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.

Synthesis and characterization of estrogen 2,3- and 3,4-quinones. Comparison of DNA adducts formed by the quinones versus horseradish peroxidase-activated catechol estrogens.

Catechol estrogens (CE) are among the major metabolites of estrone (E1) and 17 beta-estradiol (E2). Oxidation of these metabolites to semiquinones and quinones could generate ultimate carcinogenic forms of E1 and E2. The 2,3- and 3,4-quinones of E1 and E2 were synthesized by MnO2 oxidation of the corresponding CE, following the method for synthesizing E1-3,4-quinone [Abul-Hajj (1984) J.

Identification of C8-methylguanine in the hydrolysates of DNA from rats administered 1,2-dimethylhydrazine. Evidence for in vivo DNA alkylation by methyl radicals.

C8-Methylguanine was identified in the neutral hydrolysates of DNA isolated from the liver or colon tissue of rats administered 1,2-dimethylhydrazine. In all the samples examined, the biologically isolated adducts were characterized by co-elution with synthetic C8-methylguanine under different high pressure liquid chromatography conditions. The sample isolated from liver DNA was also identified by UV spectroscopy at different pH values and by mass spectrometry.

Bacterial mutagenicity of extracts of the baked and raw Agaricus bisporus mushroom.

Aqueous extracts of baked and raw Agaricus bisporus (AB) mushroom were tested for mutagenic activity in Salmonella typhimurium strains TA1535 and TA1537. The extracts were studied with and without metabolic activation by Aroclor-induced rat liver S9 mix. The extracts of the baked and raw AB exhibited a dose related mutagenic activity with and without activation in strain TA1535.

Correlation studies of anodic peak potentials and ionization potentials for polycyclic aromatic hydrocarbons.

One-electron oxidation represents one of the major metabolic pathways of bioactivation of polycyclic aromatic hydrocarbons (PAH) to ultimate carcinogens capable of binding to cellular macromolecules, thereby initiating the cancer process. Since the ionization potential (IP) is related to the ease of removal of a pi electron from an aromatic molecule, a low IP is a necessary condition for the PAH to undergo one-electron oxidation. The principal aim of this study was to provide a general and simple technique suitable for obtaining IP of PAH with satisfactory accuracy.

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.

Model adducts of benzo[a]pyrene and nucleosides formed from its radical cation and diol epoxide.

Reference adducts formed by reaction of deoxyribonucleosides with the ultimate carcinogenic forms of benzo[a]pyrene (BP), BP radical cation and BP diol epoxide, are essential for identifying the structures of adducts formed in biological systems. Electrochemical oxidation of BP in the presence of dG or dA produces adducts from BP radical cation. When 8 equiv of charge are consumed, four adducts are formed with dG: 7-(BP-6-yl)Gua, 8-(BP-6-yl)Gua, N2-(BP-6-yl)dG and 3-(BP-6-yl)dG.

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 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.

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.

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%.

Formation of 8-methylguanine as a result of DNA alkylation by methyl radicals generated during horseradish peroxidase-catalyzed oxidation of methylhydrazine.

Methylhydrazine oxidation promoted by horseradish peroxidase-H2O2 or ferricyanide led to the generation of high yields of methyl radicals and to the formation of 7-methylguanine and 8-methylguanine upon interaction with calf thymus DNA. Methyl radicals were identified by spin-trapping experiments with alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone and tert-nitrosobutane. The methylated guanine products were identified in the neutral hydrolysates of treated DNA by high pressure liquid chromatography (HPLC) analysis and spiking with authentic samples.

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.

Identification and quantitation of 7-(benzo[a]pyren-6-yl)guanine in the urine and feces of rats treated with benzo[a]pyrene.

The major identified benzo[a]pyrene (BP)-DNA adduct formed by cytochrome P-450 contains BP bound at the C-6 position to the N-7 position of guanine (BP-N7Gua). This adduct is rapidly depurinated from DNA. When rats were treated with [14C]BP, about 0.

The impact of pestivirus on an artificial breeding program for cattle.

After the introduction of pestivirus into a herd undergoing an embryo transfer and artificial insemination program, substantial post-weaning calf losses occurred. The predominant clinical feature was severe respiratory disease, in contrast to the commonly recognised mucosal disease. Thirty-one of 76 calves were affected, with a case fatality rate of 58%.

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.

Radical cations in aromatic hydrocarbon carcinogenesis.

Most carcinogens, including polycyclic aromatic hydrocarbons (PAH), require metabolic activation to produce the ultimate electrophilic species that bind covalently with cellular macromolecules to trigger the cancer process. Metabolic activation of PAH can be understood in terms of two main pathways: one-electron oxidation to yield reactive intermediate radical cations and monooxygenation to produce bay-region diol epoxides. The reason we have postulated that one-electron oxidation plays an important role in the activation of PAH derives from certain common characteristics of the radical cation chemistry of the most potent carcinogenic PAH.

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.

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.

Fluorescence line narrowing spectrometric analysis of benzo[a]pyrene-DNA adducts formed by one-electron oxidation.

Fluorescence line narrowing (FLN) was demonstrated for five benzo[a]pyrene (BP)-nucleoside adducts synthesized by one-electron oxidation of BP in the presence of guanosine, deoxyguanosine, and deoxyadenosine. The standard FLN spectra were used to prove that a major depurination adduct from the binding of BP to DNA in rat liver nuclei is 7-(benzo[a]pyren-6-yl)guanine (N7Gua). The structural characterization was performed with only 20 pg of the adduct.

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.

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.

Carcinogenicity of aromatic hydrocarbons directly applied to rat mammary gland.

To obtain some initial evidence on the mechanism(s) of activation of PAH in rat mammary gland, we studied the carcinogenicity of a series of PAH directly applied to this tissue. A series of PAH which are or are not expected to be activated by one-electron oxidation because of their low or high ionization potential (IP), respectively, were tested. The compounds were dispersed as fine powders on an exposed mammary gland of female Sprague-Dawley rats.

Tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland of fluorinated derivatives of benzo[a]pyrene and 3-methylcholanthrene.

Comparative studies of tumor-initiating activity in mouse skin and carcinogenicity in rat mammary gland were conducted with benzo[a]pyrene (BP) and 3-methylcholanthrene (MC) derivatives. SENCAR mice were initiated with BP, 6-fluorobenzo[a]pyrene (6-FBP), 6-methylBP, 7-FBP, 8-FBP, 9-FBP, 10-FBP, or 10-azaBP and promoted with tetradecanoyl phorbol acetate. The same compounds plus BP 7,8-dihydrodiol were tested by intramammillary injection in female Sprague-Dawley rats.

Tumorigenicity of 6-halogenated derivatives of benzo[a]pyrene in mouse skin and rat mammary gland.

Studies of the tumorigenicity of 6-halogenated derivatives of benzo[a]pyrene (BP) can provide evidence about the role of the 6 position in the carcinogenic activation of BP. Female Swiss and A-strain mice were treated on the skin with BP, 6-fluorobenzo[a]pyrene (6-FBP), 6-chlorobenzo[a]pyrene (6-C1BP), 6-bromobenzo[a]pyrene (6-BrBP) and 6-iodobenzo[a]pyrene (6-IBP) by repeated application, and in some cases by initiation-promotion. While BP was more potent than 6-FBP, only these two compounds exhibits tumor-initiating and carcinogenic activity in mouse skin.