Formation of benzo(a)pyrene/DNA adducts and their relationship to tumor initiation in mouse epidermis

The tumorigenicity of benzo(a)pyrene [B(a)P] applied topically as a skin tumor initiator in Sencar mice and the formation of epidermal B(a)P/deoxyribonucleoside adducts were compared over a similar range of doses (50 to 1600 nmol). The tumor-initiating activity of B(a)P, its covalent binding to mouse epidermal DNA, and the formation of the major hydrocarbon/deoxyribonucleoside adduct showed approximately parallel dose-response curves. The major hydrocarbon/deoxyribonucleoside adduct formed cochromatographed with marker adducts of (N2-(10S-[7R,8S,9R-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]y) deoxyguanosine while other minor adducts also were observed. The disappearance of DNA-bound products in the epidermis was followed for 21 days after an initiating dose of B(a)P (100 nmol) was applied topically to the mice. The half-lives of the B(a)P/deoxyribonucleoside adducts and the total radioactivity bound to the DNA were 4.5 and 5.5 days, respectively. However, in spite of the loss of measurable DNA-bound material, the tumor yield was unchanged regardless of whether promotion was begun 7 or 21 days after initiation. The results suggest a possible causal relationship between B(a)P/deoxyribonucleoside adduct formation and papilloma formation in mouse skin.     

Morphological transformation and DNA adduct formation by benz[j]aceanthrylene and its metabolites in C3H10T1/2CL8 cells: evidence for both cyclopenta-ring and bay-region metabolic activation pathways

Benz[j]aceanthrylene (B[j]A), a cyclopenta-fused polycyclic aromatic hydrocarbon related to 3-methylcholanthrene, has been studied to identify the major routes of metabolic activation in transformable C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts in culture. Previous studies have reported that the major (55% of total) B[j]A metabolite formed by C3H10T1/2 cells was (+/-)-trans-9,10-dihydro-9,10-dihydroxy-B[j]A (B[j]A-9,10-diol), the dihydrodiol in the bay-region ring, with moderate amounts (14% of total) of (+/-)-trans-1,2-dihydro-1,2-dihydroxy-B[j]A (B[j]A-1,2-diol), the cyclopenta-ring dihydrodiol. The morphological transforming activities of three potential intermediates formed by metabolism of B[j]A by C3H10T1/2 cells, (+/-)-anti-trans-9,10-dihydro-9,10-dihydroxy-B[j]A-7,8-oxide (B[j]A-diol-epoxide), B[j]A-9,10-oxide, and B[j]A-1,2-oxide as well as the two B[j]A-dihydrodiols were examined. B[j]A, B[j]A-diol-epoxide, B[j]A-1,2-oxide, and B[j]A-9,10-diol were found to have moderate to strong activities with B[j]A-diol-epoxide the most active compared to B[j]A, while B[j]A-1,2-diol was inactive. B[j]A-9,10-oxide was found to be a weak transforming agent. At 0.5 microgram/ml, the following percentage of dishes with type II or III foci were observed: B[j]A, 59%; B[j]A-diol-epoxide, 75%; B[j]A-1,2-oxide, 25%; and B[j]A-9,10-diol, 17%. DNA adducts of B[j]A, B[j]A-9,10-diol, B[j]A-diol-epoxide, B[j]A-9,10-oxide, and B[j]A-1,2-oxide in C3H10T1/2 cells were isolated, separated, identified, and quantitated using the 32P-postlabeling method. B[j]A forms two major groups of adducts: one group of adducts is the result of the interaction of B[j]A-1,2-oxide with 2'-deoxyguanosine and 2'-deoxyadenosine; the second group of adducts is a result of the interaction of B[j]A-diol-epoxide with 2'-deoxyguanosine and 2'-deoxyadenosine. Qualitative and quantitative analysis of the postlabeling data suggests that B[j]A is metabolically activated by two distinct routes, the bay-region diol-epoxide route and the cyclopenta-ring oxide route, the former being the most significant.     

Reanalysis and clarification of the structures of alpha-naphthoflavone dihydrodiols formed by uninduced and induced rat liver microsomes from Charles River CD and Sprague-Dawley rats

The structures of -naphthoflavone (ANF) dihydrodiols formedby uninduced and induced rat liver microsomes are identifiedby conversion of the metabolically formed ANF-dihydrodiols tothe corresponding phenols. Comparison of these phenols withsynthetic standards provides an unambiguous method for structuralidentification. The results of these studies are that hepaticmicrosomes from uninduced or phenobarbital, Aroclor-1254, 3-methylcholanthrene,or 5,6-benzoflavone induced Sprague-Dawley or Charles RiverCD rats each produce a major and a minor ANF-dihydrodiol identifiedas ANF-7,8-dihydrodiol and ANF-5,6-dihydrodiol, respectively.     

Lung and liver cell-mediated mutagenesis systems: specificities in the activation of chemical carcinogens

A liver and lung cell-mediated-V79 cell mutagenesis system usingintact cells as metabolic activation systems was employed tostudy the relative ability of cells from these organs to activatechemical carcinogens. Primary cultures of liver and lung cellsfrom male Sprague Dawley rats were used to metabolically activatethe chemicals and the mutation of Chinese hamster V79 cellsto ouabain resistance used to detect mutagenic intermediates.7,12-Dimethylbenz[a]anthracene and 3-methylcholanthrene, weremore active in the lung system than in the liver cell system.Benzo[a]pyrene (B[a]P) was inactive in the liver cell-mediatedsystem but mutagenic to V79 cells in the lung cell-mediatedsystem. Dimethylnitrosamine (DMN) was inactive in the presenceof lung cells but was mutagenic to V79 cells in the presenceof liver cells. Aflatoxin B₁ was mutagenic in the liver cell-mediatedsystem, but only weakly mutagenic in the lung cell-mediatedsystem. Because the mutagenicities of DMN and B[a]P were organ-specific,the metabolism of these carcinogens in the two primary cellsystems was investigated. DMN was metabolized by liver but notby lung cells, possibly accounting for its lack of mutagenicityin the lung cell system. B[a]P was extensively metabolized byboth cell types, but quantitative differences were observedwhen the metabolic products were analyzed by high pressure liquidchromatography. Comparing total organic and water soluble metabolites,lung cells produced similar amounts of 7,8- and 9,10-diols butlittle 4,5-diol, while liver cells produced equivalent totalamounts of the three diols. Lung cells produced twice the amountof B[a]P glucuronide conjugates as liver cells, while livercells produced twice the amount of B[a]P sulfate conjugatesas lung. The data suggest that intact cells from various organscan be used as metabolic activating systems in in vitro assaysand that studies into organ specificity can be investigatedby this approach.     

Three conazoles increase hepatic microsomal retinoic acid metabolism and decrease mouse hepatic retinoic acid levels in vivo

Conazoles are fungicides used in agriculture and as pharmaceuticals. In a previous toxicogenomic study of triazole-containing conazoles we found gene expression changes consistent with the alteration of the metabolism of all trans-retinoic acid (atRA), a vitamin A metabolite with cancer-preventative properties (Ward et al., Toxicol. Pathol. 2006; 34:863-78). The goals of this study were to examine effects of propiconazole, triadimefon, and myclobutanil, three triazole-containing conazoles, on the microsomal metabolism of atRA, the associated hepatic cytochrome P450 (P450) enzyme(s) involved in atRA metabolism, and their effects on hepatic atRA levels in vivo. The in vitro metabolism of atRA was quantitatively measured in liver microsomes from male CD-1 mice following four daily intraperitoneal injections of propiconazole (210 mg/kg/d), triadimefon (257 mg/kg/d) or myclobutanil (270 mg/kg/d). The formation of both 4-hydroxy-atRA and 4-oxo-atRA were significantly increased by all three conazoles. Propiconazole-induced microsomes possessed slightly greater metabolizing activities compared to myclobutanil-induced microsomes. Both propiconazole and triadimefon treatment induced greater formation of 4-hydroxy-atRA compared to myclobutanil treatment. Chemical and immuno-inhibition metabolism studies suggested that Cyp26a1, Cyp2b, and Cyp3a, but not Cyp1a1 proteins were involved in atRA metabolism. Cyp2b10/20 and Cyp3a11 genes were significantly over-expressed in the livers of both triadimefon- and propiconazole-treated mice while Cyp26a1, Cyp2c65 and Cyp1a2 genes were over-expressed in the livers of either triadimefon- or propiconazole-treated mice, and Cyp2b10/20 and Cyp3a13 genes were over-expressed in the livers of myclobutanil-treated mice. Western blot analyses indicated conazole induced-increases in Cyp2b and Cyp3a proteins. All three conazoles decreased hepatic atRA tissue levels ranging from 45-67%. The possible implications of these changes in hepatic atRA levels on cell proliferation in the mouse tumorigenesis process are discussed.     

The lack of initiating and/or promoting activity of sodium malondialdehyde on SENCAR mouse skin

Malondialdehyde (MDA), a lipid peroxidation product, has been implicated in carcinogenesis, in part for its reported mutagenic activity. It was of interest therefore, to determine its activity as either a complete carcinogen, a tumor initiator, or a tumor promoter. Using the SENCAR mouse skin model, pure sodium MDA (NaMDA) was found to lack activity in any of these categories. Furthermore, NaMDA was determined to be negative in the Chinese hamster V-79 metabolic cooperation assay for promoters.     

Inhibition of benzo(a)pyrene-induced transformation of C3H/10T1/2 cells by allylisopropylacetamide and isopropylvaleramide

Allylisopropylacetamide (AIA) and isopropylvaleramide (IVA) have been demonstrated previously to protect in vivo against the acute toxicity and adrenal necrotic effect of 7,12-dimethylbenz(a)anthracene. In the present study, the influence of these two amides on the in vitro transforming ability of two potent carcinogens, benzo(a)pyrene [B(a)P] and 7,12-dimethylbenz(a)anthracene, on C3H10T1/2 cells was investigated. Both AIA and IVA showed a dose-dependent inhibition of B(a)P-induced transformation of C3H10T1/2 cells when added simultaneously for 24 hr with the carcinogen. While pretreatment, simultaneous treatment, and posttreatment of the cells with AIA or IVA inhibited transformation, the 24-hr posttreatment was somewhat more effective. The protective effect did not appear to results from alterations in B(a)P metabolism inasmuch as aryl hydrocarbon hydroxylase activity and the metabolic products of B(a)P detected by high-pressure liquid chromatography were not changed by AIA or IVA treatment. Furthermore, AIA and IVA did not selectively kill chemically transformed C3H10T1/2 cells, as indicated by the absence of their effect on an established, chemically transformed cell line. AIA and IVA also inhibited 7,12-dimethylbenz(a)anthracene-induced transformation of C3H10T1/2 cells. These data suggest that AIA and IVA may be useful protective agents and that they presumably exert their protective effect at some stage between the activation of the carcinogen and the expression of the transformed phenotype.