Bioalkylation of benz[a]anthracene, 7-methylbenz[a]anthracene, and 12-methylbenz[a]anthracene in rat lung cytosol preparations

Benz[a]anthracene (BA) and the monomethyl meso-anthracenic or L-region derivatives 7-methylbenz[a]anthracene (7-methylBA) and 12-methylbenz[a]anthracene (12-methylBA) underwent a bioalkylation substitution reaction in rat lung ctyosol preparations, fortified with S-adenosyl-L-methionine to form the more potent carcinogen 7,12-dimethylbenz[a]anthracene. The methyl groups of the highly reactive L-region methylated metabolites also underwent enzymatic hydroxylation in rat lung cytosol preparations to yield the corresponding hydroxymethyl derivatives, 7-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, and 7,12-dihydroxymethylbenz[a]anthracene. The biooxidation reaction took place enzymatically, and exclusively, or nearly so, at the reactive methyl groups attached to the meso positions or L-region of the hydrocarbon. Bioalkylation and biooxidation reactions did not occur when the hydrocarbons were incubated with a boiled cytosol preparation, indicating the need for enzymatic activation of the L-region methyl groups. Also, the bioalkylation reaction did not occur in the absence of S-adenosyl-L-methionine. Furthermore, the S-adenosyl-L-methionine-dependent reaction was inhibited by S-adenosyl-L-homocysteine, suggesting that the reaction is catalyzed by a cytosolic S-adenosyl-L-methionine-dependent methyltransferase.     

Influence of effectors of prostaglandin metabolism on the toxicity induced by 7-hydroxymethyl-12-methylbenz(a)anthracene in cultured rat adrenal cells

1. 7,12-Dimethylbenz(a)anthracene (DMBA) and 7-hydroxymethyl-12-methylbenz(a)anthracene (7-OHM-12-MBA), but not benzo(a)pyrene (BP), selectively produce necrosis in the two inner zones of the rat adrenal cortex and are toxic to cultured rat adrenocortical cells.

2. The toxicity induced by 7-OHM-12-MBA in the adrenocortical cells was partially prevented by the inhibitor of the cyclooxygenase activity of prostaglandin H synthetase, indomethacin. In contrast, indomethacin did not influence the effect of BP and DMBA on these cells.

3. Two other effectors of the prostaglandin metabolism, 5,8,11,14-eicosatetraynoic acid (ETYA) and nordihydroguaiaretic acid (NDGA), as well as the anti-inflammatory steroids cortisol and dexamethasone, partially protected against, whereas arachidonic acid and bradykinin exacerbated, the cytotoxicity induced by 7-OHM-12-MBA.

4. These results indicate that prostaglandin metabolism may be involved in the necrotic mechanism of 7-OHM-12-MBA in rat adrenal cortex.     

Influence of effectors of prostaglandin metabolism on the toxicity induced by 7-hydroxymethyl-12-methylbenz(a)anthracene in cultured rat adrenal cells

1. 7,12-Dimethylbenz(a)anthracene (DMBA) and 7-hydroxymethyl-12-methylbenz(a)anthracene (7-OHM-12-MBA), but not benzo(a)pyrene (BP), selectively produce necrosis in the two inner zones of the rat adrenal cortex and are toxic to cultured rat adrenocortical cells. 2. The toxicity induced by 7-OHM-12-MBA in the adrenocortical cells was partially prevented by the inhibitor of the cyclooxygenase activity of prostaglandin H synthetase, indomethacin. In contrast, indomethacin did not influence the effect of BP and DMBA on these cells. 3. Two other effectors of the prostaglandin metabolism, 5,8,11,14-eicosatetraynoic acid (ETYA) and nordihydroguaiaretic acid (NDGA), as well as the anti-inflammatory steroids cortisol and dexamethasone, partially protected against, whereas arachidonic acid and bradykinin exacerbated, the cytotoxicity induced by 7-OHM-12-MBA. 4. These results indicate that prostaglandin metabolism may be involved in the necrotic mechanism of 7-OHM-12-MBA in rat adrenal cortex.     

Effect of 7-hydroxymethyl-12-methylbenz[a]anthracene and 1,3-bis-(2-chloroethyl)-1-nitrosourea on enzyme activities and oxidation of glutathione in cultured rat adrenal cells

1. The activities of enzymes participating in the regeneration of reduced glutathione (GSH), and their subcellular distribution were studied in cultured rat adrenal cells. 2. It has previously been shown that the adrenocorticolytic agent 7-hydroxymethyl-12-methylbenz[a]anthracene (7-hydroxymethyl-12-MBA) causes a drastic and selective oxidation of mitochondrial GSH in rat adrenal cells. Treatment of the adrenal cells with 7-hydroxymethyl-12-MBA, resulted in a minor decrease in the content of cytochrome c oxidase, nicotinamide nucleotide transhydrogenase, isocitrate dehydrogenase and cytosolic GSH reductase, whereas the activity of lactate dehydrogenase and citrate synthase was unaffected. None of these effects were considered to be responsible for the massive oxidation of mitochondrial GSH induced by 7-hydroxymethyl-12-MBA. 3. 1,3-Bis-(2-chloroethyl)-1-nitrosourea (BCNU) was used to obtain rat adrenal cells cultures with inactivated cytosolic and mitochondrial GSH reductase. The oxidation of mitochondrial GSH, induced by 7-hydroxymethyl-12-MBA, was not dramatically enhanced by the inactivation of GSH reductase, indicating that this enzyme was not rate-limiting in the regeneration of GSH. 4. Fractionation of rat adrenal cells with increasing concentrations of digitonin resulted in an earlier release of citrate synthase in cells treated with 7-hydroxymethyl-12-MBA compared with controls. These results may indicate damage to mitochondrial membranes as a result of 7-hydroxymethyl-12-MBA treatment.     

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.     

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.     

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.     

Metabolism and toxic effects of 7,12-dimethyl-benz[a]anthracene in isolated rat adrenal cells

Isolated rat adrenal cells in tissue culture, showing ACTH-induced corticosterone synthesis, were used as a model system for the study of adrenal metabolism and toxicity of 7,12-dimethylbenz[a]anthracene (DMBA). DMBA was metabolized at a rate of 10 pmol/min/10(6) cells and with a Km of 0.5 microM. Metabolite patterns and sensitivity to various AHH inhibitors suggest the involvement of an epoxide intermediate. In agreement with this proposal DMBA metabolite(s) were bound to cellular protein at a rate which was related to the AHH activity. Adrenal AHH was found to be insensitive to ACTH during a period of 24 h. Using ACTH-induced corticosterone synthesis as an indicator of cell damage the hepatic metabolite 7-hydroxymethyl-12-methyl-benz[a]anthracene was shown to be significantly more toxic than the parent compound DMBA. It is concluded that DMBA-dependent adrenal damage in vivo is due mainly to the liver metabolite 7-hydroxymethyl-12-methyl-benz[a]anthracene (7-OHM-12-MBA), possibly after secondary metabolic activation in the adrenal.     

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.     

Evidence for a free-radical-dependent metabolism of 7,12-dimethylbenz(a)anthracene in rat testis

Polycyclic aromatic hydrocarbons, e.g., 7,12-dimethylbenz(a)anthracene (DMBA), cause various toxic effects in rat testis. To clarify the mechanism of action of DMBA in adult rat testis microsomes and mitochondria from this organ were investigated in vitro with respect to their capacity to metabolize DMBA. Qualitatively, both preparations showed DMBA-hydroxylase activities which were influenced by cytochrome P-450 inhibitors, chelators, and free-radical scavengers, suggesting that the DMBA metabolism was accounted for by different metabolic pathways in these organelles. Metabolism of DMBA was also accompanied by a pronounced covalent binding to both microsomal and mitochondrial protein, catalyzed primarily by a free-radical mechanism involving free or loosely bound iron which may involve superoxide anion shown to be generated by testis mitochondria. With microsomes covalent binding was markedly enhanced by added horseradish peroxidase but not by hydrogen peroxide whereas the mitochondrial binding was affected neither by added horseradish peroxidase nor by hydrogen peroxide. Antibodies raised against cytochrome P-450 c from rat liver inhibited the microsomal DMBA-hydroxylase but not the mitochondrial DMBA metabolism. It is concluded that the microsomal DMBA conversion and covalent binding are due to a mixture of cytochrome P-450 and free-radical-dependent metabolic pathways whereas the corresponding mitochondrial reaction is due mainly to a free-radical-dependent pathway. However, the data do not allow for a conclusion as to the quantitative importance of these pathways. It is proposed that both pathways may be important in DMBA-dependent testis toxicity but also in polycyclic aromatic hydrocarbon-dependent testis toxicity in general.     

Biotransformation and bioactivation of 7, 12-dimethylbenz[a]anthracene (7, 12-DMBA)

As the result of rapidly developing technological advances, our understanding of the biotransformation and bioactivation of 7, 12-DMBA has increased markedly in recent years. In terms of the metabolic conversion of this polynuclear aromatic hydrocarbon to reactive mutagen/carcinogens, the "bay region" generalization appears to apply, although the candidacy of a number of other intermediary metabolites as ultimate biologically-active forms still remains viable. Large gaps remain in knowledge concerning the nonoxidative metabolic transformations of 7, 12-DMBA, and these require closing in order to further our understanding of the regulation of mechanics controlling steady-state levels of reactive intermediates. Studies on the photooxidation of the hydrocarbon have allowed a stronger appreciation of its chemical reactivity and instability and promise to help resolve many of the apparently conflicting observations of the past. 7, 12-DMBA remains a highly interesting and valuable tool in investigations of bioactivation processes as they relate to the etiology of several important pathologic conditions, including chemically induced tissue necrosis, mutagenesis, carcinogenesis, teratogenesis, atherogenesis, and, possibly, other pathogenic phenomena as well. It is hoped that this review will serve to benefit research in these areas and hasten the reduction of such pathologic phenomena in our society.     

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.     

Cellular localization and hormonal regulation of 7,12-dimethylbenz[a]anthracene mono-oxygenase activity in the rat ovary.

To identify the ovarian cell type(s) responsible for the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) and their dependence on hormonal influences, DMBA mono-oxygenase activity was determined in primary cultures of cells dispersed from rat ovaries and separated by centrifugation on discontinuous Percoll density gradients. The contents of progesterone and oestradiol in the different cell cultures were characterized. Moreover, the morphological appearance of ovarian cells obtained from untreated and pregnant mare's serum gonadotropin (PMSG)-treated rats was examined. It is concluded from experiments with immature PMSG-treated rats, that DMBA mono-oxygenase activity is localized in follicular granulosa (and/or theca) cells. This activity decreases during luteinization, and is recovered in a population of cells harvested at a higher density on the Percoll gradient. The xenobiotic-metabolizing activity was not correlated to the rate of biosynthesis of progesterone or oestradiol in isolated cells, measured in the presence or absence of human chorionic gonadotropin and/or testosterone. However, a certain dependence of DMBA metabolism on steroids and/or steroid-synthesizing enzymes could not be excluded. For example, DMBA mono-oxygenase activity was markedly increased in a cell population, tentatively identified as granulosa cells, obtained from untreated mature rat ovaries upon addition of testosterone, which is the substrate for oestrogen synthesis, to the cell culture.     

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)