Characterization of common CYP1B1 variants with different capacity for benzo[a]pyrene-7,8-dihydrodiol epoxide formation from benzo[a]pyrene.

Cytochrome P450 1B1 (CYP1B1), an extrahepatic enzyme inducible by smoking, is overexpressed in many tumors and catalyzes the metabolic activation of procarcinogens such as polycyclic aromatic hydrocarbons. In human, CYP1B1 is genetically polymorphic and five common missense mutations causing amino acid substitution have been identified. In this study, we have investigated CYP1B1 haplotypes present in a Spanish population and carried out functional analyses of the corresponding enzymes in yeast using benzo[a]pyrene as a substrate. CYP1B1*1, CYP1B1*2, CYP1B1*3, CYP1B1*4, CYP1B1*6, and CYP1B1*7, encoding combinations of the Arg48Gly, Ala119Ser, Leu432Val, Asn453Ser, and Ala443Gly amino acid substitutions, were present at frequencies of 14.3%, 25.5%, 38.8%, 18.1%, 0.4%, and 2.6%, respectively. The variant CYP1B1 forms were heterologously expressed with human reductase in Saccharomyces cerevisiae and kinetic analyses of benzo[a]pyrene metabolism were carried out. CYP1B1.7, having the amino acid substitutions Arg48Gly, Ala119Ser, Leu432Val, and Ala443Gly, exhibited a significantly decreased capacity (P < 0.001) for the formation of (+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol from benzo[a]pyrene as indicated by lower intrinsic clearance (Vmax/Km). A somewhat decreased clearance was observed for CYP1B1.4, whereas no significant differences in kinetic properties among the remaining variant enzymes were observed as compared with CYP1B1.1. Thus, genetic polymorphism in the CYP1B1 gene, as defined by the haplotypes investigated, might cause interindividual differences in susceptibility (e.g., to lung cancer induced by smoking). The results indicate the necessity to make molecular epidemiologic investigations regarding the association of the specific CYP1B1 haplotypes and cancer risk.

Evaluation of human liver slices and reporter gene assays as systems for predicting the cytochrome p450 induction potential of drugs in vivo in humans.

PURPOSE: The aim of the study was to investigate the feasibility of predicting human in vivo cytochrome P450 (CYP) induction properties of drugs using in vitro methods. METHODS: The CYP induction potential of compounds was tested in human liver slices and in reporter gene assays for the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR). RESULTS: In human liver slices, CYP activities decreased dramatically over the experimental period, whereas mRNA levels could reliably be used to investigate CYP1A, 2C9, and 3A4 induction. However, the interindividual variations and demanding experimentation limit the use of liver slices in screening programs. Reporter gene assays are robust and reliable assays, amenable to high throughput screening. Several compounds activated AhR. The relevance of this activation, however, needs to be further investigated since there are no clear reports on drugs inducing CYP1A in vivo. The results from the PXR assay could be used to correctly classify compounds with known CYP3A induction properties when relating in vivo AUCtot to PXR EC50 values. CONCLUSIONS: Liver slices are a valuable model to study the regulation of a larger number of enzymes by single compounds. The PXR reporter gene assay could be used as a reliable screening method to predict CYP3A induction in vivo.

A novel polymorphic cytochrome P450 formed by splicing of CYP3A7 and the pseudogene CYP3AP1.

The cytochrome P450 3A7 (CYP3A7) is the most abundant CYP in human liver during fetal development and first months of postnatal age, playing an important role in the metabolism of endogenous hormones, drugs, differentiation factors, and potentially toxic and teratogenic substrates. Here we describe and characterize a novel enzyme, CYP3A7.1L, encompassing the CYP3A7.1 protein with the last four carboxyl-terminal amino acids replaced by a unique sequence of 36 amino acids, generated by splicing of CYP3A7 with CYP3AP1 RNA. The corresponding CYP3A7-3AP1 mRNA had a significant expression in liver, kidney, and gastrointestinal tract, and its presence was found to be tissue-specific and dependent on the developmental stage. Heterologous expression in yeast revealed that CYP3A7.1L was a functional enzyme with a specific activity similar to that of CYP3A7.1 and, in some conditions, a different hydroxylation specificity than CYP3A7.1 using dehydroepiandrosterone as a substrate. CYP3A7.1L was found to be polymorphic due to a mutation at position -6 of the first splicing site of CYP3AP1 (CYP3A7_39256T-->A), which abrogates the pseudogene splicing. This polymorphism had pronounced interethnic differences and was in linkage disequilibrium with other functional polymorphisms described in the CYP3A locus: CYP3A7*2 and CYP3A5*1. Therefore, the resulting CYP3A haplotypes express different sets of enzymes within the population. In conclusion, a novel mechanism, consisting of the splicing of the pseudogene CYP3AP1 to CYP3A7, causes the formation of the novel CYP3A7.1L having a different tissue distribution and functional properties than the parent CYP3A7 enzyme, with possible developmental, physiological, and toxicological consequences.

Functional analysis of six different polymorphic CYP1B1 enzyme variants found in an Ethiopian population.

Cytochrome P450 1B1 (CYP1B1) is an extrahepatic enzyme of potential importance for the metabolism of estrogen and for metabolic activation of environmental carcinogens. We investigated an Ethiopian population for functional polymorphisms in the CYP1B1 gene using genomic DNA sequencing and detected three novel single nucleotide polymorphisms (SNPs). One of these (4360C-->G in exon 3) is present at a frequency of 7% and causes an Ala443Gly amino acid substitution. In addition, the four described previously missense mutations Arg48Gly, Ala119Ser, Leu432Val, and Asn453Ser were found with frequencies of 51, 50, 53, and 2%, respectively, yielding a total of 32 possible CYP1B1 haplotypes. Allele-specific PCR methods for haplotype analysis were developed and seven different CYP1B1 alleles were found: CYP1B1*1, *2, *3, *4, *5, *6, and *7 with frequencies of 8, 37, 39, 2, 0.7, 6, and 7%, respectively. The functional properties of different forms of CYP1B1, as well as of the Leu432Val + Asn453Ser and Leu432Val + Ala443Gly variants, were evaluated after heterologous expression of the corresponding cDNAs in Saccaromyces cerevisiae. The results revealed that CYP1B1.6 and CYP1B1.7, having the amino acid substitutions Arg48Gly, Ala119Ser, and Leu432Val in common, exhibited altered kinetics with significantly increased apparent K(m) and lowered V(max) values for both the 2- and 4-hydroxylation of 17 beta-estradiol, whereas the other constructs were indistinguishable from the CYP1B1.1 enzyme. The results emphasize the necessity of a complete haplotype analysis of enzyme variants for evaluation of functional consequences in vivo and for analyses of genetic polymorphisms in relation to, for example, cancer incidence.

Cytochrome P450 1A1/2 induction by antiparasitic drugs: dose-dependent increase in ethoxyresorufin O-deethylase activity and mRNA caused by quinine, primaquine and albendazole in HepG2 cells.

OBJECTIVE: To investigate the inductive effects of twenty-four antiparasitic drugs on cytochrome P450 (CYP) 1A1 and 1A2 enzyme activities. METHODS: Human hepatoma (HepG2) cells were exposed to antiparasitic drugs for 24 h, and the ethoxyresorufin O-deethylase (EROD) activity, indicative of CYP1A enzyme activity, was measured fluorometrically. In addition, the CYP1A1 and CYP1A2 mRNA expression levels were determined by means of quantitative reverse-transcriptase polymerase chain reaction. RESULTS: Quinine, albendazole and primaquine caused a dose-dependent increase in EROD activity of 5.5, 4.0 and 7.5-fold, at concentrations eliciting maximal induction, respectively. 2,3,7,8-tetrachlorodibenzo- p-dioxin, used as a positive control at a final concentration of 1.5 nM, caused a 30-fold increase in EROD activity. The induction of EROD activity was accompanied by an increase in CYP1A1 and CYP1A2 mRNA expression levels. Niclosamide, 4-chlorophenylbiguanide, dapsone, amodiaquine and desethylamodiaquine caused slight increases in EROD activity. No effect on CYP1A was observed for artemisinin, suramin, diethylcarbamazine, pyrimethamine, metrifonate, ivermectin, pyrantel artesunate, cycloguanil, atovaquone, melarsoprol, praziquantel, proguanil and dihydroartemisinin. CONCLUSIONS: Quinine, albendazole and primaquine induce CYP1A1 and CYP1A2 at the transcriptional level. Considering the plasma concentrations (C(max)) achieved in vivo after administration of a therapeutic dose, induction by quinine and albendazole might be of clinical significance. The induction by primaquine, however, may not be of pharmacological or toxicological significance as concentrations at which it occurs are much higher than those attained in vivo.

Comparative analysis of CYP3A expression in human liver suggests only a minor role for CYP3A5 in drug metabolism.

To study mechanisms behind the interindividual variability in CYP3A expression and the relative contribution of the different CYP3A enzymes to the overall CYP3A activity, we have analyzed CYP3A4, CYP3A5, CYP3A43, and PXR mRNA and CYP3A4 and CYP3A5 protein expression, catalytic activity, and polymorphism in the CYP3A5 gene in a panel of 46 Caucasian human livers. Protein quantification was performed by Western blotting using enzyme-specific antibodies directed to the C termini of CYP3A4 or CYP3A5, and carrier protein-coupled peptides as standards. The mRNA levels were determined by quantitative real-time PCR. CYP3A activity was measured by analysis of the rate of testosterone 6beta-hydroxylation. A correlation existed between all CYP3A and PXR mRNA transcripts measured. The interindividual variability in CYP3A4 and CYP3A5 mRNA expression was higher than that of CYP3A protein and activity. The CYP3A5 protein was expressed at quantifiable levels in 5 (10.9%) of the livers. Four of those were heterozygous for the CYP3A5*1 allele and had CYP3A5 protein at a mean level of 17% of that of total CYP3A, whereas one liver sample was from a CYP3A5*3 homozygote individual having lower amounts of CYP3A5. In total, CYP3A5 only contributed 2% of the overall CYP3A protein among all samples. In conclusion, our data indicate that CYP3A4, CYP3A5, CYP3A43, and PXR hepatic mRNA expression correlate, indicating common regulatory features, and that the contribution of CYP3A5 to hepatic drug metabolism in Caucasians is insignificant.