A multiplex-PCR/RFLP procedure for simultaneous CYP2E1, mEH and GSTM1 genotyping
Introduction
The metabolism of environmental chemicals is regulated by a wide variety of genes. Most chemicals are first converted into reactive electrophilic metabolites by oxidative enzymes, mainly cytochrome P450-related enzymes [1], [2]. Subsequent metabolism by enzymes, such as epoxide hydrolase and other subsets of activating CYP isoforms, leads to the formation of highly reactive metabolites that can bind to genomic DNA [3]. The second metabolic step mainly involves transferases such as glutathione S-transferases, acetyltransferases, and UDP-glucuronsyltransferases, which take part in detoxification of oxidated forms of xenobiotics [4]. Thus, the concerted action of these phase I and II enzymes is crucial in determining the final biological effect of xenobiotic exposure.
Inter-individual variation in the activities of enzymes involved in biotransformation of xenobiotics is related to genetic polymorphisms [5]. We have been elucidating the involvement of polymorphic metabolizing genes in the development of environmentally induced disease [6], [7], [8]. From our own experience and from our review of the literature, it has become obvious that if the characterization of several polymorphic genes can be performed simultaneously, the goal of understanding susceptibility to environmental disease can be achieved sooner.
It is well established that the human cytochrome P450 II E1 gene (CYP2E1) is polymorphic [9]. The PstI and RsaI polymorphic restriction sites in the 5′-flanking region of CYP2E1 are in complete linkage disequilibrium [10]. These genetic polymorphisms change the transcriptional regulation of the gene, affecting its expression [11], [12], [13], and thus, altering the metabolism of a range of chemicals such as aliphatic alcohols, ethers, halides, nitrites, carbon tetrachloride, nitrosamines, paracetamol and chloroform [14], [15], [16]. CYP2E1 polymorphism has been associated with lung cancer [8], [17], nasopharyngeal carcinoma [18] and alcoholic liver disease [19].
Microsomal epoxide hydrolase (mEH) is an enzyme involved in the first-pass metabolism of highly reactive epoxide intermediates [20]. It cleaves a range of alkene and arene oxides to form trans-dihydrodiols [21], [22]. Several polymorphisms have been described in the human mEH gene. One of them, tyrosine-113-histidine (His/Tyr), results in the reduction of enzymatic activity by approximately 40% in vitro [23], [24], which may be due to altered protein stability [25]. mEH is associated with an increased risk for hepatocellular carcinoma [26], emphysema [27], lung cancer [28] and ovarian carcinoma [29].
The glutathione S-transferase mu family includes five genes (GSTM1 to GSTM5) [30]. GSTM1, the most commonly expressed gene [20], catalyzes the inactivation of a range of chemical carcinogens including 7,12 dimethylbenz[a]anthrancene and benzo[a]pyrene [31], and styrene 7,8-oxide, and trans-stilbene oxide [32]. Inheritance of the homozygous deletion of the gene causes deficiency in enzyme activity [33] and is associated with the development of various types of cancer such as lung [34], skin [35] and bladder cancer [36].
mEH acts coordinately with CYP2E1 in biotransformation of several chemicals, e.g. benzene, butadiene and styrene, leading to formation of diol intermediates that are further metabolized by GSTs [37], [38], [39]. Some of these reactive metabolites can bind to DNA to initiate a series of events that may lead to tumor formation. Therefore, a research strategy to assess multiple polymorphic genes involved in the metabolic pathway of environmental toxicants can provide a better understanding of the role of genetic susceptibility to human disease.
In this report, we describe the development of a new multiplex-polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) procedure for simultaneous characterization of the CYP2E1, mEH and GSTM1.
Section snippets
Chemicals and reagents
All primers used for PCR were custom synthesized by the Recombinant DNA Laboratory, Sealy Center for Molecular Sciences, University of Texas Medical Branch (UTMB), Galveston, TX. All other chemicals were from commercial sources and were of the highest purity available.
Sample collection and DNA isolation
A total of 25 human genomic DNA samples, previously obtained from ongoing studies in our laboratory at UTMB, were used in the development of this procedure. The procedures for donor recruitment, DNA processing and genotyping were
Results and discussion
As is illustrated in Fig. 1, a CYP2E1 wild type allele, characterized by the absence of a PstI restriction site, is revealed by a band at 410 bp after PstI digestion. The presence of bands at 290 and 120 bp indicates a homozygous mutant allele. The heterozygous mutant allele is characterized by the presence of bands at 410, 290 and 120 bp. GSTM1 polymorphism (wild-type or null) is characterized, after PstI digestion, by the presence or absence of two bands at 150 and 65 bp. For mEH, Tth111I
Acknowledgements
This study was supported by a fellowship from the Ministry of Education, Arab Republic of Egypt to S.A.S.; by a fellowship from COLCIENCIAS-Fulbright-LASPAU to C.H.S.T.; and by a fellowship from the Ministry of Government Administration and Home Affairs, Republic of Korea to H.Y.O. Synthesis of the primers was partially supported by the NIEHS center at UTMB.
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