Cytochrome P-450h Research Articles

Monoclonal Antibodies of Differentiating Specificities as Probes of Cytochrome P450h (2C11)

A panel of 30 monoclonal antibodies against rat hepatic microsomal cytochrome P450h (2C11) has been produced, purified, and characterized. A broad range of reactivities was observed when 13 purified rat cytochrome P450 isozymes were tested for epitope relatedness in a noncompetitive enzyme-linked immunosorbent assay or on immunoblots. Several antibodies were antigen-specific, others reacted with additional members of the 2C subfamily, and other monoclonal antibodies recognized cytochromes P450 from the 2E, 2B, 2A, and 1A subfamilies. Cytochromes P450p (3A1) and P4501 (3A2) did not react with any of the antibodies. A minimum of seven spatially distinct epitopes on cytochrome P450h were defined by the panel of antibodies. Immunoblot analysis of rat microsomes illustrated the male specificity of cytochrome P450h expression which extended to extrahepatic tissues including kidney and lung. A survey of various species by immunoblot analysis with several antibodies revealed little if any epitope relatedness among microsomal proteins from rats, mice, rabbits, hamsters, squirrel monkeys, guinea pigs, or humans. All of the antibodies were screened as potential inhibitors of cytochrome P450h-mediated testosterone hydroxylation in a reconstituted system. Although most of the antibodies were noninhibitory, greater than 70% inhibition of 2a-and 16a-hydroxylation of testosterone was observed with selected antibodies. These inhibitory antibodies gave similar results when benzphetamine-demethylation was evaluated in the reconstituted system. The inhibitory antibodies were then used to assess the role of cytochrome P450h in microsomal benzphetnmine N-demethylation, since this isozyme exhibits high catalytic activity for this substrate. Only 20-25% inhibition of benzphetamine metabolism was attained in microsomal preparations from adult male rats, and the antibodies did not influence the microsomal catalytic activity of immature males or females or adult females. Thus, despite the high level of expression of cytochrome P450h in microsomes from adult male rats and the high catalytic activity of the purified protein for benzphetamine, this isozyme contributes only a small portion of the metabolism of this substrate in microsomes.

Rat liver endothelial and Kupffer cell-mediated mutagenicity of polycyclic aromatic hydrocarbons and aflatoxin B1.

The ability of isolated rat liver endothelial and Kupffer cells to activate benzo(a)pyrene (BP), trans-7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene (DDBP), trans-1,2-dihydroxy-1,2-dihydrochrysene (DDCH), and aflatoxin B1 (AFB1) to mutagenic metabolites was assessed by means of a cell-mediated bacterial mutagenicity assay and compared with the ability of parenchymal cells to activate these compounds. Endothelial and Kupffer cells from untreated rats were able to activate AFB1 and DDBP; DDBP was activated even in the absence of an NADPH-generating system. Pretreating the animals with Aroclor 1254 strongly enhanced the mutagenicity of the dihydrodiol, whereas the mutagenicity of AFB1 showed a slight increase. BP and DDCH were only activated by endothelial and Kupffer cells isolated from Aroclor 1254-pretreated rats. Parenchymal cells from untreated animals activated all four carcinogens tested; Aroclor 1254 enhanced the parenchymal cell-mediated mutagenicity of BP and DDCH but did not affect that of DDBP and clearly reduced that of AFB1. The reduced mutagenicity of AFB1 correlates with the decrease in the amount of 2 alpha-hydroxytestosterone formed when testosterone was incubated with parenchymal cell microsomes from Aroclor 1254-pretreated rats (compared with microsomes from untreated animals): the formation of 2 alpha-hydroxytestosterone is specifically catalyzed by cytochrome P-450h, a hemoprotein thought to be involved in the activation of AFB1. These results show that not only rat liver parenchymal cells, but also endothelial and Kupffer cells, activate several carcinogens to mutagenic metabolites.

Expression and catalysis of sex-specific cytochrome P450 isozymes in rat liver

Research interest in the study of cytochromes P450 has recently been shifting to the characterization of "constitutively" expressed isozymes from that of the inducible forms. Several "constitutive" cytochrome P450 isozymes have been purified from rat liver including five immunochemically related proteins designated cytochromes P450f, P450g, P450h, P450i, and P450k. These hemoproteins have been identified as distinct isozymes on the basis of spectral, electrophoretic, and catalytic properties and NH2-terminal sequence analysis. Purification and immunoquantitation studies have indicated that these isozymes are expressed in a developmental as well as sex-related manner, and are relatively refractory to induction by xenobiotics. Cytochromes P450h and P450g are male-specific proteins, cytochrome P450i is a female-specific isozyme, while cytochromes P450f and P450k are present in both male and female adult rats. In addition, the expression of cytochrome P450g was shown to segregate into two phenotypes in outbred rats. Genetic studies utilizing inbred strains have indicated that the gene responsible for inheritance of high levels of cytochrome P450g is autosomal. Although considerable progress has been made in understanding the role of gonadal hormones and growth hormone in the hepatic regulation of cytochromes P450g, P450h, and P450i in particular, the physiological significance of the "constitutive" isozymes in the liver remains largely unresolved.

Pronounced and differential effects of ionic strength and pH on testosterone oxidation by membrane-bound and purified forms of rat liver microsomal cytochrome P-450

The aim of this study was to determine the effects of ionic strength and pH on the different pathways of testosterone oxidation catalyzed by rat liver microsomes. The catalytic activity of cytochromes P-450a (IIA1), P-450b (IIB1), P-450h (IIC11) and P-450p (IIIA1) was measured in liver microsomes from mature male rats and phenobarbital-treated rats as testosterone 7α-, 16β-, 2α- and 6β-hydroxylase activity, respectively. An increase in the concentration of potassium phosphate (from 25 to 250 mM) caused a marked decrease in the catalytic activity of cytochromes P-450a (to 8%), P-450b (to 22%) and P-450h (to 23%), but caused a pronounced increase in the catalytic activity of cytochrome P-450p (up to 4.2-fold). These effects were attributed to changes in ionic strength, because similar but less pronounced effects were observed with Tris-HCl (which has ∼ 1 3 the ionic strength of phosphate buffer at pH 7.4). Testosterone oxidation by microsomal cytochromes P-450a, P-450b, P-450h and P-450p was also differentially affected by pH (over the range 6.8–8.0). The pH optima ranged from 7.1 (for P-450a and P-450h) to 8.0 (for P-450p), with an intermediate value of 7.4 for cytochrome P-450b. Increasing the pH from 6.8 to 8.0 unexpectedly altered the relative amounts of the 3 major metabolites produced by cytochrome P-450h. The decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h that accompanied an increase in ionic strength or pH could be duplicated in reconstitution systems containing purified P-450a, P-450b or P-450h, equimolar amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. This result indicated that the decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h was a direct effect of ionic strength and pH on these enzymes, rather than a secondary effect related to the increase in testosterone oxidation by cytochrome P-450p. Similar studies with purified cytochrome P-450p were complicated by the atypical conditions needed to reconstitute this enzyme. However, studies on the conversion of digitoxin to digitoxigenin bisdigitoxoside by liver microsomes, which is catalyzed specifically by cytochrome P-450p, provided indirect evidence that the increase in catalytic activity of cytochrome P-450p was also a direct effect of ionic strength and pH on this enzyme. The mechanism by which an increase in ionic strength or pH caused a decrease in testosterone oxidation by cytochromes P-450a, P-450b and P-450h may involve an impairment of the interaction between these enzymes and NADPH-cytochrome P-450 reductase, because the effects of ionic strength and pH were diminished when purified P-450a, P-450b and P-450h were incubated with saturating amounts of NADPH-cytochrome P-450 reductase. The mechanism by which an increase in ionic strength or pH caused an increase in testosterone (and digitoxin) oxidation by cytochrome P-450p is unknown, but may involve a stimulation of the interaction between NADPH-cytochrome P-450 reductase and cytochrome b 5.

Induction of cytochrome P-450 isozymes during long-term nifedipine administration

The wide substrate specificity of the microsomal monooxygenase system, catalyzing oxidation of many xenobiotics and endogenous compounds, is determined by the existence of multiple forms of cytochrome P-450 [13]. A modern approach to the characterization and identification of the individual cytochrome P-450 isozymes is to study specific reactions catalyzed by these hemoproteins. There are substrates whose metabolism is more specific for a particular cytochrome P-450 isozyme. For example, the O-de-ethylation of 7-ethoxyresorufin is catalyzed by cytochrome P-450 c [3] . The steroid hormones testosterone, androstenedione (AD), and progesterone are utilized as specific substrates by the microsomal monooxygenase of the liver, with well-marked regional and stereospecificity of metabolism [16]. The rate of formation of hydroxylated metabolites gives information on activity of different forms of cytochrome P-450. For example, the formation of 16a-OH-AD is catalyzed by cytochrome P-450h, which is characterized only of male rats. The formation of 16~-OH-AD is catalyzed by P-45%, that of 7a-OH-AD by cytochrome P-450a, and that of 6fl-OH-AD by cytochrome P-450p [15]. Thus the specificity of the reaction of metabolism of certain substrates can serve as an important criterion for the identification of many forms of cytochrome P-450. On the other hand, the immunochemical method using monospecific antibodies against individual cytochrome P-450 isozymes makes it quantitative and qualitative determination in microsomes possible. This combined approach can be used in pharmacologic investigations, especially those in which the drug influences the microsomal monooxygenase system. The calcium antagonist nifedipine, widely used in cardiologic practice, is metabolized by cytochrome P-450p in rat liver microsomes [6] or by P-450NF in human liver microsomes [14], which are induced by the synthetic steroid pregnenolone-16carbonitrile and steroids of the glucocorticoid class, such as dexamethasone and macrolide antibiotics [12]. Previously the writers described induction of a cytochrome P-450-dependent monooxygenase system after long-term administration of nifedipine to rats [7]. The aim of the present investigation was to discover what forms of cytochrome P-450 can be induced under these conditions.

Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Cytochrome P450p (IIIA1) has been purified from rat liver microsomes by several investigators, but in all cases the purified protein, in contrast to other P450 enzymes, has not been catalytically active when reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. We now report the successful reconstitution of testosterone oxidation by cytochrome P450p, which was purified from liver microsomes from troleandomycin-treated rats. The rate of testosterone oxidation was greatest when purified cytochrome P450p (50 pmol/ml) was reconstituted with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b 5, 200 μg/ml of a chloroform/methanol extract of microsomal lipid (which could not be substituted with dilauroylphosphatidylcholine), and the nonionic detergent, Emulgen 911 (50 μg/ml). Testosterone oxidation by cytochrome P450p was optimal at 200 m m potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these components was found to influence the catalytic activity of cytochrome P450p. Under these experimental conditions, purified cytochrome P450p converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P450p/min (which is comparable to the rate of testosterone oxidation catalyzed by other purified forms of rat liver cytochrome P450). The four major metabolites were 6β-hydroxytestosterone (51%), 2β-hydroxytestosterone (18%), 15β-hydroxytestosterone (11%) and 6-dehydrotestosterone (10%). The four minor metabolites were 18-hydroxytestosterone (3%), 1βhydroxytestosterone (3%), 16β-hydroxytestosterone (2%), and androstenedione (2%). With the exception of 16β-hydroxytestosterone and androstenedione, the conversion of testosterone to each of these metabolites was inhibited > 85% when liver microsomes from various sources were incubated with rabbit polyclonal antibody against cytochrome P450p. This antibody, which recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-treated rats, did not inhibit testosterone oxidation by cytochromes P450a, P450b, P450h, or P450m. The catalytic turnover of microsomal cytochrome P450p was estimated from the increase in testosterone oxidation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycin or erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). Based on this estimate, the catalytic turnover values for purified, reconstituted cytochrome P450p were 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P450p.

Identification of the cytochrome P-450 isozymes responsible for testosterone oxidation in rat lung, kidney, and testis: evidence that cytochrome P-450a (P450IIA1) is the physiologically important testosterone 7 alpha-hydroxylase in rat testis.

Previous studies have shown that several forms of cytochrome P-450 present in rat liver microsomes oxidize testosterone with a high degree of regio- and stereospecificity. The aim of this study was to characterize the pathways of testosterone oxidation catalyzed by rat extrahepatic microsomes. Lung, kidney, testis, prostate, and brain were isolated from 3- and 14-week-old-male Sprague-Dawley rats. Microsomes from lung, kidney, and testis catalyzed distinctly different pathways of testosterone oxidation, whereas microsomes from prostate and brain failed to hydroxylate testosterone directly in a time- and protein-dependent manner. Lung microsomes from immature and mature rats converted testosterone to 16 alpha-hydroxytestosterone, 16 beta-hydroxytestosterone, and androstenedione. Lung microsomes were shown by Western immunoblot to contain cytochrome P-450b (P450IIB1), which has been shown previously to catalyze these three pathways of testosterone oxidation. Antibody against cytochrome P-450b strongly inhibited (greater than 80%) androstenedione formation and completely inhibited (greater than 95%) the 16 alpha- and 16 beta-hydroxylation of testosterone catalyzed by lung microsomes (as did carbon monoxide and antibody against NADPH-cytochrome P-450 reductase). Kidney microsomes from mature male rats converted testosterone to 2 alpha-hydroxytestosterone, 16 alpha-hydroxytestosterone, and androstenedione, whereas only the latter pathway was catalyzed by kidney microsomes from immature rats. Kidney microsomes from mature male rats were shown by Western immunoblot to contain cytochrome P-450h (P450IIC11), which has been shown previously to convert testosterone to 2 alpha-hydroxytestosterone, 16 alpha-hydroxytestosterone, and androstenedione. Antibody against cytochrome P-450h completely inhibited (greater than 95%) the 2 alpha- and 16 alpha-hydroxylation of testosterone by kidney microsomes, but had little effect on androstenedione formation, which is catalyzed by 17 beta-hydroxysteroid dehydrogenase. Testicular microsomes from mature, but not immature, rats catalyzed the 7 alpha-hydroxylation of testosterone. Previous studies have shown that this reaction is catalyzed in liver microsomes by cytochrome P-450a (P450IIA1). Testicular microsomes from mature, but not immature, rats were shown by Western immunoblot to contain cytochrome P-450a. Antibody against cytochrome P-450a or NADPH-cytochrome P-450 reductase completely inhibited (greater than 95%) the 7 alpha-hydroxylation of testosterone by testicular microsomes. A 90:10 atmosphere of carbon monoxide and oxygen did not appreciably block the 7 alpha-hydroxylation of testosterone by testicular microsomes, wh

Purification of two isozymes of rat liver microsomal cytochrome P450 with testosterone 7α-hydroxylase activity

Cytochrome P450a was purified to electrophoretic homogeneity from liver microsomes from immature male Long-Evans rats treated with Aroclor 1254. Rabbit polyclonal antibody raised against cytochrome P450a cross-reacted with cytochromes P450b, P450e, and P450f (which are structurally related to cytochrome P450a). The cross-reacting antibodies were removed by passing anti-P450a over an N-octylamino-Sepharose column containing these heterologous antigens. The immunoabsorbed antibody recognized only a single protein (i.e., cytochrome P450a) in liver microsomes from immature male rats treated with Aroclor 1254 (i.e., the microsomes from which cytochrome P450a was purified). However, the immunoabsorbed antibody recognized three proteins in liver microsomes from mature male rats, as determined by Western immunoblot. As expected, one of these proteins ( M r 48,000) corresponded to cytochrome P450a. The other two proteins did not correspond to cytochromes P450b, P450e, or P450f (as might be expected if the antibody were incompletely immunoabsorbed), nor did they correspond to cytochromes P450c, P450d, P450g, P450h, P450i, P450j, P450k, or P450p. One of these proteins was designated cytochrome P450m ( M r ~49,000), the other cytochrome P450n ( M r ~50,000). Like cytochrome P450a, cytochrome P450n was present in liver microsomes from both male and female rats. However, whereas cytochrome P450a was detectable in liver microsomes from 1-week-old rats, cytochrome P450n was barely detectable until the rats were at least 3 weeks old. Furthermore, in contrast to cytochrome P450a, the levels of cytochrome P450n did not decline appreciably with age in postpubertal male rats. Cytochrome P450m was detectable only in liver microsomes from postpubertal (>4 week-old) male rats. Cytochromes P450m and P450n were isolated from liver microsomes from mature male rats and purified to remove cytochrome P450a. When reconstituted with NADPH-cytochrome P450 reductase and lipid, cytochrome P450n exhibited little testosterone hydroxylase activity, whereas cytochrome P450m catalyzed the 15α-, 18-, 6β-, and 7α-hydroxylations of testosterone at 10.8, 4.6, 2.0, and 1.9 nmol/nmol P450/min, respectively. The ability of cytochrome P450m to catalyze the 7α-hydroxylation of testosterone was not due to contamination with cytochrome P450a, which catalyzed this reaction at ~25 nmol/nmol P450a/min. Cytochrome P450m also converted testosterone to several minor metabolites, including androstenedione and 15β-, 14α-, and 16α-hydroxytestosterone. The identity of the metabolites formed by cytochrome P450m was based on cochromatography with authentic standards resolved by HPLC. Cytochromes P450a and P450m had identical N-terminal amino acid sequences for the first 20 residues, except that residue 19 was leucine in cytochrome P450a but phenylalanine in cytochrome P450m. This high degree of amino acid sequence homology explains in part the immunochemical relatedness of cytochromes P450a and P450m. The results of this study indicate that cytochromes P450a, P450m, and P450n are immunochemically related proteins present in rat liver microsomes, and that two of these proteins, namely cytochromes P450a and P450m, are structurally related isozymes that can catalyze the 7α-hydroxylation of testosterone. The results of this study also indicate that cytochromes P450a, P450m, and P450n are independently regulated as a function of the age and sex of rats.

Differential expression of multiple forms of cytochrome P-450 in vertebrates: Antibodies to purified rat cytochrome P-450s as molecular probes for the evolution of P-450 gene families I and II

Polyclonal antibodies against rat cytochrome P-450c (P-450IA1), P-450d (P-450IA2), P-450b (P-450IIB1), P-450h (P-450IIC11) and P-450j (P-450IIE1), were used to probe liver microsomes prepared from species in a number of vertebrate groups ranging from the hagfish, a very primitive vertebrate, to the male rat. Cross-reactivity in Western blots was used to examine the appearance of cytochrome P-450 isozymes structurally related to the rat forms in lower vertebrate groups and to provide information on the evolution of cytochrome P-450 gene families I and II and their regulation in lower vertebrates.

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent.

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH-cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemoproteins catalyzed the 16 alpha- and 16 beta-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase*. The activity of cytochrome P-450b and P-450b* increased up to 50% when reconstituted with reductase* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.

Polymorphisms of four hepatic cytochromes P-450 in twenty-eight inbred strains of rat.

Two-dimensional gel electrophoresis of hepatic microsomes from phenobarbital-treated animals was used to analyze electrophoretic/regulatory polymorphisms for cytochromes P-450b, P-450e, P-450g, and P-450h in 28 inbred strains of rat. Previous studies with outbred rats revealed the existence of four electrophoretic variants for P-450b, two for P-450e, and three for P-450h as well as two regulatory alleles for P-450g. With the exception of one allozymic form of P-450h, all of these alleles as well as a novel (null) allele for P-450e were found to be homozygous in at least two of the inbred strains tested. Eight phenotypes for combinations of these four cytochromes P-450 were observed. Inbred strains were identified that can be used in studies on the structure/function of unique cytochrome P-450-allozymes and in genetic crosses to map the four distinct cytochrome P-450 genes.

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

The pathways of testosterone oxidation catalyzed by purified and membrane-bound forms of rat liver microsomal cytochrome P-450 were examined with an HPLC system capable of resolving 14 potential hydroxylated metabolites of testosterone and androstenedione. Seven pathways of testosterone oxidation, namely the 2α-, 2β-, 6β-, 15β-, 16α-, and 18-hydroxylation of testosterone and 17-oxidation to androstenedione, were sexually differentiated in mature rats (male/female = 7–200 fold) but not in immature rats. Developmental changes in two cytochrome P-450 isozymes largely accounted for this sexual differentiation. The selective expression of cytochrome P-450h in mature male rats largely accounted for the male-specific, postpubertal increase in the rate of testosterone 2α-, 16α-, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2β-, 6β-, 15β-, and 18-hydroxylase activity. A variety of cytochrome P-450p inducers, when administered to mature female rats, markedly increased (up to 130-fold) the rate of testosterone 2β-, 6β-, 15β-, and 18-hydroxylation. These four pathways of testosterone hydroxylation were catalyzed by partially purified cytochrome P-450p, and were selectively stimulated when liver microsomes from troleandomycin- or erythromycin estolate-induced rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and these macrolide antibiotics. Just as the testosterone 2β-, 6β-, 15β-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7α-hydroxylase activity reflected the levels of cytochrome P-450a; 16β-hydroxylase activity the levels of cytochrome P-450b; and 2α-hydroxylase activity the levels of cytochrome P-450h. It is concluded that the regio- and stereoselective hydroxylation of testosterone provides a functional basis to study simultaneously the regulation of several distinct isozymes of rat liver microsomal cytochrome P-450.

Mapping of genes for cytochromes P-450b, P-450e, P-450g, and P-450h in the rat. Demonstration of two gene clusters with P450-b,e localized on chromosome 1.

Inbred ACI, WF, and RCS rats having characteristic markers for albino (c), hemoglobin beta-chain (Hbb), and pink-eyed dilution (p) on chromosome 1 and expressing variants for hepatic cytochromes P-450b, P-450e, P-450g, and P-450h were used in genetic mapping studies for these hemoproteins. The results of WF X (ACI X WF)F1 and RCS X (WF X RCS)F1 backcrosses revealed the existence of two gene clusters designated the P450-b,e and P450-g,h loci. The linkage map P450-b,e-p-c-Hbb on rat chromosome 1 was demonstrated and found to be congruent with Coh(P450-b,e)-p-c-Hbb on mouse chromosome 7. P450-g,h is not linked with P450-b,e and the other markers tested on rat chromosome 1. It appears that close genetic linkage, rather than common functional/regulatory properties, typify members of cytochrome P-450 families/subfamilies.

A regulatory polymorphism for rat hepatic cytochrome P-450g

Rat hepatic cytochrome P-450g is a male-specific hemoprotein found at significant levels only in adult animals. In the present study, two-dimensional gel electrophoretic and immunochemical methods were used to study a polymorphism of this isozyme and its ontogenetic regulation. Inbred ACI/Hsd and WF/Hsd rats were found to express high and low levels of cytochrome P-450g, respectively. F 1 hybrids of these strains showed additive inheritance for this trait and the responsible gene was found to be autosomal. Cytochrome P-450g and another male-specific form of the enzyme, cytochrome P-450h, were characterized by a similar time-course for their ontogenetic expressions. However, unlike cytochrome P-450g, the level of cytochrome P-450h was indistinguishable in hepatic microsomes from mature ACI/Hsd and WF/Hsd rats. Considering these results, we tentatively conclude that the gene regulating the level of cytochrome P-450g is Cisacting.

Age- and sex-related expression of cytochromes P450f and P450g in rat liver

We have previously shown that rat hepatic cytochromes P450f, P450g, P450h, and P450i possess a high degree of immunochemical and, presumably, structural relatedness. Polyclonal antibodies directed against cytochromes P450f and P450g were made monospecific by immunoabsorption against the cross-reactive proteins. The specificity of the immunoabsorbed antibodies was established by using Ouchterlony double diffusion analyses, enzyme-linked immunosorbent assays (ELISA), and immunoblots. Since factors regulating the expression of cytochromes P450fand P450g are unknown, a competitive ELISA employing the monospecific antibodies was developed to quantitate each of these isozymes in hepatic microsomes from control and treated rats. The results obtained showed that expression of cytochrome P450f is developmentally regulated in both male and female rat liver. Cytochrome P450f levels rise from less than 1% in young animals to approximately 7 and 14% of total cytochrome P450 in adult male and female rats, respectively. Cytochrome P450g is sex-specific since it is expressed only in male rat liver where it also is developmentally regulated. Levels of cytochrome P450g rise from less than 1% in 3-week-old male rats to an average value of 17% of total cytochrome P450 in 6-week-old adult animals. However, there appear to be at least two subpopulations of adult male Long Evans rats, one of which expresses low levels (<1%) of cytochrome P450g and the other high levels (⩾10%). This expression appears to be independent of serum testosterone levels. Treatment of immature and adult male rats with 20 xenobiotics that are known inducers of certain cytochrome P450 isozymes revealed that cytochromes P450f and P450g are relatively refactory to induction, although Kepone appears to be a weak inducer of cytochrome P450f.

Microheterogeneity of a male-specific rat hepatic cytochrome P-450: Existence of three allozymic forms

Preparations of hepatic cytochrome P-450h [D. E. Ryan, et al. (1984) J. Biol. Chem. 259, 1239] and cytochrome P-450 2c [ D. J. Waxman (1984) J. Biol. Chem. 259, 15481] from outbred Sprague-Dawley rats were anlayzed using two-dimensional electrophoresis and in situ peptide mapping. Both preparations consisted of the same isozyme which was previously characterized as a developmentally regulated, male-specific cytochrome P-450 active in the 16α-hydroxylation of steroids. Each preparation evidenced microheterogeneity which was shown, in part, to result from the existence of two genetically determined variant forms of cytochrome P- 450 h 2 c . Analyses of hepatic microsomes from several inbred strains of rat revealed that each was characterized by a single variant form of this isozyme, with some strains expressing a variant that was not present in Sprague-Dawley rats. Genetic crosses indicated that these electrophoretic variants represent allozymic forms of cytochrome P- 450 h 2 c which are codominantly expressed at a single autosomal locus. Additional microheterogeneity of each allozymic form of cytochrome P- 450 h 2 c was shown to result from a specific in vitro modification that may involve limited proteolysis near its C terminus by a microsome-bound protease.

Evidence for a family of four immunochemically related isozymes of cytochrome P-450 purified from untreated rats

Hepatic microsomal cytochromes P-450f, P-450g, P-450h, and P-450i have recently been purified to electrophoretic homogeneity from untreated adult rats and identified as distinct isozymes [Ryan et al. (1984) J. Biol. Chem. 259, 1239–1250; Haniu et al. (1984) Arch. Biochem. Biophys. 235, 304–311]. In Ouchterlony double-diffusion plates, as well as enzyme-linked immunosorbent assays and Western blots, purified immunoglobulin G (IgG) raised in rabbits against cytochromes P-450f or P-450g show strong cross-reactions with the heterologous protein, indicating apparent partial identity. Anti- P-450f and anti- P-450g also show strong cross-reactivity with cytochromes P-450h and P-450i but not with five previously characterized rat hepatic cytochromes, P-450 (i.e., P-450a- P-450e), indicating a high degree of immunochemical and structural relatedness among cytochromes P-450f, P-450g, P-450h, and P-450i.

NH 2-terminal sequence analyses of four rat hepatic microsomal cytochromes P-450 1

Cytochromes P-450f, P-450g, P-450h, and P-450i are four hepatic microsomal hemoproteins that have been purified from adult rats. Whereas cytochromes P-450g and P-450h appear to be male-specific hemoproteins, cytochrome P-450i is apparently a female-specific enzyme purified from untreated adult female rats. Cytochrome P-450f has been purified from adult male and female rats with equivalent recoveries. Amino-terminal sequence analyses of the first 15–20 amino acid residues of each of these cytochromes P-450 has been accomplished in the current investigation. Each protein possesses a hydrophobic leader sequence consisting of 65–87% hydrophobic amino acids, and only one charged amino acid (Asp) in the ammo-terminal region. Although differences in the amino-terminal sequences of cytochromes P-450f, P-450g, P-450h, and P-450i are identified, these hemoproteins all begin with Met-Asp, and marked structural homology is observed among certain of these enzymes. Cytochromes P-450g and P-450h, two male-specific proteins, have 11–12/15 identical residues with cytochrome P-450i, a female-specific isozyme. Cytochromes P-450f and P-450h have 16/20 identical amino-terminal residues. Only limited sequence homology is observed between the amino-terminal sequences of cytochromes P-450f-i compared to rat liver cytochromes P-450a-e. The results demonstrate that cytochromes P-450f, P-450g, P-450h, and P-450i are isozymic to each other and five additional rat hepatic microsomal cytochrome P-450 isozymes ( P-450a-e).

Characterization of three highly purified cytochromes P-450 from hepatic microsomes of adult male rats.

Three hepatic microsomal cytochromes P-450 (P-450f, P-450g, and P-450h) have been purified to electrophoretic homogeneity from both untreated and ethanol-treated adult male rats. By all criteria examined, the hemoproteins isolated from untreated rats are indistinguishable from the corresponding enzymes purified from rats administered ethanol. Highly purified cytochromes P-450f, P-450g and P-450h are characterized by minimum Mr of 51,000, 50,000, and 51,000, respectively, and unique coordinates in two-dimensional isoelectric focusing-sodium dodecyl sulfate-polyacrylamide gels. The CO-reduced spectral maxima of cytochromes P-450f and P-450g are at 447-448 nm, and the peak of cytochrome P-450h is at 451 nm. Cytochrome P-450h is a versatile catalyst exhibiting high activity toward benzphetamine, hexobarbital, and estradiol-17 beta and moderate activity toward benzo[alpha]pyrene and zoxazolamine. In contrast, cytochromes P-450f and P-450g have low metabolic activity for these substrates. The three hemoproteins catalyze the metabolism of testosterone with different regio- and stereospecificities and overall rates. Both cytochromes P-450f and P-450h catalyze the hydroxylation of testosterone at the 16 alpha-position; however, cytochrome P-450h also oxidizes the steroid at the 2 alpha- and 17 beta-position (androstenedione formation). Testosterone is oxidatively metabolized at the 6 beta-, 15 alpha- and an unknown position by cytochrome P-450g. Peptide maps, generated by proteolytic or chemical digestion of the hemoproteins, indicate that cytochromes P-450f, P-450g, and P-450h differ structurally from each other and five previously characterized rat hepatic microsomal cytochromes P-450 (P-450a, P-450b, P-450c, P-450d, and P-450e). Cytochromes P-450f, P-450g, and P-450h do not react with antibodies directed against these inducible hemoproteins by Ouchterlony immunodiffusion in the presence of detergent; however, in the absence of detergent, cytochrome P-450f cross-reacts weakly with anti-P-450b. Results of this study indicate that rat hepatic microsomal cytochromes P-450 are composed of at least four hemoproteins with CO-reduced absorbance maxima between 447-448 nm. Furthermore, a minimum of four microsomal cytochromes P-450 are now known to 16 alpha-hydroxylate testosterone.