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Purpose: To investigate the roles of CYP1B1 and MYOC mutations and characterize the phenotype of primary congenital glaucoma in Israeli patients from 3 different ethnic backgrounds., Design: Interventional case series., Methods: This institutional study included 34 Israeli primary congenital glaucoma patients (26 families) comprising 9 Jews (9 families), 17 non-Bedouin Muslim Arabs (10 families), and 8 Druze (7 families). The patients and their relatives (n = 99) were screened for CYP1B1 and MYOC mutations., Results: Mutations in the CYP1B1 gene were detected in 12 of 26 families (46%) with primary congenital glaucoma (5 Muslim Arab, 5 Druze, and 2 Jewish). The Jewish families had compound heterozygous mutations and digenic mutations (ie, an Ashkenazi family had mutations in the CYP1B1 gene [Arg368His, R48G, A119S, and L432V haplotypes] and an Ashkenazi-Sephardic family had a mutation on the CYP1B1 gene [1908delA, Sephardic] with a second missense mutation on the MYOC gene [R76K, Ashkenazi]). The Muslim Arabs and Druze tended to have a more severe phenotype than that of the Jews., Conclusion: The phenotype and spectrum of the CYP1B1 and MYOC mutation roles in the clinical characteristics of primary congenital glaucoma varied according to ethnicity. The rarity of mutations in the CYP1B1 gene among Ashkenazi primary congenital glaucoma patients indicates that a different locus may be involved in the phenotype., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: Lycopene is a carotenoid widely distributed in fruit and vegetables. Epidemiological studies suggest that lycopene consumption is associated with decreased cancer risk. Animal studies have revealed that lycopene may protect against dimethylbenz[a]anthracene (DMBA)-induced carcinogenesis in the breast. Polycylic aromatic hydrocarbons (PAH) are environmental toxicants that can be metabolized by two phase I enzymes, cytochrome P450 1A1 and 1B1. Products formed by these reactions are DNA-attacking moieties. Mutation generated by these genotoxic intermediates is believed to be an important step in cancer initiation. Some phase II detoxifying enzymes, such as uridine diphosphate (UDP)-glucuronosyltransferases (UGT), facilitate the elimination of these genotoxic moieties. In the present study, the mechanism by which lycopene prevented PAH-induced carcinogenesis in the breast was investigated in a cell culture model MCF-7., Research Methods and Procedures: The inhibitory action of lycopene on CYP1 enzymes was assessed in recombinant protein and cell culture using ethoxyresorufin-O-deethylase assay. Messenger RNA expressions of CYP1A1 and 1B1, and UGT were estimated by semi-quantitative reverse transcription-polymerase chain reaction. Cells were co-treated with tritiated DMBA and lycopene for quantifying the protection of the phytocompound against DNA lesion generated from the DMBA metabolites., Results: Lycopene inhibited recombinant CYP1A1 and CYP1B1 with estimated K(i)s in the micromolar range. In MCF-7 cells, lycopene administration slightly reduced the DMBA-induced ethoxyresorufin-O-deethylase activity by 20%. Meanwhile, a four-fold increase in microsomal UGT activity was observed., Conclusion: The present study illustrated that phase I enzyme inhibition and phase II enzyme induction were the underlying chemoprotective mechanisms of lycopene against PAH-induced toxicity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Several studies suggest an involvement of PCBs in breast cancer formation, but the results are ambiguous and the mechanisms not clear. We propose that local activation of cytochrome P450 enzymes, CYP1A1 and CYP1B1 by PCB3, may generate active metabolites which affect apoptosis and thereby promote mammary carcinogenesis. To test this hypothesis MCF-7 human breast cancer cells were exposed to 300 nM PCB3 and its hydroxylated metabolites, 4OH-PCB and 3,4diOH-PCB3. The enzyme activity for CYP1A1 was assayed using the EROD assay, and CYP1A1 and CYP1B1 protein expression by western blotting. PCB3 increased CYP1A1 activity (~1.5fold) and protein levels within 6h after exposure. No effect on CYP1B1 protein expression was observed. The effects of PCB3 and both its metabolites on staurosporine-induced apoptosis were determined by measuring DNA fragmentation using ELISA and TUNEL assays, and by measuring caspase-8 and caspase-9 activity. We found that PCB3 and both of its hydroxylated metabolites had no effect on caspase-8 and caspase-9 activity when cells were grown in medium deprived of estrogen, but reduced caspase-9 activity when cells were grown in medium supplemented with serum containing estradiol. Interestingly, a decrease of DNA fragmentation was observed upon treatment with 3,4diOH-PCB3 in both culture conditions, suggesting that 3,4diOH-PCB3 affects a caspase-independent pathway of cell death. In summary, interactions of PCB3 and its metabolites with estradiol by yet unknown mechanisms inhibit caspase 9-related apoptosis and additional, other death pathways are affected by the catechol metabolite 3,4diOH-PCB3. These anti-apoptotic effects and the change in metabolic activity may contribute to the carcinogenic effect of PCBs., (Copyright © 2009 Elsevier Ltd. All rights reserved.)

Cytochrome P450 (CYP) 1B1 catalyzes 17beta-estradiol (E(2)) to predominantly carcinogenic 4-hydroxy-E(2), whereas CYP1A1 and 1A2 convert E(2) to non-carcinogenic 2-hydroxy-E(2). Hence, selective inhibition of CYP1B1 is recognized to be beneficial for the prevention of E(2) related breast cancer. In this study, we first evaluated the structure-property relationship of 18 major flavonoids on inhibiting enzymatic activity of CYP1A1, 1A2 and 1B1 by using an ethoxyresorufin O-deethylation assay. Flavones and flavonols indicated relatively strong inhibitory effects on CYP1s compared with flavanone that does not have the double bond between C-positions 2 and 3 on the C-ring. Flavonoids used in this study selectively inhibited CYP1B1 activity. In particular, methoxy types of flavones and flavonols such as chrysoeriol and isorhamnetin showed strong and selective inhibition against CYP1B1. To understand why selective inhibition was observed, we carried out a molecular docking analysis of these methoxyflavonoids with the 2-3 double bond and CYP1s. The results suggested that chrysoeriol and isorhamnetin fit well into the active site of CYP1B1, but do not fit into the active site of CYP1A2 and 1A1 because of steric collisions between the methoxy substituent of these methoxyflavonoids and Ser-122 in CYP1A1 and Thr-124 in CYP1A2. In conclusion, our results demonstrate: (1) strong inhibitory effects of flavonoids on CYP1 activities require the 2-3 double bond on the C-ring; (2) methoxyflavonoids with the 2-3 double bond had strong and selective inhibition against CYP1B1, suggesting chemopreventive flavonoids for E(2) related breast cancer; and (3) binding specificity of these methoxyflavonoids is based on the interactions between the methoxy groups and specific CYP1s residues., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Purpose: To estimate an accurate incidence rate for CYP1B1 mutations in German patients with primary congenital glaucoma (PCG)., Design: Observational case series., Methods: Blood was obtained from 39 unrelated patients of German origin with clear clinical features of PCG and screened for mutations in the CYP1B1 gene using direct deoxyribonucleic acid sequencing. One hundred ethnically matched control subjects were screened for novel sequence variants using restriction fragment length polymorphism and denaturing high-performance liquid chromatography., Results: Sequence analysis identified 11 different mutations in 7 patients (18%). Four patients were compound heterozygotes, 2 subjects heterozygous, and 1 homozygous for CYP1B1 mutations. One deletion (c.199_206del8) and 3 missense mutations (L177P, F190L, and S282N) were novel. None of the novel missense mutations identified was found in normal controls., Conclusions: Our results indicate that only a minor proportion of German PCG patients harbor mutations in the CYP1B1 gene and are in line with similar studies from other ethnic populations in which the rate of consanguinity is low. In addition, this is the first report discussing the phenotypes of German PCG patients with and without CYP1B1 mutations.

Purpose: To determine whether there is a correlation among mutations in the cytochrome P4501B1 gene (CYP1B1), the degree of angle dysgenesis observed histologically, and disease severity in congenital glaucoma., Design: Interventional case series., Methods: Direct DNA sequencing was used to screen six unrelated children with congenital glaucoma, each set of parents, and all siblings for CYP1B1 mutations. Specimens of the anterior chamber angle obtained at trabeculectomy were examined histologically to identify abnormalities of the aqueous outflow pathway. CYP1B1 mutations were correlated with both the degree of angle dysgenesis and the patients' disease severity (age at diagnosis, difficulty in achieving intraocular pressure [IOP]) control., Results: Four (66.7%) of the six patients were compound heterozygotes for mutations in the CYP1B1 gene. Seven of the eight CYP1B1 mutations were identified, including two novel mutations (R117P, C209R) and five others previously described (G61E, R368H, R390H, E229K, 4340delG). The cases were divided on the basis of histological phenotype into categories of (1) severe goniodysgenesis highlighted by the agenesis of the canal of Schlemm (two patients), (2) moderate goniodysgenesis characterized by the presence of a band of collagenous tissue (CT) in the trabecular meshwork (TM) and/or the juxtacanalicular tissues (JXT) (three patients), and (3) mild goniodysgenesis with deposition of a mucopolysaccharide material in the JXT (one patient). CYP1B1 mutations were identified in both cases of severe angle dysgenesis and two of three cases of moderate dysgenesis. Disease severity closely correlated with the degree of angle dysgenesis., Conclusions: Most patients in our cohort had compound heterozygous CYP1B1 mutations. Specific CYP1B1 mutations may be associated with severe or moderate angle abnormalities.

Purpose: To report the spectrum of the CYP1B1 mutation in Kuwaiti patients with primary congenital glaucoma (PCG)., Design: Clinical diagnosis of PCG and laboratory based experimental study., Methods: Polymerase chain reaction-restriction polymorphism length fragment (PCR-RPLF) and direct sequencing of exon 2 and the coding region of exon 3 of CYP1B1 gene were the methods used for screening 17 PCG patients, their families, and 105 health individuals from the same ethnicity., Results: Four different mutations were detected in CYP1B1 in 70.6% of the screened patients. The most common one (47%) was homozygote Gly61Glu mutation, previously described in Saudi Arabia, Turkey, and Morocco; all patients were products of consanguineous marriages. The second common mutation was a novel missense (Ala388Thr) mutation found in three patients (17.6%) as compound heterozygote with Arg368His in one patient, and with Gly61Glu in another one while the second mutation in third patient was not detected in the CYP1B1 gene. One patient (5.8%) was homozygote for Cyt280X mutation previously reported in only one Japanese family. In addition to these mutations, a novel Val422Gly polymorphic site was found in three of the PCG patients and in 18 of the 210 tested chromosomes of healthy volunteers., Conclusions: The CYP1B1 mutation spectrum of Kuwaiti PCG patients is similar to that detected in the neighboring countries. No clear genotype-phenotype correlation detected in patients showed different types of CYP1B1 mutation.

Background: Polymorphisms which are inherited alterations in the activity of cytochrome P450 1B1 (CYP1B1), catechol O-methyltransferase (COMT), manganese superoxide dismutase (MnSOD) hold the potential to define differences in estrogen metabolism and, thereby, possibly explain inter-individual differences in cancer susceptibility associated with estrogen-mediated carcinogenesis., Methods: The CYP1B1 (L432V), COMT (V158M), MnSOD (Ala-9Val) genotypes, to examine estrogen metabolism and the influence of age of menarche/menopause, were determined by using different polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) based on genotyping assays., Results: Women who carried CYP1B1 *3 and COMT-L alleles had an earlier age at menarche than the women who carried wild alleles (chi2 = 4.57, p = 0.032), whereas I did not observe any correlation in women with all mutant alleles. Also, CYP1B1 *3 and COMT-H genotypes were common among postmenopausal women with a body mass index (BMI) > 27 kg/m2 (Fisher exact test, p = 0.044)., Conclusions: To my knowledge, this is the first genetic study on the association of these genes with susceptibility in Turkish women. Although the small sample size of each combination of estrogen metabolizing, results suggest that the CYP1B1 *3 and COMT-L alleles influence age at menarche in healthy Turkish women.

A cDNA was cloned from Japanese monkey liver mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR) using oligonucleotide primers based on the marmoset cytochrome P450 2D19 (CYP2D19) nucleotide sequence. The full-length cDNA encoded a 497 amino acid protein (designated CYP2D29) that is 96, 91, and 88% homologous to human CYP2D6, cynomolgus monkey CYP2D17, and marmoset monkey CYP2D19, respectively. Yeast cells (Saccharomyces cerevisiae AH-22 strain) transfected with pGYR1 vectors containing the CYP2D29 cDNA were cultured, and microsomal fractions were obtained. Reduced carbon monoxide-difference spectra and western blot analysis using polyclonal antibodies raised against rat CYP2D2 demonstrated that in yeast cell microsomal fractions, the level of CYP2D29 holoenzyme was similar to that of CYP2D6 holoenzyme. However, western blot analysis indicated that the level of CYP2D29 in Japanese monkey liver microsomes might be much higher than that of CYP2D6 in human liver microsomes. Japanese monkey liver microsomes exhibited much higher activities than did human liver microsomes, expressed as nmol/min/mg protein, for debrisoquine (DB) 4-hydroxylation and bufuralol (BF) 1"-hydroxylation (typical reactions catalyzed by CYP2D6), whereas recombinant CYP2D29 activity, expressed as nmol/min/nmol CYP, was similar to that of CYP2D6 for DB and BF hydroxylation. In kinetic analyses, the K(m) value of CYP2D29 for DB 4-hydroxylation was much lower than that of Japanese monkey liver microsomes, whereas the K(m) value of CYP2D6 for DB 4-hydroxylation was similar to that of human liver microsomes. In contrast, K(m) values for BF 1"-hydroxylation were similar for Japanese monkey and human liver microsomes and yeast cell microsomal fractions expressing recombinant CYP2D29 or CYP2D6. These results suggest that the properties of Japanese monkey CYP2D29 are similar to those of human CYP2D6, but their populations and/or some other factors in liver microsomes may cause the difference in microsomal DB 4-hydroxylase activities between Japanese monkeys and humans.

The aromatic hydrocarbon (Ah) receptor (AHR) is the only known cellular receptor of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and of many other widespread environmental contaminants that cause diverse toxic effects in animals and humans. Most, if not all, the biological effects of TCDD are mediated by the activation of AHR, which is a ligand-activated transcription factor required for ligand-induced expression of several detoxification genes, including those encoding for cytochrome P450 enzymes CYP1A1, CYP1A2, and CYP1B1. Environmental agents also activate several mitogen-activated protein kinase (MAPK) pathways, believed to modulate transcription factor function and to regulate gene expression. However, the contribution to TCDD toxicity resulting from cross-talk between AHR and MAPK pathways has yet to be determined. In this study, we show that TCDD and other AHR ligands induced the immediate activation of the extracellular signal-regulated kinases and the Jun N-terminal kinases, but not the p38 MAPKs. MAPK activation by TCDD did not require the AHR, since it occurred equally well in AHR-negative CV-1 cells and in Ahr (-/-) mouse embryonic fibroblasts as in AHR-positive cells. Distinct from serum factors and the tumor promoter TPA-induced MAPKs, which resulted in transcriptional activation of ELK or c-JUN, TCDD-stimulated MAPKs were critical for the induction of AHR-dependent gene transcription and CYP1A1 expression. These data indicate that AHR ligands elicit AHR-independent non-genomic events that are essential for AHR activation and function.

The cytochrome P450 3A (CYP3A)-mediated midazolam oxidation was studied in rat precision-cut liver slices (PCLS) maintained for 20hr at 4, 20 and 37 degrees, and further incubated for 8hr at 37 degrees. Either at 4 or 20 degrees, midazolam was oxidised by PCLS at similar rates to that observed in freshly cut slices. Moreover, PCLS kept a regioselectivity since 4-hydroxylation was more important than 1'-hydroxylation. Conversely, PCLS totally lost their capacity to oxidise midazolam after 20hr at 37 degrees, and both CYP3A2 protein and mRNA were not detected. CYP3A1 protein was unaffected by a temperature of 37 degrees but its mRNA was totally lost. By blocking transcription with actinomycin D, the decay of both CYP3A mRNAs followed the same profile at either 20 or 37 degrees, indicating that temperature affected the CYP3A2 protein stability. Cell functionality was not involved in such an impairment since the low values of ATP, GSH and protein synthesis rates observed at 4 and 20 degrees were rapidly restored, when PCLS were further incubated at 37 degrees. The use of rat supersomes expressing either CYP3A1 or CYP3A2, strongly supported the hypothesis that 4-hydroxymidazolam was mainly formed by CYP3A2. These results suggest that: (1) CYP3A1 protein is constitutive and largely expressed in rat liver slices; (2) regioselective midazolam oxidation appears to be mainly CYP3A2 dependent; and (3) since CYP3A isoforms have similar half-lives (about 10-14hr), the loss of CYP3A2 protein at 37 degrees might be due to a selective targeting (phosphorylation ?) leading to proteolytic disposal by the proteasome.

The constitutive expression of CYP2B mRNA in the livers of mice in the prepubertal stage was sex-independent, with CYP2B9 as the principal isoform. During the maturation stage, CYP2B10 was expressed in both sexes, whereas CYP2B9 was diminished markedly in the males, resulting in a sexually dimorphic expression in adult mice. Hypophysectomy eliminated the sexual dimorphism in the mouse CYP2B subfamily by markedly increasing the expression of both CYP2B9 and CYP2B10 in males to levels similar to those in females.

The mammalian constitutively active receptor (CAR) is a novel ligand-activated transcription factor that participates in controlling the expression of cytochrome P450 2B (CYP2B) genes in response to pharmaceutical agents (phenobarbital) and halogenated aromatic hydrocarbons (ortho-substituted PCBs). The occurrence and physiological function of this protein are as yet unknown in marine animals, where there has been a paradoxical lack of induction by PB-type chemicals. One approach to understanding CAR function is to study the evolutionary history of processes such as CAR-CYP2B coupling. In this study, CAR function was evaluated in a representative teleost fish (scup, Stenotomus chrysops). Treatment of scup with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), which is one of the most potent CYP2B inducers in mouse, caused no increase in hepatic alkoxyresorufin O-dealkylase activity nor in immunodetectable CYP2B-like protein levels. Western blot analyses of scup livers using anti-human CAR antisera revealed the occurrence of a putative CAR homologue in nuclear and cytoplasmic fractions, but no nuclear accumulation of CAR following TCPOBOP treatment, which is a first step regulating the transcriptional activation of CYP2B genes in mouse. Immunohistochemical study also showed no translocation of CAR into nucleus in the hepatocytes of TCPOBOP-treated scup. These results suggest that there may be species-specific differences in CAR activation or CAR-CYP2B coupling signaling transduction in fish from those in mouse.

The plasma cholesterol-lowering effect and mechanism thereof of a choleretic phloracetophenone or 2,4,6-trihydroxyacetophenone (THA) were investigated in hypercholesterolemic male hamsters. Intragastric administration of THA (300-600 micromol/kg) twice a day for 7 days to these animals caused a dose- and time-dependent decrease in both plasma cholesterol and triglyceride levels. THA at a dose of 400 micromol/kg reduced the cholesterol and triglyceride levels in plasma to 52% and 25% of the level in corresponding cholesterol-fed controls, respectively, with decreases in both plasma very low density lipoprotein and low density lipoprotein cholesterol but not in high density lipoprotein cholesterol. THA did not significantly alter total hepatic cholesterol content but significantly increased the excretion of both bile acids and cholesterol into the intestinal lumen for elimination. Corresponding to the increase in bile acid excretion, THA caused a seven-fold increase in hepatic cholesterol 7alpha-hydroxylase activity. These results suggest that THA exerts its cholesterol lowering effect by increasing hepatic cholesterol 7alpha-hydroxylase activity which increases hepatic conversion of cholesterol to bile acid for disposal via biliary secretion. This compound may have a potential for future development as a therapeutic agent for lowering lipids in hypercholesterolemic patients.

In this study, we performed a screening of the specificities of rat cytochrome P450 (CYP) isoforms for metabolic reactions known as the specific probes of human CYP isoforms, using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or B-lymphoblastoid cells. Among the metabolic reactions studied, diclofenac 4-hydroxylation (DFH), dextromethorphan O-demethylation (DMOD) and midazolam 4-hydroxylation were specifically catalyzed by CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. These results suggest that diclofenac 4-hydroxylation, dextromethorphan O-demethylation and midazolam 4-hydroxylation are useful as catalytic markers of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. On the other hand, phenacetin O-deethylation and 7-ethoxyresorufin O-deethylation were catalyzed both by CYP1A2 and by CYP2C6. Benzyloxyresorufin O-dealkylation and pentoxyresorufin O-dealkylation were also catalyzed by CYP1A2 in addition to CYP2B1. Bufuralol 1'-hydroxylation was extensively catalyzed by CYP2D2 but also by CYP2C6 and CYP2C11. p-Nitrophenol 2-hydroxylation and chlorzoxazone 6-hydroxylation were extensively catalyzed by CYP2E1 but also by CYP1A2 and CYP3A1. Therefore, it is necessary to conduct further study to clarify whether these activities in rat liver microsomes are useful as probes of rat CYP isoforms. In contrast, coumarin 7-hydroxylation and S- and R-mephenytoin 4'-hydroxylation did not show selectivity toward any isoforms of rat CYP studied. Therefore, activities of coumarin 7-hydroxylation and S- and R-mephenytoin 4'-hydroxylation are not able to be used as catalytic probes of CYP isoforms in rat liver microsomes. These results may provide useful information regarding catalytic probes of rat CYPs for studies using rat liver microsomal samples.

We investigated the effect of dexamethasone (DEX) on the disposition kinetics of cyclosporin A (CyA) and the mechanism of this drug interaction. Rats were treated with DEX (1 or 75mg/kg per day, i.p.) once a day for 1-7 days, and the blood concentration of CyA was measured after an i.v. or p.o. dose of CyA (10mg/kg) at 1.5hr after the last DEX treatment. In rats treated with a low dose of DEX (1mg/kg), the blood concentration of CyA after i.v. administration was unchanged compared with that of untreated rats, whereas the blood concentration after oral administration was significantly decreased, and this decrease was dependent on the duration of DEX administration. The total clearance (CL(tot)) of CyA was unchanged, but the bioavailability was significantly decreased to about one-third of that in DEX-untreated rats after 7 days of DEX treatment. At this time, the expression of mdr1a mRNA and P-gp in the liver and intestine was increased, whereas CYP3A2 was unaffected at both the mRNA and protein levels. In rats treated with a high dose of DEX (75mg/kg), the blood concentration of CyA was significantly decreased after both i.v. and p.o. administrations compared with those of untreated rats. The bioavailability of CyA was decreased, and the CL(tot) was significantly increased. The P-gp and CYP3A2 in the liver and intestine were increased at both the mRNA and protein levels. Our results indicate that the drug interaction between CyA and DEX is a consequence of modulation of P-gp and CYP3A2 gene expression by DEX, with differential dose-dependence.

In previous work it was shown that hepatic microsomes from rats treated with 3-methylcholanthrene and similar inducers had increased bilirubin-degrading activity. The activity was further stimulated by addition of 3,4-tetrachlorobiphenyl (TCB), a response specifically dependent on CYP1A1. Here, we compared the effect of adding PCBs of either planar or non-planar configuration on rate of bilirubin degradation, monooxygenase activity and NADPH/O(2) consumption by liver microsomes from animals treated with either phenobarbital or 3-methylcholanthrene/beta-naphthoflavone. We also examined the oxidation of uroporphyrinogen (hexahydro-uroporphyrin) (URO'gen) under these conditions. Polychlorinated biphenyl (PCBs) stimulated the rate of bilirubin and URO'gen oxidation with microsomes expressing high levels of either CYP2B or CYP1A, inhibiting at the same time their monooxygenase activities (PROD and EROD, respectively); however, non-planar di-ortho-substituted PCBs were preferentially active with phenobarbitone-induced microsomes, in contrast to those active with 3-methylcholanthrene/beta-naphthoflavone microsomes, where a planar configuration was required for activity. An antibody raised against CYP2B1 markedly inhibited the PCB-dependent bilirubin degradation and PROD activities of phenobarbital-induced microsomes with similar dose-response curves for the two effects. Increased microsomal utilizations of NADPH and O(2) were also caused by PCBs with both types of induced microsomes and here again PCBs of different configuration were preferentially active. It is concluded that PCBs of the appropriate configuration may interact with either CYP1A1 or CYP2B1, increase production of oxidative species by an uncoupling mechanism, and lead to oxidation of target molecules in the cell, among these uroporphyrinogen and bilirubin.

Upregulation of mouse hepatic cytochrome P450 2A5 (CYP2A5) is a process closely associated with hepatocellular damage and formation of liver tumours. 2-Aminopurine, a protein kinase inhibitor modulating cell cycle control, was recently shown to strongly induce CYP2A5 in mouse hepatocytes. The objective of this study was to determine the association between CYP2A5 induction and apoptosis in mouse primary hepatocytes. Five well-characterised CYP2A5 inducers were tested for their ability to affect apoptosis rate, determined by immunohistochemical in situ 3'-end-labelling technique, in a primary mouse hepatocyte model. Transforming growth factor beta (TGFbeta) was used as a positive (proapoptotic) control. Phenobarbital, pyrazole and the mitogen-activated protein kinase inhibitor PD98059 did not significantly affect apoptosis rate in hepatocytes. Norcocaine induced apoptosis at 6 hr (1.8-fold) and 2-aminopurine 12 hr (1.4-fold) after treatment, which is considerably earlier than peaks in the amount of CYP2A5 mRNA. TGFbeta reduced CYP2A5 marker activity, coumarin 7-hydroxylase by 74%. These results indicate that in a primary hepatocyte model (a) there is no systematic correlation between apoptosis and CYP2A5 induction; (b) phenobarbital does not significantly affect the rate of apoptosis; and (c) the induction of apoptosis caused by the chemicals tested occurs considerable earlier than elevation of CYP2A5 expression. Thus, no causal link appears to exist between induction of CYP2A5 and apoptotic rate.

Multidrug resistance-associated protein 2 (MRP2) is a drug efflux pump found at the biliary pole of hepatocytes. In the present study, we have investigated its expression in response to phenobarbital, a liver tumor promoter known to up-regulate hepatic cytochromes P450 (CYPs), such as CYP2B1/2 and CYP3A1/2. MRP2 mRNA and protein levels were found to be markedly increased in both primary rat and human hepatocytes exposed to phenobarbital. However, features of this up-regulation, especially the dose-response, were different from those of the induction of CYP2B1/2 and CYP3A1/2. In addition, hepatic MRP2 expression remained unaltered in rats treated by phenobarbital that, by contrast, increased CYP2B1/2 and CYP3A1/2 gene expression in the liver. Therefore, MRP2 and CYPs appeared differently regulated in response to phenobarbital in both in vivo and in vitro situations, suggesting that cellular and molecular mechanisms underlying up-regulation of MRP2 are, at least in part, unrelated to those operating for CYPs. Phenobarbital-related MRP2 induction in primary rat hepatocytes was associated with some phenotypic effects of the barbiturate, such as prolonged cell survival and inhibition of cell proliferation. Phenobarbital also inhibited growth of human hepatoma HepG(2) cells and increased their level of MRP2 gene expression. Such results may favor a putative relationship between phenobarbital-mediated MRP2 regulation in cultured liver parenchymal cells and alteration of cell cycle and survival.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances or suppresses the transcriptional activation of CYP1A1 by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a cell/tissue-specific manner. The basis for these effects is not known. Exposure of the immortalized human breast epithelial cell line MCF10A-Neo to TPA at the time of, or up to 12 hr prior to, the addition of TCDD strongly suppressed the transcriptional activation of CYP1A1 and CYP1B1 (IC(50) approximately 0.5 nM). A recent study (Carcinogenesis 2000;21:1303-12) demonstrated that TPA-treated MCF10A-Neo cells rapidly activate the latent transforming growth factor beta (TGFbeta) in the serum used to supplement the culture medium. The suppressive effects of TPA on CYP1A1 induction by TCDD in MCF10A-Neo cultures could be partially suppressed by: (a) co-incubation of TCDD + TPA-treated cultures with a neutralizing TGFbeta pan antibody; (b) prior removal of latent TGFbeta from the culture medium; or (c) switching cultures to serum- and growth factor-free medium immediately before the addition of TPA and TCDD. Exposure of cultures to TPA 24-48 hr prior to subsequent TPA + TCDD treatment not only inhibited the suppressive effects of TPA, but markedly enhanced CYP1A1 mRNA accumulation. TPA caused a rapid and protracted activation of extracellular signal-regulated kinases (ERKs). Pretreatment of cultures with the mitogen-activated protein kinase kinase (MEK) inhibitor PD184352 [2-(2-chloro-4-iodo-phenylamino)-N-cyclopropyl-methoxy-3,4-difluoro-benzamide] completely inhibited ERK activation by TPA. However, PD184352 did not prevent the suppressive effects of TPA on CYP1A1 activation by TCDD. These studies demonstrate that TPA initiates protein kinase C-dependent, ERK-independent processes that suppress CYP1A1 activation by TCDD in MCF10A-Neo cells. Furthermore, TGFbeta mediates a small portion of this suppressive activity.

The present study investigated the effect of dietary zinc deficiency during the developmental period on hepatic cytochrome P450 (CYP) expression in postpubertal male rats. Twenty-one-day-old weanling male Wistar rats were randomly assigned to one of the following dietary groups: zinc-adequate (31 mg zinc/kg diet); marginal zinc-deficient (3 mg zinc/kg diet); severe zinc-deficient (1 mg zinc/kg diet); or pair-fed control for either the marginal or severe zinc-deficient group. All rats were killed at 63 days of age. Compared with the corresponding pair-fed controls, marginal zinc deficiency decreased CYP2C11-mediated testosterone 2alpha- and 16alpha-hydroxylase activities by 43 and 42%, respectively, whereas severe zinc deficiency reduced each of these activities by approximately 60%. The decrease in CYP2C11 activity was accompanied by a reduction in CYP2C11 protein and mRNA levels, as assessed by immunoblot and reverse transcription-polymerase chain reaction (RT-PCR) assays, respectively. Additional RT-PCR analysis indicated that severe zinc deficiency decreased CYP3A2 and CYP3A18 mRNA levels by 49 and 43%, respectively, whereas it increased CYP2C12 (253%) and CYP3A9 (238%) mRNA expression. Plasma testosterone concentration was decreased by 67% in the marginal zinc-deficient group when compared with the corresponding pair-fed control group. By comparison, it was below the limit of quantification (0.2 ng/mL) in the severe zinc-deficient rats. Overall, these results indicate that dietary zinc deficiency during the developmental period feminized the hepatic gene expression of the sexually dimorphic CYP2C11, CYP3A2, CYP3A18, CYP2C12, and CYP3A9 in postpubertal male rats.

A higher proportion of alcoholics than non-alcoholics smoke (>80 vs 30%). In animals, chronic administration of alcohol induces tolerance to some effects of nicotine. To investigate if chronic ethanol (EtOH) induces alterations in CYP2B1/2 and nicotine C-oxidation activity, male rats (N = 4-6/group) were treated once daily with saline or EtOH (0.3, 1.0, and 3.0 g/kg, p.o./by gavage) for 7 days. A quantitative immunoblotting assay was developed to detect CYP2B1/2 in the brain, where constitutive expression is low, and in the liver. Using this method, it was determined that EtOH did not alter CYP2B1/2 protein expression significantly in six brain regions (olfactory bulbs, olfactory tubercles, frontal cortex, hippocampus, cerebellum, and brainstem). However, a dose-dependent induction of CYP2B1/2 protein expression was detected in the liver. Significant induction of 2-, 3-, and 2.7-fold were observed for the 0.3, 1.0, and 3.0 g/kg doses, respectively. Increases were also observed in CYP2B1 mRNA, which was induced by 14, 38, and 43% at the same doses. Liver microsomal nicotine C-oxidation also was increased (1.3 to 4.5-fold). CYP2B selective inactivators demonstrated that approximately 70% of nicotine C-oxidation was mediated by CYP2B1/2 in both EtOH-induced and uninduced hepatic microsomes. In summary, chronic, behaviorally relevant doses of EtOH induce CYP2B1/2 protein, mRNA, and nicotine C-oxidation activity in rat liver but not in rat brain, and these increases could contribute to cross-tolerance and co-abuse of ethanol and nicotine.

Hepatic expression of cytochrome P450 2A6 (CYP2A6) varies widely in humans and is induced during hepatitis; however, the mechanism regulating CYP2A6 has not been established. The murine orthologue Cyp2a5 is regulated post-transcriptionally by mRNA stabilization. A 43-kDa protein that binds to the 3'-untranslated region (3'-UTR) of Cyp2a5 mRNA has been identified, but its role in mRNA stabilization is unclear. We hypothesized that similar interactions occur between cytosolic proteins in human liver and CYP2A6 3'-UTR mRNA. We identified, by RNA electrophoretic mobility shift assay, an hepatic cytosolic protein that binds specifically to sequences in the 3'-UTR of CYP2A6. Complexes did not form with denatured proteins and were eliminated with proteinase K digestion. Complex formation was inhibited with a molar excess of unlabeled CYP2A6 RNA but not by non-specific competitor RNA. Protein-mRNA interactions were not affected by probe denaturation, suggesting that RNA secondary structure is not essential for binding. UV cross-linking of complexes revealed RNA-binding proteins in both human and mouse liver cytosols with molecular masses of approximately 43 kDa. Using truncated RNA probes corresponding to various lengths of CYP2A6 mRNA, the protein-binding site was localized to a 50-nucleotide region between bases 1478 and 1527 of the 3'-UTR. Complex formation with hepatic cytosolic protein from four human subjects correlated with levels of hepatic CYP2A6 microsomal protein, suggesting a possible regulatory role. Further characterization of the RNA-binding protein, the primary binding site, and the influence of this interaction on CYP2A6 mRNA stability will help to elucidate the relevance of these findings to the post-transcriptional control of CYP2A6.

This study was undertaken to assess associations between age, gender, cigarette smoke and non-workplace cadmium exposure, and liver pathology and inter-individual variation in cytochrome P450 (CYP) expression in human tissues. Autopsy specimens of twenty-eight Queensland residents whose ages ranged from 3 to 89 years were analyzed for the presence of nine CYP protein isoforms by immunoblotting. All subjects were Caucasians and their liver cadmium contents ranged from 0.11 to 3.95 microg/g wet weight, while their kidney cadmium contents were in the range of 2 to 63 microg/g wet weight. CYP1A2, CYP2A6, CYP2D6, CYP3A4, and CYP3A5 were detected in liver but not in kidney, and CYP1A1 and CYP1B1 were not found in liver or kidney. Lowered liver CYP2C8/19 protein contents were found to be associated with liver pathology. Importantly, we show elevated levels of CYP2C9 protein to be associated with cadmium accumulation in liver. No mechanism that explains this association is apparent, but there are two possibilities that require further study. One is that variation in CYP2C9 protein levels may be, in part, attributed to an individual's non-workplace exposure to cadmium, or an individual's CYP2C9 genotype may be a risk factor for cadmium accumulation. A positive correlation was found between liver CYP3A4 protein and subject age. Levels of liver CYP1A2 protein, but not other CYP forms, were increased in people more exposed to cigarette smoke, but there was no association between CYP1A2 protein and cadmium. CYP2A6 protein was found in all liver samples and CYP2A6 gene typing indicated the absence of CYP2A6 null allele (CYP2A6(D)) in this sample group, confirming very low prevalence of homozygous CYP2A6(D) in Caucasians. CYP2A6 gene types W/W, W/C, and C/C were not associated with variations in liver microsomal CYP2A6 protein. CYP2D6 protein was absent in all twenty-five kidney samples tested but was detectable in liver samples of all but two subjects, indicating the prevalence of the CYP2D6 null allele (CYP2D6(D)) in this sample group to be about 7%, typical of Caucasian populations.

The role of metabolic polymorphism in the development of physical dependence to codeine was assessed in cytochrome P450 2D2 (CYP2D2) deficient Dark Agouti and CYP2D2 intact Sprague-Dawley rats by assessment of the severity of naloxone precipitated withdrawal after codeine and morphine administration. Plasma morphine concentrations after codeine were significantly higher (P<0.01) in Sprague-Dawley than in Dark Agouti rats with metabolic ratios of 0.71 +/- 0.27 and 0.07 +/- 0.04, respectively. Withdrawal after codeine resulted in significantly greater hypothermia (3.5-4 degrees C, P<0.0001) in Sprague-Dawley animals compared to the other groups. Body weight loss was similar for all groups ranging from 6.2 +/- 0.4 to 8.2 +/- 0.6 g. When strain and treatment data were combined, a relationship between body temperature and plasma morphine concentration could be described by the inverse Hill equation (r(2)=0.76, EC(50)=556 +/- 121 ng/ml, n=2.9 +/- 1.5). These data indicate that dependence and withdrawal after codeine administration are dependent on its bioconversion to morphine.

The objective was to examine the NADPH-dependent oxygenation of prostaglandin H(2) (PGH(2)) and three PGH(2) analogues, 9,11-diazo-15-deoxy-PGH(2) (U51605), 9,11-epoxymethano-PGH(2) (U44069), and 11,9-epoxymethano-PGH(2) (U46619), by cytochromes P450, and to characterize the metabolites by mass spectrometry. CYP2C19, CYP4A11, CYP4F8, and liver and renal cortical microsomes oxidized the omega-side chain of U44069, U46619, and U51605, whereas only CYP4F8 oxidized the omega-side chain of PGH(2). PGH(2) was transformed to four stereoisomers of 5-hydroxy-PGI(1) by recombinant cytochromes P450. CYP4F8 formed the 5-hydroxy-PGI(1) isomers in small amounts compared to the 19-hydroxy metabolites of PGH(2). Isomers of 5-hydroxy-PGI(1) and 6-keto-PGF(1 alpha) were detectable when PGH(2) decomposed in the presence of hemin, hemoglobin, or heat-inactivated microsomes. 5-Hydroxy-PGI(1) is likely formed from PGH(2) in a pseudo-enzymatic reaction involving homolytic scission of the endoperoxide and formation of an ether between C-9 and C-6 and a carbon-centered radical at C-5, which reacts with molecular oxygen. CYP4F8 catalyzes 19-hydroxylation of PGH(2), but the absolute configuration of the 19-hydroxy group is unknown, whereas human seminal fluid contains (19R)-hydroxy-PGE(2). CYP4F8 was found to metabolize U51605 to 90% of the (19R)-hydroxy metabolite, providing further evidence in favor of a role of CYP4F8 in biosynthesis of (19R)-hydroxy PGE in human seminal vesicles. We conclude that omega-side chain hydroxylation of PGH(2) analogues may be catalyzed by many different cytochromes P450, but only CYP4F8 oxidizes the omega-side chain of PGH(2) efficiently.

Oxidative ring opening of troglitazone (TGZ)(1) a thiazolidine 2,4-dione derivative used for the treatment of type II diabetes mellitus, leads to the formation of a quinone metabolite. The formation of TGZ quinone was shown to be NADPH dependent and to require active microsomal enzymes. Quinone formation was not affected by co-incubation with catalase or sodium azide and was partially inhibited (25%) by superoxide dismutase (SOD). Kinetic analysis of TGZ quinone formation in human liver microsomes implied single enzyme involvement. CYP3A isoforms were characterized as the primary enzymes involved in quinone formation by several lines of evidence including: (a) troleandomycin and ketoconazole almost completely inhibited microsomal quinone formation when SOD was present, whereas other CYP inhibitors had minimal effects (<20%); (b) TGZ quinone formation was highly correlated with regard to both contents (r(2): 0.9374) and activities (r(2): 0.7951) of CYP3A4 in human liver microsomes (HLM); (c) baculovirus insect cell-expressed human CYP3A4 was able to catalyze TGZ quinone formation at a higher capacity (V(max)/K(m)) than other human CYPs with the relative contribution of CYP3A4 in HLM estimated to be 20-fold higher than that of other CYPs; (d) TGZ quinone formation was increased by 350% in liver microsomes from rats pretreated with dexamethasone (DEX); and (e) plasma concentrations of TGZ quinone were increased by 260-680% in rats pretreated with DEX. The chemical nature of the quinone metabolite suggests an atypical CYP reaction consistent with a one-electron oxidation mechanism where an intermediate phenoxy radical combines with ferryl oxygen to subsequently form the quinone metabolite.

The metabolism of tocopherols by omega- and beta-oxidation of the phytyl side chain has been inferred from the identification of the final products carboxyethyl-hydroxychromans (CEHC) and immediate precursors, alpha- and gamma-carboxymethylbutyl-hydroxychromans (CMBHCs). This hypothesis is here corroborated by the identification of a further alpha-tocopherol metabolite, alpha-carboxymethylhexyl-hydroxychroman (alpha-CMHHC), and evidence for the involvement of a P450-type cytochrome. HepG2 cells, when exposed to 100 microM all-rac-alpha-tocopherol, released alpha-CEHC, alpha-CMBHC, and alpha-CMHHC into the medium. The detection of those metabolites required pretreatment of the cells with alpha-tocopherol for 10 d. In contrast, analogous metabolites of gamma and delta-tocopherol were detectable without any preconditioning, while corresponding metabolites of RRR-alpha-tocopherol could not be detected at all. The formation of alpha-CEHC from all-rac-alpha-tocopherol was enhanced up to 5-fold by pretreatment of the HepG2 cells with rifampicin, known to induce CYP3A-type cytochromes with the capability of catalyzing omega-oxidation. In contrast, clofibrate did not reveal any effect. This observation suggests that a CYP3A-type cytochrome initiates tocopherol metabolism by omega-oxidation. It further reveals that inducible omega-oxidation is the rate-limiting step in tocopherol metabolism. It is discussed that competition of microsomal omega-oxidation with specific binding by the alpha-tocopherol transfer protein (alpha-TTP) determines the metabolic fate of the individual tocopherols.

The expression of Cyp2b9 and Cyp2b10 genes was investigated in kidney, liver, and cultured hepatocytes of adult C57BL/6NCrj mice. The constitutive expression level of CYP2B mRNA in kidney was higher in female than in male mice, as it was in the liver where more CYP2B9 than CYP2B10 was expressed in the females, and more CYP2B10 was expressed in the males. After treatment with dexamethasone (Dex), induction of CYP2B10 mRNA and protein in the kidneys was far greater in male than in female mice. In contrast to Dex, phenobarbital (PB), pregnenolone-16 alpha-carbonitrile (PCN), and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) did not induce the expression of the Cyp2b gene in the kidneys of either sex. In the liver, PB, PCN, and DDT induced both CYP2B9 and CYP2B10 in both sexes to the same extent, whereas Dex induced only CYP2B10 and simultaneously suppressed CYP2B9. Dex-inducible expression of CYP2B mRNA was decreased by 11 beta-[4-dimethylamino]phenyl-17 beta-hydroxy-17-[1-propynyl]estra-4,9-dien-3-one (RU-486), in both the kidneys and liver from male mice, and in cultured hepatocytes. However, RU-486 itself induced the expression of CYP2B mRNA in female liver and cultured hepatocytes. Interestingly, RU-486 increased PB-inducible expression of these species in cultured hepatocytes. Gonadectomy increased the expression of CYP2B mRNA in untreated male liver, but suppressed Dex-induced expression in the kidneys of both sexes. These observations suggest that (a) there are multiple regulatory pathways in the expression of Cyp2b genes, one of which used by Dex is mediated via the glucocorticoid receptor, which is different from that used by PB, and (b) sex hormones play a role in the regulation of the sex-dependent expression of Cyp2b genes in the mouse.

The overexpression of human cytochrome P450 CYP1B1 has been observed in a wide variety of malignant tumours, but the protein is undetectable in normal tissues. A number of cytochrome P450 enzymes are known to metabolise a variety of anticancer drugs, and the consequence of cytochrome P450 metabolism is usually detoxification of the drug, although bioactivation occurs in some cases. In this study, a Chinese hamster ovary cell line expressing human cytochrome P450 CYP1B1 was used to evaluate the cytotoxic profile of several anticancer drugs (docetaxel, paclitaxel, cyclophosphamide, doxorubicin, 5-fluorouracil, cisplatin, and carboplatin) commonly used clinically in the treatment of cancer. The MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to determine the levels of cytotoxicity. The key finding of this study was that on exposure to docetaxel, a significant decrease in sensitivity towards the cytotoxic effects of docetaxel was observed in the cell line expressing CYP1B1 compared to the parental cell line (P = 0.03). Moreover, this difference in cytotoxicity was reversed by co-incubation of the cells with both docetaxel and the cytochrome P450 CYP1 inhibitor alpha-naphthoflavone. This study is the first to indicate that the presence of CYP1B1 in cells decreases their sensitivity to the cytotoxic effects of a specific anticancer drug.

An 163-bp fragment of the rat cytochrome P450 gene, CYP2B2 has been shown to contain sequences that mediate phenobarbital (PB) responsiveness of this gene. In studies on this rat gene and the orthologous mouse gene, Cyp2b10, the minimal fragment required for near full PB responsiveness has varied from about 50 to 80 bp depending on the gene used and the number of copies of the PB responsive sequences assessed. Since there is a single copy of the CYP genes in the genome, we have evaluated deletion and block mutations across an 84-bp region of the PB responsive unit (PBRU), by in situ transfection in rat liver using single copies of the PBRU sequences. From the 5' end, deletions to -2243 retained more than 50% responsiveness to PB compared to the 163-bp fragment. The fragment -2237 to -2155 retained less than 20% responsiveness even though it contained the nuclear receptor (NR)-1, NR-2, and NF-1 motifs which are present in the core of the PBRU. From the 3' end, deletions from -2170 to -2194 eliminated PB responsiveness indicating that the 74-bp sequence from -2243 to -2170 is able to mediate full PB responsiveness. Block mutations within the NR-1 and NF-1 regions reduced responsiveness most dramatically, but did not abolish it, and mutations 3' of the NF-1 site modestly reduced responsiveness. Protein binding was not affected by mutations in the NR-1 region as assessed by DNase I footprinting in vitro but mutations within the NR-2 region reduced binding to the NF-1 site. Mutations of the 5' half or the 3' half of the bipartite NF-1 site, resulted in loss of protection of the NF-1 site and new footprints to the 3' or 5' side, respectively, of the NF-1 site. These results indicate that sequences in addition to the NR-1 and -2 and the NF-1 sites are required for full responsiveness to PB and suggest that proteins which bind to these sites may interact.

Numerous xenobiotics are capable of inducing their own metabolism and by enzyme induction can also lead to enhanced biotransformation of other xenobiotics. In this project, we examined the influence of pyrethroids (permethrin, cypermethrin, and fenvalerate) on the expression and activity of the phenobarbital (PB)-inducible cytochrome P450 2B1 isoform (CYP2B1) in primary rat hepatocyte cultures. Incubation of hepatocyte cultures with pyrethroids resulted in a marked CYP2B1 induction. Among the tested pyrethroids, permethrin elicited the most pronounced induction of CYP2B1 mRNA, which exceeded maximal induction achieved by PB at concentrations approximately 10-fold higher. Furthermore, permethrin induced CYP3A1 mRNA expression, while the expression of the CYP1A1 isoform, which in vivo is not responsive to PB treatment, was not significantly affected by pyrethroids. Permethrin-dependent enhancement of CYP2B1 and CYP3A1 mRNA expression was repressed by the hepatotrophic cytokine epidermal growth factor, which is known to also inhibit PB-dependent induction of CYP2B1. Several metabolites of permethrin formed by hepatocytes (3-(2',2'-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 3-phenoxybenzyl alcohol, and 3-phenoxybenzoic acid) were ineffective in inducing CYP2B1 mRNA. Furthermore, permethrin stimulated the expression of the luciferase reporter gene under control of the CYP2B1 promoter (comprising the PB-responsive enhancer module) in transiently transfected primary hepatocyte cultures. Thus, permethrin-stimulated gene expression occurred on the transcriptional level. Taken together, these results indicate that the pyrethroid permethrin is a PB-like inducer. Due to its superior potency in induction, permethrin appears as a useful substance for mechanistic studies to elucidate the mechanism of enzyme induction by phenobarbital.

A new direct injection/on-line guard cartridge extraction-tandem mass spectrometry (DI/GCE-MS-MS) method has been validated for high-throughput evaluation of cytochrome P450 (CYP) 2C19 inhibition potential using human hepatic microsomes and 96-well microtiter plates. Microsomal incubations were quenched with formic acid, centrifuged, and the resulting supernatants were injected for DI/GCE-MS-MS analysis. Due to the use of a C(18) guard cartridge (4x2.0 mm I.D.), this method exhibits several advantages such as little sample preparation, rapid on-line extraction, short analysis time (2.5 min), and minimal source contamination and performance deterioration. The DI/GCE-MS-MS method demonstrates an inter-day accuracy ranged from 0.3 to 2.4% with precision ranging from 2.0 to 3.0% for the quantification of 4-hydroxymephenytoin, a marker metabolite of S-mephenytoin mediated by CYP2C19, in microsomal incubations. The CYP2C19 inhibition assay has been validated using tranylcypromine as a known inhibitor of the isoform. The IC(50) value (43.5 microM) measured by the new method is in agreement with a reported literature value (approximately 30 microM).

Purpose: To report a novel missense mutation and DNA polymorphism of the CYP1B1gene in Japanese patients with primary congenital glaucoma., Methods: A series of 11 unrelated patients with primary congenital glaucoma was examined. Patients were followed in the Kagoshima University Hospital between 1979 and 1998. DNA was extracted from leukocytes of the patients, their families, and unrelated healthy individuals. Amplicons spanning the coding regions of the CYP1B1 gene were examined by direct sequencing and enzyme-restriction detection., Results: In the 11 unrelated patients, besides the previously reported insertional mutation (1620 ins G), a novel missense mutation was identified at codons 444 to replace arginine with glutamine (R444Q) in one patient. The novel missense mutation cosegregated in the relevant family as an autosomal recessive pattern and was not found in other patients or control individuals. In addition, five polymorphic sites were found at codons 48, 119, 330, 432, and 449. These polymorphic alleles did not cosegregate with the disease, and they were found in healthy individuals as well., Conclusions: Approximately 20% of Japanese patients with primary congenital glaucoma may be affected by mutations in the CYP1B1 gene. Further studies are justified to explore whether a relationship exists between the phenotypic expressivity of the disease and the type of mutation.

Enzymes of the cytochrome P450 3A (CYP3A) sub-family are abundant in adult liver and gut and contribute significantly to the first-pass metabolism of many orally administered drugs. The development of CYP3A enzymes with regard to their expression and activity in enterocytic and hepatic microsomes from 1-day-old through to adult male and female rats has been studied. Microsomes were prepared by calcium precipitation. Enzyme expression was assessed semi-quantitatively by Western blotting using rat polyclonal CYP3A2 and 2C11 antibodies and peptide antibodies specific to rat CYPs 3A1, 3A2, 2C12, and 2C13. The formation of 6beta-hydroxytestosterone (6OHT), determined by HPLC, was used as a measure of enzyme activity. Formation of 6OHT by enterocytic microsomes was similar for males and females and showed a sharp increase at weaning. This pattern was mirrored by levels of immunoquantifiable CYP3A2 (CYP3A9), but CYP3A1 followed a more gradual development. CYPs 2C11, 2C12, or 2C13 were not detected in gut microsomes. In contrast, CYPs 3A1, 3A2, 2C11, 2C12, and 2C13 were all expressed in hepatic microsomes. There was no surge in hepatic enzyme expression or hepatic 6OHT formation at weaning, and a marked sex difference in 6OHT formation was apparent from day 25. The surge in gut activity at weaning may be a protective mechanism against ingested toxins.

The study of drug metabolism in cultured rat hepatocytes is hampered by the rapid loss of the expression of cytochrome P450 enzymes. Nevertheless, the activity of cytochrome P450 3A (CYP3A), one of the most important isoenzymes for drug metabolism, can be elevated by chemical inducers. In the present study, we investigated in cultured rat hepatocytes the induction of all four currently identified CYP3A isoforms by dexamethasone, and compared the results obtained in vitro with the induction profile of dexamethasone in vivo. To this end, CYP3A mRNA levels were quantified with a novel, radioactive reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and CYP3A enzymatic activity was measured by a testosterone hydroxylation assay. In the RT-PCR assay, CYP3A isoforms were co-amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of radioactively labeled nucleotides. This resulted in an extremely sensitive and accurate determination of CYP3A expression levels, relative to those of GAPDH. Using this RT-PCR assay, it was found that the expression of all CYP3A isoforms in rat hepatocytes, cultured on a collagen matrix, was decreased by 80-90% within one day of cultivation. After addition of dexamethasone, at one day after isolation, CYP3A1 mRNA levels were elevated to levels comparable to those in freshly isolated hepatocytes within two days. In contrast, CYP3A2, CYP3A9, and CYP3A18 mRNA levels were not affected by dexamethasone treatment, and were hardly detectable after three days of cultivation. CYP3A enzymatic activity was also induced in cultured hepatocytes (approximately 6-fold) after addition of dexamethasone. In vivo, CYP3A1 mRNA levels increased 45-fold after dexamethasone administration. However, in contrast to the situation in cultured hepatocytes, CYP3A2 and CYP3A18 were also induced, albeit to a lesser extent (4- and 7-fold elevated mRNA levels, respectively). We conclude that the selective induction of CYP3A1 in dexamethasone-treated rat hepatocytes allows the study of biotransformation reactions by CYP3A1, without interference by any of the other CYP3A isoenzymes.

An experimental model of porphyria cutanea tarda (PCT) can be achieved in 3 weeks by a single injection of a mixture of polychlorinated biphenyls (Aroclor 1254) into iron-loaded female Fischer 344 rats maintained continuously on delta-aminolevulinic acid-supplemented drinking water. In this model, daily treatment with 5-pregnen-3 beta-ol-20-one-16 alpha-carbonitrile (pregnenolone 16 alpha-carbonitrile) attenuated uroporphyrin and heptacarboxylporphyrin accumulation and excretion by 75%. Pregnenolone 16 alpha-carbonitrile treatment had only a minor effect on hepatic iron stores, and it had no effect on the induction of CYP1A activities by Aroclor 1254. In the absence of Aroclor 1254, pregnenolone 16 alpha-carbonitrile had no effect on the accumulation and excretion of highly carboxylated porphyrins. Attenuation of porphyrin accumulation could also be demonstrated with daily troleandomycin treatment. Troleandomycin increased CYP3A-dependent erythromycin demethylase activity, but to a lesser extent than pregnenolone 16 alpha-carbonitrile. Much of the CYP3A induced by troleandomycin was sequestered as a catalytically inactive metabolic-intermediate complex. In the absence of Aroclor 1254, troleandomycin had no effect on the accumulation and excretion of highly carboxylated porphyrins, nor did troleandomycin alter the induction of CYP1A by Aroclor 1254. The results suggest that the major attenuation of hepatic accumulation and urinary excretion of uro- and heptacarboxylporphyrins in the rat PCT model by pregnenolone 16 alpha-carbonitrile and troleandomycin is due to an enhancement of CYP3A catalytic activity.

The ability of unsaturated side-chain analogs of Delta(8)-tetrahydrocannabinol (THC) to selectively inactivate mouse hepatic cytochromes P450 3A11 and 2C29 was examined. THC side-chain analogs were preincubated with mouse hepatic microsomes and NADPH for various times before dilution and determination of Delta(9)-THC metabolism specific for P450s 3A11 and 2C29. THC-enyl analogs had little or no effect on P450 3A11 but inactivated P450 2C29 in a time-dependent manner, with approximately 50% inactivation observed after a 30-min preincubation. THC-ynyl analogs were less selective in their P450 inactivation but appeared to be more effective than their corresponding enyl analogs. THC-ynyl analogs inactivated P450s 3A11 and 2C29 in a time-dependent manner and could inactive 40-80% of their activities after a 30-min preincubation. The THC-ynyl analogs were nearly as effective as cannabidiol, a well-characterized inactivator of these mouse P450s. Despite their ability to inactivate P450 in vitro, neither the THC-enyl nor the THC-ynyl analogs were very effective after in vivo administration. Unsaturated side-chain THC analogs may be useful in the development of specific P450 inactivators.

Assessing the activity of CYP3A4 is important for predicting the pharmacokinetic behavior of protease inhibitors in HIV positive patients, especially in pregnant women. The endogenous hormonal ratio of 6beta-hydroxycortisol (beta-OHF) to cortisol (F) in the urine is an index for metabolic enzyme activity of cytochrome p-450 (CYP) 3A4. Because the ratio is a unique way to assess the enzyme activity without using any exogenous probes for this isozyme, it is practical for use in pregnant women. In this paper, we describe a method using high performance liquid chromatography (HPLC) for 6beta-OHF in urine from pregnant women to estimate the ratio of 6beta-OHF/F. Urinary 6beta-OHF was measured by using C18-cartridge solid phase extraction and isocratic HPLC. Aliquots (1 ml) of urine samples spiked with internal standard, 6beta-hydroxyprednisolone (6beta-OHPSL), were alkalinized with NaOH, then applied to C18-cartridges, which were washed with water and hexane and eluted with ethyl acetate. After the effluents were dried and reconstituted in 10% acetonitrile, the samples were analyzed by HPLC using an isocratic mobile phase (acetic acid/acetonitrile/50 mM potassium dihydrogenphosphate: 0.2/9/90.8; v/v) and ultraviolet detection at 245 nm. The recoveries of 6beta-OHF from C18 cartridges were 93.2 and 93.9% when the authentic 6beta-OHF was added to the urine sample at the concentration of 50 and 300 ng/ml, respectively. Intra- and inter-day variations estimated at concentrations of 113-674 ng/ml were 2.9-5.6 and 4.9-8.1%, respectively. The method was applied to morning urine samples collected from HIV-positive pregnant women managed with protease inhibitor containing anti-retroviral regimens.

The role of specific human cytochrome P450 (CYP) isoforms in the oxidative metabolism of all-trans-retinoic acid was investigated by studies in human liver microsomes using isoform-specific chemical inhibitors and inhibitory antibodies. Studies using individual isoforms expressed in lymphoblastoid cells and correlation analysis using different microsome preparations were also performed. With expressed isoforms, evidence for a role for CYP2C8, CYP3A4, CYP2C9, and CYP1A1 in 4-hydroxylation was obtained, with the highest catalytic efficiency being observed for CYP2C8. Using inhibition studies and correlation analysis, we also concluded that CYP2C8 was the major all-trans-retinoic acid 4-hydroxylating cytochrome P450 in human liver microsomes, though CYP3A4 and, to a lesser extent CYP2C9, also made a contribution. In addition, we compared the rate of retinoic acid degredation in HepG2 cells when cultured in the absence and presence of 3-methylcholanthrene or all-trans-retinoic acid. Culture in the presence of all-trans-retinoic acid decreased the half-life twofold and resulted in an increased sensitivity of retinoic acid degredation to ketoconazole. Since no induction of either CYP1A1, CYP2C8, CYP2C9, or CYP3A4 was detected using immunoblotting and as mRNA encoding another cytochrome P450 enzyme, CYP26, has been previously demonstrated to be induced by retinoic acid treatment of HepG2 cells and to be highly sensitive to ketoconazole, this enzyme in addition to CYP2C8, CYP2C9 and CYP3A4 likely plays a role in all-trans-retinoic acid oxidation in the liver at high retinoic acid levels.

Phenobarbital (PB) was shown to induce the major PB-inducible cytochrome P450 (CYP) isoforms, CYP2B1/2, in perivenular hepatocytes by a single injection, and in midzonal and periportal hepatocytes in addition to perivenular hepatocytes by injections of the same dosage once a day for 3 days in rat livers. The present study was undertaken to determine whether the spread of enzyme induction to midzonal and periportal hepatocytes is caused by the increase in total dose of the drug by repetitive injections or by the repetitive injections of the drug themselves. Male adult rats were administered PB by a single injection (80 mg/kg) or repetitive injections (20 mg/kg once a day for 4 days; a total dose of 80 mg/kg), and the molar content of CYP2B1/2 was measured by quantitative immunohistochemistry in the cytoplasm of perivenular, midzonal, and periportal hepatocytes. In addition, the molar content of total CYP in the cytoplasm was measured by microphotometry, and the expression of CYP2B2 mRNA was examined by in situ hybridization. When animals received the single injection, the isoforms and CYP2B2 mRNA increased markedly in perivenular hepatocytes, increased somewhat in midzonal hepatocytes, and remained unchanged in periportal hepatocytes. If animals received the repetitive injections, however, although the isoforms and the mRNA increased markedly in perivenular hepatocytes, they also increased markedly in midzonal hepatocytes and somewhat in periportal hepatocytes. These findings demonstrated that the enlargement of the sublobular area in which induction of the isoforms occurred was caused by the repetitive injections of PB themselves.

The aryl hydrocarbon receptor (AhR) was cloned from the chick embryo and its function and developmental expression characterized. Chicken AhR cDNA coded for 858 amino acid protein and 396 bp of 3' UTR. The basic helix loop helix domain exhibited 87-100% amino acid identity to avian, mammalian, and amphibian AhR, and 69-74% to piscine AhR. The PAS (Per-ARNT-Sim) region was slightly less well conserved with (a) 97% identity to other avian sequences, (b) 81-86% to amphibian and mammalian AhR, and (c) 64-69% with piscine AhR. The carboxy terminus diverged the most among species with less than 53% amino acid identity between chicken and any available mammalian and piscine AhR sequences. The chicken AhR RNA and protein were 6.1 kb and 103 kDa, respectively. Chicken AhR dimerized with human AhR nuclear translocator and bound the mammalian dioxin-response element in a ligand-dependent manner. AhR protein was detected in neural ganglia; smooth, cardiac, and skeletal muscle; and epithelium involved in epithelial-to-mesenchymal transformations, such as pituitary, gastrointestinal tract, limb apical-ectodermal ridge, and kidney collecting ducts. AhR mRNA was detected in all tissues expressing protein, except myocardium. Cytochrome P4501A4 mRNA was highly induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a subset of tissues expressing AhR, including small intestine, liver, kidney, blood vessels, and outflow tract myocardium. In conclusion, the AhR sequence and function is highly conserved between birds and mammals, and although many tissues express AhR during chick embryo development, only a subset are responsive to TCDD induction of CYP1A4.

Expression of drug-metabolizing enzymes including cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO) in various tissues of Suncus murinus (Suncus) were examined. Northern blot analysis showed that mRNAs hybridizable with cDNAs for rat CYP1A2, human CYP2A6, rat CYP2B1, human CYP2C8, human CYP2D6, rat CYP2E1, human CYP3A4 and rat CYP4A1 were expressed in various tissues from Suncus. The mRNA level of CYP2A in the Suncus lung was very high. Furthermore, it was found that the level of CYP2A mRNA in the Suncus lung was higher compared to the Suncus liver. The expression level of mRNA hybridizable with cDNA for human CYP3A4 was very low. The presence of CYP3A gene in Suncus was proven by the induction of the CYP with dexamethasone. Very low expression levels of mRNAs hybridizable with cDNAs for rat FMO1, rat FMO2, rat FMO3 and rat FMO5 were also seen in Suncus liver. No apparent hybridization band appeared when human FMO4 cDNA was used as a probe. The hepatic expression of mRNAs hybridizable with cDNAs for UDP-glucuronosyltransferase 1*6, aryl sulfotransferase, glutathione S-transferase 1, carboxyesterase and microsomal epoxide hydrolase in the Suncus were observed. These results indicate that the Suncus is a unique animal species in that mRNAs for CYP3A and FMO are expressed at very low levels.

We measured rates of oxidative metabolism of two tetrachlorobiphenyl (TCB) congeners by hepatic microsomes of two marine mammal species, beluga whale and pilot whale, as related to content of selected cytochrome P450 (CYP) forms. Beluga liver microsomes oxidized 3,3',4,4'-TCB at rates averaging 21 and 5 pmol/min per mg for males and females, respectively, while pilot whale samples oxidized this congener at 0.3 pmol/min per mg or less. However, rates of 3,3',4,4'-TCB metabolism correlated with immunodetected CYP1A1 protein content in liver microsomes of both species. The CYP1A inhibitor alpha-naphthoflavone inhibited 3,3',4,4'-TCB metabolism by 40% in beluga, supporting a role for a cetacean CYP1A as a catalyst of this activity. Major metabolites of 3,3',4,4'-TCB generated by beluga liver microsomes were 4-OH-3,3',4',5-TCB and 5-OH-3,3',4,4'-TCB (98% of total), similar to metabolites formed by other species CYP1A1, and suggesting a 4,5-epoxide-TCB intermediate. Liver microsomes of both species metabolized 2,2',5,5'-TCB at rates of 0.2-1.5 pmol/min per mg. Both species also expressed microsomal proteins cross-reactive with antibodies raised against some mammalian CYP2Bs (rabbit; dog), but not others (rat; scup). Whether CYP2B homologues occur and function in cetaceans is uncertain. This study demonstrates that PCBs are metabolized to aqueous-soluble products by cetacean liver enzymes, and that in beluga, rates of metabolism of 3,3',4,4'-TCB are substantially greater than those of 2,2',5,5'-TCB. These directly measured rates generally support the view that PCB metabolism plays a role in shaping the distribution patterns of PCB residues found in cetacean tissue.

Aromatic hydrocarbon receptor-mediated transcriptional up-regulation of cytochrome P450 (CYP) enzymes of the CYP1A subfamily by polycyclic aromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (MC) is accompanied by down-regulation of rat hepatic CYP2C11 expression at the catalytic activity, protein, and mRNA levels. To gain insight into the molecular mechanism of this CYP2C11 suppression response, we have used a nuclear run-on assay to assess directly the effect of MC on the hepatic transcription rate of the CYP2C11 gene following in vivo administration of MC to adult male rats. A single intraperitoneal dose of MC (40 mg/kg) caused a 179-fold increase in the rate of CYP1A gene transcription at 6 hr, and the rate of CYP2C11 gene transcription was reduced by 51% at this time point, compared with vehicle controls. By 48 hr after MC treatment, the rates of CYP1A and CYP2C11 gene transcription were no longer significantly different from the corresponding vehicle controls. These results indicate for the first time that the suppression of hepatic CYP2C11 caused by in vivo administration of PAHs to adult male rats is at least partially due to a decrease in the rate of transcription of the CYP2C11 gene.

The present study investigated the role of growth hormone (GH) in hepatic CYP3A18 and CYP3A9 expression in prepubertal and adult male rats. For comparison, the effects of GH on CYP3A2 expression were also measured. Initial experiments demonstrated that CYP3A18 mRNA levels were greater during puberty and adulthood than during the prepubertal period, CYP3A9 mRNA was not expressed until puberty and its expression increased in adulthood, and CYP3A2 mRNA levels were relatively constant from prepuberty to adult life. Hypophysectomy, which results in the loss of multiple pituitary factors including GH, increased CYP3A2 and CYP3A18 mRNA expression 3- to 4-fold, but it did not affect CYP3A9 mRNA levels or CYP3A-mediated testosterone 2beta- or 6beta-hydroxylase activity in adult rats. GH administered as twice daily s.c. injections (0.12 microg/g body weight) to hypophysectomized or intact adult rats did not affect CYP3A18 or CYP3A9 mRNA expression. The same treatment decreased CYP3A2 mRNA and protein and testosterone 2beta- and 6beta-hydroxylase activity levels in intact but not hypophysectomized rats. However, in intact prepubertal rats, intermittent GH administration decreased CYP3A18 and CYP3A2 mRNA levels, but a higher dosage (3.6 microg/g) was required to suppress CYP3A2. Overall, the present study demonstrated that: (a) the constitutive expression of CYP3A18, CYP3A9, and CYP3A2 does not require the presence of GH, (b) CYP3A18 is more sensitive than CYP3A9 to GH modulation in adult rats; and (c) CYP3A2 is less sensitive to the suppressive influence of GH during the prepubertal period than during adult life.

We determined the inducibility, as well as the persistence of the induction, of hepatic microsomal CYP1A1 and CYP1A2 (by western blot analysis), and their catalytic activities (as measured by resorufin ether O-dealkylation) in prepubertal (25-day-old) and adult (120-day-old) offspring of timed-pregnant Sprague-Dawley rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD treatment was subcutaneous, at a low dose of 0.1 microg/kg, on gestational days 7, 14, and 20, and on lactational days 7 and 14. CYP1A1 protein was induced significantly (23-fold) in prepubertal but not in adult offspring of TCDD-exposed dams, whereas ethoxyresorufin O-deethylase (EROD) activity, which is CYP1A1-preferential, was induced less extensively (5-fold) and slightly (1.7-fold) in the prepubertal and adult offspring, respectively. Benzyloxyresorufin O-debenzylase (BROD) activity, which is CYP2B-preferential but has been reported to be catalyzed by CYP1A1, was also induced 5- and 6-fold in prepubertal and adult offspring, respectively, of TCDD-exposed dams. However, the induced BROD activity was neither inhibited by antibody against CYP1A1 nor accompanied by an elevated level of microsomal CYP2B. CYP1A2 was induced slightly only in prepubertal offspring of TCDD-treated dams. There was suggestive evidence of enhanced lipid peroxidation in hepatic microsomes from prepubertal but not adult offspring of TCDD-treated dams. These data showed that in utero plus lactational TCDD exposure effected transient induction of hepatic microsomal CYP1A1 but sustained induction of BROD activity, which may be catalyzed by enzymes other than CYP1A or CYP2B.

The anticancer alkylating agents cyclophosphamide (CPA) and ifosfamide (IFA) are prodrugs that undergo extensive P450-catalyzed metabolism to yield both active (4-hydroxylated) and therapeutically inactive but neurotoxic (N-dechloroethylated) metabolites. Whereas the human liver microsomal P450 catalysts of CPA and IFA 4-hydroxylation are well characterized, the P450 enzyme catalysts of the alternative N-dechloroethylation pathway are poorly defined. Analysis of a panel of fifteen human P450 cDNAs in the baculovirus expression system ('Supersomes') demonstrated that CYP3A4 exhibited the highest N-dechloroethylation activity toward both CPA and IFA, whereas CYP2B6 displayed high N-dechloroethylation activity toward IFA, but not CPA. The contributions of each human P450 to overall liver microsomal N-dechloroethylation were calculated using a recently described relative substrate-activity factor method, and were found to be in excellent agreement with the results of inhibition studies using the CYP3A inhibitor troleandomycin and an inhibitory monoclonal antibody to CYP2B6. With CPA as substrate, CYP3A4 was shown to catalyze >/=95% of liver microsomal N-dechloroethylation, whereas with IFA as substrate, CYP3A4 catalyzed an average of approximately 70% of liver microsomal N-dechloroethylation (range = 40-90%), with the balance of this activity catalyzed by CYP2B6 (range = 10-70%, dependent on the CYP2B6 content of the liver). Because CYP2B6 can make a significant contribution to human liver microsomal IFA N-dechloroethylation, but only a minor contribution to IFA 4-hydroxylation, the selective inhibition of hepatic CYP2B6 activity in individuals with a high hepatic CYP2B6 content may provide a useful approach to minimize the formation of therapeutically inactive but toxic N-dechloroethylated IFA metabolites.

Cyclosporine is a potent immunosuppressive drug that undergoes extensive hepatic metabolism catalyzed primarily by the cytochrome P450 (P450) 3A enzyme family. Cyclosporine alters its own metabolism by selective suppression of specific P450 isoforms after chronic therapy in rats. Modulation of hepatic P450 by chronic cyclosporine dosing is associated with increased blood concentrations leading to nephropathy. However, the relationship between cyclosporine dose and hepatic enzyme suppression is not known. The purpose of this study was to examine the effect of escalating doses of cyclosporine on P450 regulation and metabolic activity in the rat. Following 1 week of a low-salt diet, rats were given cyclosporine 5, 15, 30 or 50 mg/kg per day or an equal volume of vehicle for 2 weeks via oral gavage. At the end of the dosing period, livers were removed and hepatic microsomes prepared. Hepatic P450 proteins were measured using Western blot analysis and catalytic activity determined by in vitro testosterone hydroxylation. Cyclosporine dosing suppressed both P450 3A2 and 2C11 protein expression and catalytic activity in a dose-dependent manner. Catalytic activity of two other P450 isoforms, 2A1 and 2B1, were unchanged by cyclosporine administration. Thus, the selective suppression of hepatic microsomal P450 by cyclosporine is not only dependent on the length of therapy, but also the dose administered.