Detection of dibenzo[a,l]pyrene-induced H-ras codon 61 mutant genes in preneoplastic SENCAR mouse skin using a new PCR-RFLP method.

One of the key events in tumor initiation in mouse skin is mutational activation of the H-ras gene. Papillomas induced by the most carcinogenic environmental polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DB[a,l]P), in SENCAR mouse skin contain a specific H-ras codon 61 (CAA-->CTA) mutation. We describe here detection of these mutations in preneoplastic skin by measuring the frequency of an induced XbaI RFLP, created by the mutation. Development of the PCR-XbaI RFLP method, sensitive enough to detect 1 codon 61 mutant allele among 10,000 wild-type genes, is described. The results indicate that codon 61 mutations are induced 1 day (0.1%) after DB[a,l]P treatment on mouse skin, reach a high value (5%) by day 3, rapidly decline between days 7-9 and increase again during the clonal expansion of pre-papillomas into tumors. The detection of codon 61 mutations 1 day after DB[a,l]P exposure suggests that mutations occurred by pre-replication misrepair.

Evidence that a burst of DNA depurination in SENCAR mouse skin induces error-prone repair and forms mutations in the H-ras gene.

Treatment of SENCAR mouse skin with dibenzo[a,l]pyrene results in abundant formation of abasic sites that undergo error-prone excision repair, forming oncogenic H-ras mutations in the early preneoplastic period. To examine whether the abundance of abasic sites causes repair infidelity, we treated SENCAR mouse skin with estradiol-3,4-quinone (E(2)-3,4-Q) and determined adduct levels 1 h after treatment, as well as mutation spectra in the H-ras gene between 6 h and 3 days after treatment. E(2)-3,4-Q formed predominantly (> or =99%) the rapidly-depurinating 4-hydroxy estradiol (4-OHE(2))-1-N3Ade adduct and the slower-depurinating 4-OHE(2)-1-N7Gua adduct. Between 6 h and 3 days, E(2)-3,4-Q induced abundant A to G mutations in H-ras DNA, frequently in the context of a 3'-G residue. Using a T.G-DNA glycosylase (TDG)-PCR assay, we determined that the early A to G mutations (6 and 12 h) were in the form of G.T heteroduplexes, suggesting misrepair at A-specific depurination sites. Since G-specific mutations were infrequent in the spectra, it appears that the slow rate of depurination of the N7Gua adducts during active repair may not generate a threshold level of G-specific abasic sites to affect repair fidelity. These results also suggest that E(2)-3,4-Q, a suspected endogenous carcinogen, is a genotoxic compound and could cause mutations.

The approach to understanding aromatic hydrocarbon carcinogenesis. The central role of radical cations in metabolic activation.

Polycyclic aromatic hydrocarbons (PAH) are carcinogens requiring metabolic activation to react with cellular macromolecules, the initial event in carcinogenesis. Cytochrome P450 mediates binding of PAH to DNA by two pathways of activation. One-electron oxidation to form radical cations is the major pathway of activation for the most potent carcinogenic PAH, whereas monooxygenation to form bay-region diol epoxides is generally a minor pathway. For benzo[a]pyrene and 7,12-dimethylbenz[a]-anthracene, 80% and 99%, respectively, of the DNA adducts formed by rat liver microsomes or in mouse skin arise via the radical cation. Therefore, studies of PAH activation should begin by considering one-electron oxidation as the primary mechanism.

Radical cations in the horseradish peroxidase and prostaglandin H synthase mediated metabolism and binding of benzo[a]pyrene to deoxyribonucleic acid.

Metabolism and DNA binding studies are used to investigate mechanisms of activation for carcinogens. In this paper we describe metabolism of benzo[a]pyrene (BP) and 6-fluorobenzo[a]pyrene (6-FBP) by two peroxidases, horseradish peroxidase (HRP) and prostaglandin H synthase (PHS), which are known to catalyze one-electron oxidation. In addition, binding of BP and BP quinones to DNA was compared in the two enzyme systems. The only metabolites formed from BP or 6-FBP by either enzyme were the quinones, BP 1,6-, 3,6- and 6,12-dione. HRP metabolized BP and 6-FBP to the same extent and produced the same proportion of each dione from both compounds, approximately 40% each of BP 1,6- and 3,6-dione and 20% BP 6,12-dione. PHS formed twice as much quinones from BP as from 6-FBP and produced relatively more BP 3,6-dione from 6-FBP (46%) compared to BP (30%) and relatively less BP 6,12-dione from 6-FBP (16%) compared to BP (33%). Removal of the fluoro substituent in the metabolism of 6-FBP is consistent only with an initial one-electron oxidation of the substrate. Since BP quinones were the only products formed in HRP- and PHS-catalyzed activation of BP, their possible binding to DNA was compared to that of BP. No significant binding of BP quinones to DNA occurred with either HRP or PHS. These results, coupled with those from other chemical and biochemical experiments, demonstrate that HRP- and PHS-catalyzed one-electron oxidation of BP to its radical cation is the mechanism of formation of quinones and binding of BP to DNA.

Radical cations as precursors in the metabolic formation of quinones from benzo[a]pyrene and 6-fluorobenzo[a]pyrene. Fluoro substitution as a probe for one-electron oxidation in aromatic substrates.

Three classes of products are formed when benzo[a]pyrene (BP) is metabolized by cytochrome P-450: dihydrodiols, phenols and the quinones, BP 1,6-, 3,6- and 6,12-dione. These products have been thought to arise from attack of a catalytically-activated electrophilic oxygen atom. In this paper we report chemical and biochemical experiments which demonstrate that BP quinones arise from an initial one-electron oxidation of BP to form its radical cation. BP, 6-fluorobenzo[a]pyrene (6-FBP), 6-chlorobenzo[a]pyrene (6-ClBP), and 6-bromobenzo[a]pyrene (6-BrBP) were metabolized by uninduced and 3-methylcholanthrene-induced rat liver microsomes in the presence of NADPH or cumene hydroperoxide (CHP) as cofactor. BP and 6-FBP produced similar metabolic profiles with induced microsomes in the presence of NADPH or 2 mM CHP. With NADPH both compounds produced dihydrodiols, phenols and quinones, whereas with CHP, they yielded only quinones. Metabolism of BP and 6-FBP was also similar with uninduced microsomes and 2 mM CHP, yielding the same BP quinones. With uninduced microsomes in the presence of NADPH, BP produced all three classes of metabolites, whereas 6-FBP afforded only quinones. At a low concentration of CHP (0.10 mM), BP was metabolized to phenols and quinones, whereas 6-FBP gave only quinones. 6-ClBP and 6-BrBP were poor substrates, forming metabolites only with induced microsomes and NADPH. One-electron oxidation of BP by Mn(OAc)3 occurred exclusively at C-6 with predominant formation of 6-acetoxyBP and small amounts of BP quinones. In the one-electron oxidation of 6-FBP by Mn(OAc)3, the major products obtained were 6-acetoxyBP, a mixture of 1,6- and 3,6-diacetoxyBP, and BP quinones. Reaction of BP and 6-FBP radical cation perchlorates with water produced the same BP quinones. Conversely, electrophilic substitution of 6-FBP with bromine or deuterium ion afforded C-1 and/or C-3 derivatives with retention of the fluoro substituent at C-6. These results indicate that metabolic formation of BP quinones from BP and 6-FBP can only derive from their intermediate radical cation.

Isomer differentiation in 7, 12-dimethylbenz[a]anthracene-pyridine adducts by fast atom bombardment tandem mass spectrometry.

Three isomeric 7,12-dL-nethylbenz[α]anthracene-pyridine adduct salts, namely.. the 5-N-pyridinium-7,12-dimethylbenz[α]anthracene perchlorate, the 7-N-pyridiniummethylene-12methylbenz[ α]anthracene picrate, and the 7-methyl-12-N-pyridiniummethylenebenz[ α]anthracene picrate, were studied by fast atom bombardment tandem mass spectrometry using high energy collisional-activated dissociation (CAD). The CAD mass spectra of the molecular cations and the (M - pyridine)(+) ions allow one to distinguish positional isomers on the basis of daughter ion peak height ratios. The differences in the CAD mass spectra of the (M - pyridine)(+) ions are probably due in part to formation of isomer-specific fused-ring tropyliumions.

Matrix design for matrix-assisted laser desorption ionization: Sensitive determination of PAH-DNA adducts.

Two matrices, 4-phenyl-α-eyanocinnamic acid (PCC) and 4-benzyloxy-α-eyanocinnamic acid (BCC), were identified for the determination of polycyclic aromatic hydrocarbon (PAH) adducts of DNA bases by matrix-assisted laser desorption ionization. These matrices were designed based on the concept that the matrix and the analyte should have structural similarities. PCC is a good matrix for the desorption of not only PAH-modified DNA bases, but also PAHs themselves and their metabolites. Detections can be made at the femtomolar level.

Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[a]pyrenes: the very potent environmental carcinogen dibenzo[a, l]pyrene.

Comparative studies of tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland were conducted with several dibenzo[a]-pyrenes (DBPs). SENCAR mice were initiated with DB[a, e]P, DB[a, h]P, DB[a, i]P, DB[a, l]P and anthanthrene, and promoted with tetradecanoyl-phorbol acetate. The same compounds were tested by intramammillary injection in female Sprague-Dawley rats. Anthanthrene was inactive in both mouse skin and rat mammary gland. DB[a, e]P was a very weak tumor-initiator in mouse skin and was inactive in rat mammary gland. DB[a, h]P induced twice as many papillomas in mouse skin as DB[a, i]P, although both compounds exhibited similar tumor latencies and percentages of tumor-bearing mice. These two compounds induced similar numbers of mammary tumors, but treatment of the rats with DB[a, i]P resulted in a significantly larger number of adenocarcinomas. DB[a, l]P was toxic to both the mice and rats. Treatment of mouse skin with this compound led to an erythema, which delayed the beginning of promotion until the 3rd week after initiation. Despite this delay, papillomas began appearing 5 weeks after initiation with DB[a, l]P and the number of tumors increased rapidly. The compound was so toxic in the rats that half of the animals died in the first 9 weeks and the remaining animals were sacrificed after 15 weeks. Nonetheless, DB[a, l]P was the strongest carcinogen tested, inducing seven tumors per rat within 10 weeks. These results demonstrate that DB[a, l]P, which is present in tobacco smoke, is an extremely potent carcinogenic aromatic hydrocarbon. Furthermore, some of these compounds can serve as useful models for elucidating their mechanisms of activation.

Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis.

Long term exposure to estradiol increases the risk of breast cancer in a variety of animal species, as well as in women. The mechanisms responsible for this effect have not been firmly established. The prevailing theory proposes that estrogens increase the rate of cell proliferation by stimulating estrogen receptor-mediated transcription and thereby the number of errors occurring during DNA replication. An alternative hypothesis proposes that estradiol can be metabolized to quinone derivatives which can react with DNA and then remove bases from DNA through a process called depurination. Error prone DNA repair then results in point mutations. We postulate that these two processes, increased cell proliferation and genotoxic metabolite formation, act in an additive or synergistic fashion to induce cancer. If correct, aromatase inhibitors would block both processes whereas anti-estrogens would only inhibit receptor-mediated effects. Accordingly, aromatase inhibitors would be more effective in preventing breast cancer than use of anti-estrogens. Our studies initially demonstrated that catechol estrogen (CE) quinone metabolites are formed in MCF-7 human breast cancer cells in culture. Measurement of estrogen metabolites and conjugates involved utilization of an HPLC separation coupled with an electrochemical detector. We then utilized an animal model that allows dissociation of estrogen receptor-mediated function from that of the effects of estradiol metabolites. Wnt-1 transgenic mice harboring a knock-out of ERalpha provides a means of examining the effect of estrogen deprivation in the absence of the ER in animals with a high incidence of breast tumors. ERbeta was shown to be absent in the breast tissue of these animals by RNase protection assay. In the breast tissue of these estrogen receptor alpha knock-out (ERKO)/Wnt-1 transgenic mice, we demonstrated formation of genotoxic estradiol metabolites. The ERKO/Wnt-1 breast extracts contained picomole amounts of the 4-catechol estrogens, but not their methoxy conjugates nor the 2-CE or their methoxy conjugates. The 4-CE conjugates with glutathione or its hydrolytic products (cysteine and N-acetylcysteine) were detected in picomole amounts in both tumors and hyperplastic mammary tissue, demonstrating the formation of CE-3,4-quinones. These results are consistent with the hypothesis that mammary tumor development is primarily initiated by metabolism of estrogens to 4-CE and, then, to CE-3,4-quinones, which may react with DNA to induce oncogenic mutations. The next set of experiments examined the incidence of tumors formed in Wnt-1 transgenic mice bearing wild type ERalpha (ER+/+), the heterozygous combination of genes (ER+/ER-) or ERalpha knock-out (ER-/-). To assess the effect of estrogens in the absence of ER, half of the animals were oophorectomized on day 15 and the other half were sham operated. Castration reduced the incidence of breast tumors in all animal groups and demonstrated the dependence of tumor formation upon estrogens. A trend toward reduction in tumor number (not statistically significant at this interim analysis) occurred in the absence of functional ER since the number of tumors was markedly reduced in ERKO animals which were castrated early in life. In aggregate, our results support the concept that metabolites of estradiol may act in concert with ER mediated mechanisms to induce breast cancer.

Linear amplification mapping of polycyclic aromatic hydrocarbon-reactive sequences in H-ras gene.

Linear amplification, or primer directed single-strand DNA synthesis, is commonly used in applications such as cycle sequencing and mapping replication block sites in DNA. Although linear amplification reactions would be expected to synthesize full-length single-stranded DNA, the synthesis is often prematurely terminated. We describe the optimization of a linear amplification protocol for synthesizing a full-length (985-nt) single-stranded pBR322 segment. The enzyme activities of five DNA polymerases commonly used in PCR amplification, namely, AmpliTaq, Stoffel fragment, Tth, Pfu, and Vent, were tested either singly or in combination. The results indicate that the additive action of small amounts of proofreading DNA polymerases to a nick-translating polymerase is optimum for linear amplification. From these results, a linear amplification protocol was developed to map DNA synthesis-blocking sites generated by the reaction of (+/-) anti-benzo[a]pyrene-7,8-diol-9,10-epoxide, or anti- or syn-dibenzo[a,l]pyrene-9,10-diol-11,12-epoxide with H-ras DNA surrounding the oncogenic codon 61 region. The results indicate that the central A of H-ras codon 61 (CAA) reacts with these polycyclic aromatic hydrocarbons.

Role of human cytochrome P450 1A1, 1A2, 1B1, and 3A4 in the 2-, 4-, and 16alpha-hydroxylation of 17beta-estradiol.

The steady-state kinetics and specific activity of 2-, 4-, and 16alpha-hydroxylation of 17beta-estradiol (E(2)) were evaluated for human cytochrome P450 (CYP) 1A1, 1A2, 1B1, and 3A4 enzymes, using complementary DNA-expressed CYP isoforms. CYP1A2 showed the highest 2-hydroxylation activity, followed by CYP1A1, 1B1, and 3A4. CYP1B1 had the highest 4-hydroxylation activity, followed by CYP1A2, 1A1, and 3A4. The 16alpha-hydroxylation reaction was catalyzed mainly by CYP1A2 and, to a similar, slightly lower extent, CYP3A4 and 1A1, with a lesser contribution by CYP1B1. The E(2) 2-, 4-, and 16alpha-hydroxylation activities of human liver microsomes were 1.3 +/- 0.3, 0.5 +/- 0.06, and 0.3 +/- 0.05 nmol metabolite/min/nmol P450, respectively. The contribution of CYP1A1 and 1B1 (mainly extrahepatic) to the E(2) hydroxylation reactions, relative to CYP1A2 and 3A4 (predominantly hepatic), may be relevant to understanding the process of hormonal carcinogenesis both in liver and in extrahepatic tissues.

Structure elucidation of a 6-methylbenzo[a]pyrene-DNA adduct formed by horseradish peroxidase in vitro and mouse skin in vivo.

Activation of polycyclic aromatic hydrocarbons (PAH) by horseradish peroxidase (HRP) with H2O2 has been studied as a model system for one-electron oxidation. This peroxidase has been used to catalyze binding of 6-[14C]methylbenzo[a]pyrene (BP-6-CH3) to DNA, which was purified, hydrolyzed to deoxyribonucleosides and analyzed by high pressure liquid chromatography (HPLC). The predominant hydrocarbon-DNA adduct observed was identified as BP-6-CH3 bound at the 6-methyl group to the 2-amino group of dG, confirming that activation by HRP occurs by one-electron oxidation. When DNA from mouse skin treated in vivo with [14C]BP-6-CH3 was purified, hydrolyzed and analyzed by HPLC, a profile was observed which was qualitatively similar to that from the peroxidase system. In particular, the identified adduct with the hydrocarbon bound at the 6-methyl group to the 2-amino group of dG was obtained. These results demonstrate that one-electron oxidation is the mechanism of activation by HRP for aromatic hydrocarbons and indicate that the same mechanism may occur in mouse skin, a target tissue for hydrocarbon carcinogenesis.

Non-enzymatic ATP-mediated binding of hydroxymethyl derivatives of aromatic hydrocarbons to DNA.

ATP mediates covalent binding of hydroxymethyl derivatives of aromatic hydrocarbons to DNA. This non-enzymatic reaction has been studied with 6-[14C]hydroxymethylbenzo[alpha]pyrene (]14C]BP-6-CH2OH) and 7-[14C]-hydroxymethylbenz[alpha]anthracene ([14C]BA-7-CH2OH) at 37 degrees C in Tris buffer (pH 7.0). While ADP mediates the reaction 25-50% as well as ATP, six other possible phosphate donors including AMP were inactive as cofactors. A complex response to ATP occurred in which low binding of BP-6-CH2OH or BA-7-CH2OH was observed at concentrations of ATP below 2.5 mM, but a greater than linear response to higher concentrations of ATP was observed until ATP was saturating. Binding of the substrates to RNA was much lower than to DNA. Fluorescence spectra of BP-6-CH2OH bound to DNA were almost identical to the spectra of 6-bromomethylbenzo[alpha]pyrene bound to DNA and free 6-methylbenzo]alpha]pyrene, indicating that ATP-mediated binding of BP-6-CH2OH to DNA occurs at the 6-methyl group. The fate of ATP and ADP in the binding reaction of BP-6-CH2OH was examined by thin layer chromatography. Loss of one phosphate group occurs during the reaction. With ATP the rate of loss is about 100-fold greater than the rate of binding of BP-6-CH2OH to DNA. This implies that the binding reaction proceeds through formation of a presumed reactive and unstable phosphate ester intermediate which then inefficiently binds to DNA.

The relationship between ionization potential and horseradish peroxidase/hydrogen peroxide-catalyzed binding of aromatic hydrocarbons to DNA.

The ionization potentials (IP) of 91 alternant polycyclic aromatic hydrocarbons (PAH) were determined from the absorption maximum of the charge-transfer complex of each hydrocarbon and chloranil in chloroform. The extent of horseradish peroxidase (HRP)-catalyzed binding to DNA of 14 hydrocarbons of varying IP was measured. Only hydrocarbons with IP less than approx. 7.35 eV were significantly bound to DNA. These results provide further evidence that HRP-mediated binding of PAH to DNA occurs by one-electron oxidation and indicate that hydrocarbons must have IP less than approx. 7.35 eV to be activated by one-electron oxidation. Thus, determination of IP and HRP-catalyzed binding to DNA can offer some guidelines for selecting aromatic hydrocarbons which might undergo carcinogenic activation by this mechanism.

Mutagenicity of benzylic acetates, sulfates and bromides of polycyclic aromatic hydrocarbons.

Studies were performed to determine the direct mutagenicity of the acetates and some bromides and sulfates of hydroxymethyl polycyclic aromatic hydrocarbons in S. typhimurium strains TA98 and TA100. Benzylic acetates, bromides and sulfates were synthesized and characterized. The compounds tested were benzyl alcohol, 5-hydroxymethylchrysene, 1-hydroxymethylpyrene, 6-hydroxymethylbenzo[a]pyrene, 6-(2-hydroxyethyl)benzo[a]pyrene, 6-hydroxymethylanthanthrene, 9-hydroxymethylanthracene, 9-hydroxymethyl-10-methylanthracene, 7-hydroxymethylbenz[a]anthracene, 7-(2-hydroxyethyl)benz[a]anthracene, 12-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, 12-hydroxymethyl-7-methylbenz[a]anthracene, 1-hydroxy-3-methylcholanthrene, 2-hydroxy-3-methylcholanthrene, 3-hydroxy-3, 4-dihydrocyclopental[cd]pyrene and 4-hydroxy-3, 4-dihydrocyclopental[cd]pyrene. The benzylic sulfate esters of 6-hydroxymethylbenzo[a]pyrene and 7-hydroxymethylbenz[a]anthracene were the most mutagenic compounds, whereas the aliphatic sulfate ester of 7-hydroxyethylbenz[a]anthracene did not cause an increase in mutations above background. All meso-anthracenic benzylic acetate esters were mutagenic in both strains with various degrees of activity, whereas the corresponding non-benzylic esters were inactive, as expected. Of the non-meso-benzylic acetate esters, only the 3-acetoxy-3, 4-dihydrocyclopenta[cd]pyrene was mutagenic. In the benzylic bromide series, only the eight mesoanthracenic were mutagenic, whereas benzyl bromide and 5-bromomethylchrysene were inactive. The aliphatic bromides, 6-(2-bromoethyl)benzo[a]pyrene and 7-(2-bromoethyl)benz[a]anthracene did not display significant activity. The potencies of the acetate esters more accurately reflect the mutagenicity because the rate of solvolysis did not compete with the reactivity of the esters with bacterial DNA. In the case of benzylic sulfates and bromides, the rate of solvolysis was very rapid and could have diminished the level of mutagenicity, depending on the assay conditions. These results demonstrate that meso-anthracenic benzylic acetates, sulfates and bromides are mutagenic, whereas benzylic acetate esters attached to other carbon atoms are inactive.

Synthesis and structure determination of 6-methylbenzo[a]pyrene-deoxyribonucleoside adducts and their identification and quantitation in vitro and in mouse skin.

Activation of the moderate carcinogen 6-methylbenzo[a]pyrene (6-CH(3)BP) by one-electron oxidation to form DNA adducts was studied. Iodine oxidation of 6-CH(3)BP in the presence of dGuo produces BP-6-CH(2)-N(2)dGuo, BP-6-CH(2)-N7Gua and a mixture of 6-CH(3)BP-(1&3)-N7Gua, whereas in the presence of Ade the adducts BP-6-CH(2)-N1Ade, BP-6-CH(2)-N3Ade, BP-6-CH(2)-N7Ade and 6-CH(3)BP-(1&3)-N1Ade are obtained. Furthermore, for the first time an aromatic hydrocarbon radical cation afforded an adduct with dThd, the stable adduct BP-6-CH(2)-N3dThd. Formation of these adducts indicates that the 6-CH(3)BP radical cation has charge localized at the 6, 1 and 3 position. When 6-CH(3)BP was activated by horseradish peroxidase in the presence of DNA, two depurinating adducts were identified, BP-6-CH(2)-N7Gua (48%) and 6-CH(3)BP-(1&3)-N7Gua (23%), with 29% unidentified stable adducts. In the binding of 6-CH(3)BP catalyzed by rat liver microsomes, the same two depurinating adducts, BP-6-CH(2)-N7Gua (22%) and 6-CH(3)BP-(1&3)-N7Gua (10%), were identified, with 68% unidentified stable adducts. In 6-CH(3)BP-treated mouse skin, the two depurinating adducts, BP-6-CH(2)-N7Gua and 6-CH(3)BP-(1&3)-N7Gua, were identified. Although quantitation of these two adducts was not possible due to coelution of metabolites on HPLC, they appeared to be the major adducts found in mouse skin. These results show that 6-CH(3)BP forms depurinating adducts only with the guanine base of DNA, both in vitro and in mouse skin. The weaker reactivity of 6-CH(3)BP radical cation vs. BP radical cation could account for the weaker tumor-initiating activity of 6-CH(3)BP in comparison to that of BP.

Evidence that error-prone DNA repair converts dibenzo[a,l]pyrene-induced depurinating lesions into mutations: formation, clonal proliferation and regression of initiated cells carrying H-ras oncogene mutations in early preneoplasia.

Initiation of skin tumors in mice is associated with the formation of oncogenic mutations in the H-ras gene. Mice treated on the dorsal skin with the potent polycyclic aromatic hydrocarbon (PAH) carcinogen dibenzo[a,l]pyrene (DB[a,l]P) form papillomas carrying the H-ras codon 61 (CAA to CTA) mutations. These mutations are induced in early preneoplastic skin within 1 day after DB[a,l]P treatment (Oncogene 16 (1998) 3203-3210) and appear to be related to DB[a,l]P-Ade-depurinating adducts (Proc. Natl. Acad. Sci. U. S. A. 92 (1995) 10422-10426). The rapid kinetics of mutation induction suggests that abasic sites generated from base depurination may undergo error-prone excision repair in pre-S-phase cells to induce these mutations. Analysis of mutations in the H-ras exon 1 and 2 region in DB[a,l]P-treated early preneoplastic skin indicated great changes in mutation spectra in the preneoplastic period. The initial spectra contained abundant A-->G mutations, which frequently occurred 3' to a putative conserved sequence (TGN-doublet). These mutations appeared to be induced initially as mismatched (G.T) heteroduplexes and then converted into double-stranded mutations by one round of replication. Unlike the A-->G mutations found in DB[a, l]P-treated skin (which forms 99% depurinating adducts), A-->G mutations found in anti-DB[a,l]P-diol epoxide-treated skin (forms 97% stable adducts) did not appear to be G.T heteroduplexes. These results, therefore, suggest that under these conditions, the repair errors occurred only from abasic sites but not from stable adducts. Initiated cells carrying specific oncogenic mutations, formed presumably by misrepair, underwent rapid clonal expansion and regression (transient clonoplasia). The multiplication of initiated stem cells during transient clonoplasia may be a factor determining the tumor-initiating potential of some PAH carcinogens.