Interindividual differences in xenobiotic-metabolising enzymes : the human genetic factor

Sammanfattning: The vast number of human xenobiotic-metabolising enzymes display interindividual variability that can alter the disposition of any compound metabolised by these enzymes, including environmental chemicals and many clinically used drugs. Both genetic and non-genetic factors affect the levels and activities of these enzymes. The purpose of this study was to investigate some of the underlying genetic differences and also to examine genetic variations among different ethnic groups. Cytochrome P450 2D6 (CYP2D6) displays both interindividual and interethnic variation. The CYP2D6 alleles distributed in a Saudi Arabian population were investigated. We found a high frequency of subjects carrying duplicated CYP2D6 alleles (21%) and very few defective alleles, which is in agreement with earlier Saudi Arabian CYP2D6 phenotyping studies which reported a very low frequency of poor metabolisers. This suggests that CYP2D6 could be under selective pressure in this region possibly due to dietary reasons. It has been reported that CYP2D6 expression in easily accessible human peripheral blood mononuclear cells (PBMCs) can be used to predict whole-body metabolic activity. We examined if this enzyme is expressed in this cell population and if so, whether the expression would correlate to gene copy number. However, no correctly spliced CYP2D6 mRNA or protein was observed in mononuclear cells and the validity of using PBMCs as a surrogate marker to predict hepatic CYP2D6 activity can be questioned. The mu class glutathione S-transferase gene, GSTM1, is polymorphic in humans with roughly half of the population being homozygous deleted for this gene. In contrast, an earlier Saudi Arabian GSTM1 phenotyping study revealed that some individuals exhibited ultrarapid enzyme activities, therefore the genetic basis of this observation was investigated. By doing restriction fragment length polymorphism (RFLP) analysis and using a quantitative multiplex PCR method, the existence of a GSTM cluster containing a duplicated functional GSTM1 gene was revealed. The generation of the duplicated GSTM1 locus likely involved an unequal crossover event between homologous regions in the locus. Individuals carrying extra GSTM1 genes may gain a protective effect against some chemicals as they are likely to rapidly conjugate and inactivate carcinogenic compounds. Cytochrome P450 2A6 (CYP2A6) is the major nicotine C-oxidase in humans and can activate procarcinogens. We re-evaluated the genotyping method for the CYP2A6'3 allele and discovered a gene conversion with CYP2A7 in the 3'-flanking region of 30-40% of CYP2A6 alleles, causing misclassification of the CYP2A6 genotype. The CYP2A6'3 allele was not found using our new genotyping method which questions its existence. The structure of the CYP2A6 gene deletion was characterised and the generation of this locus likely involved an unequal crossing-over event between the 3'-flanking region of the CYP2A6 and CYP2A7 genes. A rapid PCR-based method was created to detect the allele and it was found at a frequency of 15% in Chinese but only 1% in Caucasians. Another allele, CYP2A6'5, was found to encode an enzyme with a G479L substitution that was very unstable when expressed in yeast and yielded very low protein levels and coumarin 7-hydroxylase activity. It was shown to be quite rare with a frequency of only 0-1%. An inducible enzyme that is expressed in extrahepatic tissues, can activate procarcinogens, and is a low-Km. 17ß -estradiol 4- and 2-hydroxylase is cytochrome P450 1B1 (CYP1B1). The CYP1B1.2 enzyme contains the two linked amino acid substitutions R48G and A119S compared to CYP1B1.1. To functionally characterise the two variants with respect to kinetic properties and protein stability, the alleles were expressed in yeast and in mammalian COS-1 cells. It was found that CYP1B1.1. and CYP1B1.2 share very similar properties with respect to catalytic activities and stability and that these amino acid substitutions do not alter CYP1B1 function. In conclusion, the present study has identified and characterised several novel alleles for genes encoding some of the human xenobiotic-metabolising enzymes and examined their distribution along with other known alleles in different ethnic groups. Genotyping methods were developed to detect the new alleles that can be used not only in molecular epidemiological studies but also for helping to design more efficient and safer drug treatments.