Immunosuppression following exposure to 7,12-dimethylbenz[a]anthracene (DMBA) in Ah-responsive and Ah-nonresponsive mice

Recent reports suggest that the immunotoxicity of certain polycyclic aromatic hydrocarbons is associated with the Ah locus in mice. To test whether immunosuppression mediated by 7,12-dimethylbenz[a]anthracene (DMBA) is regulated by the Ah locus, several endpoints of immune function were measured in Ah-responsive B6C3F1 and Ah-nonresponsive DBA/2N and in Ah-congenic C57BL/6J (responsive B6-AhbAhd and nonresponsive B6-AhdAhd) mice dosed sc with up to 100 micrograms/g DMBA in corn oil. Some groups of B6C3F1 and DBA/2N mice were exposed to 100 micrograms/g benzo[a]pyrene (B(a)P) or 1 nmol 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for determination of hepatic microsomal monooxygenase activity. The body weights of all mice were unaffected by DMBA exposure, but thymus weights and spleen cellularity were decreased. Antibody plaque-forming cells (PFC) measured 4 days after iv sheep erythrocyte (SRBC) immunization were suppressed 99% in B6C3F1 and 96% in DBA/2 mice. Antibody PFC after in vitro immunization to SRBC were similarly suppressed 98% in both B6-AhbAhd and B6-AhdAhd Ah-congenic mice exposed to 100 micrograms/g DMBA. Responses to the T-cell mitogens concanavalin A and phytohemagglutinin were significantly suppressed in both B6C3F1 and DBA/2N strains, as was mitogenesis to bacterial lipopolysaccharide. The unidirectional mixed lymphocyte responses of the congenic strains were suppressed 76% in B6-AhbAhd and 85% in B6-AhdAhd, cytotoxic lymphocyte generation was suppressed 68% in B6-AhbAhd and 78% in B6-AhdAhd. The overall differences between immunosuppressive responses in splenocytes from B6-AhbAhd and B6-AhdAhd congenics were not significant. Induction of cytochrome P1-450, a marker of Ah responsiveness, was determined by 7-ethoxycoumarin O-deethylase monooxygenase activity in hepatic microsomes or splenocytes. This monooxygenase activity was not significantly increased in either B6C3F1 or DBA/2 mice exposed to DMBA, whereas B(a)P and TCDD exposure significantly induced enzyme activity in B6C3F1 hepatocytes. These data suggest that DMBA has an immunosuppressive action on murine splenocytes which is independent of the Ah locus and associated induction of cytochrome P1-450 xenobiotic-metabolizing enzymes.     

Dietary modulation of 7,12-dimethylbenz[a]anthracene (DMBA)-induced adrenal toxicity in female Sprague-Dawley rats.

In this study, dietary modulation of 7,12-dimethylbenz[a]anthracene (DMBA)-induced adrenal toxicity in rats was investigated. Beginning at postnatal day (PND) 21, female Sprague-Dawley rats were fed either soy-containing NIH-31 diet or soy- and alfalfa-free 5K96 diet. On the first day of diestrus when the animals were PND 50 +/- 5, rats received either an oral dose of 80 mg/kg DMBA or sesame oil, the vehicle, and were sacrificed at 24, 36, or 48 h after treatment. Apoptosis was manifested at 24 and 36 h after DMBA treatment in the zona reticularis (ZR) and the zona fasciculata (ZF) of the adrenal cortex; this was followed by severe hemorrhagic necrosis at 48 h. DMBA-induced apoptosis, evaluated by the TUNEL assay, immunohistochemical analysis of activated caspase 3, and the ratio of expression of pro-apoptotic Bax to anti-apoptotic Bcl2, was greater in rats fed NIH-31 diet relative to rats fed 5K96 diet at 24 h after treatment. Four of six DMBA-treated rats fed 5K96 diet had severe adrenal necrosis by 48 h, whereas this lesion was present in only two of six DMBA-treated rats fed NIH-31 diet. DMBA also caused a significant decrease of serum corticosterone relative to controls at 48 h in rats fed 5K96 diet. The present study indicated that diet modulates DMBA-induced adrenal toxicity in female rats, with increased apoptosis early and reduced necrosis later in rats fed a soy-containing diet.     

Microsomal expoxide hydrolase is required for 7,12-dimethylbenz[a]anthracene (DMBA)-induced immunotoxicity in mice.

Microsomal epoxide hydrolase (mEH, EPHX1) is involved in the metabolism of chemicals to generate dihydrodiol intermediates in the presence of the cytochrome P450. We have previously shown that 7,12-dimethylbenz[a]anthracene (DMBA) can suppress both cell-mediated and humoral immune responses in wild-type (WT) C57BL/6N mice but not in CYP1B1 null mice. In the present studies, we hypothesized the critical metabolite responsible for DMBA-induced immunotoxicity is likely to be the 3,4-dihydrodiol-1,2-epoxide metabolite of DMBA, which requires mEH for formation. Mice were gavaged orally with DMBA (0, 17, 50, and 150 mg/kg) once a day for 5 days. Immune function and other assays were performed on day 7. Our data showed that unlike WT mice, DMBA treatment of mEH null mice produced no alterations in the body weight, spleen weight, or spleen cellularity. Similarly, DMBA treatments did not affect the PFC response in mEH null mice. Natural killer activity was not altered by DMBA treatment in mEH null mice. T-cell mitogenesis was partially suppressed by 50 and 150 mg/kg DMBA treatments of mEH null mice, but B-cell mitogenesis was not affected. Finally, we assessed the biodistribution of DMBA in both C57BL/6N WT and mEH null mice in spleen, thymus, and liver after 24 h and 7 days oral gavage. The concentrations of DMBA in each organ were not significantly different in WT and in mEH null mice. Collectively, these results demonstrate that mEH (EPHX1 gene) is a crucial enzyme for metabolic activation of DMBA in vivo leading to immunosuppression of spleen cells.     

Protective effect of Shemamruthaa on lipids anomalies in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinoma-bearing rats

The objective of this work was to evaluate the beneficial effect Shemamruthaa (a siddha formulation which constitutes Hibiscus rosa sinensis, Emblica officinalis, and Honey in define ratio) on lipid metabolism in 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinoma-bearing rats. Lipid profile and lipid-metabolizing enzyme were investigated in serum, liver, and kidney of DMBA-induced mammary carcinoma-bearing SD rats. The levels of total cholesterol, free cholesterol, phospholipids, triglycerides, and free fatty acids were significantly increased, whereas, the levels of ester cholesterol were significantly decreased in plasma, liver and kidney of cancer-bearing animals, and those levels were restored to near normal levels upon treatment with SM. Moreover, the activities of total lipase, cholesterol ester synthase, and cholesterol ester hydrolase were found to be increased, and lipoprotein lipase and lecithin cholesterol acyl transferase were decreased in cancer-bearing animals. In addition, the levels of very low-density lipoprotein and low-density lipoprotein were increased, and the level of high-density lipoprotein was decreased. These alterations were recouped back upon treatment with SM when compared to cancer animals. The result indicated that Shemamruthaa administration plays a beneficial role in regulating lipid profile and lipid-metabolizing enzymes in cancer-suffering animals.     

Dermatological effects of chronic exposure to 7,12-dimethylbenz[A]anthracene (DMBA) or N-methyl-N-nitrosoguanidine (MNNG) in swine

Purpose: To determine whether chronic exposure to DMBA or MNNG in combination with or without UVB exposure would induce skin carcinomas in swine. Methods: Eight gilts were exposed to 100 mJ of UVB in their left side, allowed to recuperate, and divided into two groups. Each gilt received identical high doses (DMBA 50 microM; MNNG 250 mM), low doses (DMBA 500 nM; MNNG 2.5 mM), carrier (DMSO), or nothing added treatments in the UVB and non-UVB sides. Animals were exposed weekly for 30 weeks and skin samples collected at 10, 20, and 30 weeks from initiation of exposure. An additional sample was collected 16 weeks following cessation of exposure. All samples were scored for dermal morphology, including intracellular epidermal edema, intercellular epidermal edema, papillary dermal edema, perivascular infiltrates, pyknotic stratum basale cells, collagen necrosis, and epidermal-dermal separation, and the data were analyzed by ANOVA. MNNG and UVB light had a significant effect on epidermal thickness and the number of cell layers. The greatest increase in epidermal thickness occurred from 20 weeks to 30 weeks in the UVB plus MNNG treatment. Treatment with MNNG resulted in intracellular and intercellular epidermal edema, dermal edema, and dermal inflammation at both the low and high doses of MNNG. In contrast, all the morphological evaluations of the DMBA treatments were less severe than the MNNG. Conclusion: Our findings show that although chronic exposure to MNNG and DMBA, with or without UVB exposure, caused severe to mild dermatopathological changes, neither resulted in the development of skin carcinomas. These results indicate that at least with respect to responses to DMBA and MNNG, the swine model mimics more closely the responses seen in human skin.     

Effects of morin on the pharmacokinetics of docetaxel in rats with 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors

Docetaxel is a P-glycoprotein (P-gp) substrate and metabolized via cytochrome P450 (CYP) 3A subfamily in rats. Morin is an inhibitor of both CYPs and P-gp. Hence, the effects of morin on the intravenous and oral pharmacokinetics of docetaxel were investigated using 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor rats (DMBA rats) as an animal model of human breast cancer. Docetaxel was administered intravenously (4 mg/kg) and orally (20 mg/kg) without and with morin (15 mg/kg) in DMBA rats. After the intravenous administration of docetaxel in control and DMBA rats with and without morin, the values of non-renal clearance and area under the plasma concentration-time (AUC) for docetaxel were comparable. Morin did not increase AUC or the absolute oral bioavailability (F) for docetaxel after the oral administration of docetaxel in control and DMBA rats with and without morin. The inhibition of hepatic and intestinal metabolism of docetaxel by morin and/or DMBA and the effect of intestinal P-gp inhibition by morin on the pharmacokinetics of docetaxel did not seem to be considerable in DMBA-induced mammary tumor rats.     

Anti-clastogenic potential of carnosic acid against 7,12-dimethylbenz(a)anthracene (DMBA)-induced clastogenesis

Carnosic acid, a primary phenolic compound found in the leaves of rosemary (Rosmarinus officinalis), has diverse pharmacological and biological activities. The aim of the present study was to investigate the anti-clastogenic effect of carnosic acid in DMBA-induced clastogenesis. The frequency of bone marrow micronucleated polychromatic erythrocytes (MnPCEs), chromosomal aberrations (cytogenetic end points), the status of Phase I and II detoxification enzymes, lipid peroxidation by-products and antioxidants (biochemical endpoints) were analyzed to assess the anti-clastogenic effect of carnosic acid in DMBA-induced clastogenesis. Oral pretreatment of carnosic acid for five days to DMBA-treated hamsters significantly protected DMBA-induced clastogenesis as well as biochemical abnormalities. Although the exact mechanism of anti-clastogenic effects of carnosic acid is unclear, the antioxidant potential and effect on modulation of Phase I and II detoxification enzymes could play a possible role.     

Target cells for cytochrome p450-catalysed irreversible binding of 7,12-dimethylbenz[a]anthracene (DMBA) in rodent adrenal glands

7,12-Dimethylbenz[a]anthracene (DMBA) is an adrenocorticolytic agent that causes apoplexy (haemorrhage) and massive necrosis in the adrenal cortex in rat. Several explanations regarding the origin of toxicity have been proposed. Huggins and Morii (J Exp Med 114:741-60, 1961) suggested that the cells of the inner adrenal cortex are the primary target, whereas Horváth and Kovács (Pathol Eur 8:43-59, 1973) suggested the vascular endothelium as being the origin of toxicity. In the present study, cultured precision-cut tissue slices were used to localize target cells for irreversible [(3)H]DMBA binding in rat and mouse adrenal cortex. The sites of binding were confirmed by autoradiography in vivo. Irreversible [(3)H]DMBA binding was confined to zona fasciculata/reticularis cells in rat (but not in mouse) adrenal cortex. Pronounced binding was observed in clusters of cells (focal binding), localized predominantly in zona reticularis of rat. [(3)H]DMBA binding in zona fasciculata/reticularis cells was inhibited by the cytochrome p450 1A/B (CYP1A/B) inhibitors ellipticine, alpha-naphthoflavone, and 1-ethynylpyrene. The CYP11B1-inhibitor metyrapone did not reduce [(3)H]DMBA binding. In CYP1-induced (PCB 126-treated) rats and mice, intense irreversible [(3)H]DMBA binding was found also in endothelial cells of the adrenal cortex. The endothelial binding was abolished by the CYP1 inhibitors but remained unaffected by metyrapone. We conclude that the metabolic activation in adrenal parenchymal cells is presumably catalysed by CYP1B1, whereas CYP1A1 presumably catalyses the activation in endothelial cells. We suggest that the adrenocorticolytic effect of DMBA is the result of a dual mode of action, targeting both endothelial and parenchymal cells in the rat adrenal cortex.     

Inhibition of lymphocyte activation in splenic and gut-associated lymphoid tissues following oral exposure of mice to 7,12-dimethylbenz[a]anthracene

The hypothesis that 7,12-dimethyl-benz[a]anthracene (DMBA) suppresses immune function in mice via an inhibition of lymphocyte activation was examined in these studies. Daily exposure of B6C3F1 mice to DMBA (cumulative doses of 1.4 to 140 mg/kg) via the oral route for 14 days was found to inhibit phytohemagglutinin (PHA) and lipopolysaccharide mitogen responses in lymphoid cells obtained from the spleen. Peyer's Patches, and mesenteric lymph nodes. The 14 mg/kg cumulative dose of DMBA produced no significant decrease in the number of recovered viable cells, yet mitogen responses were suppressed by approximately 50% in the spleen and mesenteric lymph nodes, and by greater than 70% in the Peyer's Patches. DMBA inhibited PHA-induced Ca+2 mobilization measured by flow cytometry in each of these three lymphoid tissues. There was no change in the percentage of T cells recovered from the spleen, mesenteric lymph nodes, or Peyer's Patches. Peyer's Patch lymphocytes obtained from the GI tract appeared to be slightly more sensitive to inhibition of mitogen responsiveness and perhaps Ca+2 mobilization, potentially due to the oral route of exposure to DMBA. These studies provide evidence that DMBA inhibits early events associated with lymphocyte activation in mice.     

Temperature-modulated carcinogenicity of 7,12-dimethylbenz[a]anthracene in rainbow trout

Temperature-modulated hepatic disposition, covalent binding of radiolabeled genotoxin to hepatic DNA, and cancer incidence in rainbow trout (Oncorhyncus mykiss) were assessed after a single exposure to 7,12-dimethylbenz[a]anthracene (DMBA). Fish (2 g) were acclimated at 10, 14, or 18 degrees C for 1 mo and then exposed to 1 ppm DMBA in their water for 20 h. Exposures were at respective acclimation temperatures, or 10 and 18 degrees C acclimated fish were shifted to 14 degrees C for DMBA exposures. After 4 but not 20 h of exposure, hepatic [(3)H]DMBA equivalents increased with temperature for fish exposed at their respective acclimation temperatures (10 or 18 degrees C). Covalent binding of [(3)H]DMBA to hepatic DNA was similar after 3 d in fish exposed at their respective acclimation temperatures. However, in fish exposed at 14 degrees C, after 3 d the concentration of [(3)H]DMBA covalently bound to hepatic DNA was higher in 10 degrees C than 18 degrees C acclimated fish. After 21 d, covalent binding of [(3)H]DMBA to hepatic DNA was less persistent in 18 degrees C than 10 degrees C acclimated, exposed, and reared fish. There were no differences between temperature-shifted groups at that time. Temperature effects on tumor incidence were assessed 9 mo after DMBA waterborne exposures in fish that were reared at (1) their respective acclimation and exposure temperatures, (2) 14 degrees C after exposure at their respective acclimation temperature, and (3) 14 degrees C after 14 degrees C exposures. Incidence of stomach, liver, and swimbladder cancer increased dramatically with rearing temperature. Differences in tumor incidence were less marked in fish reared at the same temperature (14 degrees C). A strong negative correlation between liver tumor incidence and persistence of [(3)H]DMBA equivalents covalently bound to hepatic DNA suggested increased error-prone DNA repair at warmer temperature played an important role in increased tumor incidence.     

Biooxidation of 7,12-dimethylbenz[a]anthracene in rat subcutaneous tissue

The present study demonstrates the biooxidation of 7,12-dimethylbenz[a]anthracene to the corresponding hydroxyalkyl metabolites, 7-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, and 7,12-dihydroxymethylbenz[a]anthracene in the dorsal subcutaneous tissue of the rat, in vivo, a tissue highly susceptible to the carcinogenic action of 7,12-dimethylbenz[a]anthracene.     

Suppression of the in vitro humoral immune response of mouse splenocytes by 7,12-dimethylbenz[a]anthracene metabolites and inhibition of immunosuppression by alpha-naphthoflavone

Exposure to 7,12-dimethylbenz[a]anthracene (DMBA) has been demonstrated by numerous investigators to result in suppression of both humoral and cell-mediated immune responses of mice and cultured splenocytes. The mechanism(s) of this DMBA-induced immunosuppression, however, is not well characterized. PAHs must be converted to reactive metabolites via cytochrome P450-dependent monooxygenase systems to exert their carcinogenic and mutagenic effects. Thus, we have hypothesized that immunosuppression seen upon exposure to DMBA may also be mediated by its reactive metabolites. The objective of this study was to determine if DMBA metabolites can suppress the in vitro, T-dependent humoral immune response to sheep red blood cells. Compounds were evaluated in the in vitro plaque-forming cell (PFC) response at concentrations of 10(-9) to 10(-5) M. DMBA and benzo[a]pyrene (B[a]P) were also evaluated for their ability to suppress the in vitro PFC response. Addition of either of these PAHs to splenocyte cultures produced a concentration-dependent suppression of the PFC response, in which B[a]P was found to be 17.5-fold more potent than DMBA. These results are in contrast to those found in vivo, where DMBA has been shown to be more potent than B[a]P at suppressing humoral immunity. The 3,4-diol metabolite of DMBA produced a concentration-dependent suppression (10(-8) to 10(-5) M) of the in vitro PFC response and was found to be 65-fold more potent than the parent compound DMBA. In contrast, the 5,6-diol metabolite of DMBA had no effect on the PFC response or cell viability. Both the 3-OH-DMBA and 7-hydroxymethyl-12-methyl-benz[a]anthracene (7-OHMe-12-Me-BA) metabolites were found to be immunosuppressive at concentrations of 10(-6)M. Furthermore, suppression by 7-OHMe-12-Me-BA was observed at concentrations as low as 10(-8) M. Immunosuppression by the 7-Me-12-OHMe-BA and the di-OHMe-BA metabolites was only observed at high (10(-5) M) concentrations. The cytochrome P450 inhibitor, alpha-naphthoflavone (ANF), was utilized to determine if cytochrome P450-mediated metabolism is involved in DMBA-induced suppression of the in vitro PFC response. ANF (10(-5) M) reversed the DMBA-induced immunosuppression seen at 10(-5) M and attenuated the immunosuppression at 3 x 10(-5) and 10(-4) M. The results of these studies demonstrate that several metabolites of DMBA which can be generated by the cytochrome P450-dependent monooxygenase systems are immunosuppressive in the in vitro PFC response assay. Furthermore, the cytochrome P450 inhibitor, ANF, was able to reverse DMBA-induced immunosuppression.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ellagic acid effects on the carcinogenicity, DNA-binding and metabolism of 7,12-dimethylbenz(a)anthracene (DMBA)

Naturally-occurring components of the human food supply have recently received attention as possible agents for cancer chemoprevention. The plant phenol ellagic acid has been reported to be an effective inhibitor of carcinogen metabolism and certain chemically-induced tumors. Therefore, we evaluated the efficacy of ellagic acid in inhibiting DMBA metabolism, DNA-binding and the initiation of DMBA-induced carcinogenesis in rat mammary tissue. Mammary epithelial cell aggregates were isolated from rats fed control and ellagic acid (0.4 and 0.8%) containing diets. When incubated with DMBA, aggregates from ellagic acid-fed rats exhibited a significant but modest inhibition of DMBA metabolism and DNA-binding. An inhibition of DMBA-DNA binding and DMBA metabolism in secondary cultures of mammary epithelial cells also was detected only when ellagic acid was added at 150 molar excess compared to DMBA. The feeding of ellagic acid (0.8%) to rats for 28 days prior to the administration of DMBA resulted in a 21% reduction in mammary tumor incidence at 21 weeks which was, however, not statistically significant. Together, these results indicate that, in contrast to its effects with other carcinogens in other tissues, ellagic acid is not a potent inhibitor of DMBA metabolism, DNA-binding and carcinogenicity with rat mammary tissue.     

Cytochrome P450-dependent binding of 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) in murine heart, lung, and liver endothelial cells

Autoradiography was used to investigate the cellular sites of irreversible binding of 3H-labelled 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P) in mice. Autoradiograms obtained from solvent-extracted tape-sections revealed an even distribution of DMBA- and B[a]P-derived radioactivity in control mice lacking sites of selective binding in the tissues. In mice pretreated with a cytochrome P4501A (CYP1A) inducer, beta-naphthoflavone (BNF) or 3,3',4,4', 5-pentachlorobiphenyl (PCB 126), a noticeable accumulation of bound radioactivity was observed in the pulmonary alveolar region. Increased labelling was also observed in heart tissue of induced mice. As demonstrated by microautoradiography of tissues from CYP1A-induced mice treated with 3H-DMBA or 3H-B[a]P in vivo, irreversible binding in lung tissue was present in endothelial cells of arteries and veins, in the alveolar septal walls, and in type 2 pneumocytes. In heart tissue, binding was confined to endothelial cells of arteries, capillaries and veins. In liver, binding was found in the hepatocytes as well as in endothelial cells of the portal veins, whereas no binding was seen in endothelial cells of the sinusoids, central veins, or arteries. These findings were confirmed in vitro using 3H-DMBA-exposed precision-cut slices, indicating that reactive intermediates of DMBA and B(a)P were formed in situ. The addition of the CYP1A inhibitor ellipticine abolished binding in the target endothelial cells. Increased endothelial binding in the lungs and liver of CYP1A-induced mice was concomitant with increased 7-ethoxyresorufin O-deethylase (EROD) and DMBA hydroxylase activity. In heart, endothelial binding was positively correlated with EROD, but not with DMBA hydroxylase. The results suggest that endothelial cells may be targets for CYP-dependent activation of such toxicants as polycyclic aromatic hydrocarbons. Consequently, the possibility that chemically induced endothelial dysfunction is a risk factor in the aetiology of cardiovascular disease demands consideration.     

Estrogenic status modulates the effect of soy on hepatic responses to 7,12-dimethylbenz(a)anthracene (DMBA)

We examined the influence of estradiol (E2) status and soy protein isolate (SPI) intake on the hepatic responses altered by 7,12-dimethylbenz(a)anthracene (DMBA, a polycyclic aromatic hydrocarbon [PAH]). Sprague-Dawley rats were ovariectomized (OVX) at PND50 and infused with E2 or vehicle for 14 d and gavaged with 50 mg/kg DMBA or vehicle 24 h before sacrifice at PND64. Rats were fed an AIN-93G diet made with SPI or casein as sole protein source throughout the study. Basal AhR protein levels were reduced (P < 0.05) by SPI feeding irrespective of the E2 status. However, DMBA increased (P < 0.05) AhR-induced CYP1A1 gene expression in OVX, SPI-fed rats, but reduced (P < 0.05) CYP1A1 in OVX + E2, SPI-fed rats. Chromatin-immunoprecipitation demonstrated lower (P < 0.05) DMBA-mediated recruitment of estrogen receptor alpha to the CYP1A1 promoter by SPI feeding in the presence of E2, suggesting an estrogen-like action of SPI on DMBA-mediated signaling in the absence of E2. Further, microarray analysis (Rat 230-2.0 Affymetrix-GeneChip™) revealed 231 genes common to SPI + DMBA and SPI + E2 + DMBA (normalized to E2) treatments. AhR-activated genes (CYP1A1, CYP1A2, and NQO1) were down-regulated by SPI + E2 + DMBA compared to SPI + DMBA. Unique interactions among SPI, DMBA and E2 altered the expression profile of 316 genes, not observed by either treatment alone. Our data suggest that although E2 status does not effect soy-mediated AhR degradation, it modulates the effects of soy on many genes, including CYP1A1.     

Formation of benzylic-DNA adducts resulting from 7,12-dimethylbenz[a]anthracene in vivo

Studies were undertaken to determine the formation of benzylic-DNA adducts in rats administered 7,12-dimethylbenz[a]anthracene (DMBA) and its meso-region metabolites by subcutaneous injection. Here, we show that 7-hydroxymethyl-12-methylbenz[a]anthracene (7-HMBA) and 7-sulfoxymethyl-12-methylbenz[a]anthracene (7-SMBA) gave rise to some benzylic-DNA adducts indistinguishable from adducts formed from DMBA. Adducts were analyzed by butanol enrichment-mediated 32P-postlabeling assay. Female Sprague-Dawley rats given a combined dose of 420 micromol DMBA/kg b. wt resulted in two major and up to nine minor adducts in the subcutaneous tissue, with chromatographic resemblance to benzylic-DNA adducts prepared in vitro. Subcutaneous administration of 7-HMBA, 7-SMBA, and 7-methyl-12-hydroxymethylbenz[a]anthracene (12-HMBA) (210, 42, and 210 micromol/kg b. wt, respectively) each resulted in one major and several minor benzylic-DNA adducts. From cochromatography with reference adducts, it was concluded that the benzylic DNA adduct 4, derived from the parent compound, comigrates with the major adduct from 7-HMBA and 7-SMBA, whereas adducts 2 and 3 comigrate with adducts resulting from 12-HMBA and 7-methyl-12-sulfooxymethylbenz[a]anthracene, respectively. These data suggest that 7-sulfooxymethyl- and 12-sulfooxymethy derivatives produce distinct adducts. Several major and minor diol epoxide-related DNA adducts were also detected. The diol epoxide- and benzylic-DNA adducts were found in a 2:1 ratio. The oral, intraperitoneal, and intramammiliary treatments with DMBA showed no detectable benzylic adducts in the liver and mammary glands 24 h after the last treatment, although the adduct formation was clearly evident with SMBA and/or HMBA treatments, suggesting that hydroxylation of DMBA to form HMBA may be the rate-limiting step for the meso-methyl substitution pathway. The present study clearly demonstrates the in vivo formation of benzylic-DNA adducts from DMBA. The data also reveal the involvement of the 12-methyl group of DMBA in adduct formation.     

Synthesis and mutagenicity of A-ring reduced analogues of 7,12-dimethylbenz[a]anthracene

The synthesis and mutagenicity of two derivatives of 7,12-dimethylbenz[a]anthracene (DMBA; 1), i.e., 1,2-H2DMBA (4) and 1,2,3,4-H4DMBA (5), are reported. These analogues (4 and 5) represent dihydro and tetrahydro A-ring reduced forms of DMBA, a region in the parent hydrocarbon (1) proposed to be involved in metabolism to the ultimate carcinogen. The synthesis for 4 without isolation of intermediates from the tosylhydrazone of 1,2,3,4-tetrahydrobenz[a]anthracene-4,7,12-trione (10) by successive reaction with 8 molar equiv of CH3Li, HI, and NaBH4 represents a novel approach to this hydrocarbon now available in sufficient quantity for biological studies. Interestingly, both of these reduced analogues 4 and 5 exhibited mutagenic activity in the Ames assay in the presence or absence of microsomal activation for strains TA98 and TA100. In these strains, DMBA was active only in the presence of S-9 fraction. In the plasmid-deficient strain TA1537, only tetrahydro analogue 5 exhibited mutagenic activity both in the absence and presence of S-9 fraction.