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An improved assay for the determination of benzo[ a ]pyrene monooxygenase is described. This radiometric assay is a modification of the assay of DePierre, J. W., Moron, M. S., Johannesen, K. A. M., and Ernster, L. [(1975) Anal. Biochem. 63, 470] and uses trifluoroacetic acid to convert arene oxides which have been extracted into the hexane phase into phenols. These phenols are then extracted into the basic (product) phase. This assay method gives results comparable to those obtained by high-pressure liquid chromatography under a wide variety of conditions and, therefore, is generally applicable to the determination of benzo[ a ]pyrene monooxygenase, especially in the absence of arene oxide hydrase or in the presence of arene oxide hydrase inhibitors.

The contributions of protein synthesis and formation of microtubules and microfilaments to corticotropin-stimulated steroidogenesis in rat adrenal cell suspensions has been assessed by use of a series of inhibitors to each function. Five inhibitors of protein synthesis (cycloheximide, puromycin, blastocidin S, anisomycin, and trichodermin) each exhibited time-dependent inhibition of corticotropin-stimulated steroidogenesis. For the first 30 min, steroidogenesis was more extensively inhibited than protein synthesis, after which the effectiveness of the inhibitors diminished on steroidogenesis but not on protein synthesis. The reversal effect was not observed at high levels of inhibitors. One inhibitor of microfilament formation (cytochalasin B) and four inhibitors of microtubule formation (colchicine, podophyllotoxin, vinblastine sulfate and griseofulvin) inhibited steroidogenesis without inhibiting protein synthesis and without any reversal effect with prolonged incubation. The actions of all ten inhibitors were shown to be fully reversible. Cell superfusion of adrenal cells showed that the decay of steroidogenesis upon addition of all the protein synthesis inhibitors was similar to decay upon removal of corticotropin from the medium (t1/2 = 4--6 min). Recoveries from inhibition upon removal of the inhibitors were similar to each other and comparable to initial corticotropin stimulation of the cells (lag of 3--5 min, t1/2=7--9 min). Similar kinetics of inhibition and recovery were observed for vinblastine sulfate while a direct inhibition of cytochrome P-450scc by aminoglutethimide was complete within 1 min and was rapidly reversed. Injection of each inhibitor (all classes) into hypophysectomized rats inhibited the elevation of plasma corticosterone by corticotropin. The extent of cholesterol combination with cytochrome P-450scc in adrenal mitochondria isolated from these rats was also decreased by all of the inhibitors. Decreases in plasma corticosterone correlated directly with decreases in cholesterol combination with cytochrome P-450scc (r=0.94). It is concluded that protein synthesis and steroidogenesis must be intimately coupled probably due to the requirement of a labile protein for cholesterol transport to cytochrome P-450scc. An involvement of microtubules and microfilaments in this process is clearly indicated.

Rat adrenal mitochondria exhibit a linear 2-fold accumulation of cholesterol for 20 min following either in vivo ether stress or ACTH administration, providing cholesterol metabolism is inhibited by aminoglutethimide (AMG). Additional cycloheximide (CX) pretreatment only slightly decreases this increase, but the location of accumulation shifts from the inner membrane to the outer membrane, implying a decreased cholesterol transfer from outer to inner membrane. Although the capacity of outer mitochondrial membranes was saturated after a 10-min treatment with CX, a 20-min treatment resulted in further retention of cholesterol in intact mitochondria that was not recovered in the isolated membranes. An additional pool of loosely bound cholesterol is proposed for CX mitochondria. These studies provide evidence that the CX-sensitive step of adrenal steroidogenesis attributed to loss of a labile ACTH regulatory protein (Pedersen, R.C. and Brownie, A.C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1882-1886) involves cholesterol transfer from the outer to the inner mitochondrial membrane. ACTH also enhances the PI and PE content of the outer membranes by a CX-sensitive mechanism that may contribute to intramitochondrial cholesterol transport. CX treatment does not affect cholesterol uptake by the inner membrane from phospholipid vesicles. The initial rate of endogenous metabolism in isolated inner membranes is insensitive to pretreatment (2 nmol/nmol P-450/min). The duration of this linear rate was increased 4-fold by AMG treatment while this increase was prevented by CX treatment. The kinetics indicate differences in inner membrane reactive cholesterol levels. Inner membranes also contained a fraction of unreactive cholesterol that is insensitive to pretreatment. Cholesterol-P-450scc complex formation for all pretreatments fits a single hyperbolic function of the reactive cholesterol content of the inner mitochondrial membrane (Kd = 0.025 mol cholesterol/mol phospholipid), and is activated over 5-fold upon mitochondrial disruption. All changes in inner membranes caused by CX can, therefore, be attributed solely to the restricted cholesterol access in vivo.

Conversion of benzo[ a ]pyrene (BP) to BP 7,8-dihydrodiol 9,10-oxides (DE) (measured as 7,10/8,9-tetrols) by untreated (UT) rat liver microsomes is over 10 times slower than following 3-methylcholanthrene (MC) induction. Time courses have been subjected to a kinetic analysis analogous to that previously reported for metabolism by MC-induced microsomes (J. Biol. Chem., 259 (1984) 13770–13776). Competition between BP and 7,8-dihydrodiol for P -450 is the major determinant of the rate of DE formation. Glucuronidation of quinones and phenols only increases the isolated BP metabolites including DE by 40%. This indicates far less inhibition by these products than for metabolism in MC-microsomes (4–6-fold). Thus stimulation may result from a decreased quinone-mediated oxidation of metabolites. In the presence of DNA, UT-microsomes metabolize BP to approximately equal amounts of 9-phenol-4,5-oxide (9-PO) and DE/DNA adducts. Addition of uridine diphosphoglucuronic acid (UDPGA) fails to enhance modification of DNA by DE, but formation of the 9-PO adduct is reduced as a result of lower free 9-phenol levels. The kinetic characteristics of BP metabolism by UT-microsomes are highly sensitive to the presence of very small but variable amounts (2–25 pmol/mg) of the very active cytochrome P -450 c , which is the predominant form in MC-microsomes. The major effect of elevated levels of P -450 c is an 8-fold increase in DE formation at low concentrations of BP due to a lowering of K m (7.9–2.6 μM) and an increase in the regioselectivity for DE formation from 7,8-dihydrodiol (5–15% of total BP metabolites). The formation of DE was directly correlated with the content of P -450 c ( r = 0.94). The presence of increased levels of P -450 c in UT-microsomes is probably due to previous exposure of the animals to environmental inducers and is minimized by controlled housing and feeding.

Seven different forms of cytochrome P-450 have been purified from rat liver microsomes. The major 3-methylcholanthrene (MC) inducible cytochrome P-450 (form c) exhibits the greatest activity toward both benzo[a]pyrene (BP) (58 min-1) and 7,12-dimethylbenz[a]anthracene (DMBA) (29 min-1) and forms substantially high spin, high affinity complexes (Kd = 10 nM) with both hydrocarbons. Cytochrome P-450d, a minor MC-inducible form, has far lower activity for metabolism of both polycyclic aromatic hydrocarbons (PAH), yet also forms high affinity complexes (Kd approximately 100 nM) with both PAH, retaining the full high spin state of the free cytochrome. Although two phenobarbital (PB)-induced forms (P-450's b and e) differ by only 13 amino acids, they exhibit significant differences in metabolism of PAH and in complex formation. Whereas P-450b is only active in metabolism of DMBA (9.8 min-1 versus 1.9 min-1 for BP), P-450e has low activity for both substrates (3.3 and 1.2 min-1). Nevertheless, P-450e forms a high affinity complex (Kd approximately 100 nM) with both PAH that enhances the proportion of the high spin state (from 30% to 70%). Failure to displace n-octylamine (NOA) suggests binding that is removed from the heme. P-450b remains low spin in the presence of PAH and NOA is again not displaced. In addition, the two forms can be distinguished by their regioselectivities for both PAH. P-450's a, h, and pregnenolone-16 alpha-carbonitrile (PCN) exhibit little activity toward BP or DMBA, but P-450 PCN does form a low spin complex with BP (not DMBA). Regioselectivity in metabolism of DMBA by PB-induced microsomes does not agree with that of the major constituent forms. Only the minor, less active purified forms (e and a) mediate substantial 12-hydroxylation and 3,4-epoxidation of DMBA. Thus, additional factors in microsomal reactions must contribute to these differences.

The biosynthesis of sterols involves a series of both simple and complex cytochrome P-450-catalyzed monooxygenase reactions. These processes are generally distinguished from the multiplicity of reactions catalyzed by hepatic microsomal forms of P-450 by involving P-450 isozymes that exhibit a high degree of regio- and stereospecificity. The cell specificity of these monooxygenases has been noted in the previous chapter. In this chapter, we will examine the biochemical control of these steroidogenic cytochromes P-450. This is far more stringent than for the hepatic cytochromes P-450 because of the physiological importance of steroid hormones and bile acids. While the biosynthesis of these cytochromes is under hormonal control, this response takes a minimum of 12 hr for steroidogenic cytochromes P-450 (see Chapter 10). More common fluctuations in activity are required within minutes or hours. Here we will discuss the mechanisms of steroid hydroxylation and also some of the proposed mechanisms for rapid physiological control of steroidogenic enzymes.

Rat adrenal mitochondria accumulated cholesterol during ether stress in vivo when side-chain cleavage was inhibited by aminoglutethimide (control = 14.6 vs. aminoglutethimide = 26.5 micrograms of cholesterol per mg of protein). This accumulation was insensitive to simultaneous administration of cycloheximide (24.2 micrograms/mg), but side chain cleavage in the mitochondria was greatly decreased. Outer and inner mitochondrial membrane fractions were separated by discontinuous Ficoll gradient centrifugation. Quantitation of marker enzymes for inner, outer, and microsomal enzymes indicated that outer membranes contained less than 5% inner membranes. The inner membrane fraction contained less than 7% outer membrane and included 90% of mitochondrial cytochrome P-450. Electron microscopy revealed outer membranes as circular intact ghosts, whereas inner membranes were largely intact and retained vesicular structure typical of intact adrenal cortex mitochondria. Administration of aminoglutethimide effected a 2-fold increase in inner membrane cholesterol (9.4 vs. 20.1 micrograms/mg) but simultaneous administration of cycloheximide completely blocked this increase (10.9 micrograms/mg). We conclude that: (i) in the presence of aminoglutethimide, stress stimulates accumulation of cholesterol in the inner membrane of adrenal mitochondria; and (ii) transfer of cholesterol from outer to inner membranes requires a cycloheximide-sensitive agent.

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 (~1 nmol/nmol P450e/min).

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-450sce only increases after 12 h. Mitochondrial-free cholesterol levels increased during the same time period (16.5–25 μg/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 (< 10min) cellular steroidogenesis, cholesterol SCC activity, and access of cholesterol to cytochrome P-450sce without affecting mitochondrial-free cholesterol. Exposure of confluent cultures to the potent environmental toxicant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (1O−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 μg/mg), pretreatment of cells with TCDD caused a decrease in mitochondrial cholesterol (13-8 μg/mg). The effect of TCDD was produced relatively rapidly ((t12 ∼ 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. Examination of the effects of in vivo treatment of rats with ACTH and cycloheximide on isolated rat adrenal mitochondria supports the hypothesis that ACTH stimulates highly labile regulatory protein(s) that modulate transfer of cholesterol between mitochondrial membranes and possibly also the phospholipid content of the outer membrane. Studies from several laboratories suggest that SCP2 and phosphatidyl inositides may exert additional synergistic effects on cholesterol transfer to P-450sec while fatty acid binding protein and Apo E1 may be candidates for mitochondrial-cholesterol storage and removal. Bovine adrenal cells offer an excellent model to study these processes further.

Vinblastine (antimicrotubular agent) and cytochalasin B (antimicrofilament agent) block the build up of adrenal mitochondrial cholesterol seen in the presence of AMG. ACTH stimulated steroidogenesis is inhibited in vivo by both agents via a reduction in the transfer of intra-adrenal cholesterol to adrenal mitochondria, resulting in a decrease in the synthesis of adrenal steroids. Both inhibitors also decrease ACTH stimulated formation of cholesterol cytochrome P450SCC complex in adrenal mitochondria, as determined by difference spectroscopy. The effects of these inhibitors contrast with the actions of protein synthesis inhibitors which decrease cholesterol binding to P450SCC while increasing mitochondrial cholesterol content.

Rat mammary epithelial cells (RMEC) in culture have been shown to activate polycyclic aromatic hydrocarbon (PAH) carcinogens. This study investigates the role of mammary cytochrome P-450 monooxygenases in these metabolic processes. Monooxygenation of 7,12-dimethylbenz[a]anthracene (DMBA) by RMEC in culture exhibited a 6-h lag period before reaching a constant rate. The mechanism for this time-dependent expression of DMBA monooxygenase activity was investigated in lysed cells, where both conjugation and in situ induction of P-450 are prevented. Although metabolism of DMBA by untreated RMEC lysates was undetectable (less than 1 pmol/mg cell protein/h), prior exposure of cultured cells to benz[a]anthracene (BA) induced DMBA metabolism, (approximately 100 pmol/mg cell protein/h). BA pretreatment also eliminated the lag period for metabolism of DMBA by cultured RMEC but did not prevent additional induction of DMBA monoxygenase activity by the substrate. The distribution of monooxygenated DMBA metabolites formed by BA-induced cell lysates was clearly different from that obtained with purified P-450c, the predominant PAH-inducible isozyme in rat liver. For example, the carcinogen precursor DMBA 3,4-dihydrodiol, which is not formed by P-450c, was a clearly detectable product in RMEC. The low epoxide hydratase activity of BA-induced lysate (approximately 400-fold lower compared to that in the liver) limited formation of all DMBA dihydrodiols. The formation of DMBA 3,4-dihydrodiol increased by 5-fold following addition of exogenous purified epoxide hydratase. The DMBA monooxygenase activity of BA-induced RMEC lysates was completely inhibited by alpha-naphthoflavone but was only partially inhibited (50%) by a polyclonal antibody raised against cytochrome P-450c. Anti P-450c completely inhibited formation of some of the metabolites, partially inhibited formation of others and notably stimulated formation of DMBA 3,4-dihydrodiol by 60%. A polyclonal antibody that recognized both rat hepatic P-450a and a group of P-450 isozymes related to P-450h, and which totally inhibited DMBA 3,4-dihydrodiol formation by rat liver microsomes, did not inhibit formation of any DMBA metabolite in RMEC, including DMBA 3,4-dihydrodiol. Western blot analyses of RMEC homogenates demonstrated that BA pretreatment induces P-450c, but not P-450a or any of the P-450h-related isozymes. We conclude that metabolism of DMBA by RMEC depends on induction of P-450c and at least one additional form of cytochrome P-450 which is immunochemically distinct from rat hepatic P-450a and P-450h related isozymes, but is sensitive to alpha-naphthoflavone.

Summary The acute effect of ether stress on cholesterol side chain cleavage and cytochrome P-450 has been studied in intact rat adrenal mitochondria using light absorption and EPR spectroscopy. The effect of stress is to increase the ratio of high-spin to low-spin oxidized cytochrome P-450 and this is accompanied by an increase in the rate of pregnenolone synthesis from adrenal mitochondrial cholesterol. Cycloheximide pre-treatment prevented these effects of ether stress. The action of ACTH appears to involve an increase in the association of cholesterol with cholesterol side chain cleavage cytochrome P-450.