Role of the SLCO1B1 haplotypes on the rifampicin-induced CYP3A activity

Rifampin, a member of the rifamycin class of antibiotics, is well known for its ability to activate the pregnant X receptor and induce drug-metabolizing enzymes and transporters. Available data suggest rifampin entry into hepatocytes is mediated by OATP1B1. Accordingly, it is therefore plausible that modulation of the intracellular concentration of rifampin by OATP1B1 genetic polymorphisms would influence the degree of CYP3A induction. AIM： To study the association between haplotypes of the SLCO1B1 and the rifampicin-mediated inducible CYP3A4 activity. A single-point determination of midazolam plasma concentration method was developed to assess the constitutive and inducible CYP3A4 activity. A pharmacokinetic study of a single dose of 450 mg rifampicin was conducted to evaluate the mechanism of rifampicin-midazolam interaction in different SLCO1B1 genotypic subjects.METHODS： Twenty-three healthy volunteers with different SLCO1B1 haplotypes （7 for SLCO1B1 ＊ 1a/＊ 1a, 7 for SLCO1B1 ＊ 1b/＊ 1b, 7 for SLCO1B1 ＊ 1b / ＊ 15 and 2 for SLCO1B1 ＊ 15/＊ 15） were enrolled in this study. Each was given a single oral dose of 7.5 nag naidazolam on day 0 and day 6. Rifampicin of 450 nag was given from day 1 to day 5. Plasma concentrations of midazolam were measured for up to 8 hours by LC-MS, and its pharmacokinetic parameters were analyzed. Plasma concentrations of a single oral dose of 450 nag rifampicin were measured for up to 12 hours.[第一段]     

Molecular modelling of CYP1 family enzymes CYP1A1, CYP1A2, CYP1A6 and CYP1B1 based on sequence homology with CYP102

Molecular modelling of a number of CYP1 family enzymes from rat, plaice and human is described based on amino acid sequence homology with the haemoprotein domain of CYP102, a unique bacterial P450 of known structure. The interaction of various substrates and inhibitors within the putative active sites of rat CYP1A1, human CYP1A2, a fish CYP1 enzyme CYP1A6 (from plaice) and human CYP1B1, is shown to be consistent with P450-mediated oxidation in each example or, in the case of inhibitors, mechanism of inhibition. It is reported that relatively small changes between the enzymes' active site regions assist in the rationalization of CYP1 enzyme preferences for particular substrate types, and a template of superimposed CYP1A2 substrates is shown to fit the putative active site of the human CYP1A2 enzyme.     

Characterization of CYP1A1 and CYP1A3 gene expression in rainbow trout (Oncorhynchus mykiss)

The rainbow trout CYP1A1 and CYP1A3 genes share 96% amino acid identity and have similar enzymatic activity. The expression of CYP1A1 and 1A3 genes was investigated in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated rainbow trout tissues, sac fry and cell lines. Both CYP1A1 and CYP1A3 were induced by TCDD in all the tissues examined. While CYP1A3 gene was expressed constitutively at higher levels than CYP1A1 in trout intestine, preferential expression of CYP1A1 occurred in trout liver, heart, kidney and trout sac fry. In rainbow trout gonad (RTG)-2 and rainbow trout hepatoma (RTH)-149 cell lines, CYP1A1 was constitutively expressed and induced by exposure to TCDD, but CYP1A3 message was not detected, even after TCDD treatment. Quantitative analysis of CYP1A genes expression in rainbow trout liver revealed that TCDD induced CYP1A1 expression more than 50-fold and CYP1A3 RNA levels increased at least 100-fold over untreated fish. The cell- and tissue-specific expression indicates that these closely related CYP1A genes are independently regulated and that negative regulation may play a role in CYP1A3 gene expression.     

Correlation between gene expression of aryl hydrocarbon receptor (AhR), hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1) and 1B1 (CYP1B1), and inducibility of CYP1A1 and CYP1B1 in human lymphocytes.

The relationships between gene expression of aryl hydrocarbon receptor (AhR), aryl hydrocarbon receptor nuclear translocator (Arnt), cytochromes P4501A1 (CYP1A1), 1B1 (CYP1B1), CYP1A1, and the inducibility of CYP1A1 and CYP1B1 were determined in 32 cultivated human lymphocytes. Cytochrome P450 induction was performed by incubating lymphocytes with benzanthracene. The relative gene expression levels were determined by quantitative real-time RT-PCR assay. We found that gender is an important confounding factor for gene expression in cultivated lymphocytes. AhR, CYP1A1 and CYP1B1 levels in noninduced lymphocytes were significantly higher in female nonsmokers than in male nonsmokers (p < 0.05). Nevertheless, CYP1A1 and CYP1B1 inducibility was lower in female nonsmokers. CYP1A1 inducibility was higher in male smokers than in male nonsmokers (p < 0.05). After controlling for gender and cigarette smoking, AhR levels positively correlated with CYP1B1 levels and CYP1A1 inducibility (p < 0.01 and p = 0.03, respectively). Arnt levels also correlated with CYP1B1 levels in induced lymphocytes (p < 0.01). However, AhR levels were negatively correlated with CYP1B1 inducibility. These data indicate that AhR expression associates with individual variation of CYP1A1 inducibility and CYP1B1 expression in cultivated lymphocytes. Furthermore, gender and cigarette smoking are important confounding factors for gene expression levels in cultivated lymphocytes.     

Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression

Despite widespread use of bromuconazole as a pesticide for food crops and fruits, limited studies have been done to evaluate its toxic effects. Here, we evaluated the hepatotoxic effect of bromuconazole using classical toxicological (biochemical analysis and histopathological examination) and gene-based molecular methods. Male rats were treated either orally or topically with bromuconazole at doses equal to no observed adverse effect level (NOAEL) and 1/10 LD50 for 90?d. Bromuconazole increased activities of liver enzymes (ALT, AST, ALP, and ACP), and levels of bilirubin. It also induced hepatic oxidative stress as evidenced by significant decrease in the activities of superoxide dismutase (SOD), and significant increase in levels of malondialdehyde (MDA) in liver. In addition, bromuconazole caused an increase in liver weights and necrobiotic changes (vacuolation and hepatocellular hypertrophy). It also strongly induced the expression of PXR and its downstream target CYP3A1 gene as well as the activity of CYP3A1. However, it inhibited the expression of CAR and its downstream target CYP2B1 gene without significant changing in CYP2B1 activity. Overall, the oral route showed higher hepatotoxic effect and molecular changes than the dermal route and all changes were dose dependent. This is the first investigation to report that bromuconazole-induced liver oxidative damage is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1.     

Real-time polymerase chain reaction analysis of CYP1B1 gene expression in human liver.

Procarcinogen-activating cytochrome P450 (CYP) enzymes such as CYP1B1, CYP1A1, and CYP1A2 are considered to play an important role in chemical carcinogenesis. However, conflicting data exist with respect to CYP1B1 expression in human liver. In the present study, we measured CYP1B1 mRNA and protein expression in liver samples from 12 individuals (7 nonsmokers, 4 smokers, and 1 ex-smoker) and compared the levels to those of CYP1A1 and CYP1A2. As analyzed by real-time polymerase chain reaction, CYP1B1 mRNA was present in all samples and the inter-individual variability was 16-fold. The group mean level was 5-fold greater in smokers than nonsmokers (121 +/- 46 vs. 26 +/- 5 molecules/ng double-stranded DNA, p < 0.05). By comparison, CYP1A1 mRNA was detectable in samples from 4 of 7 nonsmokers, 3 of 4 smokers, and one ex-smoker, whereas CYP1A2 mRNA was detectable in samples from 5 nonsmokers, 4 smokers, and the ex-smoker. The mean levels of CYP1A1 and CYP1A2 mRNA were 4-fold and 9-fold greater, respectively, in smokers than nonsmokers, but the differences were not statistically significant. The inter-individual variability in CYP1A1 and CYP1A2 mRNA expression was 26-fold and 500-fold, respectively. Immunoblot analysis using several antibodies and with a larger panel (n = 27) of liver microsomes showed that CYP1A1 and CYP1B1 proteins were undetectable, whereas CYP1A2 was detectable in all samples and quantifiable in 24 of 27 samples. In summary, our novel finding indicates that CYP1B1 mRNA is expressed in human liver and the levels are increased in smokers, but the protein is undetectable.     

Enhanced expression of CYP1B1 in Escherichia coli

Conditions for the optimal expression of the human CYP1B1 hemoprotein in Escherichia coli have been investigated. CYP1B1 cDNA was prepared from a retinal cDNA template and used to generate cDNA fragments with modified 5'-sequences reported to allow enhanced expression in E. coli DH5alpha. Plasmids were constructed, using the pCWori+ expression vector and were used to examine necessity for thiamine, delta-aminolevulinic acid (ALA), and IPTG. The optimal shaking speed in an orbital incubator was 150 rpm at 30 degrees C. Higher speeds resulted in increased cell death and lower speeds resulted in lower expression of cytochrome P450. IPTG was necessary for this expression system, which makes use of the lac repressor, but levels above 0.5 mM were without additional benefit. We were able to show thiamine to be unnecessary in this expression system, although included by others expressing CYP1B1. ALA has been reported to enhance expression of several different forms of cytochrome P450. We examined the dependence of CYP1B1 expression on ALA. The expression proved to be highly dependent upon this heme precursor, with levels of CYP1B1 increasing approximately 20-fold, to 920 nmol/l in the presence of up to 2.5 mM ALA. The question of whether heme synthesis and apoprotein synthesis were coupled was then investigated. It could be shown that although heme synthesis was not limiting (CYP101 holoenzyme expression in the absence of ALA was four times higher than the ALA-supported CYP1B1 holoenzyme expression), it was necessary for optimal expression of CYP1B1. CYP1B1 protein synthesis appears to be coupled to heme precursor availability, as seen by SDS-PAGE, because in the absence of heme precursor apocytochrome P450 1B1 does not accumulate.     

Comparison of the function and expression of CYP26A1 and CYP26B1, the two retinoic acid hydroxylases

All-trans-retinoic acid (atRA) is an important signaling molecule in all chordates. The cytochrome P450 enzymes CYP26 are believed to partially regulate cellular concentrations of atRA via oxidative metabolism and hence affect retinoid homeostasis and signaling. CYP26A1 and CYP26B1 are atRA hydroxylases that catalyze formation of similar metabolites in cell systems. However, they have only 40% sequence similarity suggesting differences between the two enzymes. The aim of this study was to determine whether CYP26A1 and CYP26B1 have similar catalytic activity, form different metabolites from atRA and are expressed in different tissues in adults. The mRNA expression of CYP26A1 and CYP26B1 correlated between human tissues except for human cerebellum in which CYP26B1 was the predominant CYP26 and liver in which CYP26A1 dominated. Quantification of CYP26A1 and CYP26B1 protein in human tissues was in agreement with the mRNA expression and showed correlation between the two isoforms. Qualitatively, recombinant CYP26A1 and CYP26B1 formed the same primary and sequential metabolites from atRA. Quantitatively, CYP26B1 had a lower K(m) (19nM) and V(max) (0.8 pmol/min/pmol) than CYP26A1 (K(m)=50 nM and V(max)=10 pmol/min/pmol) for formation of 4-OH-RA. The major atRA metabolites 4-OH-RA, 18-OH-RA and 4-oxo-RA were all substrates of CYP26A1 and CYP26B1, and CYP26A1 had a 2-10-fold higher catalytic activity towards all substrates tested. This study shows that CYP26A1 and CYP26B1 are qualitatively similar RA hydroxylases with overlapping expression profiles. CYP26A1 has higher catalytic activity than CYP26B1 and seems to be responsible for metabolism of atRA in tissues that function as a barrier for atRA exposure.     

Effect of TCDD exposure on CYP1A1 and CYP1B1 expression in explant cultures of human endometrium.

Endometriosis is a debilitating disease estimated to affect 10% of reproductive-age women and characterized by the growth of endometrial tissue outside of the uterus. The present study characterizes a human endometrial explant culture model for studying the direct effects of TCDD exposure by assessing the expression of CYP1A1 and CYP1B1 mRNA (Northern blotting), protein (Western blotting), and activity (7-ethoxyresorufin-O-deethylase; EROD) in explants cultured with and without TCDD. Explants were obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. The explants were cultured with 10 nM estradiol (E(2)) or 1 nM E(2) plus 500 nM progesterone (P(4)) with or without TCDD (first 24 h). The expression of CYP1A1 and CYP1B1 mRNA was greatest with 10 nM TCDD and increased up to 72 h after initial exposure. EROD activity increased up to 120 h. Explants from a secretory phase biopsy became reorganized in culture and formed a new epithelial membrane, while maintaining basic endometrial morphology and viability for up to 120 h. At 24 h, TCDD significantly increased CYP1A1 and CYP1B1 mRNA, and at 72 h, TCDD significantly increased EROD activity and CYP1B1 protein compared to explants cultured without TCDD for similar times. CYP1B1 protein also exhibited substantial constitutive expression that was similar in uncultured biopsies, where CYP1B1 protein was immunolocalized in the cytoplasm of epithelial glands, with only occasional patches of protein in the surface epithelial membrane. In explants cultured with and without TCDD exposure, CYP1B1 protein was localized in the cytoplasm of the new surface epithelial membrane and glands closest to the surface. CYP1A1 protein was not detected in uncultured biopsies or explants. Both younger age (age 30 and under) and proliferative phase were associated with higher TCDD-induced EROD activity in specimens treated with E(2):P(4). No significant endometriosis-related differences were observed for any of the biomarkers, but the detection of disease-specific change was limited by small sample size and variability in tissue-cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase and hormonal exposure.     

Association between cancer risk and drug-metabolizing enzyme gene (CYP2A6, CYP2A13, CYP4B1, SULT1A1, GSTM1, and GSTT1) polymorphisms in cases of lung cancer in Japan

Genetic polymorphisms of enzymes involved in the metabolism of carcinogens are suggested to modify an individual's susceptibility to lung cancer. The purpose of this study was to investigate the relationship between lung cancer cases in Japan and variant alleles of cytochrome P450 (CYP) 2A6 (CYP2A6*4), CYP2A13 (CYP2A13*1-*10), CYP4B1 (CYP4B1*1-*7), sulfotransferase 1A1 (SULT1A1*2), glutathione S-transferase M1 (GSTM1 null), and glutathione S-transferase T1 (GSTT1 null). We investigated the distribution of these polymorphisms in 192 lung cancer patients and in 203 age- and sex-matched cancer-free controls. The polymorphisms were analyzed using various techniques including allele-specific PCR, hybridization probe assay, multiplex PCR, denaturing high-performance liquid chromatography (DHPLC), and direct sequencing. We also investigated allele and genotype frequencies and their association with lung cancer risk, demographic factors, and smoking status. The prevalence of the CYP2A6*4/*4 genotype in lung cancer cases was 3.6%, compared with 9.4% in the controls (adjusted OR = 0.36, 95% CI = 0.15-0.88, P = 0.025). In contrast, there was no association between the known CYP2A13, CYP4B1, SULT1A1, GSTM1, and GSTT1 polymorphisms and lung cancer. These data indicate that CYP2A6 deletions may be associated with lung cancer in the Japanese population studied.     

Medical problems raised by the TCDD contamination in Seveso,Italy

The Seveso accident, where TCDD was produced and released on an urban area, raised several medical problems linked with the non-specificity of the clinical features of the effects from the exposure and the wide variety and severity of lesions and symptoms caused by this toxin in the animal experiment. The assessment of the health hazards had to rely on the detection by chemical analysis of the toxin in the environment and on the definition of an area contaminated by the toxin. Evacuation of the population was the only possible preventive measure for precluding or at least strongly reducing further exposure. Of the known clinical symptoms of the TCDD toxic effects on humans, the typical skin lesions (chloracne) appeared almost only in children and young people. The degree and intensity was mild and spontaneous healing occurred in the majority of the cases.Transient decrease of the lymphocytes and passing signs of impairment of the liver functions have been detected by the immediately established monitoring of the health condition of the population. No overt pathology of the liver, the kidney, the blood reproductive organs, the central and peripheral nervous system, the carbohydrate, fat and porphyrin metabolism has been registered up to now.Close control and thorough examination of the current pregnancies and their outcome did not reveal any embryotoxic or teratogenic effect imputable to the chemical.Comparing the picture of the medical injuries caused by the Seveso accident with that of similar accidents occurred in the past in USA and Europe, both in respect of the short as well as long term toxicity, one should conclude that the TCDD exposure in Seveso has been less severe than in the previous accidents.Paper presented at the 5th International Conference on Occupational Health in the Chemical Industry (Medichem) San Francisco, California, September 5–10, 1977