L.W. Wormhoudt

Genetic Polymorphisms of Human N-Acetyltransferase, Cytochrome P450, Glutathione-S-Transferase, and Epoxide Hydrolase Enzymes: Relevance to Xenobiotic Metabolism and Toxicity

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.

Inter-individual variability in the oxidation of 1,2-dibromoethane: use of heterologously expressed human cytochrome P450 and human liver microsomes.

1,2-Dibromoethane (1,2-DBE) is mainly used as an additive in leaded gasoline and as a soil fumigant and it is a suspected carcinogen in humans. In this study, the oxidative bioactivation of 1,2-DBE to 2-bromoacetaldehyde (2-BA) was studied using heterologously expressed human cytochrome P450 (P450) isoenzymes and human liver microsomes. Out of ten heterologously expressed human P450 isoenzymes (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C8, CYP2C9, CYP2C18, CYP3A4 and CYP3A5), only human CYP2A6, CYP2B6 and CYP2E1 metabolized 1,2-DBE, albeit with strongly differing catalytic efficiencies. The apparent Km and Vmax values were 3.3 mM and 0.17 pmol/min per pmol P450 for CYP2A6, 9.7 mM and 3.18 pmol/min per pmol P450 for CYP2B6 and 42 microM and 1.3 pmol/min per pmol P450 for CYP2E1, respectively. In all of 21 human liver samples studied, 1,2-DBE was oxidized with activities ranging from 22.2 to 1027.6 pmol/min per mg protein, thus showing a 46-fold inter-individual variability. The kinetics of the oxidative metabolism of 1,2-DBE to 2-BA in human liver microsomes were linear, indicating the involvement of primarily one single P450 isoenzyme. There was a tendency towards a positive correlation between the oxidative metabolism of 1,2-DBE in the human liver microsomes and the 6-hydroxylation of chlorzoxazone, a selective substrate for CYP2E1. Furthermore, the oxidative metabolism of 1,2-DBE was inhibited by the specific CYP2E1 inhibitors disulfiram (DS) and diethyldithiocarbamate (DDC). In contrast, a poor correlation was found between the immunochemically quantified amount of CYP2E1 and the microsomal chlorzoxazone 6-hydroxylation or the 1,2-DBE oxidation. The results indicate that CYP2E1 is probably the major P450 isoenzyme involved in the oxidative hepatic metabolism of 1,2-DBE in humans. The inter-individual variability in the oxidative bioactivation of 1,2-DBE in humans, largely due to inter-individual variability in the catalytic activity of hepatic CYP2E1, may have important consequences for the risk assessment for human exposure to 1,2-DBE.

Toxicity of fotemustine in rat hepatocytes and mechanism-based protection against it.

Fotemustine is a relatively novel DNA-alkylating 2-chloroethyl-substituted N-nitrosourea (CENU) drug, clinically used for the treatment of disseminated malignant melanoma in different visceral and non-visceral tissues. Thrombocytopenia has been observed in patients treated with fotemustine and liver and renal toxicities as well. In this study, firstly the metabolism of fotemustine was investigated in vitro and secondly the undesired cytotoxicity of fotemustine as well as different ways of protection against it. In rat hepatocytes, chosen as a model system, fotemustine was shown to cause lactate dehydrogenase (LDH) leakage, glutathione (GSH) depletion, GSSG-formation and lipid peroxidation (LPO). A reactive metabolite, DEP-isocyanate, is most likely responsible for these undesired cytotoxic effects. Based on the observed cytotoxicity mechanisms, chemoprotection with several sulfhydryl-containing nucleophiles and antioxidants was investigated. The sulfhydryl nucleophiles; GSH, N-acetyl-L-cysteine (NAC) and glutathione isopropylester (GSH-IP) protected almost completely against fotemustine-induced LDH-leakage and LPO. NAC and GSH protected partly against fotemustine-induced GSH-depletion. The antioxidant, vitamin E protected completely against fotemustine-induced LPO, but only partly against fotemustine-induced LDH-leakage and not against GSH-depletion. Ebselen, a peroxidase-mimetic organoselenium compound, did not show protective effects against the cytotoxicity of fotemustine, possibly because GSH is required for the bioactivation of ebselen. It is concluded that co-administration of sulfhydryl nucleophiles, in particular NAC and GSH-IP, possibly in combination with antioxidants, such as vitamin E, are effective against the toxicity of fotemustine in vitro. It might, therefore, be worthwhile to investigate the cytoprotective potency of these agents against undesired toxicities of fotemustine in vivo as well.

Cytochrome P450 catalyzed metabolism of 1,2-dibromoethane in liver microsomes of differentially induced rats.

The cytochrome P450 (P450) catalyzed oxidation of 1,2-dibromoethane (1,2-DBE) to 2-bromoacetaldehyde (2-BA) was measured in liver microsomes of both control and differentially induced rats. 2-BA formation was quantified by derivatization of 2-BA with adenosine (ADO), resulting in the formation of the highly fluorescent 1,N6-ethenoadenosine (epsilon-ADO), which was measured by HPLC. After microsomal incubation with 1,2DBE in the presence of ADO and removal of proteins by denaturation and centrifugation, derivatization by heating 4 h at 65 degrees C appeared necessary to ensure efficient formation of epsilon-ADO. Using this optimized derivatization method to quantitate 2-BA formation, the enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA were measured in liver microsomes prepared from untreated rats and rats pretreated with phenobarbital (PB), beta-naphtoflavone (beta NF) and pyrazole (PYR). P450 isoenzymes in PYR- and beta NF-induced microsomes showed linear enzyme kinetics while P450 isoenzymes in control and PB-induced microsomes showed non-linear enzyme kinetics. The apparent Vmax- and Km- values for the metabolism of 1,2-DBE to 2-BA were 2.5 nmol/min/mg protein and 144 microns for P450 isoenzymes in PYR-induced microsomes and 773 pmol/min/mg protein and 3.3 mM for P450 isoenzymes in beta NF-induced microsomes, respectively. Due to the non-linear enzyme kinetics of the P450 catalyzed oxidation of 1,2-DBE to 2-BA using control and PB-induced microsomes, no proper Vmax- and Km- values could be calculated. However, from Michaelis-Menten plots it was clear that the affinity of P450 isoenzymes for 1,2-DBE in control and PB-induced microsomes was in the same range when compared to beta NF-induced microsomes and thus much lower than the PYR-induced microsomes.