Immunohistochemical detection of 1,N(6)-ethenodeoxyadenosine, a promutagenic DNA adduct, in liver of rats exposed to vinyl chloride or an iron overload.

Etheno adducts in DNA bases are formed from exogenous agents such as vinyl chloride and urethane, but also via endogenous lipid peroxidation products like trans-4-hydroxy-2-nonenal. An immunohistochemical method was developed to localize the promutagenic 1,N(6)-ethenodeoxyadenosine DNA adduct in liver of rats exposed to vinyl chloride or an iron overload with or without carbon tetrachloride. Six monoclonal antibodies, previously produced through collaborative efforts, were screened for their optimal adduct recognition and low background formation. The antibody generated by clone EM-A-4 was found to be most suitable. Semi-quantitative image analysis of relative pixel intensity showed approximately 1.5 times higher adduct levels (P < 0.05) in the livers of rats treated with vinyl chloride or an iron overload when compared with untreated controls. Significantly elevated adduct levels persisted in vinyl chloride-treated rat liver 14 days after cessation of exposure, suggesting that this adduct is not rapidly eliminated from rat liver DNA. Using the new immunohistochemical method it is possible to visualize this promutagenic etheno-DNA adduct that may play a role in oxidative stress and lipid peroxidation-induced DNA damage in carcinogenesis.

Etheno-adduct-forming chemicals: from mutagenicity testing to tumor mutation spectra.

During the past 25 years, ethenobases have emerged as a new class of DNA lesions with promutagenic potential. Ethenobases were first investigated as DNA reaction products of vinyl chloride, an occupational carcinogen causing angiosarcoma of the liver (ASL). They were subsequently shown to be formed by several carcinogenic agents, including urethane (ethyl carbamate), and more recently, to occur in various tissues of unexposed humans and rodents. The endogenous source of ethenobases in DNA is thought to be a lipid peroxidation (LPO) product. Initial studies on metabolic activation, mutagenicity and carcinogenicity moved to the analyses of the formation of ethenobases in vivo and to the determination of their promutagenic properties. Quantification of etheno adducts in vivo became possible with the development of ultrasensitive techniques of analysis. To study the miscoding properties of ethenobases, the initial assays on the fidelity of replication or of transcription were replaced by site-directed mutagenesis assays in vivo. Ethenobases generate mainly base pair substitution mutations. With the advent of new techniques of molecular biology, mutations were investigated in the ras and p53 genes of tumors induced by vinyl chloride and urethane. In liver tumors induced by vinyl chloride, specific mutational patterns were found in the Ki-ras gene in human ASL, in the Ha-ras gene in hepatocellular carcinoma (HCC) in rats, and in the p53 gene in human and rat ASL. In tumors induced by urethane in mice, codon 61 of the Ha-ras gene (liver, skin) and of the Ki-ras gene (lung) seems to be a characteristic target. These tumor mutation spectra are compatible with the promutagenic properties of etheno adducts and with their formation in target tissues, suggesting that ethenobases can be initiating lesions in carcinogenesis. Another recent focus has been given to the repair of etheno adducts, and DNA glycosylases able to excise these adducts in vitro have been identified. The last two decades have brought ethenobases to light as potentially important DNA lesions in carcinogenesis. More research is needed to better understand the environmental and genetic factors that affect the formation and persistence of ethenobases in vivo.

Ras gene mutations in vinyl chloride-induced liver tumours are carcinogen-specific but vary with cell type and species.

Previous studies have shown that a high proportion (5/6) of human liver angiosarcomas (ASL) associated with exposure to vinyl chloride (VC) contains a GC-->AT mutation at the Ki-ras codon 13. This mutation, however, has not been found in 5 ASL or 2 hepatocellular carcinomas (HCC) induced in rats by VC. These 2 HCC did contain a mutation at codon 61 of the Ha-ras gene. In order to extend this study and further explore the mechanisms of tumour induction, an additional 6 ASL and 6 HCC induced in rats by VC were analysed for ras gene point mutations, as well as 10 rat and 10 murine ASL induced by vinyl fluoride (VF), and 5 ASL, 6 Kupffer cell sarcomas, 4 HCC and 2 cholangiocellular carcinomas induced by Thorotrast in rats. Tumour DNA was analysed by PCR-SSCP and direct sequencing. None of the rodent ASL contained a mutation at codon 13 of the Ki-ras gene showing that the ras gene mutational pattern is species-specific. The CAA-->CTA mutation, previously found at codon 61 of the Ha-ras gene in rat HCC, was observed in 5 further VC-induced HCC but was not detected in the Thorotrast-induced HCC, suggesting carcinogen-specificity. This mutation was also absent in VC-induced ASL, which supports the cell-specificity of the ras mutational pattern in chemically induced tumours. No predominant mutation was detected in VF- and Thorotrast-induced tumours. Thus, a given mutation in a tumour may be carcinogen-specific but also depend on the species and the cell type.

A simple and sensitive method for in vitro quantitation of abasic sites in DNA.

A novel method for the quantitation of abasic sites (AP sites) in DNA is described. As abasic sites can be generated by controlled thermal treatment of base-modified DNA, this method can be used for estimation of the extent of DNA damage resulting from exposure to genotoxic agents. The method involves use of probe molecules 1 and 2 that contain a fluorescent label linked to an aminooxy group which reacts specifically with the aldehydic function of the ring-opened form of abasic sites. The two fluorescent probes 1 and 2 were found to react with 2-deoxyribose, a model substrate, at the optimum of pH 4.0. As spontaneous depurination occurs at low pH, the reactions with abasic DNA were carried out at neutral pH with an excess concentration of the probes. Studies with alkylated, depurinated calf thymus DNA showed that the method is selective and quantitative. Good correlations were found between the level of 7-methylguanine (7-MeGua), generated in vitro in DNA by the methylating agent dimethyl sulfate, and the amount of AP sites as determined by the method presented here. In addition, similar correlations were found when the assay was used to detect abasic sites in DNA isolated from rats treated with carcinogenic alkylating agents. In each case, the level of abasic sites, as expected, is slightly higher than the level of 7-MeGua which is known to represent about 70% of the total modifications of DNA following exposure to the methylating agent. This method may be useful not only in experimental settings but also in studies of DNA damage in humans resulting from chemotherapy or exposure to environmental agents.

Etheno DNA-base adducts from endogenous reactive species.

Promutagenic etheno (epsilon) adducts in DNA are generated through reactions of DNA bases with LPO products derived from endogenous sources or from exposure to several xenobiotics. The availability of sensitive methods has made it possible to detect three epsilon-adducts in vivo, namely epsilon dA, epsilon dC and N2,3-epsilon dG. One probable endogenous source for the formation of these adducts arises from LPO products such as trans-4-hydroxy-2-nonenal (HNE), resulting in highly variable background epsilon-adduct levels in tissues from unexposed humans and rodents. The range of background levels of epsilon dAx10-8dA detected inhuman tissues was <0.05 to 25 and in rodent tissues 0.02 to 10; the corresponding values for epsilon dCx10-8dC were 0.01 to 11 and 0.03 to 24, respectively. Part of this variability may be associated with different dietary intake of antioxidants and/or omega-6 PUFAs which oxidize readily to form 4-hydroxyalkenals, as epsilon dA and epsilon dC levels in WBC-DNA of female volunteers on a high omega-6 PUFA diet were drastically elevated. Increased levels of etheno adducts were also found in the liver of cancer-prone patients suffering from hereditary metal storage diseases, i.e., Wilson's disease (WD) and primary hemochromatosis (PH) as well as in Long-Evans Cinnamon rats, an animal model for WD. Increased metal-induced oxidative stress and LPO-derive epsilon-adducts, along with other oxidative damage, may trigger this hereditary liver cancer. Epsilon-Adducts could hence be explored as biomarkers (i) to ascertain the role of LPO mediated DNA damage in human cancers associated with oxidative stress imposed by certain lifestyle patterns, chronic infections and inflammations, and (ii) to verify the reduction of these epsilon-adducts by cancer chemopreventive agents. This article summarizes recent results on the formation, occurrence and possible role of epsilon-DNA adducts in carcinogenesis and mutagenesis.

Role of etheno DNA adducts in carcinogenesis induced by vinyl chloride in rats.

Vinyl chloride, a hepatocarcinogen in humans and rodents, can form promutagenic etheno bases in DNA after metabolic activation. The formation of 1,N6-ethenoadenine (epsilon A) and 3,N4-ethenocytosine (epsilon C) was measured in adult Sprague-Dawley rats by immunoaffinity purification and 32P-postlabelling. A highly variable background was found in all tissues from untreated animals: the mean molar ratios of epsilon A:A and epsilon C:C in DNA ranged from 0.043 x 10(-8) to 31.2 x 10(-8) and from 0.062 x 10(-8) to 20.4 x 10(-8), respectively. After exposure to 500 ppm vinyl chloride by inhalation (4 h/day, 5 days/week for 8 weeks), increased levels of epsilon A were found in the liver, lung, circulating lymphocytes and testis, the mean (+/- SD) of induced levels (treated-control values) being (4.1 +/- 1.5) x 10(-8) for these tissues. No increase in the epsilon A:A ratio was observed in kidney, brain or spleen. The levels of epsilon C increased in all the tissues examined except the brain. The mean value of the induced epsilon C:C ratios was (7.8 +/- 1.2) x 10(-8) for the liver, kidney, lymphocytes and spleen, and these ratios were higher in the lung (28 x 10(-8)) and testis (19 x 10(-8)). The results suggest a variable repair capacity for epsilon A or epsilon C in different tissues. The results are discussed in relation to published studies on the accumulation and persistence of etheno bases in the liver during and after exposure to vinyl chloride and on mutation spectra in the ras and p53 genes in liver tumours induced by vinyl chloride. In addition, we show that the linear relationship established for monofunctional alkylating agents between their carcinogenic potency in rodents and their covalent binding index for promutagenic bases in hepatic DNA holds for vinyl chloride. It is concluded that etheno bases are critical lesions in hepatocarcinogenesis induced by vinyl chloride. For a better understanding of the mechanism of action of this compound, further work is needed on the role of DNA repair pathways and of endogenous lipid peroxidation products in the formation and persistence of etheno bases in vivo.

Heritable and cancer risks of exposures to anticancer drugs: inter-species comparisons of covalent deoxyribonucleic acid-binding agents.

In the past years, several methodologies were developed for potency ranking of genotoxic carcinogens and germ cell mutagens. In this paper, we analyzed six sub-classes of covalent deoxyribonucleic acid (DNA) binding antineoplastic drugs comprising a total of 37 chemicals and, in addition, four alkyl-epoxides, using four approaches for the ranking of genotoxic agents on a potency scale: the EPA/IARC genetic activity profile (GAP) database, the ICPEMC agent score system, and the analysis of qualitative and quantitative structure-activity and activity-activity relationships (SARs, AARs) between types of DNA modifications and genotoxic endpoints. Considerations of SARs and AARs focused entirely on in vivo data for mutagenicity in male germ cells (mouse, Drosophila), carcinogenicity (TD50s) and acute toxicity (LD50s) in rodents, whereas the former two approaches combined the entire database on in vivo and in vitro mutagenicity tests. The analysis shows that the understanding and prediction of rank positions of individual genotoxic agents requires information on their mechanism of action. Based on SARs and AARs, the covalent DNA binding antineoplastic drugs can be divided into three categories. Category 1 comprises mono-functional alkylating agents that primarily react with N7 and N3 moieties of purines in DNA. Efficient DNA repair is the major protective mechanism for their low and often not measurable genotoxic effects in repair-competent germ cells, and the need of high exposure doses for tumor induction in rodents. Due to cell type related differences in the efficiency of DNA repair, a strong target cell specificity in various species regarding the potency of these agents for adverse effects is found. Three of the four evaluation systems rank category 1 agents lower than those of the other two categories. Category 2 type mutagens produce O-alkyl adducts in DNA in addition to N-alkyl adducts. In general, certain O-alkyl DNA adducts appear to be slowly repaired, or even not at all, which make this kind of agents potent carcinogens and germ cell mutagens. Especially the inefficient repair of O-alkyl-pyrimidines causes the high mutational response of cells to these agents. Agents of this category give high potency scores in all four expert systems. The major determinant for the high rank positions on any scale of genotoxic of category 3 agents is their ability to induce primarily structural chromosomal changes. These agents are able to cross-link DNA. Their high intrinsic genotoxic potency appears to be related to the number of DNA cross-links per target dose unit they can induce. A confounding factor among category 3 agents is that often the genotoxic endpoints occur close to or at toxic levels, and that the width of the mutagenic dose range, i.e., the dose area between the lowest observed effect level and the LD50, is smaller (usually no more than 1 logarithmic unit) than for chemicals of the other two categories. For all three categories of genotoxic agents, strong correlations are observed between their carcinogenic potency, acute toxicity and germ cell specificity.

Formation of DNA etheno adducts in rodents and humans and their role in carcinogenesis.

Ethenobases are exocyclic adducts formed with DNA by some environmental carcinogens such as vinyl chloride or urethane. In the last few years, they have received a renewed interest due to the development of sensitive techniques of analysis that made it possible to measure their formation in vivo. This minireview summarizes the information gained recently from the work of several laboratories, including ours. Increased levels of DNA etheno adducts have been measured in target tissues from rodents exposed to vinyl chloride or urethane. Hepatic tumours caused by exposure to vinyl chloride in humans and in rats and lung tumours induced by urethane in mice exhibit base pair substitution mutations in the ras and p53 genes which seem to be exposure-specific and consistent with the promutagenic properties of ethenobases. Background levels of etheno adducts have been detected in DNA from non-exposed humans or animals, pointing to an alternative, endogenous pathway of formation. This background may be affected by dietary factors. It could arise from the reaction of trans-4-hydroxy-2-nonenal (or its epoxide 2,3-epoxy-4-hydroxynonanal), a lipid peroxidation product, with nucleic acid bases. Elevated levels of etheno adducts are found in hepatic DNA from humans and rodents with genetic predisposition to oxidative stress and lipid peroxidation in the liver, and with an associated increased risk of liver cancer. These data suggest that DNA ethenobases could serve as new biomarkers of oxidative stress/lipid peroxidation.

Metabolic activation of vinyl chloride by rat liver microsomes: low-dose kinetics and involvement of cytochrome P450 2E1.

The metabolism and pharmacokinetics of vinyl chloride (VC) have been extensively studied in rodents and humans, but the maximum velocity (Vmax) and Michaelis constant (K(m)) for the activation of VC by microsomal monooxygenases in vitro have not yet been determined. Using a new sensitive assay, the epoxidation of VC by rat liver microsomes (adult Sprague-Dawley) at concentrations from 1 ppm to 10(6) ppm in the gas phase was measured. In the assay, the reactive VC metabolites chloroethylene oxide and 2-chloroacetaldehyde were trapped with excess cAMP, yielding, 1,N6-etheno-cAMP (epsilon cAMP) which was quantitated by HPLC fluorimetry. The trapping efficiency of electrophilic VC metabolites by cAMP was close to 10%. The specificity of the method was confirmed by purification of epsilon cAMP on an immunogel. The VC concentration in the gas phase was measured by GC/flame ionization detection, while in the aqueous phase it was calculated from the partition coefficient between air and the microsomal suspension. Activation of VC by rat liver microsomes followed Michaelis-Menten kinetics with K(m) = 7.42 +/- 0.37 (+/- SD) microM and Vmax = 4674 +/- 46 pmol.mg protein-1.min-1. Inhibitor studies and immunoinhibition assays showed that VC was activated by cytochrome P450 (CYP) 2E1 down to 1 ppm in the air phase. Based on the metabolic parameters determined, the uptake of VC by rats in vivo can be accurately predicted.

p53 gene mutation pattern in rat liver tumors induced by vinyl chloride.

Vinyl chloride (VC) induces angiosarcomas of the liver (ASL) and hepatocellular carcinomas (HCCs) in humans and rodents. We examined the presence of p53 gene mutations in ASL and HCC induced by VC in Sprague Dawley rats; 25 ASL and eight HCCs were analyzed for point mutations in exons 5-8, using PCR amplification, single-strand conformation polymorphism analysis, and direct DNA sequencing. Mutations were found in 11 (44%) of the ASL and in 1 HCC. A 12-base pair deletion was found in one tumor; all others were base pair substitutions. Nine of the point mutations were observed at A:T base pairs (5 A:T --> T:A; 2 A:T --> G:C, and 2 A:T --> C:G), and of three G:C --> A:T transitions, only one was at a CpG site. In ASL, four mutations were found in exon 5, two in exon 6, and six in exon 7; the base pair substitution found in one HCC was in exon 8. One ASL exhibited two point mutations, including a silent one. Two ASL exhibited the same mutation in codon 203 and two other samples in codon 253. Codon 235 was found to be mutated in three ASL. These data show that p53 is often mutated in ASL induced by VC in rats and, as observed in ASL in humans exposed to VC, the majority of the missense mutations involved A:T base pairs. The characteristic patterns of mutations found suggest that a common mechanism operates in VC-induced p53 mutagenesis in both species, and these mutations are consistent with the formation of DNA etheno adducts by VC in the liver. The A:T --> T:A transversion observed in the first nucleotide of codon 253 in two rat ASL is equivalent to the A:T --> T:A transversion characterized previously in codon 255 in one human ASL associated with VC exposure.

Connexin 37 mutations in rat hepatic angiosarcomas induced by vinyl chloride.

Connexin genes have been shown to restore normal cell growth when transfected into certain tumorigenic cells and thus are considered to form a family of tumor suppressor genes. In this study, we have analyzed mutations of the connexin 37 (Cx37) gene in rat hepatic angiosarcomas induced by vinyl chloride. A total of 25 rat liver tumors (22 hepatic angiosarcomas and 3 hepatocellular carcinomas) were analyzed by PCR-single-strand conformation polymorphism analysis and DNA sequencing. Four mutations were detected in three tumors: (a) one GGG(Gly) to GAG(Glu) mutation at codon 168; and (b) three silent mutations, CGA(Arg) to CGC(Arg), at codon 166. In addition, we found that codon 88 is polymorphic (GAG(Glu) to GAA(Glu)). Cx37 proteins are detectable in endothelial cells of normal liver by immunohistochemical analysis, but none of the angiosarcomas showed Cx37-positive spots. These results suggest that Cx37-mediated gap junctional intercellular communication may be disturbed in most of these angiosarcomas, but mutation of the Cx37 gene is rare.

Formation and accumulation of DNA ethenobases in adult Sprague-Dawley rats exposed to vinyl chloride.

DNA ethenobases are promutagenic lesions formed by carcinogens such as vinyl chloride (VC). Their formation was investigated in 6-week old, male Sprague-Dawley rats exposed to 500 p.p.m. VC by inhalation (4 h/day, 5 days/ week) for 1, 2, 4 or 8 weeks and in 7- and 14-week old, matched control animals. 1,N6-Ethenoadenine (epsilon A) and 3, N4-ethenocytosine (epsilon C) deoxyribonucleotides were analysed by immunoaffinity purification and 32P-postlabelling. This postlabelling method was compared with a radio-immunoassay method, which yielded similar results. Background levels of ethenobases were found in DNA from the liver, lungs, kidneys and circulating lymphocytes of unexposed, control rats. In the liver, the following background molar ratios of ethenobase to parent base in DNA were detected (mean values x 10(-8)): epsilon A/A, 0.04-0.05; epsilon C/C, 0.06-0.07. In the lungs, kidneys and circulating lymphocytes, background levels of epsilon A and epsilon C ranged from 1.7 to 4.2 x 10(-8) and from 4.8 to 11.2 x 10(-8), respectively. Following a 5-day exposure to VC, a significant increase of epsilon A and epsilon C was measured in hepatic DNA from rats sacrificed immediately after treatment. Further, a dose-dependent increase of both etheno adducts was observed in liver DNA of VC-treated rats. Compared to the 5-day exposure, approximately 4-fold higher levels of epsilon A and epsilon C were observed in the liver of animals after 8 weeks of exposure. In contrast, there was an accumulation of epsilon C but not of epsilon A in lungs and kidneys. In circulating lymphocytes, no significant increase of ethenobase levels above control values was observed after 2 months of exposure to VC. Both etheno adducts were found to be persistent in liver DNA, after 2 months following the termination of VC exposure. These results further support the notion that DNA etheno-bases are critical lesions in VC-induced carcinogenesis. The possible contribution of lipid peroxidation products that also yield ethenobases, on the formation and persistence of these DNA adducts, remains to be clarified.

Detection of 1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytidine by immunoaffinity/32P-postlabelling in liver and lung DNA of mice treated with ethyl carbamate (urethane) or its metabolites.

The capacity of the chemical carcinogen ethyl carbamate (EC, urethane) and its metabolites vinyl carbamate (VC) and vinyl carbamate epoxide (VCO) to form ethenobases was studied in liver and lung DNA of 12-day-old and adult CD-1, B6C3F1, C3H/HeJ and C57BL/6J mice. Following single and multiple doses of EC, VC or VCO, the formation of 1,N6-ethenodeoxyadenosine (epsilon dA) and 3,N4-ethenodeoxycytidine (epsilon dC) was quantified by an immunoaffinity chromatography/32P-postlabelling technique. Both etheno adducts were detected in untreated control DNA samples from liver and lung in the range of 2-15 adducts/10(9) parent nucleotides. Following five repeated injections of 250 or 280 nmol/g body wt VC to adult mice, 51, 57 and 78 epsilon dA/10(9) dA and 28, 42 and 42 epsilon dC/10(9) dC (means of duplicate analyses) were detected in liver DNA of CD-1, C3H/HeJ and C57BL/6J mice respectively. In lung DNA of these VC-treated mice, the levels were 87, 49 and 58 (epsilon dA/10(9) dA) and 64, 39 and 43 (epsilon dC/10(9) dC) respectively. Under similar dose regimens, lower levels of etheno adducts were detected in B6C3F1 mice. Etheno-DNA adducts were also formed in liver and lung upon treatment with EC in adult mice, but at 3-fold lower levels as compared with VC. In 12-day-old C3H/HeJ and C57BL/6J mice, 2- to 3-fold higher etheno adduct levels were detected in liver DNA, when compared with adults, upon a single treatment with 250 nmol/g body wt VC, suggesting that young animals are more susceptible to adduct formation. Combined analysis of adduct formation in adult CD-1, C3H/HeJ and C57BL/6J mice at the higher dose showed a statistically significant increase in etheno adduct formation in the order EC > VC. The results demonstrate that EC and its activated intermediates bind to liver and lung DNA to form epsilon dA and epsilon dC, and the differences in DNA binding further support the hypothesis that metabolic activation of EC to VC is involved. Preliminary data also suggest that background levels of epsilon dA and epsilon dC in DNA are affected by the type of diet given to the animals.

DNA damage and repair in mutagenesis and carcinogenesis: implications of structure-activity relationships for cross-species extrapolation.

Previous studies on structure-activity relationships (SARs) between types of DNA modifications and tumour incidence revealed linear positive relationships between the log TD50 estimates and s-values for a series of mostly monofunctional alkylating agents. The overall objective of this STEP project was to further elucidate the mechanistic principles underlying these correlations, because detailed knowledge on mechanisms underlying the formation of genotoxic damage is an absolute necessity for establishing guidance values for exposures to genotoxic agents. The analysis included: (1) the re-calculation and further extension of TD50 values in mmol/kg body weight for chemicals carcinogenic in rodents. This part further included the checking up data for Swain-Scott s-values and the use of the covalent binding index (CBI); (2) the elaboration of genetic toxicity including an analysis of induced mutation spectra in specific genes at the DNA level, i.e., the vermilion gene of Drosophila, a plasmid system (pX2 assay) and the HPRT gene in cultured mammalian cells (CHO-9); and (3) the measurement of specific DNA alkylation adducts in animal models (mouse, rat, hamster) and mammalian cells in culture. The analysis of mechanisms controlling the expression of mammalian DNA repair genes (alkyltransferases, glycosylases) as a function of the cell type, differentiation stage, and cellular microenvironment in mammalian cells. The 3 classes of genotoxic carcinogens selected for the project were: (1) chemicals forming monoalkyl adducts upon interaction with DNA; (2) genotoxins capable of forming DNA etheno-adducts; and (3) N-substituted aryl compounds forming covalent adducts at the C8 position of guanine in DNA. In general, clear SARs and AARs (activity-activity relationships) between physiochemical parameters (s-values, O6/N7-alkylguanine ratios, CBI), carcinogenic potency in rodents and several descriptors of genotoxic activity in germ cells (mouse, Drosophila) became apparent when the following descriptors were used: TD50 estimates (lifetime doses expressed in mg/kg b.wt. or mmol/kg b.wt.) from cancer bioassays in rodents; the degree of germ-cell specificity, i.e., the ability of a genotoxic agent to induce mutations in practically all cell stages of the male germ-cell cycle of Drosophila (this project) and the mouse (literature search), as opposed to a more specific response in postmeiotic stages of both species; the Mexr-/Mexr+ hypermutability ratio, determined in a repair assay utilizing Drosophila germ cells; mutation spectra induced at single loci (the 7 loci used in the specific-locus test of the mouse (published data), and the vermilion gene of Drosophila); and doubling doses (DD) in mg/kg (mmol/kg) for specific locus test results on mice. By and large, the TD50 values, the inverse of which can be considered as measures of carcinogenic potency, were shown to be predictable from knowledge of the in vivo doses associated with the absorbed amounts of the investigated alkylators and with the second-order constant, kc, reaction at a critical nucleophilic strength, nc. For alkylating agents kc can be expressed as the second-order rate constant for hydrolysis, kH2O, and the substrate constant s:kH2OTD50 is a function of a certain accumulated degree of alkylation, here given as the (average) daily increment, ac, for 2 years exposure of the rodents. The TD*50 in mmol/kg x day) could then be written: [formula: see text] This expression would be valid for monofunctional alkylators provided the reactive species are uncharged. This is the case for most SN2 reagents. Although it appears possible to predict carcinogenic potency from measured in vivo doses and from detailed knowledge of reaction-kinetic parameter values, it is at present not possible to quantify the uncertainty of such predictions. One main reason for this is the complication due to uneven distribution in the body, with effects on the dose in target tissues. The estimation can be impro

Copper-dependent formation of miscoding etheno-DNA adducts in the liver of Long Evans cinnamon (LEC) rats developing hereditary hepatitis and hepatocellular carcinoma.

Formation of etheno-DNA adducts in the liver was investigated in Long Evans cinnamon (LEC) rats, a Long Evans strain with hereditary abnormal copper metabolism, which develop spontaneous hepatitis and later hepatocellular carcinoma. Using an ultrasensitive immunoaffinity/32P-postlabeling assay (J. Nair et al., Carcinogenesis, 16: 613-617, 1995), the etheno adducts 1,N6-ethenodeoxyadenosine (epsilon dA) and 3,N4-ethenodeoxycytidine (epsilon dC) were measured in the liver of 7-, 18-, 30-, and 87-week-old LEC rats. Levels were highest in the liver of 18-week old rats 85 +/- 17 (epsilon dA) and 85 +/- 30 (epsilon dC) adducts per 10(9) parent nucleotides, and the increase in the levels of etheno adducts was age dependent. Age-matched Long Evans agouti rats, a tumor-free sibling line of LEC rats, had much lower levels of both etheno adducts. Etheno adduct levels in LEC rats were well correlated with the hepatic copper levels, and peak adduct levels coincided with the age of commencement of fulminant hepatitis. Our results demonstrate for the first time a copper- and age-dependent formation of highly miscoding etheno-DNA adducts in the liver of LEC rats. These adducts are formed from lipid peroxidation products (F. El-Ghissassi et al., Chem. Res. Toxicol., 8: 273-283, 1995) and thus could arise in the liver of LEC rats from oxygen radicals generated by copper-catalyzed Fenton-type reactions. Etheno-DNA adducts along with other oxidative DNA base damages may thus be involved in liver carcinogenesis in LEC rats.

Formation of 1,N6-ethenoadenine and 3,N4-ethenocytosine by lipid peroxidation products and nucleic acid bases.

Lipid peroxidation (LPO) products are known to interact with DNA, yielding several types of adduct with nucleobases. In this study, we demonstrate the formation of two ethenobase adducts, 1,N6-ethenoadenine and 3,N4-ethenocytosine, by reaction of LPO products with nucleic acid bases. Rat liver microsomes were incubated at 37 degrees C for 30 min in the presence of inducers of LPO [Fe(II) or cumene hydroperoxide] and adenine or cytosine nucleotides or nucleosides, followed by further heating at 80 degrees C for 30 min to complete the reactions. The etheno adducts detected after immunoaffinity chromatography were 1,N6-etheno-cAMP and 1,N6-etheno-2'-deoxyadenosine (HPLC/fluorimetry), 3,N4-etheno-2'-deoxycytidine (competitive radioimmunoassay), and 1,N6-etheno-2'-deoxyadenosine 3'-monophosphate and 3,N4-etheno-2'-deoxycytidine 3'-monophosphate (32P-postlabeling). Incubation of arachidonic acid supplemented with Fe(II) also led to the formation of the 1,N6-etheno adduct from cAMP. LPO intermediates that may be involved are discussed. These data suggest that etheno adducts may be markers of DNA damage associated with LPO.

1,N6-ethenodeoxyadenosine and 3,N4-ethenodeoxycytine in liver DNA from humans and untreated rodents detected by immunoaffinity/32P-postlabeling.

The etheno-bridged exocyclic DNA adducts 1,N6-ethenodeoxyadenosine (epsilon dA) and 3,N4-ethenodeoxycytine (epsilon dC) can be formed by several structurally diverse carcinogens and mutagens that include vinyl chloride and urethane. In order to investigate the occurrence and persistence of these adducts in rodents exposed to such DNA-damaging agents, an ultra-sensitive detection method has been developed. It is based on immunoaffinity purification of the etheno adducts and subsequent 32P-postlabelling followed by separation as 5'-monophosphates on polyethyleneimine-cellulose-coated thin-layer plates. Normal nucleotides in the DNA samples were quantitated by HPLC. Optimal conditions for enzymatic hydrolysis of DNA are described: deoxyuridine 3'-monophosphate was used as internal standard to correct for labelling efficiency of the etheno adducts. The method had a detection limit of 25 amol of epsilon dA and epsilon dC for a 50 micrograms DNA sample. Using this technique, analysis of liver DNA from humans with unknown exposure revealed the presence of epsilon dA and epsilon dC residues in the range of 0-27 adducts per 10(9) parent bases. Liver DNA obtained from untreated mice and rats was also shown to contain similar low but variable levels of these etheno adducts. In vitro studies indicated that these promutagenic DNA lesions could arise from endogenously formed lipid peroxidation products.

New sites of methylcytosine-rich DNA detected on metaphase chromosomes.

In situ immunofluorescence detection of antibodies against 5-methylcytosine on metaphase chromosomes prepared by a new procedure allows the display of new 5-methylcytosine-rich sites as compared to previously published methods. In short-term culture lymphocytes, the immunofluorescent signals give a recurrent pattern in which four types of binding sites can be distinguished. Type I sites are the secondary constrictions and a few juxtacentromeric regions, type II sites correspond to T-bands. Both types I and II sites emit a strong fluorescence. Type III sites form an R-band pattern and emit a weaker fluorescence. Type IV sites are the short arms of acrocentrics, they emit strong but polymorphic signals. The results obtained from control experiments suggest that the pattern observed is rather the expression of an uneven distribution of 5-methylcytosine-rich sites than a consequence of the various treatments used. In a lymphoblastoid cell line known to have a reduced 5-methylcytosine content, it was possible to demonstrate a heterogeneous hypomethylation among chromosome structures, principally involving type I sites. The method opens the possibility of studying in situ on chromosomes, regional variations of methylation in pathological conditions.

Mutagenesis of ras proto-oncogenes in rat liver tumors induced by vinyl chloride.

Vinyl chloride is a DNA-damaging carcinogen which induces liver angiosarcomas in humans and animals. Activation of the Ki-ras 2 gene by a GC-->AT transition at the second base of codon 13 in human liver angiosarcomas associated with occupational exposure to vinyl chloride has been reported recently. In order to compare the molecular pathways of carcinogenesis in humans and animals, Sprague-Dawley rats were exposed to vinyl chloride and hepatic tumors, including two hepatocellular carcinomas and five liver angiosarcomas, were investigated for mutations at codons 12, 13 and 61 of the Ha-ras, Ki-ras and N-ras genes. High molecular weight DNA was amplified by the polymerase chain reaction and point mutations were analyzed by allele specific oligonucleotide hybridization, direct sequencing of polymerase chain reaction products and sequencing after cloning. None of the tumors exhibited a mutation in codons 12, 13 and 61 of the Ki-ras gene, nor in codons 12 of the Ha-ras gene or 61 of the N-ras gene. However, an activating AT-->TA transversion at base 2 of codon 61 of the Ha-ras gene was detected in the two hepatocellular carcinomas. Mutations involving codon 13 (GGC-->GAC) and codon 36 (ATA-->CTA) of the N-ras A gene were detected in two liver angiosarcomas, suggesting that the nature of the ras gene affected by a given carcinogen depends on host factors specific to cell types. Several additional base pair substitutions were found in exon 1 of the N-ras B and C sequences. NIH 3T3 transfection assays and Southern blot analysis of DNA from transformed NIH 3T3 cells confirmed the presence of a dominant activated N-ras gene. These results emphasize the differences in the molecular pathways leading to tumors in humans and rats and within a given species between different cell types.