摘要:

A pronounced variability limits the usefulness of CYP1A2 phenotyping for drug therapy, for evaluating liver function, and for assessing the role of this enzyme in carcinogenesis. To identify and quantify sources of this variation, we estimated CYP1A2 activity in 863 healthy Caucasians using caffeine clearance derived from saliva concentrations before and 5–7 h after a caffeine test dose. Data from 786 individuals were eligible for evaluation (mean age 39 years, 415 women including 94 taking oral contraceptives, 401 non-smokers). Overall geometric mean (geometric SD) caffeine clearance was 1.34 ml min-1kg b.w.-1(1.65). The effect of the following covariates was evaluated by analysis of covariance: age, sex, oral contraceptives, body height, body weight, body mass index, number of cigarettes smoked, tar exposure from smoking, several indices of dietary caffeine consumption, intake of sauerkraut, and country of residence (Germany, Bulgaria or Slovakia). Estimated changes relative to arbitrarily defined basal caffeine clearance (male, nonsmoking, German resident) exerted by significant (P< 0.05) covariates were: coffee, 1.45-fold per litre of coffee drunk daily; body mass index, 0.99-fold per kg m-2; smoking, 1.22-fold, 1.47-fold, 1.66-fold, and 1.72-fold for 1–5, 6–10, 11–20, and > 20 cigarettes smoked per day, respectively; oral contraceptives, 0.72-fold; country of residence, 0.81-fold and 0.74-fold for Bulgaria and Slovakia, respectively; female, 0.90-fold. These covariates explained 37% of overall variation. The 95% confidence interval of individual clearance was 0.46–2.20 times the predicted value. No relevant polymorphism was found for CYP1A2 activity when adjusted for covariate effects.

ISSN:0960-314X    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

We studied the phenotype and the nucleotide sequence for the cDNAs of theAldh3a1aandAldh3a1callelic forms of the dioxin-inducible cytosolic aldehyde dehydrogenase (ALDH3A1) present in inbred mouse strains. This gene is constitutively expressed in cornea, stomach, skin, urinary bladder and lungs. TheAldh3a1aallele is found in most inbred mouse strains and codes for a ‘high-activity’ corneal enzyme compared to the ‘low-activity’ encoded by theAldh3a1callele in SWR/J strain. The ‘low-activity’ variant is associated with extensive corneal clouding after a single exposure to ultraviolet light. The ALDH3A1 phenotype was examined in tissues from inbred mouse strains carrying theAldh3a1aallele including, SJL/J, C57BL/6 J/Ibg, DBA/2 J/Ibg, C3H/Ibg and theAldh3a1callele (SWR/J). Only trace levels of ALDH3A1 activity were found in all SWR/J tissues. All other strains had significant levels of ALDH3A1 activity in eye, stomach, skin, less in urinary bladder and lungs and only trace amounts in liver. However, no differences were found in corneal and stomach ALDH3A1 mRNA levels between the ‘low-’ and ‘high-activity’ variants. A 1556-bp ALDH3A1 cDNA fragment, containing the entire coding region plus 5'and 3' untranslated regions, was amplified by reverse transcriptase-polymerase chain reaction from SWR/J and DBA/2 J/Ibg mouse strains. Sequence analysis revealed 13 nucleotide changes in theAldh3a1callele. Four of these changes result in G88R, I154N, H305R and 1352V substitutions, whereas nine changes are silent. The I154N disrupts a potential α helix, which belongs to the Rossmann fold. Replacement of Arg with the more ionizable His at position 305 of a β strand might directly affect catalytic activity of the enzyme. It is likely that structural changes associated with these amino acid changes are responsible for the loss of ALDH3A1enzymatic activity in SWR/J mice.

ISSN:0960-314X    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Flavin-containing monooxygenase (FMO) activity was determined in 82 Korean volunteers by taking molar concentration ratio of theobromine and caffeine present in the 1 h urine (between 4 and 5 h) samples collected after administration of a cup of coffee containing 110 mg of caffeine. Among 82 volunteers, there were 19 women and 63 men (30 smokers and 52 nonsmokers). Volunteers were divided into two groups comprising low (0.53–2.99) and high (3.18–11.95) FMO activities separated by an antimode of 3.18. Peripheral bloods were sampled from these volunteers and their genomic DNAs were amplified by polymerase chain reaction with oligonucleotides designed from intronic sequences of humanFMO3gene. Comparing nucleotide sequences of the amplifiedFMO3gene originating from randomly selected individuals with low and high FMO activities, nine point mutations were identified in the open reading frame sequences. Among these nine mutations, three FMO3 mutant types (FMO3/Stop148, Lys158and Gly308) were selected and correlated with FMO activities observed in our Korean population. A rare FMO3/Stop148mutant allele originating from FMO3/GIy148occurred by substitution of G442T in exon 4 and yielded a premature TGA stop codon. The stop codon was detected in one individual having the second lowest FMO activity and he had the mutation in heterozygous state. In a pedigree study, he was found to have inherited the mutation from his mother who also had a heterozygous stop codon and equally low FMO activity. In our volunteers, two other common mutations were detected in exons 4 and 7. The one in exon 4 resulted from a G472A change eliminating aHinfIrestriction site and produced an amino acid substitution from Glu158 to Lys. The other mutation in exon 7 resulted from an A923G change generating aDraIIrestriction site and produced a non-conservative replacement of Glu308to Gly. Based on the secondary structure maps of FMO3 enzyme proteins for these two mutant types, FMO3/Gly308mutation transformed the helix structure into a sheet shape and indicated that dysfunctional FMO3 may be produced. FMO3/Lys158mutation did not alter the secondary structure. Approximately 80% of volunteers with homozygous and/or heterozygous mutations on either one or two of these mutations had low FMO activities. Thus, individuals with theseFMO3gene mutations may have defective metabolic activity for many clinically used drugs and dietary plant alkaloids which are oxidized primarily by hepatic FMO3.

ISSN:0960-314X    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The A/G polymorphism at nucleotide 313 in the glutathioneS-transferase P1-1 (GSTP1) gene was examined in patients with different types of smoking-related cancers (oral, lung, gastric, colorectal and urothelial cancers) and healthy control individuals. This polymorphism results in an amino acid substitution from isoleucine to valine at residue 105, which reduces catalytic activity of the enzyme. In control individuals, 23.8% of individuals hadGSTP1AGorGGgenotype. This rose to 37.3% [n= 83, odds ratio = 1.93 (1.05–3.58),P= 0.035] in oral cancer patients. No increase in the frequency of theGSTP1 AGorGGgenotype was obtained in lung, gastric, colorectal or urothelial cancers in this Japanese population. After grouping by smoking status, no consistent difference was observed between smoking patients and corresponding control individuals for the frequency of the GSTP1 A/G polymorphism for any cancer. However, a moderate risk (odds ratio = 2.78; 95% confidence interval 1.06–7.51) was associated with this polymorphism in the non-smoking group of oral cancer patients. The results suggest theGSTP1polymorphism at nucleotide 313 may be associated with susceptibility to oral squamous cell carcinoma in the Japanese population.

ISSN:0960-314X    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Following neonatal exposure tod-methamphetamine, adult rats have previously been shown to exhibit augmented acoustic startle and spatial learning deficits.d-Methamphetamine is structurally similar to several phenylethylamines that are metabolized byCYP2D6. In humans, allelic differences in theCYP2D6confer the extensive or poor metabolizer phenotype for the more than three dozen drugs that are members of theCYP2D6-mediated ‘debrisoquine/sparteine panel.’ An analogous genotype exists with theCYP2D2gene in rats. Female Dark Agouti rats show the poor metabolizer phenotype, whereas Sprague-Dawley rats show the extensive metabolizer phenotype; male Dark Agouti rats are intermediate. We sought to test the possibility that these strains might exhibit alteredd-methamphetamine-induced developmental neurotoxicity. Dark Agouti and Sprague-Dawley litters (11–20 days of age) were givend-methamphetamine or vehicle alone subcutaneously twice daily (15 mg/kg). Offspring were assessed as adults (beginning at 50 days of age) on acoustic startle, straight-channel swimming, and spatial learning and memory in a Morris hidden platform maze. Increases in d-methamphetamine-induced acoustic startle were found in both male and female Dark Agouti rats, but not Sprague-Dawley rats. In the Morris maze,d-methamphetamine-induced spatial navigation deficits were found in both strains among males, suggesting some mechanism other than theCYP2D2polymorphism. In contrast, among females only thed-methamphetamine-treated Dark Agouti rats showed deficits in spatial navigation. The maze deficits in Dark Agouti females, and enhanced acoustic startle in Dark Agouti females and males, support the hypothesis that theCYP2D2poor metabolizer phenotype confers increased vulnerability tod-methamphetamine-induced developmental neurotoxicity, indicating that the parent drug rather than a CYP2D2-mediated metabolite is responsible for this behavioural defect – which occurs in adults who had been exposed tod-methamphetamine during the neonatal period.

ISSN:0960-314X    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

We show here that DBA/2 strain mice have a complex mutation/polymorphism in the promoter region of theTrp53locus (the mouse p53 locus). This region has previously been shown to be essential for p53 expression. We further show that the DBA/2 mutation is associated with approximately fourfold lower p53 levels in thymocytes treated with the DNA-damaging agent etoposidein-vitro, and with relative resistance of these thymocytes to apoptosis induced by the DNAdamaging agent etoposide compared with C57BL/6 mice. When part of the promoter containing this mutation was inserted into a plasmid containing a luciferase reporter gene but lacking eukaryote promoter sequences and transfected into MCF-7 human breast cell line cells, the mean luciferase activity was slightly less with the DBA/2 than with the C57BL/6 promoter-reporter construct (p< 0.01). We found that DBA/2xC57BL/6 F2hybrid mice with the DBA/2 genotype at theTrp53locus were relatively resistant to tetrachlorodibenzo-p-dioxin toxicity, and this resistance was additive with resistance associated with theAhrlocus. DBA/2 mice are long-lived and do not have particularly high levels of cancer, suggesting either that they carry other compensatory tumour resistance alleles (such asAhrd), or that, while there may be a p53 protein dosage effect for acute toxicity, lower than normal levels of p53 may still be sufficient to protect against cancer. In evolutionary terms, it may be better to maintain low levels of p53 in order to avoid death from acute toxicity, even at the expense of a higher incidence of cancer in later life.

ISSN:0960-314X    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Characterization of the genetic polymorphism of thiopurineS-methyltransferase enzyme (TPMT; EC 2.1.1.67) is required because of its clinical importance for patients exposed to thiopurine drugs. A number of point mutations have already been characterized in exons and introns of theTPMTgene. Here we report the identification of a polymorphic locus within the promoter region of the gene. This polymorphism was detected by polymerase chain reaction - single strand conformation polymorphism analysis of DNA samples from 54 unrelated European individuals. A total of five alleles with length variations were distinguished through the 5'-flanking region involved in theTPMTgene expression. Sequence analysis revealed that these variations were due to a variable number of tandem repeats (VNTR), ranging from four to eight repeats. Each repeat consists of 17 or 18 bp units and contains putative binding sites for transcription factors. The most frequent alleles harbour four or five tandem repeats, a heter ozygosity rate of 0.44 was calculated, and a stable Mendelian inheritance of alleles was demonstrated. Analysis of the effect of eachVNTRallele on promoter activity of a reporter gene was further performed in various cell lines by transient transfection assay. A modulatory effect of VNTR alleles was observed inin vitro, but the repeat polymorphism did not display a significative role inTPMTgene regulationin vivo. Further studies need to be carried out to support the hypothesis that VNTR may contribute to the large interindividual variations of TPMT activity.

ISSN:0960-314X    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The functional analysis of expressed human gene variants is important in the study of genetic susceptibility to diseases, pharmacogenetic traits and for the investigation of the human genetic diversity at the molecular level. We have performed the analysis of sequence polymorphisms in the human D5dopamine receptor gene (DRD5) predicting missense and nonsense amino acid changes in the receptor protein. The amino acid substitutions in the human D5dopamine receptor are: Leu88to Phe in the putative second transmembrane domain, Ala269to Val in the third intracellular and Pro330to Gln in the third extracellular loops, Asn351to Asp in the seventh transmembrane and Ser453to Cys in theC-terminal domains and Cys335to Stop in the third extracellular loop. The two amino acid substitutions in the transmembrane domains had an effect on agonist binding to the human D5dopamine receptor. Asn351to Asp resulted in an approximately 10-fold decrease in dopamine and threefold decrease in R(+)-SKF-38393 binding affinities. Leu88to Phe resulted in a small increase in dopamine binding affinity. Antagonist binding affinities were mostly unaffected by the amino acid substitutions with the exception of Leu88to Phe, which showed small reductions in binding affinities of SCH-23390 and risperidone. The existence of functionally different variants of the human dopamine receptors might have phenotypic consequences given their importance in central nervous system physiology and pharmacology.

ISSN:0960-314X    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

Variations in glucuronidation activities among different individuals have been reported; however, genetic polymorphisms in the genes encoding phase II drug metabolizing UDP-glucuronosyltransferases have not been studied extensively. A novel UGT2B cDNA clone UGT2B4(E458) was isolated from human prostate and LNCaP cell cDNA libraries. The cDNA encoding UGT2B4(E458) is 2097 bp in length and has an open reading frame of 1584 nucleotides encoding a protein of 528 amino acids. Characterization of the UGT2B4 (E458) cDNA revealed nucleotide differences with the previously published UGT2B4 and UGT2B11 cDNAs. These variations in the UGT2B4 sequence lead to an amino acid change from aspartic acid to glutamic acid at position 458. In the previous UGT2B11 cDNA (which has subsequently been renamed UGT2B4 (L109,396, D458)), leucine residues are found at positions 109 and 396, whereas phenylalanines are present at these positions in the UGT2B4(D458) and UGT2B4(E458) enzymes. Analysing the genomic DNA of 26 unrelated Caucasian individuals demonstrated the presence of variant alleles encoding UGT2B4(D458) and UGT2B4 (E458). Stable expression of UGT2B4(E458) cDNA in HK293 cells demonstrates the presence of a 52 kDa protein, which is in agreement with other characterized UGT2B proteins. UGT2B4 (E458) conjugates hyodeoxycholic acid (HDCA) as well as 4-hydroxyestrone (4-OH-E1), androstane-3α,17β-diol (3α-diol) and androsterone (ADT). Specific reverse transcriptase - polymerase chain reaction analysis revealed expression of UGT2B4(D458) and UGT2B4(E458) transcripts in a wide range of extrahepatic tissues, including the liver, kidney, testis, mammary gland, prostate, placenta, adipose, adrenal, skin and lung. Our results suggest that UGT2B4(E458) and UGT2B(E458) are two widely expressed isoenzymes, and that polymorphism in theUGT2B4gene might be responsible for differences in UGT2B4 enzymatic properties.

ISSN:0960-314X    

出版商:OVID    

年代:1999

数据来源: OVID

摘要:

The development of CYP2D6 has been attributed to the need of earth-dwelling animals to detoxify toxic xenobiotics (phytoalexins) present in plants. This hypothesis has been extrapolated to humans, but is yet unconfirmed. Therefore, we studied two Amerindian populations as the best available model to test the effect of selection through diet on human CYP2D6 evolution. The frequency of sparteine poor metabolizers in Ngawbe was 4.4% (n= 344), while the frequency in Embera was 2.2% (n= 153). Among Ngawbe and Embera,CYP2D6*4(allelic frequencies for each tribe, respectively: 0.171; 0.14),CYP2D6*6(0.005; 0.011) andCYP2D6*10(0.175; 0.069) were detected, whileCYP2D6*3, CYP2D6*5, CYP2D6*9and CYP2D6*16 were absent. All poor metabolizers possessed eitherCYP2D6*4 or CYP2D6*6and there were no disagreements between genotypic and phenotypic data. The total frequency of mutant alleles showed no difference among Amerindians or when compared to Caucasians. It was higher than in Chinese, since the frequency ofCYP2D6*4was higher in Amerindians.Xbalrestriction fragment length polymorphisms haplotypes were very homogeneous in Amerindians, because the only fragment that hybridized with the CYP2D6 cDNA probe was the 29 kb (not 42/44 kb or 11.5/13 kb). This indicated no gene cluster recombinations that generate insertions or deletions. We propose that in earlier hominids and humans, CYP2D6 had increasingly become a vestigial characteristic unconstrained by dietary stressors, as a result of cultural survival strategies. Human CYP2D6 evolution was preferentially affected by random genetic drift, and not by adaptive or purifying selection.

ISSN:0960-314X    

出版商:OVID    

年代:1999

数据来源: OVID