Phenobarbital increases DNA adduct and metabolites formed by ochratoxin A: role of CYP 2C9 and microsomal glutathione-S-transferase

Ochratoxin A (OTA), a mycotoxin that induces nephrotoxicity and urinary tract tumors, is genotoxic and can be metabolized not only by different cytochromes P450 (CYP) but also by peroxidases involved in the arachidonic cascade, although the exact nature of the metabolites involved in the genotoxic process is still unknown. In order to establish the relation between OTA genotoxicity and the formation of metabolites, we chose three experimental models: kidney microsomes from rabbit, human bronchial epithelial cells, and microsomes from yeast that specifically express the human cytochrome P450 2C9 or 2B6 genes. OTA-DNA adducts were analyzed by (32)P postlabeling and the OTA derivatives formed were isolated by HPLC after incubation of OTA in the presence of: (1) kidney microsomes from rabbit pretreated or not with phenobarbital (PB); (2) human pulmonary epithelial cells simultaneously pretreated (or not) with PB alone or in the presence of ethacrynic acid (EA); (3) microsomes expressing CYP 2B6 and 2C9. PB pretreatment significantly increased DNA adducts formed after OTA treatment, both in the presence of kidney microsomes and bronchial epithelial cells, and induced the formation of new adducts. Ethacrynic acid, which inhibits microsomal glutathione-S-transferase, reduced DNA adduct level. DNA adducts were detected when OTA were incubated with microsomes expressing human CYP 2C9 but not with those expressing CYP 2B6. Several metabolites detected by HPLC were increased after PB treatment. Some of them could be related to DNA-adduct formation. In conclusion, OTA biotransformation, enhanced by PB pretreatment, increased DNA-adduct formation through pathways involving microsomal glutathion-S-transferase and CYP 2C9.     

Progressive glomerulonephritis and histiocytic sarcoma associated with macrophage functional defects in CYP1B1-deficient mice

The cytochrome P450 CYP1B1 enzyme metabolically activates polycyclic aromatic hydrocarbons and is a major P450 isoenzyme in human monocytes and macrophages. We have shown previously that mice deficient in CYP1B1 were resistant to induced tumors after 7,12-dimethylbenz[a]anthracene exposure. The pathology of aging CYP1B1 null mice on a B6; 129 background was studied in groups of 29 males and 30 females. By 12 months, 50% of the female mice had developed a unusual progressive glomerulonephritis while males had similar renal lesions later in life. This disease followed a sequence of proliferative, membranoproliferative and sclerotic glomerulonephritis. Anti-DNA antibodies were found in the blood of the mice along with immune deposits containing immunoglobulins in subepithelial locations of the glomerular basement membrane. The lesions were unlike those found in aging wild-type B6;129 mice or mice of other strains. We found that macrophages from CYP1B1-null mice were impaired in the phagocytosis of apoptotic, necrotic, and opsonized cells. This suggests a generalized defect in the phagocytic activity of CYP1B1-null mouse macrophages. Male mice also developed a high incidence (62-64%) of histiocytic sarcomas. Our study provides evidence that deficiency of CYP1B1 can play a role in the development of glomerular disease, normal processing of catabolic DNA and tumors of the mononuclear phagocyte system. The function of CYP1B1 in histiocytes and macrophages may involve both self-tolerance and tumor suppression.     

Association of Single Nucleotide Polymorphisms in CYP1B1 and COMT Genes with Breast Cancer Susceptibility in Indian Women

Cytochrome P450 1B1 (CYP1B1) and catechol-$O$-methyltransferase (COMT) enzymes play critical roles in estrogen metabolism. Alterations in the catalytic activity of CYP1B1 and COMT enzymes have been found associated with altered breast cancer risk in postmenopausal women in many populations. The substitution of leucine (Leu) to valine (Val) at codon 432 increases the catalytic activity of CYP1B1, however, substitution of Val to methionine (Met) at codon 158 decreases the catalytic activity of COMT. The present study was performed to evaluate the associations of CYP1B1 Leu⁴³²Val and/or COMT Val¹⁵⁸Met polymorphisms with total, premenopausal and postmenopausal breast cancer risks in Indian women. COMT and CYP1B1 polymorphisms in controls and breast cancer patients were analyzed employing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed by gel electrophoresis. Although CYP1B1 and COMT genotypes did not exhibit statistically significant association with breast cancer risks when analyzed individually, COMT wild type (Val¹⁵⁸Val) in combination with CYP1B1 heterozygous variant (Leu⁴³²Val) [OR: 0.21; 95% CI (0.05–0.82), p value; 0.021] and COMT heterozygous variant (Val¹⁵⁸Met) in combination with CYP1B1 wild type (Leu⁴³²Leu) [OR: 0.29; 95% CI (0.08–0.96), p value; 0.042] showed significant protective association with premenopausal breast cancer risk. The results demonstrate that CYP1B1 wild type in combination with COMT heterozygous or their inverse combination offer protection against breast cancer in premenopausal Indian women.     

Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not involved in the risk of recurrent pregnancy loss

The etiology of recurrent pregnancy loss (RPL) remains unclear, but it may be related to a possible genetic predisposition together with involvement of environmental factors. We examined the relation between RPL and polymorphisms in four genes, human aryl hydrocarbon (Ah) receptor, cytochrome P450 (CYP) 1A1, CYP1A2 and CYP1B1, which are involved in the metabolism of a wide range of environmental toxins and carcinogens. All cases and controls were women resident in Sapporo, Japan and the surrounding area. The Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotypes were assessed in 113 Japanese women with recurrent pregnancy loss (RPL) and 203 ethnically matched women experiencing at least one live birth and no spontaneous abortion (control). No significant differences in Ah receptor, CYP1A1, CYP1A2 and CYP1B1 genotype frequencies were found between the women with RPL and the controls [Ah receptor: Arg/Arg (reference); Arg/Lys and Lys/Lys, odds ratio (OR)=0.67; 95% confidence interval (CI)=0.40-1.11, CYP1A1: m1m1 (reference); m1m2 and m2m2, OR = 0.86; 95% CI = 0.53-1.40, CYP1A2: C/C and C/A (reference); A/A, OR = 1.16; 95% CI = 0.71-1.88, CYP1B1: Leu/Leu (reference); Leu/Val and Val/Val, OR = 1.18; 95% CI = 0.68-2.02]. The present study suggests that the Ah receptor, CYP1A1, CYP1A2 and CYP1B1 gene polymorphisms are not major genetic regulators in RPL.     

Differential regulation of CYP1A1 and CYP1B1 expression in resveratrol-treated human medulloblastoma cells

Resveratrol induces differentiation and Fas-independent apoptosis of medulloblastoma cells by a largely unknown mechanism. CYP1A1 and 1B1 are involved in resveratrol-mediated tumor suppression but their expression in medulloblastoma cells and their relevance to anti-medulloblastoma activity of resveratrol have not been described. The statuses of CYP1A1 and 1B1 in UW228-3 medulloblastoma cells without and with resveratrol treatments were elucidated in this study with ethoxyresorufin O-deethylation assay, followed by RT-PCR, immunocytochemical staining and Western blot hybridization. CYP1A1/1B1 enzymatic activity was low in UW228-3 cells but became several folds higher upon resveratrol treatments. CYP1A1 was undetectable and CYP1B1 was expressed in normally cultured cells. Accompanied by the increased fraction of apoptosis, enhanced CYP1A1 and downregulated CYP1B1 were observed in resveratrol-treated cells in time- and dose-related fashions. Our results demonstrate for the first time that in the medulloblastoma cell system, CYP1A1 upregulation is paralleled with resveratrol-induced differentiation and apoptosis, while CYP1B1 may not be an essential element in metabolic activation of resveratrol in those cells. CYP1A1 and 1B1 are resveratrol response genes and potential chemosensitive markers of medulloblastoma cells.     

Functional analysis of CYP1B1 mutations and association of heterozygous hypomorphic alleles with primary open-angle glaucoma

López-Garrido M-P, Blanco-Marchite C, Sánchez-Sánchez F, López-Sánchez E, Chaqués-Alepuz V, Campos-Mollo E, Salinas-Sánchez AS, Escribano J. Functional analysis of CYP1B1 mutations and association of heterozygous hypomorphic alleles with primary open-angle glaucoma.
Glaucoma is an inherited complex and heterogeneous disease, and one of the most prevalent causes of definitive blindness in the world. Recent reports have indicated that heterozygous mutations of the CYTOCHOROME P4501B1 ( CYP1B1 ) gene are present in 4–10% of patients with primary open-angle glaucoma (POAG). To further evaluate the role of CYP1B1 mutations in POAG we extended our previous association study and carried out a functional analysis of the mutations identified by polymerase chain reaction (PCR) DNA sequencing of the three exons of the gene in a total of 245 unrelated Spanish patients and 326 control subjects. Eight of nine different mutations identified in these patients were cloned and functionally assessed by measuring ethoxyresorufin O-deethylation activity and CYP1B1 stability in transiently transfected HEK-293T cells. All these mutants showed reduced catalytic activity, ranging from 20% to 60% of wild-type and/or decreased protein stability and, therefore, they were classified as hypomorphic alleles. No null alleles were identified in these patients. We found heterozygous hypomorphic CYP1B1 mutations in 17 (6.7%) patients and in seven controls (2.1%) showing that these mutations are associated with an increased risk of POAG (p = 0.005; odds ratio = 3.2; 95% confidence interval = 1.30–9.19). Our data suggest that hypomorphic CYP1B1 mutations are, to date, the main known genetic risk factor in POAG.     

Liver tumor-promoting effect of beta-naphthoflavone, a strong CYP 1A1/2 inducer, and the relationship between CYP 1A1/2 induction and Cx32 decrease in its hepatocarcinogenesis in the rat

Interrelationships among induction of cytochrome P-450 (CYP) 1A1/2, decrease in connexin 32 (Cx32), and liver tumor-promoting activity by beta-naphthoflavone (BNF) in the promotion stage were examined in a 2-stage liver carcinogenesis model. A total of 20 male Fischer 344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or were given the saline vehicle alone. Starting 2 weeks later, they were fed a diet containing 2%, 1%, or 0% BNF for 6 weeks. All animals were subjected to a two-thirds partial hepatectomy at week 3 and were sacrificed at week 8. Absolute and relative liver weights were significantly increased in the DEN+BNF groups as compared to the DEN-alone group. Diffuse hepatocellular hypertrophy with cytoplasmic eosinophilia, sometimes accompanied by development of adenoma-like hepatic foci, was observed in the BNF-treated rats. Remarkable induction of cytochrome CYP 1A1/2 and significant increase in CYP 2E1 were noted in the DEN+BNF groups, and positive immunohistochemical staining for both was observed diffusely. The areas of Cx32-positive spots per hepatocyte in the centrilobular areas of livers of the BNF-treated rats were significantly decreased, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form were increased in the BNF-treated groups. These results suggest that BNF is a liver tumor promoter that, unlike phenobarbital, does not induce CYP 2B1/2 isozymes, and there seems to be no direct relationship between CYP 1A1/2 induction and Cx32 reduction in BNF hepatocarcinogenesis.     

Genetic polymorphism of CYP1A2 increases the risk of myocardial infarction

Background: There is growing evidence that DNA damage caused by mutagens found in tobacco smoke may contribute to the development of coronary heart disease (CHD). In order to bind to DNA many mutagens require metabolic activation by cytochrome P450 (CYP) 1A1 or CYP1A2. The objective of this study was to determine the effects of CYP1A1 and CYP1A2 genotypes on risk of myocardial infarction (MI) and whether smoking interacts with genotype to modify risk.

Methods: Subjects (n = 873) with a first acute non-fatal MI and population based controls (n = 932) living in Costa Rica, matched for age, sex, and area of residence, were genotyped for CYP1A1*2A and CYP1A2*1F by restriction-fragment length polymorphism (RFLP)-PCR, and smoking status was determined by questionnaire.

Results: After adjusting for matching variables and potential confounders, no association was observed between CYP1A1 genotype and risk of MI. Compared to individuals with the high inducibility CYP1A2*1A/*1A genotype, the adjusted odds ratio and 95% confidence intervals for risk of MI were 1.19 (0.97 to 1.47) for the *1A/*1F genotype and 1.55 (1.10 to 2.18) for the *1F/*1F genotype. No significant interactions were observed between smoking and either CYP1A1 or CYP1A2 genotype.

Conclusions: The low inducibility genotype for CYP1A2 was associated with an increased risk of MI. This effect was independent of smoking status and suggests that a substrate of CYP1A2 that is detoxified rather than activated may play a role in CHD.

     

Mutations in CYP1B1 cause primary congenital glaucoma by reduction of either activity or abundance of the enzyme

Primary congenital glaucoma (PCG) is an autosomal recessive disorder caused predominantly by mutations in the CYP1B1 gene. A total of five frequent single nucleotide polymorphisms (SNPs) have been identified in the coding sequence of CYP1B1: rs10012C>G (p.R48G), rs1056827G>T (p.A119S), rs1056836C>G (p.V432L), rs1056837C>T (p.D449D), and rs1800440A>G (p.N453S). We performed a functional characterization of four common CYP1B1 variants presenting different coding SNP haplotypes (RAVDN, GSLDN, RALDS, and RALDN) and five CYP1B1 mutations reported for PCG patients: c.182G>A (p.G61E), c.608A>G (p.N203S), c.1033_1035del (p.L343del), c.241 T>A (p.Y81N), and c.685G>A (p.E229 K). Each mutation was embedded in its corresponding background SNP haplotype. The common variants revealed variation in enzymatic activity; among them, RAVDN showed the highest activity. Mutants p.G61E, p.N203S, and p.L343del each revealed a residual activity (<10%) of their respective haplotype. The microsomal CYP1B1 abundance relative to total protein also showed variation in common variants and a significant reduction in p.L343del, p.Y81N, and p.E229 K. The free energy of folding (DeltaDeltaG) values suggest that the lower stability of the mutants is one key property leading to the experimentally observed lower protein abundance. Our new measure of relative enzymatic activity (U/mg total protein), which combines activity and abundance values, was significantly lower for all five mutations compared to the corresponding background haplotype. We classified p.Y81N and p.E229 K not as mutations but as hypomorphic alleles, since their relative activity values are intermediate between bona fide mutations and the common variant with the lowest activity (RALDS). We propose that CYP1B1 mutations can act by either reducing enzymatic activity (p.G61E and p.N203S), reducing the abundance of the enzyme (p.Y81N and p.E229 K), or both (p.L343del).     

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.     

A peroxisome proliferator-activated receptor-alpha activator induces renal CYP2C23 activity and protects from angiotensin II-induced renal injury

Cytochrome P450 (CYP)-dependent arachidonic acid (AA) metabolites are involved in the regulation of renal vascular tone and salt excretion. The epoxygenation product 11,12-epoxyeicosatrienoic acid (EET) is anti-inflammatory and inhibits nuclear factor-kappa B activation. We tested the hypothesis that the peroxisome proliferator-activated receptor-alpha-activator fenofibrate (Feno) induces CYP isoforms, AA hydroxylation, and epoxygenation activity, and protects against inflammatory organ damage. Double-transgenic rats (dTGRs) overexpressing human renin and angiotensinogen genes were treated with Feno. Feno normalized blood pressure, albuminuria, reduced nuclear factor-kappa B activity, and renal leukocyte infiltration. Renal epoxygenase activity was lower in dTGRs compared to nontransgenic rats. Feno strongly induced renal CYP2C23 protein and AA-epoxygenase activity under pathological and nonpathological conditions. In both cases, CYP2C23 was the major isoform responsible for 11,12-EET formation. Moreover, we describe a novel CYP2C23-dependent pathway leading to hydroxy-EETs (HEETs), which may serve as endogenous peroxisome proliferator-activated receptor-alpha activators. The capacity to produce HEETs via CYP2C23-dependent epoxygenation of 20-HETE and CYP4A-dependent hydroxylation of EETs was reduced in dTGR kidneys and induced by Feno. These results demonstrate that Feno protects against angiotensin II-induced renal damage and acts as inducer of CYP2C23-mediated epoxygenase activities. We propose that CYP-dependent EET/HEET production may serve as an anti-inflammatory control mechanism.     

Cloning and expression of rat CYP2E1 in Saccharomyces cerevisiae: detection of genotoxicity of N-alkylformamides

A cDNA coding for rat cytochrome P450 2E1 was cloned into the multicopy vector pYeDP60 and expressed in haploid RSY6 and diploid RS112 yeast strains of Saccharomyces cerevisiae under control of the GAL10-CYC1 promoter. Spectral and catalytic properties of the expressed 2E1 were examined in whole cells or microsomes of both strains. The level of CYP2E1 obtained in RS112 (200 pmol/mg microsomal protein) was the highest among CYP2E1 produced in the various expression systems. The monooxygenase activity in the microsomes of both strains, measured as aniline hydroxylase, was found comparable to that of control rat hepatic microsomes. In a reconstituted system in the presence of exogenous rat P450 reductase, their activity increased about 10-fold. When exposed to the carcinogen NDMA, a known 2E1 substrate, the recombination frequency determined in the 2E1-expressing RS112 cells was enhanced, in a dose-dependent manner, up to 20-fold. The exposure of the same cells to the hepatotoxic solvents, N-methyl- and N-ethylformamide, resulted in an induction of recombination frequency, which was not observed in the void plasmid containing RS112 cells in the presence of S9 hepatic fractions from pyrazole-induced rats, as a specific exogenous metabolic activation system. These results demonstrate that the 2E1-expressing cells metabolize the two N-alkylformamides to genotoxic intermediates and, therefore, they provide an useful tool to study the bioactivation mechanism of potential P450 2E1 substrates.     

Polymorphisms of the CYP1B1 gene may be associated with the onset of natural menopause in Chinese women

The Cytochrome P450 1B1 (CYP1B1) is one of the major CYP450 enzymes catalyzing 4-hydroxylation, an important elimination step for estrogens. Relatively little is known, however, about the impact of this gene on the onset and cessation of menstruation, which are significant milestones in a woman's life and predictors of many hormone related diseases. In this report, we described the association of four SNPs in the CYP1B1 gene, Arg48Gly, Ala119Ser, Leu432Val, and Asp449Asp, with the ages of menarche and menopause, years of menstruation and total number of menstrual cycles. Included in the study were 1958 community controls from two recently completed population-based case-control studies of breast cancer and endometrial cancer. No association was observed between the CYP1B1 polymorphisms and the age of menarche among either pre- or post-menopausal women. Among the women who experienced natural menopause, the three non-synonymous SNPs were significantly associated with menopausal age, years of menstruation, and total number of menstrual cycles. The Gly and Ser alleles of Arg48Gly and Ala119Ser were associated with later menopause, more years of menstruation and more menstrual cycles, while women with allele Val at Leu432Val had a 0.9 year earlier menopause, 1.0 year shorter reproductive span, and 12.6 fewer menstrual cycles than those women without this allele. In conclusion, the results from this study suggested that CYP1B1 genetic polymorphisms may be associated with the natural onset of menopause.     

Ethanol-mediated CYP1A1/2 induction in rat skeletal muscle tissue

The causes of non-trauma-mediated rhabdomyolysis are not well understood. It has been speculated that ethanol-associated rhabdomyolysis may be attributed to ethanol induction of skeletal muscle cytochrome P450(s), causing drugs such as acetaminophen or cocaine to be metabolized to myotoxic compounds. To examine this possibility, the hypothesis that feeding ethanol induces cytochrome P450 in skeletal muscle was tested. To this end, rats were fed an ethanol-containing diet and skeletal muscle tissue was assessed for induction of CYP2E1 and CYP1A1/2 by immunohistochemical procedures; liver was examined as a positive control tissue. Enzymatic assays and Western blot analyses were also performed on these tissues. In one feeding system, ethanol-containing diets induced CYP1A1/2 in soleus, plantaris, and diaphragm muscles, with immunohistochemical staining predominantly localized to capillaries surrounding myofibers. Antibodies to CYP2E1 did not react with skeletal muscle tissue from animals receiving a control or ethanol-containing diet. However, neither skeletal muscle CYP1A1/2 nor CYP2E1 was induced when ethanol diets were administered by a different feeding system. Ethanol consumption can induce some cytochrome P450 isoforms in skeletal muscle tissue; however, the mechanism of CYP induction is apparently complex and appears to involve factors in addition to ethanol, per se.     

CYP1B1 Gene Mutations Causing Primary Congenital Glaucoma in Tunisia

Primary congenital glaucoma (PCG) is responsible for a significant proportion of childhood blindness in Tunisia. Early prevention based on genetic diagnosis is therefore required. This study sought to determine the frequency of CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1) mutations in 18 PCG patients, recruited from Central and Southern of Tunisia. Genomic DNA was extracted and the coding regions of CYP1B1 were analysed by direct sequencing. A phylogenetic network of CYP1B1 haplotypes was drawn using the median‐joining algorithm. Sequence analysis revealed a “tetra‐allelic mutation” (two novel mutations, p.F231I and p.P437A in the homozygous state) in one patient. The healthy members of his family carried those variations on the same allele. Two previously described mutations p.G61E and c.535delG were also identified in the homozygous state in seven and two probands, respectively. Seven single‐nucleotide polymorphisms were identified and used to generate haplotypes. Our results showed that the CYP1B1 mutations were present in 55% of Tunisian PCG patients’ alleles. Haplotype analysis allowed us to define the proto‐haplotype and to confirm historical migratory flows. Establishment of PCG genetic aetiology in Tunisia will improve genetic diagnosis and counselling.     

Effect of triphenyldioxane on phase I xenobiotic metabolism enzymes in the liver of rats and rabbits

We studied the effect of triphenyldioxane on phase I xenobiotic metabolism enzymes in the liver of rats and rabbits. Total cytochrome P450 content, protein concentration, and catalytic activity of CYP2B, CYP3A, and CYP2C isoforms were measured. Triphenyldioxane significantly increases specific activity of CYP2B and CYP2C in the liver of rats and rabbits, respectively. Immunoblotting analysis of microsomal enzymes in the liver of animals showed that the increase in specific activity of CYP is related to high content of apoenzymes. We showed for the first time that rats and rabbits are characterized by interspecies differences in the induction of cytochrome P450 isoforms under the influence of triphenyldioxane. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 6, pp. 646–648, June, 2006     

Role of interleukin-1beta in the modulations of cytochrome P450 and heme metabolism in rat liver.

The effect of recombinant human interleukin-1beta (IL-1beta) on the modulation of hepatic cytochrome P450 (P450) was investigated by in vivo subcutaneous dosing studies in male Sprague-Dawley rats. To assess the effect of IL-1beta on heme metabolism, we determined the delta-aminolevulinic acid synthetase (delta-ALAS) and heme oxygenase activities in the liver. IL-1beta suppressed the microsomal total P450 and heme contents and delta-ALAS activity in the liver. In contrast, microsomal heme oxygenase activity was significantly increased by the IL-1beta treatments. Western blot analysis and marker enzyme activities for individual P450 isoforms demonstrated that IL-1beta suppressed CYP2C6, 2C13, 2E1, and 3A2, whereas CYP2A, 2B1/2, 2C11, and 4A1 were not influenced by the treatments. IL-1beta inhibited both allylisopropylamide- and phenobarbital-inducible delta-ALAS activities in the liver. These results indicate that IL-1beta has differential effects on the constitutive P450, and also on delta-ALAS and heme oxygenase activities in rat liver. Thus, the modulation of hepatic P450 by IL-1beta is complex, and IL-1beta may be involved in the regulation of both apoprotein synthesis for each P450 isoform and the heme pools in the liver.     

Growth kinetics in MCF-7 cells modulate benzo[a]pyrene-induced CYP1A1 up-regulation

Pro-carcinogens, such as benzo[a]pyrene (B[a]P), that are exogenous ligands of the aromatic hydrocarbon receptor may influence the susceptibility of target-cell populations through the up-regulation of cytochrome P450 (CYP) mixed function oxidases. We examined whether the growth kinetics of MCF-7 cells might determine the level of up-regulation of CYP1A1, CYP1A2 or CYP1B1 by B[a]P, and whether this could then influence subsequent levels of DNA damage. Cell cultures manipulated to be G(0)/G(1)-phase concentrated, S-phase concentrated or G(2)/M-phase concentrated were treated with B[a]P and the expression levels of CYP1A1, CYP1A2, CYP1B1, cyclin-dependent kinase inhibitor 1A [CDKN1A (P21(WAF1/CIP1))], B-cell leukaemia/lymphoma-2 (BCL-2), and Bcl-2-associated X levels were determined. Levels of DNA damage were measured as DNA single-strand breaks (SSBs) by the alkaline single-cell gel electrophoresis (comet) assay or as DNA adducts by (32)P-postlabelling analysis. B[a]P-induced up-regulation of CYP1A1 was >100-fold in S-phase-concentrated cells, but in G(0)/G(1)-phase- or G(2)/M-phase-concentrated cultures up-regulation occurred to a significantly lower extent. Consistent with this, B[a]P-treated S-phase-concentrated cultures exhibited markedly up-regulated P21(WAF1/CIP1), higher levels of dose-related increases in DNA SSBs, and increased DNA adduct levels presumably as a result of CYP1A1-mediated activation of B[a]P to B[a]P-diol-epoxide compared with the cultures enriched for the other cell cycle phases. Growth kinetics in vitro may be an important predeterminant of susceptibility to an exogenous pro-carcinogen in short-term test systems and these findings have important implications when extrapolating such results to a particular target-cell population in vivo.     

CYP1B1基因多态性与子宫内膜异位症易感性的相关研究

目的 研究CYP1B1基因第2外显子119(G-T)、第3外显子432(C-G)多态性与子宫内膜异位症(endometriosis,Ems)易感性的关系.方法 采用等位基因特异性聚合酶链反应对55例Ems患者和45例对照组进行CYP1B1基因第2外显子119(G-T)、第3外显子432(C-G)突变分析,探讨Ems的发生与CYP1B1基因多态性之间的相关性.结果 CYP1B1基因密码子119中等位基因G、T在Ems组和对照组分布的差异有统计学意义(P＜0.05),其中等位基因T使Ems发病风险提高2.061倍;CYP1B1基因密码子119G/T各基因型分布两组间差异有统计学意义(P＜0.05),纯合突变(T/T)基因型、杂合突变(G/T)基因型与野生型(G/G)基因型相比,患Ems的危险度分别为2.625倍和3.214倍.以CYP1B1联合野生型GG和CC个体的OR值为1相比,CYP1B基因密码子119杂合型突变(Ala/Ser)合并密码子432野生型个体的OR值为2.976,95%CI:1.129～7.848,P＜0.05.结论 CYP1B1基因第2外显子119(G-T)突变等位基因与Ems的发生有一定关系,突变基因型增加了Ems的发病风险;CYP1B1基因第2外显子杂合型突变(Ala/Ser)联合密码子432野生型能增加Ems的发病风险.     

Flavonoids inhibit genetic toxicity produced by carcinogens in cells expressing CYP1A2 and CYP1A1.

The effects of the flavonoids quercetin, apigenin and chrysin (10 microM) on the genetic toxicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and benzo[a]pyrene (BaP) was investigated at sub-cytotoxic concentrations in Chinese hamster V79 cells expressing human or rat cytochromes P450. In V79 r1A2-NH and V79 h1A1-MZ cells, none of the flavonoids increased DNA strand breaks (SB) (measured by the Comet assay) or produced detectable DNA adducts (measured by 32P-post-labelling). Neither IQ nor BaP produced DNA damage in the absence of expressed CYP1A2 or CYP1A1, respectively. DNA damage measured as SB and DNA adducts was detectable in V79 r1A2-NH cells expressing rat CYP1A2 when treated with IQ (2.5-50 microM) and this was inhibited by quercetin. Likewise, DNA damage (SB and DNA adducts) was elevated in V79 h1A1-MZ cells expressing human CYP1A1 when treated with BaP (0.1-0.5 microM) and this was inhibited by chrysin and apigenin, but not by quercetin. The specificity of CYP1A1 inhibition by chrysin and apigenin and CYP1A2 inhibition by quercetin was confirmed by ethylresorufin O-deethylase assay.