Cytochrome P450 1B1 Gene Research Articles

Transcriptional Determination of Sexually Dimorphic Expression of Nuclear Receptor Constitutively Active Receptor (CAR) in Wistar Rats

The mammalian constitutively active receptor (CAR) is a ligand-activated transcription factor that participates in controlling the expression of cytochrome P450 2B (CYP2B) genes in response to phenobarbital (PB) in rodents. CYP2B forms are highly inducible in liver and intestine in male Wistar rats. In contrast, PB-dependent increases in CYP2B activity are rarely observed in female Wistar rats. The profiles of CAR mRNA expression measured by RT-PCR in various organs in male and female Wistar rats were consistent with those of PB-dependent expression of CYP2B genes. The sex-related dimorphic PB-responses of CYP2B genes in Wistar rats might be determined transcriptionally as are the organospecific PB-responses in both males and females.

Transcriptional regulation of CYP2B1 induction in primary rat hepatocyte cultures: repression by epidermal growth factor is mediated via a distal enhancer region.

Phenobarbital (PB) alters expression of numerous hepatic genes, including genes involved in xenobiotic metabolism. Phenobarbital-dependent induction of cytochrome P-450 2B1 (CYP2B1) is subject to regulation by cytokines [e.g., by epidermal growth factor (EGF)], hormones [e.g., by growth hormone (GH)], or the cellular redox status. To investigate mechanisms involved in regulation of CYP2B1 transcription, we performed promoter activation studies using primary rat hepatocyte cultures transiently transfected with individual CYP2B1 promoter-luciferase reporter gene constructs. The 2679-bp native 5'-flanking region of the CYP2B1 gene conferred reporter gene activation by PB and the potent PB-like inducer permethrin (PM). Furthermore, this region mediated EGF- and GH-dependent repression of gene activation by PB-like inducers. A wide promoter mapping strategy with constructs bearing internal CYP2B1 promoter deletions led to identification of a distal responsive CYP2B1 enhancer region at -2230 to -2170, encompassing the section equivalent to the 51-bp PB-responsive enhancer module situated in the distal mouse Cyp2b10-5'-flanking region. The distal CYP2B1 enhancer region conferred gene activation by PM, repression of PM-dependent activation by EGF, and enhancement of activation by the antioxidant N-acetylcysteine (NAC). Mutational analyses of the region at -2230 to -2170 suggested that the mechanisms of PB-dependent induction of CYP2B1 and the modulating effects by EGF or NAC are closely related.

Evaluation of a real-time polymerase chain reaction method for the quantification of CYP1B1 gene expression in MCF-7 human breast carcinoma cells

Introduction: Cytochrome P450 1B1 (CYP1B1) catalyzes the bioactivation of numerous procarcinogens and it is expressed in tumor cells, including human breast cancer cells. To study CYP1B1 gene expression, it is important to have an accurate, precise, reproducible, specific, and quantitative method. Methods: MCF-7 human breast carcinoma cells were treated with β-naphthoflavone (BNF; 50 μM), emodin (0.1–3 μM), trans-resveratrol (2.5–20 μM), or 0.1% dimethylsulfoxide (DMSO; vehicle control). Total cellular RNA was isolated and reverse transcribed. cDNA samples were quantified by a fluorescence assay and a constant amount (1 ng) was amplified in a real-time DNA thermal cycler (LightCycler). Results: Melting curve analysis and agarose gel electrophoresis of the amplicons resulted in a single peak and a single band, respectively. The identity of the amplicon was confirmed to be CYP1B1 by sequencing analysis. The standard curve for the real-time PCR amplification of CYP1B1 cDNA was log-linear for at least four orders of magnitude. The limit of quantitation (LOQ) of the assay was 100 copies. At the LOQ, the assay had an accuracy of 8% and a precision of 10%. The intraday ( n=4) variability (expressed as percent coefficient of variation) was 9% for a sample with low CYP1B1 mRNA expression (cells treated with 0.1% DMSO; i.e., Sample A) and 3% for a sample with elevated CYP1B1 mRNA expression (cells treated with BNF; i.e., Sample B). The interday ( n=4) variability was 16% for Sample A and 15% for Sample B. Emodin increased CYP1B1 mRNA expression in cultured MCF-7 cells (maximal effect of ninefold induction achieved at 1 μM), whereas trans-resveratrol suppressed it (IC 50=6.6±1.0 μM, mean±S.E.M., n=3). Discussion: An accurate, precise, reproducible, and specific method is described for the real-time PCR quantification of CYP1B1 gene expression in MCF-7 human breast carcinoma cells.

Novel cytochrome P450 1B1 (CYP1B1) mutations in patients with primary congenital glaucoma in France.

The CYP1B1 gene (GenBank: U56438), a member of the cytochrome P450 gene family, has been shown to be mutated in patients with primary congenital glaucoma (PCG), a rare but severely blinding form of glaucoma. Here, we have investigated CYP1B1 mutations in 31 unrelated French PCG patients. Mutations were found in 15 (48%) patients. Six of these mutations were novel. One, g.3979delA, caused a frameshift followed by a stop codon at residue 59. Two mutations, g.4547C>T (p.Q248X) and g.8167C>T (p.R444X), created a stop codon. Three other mutations, g.4499G>C (p.G232R), g.8033T>G (p.I399S), (p.N423Y), induced a significant amino acid change. Seven patients, who were of French descent, were compound heterozygotes. Six patients, whose families came from North Africa or from Portugal, carried a homozygous mutation reflecting their geographic origin. Intriguingly, one mutation, p.E229K, was present in heterozygous state in two unrelated patients. All together, these findings demonstrate the major role and the diversity of CYP1B1 mutations in French PCG patients.

Cytochrome P450 1B1 gene polymorphisms and postmenopausal breast cancer risk.

Cytochrome P450 1B1 (CYP1B1) is active in the metabolism of estrogens to reactive catechols and of different procarcinogens. Several studies have investigated the relationship between genetic polymorphisms of CYP1B1 and breast cancer risk, however, with inconsistent results. We investigated such an association in postmenopausal Swedish women, with special emphasis on long-term menopausal hormone users, in a large population-based case-control study. We genotyped 1521 cases and 1498 controls for the CYP1B1 single nucleotide polymorphisms (SNPs) m2, m3 and m4 and reconstructed haplotypes. The frequencies of CYP1B1*1, CYP1B1*2, CYP1B1*3 and CYP1B1*4 alleles among controls were estimated to be 0.087, 0.293, 0.444 and 0.175, respectively. It thus appeared that very few haplotypes contained combinations of SNPs at two or three loci and that single SNP genotype data effectively represented haplotypes. Odds ratios (OR) and 95% confidence intervals (CI) were calculated from logistic regression models. We found no overall association between any CYP1B1 genotype and breast cancer risk. The data indicated, however, that women who had used menopausal hormones for 4 years or longer, and carried the CYP1B1*3/*3 genotype may be at increased risk of breast cancer, OR 2.0 (95% CI 1.1-3.5), compared with long-term users without this genotype. We explored the effect of CYP1B1 genotype on breast cancer risk in subgroups defined by body mass index, family history, smoking and catechol-O-methyl transferase genotype, but found no convincing evidence for interaction. In summary, our results strongly indicate that the studied CYP1B1 gene polymorphisms do not influence breast cancer risk overall but may modify the risk after long-term menopausal hormone use.

Differential expression of the tetracycline-controlled transactivator-driven human CYP1B1 gene in double-transgenic mice is due to androgens: application for detecting androgens and antiandrogens

Differential expression of the tetracycline-controlled transactivator (tTA)-driven human cytochrome P450 (CYP) 1B1 gene was found in the livers of male mice, at high levels in neonates, but at low levels in adults. The goals of this study were to determine whether the differential expression of the tTA-driven human CYP1B1 (hCYP1B1) gene in neonates and adults was testosterone dependent and whether flutamide, a representative potent antiandrogen, led to the induction of hCYP1B1. This was tested by treating castrated transgenic mice with testosterone propionate and musk extracts. It was concluded that: (i) the levels of expression of both tTA and hCYP1B1 gradually declined, with clear changes being apparent between 2 and 4 weeks of age, (ii) castration of adult males resulted in the increased expressions of both tTA and hCYP1B1 to levels similar to those found in adult females, (iii) treatment of castrated male and adult female mice with testosterone propionate and musk extracts led to the restoration of the levels of expression of hCYP1B1 in the adult males, and (iv) treatment of adult males with flutamide caused an increase in the levels of expression of hCYP1B1 in the adult females, as indicated by the antiandrogenic activity. Thus, the differential expression of the tTA-driven hCYP1B1 gene in the transgenic mice was caused by androgen, and it is possible that castrated male and adult female mice expressing the tTA-controlled hCYP1B1 could be used as the basis for a strategy for the detection of androgens and antiandrogens.

A novel frameshift founder mutation in the cytochrome P450 1B1 (CYP1B1) gene is associated with primary congenital glaucoma in Morocco.

Primary congenital glaucoma (PCG) is a heterogeneous autosomal recessive disorder caused by unknown developmental defect(s) of the anterior chamber of the eye. A member of the cytochrome P450 gene family, CYP1B1, was found to be mutated in PCG patients in different populations, albeit to a variable extent. In this study, CYP1B1 mutations were searched for in 32 unrelated PCG patients from Morocco. Two mutations were detected in 11 (34%) patients. One, 4339delG, is novel and causes a frameshift at residue 179. The other, G61E, was previously found in patients from Turkey and Saudi Arabia. Seven patients were homozygous for 4339delG and two other patients for G61E, whereas the two remaining patients were compound heterozygotes. The close association of 4339delG with a rare allele of D2S177, a microsatellite marker located 270 kb upstream of CYP1B1, strongly suggested a founder effect for 4339delG. The occurrence of this mutation was tentatively dated at between 900 and 1700 years ago. Typing 4339delG and G61E mutations should help to prevent blindness resulting from a delayed diagnosis of PCG in Morocco.

Cytochrome P450 CYP1B1 and catechol O-methyltransferase (COMT) genetic polymorphisms and breast cancer susceptibility in a Turkish population.

Epidemiological studies indicate that most risk factors for breast cancer are related to reproductive and hormonal factors. Estrogen has been proposed to trigger breast cancer development via an initiating mechanism involving its metabolite, catechol estrogen (CE). Because of the important role of cytochrome P450 1B1 ( CYP1B1) and catechol O-methyltransferase ( COMT) in mammary estrogen and carcinogen metabolism, we examined the CYP1B1 and COMT genes to determine whether genetic variations could account for inter-individual differences in breast cancer. In this case-control study, we determined CYP1B1 and COMT genotypes in 84 breast cancer patients and 103 healthy unrelated women controls from a Turkish population. In the case of CYP1B1, we genotyped CYP1B1*3 (L432 V) allele. We found that carriers of the CYP1B1*3 allele were more frequent among breast cancer patients with adjusted odds ratio (OR) for age, age at menarche, age at first full-term pregnancy, body mass index (BMI) and smoking status of 2.32 (95% confidence interval 1.26-4.25) associated with the allele. However, this allele appeared to be a significant factor for susceptibility only in patients with a BMI greater than 24 kg/m(2). Menopausal status did not appear to affect susceptibility. In the case of COMT, there was no significant difference in susceptibility for breast cancer development between patients with low activity COMT-L (V158 M) allele and high activity COMT-H allele, and susceptibility was not affected by menopausal status, BMI or CYP1B1 genotype. We conclude that the CYP1B1* 3 allele appears to be a factor for susceptibility to breast cancer in Turkish women especially those with a BMI greater than 24 kg/m(2).

Species-specific responses of constitutively active receptor (CAR)–CYP2B coupling: lack of CYP2B inducer-responsive nuclear translocation of CAR in marine teleost, scup ( Stenotomus chrysops)

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.

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.

Genetic modeling of estrogen metabolism as a risk factor of hormone-dependent disorders

Estradiol is a pleiotropic hormone, involved in the etiology of a wide variety of diseases. Over the last decade individual genetic variability of the estradiol metabolism has been described as a significant contributor to disease susceptibility with variations depending on ethnic background. Among others, genetic variations of genes encoding cytochrome P450 (CYP) enzymes play an important role in this regard. Mutant alleles of the CYP 1A1 gene are major modulators of lung cancer risk among smokers, mediate gender differences in lung cancer susceptibility, and have been associated with an elevated risk for breast, prostate, colorectal, and oral squamous cell cancer. Variants of the CYP 1B1 gene modulate the risk for prostate, ovarian, lung, and breast cancer. Also, mutations in the CYP 1B1 gene are the major genetic determinant of congenital glaucoma. Mutant CYP 17 alleles are associated with serum and plasma levels of steroid hormones, use of hormone replacement therapy, and endometrial, prostate, and breast cancer. Available data indicate that the protective effect of a later age at menarche is limited to mutant CYP 17 allele carriers. Among women with the Polycystic Ovary (PCO) syndrome, mutant CYP 17 alleles are sufficient to aggravate the clinical presentation of the disease. Molecular variants of the CYP 19 gene are associated with an increased risk for breast cancer, advanced disease stage, and tumor aromatase production. Carriage of a mutant catechol- O-methyltransferase (COMT) allele is associated with breast cancer, neurologic disorders such as Parkinson's disease, and modulates behavior among patients with schizophrenia, alcoholics and the general population. In summary, the available evidence points to estrogen metabolising genes as strong hereditary determinants of the susceptibility to benign and malignant conditions.

Intratumoral activation of cyclophosphamide by retroviral transfer of the cytochrome P450 2B1 in a pancreatic tumor model. Combination with the HSVtk/GCV system.

Pancreatic cancer is one of the most aggressive human tumors and the development of new therapeutic approaches is particularly urgent since current therapies are not effective. The use of pro-drug-activating genes is a possible approach for cancer gene therapy. The present study evaluated the efficiency of the cytochrome P4502B1 (CYP2B1) suicide gene that encodes the enzyme responsible for activating the pro-drug cyclophosphamide (CPA), in pancreatic tumor cells invitro and in vivo. The effects on tumor growth of the combination of two suicide systems, CYP2B1/CPA and herpes simplex virus thymidine kinase gene/ganciclovir (HSVtk/GCV), were also studied. Retroviral CYP2B1 transfer followed by CPA treatment highly sensitized pancreatic tumor cells NP-9, NP-18, and NP-31, and led to stabilization of tumor growth in a pancreatic tumor model. Differences in tumor volume at the end of the treatment were statistically significant when compared with animals injected with CPA alone. The combination of both suicide systems CYP2B1/CPA and HSVtk/GCV in vitro resulted in a potentiation of the killing effect. However, no potentiation was achieved in vivo, although retardation in tumor growth was evident. The results show that in situ transduction of pancreatic tumor cells with the CYP2B1 gene by retroviral vectors clearly increases the sensitivity to CPA. Moreover, they suggest that in order to achieve a potentiation on cell killing when the two suicide systems HSVtk/GCV and CYP2B1/CPA are combined, co-expression of both genes in the same tumor cell would be necessary.

Inducer effect on the complex formation between rat liver nuclear proteins and cytochrome P450 2B gene regulatory elements.

DNA gel retardation assay has been applied to the investigation of complexes between rat liver nuclear proteins and Barbie box positive regulatory element of cytochrome P450 2B (CYP2B) genes. The intensities of B1 and B2 bands detected in the absence of an inducer increased after 30 min protein incubation with phenobarbital (PB) or triphenyldioxane (TPD), but not with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOPOB). In addition, a new complex (B3 band) was for the first time detected under induction by PB, TPD, and TCPOPOB. Increase in the incubation time up to 2 h facilitated the formation of other new complexes (B4 and B5 bands), which were detected only in the presence of TPD. The use of [3H]TPD in hybridization experiments revealed that this inducer, capable of binding to Barbie box DNA, is also present in B4 and B5 complexes. It is probable that the investigated compounds activate the same proteins at the initial induction steps, which correlates with the formation of B1, B2, and B3 complexes. The further induction step might be inducer-specific, as indicated by the formation of B4 and B5 complexes in the presence of TPD only. Thus, the present data suggest the possibility of specific gene activation signaling pathways that are dependent on a particular inducer.

Genotyping human cytochrome: P450 1B1 variants

This chapter describes the methods used in laboratory to genotype the four common single nucleotide polymorphisms found in the coding region of human cytochrome P450 1B1 (CYP1B1). Human cytochrome P450 1B1 (CYP1B1) was first isolated by differential hybridization as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-responsive cDNA clone from a human keratinocyte cell line treated with TCDD. Initial characterization of the human CYP1B1 gene described the DNA sequence of a 12-kb genomic clone corresponding to the entire 5.1-kb CYP1B1 cDNA and containing 3.0 kb of upstream DNA. Comparison of these sequences revealed the location of three exons (371, 1044, and 3707 bp) and two introns (390 and 3032 bp), with the CYP1B1 open reading frame spanning exons 2 and 3. Genotyping by restriction fragment length polymorphism analysis will soon be replaced by more rapid, high-throughput assays based on electrochemical or fluorescent detection methods, including oligonucleotide microarrays. In the interim, the procedures described in the chapter should facilitate CYP1B1 genotyping of banked DNA samples.

Long-term enhancement of cytochrome P450 2B1/2 expression in rat hepatocyte spheroids through adenovirus-mediated gene transfer.

Tissue-like structures of cells organized in vitro have a great potential for a number of clinical and biomedical applications. Cell functions may be modulated with gene delivery, improving the characteristics of these structures. Hepatocytes that self-assemble into spheroids can be transduced through adenovirus-mediated gene transfer. An adenoviral vector (AdGFP) was employed to deliver a gene encoding for green fluorescent protein (GFP) in rat hepatocyte spheroids. GFP fluorescence was detected for at least one month. Furthermore, the rat cytochrome P450 2B1 gene (CYP2B1) was transferred through infection with a recombinant adenovirus (AdCYP2B1) in hepatocyte spheroids cultured in suspension. The CYP2B1/2 mRNA and apoprotein levels were continuously higher for over 23 days compared to phenobarbital-induced and control cultures. P450-catalyzed pentoxyresorufin-O-dealkylation activity was also high in the AdCYP2B1-infected spheroids. In these spheroid cultures, albumin and urea levels were similar to those in uninfected spheroid cultures, indicating that expression of the CYP2B1 transgene did not impair these liver-specific functions. Hepatocyte spheroids transduced by recombinant adenoviral vectors can be efficiently used for drug metabolism studies, in implantation, and in bioartificial liver devices.

Cytochrome P450 1B1 gene mutations in Japanese patients with primary congenital glaucoma

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.

Human CYP1B1 Leu432Val gene polymorphism: ethnic distribution in African-Americans, Caucasians and Chinese; oestradiol hydroxylase activity; and distribution in prostate cancer cases and controls.

Cytochrome P4501B1 (CYP1B1) is involved in the activation of many carcinogens and in the metabolism of steroid hormones, including 17beta-oestradiol (E2) and testosterone. We report a significant difference in the allele frequencies of two point mutations in the coding region of the CYP1B1 gene among Caucasian (n = 189), African-American (n = 52) and Chinese (Linxian) (n = 109) populations. A (C to G) transversion at position 1666 in exon 3, which results in an amino acid substitution of Leu432 to Val, was present in African-Americans with an allele frequency for Va1432 of 0.75, in Caucasians of 0.43, and in Chinese of 0.17. A (C to T) transition at position 1719 in exon 3, with no amino acid change (Asp449), appeared to be closely linked with the Val432 variant. Results using human lung microsomal preparations from individuals with the CYP1B1Val/Val and CYP1B1Leu/Leu genotypes indicate that Val432 variant may be a high activity allele and thus may contribute to the interindividual differences in CYP1B1 activity. Because CYP1B1 is involved in hormone and carcinogen metabolism, and given the disparate rates of prostate cancer among ethnic groups, we also evaluated the association of the CYP1B1 Leu432Val polymorphism with prostate cancer risk in a pilot case-control study. Among Caucasians, 34% of men with cancer (n = 50) were homozygous for the Val432 polymorphism, while only 12% of matched control subjects (n = 50) had this genotype. These preliminary data indicate that genetic polymorphisms in CYP1B1 might play an important role in human prostate carcinogenesis.

Trans-resveratrol modulates the catalytic activity and mRNA expression of the procarcinogen-activating human cytochrome P450 1B1

The present study was performed to determine if trans-resveratrol (3,5,4'-trihydroxy-trans-stilbene) modulates the catalytic activity and gene expression of cytochrome P450 1B1 (CYP1B1). In vitro, trans-resveratrol decreased human recombinant CYP1B1-catalyzed 7-ethoxyresorufin O-dealkylation activity, with an IC50value of 1.4 ± 0.2 µM (mean ± SEM). Enzyme kinetic analysis indicated that trans-resveratrol inhibited CYP1B1 enzyme activity by a mixed-type inhibition and the apparent Kiwas 0.75 ± 0.06 µM. To determine if trans-resveratrol modulates constitutive CYP1B1 gene expression, cultured MCF-7 human breast carcinoma cells were treated with trans-resveratrol. As indicated by RT-PCR analysis, treatment of MCF-7 cells with 10 µM trans-resveratrol decreased relative CYP1B1 mRNA levels after 5 h, but not after 1.5 or 3 h, of exposure. trans-Resveratrol treatment at 5, 7.5, 10, or 20 µM for 5 h produced a concentration-dependent decrease in CYP1B1 mRNA levels. The extent of suppression was ~50% at 20 µM concentration. The suppressive effect was not a consequence of a toxic response to the compound as assessed by a cell proliferation assay. Overall, our novel finding that trans-resveratrol inhibits the catalytic activity and suppresses the constitutive gene expression of CYP1B1 leads to the possibility that this nutraceutical confers protection against toxicity and carcinogenicity induced by compounds that undergo CYP1B1-catalyzed bioactivation.Key words: cytochrome P450, CYP1B1, 7-ethoxyresorufin, nutraceutical, trans-resveratrol.

Novel compound heterozygous mutations in the cytochrome P4501B1 gene (CYP1B1) in a Japanese patient with primary congenital glaucoma

Novel compound heterozygous mutations in the cytochrome P4501B1 gene (CYP1B1) in a Japanese patient with primary congenital glaucoma

Characterization and Functional Analysis of Two Common Human Cytochrome P450 1B1 Variants

Cytochrome P450 1B1 (CYP1B1) is a human extrahepatic P450 that activates procarinogens, metabolizes 17β-estradiol, and may well have a role in the pathogenesis of some forms of cancer. Besides rare deleterious mutations reported for the CYP1B1 gene, six single-nucleotide polymorphisms have been reported, of which four cause amino acid exchanges. We have expressed two of the common CYP1B1 alleles in yeast cells and mammalian COS-1 cells in order to functionally characterize the alleles with respect to kinetic properties and protein stability. The CYP1B1.2 variant contains the two linked amino acid substitutions R48G and A119S compared to CYP1B1.1. The kinetic parameters of two structurally unrelated CYP1B1 substrates for the two variants were examined. No kinetic differences were seen of 17β-estradiol hydroxylation activities between the two CYP1B1 variants and an only minor increase in the apparent Km for ethoxyresorufin was observed for CYP1B1.2. It therefore appears that they have very similar catalytic properties and the substitutions do not appear to alter CYP1B1 catalytic function. The two CYP1B1 variants were similarly stable when expressed in mammalian COS-1 cells, indicating that the substitutions have no effect on protein folding or stability. The combined results indicate that these two CYP1B1 variants show very similar properties with respect to catalytic activities and protein stability and do not alter CYP1B1 function.