Covalent binding of catechol estrogens to glutathione catalyzed by horseradish peroxidase, lactoperoxidase, or rat liver microsomes.

Oxidation of catechol estrogens (CE) leads to the reactive electrophilic CE quinones. Reaction of CE-3,4-quinones with DNA has been implicated in tumor initiation. One pathway to prevent this reaction is conjugation of CE quinones with glutathione (GSH). Four CE, 4-hydroxy estrone (4-OHE1), 4-hydroxyestradiol (4-OHE2), 2-OHE1, and 2-OHE2, were conjugated with GSH after oxidation catalyzed by horseradish peroxidase (HRP), lactoperoxidase (LP), or rat liver microsomal cytochrome P450. This reaction is a free-radical chain autoxidation that produces very high yields of products. Six mono-GSH conjugates, 4-OHE1(E2)-2-SG, 2-OHE1(E2)-1-SG, and 2-OHE1(E2)-4-SG, and four di-GSH conjugates, 4-OHE1(E2)-1,2-bisSG and 2-OHE1(E2)-1,4-bisSG, were identified and quantified. These di-GSH conjugates were also obtained quantitatively from oxidation of mono-GSH conjugates by the same enzymes. HRP and LP gave very similar product profiles. Phenobarbital- and 3-methylcholanthrene-induced microsomes with either NADPH or cumene hydroperoxide as cofactor oxidized 4-OHE2 to form similar amounts of GSH conjugates. Enzymatic oxidation of 2-OHE1(E2) in the presence of GSH produced more 2-OHE1(E2)-4-SG than the 1-isomer. This contrasts with the direct reaction of E1(E2)-2,3-Q and GSH, in which the 1-isomer is formed more abundantly than the 4-isomer (Cao, K., Devanesan, P. D., Ramanathan, R., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1998) Chem. Res. Toxicol. 11, 909-916). Competitive enzymatic oxidation of equimolar 4-OHE2 and 2-OHE2 in the presence of an equimolar amount of GSH yielded more 2-OHE2 conjugates than 4-OHE2 conjugates, despite E2-3,4-Q being more reactive with GSH than E2-2,3-Q. These results suggest that 2-OHE2 is a better substrate than 4-OHE2 in the catalytic oxidation to quinones, despite the greater reactivity of E2-3,4-Q, compared to E2-2,3-Q, with GSH.

Metabolic activation and formation of DNA adducts of hexestrol, a synthetic nonsteroidal carcinogenic estrogen.

Hexestrol (HES), a synthetic nonsteroidal estrogen, is carcinogenic in Syrian golden hamsters. The major metabolite of HES is its catechol, 3'-OH-HES, which can be metabolically converted to the electrophilic catechol quinone, HES-3',4'-Q, by peroxidases and cytochrome P450. Standard adducts were synthesized by reacting HES-3',4'-Q with dG and dA to produce the adducts 3'-OH-HES-6'(alpha, beta)-N7Gua and HES-3',4'-Q-6'-N6dA, respectively. When HES-3',4'-Q was reacted with calf thymus DNA, 3'-OH-HES-6'(alpha,beta)-N7Gua was identified by HPLC and tandem mass spectrometry as the depurinating adduct, with minor amounts of stable adducts. 3'-OH-HES was bound to DNA after activation by horseradish peroxidase, lactoperoxidase, or rat liver microsomes. The depurinating adduct 3'-OH-HES-6'(alpha, beta)-N7Gua was identified in these systems at levels of 65, 41, and 11 micromol/mol of DNA-P, respectively. Unidentified stable adducts were observed in much lower amounts and were quantified by the 32P-postlabeling method. Similarly to 3'-OH-HES, the catechol metabolites of the natural steroidal estrogens estrone (E1) and estradiol (E2), namely, 2-OHE1, 4-OHE1, 2-OHE2, and 4-OHE2, can be oxidized to their corresponding quinones by peroxidases and cytochrome P450. The quinones of the carcinogenic 4-OHE1 and 4-OHE2 have chemical and biochemical properties similar to those of HES-3',4'-Q. The results suggest that formation of HES-3',4'-Q may be a critical event in tumor initiation by HES and that HES is an excellent model compound to corroborate the hypothesis that estrogen-3,4-quinones are ultimate carcinogenic metabolites of the natural steroidal estrogens E1 and E2.

Molecular origin of cancer: catechol estrogen-3,4-quinones as endogenous tumor initiators.

Cancer is a disease that begins with mutation of critical genes: oncogenes and tumor suppressor genes. Our research on carcinogenic aromatic hydrocarbons indicates that depurinating hydrocarbon-DNA adducts generate oncogenic mutations found in mouse skin papillomas (Proc. Natl. Acad. Sci. USA 92:10422, 1995). These mutations arise by mis-replication of unrepaired apurinic sites derived from the loss of depurinating adducts. This relationship led us to postulate that oxidation of the carcinogenic 4-hydroxy catechol estrogens (CE) of estrone (E1) and estradiol (E2) to catechol estrogen-3,4-quinones (CE-3, 4-Q) results in electrophilic intermediates that covalently bind to DNA to form depurinating adducts. The resultant apurinic sites in critical genes can generate mutations that may initiate various human cancers. The noncarcinogenic 2-hydroxy CE are oxidized to CE-2,3-Q and form only stable DNA adducts. As reported here, the CE-3,4-Q were bound to DNA in vitro to form the depurinating adduct 4-OHE1(E2)-1(alpha,beta)-N7Gua at 59-213 micromol/mol DNA-phosphate whereas the level of stable adducts was 0.1 micromol/mol DNA-phosphate. In female Sprague-Dawley rats treated by intramammillary injection of E2-3,4-Q (200 nmol) at four mammary glands, the mammary tissue contained 2.3 micromol 4-OHE2-1(alpha, beta)-N7Gua/molDNA-phosphate. When 4-OHE1(E2) were activated by horseradish peroxidase, lactoperoxidase, or cytochrome P450, 87-440 micromol of 4-OHE1(E2)-1(alpha, beta)-N7Gua was formed. After treatment with 4-OHE2, rat mammary tissue contained 1.4 micromol of adduct/mol DNA-phosphate. In each case, the level of stable adducts was negligible. These results, complemented by other data, strongly support the hypothesis that CE-3,4-Q are endogenous tumor initiators.

Synthesis of depurinating DNA adducts formed by one-electron oxidation of 7H-dibenzo[c,g]carbazole and identification of these adducts after activation with rat liver microsomes.

It is hypothesized that 7H-dibenzo[c,g]carbazole (DBC) is metabolically activated by one-electron oxidation in accordance with its propensity to be easily oxidized to its radical cation. Iodine oxidation of DBC produces a radical cation that subsequently binds to nucleophilic groups of dG or Ade. Oxidation of DBC in the presence of dG products three adducts: DBC-5-N7Gua, DBC-6-N7Gua, and DBC-6-C8Gua, whereas in the presence of Ade, four adducts are obtained: DBC-5-N7Ade, DBC-5-N3Ade, DBC-5-N1Ade, and DBC-6-N3Ade. Formation of these adducts demonstrates that the DBC radical cation reacts at C-5 or C-6 with the reactive nucleophiles N-7 and C-8 of dG and N-7, N-3, and N-1 of Ade. Formation DNA adducts by DBC was studied by using horesradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes for activation. Identification of the biologically-formed depurinating adducts was achieved by comparison of their retention times on HPLC in two different solvent systems and by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Quantitation of the adducts formed by rat liver microsomes shows that 96% are depurinating adducts, DBC-5-N7Gua (11%), DBC-6-N7Gua (32%), and DBC-5-N7Ade (53%), and 4% are unidentified stable adducts. Activation of DBC by horseradish peroxidase affords 32% stable unidentified adducts and 68% depurinating adducts: 19% DBC-5-N7Gua, 13% DBC-6-N7Gua, 27% DBC-5-N7Ade, and 9% DBC-5-N3Ade. Thus, activation of DBC by cytochrome P450 predominantly forms depurinating adducts by one-electron oxidation.

Depurinating and stable benzo[a]pyrene-DNA adducts formed in isolated rat liver nuclei.

Polycyclic aromatic hydrocarbons are bound to DNA by two major pathways, one-electron oxidation and monooxygenation, to form adducts that are stable in DNA under normal conditions of isolation and depurinating adducts that are released from DNA by cleavage of the bond between the purine base and deoxyribose. Isolated rat liver nuclei have been used as an in vitro model for studying covalent binding of aromatic hydrocarbons to DNA, but the depurinating adducts formed by nuclei have not been identified or compared to those formed by the more commonly used rat liver microsomes. To examine the profiles of stable and depurinating adducts, nuclei from the livers of 3-methylcholanthrene-induced male MRC Wistar rats were incubated with [3H]benzo[a]pyrene (BP) and NADPH. Three depurinating adducts, 8-(BP-6-yl)Gua, 7-(BP-6-yl)Gua, and 7-(BP-6-yl)Ade, were obtained from the nuclei, as seen previously with rat liver microsomes or in mouse skin. The profile of stable adducts analyzed by the 32P-postlabeling method was qualitatively similar to that found in the microsomal activation of BP or in mouse skin treated with BP. Low-temperature fluorescence studies of the nuclear DNA revealed the presence of stable BP adducts originating from syn- and anti-BP diol epoxide.

Expanded analysis of benzo[a]pyrene-DNA adducts formed in vitro and in mouse skin: their significance in tumor initiation.

This paper reports expanded analyses of benzo[a]pyrene (BP)-DNA adducts formed in vitro by activation with horseradish peroxidase (HRP) or 3-methylcholanthrene-induced rat liver microsomes and in vivo in mouse skin. The adducts formed by BP are compared to those formed by BP-7,8-dihydrodiol and anti-BP diol epoxide (BPDE). First, activation of BP by HRP produced 61% depurinating adducts: 7-(benzo[a]pyrene-6-yl)guanine (BP-6-N7Gua), BP-6-C8Gua, BP-6-N7Ade, and the newly identified BP-6-N3Ade. As a standard, the last adduct was synthesized along with BP-6-N1Ade by electrochemical oxidation of BP in the presence of adenine. Second, identification and quantitation of BP-DNA adducts formed by microsomal activation of BP showed 68% depurinating adducts: BP-6-N7Ade, BP-6-N7Gua, BP-6-C8Gua, BPDE-10-N7Ade, and the newly detected BPDE-10-N7Gua. The stable adducts were mostly BPDE-10-N2dG (26%), with 6% unidentified. BPDE-10-N7Ade and BPDE-10-N7Gua were the depurinating adducts identified after microsomal activation of BP-7, 8-dihydrodiol or direct reaction of anti-BPDE with DNA. In both cases, the predominant adduct was BPDE-10-N2dG (90% and 96%, respectively). Third, when mouse skin was treated with BP for 4 h, 71% of the total adducts were the depurinating adducts BP-6-N7Gua, BP-6-C8Gua, BP-6-N7Ade, and small amounts of BPDE-10-N7Ade and BPDE-10-N7Gua. These newly detected depurinating diol epoxide adducts were found in larger amounts when mouse skin was treated with BP-7,8-dihydrodiol or anti-BPDE. The stable adduct BPDE-10-N2dG was predominant, especially with anti-BPDE. Comparison of the profiles of DNA adducts formed by BP, BP-7,8-dihydrodiol, and anti-BPDE with their carcinogenic potency indicates that tumor initiation correlates with the levels of depurinating adducts, but not with stable adducts. Furthermore, the levels of depurinating adducts of BP correlate with mutations in the Harvey-ras oncogene in DNA isolated from mouse skin papillomas initiated by this compound [Chakravarti et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10422-10426]. The depurinating adducts formed by BP in mouse skin appear to be the key adducts leading to tumor initiation.

32P-postlabeling analysis of the DNA adducts of 6-fluorobenzo[a]pyrene and 6-methylbenzo[a]pyrene formed in vitro.

Studies of benzo[a]pyrene (BP) and selected derivatives are part of the strategy to elucidate mechanisms of tumor initiation by polycyclic aromatic hydrocarbons. Substitution of BP at C-6 with fluorine to form 6-fluorobenzo[a]pyrene (6-FBP) or a methyl group to form 6-methylbenzo[a]pyrene (6-CH3BP) decreases tumorigenicity compared to BP. BP, 6-FBP, and 6-CH3BP formed adducts with DNA when (1) they were activated by 3-methylcholanthrene-induced rat liver microsomes, (2) they were activated by horseradish peroxidase (HRP), (3) their 7,8-dihydrodiols were activated by microsomes, or (4) the radical cation of BP, 6-FBP, or 6-CH3-BP was directly reacted with DNA. With microsomes, 6.5 mumol of [3H]6-FBP/mol of DNA-P and 10 mumol of [14C]6-CH3BP/mol of DNA-P were bound vs 15 mumol of [3H]BP. With microsomes, two major 6-FBP adducts and some minor adducts were obtained. One major adduct coincided with that from 6-FBP-7,8-dihydrodiol. With microsomes, the minor 6-FBP adducts coincided with the adducts obtained from 6-FBP radical cation plus DNA and the major adduct of HRP-activated 6-FBP. With microsomes, 6-CH3BP showed adducts similar to some formed with HRP and one from 6-CH3BP radical cation. 6-CH3BP-7,8-dihydrodiol produced a small amount of one adduct that did not coincide with any from 6-CH3BP. The adducts of 6-FBP appear to be formed mostly through the diolepoxide pathway, whereas those of 6-CH3BP appear to arise mostly via one-electron oxidation.

Identification and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin.

The DNA adducts of benzo[a]pyrene (BP) formed in vitro were previously identified and quantitated. In this paper, we report the identification and quantitation of the depurination adducts of BP, 8-(benzo[a]pyren-6-yl)guanine (BP-6-C8Gua), BP-6-N7Gua, and BP-6-N7Ade, formed in mouse skin by one-electron oxidation, as well as the major stable adduct formed via the diolepoxide pathway, BP diolepoxide bound at C-10 to the 2-amino of dG (BPDE-10-N2dG). Identification of the depurination adducts was achieved by HPLC and fluorescence line narrowing spectroscopy. The depurination adducts, BP-6-C8Gua (34%), BP-6-N7Gua (10%), and BP-6-N7Ade (30%), constituted 74% of the adducts found in mouse skin 4 h after treatment with BP. The stable adduct BPDE-10-N2dG accounted for 22% of the adducts. Treatment of the skin with BP-7,8-dihydrodiol or BP diolepoxide yielded almost exclusively the stable adduct BPDE-10-N2dG. When BP or BP-7,8-dihydrodiol was bound to RNA or denatured DNA in reactions catalyzed by rat liver microsomes, no depurination adducts were detected. The profiles of stable adducts were similar both qualitatively and quantitatively with native or denatured DNA. With activation of BP by horseradish peroxidase, the profiles of stable adducts differed with native and denatured DNA. The total amount of adducts with denatured DNA was only 25% of the amount detected with native DNA. No depurination adducts were detected with denatured DNA or RNA in the peroxidase system.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and quantitation of 7,12-dimethylbenz[a]anthracene-DNA adducts formed in mouse skin.

Identification and quantitation of the depurination and stable DNA adducts of 7,12-dimethylbenz[a]anthracene (DMBA) formed by cytochrome P450 in rat liver microsomes previously established one-electron oxidation as the predominant mechanism of activation of DMBA to bind to DNA. In this paper we report the identification and quantitation of the depurination and stable DMBA-DNA adducts formed in mouse skin. The depurination adducts, which constitute 99% of all the adducts detected, are DMBA bound at the 12-methyl group to the N-7 of adenine or guanine, namely, 7-methylbenz[a]anthracene (MBA)-12-CH2-N7Ade and 7-MBA-12-CH2-N7Gua. The depurination adducts were identified by HPLC and fluorescence line narrowing spectroscopy. The stable DNA adducts were analyzed by the 32P-postlabeling method. Almost 4 times as much of the depurination adduct 7-MBA-12-CH2-N7Ade (79%) was formed compared to 7-MBA-12-CH2-N7Gua (20%). The stable adducts accounted for only 1% of all the adducts detected and 80% of these were formed from DMBA diolepoxide. The binding of DMBA to DNA specifically at the 12-CH3 group is consistent with the results of carcinogenicity experiments in which this group plays a key role. When DMBA was bound to RNA or denatured DNA in reactions catalyzed by microsomes or by horseradish peroxidase (HRP), no depurination DNA adducts of DMBA were detected. The amount of stable DNA adducts observed with denatured DNA was 70% lower in the HRP system and 30% lower in the microsomal system compared to native DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of metabolic activation of the potent carcinogen 7,12-dimethylbenz[a]anthracene.

The DNA adducts of 7,12-dimethylbenz[a]anthracene (DMBA) previously identified in vitro and in vivo are stable adducts formed by reaction of the bay-region diol epoxides of DMBA with dG and dA. In this paper we report identification of several new DMBA-DNA adducts formed by one-electron oxidation, including two adducts lost from DNA by depurination, DMBA bound at the 12-methyl to the N-7 of adenine (Ade) or guanine (Gua) [7-methylbenz[a]anthracene (MBA-12-CH2-N7Ade or 7-MBA-12-CH2-N7Gua, respectively]. The in vitro systems used to study DNA adduct formation were DMBA activated by horseradish peroxidase or 3-methyl-cholanthrene-induced rat liver microsomes. The biologically-formed depurination adducts were identified by high-pressure liquid chromatography and by fluorescence line narrowing spectroscopy. Stable DMBA-DNA adducts were analyzed by the 32P-postlabeling method. Quantitation of DMBA-DNA adducts formed by microsomes showed about 99% as depurination adducts: 7-MBA-12-CH2-N7Ade (82%) and 7-MBA-12-CH2-N7Gua (17%). Stable adducts (1.4% of total) included one adduct spot that may contain adduct(s) formed from the diol epoxide (0.2%) and unidentified adducts (1.2%). Activation of DMBA by horseradish peroxidase afforded 56% of stable unidentified adducts and 44% of depurination adducts, with 36% of 7-MBA-12-CH2-N7Ade and 8% of 7-MBA-12-CH2-N7Gua. Adducts containing the bond to the DNA base at the 7-CH3 group of DMBA were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro.

The two DNA adducts of benzo[a]pyrene (BP) previously identified in vitro and in vivo are the stable adduct formed by reaction of the bay-region diol epoxide of BP (BPDE) at C-10 with the 2-amino group of dG (BPDE-10-N2dG) and the adduct formed by reaction of BP radical cation at C-6 with the N-7 of Gua (BP-6-N7Gua), which is lost from DNA by depurination. In this paper we report identification of several new BP-DNA adducts formed by one-electron oxidation and the diol epoxide pathway, namely, BP bound at C-6 to the C-8 of Gua (BP-6-C8Gua) and the N-7 of Ade (BP-6-N7Ade) and BPDE bound at C-10 to the N-7 of Ade (BPDE-10-N7Ade). The in vitro systems used to study DNA adduct formation were BP activated by horseradish peroxidase or 3-methylcholanthrene-induced rat liver microsomes, BP 7,8-dihydrodiol activated by microsomes, and BPDE reacted with DNA. Identification of the biologically-formed depurination adducts was achieved by comparison of their retention times on high-pressure liquid chromatography in two different solvent systems and by comparison of their fluorescence line narrowing spectra with those of authentic adducts. The quantitation of BP-DNA adducts formed by rat liver microsomes showed 81% as depurination adducts: BP-6-N7Ade (58%), BP-6-N7Gua (10%), BP-6-C8Gua (12%), and BPDE-10-N7Ade (0.5%). Stable adducts (19% of total) included BPDE-10-N2dG (15%) and unidentified adducts (4%). Microsomal activation of BP 7,8-dihydrodiol yielded 80% stable adducts, with 77% as BPDE-10-N2dG and 20% of the depurination adduct BPDE-10-N7Ade. The percentage of BPDE-10-N2dG (94%) was higher when BPDE was reacted with DNA, and only 1.8% of BPDE-10-N7Ade was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative dose-response tumorigenicity studies of dibenzo[alpha,l]pyrene versus 7,12-dimethylbenz[alpha]anthracene, benzo[alpha]pyrene and two dibenzo[alpha,l]pyrene dihydrodiols in mouse skin and rat mammary gland.

Comparative studies were conducted of the tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of dibenzo[a,l]pyrene (DB[a,l]P) versus 7,12-dimethyl-benz[a]anthracene (DMBA), the most potent recognized carcinogenic polycyclic aromatic hydrocarbon (PAH); benzo[a]pyrene (B[a]P), the most potent recognized carcinogenic environmental PAH; DB[a,l]P 8,9-dihydrodiol, the K-region dihydrodiol; and DB[a,l]P 11,12-dihydrodiol, precursor to the bay-region diolepoxide. The tumor-initiating activity of DB[a,l]P and B[a]P was compared in the skin of female SENCAR mice at doses of 300, 100 and 33.3 nmol. The mice were promoted with 12-O-tetradecanoylphorbol-13-acetate (TPA) twice-weekly for 13 weeks. DB[a,l]P at all doses induced significantly more tumors than B[a]P at the corresponding dose, with a significantly shorter latency. Subsequently, the tumor-initiating activity of DB[a,l]P was compared in the skin of female SENCAR mice to that of DMBA, B[a]P, DB[a,l]P 8,9-dihydrodiol and DB[a,l]P 11,12-dihydrodiol at doses of 100, 20 and 4 nmol. The mice were promoted with TPA twice-weekly for 24 weeks. In addition, groups of mice were initiated with 100 nmol of DB[a,l]P, DMBA, B[a]P, DB[a,l]P 8,9-dihydrodiol or DB[a,l]P 11,12-dihydrodiol and kept without promotion. This experiment showed that in the mouse skin, DB[a,l]P and DB[a,l]P 11,12-dihydrodiol displayed similar tumor-initiating activity with a response inversely proportional to the dose, presumably due to the toxicity of the compounds. At the high dose they elicited tumors earlier than DMBA, though DMBA produced a much higher tumor multiplicity. At the low dose, DMBA, DB[a,l]P and DB[a,l]P 11,12-dihydrodiol exhibited similar tumorigenicities. DB[a,l]P 8,9-dihydrodiol was a marginal tumor initiator. Once again, DB[a,l]P was by far a much stronger tumor initiator than B[a]P. Female Sprague-Dawley rats were treated with 1.0 or 0.25 mumol of DB[a,l]P, DMBA or B[a]P by intramammillary injection at eight teats. DB[a,l]P at both doses was a more potent carcinogen than DMBA at the corresponding dose in the rat mammary gland. B[a]P was a marginal mammary carcinogen, eliciting only a few fibrosarcomas. Thus, these data suggest that DB[a,l]P is the strongest PAH carcinogen ever tested.

A monoclonal antibody to rat liver cytochrome P450 IIC11 strongly and regiospecifically inhibits constitutive benzo[a]pyrene metabolism and DNA binding.

The monoclonal antibody MAb 1-68-11, prepared to constitutive cytochrome P450 IIC11 (2c/RLM5) from male Sprague-Dawley rat liver, was used to study the contribution of the class of cytochrome P450s epitopically related to P450 IIC11 to the regiospecific metabolism of benzo[a]pyrene (BP) and its binding to DNA. The effect of MAb 1-68-11 was determined on the conversion of BP to BP-9,10-dihydrodiol, BP-7,8-dihydrodiol, BP-4,5-dihydrodiol, BP phenols, and BP quinones, and on the P450-dependent DNA binding catalyzed by P450 in microsomes from uninduced male and female Wistar and Sprague-Dawley rat livers, as well as 3-methylcholanthrene- and phenobarbital (PB)-induced male Wistar rat livers. In liver microsomes from untreated male rats, MAb 1-68-11 inhibited BP-9,10-dihydrodiol formation by 80%; in liver microsomes from untreated female rats, the inhibition was 100%. BP-7,8-dihydrodiol formation was inhibited from 38 to 77% in microsomes from males and 50% in those from females. In microsomes from PB-induced rats, inhibition of the 9,10-dihydrodiol and the 7,8-dihydrodiol was 90% and 73%, respectively, whereas BP-4,5-dihydrodiol formation was enhanced 80%. In microsomes from 3-methylcholanthrene-treated rats, no inhibition of MAb 1-68-11 was observed on either the metabolism of BP or its binding to DNA. In contrast, the binding of BP to DNA was completely inhibited by MAb 1-68-11 in microsomes from uninduced male Wistar rats and 70% in PB-induced microsomes. 32P-postlabeling analysis showed that formation of the major stable adduct, BP diol epoxide bound at C-10 to the 2-amino of deoxyguanosine, was strongly inhibited in uninduced and PB-induced microsomes. Formation of the major labile BP-DNA adduct 7-(benzo[a]pyren-6-yl) guanine (BP-N7Gua) was inhibited about 60% in microsomes from untreated male Wistar rats. These results show that MAb 1-68-11 regiospecifically inhibits cytochrome P450 IIC11 and epitopically related P450s that metabolize BP at the 7,8 and 9,10 positions. MAb 1-68-11 also inhibits enzyme-catalyzed binding of BP to DNA in the specific formation of BP-N7Gua and adducts detected by the 32P-postlabeling technique.

Metabolism and mutagenicity of dibenzo[a,e]pyrene and the very potent environmental carcinogen dibenzo[a,l]pyrene.

Dibenzo[a,l]pyrene (DB[a,l]P) is one of the most potent carcinogens ever tested in mouse skin and rat mammary gland. DB[a,l]P is present in cigarette smoke and, presumably, in other environmental pollutants. Metabolism and mutagenicity studies of this compound compared to the weak carcinogen dibenzo[a,e]pyrene (DB[a,e]P) can provide preliminary evidence on its mechanism of carcinogenesis. The mutagenicity of DB[a,l]P, DB[a,e]P, and benzo[a]pyrene (BP) was compared in the Ames assay with Aroclor-induced rat liver S-9. BP was the strongest mutagen. In strain TA100, DB[a,l]P and DB[a,e]P were marginally mutagenic. In strain TA98 both compounds were mutagenic, and DB[a,l]P induced more than twice as many revertants as DB[a,e]P. The mutagenicity of DB[a,l]P does not correlate with its carcinogenicity, since DB[a,l]P is a much stronger carcinogen, but a much weaker mutagen, than BP. The NADPH-supported metabolism of DB[a,e]P and DB[a,l]P was conducted with uninduced and 3-methylcholanthrene-induced rat liver microsomes. Metabolites were analyzed by reverse-phase HPLC and identified by NMR, UV, and mass spectrometry. Uninduced microsomes produced only traces of metabolites with either compound. The major metabolites of DB[a,l]P with induced microsomes were DB[a,l]P 8,9-dihydrodiol, DB[a,l]P 11,12-dihydrodiol, 7-hydroxyDB[a,l]P, and a DB[a,l]P dione. The metabolites of DB[a,e]P with induced microsomes were DB[a,e]P 3,4-dihydrodiol, 3-hydroxyDB[a,e]P, 7-hydroxyDB[a,e]P, and 9-hydroxyDB[a,e]P. Some of these metabolites are very useful in assessing possible pathways of activation in the initiation of cancer.

Binding of benzo[a]pyrene to DNA by cytochrome P-450 catalyzed one-electron oxidation in rat liver microsomes and nuclei.

To investigate whether cytochrome P-450 catalyzes the covalent binding of substrates to DNA by one-electron oxidation, the ability of both uninduced and 3-methylcholanthrene (MC) induced rat liver microsomes and nuclei to catalyze covalent binding of benzo[a]pyrene (BP) to DNA and formation of the labile adduct 7-(benzo[a]pyren-6-yl)guanine (BP-N7Gua) was investigated. This adduct arises from the reaction of the BP radical cation at C-6 with the nucleophilic N-7 of the guanine moiety. In the various systems studied, 1-9 times more BP-N7Gua adduct was isolated than the total amount of stable BP adducts in the DNA. The specific cytochrome P-450 inhibitor 2-[(4,6-dichloro-o-biphenyl)oxy]ethylamine hydrobromide (DPEA) reduced or eliminated BP metabolism, binding of BP to DNA, and formation of BP-N7Gua by cytochrome P-450 in both microsomes and nuclei. The effects of the antioxidants cysteine, glutathione, and p-methoxythiophenol were also investigated. Although cysteine had no effect on the microsome-catalyzed processes, glutathione and p-methoxythiophenol inhibited BP metabolism, binding of BP to DNA, and formation of BP-N7Gua by cytochrome P-450 in both microsomes and nuclei. The decreased levels of binding of BP to DNA in the presence of glutathione or p-methoxythiophenol are matched by decreased amounts of BP-N7Gua adduct and of stable BP-DNA adducts detected by the 32P-postlabeling technique. This study represents the first demonstration of cytochrome P-450 mediating covalent binding of substrates to DNA via one-electron oxidation and suggests that this enzyme can catalyze peroxidase-type electron-transfer reactions.

32P-postlabeling analysis of benzo[a]pyrene-DNA adducts formed in vitro and in vivo.

Benzo[a]pyrene (BP) was bound to DNA by horseradish peroxidase, rat liver microsomes, and rat liver nuclei in vitro and in mouse skin in vivo. The BP-DNA adducts formed were analyzed by the 32P-postlabeling technique. Activation by microsomes and nuclei resulted in the detection of five adducts, including a major adduct (55%) which cochromatographed with the adduct (+/-)-10 beta-deoxyguanosin-N2-yl-7 beta, 8 alpha, 9 alpha-trihydroxy-7,8,9,10-tetrahydro-BP (BPDE-N2dG) formed by reaction of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydro-BP (BPDE) with DNA or by microsomal activation of BP 7,8-dihydrodiol. Activation by horseradish peroxidase, which catalyzes one-electron oxidation, produced seven adducts, including a major one (30%) that coeluted with an adduct observed with microsomal (2%) and nuclear (14%) activation. The pattern of adducts formed in mouse skin treated with BP in vivo for 4 or 24 h contained four of the same adducts observed with nuclei or microsomes in vitro, and the predominant adduct detected (86%) was BPDE-N2dG. The adduct common to horseradish peroxidase, microsomes, and nuclei was also detected in mouse skin DNA (2%). These results demonstrate that multiple BP-DNA adducts are formed in these in vitro and in vivo systems and suggest that at least one adduct is formed in common in all of the systems. Thus, it appears that stable BP adducts can be formed in mouse skin DNA by both monooxygenation and one-electron oxidation.

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.

Spin probes as mechanistic inhibitors and active site probes of thymidylate synthetase.

C-4- and C-5-substituted analogues of dUMP were examined as inhibitors of thymidylate synthetase and as topographical probes of its active site by electron spin resonance (ESR). The C-5-substituted spin-labeled analogues pDUAP (2) and a pDUTT (3) as well as the unlabeled AAdUMP (1) were competitive inhibitors with Ki's of 9.2, 89, and 7.9 microM, respectively. The C-4-spin-labeled pls4dU (4) displayed no inhibition activity. Scatchard plots as determined by ESR gave similar association constants for 2 (Kassoc = 1.9 X 10(5) M-1) and for 3 (Kassoc = 2.4 X 10(5) M-1). Both of these values are similar to the Kassoc of FdUMP indicating that the bulky substituent in position 5 does not interfere with the formation of the binary complex. The enzyme-C-5-spin-labeled nucleotide complexes indicate the presence of similarly immobilized spin labels by ESR, whereas no binding and immobilization were noticed with the C-4-spin-labeled nucleotide. A model for the active-site geometry of the enzyme was derived which suggests that the C-5 substituents point toward the opening of the binding cavity whose depth is at least 12 A. Also, the approximate 10-fold increased inhibitory activity of 2 as compared to that of 3 may be attributed to the significant electron withdrawing properties of the C-5 substituent in 2. Finally, the set of probes used for the binding and inhibition of thymidylate synthetase gives direct experimental evidence that an electron-withdrawing C-5 substituent primarily affects the formation of the ternary complex and will not substantially influence the stability of the binary complex.

Protein synthesis and competitive ESR binding studies with E. coli ribosomes and spin-labeled polynucleotides.

Enzymatically prepared spin labeled copolymers of (U)n were tested for their ability to direct polyphenylalanine synthesis in vitro using E. coli B enzymes and ribosomes. Spin labeling of the C5 position using (RUGT,U)n (1:100) or (RUTT,U)n (1:100) did not alter the amount of polyphenylalanine formed in comparison to (U)n. In contrast, the C4 spin labeled copolymer (ls4U,U)n (1:100) reduced phenylalanine incorporation by 70-75% of the (U)n control levels. ESR monitoring of competitive ribosome binding to equimolar mixtures of polynucleotides was demonstrated with the macromolecular probe (DUTT,dT)n (1:100), the DNA analogue of (RUTT,U)n. The ESR competition approach showed that the affinity of the ribosomes was essentially the same for (dT)n, (A,U,G)n, and (A,U,G)n + tRNArmet.