The frequency and effects of cytochrome P450 (CYP) 2C9 polymorphisms in patients receiving warfarin

BACKGROUND: Warfarin sodium (warfarin) is commonly prescribed in surgical practice. Warfarin use is complicated by an unpredictable dose response that may be due in part to genetically determined differences in metabolic capacity. To better understand the interaction between genotype and response to warfarin therapy, we determined the frequency and functional effects of polymorphisms of the predominant cytochrome P450 subfamily responsible for warfarin metabolism (eg, CYP2C9) in an ethnically defined U.S. patient population. DESIGN: Patients requiring chronic anticoagulation with warfarin sodium (warfarin) were recruited over an 11-month period (June 1999 through May 2000) from the inpatient and outpatient divisions of a tertiary care medical center in this prospective observational study. Clinical and demographic information was collected and CYP2C9 genotype was determined. RESULTS: One hundred fifty-three patients receiving warfarin therapy for at least four weeks and comprising two ethnic groups [33 African Americans (22%) and 120 Caucasians (78%)] were genotyped. The mean weekly warfarin dose (+/-SEM) for all patients [36.9 (+/- 1.5) mg] was not influenced by gender [85 males (56%), 68 females (44%)] or ethnicity (p>0.05 for both), but was significantly affected by age (p = 0.006 for weight adjusted warfarin dose). The frequencies of CYP polymorphisms were as follows: 2C9*2 (24/153) 15.7%, 2C9*3 (23/153) 15.0%. There were no gender differences in polymorphism frequency (CYP2C9*2 frequency = (13/ 85) 15.3% in males, (12/68) 17.6% in females, p=0.74; CYP2C9*3 frequency = (15/85) 17.6% in males and (8/68) 11.8% in females, p = 0.38). CYP polymorphisms were much less common in African Americans than Caucasians [(5/33) 15.2% versus (47/120) 39.2%, respectively p = 0.05)]. Patients with CYP polymorphisms (2C9*2, 2C9*3) had significantly lower warfarin doses compared to patients with wild-type genotypes [30.6 (+/- 2.5) mg versus 40.1 (+/- 1.7) mg, p = 0.0021] . Stepwise backward regression analysis suggested a moderate ability to predict warfarin dose based on CYP genotype (r2 = 0.26), p < 0.01). CONCLUSIONS: CYP2C9 polymorphisms are common, associated with significant reductions in warfarin dose, and partly account for interpatient variability in warfarin sensitivity. As interactions between genetic factors and other variables that influence warfarin effect are more completely understood, CYP analysis may prove a useful adjunct for increasing the safety and efficacy of this agent.     

The effect of selective inhibition of inducible nitric oxide synthase on cytochrome P450 after liver transplantation in a rat model

BACKGROUND: Activity levels of cytochrome P450 (CYP) provide markers for liver function and graft rejection episodes after orthotopic liver transplantation (OLT). Some in vitro studies have shown decreased CYP activation of inducible nitric oxide synthase (iNOS) in rejecting liver grafts. The aim of this study was to evaluate CYP isoenzyme activity changes in vivo and to examine histopathologic aspects during inhibition of iNOS after treatment with aminoguanidine (AG) using OLT in the rat. MATERIALS AND METHODS: Thirty DA-(RT1av1) rats that served as donors and LEWIS-(RT(1)) rats as recipients were divided into three groups: group I (controls, syngeneic rats; n = 6), group II (allogeneic rats without immunosupression; n = 11), and group III (allogeneic rats with AG treatment; n = 13). On postoperative days 5, 8, and 10 we performed laboratory investigations and liver biopsies for histopathologic investigations. RESULTS: On postoperative day 5, activities of CYP-1A1 and -3A4 were significantly lower (P = .022) in group III and the activity of CYP-1A2 higher (P < .05) compared with group II. At postoperative days 8 and 10, the activities of all CYP isoenzymes were significant higher in AG-treated rats (group III) in contrast with group II after allogeneic OLT without immunosuppression. Histopathologic findings revealed less distinct rejection signs in group III specimens after AG treatment compared with group II. CONCLUSION: Summarizing our results, we concluded that AG treatment led to increased CYP activity and less distinction of graft rejection after OLT in rats.     

CYP2C19遗传多态性与膀胱癌易感性关系的研究

目的：观察ＣＹＰ２Ｃ１９遗传多态性与膀胱癌易感性之间的关系。方法：根据ＣＹＰ２Ｃ１９两个突变位点和ＡＳＡ原理设计ＰＣＲ引物,对５０例膀胱癌患者和７０例正常对照者进行ＣＹＰ２Ｃ１９基因分析。结果：膀胱癌组ＣＹＰ２Ｃ１９慢代谢者２例,正常对照慢代谢者１１例;两组间比较差别有显著性意义（Ｐ＜０．０５）。结论：ＣＹＰ２Ｃ１９可能参与膀胱癌前致癌物的活化。     

The effect of anesthesia by diethyl ether or isoflurane on activity of cytochrome P450 2E1 and P450 reductases in rat liver

In this study we sought to determine whether exposure to the anesthetics diethyl ether and isoflurane influences the activity of hepatic cytochrome P450 2E1 and P450 reductases in the rat. Rats were fed a purified diet for 6 wk before anesthesia with 1 of 3 anesthetics: carbon dioxide, diethyl ether, or isoflurane. Cytochrome P450 2E1 and P450 reductases were measured in liver microsomes. No significant differences in enzyme activities were found among the groups. These results indicate that diethyl ether and isoflurane can be used to kill rats without inducing P450 enzymes. IMPLICATIONS: Rats were anesthetized with ether, isoflurane, or carbon dioxide and liver P450 enzymes were quantified by spectrophotometry. Based on the results of this study, rats can be anesthetized with isoflurane or diethyl ether for a short period without a change in the activity of P450 enzymes.     

Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethanol liquid diet or a non-ethanol liquid diet, with or without CMZ for one and ten months. A single intraperitoneal injection of diethylnitrosamine (DEN, 20 mg/kg) was given to initiate hepatic carcinogenesis. CYP2E1 expression, inflammatory proteins, cell proliferation, protein-bound 4-HNE, etheno-DNA adducts, 8-hydroxy-2''-deoxyguanosine (8-OHdG), retinoid concentrations, and hepatic carcinogenesis were examined. Ethanol feeding for 1 month with DEN resulted in significantly increased hepatic CYP2E1 levels and increased nuclear accumulation of NF-κB protein and TNF-α expression, which were associated with increased cyclin D1 expression and p-GST positive altered hepatic foci. All of these changes induced by ethanol feeding were significantly inhibited by the one month CMZ treatment. At 10-months of treatment, hepatocellular adenomas were detected in ethanol-fed rats only, but neither in control rats nor in animals receiving ethanol and CMZ. The 8-OHdG formation was found to be significantly increased in ethanol fed animals and normalized with CMZ treatment. In addition, alcohol-reduced hepatic retinol and retinoic acid concentrations were restored by CMZ treatment to normal levels in the rats at 10 months of treatment. These data demonstrate that the inhibition of ethanol-induced CYP2E1 as a key pathogenic factor can counteract the tumor-promoting action of ethanol by decreasing TNF-α expression, NF-κB activation, and oxidative DNA damage as well as restoring normal hepatic levels of retinoic acid in DEN-treated rats.Key Words: Ethanol, hepatic carcinogenesis, cytochrome P450 2E1, NF-κB, cell proliferation     

Effect of atorvastatin and fluvastatin on the metabolism of midazolam by cytochrome P450 in vitro

We have investigated the effects of the statins atorvastatin and fluvastatin on the cytochrome P450 3A4 enzyme (CYP 3A4)-mediated metabolism of midazolam in vitro, using pooled human liver microsomes. Midazolam was metabolised by human hepatic microsomes with a Michaelis-Menten constant (K(m)) of 5.25 (SD 1.2) micromol.l(-1). Atorvastatin was a moderate competitive inhibitor of CYP 3A4 with an inhibitory constant (K(i)) of 12.4 (95% CI 4.65-20.06) micromol.l(-1). Fluvastatin was a weak non-competitive inhibitor of CYP 3A4 with a K(i) of 94.3 (95% CI 55.01-133.5) micromol.l(-1). Both atorvastatin and fluvastatin inhibit the CYP 3A4-mediated metabolism of midazolam in vitro.     

Inhaled budesonide induced Cushing's syndrome in cystic fibrosis patients, due to drug inhibition of cytochrome P450.

Two CF patients developed Cushing's syndrome during administration of inhaled budesonide (400 microg/d) with oral itraconazole in one and with clarithromycin in the other patient. Clinical features appeared respectively after 2 and 6 weeks of drug co-administration, with prolonged adrenal suppression, and a slow recovery after ceasing the drugs. Inhibitors of the cytochrome P450 interfere with the metabolism of corticosteroids. Combination of these drugs even with moderate doses of budesonide should be closely monitored.     

Polymorphism of cytochrome P450 2B6 and prostate cancer risk: A significant association in a Japanese population

Objectives:   To explore whether Lys262Arg polymorphism of the Cytochrome P450 2B6 (CYP2B6) gene could act as a genetic marker for prostate cancer risk among Japanese men.
Methods:   A total of 350 patients with sporadic prostate cancer and 328 controls were examined. A single nucleotide polymorphism with non-synonymous amino acid change located at Lys262Arg of the CYP2B6 gene was genotyped using a TaqMan assay.
Results:   The frequency of the Arg/Arg genotype among prostate cancer patients was significantly higher than that among the controls ( P  = 0.027). The frequency of the G allele of the Lys262Arg polymorphism was also significantly higher in prostate cancer patients than in the controls (30.4% vs 24.8%, P  = 0.025). Patients with the Lys/Arg plus Arg/Arg genotypes carried a low Gleason score more frequently than those with the Lys/Lys genotype ( P  = 0.042). The frequency of the G allele of the Lys262Arg polymorphism was significantly higher in the low Gleason score group than that in the high Gleason score group (34.3% vs 26.8%, P  = 0.038).
Conclusions:   Lys262Arg polymorphism of the CYP2B6 gene may be a genetic marker for evaluating the risk of sporadic prostate cancer in native Japanese men.     

Cytochrome P4502B6 and 2C9 do not metabolize midazolam: kinetic analysis and inhibition study with monoclonal antibodies

We determined the contribution of cytochrome P450 (CYP) isoformsto the metabolism of midazolam by kinetic analysis of humanliver microsomes and CYP isoforms and by examining the effectof chemical inhibitors and monoclonal antibodies against CYPisoforms in vitro. Midazolam was metabolized to 1'-hydroxymidazolam(1'-OH MDZ) by human liver microsomes with a Michaelis–Mentenconstant (K_m) of 4.1 (1.0) (mean (SD)) µmol litre^–1and a maximum rate of metabolism (V_max) of 5.5 (1.1) nmol min^–1mg protein^–1 (n=6). Of the nine representative human liverCYP isoforms, CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and3A5, three (CYP2B6, 3A4 and 3A5) showed midazolam 1'-hydroxylationactivity, with K_ms of 40.7, 1.7 and 3.0 µmol litre^–1,respectively, and V_max values of 12.0, 3.3 and 13.2 nmol min^–1nmol P450^–1, respectively (n=4). Midazolam 1'-hydroxylationactivity of human liver microsomes correlated significantlywith testosterone 6ß-hydroxylation activity, a markerof CYP3A activity (r²=0.77, P=0.0001), but not with S-mephenytoinN-demethylation activity, a marker of CYP2B6 activity (r²<0.01,P=0.84) (n=11). Troleandomycin and orphenadrine, chemical inhibitorsof CYP isoforms, inhibited the formation of 1'-OH MDZ by humanliver microsomes. Monoclonal antibody against CYP3A4 inhibitedthe formation of 1'-OH MDZ by 79%, whereas monoclonal antibodyagainst CYP2B6 had no effect on midazolam 1'-hydroxylation byhuman liver microsomes (n=5). These results indicate that onlyCYP3A4, but not CYP2B6 or CYP2C, is involved in the metabolismof midazolam in vitro. Br J Anaesth 2001; 86: 540–4     

Expression and inducibility of cytochrome P450 isoforms in 1-year-old intrasplenic liver cell transplants in rats

Syngenic fetal liver tissue suspensions were transplanted into the spleens of 60- to 90-day-old male Fischer 344 inbred rats. Transplant recipients were compared with age-matched control rats. One year after surgery, the animals were treated orally with beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the respective solvents 24 or 48 h before being killed. Expression of cytochrome P450 (P450) isoforms in spleens and orthotopic livers was assessed by immunohistochemistry and P450-dependent monooxygenase functions by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), pentoxyresorufin O-depentylation (PROD) and ethylmorphine N-demethylation (EMND). Spleens of control animals displayed almost no expression of P450 isoforms and P450-mediated monooxygenase functions. Similar to liver, in the transplanted hepatocytes no P450 1A1 but distinct P450 2B1 and 3A2 expression was observed. Furthermore, the transplant-containing spleens displayed significant EROD, ECOD, PROD and EMND activities. Similar to normal liver, BNF treatment enhanced P450 1A1 and 2B1, PB induced P450 2B1 and 3A2, and DEX induced P450 3A2 expression in the transplanted hepatocytes. Correspondingly, in the transplant-containing spleens EROD, ECOD and PROD activities were significantly enhanced following BNF treatment, EROD, ECOD, PROD and EMND activities after PB administration, and EMND activity by DEX treatment. These results demonstrate that hepatocytes originating from fetal liver tissue suspensions can survive in the spleen at least for 1 year. They have differentiated into adult hepatocytes and even 1 year after transplantation express different P450 isoforms which are inducible by BNF, PB and DEX, corresponding to normal adult liver.     

Clinical versatility of porcine hepatocytes in the light of interspecies differences in cytochrome P450 regulation and expression

Abstract: Background and aims: In fulminant hepatic failure, the clinical use of bioartifical liver support with porcine hepatocytes is the subject of a controversial debate. Cytochrome P450 (CYP) metabolic functions have relevant implications for drug metabolism and detoxification. In this study, we investigate interspecies differences in CYP gene expression between human and porcine primary hepatocytes and the impact of interleukin 6 (IL‐6) and tumor necrosis factor alpha (TNF‐α) exposition mimicking cytokine release in fulminant hepatic failure. Methods: Primary hepatocyte cultures were isolated from human resection specimens and from German landrace pigs. Cell cultures (single and co‐cultures) were exposed to porcine vs. human IL‐6 and TNF‐α, respectively. Changes in quantitative CYP gene expression were investigated by semi‐quantitative RT‐PCR. Results: Significant differences in species‐specific CYP gene expression by human and porcine hepatocytes were found after exposure to species‐identical IL‐6 (10 ng/ml) for CYP 1A1, CYP 2C, CYP 3A (P = 0.002, 0.022, 0.017, respectively) or species‐identical TNF‐α (30ng/ml) for CYP 1A2 and CYP 2A (P = 0.037, 0.023, respectively). In single vs. co‐culture, human hepatocytes demonstrated stronger repression of CYP 1A1, 2C8 and 3A4 expression after dosage with human IL‐6 (10ng/ml) (P = 0.022, 0.031, 0.014, respectively). Conclusion: Our findings demonstrate significant species‐specific differences in CYP gene expression and regulation when high doses of IL‐6 and TNF‐α are used (10 and 30 ng/ml, respectively). These findings may point to species‐specific physiological incompatibilities of porcine hepatocytes and thus limit their clinical versatility.     

Renal matrix metalloproteinase activity is unaffected by experimental ischemia-reperfusion injury and matrix metalloproteinase inhibition does not alter outcome of renal function

BACKGROUND: Ischemia-reperfusion injury can lead to organ damage, such as delayed graft function in kidney transplantation. Reactive oxygen species that play a key role in this disorder may directly activate latent matrix metalloproteinases (MMP). In the kidney, little is known about the role of MMP in ischemia-reperfusion. Therefore, the aim of our study was to analyze activity/expression of MMP and to assess their functional role by the use of the MMP inhibitor BB-94 (Batimastat). METHODS: Renal ischemia was induced by left renal pedicle occlusion for 60 min, preceded by right nephrectomy. Thirty-two female Sprague-Dawley rats were analyzed: sham-operated rats (n = 8), treated sham-operated rats (n = 4), ischemic rats (n = 12), and treated ischemic rats (n = 8). Batimastat therapy (30 mg/kg body weight/day) was initiated 2 days prior to induction of ischemia. Animals were sacrificed 12 h (n = 8) and 24 h (n = 24) after ischemia for analyses of MMP activity/expression and of plasma creatinine levels. RESULTS: We found no evidence for an alteration in the activity or expression of MMP as a result of renal ischemia-reperfusion. Importantly, plasma creatinine levels significantly increased to a mean of 374 +/- 61 micromol/l in ischemic rats after 24 h, almost identical to the BB-94-treated ischemic rats (384 +/- 36 micromol/l). The creatinine levels in sham-operated rats remained within normal limits. CONCLUSION: MMP play no role during the early phase of experimental renal ischemia-reperfusion injury.     

Propofol metabolism is enhanced after repetitive ketamine administration in rats: the role of cytochrome P-450 2B induction

Background. In a series of ex vivo and in vivo studies we investigatedthe ability of repetitive ketamine administration to alter themetabolism and anaesthetic effect of propofol and the role ofketamine-mediated P-450 2B induction in rats. Methods. Male Wistar rats were pretreated with 80 mg kg^–1ketamine i.p. twice daily for 4 days. Pentoxyresorufin O-dealkylation(PROD), P-450 2B protein and mRNA were determined. Residualpropofol concentration was measured after incubating hepaticmicrosomes with 100 µM propofol. Sleeping times inducedby i.p. 80 mg kg^–1 propofol were determined. Orphenadrine,a P-450 2B inhibitor, was added in both ex vivo and in vivostudies. Finally, serial whole blood propofol concentrationswere determined after i.v. infusion of 15 mg kg^–1 propofol. Results. Ketamine pretreatment produced 5.4-, 3.4- and 1.7-foldincreases in hepatic PROD activity, P-450 2B protein and mRNA,respectively. Residual propofol concentration was 46% lowerafter incubation with microsomes from ketamine-pretreated ratsthan in the control group. The addition of orphenadrine to ketamine-pretreatedmicrosomes produced an increase in residual propofol concentrationin a concentration-dependent manner. Ketamine pretreatment reducedpropofol sleeping time to 12% of the control, which was reversedby orphenadrine. The whole blood propofol concentration in ketamine-pretreatedrats was significantly lower than that of control rats at 1,2, 4 and 8 min after cessation of propofol infusion. Conclusions. Repetitive ketamine administration enhances propofolmetabolism and reduces propofol sleeping time in rats. We suggestthat P-450 2B induction may produce ketamine–propofolinteraction in anaesthetic practice.