Proteomic profiling of rectal cancer reveals acid ceramidase is implicated in radiation response.

BACKGROUND: Neoadjuvant chemoradiotherapy (CRT) is used in locally advanced rectal cancer when tumours threaten the circumferential resection margin, with varying response to treatment. This experimental study aimed to identify significantly differentially expressed proteins between patients responding and not responding to CRT, and to validate any proteins of interest. METHODS: Mass spectrometry (with isobaric tagging for relative quantification) analysis of rectal cancers pre- and post-CRT, and at resection. Validation of proteins of interest was performed by assessing tissue microarray (TMA) immunohistochemistry expression in a further 111 patients with rectal cancer. RESULTS: Proteomic data are available via ProteomeXchange with identifier PXD008436. Reduced abundance of contributing peptide ions for acid ceramidase (AC) (log fold change -1.526, p = 1.17E-02) was observed in CRT responders. Differential expression of AC was confirmed upon analysis of the TMAs. Cancer site expression of AC in stromal cells from post-CRT resection specimens was observed to be relatively low in pathological complete response (p = 0.003), and relatively high with no response to CRT (p = 0.017). CONCLUSION: AC may be implicated in the response of rectal cancer to CRT. We propose its further assessment as a novel potential biomarker and therapeutic target. SIGNIFICANCE: There is a need for biomarkers to guide the use of chemoradiotherapy in rectal cancer, as none are in routine clinical use. We have determined acid ceramidase may have a role in radiation response, based on novel proteomic profiling and validation in a wider dataset using tissue microarrays. The ability to predict or improve response would positively select those patients who will derive benefit, prevent delays in the local and systemic management of disease in non-responders, and reduce morbidity associated with chemoradiotherapy.

Exome sequencing of synchronously resected primary colorectal tumours and colorectal liver metastases to inform oncosurgical management.

BACKGROUND: Next generation sequencing technology has facilitated mapping of the colorectal cancer genotype and furthered our understanding of metastogenesis. The aim of this study was to investigate for conserved and different mutations in the exomes of synchronously resected primary colorectal tumour and liver metastases. This information could potentially be utilised to guide the treatment of advanced disease with the help of biological information from the primary tumour. METHODS: We performed exome sequencing of synchronously resected primary colorectal cancer and colorectal liver metastases as well as normal colonic mucosa and liver parenchyma, from four patients who had received neo-adjuvant chemotherapy, at a depth of 50X using the Ion Proton platform. Raw data was mapped to the reference genome prior to variant calling, annotation and downstream analysis. RESULTS: Exome sequencing identified 585 non-synonymous missense single nucleotide variants (SNVs), of which 215 (36.8%) were unique to the primary tumour, 226 (38.6%) unique to the metastasis and 81 (13.8%) present in patient matched pairs. SNVs identified in the ErbB pathway appear to be concordant between primary and metastatic tumours. CONCLUSION: Only 13.8% of the metastatic exome can be predicted by the genotype of the primary tumour. We have demonstrated concordance of a number of SNVs in the ErbB pathway, which may inform selection of therapeutic agents in advanced colorectal cancer.

Adaptation to acetaminophen exposure elicits major changes in expression and distribution of the hepatic proteome.

Acetaminophen overdose is the leading cause of acute liver failure. One dose of 10-15 g causes severe liver damage in humans, whereas repeated exposure to acetaminophen in humans and animal models results in autoprotection. Insight of this process is limited to select proteins implicated in acetaminophen toxicity and cellular defence. Here we investigate hepatic adaptation to acetaminophen toxicity from a whole proteome perspective, using quantitative mass spectrometry. In a rat model, we show the response to acetaminophen involves the expression of 30% of all proteins detected in the liver. Genetic ablation of a master regulator of cellular defence, NFE2L2, has little effect, suggesting redundancy in the regulation of adaptation. We show that adaptation to acetaminophen has a spatial component, involving a shift in regionalisation of CYP2E1, which may prevent toxicity thresholds being reached. These data reveal unexpected complexity and dynamic behaviour in the biological response to drug-induced liver injury.

Evaluation of a novel tissue stabilization gel to facilitate clinical sampling for translational research in surgical trials.

BACKGROUND: The aim was to establish the feasibility of using a tissue stabilization gel (Allprotect™) as an alternative to liquid nitrogen to facilitate collection of clinical samples for translational research. METHODS: Tumour samples from patients undergoing surgery for primary or metastatic colorectal cancer were either snap-frozen in liquid nitrogen or stored in Allprotect™ under a number of different conditions. Sample integrity was compared across different storage conditions by assessing biomolecule stability and function. DNA quality was assessed spectrophotometrically and by KRas genotyping by pyrosequencing. Total RNA retrieval was determined by nanodrop indices/RNA integrity numbers, and quality assessed by reverse transcription-PCR for two representative genes (high-mobility group box 1, HMGB1; carboxylesterase 1, CES1) and two microRNAs (miR122 and let7d). Western blot analysis of HMGB1 and CES1 was used to confirm protein expression, and the metabolic conversion of irinotecan to its active metabolite, SN-38, was used to assess function. RESULTS: Under short-term storage conditions (up to 1 week) there was no apparent difference in quality between samples stored in Allprotect™ and those snap-frozen in liquid nitrogen. Some RNA degradation became apparent in tissue archived in Allprotect™ after 1 week, and protein degradation after 2 weeks. CONCLUSION: In hospitals that do not have access to liquid nitrogen and -80°C freezers, Allprotect™ provides a suitable alternative for the acquisition and stabilization of clinical samples. Storage proved satisfactory for up to 1 week, allowing transfer of samples without the need for specialized facilities. Surgical relevance Access to clinical material is a fundamental component of translational research that requires significant infrastructure (research personnel, liquid nitrogen, specialized storage facilities). The aim was to evaluate a new-to-market tissue stabilization gel (Allprotect™), which offers a simple solution to tissue preservation without the need for complex infrastructure. Allprotect™ offers comparable DNA, RNA and protein stabilization to tissue snap-frozen in liquid nitrogen for up to 1 week. Degradation of biomolecules beyond this highlights its role as a short-term tissue preservative. Allprotect™ has the potential to increase surgeon participation in translational research and surgical trials requiring tissue collection.

Hepatic activation of irinotecan predicts tumour response in patients with colorectal liver metastases treated with DEBIRI: exploratory findings from a phase II study.

PURPOSE: The response of colorectal liver metastases to the cytotoxic agent irinotecan varies widely. Attempts to correlate tumour metabolism with response have been mixed. This study investigated the hepatic metabolism of irinotecan as a potential predictor of tumour response to irinotecan-eluting beads (DEBIRI). METHODS: Ten patients with colorectal liver metastases were treated with 200 mg irinotecan (as DEBIRI) as part of the PARAGON II study. Hepatic expression of key metabolising enzymes was measured using mass spectrometry-based proteomics. Serum drug concentrations and hepatic irinotecan metabolism were characterised and correlated with tumour response. RESULTS: Serum concentrations of irinotecan metabolites did not correlate with hepatic metabolism or pathological response. There was a strong correlation between hepatic CES-2 expression and activation of irinotecan (r (2) = 0.96, p < 0.001). Patients with a UGT1A1*28 6/7 SNP showed no difference in drug metabolism or pathological response. Hepatic CES-2 mediated activation of irinotecan clearly correlated with tumour replacement by fibrosis (r (2) = 0.54, p = 0.01). CONCLUSION: This study provides the first evidence that hepatic activation of irinotecan predicts tumour response. Delivery of liver-targeted irinotecan to normal liver tissue rather than tumour may be a more rational approach to maximise response.

iTRAQ reveals candidate pancreatic cancer serum biomarkers: influence of obstructive jaundice on their performance.

BACKGROUND: The aims of our study were to identify serum biomarkers that distinguish pancreatic cancer (pancreatic ductal adenocarcinoma, PDAC) patients from benign pancreatic disease patients and healthy subjects, and to assess the effects of jaundice on biomarker performance. METHODS: Isobaric tags for relative and absolute quantification were used to compare pooled serum and pancreatic juice samples from a test set of 59 and 25 subjects, respectively. Validation was undertaken in 113 independent subjects. RESULTS: Candidate proteins Complement C5, inter-α-trypsin inhibitor heavy chain H3, α1-ß glycoprotein and polymeric immunoglobulin receptor were elevated in cancer, as were the reference markers CA19-9 and Reg3A. Biliary obstruction had a significant effect on the performance of the markers, in particular within the PDAC group where the presence of jaundice was associated with a significant increase in the levels of all six proteins (P<0.01). Consequently, in the absence of jaundice, proteins showed reduced sensitivity for PDAC patients over benign subjects and healthy controls (HCs). Similarly, in the presence of jaundice, markers showed reduced specificity for PDAC patients over benign subjects with jaundice. Combining markers enabled improved sensitivity for non-jaundiced PDAC patients over HCs and improved specificity for jaundiced PDAC patients over jaundiced benign disease subjects. CONCLUSIONS: The presence-absence of jaundice in the clinical scenario severely impacts the performance of biomarkers for PDAC diagnosis and has implications for their clinical translation.

Human pluripotent stem cells for modeling toxicity.

The development of xenobiotics, driven by the demand for therapeutic, domestic and industrial uses continues to grow. However, along with this increasing demand is the risk of xenobiotic-induced toxicity. Currently, safety screening of xenobiotics uses a plethora of animal and in vitro model systems which have over the decades proven useful during compound development and for application in mechanistic studies of xenobiotic-induced toxicity. However, these assessments have proven to be animal-intensive and costly. More importantly, the prevalence of xenobiotic-induced toxicity is still significantly high, causing patient morbidity and mortality, and a costly impediment during drug development. This suggests that the current models for drug safety screening are not reliable in toxicity prediction, and the results not easily translatable to the clinic due to insensitive assays that do not recapitulate fully the complex phenotype of a functional cell type in vivo. Recent advances in the field of stem cell research have potentially allowed for a readily available source of metabolically competent cells for toxicity studies, derived using human pluripotent stem cells harnessed from embryos or reprogrammed from mature somatic cells. Pluripotent stem cell-derived cell types also allow for potential disease modeling in vitro for the purposes of drug toxicology and safety pharmacology, making this model possibly more predictive of drug toxicity compared with existing models. This article will review the advances and challenges of using human pluripotent stem cells for modeling metabolism and toxicity, and offer some perspectives as to where its future may lie.

alpha(1)-Adrenoceptor antagonists prevent paracetamol-induced hepatotoxicity in mice.

BACKGROUND AND PURPOSE: Paracetamol, a major cause of acute liver failure (ALF) represents a significant clinical problem. Adrenoceptor stimulation or antagonism can modulate chemical-induced hepatotoxicity. We investigated the role of endogenous catecholamines and alpha(1)-adrenoceptors in the development of paracetamol- induced hepatotoxicity. EXPERIMENTAL APPROACH: Paracetamol (3.5 mmol kg(-1)) was administered to male CD-1 mice, with and without alpha(1)-adrenoceptor antagonists (prazosin, doxazosin, terazosin and tamsulosin; 35.7 micromol kg(-1)). Serum transaminases and hepatic glutathione (GSH) levels were assessed as markers of hepatic damage. Paracetamol bioactivation was assessed by covalent binding, hepatic and urinary conjugate formation and uridine glucuronosyltransferase activity. Plasma catecholamines levels and hepatic congestion were also analysed. KEY RESULTS: Plasma catecholamine levels were significantly elevated 5 h post paracetamol administration. Prazosin prevented hepatotoxicity when administered 1 h before a toxic paracetamol insult and importantly, when administered up to 1 h post paracetamol injection. Prazosin had no effect on paracetamol-induced depletion of hepatic GSH, paracetamol bioactivation or paracetamol-induced transcription of defence genes. Paracetamol toxicity is associated with marked accumulation of erythrocytes within hepatic sinusoids and prazosin completely prevented this accumulation. CONCLUSION AND IMPLICATIONS: Paracetamol-induced hepatocellular damage is associated with increased circulating catecholamines. alpha(1)-Adrenoceptor antagonists conferred complete protection from paracetamol -induced hepatotoxicity. Protection was associated with absence of hepatic erythrocyte accumulation. Increased catecholamine levels may contribute to the pathophysiology of paracetamol-induced hepatotoxicity by compromising hepatic perfusion. Protection against paracetamol toxicity by alpha(1) antagonists in mice has implications for therapeutic management of patients presenting with paracetamol overdose and ALF.

Are chemically reactive metabolites responsible for adverse reactions to drugs?

Low molecular weight organic chemicals can be transformed by normal drug-metabolising systems into short-lived metabolites that are inherently reactive towards cellular macromolecules. There is direct evidence that the formation of such chemically reactive metabolites may lead to mutagenesis, carcinogenicity, apoptosis and necrosis in both cell and animal models. A number of drugs associated with non-pharmacological drug toxicities in man have been shown to undergo bioactivation either in vivo or in vitro. We have therefore examined the evidence for the role of reactive metabolites in the three most common drug-induced toxicities: hepatotoxicity, skin reactions and blood dyscrasias.

Metabolism of fluorine-containing drugs.

This article reviews current knowledge of the metabolism of drugs that contain fluorine. The strategic value of fluorine substitution in drug design is discussed in terms of chemical structure and basic concepts in drug metabolism and drug toxicity.

Allele and genotype frequencies of polymorphic cytochromes P4502D6, 2C19 and 2E1 in aborigines from western Australia.

The polymorphisms of the important xenobiotic metabolizing enzymes CYP2D6, CYP2C19 and CYP2E1 have been studied extensively in a large number of populations and show significant heterogeneity in the frequency of different alleles/genotypes and in the prevalence of the extensive and poor metabolizer phenotypes. Understanding of inter-ethnic differences in genotypes is important in prediction of either beneficial or adverse effects from therapeutic agents and other xenobiotics. Since no data were available for Australian Aborigines, we investigated the frequencies of alleles and genotypes for CYP2D6, CYP2C19 and CYP2E1 in a population living in the far north of Western Australia. Because of its geographical isolation, this population can serve as a model to study the impact of evolutionary forces on the distribution of different alleles for xenobiotic metabolizing enzymes. Twelve CYP2D6 alleles were analysed. The wild-type allele *1 was the most frequent (85.81%) and the non-functional alleles (*4, * 5, * 16) had an overall frequency of less than 10%. Only one subject (0.4%) was a poor metabolizer for CYP2D6 because of the genotype *5/*5. For CYP2C19, the frequencies of the *1 (wild-type) and the non-functional (*2 and *3) alleles were 50.2%, 35.5% and 14.3%, respectively. The combined CYP2C19 genotypes (*2/*2, *2/*3 or *3/*3) correspond to a predicted frequency of 25.6% for the CYP2C19 poor metabolizer phenotype. For CYP2EI, only one subject had the rare c2 allele giving an overall allele frequency of 0.2%. For CYP2D6 and CYP2C19, allele frequencies and predicted phenotypes differed significantly from those for Caucasians but were similar to those for Orientals indicating a close relationship to East Asian populations. Differences between Aborigines and Orientals in allele frequencies for CYP2D6* 10 and CYP2E1 c2 may have arisen through natural selection, or genetic drift, respectively.

Metabolic activation in drug allergies.

Drug allergies are a major problem in the clinic and during drug development. At the present time, it is not possible to predict the potential of a new chemical entity to produce an allergic reaction (hypersensitivity) in patients in preclinical development. Such adverse reactions, because of their idiosyncratic nature, only become apparent once the drug has been licensed. Our present chemical understanding of drug hypersensitivity is based on the hapten hypothesis, in which covalent binding of the drug (metabolite) plays a central role in drug immunogenicity and antigenicity. If this theory is correct, then it should be possible to develop in vitro systems to assess the potential of drugs to bind to critical proteins, either directly or indirectly after metabolic activation to protein-reactive metabolites (bioactivation) and initiate hypersensitivity. The purpose of this review is to assess critically the evidence to support the hapten mechanism, and also to consider alternative mechanisms by which drugs cause idiosyncratic toxicity.

Reactive metabolites and their role in drug reactions.

Adverse drug reactions are a major clinical problem and often preclude drug administration. Drug hypersensitivity (or allergy) represents one of the most severe and unpredictable reactions associated with drug therapy. Our current understanding of drug hypersensitivity is based on the hapten hypothesis of immune recognition of drugs by T cells. The onset of hypersensitivity involves drug bioactivation, covalent binding, followed by uptake, antigen processing and T cell proliferation. There is convincing evidence that drugs associated with a high incidence of hypersensitivity are converted to protein reactive intermediates by the normal processes of drug metabolism and stimulate a cellular immune response in sensitive individuals. Until recently, however, there has been little evidence to relate the formation of a reactive metabolite to the initiation of a cellular immune response. The purpose of this review is to detail recent advances in our understanding of the complex mechanisms of drug hypersensitivity, and using severe skin reactions as an example, assess recent evidence that supports the hapten hypothesis of drug hypersensitivity.

Advances in molecular toxicology-towards understanding idiosyncratic drug toxicity.

Idiosyncratic drug toxicity is a major complication of drug therapy and drug development. Such adverse drug reactions (ADRs) include anaphylaxis, blood dyscrasias, hepatotoxicity and severe cutaneous reactions. They are usually serious and can be fatal. At present, prediction of idiosyncratic ADRs at the preclinical stage of drug development is not possible because there are no suitable animal models and we do not understand the basic mechanisms involved in the toxicity when it does occur in man. Many idiosyncratic reactions appear to have an immunological aetiology. For example, there is increasing evidence for the role of T lymphocytes in severe skin reactions. Nevertheless, the sequence of events by which a simple chemical can elicit severe tissue damage remains poorly understood and alternative novel mechanisms of toxicity must also be explored. The purpose of this article will be to review the currently accepted mechanisms of idiosyncratic drug toxicity at the chemical and the molecular levels. In particular, we will consider how recent advances in cellular immunology and molecular biology can improve our understanding of both the chemical and clinical aspects of drug hypersensitivity. Recent advances in the role of both inter- and intra-cellular signalling in the regulation of the immune response to drugs and their metabolites will be discussed. The long-term aim of such research is to provide test systems for the evaluation of drug safety and patient susceptibility to idiosyncratic drug toxicity.

Hepatocellular response to chemical stress in CD-1 mice: induction of early genes and gamma-glutamylcysteine synthetase.

Exposure of cells to toxic chemical species can result in reduced glutathione (GSH) depletion, generation of free radicals, and/or binding to critical cell determinants. Chemical stress is usually followed by a concerted cellular response aimed at restoring homeostasis, although the precise initial stimulus for the response is unclear. We have focused on one component of this stress response, the up-regulation of gamma-glutamylcysteine synthetase (gamma-GCS) and the preceding molecular events involved in its regulation in an in vivo mouse model. Male CD-1 mice received buthionine sulphoximine (BSO; 7.2 mmol/kg), diethyl maleate (DEM; 4.2 mmol/kg), paracetamol (APAP; 3.5 and 1.0 mmol/kg), or carbon tetrachloride (CCl(4); 1.0 and 0.2 mmol/kg). Biochemical (serum transaminase and hepatic GSH levels) and molecular (c-jun and c-fos messenger RNA [mRNA] levels and activator protein 1 [AP-1] DNA binding activity) parameters were measured, as well as the consequent effects on gamma-GCS levels and activity. All compounds produced GSH depletion, but only the higher doses of APAP and CCl(4) caused liver damage. DEM, APAP, and CCl(4) increased c-jun and c-fos mRNA levels, together with an increase in AP-1 binding; BSO failed to induce AP-1 despite an increase in c-fos. Interestingly, the effects on gamma-GCS varied markedly according to the compound: BSO and DEM increased gamma-GCS enzyme activity, although only DEM, but not BSO, resulted in an increase in gamma-GCS(h) mRNA and protein. In contrast, APAP and CCl(4) both increased gamma-GCS(h) mRNA and protein; however, there was a marked dose-dependent decrease in gamma-GCS activity. These data indicate that the effect of chemical stress on the liver is compound specific and is not merely dependent on depletion of GSH.

Cellular disposition of sulphamethoxazole and its metabolites: implications for hypersensitivity.

1. Bioactivation of sulphamethoxazole (SMX) to chemically-reactive metabolites and subsequent protein conjugation is thought to be involved in SMX hypersensitivity. We have therefore examined the cellular metabolism, disposition and conjugation of SMX and its metabolites in vitro. 2. Flow cytometry revealed binding of N-hydroxy (SMX-NHOH) and nitroso (SMX-NO) metabolites of SMX, but not of SMX itself, to the surface of viable white blood cells. Cellular haptenation by SMX-NO was reduced by exogenous glutathione (GSH). 3. SMX-NHOH and SMX-NO were rapidly reduced back to the parent compound by cysteine (CYS), GSH, human peripheral blood cells and plasma, suggesting that this is an important and ubiquitous bioinactivation mechanism. 4. Fluorescence HPLC showed that SMX-NHOH and SMX-NO depleted CYS and GSH in buffer, and to a lesser extent, in cells and plasma. 5. Neutrophil apoptosis and inhibition of neutrophil function were induced at lower concentrations of SMX-NHOH and SMX-NO than those inducing loss of membrane viability, with SMX having no effect. Lymphocytes were significantly (P<0.05) more sensitive to the direct cytotoxic effects of SMX-NO than neutrophils. 6. Partitioning of SMX-NHOH into red blood cells was significantly (P<0.05) lower than with the hydroxylamine of dapsone. 7. Our results suggest that the balance between oxidation of SMX to its toxic metabolites and their reduction is an important protective cellular mechanism. If an imbalance exists, haptenation of the toxic metabolites to bodily proteins including the surface of viable cells can occur, and may result in drug hypersensitivity.

The effect of genetic polymorphisms in CYP2C9 on sulphamethoxazole N-hydroxylation.

Sulphamethoxazole undergoes CYP2C9-mediated bioactivation to a hydroxylamine. In this study, we investigated the effect of the CYP2C9Arg144 to Cys (CYP2C9*2) and CYP2C9Ile359 to Leu (CYP2C9*3) polymorphisms on sulphamethoxazole N-hydroxylation. Human livers were genotyped using polymerase chain reaction amplification and restriction fragment length polymorphism analysis. Formation of sulphamethoxazole hydroxylamine and methylhydroxy tolbutamide in microsomes prepared from cell lines and the genotyped human livers was determined by high-pressure liquid chromatography. Microsomes prepared from the cell line expressing the allelic variants CYP2C9-Cys144 and CYP2C9-Leu359 displayed a threefold and 20-fold decrease in intrinsic clearance (Cl(int)) for sulphamethoxazole, respectively, when compared with the wild-type, CYP2C9-Arg144. A significant decrease (P < 0.05) in Cl(int) was also observed with tolbutamide for both mutations. Of the 26 human livers genotyped, 61.5% were homozygous wild-type, 26.9% were heterozygotes for CYP2C9*2 and 15.4% were heterozygotes for CYP2C9*3. No homozygous mutant livers were detected. There was a good correlation between sulphamethoxazole N-hydroxylation and tolbutamide methyl hydroxylation (r = 0.825). However, there was no difference in the kinetic parameters for either sulphamethoxazole N-hydroxylation or tolbutamide methyl hydroxylation between the wild type livers (n = 6) and either the livers heterozygous for the CYP2C9*2 (n = 5) or the livers heterozygous for the CYP2C9*3 mutation (n = 3). The CYP2C9*2 and CYP2C9*3 polymorphisms may have some influence on the bioactivation of sulphamethoxazole, particularly in individuals who are homozygous mutants, and this could act as a protective factor against sulphamethoxazole hypersensitivity. However, given the rarity of homozygous mutants, it is likely that other metabolic and immunological risk factors will dominate individual susceptibility.

Expression of cytochrome P4502E1 in human liver: assessment by mRNA, genotype and phenotype.

Cytochrome P4502E1 (CYP2E1) is constitutively expressed in human liver and is responsible for the metabolic bioactivation of a wide variety of xenobiotics, including a number of protoxins and procarcinogens. CYP2E1 expression is regulated at several levels including pre-transcriptional, transcriptional and post-transcriptional levels, and any variation in enzyme concentration and hence activity may represent increased risk of toxicity or carcinogenicity. We have investigated variability in the levels of CYP2E1 mRNA, protein and functional activity in a human liver bank, and attempted to relate these parameters to the RsaI restriction fragment length polymorphism in the 5'-flanking region. Variation in CYP2E1 mRNA (18-fold) was greater than the variation seen in CYP2E1 protein (twofold) and functional activity (fourfold) determined using two probe substrates, chlorzoxazone and p-nitrophenol. Although protein and functional activity showed a significant correlation (r = 0.93 and r = 0.83 for chlorzoxazone and p-nitrophenol, respectively), there was no correlation between any of these parameters and mRNA levels. Also, the variation in CYP2E1 activity could not be directly accounted for by the RsaI polymorphism in our samples. In conclusion, our results are consistent with a complex regulation of CYP2E1 and the fact that it is highly conserved in the human population. The absence of a relationship between the RsaI polymorphism and CYP2E1 activity is consistent with other studies performed in Caucasians, but does not exclude an effect of this polymorphism on inducibility of CYP2E1.