) is one of the exocyclic DNA adducts produced by endogenous processes (e.g. lipid peroxidation) and exposure to bioactivated vinyl monomers such as vinyl chloride, which is a known human carcinogen. Existing studies exploring the miscoding potential of this lesion are quite indirect because of the lability of the glycosidic bond. We utilized a 2′-fluoro isostere approach to stabilize this lesion and synthesized oligonucleotides containing 2′-fluoro-N2,3-ϵ-2′-deoxyarabinoguanosine to investigate the miscoding potential of N2,3-ϵG by Y-family human DNA polymerases (pols). In primer extension assays, pol η and pol κ replicated through N2,3-ϵG, whereas pol ι and REV1 yielded only 1-base incorporation. Steady-state kinetics revealed that dCTP incorporation is preferred opposite N2,3-ϵG with relative efficiencies in the order of pol κ > REV1 > pol η ≈ pol ι, and dTTP misincorporation is the major miscoding event by all four Y-family human DNA pols. Pol ι had the highest dTTP misincorporation frequency (0.71) followed by pol η (0.63). REV1 misincorporated dTTP and dGTP with much lower frequencies. Crystal structures of pol ι with N2,3-ϵG paired to dCTP and dTTP revealed Hoogsteen-like base pairing mechanisms. Two hydrogen bonds were observed in the N2,3-ϵG:dCTP base pair, whereas only one appears to be present in the case of the N2,3-ϵG:dTTP pair. Base pairing mechanisms derived from the crystal structures explain the slightly favored dCTP insertion for pol ι in steady-state kinetic analysis. Taken together, these results provide a basis for the mutagenic potential of N2,3-ϵG. PMID:22910910

Competitive Deprotonation and Superoxide [O₂⁻•)] Radical-Anion Adduct Formation Reactions of Carboxamides under Negative-Ion Atmospheric-Pressure Helium-Plasma Ionization (HePI) Conditions.

PubMed

Hassan, Isra; Pinto, Spencer; Weisbecker, Carl; Attygalle, Athula B

2016-03-01

Carboxamides bearing an N-H functionality are known to undergo deprotonation under negative-ion-generating mass spectrometric conditions. Herein, we report that N-H bearing carboxamides with acidities lower than that of the hydroperoxyl radical (HO-O(•)) preferentially form superoxide radical-anion (O2(-•)) adducts, rather than deprotonate, when they are exposed to the glow discharge of a helium-plasma ionization source. For example, the spectra of N-alkylacetamides show peaks for superoxide radical-anion (O2(-•)) adducts. Conversely, more acidic amides, such as N-alkyltrifluoroacetamides, preferentially undergo deprotonation under similar experimental conditions. Upon collisional activation, the O2(-•) adducts of N-alkylacetamides either lose the neutral amide or the hydroperoxyl radical (HO-O(•)) to generate the superoxide radical-anion (m/z 32) or the deprotonated amide [m/z (M - H)(-)], respectively. For somewhat acidic carboxamides, the association between the two entities is weak. Thus, upon mildest collisional activation, the adduct dissociates to eject the superoxide anion. Superoxide-adduct formation results are useful for structure determination purposes because carboxamides devoid of a N-H functionality undergo neither deprotonation nor adduct formation under HePI conditions.

Formation of DNA adducts and induction of mutagenic effects in rats following 4 weeks inhalation exposure to ethylene oxide as a basis for cancer risk assessment.

PubMed

van Sittert, N J; Boogaard, P J; Natarajan, A T; Tates, A D; Ehrenberg, L G; Törnqvist, M A

2000-01-17

Ethylene oxide (EO) is mutagenic in various in vitro and in vivo test systems and carcinogenic in rodents. EO forms different adducts upon reaction with DNA, N7-(2-hydroxyethyl)guanine (N7-HEG) being the main adduct. The major objectives of this study were: (a) to determine the formation and persistence of N7-HEG adducts in liver DNA of adult male rats exposed to 0, 50, 100 and 200 ppm by inhalation (4 weeks, 5 days/week, 6 h/day) and (b) to assess dose-response relationships for Hprt gene mutations and various types of chromosomal changes in splenic lymphocytes.N7-HEG adducts were measured 5, 21, 35 and 49 days after cessation of exposure. By extrapolation, the mean concentrations of N7-HEG immediately after cessation of exposure ('day 0') to 50, 100 and 200 ppm were calculated as 310, 558 and 1202 adducts/10(8) nucleotides, respectively, while the mean concentration in control rats was 2.6 adducts/10(8) nucleotides. At 49 days, N7-HEG values had returned close to background levels. The mean levels of N-(2-hydroxyethylvaline) adducts in haemoglobin were also determined and amounted 61.7, 114 and 247 nmol/g globin, respectively. Statistically significant linear relationships were found between mean N7-HEG levels ('day 0') and Hprt mutant frequencies at expression times 21/22 and 49/50 days and between mean N7-HEG ('day 0') and sister-chromatid exchanges (SCEs) or high frequency cells (HFC) measured 5 days post-exposure. At day 21 post-exposure, SCEs and HFCs in-part persisted and were significantly correlated with persistent N7-HEG adducts. No statistically significant dose effect relationships were observed for induction of micronuclei, nor for chromosome breaks or translocations. In conclusion, this study indicates that following sub-chronic exposure, EO is only weakly mutagenic in adult rats. Using the data of this study to predict cancer risk in man resulting from low level EO exposures in conjunction with other published data, i.e., those on (a) genotoxic

One-milliliter wet-digestion for inductively coupled plasma mass spectrometry (ICP-MS): determination of platinum-DNA adducts in cells treated with platinum(II) complexes.

PubMed

Yamada, Kanae; Kato, Naoyuki; Takagi, Akimitsu; Koi, Minoru; Hemmi, Hiromichi

2005-08-01

Platinum (Pt)-DNA adducts formed by the anti-tumor agent cisplatin are recognized by the DNA mismatch repair (MMR) system. To investigate the involvement of MMR proteins including hMLH1 in the removal of these adducts, we developed a mL-scale wet-digestion method for inductively coupled plasma mass spectrometry (ICP-MS). The detection limit was 0.01 ng mL(-1) Pt, which corresponded to 2 pg Pt/microg DNA when 10 microg of DNA was used. The mean relative errors were 5.4% or better for a dynamic range of 0.01-10 ng mL(-1) Pt. DNA (approximately 500 microg) had no matrix effect. To improve the accuracy, DNA preparations were treated with ribonuclease and the apparent reduction in the concentration of Pt was corrected using cellular DNA levels, which were determined with Hoechst 33258. No significant differences were observed, in terms of the formation of Pt-DNA adducts or their removal over 6 h, between hMLH1-deficient HCT116 cells, a human colorectal cancer cell line, and hMLH1-complemented HCT116+ch3 cells (n=5; P>0.05), indicating that the hMLH1-dependent DNA repair systems contribute to neither the formation nor the removal of the adducts at detectable levels. In addition, approximately 19% of the adducts were removed within 6 h in both cell lines. A time course analysis (~24 h) suggested that the removal of cisplatin-generated Pt-DNA adducts follows first-order kinetics (t(1/2)=32 h). The amount of Pt-DNA adduct formed by oxaliplatin in 1 h was 56% (ratio of means) of that generated by an equimolar concentration of cisplatin in HCT116. The proposed procedure could be useful for determining Pt-DNA adducts formed by Pt(II) complexes.

Quantitative detection of 4-hydroxyequilenin-DNA adducts in mammalian cells using an immunoassay with a novel monoclonal antibody.

PubMed

Okahashi, Yumiko; Iwamoto, Takaaki; Suzuki, Naomi; Shibutani, Shinya; Sugiura, Shigeki; Itoh, Shinji; Nishiwaki, Tomohisa; Ueno, Satoshi; Mori, Toshio

2010-07-01

Estrogen-DNA adducts are potential biomarkers for assessing the risk and development of estrogen-associated cancers. 4-Hydroxyequilenin (4-OHEN) and 4-hydroxyequilin (4-OHEQ), the metabolites of equine estrogens present in common hormone replacement therapy (HRT) formulations, are capable of producing bulky 4-OHEN-DNA adducts. Although the formation of 4-OHEN-DNA adducts has been reported, their quantitative detection in mammalian cells has not been done. To quantify such DNA adducts, we generated a novel monoclonal antibody (4OHEN-1) specific for 4-OHEN-DNA adducts. The primary epitope recognized is one type of stereoisomers of 4-OHEN-dA adducts and of 4-OHEN-dC adducts in DNA. An immunoassay with 4OHEN-1 revealed a linear dose-response between known amounts of 4-OHEN-DNA adducts and the antibody binding to those adducts, with a detection limit of approximately five adducts/10(8) bases in 1 microg DNA sample. In human breast cancer cells, the quantitative immunoassay revealed that 4-OHEN produces five times more 4-OHEN-DNA adducts than does 4-OHEQ. Moreover, in a mouse model for HRT, oral administration of Premarin increased the levels of 4-OHEN-DNA adducts in various tissues, including the uterus and ovaries, in a time-dependent manner. Thus, we succeeded in establishing a novel immunoassay for quantitative detection of 4-OHEN-DNA adducts in mammalian cells.

MEASUREMENT OF HEMOGLOBIN AND ALBUMIN ADDUCTS OF NAPHTHALENE-1,2-OXIDE, 1,2-NAPHTHOQUINONE AND 1,4-NAPHTHOQUINONE AFTER ADMINISTRATION OF NAPHTHALENE TO F344 RATS

EPA Science Inventory

Naphthalene-1,2-oxide (NPO), 1,2-naphthoquinone (1,2-NPQ) and 1,4-naphthoquinone (1,4-NPQ) are the major metabolites of naphthalene that are thought to be responsible for the cytotoxicity and genotoxicity of this chemical. We measured cysteinyl adducts of these metabolites in ...

Advanced glycation end product free adducts are cleared by dialysis.

PubMed

Agalou, S; Ahmed, N; Thornalley, P J; Dawnay, A

2005-06-01

Plasma advanced glycation end product (AGE) free adducts are increased up to 50-fold among patients on dialysis. We examined the ability of hemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD) to clear these compounds. The AGE free adducts Nepsilon-carboxymethyl-lysine (CML) and Nepsilon-(1-carboxyethyl)lysine (CEL) and the hydroimidazolones derived from glyoxal (G-H1), methylglyoxal (MG-H1), and 3-deoxyglucosone (3DG-H) were determined by LC-MS/MS and pentosidine by HPLC with fluorimetric detection in ultrafiltrates of plasma, urine, or PD effluent as appropriate from patients on HD (n = 8) or PD (n = 8), and from healthy controls (n = 8). Among patients on HD, all free AGEs predialysis were significantly higher than in controls and were decreased with dialysis. The removal of MG-H1 and 3DG-H was comparable to that of urea, whereas that of CML and pentosidine was some 20% higher; in contrast, the removal of CEL and G-H1 was 25% lower. Among patients on CAPD, free AGEs in PD effluent increased with increasing dwell time. The combined renal and peritoneal 24-h excretion rates of CML (4.7 micromol), CEL (6.5 micromol), 3DG-H (16.6 micromol), and pentosidine (0.08 micromol) were twofold higher than the amount excreted in healthy controls, whereas MG-H1 was ninefold higher (59 micromol); the combined clearances of all free AGEs except pentosidine were lower than in healthy controls. Impaired renal clearance contributes to increased plasma free AGEs in uremia, but the increased excretion rate among patients on PD demonstrates that there was also an increased synthesis of free AGEs. Both HD and PD are able to remove free AGEs.

Reduced nucleotide excision repair and GSTM1-null genotypes influence anti-B[a]PDE-DNA adduct levels in mononuclear white blood cells of highly PAH-exposed coke oven workers.

PubMed

Pavanello, Sofia; Pulliero, Alessandra; Siwinska, Ewa; Mielzynska, Danuta; Clonfero, Erminio

2005-01-01

It is important to identify the potential genetic-susceptible factors that are able to modulate individual responses to exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). In the present study we evaluated the influence of four polymorphisms of nucleotide excision repair (NER) genes [xeroderma pigmentosum-C (XPC)-PAT +/-, xeroderma pigmentosum-A (XPA) 5' non-coding region-A23G, XPD-exon 23 A35931C Lys751Gln, xeroderma pigmentosum-D (XPD)-exon 10 G23591A Asp312Asn] and that of glutathione S-transferase mu1 (GSTM1-active or -null) on benzo[a]pyrene diol epoxide (B[a]PDE)-DNA adduct levels from the lympho-monocyte fraction (LMF) of highly PAH benzo[a]pyrene (B[a]P)-exposed Polish coke oven workers (n = 67, 67% current smokers) with individual urinary post-shift excretion of 1-pyrenol exceeding the proposed biological exposure index (BEI) (2.28 micromol/mol creatinine). The bulky (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-B[a]PDE)-DNA adduct levels were detected by high-performance liquid chromatography (HPLC)/fluorescence analysis and genotypes by polymerase chain reaction. We found that workers with the low DNA repair capacity of XPC-PAT+/+ and XPA-A23A genotypes had significantly increased anti-B[a]PDE-DNA adduct levels (Mann-Whitney U-test, z = 2.24, P = 0.02 and z = 2.65, P = 0.01). Moreover, DNA adducts were also raised in workers without GSTM1 activity (GSTM1-null genotype) (Mann-Whitney U-test, z = 2.25, P = 0.0246). Workers with unfavourable XPC-PAT+/+ and XPA-A23A NER genotypes, alone (approximately 65% of workers) or combined with GSTM1-null genotype (approximately 75% of workers) were in the tertile with the highest adduct level, i.e. >4.11 adducts/10(8) nt (chi2 = 5.85, P = 0.0156 and chi2 = 5.40, P = 0.01). The increase in anti-B[a]PDE-DNA adduct levels (ln values) was significantly related in a multiple linear regression analysis to PAH exposure (i.e. urinary post-shift excretion of 1-pyrenol) (t = 2.61, P

Quantitative comparison between in vivo DNA adduct formation from exposure to selected DNA-reactive carcinogens, natural background levels of DNA adduct formation and tumour incidence in rodent bioassays.

PubMed

Paini, Alicia; Scholz, Gabriele; Marin-Kuan, Maricel; Schilter, Benoît; O'Brien, John; van Bladeren, Peter J; Rietjens, Ivonne M C M

2011-09-01

This study aimed at quantitatively comparing the occurrence/formation of DNA adducts with the carcinogenicity induced by a selection of DNA-reactive genotoxic carcinogens. Contrary to previous efforts, we used a very uniform set of data, limited to in vivo rat liver studies in order to investigate whether a correlation can be obtained, using a benchmark dose (BMD) approach. Dose-response data on both carcinogenicity and in vivo DNA adduct formation were available for six compounds, i.e. 2-acetylaminofluorene, aflatoxin B1, methyleugenol, safrole, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline and tamoxifen. BMD(10) values for liver carcinogenicity were calculated using the US Environmental Protection Agency BMD software. DNA adduct levels at this dose were extrapolated assuming linearity of the DNA adduct dose response. In addition, the levels of DNA adducts at the BMD(10) were compared to available data on endogenous background DNA damage in the target organ. Although for an individual carcinogen the tumour response increases when adduct levels increase, our results demonstrate that when comparing different carcinogens, no quantitative correlation exists between the level of DNA adduct formation and carcinogenicity. These data confirm that the quantity of DNA adducts formed by a DNA-reactive compound is not a carcinogenicity predictor but that other factors such as type of adduct and mutagenic potential may be equally relevant. Moreover, comparison to background DNA damage supports the notion that the mere occurrence of DNA adducts above or below the level of endogenous DNA damage is neither correlated to development of cancer. These data strongly emphasise the need to apply the mode of action framework to understand the contribution of other biological effect markers playing a role in carcinogenicity.

Intercalation of aflatoxin B sub 1 in two oligodeoxynucleotide adducts: Comparative sup 1 H NMR analysis of d(ATC sup AFB GAT)ter dot d(ATCGAT) and d(AT sup ATB GCAT) sub 2

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

Gopalakrishnan, S.; Harris, T.M.; Stone, M.P.

8,9-Dihydro-8-(N7-guanyl-(d(ATCGAT)))-9-hydroxyaflatoxin B{sub 1}{center dot}d(ATCGAT) and 8,9-dihydro-8-(N7-guanyl-(d(ATGCAT)))-9-hydroxyafltoxin B{sub 1}{center dot}8,9-dihydro-8-(N7-guanyl-(d(ATGCAT)))-9-hydroxyaflatoxin B{sub 1} were prepared by direct addition of aflatoxin B{sub 1} 8,9-expoxide to d(ATCGAT){sub 2} and d(ATGCAT){sub 2}, respectively. {sup 1}H NOE experiments, nonselective {sup 1}H T{sub 1} relaxation measurements, and {sup 1}H chemical shift perturbations demonstrate that in both modified oligodeoxynucleotides the aflatoxin moiety is intercalated above the 5{prime}-face of the modified guanine. The oligodeoxynucleotides remain right-handed, and perturbation of the B-DNA structure is localized adjacent to the adducted guanine. Aflatoxin-oligodeoxynucleotide {sup 1}H NOEs are observed between aflatoxin and the 5{prime}-neighbor base pair and include both the major groove andmore » the minor groove. The protons at C8 and C9 of the aflatoxin terminal furan ring exhibit slower spin-lattice relaxation as compared to other oligodeoxynucleotide protons, which supports the conclusion that they face into the major groove. Increased shielding is observed for aflatoxin protons. The difference in reaction stoichiometry is consistent with an intercalated transition-state complex between aflatoxin B{sub 1} 8,9-epoxide and B-DNA. Intercalation provides excellent positioning for nucleophilic attack by guanine N7 on aflatoxin B{sub 1} 8,9-epoxide, which probably accounts for the observed efficiency of adduct formation despite the relatively low DNA binding affinity observed for aflatoxin B{sub 1}.« less

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

Coke oven workers study: the effect of exposure and GSTM1 and NAT2 genotypes on DNA adduct levels in white blood cells and lymphocytes as determined by 32P-postlabelling.

PubMed

Binková, B; Topinka, J; Mracková, G; Gajdosová, D; Vidová, P; Stávková, Z; Peterka, V; Pilcík, T; Rimár, V; Dobiás, L; Farmer, P B; Srám, R J

1998-08-07

The DNA adduct levels in total white blood cells (WBC) and lymphocytes (LYM) isolated from the blood of the same individuals were evaluated using the 32P-postlabelling assay for bulky aromatic adducts. In this study, 68 male coke oven workers and 56 machines workers as a matched control were enrolled. Personal monitors were used to evaluate exposure to eight carcinogenic PAHs, including B[alpha]P, during an 8-h working shift. The exposure among coke even workers ranged widely from 0.6 to 547 micrograms/m3 and from 2 to 62,107 ng/m3, for carcinogenic PAHs and B[alpha]P, respectively. The respective values in controls were from 0.07-1.64 microgram/m3 and from 1-63 ng/m3. A significant correlation between WBC- and LYM-DNA adduct levels was found (r = 0.591, P < 0.001). DNA adduct levels in both WBC and LYM were significantly elevated in coke oven workers as compared with controls, but adduct levels were generally low (WBC: medians 2.61 vs. 1.83 LYM: 2.47 vs. 1.65 adducts/10(8) nucleotides). LYM-DNA adduct levels were significantly higher for smokers as compared with nonsmokers in both the exposed and control groups. No such differences in WBC-DNA adduct levels were observed. Positive significant correlations were found at the individual level between DNA adducts in both cell types and carcinogenic PAHs and/or B[alpha]P in the inhaled air (r = 0.38-0.45, P < 0.001). A significant correlation at the individual level between LYM-DNA adducts and urinary cotinine was also observed (r = 0.37, P < 0.001). No differences in DNA adduct levels could be attributed to GSTM1 or NAT2 genotype in either group. Nor was there any clear association of DNA adduct levels with combined GSTM1/NAT2 genotypes. The effect of personal exposure to carcinogenic PAHs on DNA adduct levels in both cell types was also investigated using a logistic regression model with adjustment for possible modulating effect of confounders (smoking, GSTM1, NAT2, age, plasma levels of vitamins A and E, body mass

DNA Polymerases η and ζ Combine to Bypass O(2)-[4-(3-Pyridyl)-4-oxobutyl]thymine, a DNA Adduct Formed from Tobacco Carcinogens.

PubMed

Gowda, A S Prakasha; Spratt, Thomas E

2016-03-21

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are important human carcinogens in tobacco products. They are metabolized to produce a variety 4-(3-pyridyl)-4-oxobutyl (POB) DNA adducts including O(2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O(2)-POB-dT), the most abundant POB adduct in NNK- and NNN-treated rodents. To evaluate the mutagenic properties of O(2)-POB-dT, we measured the rate of insertion of dNTPs opposite and extension past O(2)-POB-dT and O(2)-Me-dT by purified human DNA polymerases η, κ, ι, and yeast polymerase ζ in vitro. Under conditions of polymerase in excess, polymerase η was most effective at the insertion of dNTPs opposite O(2)-alkyl-dTs. The time courses were biphasic suggesting the formation of inactive DNA-polymerase complexes. The kpol parameter was reduced approximately 100-fold in the presence of the adduct for pol η, κ, and ι. Pol η was the most reactive polymerase for the adducts due to a higher burst amplitude. For all three polymerases, the nucleotide preference was dATP > dTTP ≫ dGTP and dCTP. Yeast pol ζ was most effective in bypassing the adducts; the kcat/Km values were reduced only 3-fold in the presence of the adducts. The identity of the nucleotide opposite the O(2)-alkyl-dT did not significantly affect the ability of pol ζ to bypass the adducts. The data support a model in which pol η inserts ATP or dTTP opposite O(2)-POB-dT, and then, pol ζ extends past the adduct.

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

PubMed

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

2014-05-08

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

Cisplatin Intrastrand Adducts Sensitize DNA to Base Damage by Hydrated Electrons

PubMed Central

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

2015-01-01

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

Effect of Watson-Crick and Hoogsteen base pairing on the conformational stability of C8-phenoxyl-2'-deoxyguanosine adducts.

PubMed

Millen, Andrea L; Churchill, Cassandra D M; Manderville, Richard A; Wetmore, Stacey D

2010-10-14

Bulky DNA addition products (adducts) formed through attack at the C8 site of guanine can adopt the syn orientation about the glycosidic bond due to changes in conformational stability or hydrogen-bonding preferences directly arising from the bulky group. Indeed, the bulky substituent may improve the stability of (non-native) Hoogsteen pairs. Therefore, such adducts often result in mutations upon DNA replication. This work examines the hydrogen-bonded pairs between the Watson-Crick and Hoogsteen faces of the ortho or para C8-phenoxyl-2'-deoxyguanosine adduct and each natural (undamaged) nucleobase with the goal to clarify the conformational preference of this type of damage, as well as provide insight into the likelihood of subsequent mutation events. B3LYP/6-311+G(2df,p)//B3LYP/6-31G(d) hydrogen-bond strengths were determined using both nucleobase and nucleoside models for adduct pairs, as well as the corresponding complexes involving natural 2'-deoxyguanosine. In addition to the magnitude of the binding strengths, the R(C1'···C1') distances and ∠(N9C1'C1') angles, as well as the degree of propeller-twist and buckle distortions, were carefully compared to the values observed in natural DNA strands. Due to structural changes in the adduct monomer upon inclusion of the sugar moiety, the monomer deformation energy significantly affects the relative hydrogen-bond strengths calculated with the nucleobase and nucleoside models. Therefore, we recommend the use of at least a nucleoside model to accurately evaluate hydrogen-bond strengths of base pairs involving flexible, bulky nucleobase adducts. Our results also emphasize the importance of considering both the magnitude of the hydrogen-bond strength and the structure of the base pair when predicting the preferential binding patterns of nucleobases. Using our best models, we conclude that the Watson-Crick face of the ortho phenoxyl adduct forms significantly more stable complexes than the Hoogsteen face, which

Crystal structures of eight 3D molecular adducts derived from bis-imidazole, bis(benzimidazole), and organic acids

NASA Astrophysics Data System (ADS)

Ding, Aihua; Jin, Shouwen; Jin, Shide; Hu, KaiKai; Lin, Zhihao; Liu, Hui; Wang, Daqi

2018-01-01

Cocrystallization of the bis(imidazole)/bis(benzimidazole) with a series of organic acids gave a total of eight molecular adducts with the compositions: (3,6-bis(imidazole-1-yl)pyridazine): (trichloroacetic acid)2(1) [(H2L1)2+ · (tca-)2, L1 = 3,6-bis(imidazole-1-yl)pyridazine, tca- = trichloroacetate], (bis(N-imidazolyl)methane): (suberic acid) (2) [(L2) · (H2suba), L2 = bis(N-imidazolyl)methane, H2suba = suberic acid], bis(N-imidazolyl)methane: (3-nitrophthalic acid): 3H2O (3) [(H2L2)2+ · (3-Hnpa-)2 · 3H2O, 3-Hnpa- = 3-nitro hydrogenphthalate], (bis(N-imidazolyl)butane)0.5: (4-nitrophthalic acid): H2O (4) [(H2L3)0.5+ · (4-Hnpa-)- · H2O, L3 = bis(N-imidazolyl)butane, 4-Hnpa- = 4-nitro hydrogenphthalate], (1-(3-(1H-benzimidazol-1-yl)propyl)-1H-benzimidazole): (3,5-dinitrosalicylic acid) (5) [(HL4) · (3,5-dns-), L4 = 1-(3-(1H-benzimidazol-1-yl)propyl)-1H-benzimidazole, 3,5-dns- = 3,5-dinitrosalicylate], (1-(3-(1H-benzimidazol-1-yl)propyl)-1H-benzimidazole): (3-nitrophthalic acid) (6) [(H2L4) · (3-npa2-), L4 = 1-(3-(1H-benzimidazol-1-yl)propyl)-1H-benzimidazole, 3-npa2-=3-nitrogenphthalate], (bis(N-imidazolyl)butane): (pamoic acid) (7) [(H2L3) · (pam), pam = pamoate], and (3,6-bis(imidazole-1-yl)pyridazine): (1,5-naphthalenedisulfonic acid) [(H2L1)2+ · (npda)2- = 1,5-naphthalenedisulfonate] (8). The eight adducts have been characterized by X-ray diffraction technique, infrared spectrum, and elemental analysis, and the melting points of all adducts were also reported. And their structural and supramolecular aspects are fully analyzed. The result reveals that among the eight investigated crystals both the end ring N in the bis(imidazole) moieties are protonated when the organic acids are deprotonated except 2, and 5, and the crystal packing is interpreted in terms of the strong ionic Nsbnd H⋯O H-bond between the imidazolium and the deprotonated acidic groups. Except the Nsbnd H⋯O H-bond, the Osbnd H⋯O H-bonds were also found at the salts 3, 4

Cresyl saligenin phosphate makes multiple adducts on free histidine, but does not form an adduct on histidine 438 of human butyrylcholinesterase.

PubMed

Liyasova, Mariya S; Schopfer, Lawrence M; Lockridge, Oksana

2013-03-25

Cresyl saligenin phosphate (CBDP) is a suspected causative agent of "aerotoxic syndrome", affecting pilots, crew members and passengers. CBDP is produced in vivo from ortho-containing isomers of tricresyl phosphate (TCP), a component of jet engine lubricants and hydraulic fluids. CBDP irreversibly inhibits butyrylcholinesterase (BChE) in human plasma by forming adducts on the active site serine (Ser-198). Inhibited BChE undergoes aging to release saligenin and o-cresol. The active site histidine (His-438) was hypothesized to abstract o-hydroxybenzyl moiety from the initial adduct on Ser-198. Our goal was to test this hypothesis. Mass spectral analysis of CBDP-inhibited BChE digested with Glu-C showed an o-hydroxybenzyl adduct (+106 amu) on lysine 499, a residue far from the active site, but not on His-438. Nevertheless, the nitrogen of the imidazole ring of free L-histidine formed a variety of adducts upon reaction with CBDP, including the o-hydroxybenzyl adduct, suggesting that histidine-CBDP adducts may form on other proteins. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Cresyl saligenin phosphate makes multiple adducts on free histidine, but does not form an adduct on histidine 438 of human butyrylcholinesterase

PubMed Central

Liyasova, Mariya S.; Schopfer, Lawrence M.; Lockridge, Oksana

2012-01-01

Cresyl saligenin phosphate (CBDP) is a suspected causative agent of “aerotoxic syndrome”, affecting pilots, crew members and passengers. CBDP is produced in vivo from ortho-containing isomers of tricresyl phosphate (TCP), a component of jet engine lubricants and hydraulic fluids. CBDP irreversibly inhibits butyrylcholinesterase (BChE) in human plasma by forming adducts on the active site serine (Ser-198). Inhibited BChE undergoes aging to release saligenin and o-cresol. The active site histidine (His-438) was hypothesized to abstract o-hydroxybenzyl moiety from the initial adduct on Ser-198. Our goal was to test this hypothesis. Mass spectral analysis of CBDP-inhibited BChE digested with Glu-C showed an o-hydroxybenzyl adduct (+106 amu) on lysine 499, a residue far from the active site, but not on His-438. Nevertheless, the nitrogen of the imidazole ring of free L-histidine formed a variety of adducts upon reaction with CBDP, including the o-hydroxybenzyl adduct, suggesting that histidine-CBDP adducts may form on other proteins. PMID:22898212

B(a)P adduct levels and fertility: A cross-sectional study in a Sicilian population

PubMed Central

Conti, Gea Oliveri; Calogero, Aldo Eugenio; Giacone, Filippo; Fiore, Maria; Barchitta, Martina; Agodi, Antonella; Ferrante, Margherita

2017-01-01

Benzo(a)pyrene (BaP) is a carcinogenic polycyclic aromatic hydrocarbon for human tissues. Still today it is not fully investigated if BaP can affect negatively the male fertility through the BaP-DNA adducts production. In the present study, BaP Tetrol I-1 (TI-1) and BaP Tetrol II-2 (TII-2) BaP-DNA adducts were investigated in spermatozoa of a Sicilian male population. Semen samples from 86 volunteers in two eastern Sicilian cities (Regalbuto and Melilli) were collected. The quality of semen was evaluated in all samples according to the World Health Organization (WHO) guidelines. We analyzed BaP-DNA adducts in extracted sperm cell DNA using the modified high-performance liquid chromatography-fluorescence method to detects both Tetrols. Differences between Tetrol levels were assessed by the Wilcoxon signed-rank test and the Mann-Whitney U test, as appropriate. Correlation between semen quality parameters and Tetrol concentrations were analyzed using the Spearman's correlation coefficient. Σ(TI-1+TII-2) were significantly higher in spermatozoa of volunteers from Regalbuto. Furthermore, a greater dispersion of the levels of adducts was observed in these specimens. TI-1 adducts were higher than TII-2 in Melilli samples (95% CI) and TII-2 were higher than TI-1 in Regalbuto semen samples (95% CI). A significant inverse correlation between sperm progressive motility and both TI-1 and TII-2 adducts was observed. The present study showed that BaP negatively affects male fertility by TI-1 and TII-2 DNA-adduct production. These results suggest that DNA adducts could be used as biomarker to assess BaP exposure by air pollution. Further studies are needed to confirm if these findings could affect male fertility because of the growing impairment of this function observed in recent years. PMID:28350051

Malondialdehyde-acetaldehyde adducts (MAA) and anti-MAA antibody in rheumatoid arthritis

PubMed Central

Thiele, Geoffrey M.; Duryee, Michael J.; Anderson, Daniel R.; Klassen, Lynell W.; Mohring, Stephen M.; Young, Kathleen A.; Benissan-Messan, Dathe; Sayles, Harlan; Dusad, Anand; Hunter, Carlos D.; Sokolove, Jeremy; Robinson, William; O’Dell, James R.; Nicholas, Anthony P.; Tuma, Dean; Mikuls, Ted R.

2017-01-01

Objective As a product of oxidative stress associated with tolerance loss in other disease states, we investigated the presence of malondialdehyde-acetaldehyde (MAA) adducts and circulating anti-MAA antibody in rheumatoid arthritis (RA). Methods Synovial tissues from RA and osteoarthritis patients were examined for the presence of MAA-modified and citrullinated proteins. Anti-MAA antibody isotypes were measured in RA cases (n = 1720) and healthy controls (n = 80) by ELISA. Antigen-specific anti-citrullinated protein antibody (ACPA) was measured in RA cases using a multiplex antigen array. Anti-MAA isotype concentrations were compared in a subset of cases (n = 80) and matched controls (n = 80). Associations of anti-MAA antibody isotypes with disease characteristics, including ACPA, were examined in all RA cases. Results MAA adducts were increased in RA synovial tissues relative to osteoarthritis and co-localized with citrullinated protein. Anti-MAA antibody isotypes were increased in RA cases vs. controls (p < 0.001). Among RA cases, anti-MAA antibody isotypes were associated with ACPA and RF positivity (p < 0.001) in addition to select measures of disease activity. Higher anti-MAA antibody concentrations were associated with a higher number of positive antigen-specific ACPA analytes in high titer (p < 0.001) and a higher ACPA score (p < 0.001) independent of other covariates. Conclusion MAA adduct formation is increased in RA and appears to result in robust antibody responses that are strongly associated with ACPA. These results support speculation that MAA formation may be a co-factor that drives tolerance loss resulting in the autoimmune responses characteristic of RA. PMID:25417811

Studies on the reaction mechanism of lactate oxidase. Formation of two covalent flavin-substrate adducts on reaction with glycollate.

PubMed

Massey, V; Ghisla, S; Kieschke, K

1980-04-10

L-Lactate oxidase from Mycobacterium smegmatis catalyzes the oxidative decarboxylation of glycollate, with formate, CO2, and H2O as the major products. In addition, some "uncoupling" of the normal reaction occurs, with glyoxylate and H2O adition, some "uncoupling" of the normal reaction occurs, with glyoxylate and H2O2 as products. Glyoxylate is also a substrate (presumably as its hydrate); in this case, the reaction products are oxalate and H2O2. Evidence is presented that the enzyme recognizes glycollate as a prochiral substrate, differentiating between the Re- and Si-faces of the alpha carbon atom. Two highly fluorescent species are formed concomitantly from the reaction with glycollate; they are proposed to be covalent alpha-glycollyl adducts to the reduced flavin position N(5). One of these adducts is labile and in rapid equilibrium with oxidized enzyme and glycollate, and with the complex of reduced enzyme and glyoxylate; this adduct is a catalytically competent intermediate. The other adduct is comparatively stable (t 1/2 for decay = 20 min at 25 degrees C) and does not react with O2. It is formed at a rate approximately 1% that of the catalytic adduct, but because of its lack of reaction with O2 and its stability, it gradually accumulates during catalytic turnover, resulting in catalytically incompetent enzyme. An isotope effect of approximately 4 is found in the reduction of oxidized enzyme flavin and in the formation of the labile fluorescent adduct, when alpha-2H2-glycollate or (R)-glycollate-2-d is used, but not with the (S)-glycollate-2-d enantiomer. It is concluded that the catalytic adduct is formed by hydrogen abstraction from the Re-face of glycollate.

Virucidal properties of metal oxide nanoparticles and their halogen adducts.

PubMed

Häggström, Johanna; Balyozova, Denitza; Klabunde, Kenneth J; Marchin, George

2010-04-01

Selected metal oxide nanoparticles are capable of strongly adsorbing large amounts of halogens (Cl(2), Br, I(2)) and mixed halogens. These solid adducts are relatively stable thermally, and they can be stored for long periods. However, in the open environment, they are potent biocides. Herein are described studies with a number of bacteriophage MS2, phiX174, and PRD-1 (virus examples). PRD-1 is generally more resistant to chemical disinfection, but in this paper it is shown to be very susceptible to selected interhalogen and iodine adducts of CeO(2), Al(2)O(3), and TiO(2) nanoparticles. Overall, the halogen adducts of TiO(2) and Al(2)O(3) were most effective. The mechanism of disinfection by these nanoparticles is not completely clear, but could include abrasive properties, as well as oxidative powers. A hypothesis that nanoparticles damage virons or stick to them and prevent binding to the host cell is a consideration that needs to be explored. Herein are reported comparative biocidal activities of a series of adducts and electron microscope images of before and after treatment.

Some reactions of the hydroxyl adduct of adenine

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

Vanhemmen, J.J.

1975-01-01

The chemical reactions of purine derivatives resulting from pulse radiolysis were studied. Some reactions of the hydroxyl adduct of adenine are described and one of these reactions was compared with similar reactions of hydroxyl adducts of other purine derivatives. Evidence is given that in various purines opening of the imidazole ring is due to unimolecular rearrangements of the hydroxyl adducts. (GRA)

Unusual analyte-matrix adduct ions and mechanism of their formation in MALDI TOF MS of benzene-1,3,5-tricarboxamide and urea compounds.

PubMed

Lou, Xianwen; Fransen, Michel; Stals, Patrick J M; Mes, Tristan; Bovee, Ralf; van Dongen, Joost J L; Meijer, E W

2013-09-01

Analyte-matrix adducts are normally absent under typical matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) conditions. Interestingly, though, in the analysis of several types of organic compounds synthesized in our laboratory, analyte-matrix adduct ion peaks were always recorded when common MALDI matrices such as 4-hydroxy-α-cyanocinnamic acid (CHCA) were used. These compounds are mainly those with a benzene-1,3,5-tricarboxamide (BTA) or urea moiety, which are important building blocks to make new functional supramolecular materials. The possible mechanism of the adduct formation was investigated. A shared feature of the compounds studied is that they can form intermolecular hydrogen bonding with matrices like CHCA. The intermolecular hydrogen bonding will make the association between analyte ions and matrix molecules stronger. As a result, the analyte ions and matrix molecules in MALDI clusters will become more difficult to be separated from each other. Furthermore, it was found that analyte ions were mainly adducted with matrix salts, which is probably due to the much lower volatility of the salts compared with that of their corresponding matrix acids. It seems that the analyte-matrix adduct formation for our compounds are caused by the incomplete evaporation of matrix molecules from the MALDI clusters because of the combined effects of enhanced intermolecular interaction between analyte-matrix and of the low volatility of matrix salts. Based on these findings, strategies to suppress the analyte-matrix adduction are briefly discussed. In return, the positive results of using these strategies support the proposed mechanism of the analyte-matrix adduct formation.

Evaluating Metabolite-Related DNA Oxidation and Adduct Damage from Aryl Amines Using a Microfluidic ECL Array.

PubMed

Bist, Itti; Bhakta, Snehasis; Jiang, Di; Keyes, Tia E; Martin, Aaron; Forster, Robert J; Rusling, James F

2017-11-21

Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in S N 2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu 2+ and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis(2,2'-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)Os(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 10 6 nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD 50 and Comet assay results.

Antibody recognition of melphalan adducts characterized using immobilized DNA: enhanced alkylation of G-Rich regions in cells compared to in vitro.

PubMed

McCartney, H; Martin, A M; Middleton, P G; Tilby, M J

2001-01-01

The bifunctional alkylating agent, melphalan, forms adducts on DNA that are recognized by two previously described monoclonal antibodies, MP5/73 and Amp4/42. Immunoreactivity to MP5/73 was lost when alkylated DNA was exposed to alkaline pH, while Amp4/42 only recognized the structures formed after the alkali treatment. Competitive enzyme-linked immunoadsorbent assays (ELISAs) indicated that in 0.01 and 0.1 M NaOH, loss of immunoreactivity to MP5/73 occurred with half-lives that were at least 2-fold longer than half-lives for gain of immunoreactivity to Amp4/42. This supported previously published evidence that Amp4/42 did not simply recognize all the products formed by alkali treatment of adducts that were initially recognized by MP5/73. Adducts recognized by MP5/73 on RNA were considerably more stable at 100 degrees C and pH 7 than adducts on DNA. This was consistent with the hypothesis that immunorecognition involved N7 guanine adducts and ruled out the involvement of phosphotriesters in immunoreactivity. Synthetic oligodeoxyribonucleotides, covalently immobilized onto 96-well plates, were reacted with melphalan and incubated for various periods with alkali, and then the levels of adducts recognized by each antibody in replicate wells were assayed by a direct binding ELISA. Adducts formed on oligodeoxyguanylic acid were recognized very weakly by Amp4/42, unlike other DNA sequences that were tested. Retention of immobilized DNA during alkali treatment was confirmed by immunoassay of cisplatin adducts. Poor recognition by Amp4/42 of adducts in oligodeoxyguanylic acid was confirmed by a competitive ELISA. Amp4/42, unlike MP5/73, efficiently recognized adducts resulting from alkylation of DNA with chlorambucil. It is concluded that the two antibodies recognized melphalan adducts in different DNA sequence environments and that this explains (a) the different alkali stability of immunoreactive adducts and (b) previous results which showed that, in DNA from melphalan

Polycyclic aromatic hydrocarbons and PAH-related DNA adducts.

PubMed

Ewa, Błaszczyk; Danuta, Mielżyńska-Švach

2017-08-01

Investigations on the impact of chemicals on the environment and human health have led to the development of an exposome concept. The exposome refers to the totality of exposures received by a person during life, including exposures to life-style factors, from the prenatal period to death. The exposure to genotoxic chemicals and their reactive metabolites can induce chemical modifications of DNA, such as, for example, DNA adducts, which have been extensively studied and which play a key role in chemically induced carcinogenesis. Development of different methods for the identification of DNA adducts has led to adopting DNA adductomic approaches. The ability to simultaneously detect multiple PAH-derived DNA adducts may allow for the improved assessment of exposure, and offer a mechanistic insight into the carcinogenic process following exposure to PAH mixtures. The major advantage of measuring chemical-specific DNA adducts is the assessment of a biologically effective dose. This review provides information about the occurrence of the polycyclic aromatic hydrocarbons (PAHs) and their influence on human exposure and biological effects, including PAH-derived DNA adduct formation and repair processes. Selected methods used for determination of DNA adducts have been presented.

Separation of {sup 32}P-postlabeled DNA adducts of polycyclic aromatic hydrocarbons and nitrated polycyclic aromatic hydrocarbons by HPLC

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

King, L.C.; Gallagher, J.E.; Lewtas, J.

The {sup 32}P-postlabeling assay, thin-layer chromatography, and reverse-phase high-pressure liquid chromatography (HPLC) were used to separate DNA adducts formed from 10 polycyclic aromatic hydrocarbons (PAHs) and 6 nitrated polycyclic aromatic hydrocarbons (NO{sub 2}-PAHs). The PAHs included benzo[j]fluoranthene, benzo[k]fluoranthene, indeno[1,2,3-cd]pyrene, benzo[a]pyrene, chrysene, 6-methylchrysene, 5-methylchrysene, and benz[a]anthracene. The NO{sub 2}-PAHs included 1-nitropyrene, 2-nitrofluoranthene, 3-nitrofluoranthene, 1,6-dinitropyrene, 1,3-dinitropyrene, and 1,8-dinitropyrene. Separation of seven of the major PAH-DNA adducts was achieved by an initial PAH HPLC gradient system. The major NO{sub 2}-PAH-DNA adducts were not all separated from each other using the initial PAH HPLC gradient but were clearly separated from the PAH-DNA adducts. Amore » second NO{sub 2}-PAH HPLC gradient system was developed to separate NO{sub 2}-PAH-DNA adducts following one-dimensional TLC and HPLC analysis. HPLC profiles of NO{sub 2}-PAH-DNA adducts were compared using both adduct enhancement versions of the {sup 32}P-postlabeling assay to evaluate the use of this technique on HPLC to screen for the presence of NO{sub 2}-PAH-DNA adducts. To demonstrate the application of these separation methods to a complex mixture of DNA adducts, the chromatographic mobilities of the {sup 32}P-postlabeled DNA adduct standards (PAHs and NO{sub 2}-PAHs) were compared with those produced by a complex mixture of polycyclic organic matter (POM) extracted from diesel emission particles. The diesel-derived adducts did not elute with the identical retention time of any of the PAH or NO{sub 2}-PAH standards used in this study. HPLC analyses of the NO{sub 2}-PAH-derived adducts (butanol extracted) revealed the presence of multiple DNA adducts.« less

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

2005-02-07

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.

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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

DOE PAGES

Pirovano, Paolo; Farquhar, Erik R.; Swart, Marcel; ...

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 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. Moreover, this rare examplemore » 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.« less

Accommodation of an N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adduct in the active site of human DNA polymerase iota: Hoogsteen or Watson-Crick base pairing?

PubMed

Donny-Clark, Kerry; Shapiro, Robert; Broyde, Suse

2009-01-13

Bypass across DNA lesions by specialized polymerases is essential for maintenance of genomic stability. Human DNA polymerase iota (poliota) is a bypass polymerase of the Y family. Crystal structures of poliota suggest that Hoogsteen base pairing is employed to bypass minor groove DNA lesions, placing them on the spacious major groove side of the enzyme. Primer extension studies have shown that poliota is also capable of error-free nucleotide incorporation opposite the bulky major groove adduct N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF). We present molecular dynamics simulations and free energy calculations suggesting that Watson-Crick base pairing could be employed in poliota for bypass of dG-AAF. In poliota with Hoogsteen-paired dG-AAF the bulky AAF moiety would reside on the cramped minor groove side of the template. The Hoogsteen-capable conformation distorts the active site, disrupting interactions necessary for error-free incorporation of dC opposite the lesion. Watson-Crick pairing places the AAF rings on the spacious major groove side, similar to the position of minor groove adducts observed with Hoogsteen pairing. Watson-Crick-paired structures show a well-ordered active site, with a near reaction-ready ternary complex. Thus our results suggest that poliota would utilize the same spacious region for lesion bypass of both major and minor groove adducts. Therefore, purine adducts with bulk on the minor groove side would use Hoogsteen pairing, while adducts with the bulky lesion on the major groove side would utilize Watson-Crick base pairing as indicated by our MD simulations for dG-AAF. This suggests the possibility of an expanded role for poliota in lesion bypass.

Analysis of tamoxifen-DNA adducts in endometrial explants by MS and 32P-postlabeling.

PubMed

Beland, Frederick A; Churchwell, Mona I; Hewer, Alan; Phillips, David H; Gamboa da Costa, Gonçalo; Marques, M Matilde

2004-07-23

The nonsteroidal antiestrogen tamoxifen increases the risk of endometrial cancer; however, the mechanism for the induction of these tumors is not known. Recently, Sharma et al. [Biochem. Biophys. Res. Commun. 307 (2003) 157], using high performance liquid chromatography (HPLC) with online postcolumn photochemical activation and fluorescence detection, reported the presence of (E)-alpha-(deoxyguanosin- N2-yl)tamoxifen in DNA from human endometrial explants incubated with tamoxifen. Inasmuch as the methodology used by these investigators does not allow unambiguous characterization of tamoxifen-DNA adducts, we have used two additional techniques (HPLC coupled with electrospray ionization tandem mass spectrometry and 32P-postlabeling analyses) to assay for the presence of tamoxifen-DNA adducts in the human endometrial explant DNA. Tamoxifen-DNA adducts were not detected by either method.

Microdose-Induced Drug–DNA Adducts as Biomarkers of Chemotherapy Resistance in Humans and Mice

DOE PAGES

Zimmermann, Maike; Wang, Si-Si; Zhang, Hongyong; ...

2016-11-30

Here, we report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [ 14C]carboplatin (1% of the therapeutic dose). Carboplatin–DNA adducts were quantified by accelerator mass spectrometry in blood and tumor samples collected within 24 hours, and compared with subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice weremore » dosed with [ 14C]carboplatin or [ 14C]gemcitabine and the resulting drug–DNA adduct levels were compared with tumor response to chemotherapy. At least one of the drugs had to induce high drug–DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug–DNA adducts as predictive biomarkers.« less

Microdose-Induced Drug–DNA Adducts as Biomarkers of Chemotherapy Resistance in Humans and Mice

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

Zimmermann, Maike; Wang, Si-Si; Zhang, Hongyong

Here, we report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [ 14C]carboplatin (1% of the therapeutic dose). Carboplatin–DNA adducts were quantified by accelerator mass spectrometry in blood and tumor samples collected within 24 hours, and compared with subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice weremore » dosed with [ 14C]carboplatin or [ 14C]gemcitabine and the resulting drug–DNA adduct levels were compared with tumor response to chemotherapy. At least one of the drugs had to induce high drug–DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug–DNA adducts as predictive biomarkers.« less

Microdose-Induced Drug-DNA Adducts as Biomarkers of Chemotherapy Resistance in Humans and Mice.

PubMed

Zimmermann, Maike; Wang, Si-Si; Zhang, Hongyong; Lin, Tzu-Yin; Malfatti, Michael; Haack, Kurt; Ognibene, Ted; Yang, Hongyuan; Airhart, Susan; Turteltaub, Kenneth W; Cimino, George D; Tepper, Clifford G; Drakaki, Alexandra; Chamie, Karim; de Vere White, Ralph; Pan, Chong-Xian; Henderson, Paul T

2017-02-01

We report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [ 14 C]carboplatin (1% of the therapeutic dose). Carboplatin-DNA adducts were quantified by accelerator mass spectrometry in blood and tumor samples collected within 24 hours, and compared with subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice were dosed with [ 14 C]carboplatin or [ 14 C]gemcitabine and the resulting drug-DNA adduct levels were compared with tumor response to chemotherapy. At least one of the drugs had to induce high drug-DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug-DNA adducts as predictive biomarkers. Mol Cancer Ther; 16(2); 376-87. ©2016 AACR. ©2016 American Association for Cancer Research.

Microdose-induced Drug-DNA Adducts as Biomarkers of Chemotherapy Resistance in Humans and Mice

PubMed Central

Zimmermann, Maike; Wang, Si-Si; Zhang, Hongyong; Lin, Tzu-yin; Malfatti, Michael; Haack, Kurt; Ognibene, Ted; Yang, Hongyuan; Airhart, Susan; Turteltaub, Kenneth W.; Cimino, George D.; Tepper, Clifford G.; Drakaki, Alexandra; Chamie, Karim; de Vere White, Ralph; Pan, Chong-xian; Henderson, Paul T.

2017-01-01

We report progress on predicting tumor response to platinum-based chemotherapy with a novel mass spectrometry approach. Fourteen bladder cancer patients were administered one diagnostic microdose each of [14C]carboplatin (1% of the therapeutic dose). Carboplatin-DNA adducts were quantified by accelerator mass spectrometry (AMS) in blood and tumor samples collected within 24 hours, and compared to subsequent chemotherapy response. Patients with the highest adduct levels were responders, but not all responders had high adduct levels. Four patient-derived bladder cancer xenograft mouse models were used to test the possibility that another drug in the regimen could cause a response. The mice were dosed with [14C]carboplatin or [14C]gemcitabine and the resulting drug-DNA adduct levels were compared to tumor response to chemotherapy. At least one of the drugs had to induce high drug-DNA adduct levels or create a synergistic increase in overall adducts to prompt a corresponding therapeutic response, demonstrating proof-of-principle for drug-DNA adducts as predictive biomarkers. PMID:27903751

NITRO MUSK ADDUCTS OF RAINBOW TROUT ...

EPA Pesticide Factsheets

Rainbow trout and other fish species can serve as 'sentinel' species for the assessment of ecological status and the presence of certain environmental contaminants. As such they act as bioindicators of exposure. Here we present seminal data regarding dose-response and toxicokinetics of trout hemoglobin adduct formation from exposure to nitro musks that are frequently used as fragrance ingredients in formulations of personal care products. Hemoglobin adducts serve as biomarkers of exposure of the sentinel species as we have shown in previous studies of hemoglobin adducts formed in trout and environmental carp exposed to musk xylene (MX) and musk ketone (MK). Gas chromatography-electron capture negative ion chemical ionization-mass spectrometry (GC-NICI-MS) employing selected ion monitoring is used to measure 4-amino-MX (4-AMX), 2-amino-MX (2-AMX), and 2-amino-MK (2-AMK) released by alkaline hydrolysis from the sulfinamide adducts of hemoglobin. Dose-response and toxicokinetics were investigated using this sensitive method for analysis of these metabolites. In the dose-response investigation, the concentrations of 4-AMX and 2-2AMX are observed to pass through a maximum at 0.10 mg/g. In the case of 2-AMK, the adduct concentration is almost the same at dosages in the range of 0.030 to 0.10 mg/g. For toxicokinetics, the concentration of the metabolites in the Hb reaches a maximum in the 3-day sample after administration of MX or MK. Further elimination of the metabo

DNA BINDING AND ADDUCT FORMATION OF AFLATOXIN B1 IN CULTURED HUMAN AND ANIMAL TRACHEOBRONCHIAL AND BLADDER TISSUES

EPA Science Inventory

DNA binding and adduct formation of aflatoxin B1 (AFB1) was studied in cultured bladder and tracheobronchial explants from human, monkey, dog, hamster and rat. Explants were exposed to (3H)AFB1 (1 micrometer final concentration) in PFHR-4 medium (pH 7.4) without serum for 24 h, a...

Equilibrium Dynamics of β-N-Methylamino-L-Alanine (BMAA) and Its Carbamate Adducts at Physiological Conditions

PubMed Central

Zimmerman, David; Goto, Joy J.; Krishnan, Viswanathan V

2016-01-01

Elevated incidences of Amyotrophic Lateral Sclerosis/Parkinsonism Dementia complex (ALS/PDC) is associated with β-methylamino-L-alanine (BMAA), a non-protein amino acid. In particular, the native Chamorro people living in the island of Guam were exposed to BMAA by consuming a diet based on the cycad seeds. Carbamylated forms of BMAA are glutamate analogues. The mechanism of neurotoxicity of the BMAA is not completely understood, and BMAA acting as a glutamate receptor agonist may lead to excitotoxicity that interferes with glutamate transport systems. Though the interaction of BMAA with bicarbonate is known to produce carbamate adducts, here we demonstrate that BMAA and its primary and secondary adducts coexist in solution and undergoes a chemical exchange among them. Furthermore, we determined the rates of formation/cleavage of the carbamate adducts under equilibrium conditions using two-dimensional proton exchange NMR spectroscopy (EXSY). The coexistence of the multiple forms of BMAA at physiological conditions adds to the complexity of the mechanisms by which BMAA functions as a neurotoxin. PMID:27513925

Biomonitoring Human Albumin Adducts: The Past, the Present, and the Future

PubMed Central

2016-01-01

Serum albumin (Alb) is the most abundant protein in blood plasma. Alb reacts with many carcinogens and/or their electrophilic metabolites. Studies conducted over 20 years ago showed that Alb forms adducts with the human carcinogens aflatoxin B1 and benzene, which were successfully used as biomarkers in molecular epidemiology studies designed to address the role of these chemicals in cancer risk. Alb forms adducts with many therapeutic drugs or their reactive metabolites such as β-lactam antibiotics, acetylsalicylic acid, acetaminophen, nonsteroidal anti-inflammatory drugs, chemotherapeutic agents, and antiretroviral therapy drugs. The identification and characterization of the adduct structures formed with Alb have served to understand the generation of reactive metabolites and to predict idiosyncratic drug reactions and toxicities. The reaction of candidate drugs with Alb is now exploited as part of the battery of screening tools to assess the potential toxicities of drugs. The use of gas chromatography-mass spectrometry, liquid chromatography, or liquid chromatography-mass spectrometry (LC-MS) enabled the identification and quantification of multiple types of Alb xenobiotic adducts in animals and humans during the past three decades. In this perspective, we highlight the history of Alb as a target protein for adduction to environmental and dietary genotoxicants, pesticides, and herbicides, common classes of medicinal drugs, and endogenous electrophiles, and the emerging analytical mass spectrometry technologies to identify Alb-toxicant adducts in humans. PMID:27989119

Tyrosine-lipid peroxide adducts from radical termination: para coupling and intramolecular Diels-Alder cyclization.

PubMed

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

2010-12-15

Free radical co-oxidation of polyunsaturated lipids with tyrosine or phenolic analogues 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 spectroscopy as well as by mass spectrometry (MS). 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 (13)C 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 (13)C chemical shifts at ~198 ppm. All of the NMR HMBC and HSQC correlations support the structure assignments of the primary and Diels-Alder adducts, as does MS collision-induced dissociation data. Kinetic rate constants and activation parameters for the IMDA reaction were determined, and the primary adducts were reduced with cuprous ion to give a phenol-derived 4-hydroxycyclohexa-2,5-dienone. No products from adduction of peroxyls at the phenolic ortho position were found in either the primary or 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 cross-links via interprotein Michael adducts.

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

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

[Formation of pyrrole adducts in 2,5-hexanedione-containing human serum cultured in vitro].

PubMed

Zhu, Ming-xing; Yin, Hong-yin; Xie, Ke-qin

2013-08-01

To investigate the relationship between formation of pyrrole adducts and concentration of 2, 5-hexanedione (2, 5-HD) and to provide an experimental basis for the study on toxicity of n-hexane. Serum samples were collected from normal persons and were then filtered and sterilized. They were mixed with 2,5-HD to obtain sera with final 2, 5-HD concentrations of 10, 25, 50, 100, and 200 mg/L, and blank serum was also prepared. The sera were cultured at 37°C and taken at different time points. Colorimetry was used to quantify the pyrrole adducts formed in sera, and gas chromatography was used to measure the remaining 2, 5-HD levels in sera. The content of pyrrole adducts increased as the culture proceeded and was dependent on the dose of 2, 5-HD; at the end of the experiment, the content of pyrrole adducts differed significantly across all concentration groups (P < 0.5). The concentrations of 2,5-HD decreased as the culture proceeded; at the end of the experiment, the concentrations of 2, 5-HD, from the highest to the lowest, decreased by 29%, 55%, 22%, 44%, and 40%, respectively. The decrease in 2, 5-HD had a positive correlation with the increase in pyrrole adducts, and the correlation coefficients for 200∼10 mg/L 2, 5-HD were 0.865, 0.697, 0.835, 0.823, and 0.814, respectively. The content of formed pyrrole adducts increases as the concentration of 2,5-HD rises; there is a positive correlation between the decrease in 2, 5-HD and the increase in pyrrole adducts in human serum.

DNA adducts and liver DNA replication in rats during chronic exposure to N-nitrosodimethylamine (NDMA) and their relationships to the dose-dependence of NDMA hepatocarcinogenesis.

PubMed

Souliotis, Vassilis L; Henneman, John R; Reed, Carl D; Chhabra, Saranjit K; Diwan, Bhalchandra A; Anderson, Lucy M; Kyrtopoulos, Soterios A

2002-03-20

Exposure of rats to the hepatocarcinogen N-nitrosodimethylamine (NDMA) (0.2-2.64 ppm in the drinking water) for up to 180 days resulted in rapid accumulation of N7- and O6-methylguanine in liver and white blood cell DNA, maximum adduct levels being reached within 1-7 days, depending on the dose. The levels of both adducts remained constant up to treatment day 28, subsequently declining slowly to about 40% of maximal levels for the liver and 60% for white blood cells by day 180. In order to elucidate the role of DNA replication in NDMA hepatocarcinogenesis, changes in liver cell labeling index (LI) were also measured on treatment days 21, 120 and 180. Although the time- and dose-dependence of the observed effects were complex, a clear trend towards increased rates of hepatocyte LI, as indicated by BrdU incorporation, with increasing NDMA doses was evident, particularly above 1 ppm, a concentration above which NDMA hepatocarcinogenicity is known to increase sharply. In contrast, no increase in Kupffer cell DNA replication was found at any of the doses employed, in accordance with the low susceptibility of these cells to NDMA-induced carcinogenesis. No significant increase in the occurrence of necrotic or apoptotic cells was noted under the treatment conditions employed. These results suggest that, in addition to the accumulation of DNA damage, alterations in hepatocyte DNA replication during the chronic NDMA exposure may influence the dose-dependence of its carcinogenic efficacy.

DNA adduct formation among workers in a Thai industrial estate and nearby residents.

PubMed

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

2008-01-25

The genotoxic effects of air pollutant exposures have been studied in people living and working in Map Ta Phut, Rayong province, Thailand, a site where is located the Map Ta Phut Industrial Estate (MIE) one of the largest steel, refinery and petrochemical complex in the South-Eastern Asia. This was done by the conduction of a transversal study aimed to compare the prevalence of bulky DNA adducts in groups of subjects experiencing various degree of air pollution. DNA adduct analysis was performed in the leukocytes of 201 volunteers by the (32)P-postlabelling assay: 79 were workers in the MIE complex, including 24 refinery workers, 40 steel workers and 15 tinplate workers, 72 were people residing downwind in the MIE area and 50 were residents in a control district of the same Rayong province but without industrial exposures. The groups of workers were analyzed separately to evaluate if DNA adduct formation differs by the type of industry. The levels of bulky DNA adducts were 1.17+/-0.17 (SE) adducts/10(8) nucleotides in refinery workers, 1.19+/-0.19 (SE) in steel workers, 0.87+/-0.17 (SE) in tinplate workers, 0.85+/-0.07 (SE) in MIE residents and 0.53+/-0.05 (SE) in district controls. No effects of smoking habits on DNA adducts was found. The multivariate regression analysis shows that the levels of DNA adducts were significantly increased among the individuals living near the MIE industrial complex in respect to those resident in a control district (p<0.05). In the groups of occupationally exposed workers, the highest levels of DNA adducts were found among the workers experiencing an occupational exposure to polycyclic aromatic hydrocarbons, e.g. the steel factory and refinery workers. When we have evaluated if the levels of DNA adducts of the PAH exposed workers were different from those of the MIE residents, a statistical significantly difference was found (p<0.05). Our present study indicates that people living near point sources of industrial air pollution can

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..., ethylene oxide adduct. (a) Chemical substances and significant new uses subject to reporting. (1) The chemical substance identified generically as fatty acid, ester with styrenated phenol, ethylene oxide...

Structure and Oxidation of Pyrrole Adducts Formed between Aflatoxin B2a and Biological Amines.

PubMed

Rushing, Blake R; Selim, Mustafa I

2017-06-19

Aflatoxin B 2a has been shown to bind to proteins through a dialdehyde intermediate under physiological conditions. The proposed structure of this adduct has been published showing a Schiff base interaction, but adequate verification using structural elucidation instrumental techniques has not been performed. In this work, we synthesized the aflatoxin B 2a amino acid adduct under alkaline conditions, and the formation of a new product was determined using high performance liquid chromatography-time-of-flight mass spectrometry. The resulting accurate mass was used to generate a novel proposed chemical structure of the adduct in which the dialdehyde forms a pyrrole ring with primary amines rather than the previously proposed Schiff base interaction. The pyrrole structure was confirmed using 1 H, 13 C, correlation spectroscopy, heteronuclear single quantum correlation, and heteronuclear multiple bond correlation NMR and tandem mass spectrometry. Reaction kinetics show that the reaction is overall second order and that the rate increases as pH increases. Additionally, this study shows for the first time that aflatoxin B 2a dialdehyde forms adducts with phosphatidylethanolamines and does so through pyrrole ring formation, which makes it the first aflatoxin-lipid adduct to be structurally identified. Furthermore, oxidation of the pyrrole adduct produced a product that was 16 m/z heavier. When the aflatoxin B 2a -lysine (ε) adduct was oxidized, it gave a product with an accurate mass, mass fragmentation pattern, and 1 H NMR spectrum that match aflatoxin B 1 -lysine, which suggest the transformation of the pyrrole ring to a pyrrolin-2-one ring. These data give new insight into the fate and chemical properties of biological adducts formed from aflatoxin B 2a as well as possible interferences with known aflatoxin B 1 exposure biomarkers.

DNA adducts of the tobacco carcinogens 2-amino-9H-pyrido[2,3-b]indole and 4-aminobiphenyl are formed at environmental exposure levels and persist in human hepatocytes.

PubMed

Nauwelaërs, Gwendoline; Bellamri, Medjda; Fessard, Valérie; Turesky, Robert J; Langouët, Sophie

2013-09-16

Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25-100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of AαC and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with AαC and 4-ABP, whereas adduct formation was ∼5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a 10,000-fold concentration range (1 nM-10 μM). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes suggest that AαC can contribute to DNA damage

DNA Adducts of the Tobacco Carcinogens 2-Amino-9H-pyrido[2,3-b]indole and 4-Aminobiphenyl are Formed at Environmental Exposure levels and Persist in Human Hepatocytes

PubMed Central

Nauwelaërs, Gwendoline; Bellamri, Medjda; Fessard, Valérie; Turesky, Robert J.; Langouët, Sophie

2013-01-01

Aromatic amines and structurally related heterocyclic aromatic amines (HAAs) are produced during the combustion of tobacco or during the high-temperature cooking of meat. Exposure to some of these chemicals may contribute to the etiology of several common types of human cancers. 2-Amino-9H-pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in mainstream tobacco smoke: it arises in amounts that are 25–100 times greater than the levels of the arylamine, 4-aminobiphenyl (4-ABP), a human carcinogen. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a prevalent HAA formed in cooked meats. AαC and MeIQx are rodent carcinogens; however, their carcinogenic potency in humans is unknown. A preliminary assessment of the carcinogenic potential of these HAAs in humans was conducted by examining the capacity of primary human hepatocytes to form DNA adducts of AαC and MeIQx, in comparison to 4-ABP, followed by the kinetics of DNA adduct removal by cellular enzyme repair systems. The principal DNA adducts formed were N-(deoxyguanosin-8-yl) (dG-C8) adducts. Comparable levels of DNA adducts were formed with AαC and 4-ABP, whereas adduct formation was ~5-fold lower for MeIQx. dG-C8-AαC and dG-C8-4-ABP were formed at comparable levels in a concentration-dependent manner in human hepatocytes treated with procarcinogens over a ten thousand-fold concentration range (1 nM – 10 µM). Pretreatment of hepatocytes with furafylline, a selective inhibitor of cytochrome P450 1A2, resulted in a strong diminution of DNA adducts signifying that P450 1A2 is a major P450 isoform involved in bioactivation of these procarcinogens. The kinetics of adduct removal varied for each hepatocyte donor. Approximately half of the DNA adducts were removed within 24 h of treatment; however, the remaining lesions persisted over 5 days. The high levels of AαC present in tobacco smoke and its propensity to form persistent DNA adducts in human hepatocytes, suggests that AαC can contribute to

Increasing Ubiquitin Ion Resistance to Unfolding in the Gas Phase Using Chloride Adduction: Preserving More "Native-Like" Conformations Despite Collisional Activation.

PubMed

Wagner, Nicole D; Kim, Doyong; Russell, David H

2016-06-07

Electrospray ionization (ESI) of ubiquitin from acidified (0.1%) aqueous solution produces abundant ubiquitin-chloride adduct ions, [M + nH + xCl]((n - x)+), that upon mild heating react via elimination of neutral HCl. Ion mobility collision cross section (CCS) measurements show that ubiquitin ions retaining chloride adducts exhibit CCS values similar to those of the "native-state" of the protein. Coupled with results from recent molecular dynamics (MD) simulations for the evolution of a salt-containing electrospray droplet, this study provides a more complete picture for how the presence of salts affects the evolution of protein conformers in the final stages of dehydration of the ESI process and within the instrument.

Selective borane reduction of phosphinoferrocene carbaldehydes to phosphinoalcohol-borane adducts. The coordination behaviour of 1-(diphenylphosphino)-1'-(methoxymethyl)ferrocene, a new ferrocene O,P-hybrid donor prepared from such an adduct.

PubMed

Stěpnička, Petr; Císařová, Ivana

2013-03-14

The reduction of ferrocene phosphino-aldehydes, R(2)PfcCHO (R = Ph, 2; Cy, 3; fc = ferrocene-1,1'-diyl, Cy = cyclohexyl) and (S(p))-[Fe(η(5)-C(5)H(3)-1-CHO-2-PPh(2))(η(5)-C(5)H(5))] ((S(p))-4), with BH(3)·THF or BH(3)·SMe(2) in THF at 0 °C selectively afforded the corresponding phosphinoalcohol-borane adducts, R(2)PfcCH(2)OH·BH(3) (R = Ph, 5; Cy, 6) and (S(p))-[Fe(η(5)-C(5)H(3)-1-CH(2)OH-2-PPh(2))(η(5)-C(5)H(5))]·BH(3) ((S(p))-7), in quantitative yields. In contrast, the reactions performed at elevated temperatures favoured the formation of methyl derivatives (e.g., Ph(2)PfcCH(3)·BH(3) (8)) resulting from overreduction (deoxygenation). The crystal structures of 3, 5, (S(p))-7, 8 and Cy(2)PfcBr (9) have been determined by single-crystal X-ray diffraction analysis. The crystal assemblies of adducts 5 and (S(p))-7 are built up by means of C-H...O contacts, O-H...HB dihydrogen bonds and other soft interactions but, surprisingly, not via the conventional O-H...O hydrogen bonds. Adduct 5 was smoothly deprotected to give the corresponding free phosphine, Ph(2)PfcCH(2)OH (1), and was further used for the preparation of a hybrid phosphinoether ligand, Ph(2)PfcCH(2)OMe (11). The latter compound was studied as a donor for Group 8-10 metal ions and for Cu(i), whereupon the following complexes were isolated and structurally characterised: [(η(6)-p-cymene)RuCl(2)(11-κP)] (12*), [(η(6)-p-cymene)RuCl(11-κP)(MeCN)][SbF(6)] (13*), [RhCl(cod)(11-κP)] (cod = η(2):η(2)-cycloocta-1,5-diene; 14), trans-[PdCl(2)(11-κP)(2)] (trans-15*), [PdCl(μ-Cl)(11-κP)](2) (16*), cis- and trans-[PtCl(2)(11-κP)(2)] (cis-17 and trans-17*), and [Cu(CF(3)SO(3)-κO)(11-κP)(H(2)O)] (18) (the asterisk indicates that the crystal structure was determined). In all these compounds, ligand 11 behaves as a P-monodentate donor while its ether group remains uncoordinated. This probably reflects structural flexibility of 11 resulting from the presence of the methylene linker and also distinguishes

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.

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

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

PubMed

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, we exposed male Sprague-Dawley rats daily to fresh mainstream smoke from the University 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 32P-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, trachea, and larynx were increased by 10- to 20-fold in the smoke-exposed group. Five-fold increase was observed in the bladder tissue, but differences were not present in the liver DNA of control and smoke-exposed groups. These data suggest selective formation of DNA adducts in the tissues.

Acetaminophen Adducts Detected in Serum of Pediatric Patients With Acute Liver Failure.

PubMed

Alonso, Estella M; James, Laura P; Zhang, Song; Squires, Robert H

2015-07-01

Previous studies in patients with acute liver failure identified acetaminophen (APAP) protein adducts in the serum of 12% and 19% of children and adults, respectively, with acute liver failure of indeterminate etiology. This article details the testing of APAP adducts in a subset (n = 393) of patients with varied diagnoses in the Pediatric Acute Liver Failure Study Group (PALFSG). Serum samples were available from 393 participants included in the PALFSG registry. Adduct measurement was performed using validated methods. Participants were grouped by diagnostic category as known APAP overdose, known other diagnosis, and indeterminate etiology. Demographic and clinical characteristics and participant outcomes were compared by adduct status (positive or negative) within each group. APAP adduct testing was positive in 86% of participants with known APAP overdose, 6% with other known diagnoses, and 11% with an indeterminate cause of liver failure. Adduct-positive participants were noted to have marked elevation of serum alanine aminotransferase and aspartate aminotransferase coupled with total serum bilirubin that was significantly lower than adduct-negative patients. In the indeterminate group, adduct-positive patients had different outcomes than adduct-negative patients (P = 0.03); spontaneous survival was 16 of 21 (76%) in adduct-positive patients versus 75 of 169 (44%) in adduct-negative patients. Prognosis did not vary by adduct status in patients with known diagnoses. Furthermore, study is needed to understand the relation of APAP exposure, as determined by the presence of APAP adducts, to the clinical phenotype and outcomes of children with acute liver failure.

Associations of serum aflatoxin B1-lysine adduct level with socio-demographic factors and aflatoxins intake from nuts and related nut products in Malaysia.

PubMed

Leong, Yin-Hui; Rosma, Ahmad; Latiff, Aishah A; Izzah, A Nurul

2012-04-01

Aflatoxins are one of the major risk factors in the multi-factorial etiology of human hepatocellular carcinoma. Therefore, the information on aflatoxins exposure is very important in the intervention planning in order to reduce the dietary intake of aflatoxins, especially among the children. This study investigated the relationship between aflatoxin B(1) (AFB(1)) lysine adduct levers in serum and socio-demographic factors and dietary intake of aflatoxins from nuts and nut products in Penang, Malaysia. A cross-sectional field study was conducted in five districts of Penang. A survey on socio-demographic characteristics was administered to 364 healthy adults from the three main ethnic groups (Malay, Chinese and Indian). A total of 170 blood samples were successfully collected and tested for the level of AFB(1)-lysine adduct. 97% of the samples contained AFB(1)-lysine adduct above the detection limit of 0.4 pg/mg albumin and ranged from 0.20 to 23.16 pg/mg albumin (mean±standard deviation=7.67±4.54 pg/mg albumin; median=7.12 pg/mg albumin). There was no significant association between AFB(1)-lysine adduct levels with gender, district, education level, household number and occupation when these socio-demographic characteristics were examined according to high or low levels of AFB(1)-lysine. However, participants in the age group of 31-50 years were 3.08 times more likely to have high AFB(1) levels compared to those aged between 18 and 30 years (P=0.026). Significant difference (P=0.000) was found among different ethnic groups. Chinese and Indian participants were 3.05 and 2.35 times more likely to have high AFB(1) levels than Malay. The result of AFB(1)-lysine adduct suggested that Penang adult population is likely to be exposed to AFB(1) but at a level of less than that needed to cause direct acute illness or death. Copyright © 2011 Elsevier GmbH. All rights reserved.

DNA adducts in coal miners: association with exposures to diesel engine emissions.

PubMed

Shu-Xin Qu James Leigh Hamilton Koelmeyer And Neill H Stacey

1997-01-01

The potential carcinogenic effects of exposure to diesel engine emissions (DEE) are of growing concern. Due to the use of diesel equipment in underground mines, DNA adducts in peripheral blood mononuclear cells have been measured using the (32)P-postlabelling technique in workers from two coal mines (A, B)in NSW, Australia, before and after a period of more intense exposure (long wall change out, LWCO). DNA adducts were readily detected in all workers. At Mine A, in the 89 participants before LWCO, no significant difference was found among the groups categorized by exposure levels. However, significantly higher concentrations of total DNA adducts were observed in the specific job categories, 'miners and loadmen', and 'machinemen, drivers and shiftmen' and in the smoking group. On comparing total DNA adducts before and after LWCO in a small number of workers, a significant increase was also found. At Mine B, before or after LWCO, the total DNA adduct levels showed no significant difference among groups categorized by exposure conditions, smoking status, job categories and job time length. However, the total DNA adducts for the 61 subjects were significantly increased (geometric means) from 297 to 389 amol lg(-1) DNA after LWCO (p < 0.0001, paired t test). Some individual adducts were also elevated to a greater extent (p < 0.05, paired non-parametric test, Wilcoxon signed rank test). Furthermore, using generalized estimating equations for adjusting all factors across the observation period, no particular factor showed any significant interactive effects. Given the association of exposure to DEE with lung cancer and the apparent increase in adducts during a period of intense DEE exposures it would be prudent to pay particular attention to keeping exposures as low as possible, especially during LWCO operations.

Intramolecular and intermolecular N-H...C(5)H(5)(-) hydrogen bonding in magnesocene adducts of alkylamines. Implications for chemical vapor deposition using cyclopentadienyl source compounds.

PubMed

Xia, Aibing; Heeg, Mary Jane; Winter, Charles H

2002-09-25

Magnesocene adducts of alkylamines were prepared and characterized. Treatment of 3-amino-2,4-dimethylpentane, isopropylamine, tert-butylamine, benzylamine, or N-isopropylbenzylamine with magnesocene at ambient temperature in toluene afforded the amine adducts Cp2Mg(NH2CH(CH(CH3)2)2) (91%), Cp2Mg(NH2iPr) (80%), Cp2Mg(NH2tBu) (67%), Cp2Mg(NH2CH2Ph) (80%), and Cp2Mg(NH(CH(CH3)2)(CH2C6H5)) (91%). These adducts are stable at ambient temperature, and Cp2Mg(NH2CH(CH(CH3)2)2) can be sublimed at 60 degrees C/0.05 Torr without any evidence for reversion to magnesocene. The solid-state structure of Cp2Mg(NH2CH(CH(CH3)2)2) contains eta5- and eta2-cyclopentadienyl ligands, and the hydrogen atoms on the coordinated amine nitrogen atom participate in intramolecular and intermolecular hydrogen bonding to the eta2-cyclopentadienyl ligand. The observed hydrogen bonding is relevant to the path by which cyclopentadiene is eliminated from metal cyclopentadienyl CVD source compounds during film growth employing acidic element hydrides as co-reactants.

Preparation of SRN1-type coupling adducts from aliphatic gem-dinitro compounds in ionic liquids.

PubMed

Kamimura, Akio; Toyoshima, Seiichi

2012-04-25

S(RN)1-type coupling adducts are readily prepared by the reaction between a-sulfonylesters or a-cyanosulfones and gem-dinitro compounds in ionic liquids. The reactions progress smoothly and recovered ionic liquids can be used for several iterations, as long as they are washed with water to remove alkali metallic salts. The reaction rate is slower than the corresponding S(RN)1 reaction in DMSO, but no acceleration on irradiation or no inhibition in the presence of m-DNB are observed.

Malondialdehyde-acetaldehyde (MAA) adducted protein inhalation causes lung injury

PubMed Central

Wyatt, T. A.; Kharbanda, K. K.; McCaskill, M. L.; Tuma, D. J.; Yanov, D.; DeVasure, J.; Sisson, J. H.

2011-01-01

In addition to cigarette smoking, alcohol exposure is also associated with increased lung infections and decreased mucociliary clearance. However, little research has been conducted on the combination effects of alcohol and cigarette smoke on lungs. Previously, we have demonstrated in a mouse model that the combination of cigarette smoke and alcohol exposure results in the formation of a very stable hybrid malondialdehyde-acetaldehyde (MAA)-adducted protein in the lung. In in vitro studies, MAA-adducted protein stimulates bronchial epithelial cell interleukin-8 via the activation of protein kinase C epsilon (PKCε). We hypothesized that direct MAA-adducted protein exposure in the lungs would mimic such a combination of smoke and alcohol exposure leading to airway inflammation. To test this hypothesis, C57BL/6J female mice were intranasally instilled with either saline, 30 µL of 50 µg/mL BSA-MAA, or unadducted BSA for up to 3 wk. Likewise, human lung surfactant proteins A and D (SPA, SPD) were purified from human pulmonary proteinosis lung lavage fluid and successfully MAA-adducted in vitro. Similar to BSA-MAA, SPD-MAA was instilled into mouse lungs. Lungs were necropsied and assayed for histopathology, PKCε activation, and lung lavage chemokines. In control mice instilled with saline, normal lungs had few inflammatory cells. No significant effects were observed in un-adducted BSA- or SPD-instilled mice. However, when mice were instilled with BSA-MAA or SPD-MAA for 3 wk, a significant peribronchiolar localization of inflammatory cells was observed. Both BSA-MAA and SPD-MAA stimulated increased lung lavage neutrophils and caused a significant elevation in the chemokine, KC, which is a functional homologue to human interleukin-8. Likewise, MAA-adducted protein stimulated the activation of airway and lung slice PKCε. These data support that MAA-adducted protein induces a pro-inflammatory response in the lungs and that lung surfactant protein is a biologically

INVESTIGATION OF THE USE OF ISOCYANATE ADDUCTS IN URETHANE FOAM

DTIC Science & Technology

The fea ibility of a one-can, delayed action foaming system was successfully d monstra e . A literary arch revealed that y co poun s po e ing...ctive ydrogens may react with organic isocyanates to produce heat-s nsitive adducts. T HESE D UCT YI LD BACK THE ORIGINAL ISOCY N TE AND THE

Effect of combined actions of hip adduction/abduction on the force generation and maintenance of pelvic floor muscles in healthy women

PubMed Central

Amorim, Amanda C.; Cacciari, Licia P.; Passaro, Anice C.; Silveira, Simone R. B.; Amorim, Cesar F.; Loss, Jefferson F.

2017-01-01

Pelvic floor muscle (PFM) force and coordination are related to urinary incontinence severity and to sexual satisfaction. Health professionals frequently combine classic PFM exercises with hip adduction/abduction contraction to treat these disorders, but the real benefits of this practice are still unknown. Based on a theoretical anatomy approach whereby the levator ani muscle is inserted into the obturator internus myofascia and in which force generated by hip movements should increase the contraction quality of PFMs, our aim was to investigate the effects of isometric hip adduction and abduction on PFM force generation. Twenty healthy, nulliparous women were evaluated using two strain-gauge dynamometers (one cylinder-like inside the vaginal cavity, and the other measuring hip adduction/abduction forces around both thighs) while performing three different tasks: (a) isolated PFM contraction; (b) PFM contraction combined with hip adduction (30% and 50% maximum hip force); and (c) PFM contraction combined with hip abduction (30% and 50% maximum hip force). Data were sampled at 100Hz and subtracted from the offset if existent. We calculated a gradient between the isolated PFM contraction and each hip condition (Δ Adduction and Δ Abduction) for all variables: Maximum force (N), instant of maximum-force occurrence (s), mean force in an 8-second window (N), and PFM force loss (N.s). We compared both conditions gradients in 30% and 50% by paired t-tests. All variables did not differ between hip conditions both in 30% and 50% of maximum hip force (p>.05). PFM contraction combined with isometric hip abduction did not increase vaginal force in healthy and nulliparous women compared to PFM contraction combined with isometric hip adduction. Therefore, so far, the use of hip adduction or abduction in PFM training and treatments are not justified for improving PFM strength and endurance. PMID:28542276

Effect of combined actions of hip adduction/abduction on the force generation and maintenance of pelvic floor muscles in healthy women.

PubMed

Amorim, Amanda C; Cacciari, Licia P; Passaro, Anice C; Silveira, Simone R B; Amorim, Cesar F; Loss, Jefferson F; Sacco, Isabel C N

2017-01-01

Pelvic floor muscle (PFM) force and coordination are related to urinary incontinence severity and to sexual satisfaction. Health professionals frequently combine classic PFM exercises with hip adduction/abduction contraction to treat these disorders, but the real benefits of this practice are still unknown. Based on a theoretical anatomy approach whereby the levator ani muscle is inserted into the obturator internus myofascia and in which force generated by hip movements should increase the contraction quality of PFMs, our aim was to investigate the effects of isometric hip adduction and abduction on PFM force generation. Twenty healthy, nulliparous women were evaluated using two strain-gauge dynamometers (one cylinder-like inside the vaginal cavity, and the other measuring hip adduction/abduction forces around both thighs) while performing three different tasks: (a) isolated PFM contraction; (b) PFM contraction combined with hip adduction (30% and 50% maximum hip force); and (c) PFM contraction combined with hip abduction (30% and 50% maximum hip force). Data were sampled at 100Hz and subtracted from the offset if existent. We calculated a gradient between the isolated PFM contraction and each hip condition (Δ Adduction and Δ Abduction) for all variables: Maximum force (N), instant of maximum-force occurrence (s), mean force in an 8-second window (N), and PFM force loss (N.s). We compared both conditions gradients in 30% and 50% by paired t-tests. All variables did not differ between hip conditions both in 30% and 50% of maximum hip force (p>.05). PFM contraction combined with isometric hip abduction did not increase vaginal force in healthy and nulliparous women compared to PFM contraction combined with isometric hip adduction. Therefore, so far, the use of hip adduction or abduction in PFM training and treatments are not justified for improving PFM strength and endurance.

In vitro screening of 50 highly prescribed drugs for thiol adduct formation--comparison of potential for drug-induced toxicity and extent of adduct formation.

PubMed

Gan, Jinping; Ruan, Qian; He, Bing; Zhu, Mingshe; Shyu, Wen C; Humphreys, W Griffith

2009-04-01

Reactive metabolite formation has been associated with drug-induced liver, skin, and hematopoietic toxicity of many drugs that has resulted in serious clinical toxicity, leading to clinical development failure, black box warnings, or, in some cases, withdrawal from the market. In vitro and in vivo screening for reactive metabolite formation has been proposed and widely adopted in the pharmaceutical industry with the aim of minimizing the property and thus the risk of drug-induced toxicity (DIT). One of the most common screening methods is in vitro thiol trapping of reactive metabolites. Although it is well-documented that many hepatotoxins form thiol adducts, there is no literature describing the adduct formation potential of safer drugs that are widely used. The objective of this study was to quantitatively assess the thiol adduct formation potential of 50 drugs (10 associated with DIT and 40 not associated) and document apparent differences in adduct formation between toxic and safer drugs. Dansyl glutathione was used as a trapping agent to aid the quantitation of adducts following in vitro incubation of drugs with human liver microsomes in the presence and absence of NADPH. Metabolic turnover of these drugs was also monitored by LC/UV. Overall, 15 out of the 50 drugs screened formed detectable levels of thiol adducts. There were general trends toward more positive findings in the DIT group vs the non-DIT group. These trends became more marked when the relative amount of thiol adducts was taken into account and improved further when dose and total daily reactive metabolite burdens were considered. In conclusion, there appears to be a general trend between the extent of thiol adduct formation and the potential for DIT, which would support the preclinical measurement and minimization of the property through screening of thiol adduct formation as part of an overall discovery optimization paradigm.

Detection of endogenous DNA adducts, O-carboxymethyl-2'-deoxyguanosine and 3-ethanesulfonic acid-2'-deoxycytidine, in the rat stomach after duodenal reflux.

PubMed

Terasaki, Masaru; Totsuka, Yukari; Nishimura, Koichi; Mukaisho, Ken-Ichi; Chen, Kuan-Hao; Hattori, Takanori; Takamura-Enya, Takeji; Sugimura, Takashi; Wakabayashi, Keiji

2008-09-01

The endogenous DNA adducts O(6)-carboxymethyl-deoxyguanosine (O(6)-CM-dG) and 3-ethanesulfonic acid-deoxycytidine (3-ESA-dC) are produced from N-nitroso bile acid conjugates, such as N-nitrosoglycocholic acid (NO-GCA) and N-nitrosotaurocholic acid (NO-TCA), respectively. Formation of these DNA adducts in vivo was here analyzed by 32P-postlabeling in the glandular stomach of rats subjected to duodenal content reflux surgery. In this model, all duodenal contents, including bile acid conjugates, flow back from the jejunum into the gastric corpus. The levels of O(6)-CM-dG found at 4 and 8 weeks after surgery were 40.9 +/- 9.4 and 56.3 +/- 3.2 per 10(8) nucleotides, respectively, whereas the sham operation groups had values of 5.8 +/- 2.3 and 5.9 +/- 0.5 per 10(8) nucleotides. Moreover, adduct spots corresponding to 3-ESA-dC were detected in both duodenal reflux and sham operation groups and levels in the duodenal reflux groups were around four-fold elevated at 11.2 +/- 1.0 and 8.9 +/- 1.0 per 10(8) nucleotides after 4 and 8 weeks, respectively. When the duodenal reflux animals were treated with a nitrite trapping agent, thiazolidine- 4-carboxylic acid (thioproline, TPRO), the levels of O(6)-CM-dG and 3-ESA-dC were reduced to the same levels as in the sham operation animals. These observations suggest that NO-TCA and NO-GCA are formed by nitrosation of glycocholic acid and taurocholic acid, respectively, and these nitroso compounds produce DNA adducts in the glandular stomach of rats subjected to duodenal content reflux surgery.

Rotational Investigation of the Adducts of Formic Acid with Alcohols, Ethers and Esters

NASA Astrophysics Data System (ADS)

Evangelisti, Luca; Spada, Lorenzo; Li, Weixing; Caminati, Walther

2016-06-01

Mixtures of formic acid with methyl alcohol, with isopropyl alcohol, with tert-butyl alcohol, with dimethylether and with isopropylformiate have been supersonically expanded as pulsed jets. The obtained cool plumes have been analyzed by Fourier transform microwave spectroscopy. It has been possible to assign the rotational spectra of the 1:1 adducts of formic acid with tert-butyl alcohol, with dimethyl ether and with isopropylformiate. The conformational shapes and geometries of these adducts, as well as the topologies of their itermolecular hydrogen bonds will be presented. An explanation is given of the failure of the assignments of the rotational spectra of the adducts of formic acid with methyl alcohol and isopropyl alcohol.

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. © The Author(s) 2016. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Cytochrome P450 1b1 in polycyclic aromatic hydrocarbon (PAH)-induced skin carcinogenesis: Tumorigenicity of individual PAHs and coal-tar extract, DNA adduction and expression of select genes in the Cyp1b1 knockout mouse

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

Siddens, Lisbeth K.; Superfund Research Center, Oregon State University, Corvallis, OR 97331; Bunde, Kristi L.

FVB/N mice wild-type, heterozygous or null for Cyp 1b1 were used in a two-stage skin tumor study comparing PAH, benzo[a]pyrene (BaP), dibenzo[def,p]chrysene (DBC), and coal tar extract (CTE, SRM 1597a). Following 20 weeks of promotion with TPA the Cyp 1b1 null mice, initiated with DBC, exhibited reductions in incidence, multiplicity, and progression. None of these effects were observed with BaP or CTE. The mechanism of Cyp 1b1-dependent alteration of DBC skin carcinogenesis was further investigated by determining expression of select genes in skin from DBC-treated mice 2, 4 and 8 h post-initiation. A significant reduction in levels of Cyp 1a1,more » Nqo1 at 8 h and Akr 1c14 mRNA was observed in Cyp 1b1 null (but not wt or het) mice, whereas no impact was observed in Gst a1, Nqo 1 at 2 and 4 h or Akr 1c19 at any time point. Cyp 1b1 mRNA was not elevated by DBC. The major covalent DNA adducts, dibenzo[def,p]chrysene-(±)-11,12-dihydrodiol-cis and trans-13,14-epoxide-deoxyadenosine (DBCDE-dA) were quantified by UHPLC-MS/MS 8 h post-initiation. Loss of Cyp1 b1 expression reduced DBCDE-dA adducts in the skin but not to a statistically significant degree. The ratio of cis- to trans-DBCDE-dA adducts was higher in the skin than other target tissues such as the spleen, lung and liver (oral dosing). These results document that Cyp 1b1 plays a significant role in bioactivation and carcinogenesis of DBC in a two-stage mouse skin tumor model and that loss of Cyp 1b1 has little impact on tumor response with BaP or CTE as initiators. - Highlights: • Cyp1b1 null mice exhibit lower skin cancer sensitivity to DBC but not BaP or CTE. • Cyp1b1 expression impacts expression of other PAH metabolizing enzymes. • cis/trans-DBCDE-dA ratio significantly higher in the skin than the spleen, lung or liver • Potency of DBC and CTE in mouse skin is higher than predicted by RPFs.« less

Accommodation of an N-(deoxyguanosin-8-yl)-2-acetylaminofluorene adduct in the active site of human DNA polymerase ι: Hoogsteen or Watson-Crick base pairing?†

PubMed Central

Donny-Clark, Kerry; Shapiro, Robert; Broyde, Suse

2009-01-01

Bypass across DNA lesions by specialized polymerases is essential for maintenance of genomic stability. Human DNA polymerase ι (polι) is a bypass polymerase of the Y family. Crystal structures of polι suggest that Hoogsteen base pairing is employed to bypass minor groove DNA lesions, placing them on the spacious major groove side of the enzyme. Primer extension studies have shown that polι is also capable of error-free nucleotide incorporation opposite the bulky major groove adduct N-(deoxyguanosin-8-yl)-2-acetyl-aminofluorene (dG-AAF). We present molecular dynamics simulations and free energy calculations suggesting that Watson-Crick base pairing could be employed in polι for bypass of dG-AAF. In polι with Hoogsteen paired dG-AAF the bulky AAF moiety would reside on the cramped minor groove side of the template. The Hoogsteen-capable conformation distorts the active site, disrupting interactions necessary for error-free incorporation of dC opposite the lesion. Watson-Crick pairing places the AAF rings on the spacious major groove side, similar to the position of minor groove adducts observed with Hoogsteen pairing. Watson-Crick paired structures show a well-ordered active site, with a near reaction-ready ternary complex. Thus our results suggest that polι would utilize the same spacious region for lesion bypass of both major and minor groove adducts. Therefore, purine adducts with bulk on the minor groove side would use Hoogsteen pairing, while adducts with the bulky lesion on the major groove side would utilize Watson-Crick base pairing as indicated by our MD simulations for dG-AAF. This suggests the possibility of an expanded role for polι in lesion bypass. PMID:19072536

[Determination of normal reference value of pyrrole adducts in urine in young people in a university in Shandong, China].

PubMed

Wang, Hui; Wang, Yiping; Zhou, Zhenwei; Wang, Shuo; Yin, Hongyin; Xie, Keqin

2015-06-01

To determine the normal reference value of pyrrole adducts in urine in young people in a university in Shandong, China, and to provide a reliable basis for the clinical diagnosis of n-hexane poisoning. A total of 240 college students were randomly selected. After excluding 32 ineligible students, 208 subjects were included in this study, consisting of 104 males and 104 females, with a mean age of 21?3 years (range: 18 to 24 years). Morning urine was collected from each subject. The content of pyrrole adducts was determined by chromatometry. The content of pyrrole adducts in both male and female obeyed a positively skewed distribution. The median level of pyrrole adducts in male subjects was 0.88 nmol/ml, and the reference value was 0.14-3.92 nmol/ml. The median level of pyrrole adducts in female subjects was 0.93 nmol/ ml, and the reference value was 0.09-3.27 nmol/ml. Student's t test identified no statistical difference in pyrrole adduct level between male and female subjects (t=0.15, P>0.05). The median level of pyrrole adducts in normal young people is 0.91 nmol/ml, and the reference value is 0.11-3.95 nmol/ml.

Single d(ApG)/cis-diamminedichloroplatinum(II) adduct-induced mutagenesis in Escherichia coli

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

Burnouf, D.; Fuchs, R.P.P.; Gauthier, C.

1990-08-01

The mutation spectrum induced by the widely used antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP) showed that cisDDP(d(ApG)) adducts, although they account for only 25% of the lesions formed are {approx}5 times more mutagenic than the major GG adduct. The authors report the construction of vectors bearing a single cisDDP(d(ApG)) lesion and their use in mutagenesis experiments in Escherichia coli. The mutagenic processing of the lesion is found to depend strictly on induction of the SOS system of the bacterial host cells. In SOS-induced cells, mutation frequencies of 1-2% were detected. All these mutations are targeted to the 5{prime} base of the adduct.more » Single A {yields} T transversions are mainly observed (80%), whereas A {yields} G transitions account for 10% of the total mutations. Tandem base-pair substitutions involving the adenine residue and the thymine residue immediately 5{prime} to the adduct occur at a comparable frequency (10%). No selective loss of the strand bearing the platinum adduct was seen, suggesting that, in vivo, cisDDP(d(ApG)) adducts are not blocking lesions. The high mutation specificity of cisDDP-(d(ApG))-induced mutagenesis is discussed in relation to structural data.« less

The influence of ochratoxin A on DNA adduct formation by the carcinogen aristolochic acid in rats.

PubMed

Stiborová, Marie; Bárta, František; Levová, Kateřina; Hodek, Petr; Frei, Eva; Arlt, Volker M; Schmeiser, Heinz H

2015-11-01

Exposure to the plant nephrotoxin and carcinogen aristolochic acid (AA) leads to the development of AA nephropathy, Balkan endemic nephropathy (BEN) and upper urothelial carcinoma (UUC) in humans. Beside AA, exposure to ochratoxin A (OTA) was linked to BEN. Although OTA was rejected as a factor for BEN/UUC, there is still no information whether the development of AA-induced BEN/UUC is influenced by OTA exposure. Therefore, we studied the influence of OTA on the genotoxicity of AA (AA-DNA adduct formation) in vivo. AA-DNA adducts were formed in liver and kidney of rats treated with AA or AA combined with OTA, but no OTA-related DNA adducts were detectable in rats treated with OTA alone or OTA combined with AA. Compared to rats treated with AA alone, AA-DNA adduct levels were 5.4- and 1.6-fold higher in liver and kidney, respectively, of rats treated with AA combined with OTA. Although AA and OTA induced quinone oxidoreductase (NQO1) activating AA to DNA adducts, their combined treatment did not lead to either higher NQO1 enzyme activity or higher AA-DNA adduct levels in ex vivo incubations. Oxidation of AA I (8-methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxole-5-carboxylic acid) to its detoxification metabolite, 8-hydroxyaristolochic acid, was lower in microsomes from rats treated with AA and OTA, and this was paralleled by lower activities of cytochromes P450 1A1/2 and/or 2C11 in these microsomes. Our results indicate that a decrease in AA detoxification after combined exposure to AA and OTA leads to an increase in AA-DNA adduct formation in liver and kidney of rats.

DNA adducts induced by in vitro activation of extracts of diesel and biodiesel exhaust particles.

PubMed

Ross, Jeffrey A; Nelson, Garret B; Mutlu, Esra; Warren, Sarah H; Gilmour, M Ian; DeMarini, David M

2015-01-01

Biodiesel and biodiesel-blend fuels offer a renewable alternative to petroleum diesel, but few data are available concerning the carcinogenic potential of biodiesel exhausts. We compared the formation of covalent DNA adducts by the in vitro metabolic activation of organic extracts of diesel-exhaust particles (DEP) from petroleum diesel and soy biodiesel and correlated DNA adduct levels and mutagenicity in Salmonella TA100. We examined two different DEP from petroleum diesel (C-DEP and B0), one from soy bean oil biodiesel (B100) and one from combustion of a blend of 20% B100 and 80% B0 (B20) for in vitro DNA adduct-forming potential under oxidative or nitroreductive conditions in the presence of calf thymus DNA as well as in vivo in Salmonella TA100. The modified DNA was hydrolyzed and analyzed by (32)P-postlabeling using either butanol extraction or nuclease P1 pre-enrichment. Multiple DNA adducts were produced with chromatographic mobilities consistent with PAH and nitro-PAH adducts. The types and quantities of DNA adducts produced by the two independent petroleum diesel DEP were similar, with both polycyclic aromatic hydrocarbon (PAH)- and nitro-PAH-derived adducts formed. Relative potencies for S9-mediated DNA adduct formation, either per mass of particulate or per MJ(th) energy consumed were B100 > B0 > B20. Soy biodiesel emissions induced DNA damage in the form of presumptive PAH and nitro-PAH DNA adducts that correlated with mutagenicity in Salmonella. B20 is the soy biodiesel used most commonly in the US, and it produced the lowest DNA adduct-emission factor, ∼50% that of petroleum diesel.

Formation mechanism of glyoxal-DNA adduct, a DNA cross-link precursor.

PubMed

Vilanova, B; Fernández, D; Casasnovas, R; Pomar, A M; Alvarez-Idaboy, J R; Hernández-Haro, N; Grand, A; Adrover, M; Donoso, J; Frau, J; Muñoz, F; Ortega-Castro, J

2017-05-01

DNA nucleobases undergo non-enzymatic glycation to nucleobase adducts which can play important roles in vivo. In this work, we conducted a comprehensive experimental and theoretical kinetic study of the mechanisms of formation of glyoxal-guanine adducts over a wide pH range in order to elucidate the molecular basis for the glycation process. Also, we performed molecular dynamics simulations to investigate how open or cyclic glyoxal-guanine adducts can cause structural changes in an oligonucleotide model. A thermodynamic study of other glycating agents including methylglyoxal, acrolein, crotonaldehyde, 4-hydroxynonenal and 3-deoxyglucosone revealed that, at neutral pH, cyclic adducts were more stable than open adducts; at basic pH, however, the open adducts of 3-deoxyglucosone, methylglyoxal and glyoxal were more stable than their cyclic counterparts. This result can be ascribed to the ability of the adducts to cross-link DNA. The new insights may contribute to improve our understanding of the connection between glycation and DNA cross-linking. Copyright © 2017 Elsevier B.V. All rights reserved.

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...

Endogenous 5-methylcytosine protects neighboring guanines from N7 and O6-methylation and O6-pyridyloxobutylation by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

PubMed

Ziegel, Rebecca; Shallop, Anthony; Upadhyaya, Pramod; Jones, Roger; Tretyakova, Natalia

2004-01-20

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously

IDENTIFICATION OF STEREOCHEMICAL CONFIGURATIONS OF CYCLOPENTA[CD]PYRENE-DNA ADDUCTS IN STRAIN A/J MOUSE LUNG AND C3H10T1/2CL8 CELLS

EPA Science Inventory

Identification of Sterochemical Configurations of Cyclopent A[cd]Pyrene DNA Adducts in Strain A/J Mouse Lung and C3H10T1/2CL8 Cells.

Four major and several minor DNA adducts were resolved by 32P-postlabeling analysis of DNA from strain A/J mouse lung and C3H10T1/2CL8 (C3H...

Formation of diastereomeric benzo[a]pyrene diol epoxide-guanine adducts in p53 gene-derived DNA sequences.

PubMed

Matter, Brock; Wang, Gang; Jones, Roger; Tretyakova, Natalia

2004-06-01

(-)-trans-N(2)-BPDE-dG, 2.1 and 8.5% for (-)-cis-N(2)-BPDE-dG, and 0.5 and 8.3% for (+)-cis-N(2)-BPDE-dG. The relative yields of the minor N(2)-BPDE-dG stereoisomers were elevated at the sites of inefficient adduction, while the major (+)-trans-BPDE lesion was even more dominant at the frequently adducted sites. The introduction of 5-methyl groups at adjacent cytosine bases increased the yields of N(2)-BPDE-dG diastereomers, probably a result of favorable hydrophobic interactions between BPDE and 5-methylcytosine. The targeted formation of N(2)-BPDE-dG at (Me)CG dinucleotides within the p53 gene is consistent with the high prevalence of G --> T transversions at these sites in smoking-induced lung cancer.

An isotope-dilution UPLC-MS/MS technique for the human biomonitoring of the internal exposure to glycidol via a valine adduct at the N-terminus of hemoglobin.

PubMed

Hielscher, Jan; Monien, Bernhard H; Abraham, Klaus; Jessel, Sönke; Seidel, Albrecht; Lampen, Alfonso

2017-08-01

Fatty acid esters of glycidol (glycidyl esters) are processing contaminants generated as a byproduct of the industrial deodorization of vegetable oils and fats. Oral intake of glycidyl esters leads to the release of glycidol in the gastrointestinal tract. Glycidol is carcinogenic, genotoxic and teratogenic in rodents. It is rated as probably carcinogenic to humans (IARC group 2A). The determination of internal exposure of glycidol may support the assessment of the possible human health risks related to glycidyl ester intake. For this purpose, hemoglobin adducts of glycidol may be suitable biomarkers reflecting the cumulative exposure of up to four months. We applied a modified Edman degradation to assess the glycidol adduct at the N-terminal valine, N-(2,3-dihydroxypropyl)-valine (2,3-diHOPr-Val), of hemoglobin. The modified valine was cleaved with fluorescein-5-isothiocyanate (FITC), resulting in the formation of N-(2,3-dihydroxypropyl)-valine fluorescein thiohydantoin (DHP-Val-FTH). An isotope-dilution technique was developed for the quantification of the thiohydantoin analyte by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and DHP-Val-d 7 -FTH as reference standard. The limit of detection was 4 fmol DHP-Val-FTH per injection corresponding to 0.7pmol 2,3-diHOPr-Val/g hemoglobin. The adduct levels in blood samples of 12 non-smoking participants were in the range of 2.2-4.9pmol 2,3-diHOPr-Val/g hemoglobin. The current work presents the first isotope-dilution technique using UPLC-MS/MS for the quantification of 2,3-diHOPr-Val at the N-terminus of hemoglobin as a sensitive and convenient alternative to earlier GC-MS methods. Copyright © 2017 Elsevier B.V. All rights reserved.

Lack of contribution of covalent benzo[a]pyrene-7,8-quinone-DNA adducts in benzo[a]pyrene-induced mouse lung tumorigenesis.

PubMed

Nesnow, Stephen; Nelson, Garret; Padgett, William T; George, Michael H; Moore, Tanya; King, Leon C; Adams, Linda D; Ross, Jeffrey A

2010-07-30

Benzo[a]pyrene (B[a]P) is a potent human and rodent lung carcinogen. This activity has been ascribed in part to the formation of anti-trans-7,8-dihydroxy-7,8-dihydroB[a]P-9,10-epoxide (BPDE)-DNA adducts. Other carcinogenic mechanisms have been proposed: (1) the induction of apurinic sites from radical cation processes, and (2) the metabolic formation of B[a]P-7,8-quinone (BPQ) that can form covalent DNA adducts or reactive oxygen species which can damage DNA. The studies presented here sought to examine the role of stable BPQ-DNA adducts in B[a]P-induced mouse lung tumorigenesis. Male strain A/J mice were injected intraperitoneally once with BPQ or trans-7,8-dihydroxy-7,8-dihydroB[a]P (BP-7,8-diol) at 30, 10, 3, or 0mg/kg. Lungs and livers were harvested after 24h, the DNA extracted and subjected to (32)P-postlabeling analysis. Additional groups of mice were dosed once with BPQ or BP-7,8-diol each at 30 mg/kg and tissues harvested 48 and 72 h later, or with B[a]P (50mg/kg, a tumorigenic dose) and tissues harvested 72 h later. No BPQ or any other DNA adducts were observed in lung or liver tissues 24, 48, or 72 h after the treatment with 30 mg/kg BPQ. BP-7,8-diol gave BPDE-DNA adducts at all time points in both tissues and B[a]P treatment gave BPDE-DNA adducts in the lung. In each case, no BPQ-DNA adducts were detected. Mouse body weights significantly decreased over time after BPQ or BP-7,8-diol treatments suggesting that systemic toxicity was induced by both agents. Model studies with BPQ and N-acetylcysteine suggested that BPQ is rapidly inactivated by sulfhydryl-containing compounds and not available for DNA adduction. We conclude that under these treatment conditions BPQ does not form stable covalent DNA adducts in the lungs or livers of strain A/J mice, suggesting that stable BPQ-covalent adducts are not a part of the complex of mechanisms involved in B[a]P-induced mouse lung tumorigenesis. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Reactive aldehyde metabolites from the anti-HIV drug abacavir: amino acid adducts as possible factors in abacavir toxicity.

PubMed

Charneira, Catarina; Godinho, Ana L A; Oliveira, M Conceição; Pereira, Sofia A; Monteiro, Emília C; Marques, M Matilde; Antunes, Alexandra M M

2011-12-19

bionucleophiles occurs exclusively via Schiff base formation, whereas soft sulfur nucleophiles react by Michael-type 1,4-addition to the α,β-unsaturated system. The synthetic phenylthiohydantoin adduct was subsequently used as standard for LC-ESI-MS monitoring of N-terminal valine adduct formation, upon modification of human hemoglobin in vitro with the conjugated abacavir aldehyde, followed by reduction and Edman degradation. The same postmodification strategy was applied to investigate the products formed by incubation of abacavir with rat liver cytosol, followed by trapping with ethyl valinate. In both instances, the major adduct detected corresponded to the synthetic phenylthiohydantoin standard. These results suggest that abacavir metabolism to the carboxylate(s) via aldehyde intermediate(s) could be a factor in the toxic events elicited by abacavir administration. Furthermore, the availability of a reliable and fully characterized synthetic standard of the abacavir adduct with the N-terminal valine of hemoglobin and its easy detection in the model hemoglobin modifications support the usefulness of this adduct as a prospective biomarker of abacavir toxicity in humans. © 2011 American Chemical Society

Raman spectroscopic evaluation of DNA adducts of a platinum containing anticancer drug.

PubMed

Jangir, Deepak K; Mehrotra, Ranjana

2014-09-15

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. Copyright © 2014 Elsevier B.V. All rights reserved.

Synthesis of densely substituted trans-configured 4-acylated piperidine-2,4-diones as 3:1 adducts of imines and ketenes.

PubMed

Cabrera, José; Hellmuth, Tina; Peters, René

2010-06-18

An operationally simple method is described to form densely substituted diastereomerically pure trans-configured and potentially biologically interesting 5,6-dihydropyridone derivatives as 3:1 adducts of ketenes formed in situ from acyl bromides and aromatic imines.

Efficient CO2 capture by tertiary amine-functionalized ionic liquids through Li+-stabilized zwitterionic adduct formation

PubMed Central

Yang, Zhen-Zhen

2014-01-01

Summary Highly efficient CO2 absorption was realized through formation of zwitterionic adducts, combining synthetic strategies to ionic liquids (ILs) and coordination. The essence of our strategy is to make use of multidentate cation coordination between Li+ and an organic base. Also PEG-functionalized organic bases were employed to enhance the CO2-philicity. The ILs were reacted with CO2 to form the zwitterionic adduct. Coordination effects between various lithium salts and neutral ligands, as well as the CO2 capacity of the chelated ILs obtained were investigated. For example, the CO2 capacity of PEG150MeBu2N increased steadily from 0.10 to 0.66 (mol CO2 absorbed per mol of base) through the formation of zwitterionic adducts being stabilized by Li+. PMID:25246955

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

Evidence for phosphorus bonding in phosphorus trichloride-methanol adduct: a matrix isolation infrared and ab initio computational study.

PubMed

Joshi, Prasad Ramesh; Ramanathan, N; Sundararajan, K; Sankaran, K

2015-04-09

The weak interaction between PCl3 and CH3OH was investigated using matrix isolation infrared spectroscopy and ab initio computations. In a nitrogen matrix at low temperature, the noncovalent adduct was generated and characterized using Fourier transform infrared spectroscopy. Computations were performed at B3LYP/6-311++G(d,p), B3LYP/aug-cc-pVDZ, and MP2/6-311++G(d,p) levels of theory to optimize the possible geometries of PCl3-CH3OH adducts. Computations revealed two minima on the potential energy surface, of which, the global minimum is stabilized by a noncovalent P···O interaction, known as a pnictogen bonding (phosphorus bonding or P-bonding). The local minimum corresponded to a cyclic adduct, stabilized by the conventional hydrogen bonding (Cl···H-O and Cl···H-C interactions). Experimentally, 1:1 P-bonded PCl3-CH3OH adduct in nitrogen matrix was identified, where shifts in the P-Cl modes of PCl3, O-C, and O-H modes of CH3OH submolecules were observed. The observed vibrational frequencies of the P-bonded adduct in a nitrogen matrix agreed well with the computed frequencies. Furthermore, computations also predicted that the P-bonded adduct is stronger than H-bonded adduct by ∼1.56 kcal/mol. Atoms in molecules and natural bond orbital analyses were performed to understand the nature of interactions and effect of charge transfer interaction on the stability of the adducts.

Malondialdehyde-Deoxyguanosine Adduct Formation in Workers of Pathology Wards. The Role of Air Formaldehyde Exposure

PubMed Central

Romanazzi, Valeria; Munnia, Armelle; Piro, Sara; Allione, Alessandra; Ricceri, Fulvio; Guarrera, Simonetta; Pignata, Cristina; Matullo, Giuseppe; Wang, Poguang; Giese, Roger W.; Peluso, Marco

2010-01-01

Background Formaldehyde is a ubiquitous pollutant to which humans are exposed. Pathologists can experience high formaldehyde exposure levels. Formaldehyde – among other properties – induce oxidative stress and free radicals, which react with DNA and lipids, leading to oxidative damage and lipid peroxidation, respectively. We measured the levels of air-formaldehyde exposure in a group of Italian pathologists and controls. We analyzed the effect of formaldehyde exposure on leukocyte malondialdehyde-deoxyguanosine adducts (M1-dG), a biomarker of oxidative stress and lipid peroxidation. We studied the relationship between air-formaldehyde and M1-dG adducts. Methods Air-formaldehyde levels were measured by personal air samplers. M1-dG adducts were analyzed by 32P-postlabelling assay. Results Reduction rooms pathologists were significantly exposed to air-formaldehyde in respect to controls and to the pathologists working in other laboratory areas (p<0.001). A significant difference for M1-dG adducts between exposed pathologists and controls was found (p=0.045). The effect becomes stronger when the evaluation of air-formaldehyde exposure was based on personal samplers (p=0.018). Increased M1dG adduct levels were only found in individuals exposed to air-formaldehyde concentrations higher than 66 μg/m3. When the exposed workers and controls were subgrouped according to smoking, M1-dG tended to increase in all the subjects but a significant association between M1-dG and air-formaldehyde was only found in not smokers (p= 0.009). Air formaldehyde played a role positive but not significant (r = 0.355, p = 0.075, Pearson correlation) in the formation of M1-dG, only in not smokers. Conclusions Working in the reduction rooms and to be exposed to air-formaldehyde concentrations higher than 66 μg/m3 is associated with increased levels of M1-dG adducts. PMID:20707408

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

PubMed

Batal, Mohamed; Boudry, Isabelle; Mouret, Stéphane; Wartelle, Julien; Emorine, Sandy; Bertoni, Marine; Bérard, Izabel; Cléry-Barraud, Cécile; Douki, Thierry

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 6h 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. © 2013.

Protein adducts as biomarkers of exposure to aromatic diisocyanates in workers manufacturing polyurethane (PUR) foam.

PubMed

Säkkinen, Kirsi; Tornaeus, Jarkko; Hesso, Antti; Hirvonen, Ari; Vainio, Harri; Norppa, Hannu; Rosenberg, Christina

2011-04-01

This work was undertaken to investigate the usefulness of diisocyanate-related protein adducts in blood samples as biomarkers of occupational exposure to toluene diisocyanate (TDI; 2,4- and 2,6-isomers) and 4,4'-methylenediphenyl diisocyanate (MDI). Quantification of adducts as toluene diamines (TDAs) and methylenedianiline (MDA) was performed on perfluoroacylated derivatives by gas chromatography-mass spectrometry (GC-MS/MS) in negative chemical ionisation mode. TDI-derived adducts were found in 77% of plasma and in 59% of globin samples from exposed workers manufacturing flexible polyurethane foam. The plasma levels ranged from 0.003 to 0.58 nmol mL(-1) and those in globin from 0.012 to 0.33 nmol g(-1). The 2,6-isomer amounted to about two-thirds of the sum concentration of TDA isomers. MDI-derived adducts were detected in 3.5% of plasma and in 7% of globin samples from exposed workers manufacturing rigid polyurethane foam. A good correlation was found between the sum of TDA isomers in urine and that in plasma. The relationship between globin adducts and urinary metabolites was ambiguous. Monitoring TDI-derived TDA in plasma thus appears to be an appropriate method for assessing occupational exposure. Contrary to TDI exposure, adducts in plasma or globin were not useful in assessing workers' exposure to MDI. An important outcome of the study was that no amine-related adducts were detected in globin samples from TDI- or MDI-exposed workers, alleviating concerns that TDI or MDI might pose a carcinogenic hazard. Further studies are nevertheless required to judge whether diisocyanates per se could be such a hazard.

Complexation of rhodium(II) tetracarboxylates with aliphatic diamines in solution: 1H and 13C NMR and DFT investigations.

PubMed

Jaźwiński, Jarosław; Sadlej, Agnieszka

2013-10-01

The complexation of rhodium(II) tetraacetate, tetrakistrifluoroaceate and tetrakisoctanoate with a set of diamines (ethane-1,diamine, propane-1,3-diamine and nonane-1,9-diamine) and their N,N'-dimethyl and N,N,N',N'-tetramethyl derivatives in chloroform solution has been investigated by (1) H and (13) C NMR spectroscopy and density functional theory (DFT) modelling. A combination of two bifunctional reagents, diamines and rhodium(II) tetracarboxylates, yielded insoluble coordination polymers as main products of complexation and various adducts in the solution, being in equilibrium with insoluble material. All diamines initially formed the 2 : 1 (blue), (1 : 1)n oligomeric (red) and 1 : 2 (red) axial adducts in solution, depending on the reagents' molar ratio. Adducts of primary and secondary diamines decomposed in the presence of ligand excess, the former via unstable equatorial complexes. The complexation of secondary diamines slowed down the inversion at nitrogen atoms in NH(CH3 ) functional groups and resulted in the formation of nitrogenous stereogenic centres, detectable by NMR. Axial adducts of tertiary diamines appeared to be relatively stable. The presence of long aliphatic chains in molecules (adducts of nonane-1,9-diamines or rhodium(II) tetrakisoctanoate) increased adduct solubility. Hypothetical structures of the equatorial adduct of rhodium(II) tetraacetate with ethane-1,2-diamine and their NMR parameters were explored by means of DFT calculations. Copyright © 2013 John Wiley & Sons, Ltd.

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.

Gender differences in hip adduction motion and torque during a single-leg agility maneuver.

PubMed

Hewett, Timothy E; Ford, Kevin R; Myer, Gregory D; Wanstrath, Kim; Scheper, Melia

2006-03-01

The purpose of this study was to identify gender differences in hip motion and kinetics during a single leg bidirectional deceleration maneuver. The rationale for the development of this maneuver was to test dynamic hip control during the deceleration of three different types of single-leg landings. The hypothesis was that female athletes would display increased hip adduction angles and moments during the maneuver compared to male athletes. Thirty-six collegiate soccer players (19 female, 17 male) volunteered to participate. Subjects were instructed to start the maneuver balancing on one foot, to hop through an agility-speed ladder on the same leg "up two boxes, back one, and then up one and hold it." Hip kinematics and kinetics during all three landings were examined. Females demonstrated significantly greater hip adduction angles at initial contact during all three landings and greater maximal hip adduction during landings 1 and 2 compared to male athletes. Females also exhibited significantly increased external hip adduction moments during landing 1, however, no differences were found between genders during landings 2 and 3. Copyright 2006 Orthopaedic Research Society.

32P-postlabeling analysis of dibenz[a,j]acridine DNA adducts in mice: preliminary determination of initial genotoxic metabolites and their effect on biomarker levels.

PubMed

Roh, J; Schamer, M; Reilman, R; Xue, W; Warshawsky, D; Talaska, G

1993-01-01

N-Heterocyclic aromatics (NHA) are widely occurring environmental pollutants formed during the pyrolysis of nitrogen-containing organic chemicals. NHA are found in significant amounts in tobacco condensates, synthetic fuels, gasoline engine exhaust, and effluents from the heating of coal. Dibenz[a,j]acridine (DBA) is an example of NHA. The potency of many carcinogenic compounds is related, at least in part, to the efficiency of their biological activation. We undertook studies to determine which initial metabolites of DBA lead to the formation of high levels of carcinogen-DNA adducts in vivo. DBA and its metabolites, trans-DBA-1,2-dihydrodiol (DBA-1,2-DHD), trans-DBA-3,4-dihydrodiol (DBA-3,4-DHD), and trans-DBA-5,6-dihydrodiol (DBA-5,6-DHD), were applied to the skin of mice. DNA was isolated using enzyme-solvent extraction method. DNA was 32P-postlabeled under conditions of limiting [32P]ATP. In skin, DBA produced two distinct adducts. The same two adducts were seen when DBA-3,4-DHD was applied. In addition the total adduct level elicited by DBA-3,4-DHD was higher than that of parent compound. Two adducts were seen when DBA-5,6DHD was applied, but these were very different from adducts seen with DBA. These results suggested that activation of DBA to DNA-binding compounds in skin includes initial formation of DBA-3,4-DHD. The data support development of biomarkers for the exposure and effect of this compound, and also suggest that specific metabolic susceptibility markers might be able to predict populations at increased risk.

How Y-Family DNA polymerase IV is more accurate than Dpo4 at dCTP insertion opposite an N2-dG adduct of benzo[a]pyrene.

PubMed

Sholder, Gabriel; Creech, Amanda; Loechler, Edward L

2015-11-01

To bypass DNA damage, cells have Y-Family DNA polymerases (DNAPs). One Y-Family-class includes DNAP κ and DNAP IV, which accurately insert dCTP opposite N(2)-dG adducts, including from the carcinogen benzo[a]pyrene (BP). Another class includes DNAP η and DNAP V, which insert accurately opposite UV-damage, but inaccurately opposite BP-N(2)-dG. To investigate structural differences between Y-Family-classes, regions are swapped between DNAP IV (a κ/IV-class-member) and Dpo4 (a η/V-class-member); the kinetic consequences are evaluated via primer-extension studies with a BP-N(2)-dG-containing template. Four key structural elements are revealed. (1) Y-Family DNAPs have discreet non-covalent contacts between their little finger-domain (LF-Domain) and their catalytic core-domain (CC-Domain), which we call "non-covalent bridges" (NCBs). Arg37 and Arg38 in DNAP IV's CC-Domain near the active site form a non-covalent bridge (AS-NCB) by interacting with Glu251 and Asp252, respectively, in DNAP IV's LF-Domain. Without these interactions dATP/dGTP/dTTP misinsertions increase. DNAP IV's AS-NCB suppresses misinsertions better than Dpo4's equivalent AS-NCB. (2) DNAP IV also suppresses dATP/dGTP/dTTP misinsertions via a second non-covalent bridge, which is ∼8Å from the active site (Distal-NCB). Dpo4 has no Distal-NCB, rendering it inferior at dATP/dGTP/dTTP suppression. (3) dCTP insertion is facilitated by the larger minor groove opening near the active site in DNAP IV versus Dpo4, which is sensible given that Watson/Crick-like [dCTP:BP-N(2)-dG] pairing requires the BP-moiety to be in the minor groove. (4) Compared to Dpo4, DNAP IV has a smaller major groove opening, which suppresses dGTP misinsertion, implying BP-N(2)-dG bulk in the major groove during Hoogsteen syn-adduct-dG:dGTP pairing. In summary, DNAP IV has a large minor groove opening to enhance dCTP insertion, a plugged major groove opening to suppress dGTP misinsertion, and two non-covalent bridges (near and distal

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells

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

Ganesan, Shanthi, E-mail: shanthig@iastate.edu; Keating, Aileen F., E-mail: akeating@iastate.edu

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 6more » 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.« less

Regiochemically controlled synthesis of a β-4-β' [70]fullerene bis-adduct

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

Cerón, Maira R.; Castro, Edison; Neti, Venkata S. Pavan K.

2016-12-22

A β-4-β' C 70 bis-adduct regioisomer and an uncommon mono-adduct β-malonate C 70 derivative were synthesized by using a Diels–Alder cycloaddition followed by an addition–elimination of bromo-ethylmalonate and a retro-Diels–Alder cycloaddition reaction. Here, we also report the regioselective synthesis and spectroscopic characterization of C s-symmetric tris- and C 2v-symmetric tetra-adducts of C 70, which are the precursors of the mono- and bis-adduct final products.

Formation of PAH-DNA adducts after in vivo and vitro exposure of rats and lung cells to different commercial carbon blacks.

PubMed

Borm, Paul J A; Cakmak, Gonca; Jermann, Erich; Weishaupt, Christel; Kempers, Pascal; van Schooten, Frederik Jan; Oberdörster, Günter; Schins, Roel P F

2005-06-01

The current study was designed to test the possible release and bioavailability of polycyclic aromatic hydrocarbons (PAHs) from a set of commercial carbon blacks (CBs) as well as the ability of these PAHs to form bulky DNA adducts. In four commercial CBs (Printex 90, Sterling V, N330, Lampblack 101), leaching of PAH was examined through (1) release of parent PAHs in saline with or without surfactant, and (2) PAH adducts in lung epithelial cells (A549) or in rat lungs after exposure to two CBs (Printex 90, Sterling V) for 13 weeks (50 mg/m(3)). In vitro experiments were done with original and extracted particles, as well as organic extracts of CB in DMSO. As positive controls, B[a]P (0.03 microM) and a mixture of 16 PAHs (0.1 microM) were used. No leaching of PAHs was measured in saline or surfactant-containing saline. In vitro incubations with CB particles (30-300 microg/cm(2)) revealed no adduct spots except for Sterling V. However, the spot was not concentration dependent and remains unidentified. Lung DNA from rats after inhalation of Printex 90 or Sterling V showed no spots related to PAH-DNA adduct formation compared to sham-exposed rats. The results suggest that PAHs are very tightly bound to these CBs. Only using organic extracts or particles of low-surface Sterling V, with high PAH content, PAHs may become available to form PAH-DNA adducts. However, the in vitro conditions showing this effect will not be encountered in vivo and renders this mechanism in particle-induced lung cancer at in vivo exposures highly unlikely.

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...

Spectral characterization of guanine C4-OH adduct: a radiation and quantum chemical study.

PubMed

Phadatare, Suvarna D; Sharma, Kiran Kumar K; Rao, B S M; Naumov, S; Sharma, Geeta K

2011-11-24

The reaction of hydroxyl radical ((•)OH) with guanine was investigated under restricted pH condition (pH 4.6) using pulse radiolysis technique. The time-resolved optical transient absorption spectra showed two peaks centered at 300 and 330 nm at 4 μs after the pulse which exhibited different reactivity toward molecular oxygen (O(2)). The peak at 300 nm was found to be relatively more stable than the peak at 330 nm. The peak corresponding to 330 nm decayed within 20 μs having a first order rate constant 4-7 × 10(4) s(-1) and was pH dependent. On longer time scale, the species decayed by a bimolecular process. The presence of O(2) did not affect its decay rate constant. The (•)OH reacts with guanine at pH 4.6 with a diffusion-controlled second order rate constant of ≥1 × 10(10) mol(-1) dm(3) s(-1). The reaction of Br(2)(•-), O(2)(•-), and 2-hydroxy-2-propyl radical with guanine was also investigated to differentiate among the one-electron oxidized, one-electron reduced species of guanine and the guanine-OH adducts formed in the reaction of (•)OH at pH 4.6. On the basis of the spectral characteristics and reactivity toward O(2), two guanine-OH adduct species were identified (i) the C4-OH adduct species absorbing at 330 nm which has not been reported so far and (ii) the C8-OH adduct species absorbing at 300 nm in agreement with the known literature absorption features. Quantum chemical calculations using BHandHLYP with 6-31+G(d,p) basis set and excited state calculations using TDDFT for all possible transients complement the assignment of the observed spectral peak at 330 nm to the C4-OH adduct of guanine. Furthermore, steady state radiolysis revealed the formation of 8-hydroxy-guanine whose precursor is known to be the C8-OH adduct species. © 2011 American Chemical Society

Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells.

PubMed

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 6μM) for 24 or 48h. Cell viability was reduced (P<0.05) after 48h of exposure to 3 or 6μM PM. The NOR-G-OH DNA adduct was detected after 24h of 6μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48h 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. Copyright © 2014 Elsevier Inc. All rights reserved.

40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

Code of Federal Regulations, 2014 CFR

2014-07-01

... epoxy adduct. 721.1850 Section 721.1850 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

Code of Federal Regulations, 2012 CFR

2012-07-01

... epoxy adduct. 721.1850 Section 721.1850 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

Code of Federal Regulations, 2013 CFR

2013-07-01

... epoxy adduct. 721.1850 Section 721.1850 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

40 CFR 721.1850 - Toluene sulfonamide bis-phe-nol A epoxy adduct.

Code of Federal Regulations, 2011 CFR

2011-07-01

... epoxy adduct. 721.1850 Section 721.1850 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.1850 Toluene sulfonamide bis-phe-nol A epoxy adduct. (a) Chemical... as toluene sulfonamide bisphenol A epoxy adduct (PMN P-90-113) is subject to reporting under this...

Conformations of stereoisomeric base adducts to 4-hydroxyequilenin.

PubMed

Ding, Shuang; Shapiro, Robert; Geacintov, Nicholas E; Broyde, Suse

2003-06-01

Exposure to estrogen through estrogen replacement therapy increases the risk of women developing cancer in hormone sensitive tissues. Premarin (Wyeth), which has been the most frequent choice for estrogen replacement therapy in the United States, contains the equine estrogens equilin and equilenin as major components. 4-Hydroxyequilenin (4-OHEN) is a phase I metabolite of both of these substances. This catechol estrogen autoxidizes to potent cytotoxic quinoids that can react with dG, dA, and dC to form unusual stereoisomeric cyclic adducts (Bolton, J. L., et al. (1998) Chem. Res. Toxicol. 11, 1113-1127). Like other bulky DNA adducts, these lesions may exhibit different susceptibilities to DNA repair and mutagenic potential, if not repaired in a structure-dependent manner. To ultimately gain insights into structure-function relationships, we computed conformations of stereoisomeric guanine, adenine, and cytosine base adducts using density functional theory. We find near mirror image conformations in stereoisomer adduct pairs for each modified base, suggesting opposite orientations with respect to the 5' --> 3' direction of the modified strand when the stereoisomer pairs are incorporated into duplex DNA. Such opposite orientations could cause stereoisomer pairs of lesions to respond differently to DNA replication and repair enzymes.

Crosslinking reactions of 4-amino-6-oxo-2-vinylpyrimidine with guanine derivatives and structural analysis of the adducts

PubMed Central

Kusano, Shuhei; Ishiyama, Shogo; Lam, Sik Lok; Mashima, Tsukasa; Katahira, Masato; Miyamoto, Kengo; Aida, Misako; Nagatsugi, Fumi

2015-01-01

DNA interstrand crosslinks (ICLs) are the primary mechanism for the cytotoxic activity of many clinical anticancer drugs, and numerous strategies for forming ICLs have been developed. One such method is using crosslink-forming oligonucleotides (CFOs). In this study, we designed a 4-amino-6-oxo-2-vinylpyrimidine (AOVP) derivative with an acyclic spacer to react selectively with guanine. The AOVP CFO exhibited selective crosslinking reactivity with guanine and thymine in DNA, and with guanine in RNA. These crosslinking reactions with guanine were accelerated in the presence of CoCl2, NiCl2, ZnCl2 and MnCl2. In addition, we demonstrated that the AOVP CFO was reactive toward 8-oxoguanine opposite AOVP in the duplex DNA. The structural analysis of each guanine and 8-oxoguanine adduct in the duplex DNA was investigated by high-resolution NMR. The results suggested that AOVP reacts at the N2 amine in guanine and at the N1 or N2 amines in 8-oxoguanine in the duplex DNA. This study demonstrated the first direct determination of the adduct structure in duplex DNA without enzyme digestion. PMID:26245348

Validation of a food frequency questionnaire measurement of dietary acrylamide intake using hemoglobin adducts of acrylamide and glycidamide

PubMed Central

Wilson, Kathryn M.; Vesper, Hubert W.; Tocco, Paula; Sampson, Laura; Rosén, Johan; Hellenäs, Karl-Erik; Törnqvist, Margareta; Willett, Walter C.

2011-01-01

Objective Acrylamide, a probable human carcinogen, is formed during high-heat cooking of many common foods. The validity of food frequency questionnaire (FFQ) measures of acrylamide intake has not been established. We assessed the validity of acrylamide intake calculated from an FFQ using a biomarker of acrylamide exposure. Methods We calculated acrylamide intake from an FFQ in the Nurses' Health Study II. We measured hemoglobin adducts of acrylamide and its metabolite, glycidamide, in a random sample of 296 women. Correlation and regression analyses were used to assess the relationship between acrylamide intake and adducts. Results The correlation between acrylamide intake and the sum of acrylamide and glycidamide adducts was 0.31 (95% CI: 0.20 – 0.41), adjusted for laboratory batch, energy intake, and age. Further adjustment for BMI, alcohol intake, and correction for random within-person measurement error in adducts gave a correlation of 0.34 (CI: 0.23 – 0.45). The intraclass correlation coefficient for the sum of adducts was 0.77 in blood samples collected 1 to 3 years apart in a subset of 45 women. Intake of several foods significantly predicted adducts in multiple regression. Conclusions Acrylamide intake and hemoglobin adducts of acrylamide and glycidamide were moderately correlated. Within-person consistency in adducts was high over time. PMID:18855107

Effect of scapular stabilization during horizontal adduction stretching on passive internal rotation and posterior shoulder tightness in young women volleyball athletes: a randomized controlled trial.

PubMed

Salamh, Paul A; Kolber, Morey J; Hanney, William J

2015-02-01

To evaluate the effect of scapular stabilization during horizontal adduction stretching (cross-body) on posterior shoulder tightness (PST) and passive internal rotation (IR). Randomized controlled trial with single blinding. Athletic club. Asymptomatic volleyball players who are women with glenohumeral internal rotation deficit (N=60). Subjects were randomly assigned to either horizontal adduction stretching with manual scapular stabilization (n=30) or horizontal adduction stretching without stabilization (n=30). Passive stretching was performed for 3- to 30-second holds in both groups. Range of motion measurements of PST and IR were performed on the athlete's dominant shoulder prior to and immediately after the intervention. Baseline mean angular measurements of PST and IR for all athletes involved in the study were 62°±14° and 40°±10°, respectively, with no significant difference between groups (P=.598 and P=.734, respectively). Mean PST measurements were significantly different between groups after the horizontal adduction stretch, with a mean angle of 83°±17° among the scapular stabilization group and 65°±13° among the nonstabilization group (P<.001). Measurements of IR were also significantly different between groups, with a mean angle of 51°±14° among the scapular stabilization group and 43°±9° among the nonstabilization group (P=.006). Horizontal adduction stretches performed with scapular stabilization produced significantly greater improvements in IR and PST than horizontal adduction stretching without scapular stabilization. Copyright © 2015 American Congress of Rehabilitation Medicine. Published by Elsevier Inc. All rights reserved.

Simultaneous quantification of soman and VX adducts to butyrylcholinesterase, their aged methylphosphonic acid adduct and butyrylcholinesterase in plasma using an off-column procainamide-gel separation method combined with UHPLC-MS/MS.

PubMed

Liu, Chang-Cai; Huang, Gui-Lan; Xi, Hai-Ling; Liu, Shi-Lei; Liu, Jing-Quan; Yu, Hui-Lan; Zhou, Shi-Kun; Liang, Long-Hui; Yuan, Ling

2016-11-15

This work describes a novel and sensitive non-isotope dilution method for simultaneous quantification of organophosphorus nerve agents (OPNAs) soman (GD) and VX adducts to butyrylcholinesterase (BChE), their aged methylphosphonic acid (MeP) adduct and unadducted BChE in plasma exposed to OPNA. OPNA-BChE adducts were isolated with an off-column procainamide-gel separation (PGS) from plasma, and then digested with pepsin into specific adducted FGES * AGAAS nonapeptide (NP) biomarkers. The resulting NPs were detected by UHPLC-MS/MS MRM. The off-column PGS method can capture over 90% of BChE, MeP-BChE, VX-BChE and GD-BChE from their respective plasma materials. One newly designed and easily synthesized phosphorylated BChE nonapeptide with one Gly-to-Ala mutation was successfully reported to serve as internal standard instead of traditional isotopically labeled BChE nonapeptide. The linear range of calibration curves were from 1.00-200ngmL -1 for VX-NP, 2.00-200ngmL -1 for GD-NP and MeP-NP (R 2 ≥0.995), and 3.00-200ngmL -1 for BChE NP (R 2 ≥0.990). The inter-day precision had relative standard deviation (%RSD) of <8.89%, and the accuracy ranged between 88.9-120%. The limit of detection was calculated to be 0.411, 0.750, 0.800 and 1.43ngmL -1 for VX-NP, GD-NP, MeP-NP and BChE NP, respectively. OPNA-exposed quality control plasma samples were characterized as part of method validation. Investigation of plasma samples unexposed to OPNA revealed no baseline values or interferences. Using the off-column PGS method combined with UHPLC-MS/MS, VX-NP and GD-NP adducts can be unambiguously detected with high confidence in 0.10ngmL -1 and 0.50ngmL -1 of exposed human plasma respectively, only requiring 0.1mL of plasma sample and taking about four hours without special sample preparation equipment. These improvements make it a simple, sensitive and robust PGS-UHPLC-MS/MS method, and this method will become an attractive alternative to immunomagnetic separation (IMS) method and

Gas-Phase Anionic σ-Adduct (Trans)formations in Heteroaromatic Systems1

NASA Astrophysics Data System (ADS)

Zimnicka, Magdalena; Danikiewicz, Witold

2015-07-01

Anions of nitroderivatives of thiophene and furan were subjected to the reactions with selected C-H acids in the gas phase. Various structures and reaction pathways were proposed for the observed ionic products. In general, the reactions of heteroaromatic anions with C-H acids may be divided into three groups, depending on the proton affinity difference between C-H acid's conjugate base and heteroaromatic anion (ΔPA). The proton transfer from C-H acid to heteroaromatic anion is a dominant process in the reactions for which ΔPA < 0 kcal mol-1, whereas the reactions with high ΔPA (ΔPA > 16 kcal mol-1) do not lead to any ionic products. The formation of σ-adducts and products of their further transformations according to the VNS, SNAr, cine, and tele substitution mechanisms have been proposed for reactions with moderate ΔPA. The other possible mechanisms as SN2 reaction, nucleophilic addition to the cyano group, ring-opening pathway, and halogenophilic reaction have also been discussed to contribute in the reactions between heteroaromatic anions and C-H acids.

Quantification of Hemoglobin and White Blood Cell DNA Adducts of the Tobacco Carcinogens 2-Amino-9H-pyrido[2,3-b]indole and 4-Aminobiphenyl Formed in Humans by Nanoflow Liquid Chromatography/Ion Trap Multistage Mass Spectrometry.

PubMed

Cai, Tingting; Bellamri, Medjda; Ming, Xun; Koh, Woon-Puay; Yu, Mimi C; Turesky, Robert J

2017-06-19

Aromatic amines covalently bound to hemoglobin (Hb) as sulfinamide adducts at the cysteine 93 residue of the Hb β chain have served as biomarkers to assess exposure to this class of human carcinogens for the past 30 years. In this study, we report that 2-amino-9H-pyrido[2,3-b]indole (AαC), an abundant carcinogenic heterocyclic aromatic amine formed in tobacco smoke and charred cooked meats, also reacts with Hb to form a sulfinamide adduct. A novel nanoflow liquid chromatography/ion trap multistage mass spectrometry (nanoLC-IT/MS 3 ) method was established to assess exposure to AαC and the tobacco-associated bladder carcinogen 4-aminobiphenyl (4-ABP) through their Hb sulfinamide adducts. Following mild acid hydrolysis of Hb in vitro, the liberated AαC and 4-ABP were derivatized with acetic anhydride to form the N-acetylated amines, which were measured by nanoLC-IT/MS 3 . The limits of quantification (LOQ) for AαC- and 4-ABP-Hb sulfinamide adducts were ≤7.1 pg/g Hb. In a pilot study, the mean level of Hb sulfinamide adducts of AαC and 4-ABP were, respectively, 3.4-fold and 4.8-fold higher in smokers (>20 cigarettes/day) than nonsmokers. In contrast, the major DNA adducts of 4-ABP, N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl, and AαC, N-(2'-deoxyguanosin-8-yl)-2-amino-9H-pyrido[2,3-b]indole, were below the LOQ (3 adducts per 10 9 bases) in white blood cell (WBC) DNA of smokers and nonsmokers. These findings reaffirm that tobacco smoke is a major source of exposure to AαC. Hb sulfinamide adducts are suitable biomarkers to biomonitor 4-ABP and AαC; however, neither carcinogen binds to DNA in WBC, even in heavy smokers, at levels sufficient for biomonitoring.

Pixantrone can be activated by formaldehyde to generate a potent DNA adduct forming agent

PubMed Central

Evison, Ben J.; Mansour, Oula C.; Menta, Ernesto; Phillips, Don R.; Cutts, Suzanne M.

2007-01-01

Mitoxantrone is an anti-cancer agent used in the treatment of breast and prostate cancers. It is classified as a topoisomerase II poison, however can also be activated by formaldehyde to generate drug–DNA adducts. Despite identification of this novel form of mitoxantrone–DNA interaction, excessively high, biologically irrelevant drug concentrations are necessary to generate adducts. A search for mitoxantrone analogues that could potentially undergo this reaction with DNA more efficiently identified Pixantrone as an ideal candidate. An in vitro crosslinking assay demonstrated that Pixantrone is efficiently activated by formaldehyde to generate covalent drug–DNA adducts capable of stabilizing double-stranded DNA in denaturing conditions. Pixantrone–DNA adduct formation is both concentration and time dependent and the reaction exhibits an absolute requirement for formaldehyde. In a direct comparison with mitoxantrone–DNA adduct formation, Pixantrone exhibited a 10- to 100-fold greater propensity to generate adducts at equimolar formaldehyde and drug concentrations. Pixantrone–DNA adducts are thermally and temporally labile, yet they exhibit a greater thermal midpoint temperature and an extended half-life at 37°C when compared to mitoxantrone–DNA adducts. Unlike mitoxantrone, this enhanced stability, coupled with a greater propensity to form covalent drug–DNA adducts, may endow formaldehyde-activated Pixantrone with the attributes required for Pixantrone–DNA adducts to be biologically active. PMID:17483512

IDENTIFICATION OF STEROCHEMICAL CONFIGERATION OF CYCLOPENTA[CD]PYRENE-DNA ADDUCTS IN STRAIN A/J MOUSE LUNG AND C3H10T1/2CL8

EPA Science Inventory

The definitive identification of stereochemical configurations of DNA adducts detected by 32P-postlabeling requires co-chromatography of adducts with synthetic chromatographic standards. Four major and several minor DNA adducts are formed by cyclopenta[cd]pyrene (CPP) in strain A...

Detection of Acetaminophen-Protein Adducts in Decedents with Suspected Opioid-Acetaminophen Combination Product Overdose.

PubMed

Thomas, Karen C; Wilkins, Diana G; Curry, Steven C; Grey, Todd C; Andrenyak, David M; McGill, Lawrence D; Rollins, Douglas E

2016-09-01

Acetaminophen overdose is a leading cause of drug-induced liver failure in the United States. Acetaminophen-protein adducts have been suggested as a biomarker of hepatotoxicity. The purpose of this study was to determine whether protein-derived acetaminophen-protein adducts are quantifiable in postmortem samples. Heart blood, femoral blood, and liver tissue were collected at autopsy from 22 decedents suspected of opioid-acetaminophen overdose. Samples were assayed for protein-derived acetaminophen-protein adducts, acetaminophen, and selected opioids found in combination products containing acetaminophen. Protein-derived APAP-CYS was detected in 17 of 22 decedents and was measurable in blood that was not degraded or hemolyzed. Heart blood concentrations ranged from 11 ng/mL (0.1 μM) to 7817 ng/mL (28.9 μM). Protein-derived acetaminophen-protein adducts were detectable in liver tissue for 20 of 22 decedents. Liver histology was also performed for all decedents, and no evidence of centrilobular hepatic necrosis was observed. © 2016 American Academy of Forensic Sciences.

XPC genotypes/diplotypes play no independent or interaction role with PAH-DNA adducts for breast cancer risk

PubMed Central

Shen, Jing; Gammon, Marilie D.; Terry, Mary Beth; Teitelbaum, Susan L.; Eng, Sybil M.; Neugut, Alfred I.; Santella, Regina M.

2008-01-01

Xeroderma pigmentosum complementation group C (XPC) is an important DNA nuclear excision repair (NER) gene that recognizes the damage caused by variety of bulky DNA adducts. We evaluated the association of two common non-synonymous polymorphisms in XPC (Ala499Val and Lys939Gln) with breast cancer risk in the Long Island Breast Cancer Study Project (LIBCSP), a population-based case-control study. Genotyping of 1,067 cases and 1,110 controls was performed by a high throughput assay with fluorescence polarization. There were no overall associations between XPC polymorphisms and breast cancer risk. A diplotype CC-CC was significantly associated with increased breast cancer risk compared with diplotype CA-CA (OR = 1.4, 95%CI: 1.0–1.9), but was not significant when compared with all other diplotypes combined (OR = 1.22, 95%CI: 0.97–1.53). No modification effects were observed for XPC genotypes by cigarette smoking status, smoking pack years or polycyclic aromatic hydrocarbons (PAH) DNA adducts. The increase in breast cancer risk was slightly more pronounced among women with detectable PAH-DNA adducts and carrying the diplotype CC-CC (OR = 1.6, 95%CI: 1.1–2.2) compared to women with non detectable PAH-DNA adducts carrying other diplotypes combined, but no statistically significant interaction was observed (P interaction = 0.69). These data suggest that XPC have neither independent effects nor interactions with cigarette smoking and PAH-DNA adducts for breast cancer risk. Further studies with multiple genetic polymorphisms in NER pathway are warranted. PMID:18053706

Evidence for the presence of mutagenic arylamines in human breast milk and DNA adducts in exfoliated breast ductal epithelial cells.

PubMed

Thompson, Patricia A; DeMarini, David M; Kadlubar, Fred F; McClure, Gail Y; Brooks, Lance R; Green, Bridgett L; Fares, Manal Y; Stone, Angie; Josephy, P David; Ambrosone, Christine B

2002-01-01

Aromatic and heterocyclic amines are ubiquitous environmental mutagens present in combustion emissions, fried meats, and tobacco smoke, and are suspect human mammary carcinogens. To determine the presence of arylamines in breast tissue and fluid, we examined exfoliated breast ductal epithelial cells for DNA adducts and matched human milk samples for mutagenicity. Breast milk was obtained from 50 women who were 4-6 weeks postpartum, and exfoliated epithelial-cell DNA was evaluated for bulky, nonpolar DNA adducts by (32)P-postlabeling and thin-layer chromatography. Milk was processed by acid hydrolysis, and the extracted organics were examined in the standard plate-incorporation Ames Salmonella assay using primarily strain YG1024, which detects frameshift mutations and overexpresses aryl amine N-acetyltransferase. DNA adducts were identified in 66% of the specimens, and bulky adducts migrated in a pattern similar to that of 4-aminobiphenyl standards. The distribution of adducts did not vary by NAT2 genotype status. Of whole milk samples, 88% (22/25) had mutagenic activity. Among the samples for which we had both DNA adduct and mutagenicity data, 58% (14/19) of the samples with adducts were also mutagenic, and 85% (11/13) of the mutagenic samples had adducts. Quantitatively, no correlation was observed between the levels of adducts and the levels of mutagenicity. Separation of the milk showed that mutagenic activity was found in 69% of skimmed milk samples but in only 29% of the corresponding milk fat samples, suggesting that the breast milk mutagens were moderately polar molecules. Chemical fractionation showed that mutagenic activity was found in 67% (4/6) of the basic fractions but in only 33% (2/6) of acidic samples, indicating that the mutagens were primarily basic compounds, such as arylamines. Although pilot in nature, this study corroborates previous findings of significant levels of DNA adducts in breast tissue and mutagenicity in human breast milk and

Exposure to meat-derived carcinogens and bulky DNA adduct levels in normal-appearing colon mucosa.

PubMed

Ho, Vikki; Brunetti, Vanessa; Peacock, Sarah; Massey, Thomas E; Godschalk, Roger W L; van Schooten, Frederik J; Ashbury, Janet E; Vanner, Stephen J; King, Will D

2017-09-01

Meat consumption is a risk factor for colorectal cancer. This research investigated the relationship between meat-derived carcinogen exposure and bulky DNA adduct levels, a biomarker of DNA damage, in colon mucosa. Least squares regression was used to examine the relationship between meat-derived carcinogen exposure (PhIP and meat mutagenicity) and bulky DNA adduct levels in normal-appearing colon tissue measured using 32 P-postlabelling among 202 patients undergoing a screening colonoscopy. Gene-diet interactions between carcinogen exposure and genetic factors relevant to biotransformation and DNA repair were also examined. Genotyping was conducting using the MassARRAY ® iPLEX ® Gold SNP Genotyping assay. PhIP and higher meat mutagenicity exposures were not associated with levels of bulky DNA adducts in colon mucosa. The XPC polymorphism (rs2228001) was found to associate with bulky DNA adduct levels, whereby genotypes conferring lower DNA repair activity were associated with higher DNA adduct levels than the normal activity genotype. Among individuals with genotypes associated with lower DNA repair (XPD, rs13181 and rs1799179) or detoxification activity (GSTP1, rs1695), higher PhIP or meat mutagenicity exposures were associated with higher DNA adduct levels. Significant interactions between the XPC polymorphism (rs2228000) and both dietary PhIP and meat mutagenicity on DNA adduct levels was observed, but associations were inconsistent with the a priori hypothesized direction of effect. Exposure to meat-derived carcinogens may be associated with increased DNA damage occurring directly in the colon among genetically susceptible individuals. Copyright © 2017 Elsevier B.V. All rights reserved.

Differences in Butadiene Adduct Formation between Rats and Mice Not Due to Selective Inhibition of CYP2E1 by Butadiene Metabolites

PubMed Central

Pianalto, Kaila M.; Hartman, Jessica H.; Boysen, Gunnar; Miller, Grover P.

2013-01-01

CYP2E1 metabolizes 1,3-butadiene (BD) into genotoxic and possibly carcinogenic 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EB-diol). The dose response of DNA and protein adducts derived from BD metabolites increase linearly at low BD exposures and then saturate at higher exposures in rats, but not mice. It was hypothesized that differences in adduct formation between rodents reflect more efficient BD oxidation in mice than rats. Herein, we assessed whether BD-derived metabolites selectively inhibit rat but not mouse CYP2E1 activity using B6C3F1 mouse and Fisher 344 rat liver microsomes. Basal CYP2E1 activities toward 4-nitrophenol were similar between rodents. Through IC50 studies, EB was the strongest inhibitor (IC50 54 μM, mouse; 98 μM, rat), BD-diol considerably weaker (IC50 1200 μM, mouse; 1000 μM, rat), and DEB inhibition nonexistent (IC50 >25 mM). Kinetic studies showed that in both species EB and BD-diol inhibited 4-nitrophenol oxidation through two-site mechanisms in which inhibition constants reflected trends observed in IC50 studies. None of the reactive epoxide metabolites inactivated CYP2E1 irreversibly. Thus, there was no selective inhibition or inactivation of rat CYP2E1 by BD metabolites relative to mouse Cyp2e1, and it can be inferred that CYP2E1 activity toward BD between rodent species would similarly not be impacted by the presence of BD metabolites. Inhibition of CYP2E1 by BD metabolites is then not responsible for the reported species difference in BD metabolism, formation of BD-derived DNA and protein adducts, mutagenicity and tumorigenesis. PMID:24021170

Differences in butadiene adduct formation between rats and mice not due to selective inhibition of CYP2E1 by butadiene metabolites.

PubMed

Pianalto, Kaila M; Hartman, Jessica H; Boysen, Gunnar; Miller, Grover P

2013-11-25

CYP2E1 metabolizes 1,3-butadiene (BD) into genotoxic and possibly carcinogenic 1,2-epoxy-3-butene (EB), 1,2:3,4-diepoxybutane (DEB), and 1,2-epoxy-3,4-butanediol (EB-diol). The dose response of DNA and protein adducts derived from BD metabolites increases linearly at low BD exposures and then saturates at higher exposures in rats, but not mice. It was hypothesized that differences in adduct formation between rodents reflect more efficient BD oxidation in mice than rats. Herein, we assessed whether BD-derived metabolites selectively inhibit rat but not mouse CYP2E1 activity using B6C3F1 mouse and Fisher 344 rat liver microsomes. Basal CYP2E1 activities toward 4-nitrophenol were similar between rodents. Through IC50 studies, EB was the strongest inhibitor (IC50 54μM, mouse; 98μM, rat), BD-diol considerably weaker (IC50 1200μM, mouse; 1000μM, rat), and DEB inhibition nonexistent (IC50>25mM). Kinetic studies showed that in both species EB and BD-diol inhibited 4-nitrophenol oxidation through two-site mechanisms in which inhibition constants reflected trends observed in IC50 studies. None of the reactive epoxide metabolites inactivated CYP2E1 irreversibly. Thus, there was no selective inhibition or inactivation of rat CYP2E1 by BD metabolites relative to mouse Cyp2e1, and it can be inferred that CYP2E1 activity toward BD between rodent species would similarly not be impacted by the presence of BD metabolites. Inhibition of CYP2E1 by BD metabolites is then not responsible for the reported species difference in BD metabolism, formation of BD-derived DNA and protein adducts, mutagenicity and tumorigenesis. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Variations in protein/flavin hydrogen bonding in a LOV domain produce non-Arrhenius kinetics of adduct decay†

PubMed Central

Zoltowski, Brian D.; Nash, Abigail I.; Gardner, Kevin H.

2011-01-01

Light Oxygen Voltage (LOV) domains utilize a conserved blue light-dependent mechanism to control a diverse array of effector domains in biological and engineered proteins. Variations in the kinetics and efficiency of LOV photochemistry fine tune various aspects of the photic response. Characterization of the kinetics of a key aspect of this photochemical mechanism in EL222, a blue-light responsive DNA binding protein from Erythrobacter litoralis HTCC2594, reveals unique non-Arrhenius behavior in the rate of dark state cleavage of the photochemically-generated adduct. Sequence analysis and mutagenesis studies establish that this effect stems from a Gln to Ala mutation unique to EL222 and homologous proteins from marine bacteria. Kinetic and spectroscopic analyses reveal that hydrogen bonding interactions between the FMN N1, O2 and ribityl hydroxyls with the surrounding protein regulate photocycle kinetics and stabilize the LOV active site from temperature-induced alteration in local structure. Substitution of residues interacting with the N1-O2 locus modulates adduct stability, structural flexibility and sequestration of the active site from bulk solvent without perturbation of light-activated DNA binding. Together, these variants link non-Arrhenius behavior to specific alteration of an H-bonding network, while affording tunability of photocycle kinetics. PMID:21923139

Fabrication and electrochemical properties of insoluble fullerene-diamine adduct thin-films as buffer layer by alternate immersion process

NASA Astrophysics Data System (ADS)

Saito, Jo; Akiyama, Tsuyoshi; Suzuki, Atsushi; Oku, Takeo

2017-01-01

Insoluble fullerene-diamine adduct thin-films consisting of C60 and 1,2-diaminoethane were easily fabricated on an electrode by an alternate immersion process. Formation of the C60-diamine adduct films were confirmed using transmission absorption spectroscopy and atomic force microscopy. An inverted-type organic solar cells were fabricated by using the C60-diamine adduct film as the electron transport layer. The resultant photoelectric conversation performance of the solar cells suggested that photocurrent is generated via the photoexcitation of polythiophene. The result suggests that the present insoluble fullerene-diamine adduct films worked as buffer layer for organic thin-film solar cells.

Context Matters: Contribution of Specific DNA Adducts to the Genotoxic Properties of the Tobacco-Specific Nitrosamine NNK.

PubMed

Peterson, Lisa A

2017-01-17

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in laboratory animals. It is classified as a Group 1 human carcinogen by the International Agency for Cancer Research. NNK is bioactivated upon cytochrome P450 catalyzed hydroxylation of the carbon atoms adjacent to the nitrosamino group to both methylating and pyridyloxobutylating agents. Both pathways generate a spectrum of DNA damage that contributes to the overall mutagenic and toxic properties of this compound. NNK is also reduced to form 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also carcinogenic. Like NNK, NNAL requires metabolic activation to DNA alkylating agents. Methyl hydroxylation of NNAL generates pyridylhydroxybutyl DNA adducts, and methylene hydroxylation leads to DNA methyl adducts. The consequence of this complex metabolism is that NNK generates a vast spectrum of DNA damage, any form of which can contribute to the overall carcinogenic properties of this potent pulmonary carcinogen. This Perspective reviews the chemistry and genotoxic properties of the collection of DNA adducts formed from NNK. In addition, it provides evidence that multiple adducts contribute to the overall carcinogenic properties of this chemical. The adduct that contributes to the genotoxic effects of NNK depends on the context, such as the relative amounts of each DNA alkylating pathway occurring in the model system, the levels and genetic variants of key repair enzymes, and the gene targeted for mutation.

Human urinary bladder epithelial cells lacking wild-type p53 function are deficient in the repair of 4-aminobiphenyl-DNA adducts in genomic DNA.

PubMed

Swaminathan, Santhanam; Torino, Jennifer L; Burger, Melissa S

2002-01-29

The effect of the tumor suppressor gene TP53 on repair of genomic DNA damage was examined in human urinary bladder transitional cell carcinoma (TCC) cell lines. Utilizing TCC10 containing wild-type p53 (wt-p53) as the parental line, an isogenic set of cell lines was derived by retroviral infection that expressed a transdominant mutant p53 (Arg --> His at codon 273, TDM273-TCC10), or the human papilloma virus 16-E6 oncoprotein (E6-TCC10). 32P-postlabeling analyses were performed on DNA from TCC cultures obtained after treatment with N-hydroxy-4-aminobiphenyl (N-OH-ABP), N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) and N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP). The major adduct was identified as N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) with all three chemicals. The amount of adducts in urothelial DNA ranged between 0.1 and 20 per 10(6) nucleotides, N-OAc-AABP yielding the highest levels, followed by N-OH-ABP and N-OH-AABP. To determine, if the functional status of p53 affects the rate of repair of dG-C8-ABP in genomic DNA, TCC10 and the TDM273-TCC10 and E6-TCC10 isotypes were exposed to N-OH-AABP for 12h and the DNA damage was allowed to repair up to 24h. The adduct levels were quantified and compared between the TCC10 isotypes. The amounts of dG-C8-ABP that remained in genomic DNA from E6-TCC10 and TDM273-TCC10 were approximately two-fold higher, as compared to the parental TCC10. At the dose used for DNA repair studies, N-OH-AABP or N-OAc-AABP did not induce apoptosis in TCC10. However, N-OAc-AABP at high doses (>5 microM) induced apoptosis, as evidenced by DNA fragmentation analyses. Furthermore, N-OAc-AABP-mediated apoptosis was independent of the functional status of wt-p53, since both E6-TCC10 and the parental TCC10 exhibited DNA fragmentation following treatment. These results suggest that p53 might modulate the repair of DNA adducts generated from the human bladder carcinogen ABP in its target human uroepithelial cells. This implies that in p53

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

Crystallographic, Spectroscopic, and Computational Analysis of a Flavin-C4a-Oxygen Adduct in Choline Oxidase

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

Orville, A.M.; Lountos, G. T.; Finnegan, S.

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. Wemore » 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.« less

Crystallographic, Spectroscopic, and Computational Analysis of a Flavin C4a-Oxygen Adduct in Choline Oxidase

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

Orville, A.; Lountos, G; Finnegan, S

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. Wemore » 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.« less

Nitro-fatty acid metabolome: saturation, desaturation, beta-oxidation, and protein adduction.

PubMed

Rudolph, Volker; Schopfer, Francisco J; Khoo, Nicholas K H; Rudolph, Tanja K; Cole, Marsha P; Woodcock, Steven R; Bonacci, Gustavo; Groeger, Alison L; Golin-Bisello, Franca; Chen, Chen-Shan; Baker, Paul R S; Freeman, Bruce A

2009-01-16

Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via beta-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO2 hydration or nitric oxide (*NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added beta-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet

Formation of monofunctional cisplatin-DNA adducts in carbonate buffer.

PubMed

Binter, Alexandra; Goodisman, Jerry; Dabrowiak, James C

2006-07-01

Carbonate in its various forms is an important component in blood and the cytosol. Since, under conditions that simulate therapy, carbonate reacts with cisplatin to form carbonato complexes, one of which is taken up and/or modified by the cell [C.R. Centerwall, J. Goodisman, D.J. Kerwood, J. Am. Chem. Soc., 127 (2005) 12768-12769], cisplatin-carbonato complexes may be important in the mechanism of action of cisplatin. In this report we study the binding of cisplatin to pBR322 DNA in two different buffers, using gel electrophoresis. In 23.8mM HEPES, N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid, 5mM NaCl, pH 7.4 buffer, cisplatin produces aquated species, which react with DNA to unwind supercoiled Form I DNA, increasing its mobility, and reducing the binding of ethidium to DNA. This behavior is consistent with the formation of the well-known intrastrand crosslink on DNA. In 23.8mM carbonate buffer, 5mM NaCl, pH 7.4, cisplatin forms carbonato species that produce DNA-adducts which do not significantly change supercoiling but enhance binding of ethidium to DNA. This behavior is consistent with the formation of a monofunctional cisplatin adduct on DNA. These results show that aquated cisplatin and carbonato complexes of cisplatin produce different types of lesions on DNA and they underscore the importance of carrying out binding studies with cisplatin and DNA using conditions that approximate those found in the cell.

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...

Structural and thermodynamic insight into E. coli UvrABC mediated incision of cluster di-acetylaminofluorene adducts on the NarI sequence

PubMed Central

Jain, Vipin; Hilton, Benjamin; Lin, Bin; Jain, Anshu; MacKerell, Alexander D.; Zou, Yue; Cho, Bongsup P.

2014-01-01

Cluster DNA damage refers to two or more lesions in a single turn of the DNA helix. Such clustering may occur with bulky DNA lesions, which may be responsible for their sequence dependent repair and mutational outcomes. Here we prepared three 16-mer cluster duplexes in which two fluoroacetylaminofluorene adducts (dG-FAAF) are separated by none, one and two nucleotides in the E. coli NarI mutational hot spot (5'-CTCTCG1G2CG3CCATCAC-3'): i.e. 5'-- CG1*G2*CG3CC--3', 5'--CG1G2*CG3*CC--3', and 5'--CG1*G2CG3*CC--3' [G*=dG-FAAF], respectively. We conducted spectroscopic, thermodynamic, and molecular dynamics studies of these di-FAAF duplexes and the results were compared with those of the corresponding mono- FAAF adducts in the same NarI sequence (Nucleic Acids Res. 2012, 3939–3951). Our nucleotide excision repair results showed greater reparability of the di-adducts in comparison to the corresponding mono-adducts. Moreover, we observed dramatic flanking base sequence effects on their repair efficiency in the order of NarI-G2G3 > -G1G3 > -G1G2. The NMR/CD/UV-melting and MD-simulation results revealed that in contrast to the mono-adducts, di-adducts produced synergistic effect on duplex destabilization. In addition, dG-FAAF at G2G3 and G1G3 destack the neighboring bases with greater destabilization occurring with the former. Overall, the results indicate the importance of base stacking and related thermal/thermodynamic destabilization in the repair of bulky cluster arylamine DNA adducts. PMID:23841451

Reoriention of diprotonated DABCO (1,4-Diazabicyclo[2.2.2]octane) cation and proton transfer in organic ferroelectric adduct DABCO-2(2-Chlorobenzoic acid)

NASA Astrophysics Data System (ADS)

Asaji, Tetsuo

2018-05-01

Temperature dependences of 1H NMR as well as 35Cl NQR spin-lattice relaxation times T1 were investigated of a ferroelectric molecular adduct with Tc = 323 K, in which 1,4-diazabicyclo[2.2.2]octane (DABCO) is sandwiched between two 2-chlorobenzoic acid (2-ClBA). The NQR frequencies clearly show that proton transfer from 2-ClBA to DABCO is occurred and the molecular adduct consists of diprotonated DABCO cation and two 2-chlorobenzoate anions. The correlation time of reorientational motion of the diprotonated DABCO molecule was determined as a function of temperature. The activation energy Ea of the motion was estimated as 22 kJ mol-1 below Tc. The steep decrease of the NQR T1 with Ea = 50 kJ mol-1, observed above ca. 280 K in the ferroelectric phase, suggests a slow fluctuation of electric field gradient at chlorine nucleus.

Synthesis of 1,4-amino alcohols by Grignard reagent addition to THF and N-tosyliminobenzyliodinane.

PubMed

Tejo, Ciputra; See, Yang Feng Anders; Mathiew, Mitch; Chan, Philip Wai Hong

2016-01-21

The synthesis of 1,4-amino alcohols from THF treated with N-tosyliminobenzyliodinane (PhINTs) followed by a Grignard reagent under mild reaction conditions at room temperature is described herein. Various Grignard reagents were shown to be compatible, furnishing the corresponding 4-substituted-N-1,4-tosylamino alcohols in good to excellent yields. A partial or full detosylation of the N-tosyl-1,4-amino alcohol was observed in instances involving a sterically bulky Grignard reagent, leading to the deprotected 1,4-amino alcohol product in moderate to good yields. The synthetic utility of this protocol was demonstrated by the synthesis of a 5-substituted-N-tosyl-1,5-amino alcohol from THP and the conversion of two examples to their corresponding γ-lactam and pyrrolidine adducts.

Mass spectrometry of cis-diamminedichloroplatinum(II) adducts with the dinucleosidemonophosphates d(ApG), d(GpG) and d(TpC) in an ion trap.

PubMed

Hagemeister, Timo; Linscheid, Michael

2002-07-01

The detection and fragmentation behaviour of adducts of the chemotherapeutic cis-diamminedichloroplatinum(II) (cisplatin) with the dinucleosidemonophosphates d(ApG), d(GpG) and d(TpC) as model compounds for DNA adducts in an ion trap with electrospray ionization were studied. Mainly the monofunctional adduct, the bifunctional adduct and the bifunctional adduct with platinum bridging two dinucleosidemonophosphates were detected. In addition, several more complex adducts were seen resulting from reactions among these species. Adduct formation was low in the case of d(TpC). Fragmentation could be controlled strongly by varying the temperature of the transfer capillary; furthermore, tandem mass spectrometric (MS/MS) experiments on both the monofunctional and the bifunctional adducts were performed. For the adducts of d(ApG) and d(GpG) losses of NH(3) and HCl were the most dominant reactions, followed by the losses of one, then another two units of 98 amu from the sugar-phosphate backbone, whereas d(TpC)-Pt predominantly forms the dinucleosidemonophosphate. In the gas phase, the conversion of the monofunctional into the bifunctional adducts through binding to another site in the dinucleotide accompanied by loss of NH(3) or HCl could also be observed. The removal of a ligand from the coordination sphere of the square-planar platinum complexes appeared to be the crucial step for the induction of further fragmentation of the dinucleotide ligand. MS(n) experiments of the bifunctional adducts of d(ApG) and d(GpG) revealed different fragmentation pathways involving the loss of phosphoric acid, metaphosphoric acid, deoxyribose units (intact or dehydrated) and the nucleobases in different orders, leaving characteristic binding site-determining fragments. Fragmentation of these ions was also performed, mainly resulting in fragmentation of the bases. The study confirmed the remarkable stability of the platinum-guanine bond compared with other nucleobases. Copyright 2002 John

Determination of phospholipid regiochemistry by Ag(I) adduction and tandem mass spectrometry.

PubMed

Yoo, Hyun Ju; Håkansson, Kristina

2011-02-15

Collision-activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) of Ag-adducted phospholipids were investigated as structural tools. Previously, determination of the acyl chains at the two phospholipid esterification sites has been performed based on the R(1)COO(-)/R(2)COO(-) ratio in negative ion mode CAD tandem mass spectrometry. However, the observed product ion ratio is dependent on the extent of unsaturation of the fatty acyl group at sn-2 as well as on the total chain length. Similarly, in positive ion mode CAD with/without alkaline or alkaline earth metal adduction, the ratio of product ions resulting from either R(1)COOH or R(2)COOH neutral losses is dependent on the nature of the phospholipid polar headgroup. Ag(+) ion chromatography, in which silver ions are part of the stationary phase, can provide information on double bond number/distribution as well as double bond configuration (cis/trans) because of interaction between Ag(+) ions and olefinic π electrons of fatty acids and lipids. We hypothesized that interactions between double bonds and Ag(+) may be utilized to also reveal phospholipid esterification site information in tandem mass spectrometry. CAD and IRMPD of Ag-adducted phospholipids with unsaturated fatty acids (R(x)COOH, x = 1 or 2) provided characteristic product ions, [R(x)COOH + Ag](+), and their neutral losses. The characteristic product ions and their abundances do not depend on the type of polar headgroup or the number of double bonds of unsaturated acyl chains. Tandem mass spectrometry of Cu-adducted phospholipids was also performed for comparison based on the Lewis acid and base properties of Cu(+) and phospholipid double bonds, respectively.

DNA adducts of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 4-aminobiphenyl are infrequently detected in human mammary tissue by liquid chromatography/tandem mass spectrometry

PubMed Central

Gu, Dan; Turesky, Robert J.; Tao, Yeqing; Langouët, Sophie A.; Nauwelaërs, Gwendoline C.; Yuan, Jian-Min; Yee, Douglas; Yu, Mimi C.

2012-01-01

Some epidemiological investigations have revealed that frequent consumption of well-done cooked meats and tobacco smoking are risk factors for breast cancer in women. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine that is formed in well-done cooked meat, and 4-aminobiphenyl (4-ABP) is an aromatic amine that arises in tobacco smoke and occurs as a contaminant in the atmosphere. Both compounds are rodent mammary carcinogens, and putative DNA adducts of PhIP and 4-ABP have been frequently detected, by immunohistochemistry (IHC) or 32P-post-labeling methods, in mammary tissue of USA women. Because of these findings, PhIP and 4-ABP have been implicated as causal agents of human breast cancer. However, the biomarker data are controversial: both IHC and 32P-post-labeling are non-selective screening methods and fail to provide confirmatory spectral data. Consequently, the identities of the lesions are equivocal. We employed a specific and sensitive liquid chromatography/mass spectrometry (MS) method, to screen tumor-adjacent normal mammary tissue for DNA adducts of PhIP and 4-ABP. Only 1 of 70 biopsy samples obtained from Minneapolis, Minnesota breast cancer patients contained a PhIP-DNA adduct. The level was three adducts per 109 nucleotides, a level that is 100-fold lower than the mean level of PhIP adducts reported by IHC or 32P-post-labeling methods. The occurrence of 4-ABP-DNA adducts was nil in those same breast tissues. Our findings, derived from a specific mass spectrometry method, signify that PhIP and 4-ABP are not major DNA-damaging agents in mammary tissue of USA women and raise questions about the roles of these chemicals in breast cancer. PMID:22072616

Structure of 7,12-dimethylbenz(a)anthracene-guanosine adducts.

PubMed

Jeffrey, A M; Blobstein, S H; Weinstein, I B; Beland, F A; Harvey, R G; Kasai, H; Nakanishi, K

1976-07-01

Arene oxides have been proposed as the reactive intermediates in the process of carcinogenesis induced by polycyclic aromatic hydrocarbons. The present study defines the structures of four guanosine adducts formed by the reaction of 7,12-dimethylbenz[a]anthracene-5,6-oxide with polyguanylic acid. The modified polymer was hydrolyzed to nucleotides and the hydrophobic guanosine adducts separated from unmodified guanosine by LH-20 column chromatograhy. The adducts were further resolved into four components (I-IV) by reverse phase high pressure liquid chromatography. Analysis of the ultraviolet, circular dichroism, mass, and proton magnetic resonance spectra of these compounds, or their acetate and free base derivatives, indicates that in all four compounds the aromatic hydrocarbon is present on the 2 amino group of guanine. Compounds I and IV, and II and III constitute diastereoisomeric pairs, respectively. In the I and IV pair, the adducts result from addition at the 6 position of the original dimethylbenz[a]anthracene oxide, whereas in the II and III pair, the addition occurs at the 5 position. Indirect evidence suggests that trans opening of the oxide occurred in all cases but this remains to be established.

Structure of 7,12-dimethylbenz(a)anthracene-guanosine adducts.

PubMed Central

Jeffrey, A M; Blobstein, S H; Weinstein, I B; Beland, F A; Harvey, R G; Kasai, H; Nakanishi, K

1976-01-01

Arene oxides have been proposed as the reactive intermediates in the process of carcinogenesis induced by polycyclic aromatic hydrocarbons. The present study defines the structures of four guanosine adducts formed by the reaction of 7,12-dimethylbenz[a]anthracene-5,6-oxide with polyguanylic acid. The modified polymer was hydrolyzed to nucleotides and the hydrophobic guanosine adducts separated from unmodified guanosine by LH-20 column chromatograhy. The adducts were further resolved into four components (I-IV) by reverse phase high pressure liquid chromatography. Analysis of the ultraviolet, circular dichroism, mass, and proton magnetic resonance spectra of these compounds, or their acetate and free base derivatives, indicates that in all four compounds the aromatic hydrocarbon is present on the 2 amino group of guanine. Compounds I and IV, and II and III constitute diastereoisomeric pairs, respectively. In the I and IV pair, the adducts result from addition at the 6 position of the original dimethylbenz[a]anthracene oxide, whereas in the II and III pair, the addition occurs at the 5 position. Indirect evidence suggests that trans opening of the oxide occurred in all cases but this remains to be established. PMID:821053

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

Code of Federal Regulations, 2010 CFR

2010-07-01

... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Ethylene oxide adduct of fatty acid... 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) is...

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

Code of Federal Regulations, 2011 CFR

2011-07-01

... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Ethylene oxide adduct of fatty acid... 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) is...

8,9-dihydroxy-8,9-dihydrodibenzo[a,l]pyrene is a potent morphological cell-transforming agent in C3H10T(1)/(2)Cl8 mouse embryo fibroblasts in the absence of detectable stable covalent DNA adducts.

PubMed

Nesnow, S; Davis, C; Padgett, W T; Adams, L; Yacopucci, M; King, L C

2000-06-01

The comparative genotoxic effects of racemic trans-8,9-dihydroxy-8, 9-dihydrodibenzo[a,l]pyrene (trans-DB[a,l]P-8,9-diol), the metabolic K-region dihydrodiol of dibenzo[a,l] pyrene (DB[a,l]P) (dibenzo[def, p]chrysene) and DB[a,l]P in transformable mouse embryo C3H10T(1)/(2)Cl8 (C3H10T(1)/(2)) fibroblasts was investigated. The C3H10T(1)/(2) mouse embryo morphological cell-transforming activities of these polycyclic aromatic hydrocarbons (PAHs) were assayed using concentration-response studies. At concentrations of 33 nM and above both trans-DB[a,l]P-8,9-diol and DB[a,l]P produced significant (and similar) numbers of type II and III foci per dish and numbers of dishes with type II and II foci. Concomitant cytotoxicity studies revealed a reduction in colony survival of approximately 25% up to 198 nM for both PAHs. DNA adducts of trans-DB[a,l]P-8,9-diol and DB[a,l]P in C3H10T(1)/(2) cells were analyzed by a (32)P-post-labeling TLC/HPLC method. No adducts were observed in the DNA of C3H10T(1)/(2) cells treated with trans-DB[a, l]P-8,9-diol at concentrations that induced morphological cell transformation. Under the same exposure and chromatographic conditions, DNA adducts of deoxyadenosine and deoxyguanosine derived from the fjord region anti-DB[a,l]P-11,12-diol-13,14-epoxide and syn-DB[a,l]P-11,12-diol-13,14-epoxide were observed in the DNA of DB[a,l]P-treated cells. These results indicate that trans-DB[a,l]P-8, 9-diol has intrinsic genotoxic activity equal to that of DB[a,l]P, based on morphological cell transformation of mouse embryo fibroblasts. The activity of trans-DB[a,l]P-8,9-diol is apparently not associated with the formation of observable stable covalent DNA adducts. These results suggest that under appropriate conditions, trans-DB[a,l]P-8,9-diol may serve as an intermediate in the genotoxicity of DB[a,l]P.

Hemoglobin Adducts of Benzene Oxide in Neonatal and Adult Dried Blood Spots

PubMed Central

Funk, William E.; Waidyanatha, Suramya; Chaing, Shu H.; Rappaport, Stephen M.

2010-01-01

Adducts of reactive chemicals with hemoglobin (Hb) or human serum albumin can be used as biomarkers of internal doses of carcinogens. Since dried blood spots (DBS) are easier to collect and store than conventional venous blood samples, they encourage applications of biomarkers of exposure in large epidemiology studies. Also, neonatal DBS can be used to investigate chemical exposures in utero. Here, we report a simple method to isolate Hb from DBS with high recovery and purity using the addition of ethanol to aqueous DBS extracts. To prove the concept that DBS-derived proteins can be used to assay for adducts, we measured Hb adducts of benzene oxide, a reactive metabolite of the ubiquitous air pollutant, benzene, in 9 neonatal and 9 adult DBS (from volunteer subjects), using a gas chromatography-mass spectrometry method that we had previously developed. For comparison, benzene oxide-Hb adducts (BO-Hb) were measured in the same 9 adult subjects, using Hb that had been isolated and purified using our conventional method for venous blood. The geometric mean BO-Hb levels in all DBS samples ranged from 27.7 to 33.1 pmol/g globin. Neither of the comparisons of mean (logged) BO-Hb levels between sources (adult conventional vs. adult DBS and adult DBS vs. newborn DBS) showed a significant difference. Based upon the estimated variance of the BO-Hb levels, we had 80% power to detect a 1.7-fold difference in geometric mean levels of BO-Hb in our samples of 9 subjects. PMID:18708378

Significant interactions between maternal PAH exposure and haplotypes in candidate genes on B[a]P-DNA adducts in a NYC cohort of non-smoking African-American and Dominican mothers and newborns

PubMed Central

Tang, Deliang

2014-01-01

Polycyclic aromatic hydrocarbons (PAH) are a class of chemicals common in the environment. Certain PAH are carcinogenic, although the degree to which genetic variation influences susceptibility to carcinogenic PAH remains unclear. Also unknown is the influence of genetic variation on the procarcinogenic effect of in utero exposures to PAH. Benzo[a]pyrene (B[a]P) is a well-studied PAH that is classified as a probable human carcinogen. Within our New York City-based cohort, we explored interactions between maternal exposure to airborne PAH during pregnancy and maternal and newborn haplotypes (and in one case, a single-nucleotide polymorphism) in key B[a]P metabolism genes on B[a]P-DNA adducts in paired cord blood samples. The study subjects included non-smoking African-American (n = 132) and Dominican (n = 235) women with available data on maternal PAH exposure, paired cord adducts and genetic data who resided in the Washington Heights, Central Harlem and South Bronx neighborhoods of New York City. We selected seven maternal and newborn genes related to B[a]P metabolism, detoxification and repair for our analyses: CYP1A1, CYP1A2, CYP1B1, GSTM3, GSTT2, NQO1 and XRCC1. We found significant interactions between maternal PAH exposure and haplotype on cord B[a]P-DNA adducts in the following genes: maternal CYP1B1, XRCC1 and GSTM3, and newborn CYP1A2 and XRCC1 in African-Americans; and maternal XRCC1 and newborn NQO1 in Dominicans. These novel findings highlight differences in maternal and newborn genetic contributions to B[a]P-DNA adduct formation, as well as ethnic differences in gene–environment interactions, and have the potential to identify at-risk subpopulations who are susceptible to the carcinogenic potential of B[a]P. PMID:24177223

Detection in vivo of a Novel Endogenous Etheno DNA Adduct Derived from Arachidonic Acid and the Effects of Antioxidants on Its Formation

PubMed Central

Cruz, Idalia M.; Pondicherry, Sharanya R.; Fernandez, Aileen; Schultz, Casey L.; Yang, Peiying; Pan, Jishen; Desai, Dhimant; Krzeminski, Jacek; Amin, Shantu; Christov, Plamen P.; Hara, Yukihiko; Chung, Fung-Lung

2014-01-01

Previous studies showed that the 7-(1′,2′-dihydroxyheptyl) substituted etheno DNA adducts are products from reactions with epoxide of (E)-4-hydroxy-2-nonenal (HNE), an oxidation product of ω-6 polyunsaturated fatty acids (PUFAs). In this work, we report the detection of 7-(1′,2′-dihydroxyheptyl)-1,N6-ethenodeoxyadenosine (DHHedA) in rodent and human tissues by two independent methods: a 32P-postlabeling/HPLC method and an isotope dilution liquid chromatography electrospray ionization tandem mass spectrometry method (ID-LC-ESI-MS/MS), demonstrating for the first time that DHHedA is a background DNA lesion in vivo. We showed that DHHedA can be formed upon incubation of arachidonic acid (AA) with deoxyadenosine (dA), supporting the notion that ω-6 PUFAs are the endogenous source of DHHedA formation. Because cyclic adducts are derived from the oxidation of PUFAs, we subsequently examined the effects of antioxidants, α-lipoic acid, Polyphenon E and vitamin E, on the formation of DHHedA and γ-hydroxy-1,N2-propanodeoxyguanosine (γ-OHPdG), a widely studied acrolein-derived adduct arising from oxidized PUFAs, in the livers of Long Evans Cinnamon (LEC) rats. LEC rats are inflicted with elevated lipid peroxidation and prone to the development of hepatocellular carcinomas. The results showed that while the survival of LEC rats increased significantly by α-lipoic acid, none of the antioxidants inhibited the formation of DHHedA and only Polyphenon E decreased the formation of γ-OHPdG. In contrast, vitamin E caused a significant increase in the formation of both γ-OHPdG and DHHedA in the livers of LEC rats. PMID:24816294

Eccentric and Isometric Hip Adduction Strength in Male Soccer Players With and Without Adductor-Related Groin Pain

PubMed Central

Thorborg, Kristian; Branci, Sonia; Nielsen, Martin Peter; Tang, Lars; Nielsen, Michael Bachmann; Hölmich, Per

2014-01-01

Background: Adductor-related pain is the most common clinical finding in soccer players with groin pain and can be a long-standing problem affecting physical function and performance. Hip adductor weakness has been suggested to be associated with this clinical entity, although it has never been investigated. Purpose: To investigate whether isometric and eccentric hip strength are decreased in soccer players with adductor-related groin pain compared with asymptomatic soccer controls. The hypothesis was that players with adductor-related groin pain would have lower isometric and eccentric hip adduction strength than players without adductor-related groin pain. Study Design: Cross-sectional study; Level of evidence, 3. Methods: Male elite and subelite players from 40 teams were contacted. In total, 28 soccer players with adductor-related groin pain and 16 soccer players without adductor-related groin pain (asymptomatic controls) were included in the study. In primary analysis, the dominant legs of 21 soccer players with adductor-related groin pain (≥4 weeks duration) were compared with the dominant legs of 16 asymptomatic controls using a cross-sectional design. The mean age of the symptomatic players was 24.5 ± 2.5 years, and the mean age of the asymptomatic controls was 22.9 ± 2.4 years. Isometric hip strength (adduction, abduction, and flexion) and eccentric hip strength (adduction) were assessed with a handheld dynamometer using reliable test procedures and a blinded assessor. Results: Eccentric hip adduction strength was lower in soccer players with adductor-related groin pain in the dominant leg (n = 21) compared with asymptomatic controls (n = 16), namely 2.47 ± 0.49 versus 3.12 ± 0.43 N·m/kg, respectively (P < .001). No other hip strength differences were observed between symptomatic players and asymptomatic controls for the dominant leg (P = .35-.84). Conclusion: Large eccentric hip adduction strength deficits were found in soccer players with adductor

Association between plasma BPDE‐Alb adduct concentrations and DNA damage of peripheral blood lymphocytes among coke oven workers

PubMed Central

Wang, Hong; Chen, Weihong; Zheng, Hongyan; Guo, Liang; Liang, Huashan; Yang, Xiaobo; Bai, Yun; Sun, Jianya; Su, Yougong; Chen, Yongwen; Yuan, Jing; Bi, Yongyi; Wei, Qingyi; Wu, Tangchun

2007-01-01

Objectives Coke oven emissions (COE) containing polycyclic aromatic hydrocarbons (PAHs) can induce both benzo[a]pyrene‐r‐7, t‐8, t‐9,c‐10‐tetrahydotetrol‐albumin (BPDE‐Alb) adducts and DNA damage. However, the relation between these biomarkers for early biological effects is not well documented in coke oven workers. Methods In this study, the authors recruited 207 male workers exposed to COE and 102 controls not exposed to COE in the same steel plant in northern China. They measured BPDE‐Alb adduct concentrations in plasma with reverse‐phase high performance liquid chromatography and DNA damage in peripheral blood lymphocytes with alkaline comet assay. Results The results showed that the median concentration of BPDE‐Alb adducts in the exposed group (34.36 fmol/mg albumin) was significantly higher than that in the control group (21.90 fmol/mg albumin, p = 0.012). The mean Olive tail moment (Olive TM) of DNA damage in the exposed and control groups were 1.20 and 0.63, respectively (p = 0.000). Multivariate logistic regression analysis revealed that the odds ratio (OR) for BPDE‐Alb adduct and Olive TM associated with the exposure were 1.72 (95% CI 1.06 to 2.81) and 1.96 (95% CI 1.20 to 3.19), respectively. These results show significant correlations between the concentrations of BPDE‐Alb adduct and Olive TM levels in exposed group (r = 0.235, p = 0.001) but not in control group (r = 0.093, p = 0.353). Conclusion The results suggest that occupational exposure to COE may induce both BPDE–Alb adducts and DNA damage in the lymphocytes of coke oven workers and that these two markers are useful for monitoring exposure to COE in the workplace. PMID:17449561

Interaction of benzo[a]pyrene diol epoxide isomers with human serum albumin: Site specific characterisation of adducts and associated kinetics

NASA Astrophysics Data System (ADS)

Motwani, Hitesh V.; Westberg, Emelie; Törnqvist, Margareta

2016-11-01

Carcinogenicity of benzo[a]pyrene {B[a]P, a polycyclic aromatic hydrocarbon (PAH)} involves DNA-modification by B[a]P diol epoxide (BPDE) metabolites. Adducts to serum albumin (SA) are not repaired, unlike DNA adducts, and therefore considered advantageous in assessment of in vivo dose of BPDEs. In the present work, kinetic experiments were performed in relation to the dose (i.e. concentration over time) of different BPDE isomers, where human SA (hSA) was incubated with respective BPDEs under physiological conditions. A liquid chromatography (LC) tandem mass spectrometry methodology was employed for characterising respective BPDE-adducts at histidine and lysine. This strategy allowed to structurally distinguish between the adducts from racemic anti- and syn-BPDE and between (+)- and (-)-anti-BPDE, which has not been attained earlier. The adduct levels quantified by LC-UV and the estimated rate of disappearance of BPDEs in presence of hSA gave an insight into the reactivity of the diol epoxides towards the N-sites on SA. The structure specific method and dosimetry described in this work could be used for accurate estimation of in vivo dose of the BPDEs following exposure to B[a]P, primarily in dose response studies of genotoxicity, e.g. in mice, to aid in quantitative risk assessment of PAHs.

Magnetic Resonance Imaging of Optic Nerve Traction During Adduction in Primary Open-Angle Glaucoma With Normal Intraocular Pressure

PubMed Central

Demer, Joseph L.; Clark, Robert A.; Suh, Soh Youn; Giaconi, JoAnn A.; Nouri-Mahdavi, Kouros; Law, Simon K.; Bonelli, Laura; Coleman, Anne L.; Caprioli, Joseph

2017-01-01

Purpose We used magnetic resonance imaging (MRI) to ascertain effects of optic nerve (ON) traction in adduction, a phenomenon proposed as neuropathic in primary open-angle glaucoma (POAG). Methods Seventeen patients with POAG and maximal IOP ≤ 20 mm Hg, and 31 controls underwent MRI in central gaze and 20° to 30° abduction and adduction. Optic nerve and sheath area centroids permitted computation of midorbital lengths versus minimum paths. Results Average mean deviation (±SEM) was −8.2 ± 1.2 dB in the 15 patients with POAG having interpretable perimetry. In central gaze, ON path length in POAG was significantly more redundant (104.5 ± 0.4% of geometric minimum) than in controls (102.9 ± 0.4%, P = 2.96 × 10−4). In both groups the ON became significantly straighter in adduction (28.6 ± 0.8° in POAG, 26.8 ± 1.1° in controls) than central gaze and abduction. In adduction, the ON in POAG straightened to 102.0% ± 0.2% of minimum path length versus 104.5% ± 0.4% in central gaze (P = 5.7 × 10−7), compared with controls who straightened to 101.6% ± 0.1% from 102.9% ± 0.3% in central gaze (P = 8.7 × 10−6); and globes retracted 0.73 ± 0.09 mm in POAG, but only 0.07 ± 0.08 mm in controls (P = 8.8 × 10−7). Both effects were confirmed in age-matched controls, and remained significant after correction for significant effects of age and axial globe length (P = 0.005). Conclusions Although tethering and elongation of ON and sheath are normal in adduction, adduction is associated with abnormally great globe retraction in POAG without elevated IOP. Traction in adduction may cause mechanical overloading of the ON head and peripapillary sclera, thus contributing to or resulting from the optic neuropathy of glaucoma independent of IOP. PMID:28829843

Hydrogen bonded binary molecular adducts derived from exobidentate N-donor ligand with dicarboxylic acids: Acid⋯imidazole hydrogen-bonding interactions in neutral and ionic heterosynthons

NASA Astrophysics Data System (ADS)

Kathalikkattil, Amal Cherian; Damodaran, Subin; Bisht, Kamal Kumar; Suresh, Eringathodi

2011-01-01

Four new binary molecular compounds between a flexible exobidentate N-heterocycle and a series of dicarboxylic acids have been synthesized. The N-donor 1,4-bis(imidazol-1-ylmethyl)benzene (bix) was reacted with flexible and rigid dicarboxylic acids viz., cyclohexane-1,4-dicarboxylic acid (H 2chdc), naphthalene-1,4-dicarboxylic acid (H 2npdc) and 1H-pyrazole-3,5-dicarboxylic acid (H 2pzdc), generating four binary molecular complexes. X-ray crystallographic investigation of the molecular adducts revealed the primary intermolecular interactions carboxylic acid⋯amine (via O-H⋯N) as well as carboxylate⋯protonated amine (via N-H +⋯O -) within the binary compounds, generating layered and two-dimensional sheet type H-bonded networks involving secondary weak interactions (C-H⋯O) including the solvent of crystallization. Depending on the differences in p Ka values of the selected base/acid (Δp Ka), diverse H-bonded supramolecular assemblies could be premeditated. This study demonstrates the H-bonding interactions between imidazole/imidazolium cation and carboxylic acid/carboxylate anion in providing sufficient driving force for the directed assembly of binary molecular complexes. In the two-component solid form of hetero synthons involving bix and dicarboxylic acid, only H 2chdc exist as cocrystal with bix, while all the other three compounds crystallized exclusively as salt, in agreement with the Δp Ka values predicted for the formation of salts/cocrystals from the base and acid used in the synthesis of supramolecular solids.

Biomarker analysis of hemoglobin adducts of acrylamide and glycidamide enantiomers for mid-term internal exposure assessment by isotope dilution ultra-high performance liquid chromatography tandem mass spectrometry.

PubMed

Zhang, Yu; Wang, Qiao; Zhang, Gong; Jia, Wei; Ren, Yiping; Wu, Yongning

2018-02-01

Hemoglobin (Hb) adducts of acrylamide (AA) and its oxidative metabolite glycidamide (GA) are important biomarkers for evaluating the mid-term exposure of acrylamide toxicity in vivo. Taking pentafluoro-2-methylphenyl isothiocyanates of N-(2-carbamoylethyl)valine (AAVal-PFPTH) and N-(2-carbamoyl-2-hydroxyethy)valine (GAVal-PFPTH) as target analytes, we developed an isotope dilution ultra-high performance liquid chromatograph tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous determination of AA and GA hemoglobin (Hb) adducts under the electroscopy ionization negative (ESI‾) mode in the present work. Among them, the enantiomer pair of GA-Hb adducts was firstly identified and successfully separated at baseline level. The method achieved high sensitivity with the LOD and LOQ ranging 1.43-5.05pmol/g Hb and 4.78-16.82pmol/g Hb, respectively. The recovery rates with low, intermediate and high spiking levels were calculated as 97.0-105.2%, 97.4-106.4% and 100.3-111.2%, respectively. Acceptable within-laboratory reproducibility (RSD < 13.7%) substantially supported the robustness of current UHPLC-MS/MS method, which was successfully applied to measure the hemoglobin adducts of acrylamide and glycidamide enantiomers in blood of both rats and humans. A linear exposure assessment model was developed for estimating the daily exposure to acrylamide in humans via considering acrylamide hemoglobin adducts as variables, indicating a novel connect between biomarker-based internal exposure and dietary-based external exposure. Overall, the present instrumental analysis and related internal exposure assessment model provide a substantially methodological support for profiling the internal biological exposure and estimating the external dietary exposure to acrylamide. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection and characterization of human serum antibodies to polycyclic aromatic hydrocarbon diol-epoxide DNA adducts.

PubMed Central

Newman, M J; Light, B A; Weston, A; Tollurud, D; Clark, J L; Mann, D L; Blackmon, J P; Harris, C C

1988-01-01

The presence of serum antibodies to the diol-epoxide DNA adducts of representative polycyclic aromatic hydrocarbons (PAH), chrysene, benz[a]anthracene and benzo[a]pyrene, was determined by ELISA using serum samples obtained from normal healthy individuals. Antibodies that reacted against PAH adducted-DNA, but not against PAH-adducted protein, were found in the serum of approximately 40% of the test individuals. Specificity analysis of the antibodies demonstrated that serological cross-reactions between the benzo[a]pyrene and the chrysene diol-epoxide adducts were present. Similar cross-reactivity between the benz[a]anthracene and the chrysene adducts was observed. Sera containing antibodies that were apparently specific for each of the three PAH-DNA adducts were also identified. The presence of antibodies to PAH-DNA adducts indicates both past exposure to these carcinogenic PAH and their metabolic activation to the DNA damaging metabolites. These antibodies may prove to be useful in both retrospective and prospective epidemiological studies of various diseases associated with PAH exposure. PMID:3392204

Detection of Dichlorvos Adducts in a Hepatocyte Cell Line

DTIC Science & Technology

2014-06-30

form adducts to DDVP.16,20−22 Two distinct DDVP adducts are formed by covalent bonding to the hydroxyl group of tyrosine, serine, and threonine ...variable modifications on methionine (oxidation) and on serine, tyrosine, or threonine (phosphorylation, O-methylphosphate, or O-dimethylphosphate). Only...modifications on tyrosine, serine, or threonine residues with a mass of 94 or 108 amu corresponding to O-methyl- or O-dimethyl-phosphate groups. We found 53

Metabolic activation of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine and DNA adduct formation depends on p53: Studies in Trp53(+/+),Trp53(+/-) and Trp53(-/-) mice.

PubMed

Krais, Annette M; Speksnijder, Ewoud N; Melis, Joost P M; Singh, Rajinder; Caldwell, Anna; Gamboa da Costa, Gonçalo; Luijten, Mirjam; Phillips, David H; Arlt, Volker M

2016-02-15

The expression of the tumor suppressor p53 can influence the bioactivation of, and DNA damage induced by, the environmental carcinogen benzo[a]pyrene, indicating a role for p53 in its cytochrome P450 (CYP)-mediated biotransformation. The carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which is formed during the cooking of food, is also metabolically activated by CYP enzymes, particularly CYP1A2. We investigated the potential role of p53 in PhIP metabolism in vivo by treating Trp53(+/+), Trp53(+/-) and Trp53(-/-) mice with a single oral dose of 50 mg/kg body weight PhIP. N-(Deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP-C8-dG) levels in DNA, measured by liquid chromatography-tandem mass spectrometry, were significantly lower in liver, colon, forestomach and glandular stomach of Trp53(-/-) mice compared to Trp53(+/+) mice. Lower PhIP-DNA adduct levels in the livers of Trp53(-/-) mice correlated with lower Cyp1a2 enzyme activity (measured by methoxyresorufin-O-demethylase activity) in these animals. Interestingly, PhIP-DNA adduct levels were significantly higher in kidney and bladder of Trp53(-/-) mice compared to Trp53(+/+) mice, which was accompanied by higher sulfotransferase (Sult) 1a1 protein levels and increased Sult1a1 enzyme activity (measured by 2-naphthylsulfate formation from 2-naphthol) in kidneys of these animals. Our study demonstrates a role for p53 in the metabolism of PhIP in vivo, extending previous results on a novel role for p53 in xenobiotic metabolism. Our results also indicate that the impact of p53 on PhIP biotransformation is tissue-dependent and that in addition to Cyp1a enzymes, Sult1a1 can contribute to PhIP-DNA adduct formation. © 2015 The Authors International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.

Mass Spectrometric Evidence of Malonaldehyde and 4-Hydroxynonenal Adductions to Radical-Scavenging Soy Peptides

PubMed Central

Zhao, Jing; Chen, Jing; Zhu, Haining; Xiong, Youling L.

2012-01-01

Antioxidative peptides in food systems are potential targets of lipid oxidation-generated reactive aldehydes, such as malonaldehyde (MDA) and 4-hydroxynonenal (HNE). In this study, covalent modifications on radical-scavenging peptides prepared from soy protein hydrolysate by MDA and HNE were characterized by liquid chromatography–electrospray ionization-mass spectrometry (LC-ESI-MS/MS). MS/MS analyses detected the formation of Schiff base type adducts of MDA on the side chain groups of lysine, histidine, arginine, glutamine, and asparagine residues as well as the N-termini of peptides. MDA also formed a fluorescent product with lysine residues. HNE adducted on lysine residues through Schiff base formation and on histidine, arginine, glutamine, and asparagine residues mainly through Michael addition. In spite of the extensive MDA modification, peptide cross-linking by this potential mechanism was undetectable. PMID:22946674

Identification of N-(deoxyguanosin-8-yl)-4-azobiphenyl by (32)P-postlabeling analyses of DNA in human uroepithelial cells exposed to proximate metabolites of the environmental carcinogen 4-aminobiphenyl.

PubMed

Hatcher, James F; Swaminathan, Santhanam

2002-01-01

DNA adducts formed in human uroepithelial cells (HUC) following exposure to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP), were analyzed by the (32)P-postlabeling method. Two adducts detected by (32)P-postlabeling were previously identified as the 3',5'-bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (dA-C8-ABP) (Frederickson S et al. [1992] Carcinogenesis 13: 955-961; Hatcher and Swaminathan [1995b] Carcinogenesis 16: 295-301). In contrast to the dG-C8-ABP adduct, which was 3'-dephosphorylated by nuclease P1, dA-C8-ABP was resistant to nuclease P1, thus providing an enrichment step before postlabeling. Autoradiography of the two-dimensional thin-layer chromatogram of the postlabeled products obtained following nuclease P1 digestion revealed several minor adducts, one of which has been identified in the present study. Postlabeling analyses following nuclease P1 digestion of the products obtained from the reaction of N-acetoxy-4-aminobiphenyl with deoxyguanosine-3'-monophosphate (dGp) demonstrated the presence of this minor adduct. The 3'-monophosphate derivative of the adduct was subsequently chromatographically purified and subjected to spectroscopic analyses. Based on proton NMR and mass spectroscopic analyses of the synthetic product, the chemical structure of the adduct has been identified as N-(deoxyguanosin-N(2)-yl)-4-azobiphenyl (dG-N==N-ABP). (32)P-Postlabeling analysis of the nuclease P1-enriched DNA hydrolysate of HUCs treated with N-OH-ABP or N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) showed the presence of the dG-N==N-ABP adduct. It was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl-CoA, or incubated with HUC microsomes and N-OH-AABP. These results demonstrate that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP and N-OH-AABP are bioactivated by

Optic Nerve Sheath Tethering in Adduction Occurs in Esotropia and Hypertropia, But Not in Exotropia

PubMed Central

Suh, Soh Youn; Clark, Robert A.; Demer, Joseph L.

2018-01-01

Purpose Repetitive strain to the optic nerve (ON) due to tethering in adduction has been recently proposed as an intraocular pressure-independent mechanism of optic neuropathy in primary open-angle glaucoma. Since strabismus may alter adduction, we investigated whether gaze-related ON straightening and associated globe translation differ in horizontal and vertical strabismus. Methods High-resolution orbital magnetic resonance imaging was obtained in 2-mm thick quasi-coronal planes using surface coils in 25 subjects (49 orbits) with esotropia (ET, 19 ± 3.6Δ SEM), 11 (15 orbits) with exotropia (XT, 33.7 ± 7.3Δ), 7 (12 orbits) with hypertropia (HT, 14.6 ± 3.2Δ), and 31 normal controls (62 orbits) in target-controlled central gaze, and in maximum attainable abduction and adduction. Area centroids were used to determine ON path sinuosity and globe positions. Results Adduction angles achieved in ET (30.6° ± 0.9°) and HT (27.2° ± 2.3°) did not significantly differ from normal (28.3° ± 0.7°), but significantly less adduction was achieved in XT (19.0° ± 2.5°, P = 0.005). ON sheath tethering in adduction occurred in ET and HT similarly to normal, but did not in XT. The globe translated significantly less than normal, nasally in adduction in XT and temporally in abduction in ET and HT (P < 0.02, for all). Globe retraction did not occur during abduction or adduction in any group. Conclusions Similar to normal subjects, the ON and sheath become tethered without globe retraction in ET and HT. In XT, adduction tethering does not occur, possibly due to limited adduction angle. Thus, therapeutic limitation of adduction could be considered as a possible treatment for ON sheath tethering.

Evaluation of serum and liver toxicokinetics for furan and liver DNA adduct formation in male Fischer 344 rats.

PubMed

Churchwell, M I; Scheri, R C; Von Tungeln, L S; Gamboa da Costa, G; Beland, F A; Doerge, D R

2015-12-01

Furan is a food processing contaminant found in many common cooked foods that induces liver toxicity and liver cancer in animal models treated with sufficient doses. The metabolism of furan occurs primarily in the liver where CYP 2E1 produces a highly reactive bis-electrophile, cis-2-butene-1,4-dial (BDA). BDA reacts with nucleophilic groups in amino acids and DNA in vitro to form covalent adducts. Evidence for BDA-nucleoside adduct formation in vivo is limited but important for assessing the carcinogenic hazard of dietary furan. This study used controlled dosing with furan in Fischer 344 rats to measure serum and liver toxicokinetics and the possible formation of BDA-nucleoside adducts in vivo. After gavage exposure, furan concentrations in the liver were consistently higher than those in whole blood (∼6-fold), which is consistent with portal vein delivery of a lipophilic compound into the liver. Formation of BDA-2'-deoxycytidine in furan-treated rat liver DNA was not observed using LC/MS/MS after single doses as high as 9.2 mg/kg bw or repeated dosing for up to 360 days above a consistent background level (1-2 adducts per 10(8) nucleotides). This absence of BDA-nucleoside adduct formation is consistent with the general lack of evidence for genotoxicity of furan in vivo. Published by Elsevier Ltd.

Nitro-fatty Acid Metabolome: Saturation, Desaturation, β-Oxidation, and Protein Adduction*

PubMed Central

Rudolph, Volker; Schopfer, Francisco J.; Khoo, Nicholas K. H.; Rudolph, Tanja K.; Cole, Marsha P.; Woodcock, Steven R.; Bonacci, Gustavo; Groeger, Alison L.; Golin-Bisello, Franca; Chen, Chen-Shan; Baker, Paul R. S.; Freeman, Bruce A.

2009-01-01

Nitrated derivatives of fatty acids (NO2-FA) are pluripotent cell-signaling mediators that display anti-inflammatory properties. Current understanding of NO2-FA signal transduction lacks insight into how or if NO2-FA are modified or metabolized upon formation or administration in vivo. Here the disposition and metabolism of nitro-9-cis-octadecenoic (18:1-NO2) acid was investigated in plasma and liver after intravenous injection in mice. High performance liquid chromatography-tandem mass spectrometry analysis showed that no 18:1-NO2 or metabolites were detected under basal conditions, whereas administered 18:1-NO2 is rapidly adducted to plasma thiol-containing proteins and glutathione. NO2-FA are also metabolized via β-oxidation, with high performance liquid chromatography-tandem mass spectrometry analysis of liver lipid extracts of treated mice revealing nitro-7-cis-hexadecenoic acid, nitro-5-cis-tetradecenoic acid, and nitro-3-cis-dodecenoic acid and corresponding coenzyme A derivatives of 18:1-NO2 as metabolites. Additionally, a significant proportion of 18:1-NO2 and its metabolites are converted to nitroalkane derivatives by saturation of the double bond, and to a lesser extent are desaturated to diene derivatives. There was no evidence of the formation of nitrohydroxyl or conjugated ketone derivatives in organs of interest, metabolites expected upon 18:1-NO2 hydration or nitric oxide (•NO) release. Plasma samples from treated mice had significant extents of protein-adducted 18:1-NO2 detected by exchange to added β-mercaptoethanol. This, coupled with the observation of 18:1-NO2 release from glutathione-18:1-NO2 adducts, supports that reversible and exchangeable NO2-FA-thiol adducts occur under biological conditions. After administration of [3H]18:1-NO2, 64% of net radiolabel was recovered 90 min later in plasma (0.2%), liver (18%), kidney (2%), adipose tissue (2%), muscle (31%), urine (6%), and other tissue compartments, and may include metabolites not yet

Fast Fourier Transform IR Characterization of Epoxy GY Systems Crosslinked with Aliphatic and Cycloaliphatic EH Polyamine Adducts