Characterization Of DNA Adducts Research Articles

Unlocking The Mysteries of DNA Adducts with Artificial Intelligence.

Cellular genome is considered a dynamic blueprint of a cell since it encodes genetic information that gets temporally altered due to various endogenous and exogenous insults. Largely, the extent of genomic dynamicity is controlled by the trade-off between DNA repair processes and the genotoxic potential of the causative agent (genotoxins or potential carcinogens). A subset of genotoxins form DNA adducts by covalently binding to the cellular DNA, triggering structural or functional changes that lead to significant alterations in cellular processes via genetic (e. g., mutations) or non-genetic (e. g., epigenome) routes. Identification, quantification, and characterization of DNA adducts are indispensable for their comprehensive understanding and could expedite the ongoing efforts in predicting carcinogenicity and their mode of action. In this review, we elaborate on using Artificial Intelligence (AI)-based modeling in adducts biology and present multiple computational strategies to gain advancements in decoding DNA adducts. The proposed AI-based strategies encompass predictive modeling for adduct formation via metabolic activation, novel adducts' identification, prediction of biochemical routes for adduct formation, adducts' half-life predictions within biological ecosystems, and, establishing methods to predict the link between adducts chemistry and its location within the genomic DNA. In summary, we discuss some futuristic AI-based approaches in DNA adduct biology.

Synthesis and Characterization of DNA Adducts from the HIV Reverse Transcriptase Inhibitor Nevirapine

Nevirapine (NVP) is a non-nucleoside reverse transcriptase inhibitor used against the human immunodeficiency virus type 1 (HIV-1), mostly to prevent mother-to-child HIV transmission in developing countries. One of the limitations of nevirapine use is severe hepatotoxicity, which raises concerns about its administration, particularly in the perinatal and pediatric settings. Nevirapine metabolism involves oxidation of the 4-methyl substituent to 12-hydroxy-NVP and the formation of phenolic derivatives. Further metabolism of 12-hydroxy-NVP by phase II esterification may produce electrophilic derivatives capable of reacting with DNA to yield covalent adducts, which could potentially be involved in the initiation of mutagenic and carcinogenic events. In the present study, we have investigated the reactivity of the synthetic model electrophile, 12-mesyloxy-NVP, toward 2'-deoxynucleosides and DNA. Parallel synthetic studies were conducted with 12-bromo-NVP and 3',5'- O-bis( tert-butyldimethylsilyl)-2'-deoxynucleosides, using palladium(0) catalysis. Multiple adducts from deoxyguanosine, deoxyadenosine, and deoxycytidine were isolated in the reactions with 12-mesyloxy-NVP and characterized by NMR and MS. The adduct structures consistently involved binding through C12 of NVP and N7 or N9 of deoxyguanosine; N1, N3, or N9 of deoxyadenosine; and N3 of deoxycytidine. Reactions conducted under palladium(0) catalysis yielded adducts through O (6) and N1 of deoxyguanosine, N1 of deoxyadenosine, and N3 of deoxycytidine. At least seven deoxynucleoside-NVP adducts were present in DNA reacted with 12-mesyloxy-NVP and enzymatically hydrolyzed. Four of these adducts were identified as 12-(deoxyadenosin-N1-yl)nevirapine, 12-(deoxycytidin-N3-yl)nevirapine, 12-(deoxyguanosin- O(6)-yl)nevirapine, and 12-(deoxyadenosin- N(6)-yl)nevirapine. One depurinating adduct, 12-(guanin-N7-yl)nevirapine, was identified in the thermal neutral DNA hydrolysate. If formed in vivo, some of these adducts would have considerable mutagenic potential. Our results thus suggest that NVP metabolism to 12-hydroxy-NVP may be a factor in NVP hepatocarcinogenicity.

DNA damage and acute toxicity caused by the urban air pollutant 3‐nitrobenzanthrone in rats; Characterization of DNA adducts in eight different tissues and organs with synthesized standards

3-Nitrobenzanthrone (3-NBA) is an urban air pollutant and rat lung carcinogen that is among the most potent mutagens yet tested in the Salmonella reversion assay. In the present study, 1 mg 3-NBA was administered orally to female F344 rats and DNA adduct formation was examined in liver, lung, kidney and five sections of the gastrointestinal (GI) tract at 6 hr, and 1, 2, 3, 5, and 10 days after administration. The DNA adduct patterns, analyzed by (32)P-postlabelling followed by HPLC separation, were similar in all tissues and organs. Five of the adduct peaks cochromatographed with synthesized DNA adduct standards. Three of these unequivocally determined standards, dGp-C8-N-ABA, dGp-N2-C2-ABA, and dAp-N6-C2-ABA, were of the nonacetylated type, suggesting that at least part of the pathway for activation of 3-NBA proceeds through O-acetylation of the hydroxylamine intermediate. The two other DNA adduct standards, dGp-C8-C2-N-Ac-ABA, and dGp-N2-C2-N-Ac-ABA, were of the acetylated type, but there was some ambiguity in the characterization of these DNA adducts, since they varied inconsistently between samples and they also aligned with peaks found in controls. At 6 hr after treatment, the level of DNA adducts was highest in glandular stomach (relative adduct labeling (RAL), approximately 70 adducts/10(8) normal nucleotides (NN)); adduct levels in this organ decreased at 24 hr, but increased afterwards. DNA adduct levels in the majority of organs were characterized by an early increase (from 6 hr to 3 days), which was followed by a decrease at 5 days and a maximum level 10 days after administration (RAL approximately 120 adducts/10(8) NN for the lung, kidney and glandular stomach, approximately 80 adducts/10(8) NN for the forestomach and ceacum, and approximately 40 adducts/10(8) NN for the liver, small intestine, and colon). This pattern was consistent with pathological observations during autopsy showing high levels of tissue damage in the GI tract; the tissue damage included hemorrhages, loss of villous surface structure in the small intestine, as well as intestine fragility and oedema of the adipose tissue around the GI-tract. Tissue damage decreased and DNA adduct levels increased at 10 days after administration. These observations suggest that 3-NBA not only exerts acute toxic effects, but that the bioavailability is affected by storage in tissues and later becomes available, resulting in the increased DNA adduct levels at the later time points of collection.

Characterization of DNA adducts from lung tissue of asphalt fume-exposed mice by nanoflow liquid chromatography quadrupole time-of-flight mass spectrometry

A bioanalytical method based on nanoflow liquid chromatography coupled to a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometry was developed to characterize selected polyaromatic hydrocarbon (PAH)–DNA adducts. The collision-induced dissociation of analytes results in characteristic fragmentation patterns that can be utilized to identify the DNA adducts. In the experiment, 32 B6C3F1 mice were exposed daily (4 h/day) to asphalt fume in a whole-body inhalation chamber for 10 days; 16 nonexposed mice served as controls. The asphalt fume was generated at 180 °C and the concentrations of PAHs in the animal exposure chamber ranged from 152 to 198 mg/m 3. The DNA adducts N 2-deoxyguanosine-benzo( a)pyrene-7,8-dihydrodiol-9,10-epoxide ( N 2-dG-BPDE); N 6-deoxyadenosine-benzo( a)pyrene-7,8-dihydrodiol-9,10-epoxide ( N 6-dA-BPDE), and N 4-deoxycytidine-benzo( a)pyrene-7,8-dihydrodiol-9,10-epoxide ( N 4-dC-BPDE) were identified. The concentrations of N 2-dG-BPDE, N 6-dA-BPDE, and N 4-dC-BPDE adducts were determined to be 1.17, 0.97, and 0.68 pmol/mg DNA, respectively, in the lung tissue of exposed mice using the nanoflow technique. The total DNA adducts in exposed lung tissue was determined to be 8.35 pmol/mg DNA by 32P-postlabeling assay. In total, the results indicated that PAH–DNA adducts were significantly elevated ( p<0.001) in the lung tissue of asphalt-fume-exposed mice relative to tissue from control animals.

Characterization of DNA adducts and tetraols derived from anti-benzo[ghi]fluoranthane-3,4-dihydrodiol-5,5a-epoxide.

A total of seven DNA adducts and two racemic tetraols derived from anti-benzo[ghi]fluoranthene-3,4-dihydrodiol-5,5a-epoxide (anti-B[ghi]FDE, 2) were characterized by analyses of UV, (1)H NMR, CD, and MALDI mass spectra. The structure of 2 is the first example of a diolepoxide in which a fully fused cyclopentane ring is covalently linked to the saturated ring bearing the epoxide function. Compound 2 is also a conformationally rigid structure analogue of the extensively studied anti-benzo[c]phenanthrene-3,4-dihydrodiol-1,2-epoxide (anti-BcPDE), thus serving as a model for probing the diolepoxide-DNA interaction [Chang et al. (2002) Chem. Res. Toxicol. 15, 198-208 (following paper in this issue)]. The most abundant adducts are formed from trans- or cis-openings of the epoxide by the amino groups of either deoxyguanosine or deoxyadenosine. Adducts of minor abundance formed by the attachment of the diolepoxide to the amino group of deoxycytidine N(4) and guanine N(7) were also isolated. Post-source decay MALDI spectra of the (M + H)(+) molecule ions are consistent with the assigned adduct structures. The lack of a typical benzylic proton at the site of deoxynucleoside attachment necessitated a new NMR assignment strategy. Despite the steric constraint, the epoxide ring opening of 2 occurred exclusively at the dibenzylic C5a, not at C5. The assignments on the trans- and cis-epoxide opening were made based on the molecular modeling structures, i.e., the pseudoaxial H5 in cis-adducts is placed directly under the strong influence of a shielding cone of the aromatic ring system, while the same proton in trans-adducts adopts a pseudoequatorial conformation, thereby protruding away from the aromatic ring system. The absolute configuration at the site of deoxynucleoside attachment (C5a) was tentatively assigned on the basis of the empirical rules that have been established for deoxynucleoside-adducts derived from traditional alternant PAH diolepoxides.

Reversed-phase high-performance liquid chromatographic separation of32P-labeled nucleotide adducts formed by food-derived carcinogenic aminoimidazoazarenes

Carcinogenic heterocyclic amines are formed on the surface of grilled or fried meat and fish. These heterocyclic amines have been shown to form the covalent DNA adducts considered to be the first stage of the process of carcinogenesis. In this paper we describe the separation of DNA adducts formed by six food-derived mutagenic aminoimidazoazarenes.32P-Postlabeling is the most sensitive technique for analysis of DNA adducts formed by covalent bonding of chemical carcinogens to DNA. An improved method developed by combining ion-exchange thin-layer chromatography with reversed-phase high-performance liquid chromatography enabled the resolution of the32P-labeled monophosphate nucleotides modified with reactive derivatives of these food mutagens. Optimum conditions for the separation were obtained by use of a Zorbax phenyl-modified silica gel column and a gradient system consisting of acetonitrile and a phosphate buffer of high ionic strength (0.5m) at pH 2.0. Sub-femtomole quantities of labeled adducts were detected with a radioactivity flow detector coupled to the HPLC equipment. This method can be used for the characterization of DNA adducts detected in human tissues and for analysis of mixtures of DNA adducts formed in experiments on combination genotoxic effects of mixtures of food-derived heterocyclic amines.

Characterization of DNA adducts in Chinese hamster ovary cells treated with mutagenic doses of 1- and 3-nitrosobenzo[ a]pyrene and the trans-7,8-diol- anti-9,10-epoxides of 1- and 3-nitrobenzo[ a]pyrene

The environmental contaminants 1- and 3-nitrobenzo[ a]pyrene (1- and 3-nitro-BaP) are mutagens in Chinese hamster ovary (CHO) cells with exogenous metabolic activation. Previous studies demonstrated the potent direct-acting mutagenicity of the oxidized metabolites, trans-7,8-dihydroxy- anti-9,10-epoxy-7,8,9,10-tetrahydro-1-nitrobenzo[ a]pyrene (1-NBaPDE) and trans-7,8-dihydroxy- anti-9,10-epoxy-7,8,9,10-tetrahydro-3-nitrobenzo[ a]pyrene (3-NBaPDE), and the partially nitroreduced metabolites, 1- and 3-nitrosobenzo[ a]pyrene (1- and 3-NO-BaP). In this study, we have identified the major adduct formed by incubation of calf thymus DNA with 1-NBaPDE and used this standard in conjunction with other adduct standards to characterize the 32P-postlabeled DNA adducts produced by 1- and 3-nitro-BaP metabolites in CHO cultures. The major adduct from 1-NBaPDE exposure was 10-(deoxyguanosin- N 2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-1-nitrobenzo[ a]pyrene; from 3-NBaPDE, 10-(deoxyguanosin- N 2-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydro-3-nitrobenzo[ a]pyrene; from 1-NO-BaP, 6-(deoxyguanosin- N 2-yl)-1-aminobenzo[ a]pyrene; and from 3-NO-BaP, 6-(deoxyguanosin- N 2-yl)-3-aminobenzo[ a]pyrene. For comparison, the adducts formed by trans-7,8-dihydroxy- anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[ a]pyrene and the related nitroreduced derivative 6-nitrosobenzo[ a]pyrene were also examined. The nitrobenzo[ a]pyrene DNA adducts described in this study are proposed to be involved in the mutagenicity of 1- and 3-nitro-BaP upon either oxidative or reductive metabolism.

Characterization of DNA Adducts Formed by the Four Configurationally Isomeric 5,6-Dimethylchrysene 1,2-Dihydrodiol 3,4-Epoxides

The DNA adducts formed from the racemic syn and anti dihydrodiol epoxides of 5,6-dimethylchrysene were characterized through various spectroscopic methods. Substantial reaction with the amino groups of both deoxyadenosine and deoxyguanosine residues were detected with both the syn and anti derivatives. The chemical shifts and coupling constants for the cis and trans opened adducts from the syn dihydrodiol epoxide were distinctly different, whereas for the anti dihydrodiol epoxide these properties were fairly similar for cis and trans adducts. In the latter case, assignment of trans and cis configurations was less obvious, and the finding that trans adducts have always predominated over cis adducts for all dihydrodiol epoxides studied to date was helpful in making these assignments. The preferential formation of cis adducts in DNA by the syn dihydrodiol epoxide is more like the chemistry of the dihydrodiol epoxide of benzo[c]phenanthrene than of benzo[g]chrysene, although both of these, like 5,6-dimethylchrysene, are non-planar compounds.

Detection and characterization of DNA adducts at the femtomole level by desorption ionization mass spectrometry.

Current methodologies for the detection and isolation of carcinogen-DNA adducts have advanced beyond the capabilities of the methods used to elucidate their structures. This difficulty seriously limits the potential use of DNA-carcinogen adducts in human dosimetry. We have investigated two general strategies for the analysis of model arylamine-nucleoside adducts using desorption ionization mass spectrometry (MS). Using fast atom bombardment MS-MS with constant neutral loss scans, we can identify the protonated molecule of derivatized adducts in samples as small as 1 pmole, and then apply daughter ion MS-MS scans to obtain structure-specific fragmentation. Using this strategy we have differentiated adducts having the same carcinogen and different bases [e.g., N-(deoxyadenosin-8-yl)-4-aminobiphenyl and N-(deoxyguanosin-8-yl)-4- aminobiphenyl] or the same base and different carcinogens [e.g., N-(deoxyguanosin-8-yl)-4- aminobiphenyl and N-(deoxyguanosin-8-yl)-2-aminofluorene]. In the second approach we used laser desorption time-of-flight MS to obtain spectra from adduct samples as small as 20 fmole. These data indicate that MS can be used for the analysis of very low (picomole-femtomole) levels of nucleoside adducts, including isomers, and that desorption ionization MS and MS-MS have significant potential for applications in human dosimetry.

Detection and Characterization of DNA Adducts at the Femtomole Level by Desorption Ionization Mass Spectrometry

Current methodologies for the detection and isolation of carcinogen-DNA adducts have advanced beyond the capabilities of the methods used to elucidate their structures. This difficulty seriously limits the potential use of DNA-carcinogen adducts in human dosimetry. We have investigated two general strategies for the analysis of model arylamine-nucleoside adducts using desorption ionization mass spectrometry (MS). Using fast atom bombardment MS-MS with constant neutral loss scans, we can identify the protonated molecule of derivatized adducts in samples as small as 1 pmole, and then apply daughter ion MS-MS scans to obtain structure-specific fragmentation. Using this strategy we have differentiated adducts having the same carcinogen and different bases [e.g., N-(deoxyadenosin-8-yl)-4-aminobiphenyl and N-(deoxyguanosin-8-yl)-4- aminobiphenyl] or the same base and different carcinogens [e.g., N-(deoxyguanosin-8-yl)-4- aminobiphenyl and N-(deoxyguanosin-8-yl)-2-aminofluorene]. In the second approach we used laser desorption time-of-flight MS to obtain spectra from adduct samples as small as 20 fmole. These data indicate that MS can be used for the analysis of very low (picomole-femtomole) levels of nucleoside adducts, including isomers, and that desorption ionization MS and MS-MS have significant potential for applications in human dosimetry.

Characterization of DNA adducts at the bay region of dibenz[a,h]anthracene formed in vitro.

Bay region diolepoxide-DNA adducts of dibenz[a,h]anthracene (DBA) formed in vitro were identified and their absolute stereochemistry was assigned. After activation of [5,12-14C]DBA with liver microsomes obtained from Aroclor 1254 treated male Sprague-Dawley rats in the presence of calf thymus DNA for 1 h, the amount of DNA adducts was found to be 9.9 +/- 2.4 pmol/mg DNA, calculated on the basis of the portion of radioactivity eluted from the HPLC reversed-phase column with a water/acetonitrile gradient. Bay region diolepoxide-DNA adducts represented 27.5% of radioactivity associated with DNA adducts. The absolute configuration of the various adducts was determined from the reaction of the (+)- and (-)-3,4-dihydrodiol after metabolic activation and the reaction of the anti- and syn-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,h]anthracen e with DNA or with the individual deoxyribonucleotides. The main bay region adduct was identified as a deoxyguanosine adduct of (anti)-3S,4R-dihydroxy-1R,2S-epoxy-1,2,3,4-tetrahydrodibenz [a,h]anthracene, a metabolite of (-)-3,4-dihydroxy-3,4-dihydrodi- benz[a,h]anthracene. Anti bay region diolepoxide-deoxyguanosine adducts of DBA contributed to 17.7% and syn diolepoxide-derived deoxyguanosine adducts to 5.8% of adduct-associated radioactivity. The amount of bay region deoxyadenosine adducts was calculated to be 4%. For six of probably eight different deoxyadenosine adducts absolute stereochemistry could be assigned. 32P-Postlabelling experiments revealed a binding of 23 +/- 6 pmol/mg DNA for (-)-3,4-dihydrodiol and of 1.5 +/- 0.4 pmol/mg DNA for (+)-3,4-dihydrodiol of DBA.

In vitro reaction of ethylene oxide with DNA and characterization of DNA adducts

Ethylene oxide (EO) is a direct-acting S N2 alkylating agent and a rodent and probable human carcinogen. In vitro reactions of EO with calf thymus DNA in aqueous solution at neutral pH and 37°C for 10 h resulted in the following 2-hydroxyethyl (HE) adducts (nmol/mg DNA): 7-HE-Gua (330), 3-HE-Ade (39), 1-HE-Ade (28), N 6-HE-dAdo (6.2), 3-HE-Cyt (3.1), 3-HE-Ura (0.8) and 3-HE-dThd (2.0). Reference (marker) compounds were synthesized from reactions of EO with 2′-deoxyribonucleosides and DNA bases, isolated by paper and high performance liquid chromatography and characterized on the basis of chemical properties and UV, NMR and mass spectra. In agreement with our earlier studies with propylene oxide (PO) (Chem.-Biol. Interact., 67 (1988) 275–294) and glycidol (Cancer Biochem. Biophys., 11 (1990) 59–67), alkylation at N-3 of dCyd by EO under physiological conditions resulted in the rapid hydrolytic deamination of 3-HE-dCyd to 3-HE-dUrd. The hydroxyl group on the alkyl side chain which forms after epoxide alkylation is mechanistically involved in this rapid hydrolytic deamination. These results may provide important insights into the mechanisms of mutagenicity and carcinogenicity exhibited by EO and other S N2 aliphatic epoxides.

Biomonitoring of human exposure to genotoxic environmental chemicals: HPLC and mass spectrometry methods for the direct characterization of DNA adducts

Biomonitoring of human exposure to genotoxic environmental chemicals: HPLC and mass spectrometry methods for the direct characterization of DNA adducts