Immortalized hepatocyte cell lines show only a weak resemblance to primary hepatocytes in terms of gene expression and function, limiting their value in predicting drug-induced liver injury (DILI). Furthermore, primary hepatocytes cultured on two-dimensional tissue culture plastic surfaces rapidly dedifferentiate losing their hepatocyte functions and metabolic competence. We have developed a three-dimensional in vitro model using extracellular matrix-based hydrogel for long-term culture of the human hepatoma cell line HepG2. HepG2 cells cultured in this model stop proliferating, self-organize and differentiate to form multiple polarized spheroids. These spheroids re-acquire lost hepatocyte functions such as storage of glycogen, transport of bile salts and the formation of structures resembling bile canaliculi. HepG2 spheroids also show increased expression of albumin, urea, xenobiotic transcription factors, phase I and II drug metabolism enzymes and transporters. Consistent with this, cytochrome P450-mediated metabolism is significantly higher in HepG2 spheroids compared to monolayer cultures. This highly differentiated phenotype can be maintained in 384-well microtiter plates for at least 28 days. Toxicity assessment studies with this model showed an increased sensitivity in identifying hepatotoxic compounds with repeated dosing regimens. This simple and robust high-throughput-compatible methodology may have potential for use in toxicity screening assays and mechanistic studies and may represent an alternative to animal models for studying DILI.

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Previously, we've described a site-directed triple mutant of cytochrome P450 BM3 (BM3) that is able to convert various drugs (van Vugt-Lussenburg, B. M. A., et al. Biochem. Biophys. Res. Commun. 2006, 346, 810-818). In the present study, random mutagenesis was used to improve the activity of this mutant. With three generations of error-prone PCR, mutants were obtained with 200-fold increased turnover toward drug substrates dextromethorphan and 3,4-methylenedioxymethylamphetamine. The initial activities of these mutants were up to 90-fold higher than that of human P450 2D6. These highly active drug metabolizing enzymes have great potential for biotechnology. Using sequencing analysis, the mutations responsible for the increase in activity were determined. The mutations that had the greatest effects on the activity were F81I, E267V, and particularly L86I, which is not located in the active site. Computer modeling studies were used to rationalize the effects of the mutations. This study shows that random mutagenesis can be used to identify novel critical residues, and to increase our insight into P450s.

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In this review, an overview is presented of the current knowledge of genetic polymorphisms of four of the most important enzyme families involved in the metabolism of xenobiotics, that is, the N-acetyltransferase (NAT), cytochrome P450 (P450), glutathione-S-transferase (GST), and microsomal epoxide hydrolase (mEH) enzymes. The emphasis is on two main topics, the molecular genetics of the polymorphisms and the consequences for xenobiotic metabolism and toxicity. Studies are described in which wild-type and mutant alleles of biotransformation enzymes have been expressed in heterologous systems to study the molecular genetics and the metabolism and pharmacological or toxicological effects of xenobiotics. Furthermore, studies are described that have investigated the effects of genetic polymorphisms of biotransformation enzymes on the metabolism of drugs in humans and on the metabolism of genotoxic compounds in vivo as well. The effects of the polymorphisms are highly dependent on the enzyme systems involved and the compounds being metabolized. Several polymorphisms are described that also clearly influence the metabolism and effects of drugs and toxic compounds, in vivo in humans. Future perspectives in studies on genetic polymorphisms of biotransformation enzymes are also discussed. It is concluded that genetic polymorphisms of biotransformation enzymes are in a number of cases a major factor involved in the interindividual variability in xenobiotic metabolism and toxicity. This may lead to interindividual variability in efficacy of drugs and disease susceptibility.

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The conjugation of reactive drug metabolites to GSH is considered an important detoxification mechanism that can be spontaneous and/or mediated by glutathione S-transferases (GSTs). In case GSTs play an important role in GSH conjugation, genetically determined deficiencies in GSTs may be a risk factor for adverse drug reactions (ADRs) resulting from reactive drug metabolites. So far, the role of GSTs in the detoxification of reactive intermediates of clozapine, a drug-causing idiosyncratic drug reactions (IDRs), has not been studied. In the present study, we studied the ability of four recombinant human GSTs (hGST A1-1, hGST M1-1, hGST P1-1, and hGST T1-1) to catalyze the GSH conjugation of reactive metabolites of clozapine, formed in vitro by human and rat liver microsomes and drug-metabolizing P450 BM3 mutant, P450 102A1M11H. Consistent with previous studies, in the absence of GSTs, three GSH conjugates were identified derived from the nitrenium ion of clozapine. In the presence of three of the GSTs, hGST P1-1, hGST M1-1, and hGST A1-1, total GSH conjugation was strongly increased in all bioactivation systems tested. The highest activity was observed with hGST P1-1, whereas hGST M1-1 and hGST A1-1 showed slightly lower activity. Polymorphic hGST T1-1 did not show any activity in catalyzing GSH conjugation of reactive clozapine metabolites. Interestingly, the addition of hGSTs resulted in major changes in the regioselectivity of GSH conjugation of the reactive clozapine metabolite, possibly due to the different active site geometries of hGSTs. Two GSH conjugates found were completely dependent on the presence of hGSTs. Chlorine substitution of the clozapine nitrenium ion, which so far was only observed in in vivo studies, appeared to be the major pathway of hGST P1-1-catalyzed GSH conjugation, whereas hGST A1-1 and hGST M1-1 also showed significant activity. The second GSH conjugate, previously also only found in in vivo studies, was also formed by hGST P1-1 and to a small extent by hGST A1-1. These results demonstrate that human GSTs may play a significant role in the inactivation of reactive intermediates of clozapine. Therefore, further studies are required to investigate whether genetic polymorphisms of hGST P1-1 and hGST M1-1 contribute to the interindividual differences in susceptibility to clozapine-induced adverse drug reactions.