Peroxisome proliferator activated receptor alpha regulates a male-specific cytochrome P450 in mouse liver

We set out to find if the strain-specific, male-specific hepatic expression of Cyp4a protein in mouse was due to expression of Cyp4a12 and to understand the genetic basis for reported differences in expression. 12-Lauric acid hydroxylase (LAH) activity was found to show higher levels in male ddY, but not C57Bl/6, mouse liver microsomes. The expression of Cyp4a12 mRNA was studied using RNAase protection assays in male and female liver and kidney of nine mouse strains. Cyp4a12 was found to be highly expressed in male liver and kidney, but at much lower levels in female liver and kidney, in all strains studied. Western blotting with an antibody specific for Cyp4a12 confirmed that Cyp4a12 was expressed in a male specific fashion in C57Bl/6 mouse liver. RNAase protection analysis for Cyp4a10 and 14 in ddY mice revealed that neither of these genes showed male-specific expression. To further investigate genetic factors that control male-specific Cyp4a12 expression, PPARalpha+/+ and -/- mice were studied, showing that total P450 and 12-LAH activity was male-specific in +/+, but not -/- mice. RNAase protection assays were used to confirm that Cyp4a12 was lower in -/- mice. However, the male-specific Slp and MUP-1 genes retained hepatic male-specific levels of expression in +/+ and -/- mice, showing that the decrease in Cyp4a12 was not a general effect on male-specific expression. Thus, PPARalpha has a specific effect on constitutive expression of Cyp4a12.     

Cyp1a2 protects against reactive oxygen production in mouse liver microsomes

H(2)O(2) production was evaluated in liver microsomes prepared from Cyp1a1/1a2(+/+) wild-type and Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice pretreated with 5 microg dioxin (TCDD)/kg body wt or vehicle alone. NADPH-dependent H(2)O(2) production in TCDD-induced microsomes from wild-type mice was about one-third of that in noninduced microsomes. In Cyp1a2(-/-) mice, H(2)O(2) production was the same for induced and noninduced microsomes, with levels significantly higher than those in wild-type mice. Cyp1a1(-/-) microsomes displayed markedly lower levels of H(2)O(2) production in both induced and noninduced microsomes, compared with those in wild-type and Cyp1a2(-/-) microsomes. The CYP1A2 inhibitor furafylline in vitro exacerbated microsomal H(2)O(2) production proportional to the degree of CYP1A2 inhibition, and the CYP2E1 inhibitor diethyldithiocarbamate decreased H(2)O(2) production proportional to the degree of CYP2E1 inhibition. Microsomal H(2)O(2) production was strongly correlated to NADPH-stimulated production of thiobarbituric acid-reactive substances, as well as to decreases in microsomal membrane polarization anisotropy, indicative of peroxidation of unsaturated membrane lipids. Our results suggest that possibly acting as an "electron sink," CYP1A2 might decrease CYP2E1-and CYP1A1-mediated H(2)O(2) production and oxidative stress. In this regard, CYP1A2 may be considered an antioxidant enzyme.     

Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation.

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7 alpha- and/or 15 alpha-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (greater than 70%) in human and monkey, but only a minor metabolite (less than 1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme (Km 0.2-0.6 microM), which was markedly inhibited (greater than 95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied approximately 17-fold among liver microsomes from 22 human subjects. This variation was highly correlated (r2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with beta-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold among the human liver microsomal samples, but this variation correlated poorly (r2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cytochrome P450 Cyp4x1 is a major P450 protein in mouse brain

A novel cytochrome P450, CYP4x1, was identified in EST databases on the basis of similarity to a conserved region in the C-helix of the CYP4A family. The human and mouse CYP4x1 cDNAs were cloned and found to encode putative cytochrome P450 proteins. Molecular modelling of CYP4x1 predicted an unusual substrate binding channel for the CYP4 family. Expression of human CYP4x1 was detected in brain by EST analysis, and in aorta by northern blotting. The mouse cDNA was used to demonstrate that the Cyp4x RNA was expressed principally in brain, and at much lower levels in liver; hepatic levels of the Cyp4x1 RNA were not affected by treatment with the inducing agents phenobarbital, dioxin, dexamethasone or ciprofibrate, nor were the levels affected in PPARalpha-/- mice. A specific antibody for Cyp4x1 was developed, and shown to detect Cyp4x1 in brain; quantitation of the Cyp4x1 protein in brain demonstrated approximately 10 ng of Cyp4x1 protein.mg(-1) microsomal protein, showing that Cyp4x1 is a major brain P450. Immunohistochemical localization of the Cyp4x1 protein in brain showed specific staining of neurons, choroids epithelial cells and vascular endothelial cells. These data suggest an important role for Cyp4x1 in the brain.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

Dimemorfan N-demethylation by mouse liver microsomal cytochrome P450 enzymes

Dimemorfan (d-3-methyl-N-methylmorphinan), an analogue of dextromethorphan, is commonly used as a non-opioid antitussive. To clarify the contribution of cytochrome P450 (P450) in dimemorfan N-demethylation, effects of selective inducers and inhibitors were studied in ICR mice. Phenobarbital (PB)- and dexamethasone (Dex)-treatments caused 5-fold increases of liver microsomal dimemorfan N-demethylation activity. In untreated mouse liver microsomes, demethylation activity was strongly inhibited by a CYP3A inhibitor, ketoconazole. In PB-and Dex-treated mouse liver microsomes, ketoconazole caused strong inhibition, whereas orphenadrine caused a decrease of less than 20%. Pretreatment of control mouse liver microsomes with anti-CYP3A inhibited demethylation activity, whereas pre-treatment with anti-CYP2B had no effect. In PB-and Dex-treated mouse liver microsomes, the demethylation activity was inhibited by both anti-CYP3A and anti-CYP2B. In control mice, the intrinsic clearance of dimemorfan from N-demethylation was 5.8 microl min(-1)mg protein(-1). In PB- and Dex-treated mice, the correlation coefficient of fitting using one-enzyme and two-enzyme models were similar. The intrinsic clearances of induced mouse liver microsomes were similar. These results revealed that CYP3A played a major role in hepatic demethylation in untreated mice. Both CYP3A and CYP2B were involved in this demethylation in PB- and Dex-treated mice.     

Schisandrin B induced antioxidant response is partly mediated by cytochrome P-4502E1 catalyzed reaction in mouse liver

In order to explore the role of cytochrome P-450 (CYP) 2E1 in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effects of Sch B treatment on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression were compared between wild-type and cyp2e1 knock-out C57B/6N mice. Cyp2e1 knock-out mice exhibited a significantly smaller degree of Sch B-induced enhancement in hepatic mtGAS when compared with the wild-type counterpart. But Hsp25/70 expression induced by Sch B was not affected. Sch B-induced enhancement of mtGAS was corroborated by the increase in hepatic mitochondrial antioxidant capacity, as assessed by in vitro measurement of oxidant production, with the enhancing effect being slightly reduced in the knock-out mice. Using liver microsomes prepared from wild-type and knock-out mice as a source of CYP, Sch B was found to be a good co-substrate for the CYP-catalyzed reaction, with the rate of NADPH oxidation observable in microsomes prepared from knock-out mice being slower. The CYP-catalyzed reaction with Sch B was associated with a concomitant production of oxidant species, with the extent of oxidant production being reduced in cyp2e1 knock-out mouse microsomes. Taken together, the results indicate that CYP2E1 is partly responsible for the hepatic metabolism of Sch B that may trigger the antioxidant response in vivo.     

Purification and characterization of a microsomal cytochrome P-450 with high activity of coumarin 7-hydroxylase from mouse liver

Phenobarbital-induced coumarin 7-hydroxylase is high in DBA/2J and low in C57BL/6N inbred mice; this genetic difference is encoded by the Coh locus on chromosome 7. The aim of this study was to develop an antibody specific for this cytochrome P-450 polymorphism. P-450 fractions, highly specific for phenobarbital-inducible coumarin 7-hydroxylase activity, were purified from DBA/2J and C57BL/6N mouse liver microsomes. Both proteins are 49 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Soret peaks of the reduced cytochrome . CO complexes are 451 nm. Reconstituted DBA/2J coumarin 7-hydroxylase activity exhibits a V twice as high as, and a Km value 10-fold less than, the reconstituted C57BL/6N activity. Antibodies were raised in rabbit. By Ouchterlony immunodiffusion, both antibodies show 100% cross-reactivity with DBA/2J and C57BL/6N microsomes and purified antigens. Yet, DBA/2J but not C57BL/6N 7-hydroxylase activity is inhibited by the antibody to DBA/2J P-450. Both DBA/2J and C57BL/6N activities are blocked by the antibody to C57BL/6N P-450. Neither antibody has any effect on liver microsomal d-benzphetamine N-demethylase, ethylmorphine N-demethylase, aminopyrine N-demethylase, 7-ethoxycoumarin O-deethylase, acetanilide 4-hydroxylase, or aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity. The DBA/2J protein most specific for phenobarbital-induced coumarin 7-hydroxylation is designated 'P-450Coh'. Anti-(P-450Coh) precipitates a relatively minor 49-kDa protein from detergent-solubilized microsomes and from in vitro translation of poly(A+)-enriched total RNA of phenobarbital-treated DBA/2J mouse liver, whereas the major phenobarbital-induced P-450 proteins exhibit a molecular mass of about 51 kDa. The immunoprecipitated translation products correspond to a messenger RNA of 2100 +/- 100 nucleotides.     

Highly sensitive high-performance liquid chromatographic assay for coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human liver cytochrome P450 enzymes

A highly sensitive method for the determination of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation by human cytochrome P450 (P450 or CYP) enzymes was developed using high-performance liquid chromatography (HPLC). The newly developed HPLC method was found to be about 100-fold more sensitive than the previous spectrofluorimetric method in detecting the metabolite 7-hydroxycoumarin (umbelliferone). With this high sensitivity, the kinetics of coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation catalyzed by human liver microsomal and recombinant P450 enzymes were determined more precisely. With 36 different substrate concentrations in these two reactions, coumarin 7-hydroxylation was found to be catalyzed mainly by a single enzyme CYP2A6 and 7-ethoxycoumarin was oxidized by at least two enzymes CYP2E1 and CYP1A2 in human liver microsomes.     

Identification of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes

Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4+/-1.0 microM and 200.5+/-83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r = 0.763, p < 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r = 0.638, p < 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while alpha-naphthoflavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, alpha-naphthoflavone inhibited the chlorpromazine 7-hydroxylase activity to 55-65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited significant activity for the chlorpromazine 7-hydroxylation. The Km values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2.     

Cyp3a11 is not essential for the formation of murine bile acids

Humans and mice differ substantially in their bile acid profiles as mice in addition to cholic acid (CA) predominantly synthesize 6β-hydroxylated muricholic acids (MCAs) whereas humans produces chenodeoxycholic acid (CDCA) and CA as primary bile acids. Identifying the gene performing 6β-hydroxylation would be useful for ‘humanizing’ the bile acid profile in mice for studies of the interaction between bile acids, gut microbiota, and host metabolism. We investigated the formation of MCAs in primary murine hepatocytes and found that αMCA is synthesized from CDCA and βMCA from UDCA. It is commonly assumed that the P450-enzyme CYP3A11 catalyzes 6β-hydroxylation of bile acids, thus we hypothesized that mice without the Cyp3a11 gene would lack MCAs. To test this hypothesis, we analyzed bile acid profiles in Cyp3a deficient mice, which lack 7 genes in the Cyp3a gene cluster including Cyp3a11, and compared them with wild-type littermate controls. Bile acid composition in liver, gallbladder, caecum and serum from Cyp3a knock out mice and wild-type littermate controls was analyzed with UPLC-MS/MS and revealed no major differences in bile acid composition. We conclude that Cyp3a11 is not necessary for 6β-hydroxylation and the formation of MCAs.     

Hepatic bile acid metabolism and expression of cytochrome P450 and related enzymes are altered in Bsep −/− mice

The bile salt export pump (BSEP/Bsep; gene symbol ABCB11/Abcb11) translocates bile salts across the hepatocyte canalicular membrane into bile in humans and mice. In humans, mutations in the ABCB11 gene cause a severe childhood liver disease known as progressive familial intrahepatic cholestasis type 2. Targeted inactivation of mouse Bsep produces milder persistent cholestasis due to detoxification of bile acids through hydroxylation and alternative transport pathways. The purpose of the present study was to determine whether functional expression of hepatic cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) is altered by Bsep inactivation in mice and whether bile acids regulate CYP and mEH expression in Bsep ^?/? mice. CYP expression was determined by measuring protein levels of Cyp2b, Cyp2c and Cyp3a enzymes and CYP-mediated activities including lithocholic acid hydroxylation, testosterone hydroxylation and alkoxyresorufin O-dealkylation in hepatic microsomes prepared from female and male Bsep ^?/? mice fed a normal or cholic acid (CA)-enriched diet. The results indicated that hepatic lithocholic acid hydroxylation was catalyzed by Cyp3a/Cyp3a11 enzymes in Bsep ^?/? mice and that 3-ketocholanoic acid and murideoxycholic acid were major metabolites. CA feeding of Bsep ^?/? mice increased hepatic Cyp3a11 protein levels and Cyp3a11-mediated testosterone 2β-, 6β-, and 15β-hydroxylation activities, increased Cyp2b10 protein levels and Cyp2b10-mediated benzyloxyresorufin O-debenzylation activity, and elevated Cyp2c29 and mEH protein levels. We propose that bile acids upregulate expression of hepatic Cyp3a11, Cyp2b10, Cyp2c29 and mEH in Bsep ^?/? mice and that Cyp3a11 and multidrug resistance-1 P-glycoproteins (Mdr1a/1b) are vital components of two distinct pathways utilized by mouse hepatocytes to expel bile acids.     

Participation of a cytochrome P450 enzyme from the 2C subfamily in progesterone 21-hydroxylation in sheep liver.

Progesterone 21-hydroxylation in hepatic microsomes from adult male sheep is a quantitatively important metabolic pathway (0.27 +/- 0.08 nmol deoxycorticosterone formed/min/mg protein; representing 13-25% of total progesterone conversion). This study was undertaken to determine whether the ovine hepatic progesterone 21-hydroxylase may be another member of the P450 2C subfamily, normally associated with progesterone 21-hydroxylation in rodent liver. An IgG preparation raised in rabbits against purified rat liver microsomal cytochrome P450 2C6 was found to recognize a single antigen (MW 52 kDa) in sheep liver microsomes. This protein was present in sheep liver (apparent concentration 16 +/- 4 ng/micrograms microsomal protein) representing approx. 28% of the corresponding content of P450 2C6 in untreated rat liver. Preincubation of the anti-P450 2C6 IgG with hepatic microsomes was found to decrease the rate of progesterone 21-hydroxylation to 50-80% of uninhibited control. Taken together, from these findings it is apparent that a P450 enzyme, most likely from the 2C subfamily, catalyses deoxycorticosterone formation from progesterone in sheep liver and that this is a quantitatively important pathway of progesterone hydroxylation in these fractions.     

Mitochondrial reactive oxygen production is dependent on the aromatic hydrocarbon receptor

2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin; TCDD) is a pervasive environmental contaminant that induces hepatic and extrahepatic oxidative stress. We have previously shown that dioxin increases mitochondrial respiration-dependent reactive oxygen production. In the present study we examined the dependence of mitochondrial reactive oxygen production on the aromatic hydrocarbon receptor (AHR), cytochrome P450 1A1 (CYP1A1), and cytochrome P450 1A2 (CYP1A2), proteins believed to be important in dioxin-induced liver toxicity. Congenic Ahr(-/-), Cyp1a1(-/-) and Cyp1a2(-/-) knockout mice, and C57BL/6J inbred mice as their Ahr/Cyp1a1/Cyp1a2(+/+) wild-type (wt) counterparts, were injected intraperitoneally with dioxin (15 microg/kg body weight) or corn-oil vehicle on 3 consecutive days. Liver mitochondria were examined 1 week following the first treatment. The level of mitochondrial H(2)O(2) production in vehicle-treated Ahr(-/-) mice was one fifth that found in vehicle-treated wt mice. Whereas dioxin caused a rise in succinate-stimulated mitochondrial H(2)O(2) production in the wt, Cyp1a1(-/-), and Cyp1a2(-/-) mice, this increase did not occur with the Ahr(-/-) knockout. The lack of H(2)O(2) production in Ahr(-/-) mice was not due to low levels of Mn(2+)-superoxide dismutase (SOD2) as shown by Western immunoblot analysis, nor was it due to high levels of mitochondrial glutathione peroxidase (GPX1) activity. Dioxin decreased mitochondrial aconitase (an enzyme inactivated by superoxide) by 44% in wt mice, by 26% in Cyp1a2(-/-) mice, and by 24% in Cyp1a1(-/-) mice; no change was observed in Ahr(-/-) mice. Dioxin treatment increased mitochondrial glutathione levels in the wt, Cyp1a1(-/-), and Cyp1a2(-/-) mice, but not in Ahr(-/-) mice. These results suggest that both constitutive and dioxin-induced mitochondrial reactive oxygen production is associated with a function of the AHR, and these effects are independent of either CYP1A1 or CYP1A2.     

For dioxin-induced birth defects, mouse or human CYP1A2 in maternal liver protects whereas mouse CYP1A1 and CYP1B1 are inconsequential

Dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) induces cleft palate and hydronephrosis in mice, when exposed in utero; these effects are mediated by the aryl hydrocarbon receptor. The Cyp1a1, Cyp1a2, and Cyp1b1 genes are up-regulated by the aryl hydrocarbon receptor. To elucidate their roles in dioxin-induced teratogenesis, we compared Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) knock-out mice with Cyp1(+/+) wild-type mice. Dioxin was administered (25 microg/kg, gavage) on gestational day 10, and embryos were examined on gestational day 18. The incidence of cleft palate and hydronephrosis was not significantly different in fetuses from Cyp1a1(-/-), Cyp1b1(-/-), and Cyp1(+/+) wild-type mice. To fetuses carried by Cyp1a2(-/-) dams, however, this dose of dioxin was lethal; this effect was absolutely dependent on the maternal Cyp1a2 genotype and independent of the embryonic Cyp1a2 genotype. Dioxin levels were highest in adipose tissue, mammary gland, and circulating blood of Cyp1a2(-/-) mothers, compared with that in the Cyp1(+/+) mothers, who showed highest dioxin levels in liver. More dioxin reached the embryos from Cyp1a2(-/-) dams, compared with that from Cyp1(+/+) dams. Fetuses from Cyp1a2(-/-) dams exhibited a approximately 6-fold increased sensitivity to cleft palate, hydronephrosis, and lethality. Using the humanized hCYP1A1_1A2 transgenic mouse (expressing the human CYP1A1 and CYP1A2 genes in the absence of mouse Cyp1a2 gene), the teratogenic effects of dioxin reverted to the wild-type phenotype. These data indicate that maternal mouse hepatic CYP1A2, by sequestering dioxin and thus altering the pharmacokinetics, protects the embryos from toxicity and birth defects; substitution of the human CYP1A2 trans-gene provides the same protection. In contrast, neither CYP1A1 nor CYP1B1 appears to play a role in dioxin-mediated teratogenesis.     

Identification of a novel rat microsomal vitamin D3 25-hydroxylase

Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1alpha-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates. In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D3 and 1alpha-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50-60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1alpha-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver.     

Characterization of hepatic drug-metabolizing activities of Bama miniature pigs (Sus scrofa domestica): comparison with human enzyme analogs

We used various substrates and selective inhibitors of human cytochrome P450 (CYP) isozymes as probes to study the metabolism of liver microsomes from Chinese Bama miniature pigs. Nifedipine oxidation (NOD) and testosterone 6beta-hydroxylation (6beta-OHT) activities were similar between human liver microsomes and those from Bama miniature pigs. However, compared with those from humans, liver microsomes from Bama miniature pigs showed decreased phenacetin O-deethylation, coumarin 7-hydroxylation, and chlorzoxazone 6-hydroxylation activities, whereas dextromethorphan O-demethylation activity was increased. Ketoconazole selectively inhibited NOD and 6beta-OHT activities in microsomes from Bama pigs, and 8-methoxypsoralen and tranylcypromine inhibited coumarin 7-hydroxylation in pig microsomes. However, furafylline and quinidine failed to selectively inhibit phenacetin O-deethylation and dextromethorphan O-demethylation in microsomes from Bama pigs, whereas chlormethiazole more efficiently inhibited coumarin 7-hydroxylation activity than chlorzoxazone 6-hydroxylation in pig microsomes. Our results suggest that liver microsomes from Chinese Bama miniature pigs are similar to those from humans in regard to metabolism of nifedipine and testosterone (both are probe substrates for human CYP3A4). In addition, chemical inhibitors used as specific probes for human P450 enzymes did not always show the same selectivity toward corresponding enzyme activities in liver microsomes from Bama pigs. However, ketoconazole (a potent inhibitor of human CYP3A4) could be used as a selective inhibitor probe for the NOD and 6beta-OHT activities in liver microsomes from Chinese Bama miniature pigs.     

A transgenic mouse expressing human CYP4B1 in the liver

The human CYP4B1 protein was expressed in the liver of a transgenic mouse line under the control of the promoter of the human apolipoprotein E (apo E) gene. Hepatic microsomes of transgenic mice catalyzed omega-hydroxylation of lauric acid and also activated 2-aminofluorene (2-AF), which is a typical substrate for CYP4B1, to mutagenic compounds detected by an umu gene expression assay. These activities observed in transgenic mouse were efficiently inhibited by CYP4B1 antibody. However, such inhibition was not observed in control mice. This is the first report to indicate catalytic activities of human CYP4B1. For further characterization of human CYP4B1, a fusion protein of CYP4B1 and NADPH-P450 reductase was expressed in yeast cells. It was able to activate 2-AF and was also able to catalyze omega-hydroxylation of lauric acid. This transgenic mouse line and the recombinant fusion protein provide a useful tool to study human CYP4B1 and its relation to chemical toxicity and carcinogenesis.