Search

Carcinogenic arylamines typically undergo metabolic activation via N-hydroxylation followed in most instances by O-esterification. In this study, the ability of methyl-, dimethyl-, and ethylaniline constituents of tobacco smoke to undergo oxidation at the nitrogen atom was analyzed. In addition, the mutagenicity of the corresponding N-hydroxyalkylanilines and the conformational properties of the DNA adducts generated from their N-acyloxy derivatives were investigated. All the arylamines underwent irreversible electrochemical N-oxidation at potentials higher than those observed for the oxidation of carcinogenic polynuclear aromatic amines. There were minor differences in the oxidation potentials, which were consistent with the position and electron-donating abilities of the alkyl substituents; however, the differences appeared to be too small to account for the range of genotoxic effects among the alkylanilines. N-Hydroxyarylamines containing p-alkyl substituents had increased mutagenicities in Salmonella typhimurium TA100, which was attributed to their higher efficiencies of adduct formation. Increased mutagenicities were also observed upon o-alkyl substitution; however, this property was not related to a greater ability of the ortho-substituted species to form DNA adducts, which suggested that adducts from ortho-substituted alkylanilines may be intrinsically more mutagenic than their meta- and para-substituted analogues. In all instances, N-(acyloxy)-arylamines generated from the N-hydroxyarylamines reacted with dG, dG nucleotides, and DNA to yield C8-substituted dG derivatives as the major adducts. The alkylaniline-dG adducts displayed distinct conformational trends that were determined by the location of the alkyl substituents. Spectroscopic data indicated higher percentages of low-energy syn conformers in the adducts that contained alkyl groups ortho to the arylamine nitrogen as opposed to adducts not bearing ortho substituents. The data strongly suggest that the conformational properties of the DNA adducts, in particular their ability to adopt syn conformations, may be determinant factors for the genotoxic responses elicited by certain alkylanilines (e.g., 2-methylaniline and 2,6-dimethylaniline).

The spectrum of mutations induced by the naturally occurring DNA adduct pyrimido[1,2-alpha]purin-10(3H)-one (M1G) was determined by site-specific approaches using M13 vectors replicated in Escherichia coli. M1G was placed at position 6256 in the (-)-strand of M13MB102 by ligating the oligodeoxynucleotide 5'-GGT(M1G)TCCG-3' into a gapped-duplex derivative of the vector. Unmodified and M1G-modified genomes containing either a cytosine or thymine at position 6256 of the (+)-strand were transformed into repair-proficient and repair-deficient E. coli strains, and base pair substitutions were quantitated by hybridization analysis. Modified genomes containing a cytosine opposite M1G resulted in roughly equal numbers of M1G-->A and M1G-->T mutations with few M1G-->C mutations. The total mutation frequency was approximately 1%, which represents a 500-fold increase in mutations compared with unmodified M13MB102. Transformation of modified genomes containing a thymine opposite M1G allowed an estimate to be made of the ability of M1G to block replication. The (-)-strand was replicated >80% of the time in the unadducted genome but only 20% of the time when M1G was present. Correction of the mutation frequency for the strand bias of replication indicated that the actual frequency of mutations induced by M1G was 18%. Experiments using E. coli with different genetic backgrounds indicated that the SOS response enhances the mutagenicity of M1G and that M1G is a substrate for repair by the nucleotide excision repair complex. These studies indicate that M1G, which is present endogenously in DNA of healthy human beings, is a strong block to replication and an efficient premutagenic lesion.

Dibenzo[a,l]pyrene (DB[a,l]P) represents the most potent carcinogenic polycyclic aromatic hydrocarbon (PAH) yet discovered. Like other PAHs, DB[a,l]P requires metabolic activation to exert its mutagenic and/or carcinogenic activity. In the human mammary carcinoma cell line MCF-7, DB[a,l]P is stereoselectively metabolized to the (-)-anti- and (+)-syn-DB[a,l]P-11,12-diol 13,14-epoxides (DB[a,l]PDE) which both bind extensively to deoxyadenosine residues in DNA. To further characterize the underlying mechanism of its strong carcinogenicity, the relationship between DNA binding and mutagenicity of DB[a,l]P was determined. Racemic DB[a,l]P-11,12-dihydrodiol and the two individual (+)- and (-)-enantiomers, the metabolic precursors of the stereoisomeric fjord region dihydrodiol epoxides, were also investigated. Induction of mutations at the HPRT locus was measured in a MCF-7 cell-mediated Chinese hamster V79 cell mutation assay. The parent hydrocarbon, (+/-)-DB[a,l]P-11,12-dihydrodiol, and (-)-DB[a,l]P-11,12-dihydrodiol were highly mutagenic under the assay conditions. In contrast, (+)-DB[a,l]P-(11S,12S)-dihydrodiol was not mutagenic using MCF-7 cells as the metabolic activating system. Analysis of DNA adducts in the same experiments revealed that MCF-7 cells treated with (-)-DB[a,l]P-11,12-dihydrodiol formed exclusively (-)-anti-DB[a,l]-PDE adducts whereas cells treated with (+)-DB[a,l]P-11,12-dihydrodiol did not contain detectable levels of DNA adducts. These results suggest that specific cytochrome P450 enzymes may have high stereoselectivity for activation of the two DB[a,l]P-11,12-dihydrodiol enantiomers, and this may play an important role in the metabolic activation of the strong carcinogen DB[a,l]P in human cells.

Mutagenic effect of polycyclic aromatic hydrocarbon (PAH)-DNA adduct was studied to explore mechanism of carcinogenecity of PAH. Adduct was formed by chemical group protection and nucleophilic displacement methods with modification of deoxyadenosine by PAH-dihydrodiol epoxide. Partial analogous sequence of human protooncogene ras (H-ras) containing mutagenic hotspot 61 codon (CAG) was synthesized by a DNA assembler and DNA was duplicated with a template of partial analogous sequence containing adduct protooncogene. Results showed that PAH-DNA adduct could obviously affect the process of DNA duplication, cause interruption of duplication of DNA or translesional bypass, lower fidelity of duplication, and induce misincorporation of bases in corresponding locus of the adduct, with point mutation of transversion (from A-T to T-A).

The possible correlation between alterations in cytokinetic response to cisplatin (CP) treatment and drug resistance in human ovarian carcinoma cell lines was examined. Using dual parameter flow cytometry, we performed detailed time-course and dose-response analysis of cell cycle modifications in the parental A2780 and resistant A2780/CP cells exposed to CP. The data suggested that drug treatment resulted in similar types of cell cycle alterations in cells with different CP sensitivity. Rapid normalization of the cytokinetic pattern in both cell lines at low doses of CP was observed. At higher drug concentrations reversible S phase delay predominated, accompanied by blocks in both G1/S and G2/M and followed by complete normalization of cytokinetic patterns in the surviving cells. CP treatment by lethal doses resulted in almost complete S phase block. The surviving cells at 72 h accumulated in G2 phase. CP-induced cell cycle perturbations, among which the most pronounced were alterations in the S phase populations, correlated with the level of DNA damage, but not with cell survival in these cell lines. However, at identical levels of DNA damage, the resistant A2780/CP cell line demonstrated decreased p53 induction and decreased apoptosis compared to the parental cell line. Thus, at equivalent levels of DNA damage, resistance in this model system correlated with a diminished p53-dependent apoptotic pathway rather than with differences in cell cycle response.

Chronic degenerative diseases are the leading causes of death in developed countries. Their control is exceedingly difficult due to their multiplicity and diversity, the interconnection with a network of multiple risk factors and protective factors, the long latency and multistep pathogenesis, and the multifocal localization. Adducts to nuclear DNA are biomarkers evaluating the biologically effective dose, reflecting an enhanced risk of developing a mutation-related disease more realistically than the external exposure dose. The localization and accumulation of these promutagenic lesions in different organs are the composite result of several factors, including (a) toxicokinetics (first-pass effect); (b) local and distant metabolism; (c) efficiency and fidelity of DNA repair; and (d) cell proliferation rate. The last factor will affect not only the dilution of DNA adducts but also the possible evolution towards either destructive processes, such as emphysema or cardiomyopathies, or proliferative processes, such as benign or malignant tumors at various sites. They also include heart tumors affecting fetal myocytes after transplacental exposure to DNA-binding agents, blood vessel tumors, and atherosclerotic plaques. In this article, particular emphasis is given to molecular alterations in the heart, which is the preferential target for the formation of DNA adducts in smokers, and in human aorta, where an extensive molecular epidemiology project is documenting the systematic presence of adducts to the nuclear DNA of smooth muscle cells from atherosclerotic lesions, and their significant correlation with known atherogenic risk factors. Exocyclic DNA adducts resulting from lipid peroxidation, and age-related indigenous adducts (I-compounds) may also originate from endogenous sources, chronic infections and infestations, and inflammatory processes. Type II I-compounds are bulky DNA lesions resulting from oxidative stress, whereas type II-compounds are presumably normal DNA modifications, which display positive correlations with median life span and are decreased in cancer and other pathological conditions. Profiles of type II-compounds strongly depend on diet and are related to the antidegenerative effects of caloric/ dietary restriction. Even broader is the possible meaning of adducts to mitochondrial DNA, which have been detected in rodents exposed to genotoxic agents and complex mixtures, as well as in untreated rodents, in larger amounts when compared to the nuclear DNA of the same cells. Mutations in mitochondrial DNA increase the number of oxidative phosphorylation-defective cells, especially in energy-requiring postmitotic tissues such as brain, heart and skeletal muscle, thereby playing an important role in aging and a variety of chronic degenerative diseases. A decreased formation of DNA adducts is an indicator of reduced risk of developing the associated disease. Therefore, these molecular dosimeters can be used as biomarkers in the prevention of chronic degenerative diseases, pursued either by avoiding exposure to adduct-forming agents or by using chemopreventive agents. Interventions addressed to the human organism by means of dietary measures or pharmacological agents have encountered a broad consensus in the area of cardiovascular diseases, and are deserving a growing interest also in cancer prevention. The efficacy of chemopreventive agents can be assessed by evaluating inhibition of nuclear DNA or mitochondrial DNA adduct formation in vitro, in animal models, and in phase II clinical trials in high-risk individuals.

Sensitive methods for quantifying DNA adducts from (i) benzo[a]pyrene (BP), (ii) alkylation exposure, and (iii) etheno(epsilon)-DNA adduct-forming chemicals were developed and applied to humans and animal models. The aims were to identify hitherto unknown sources and mechanisms of exogenous and endogenous DNA damage, to examine the effect of drug polymorphism on BP adduct levels, and to develop QSAR between tumorigenic potency, heritable genetic damage and structural elements of alkylating carcinogens (Vogel and Nivard (1994) Mutation Res., 395, 13-32). (i) BP-DNA adducts: An HPLC/fluorimetry assay suitable for measuring (+)-anti-BP-diol-epoxide (BPDE) adducts in human tissues and white blood cells (WBC) was developed (Alexandrov et al. (1992) Cancer Res., 52, 6248-6253). In smokers, a positive correlation was found between pulmonary CYP1A1-related catalytic activity (AHH) and the level of lung BPDE-DNA adducts. In coke oven workers, an enhancing effect of smoking on BPDE-adduct levels in WBC was demonstrated (Rojas et al. (1995) Carcinogenesis, 16, 1373-1376). (ii) 3-Alkyladenines (3-alkAde): Alkylating carcinogens form 3-alkAde adducts in DNA which depurinate to yield 3-alkAde in urine, for which a detection method was developed (Friesen et al. (1991) Chem. Res. Toxicol., 4, 102-106; Prevost et al. (1990) Carcinogenesis, 11, 1747-1751), using immunoaffinity purification and GC-MS analysis. The usefulness of 3-alkAde analysis for the determination of the whole-body dose of alkylating agents derived from exogenous and endogenous sources was demonstrated. (iii) Etheno-DNA adduct-forming agents: Etheno(epsilon)-DNA base adducts (epsilon A, epsilon dC, epsilon dG) are promutagenic DNA lesions that are formed by occupational (vinyl halides) and environmental (urethane) carcinogens. An ultrasensitive detection method was developed (Nair et al. (1995) Carcinogenesis, 16, 613-617), based on immunoaffinity purification and 32P-postlabelling of epsilon-nucleoside 3'-monophosphates. Liver DNA from unexposed rats, mice and from human samples contained background levels of epsilon dA and epsilon dC (Bartsch et al. (1994) Drug. Metab. Rev., 26, 349-371). As formation of epsilon dA and epsilon dC adducts by lipid peroxidation products was demonstrated (El Ghissassi et al. (1995) Chem. Res. Toxicol., 8, 278-283), they may serve as markers for oxidative stress. Results from testing this hypothesis are presented.

Usually, a particular mutagen/carcinogen forms adducts at many sites in DNA, making it impossible to determine which type of adduct causes which mutation and why. Adduct site-specific mutagenesis studies, in which a single adduct is built into a vector, can be used to overcome this problem. The adduct can be situated in double-stranded DNA, single-stranded DNA or in a single-stranded gap, and the benefit and concerns associated with each are addressed. An adduct site-specific study is most useful when it is compared to a mutagenesis study with its corresponding mutagen/carcinogen. Mutations induced by a particular mutagen/carcinogen can be influenced by DNA sequence context, mutagen/carcinogen dose (and other changes in conditions), level of SOS induction, cell type and other factors. Thus, it is important to match the conditions of the adduct study versus the mutagen/carcinogen study as closely as possible. DNA sequence context can profoundly affect the quantitative and qualitative pattern of adduct mutagenesis, which is addressed. In vitro studies with DNA polymerases, frameshift mutagenesis and semi-targeted mutagenesis, whereby a mutation is induced near but not at the site of the adduct, are each discussed. Finally, the relationship between structural studies on adducts and mutagenesis is considered.

Mitomycin C (MC) forms interstrand and intrastrand cross-link adducts and monoalkylation products (monoadducts) with DNA. Each of the three types of adducts was incorporated site-specifically into both a 15-mer and a 21-mer oligodeoxyribonucleotide duplex. The adduct-containing duplexes were 32P-phosphorylated and ligated to form multimers, which were then analyzed for anomalous electrophoretic mobility by nondenaturing polyacrylamide gel electrophoresis, using the method of Koo and Crothers [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in order to detect DNA curvature caused by the adducts. The intrastrand cross-link adduct was found to induce a 14.6 +/- 2.0 degrees DNA bend per lesion (minimum value) while no DNA bending was detected for either the interstrand cross-link or the monoadduct. Molecular mechanics modeling indicated that the possible origin of the bend lies in a considerable deviation from parallel of the normals to the best planes of the intrastrand cross-linked guanines, due to a shorter than normal distance between their N2 atoms forced upon them by the cross-link. The observed bending by the MC intrastrand lesion may be the cause of the increased flexibility of MC-modified DNA, localized to distinct regions, as observed in earlier work by hydrodynamic methods and electron microscopy. The MC adduct-caused DNA bend may serve as a recognition site for certain DNA-binding proteins.

The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo. An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct was inserted into the single-stranded genome of bacteriophage M13. Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFB1-N7-Gua of 4%. The predominant mutation was G --> T, identical to the principal mutation in human liver tumors believed to be induced by aflatoxin. The G --> T mutations of AFB1-N7-Gua, unlike those (if the AFB1-N7-Gua-derived apurinic site, were much more strongly dependent on MucAB than UmuDC, a pattern matching that in intact cells treated with the toxin. It is concluded that the AFB1-N7-Gua adduct, and not the apurinic site, has genetic requirements for mutagenesis that best explain mutations in aflatoxin-treated cells. While most mutations were targeted to the site of the lesion, a significant fraction (13%) occurred at the base 5' to the modified guanine. In contrast, the apurinic site-containing genome gave rise only to targeted mutations. The mutational asymmetry observed for AFB1-N7-Gua is consistent with structural models indicating that the aflatoxin moiety of the aflatoxin guanine adduct is covalently intercalated on the 5' face of the guanine residue. These results suggest a molecular mechanism that could explain an important step in the carcinogenicity of aflatoxin B1.

N-(2-Chloroethyl)nitrosoureas (CNU) are clinically used anticancer drugs whose cytotoxicity is associated with the generation of DNA interstrand cross-links. While studying the sequence selectivity for a series of CNU, a dramatic increase in the formation of N7-alkyldeoxyguanosine was observed when Tris buffer was used rather than phosphate or cacodylate buffers. Moreover, the formation of N7-alkyldeoxyguanosine lesions continues in Tris long after all of the CNU has hydrolyzed. These effects are not seen with the monofunctional alkylating analogues, e.g., N-methyl- and N-(2-hydroxyethyl)-N-nitrosourea. In order to determine if the nature of the CNU-mediated DNA damage was altered by Tris, studies were initiated on the following: (1) alkylation of N7-G in end-labeled DNA restriction fragments; (2) covalent modification of DNA with [ethyl-3H]-N-(2-chloroethyl)-N-nitrosourea; and (3) cytotoxicity in L1210 cells. The data presented demonstrate that Tris increases the yield of the "normal" CNU monofunctional cross-linked adducts, i.e., N7-(2-hydroxyethyl)deoxyguanosine, N7-(2-chloroethyl)deoxyguanosine, O6-(2-chloroethyl)deoxyguanosine, and bifunctional adducts, i.e., 1-(deoxycytid-3-yl)-2-(deoxyguanosin-1-yl)ethane and 1,2-bis(deoxyguanosin-7-yl)ethane. In addition, CNU appears to react with Tris to give a long-lived alkylating intermediate that affords large amounts of DNA adducts not seen with CNU in the absence of Tris. However, in vivo toxicity of CNU in L1210 cells is not affected by the presence of Tris, indicating that the reaction pathway(s) responsible for cross-linking is not significantly sensitive to the nature of the buffer.

The gene 4 protein of bacteriophage T7 provides the essential helicase and primase activities for the replication of the T7 genome. In addition, it also displays a DNA-dependent deoxyribonucleoside triphosphatase activity, the preferred substrate of which is dTTP. Previous investigations have demonstrated that the translocation of the gene 4 protein along single-stranded DNA is blocked by the presence of benzo[a]pyrene-DNA adducts and that the gene 4 protein is likely to be sequestered at the sites of these adducts. In the present study, we directly show that the helicase activity of the gene 4 protein is also profoundly inhibited by the benzo[a]pyrene-DNA adducts. The inhibitory effects of these adducts are strand-specific in that they block the DNA helicase activity of the gene 4 protein only when they are located in the DNA strand where the gene 4 protein translocates when it unwinds double-stranded DNA. Consistent with the hypothesis that the gene 4 protein is sequestered at the adduct site, we also show that the complexes formed by the gene 4 protein and benzo[a]pyrene-modified DNA are far more stable than those formed by the gene 4 protein and unmodified DNA.

The process of multistage carcinogenesis lends itself to the concept that the effects of carcinogens are mediated through dose-related, multi-hit, linear changes. Multiple in vitro model systems have been developed that are designed to examine the cellular changes associated with the progression of cells through the different stages in the process; however, these systems may have inherent limitations due to the cell lines used for these studies, the manner of assessing the effects of the carcinogens, and the subsequent growth and differentiation of the exposed cells. Each of these variables results in increasing levels of uncertainty relative to the correlation of the events with the actual process of human tumor development. Therefore, the prediction of the ultimate effect of any carcinogen is difficult. Moreover, relationships between individual biological endpoints resulting from carcinogen treatment appear at best to be approximations. The presence of an activated carcinogen inside the cell can give rise to multiple outcomes, only some of which may be critical events. For example, site-specific modification of the 12th and 13th codons of H-ras is different than that in the adjacent 14th and 15th codons. It is interesting to speculate what effect these differences might have on a biological outcome, e.g., transformation to anchorage-independent growth. The use of different model systems to examine the effects of activated carcinogens also creates additional problems. Comparisons of in vitro transformed cells with similar cells isolated from human tumors indicate that the culture environment appears to influence the expression of a particular phenotype, in that human tumor cells in culture express many of the same parameters as those found in cells transformed with carcinogens in vitro. If the process of transformation is linear, then less aggressive phenotypes should progress to a more aggressive transformed stage. However, in carcinogen-transformed human cells, the populations exhibit phenotypic diversity in that many of the transformed cells differentiate and fail to continue to divide in culture. Historically, we have assumed only a limited role for epigenetic modulation of molecular changes that occur during progression; however, our data suggest quite strongly that nonmalignant tumor populations can be converted to a more malignant phenotype without additional mutations taking place and, conversely, malignant populations can be downregulated to a nontumorigenic phenotype. Tumor cell plasticity is not only a fundamental characteristic of diverse types of human tumors, but also appears as an integral characteristic of carcinogen-transformed cells in vitro.

The toxicity and mutagenicity of three DNA adducts formed by the anticancer drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) were investigated in Escherichia coli. The adducts studied were cis-[Pt(NH3)2(d(GpG))] (G*G*), cis-[Pt(NH3)2(d(ApG))] (A*G*) and cis-[Pt(NH3)2(d(GpTpG))] (G*TG*), which collectively represent approximately 95% of the DNA adducts reported to form when the drug damages DNA. Oligonucleotide 24-mers containing each adduct were positioned at a known site within the viral strand of single stranded M13mp7L2 bacteriophage DNA. Following transfection into E. coli DL7 cells, the genomes containing the G*G*, A*G* and G*TG* adducts had survival levels of 5.2 +/- 1.2, 22 +/- 2.6 and 14 +/- 2.5% respectively, compared to unmodified genomes. Upon SOS induction, the survival of genomes containing the G*G* and A*G* adducts increased to 31 +/- 5.4 and 32 +/- 4.9% respectively. Survival of the genome containing the G*TG* adduct did not increase upon SOS induction. In SOS induced cells, the G*G* and A*G* adducts gave rise predominantly to G-->T and A-->T transversions respectively, targeted to the 5' modified base. In addition, A-->G transitions were detected for the A*G* adduct and low levels of tandem mutations at the 5' modified base as well as the adjacent 5' base were also observed for both adducts. The A*G* adduct was more mutagenic than the G*G* adduct, with a mutation frequency of 6% compared to 1.4% for the latter adduct. No cis-[Pt(NH3)2)2+ intrastrand crosslink-specific mutations were observed for the G*TG* adduct.

Possible inhibitory mechanisms of indole-3-carbinol (I3C) against aflatoxin B1 (AFB1), a potent hepatocarcinogen, were examined in rainbow trout. In the Salmonella assay using a trout post-mitochondrial activation system, I3C itself was not an antimutagen against AFB1. The study also evaluated: the antimutagenic ability of I3C oligomers; an acid reaction mixture (RXM) of I3C, generated at low pH to simulate I3C products formed under acidic conditions of the stomach; 3,3-diindolylmethane (I33'), the major derivative of I3C found in trout liver; and 5,6,11,12,17,18- hexahydrocyclononal [1,2-b:4,5-b':7,8-b"]triindole , the cyclic trimer of I3C (CT), a derivative of I3C in liver and one of the major components of RXM. Concentrations of 3.5 microM and greater of I33', CT or RXM showed about 80% inhibition compared with the control. Higher concentrations (70 microM) of the various I3C oligomers also inhibited (to a maximum of 55%) mutagenesis of synthetic AFB1-8,9-epoxide added to the Salmonella assay, in the absence of activating enzymes. I33' inhibited total microsome catalysed AFB1-DNA binding in vitro in an apparently non-competitive manner (Kis = 27.6 +/- 9.4 microM, Kii = 37.5 +/- 32.2 microM). These results suggest that the anticarcinogenic effect of I3C against AFB1 in rainbow trout, and perhaps other species, is due in part to inhibition of AFB1 bioactivation enzymes and to scavenging of the activated AFB1-8,9-epoxide.

Micromass culture systems of mesencephalon (CNS) and limb bud (LB) regions were used to evaluate the impact of the O6-methylguanine-DNA methyltransferase (MGMT) inhibitor, O6-benzylguanine (BG), on ethylnitrosourea (ENU) and methylnitrosourea (MNU)-induced in vitro toxicity. These primary rat embryo cells were preincubated with BG (2.5 microM for CNS; 10 microM for LB) for 24 hr and then ENU (0-1708 microM) or MNU (0-1600 microM) was added. Five days after culture initiation, cell survival was assessed using neutral red uptake and cell differentiation was assessed using selective staining (Alcian blue in LB cultures and hematoxylin in CNS) and image analysis. In both LB and CNS cultures, preexposure to BG resulted in a significant increase in inhibition of differentiation following MNU exposure but no increase in cytotoxicity. There were no statistically significant changes in the toxicity induced by ENU in these differentiating cells consequent to BG preexposure. Our observations lend support to the idea of a specific role for DNA adducts in defining MNU's toxic effects on cell viability and differentiation.

In order to examine the effect of adenine N6 adducts of styrene oxide (SO) on DNA replication, 33-mer templates were constructed bearing site-specific and stereospecific SO modifications. Both R- and S-SO adducts were introduced at four different base positions within a sequence containing codons 60-62 from the human N-ras gene. The resulting eight templates were replicated in primer extension assays using the Klenow fragment, Sequenase 2.0, T4 polymerase holoenzyme, polymerase alpha, and polymerase beta. Replication of the damaged templates was analyzed under conditions defining single and/or multiple encounters between the polymerase and the substrate. Polymerization by all five enzymes was sensitive to both the local sequence context and the chirality of the SO adduct. For example, R-SO lesions placed at the third position of N-ras codon 61 were readily bypassed, whereas stereochemically-identical lesions in other sequence contexts were often poor substrates for replication. Similarly, R- and S-SO adducts introduced within identical sequences were often bypassed nonequivalently. Significantly, the degree of adduct-directed termination and translesion synthesis during replication was also dependent on the choice of polymerase. Although SO adducts directed termination either opposite the lesion or 1 base 3' to the damage using all five polymerases, templates that were poor substrates for bypass synthesis with one enzyme were often read-through much more efficiently when a different polymerase was used. Thus, the activities of these enzymes on the SO-modified substrates produced replication profiles, or "fingerprints", that were unique to each polymerase.

Pharmacokinetic models have been developed to assist in extrapolating results from rodent bioassays. However, in numerous circumstances, it is necessary to combine such models with cellular response models to fully define interspecies and dose extrapolations. Interactions between pharmacokinetic target tissue end points (DNA adduct formation) and cellular proliferation in liver and urinary bladder carcinogenesis is illustrated with the results from the ED01 study involving 2-acetylaminofluorene administered to female mice. The interaction of genotoxic and cell proliferative effects are also illustrated in a co-carcinogenesis study with low doses of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide and high doses of sodium saccharin. The application of such interactions to humans is illustrated for the case of cigarette smoke-induced bladder cancer.