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Toxicological Sciences : An Official... Feb 2004Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent.... (Comparative Study)
Comparative Study
Chemical-DNA adducts provide an integrated measure of exposure, absorption, bioactivation, detoxification, and DNA repair following exposure to a genotoxic agent. Benzo[a]pyrene (BaP), a prototypical polycyclic aromatic hydrocarbon (PAH), can be bioactivated by cytochrome P-450s (CYPs) and epoxide hydrolase to genotoxic metabolites which form covalent adducts with DNA. In this study, we utilized precision-cut rat liver and lung slices exposed to BaP to investigate tissue-specific differences in chemical absorption and formation of DNA adducts. To investigate the contribution of bioactivating CYPs (such as CYP1A1 and CYP1B1) on the formation of BaP-DNA adducts, animals were also pretreated in vivo with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) prior to in vitro incubation of tissue slices with BaP. Furthermore, the tissue distribution of BaP and BaP-DNA adduct levels from in vivo studies were compared with those from the in vitro tissue slice experiments. The results indicate a time- and concentration-dependent increase in tissue-associated BaP following exposure of rat liver and lung tissue slices to BaP in vitro, with generally higher levels of BaP retained in lung tissue. Furthermore, rat liver and lung slices metabolized BaP to reactive intermediates that formed covalent adducts with DNA. Total BaP-DNA adducts increased with concentration and incubation time. Adduct levels (fmol adduct/microg DNA) in lung slices were greater than liver at all doses. Liver slices contained one major and two minor adducts, while lung slices contained two major and 3 minor adducts. The tissue-specific qualitative profile of these adducts in tissue slices was similar to that observed from in vivo studies, further validating the use of this model. Pretreatment of animals with TCDD prior to in vitro incubation with BaP potentiated the levels of DNA adduct formation. TCDD pretreatment altered the adduct distribution in lung but not in liver slices. Together, the results suggest that tissue-specific qualitative and quantitative differences in BaP-DNA adducts could contribute to the lung being a target tissue for BaP carcinogenesis. Furthermore, the results validate the use of precision-cut tissue slices incubated in dynamic organ culture as a useful model for the study of chemical-DNA adduct formation.
Topics: Animals; Aryl Hydrocarbon Hydroxylases; Benzo(a)pyrene; Carcinogens, Environmental; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; DNA Adducts; Liver; Lung; Male; Models, Animal; Organ Culture Techniques; Phosphorus Radioisotopes; Polychlorinated Dibenzodioxins; Rats; Rats, Sprague-Dawley; Tritium
PubMed: 14691214
DOI: 10.1093/toxsci/kfh030 -
Carcinogenesis Jun 19951,2,3-Trichloropropane (TCP) is a multispecies, multisite carcinogen which has been found to be an environmental contaminant. In this study, we have characterized and...
1,2,3-Trichloropropane (TCP) is a multispecies, multisite carcinogen which has been found to be an environmental contaminant. In this study, we have characterized and measured DNA adducts formed in vivo following exposure to TCP. [14C]TCP was administered to male B6C3F1 mice and Fischer-344 rats by gavage at doses used in the NTP carcinogenesis bioassay. Both target and nontarget organs were examined for the formation of DNA adducts. Adducts were hydrolyzed from DNA by neutral thermal or mild acid hydrolysis, isolated by HPLC, and detected and quantitated by measurement of radioactivity. The HPLC elution profile of radioactivity suggested that one major DNA adduct was formed. To characterize this adduct, larger yields were induced in rats by intraperitoneal administration of TCP (300 mg/kg). The DNA adduct was isolated by HPLC based on coelution with the radiolabeled adduct, and compared to previously identified adducts. The isolated adduct coeluted with S-[1-(hydroxymethyl)-2-(N7-guanyl)-ethyl]glutathione, an adduct derived from the structurally related carcinogen 1,2-dibromo-3-chloropropane (DBCP). Analysis by electrospray mass spectrometry suggested that the TCP-induced adduct and the DBCP-derived adduct were identical. The 14C-labeled DNA adduct was distributed widely among the organs examined. Adduct levels varied depending on species, organ, and dose. In rat organs, adduct concentrations for the low dose ranged from 0.8 to 6.6 mumol per mol guanine and from 7.1 to 47.6 mumol per mol guanine for the high dose. In the mouse, adduct yields ranged from 0.32 to 28.1 mumol per mol guanine for the low dose and from 12.2 to 208.1 mumol per mol guanine for the high dose. The relationship between DNA adduct formation and organ-specific tumorigenesis was unclear. Although relatively high concentrations of DNA adducts were detected in target organs, several nontarget sites also contained high adduct levels. Our data suggest that factors in addition to adduct formation may be important in TCP-induced carcinogenesis.
Topics: Animals; Carcinogens; Chromatography, High Pressure Liquid; DNA Adducts; Glutathione; Male; Mass Spectrometry; Mice; Oxidation-Reduction; Propane; Rats; Rats, Inbred F344
PubMed: 7788863
DOI: 10.1093/carcin/16.6.1419 -
Journal of Biological Inorganic... Mar 2020Arylamines are known to form covalent-DNA adducts upon metabolic activation. These covalent adducts adopt different conformational attributes, viz., major groove (B),...
Arylamines are known to form covalent-DNA adducts upon metabolic activation. These covalent adducts adopt different conformational attributes, viz., major groove (B), stacked (S), and minor groove (W), and lead to different types of mutations. The conformation depends on the flanking and next flanking bases at the 3' position of the adduct. Early detection of these conformations by simple probes is an ideal and challenging task. Here, we have reported two Ir(III)-based cyclometalated complexes, viz., [Ir(ppy)(imiphen)] (1) (ppy: 2-phenylpyridine; imiphen: 2-(1H-imidazol-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and [Ir(ppy)(furphen)] (2) (furphen: 2-(furan-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline) and its interaction with N-acetyl-2-aminofluorene-dG (AAF-dG). The sequences used in this work are NarI sequence (-CGGCGCX-) in which Gs are modified with AAF and X is either C or T. Luminescence studies reveal that the Ir(III) complexes bind to AAF-dG adduct with high specificity toward G and G compared to G and unmodified control. The selectivity also depends on the next flanking base as cytosine favors G, while thymine favors G in complex 1 and vice versa for complex 2. The quenching studies confirm that Ir(III) complexes bind with AAF-dG sequences through the minor groove. The outcome of this work reveals that the switch-on effect by the complexes can be utilized for determining the conformational heterogeneity of the adduct and also for similar covalent-DNA adducts.
Topics: Amines; Coordination Complexes; DNA Adducts; Iridium; Molecular Conformation
PubMed: 32052177
DOI: 10.1007/s00775-020-01762-7 -
Regulatory Toxicology and Pharmacology... Dec 2000The main achievements in the DNA adduct field in the 1990s have been technical innovations of methods for specific adducts reaching sensitivities required for low levels... (Review)
Review
The main achievements in the DNA adduct field in the 1990s have been technical innovations of methods for specific adducts reaching sensitivities required for low levels encountered in humans. Over 20 specific adducts or closely related groups of adducts have been determined in humans. The sources of the DNA-binding agents are endogenous and exogenous or both. In some of these studies adduct levels have been correlated to metabolic or DNA repair genotypes. An example of DNA adduct studies in human target tissue is taken on UV photoproducts in skin in situ. Adduct-induced mutations, specific mutation spectra, and their relationship to cancer are discussed. The quantitative adduct techniques will enable comparisons of endogenous and exogenous adduct levels and will give important clues to the etiology of human cancer. Furthermore, adducts will provide an intermediary tool for genotyping studies, both for metabolic enzyme and for DNA repair system genotypes. As the common polymorphisms are likely to cause at most moderate increases in the risk of cancer, the intermediary adduct endpoint is a necessary proof of causal relationships. The present and future biomonitoring studies will cover many endpoints to link the mechanistic steps from DNA adducts to cancer via mutations and modulating host susceptibility factors.
Topics: Animals; Carcinogens; DNA Adducts; DNA Damage; Humans; Mutation; Neoplasms; Ultraviolet Rays
PubMed: 11162720
DOI: 10.1006/rtph.2000.1431 -
Chemical Research in Toxicology May 2018Air pollution is a major environmental risk for human health. Acetaldehyde is present in tobacco smoke and vehicle exhaust. In this study, we show that [C]-acetaldehyde...
Air pollution is a major environmental risk for human health. Acetaldehyde is present in tobacco smoke and vehicle exhaust. In this study, we show that [C]-acetaldehyde induces DNA modification with the formation of isotopically labeled 1, N-propano-2'-deoxyguanosine adducts in the brain and lungs of rats exposed to concentrations of acetaldehyde found in the atmosphere of megacities. The adduct, with the addition of two molecules of isotopically labeled acetaldehyde [C]-1, N-propano-dGuo, was detected in the lung and brain tissues of exposed rats by micro-HPLC/MS/MS. Structural confirmation of the products was unequivocally performed by nano-LC/ESI-HRMS analyses. DNA modifications induced by acetaldehyde have been regarded as a key factor in the mechanism of mutagenesis and may be involved in the cancer risks associated with air pollution.
Topics: Acetaldehyde; Animals; Brain; Carbon Isotopes; DNA Adducts; Lung; Male; Molecular Structure; Rats; Rats, Wistar
PubMed: 29707942
DOI: 10.1021/acs.chemrestox.8b00016 -
International Journal of Biological... May 2017DNA nucleobases undergo non-enzymatic glycation to nucleobase adducts which can play important roles in vivo. In this work, we conducted a comprehensive experimental and...
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.
Topics: Aldehydes; DNA; DNA Adducts; DNA Damage; Glycosylation; Glyoxal; Guanine; Kinetics
PubMed: 28192135
DOI: 10.1016/j.ijbiomac.2017.01.140 -
Mutation Research Aug 1996We examined the binding of various steroid hormones to DNA in vitro by means of 32P-postlabeling. Seventeen steroid hormones and cholesterol (CS) were incubated with...
We examined the binding of various steroid hormones to DNA in vitro by means of 32P-postlabeling. Seventeen steroid hormones and cholesterol (CS) were incubated with human liver DNA at 37 degrees C for 1 h under aerobic conditions in the absence of catalysis. The reaction mixtures were analyzed by the nuclease P-1 version of 32P-postlabeling. The results showed that cortexolone (CX), prednisolone (PS), cortisone (CN), cortisol (CL), tetrahydrocortisol (TC), corticosterone (CC), 11-deoxycorticosterone (DC), dexamethasone (DX), dihydrocortisol (DL), and aldosterone (AL) covalently bound with DNA. However, progesterone (PG), 17 alpha-hydroxyprogesterone (HG), estrone (E1), estradiol (E2), estriol (E3), testosterone (TS), cortol (CR) and the original compound for biosynthesis, CS, did not form adducts. In absence of DNA, the steroids themselves did not give rise to any spot on TLC under the same conditions. The dose-responses of DNA binding by DC, DL, CC, CL and CN were linear. The relative adduct labeling of reactive steroids at a concentration of 2 mM were as follows: 68.8 (CX), 53.2 (PS), 39.6 (CN), 29.9 (CL), 20.9 (TC), 12.9 (CC), 12.3 (DC), 7.5 (DX), 4.7 (DL), 1.2 (AL) adducts per 10(8) nucleotides. Reactive and nonreactive steroids were distinguishable by the presence or absence of the carbonyl group (-CO-CH2OH) at carbon seventeen (C17) of the cholesterol skeleton. This implies that the electrophilic carbonyl or a neighboring group perhaps involved in the formation of covalent bond with DNA. To investigate the nature of target base(s) of these DNA reactive steroids, mononucleotides of all four bases of DNA were reacted with CN, CL, CC and cochromatographed with the obtained spots of DNA reactions. The results of which stated that these steroids and guanine reaction gave the same spots as observed in DNA reaction, indicating guanine is the main target of these DNA reactive steroids. Hep G2 human hepatocellular carcinoma cells were used as an alternative model. Although nine steroids (CL, DL, TC, PS, DX, PG, E2, TX, CR) did not react with intracellular DNA under our experimental conditions, our findings suggested that some hormonal steroids can form covalent DNA adducts in vivo.
Topics: Carcinogens; DNA Adducts; Guanine; Humans; Liver; Steroids; Tumor Cells, Cultured
PubMed: 8830806
DOI: 10.1016/s0165-1218(96)90126-3 -
Chemical Research in Toxicology Oct 2003Acrylamide (AA) is a high production volume chemical with many industrial uses; however, recent findings of ppm levels in starchy foods cooked at high temperature have...
Acrylamide (AA) is a high production volume chemical with many industrial uses; however, recent findings of ppm levels in starchy foods cooked at high temperature have refocused worldwide attention on the neurotoxicity, germ cell mutagenicity, and carcinogenicity of AA. Oxidative metabolism of AA to its epoxide metabolite, glycidamide (GA), has been observed in experimental animals and humans and may be associated with many of the toxic effects of AA exposure, including formation of N7-(2-carbamoyl-2-hydroxyethyl)guanine (N7-GA-Gua) in vivo. This paper describes the characterization of two new GA-derived DNA adducts formed in vitro, N3-(2-carbamoyl-2-hydroxyethyl)adenine (N3-GA-Ade) and N1-(2-carboxy-2-hydroxyethyl)-2'-deoxyadenosine. A sensitive method for quantification of N7-GA-Gua and N3-GA-Ade, based on LC with tandem mass spectrometry and isotope dilution, was developed and validated for use in measuring DNA adduct formation in selected tissues of adult and whole body DNA of 3 day old neonatal mice treated with AA and GA. In adult mice, DNA adduct formation was observed in liver, lung, and kidney with levels of N7-GA-Gua around 2000 adducts/10(8) nucleotides and N3-GA-Ade around 20 adducts/10(8) nucleotides. Adduct levels were modestly higher in adult mice dosed with GA as opposed to AA; however, treatment of neonatal mice with GA produced 5-7-fold higher whole body DNA adduct levels than with AA, presumably reflective of lower oxidative enzyme activity in newborn mice. DNA adduct formation from AA treatment in adult mice showed a supralinear dose-response relationship, consistent with saturation of oxidative metabolism at higher doses. These results increase our understanding of the mutagenic potential of GA and provide further evidence for a genotoxic mechanism in AA carcinogenesis.
Topics: Acrylamide; Aging; Animals; Chromatography, Liquid; DNA Adducts; Dose-Response Relationship, Drug; Epoxy Compounds; Male; Mass Spectrometry; Mice; Molecular Structure; Nucleosides; Reproducibility of Results; Salmon; Testis
PubMed: 14565774
DOI: 10.1021/tx034108e -
Mutation Research Dec 2006Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of... (Comparative Study)
Comparative Study
Aristolochic acid (AA) is a potent nephrotoxin and carcinogen and is the causative factor for Chinese herb nephropathy. AA has been associated with the development of urothelial cancer in humans, and kidney and forestomach tumors in rodents. To investigate the molecular mechanisms responsible for the tumorigenicity of AA, we determined the DNA adduct formation and mutagenicity of AA in the liver (nontarget tissue) and kidney (target tissue) of Big Blue rats. Groups of six male rats were gavaged with 0, 0.1, 1.0 and 10.0 mg AA/kg body weight five times/week for 3 months. The rats were sacrificed 1 day after the final treatment, and the livers and kidneys were isolated. DNA adduct formation was analyzed by 32P-postlabeling and mutant frequency (MF) was determined using the lambda Select-cII Mutation Detection System. Three major adducts (7-[deoxyadenosin-N6-yl]-aristolactam I, 7-[deoxyadenosin-N6-yl]-aristolactam II and 7-[deoxyguanosin-N2-yl]-aristolactam I) were identified. There were strong linear dose-responses for AA-induced DNA adducts in treated rats, ranging from 25 to 1967 adducts/10(8) nucleotides in liver and 95-4598 adducts/10(8) nucleotides in kidney. A similar trend of dose-responses for mutation induction also was found, the MFs ranging from 37 to 666 x 10(-6) in liver compared with the MFs of 78-1319 x 10(-6) that we previously reported for the kidneys of AA-treated rats. Overall, kidneys had at least two-fold higher levels of DNA adducts and MF than livers. Sequence analysis of the cII mutants revealed that there was a statistically significant difference between the mutation spectra in both kidney and liver of AA-treated and control rats, but there was no significant difference between the mutation spectra in AA-treated livers and kidneys. A:T-->T:A transversion was the predominant mutation in AA-treated rats; whereas G:C-->A:T transition was the main type of mutation in control rats. These results indicate that the AA treatment that eventually results in kidney tumors in rats also results in significant increases in DNA adduct formation and cII MF in kidney. Although the same treatment does not produce tumors in rat liver, it does induce DNA adducts and mutations in this tissue, albeit at lower levels than in kidney.
Topics: Animals; Aristolochic Acids; DNA Adducts; Kidney; Liver; Mutagens; Mutation; Rats; Rats, Inbred Strains
PubMed: 17010389
DOI: 10.1016/j.mrfmmm.2006.08.004 -
Methods in Molecular Biology (Clifton,... 2020In DNA adduct analysis, the P-postlabeling technique is a powerful tool due to its high detection sensitivity. It is performed by enzymatic digestion of DNA samples,...
In DNA adduct analysis, the P-postlabeling technique is a powerful tool due to its high detection sensitivity. It is performed by enzymatic digestion of DNA samples, enrichment of the adduct nucleotides, and then 5'-labeling with P. This method is particularly useful for detection of bulky adducts. An additional advantage is that only a small amount of DNA is required for detecting DNA adducts. This chapter describes the experimental procedure for separation and detection of DNA adducts by polyacrylamide gel electrophoresis, which is an attractive method for visually assessing differences in adduct formation between samples.
Topics: Animals; Cell Line; DNA Adducts; Electrophoresis, Polyacrylamide Gel; Humans; Isotope Labeling; Phosphorus Radioisotopes
PubMed: 31989527
DOI: 10.1007/978-1-0716-0323-9_18