Localization of DNA adducts induced by N-acetoxy-N-2-acetylaminofluorene in Chinese hamster ovary cells using electron microscopy and colloidal gold.

DNA adduct induction by N-acetoxy-N-2-acetylaminofluorene (N-Ac-AAF) has been investigated in Chinese hamster ovary (CHO) cells using immunoelectron microscopy. The major RNA and DNA adducts, N-(guanosin-8-yl)-2-aminofluorene (G-C8-AF) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), were localized with a rabbit anti-G-C8-AF antiserum and colloidal gold cytochemistry. Appropriate controls, including incubation of untreated cells with normal rabbit serum and immunogen-absorbed serum, demonstrated that colloidal gold deposits were indicative of the presence of adducts. The localization of gold particles in close association with nuclear chromatin revealed high concentration of adducts in DNA and RNA of nuclei. Morphometric evaluation of adduct formation in organelles of from different carcinogen exposures showed that 85-88% of total adducts were concentrated in nuclei. DNA adducts remaining in nuclei after RNAse treatment appeared to concentrate in heterochromatic areas, and these areas contained 59% of bound gold particles by morphometry. A total of 137-178 particles were found in nuclei of treated cells vs. 15-26 in the surrounding cytoplasm. Treated cells incubated with normal rabbit serum or specific adduct-absorbed serum showed 19-34 particles for all cellular compartments.     

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

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 acyltransferases to reactive arylnitrenium ions that covalently interact at the N2 position of deoxyguanosine in DNA.     

Analysis of rat lymphocyte activation of benzo[a]pyrene, 2-acetylaminofluorene, and several of their metabolites to mutagenic and DNA-damaging species in vitro.

Rat lymphocytes are a potentially useful and convenient cell system for monitoring the genotoxic effects of chemicals in vivo, but little is known about the ability of these cells to metabolize promutagens to genotoxic species. In this study, Fischer 344 rat lymphocytes were treated in vitro with benzo[a]pyrene (BaP), 2-acetylaminofluorene (2-AAF), and several of their metabolites, and DNA damage was measured using nucleoid sedimentation analysis. Of the BaP derivatives, BaP 4,5-oxide and BaP 7,8-diol-9, 10-epoxide decreased lymphocyte nucleoid sedimentation, whereas BaP and BaP 7,8-dihydrodiol had little effect. Among the 2-AAF derivatives, N-acetoxy-2-AAF, N-hydroxy-2-AAF, and N-hydroxy-2-aminofluorene damaged rat lymphocyte nucleoids, whereas 2-AAF, 2-aminofluorene, and fluorene produced little detectable damage. The decrease in nucleoid sedimentation produced by N-hydroxy-2-AAF was not inhibited by paraoxon, salicylamide, or 2-aminofluorene, whereas paraoxon inhibited damage produced by N-acetoxy-2-AAF. In co-culture assays, rat lymphocytes increased the mutagenicity of N-hydroxy-2-AAF in Salmonella typhimurium strain TA98, but mediated little or no mutagenic response with BaP, BaP 7,8-dihydrodiol, and 2-AAF. Also, human lymphocytes, but not rat lymphocytes, mediated a positive mutagenic response with BaP 7,8-dihydrodiol in Chinese hamster ovary UV5 cells. Although rat lymphocytes may metabolize certain proximal genotoxic chemicals to DNA-damaging species (e.g., N-hydroxy-2-AAF), these data suggest that in vivo lymphocyte DNA damage is more likely to result from lymphocytes encountering reactive chemical derivatives produced by other cells. It is also clear that differences exist between the ability of human and rat lymphocytes to activate promutagens, and this may impact on the use of the rat model to predict the genotoxicity of chemicals in humans.     

DNA damage in human transitional cell carcinoma cells after exposure to the proximate metabolite of the bladder carcinogen 4-aminobiphenyl

The DNA damage induced by N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), the proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP), was examined in human transitional cell carcinoma (TCC) cells after exposure to the chemical in vitro. 32P-postlabeling analysis of TCC cultures exposed to N-OH-AABP revealed a minor adduct identified as 3-(deoxyguanosin-N2-yl)-4-acetylaminobiphenyl (dG-N2-AABP) based on comparison of the HPLC and TLC mobility of the product with the synthetic standard. An adduct with the same chromatographic properties was also detected on postlabeling analyses of calf thymus DNA bound to N-OH-AABP by incubation with horseradish peroxidase and hydrogen peroxide. Detection of dG-N2-AABP, which contains the acetyl moiety, suggests that N-acetoxy-4-acetylamino-biphenyl might be formed as a reactive intermediate and could conceivably arise by a free-radical-mediated reaction of N-OH-AABP with endogenous peroxidases. The radical intermediates could also form reactive oxygen species (ROS). To test this possibility, TCC cultures were exposed to N-OH-AABP and the formation of ROS was measured using 2,7-dichlorofluorescein (DCF) fluorescence assay. TCC cultures exposed to N-OH-AABP showed a dose-dependent increase in the ratio of DCF/DNA fluorescence compared to the untreated controls. Formation of ROS was inhibited by butylated hydroxyanisole (BHA). Furthermore, oxidative DNA damage resulting from ROS was monitored by measurement of 8-oxoguanine products by immunochemical staining and the TCC cells treated with N-OH-AABP revealed a characteristic staining. These results suggest that N-OH-AABP caused oxidative DNA damage as well as bulky covalent adducts in urothelial DNA, possibly involving endogenous peroxidases. These findings show that human uroepithelial cells, which are the target cell types in vivo for arylamine-induced cancers, are metabolically capable of activating these proximate carcinogenic metabolites of arylamines, and these reactions might play a determinate role in the genotoxicity of these environmental carcinogens.     

The induction of DNA adducts in mammalian cells exposed to 1-nitropyrene and its nitro-reduced derivatives

1-Nitropyrene, 1-nitrosopyrene and 1-aminopyrene were investigatedfor their ability to induce covalently bound DNA adducts incalf thymus DNA and Chinese hamster lung fibroblasts. Xanthineoxidase catalysed the induction of one major and one minor DNAadduct in 1-nitropyrene- or 1-nitrosopyrene-treated calf thymusDNA, whilst 1-aminopyrene was inactive. These compounds didnot form detectable DNA adducts in the absence of xanthine oxidase.The major DNA adduct produced by 1-nitropyrene and 1-nitrosopyrenein calf thymus DNA co-migrated on h.p.l.c., and the structurewas consistent with that previously described by others as N-(deoxyguanosin-8-yl)-1-aminopyrene.The compounds were investigated for their ability to form DNAadducts in Chinese hamster lung fibroblasts. 1-Nitropyrene (5.2pmol/mg DNA/h) and 1-nitrosopyrene (129 pmol/mg DNA/h) formeda single DNA adduct in Chinese hamster lung cells which co-elutedon h.p.l.c. with the C-8 deoxyguanosine adduct isolated from1-nitropyrene-treated calf thymus DNA. 1-Nitrosopyrene was themost efficient compound investigated for the production of theC-8 guanine adducts. In contrast, 1-aminopyrene (14.7 pmol/mgDNA/h) induced the formation of a DNA adduct which did not co-elutewith the C-8 guanine adduct. The data presented here suggestthat 1-nitropyrene and 1-aminopyrene are metabolized to reactiveintermediates which form different DNA adducts in Chinese hamsterlung fibroblasts. ¹To whom correspondence should be addressed     

Effect of the co-carcinogen benzo[e] pyrene on microsome-mediated chemical mutagenesis in salmonella typhimurium

Chemical agents that possess the ability to alter tumorigenicity of carcinogens (administered at subthreshold dose) constitute a major health hazard. We have employed the Ames Salmonella assay to examine the effect of co-carcinogenic benzo[e] pyrene (B[e]P) on microsome-mediated chemical mutagenesis. B[e]P enhanced the mutagenic activity induced by 2-acetylaminofluorene (2-AAF) and benzo[a] pyrene (B[a]P) in strains TA1538 and TA98. Enhancement was also noted with N-hydroxy-2-AAF (the proximal metabolite of 2-AAF) but not with an ultimate carcinogenic form (N-acetoxy-2-AAF). These results suggest the use of this approach to detect chemical agents that possess the ability to alter the activity of mutagenic or carcinogenic chemicals.     

Effect of the co-carcinogen benzo[e]pyrene on microsome-mediated chemical mutagenesis in Salmonella typhimurium.

Chemical agents that possess the ability to alter tumorigenicity of carcinogens (administered at subthreshold dose) constitute a major health hazard. We have employed the Ames Salmonella assay to examine the effect of co-carcinogenic benzo[e]pyrene (B[e]P) on microsome-mediated chemical mutagenesis. B[e]P enhanced the mutagenic activity induced by 2-acetylaminofluorene (2-AAF) and benzo[a]pyrene (B[a]P) in strains TA1538 and TA98. Enhancement was also noted with N-hydroxy-2-AAF (the proximal metabolite of 2-AAF) but not with an ultimate carcinogenic form (N-acetoxy-2-AAF). These results suggest the use of this approach to detect chemical agents that possess the ability to alter the activity of mutagenic or carcinogenic chemicals.     

An ELISA for detection of DNA-bound carcinogen using a monoclonal antibody to N-acetoxy-2-acetylaminofluorene-modified DNA

We have produced and characterized a murine monoclonal antibody that recognizes DNA modified with N-acetoxy-2-acetylaminofluorene. The effectiveness of competitive binding inhibitors in an ELISA indicates that this antibody binds most strongly to N-(guanin-8-yl)-2-acetylaminofluorene. It also binds to N-(guanin-8-yl)-2-aminofluorene, albeit some 20-fold less avidly. Thus the monoclonal antibody displays specificity generally similar to the polyclonal antisera elicited by other laboratories but having the advantage of stable and precisely defined specificities. We employed a biotin-streptavidin ELISA to demonstrate that the antibody can detect less than 10 fmol of N-(guanin-8-yl)-2-acetylaminofluorene. N-(guanin-8-yl)-2-acetylaminofluorene is a more effective competitive binding inhibitor of the antibody than is N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene or calf thymus DNA modified with N-acetoxy-2-acetylaminofluorene. Thus the antibody should be most useful in quantifying the persistence of N-acetoxy-2-acetylaminofluorene adducts in DNA hydrolyzed with trifluoroacetic acid.     

Role of Human N-Acetyltransferase 2 Genetic Polymorphism on Aromatic Amine Carcinogen-Induced DNA Damage and Mutagenicity in a Chinese Hamster Ovary Cell Mutation Assay

Carcinogenic aromatic amines such as 4-aminobiphenyl (ABP) and 2-aminofluorene (AF) require metabolic activation to form electrophilic intermediates that mutate DNA leading to carcinogenesis. Bioactivation of these carcinogens includes N-hydroxylation catalyzed by CYP1A2 followed by O-acetylation catalyzed by arylamine N-acetyltransferase 2 (NAT2). To better understand the role of NAT2 genetic polymorphism in ABP- and AF-induced mutagenesis and DNA damage, nucleotide excision repair-deficient (UV5) Chinese hamster ovary (CHO) cells were stably transfected with human CYP1A2 and either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles. ABP and AF both caused significantly (P < 0.001) greater mutagenesis measured at the hypoxanthine phosphoribosyl transferase (hprt) locus in the UV5/CYP1A2/NAT2*4 acetylator cell line compared to the UV5, UV5/CYP1A2, and UV5/CYP1A2/NAT2*5B cell lines. ABP- and AF-induced hprt mutant cDNAs were sequenced and over 80% of the single-base substitutions were at G:C base pairs. DNA damage also was quantified by γH2AX in-cell western assays and by identification and quantification of the two predominant DNA adducts, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) and N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) by liquid chromatography-mass spectrometry. DNA damage and adduct levels were dose-dependent, correlated highly with levels of hprt mutants, and were significantly (P < 0.0001) greater in the UV5/CYP1A2/NAT2*4 rapid acetylator cell line following treatment with ABP or AF as compared to all other cell lines. Our findings provide further clarity on the importance of O-acetylation in CHO mutagenesis assays for aromatic amines. They provide evidence that NAT2 genetic polymorphism modifies aromatic amine-induced DNA damage and mutagenesis that should be considered in human risk assessments following aromatic amine exposures. Environ. Mol. Mutagen. 61:235–245, 2020. © 2019 Wiley Periodicals, Inc.     

Quantitation and immunohistochemical localization of DNA adducts in rat embryos and associated yolk sac membranes exposed in vitro to N-acetoxy-2-acetylaminofluorene (N-Ac-AAF).

Specific antibodies and radioimmunoassay (RIA) were used to measure the levels of acetylated and deacetylated C-8 substituted deoxyguanosine adducts in day 11 rat embryos and their associated yolk sacs after exposure of whole rat conceptuses in vitro to the teratogen N-acetoxy-2-acetylaminofluorene (N-Ac-AAF). The deacetylated adduct predominates in both the embryo and the associated yolk sac, and a dose response for adduct formation was observed when adducts were quantitated by RIA. Immunohistochemical localization of the deacetylated adducts revealed that adducts were confined to the nuclei in all tissues examined and that the abundance of adducts varied within and between tissues. Our initial findings indicate that specific DNA adduct antibodies may be useful in the study of teratogenesis induced by a wide variety of agents that modify DNA.     

Analysis of solvent control and 1-nitrosopyrene-induced Chinese hamster ovary cell mutants by Southern and northern blots and the polymerase chain reaction.

1-Nitrosopyrene, a metabolite of the tumorigenic environmental pollutant 1-nitropyrene, is a potent mutagen at the hprt locus in Chinese hamster ovary (CHO) cells. A single DNA adduct, N-(deoxyguanosin-8-yl)-1-aminopyrene, is produced in CHO cells treated with 1-nitrosopyrene, and this adduct is found in rats and mice exposed to 1-nitropyrene. In this study, the structure of the hprt gene and the structure and amount of hprt mRNA were analyzed in 43 CHO cell mutants (16 isolated from solvent control cultures and 27 isolated from 1-nitrosopyrene-treated cultures). Pstl- and BamHl-digested DNA from the mutants were subjected to Southern blot analysis using a hamster hprt cDNA probe. None of the 1-nitrosopyrene-induced mutants and only one of the control mutants displayed hybridization patterns that were different from the parent CHO cells. Northern blot analysis revealed that two control mutants had truncated hprt mRNAs, while 56% of the control mutants and 78% of the induced mutants had reduced levels of hprt mRNA. Using polymerase chain reaction amplification of cDNA synthesized from RNA, the hprt protein-coding region could be amplified from 23 of the 1-nitrosopyrene-induced mutants and 11 of the control mutants. The amplification products from 3 of the control mutants and 5 of the induced mutants were smaller than that found with RNA from parental CHO cells. These results indicate that the mutagenic DNA damage produced by 1-nitrosopyrene in CHO cells does not cause major structural alterations in the hprt gene and suggest that 1-nitrosopyrene acts as a point mutagen. A large number of both control and 1-nitrosopyrene-induced mutants exhibited a marked reduction in hprt mRNA concentration or possessed truncated mRNA hprt protein coding sequence. These alterations may contribute to the 6-thioguanine-resistant phenotype.     

A new modification of the 32P-post-labeling method to recover IQ-DNA adducts as mononucleotides

To obtain accurate estimates of DNA adduct levels yielded by genotoxic compounds, it is essential to completely digest adducted nucleotides to mononucleotides. We previously developed a suitable method, called modified method I, to obtain DNA adducts of heterocyclic amines as 32P-labeled-mononucleoside adduct 5'-phosphate forms, by use of nuclease P1 (NP1) and phosphodiesterase I (PDEI) to digest adducted oligonucleotides. In this study, we applied method I to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-DNA adduct analysis and found that one of the IQ-DNA adducts, 5-(deoxyguanosin-N2-yI)-2-amino-3-methylimidazo[4,5-f]quinoline 3',5'-diphosphate (pdGp-N2-IQ), was resistant to the 3'-phosphatase activity of NP1, but sensitive to that of T4 polynucleotide kinase (PNK). DNA obtained from the liver of rats fed IQ was 32P-labeled by the standard method and the 32P-labeled nucleotides obtained were incubated with PNK and NP1 to remove 3'-phosphate groups and then digested with PDEI. Three spots were obtained. One major spot was identified as N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline 5'-phosphate (pdG-C8-IQ) and a second abundant adduct as pdG-N2-IQ. The third spot, of which the structure is unknown, was minor. The new method is called modified method II. Modified method II could be applicable to a wide variety of chemicals.     

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

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 indicates that breast milk mutagens may be moderately polar basic compounds, such as arylamines.     

Uranyl acetate induces hprt mutations and uranium-DNA adducts in Chinese hamster ovary EM9 cells

Questions about possible adverse health effects from exposures to uranium have arisen as a result of uranium mining, residual mine tailings and use of depleted uranium in the military. The purpose of the current study was to measure the toxicity of depleted uranium as uranyl acetate (UA) in mammalian cells. The activity of UA in the parental CHO AA8 line was compared with that in the XRCC1-deficient CHO EM9 line. Cytotoxicity was measured by clonogenic survival. A dose of 200 microM UA over 24 h produced 3.1-fold greater cell death in the CHO EM9 than the CHO AA8 line, and a dose of 300 microM was 1.7-fold more cytotoxic. Mutagenicity at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus was measured by selection with 6-thioguanine. A dose of 200 microM UA produced approximately 5-fold higher averaged induced mutant frequency in the CHO EM9 line relative to the CHO AA8 line. The generation of DNA strand breaks was measured by the alkaline comet assay at 40 min and 24 h exposures. DNA strand breaks were detected in both lines; however a dose response may have been masked by U-DNA adducts or crosslinks. Uranium-DNA adducts were measured by inductively coupled plasma optical emission spectroscopy (ICP-OES) at 24 and 48 h exposures. A maximum adduct level of 8 U atoms/10(3) DNA-P for the 300 microM dose was found in the EM9 line after 48 h. This is the first report of the formation of uranium-DNA adducts and mutations in mammalian cells after direct exposure to a depleted uranium compound. Data suggest that uranium could be chemically genotoxic and mutagenic through the formation of strand breaks and covalent U-DNA adducts. Thus the health risks for uranium exposure could go beyond those for radiation exposure.     

The carcinogenic air pollutant 3-nitrobenzanthrone induces GC to TA transversion mutations in human p53 sequences

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and a suspected human carcinogen present in particulate matter of diesel exhaust and ambient air pollution. Employing an assay with human p53 knock-in (Hupki) murine embryonic fibroblasts (HUFs), we examined p53 mutations induced by 3-NBA and its active metabolite, N-hydroxy-3-aminobenzanthrone (N-OH-3-ABA). Twenty-nine immortalized cultures (cell lines) from 89 HUF primary cultures exposed at passage 1 for 5 days to 2 microM 3-NBA harboured 22 different mutations in the human DNA-binding domain sequence of the Hupki p53 tumour suppressor gene. The most frequently observed mutation was GC to TA transversion (46%), corroborating previous mutation studies with 3-NBA, and consistent with the presence of persistent 3-NBA-guanosine adducts found in DNA of exposed rodents. Six of the transversions found solely in 3-NBA-treated HUFs have not been detected thus far in untreated HUFs, but have been found repeatedly in human lung tumours. (32)P-post-labelling adduct analysis of DNA from HUF cells treated with 2 microM 3-NBA for 5 days showed a pattern similar to that found in vivo, indicating the metabolic competence of HUF cells to metabolize 3-NBA to electrophilic intermediates. Total DNA binding was 160 +/- 56 per 10(7) normal nucleotides with N(2)-guanosine being the major adduct. In contrast, identical treatment with N-OH-3-ABA resulted in a 100-fold lower level of specific DNA adducts and no carcinogen-specific mutation pattern in the Hupki assay. This indicates that the level of DNA adduct formation by the mutagen is critical to obtain specific mutation spectra in the assay. Our results are consistent with previous experiments in Muta Mouse and are compatible with the possibility that diesel exhaust exposure contributes to mutation load in humans and to lung cancer risk.     

Mutagenicity of a single 1-nitropyrene-DNA adduct N-(deoxyguanosin-8-yl)-1-aminopyrene in Escherichia coli located in a GGC sequence.

1-Nitropyrene, a common environmental pollutant, forms a major DNA adduct, N-(deoxyguanosin-8-yl)-1-amino- pyrene (dG(AP)). Mutational spectra of randomly introduced dG(AP) in Escherichia coli included different types of mutations, which depended on the base sequence surrounding the adduct. In earlier works we investigated the DNA sequence context effects of the adduct in repetitive CpG and non-repetitive CpGpC sequences. In the current work this adduct was incorporated into a non-repetitive GpGpC sequence in single-stranded M13mp7L2 DNA with the adduct located at either the 5' or 3' G. Potent genotoxicity of dG(AP) was evident from a significant reduction in the population of progeny phage following replication of these constructs in repair-competent E.coli cells. However, progeny derived from the 3'-G(AP) construct were much larger than those from the 5'-G(AP) construct. We suspect that a more facile translesion synthesis past the adduct at the 3' G relative to that at the 5' G, presumably due to a difference in conformation of dG(AP) in these two sites, might be responsible for this effect. With both adducted constructs, >95% of the progeny did not show any mutations at or near the adduct site, indicating highly efficient error-free translesion synthesis. However, a small population of mutants with one base deletions and base substitutions were detected. While the adduct induced -1 frameshifts (<1%) in each G site, base substitutions (1-2%), exhibiting predominantly G-->C transversions, were detected only when the adduct was located at the 5' G. A comparison of the data from this study with a prior study in the CpGpC sequence suggests that dG(AP) mutagenesis is highly sensitive to the local DNA sequence and that a 5'-pyrimidine base might be important for targeted base substitutions by this adduct in E.coli.     

Genotoxicity of m-phenylenediamine and 2-aminofluorene in Salmonella typhimurium and human lymphocytes with and without plant activation

The promutagenic arylamines, m-phenylenediamine (mPDA) and 2-aminofluorene (2-AF), were evaluated for their genotoxicity in Salmonella typhimurium strain YG 1024 and in human lymphocytes. These agents were assayed with and without TX1MX plant activation mix. Both arylamines without activation were refractory in S. typhimurium, demonstrating that plant activation was required for the generation of their ultimate mutagenic metabolites. However, using the alkaline single-cell gel/ Comet assay, both mPDA and 2-AF directly induced DNA damage in human lymphocytes. This effect was reduced when the human cells were treated with the arylamine plus TXIMX. mPDA with or without plant activation was not toxic to the exposed cells. However, at concentrations over 80 μM, 2-AF was toxic to lymphocytes. This toxic response was eliminated by incubation with TX1MX. mPDA and 2-AF were plant-activated into mutagens for S. typhimurium. However, these plant-activated products had a reduced genotoxic potency in human lymphocytes. © 1995 Wiley-Liss, Inc.     

DNA adduct level induced by 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline in Big Blue TMmice does not correlate with mutagenicity

In previous experiments we analyzed the mutant frequency (MF)and mutational types in various organs of lacl transgenic micewhich were fed a diet containing 300 p.p.m.2-aminon-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ), a food-borne mutagen/carcinogen.To clarify the relationshipsbetween mutational type and adduct molecular species and betweenadduct level and MF we analyzed adducts in the same DNA samples.TheDNA adduct in the liver, heart, colon, forestomach and bonemarrow was determined by the modified intensification methodof ³²P-post-labeling at time points 1,4 and 12 weeks.Only asingle spot corresponding to N²-(deoxyguanosin-8-yl)MeIQ 5’-monophosphatewas detected in DNA from all organs at all time points examined,with a recovery of 48%.The difference in mutation type betweenbone marrow and other organs detected in the previous experimentwas not explained by adduct molecular species. At 4 and 12 weeksadministration the adduct levels were highest in the liver andthen heart, colon, forestomach and bone marrow in decreasingorder, with values of 28.3, 8.4, 33, 1.3 and 0.4 molecule/10⁷nudeotides at 12 weeks.Based on our previously reported data,lacl MF was highest in the colon and those in the liver, bonemarrow and forestomach were 10–60% of that of the colon;no increase in MF was detected in the heart, where no DNA replicationis expected except for vascular endothelial cells. There wasno direct relationship between MF and adduct level. The MF maybe the product of adduct level and cell proliferation rate. ⁴To whom correspondence should be addressed. Tel: +81 3 3542 2511 exL 4520; Fax: +81 3 5565 1753; Email:mnaga{at}nccg.jp     

Aristolactam I a metabolite of aristolochic acid I upon activation forms an adduct found in DNA of patients with Chinese herbs nephropathy.

Aristolochic acid (AA) a naturally occuring nephrotoxin and carcinogen is implicated in a unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN). We identified AA-specific DNA adducts in kidneys and in a ureter obtained from CHN patients after renal transplantation. AA is a plant extract of aristolochia species containing AA I as the major component. Aristolactams are the principal detoxication metabolites of AA, which were detected in urine and faeces from animals and humans. They are activated by cytochrome P450 (P450) and peroxidase to form DNA adducts. Using the 32P-postlabelling assay we investigated the formation of DNA adducts by aristolactam I in these two activation systems. A combination of two independent chromatographic systems (ion-exchange chromatography TLC and reversed-phase HPLC) with reference compounds was used for the identification of adducts. Aristolactam I activated by peroxidase led to the formation of several adducts. Two major adducts were identical to adducts previously observed in vivo. 7-(deoxyguanosin-N2-yl)aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI) were formed in DNA during the peroxidase-mediated one-electron oxidation of aristolactam I. Aristolactam I activated by P450 led to one major adduct and four minor ones. Beside the principal AA-DNA adducts identified recently in the ureter of one patient with CHN, an additional minor adduct was detected, which was found to have indistinguishable chromatographic properties on TLC and HPLC from the major adduct formed from aristolactam I by P450 activation. Thus, this minor AA-adduct might be evolved from the AAI detoxication metabolite (aristolactam I) by P450 activation. These results indicate a potential carcinogenic effect of aristolactam I in humans.     

Induction of DNA synthesis in mouse liver following increases of DNA adduct levels elicited by very low cumulative doses of the genotoxic hepatocarcinogen 7H-dibenzo[c,g]carbazole.

The purpose of this work was to investigate the administration of very low but repeated doses of a genotoxic carcinogen and an eventual correlation with cellular DNA synthesis. The compound 7H-dibenzo[c,g]carbazole is a genotoxic carcinogen in the mouse liver and was administered topically at the dose of 13.35 microg per animal every 2 days to give a total of 13 applications. Animals were sacrificed 48 hours after every 2 applications until the 10th treatment, then 48 hours after every treatment. Postulated genotoxic effects such as DNA adduct formation were detected by the 32P-post labeling assay. Liver sections were examined for microscopic changes and DNA synthesis. Results showed an increase of the total DNA adduct level in the liver throughout the study with a slowing down in the level after the sixth application of the compound. This change could correspond to the onset of DNA synthesis and to the moderate hepatocellular apoptosis which was observed. The DNA synthesis, which was considered to be secondary to the cytotoxicity induced by the high level of DNA adducts altering normal cellular activity, could also be the opportunity to fix the DNA adducts into heritable mutations. These results raise the question regarding the risk assessment in humans exposed regularly to very low doses of chemicals in the environment: for non-proliferating tissue, the regular accumulation of DNA adducts could remain silent until a "threshold level" is reached from which stimulation of the DNA synthesis may fix the DNA adducts into heritable mutations, eventually leading to tumors.