Generation of specific antibodies and their use to characterize sex differences in four rat P450 3A enzymes following vehicle and pregnenolone 16alpha-carbonitrile treatment

The purpose of this study was to identify isozyme-specific antibodies and use them to determine the expression levels of four P450 3A enzymes in the livers of vehicle- and pregnenolone 16alpha-carbonitrile (PCN)-treated rats of both sexes, since previous work on mRNA levels has shown considerable sexual dimorphism. Using Western blot analysis with four isozyme-specific antibodies, we show that P450 3A1, 3A2, and 3A9 were expressed in vehicle-treated adult female rats at very low levels whereas P450 3A18 was not detected. PCN treatment of females strongly induced the expression of P450 3A1 in the livers with protein product increases of 214-, 3-, and 5-fold for P450 3A1, 3A2, and 3A9, respectively, and P450 3A18 was induced to 3.7 pmol/mg protein. In contrast, all four P450 3As were detected in livers of vehicle-treated males, in the order of 3A2 > 3A18 > 3A9 approximately = 3A1. The protein product increases induced by PCN treatment of male rats were 92-, 3-, 6-, and 16-fold for P450 3A1, 3A2, 3A9, and 3A18, respectively.     

Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1

We recently reported that antibody against purified P450 3A1 (P450p) recognizes two electrophoretically distinct proteins (50 and 51 kDa) in liver microsomes from male and female rats, as determined by Western immunoblotting. Depending on the source of the liver microsomes, the 51-kDa protein corresponded to 3A1 and/or 3A2 which could not be resolved by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. The other protein (50 kDa) appears to be another member of the P450 IIIA gene family. Both proteins were markedly intensified in liver microsomes from male or female rats treated with pregnenolone-16α-carbonitrile, dexamethasone, troleandomycin, or chlordane. In contrast, treatment of male or female rats with phenobarbital intensified only the 51-kDa protein. Treatment of male rats with Aroclor 1254 induced the 51-kDa protein, but suppressed the 50-kDa form. In addition to their changes in response to inducers, the 50- and 51-kDa proteins also differed in their developmental expression. For example, the 50-kDa protein was not expressed until weaning (3 weeks), whereas the 51-kDa protein was expressed even in 1-week-old rats. At puberty (between weeks 5 and 6), the levels of the 50-kDa and 51-kDa proteins markedly declined in female but not in male rats, which introduced a large sex difference (male > female) in the levels of both proteins. Changes in the level of the 51-kDa protein were paralleled by changes in the rate of testosterone 2β, 6β-, and 15β-hydroxylation. In male rats, the marked increase in the levels of the 50-kDa protein between weeks 2 and 3 coincided with a three- to four fold increase in the rate of testosterone 2β-, 6β-, and 15β-hydroxylation, which suggests that the 50-kDa protein catalyzes the same pathways of testosterone oxidation as the 51-kDa protein. However, this developmental increase in testosterone oxidation may have resulted from an activation of the 51-kDa 3A protein. These results indicate that the two electrophoretically distinct proteins recognized by antibody against P450 3A1 are regulated in a similar but not identical manner, and suggest that the 51-kDa 3A protein is the major microsomal enzyme responsible for catalyzing the 2β-, 6β-, and 15β-hydroxylation of testosterone.     

Cytochrome P450s of the 4A Subfamily in the Brain

Abstract: Members of the P450 4A subfamily are key enzymes in the synthesis and degradation of metabolites of arachidonic acid, which are of physiological importance in the brain. In the rat, four members of this subfamily, 4A1, 4A2, 4A3, and 4A8, have been described. In this study, the expression of members of the 4A subfamily in the rat brain has been examined by PCR amplification, by western and northern blotting, and by protein N-terminal sequencing. With PCR all four members of the subfamily were detectable in the liver and kidney. P450 4A1 was found exclusively in the liver and kidney, whereas P450 4A2 was detectable in all the tissues tested, including the lung, seminal vesicles, prostate, cerebral cortex, hypothalamic preoptic area, cerebellum, and brainstem. The tissue distribution of P450 4A3 was similar to that of 4A2 except that it was not detectable in seminal vesicles. A P450 4A8-specific fragment was amplified from the kidney, liver, and prostate and weakly from the cerebral cortex but not from other brain regions. Despite the evidence of their presence by PCR, no members of the 4A family were detectable on northern blots with mRNA from the brain. On western blots a P450 4A-specific antiserum recognized a band in P450 fractions prepared from the brain. The intensity of the signal with 30 pmol of P450 from the brain was similar to that with 10 pmol of liver microsomal P450. The brain P450 was extracted from 1 g of brain, whereas the 10 pmol of liver P450 is the equivalent of 1 mg of liver. This suggests a brain content of 4A P450 that is 0.1% of that in the liver. N-terminal sequencing of the protein bands in the brain P450 fraction revealed the presence of both P450 4A8 and 4A3. These data show the presence in the brain of forms of P450 whose level of mRNA is too low to be detected on northern blots. The specificity of tissue distribution shows that this is not just a nonspecific background level of expression and suggests a role of brain P450 in the synthesis and degradation of arachidonic acid metabolites.     

Cytochrome P450 3A9 catalyzes the metabolism of progesterone and other steroid hormones

The catalytic requirements and the role of P450 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P450 3A9 protein. The optimal steroid hormone hydroxylase activities of P450 3A9 required cholate but not cytochrome b₅. P450 3A9 was active in the hydroxylation reactions of testosterone, androstenedione, progesterone and dehydroepiandrosterone (DHEA). No activity of P450 3A9 toward cortisol was detectable under our reconstitution conditions. Among all the steroid hormones examined, female-specific P450 3A9 seemed to catalyze most efficiently the metabolism of progesterone, one of the major female hormones, to form three mono-hydroxylated products, 6-, 16-, and 21-hydroxyprogesterone. Our data also showed that P450 3A9 can catalyze the formation of a dihydroxy product, 4-pregnen-6, 21-diol-3, 20-dione, from progesterone with a turnover number, 1.3 nmol/min/nmol P450. Based on the V_max/K_m values for P450 3A9 using either 21-hydroxprogesterone or 6-hydroxyprogesterone as a substrate, 4-pregnen-6, 21-diol-3, 20-dione may be formed either by 6-hydroxylation of 21-hydroxprogesterone or 21-hydroxylation of 6-hydroxyprogesterone. As a major isoform of CYP3A expressed in rat brain, the activities of P450 3A9 toward two major neurosteroids, progesterone and DHEA suggested a possible role for P450 3A9 in the metabolism of neurosteroids.     

7,8-benzoflavone binding to human cytochrome P450 3A4 reveals complex fluorescence quenching,suggesting binding at multiple protein sites

Human cytochrome P450 (P450) 3A4 is involved in the metabolism of one-half of marketed drugs and shows cooperative interactions with some substrates and other ligands. The interaction between P450 3A4 and the known allosteric effector 7,8-benzoflavone (α-naphthoflavone, αNF) was characterized using steady-state fluorescence spectroscopy. The binding interaction of P450 3A4 and αNF effectively quenched the fluorescence of both the enzyme and ligand. The Hill Equation and Stern–Volmer fluorescence quenching models were used to evaluate binding of ligand to enzyme. P450 3A4 fluorescence was quenched by titration with αNF; at the relatively higher [αNF]/[P450 3A4] ratios in this experiment, two weaker quenching interactions were revealed (K_d 1.8–2.5 and 6.5 μM). A range is given for the stronger interaction since αNF quenching of P450 3A4 fluorescence changed the protein spectral profile: quenching of 315 nm emission was slightly more efficient (K_d 1.8 μM) than the quenching of protein fluorescence at 335 and 355 nm (K_d 2.5 and 2.1 μM, respectively). In the reverse titration, αNF fluorescence was quenched by P450 3A4; at the lower [αNF]/[P450 3A4] ratios here, two strong quenching interactions were revealed (K_d 0.048 and 1.0 μM). Thus, four binding interactions of αNF to P450 3A4 are suggested by this study, one of which may be newly recognized and which could affect studies of drug oxidations by this important enzyme.     

Transient induction of cytochrome P450 1A1 mRNA by culture medium component in primary cultures of adult rat hepatocytes

Summary Because the metabolic environment can alter gene expression in cultured cells, we examined the effects of change of medium on the levels of several cytochrome P450 mRNAs in primary cultures of rat hepatocytes maintained on Matrigel. The amounts of P450 1A2, 2B1/2, or 3A1 mRNA were unaffected by changing the medium. In contrast, P450 1A1 mRNA levels were increased 1 to 2 h after media change, reached maximum levels by 6 h, and declined to baseline by 24 h. Supplementing day-old media with components of the medium revealed that only addition of amino acids resulted in 1A1 mRNA induction. From the results of direct additions and omissions, we showed that tryptophan, but not histidine, was largely responsible for the 1A1 mRNA induction. Moreover, mild photoactivation of the tryptophan resulted in a substantially increased magnitude of 1A1 mRNA induction. The time course for 1A1 mRNA induction by treatment with photoactivated tryptophan was identical to that observed after medium change. Treatment of hepatocyte cultures with β-naphthoflavone, which is metabolized by 1A1, also resulted in a transient 1A1 mRNA induction time-course profile over a 24-h period, whereas treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin, which is relatively stable to metabolic transformation, produced sustained elevations of 1A1 mRNA, suggesting that the transient response to tryptophan also may involve metabolism of the inducer. Our results extend previous data showing that oxidized products of tryptophan induce 1A1, and suggest that the transient nature of the induction may be due to elimination of the activated tryptophan molecule.     

Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constitutive male-specific form of cytochrome P450.

A cDNA clone for rat hepatic cytochrome P450 2c (gene product IIC11) was isolated and used to study the sex specificity, expression during development, and hormonal regulation of the mRNA encoding this protein in rat liver. P450 2c mRNA levels were about 16-fold higher in males than in females and were only slightly increased in male rats after administration of phenobarbital, a drug that dramatically raises the levels of mRNAs encoding several other members of the P450 II family. In contrast to the mRNA encoding P450 f (gene product IIC7), which increases gradually over the first 6 weeks of life, P450 2c mRNA showed a dramatic increase at puberty, between 4.5-5.5 weeks of life. The roles of sex steroids and GH in controlling this male-specific, developmentally regulated mRNA were then examined. A dependence on adult androgen was demonstrated by the 2- to 4-fold decrease in P-450 2c mRNA levels after castration of adult male rats and their restoration to normal by administration of the synthetic androgen methyltrienolone. Prolonged treatment (15 days) of ovariectomized female rats with this androgen also increased the levels of P450 2c mRNA and its encoded testosterone 16 alpha-hydroxylase to those of intact males. In male rats treated with estradiol valerate, mRNAs for P450 2c and alpha 2u-globulin, a major male-specific hepatic secretory protein that is under complex hormonal control, fell to negligible levels. None of these hormonal perturbations had a detectable effect on the levels of PB-1 (gene product IIC6) mRNA, which is not expressed in a sex-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for the involvement of a distinct form of cytochrome p450 3a in the oxidation of digitoxin by rat liver microsomes

The preceding paper (B. Gemzik, D. Greenway, C. Nevins, and A. Parkinson (1992). Regulation of two electrophoretically distinct proteins recognized by antibody against rat liver cytochrome P450 3A1. J. Biochem. Toxicol, 7 (43–52).) described the regulation of two rat liver microsomal proteins (50- and 51-kDa) recognized by antibody against P450 3A1. It was also shown that changes in the levels of the 51-kDa 3A protein were usually paralleled by changes in the rate of testosterone 2β-, 6β-, and 15β-hydroxylation. The present study demonstrates that age- and sex-dependent changes in the 50-kDa protein were paralleled by changes in the rate of digitoxin oxidation to digitoxigenin bisdigitoxoside. Induction or suppression of the 50-kDa protein by treatment of rats with various xenobiotics were also paralleled by changes in the rate of digitoxin oxidation. These results suggest that, contrary to previous assumptions, the conversion of digitoxin to digitoxigenin bisdigitoxoside and the conversion of testosterone to 2β-, 6β- and 15β-hydroxytestosterone are primarily catalyzed by different forms of P450 3A. Further evidence for this coclusion was obtained from studies in which the suicide inhibitor, chloramphenicol, was administered to mature female rats previously treated with pregnenolone-16α-carbonitrile (PCN), which induces both the 50-kDa and the 51-kDa protein. Treatment of mature female rats with PCN alone caused a marked increase (16- to 18-fold) in the 6β-hydroxylation of testosterone and the rate of digitoxin oxidation. Treatment of PCN-induced rats with chloramphenicol caused a ～70% decrease in liver microsomal testosterone 6β-hydroxylation, but had no effect on the rate of conversion of digitoxin to digitoxigenin bisdigitoxoside. The oxidation of testosterone by purified 3A1 (a 51-kDa protein) was also inhibited by chloramphenicol in a time- and reduced nicotinamite adenine dinucleotide phosphate (NADPH)-dependent manner. In addition to testosterone and chloramphenicol, purified 3A1 also metabolized trole-andomycin, but it was unable to convert digitoxin to digitoxigenin bisdigitoxoside. Testosterone inhibited the microsomal oxidation of digitoxin, but digitoxin did not inhibit testosterone oxidation. This suggests that testosterone is a substrate for the 3A enzyme that metabolizes digitoxin, but that this form of P450 3A does not contribute significantly to testosterone oxidation by rat liver microsomes. We propose that the 2SbT-, 6β-, and 15β-hydroxylation of testosterone by rat liver microsomes is primarily catalyzed by the 51-kDa 3A proteins (either 3A1 or 3A2 depending on the source of microsomes), whereas digitoxin oxidation is primarily catalyzed by the 50-kDa protein.     

Effect of a newly developed ketolide antibiotic, telithromycin, on metabolism of theophylline and expression of cytochrome P450 in rats

The effects of a newly-developed ketolide antibiotic, telithromycin, on the metabolism of theophylline and the expression of hepatic cytochrome P450 (CYP) 1A2 and CYP3A2 were investigated in rats. Telithromycin at a high dose (100 mg/kg of body weight) was injected intraperitoneally once a day for 3 days. Twenty-four hours (day 4) after the final administration of telithromycin, theophylline (10 mg/kg) was administered intravenously. The presence of telithromycin significantly delayed the disappearance of theophylline from plasma. Parameters related to the pharmacokinetic interaction between theophylline and telithromycin were examined by noncompartmental methods. A significant decrease in the systemic clearance of theophylline was observed in the presence of telithromycin. Pretreatment with telithromycin significantly decreased the metabolic clearance of the major metabolites, 1-methyluric acid and 1,3-dimethyluric acid, with no change in the renal clearance of theophylline, suggesting that the decreased systemic clearance of theophylline by telithromycin is due to reduction of their metabolic clearance. Pretreatment with telithromycin significantly decreased the activity of 7-ethoxyresorufin O-deethylation and testosterone 6 beta-hydroxylation, suggesting that telithromycin decreases the activity of hepatic CYP1A2 and CYP3A2. Western blot analysis revealed that telithromycin significantly decreased the protein levels of CYP1A2 and CYP3A2 in the liver, which could explain the observed decreases in the systemic clearance of theophylline and metabolic clearance of 1-methyluric acid and 1,3-dimethyluric acid. The present study suggests that telithromycin at the dose used in this study alters the pharmacokinetics and metabolism of theophylline, due to reductions in the activity and expression of hepatic CYP1A2 and CYP3A2.     

Induction of Two Forms of Eel Cytochrome P450 1A Genes by 3-Methylcholanthrene

In eel (Anguilla japonica), exposure to polyaromatic hydrocarbons such as 3-methylcholanthrene leads to induction of two CYP1A enzymes, CYP1A1 and CYP1A6. We studied the time course and tissue specificity of induction of messenger RNAs for CYP1A1 and CYP1A6 in eel by administering 3-methylcholanthrene intraperitoneally. In both cases, the drug induced a rapid increase of mRNAs and biphasic expression. In the liver, mRNA levels of CYP1A1 and CYP1A6 increased 22-fold at 3 hours and 27-fold at 6 hours after the administration, respectively, showing initial peaks in the induction. After the initial inductions, mRNA levels decreased unexpectedly. Following these temporary decreases, the mRNA levels again increased and reached levels that were 35 and 41 times the basal levels at 24 hours after administration, respectively. CYP1A1 and CYP1A6 resembled each other also in the tissue specificity of gene expression; the expression levels were liver ≫ gill > intestine > kidney. The rapid induction, the biphasic expression, and the tissue-specific expression were common features of gene expression in CYP1A1 and CYP1A6 and may come from common structures of the regulatory regions of the two genes. Received December 7, 1998; accepted February 15, 1999     

Heterologous expression, purification, and properties of human cytochrome P450 27C1

Cytochrome P450 (P450) 27C1 is one of the "orphan" P450 enzymes without a known biological function. A human P450 27C1 cDNA with a nucleotide sequence modified for Escherichia coli usage was prepared and modified at the N-terminus, based on the expected mitochondrial localization. A derivative with residues 3-60 deleted was expressed at a level of 1350nmol/L E. coli culture and had the characteristic P450 spectra. The identity of the expressed protein was confirmed by mass spectrometry of proteolytic fragments. The purified P450 was in the low-spin iron state, and the spin equilibrium was not perturbed by any of the potential substrates vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol. P450s 27A1 and 27B1 are known to catalyze the 25-hydroxylation of vitamin D(3) and the 1alpha-hydroxylation of 25-hydroxy vitamin D(3), respectively. In the presence of recombinant human adrenodoxin and adrenodoxin reductase, recombinant P450 27C1 did not catalyze the oxidation of vitamin D(3), 1alpha- or 25-hydroxy vitamin D(3), or cholesterol at detectable rates. P450 27C1 mRNA was determined to be expressed in liver, kidney, pancreas, and several other human tissues.     

Differential effect of phenobarbital and β-naphthoflavone on the mRNAs coding for cytochrome P450 and NADPH cytochrome P450 reductase

The induction in rat liver of a specific variant(s) of cytochrome P450 (PB-P450) by phenobarbital and its repression by β-naphthoflavone occur through corresponding changes in the levels of mRNA coding for the protein(s). The level of translatable mRNA coding for NADPH-cytochrome P450 reductase in rat liver increases on treatment with phenobarbital but not β-naphthoflavone.     

Differential effect of phenobarbital and beta-naphthoflavone on the mRNAs coding for cytochrome P450 and NADPH cytochrome P450 reductase

The induction in rat liver of a specific variant(s) of cytochrome P450 (PB-P450) by phenobarbital and its repression by β-naphthoflavone occur through corresponding changes in the levels of mRNA coding for the protein(s). The level of translatable mRNA coding for NADPH-cytochrome P450 reductase in rat liver increases on treatment with phenobarbital but not β-naphthoflavone.     

Dax1 suppresses P450arom expression in medaka ovarian follicles

Dax1 is a member of an unusual orphan nuclear receptor family, and is known to regulate P450arom in mammals and is involved in sex differentiation in some vertebrates. To investigate whether Dax1 is involved in the regulation of the steroidogenic pathway for estrogen biosynthesis in medaka ovarian follicles, we isolated Dax1 cDNA from adult medaka ovaries and analyzed its expression pattern in medaka gonads. In adult ovaries, Dax1 mRNA was detected only in postvitellogenic follicles and was not detected in previtellogenic and vitellogenic follicles. In adult testis, Dax1 mRNA was not detected. We compared the expression pattern of Dax1 with that of Foxl2, Ad4BP/Sf-1, P450c17, and P450arom by in situ hybridization using adjacent sections. In contrast to Dax1 expression, these genes were co-expressed in vitellogenic follicles but were not detected in postvitellogenic follicles. Thus, in medaka ovarian follicles, Dax1 did not show any overlapping expression patterns against Foxl2, Ad4BP/Sf-1, P450c17, and P450arom. Moreover, co-transfection experiments demonstrated that Dax1 inhibits Ad4BP/Sf-1- and Foxl2-mediated P450arom expression. On the other hand, during early sex differentiation, Dax1 mRNA was not detected in both males and females. Our results suggest that Dax1 down-regulates Ad4BP/Sf-1- and Foxl2-mediated P450arom expression in medaka ovarian follicles.     

Cytochrome P450 1A-dependent enzyme activities in the liver of dab (Limanda limanda): kinetics, seasonal changes and detection limits

Concentrations of total cytochrome P450 and cytochrome P450 1A (CYP 1A) and activities of ethoxycoumarin O-deethylase (ECOD), ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase (PROD) were measured in the liver of prespawning, spawning and postspawning dab (Limanda limanda) from the German Bight. Between all P450-dependent parameters measured significant correlations were found. Generally, during prespawning and spawning season higher values were measured in the liver of males compared to females, but the ratio between sexes changed during spawning time, when concentrations and activities in the liver of males decreased and increased in the liver of females. The activity and the signal-to-noise ratio decrease in the order EROD, ECOD and PROD. This decrease is accompanied by an increase in K_m. The findings indicate that the different activities can be attributed to the strongly overlapping substrate specificity and the different enzyme affinities of one enzyme, CYP 1A, towards the three substrates. A biphasic kinetic of ECOD indicates that in addition to CYP 1A a second isozyme catalyses the O-deethylation of ethoxycoumarin in the liver of dab. Interestingly, the ratio between EROD activity and CYP 1A concentration varied seasonally but did not differ significantly between sexes.     

Cytochrome P450 52A3: Modelling of 3D Structure and Surface Mutations

Abstract

Earlier W.-H. Schunck et al. [1] have prepared a water soluble enzymatically active fragment of cytochrome P450 52A3 (CYP52A3) which is lack of 66 amino acid residues, existed as a dimer in aqueous solution. Now we propose 3D structure of the fragment, which is based on multiple sequence alignment of the CYP52A3 with its homologues proteins of known 3D structure: CYP101, 102, 107A1 and 108. The structural model have been optimised and used as a prototype for computer simulation of point mutations. These mutations should bring some changes in the surface properties, interfering dimer formation. For this aim the point of 22 hydrophobic amino acid residues have been sequentially replaced with that of charged amino acids (GLU, ASP, ARG and LYS). The scoring of “mutants” was conducted based on the changes of protein surface hydrophobicity and protein-solvent interaction energy. An analysis of the surface hydrophobicity and protein-solvent interactions permit to select most sensitive three sites (171, 352 and particularly 164 amino acid residues). The dimerization of the following “mutant” fragments must be investigated experimentally.     

Cysteine 98 in CYP3A4 contributes to conformational integrity required for P450 interaction with CYP reductase

Previously human cytochrome P450 3A4 was efficiently and specifically photolabeled by the photoaffinity ligand lapachenole. One of the modification sites was identified as cysteine 98 in the B-C loop region of the protein [B. Wen, C.E. Doneanu, C.A. Gartner, A.G. Roberts, W.M. Atkins, S.D. Nelson, Biochemistry 44 (2005) 1833-1845]. Loss of CO binding capacity and subsequent decrease of catalytic activity were observed in the labeled CYP3A4, which suggested that aromatic substitution on residue 98 triggered a critical conformational change and subsequent loss of enzyme activity. To test this hypothesis, C98A, C98S, C98F, and C98W mutants were generated by site-directed mutagenesis and expressed functionally as oligohistidine-tagged proteins. Unlike the mono-adduction observed in the wild-type protein, simultaneous multiple adductions occurred when C98F and C98W were photolabeled under the same conditions as the wild-type enzyme, indicating a substantial conformational change in these two mutants compared with the wild-type protein. Kinetic analysis revealed that the C98W mutant had a drastic 16-fold decrease in catalytic efficiency (V(max)/K(m)) for 1'-OH midazolam formation, and about an 8-fold decrease in catalytic efficiency (V(max)/K(m)) for 4-OH midazolam formation, while the C98A and C98S mutants retained the same enzyme activity as the wild-type enzyme. Photolabeling of C98A and C98S with lapachenole resulted in monoadduction of only Cys-468, in contrast to the labeling of Cys-98 in wild-type CYP3A4, demonstrating the marked selectivity of this photoaffinity ligand for cysteine residues. The slight increases in the midazolam binding constants (K(s)) in these mutants suggested negligible perturbation of the heme environment. Further activity studies using different P450:reductase ratios suggested that the affinity of P450 to reductase was significantly decreased in the C98W mutant, but not in the C98A and C98S mutants. In addition, the C98W mutant exhibited a 41% decrease in the maximum electron flow rate between P450 and reductase as measured by reduced nicotinamide adenine dinucleotide phosphate consumption at a saturating reductase concentration. In conclusion, our data strongly suggest that cysteine 98 in the B-C loop region significantly contributes to conformational integrity and catalytic activity of CYP3A4, and that this residue or residues nearby might be involved in an interaction with P450 reductase.     

Regulation of the expression of two female-predominant CYP3A mRNAs (CYP3A41 and CYP3A44) in mouse liver by sex and growth hormones

A second female-predominant murine CYP3A, CYP3A44, was isolated from liver and its mRNA expression was compared with that of the previously described CYP3A41. The expression of CYP3A44 was relatively constant after birth in females, whereas it gradually declined in males after 5 weeks of age. The expression of CYP3A41 increased with age in females after 3 weeks of age, whereas it gradually declined in males after 5 weeks of age. Hypophysectomy and growth hormone replacement indicated that expression of both CYP3A mRNAs in females was dependent on the feminine plasma growth hormone profile. Estradiol induced the expression of both mRNAs and the effect was dependent on the presence of the pituitary gland. These observations suggest that endocrine control of expression might be similar, but not identical, for two female-predominant CYP3A mRNAs.     

The effects of diabetes with hyperlipidemia on P450 expression in APA hamster livers

The effect of chronic hyperglycemia and hyperlipidemia induced by streptozotocin (SZ) on the expression of P450 in the liver of APA hamsters was studied in this experiment. No effect on the total activity of P450 was seen in SZ-induced diabetic hamsters throughout the experimental period. At 1 and 6 months after SZ-injection, the levels of CYP1A, 2C6, and 3A of SZ-injected hamsters were much lower than those of age-matched control hamsters. CYP2B expression tended to decrease and CYP2E1 and 4A expression tended to increase in SZ-injected hamsters, although the results were not significant. At 3 months after SZ-injection, however, no significant difference between SZ-injected and normal hamsters was seen in these P450 isozymes. On the other hand, CYP2C11 expression was slightly depressed in SZ1M and SZ6M, and almost equivalent to control hamsters in SZ3M. Immunohistochemistry by the use of each isozyme antibody revealed that SZ-induced diabetes affected the localization of CYP2C6, 3A, and 4A in the hepatic acinus. The expression of CYP2C6 and 3A was depressed mainly in the periportal region of the acinus, and CYP4A expression was induced mainly in the perivenous region by SZ-induced diabetes. On the other hand, the expression pattern of CYP1A, 2B, 2C11, and 2E1 were not affected. These results demonstrate that the effects of SZ-induced diabetes on hepatic P450 differ for each isozyme in APA hamsters and also differ from those of other experimental diabetic animals, including golden hamsters.