Induction of renal cytochrome P-450 in hepatic microsomes of diabetic rats

We purified two forms of cytochrome P-450 which was induced in hepatic microsomes of diabetic male rates treated with streptozotocin. One of these corresponded to P-450j. The other form, designated P450 DM-2, had a minimum molecular weight 53000 and a CO-reduced absorption maximum at 452 nm. The P450 DM-2 efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was not efficient in metabolizing aminopyrine, 7-ethoxycoumarin, aniline, N-nitrosodimethylamine, or testosterone. The NH2-terminal sequence of P450 DM-2 was identical to that of P450 K-5, the major renal cytochrome P-450. Both forms gave very similar electrophoretic patterns of proteolytic digests. P450 DM-2 and P450 K-5 are closely related forms.     

Characterization of three cytochrome P450s purified from renal microsomes of untreated male rats and comparison with human renal cytochrome P450

Three renal cytochrome P450s (P450 K-2, K-4, and K-5) were purified from renal microsomes of untreated male rats. Also, the human renal cytochrome P450 (P450 HK) was partially purified from renal microsomes and its properties were compared with those of the rat renal cytochrome P450s. The molecular weight of P450 K-2, K-4, and K-5 was 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of P450 K-2, K-4, and K-5 were at 449, 451, and 452 nm, respectively. NH2-terminal sequence analysis of P450 K-2, K-4, and K-5 showed that these forms were different from hepatic cytochrome P450s purified previously. P450 K-2, K-4, and K-5 catalyzed the O-dealkylation of 7-ethoxycoumarin but were not efficient in the hydroxylation of testosterone. Aminopyrine was metabolized by P450 K-2 and K-4 but not by P450 K-5. Lauric acid was metabolized efficiently by all of these forms in the presence of cytochrome b5. The regiospecificity of these forms toward lauric acid was different. P450 K-2 hydroxylated lauric acid only at the (omega-1)-position, not at the omega-position. P450 K-4 and K-5 hydroxylated lauric acid at both the omega- and (omega-1)-positions. The ratios of omega/(omega-1)-hydroxylation activity of P450 K-4 and K-5 were 2.5 and 7.8, respectively. Human P450 HK was purified 220-fold and its specific content was 2.0 nmol/mg of protein. The Soret maxima of P450 HK were at 418 nm for the oxidized form, 416 nm for the reduced form, and 450 nm for the CO-reduced form. P450 HK catalyzed the O-dealkylation of 7-ethoxycoumarin but was not efficient in aminopyrine N-demethylation or testosterone hydroxylation. P450 HK had high lauric acid omega- and (omega-1)-hydroxylation activities in the presence of cytochrome b5, especially omega-hydroxylation. These properties resembled those of P450 K-5 most closely. Anti-P450 K-5 antibody cross-reacted with P450 HK as well as P450 K-5 and only one band was stained on immunostained Western blotting for partially purified P450 HK. The molecular weight of P450 HK was 52,000 on Western blotting.     

Hormonal regulation of rat renal cytochrome P450s by androgen and the pituitary.

The hormonal regulation of rat renal cytochrome P450s, P450 4A2 (K-5) and K-2, was investigated. The level of P450 4A2 in male rats was five times that in female rats and accounted for some 90% of total cytochrome P450, measured photometrically. Lauric acid omega- and (omega-1)-hydroxylation activities of renal microsomes of male rats were also higher than those of female rats. The sex differences in lauric acid hydroxylation activity seemed to arise from the differences in P450 4A2 concentrations, according to an immunochemical study. P450 K-2 was a female-dominant form in rat kidneys. The level of P450 K-2 in renal microsomes of male rats was one-tenth that of P450 4A2. Castration of male rats decreased the levels of P450 4A2 and treatment of castrated male rats with testosterone reversed the decrease. The castration of male rats decreased the lauric acid hydroxylation of the renal microsomes to the level of female rats. The administration of testosterone to castrated male rats reversed the decrease. Hypophysectomy of male rats decreased the level of P450 4A2 and the administration of growth hormone reversed the decrease when intermittent injections mimicking the male secretory pattern were given, although continuous administration mimicking the female secretory pattern did not. Castration of male rats did not affect the level of P450 K-2, but testosterone decreased its level. Hypophysectomy of male rats increased the level of P450 K-2 and growth hormone decreased its level in hypophysectomized rats. These results suggested that the expression of P450 4A2 was regulated by androgen or growth hormone and regulation of P450 4A2 was different from that of P450 K-2. To explore the regulation of renal cytochrome P450 further, testosterone was given to control (intact) or hypophysectomized adult female rats. P450 4A2 was induced in the kidneys of both control and hypophysectomized female rats to close to the level of male rats. Thus, P450 4A2 was directly regulated by testosterone as well as growth hormone, and the regulation of the male-dominant form in rat kidneys was different from that of the male-specific form in the rat liver, which is regulated mostly by growth hormone.     

Purification and NH2-terminal sequence of cytochrome P-450 from kidney microsomes of untreated male rats

The major form of cytochrome P-450, P-450K-5, was purified from kidney microsomes of untreated male rats with high-performance liquid chromatography with anion-exchange and hydroxylapatite columns. The monomeric molecular weight of P-450K-5 was 52000 on SDS-polyacrylamide gel electrophoresis and the CO-reduced absorption maximum was at 452 nm. P-450K-5 catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was inefficient in the N-demethylation of benzphetamine and the O-dealkylation of 7-ethoxycoumarine. The NH2-terminal sequence of P-450K-5 was quite different from cytochrome P-450s purified from rat hepatic microsomes.     

Omega- and (omega-1)-hydroxylation of lauric acid and arachidonic acid by rat renal cytochrome P-450

We resolved four cytochrome P-450s, designated as P450 K-2, K-3, K-4, and K-5, from the renal microsomes of untreated male rats by high-performance liquid chromatography (HPLC) and investigated the lauric acid and arachidonic acid hydroxylation activities of these fractions. P450 K-4 and K-5 had high omega- and (omega-1)-hydroxylation activities toward lauric acid. The ratio of the omega-/(omega-1)-hydroxylation activity of P450 K-4 and K-5 was 3 and 6, respectively. Also, P450 K-4 and K-5 effectively catalyzed the omega- and (omega-1)-hydroxylation of arachidonic acid. P450 K-3 was not efficient in the hydroxylation of either lauric acid or arachidonic acid. P450 K-2 had low omega- and (omega-1)-hydroxylation activities toward arachidonic acid, and efficiently catalyzed the hydroxylation of lauric acid at the (omega-1)-position only, not at the omega-position.     

Changes in the amount of cytochrome P450s in rat hepatic microsomes with starvation

The effects of starvation on the composition of 12 different cytochrome P450s in rat hepatic microsomes were studied with a specific antibody. Changes in the metabolic activity of the microsomes were studied at the same time. P450 DM (P450j) was induced 2.5-fold by a 48-h starvation and its increase reflected the increase of metabolic activity of hepatic microsomes toward aniline, 7-ethoxycoumarin, and N-nitrosodimethylamine. P450 K-5, the major renal cytochrome P450 in untreated male rat, was also induced 2.5-fold by a 48-h starvation. P450 UT-2 (P450h) and P450 UT-5 (P450g), typical male-specific forms, decreased with starvation. P450 UT-2 had high testosterone 2 alpha- and 16 alpha-hydroxylation activities. These activities of hepatic microsomes were reduced with the decrease in P450 UT-2. P450 PB-1, testosterone 6 beta-hydroxylase, was increased time-dependently by starvation. P450 UT-4 (RLM2), a minor male-specific form, was not changed by starvation. P450 PB-2 (P450k), present in both sexes, was changed little by starvation. P450 PB-4 (P450b) and P450 PB-5 (P450e) are strongly induced in rat liver by phenobarbital in coordinate fashion. Starvation increased P450 PB-4 12-fold but reduced P450 PB-5 to 22% of the control level. P450 MC-1 (P450d) was decreased by starvation. P450 MC-5 (P450c) was barely detected in control rats and was not changed by starvation. P450 IF-3 (P450a), rich in immature rats, was increased by starvation, accompanied by an increase in testosterone 7 alpha-hydroxylation activity in the hepatic microsomes. We further investigated whether new cytochrome P450s appeared upon starvation by comparison of chromatographic profiles of cytochrome P450 from starved rats with those of cytochrome P450 from control rats using HPLC. Three new cytochrome P450s were detected in the starved rats. These cytochrome P450s were purified to homogeneity. One of them was P450 DM, judging from spectral properties, catalytic activity, and the NH2-terminal sequence. The two other forms were designated P450 3b and 4b. The minimum molecular weights of P450 3b and 4b were 53,000 and 52,000, respectively, and their CO-reduced absorption maxima were at 449 and 452 nm, respectively. P450 3b metabolized aminopyrine, N-nitrosodimethylamine, 7-ethoxycoumarin, and lauric acid, but with low activity. P450 4b was efficient in lauric acid omega- and (omega-1)-hydroxylation only. The spectral properties, catalytic activity, peptide map, and NH2-terminal sequence of P450 4b agreed with those of P450 K-5. P450 3b was a new cytochrome P450, judged by these criteria.     

Purification and characterization of rat pulmonary cytochrome P-450

The pulmonary cytochrome P-450, P450 L-2, was purified 460-fold from pulmonary microsomes of untreated male rats. Its specific content was 10.6 nmol/mg of protein. The monomeric molecular weight was 54,000 on SDS-polyacrylamide gel electrophoresis. The CO-reduced absorption maximum of P450 L-2 was at 451 nm, and the oxidized heme iron appeared to be in the low-spin state, as deduced from the Soret maximum at 421 nm. P450 L-2 had high lauric acid omega- and (omega-1)-hydroxylation activities, but low prostaglandin A1 omega- and (omega-1)-hydroxylation activities. It catalyzed the O-dealkylation of 7-ethoxycoumarin, but was not efficient in the hydroxylation of testosterone or the N-demethylation of aminopyrine. The NH2-terminal amino acid sequence of P450 L-2 was V-L-N-F-L-X-P-X-L (X being an unidentified residue). The catalytic properties of P450 L-2 resembled those of P450 K-5, the major rat renal cytochrome P-450. However, anti-P450 K-5 antibody did not cross-react with P450 L-2, and these forms had different NH2-terminal sequences. To judge from the results of NH2-terminal sequence analysis, P450 L-2 seems to be placed in the IVB gene family. Also, P-450 IIB1 was detected by immunoblotting in one of the peaks on ion-exchange HPLC during the purification of P450 L-2, suggesting the presence of P-450 IIB1 in rat pulmonary microsomes.     

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

Constitutive testosterone 6 beta-hydroxylase in rat liver

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 2 alpha-, 2 beta-, 6 beta-, 15 beta-, 16 alpha-, 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 2 alpha-, 16 alpha, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2 beta-, 6 beta-, 15 beta-, 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 2 beta-, 6 beta-, 15 beta-, 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 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7 alpha-hydroxylase activity reflected the levels of cytochrome P-450a; 16 beta-hydroxylase activity the levels of cytochrome P-450b; and 2 alpha-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.     

Expression of four phenobarbital-inducible cytochrome P-450s in liver, kidney, and lung of rats

Specific antibodies were prepared against cytochromes P450 PB-1, PB-2, PB-4, and PB-5 purified from hepatic microsomes of male rats treated with phenobarbital. With these antibodies, the levels of these four cytochrome P450s in hepatic, renal, and pulmonary microsomes of male rats that were untreated, treated with phenobarbital, or treated with 3-methylcholanthrene were examined. P450 PB-1 and PB-2 were present in moderate amounts in hepatic microsomes of untreated male rats and were induced 2- to 3-fold with phenobarbital. Also, the expression of these forms was suppressed by 3-methylcholanthrene. These forms were not detected in the renal or pulmonary microsomes of untreated rats or rats treated with phenobarbital or 3-methylcholanthrene. P450 PB-4 and PB-5 were found in the hepatic microsomes of untreated male rats at a low level but were induced with phenobarbital more than 50-fold. P450 PB-4 and PB-5 were not detected in renal microsomes; only P450 PB-4 or a closely related form was present in the pulmonary microsomes of untreated male rats, and its level was not changed by phenobarbital treatment. The constitutive presence of P450 PB-4 in pulmonary microsomes was confirmed by the investigation of testosterone metabolism. Purified P450 PB-4 had high testosterone 16 alpha- and 16 beta-hydroxylation activity in a reconstituted system. The testosterone 16 beta-hydroxylation activity of hepatic microsomes was induced with phenobarbital, and more than 90% of the testosterone 16 beta-hydroxylation activity of hepatic microsomes from rats treated with phenobarbital was inhibited by anti-P450 PB-4 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isozyme specificity of testosterone 7 alpha-hydroxylation in rat hepatic microsomes: is cytochrome P-450a the sole catalyst?

In the present study we show that monospecific antibody against cytochrome P-450a completely inhibits testosterone 7 alpha-hydroxylation in hepatic microsomes of untreated male or female rats or rats of either sex treated with dexamethasone. These data are in contrast with those of K. Nagata et al. (1987, J. Biol. Chem. 262, 2787-2793) who recently reported that an antibody prepared against cytochrome P-450a completely inhibited testosterone 7 alpha-hydroxylase activity in microsomes from untreated or 3-methylcholanthrene-treated rats but only inhibited 50% of the activity in microsomes from dexamethasone-treated rats. They proposed that dexamethasone treatment of rats induced another testosterone 7 alpha-hydroxylase in rat liver. The discrepancy in the two sets of data was due, at least in part, to the use of a chromatography system by Nagata et al. that is incapable of resolving a number of testosterone metabolites. Dexamethasone treatment of rats leads to a marked increase in the production of several testosterone metabolites, including 15 beta-hydroxytestosterone which is cochromatographic with 7 alpha-hydroxytestosterone in their chromatography system. Our results indicate that cytochrome P-450a accounts for all of the testosterone 7 alpha-hydroxylase activity in microsomes from dexamethasone-treated rats, and that testosterone 7 alpha-hydroxylation continues to be a useful marker for monitoring cytochrome P-450a in rat hepatic microsomes.     

Renal corticosterone 6β-hydroxylase in the spontaneously hypertensive rat

Excess 6β-OH-corticosterone production by family 3A cytochromes P-450 may play a role in genesis of hypertension in the spontaneously hypertensive rat (SHR), by producing a renal defect in Na⁺ excretion. Renal cytochromes P-450 may be a causal factor in this genetic model. Since family 3A P-450 is present in rat kidney (collecting duct), the renal family 3A catalytic (6β-OHase) and immunoreactive activities were compared in SHR and normotensive control (Wistar-Kyoto; WKY) rats. Corticosterone 6β-hydroxulation is markedly higher in SHR than in WKY renal microsomal preparations. Western blot analysis with antibodies to rat and rabbit liver family 3A isoforms demonstrated related proteins. Densitometry revealed greater relative intensity of staining in SHR compared to WKY with both antibodies. Both antibodies inhibited corticosterone 6β-hydroxylation by SHR renal microsomes. Increased renal 6β-OH-corticosterone production by increased renal family 3A cytochromes P-450 may play a role in the blood pressure elevation in SHR.     

Genetic regulation of testosterone 15 alpha-hydroxylase (cytochrome P-450(15)alpha) in renal microsomes of female mice

P-450(15)alpha is a form of cytochrome P-450 purified from liver microsomes of female 129/J mice that is specific for oxidation of testosterone to its 15 alpha-hydroxylated product. Testosterone 15 alpha-hydroxylase activity that was inhibited by anti-P-450(15)alpha antibody was approximately 50 times higher in renal microsomes from 129/J than in BALB/cJ females. Western blots of renal microsomes using anti-P-450(15)alpha antibody showed the presence of immunoreactive protein with a molecular weight identical with that of hepatic P-450(15)alpha in 129/J but not in BALB/cJ female mice. To investigate the genetic basis for the strain differences in this activity, the distribution of P-450(15)alpha-dependent testosterone 15 alpha-hydroxylase activity in renal microsomes from individual females of 129/J and BALB/cJ, of F1 offspring of these strains, and of F1 back-crosses to the progenitor strains were determined. The results were consistent with a sex-related autosomal dominant regulation of the higher activity in 129/J females by a single locus, designated Rsh (regulation of steroid hydroxylase). The amounts of immunochemically cross-reactive P-450(15)alpha protein were linearly correlated with testosterone 15 alpha-hydroxylase activities in renal microsomes from Rsh heterozygotes and homozygotes. At least twice as much mRNA, which hybridized with the cDNA clone for hepatic P-450(15)alpha, was detected in 129/J and 129CF1/J compared to BALB/cJ female kidneys. The evidence suggests a pretranslational regulation of the P-450(15)alpha isozyme in the female mouse kidney by the Rsh locus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-dependent expression of cytochrome P-450s in rat liver

Age-related changes in the levels of multiple forms of cytochrome P-450 as well as in the testosterone hydroxylation activities of hepatic microsomes of male and female rats of different ages from 1 week to 104 weeks (24 months) were investigated. The total cytochrome P-450 measured photometrically did not change much with age in either male and female rats. Testosterone 2 alpha-, 2 beta-, 6 beta-, 15 alpha-, 16 beta-hydroxylation activities of male rats were much higher than those in female rats and were induced developmentally. These activities in male rats declined with aging to the very low level in female rats by 104 weeks of age. Testosterone 7 alpha-hydroxylation activity was maximum at 3 weeks of age in rats of both sexes. The levels of individual cytochrome P-450s were measured by immunoblotting. P450IA1 and IA2 (3-methylcholanthrene-inducible forms) and P450IIB1 and IIB2 (phenobarbital-inducible forms) were detected at low levels in rats of both sexes at all ages. P450IIA2, IIC11 and IVA2 were detected in male rats only and were induced developmentally. These male-specific forms disappeared in male rat liver at 104 weeks of age. P450IIC12, a typical female-specific form, was induced developmentally in female rats and was also detected in male rats at 3 and 104 weeks of age. P450IIIA2 (testosterone 6 beta-hydroxylase) was induced developmentally in male rats, but disappeared when the rats were 104 weeks of age. In female rats, P450IIIA2 was detected only at 1 and 3 weeks of age. P450IIA1, IIC6, IIE1 and IVA3 were detected in rats of both sexes at any age. P450IIC6 and IVA3 were induced developmentally and detected at a similar level in rats of both sexes. The level of P450IIA1 was maximum at 3 weeks of age in rats of both sexes. The changes in the level of P450IIE1 during aging were small compared with the changes in other cytochrome P-450s used in this study. These observations provide concrete evidence to our earlier hypothesis that each of the forms of cytochrome P-450 in male rats alter with aging in different patterns resulting in a practical feminization of over-all cytochrome P-450 composition at old age.     

Regulation of three forms of cytochrome P-450 and epoxide hydrolase in rat liver microsomes. Effects of age, sex, and induction

A procedure for the preparation of monospecific antibody directed against rat liver microsomal cytochrome P-45-a is described. This antibody, together with monospecific antibodies to cytochromes P-450b and P-450c, has been used to show that these three forms of cytochrome P-450 are distinct and share no common antigenic determinants. These antibodies (a) give single immunoprecipitin bands with detergent-solubilized microsomes; (b) do not cross-react with the purified heterologous antigens in Ouchterlony double diffusion analyses; (c) have no effect on catalytic activity of the heterologous antigens but completely inhibit the enzymatic activity of the homologous antigens; and (d) remove only the homologous antigen from detergent-solubilized microsomes when covalently bound to a solid support. With radial immunodiffusion assay, we have quantitated these three forms of cytochrome P-450 in liver microsomes after treatment of rats with seven different inducers of cytochrome P-450. The levels of these cytochrome P-450 isozymes vary independently and are also regulated by the age and sex of the animal. The antibodies have also been used to assess the contribution of cytochromes P-450a, P-450b, and P-450c in the metabolism of xenobiotics by rat liver microsomes. A large proportion of benzo(a)pyrene metabolism and testosterone 16 alpha-hydroxylation in microsomes from untreated rats is not catalyzed by cytochromes P-450a, P-450b, and P-450c. Epoxide hydrolase, another microsomal enzyme involved in the metabolism of xenobiotics, was also quantitated by radial immunodiffusion after prior treatment of rats with microsomal enzyme inducers. The inductions of epoxide hydrolase varies independently of the induction of cytochromes P-450a, P-450b, and P-450c.     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

Decrease in the levels of a constitutive cytochrome P-450 (RLM5) in hepatic microsomes of diabetic rats

Cytochrome P-450 dependent hydroxylation of testosterone has been measured in hepatic microsomes of control, diabetic and insulin-treated diabetic rats. The observed decrease in testosterone 16 alpha-hydroxylase activity in diabetes, an activity previously shown to be largely due to RLM5, was accompanied by a dramatic decrease in immunodetectable RLM5. Diabetic rats which received insulin had elevated testosterone 16 alpha-hydroxylase activity relative to the diabetic animals, which was accompanied by a corresponding increase in the levels of RLM5. These results provide evidence that specific constitutive cytochrome P-450 enzymes are altered in the diabetic state and that these changes are not permanent since they can be overcome, at least partially, by insulin replacement therapy.     

Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily

Polyclonal antibody has been shown previously to react identically with cytochromes P-450b and P-450e purified from Long Evans rats and a strain variant of cytochrome P-450b purified from Holtzman rats (P-450bH). In the present study, an array of 12 different monoclonal antibodies produced against cytochrome P-450b has been used to distinguish among these closely related phenobarbital-inducible rat hepatic cytochromes P-450. In immunoblots and enzyme-linked immunosorbent assays, 10 monoclonal antibodies bind to cytochromes P-450b, P-450e, and P-450bH; one monoclonal antibody (B50) recognizes cytochromes P-450b and P-450bH but not cytochrome P-450e; and one monoclonal antibody (B51) is specific for cytochrome P-450b. In addition, one monoclonal antibody (BEF29) reacts strongly with cytochrome P-450f, and another antibody (BEA33) reacts weakly with cytochrome P-450a. No cross-reactions with cytochromes P-450c, P-450d, and P-450g-P-450j were detected with any of the monoclonal antibodies in these assays. Six spatially distinct epitopes on cytochrome P-450b were identified, and differences in antibody reactivity provided evidence for three additional overlapping epitopes. Several monoclonal antibodies are potent inhibitors of testosterone and benzphetamine metabolism supported by cytochrome P-450b in a reconstituted system. B50 and BE52 do not inhibit metabolism of the two substrates by microsomes from untreated rats, but inhibit benzphetamine N-demethylation and testosterone metabolism to 16 alpha- and 16 beta-hydroxytestosterone as well as androstenedione formation 67-94% by microsomes from phenobarbital-treated rats. No other pathways of testosterone metabolism are inhibited by these monoclonal antibodies. The differential inhibition of microsomal metabolism of benzphetamine and testosterone by these monoclonal antibodies is a reflection of the content and inducibility of cytochromes P-450b and P-450e as well as other cytochrome P-450 isozymes.     

Regiospecificity in the hydroxylation of lauric acid by rainbow trout hepatic cytochrome P450 isozymes

The catalytic activity of two hepatic cytochrome P450 isozymes from untreated rainbow trout towards lauric acid was investigated. In a reconstituted system, cytochrome P450 LMC1 and P450 LMC2 were found to catalyze exclusively the omega- and (omega-1)-hydroxylation of lauric acid, respectively. Microsomal enzyme inhibition studies with polyclonal antibodies raised against the individual P450 isozymes showed that P450 LMC1 and LMC2, respectively, accounted for most if not all the omega- and (omega-1)-lauric acid hydroxylase activity of trout liver microsomes. The polyclonal antibodies were highly specific in that they only inhibited the enzyme activity of the P450 used as the immunogen. These results illustrate that as in mammals, omega- and (omega-1)-hydroxylation of lauric acid by trout liver microsomes can be carried out separately by distinct isozymes of cytochrome P450.