Expression and function of three cytochrome P-450 isozymes in rat extrahepatic tissues

Western blots using a polyclonal and a monoclonal antibody raised against rat liver cytochrome P-450b indicate tissue-specific expression of low levels of cytochrome P-450's b and e. P-450b and P-450e were expressed very selectively in, respectively, lung and adrenal microsomes of untreated rats but neither isozyme was detected in the corresponding kidney or small intestine microsomes. The regioselectivity of microsomal metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) as well as the sensitivity to inhibition by anti P-450b/e IgG established that low levels of "b-like" P-450's are functional in lung and adrenal microsomes from uninduced rats, but not in microsomes from the kidney or small intestine. Functional P-450c was also detected at low levels in liver, lung, kidney, and adrenals of untreated rats. Among the extrahepatic tissues examined, DMBA metabolism was the highest in rat adrenal microsomes. However, only 30% of this activity was due to P-450's b, e, or c. Phenobarbital (PB) treatment of rats increased microsomal DMBA metabolism in all extrahepatic tissues examined. The selectivity of this increase for 12-methyl hydroxylation of DMBA and the near complete inhibition by anti-P-450b/e are consistent with induction of P-450e even though P-450b was preferentially induced in each of the extrahepatic tissues examined. The levels of expression of P-450b were increased by PB in all sets of adrenal, lung, and intestinal microsomes and in three out of six sets of kidney microsomes. The levels of P-450e were also increased by PB in all sets of adrenal microsomes. Following PB treatment, P-450e became immunoquantifiable (greater than 2 pmol/mg protein) in three of six sets of lung and kidney microsomes but remained below detection in all sets of intestinal microsomes. Based on the activity of purified P-450e, undetectable levels (less than 1 pmol/mg protein) could account for increased DMBA metabolism in this tissue. The high constitutive level of P-450b in the lung (approximately 40 pmol/mg), was remarkably inactive in DMBA metabolism and was only slightly increased by PB treatment (50%). In contrast, PB treatment caused a 2.5- to 10-fold increase in 12-methyl hydroxylation of DMBA that was highly sensitive to anti-P-450b/e. A protein comigrating with P-450e was well above detection (6-7 pmol/mg) in two of six preparations of lung microsomes that showed highest induction of this activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

ACTH regulation of cholesterol movement in isolated adrenal cells

Confluent bovine adrenal cell primary cultures respond to stimulation by adrenocorticotropin (ACTH) to produce steroids (initially predominantly cortisol and corticosterone) at about one-tenth of the output of similarly stimulated rat adrenal cells. The early events of steroidogenesis, following ACTH stimulation, have been investigated in primary cultures of bovine adrenal cortical cells. Steroidogenesis was elevated 4-6-fold within 5 min of exposure to 10(-7) M ACTH and increased linearly for 12 h and declined thereafter. Cholesterol side-chain cleavage (SCC) activity was increased 2.5-fold in mitochondria isolated from cells exposed for 2 h to ACTH and 0.5 mM aminoglutethimide (AMG), even though cytochrome P-450scc only increases after 12 h. Mitochondrial-free cholesterol levels increased during the same time period (16.5-25 micrograms/mg of protein), but then both cholesterol levels and SCC activity declined in parallel. More prolonged exposure to ACTH prior to addition of AMG caused the elevation in mitochondrial cholesterol to more than double, possibly due to enhanced binding capacity. Early ACTH-induced effects on cellular steroidogenesis result from these changes in mitochondrial-free cholesterol. The maximum rate of cholesterol transport to mitochondria in AMG-blocked cells was consistent with the maximum rate of cellular steroidogenesis. Cycloheximide (0.2 mM) rapidly blocked (less than 10 min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450scc without affecting mitochondrial-free cholesterol. Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8) M), for 24 h prior to ACTH addition decreased the rates of ACTH- and cAMP-stimulated steroidogenesis but did not affect the basal rate. In both cases, the effectiveness of TCDD increased with time of exposure to the stimulant. Although cholesterol accumulated in the presence of ACTH and AMG (13-28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13-8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). Since even in the absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h) TCDD pretreatment may increase the presence of a protein(s) that cause this mitochondrial-cholesterol depletion following stimulation by ACTH or cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Selective potent restriction of P450b- but not P450e-dependent 7,12-dimethylbenz[a]anthracene metabolism by the microsomal environment

The prototypic members of the rat liver cytochrome P450IIB subfamily, P450b and P450e, differ by only 13 amino acids and yet purified P450b is considerably more active than P450e for all known substrates. A unique regioselectivity difference between cytochromes P450b and P450e for the metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) and a genetic deficiency in P450e expression in the Marshall (M520/N) rat strain have been exploited to determine the microsomal contributions of the respective forms toward the metabolism of DMBA. The total contribution to metabolism by each isozyme has been assessed based on the sensitivity to rabbit anti-P450b/e IgG and comparison with microsomal P450b and P450e content as measured by Western blots. Liver microsomes from untreated M520/N rats do not express detectable levels of P450e but express P450b at a level that is 2-fold higher than that of P450e in liver microsomes from untreated F344 rats (50 pmol/mg). However, only 4% of the constitutive DMBA metabolizing activity of liver microsomes from the M520/N rat strain could be inhibited by anti-P450b/e IgG. A 30-fold induction of hepatic P450b by phenobarbital (PB) was also completely ineffective in increasing P450b-dependent DMBA metabolism. PB treatment had no appreciable effect on either the levels of expression of P450b protein or P450b-dependent DMBA metabolism, in M520/N lung and adrenal microsomes. In contrast, PB treatment of F344 rats considerably increased P450b/e-dependent metabolism by liver, lung, and adrenal microsomes. The regioselectivity of the anti-P450b/e-sensitive metabolism (predominantly 12-methyl hydroxylation), however, indicated a much greater contribution from P450e than P450b in every tissue examined despite a several fold higher expression of P450b than of P450e. P450b was expressed constitutively in lung microsomes from both strains but again failed to exhibit appreciable DMBA metabolizing activity. Based on these activities and microsomal P450b contents, P450b consistently exhibited turnover numbers (0.02-0.15 nmol/nmol P450b/min) that were at least 10-fold lower than those of pure P450b. In contrast, the calculated turnover numbers for microsomal P450e were consistently comparable to those of pure P450e (approximately 1 nmol/nmol P450e/min).     

Cell-specific metabolic activation of 7,12-dimethylbenz[a]anthracene in rat testis

The binding of metabolites of the polycyclic aromatic hydrocarbon (PAH) 7,12-dimethylbenz[a]anthracene (DMBA) to protein in rat testis seminiferous tubules was studied. Treatment of cultured seminiferous tubule segments with DMBA resulted in very little binding to protein, suggesting that the seminiferous epithelium from rat testis lacks the cytochrome P-450-dependent monooxygenase(s) required for DMBA metabolism. In contrast, Leydig cells from rat testis contain monooxygenase systems which catalyze the metabolism of PAH, such as DMBA. This metabolic activation of DMBA was localized in both mitochondria and microsomes derived from Leydig cells and was decreased by inhibitors of the cytochrome P-450 system and by free radical scavengers, suggesting that the metabolism involved both cytochrome P-450 and free radical-dependent pathways. In the presence of whole Leydig cells or microsomes prepared from Leydig cells, the covalent binding of DMBA metabolites to protein of rat testis seminiferous tubules was increased 5- and 13-fold, respectively. These results suggest that DMBA is metabolized primarily in rat testis Leydig cells and that part of the produced metabolites find their way to the seminiferous epithelium, where they undergo further metabolism producing reactive metabolites, possibly cation radicals and diolepoxides, which interfere with the functions of spermatogonia and spermatocytes by modifying key proteins covalently.     

Inhibition of ACTH action on cultured bovine adrenal cortical cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin through a redistribution of cholesterol

The conversion of cholesterol to cortisol by cultured bovine adrenal cortical cells is stimulated 6-fold by adrenocorticotropin and is limited by the movement of cholesterol to the mitochondria (DiBartolomeis, M.J., and Jefcoate, C.R. (1984) J. Biol. Chem. 259, 10159-10167). Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (10(-8)M), for 24 h prior to adrenocorticotropin (ACTH) addition decreased the rate of ACTH-stimulated steroidogenesis but did not affect the basal rate. TCDD was more effective against stimulation at 10(-11) M ACTH (4-fold) than at 10(-7) M ACTH (10%), consistent with an increase in EC50 for ACTH. Stimulation of bovine adrenal cortical cells by cAMP was similarly decreased by TCDD. In both cases the effectiveness of TCDD increased with time of exposure to the stimulant. The transfer of cholesterol to mitochondria in intact cells was quantitated by means of the 2-h accumulation of mitochondrial cholesterol in the presence of aminoglutethimide, an inhibitor of cholesterol side chain cleavage. Although cholesterol accumulated in the presence of ACTH (13 to 28 micrograms/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13 to 8 micrograms/mg). The effect of TCDD was produced relatively rapidly (t1/2 approximately 4 h). In absence of TCDD, the mitochondria of ACTH-stimulated cells also eventually lose cholesterol (after 2 h). It is concluded that TCDD pretreatment may increase the presence of a protein(s) that cause mitochondrial cholesterol depletion when the cells are stimulated by ACTH or cAMP. TCDD-enhanced cholesterol efflux from mitochondria diminishes cholesterol side chain cleavage when mitochondrial cholesterol is sufficiently depleted (after 2-4 h).     

Metabolism and toxicity of xenobiotics in the adrenal cortex,with particular reference to 7,12-dimethylbenz(a) anthracene

The adrenal cortex contains high amounts of detoxifying enzymes, as well as generators and protectors of reactive oxygen species. The high content of cytochrome P-450 enzymes in the adrenal cortex together with its remarkable tendency to accumulate hydrophobic substances probably contributes to the extraordinary vulnerability of the gland to a number of xenobiotics. The best studied adreno-corticolytic compounds are the potent carcinogen 7, 12-dimethylbenz(a)anthracene (DMBA) and its liver metabolite 7-hydroxymethyl-12-methylbenz (a)anthracene (7-OHM-12-MBA). Adrenocorticolysis generated by these agents in vivo as well as in vitro demonstrates high regioselective requirements and is strongly influenced by the presence of ACTH, steroids, cytochrome P-450 inhibitors and antioxi-dants. Furthermore, 7-OHM-12-MBA has been demonstrated to uniquely generate selective and massive oxidation of mitochondrial glutathione in cultured rat adrenal cells. The DMBA-induced adrenocorticolysis is thoroughly discussed in this review with particular emphasis on the metabolism of DMBA and the influence of various effectors. A working hypothesis involving a possible peroxidative mechanism is also presented.     

Metabolism and toxicity of xenobiotics in the adrenal cortex, with particular reference to 7,12-dimethylbenz(a)anthracene

The adrenal cortex contains high amounts of detoxifying enzymes, as well as generators and protectors of reactive oxygen species. The high content of cytochrome P-450 enzymes in the adrenal cortex together with its remarkable tendency to accumulate hydrophobic substances probably contributes to the extraordinary vulnerability of the gland to a number of xenobiotics. The best studied adrenocorticolytic compounds are the potent carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) and its liver metabolite 7-hydroxymethyl-12-methylbenz(a)anthracene (7-OHM-12-MBA). Adrenocorticolysis generated by these agents in vivo as well as in vitro demonstrates high regioselective requirements and is strongly influenced by the presence of ACTH, steroids, cytochrome P-450 inhibitors and antioxidants. Furthermore, 7-OHM-12-MBA has been demonstrated to uniquely generate selective and massive oxidation of mitochondrial glutathione in cultured rat adrenal cells. The DMBA-induced adrenocorticolysis is thoroughly discussed in this review with particular emphasis on the metabolism of DMBA and the influence of various effectors. A working hypothesis involving a possible peroxidative mechanism is also presented.     

Purification and immunological characterization of a novel cytochrome P450 from C3H/10T1/2 cells

The major polycyclic aromatic hydrocarbon-metabolizing cytochrome P450 in the mouse embryo fibroblast-derived C3H/10T1/2 CL8 cell line (P450-EF) has been partially purified from benz[a]anthracene (BA)-induced 10T1/2 cells (40 pmol P450/mg). The purification of P450-EF was carried out by sequential chromatography of solubilized microsomes over hydrophobic aminohexyl-Sepharose 4B, anion exchange DE-52 cellulose, and cation exchange carboxymethyl trisacryl columns. The final preparation (1700 pmol/mg) appeared as a single major 55-kDa band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reconstitution of detergent-free partially purified P450-EF yielded a relatively high turnover for 7,12-dimethylbenz[a]anthracene (DMBA) metabolism (5.4 nmol/nmol/min). Polyclonal antibodies to purified P450-EF (anti-P450-EF), raised in, respectively, rabbit and chicken, detected a single 55-kDa band in 10T1/2 cell microsomes that was highly inducible by BA (approximately 20-fold) and TCDD (approximately 5-fold). Rabbit anti-P450-EF was much more effective than the corresponding chicken antibody at binding denatured P450-EF protein on Western blots. Conversely, only the chicken antibody was effective at inhibiting DMBA metabolism catalyzed by microsomal P450-EF. This antibody did not inhibit P450IA1-mediated DMBA metabolism. Rabbit anti-P450-EF recognized very weakly (less than 1% of homologous protein response) pure P450IA1, IIB1, IIC7, IIE1, and IIIA1 proteins on Western blots but exhibited substantial cross-reactivity (approximately 10%) with pure P450IIA1 and very strong cross-reactivity (approximately 75%) with a hormonally regulated rat adrenal P450. Polyclonal antibodies to several major P450 subfamilies either did not recognize P450-EF (anti-P450IA, IIB, and IIC) or recognized it very weakly (anti-P450IIA1). P450-EF is probably distantly related to the P450IIA subfamily and may belong to a new P450 subfamily.     

Induction of synthesis of bovine adrenocortical cytochromes P-450scc, P-45011 beta, P-450C21, and adrenodoxin by prostaglandins E2 and F2 alpha and cholera toxin

To further elucidate the mechanisms by which ACTH (adrenocorticotropin) exerts its long-term action to maintain normal levels of adrenocortical cytochromes P-450 and related enzymes, the abilities of cholera toxin and prostaglandins E2 and F2 alpha to induce the synthesis of cytochromes P-450scc, P-45011 beta, and P-450C21 and adrenodoxin have been examined. These effectors stimulate the production of cyclic AMP and thus steroidogenesis in the adrenal cortex. Using bovine adrenocortical cells in primary monolayer culture, we have shown that treatment with cholera toxin results in increased synthesis of cytochromes P-450scc and P-45011 beta and adrenodoxin, similar to the effect observed upon ACTH treatment. Prostaglandins E2 and F2 alpha are less effective at inducing the synthesis of the mitochondrial cytochromes P-450, and do not seem to induce the synthesis of adrenodoxin. Furthermore, cholera toxin was found to be less effective at inducing the synthesis of microsomal cytochrome P-450C21 than ACTH, and no more effective than the prostaglandins. Thus, while it appears that elevation of cyclic AMP levels is a necessary step leading to increased synthesis of adrenocortical forms of cytochrome P-450, the detailed mechanism of this induction will be found to be different for each of the different enzymes.     

Differential expression and function of three closely related phenobarbital-inducible cytochrome P-450 isozymes in untreated rat liver

The levels of expression of cytochromes P-450b and P-450e (both inducible by phenobarbital (PB) and differing by only 14 of 491 amino acids) in liver microsomes from untreated male rats were separately quantitated by Western blotting with a polyclonal antibody raised against P-450b that is equally effective against P-450e (anti P-450b/e). A protein with mobility identical to P-450e was detected in all microsomal samples. Microsomes from uninduced livers of individual male rats from five different strains exhibited only minor interstrain and interindividual variability in the expression of P-450e (17 +/- 5 pmol P-450e/mg microsomal protein) with the exception of the Brown Norway strain (8.5 +/- 0.5 pmol P-450e/mg). Expression of P-450b varied widely from undetectable levels (less than 2 pmol/mg) in most Sprague-Dawley rats to about 50% of P-450e levels in Fischer and Brown Norway strains. Anti P-450b/e inhibited total metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) by uninduced microsomes, to an extent dependent on rat strain (15-30%), predominantly through inhibition of formation of 12-hydroxymethyl-7-methyl BA (12HOMMBA) (65-85%), the major metabolite of purified P-450e. A specific activity for P-450e-dependent DMBA metabolism was calculated from four sets of microsomes where the P-450b content was either undetectable or very low (0.7-1.0 nmol/nmol P-450e/min-1). Comparable calculated activities were, however, obtained from other untreated rat liver microsomes where P-450b levels were significant. Polymorphism in P-450b was detected but did not affect total P-450b expression or the sensitivity of DMBA metabolism to anti P-450b/e. A fourth band of greater mobility than P-450b (apparent Mr less than 50,000), was also recognized by anti P-450b/e. The intensity of this band did not vary among individual rats or among the different strains and therefore did not correlate with the sensitivity of microsomal DMBA metabolism to anti P-450b/e. A monoclonal antibody (MAb) against P-450b (2-66-3) recognized P-450's b, b2, and e on Western blots but did not react with this higher mobility band. MAb 2-66-3 and two other MAbs produced against P-450b inhibited 12-methylhydroxylation of DMBA by untreated rat liver microsomes to the same extent as anti P-450b/e. Following PB induction, P-450b was induced to about double the level of P-450e in most rat strains examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of aryl hydrocarbon hydroxylase activity in normal and neoplastic human breast epithelium

Studies were conducted to determine whether normal and/or neo-plastic (MCF-7) human breast epithelial cells contain the microsomal aryl hydrocarbon hydroxylase (AHH) which catalyses the conversion of polycyclic aromatic hydrocarbons (PAH) to carcinogenic intermediates. Low constitutive levels of AHH activity were found in homogenates of both normal human breast epithelial and MCF-7 cells. The addition of 7,12-dimethylbenz(a)anthracene (DMBA) to the culture medium of either cell type significantly increased AHH activity. Peak induction of hydroxylase activity occurred following the $\text{in}$$\text{vitro}$ addition of 10 μM DMBA. A time course of DMBA-induced AHH activity in both normal human breast epithelium and MCF-7 cells revealed maximal induction 16 hr after 10 μM DMBA was added to the culture medium. Benzo(a)pyrene (BP), 3-methylcholanthrene (MCA) and benz(a)anthracene (BA) also induced AHH activity in normal and MCF-7 cells. For example, the addition of 10 μM BP to the culture medium of either normal human breast epithelial or MCF-7 cells for 16 hr increased AHH activity 13.8 and 65.3-fold, respectively. For all PAH, the magnitude of AHH induction was substantially greater in MCF-7 than normal breast epithelial cells. Finally, α-naphthoflavone inhibited BA-induced AHH activity in MCF-7 cells. The study demonstrates the presence of a PAH-inducible AHH enzyme(s) in normal human breast epithelial cells grown in primary culture and in the human breast tumor cell line, MCF-7.     

Danazol inhibits human adrenal 21-and 11β-hydroxylation invitro

The effects of danazol on steroidogenesis $\text{in}$$\text{vitro}$ in the 16–20 week old human fetal adrenal were examined by studying: 1) danazol binding to adrenal microsomal and mitochondrial cytochrome P-450, and 2) enzyme kinetics of danazol inhibition of the adrenal microsomal 21-hydroxylase and the mitochondrial llβ-hydroxylase. The addition of danazol to preparations of adrenal microsomes or mitochondria elicited a type I cytochrome P-450 binding spectrum. Danazol bound to microsomal cytochrome P-450 with a high affinity apparent spectral dissociation constant (Kg) of 1 μM and with a lower affinity K's of 10 μM. Danazol bound to mitochondrial cytochrome P-450 with a Kg of 5 μM. In addition, danazol competitively inhibited the microsomal 21-hydroxylase (apparent enzymatic inhibition constant K_I = 0.8 μM) and the mitochondrial 11β-hydroxylase (K_I = 3 μM). These findings demonstrate that low concentrations of danazol directly inhibit steroidogenesis in the human fetal adrenal $\text{in}$$\text{vitro}$.     

Spironolactone inhibition of cortisol production by guinea pig adrenocortical cells

Prior investigations with adrenal subcellular fractions demonstrated that the diuretic, spironolactone (SL), was converted to a reactive metabolite by adrenal microsomes, resulting in the degradation of microsomal cytochrome(s) P-450. Studies were done to evaluate the effects of SL and 7 alpha-thio-SL, a putative intermediate in the activation pathway, on cortisol production by intact guinea pig adrenocortical cells. Preincubation of adrenal cells with SL or 7 alpha-thio-SL caused time-dependent and concentration-dependent decreases in subsequent ACTH-stimulated cortisol production. 7 alpha-Thio-SL was a far more potent inhibitor than SL. In the absence of a preincubation period, neither SL nor 7 alpha-thio-SL affected cortisol production. The results indicate that the effects of SL on adrenal microsomal cytochrome(s) P-450 compromise steroid synthesis by intact adrenal cells and lend support to the hypothesis that metabolism of the drug is required for the inhibition of steroidogenesis.     

Differential control of adrenal drug and steroid metabolism in the guinea pig.

Studies were carried out to compare the effects of several physiological variables on adrenal microsomal drug (ethylmorphine demethylation) and steroid (21-hydroxylation) metabolism in guinea pigs. The rate of adrenal ethylmorphine (EM) metabolism increased with maturation in males but not females, resulting in a sex difference (M > F) in adrenal enzyme activity in adult guinea pigs. Twenty-one hydroxylase activity, in contrast, was similar in adrenals from males and females. The concentration of adrenal microsomal cytochrome P-450 was unaffected by age or sex. ACTH administration decreased adrenal EM demethylase activity but did not affect 21-hydroxylation. Testosterone, when given to female guinea pigs, increased the rate of EM metabolism and decreased 21-hydroxylase activity. Various compounds known to interact with adrenal microsomal cytochrome P-450 had divergent effects on EM metabolism and 21-hydroxylation $\text{in}$$\text{vitro}$. Prostaglandins E₁ and F_2α, spironolactone, and canrenone inhibited EM demethylation but not 21-hydroxylation. Simple aromatic hydrocarbons (benzene, toluene), in contrast, inhibited 21-hydroxylation but did not affect EM metabolism. The results indicate that adrenal drug and steroid metabolism are independently regulated and that different terminal oxidases (cytochrome P-450) are probably involved in adrenal 21-hydroxylation and EM demethylation.     

Suppression of cAMP-mediated signal transduction in mouse adrenocortical cells which express apolipoprotein E.

We have reported previously that expression of the human apolipoprotein E (apoE) gene in mouse Y1 adrenocortical cells suppresses basal and adrenocorticotropin (ACTH)-stimulated steroidogenesis. To understand the mechanism of this suppression, we have examined the integrity of cAMP regulated events required for adrenal steroidogenesis. Both acute and chronic responses to ACTH or cAMP are suppressed in Y1 cells which express apoE (Y1-E cells) as compared with parental Y1 cells. Acute morphologic changes in response to cAMP and acute induction of steroidogenesis by cAMP are suppressed in the Y1-E cell lines. Constitutive expression of P450-cholesterol side chain cleavage enzyme mRNA, the rate-limiting enzyme in steroid hormone synthesis, is reduced up to 11-fold in the Y1-E cell lines. The level of mRNA encoding P450-cholesterol side chain cleavage correlates directly with the reduction in basal steroid production observed in the individual Y1-E cell lines. Expression of P450-11 beta-hydroxylase mRNA, although readily detectable in Y1 parent cells, is absent or reduced in the Y1-E cell lines. Inhibition of cAMP-regulated gene expression is not restricted to genes required for steroid synthesis, since cAMP induction of ornithine decarboxylase mRNA is also inhibited in the Y1-E cell lines. These data indicate that suppression of steroidogenesis in Y1-E cells is due, at least in part, to inhibition of cAMP-regulated gene expression. These effects are not due to a defective cAMP-dependent protein kinase, since kinase activity in vitro and activation in vivo are unaltered in the Y1-E cell lines. These results suggest that expression of apoE in Y1 cells blocks cAMP-mediated signal transduction at a point distal to activation of cAMP-dependent protein kinase.     

Relevance of c-fos proto-oncogene induction for the steroidogenic response to ACTH,dcAMP and phorbol ester in adrenocortical cells

We previously reported that ACTH, but not dibutyryl cAMP, rapidly induces the c-fos proto-oncogene in Y-1 adrenocortical cells.Here we show that PMA induces c-fos with similar kinetics when compared with ACTH (0.5–1 h peak) but reaches only 60% of the maximal ACTH induction and dcAMP is a weak c-fos inducer (15% of ACTH). However, combination of PMA and dcAMP has a synergistic effect leading to maximal c-fos induction. c-fos expression may play a role in the RNA synthesis-dependent corticosteroidogenesis response and/or growth regulation by ACTH.We also show that, in contrast to dcAMP, PMA is a poor steroidogenesis stimulator (15 to 17% of maximum ACTH-stimulated level), its activity being completely dependent on RNA synthesis. Combination of dcAMP and PMA yields an additive steroidogenesis stimulation, an effect that is also dependent on RNA synthesis. Although no strict correlation was found between c-fos induction and early steroidogenesis stimulation, particularly with respect to cAMP derivatives, the results suggest that a PKC pathway is likely to cooperate with the classical cAMP-PKA pathway in adrenal cells' RNA-dependent steroidogenesis.Abbreviations ACTH Adrenocorticotropic Hormone - PMA Phorbol-12-Myrystate-13-Acetate - dcAMP dibutyryl cyclic AMP - DME Dulbecco's Modified Eagle's minimal medium - FCS Fetal Calf Serum     

Arachidonic acid metabolism by dioxin-induced cytochrome P-450: a new hypothesis on the role of P-450 in dioxin toxicity

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD) is a highly potent inducer of cytochrome P-450. The role of the induced P-450 in TCDD toxicity has been obscure as P-450 neither detoxifies TCDD nor activates it to genotoxic or cytotoxic metabolites. We show, using a chick embryo model, that TCDD causes major increases in the NADPH dependent metabolism of arachidonic acid (AA), a predominant cell membrane fatty acid, that it does so with extremely high potency (ED50, 6.3 pmol per egg) and that this metabolism is catalyzed by TCDD-induced cytochrome P-450 species. Thus, TCDD treatment increased by six to ten fold the P-450 mediated hepatic microsomal metabolism of AA to epoxides and monohydroxyeicosatetraenoic acids, products whose diverse biological activities suggest links to TCDD's toxic effects. In contrast only x and x-1 hydroxy AA, inactive products, were significantly formed by the controls. These findings open a new perspective on how P-450 induction could be related to the diverse toxic effects of TCDD. They lead to the novel hypothesis that TCDD-induced cytochrome P-450 metabolizes an endogenous fatty acid to reactive products that in turn mediate or modulate varied manifestations of TCDD toxicity.