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CHARACTERISATION AND PCR-BASED DETECTION OF A CYP2A6 GENE DELETION FOUND AT A HIGH FREQUENCY IN A CHINESE POPULATION

Cytochrome P450 2A6 is an important human hepatic P450 which activates pre‐carcinogens, oxidises some drugs and constitutes the major nicotine C‐oxidase. In fact, results have been presented in the literature which suggested a relationship between the distribution of defective CYP2A6 alleles and smoking behaviour as well as cigarette consumption. In the present report, we describe the structure of a novel CYP2A locus where the whole CYP2A6 gene has been deleted, resulting in an abolished cytochrome P450 2A6‐dependent metabolism. The origin of this locus is apparently due to an unequal crossover event between the 3′‐flanking region of the CYP2A6 and CYP2A7 genes. A rapid PCR‐based method for the detection of the CYP2A6del allele was developed and the allele frequency was 15.1% among 96 Chinese subjects, but only 1.0% in Finns (n=100) and 0.5% in Spaniards (n=100). In the Chinese population, we did not detect any CYP2A6*2 alleles using an improved genotyping procedure, in contrast to the 11–20% previously reported. It is concluded that genotyping for the CYP2A6del allele is of great importance in studies correlating, for example, smoking behaviour, pre‐carcinogen activation or drug metabolism to the CYP2A6 genotype, in particular when oriental populations are investigated.

Genotyping of human cytochrome P450 2A6 (CYP2A6), a nicotine C-oxidase

Cytochrome P450 2A6 (CYP2A6) is a polymorphic enzyme responsible for the oxidation of certain precarcinogens and drugs and is the major nicotine C‐oxidase. The role of CYP2A6 for nicotine elimination was emphasised recently by the finding that smokers carrying defective CYP2A6 alleles consumed fewer cigarettes [Pianezza et al. (1998) Nature 393, 750]. The method used for CYP2A6 genotyping has, however, been found to give erroneous results with respect to the coumarin hydroxylase phenotype, a probe reaction for the CYP2A6 enzyme. The present study describes an allele‐specific PCR genotyping method that identifies the major defective CYP2A6 allele and accurately predicts the phenotype. An allele frequency of 1–3% was observed in Finnish, Spanish, and Swedish populations, much lower than described previously.

Ten percent of North Spanish individuals carry duplicated or triplicated CYP2D6 genes associated with ultrarapid metabolism of debrisoquine.

Ten percent of North Spanish subjects carry duplicated or triplicated CYP2D6 genes associated with ultrarapid metabolism of debrisoquine.

Frequency distribution of C3435T mutation in exon 26 of the MDR1 gene in a Spanish population.

P-glycoprotein (PGP) is a transmembrane efflux transporter with an important role in drug therapy. The level of PGP expression leads to relevant consequences in terms of efficacy and toxicity by modulating drug disposition. A single nucleotide polymorphism (SNP) in exon 26 of the MDR1 gene C3435T was recently associated to PGP levels and substrate uptake. Persons who were homozygous for the T-allele had significantly decreased PGP expression compared with C/C persons. Due to this fact and bearing in mind the important differences among populations regarding the frequencies of persons carrying mutations affecting drug disposition, the authors wanted to study the prevalence of this genetic trait in their population. DNA samples from 408 persons were assayed by a PCR-RFLP method. The results showed that the distributions of the C/C, T/T, and C/T genotypes in the Spanish population were 26%, 22%, and 52%, respectively. With regard the C-allele frequency, which has been studied in several populations, the result in their population was 52%, significantly lower (P < 0.0001) than that found in African populations and similar to several Asian and Caucasian (UK) populations (P > 0.05). By contrast, the C-allele frequency in southwest Asian, German, and Portuguese populations was significantly lower than in the Spanish population (P < 0.001, P < 0.01, and P < 0.05, respectively). The great differences found between their population and others, such as the African and southwest Asian populations, could have important therapeutic implications when drugs that are a substrate of PGP are used.

Sister chromatid exchanges, proliferating rate index, and micronuclei in biomonitoring of internal exposure to vinyl chloride monomer in plastic industry workers

The frequency of micronuclei (MN), sister chromatid exchange (SCEs), and the proliferating rate index in peripheral blood lymphocytes from 93 individuals were measured. Fifty-two of the individuals were workers in the plastics industry where they were exposed to vinyl chloride monomer while the remaining 41 individuals served as a control group. In our results, an increase of SCEs and MN, as well as inhibited cell kinetics, was observed in the group of exposed workers. Of the tests used, SCE was found to be the most sensitive endpoint for indicating a biological response. However, since methods for restricting the MN analysis to only cells at risk (i.e., second generation interphase cells) were not used, this statement requires verification.

CYP3A5*3 and CYP3A4*1B allele distribution and genotype combinations: differences between Spaniards and Central Americans.

The aim of this study was to detect genotypic differences between three populations of healthy volunteers from Northern Spain (204 subjects), Nicaragua (120 subjects), and El Salvador (112 subjects) regarding CYP3A4*1B and CYP3A5*3 polymorphisms. No significant differences were found by comparing allelic frequencies between the two Central American populations. The CYP3A5*3 allele frequency was significantly different (P < 0.01) between Central Americans (76%) and Spaniards (91%). By contrast, CYP3A4*1B allele was more prevalent among Central Americans (12.5%) than among North Spaniards (4%) (P < 0.01). Analysis of CYP3A4-3A5 genotype combinations revealed that individuals carrying CYP3A4*1B/CYP3A5*1 were more represented in Central Americans (16.9%) than in Spaniards (5.4%), suggesting a marked linkage disequilibrium. These data are compatible with a higher CYP3A enzyme activity in Central Americans as opposed to Spaniards and other white groups, which could imply differences in dose requirements for drugs metabolized by CYP3A and should be considered in allele-disease association studies.

CYP2A6 activity in a healthy Spanish population: effect of age, sex, smoking, and oral contraceptives

This study was performed to assess the influence of age, sex, smoking, and contraceptive use on CYP2A6 activity. In the metabolism of caffeine, the conversion of 1,7 dimethylxanthine (17X) to 1,7 dimethiylurate (17U) is catalyzed primarily by CYP2A6. CYP2A6 phenotype was determined by the urinary ratio 17U:17X in the interval of 4–5 h after caffeine intake in 179 healthy white Spaniards (102 women and 76 men). There were 99 non-smokers and 80 smokers. Among women, 26 were taking oral contraceptives. The age was the most important predictive factor of CYP2A6 activity (P < 0.001) with older subjects having higher activity. The influence of the gender was more modest (P = 0.07) with women exhibiting borderline increased values of the CYP2A6 marker than men. Tobacco smoking did not affect CYP2A6 activity. However, the CYP2A6 marker resulted to be strongly related to the use of oral contraceptives. The women users of oral contraceptives had higher values of CYP2A6 marker than both women not taking oral contraceptives and men (P < 0.001 in both comparisons). The results indicate that age, oral contraceptive use, and possibly gender should be controlled in epidemiological studies dealing with CYP2A6 activity and its relationship with xenobiotics exposure and genetic or pathological factor.

Micronucleus assay in biomonitoring of patients undergoing excretory urography with diatrizoate and ioxaglate

In order to perform biological monitoring of exposure to radiation and contrast media, we evaluated the micronucleus count (MN) and the mitotic index (MI) in peripheral blood lymphocytes from patients undergoing excretory urography with diatrizoate (20 patients) and ioxaglate (20 patients). Three samples were taken for each patient: A (before exploration), B (immediately after exploration) and C (7 days later). There were no significant differences in the radiation doses received, nor in the dose of contrast agent, between both groups. The micronucleus count increased significantly in sample B in both groups, the increase being more statistically significant in the diatrizoate group (p less than 0.01) than in the ioxaglate group (p less than 0.05). One week later, the MN were still slightly high (p less than 0.05) in the diatrizoate group only. These results suggest a clastogenic effect which depends, to a great extent, on the nature of the contrast medium.

Haplotype structure and allele frequencies of CYP2B6 in Spaniards and Central Americans

This study was aimed to investigate the potential differences in allele frequencies of the CYP2B6 gene between Spaniards and Central Americans. Three single nucleotide polymorphisms of the CYP2B6 gene 516 G>T, 785 A>G and 1459 C>T were assayed by a polymerase chain reaction in 180 Spaniards and 182 Central Americans. The allele frequencies for CYP2B6*1, CYP2B6*4, CYP2B6*5, CYP2B6*6, CYP2B6*9 in Spaniards and Central Americans were 0.593 and 0.642, 0.062 and 0.073, 0.113 and 0.030, 0.215 and 0.230, 0.014 and 0.023, respectively. CYP2B6*5 was less prevalent among Central Americans than in Spaniards (P < 0.001). In comparison to other previously studied populations, the CYP2B6*5 allele frequency among Spaniards was similar to other Caucasian or African groups, and higher than that in Asian populations. The CYP2B6*5 allele frequency in Central Americans was lower than that in Africans or Caucasian groups and higher than in Asians. The results indicate the presence of ethnic differences in CYP2B6 genetic variants between Spaniards and Central Americans, and support the need for further investigations to explore whether these differences significantly alter the efficacy or toxicity of CYP2B6 substrate drugs.

Six mutagenicity assays in exposure biomonitoring of patients receiving carbamazepine for epilepsy or trigeminal neuralgia

The mutagenic potential of carbamazepine (CBZ) therapy has been studied in 37 patients undergoing long-term treatment with this drug. Of the total group, 23 patients suffered from epilepsy and 14 from trigeminal neuralgia. Thirty-one healty subjects served as controls. Six mutagenicity assays with different end-points were performed. The possible cytogenetic alterations were evaluated by analyzing sister-chromatid exchange frequencies (SCE), chromosome aberrations (CA), micronuclei (MN), proliferation indices (PRI), and mitotic indices. The Salmonella assay with and without microsomal activation served to measure urinary mutagenicity. The results show that CBZ leads to an increase in SCE (p < 0.01) and PRI (p < 0.05) but had no effect on the other cytogenetic parameters. CBZ was negative in the urine mutagenicity test. Plasma levels of total CBZ, free CBZ and CBZ-10,11-epoxide did not correlate with the cytogenetic alterations. Even though folic acid and gamma-glutamyltranspeptidase were significantly different in patients and controls, there was no significant association between these values and SCE or PRI. Patients with epilepsy and those with trigeminal neuralgia did not differ with respect to the end-points analyzed.