Showing papers in "Pharmacogenetics in 1995"

Nomenclature for N-acetyltransferases.

Abstract: A consolidated classification system is described for prokaryotic and eukaryotic N-acetyltransferases in accordance with the international rules for gene nomenclature. The root symbol (NAT) specifically identifies the genes that code for the N-acetyltransferases, and NAT* loci encoding proteins with similar function are distinguished by Arabic numerals. Allele characters, denoted by Arabic numbers or by a combination of Arabic numbers and uppercase Latin letters, are separated from gene loci by an asterisk, and the entire gene-allele symbols are italicized. Alleles at the different NAT* loci have been numbered chronologically irrespective of the species of origin. For designation of genotypes at a single NAT* locus, a slash serves to separate the alleles; in phenotype designations, which are not italicized, alleles are separated by a comma.

Dopamine D2 receptor gene variants: association and linkage studies in impulsive-addictive-compulsive behaviour

Abstract: Drug and alcohol seeking behaviour has become a great global problem affecting millions of inhabitants with a cost to society in the billions. Dopaminergic reward pathways have frequently been implicated in the etiology of addictive behaviour. While other neurotransmitters have also been implicated, to date the only molecular genetic defect which has been found to associate with alcoholism, drug dependency, obesity, smoking, pathological gambling, attention-deficit-hyperactivity disorder (ADHD), Tourette syndrome, as well as other related compulsive behaviours, are the variants of the dopamine D2 receptor gene (DRD2). In this review of the available data on the subject, we report a number of independent meta-analyses that confirm an association of DRD2 polymorphisms and impulsive-additive-compulsive behaviour (IACB), which we have termed "Reward Deficiency Syndrome". While we agree that Meta-analyses of all exant studies support an association of variants of DRD2 and IACB, correct negative findings with alcoholism may be due to differences in assessing controls and inclusion/exclusion criteria for selection of diseased probands.

Role of cytochrome P4501A2 in chemical carcinogenesis: implications for human variability in expression and enzyme activity.

Abstract: Cytochrome P4501A2 (CYP1A2) has been identified as a key factor in the metabolic activation of numerous chemical carcinogens, including aflatoxin B1, various heterocyclic and aromatic amines, and certain nitroaromatic compounds. In addition, CYP1A2 contributes to the inactivation of several common drugs and dietary constituents, including acetaminophen and caffeine. Two xenobiotic-responsive-element (XRE)-like sequences and an antioxidant response element (ARE) have been identified in the regulatory region of the CYP1A2 gene; however, the functionality of the ARE remains to be demonstrated. Based on in vivo phenotyping assays, substantial interindividual variability in CYP1A2 activity has been reported. Some population-based studies have reported either bi- or tri-modal distributions in CYP1A2 phenotype, suggesting a genetic basis for the large interindividual differences in CYP1A2 activity. However, despite the polymodal distributions reported for CYP1A2 activity, a distinct functional genetic polymorphism in the gene has not been identified. Potential mechanisms contributing to the large interindividual variability in CYP1A2 activity are discussed. A thorough understanding of the functions and regulation of the CYP1A2 gene may ultimately lead to new methods for preventing or intervening in the development of certain chemically-related human cancers.

Detection of the poor metabolizer-associated CYP2D6(D) gene deletion allele by long-PCR technology

Abstract: The cytochrome P450 enzyme debrisoquine 4-hydroxylase metabolizes many different classes of commonly used drugs, such as antidepressants and neuroleptics. Deficient hydroxylation of debrisoquine, known as the poor metabolizer (PM) phenotype, affects 5- 10% of Caucasians and may lead to adverse react

Use of omeprazole as a probe drug for CYP2C19 phenotype in Swedish Caucasians: comparison with S-mephenytoin hydroxylation phenotype and CYP2C19 genotype

Abstract: A single oral dose of omeprazole (20 mg) was given orally to 160 healthy Caucasian Swedish subjects and tested as a probe for CYP2C19. The study was nonrandomized and included seven subjects previously classified as poor metabolizers (PM) of S-mephenytoin. The ratio between the plasma concentrations of omeprazole and hydroxyomeprazole (metabolic ratio; MR) was determined by HPLC in a blood sample drawn 3 h after drug intake. In 17 subjects the test was repeated and the MRs of omeprazole on the two occasions were correlated (rs = 0.85; p < 0.0001). There was a significant correlation between the MR of omeprazole and the S/R mephenytoin ratio among 141 subjects, in whom both ratios were determined (rs = 0.63, p < 0.001). All seven PMs of S-mephenytoin had higher MRs of omeprazole (7.1-23.8) than extensive metabolizers (EM) (0.1-4.9). All 160 subjects and another 15 Caucasian Swedish PMs previously phenotyped with mephenytoin were analysed with respect to the presence of the CYP2C19m1 allele by PCR amplification of the intron 4/exon 5 junction followed by Sma I digestion. EMs heterozygous for the CYP2C19m1 gene had MRs of omeprazole and S/R ratios of mephenytoin that were higher than those of subjects who were homozygous for the wild-type allele (p = 0.0001). Nineteen of the 22 PMs were homozygous for the CYP2C19m1 gene. Three were heterozygous for this allele. Thus, 41 of the 44 alleles (93%) of PMs were defective CYP2C19m1. One of the remaining three PM alleles was subsequently found to contain the CYP2C19m2 mutation, which has earlier been shown to be associated with the PM phenotype in Oriental populations. In conclusion, the phenotype determined by omeprazole correlated with that of mephenytoin, and was in good agreement with the genotype.

The pharmacogenetics of codeine hypoalgesia.

Abstract: Codeine is an old drug that is still widely used to treat mild and moderate pain. It is mainly metabolised by glucuronidation, but minor pathways are N-demethylation to norcodeine and O-demethylation to morphine. The latter pathway depends on the genetically polymorphic CYP2D6 which is absent in 7% of the white population (PM) and present in the remainder (EM). Lack of influence of codeine on experimental pain in PM as well as in EM treated with the CYP2D6 blocker quinidine, who are both practically unable to convert codeine to morphine, has supported an old hypothesis that codeine acts through metabolically formed morphine. Possibly, local codeine O-demethylation in the CNS is of major importance for its hypoalgesic effect. Such a local morphine formation from codeine, which supposedly is also catalysed by CYP2D6, could explain why the hypoalgesic effect of codeine stems from morphine despite relatively low plasma levels of morphine after standard hypoalgesic doses of codeine. Dependence of codeine hypoalgesia on morphine formation via CYP2D6 makes this effect liable to interaction with drugs that are inhibitors of CYP2D6. Examples of potent inhibitors of CYP2D6 are quinidine, some selective serotonin reuptake inhibitors and some neuroleptics. Less potent inhibitors, such as tricyclic antidepressants, will probably also reduce the pain relieving effect of codeine, since codeine has a low affinity for CYP2D6. Biosynthesis of morphine in humans may also include steps catalyse by CYP2D6. Experimental studies in large groups of EM and PM indicate that this may lead to interphenotype differences in pain tolerance.

Detection of CYP2C9 polymorphism based on the polymerase chain reaction in Chinese.

Abstract: Cytochrome P450 (CYP) 2C9 catalyses the metabolism of a wide range of drugs Previous studies have shown the differences in the amino acid composition among CYP2C9 variants at Cys144/Arg, Tyr358/Cys, Leu359/Ile, and Gly417/Asp PCR-endonuclease digestion methods have been developed to detect these four possible polymorphisms The T416-->C mutation in exon 3 of CYP2C9 (Cys144-->Arg) creates an Ava II site In the 135 subjects we tested, all leukocyte DNA samples showed a complete Ava II digestion indicating homozygous C416 (Arg144) A Tyr358-->Cys mutation will create a Nsi I site at codon 1057-1063 in exon 7 In 40 subjects tested, all samples showed negative results DNA sequencing on a few samples showed Tyr358Ile359 A mismatched PCR primer pair was then designed to detect codon C1061-->A (Leu359-->Ile) mutation In 115 subjects tested, 111 samples showed a complete Nsi I digestion (Ile359) and four samples showed heterozygous results Another mismatched PCR primer pair was used to confirm the C1061 codon in heterozygous subjects The four heterozygous subjects showed partial digestion with endonuclease Kpn I, which confirmed the heterozygous Ile/Leu at amino acid 359 The G1236-->A mutation in exon 8 of CYP2C9 (Gly417-->Asp) creates a Hph I site In all 46 subjects, homozygous G1236 (Gly417) was found Most Chinese subjects actually have Arg144 Tyr358 Ile359 Gly417 in CYP2C9 as previously reported human-2 Furthermore, we found an A-->T (+12 position in intron 2) mutation in our CYP2C9 sequencing process The mutation creates a NIa III site(ABSTRACT TRUNCATED AT 250 WORDS)

Cytochrome P450 2E1 genotype and chlorzoxazone metabolism in healthy and alcoholic Caucasian subjects.

Abstract: Susceptibility to cancer or ethanol-related liver diseases may be associated with a large variability in cytochrome P450 2E1 activity. This variability may be of genetic origin or reflect environmental factors. To test the role of genetics, the phenotype and genotype of this enzyme were determined in 42 non-alcoholic and 74 alcoholic patients hospitalized for detoxification treatment. Chlorzoxazone metabolism was used to assess CYP2E1 phenotype. Restriction length fragment polymorphisms with Rsa I or Pst I, and Dra I endonucleases were used to determine the two mutant alleles, Pst I/Rsa I-c2 and Dra I-C. A significant gender difference in basal CYP2E1 activity was observed in non-smoking controls (p < 0.05) but not in alcoholics or smokers. Subjects heterozygous for the C or c2 mutated allele did not show any difference in CYP2E1 activity at the basal level, compared with the wild type homozygotes. Conversely, patients with the mutated genotype appeared less inducible than the others after ethanol induction (p < 0.01).

Identification of an NAD(P)H:quinone oxidoreductase polymorphism and its association with lung cancer and smoking.

Abstract: The enzyme NAD(P)H:quinone oxidoreductase (NQO1) catalyses bioreduction and bioactivation reactions. A mutation in the NQO1 gene had previously been demonstrated in a cancer cell line with reduced NQO1 activity. In this study, several regions of the NQO1 locus were examined for constitutional variation at the DNA level. The previously described mutation in exon 6 was detected by the single-strand conformation polymorphism technique. This was confirmed by sequencing to result from a C-->T substitution. Genotype analysis in the Centre d'Etude Polymorphisme Humain (CEPH) reference panel revealed two alleles with frequencies of 0.87 and 0.13 and demonstrated Mendelian transmission. Genotype distributions were consistent with Hardy-Weinberg equilibrium. Linkage analysis mapped the gene locus to chromosome 16q. NQO1 was felt to be a candidate gene for the susceptibility to lung cancer, given its potential role in protection against carcinogenic compounds. The frequency of NQO1 variants was examined in 150 lung cancer cases and in two reference populations. The allele distribution in CEPH parent controls was significantly different from cases (chi 2 = 5.52, p = 0.019), but no difference was noted between cases and a healthy local reference population. When the local reference distribution was stratified on smoking status, a significant difference was observed (chi 2 = 3.88, p = 0.048).(ABSTRACT TRUNCATED AT 250 WORDS)

Susceptibility mutations in the mitochondrial small ribosomal RNA gene in aminoglycoside induced deafness.

Abstract: Aminoglycoside induced deafness has been linked recently to a predisposing homoplasmic mutation in the 3' end of the small ribosomal RNA (rRNA) gene of the human mitochondria (1555 A-->G) that makes the mitochondrial rRNA structurally more similar to its bacterial counterpart. This mitochondrial DNA mutation was consistently found in families in which the susceptibility to develop ototoxic deafness was inherited through the maternal lineage. However, the 1555 A-->G mutation was rarely found in sporadic patients in China, where a significant proportion of the population has been exposed to aminoglycosides. To further characterize the mutations predisposing to aminoglycoside ototoxicity, we analysed the 12S rRNA gene in 35 Chinese sporadic patients without the 1555 A-->G mutation. Using single stranded conformational polymorphism (SSCP) analysis, heteroduplex (HD) analysis, sequencing, and allele specific oligonucleotide hybridization, we found three out of 35 sporadic patients with unique sequence changes in the 12S rRNA gene. Two of the patients had homoplasmic mutations. One patient displayed localized heteroplasmy around nt 961, with an absence of the thymidine at this position and different populations of mitochondrial DNA with varying numbers of inserted cytosines. The description of these putative susceptibility mutations, in particular the heteroplasmic mutation around nt 961, provides further support for the important role of the mitochondrial 12S rRNA in genetic predisposition to aminoglycoside induced ototoxic deafness.

Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1

Abstract: Chlorzoxazone, a muscle-relaxing drug, is metabolized by carbon-hydroxylation at position 6. Chlorzoxazone has been suggested as an in vivo probe for CYP2E1. We studied the specificity of such a substrate using vaccinia virus expressed human P450 forms and the effect of inhibitors for chlorzoxazone metabolism by human liver microsomes. The 6-hydroxylation of chlorzoxazone was mediated by CYP1A2 as well as by CYP2E1. The Km value of CYP1A2 and CYP2E1 for the reaction was 5.69 microM and 232 microM, respectively. However, the Vmax value of CYP2E1 for the reaction was approximately 8.5-fold higher than that of CYP1A2. The CYP1A inhibitor, alpha-naphthoflavone, as well as the CYP2E1 inhibitor, diethyldithiocarbamate, decreased chlorzoxazone 6-hydroxylation at a low substrate concentration by human liver microsomes. Our results raise questions about the suitability of chlorzoxazone as an in vivo probe for hepatic CYP2E1 activity. In human liver microsomal samples, the Km = 40 microM was different from either the Km of CYP1A2 or CYP2E1. We think that this discrepancy is due to the co-expression of similar levels of CYP1A2 and CYP2E1 in human liver. Furthermore, it is suggested that the role of CYP2E1 in 6-hydroxychlorzoxazone formation at the physiological chlorzoxazone concentration of 30-60 microM is almost the same when compared to that of CYP1A2.

Polymorphisms of human Ah receptor gene are not involved in lung cancer.

Abstract: The Ah receptor (Ahr) is a ligand-dependent transcription factor that positively regulates inducible expression of the CYP1A1 gene. Based on the sequence information of the human Ahr and the intron-exon junctions of the mouse counterpart, an analysis of single-strand conformational polymorphism (SSCP) was carried out to detect subtle base differences in the coding region of the gene among individuals. We found that the Ahr protein has at least two forms of variants in a Japanese gene pool, and that these variants can be ascribed to one amino acid replacement of Arg by Lys at codon 554. The frequencies of Arg-coded and Lys-coded alleles were 0.57 and 0.43, respectively. We found, however, that this germ line polymorphism of the Ahr gene did not show a significant association with aryl hydrocarbon hydroxylase (AHH) inducibility nor with lung cancer incidence.

The CYP2A Gene Subfamily: Species Differences, Regulation, Catalytic Activities and Role in Chemical Carcinogenesis

Abstract: The CYP2A subfamily has been characterized in several mammalian species including mouse, rat, rabbit, hamster, cattle and human. Marked species differences have been demonstrated in the catalytic activities and regulation of this subfamily. In humans, the CYP2A genes are found as a cluster on the long arm of chromosome 19 with the CYP2B and CYP2F genes. Marked interindividual differences in expression of the CYP2A6 gene was found in livers analyzed in vitro and in humans phenotyped in vivo by using coumarin, a specific substrate for the enzyme. Efforts are underway to determine the existence of mutant and variant CYP2A6 alleles in the human population. Since CYP2A6 is able to metabolically-activate chemical carcinogens and is expressed in extrahepatic tissue, it will be of interest to determine whether genetic differences in expression of the gene is associated with cancer risk.

Genetic analysis of the CYP2D locus in relation to debrisoquine hydroxylation capacity in Korean, Japanese and Chinese subjects.

Abstract: The CYP2D6 genotype and the debrisoquine and mephenytoin hydroxylation phenotypes were studied in 63 Oriental subjects including 21 Chinese, 21 Japanese and 21 Koreans. All subjects were extensive metabolizers of debrisoquine. The incidence of the S-mephenytoin poor metabolizer phenotype was 14% in

A multifamily study on the relationship between CYP2C19 genotype and s-mephenytoin oxidation phenotype.

Abstract: It has recently been shown that the most common mutation (named m1) in both Caucasian and Japanese poor metabolizers (PM) of S-mephenytoin is a single base pair mutation (G-->A) in exon 5 of the CYP2C19 gene. In Japanese, a second defective allele of CYP2C19 named m2 consists of a G-->A mutation in exon 4. In the present study, we have investigated the inheritance of the CYP2C19 wild type allele (wt) and the two defective alleles (m1 and m2) in families of 11 Danish PM probands. The study was carried out for two principal reasons. First, we wanted to confirm the autosomal recessive inheritance of the defective alleles, and second, we wanted to examine the specificity and sensitivity of the CYP2C19 genotyping test. Individuals were phenotyped by measuring the ratio of S/R mephenytoin excreted in the urine after administration of mephenytoin, and genotyping was carried out by a PCR-based DNA amplification procedure. The genotypes of nine of the 11 probands were consistent with their phenotypes. Eight were homozygous m1/m1, and one was heterozygous m1/m2. The genotypes of two putative PM probands (wt/m1) were not consistent with their phenotypes. On the basis of extended phenotyping (additional late urine collections (24-36 h) and acidification of urine), one of these could probably be reclassified as an extensive metabolizer (EM) while the other was considered to be a true PM. This suggests the presence of an additional unknown mutant allele in the latter. Seven of the 41 phenotyped relatives in the 11 families were phenotyped as PMs, and with the exception of the father of family 10, their genotypes (m1/m1) were consistent with their phenotypes. Extended phenotyping (acidification of urine) suggested that the father of family 10 in fact is an EM and hence that his genotype (wt/m1) is concordant with his phenotype. Thus, the specificity of genotyping tests for PM was 100%, while the sensitivity was 15/16 or 94%. Our study provides unequivocal evidence for autosomal recessive inheritance of the PM trait.

Modulation of xenobiotic-inducible cytochrome P450 gene expression by dexamethasone in primary rat hepatocytes

Abstract: Our previous studies (Sidhu JS et al. Arch Biochem Biophys 1993: 301, 103-113; Sidhu JS et al. In Vitro Toxicol 1994: 7, 225-242) demonstrated the importance of culturing primary rat hepatocytes with an overlay of extracellular matrix (ECM), together with optimal media formulations (WilliamsE or Che

Conjugation of carcinogens by theta class glutathione s-transferases: mechanisms and relevance to variations in human risk.

Abstract: Conjugation of chemicals with glutathione (GSH) can lead to decreased or increased toxicity. A genetic deficiency in the GSH S-transferase mu class gene M1 has been hypothesized to lead to greater risk of lung cancer in smokers. Recently a gene deletion polymorphism involving the human theta enzyme T1 has been described: the enzyme is present in erythrocytes and can be readily assayed. A rat theta class enzyme, 5-5, has structural and catalytic similarity and the protein was expressed in the Salmonella typhimurium tester strain TA1535. Expression of the cDNA vector increased the mutagenicity of ethylene dibromide and several methylene dihalides. Mutations resulting from the known GSH S-transferase substrate 1,2-epoxy-3-(4'nitrophenoxy)propane were decreased in the presence of the transferase. Expression of transferase 5-5 increased mutations when 1,2,3,4-diepoxybutane (butadiene diepoxide), 4-bromo-1,2-epoxybutane, or 1,3-dichloracetone were added. The latter compound is a model for the putative 1,2-dibromo-3-chloropropane oxidation product 1-bromo-3-chloroacetone. These genotoxicity and genotyping assays may be of use in further studies of the roles of GSH S-transferase theta enzymes in bioactivation and detoxication and any changes in risk due to polymorphism.

Cancer genes: single and susceptibility: exposing the difference.

Abstract: 'Single' genes, necessary and sufficient to cause cancer, are contrasted with 'susceptibility' genes that are neither, but may act in the presence of specific environmental exposures to alter the chances of cancer in the host. The former are rare, are of high absolute and relative risks, have minimal dependence on exposures and therefore have low population attributable risks. A small number of such genes are well established in the literature and typically exhibit familial aggregations of disease that serve as a starting point for genetic studies. 'Susceptibility' genes, as typified by the pharmacogenetic model, are common, have low relative and absolute risk, are strongly dependent upon exposure, and may have potentially high population attributable risks. Mechanistic and epidemiologic data are suggestive but currently fall short of confirmation for these associations. Familial aggregation is not a prominent feature and epidemiological study designs with careful exposure assessment is the investigative method of choice. Both approaches require interdisciplinary expertise and benefit from advances in molecular biology.

Genetic polymorphism of cytochrome P4502E1 and risk of alcoholic liver disease in Caucasians.

Abstract: Genetic factors may be of importance in determining inter-individual susceptibility to alcoholic liver disease (ALD). Among the candidate genes which have been considered to be important are those which code for enzymes involved in alcohol metabolism. Cytochrome P4502E1 (CYP2E1) metabolizes alcohol to acetaldehyde and the hydroxyethyl radical, and is also inducible by alcohol. A Rsa I restriction fragment length polymorphism (RFLP) in the 5'-flanking region of the CYP2E1 gene has been identified by other investigators, studies showing that the mutant allele (termed c2) shows greater transcriptional activity, higher protein levels and increased activity compared with the wild-type allele (c1). We have used PCR-RFLP analysis to determine whether the frequency of these alleles differed in 95 Caucasian patients with ALD compared with 205 control subjects (comprising 58 alcoholics with no liver disease, 47 patients with non-alcoholic liver disease and 100 healthy volunteers). In controls, the frequency (0.024) of the c2 allele was similar to that previously reported in other Caucasian populations. The c2 allele frequency in patients with ALD (0.1), however, was significantly (p = 0.0003; odds ratio (OR) 4.5, 95% CI 1.9-10.9) higher than in control subjects. The findings indicate that Caucasians carrying the Rsa I c2 allele of the CYP2E1 gene may be at higher risk of developing ALD if they abuse alcohol.

Higher activity of polymorphic thiopurine S-methyltransferase in erythrocytes from neonates compared to adults.

Abstract: Thiopurine S-methyltransferase (TPMT) catalyses the S-methylation of aromatic and heterocyclic sulfhydryl compounds, including thiopurine antimetabolites (i.e. mercaptopurine and thioguanine). The activity of TPMT in erythrocytes and other tissues exhibits genetic polymorphism, which is inherited as an autosomal codominant trait. Although inheritance is the principal determinant of TPMT activity, other factors (e.g. renal function, race and thiopurine therapy) have been shown to influence erythrocyte TPMT activity. Because the TPMT polymorphism has not been established in early erythrocyte populations, and the activity of many enzymes differs in neonates, we determined the activity of TPMT in erythrocytes obtained from 60 full-term newborns. Median peripheral blood TPMT activity was 25.3 U per ml pRBC (range 9-52.8 U per ml pRBC), which was > 50% higher than race matched healthy adults (p < 0.001). Western blot analysis demonstrated higher TPMT protein content in erythrocytes from newborns compared to adults, and revealed a significant correlation between TPMT protein and TPMT activity in erythrocytes (rs = 0.63, p = 0.03). Although erythrocyte TPMT activity was significantly higher in newborns, the distribution of activity in newborns was consistent with the genetic polymorphism previously observed in adults.

An efficient strategy for detection of known and new mutations of the CYP2D6 gene using single strand conformation polymorphism analysis.

Abstract: To detect mutations in the cytochrome P450 CYP2D6 gene (CYP2D6), we developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The efficiency of the method was evaluated by analysing DNA samples from extensive metabolizers (EM) and poor metabolizers (PM) of debrisoquine. Haplotypes, alleles and mutations of CYP2D6 had previously been characterized in each individual using PCR assays, Xba I restriction fragment length polymorphism (RFLP) and sequencing. PCR-SSCP results were in complete agreement with those obtained using established methods. All previously characterized mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments allowing their identification. We further tested the efficiency of PCR-SSCP for detecting new CYP2D6 mutations. DNA from a PM subject presumed to carry an unknown non-functional mutant allele of CYP2D6 was amplified and bands with aberrant migration patterns were observed on SSCP gels. Sequence analysis of the corresponding DNA fragments revealed the causative mutations. In this way, a novel non-functional allele of the gene, carrying three previously reported mutations and a new mutation in the third exon which results in a premature termination codon, was characterized. Finally, CYP2D6 SSCP analysis was performed on DNA amplified with fluorescent primers and an automated DNA sequencer was used for SSCP analysis of products. We conclude that the PCR-SSCP approach is a powerful method of identifying simultaneously known and new mutations of the CYP2D6 gene.

Lung cancer and mutations at the polymorphic NAT2 gene locus

Abstract: The prevalence of seven point mutations on the polymorphic NAT2 gene was studied in DNA from 108 patients with histologically proved bronchogenic carcinoma and 243 healthy controls. By means of a mutation-specific polymerase chain reaction analysis, the wild-type and fourteen mutant allelic variants of the NAT2 gene were identified. The prevalence for the poor acetylator genotypes in patients and control subjects were similar, however the frequency of mutant alleles was higher in patients with lung cancer. This was attributable to an increase in the prevalence of the allelic variants 590A and 341C + 481T + 803G among patients (p < 0.05 and 0.06, respectively). These allelic variants were at increased frequency in patients with adenocarcinoma, squamous cell or small cell lung cancer. Subjects poor acetylators that are homozygous for the allelic variant 341C + 481T + 803G seem to be at increased risk to develop lung cancer (odds ratio; 95% CI = 1.75; 0.99-3.12). We conclude that the acetylator status is not a major factor in lung cancer risk, however the presence of the 341C + 481T + 803G and the 590A alleles of the polymorphic NAT2 gene may be a secondary risk factor for the development of lung cancer.

Glutathione S transferase-theta (GSTT1) genetic polymorphism among Chinese, Malays and Indians in Singapore.

Abstract: Glutathione S-transferase-theta (GSTT1) is subject to a genetic polymorphism where approximately 50% of a Caucasian population are homozygous for the null allele Because of the possible association of the polymorphism with increased cancer risk in individuals, we genotyped by polymerase chain reaction 187 normal Chinese, 167 normal Malays and 152 normal Indians from Singapore and Malaysia The proportion of Chinese, Malays and Indians with the null genotype were 58%, 38% and 16% respectively and mirrored previously reported frequencies of the GSTM1 null genotype in these populations The frequency of the combined GSTM1 and GSTT1 null genotypes among Chinese, Malays and Indians were 37%, 22% and 5% respectively The similarity with predicted frequencies indicated no interaction between the two genetic polymorphisms

CYP1A1 and CYP2E1 polymorphism and lung cancer, case-control study in Rio de Janeiro, Brazil.

Abstract: Msp I polymorphism and exon 7 Ile-Val polymorphism of CYP1A1, and Rsa I polymorphism of CYP2E1 were studied in lung cancer patients and controls in Rio de Janeiro, Brazil. Of the three polymorphisms studied, only the exon 7 polymorphism of CYP1A1 (Val-containing genotypes) had a distribution which was statistically significant in the patients and controls. The contribution of Val containing genotypes of CYP1A1 exon 7 was greater in the subpopulation of squamous cell carcinoma patients with a lower life-time smoking consumption (OR, 2.92 vs 1.97). This association is consistent with the previous findings by Kawajiri et al. and the first observation of the positive association of this locus with lung cancer in a Western population (Kawajiri K, Nakachi K, Imai K, Yoshii A, Shimada N, Watanabe J. FEBS Let 1990; 263, 131-133). Furthermore, together with the lack of association of Msp I polymorphism in the non-coding region of CYP1A1, the locus truly responsible for lung cancer risk among pleural polymorphisms of CYP1A1 appeared to be exon 7 Ile-Val polymorphism. In the future, investigations of multiple markers in different ethnic populations may reveal cancer risk markers common to all mankind.

Primary structures and properties of two related forms of aryl sulfotransferases in human liver.

Abstract: cDNAs and genes of ST1A2 and ST1A3 section were isolated from human liver cDNA and genomic libraries and shown to encode arylsulfotransferases. Studies on their mRNAs and proteins expressed indicate the existence of two distinct phenol-sulfating enzymes which are involved in the metabolic activation of carcinogenic arylamines in human livers.

Interindividual variability in the glucuronidation of (S) oxazepam contrasted with that of (R) oxazepam.

Abstract: Although conjugation with glucuronic acid is a major process for converting many xenobiotics into hydrophilic, excretable metabolites, relatively little has been reported concerning interindividual variability of glucuronidation in human populations. Oxazepam, a therapeutically active metabolite of diazepam, is one of a number of C3-hydroxylated benzodiazepines for which glucuronide conjugation is the predominant pathway of biotransformation. The drug is normally formulated as a racemic mixture of inactive (R) and active (S) enantiomers. In the present study we have investigated the use of oxazepam as a potential probe drug for studying the variability of glucuronide conjugation, and for demonstrating the extent to which genetic factors may be responsible. In preliminary studies we determined oxazepam pharmacokinetics metabolite profiles after administration of racemic (R,S) oxazepam to eleven human volunteers. The (S) glucuronide was preferentially formed and excreted in nine of the eleven subjects. The ratios of (S) to (R) glucuronide metabolites (S/R ratios) were 3.87 +/- 0.79 (mean +/- SD) and 3.52 +/- 0.60 in urine and plasma, respectively. However, both ratios were significantly lower in two subjects (p < 0.01). In these two atypical subjects, the half-life of (R,S) oxazepam was also markedly longer (14.7 and 15.9 h) than in the other subjects (8.1 +/- 3.2 h). A good correlation (rs = 0.90) between the S/R-glucuronide ratio in urine and the plasma clearance of (R,S) oxazepam suggested that a low S/R ratio may be a marker of poor elimination of oxazepam. In further investigations, the drug was administered to 66 additional subjects. The S/R-glucuronide ratio in 8 h pooled urine was bimodally distributed, with 10% of all subjects possessing ratios below an apparent antimode of 1.9. A survey of the in vitro formation of oxazepam glucuronides by microsomes from 37 human livers also showed that 10% of the livers displayed an abnormally high apparent Michaelis constant (Km) for the formation of the (S) glucuronide, but not of the (R) glucuronide. These results suggest that the glucuronidation of the pharmacologically active (S) enantiomer of oxazepam is decreased in a significant percentage (10%) of Caucasian individuals. The observed in vitro differences in apparent kinetics of the S-glucuronidation reaction may reflect defects at the genetic level, leading to structural changes in the isozyme(s) of UDP-glucuronyltransferase that catalyse this reaction.

Phenotyping of CYP2C19 with enantiospecific HPLC-quantification of R- and S-mephenytoin and comparison with the intron4/exon5 G-->A-splice site mutation.

Abstract: S-Mephenytoin 4'-hydroxylase (CYP2C19) is a genetically polymorphic cytochrome P450. A modified method for CYP2C19 phenotyping was evaluated in 174 healthy German volunteers and the results were compared with genotyping for the intron4/exon5 G-->A splice site mutation (m1) of CYP2C19, associated with the poor metabolizer (PM) phenotype. A smaller than usual test-dose of 50 mg (R,S)-mephenytoin was used and urine samples were collected from 0 to 5 h and from 5 to 8 h after administration. Trait measurements included the mephenytoin S/R enantiomeric ratio and the hydroxylation index (i.e. the molar ratio of 4'-hydroxy-mephenytoin urinary recovery to the administered S-mephenytoin dose). S- and R-mephenytoin were quantified by isocratic HPLC with a Chiraspher column and 80% n-hexane and 20% dioxane as the mobile phase. All individuals from whom DNA was available (n = 140, including six phenotypically identified PMs) were analysed for the m1 mutation. The population frequency of this CYP2C19 mutation was 0.15. Four individuals were homozygous for m1 having S/R ratios of 0.9 or greater in both intervals of urine collection. Thus, individuals with an S/R ratio > or = 0.9 were classified as PMs and seven of all 174 phenotyped individuals were PMs (4%; 95% confidence limits: 1.6-8.1%). Heterozygous carriers of m1 (n = 34) had a median S/R ratio (5-8 h urine) of 0.06 compared to 0.01 in individuals without this mutation (n = 102; p = 0.0005, Mann-Whitney U-test). No such gene-dose relation was apparent with the hydroxylation index.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic polymorphisms of drug-metabolizing enzymes and lung cancer susceptibility.

Abstract: A close association of smoking-associated lung cancer incidence with the Msp 1 and 1le-Val polymorphisms of CYP1A1 gene was found in a Japanese population in terms of genotype frequency comparison and cigarette dose response. A synergistic increase in susceptibility to lung cancer was observed when the susceptible genotypes of CYP1A1 were combined with a deficient GSTM1 genotype. Individual difference in expression levels of Ahr and Arnt mRNAs was observed, and the expression levels of CYP1A1 appeared to associate with those of transcriptional factors. The Ahr protein has two different structures, ascribed to one amino acid replacement at codon 554 of Arg by Lys. However, this germ line polymorphism did not show a significant association with AHH inducibility nor lung cancer incidence. The p53 gene alterations in lung cancer tissues were more frequently observed among the patients with a susceptible allele of CYP1A1 gene.