CYP2D6 genotype predicts tamoxifen discontinuation and drug response: a secondary analysis of the KARISMA trial.

BACKGROUND: Guidelines regarding whether tamoxifen should be prescribed based on women's cytochrome P450 2D6 (CYP2D6) genotypes are conflicting and have caused confusion. This study aims to investigate if CYP2D6 metabolizer status is associated with tamoxifen-related endocrine symptoms, tamoxifen discontinuation, and mammographic density change. PATIENTS AND METHODS: We used data from 1440 healthy women who participated the KARISMA dose determination trial. Endocrine symptoms were measured using a modified Functional Assessment of Cancer Therapy - Endocrine Symptoms (FACT-ES) questionnaire. Change in mammographic density was measured and used as a proxy for tamoxifen response. Participants were genotyped and categorized as poor, intermediate, normal, or ultrarapid CYP2D6 metabolizers. RESULTS: The median endoxifen level per mg oral tamoxifen among poor, intermediate, normal and ultrarapid CYP2D6 metabolizers were 0.18 ng/ml, 0.38 ng/ml, 0.56 ng/ml and 0.67 ng/ml, respectively. Ultrarapid CYP2D6 metabolizers were more likely than other groups to report a clinically relevant change in cold sweats, hot flash, mood swings, being irritable, as well as the overall modified FACT-ES score, after taking tamoxifen. The 6-month tamoxifen discontinuation rates among poor, intermediate, normal, and ultrarapid CYP2D6 metabolizers were 25.7%, 23.6%, 28.6%, and 44.4%, respectively. Among those who continued and finished the 6-month tamoxifen intervention, the mean change in dense area among poor, intermediate, normal, and ultrarapid CYP2D6 metabolizers were -0.8 cm2, -4.5 cm2, -4.1 cm2, and -8.0 cm2 respectively. CONCLUSIONS: Poor CYP2D6 metabolizers are likely to experience an impaired response to tamoxifen, measured through mammographic density reduction. In contrast, ultrarapid CYP2D6 metabolizers are at risk for exaggerated response with pronounced adverse effects that may lead to treatment discontinuation.

Poor Correlation between Meropenem and Piperacillin Plasma Concentrations and Delivered Dose of Continuous Renal Replacement Therapy.

There is insufficient data on the relationship between antibiotic dosing and plasma concentrations in patients treated with continuous renal replacement therapy (CRRT). In this prospective observational study, we explored the variability in plasma concentrations of meropenem and piperacillin in critically ill patients treated with CRRT and the correlation between concentrations and CRRT intensity. Antibiotic concentrations were measured at the middle and end of the dosing interval and repeated after 2 to 3 days when feasible. Measured concentrations were compared to the clinical susceptible breakpoints for Pseudomonas aeruginosa, 16 and 2 mg/liter for piperacillin and meropenem, respectively. CRRT intensity was estimated by delivered, time-averaged, total effluent flow (Qeff), corrected for predilution. Concentrations were also compared between patients with different residual diuresis. We included 140 meropenem concentrations from 98 patients and 47 piperacillin concentrations from 37 patients. Concentrations at the middle of the dosing interval were above target at all occasions for both antibiotics. For meropenem, 6.5% of trough concentrations were below target, and for piperacillin, 22%. Correlations between Qeff and antibiotic concentrations or the concentration half-life (t1/2) were either statistically not significant or weak. Meropenem concentrations and t1/2 values differed between patients with different residual diuresis. Thus, when treating intensive care patients with CRRT and recommended doses of meropenem or piperacillin, both low, suboptimal plasma concentrations and unnecessarily high, potentially toxic, plasma concentrations are common. Plasma concentrations cannot be predicted from CRRT intensity. Residual diuresis is associated with lower meropenem concentrations, but the correlation is weak. Concentration measurement is probably the most useful approach to avoid suboptimal treatment.

Pharmacokinetics of Caspofungin in Critically Ill Patients in Relation to Liver Dysfunction: Differential Impact of Plasma Albumin and Bilirubin Levels.

Caspofungin has a liver-dependent metabolism. Reduction of the dose is recommended based on Child-Pugh (C-P) score. In critically ill patients, drug pharmacokinetics (PK) may be altered. The aim of this study was to investigate the prevalence of abnormal liver function tests, increased C-P scores, their effects on caspofungin PK, and whether pharmacokinetic-pharmacodynamic (PK/PD) targets were attained in patients with suspected candidiasis. Intensive care unit patients receiving caspofungin were prospectively included. PK parameters were determined on days 2, 5, and 10, and their correlations to the individual liver function tests and the C-P score were analyzed. Forty-six patients were included with C-P class A (n = 5), B (n = 40), and C (n = 1). On day 5 (steady state), the median and interquartile range for area under the curve from 0 to 24 h (AUC0-24), clearance (CL), and central volume of distribution (V1) were 57.8 (51.6 to 69.8) mg·h/liter, 0.88 (0.78 to 1.04) liters/h, and 11.9 (9.6 to 13.1) liters, respectively. The C-P score did not correlate with AUC0-24 (r = 0.03; P = 0.84), CL (r = -0.07; P = 0.68), or V1 (r = 0.19; P = 0.26), but there was a bilirubin-driven negative correlation with the elimination rate constant (r = -0.46; P = 0.004). Hypoalbuminemia correlated with low AUC0-24 (r = 0.45; P = 0.005) and was associated with higher clearance (r = -0.31; P = 0.062) and somewhat higher V1 (r = -0.15; P = 0.37), resulting in a negative correlation with the elimination rate constant (r = -0.34; P = 0.042). For Candida strains with minimal inhibitory concentrations of ≥0.064 µg/ml, PK/PD targets were not attained in all patients. The caspofungin dose should not be reduced in critically ill patients in the absence of cirrhosis, and we advise against the use of the C-P score in patients with trauma- or sepsis-induced liver injury.

Standard dosing of piperacillin and meropenem fail to achieve adequate plasma concentrations in ICU patients.

BACKGROUND: Controversies remain regarding optimal dosing and the need for plasma concentration measurements when treating intensive care patients with beta-lactam antibiotics. METHODS: We studied ICU patients treated with either antibiotic, excluding patients on renal replacement therapy. Antibiotic concentrations were measured at the mid and end of the dosing interval, and repeated after 2-3 days when feasible. Glomerular filtration rate (GFR) was estimated from plasma creatinine and cystatin C, GFR calculated from cystatin C (eGFR) and measured creatinine clearance (CrCl). Measured concentrations were compared to the clinical susceptible breakpoints for Pseudomonas aeruginosa, 16 and 2 mg/l for piperacillin and meropenem respectively. RESULTS: We analysed 33 and 31 paired samples from 20 and 19 patients treated with piperacillin-tazobactam and meropenem respectively. Antibiotic concentrations at the mid and end of the dosing interval were for piperacillin, 27.0 (14.7-52.9) and 8.6 (2.7-30.3); and for meropenem, 7.5 (4.7-10.2) and 2.4 (1.0-3.5). All values median (interquartile range) and concentrations in mg/l. The percentage of measured concentrations below the breakpoint at the mid and end of the dosing interval were for piperacillin, 27% and 61%; and for meropenem, 6% and 48%. Lower estimates of GFR were associated with higher concentrations but concentrations varied greatly between patients with similar GFR. The correlation with terminal concentration half-life was similar for eGFR and CrCl. CONCLUSIONS: With standard doses of meropenem and piperacillin-tazobactam, plasma concentrations in ICU patients vary > 10-fold and are suboptimal in a significant percentage of patients. The variation is large also between patients with similar renal function.

UGT1A4*3 encodes significantly increased glucuronidation of olanzapine in patients on maintenance treatment and in recombinant systems.

Olanzapine, a world leader in antipsychotic drugs, is used in the treatment of schizophrenia and bipolar disorder. There is considerable interpatient variability in its hepatic clearance. Polymorphic glucuronidation of olanzapine by uridine diphosphate glucuronosyltransferase 1A4 (UGT1A4) was investigated retrospectively in patient samples taken for routine therapeutic drug monitoring (TDM) and in recombinant metabolic systems in vitro. Multivariate analyses revealed that patients who were heterozygous as well as those who were homozygous for the UGT1A4*3 allelic variant had significantly higher concentrations of the major metabolite olanzapine 10-N-glucuronide in serum (+38% (P = 0.011) and +246% (P < 0.001), respectively). This finding was in line with the significant increases in glucuronidation activity of olanzapine observed with recombinant UGT1A4.3 (Val-48) as compared with UGT1A4.1 (Leu-48) (1.3-fold difference, P < 0.001). By contrast, serum concentrations of the parent drug were not significantly influenced by UGT1A4 genotype. Our findings therefore indicate that UGT1A4-mediated metabolism is not a major contributor to interpatient variability in olanzapine levels. However, with respect to other drugs for which UGT1A4 has a dominant role in clearance, increased glucuronidation encoded by UGT1A4*3 might impact the risk for subtherapeutic drug exposure.

Drug-drug interactions that reduce the formation of pharmacologically active metabolites: a poorly understood problem in clinical practice.

Drug-drug interactions can lead to reduced efficacy of medical treatment. Therapeutic failure may for instance result from combined treatment with an inhibitor of the specific pathway that is responsible for the generation of pharmacologically active drug metabolites. This problem may be overlooked in clinical practice. Several examples of drugs will be discussed -clopidogrel, losartan, tamoxifen and codeine - to illustrate differences in the potential impact on drug treatment in clinical practice. We conclude that the combined use of cytochrome P450-blocking serotonin reuptake inhibitors and tamoxifen or codeine should be avoided, whereas the situation is much more complex regarding the use of proton pump inhibitors together with clopidogrel, and the evidence regarding cytochrome P450 inhibitor-dependent activation of losartan is inconclusive.

Clinically significant CYP2C inhibition by noscapine but not by glucosamine.

Noscapine and glucosamine reportedly interact with warfarin. We investigated the effects of these drugs on various cytochrome P450 (CYP) activity markers. Twelve healthy subjects were phenotyped at baseline and during separate treatments with noscapine and glucosamine. Whereas glucosamine had no significant effect on CYP activity, noscapine caused marked inhibition of CYP2C9 (4.9-fold increase in urinary losartan/E3174 ratio) and CYP2C19 (3.6-fold increase in the plasma omeprazole/5-hydroxyomeprazole ratio). Noscapine-dependent inhibition of CYP2C9 may explain the interaction with warfarin.

The convergence of conventional therapeutic drug monitoring and pharmacogenetic testing in personalized medicine: focus on antidepressants.

The development and prospects of conventional therapeutic drug monitoring (TDM) and pharmacogenetic testing as aids in personalized treatment with antidepressants and antipsychotics are described. Our own experience is discussed in relation to international guidelines for rational TDM. Emphasis is put on the usefulness of TDM combined with genotyping of cytochrome P450 2D6 (CYP2D6), the key enzyme involved in the polymorphic metabolism of the majority of antidepressants (both tricyclics and selective serotonin reuptake inhibitors) and antipsychotic drugs. This combination of methods is particularly useful in verifying concentration-dependent adverse drug reactions (ADRs) due to poor metabolism, 'and diagnosing pharmacokinetic reasons (ultrarapid metabolism (UM)) for drug failure. This is because ADRs may mimic the psychiatric illness itself and therapeutic failure due to UM may be mistaken for poor compliance with the prescription.

Role of CYP2C9 polymorphism in losartan oxidation.

Losartan, an angiotensin II receptor antagonist, is oxidized by hepatic cytochromes P450 to an active carboxylic acid metabolite, E-3174. The aim of the present investigation was to study the contribution of CYP2C9 and CYP3A4 in losartan oxidation in vitro and to evaluate the role of CYP2C9 polymorphism. Kinetic properties of different genetic CYP2C9 variants were compared both in a yeast expression system and in 25 different samples of human liver microsomes where all known genotypes of CYP2C9 were represented. Microsomes were incubated with losartan (0.05-50 microM), and the formation of E-3174 was analyzed by high-performance liquid chromatography to estimate V(max), K(m), and intrinsic clearance for all individual samples. Sulfaphenazole, a CYP2C9 inhibitor, blocked the formation of E-3174 at low losartan concentrations (<1 microM), whereas the inhibitory effect of triacetyloleandomycin, a CYP3A4 inhibitor, was significant only at high concentrations of losartan (>25 microM). In comparison to the CYP2C9.1 variant, oxidation of losartan was significantly reduced in yeast expressing the rare CYP2C9.2 or CYP2C9.3 variants. Moreover, the rate of losartan oxidation was lower in liver microsomes from individuals hetero- or homozygous for the CYP2C9*3 allele, or homozygous for the CYP2C9*2 allele. The difference between the common and rare CYP2C9 variants was mainly explained by a lower V(max), both in yeast and human liver microsomes. In summary, these in vitro results indicate that CYP2C9 is the major human P450 isoenzyme responsible for losartan oxidation and that the CYP2C9 genotype contributes to interindividual differences in losartan oxidation and activation.

The role of CYP2C9 genotype in the metabolism of diclofenac in vivo and in vitro.

INTRODUCTION: The polymorphic cytochrome P450 enzyme 2C9 (CYP2C9) catalyses the metabolism of many drugs including S-warfarin, acenocoumarol, phenytoin, tolbutamide, losartan and most of the non-steroidal anti-inflammatory drugs. Diclofenac is metabolised to 4'-hydroxy (OH), the major diclofenac metabolite, 3'-OH and 3'-OH-4'-methoxy metabolites by CYP2C9. The aim of the present study was to clarify the impact of the CYP2C9 polymorphism on the metabolism of diclofenac both in vivo and in vitro. SUBJECTS, MATERIALS AND METHODS: Twenty healthy volunteers with different CYP2C9 genotypes [i.e. CYP2C9*1/ *1 (n = 6), *1/*2 (n = 3), *1,/*3 (n = 5), *2/*3 (n = 4), *21*2 (n = 1), *31*3 (n = 1)] received a single 50-mg oral dose of diclofenac. Plasma pharmacokinetics [peak plasma concentration (Cmax), half-life (t 1/2) and area under the plasma concentration-time curve (AUCtotal)] and urinary recovery of diclofenac and its metabolites were compared between the genotypes. Diclofenac 4'-hydroxylation was also analysed in vitro in 16 different samples of genotyped [i.e. CYP2C9*1/*1 (n = 7), *1/*2 (n=2), *1/*3 (n = 2), *2/*3 (n = 2), *2/*2 (n = 2), *31/*3 (n = 1)] human liver microsomes. RESULTS: Within each genotype group, a high variability was observed in kinetic parameters for diclofenac and 4'-OH-diclofenac (6- and 20-fold, respectively). No significant differences were found between the different genotypes either in vivo or in human liver microsomes. No correlation was found between the plasma AUC ratio of diclofenac/4'-OH-diclofenac and that of losartan/ E-3174, previously determined in the same subjects. CONCLUSION: No relationship was found between the CYP2C9 genotype and the 4'-hydroxylation of diclofenac either in vivo or in vitro. This, together with the lack of correlation between losartan oxidation and diclofenac hydroxylation in vivo raises the question about the usefulness of diclofenac as a CYP2C9 probe.

Validation of methods for CYP2C9 genotyping: frequencies of mutant alleles in a Swedish population.

Cytochrome P450 2C9 (CYP2C9) catalysis the metabolism of important drugs such as phenytoin, S-warfarin, tolbutamide, losartan, torasemide, and nonsteroidal anti-inflammatory drugs. A functional polymorphism of the CYP2C9 gene has been described. The variant alleles include CYP2C9*2 having a point mutation in exon 3 causing an Arg144Cys exchange, and CYP2C9*3 with a point mutation in exon 7 resulting in an Ile359Leu exchange. Genotyping of these variant forms was carried out in 430 Swedish healthy volunteers and three different methods were compared. Sequence analysis of the different PCR products revealed that other genes in the CYP2C locus were co-amplified in one of the methods applied, whereas the other two methods were specific for CYP2C9. The frequencies of the CYP2C9*1, CYP2C9*2 and CYP2C9*3 alleles in the population examined were found to be 0.819, 0.107, and 0.074, respectively. The need for careful evaluation of the genotyping procedure by sequence analysis of PCR products is emphasised.

Amyloid fibril formation by pulmonary surfactant protein C.

Lung surfactant protein C (SP-C) is a lipopeptide that contains two fatty acyl (palmitoyl) chains bound via intrinsically labile thioester bonds. SP-C can transform from a monomeric alpha-helix into beta-sheet aggregates, reminiscent of structural changes that are supposed to occur in amyloid fibril formation. SP-C is here shown to form amyloid upon incubation in solution. Furthermore, one patient with pulmonary alveolar proteinosis (PAP, a rare disease where lung surfactant proteins and lipids accumulate in the airspaces) and six healthy controls have been studied regarding presence and composition of amyloid fibrils in the cell-free fraction of bronchoalveolar lavage (BAL) fluid. Abundant amyloid fibrils were found in BAL fluid from the patient with PAP and, in low amounts, in three of the six healthy controls. SDS-insoluble fibrillar material associated with PAP mainly consists of SP-C, in contrast to the fibrils found in controls. Fibrillated SP-C has to a significant extent lost the palmitoyl groups, and removal of the palmitoyl groups in vitro increases the rate of fibril formation.

Expression of autoantibodies to specific cytochromes P450 in a case of disulfiram hepatitis.

BACKGROUND/AIMS: Immunological mechanisms are involved in many adverse drug reactions. In certain forms of drug-induced hepatitis, patients have been reported to express specific autoantibodies to hepatic drug-metabolising enzymes. The alcohol deterrent disulfiram is associated with a low frequency of severe liver toxicity, including hepatitis, but the mechanism of the toxicity is unknown. We investigated whether autoantibodies to cytochrome P450 enzymes were expressed in the serum of a 28-year-old male patient, who developed hepatitis after 7 weeks of disulfiram treatment and in whom possible causes of hepatitis other than disulfiram had been ruled out. METHODS: Patient serum IgG reactivity was analysed by immunoblotting or ELISA against test antigens consisting of recombinant/purified human or rat liver P450 enzymes, or isolated rat liver microsomes. RESULTS: A significant serum reactivity was found in immunoblotting against human cytochromes P450 1A2 and rat P450 3A1, using serum dilutions of up to 1:900 and 1:2400, respectively. In contrast, the reactivity against cytochromes P450 2E1, 2C9, 2D6, 3A4, and rat liver P450 reductase was either very low or undetectable. ELISA reactivity was low in general, indicating that the P450 epitopes were not surface exposed. Immunoblotting of rat liver microsomes revealed that autoantibodies recognised one major polypeptide corresponding to P450 3A. Autoantibody titres remained stable for at least 6 months after acute hepatitis. A similar reactivity was not found in any of ten control sera. CONCLUSIONS: The expression of autoantibodies directed against specific cytochromes P450 in a case of disulfiram hepatitis suggests that immunological mechanisms are involved in this adverse drug reaction, and that these P450 proteins should be evaluated as possible diagnostic test antigens in disulfiram hepatotoxicity.

Immunochemical identification of hepatic protein adducts derived from estragole.

Hepatic protein adducts derived from the allylbenzene food flavor estragole, which is hepatocarcinogenic when given to rodents at high doses, have been identified using immunochemical approaches. Male Fischer 344 rats were given estragole orally and hepatic protein adducts were detected by immunoblotting, using antisera raised by immunizing rabbits with 4-methoxycinnamic acid-modified rabbit serum albumin. A major 155-kDa adduct was expressed in livers of animals that had been treated with estragole at 100, 300, or 500 mg/kg. Levels of expression of the adduct increased disproportionately with respect to dose, and other adducts (170, 100, 44, and 35 kDa) were detected also in the high-dose group. Rats given estragole for 5 days, at 300 mg/kg/day, expressed predominantly 155- and 44-kDa adducts. The 155-, 100-, 44-, and 35-kDa adducts were detected in greatest abundance in liver microsomal fractions, while the 170-kDa adduct was most abundant in the nuclear fraction. Interestingly, whereas the 170-, 155-, 100-, and 35-kDa adducts were detected in cytosolic fractions, relatively low levels of the 44-kDa adduct were detected in nuclear fractions but not in cytosolic fractions. The various adducts were solubilized when microsomal fractions were extracted with sodium carbonate and were digested by trypsin. This implies that the target proteins are peripheral membrane proteins bound to the outer surface of microsomal membranes. Experiments undertaken with isolated rat hepatocytes and with V79 cells transfected with human monoamine phenol sulfotransferase cDNA revealed that adduct formation required 1'-hydroxylation of estragole, followed by sulfation. The pattern of adducts expressed when the transfected V79 cells were incubated with 1'-hydroxyestragole was very similar to that expressed in livers of estragole-treated rats. These cells should constitute a valuable in vitro model system for investigation of toxicological consequences arising from estragole-induced protein adduct formation.

Interindividual variability in P450-dependent generation of neoantigens in halothane hepatitis.

Halothane hepatitis occurs because susceptible patients mount immune responses to trifluoroacetylated protein antigens, formed following cytochrome P450-mediated bioactivation of halothane to trifluoroacetyl chloride. In the present study, an in vitro approach has been used to investigate the cytochrome P450 isozyme(s) which catalyze neoantigen formation and to explore the protective role of non-protein thiols (cysteine and reduced glutathione). Significant levels of trifluoroacetyl protein antigens were generated when human liver microsomes, and also microsomes from livers of rats pre-treated with isoniazid, phenobarbital or beta-naphtoflavone, were incubated with halothane plus a nicotinamide adenine dinucleotidephosphate (NADPH) generating system. Immunoblotting studies revealed that the major trifluoroacetyl antigens expressed in vitro exhibited molecular masses of 50-55 kDa and included 60 and 80 kDa neoantigens recognized by antibodies from patients with halothane hepatitis. Much lower concentrations of halothane were required to produce maximal antigen generation in isoniazid-induced rat microsomes, as compared with phenobarbital or isosafrole-induced microsomes (0.5 vs 12.5 microl/ml). In isoniazid-induced microsomes, antigen generation was inhibited > 90% by the nucleophiles cysteine and glutathione and by the CYP2E1-selective inhibitors diallylsulfide and p-nitrophenol, but was unaffected by inhibitors of other P450 isozymes (furafylline, sulfaphenazole or triacetyloleandomycin). Neoantigen formation in six human liver microsomal preparations was inhibited in the presence of diallylsulfide, but not by furafylline, sulfaphenazole or triacetyloleandomycin, and exhibited marked variability which correlated with CYP2E1 levels. These results suggest that the balance between metabolic bioactivation by CYP2E1 and detoxication of reactive metabolites by cellular nucleophiles could be an important metabolic risk factor in halothane hepatitis.

Enzyme-specific transport of rat liver cytochrome P450 to the Golgi apparatus.

It has been reported that cytochrome P450 is expressed in the plasma membrane of hepatocytes isolated from human and rat. Cytochrome P450s expressed on the cell surface are potential targets for the immune response of drug-induced and autoimmune hepatitis. However, the mechanisms behind transport of cytochrome P450 to the plasma membrane are obscure. The present investigation aimed at identifying cytochrome P450 expressed in the Golgi apparatus. Golgi membrane fractions from rat liver were prepared and characterized: one enriched with cis-Golgi, one highly enriched with trans-Golgi, and one intermediate Golgi fraction representing medial-Golgi. In these three fractions, significant amounts of cytochrome P450 and NADPH cytochrome P450 reductase were present, which could not be accounted for by contamination with endoplasmic reticulum. A marked difference between the relative content of different cytochrome P450 enzymes was found. CYP4A1 was found at the highest concentration, CYP2E1 at an intermediary level, and CYP1A2 at low levels, whereas no Golgi-specific CYP3A1 was detectable. It was also shown that the CYP2E1 present in the Golgi fractions was catalytically active. It is suggested that various forms of hepatic cytochrome P450 are transported to the plasma membrane through the Golgi apparatus in an enzyme-specific manner.

Cytochrome P450 2E1 is a cell surface autoantigen in halothane hepatitis.

Recent studies have shown that cytochrome P450 2E1 (CYP2E1) is a major catalyst of formation of trifluoroacetylated proteins, which have been implicated as target antigens in the mechanism of halothane hepatitis. In the present investigation, trifluoroacetylated CYP2E1 was detected immunochemically in livers of rats treated with halothane. Furthermore, high levels of autoantibodies that recognized purified rat CYP2E1 but not purified rat CYP3A were detected by enzyme-linked immunosorbent assay in 14 of 20 (70%) sera from patients with halothane hepatitis. Only very low levels of such antibodies were detected in sera from healthy controls, from patients anesthetized with halothane without developing hepatitis, or from patients with other liver diseases. The intracellular distribution of CF3CO-adducts was studied in highly differentiated FGC4 rat hepatoma cell cultures. High levels of adducts were found after 22-hr culture in the presence of halothane, and their generation was dependent on the expression of CYP2E1. Adducts were predominantly located in the endoplasmic reticulum but also, to a minor extent, on the cell surface, as detected by immunofluorescence. A very similar distribution was found for CYP2E1 in FGC4 cells, and immunoprecipitation experiments performed in cultures of FGC4-related Fao hepatoma cells suggest that surface immunoreactivity originates from a small fraction of intact CYP2E1 apoprotein. Human CYP2E1, expressed in V79 cells after cDNA transfection, was also detected to a minor extent in the plasma membrane, whereas no immunofluorescence was evident in parental V79 cells. It is suggested that immune responses to cell surface CYP2E1 could be involved in the pathogenesis of halothane hepatitis.

Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics.

BACKGROUND & AIMS: We have previously reported that alcoholics have increased titers of immunoglobulins reacting with protein adducts of hydroxyethyl free radicals. Because hydroxyethyl radicals are produced during ethanol metabolism by liver microsomes, the aim of this study was to determine whether such antibodies recognize microsomal proteins complexed with hydroxyethyl radicals. METHODS: Liver microsomal proteins reacting with the anti-hydroxyethyl radical antibodies were characterized by an enzyme-linked immunosorbent assay and Western blotting. RESULTS: Alcoholic cirrhotics, but not patients with nonalcoholic cirrhosis or healthy subjects, had increased serum levels of immunoglobulin G and A directed against antigens produced in microsomes incubated with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and ethanol. Such immunoreactivity was completely blocked when microsomes were incubated with ethanol in the presence of the spin-trapping agent 4-pyridyl-1-oxide-t-butyl nitrone or by preincubating the sera with hydroxyethyl radical-bound human albumin. Immunoblotting of proteins from human liver microsomes incubated with NADPH and ethanol showed that 86% of the sera from alcoholic cirrhotics reacted with a 52-kilodalton protein, whereas variable reactivity was observed with proteins of 78, 60, and 40 kilodaltons, respectively, The 52-kilodalton protein was identified by immunoblotting and immunoprecipitation as ethanol-inducible cytochrome P4502E1. CONCLUSIONS: Antibodies from alcoholic cirrhotics specifically recognized hydroxyethyl radical-cytochrome P4502E1 adducts, suggesting the possible implication of these antigens in the development of autoimmune reactions in alcoholic liver disease.

Stable expression of human cytochrome P450 2E1 in V79 Chinese hamster cells.

A V79 Chinese hamster cell line was constructed for stable expression of human cytochrome P450 2E1 (CYP2E1) by integration of a SV40 Early promoter recombinant CYP2E1 cDNA into the chromosomal DNA. The cDNA encoded CYP2E1 was effectively expressed and enzymatically active, as shown by hydroxylation of chlorzoxazone and of p-nitrophenol, at rates of about 70 pmol x mg-1 total protein x min-1. CYP2E1 content and activity was increased upon cultivation in the presence of ethanol indicating a substrate mediated stabilization effect. A similar stabilizing effect was also observed for inhibitors of CYP2E1, e.g. imidazole, 4-methylpyrazole, and isoniazid. The feasibility of the newly established cell line V79MZh2E1 for toxicological studies was shown by CYP2E1-mediated activation of N-nitrosodimethylamine and p-nitrophenol and a dose-dependent cytotoxic and mutagenic effect.