Relative importance of maternal and embryonic microsomal epoxide hydrolase in 7,12-dimethylbenz[a]anthracene-induced developmental toxicity

Microsomal epoxide hydrolase (mEH) catalyzes the hydrolysis of epoxide intermediates derived from drugs and environmental chemicals. The response of in vivo (embryo) and in vitro (embryo fibroblast) tests were analyzed using mEH-null and wild-type mice to determine the relative role of maternal and embryonic mEH in the developmental toxicity induced by 7,12-dimethylbenz[a]anthracene (DMBA). Embryos derived from DMBA-treated [50mg/kg, daily from gestational day (GD) 11 to GD 15] dams were analyzed. Although weight (P=0.0009) and crown-rump length (P=0.0003) of wild-type fetuses on GD 18 were significantly lower than those of mEH-null fetuses, respectively, no significant difference was found between mEH-null and heterozygous fetuses of mEH-null dams. Cell viability was decreased to 50% in wild-type mouse embryo fibroblasts (MEFs) treated with 3 microM DMBA, but no significant decrease was found in mEH-null MEFs. DMBA-3,4-diol produced a significant decrease in cell viability and suppressed the proliferation of wild-type MEFs at a 10-fold lower concentration than did DMBA. Although mEH protein was expressed in liver microsomes from wild-type embryos (GD 15), DMBA-3,4-diol was not detected among the DMBA metabolites. However, it was detected in the serum of wild-type pregnant mice treated with DMBA, but not in that of mEH-null mice. These results suggest that maternal mEH plays a major role in DMBA-induced developmental toxicity, and embryonic mEH is less involved in the toxicity.     

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.     

Oxidative metabolites of 7,12-dimethylbenz[a]anthracene. Further investigation of the K-region epoxide

Metabolism of 7,12-dimethylbenz[a]anthracene in rat liver 10,000 g supernatant fraction has been examined by combined gas chromatography-mass spectrometry subsequent to high-pressure liquid-chromatographic separation of the incubation mixture. In addition to the mono-, di-, and trihydroxylated oxidative metabolites usually associated with the biotransformation of polycyclic aromatic hydrocarbons, we observed the formation of a methanolysis product from the K-region epoxide intermediate during the workup procedure. The identification of this compound provides a direct evidence for the presence of the K-region epoxide in the metabolism mixture, and it serves as a convenient means to assay for this metabolite. Moreover, we detected the methanolysis derivative only from the K-region epoxide but none from the non-K-region counterparts. This finding suggests that the non-K-region epoxides undergo facile enzymatic and nonenzymatic hydrolysis and/or rearrangement reactions as soon as they are produced. On the other hand, the K-region epoxide possesses greater stability. It can remain longer in a metabolism mixture and react with methanol subsequently.     

Acute 7,12-dimethylbenz[a]anthracene exposure causes differential concentration-dependent follicle depletion and gene expression in neonatal rat ovaries

Chronic exposure to the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA), generated during combustion of organic matter including cigarette smoke, depletes all ovarian follicle types in the mouse and rat, and in vitro models mimic this effect. To investigate the mechanisms involved in follicular depletion during acute DMBA exposure, two concentrations of DMBA at which follicle depletion has (75 nM) and has not (12.5 nM) been observed were investigated. Postnatal day four F344 rat ovaries were maintained in culture for four days before a single exposure to vehicle control (1% DMSO; CT) or DMBA (12 nM; low-concentration or 75 nM; high-concentration). After four or eight additional days of culture, DMBA-induced follicle depletion was evaluated via follicle enumeration. Relative to control, DMBA did not affect follicle numbers after 4 days of exposure, but induced large primary follicle loss at both concentrations after 8 days; while, the low-concentration DMBA also caused secondary follicle depletion. Neither concentration affected primordial or small primary follicle number. RNA was isolated and quantitative RT-PCR performed prior to follicle loss to measure mRNA levels of genes involved in xenobiotic metabolism (Cyp2e1, Gstmu, Gstpi, Ephx1), autophagy (Atg7, Becn1), oxidative stress response (Sod1, Sod2) and the phosphatidylinositol 3-kinase (PI3K) pathway (Kitlg, cKit, Akt1) 1, 2 and 4 days after exposure. With the exception of Atg7 and cKit, DMBA increased (P < 0.05) expression of all genes investigated. Also, BECN1 and pAKT^Thr308 protein levels were increased while cKIT was decreased by DMBA exposure. Taken together, these results suggest an increase in DMBA bioactivation, add to the mechanistic understanding of DMBA-induced ovotoxicity and raise concern regarding female low concentration DMBA exposures.     

Impact of 7,12-dimethylbenz[a]anthracene exposure on connexin gap junction proteins in cultured rat ovaries

7,12-Dimethylbenz[a]anthracene (DMBA) destroys ovarian follicles in a concentration-dependent manner. The impact of DMBA on connexin (CX) proteins that mediate communication between follicular cell types along with pro-apoptotic factors p53 and Bax were investigated. Postnatal day (PND) 4 Fisher 344 rat ovaries were cultured for 4 days in vehicle medium (1% DMSO) followed by a single exposure to vehicle control (1% DMSO) or DMBA (12.5 nM or 75 nM) and cultured for 4 or 8 days. RT-PCR was performed to quantify Cx37, Cx43, p53 and Bax mRNA level. Western blotting and immunofluorescence staining were performed to determine CX37 or CX43 level and/or localization. Cx37 mRNA and protein increased (P < 0.05) at 4 days of 12.5 nM DMBA exposure. Relative to vehicle control-treated ovaries, mRNA encoding Cx43 decreased (P < 0.05) but CX43 protein increased (P < 0.05) at 4 days by both DMBA exposures. mRNA expression of pro-apoptotic p53 was decreased (P < 0.05) but no changes in Bax expression were observed after 4 days of DMBA exposures. In contrast, after 8 days, DMBA decreased Cx37 and Cx43 mRNA and protein but increased both p53 and Bax mRNA levels. CX43 protein was located between granulosa cells, while CX37 was located at the oocyte cell surface of all follicle stages. These findings support that DMBA exposure impacts ovarian Cx37 and Cx43 mRNA and protein prior to both observed changes in pro-apoptotic p53 and Bax and follicle loss. It is possible that such interference in follicular cell communication is detrimental to follicle viability, and may play a role in DMBA-induced follicular atresia.     

Expression and activity of microsomal epoxide hydrolase in follicles isolated from mouse ovaries.

Microsomal epoxide hydrolase (mEH) is involved in the detoxification of xenobiotics that are or can form epoxide metabolites, including the ovotoxicant, 4-vinylcyclohexene (VCH). This industrial chemical is bioactivated by hepatic CYP450 to the diepoxide metabolite, VCD, which destroys mouse small preantral follicles (F1). Since ovarian mEH may play a role in VCD detoxification, these studies investigated the expression and activity of mEH in isolated ovarian fractions. Mice were given 1 or 15 daily doses (ip) of VCH (7.4 mmol/kg/day) or VCD (0.57 mmol/kg/day); 4 h following the final dose, ovaries were removed, distinct populations of intact follicles (F1, 25-100 microm; F2, 100-250 microm; F3, > 250 microm) and interstitial cells (Int) were isolated, and total RNA and protein were extracted. Real-time polymerase chain reaction and the substrate cis-stilbene oxide (CSO; 12.5 microM) were used to evaluate expression and specific activity of mEH, respectively. Confocal microscopy evaluated ovarian distribution of mEH protein. Expression of mRNA encoding mEH was increased in F1 (410 +/- 5% VCH; 292 +/- 5% VCD) and F2 (1379 +/- 4% VCH; 381 +/- 11% VCD) follicles following repeated dosing with VCH or VCD. Catalytic activity of mEH increased in F1 follicles following repeated dosing with VCH/VCD (381 +/- 11% VCH; 384 +/- 27% VCD). Visualized by confocal microscopy, mEH protein was distributed throughout the ovary with the greatest staining intensity in the interstitial cells and staining in the theca cells that was increased by dosing (56 +/- 0.8% VCH; 29 +/- 0.9% VCD). We conclude that mEH is expressed and is functional in mouse ovarian follicles. Additionally,in vivo dosing with VCH and VCD affects these parameters.     

Male mice deficient in microsomal epoxide hydrolase are not susceptible to benzene-induced toxicity.

Enzymes involved in benzene metabolism are likely genetic determinants of benzene-induced toxicity. Polymorphisms in human microsomal epoxide hydrolase (mEH) are associated with an increased risk of developing leukemia, specifically those associated with benzene. This study was designed to investigate the importance of mEH in benzene-induced toxicity. Male and female mEH-deficient (mEH-/-) mice and background mice (129/Sv) were exposed to inhaled benzene (0, 10, 50, or 100 ppm) 5 days/week, 6 h/day, for a two-week duration. Total white blood cell counts and bone marrow cell counts were used to assess hematotoxicity and myelotoxicity. Micronucleated peripheral blood cells were counted to assess genotoxicity, and the p21 mRNA level in bone marrow cells was used as a determinant of the p53-regulated DNA damage response. Male mEH-/- mice did not have any significant hematotoxicity or myelotoxicity at the highest benzene exposure compared to the male 129/Sv mice. Significant hematotoxicity or myelotoxicity did not occur in the female mEH-/- or 129/Sv mice. Male mEH-/- mice were also unresponsive to benzene-induced genotoxicity compared to a significant induction in the male 129/Sv mice. The female mEH-/- and 129/Sv mice were virtually unresponsive to benzene-induced genotoxicity. While p21 mRNA expression was highly induced in male 129/Sv mice after exposure to 100-ppm benzene, no significant alteration was observed in male mEH-/- mice. Likewise, p21 mRNA expression in female mEH-/- mice was not significantly induced upon benzene exposure whereas a significant induction was observed in female 129/Sv mice. Thus mEH appears to be critical in benzene-induced toxicity in male, but not female, mice.     

Antigenotoxic effect of genistein against 7,12-dimethylbenz[a]anthracene induced genotoxicity in bone marrow cells of female wistar rats

Carcinogen induced mutation in somatic cells leads to genetic instability, which is considered as an important facet of carcinogenesis. Agents that inhibit DNAadduct formation, stimulate DNArepair mechanisms, and possess antioxidant functions are considered as antigenotoxic agents. Genistein, the major isoflavone of soy products, protects animals against experimentally induced mammary and prostate cancers. 7,12-dimethylbenz[a]anthracene (DMBA), a potent site-specific carcinogen, induce mutations in DNA through its active metabolite, dihydrodiol epoxide, is a crucial step in cancer initiation. The antigenotoxic effect of genistein against DMBA-induced genotoxicity has been investigated in the present study by analyzing the frequency of micronucleated polychromatic erythrocytes (MnPCEs) and chromosomal aberrations as cytogenetic end-points. The status of lipid peroxidation, antioxidants and detoxication agents were used as biochemical end-points to assess the antigenotoxic effect of genistein. Elevated MnPCEs frequency, marked chromosomal aberrations and enhanced status of lipid peroxidation, antioxidants and detoxication agents were observed in DMBAtreated animals. Oral pretreatment of genistein (20 mg/kg b.w.) for 5 days to DMBAtreated animals significantly reduced the frequency of micronucleus formation and chromosomal abnormalities as well as reversed the status of biochemical variables. Our results suggest that genistein has potent antigenotoxic effect against DMBA-induced genotoxicity.     

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.     

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.     

Tangeretin ameliorates oxidative stress in the renal tissues of rats with experimental breast cancer induced by 7,12-dimethylbenz[a]anthracene

Tangeretin, a citrus polymethoxyflavone, is an antioxidant modulator which has been shown to exhibit a surfeit of pharmacological properties. The present study was hypothesized to explore the therapeutic activity of tangeretin against 7,12-dimethylbenz[a]anthracene (DMBA) induced kidney injury in mammary tumor bearing rats. Recently, we have reported the chemotherapeutic effect of tangeretin in the breast tissue of DMBA induced rats. Breast cancer was induced by “air pouch technique” with a single dose of 25 mg/kg of DMBA. Tangeretin (50 mg/kg/day) was administered orally for four weeks. The renoprotective nature of tangeretin was assessed by analyzing the markers of oxidative stress, proinflammatory cytokines and antioxidant competence in DMBA induced rats. Tangeretin treatment revealed a significant decline in the levels of lipid peroxides, inflammatory cytokines and markers of DNA damage, and a significant improvement in the levels of enzymatic and non-enzymatic antioxidants in the kidney tissue. Similarly, mRNA, protein and immunohistochemical analysis substantiated that tangeretin treatment notably normalizes the renal expression of Nrf2/Keap1, its downstream regulatory proteins and the inflammatory cytokines in the DMBA induced rats. Histological and ultrastructural observations also evidenced that the treatment with tangeretin effectively protects the kidney from DMBA-mediated oxidative damage, hence, proving its nephroprotective nature.     

7,12-dimethylbenz[a]anthracene interferes with the development of cultured mouse mandibular molars.

Clinical studies suggest that maternal smoking during pregnancy can reduce the crown size of the child's teeth. Delayed dental age compared with chronological age has also been reported in children whose parents smoke. Among the main components of tobacco smoke are nonhalogenated polycyclic aromatic hydrocarbons (PAHs), many of which are highly toxic. Humans are exposed to PAH compounds mainly via tobacco smoke and diet. The aim of our study was to investigate the effect of PAHs on tooth formation and the function of tooth-forming cells. We exposed mouse (NMRI) E18 mandibular first and second molar explants to 7,12-dimethylbenz[a]anthracene (DMBA), a toxic PAH compound, in organ culture for 7 or 12 days. DMBA concentrations used were 0.1, 0.5, 1, and 2 microM. The mesiodistal width of each first molar (12-day culture) was measured in stereomicroscopic images, and the teeth were analysed histologically. DMBA exposure significantly reduced the mesiodistal width of the first molars. DMBA impaired or delayed amelogenesis and dentinogenesis in both molars at the lowest concentration of 0.1 microM. DMBA affected enamel formation more severely than dentin formation and occasionally prevented amelogenesis completely. Elongation and polarization of ameloblasts were impaired, and blood vessel architecture of the dental papilla (future pulp) was altered. Cusps were thin and sharp. In line with the finding that maternal smoking during pregnancy has an adverse effect on child's tooth development, this study shows the toxic influence of PAHs on tooth development in vitro.     

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.     

Effect of milk on the 7,12-dimethylbenz[a]-anthracene-induced mammary tumor model in rat.

Milk may be one of the risk factors in the development of breast cancer from epidemiological investigations. Our study investigated the hormones and main ingredients in milk and assessed the effects of milk on the development of 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in rats by comparing differences among three groups: commercial milk (C), traditional milk (T) or water (W). During the 20-weeks experiment the C and T groups showed higher incidences of mammary tumors than the W group. After excluding potential confounding factors including fat and calcium, the C group was found to score higher than the T group in the indices of tumorigenesis. These findings suggested that DMBA-induced mammary tumors are more prevalent in milk-fed groups due in part to the contribution of estrogen and progesterone in milk.     

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)     

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.     

Suppression of splenic lymphocyte function by 7,12-dimethylbenz[a]anthracene (DMBA) in vitro

The effects of the immunosuppressive polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) were studied directly by in vitro exposure of splenic lymphocytes. On the basis of evidence from prior studies that DMBA immunotoxicity in vivo may not be dependent upon induction of the Ah locus in mice, splenocytes from Ah-responsive B6C3F1, Ah-nonresponsive DBA/2N, and in C57BL/6J Ah-congenic mice (responsive B6-Ah(b)Ah(d) and nonresponsive B6-Ah(d)Ah(d) were exposed to xenobiotic in culture. For some experiments, B6C3F1 mice were pretreated with 200 nmol 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) to induce Ah-associated enzymatic activity prior to in vitro splenocyte exposure to DMBA. Humoral immunity assessed as splenic antibody plaque-forming cells measured after a 5-day in vitro immunization to sheep erythrocytes (SRBC) was suppressed up to 99% by continuous exposure to 20 microM DMBA, and was comparable between control mice having basal levels of hepatic monooxygenase activity and Ah-induced mice (TCDD-treated) having elevated enzyme activity. Similarly, cytotoxic T-lymphocyte generation against P815 target cells was suppressed up to 88 and 86% in 40 microM DMBA-exposed splenocytes from Ah-induced and noninduced mice, respectively. The mixed lymphocyte responsiveness (MLR) of B6C3F1, DBA/2N, B6-Ah(b)Ah(d), and B6-Ah(d)Ah(d) splenocytes exposed in vitro to 40 microM DMBA was suppressed 54, 72, 51, and 29%, respectively. However, the degree of suppression was not significantly different between the strains. The secretion of interleukin 2 (IL2) was also suppressed in splenocytes from both strains exposed to 40 microM DMBA in vitro. Studies which included benzo[a]pyrene (BaP) as a control xenobiotic known to demonstrate Ah dependence showed that the MLR of splenic lymphocytes from Ah-congenic mice was comparably suppressed following 40 microM DMBA exposure, whereas exposure to 40 microM BaP resulted in suppression of the MLR only in B6-Ah(b)Ah(d) splenocytes. In addition, mitogen-stimulated proliferation was inhibited in both B6C3F1 and DBA/2N splenocytes exposed to 40 microM DMBA, whereas 40 microM BaP inhibited only B6C3F1 splenocyte proliferation to LPS. These data suggest that DMBA may act on immunocytes by mechanisms largely independent of the Ah locus and associated metabolic processes.     

Metabolism of 7,12-dimethylbenz[a]anthracene in hepatic microsomal membranes from rats treated with isoenzyme-selective inducers of cytochromes P450

Previous work has shown that member(s) of the cytochrome P450IIC sub-family play significant roles in the formation of diols of 7,12-dimethylbenz[a]anthracene (DMBA) and are particularly important in formation of the proximate carcinogen (DMBA-3,4-diol). To further characterize the role of members of this subfamily in DMBA-diol formation and to assess the part played by other P450s, DMBA metabolism has been investigated in microsomes prepared from animals pre-treated with isoenzyme selective inducers. The rates of formation of DMBA-diols in membranes from phenobarbital-treated rats were very low when NADH was used as reductant and rates were not altered when NADPH and NADH were used in combination rather than using NADPH alone. This suggests that cytochrome b5 is not involved in DMBA-diol formation in these membranes. Treatment of animals with clofibrate, pyrazole and dexamethasone produced regio-selective alterations in the rates of formation of DMBA-diols at the -3,4-, -5,6- and -8,9- positions. However, none of the inducers caused increases in the rates of DMBA-diol formation of any great magnitude suggesting that the isoforms which are the major induced proteins (P450IVA1, P450IIE1 and P450IIIA1) do not play a significant role in diol formation. The content of other P450s in these membrane are also altered and these were investigated by Western blot using antibodies to P450IIC6, P450IIB1 and P450IIIA1. The results of the Western blots show that the effects of the inducing agents on DMBA-diol formation can be explained by alterations of members of the P450IIC and P450IIB subfamilies.