Ana Fanlo

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.

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.

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.

Influence of the urine flow rate on some caffeine metabolite ratios used to assess CYP1A2 activity

Five established metabolite ratios (MRs) to measure P450 CYP1A2 activity—MR1 (17X + 17U)/137X, MR2 (AFMU + 1X + 1U)/17U, MR3 (17X/137X), MR4 (AFMU + 1X + 1U + 17X + 17U)/137X, and MR5 (AFMU + 1X + 1U)/17X—were calculated in urine 4–5 hours after caffeine intake. First, to assess the potential of omeprazole to induce CYP1A2 activity, a caffeine test was performed in 27 subjects on two occasions: before and after 14 days on omeprazole (20 mg/day). Samples of urine were analyzed by high-performance liquid chromatography (HPLC) to quantify caffeine and metabolites used to calculate the different caffeine MRs. MR1, MR3, and MR4 were enhanced after treatment; the percentage of change was inversely associated with that of the urine flow, with r values of −0.48, −0.49, and −0.47, respectively. However, MR2 or MR5 were not modified. To determine the reason for these contradictory results, the authors analyzed data of metabolites, ratios, and their components (numerators and denominators) from 152 subjects (who underwent one caffeine test) and their relationship with the urinary flow. Caffeine concentration in urine was the only compound nondependent on the urine flow. Consistently, ratios containing caffeine (MR1, MR3, and MR4) were highly influenced by the rate of urine excretion, since the flow dependence of their numerators is not canceled out by that of caffeine in their denominators. The dependency of the caffeine excretion on renal factors may explain the opposite results found with the different ratios in the aforementioned prospective study of drug interaction, the absence of closer correlations of the five MRs to each other, the discrepancies about the type of frequency distribution of the different MRs (either normal or multimodal), and the higher sensitivity of MR2 to detect gender differences in CYP1A2 activity found in this study. In summary, the data clearly emphasize the need for a strict control of the liquid intake to avoid high urine flows when MRs containing caffeine are used to assess CYP1A2 activity, especially in studies of drug interactions.

Urinary Mutagenicity, CYP1A2 and NAT2 Activity in Textile Industry Workers

Urinary Mutagenicity, CYP1A2 and NAT2 Activity in Textile Industry Workers: Ana Fanlo, et al. Department of Pharmacology and Physiology, University of Zaragoza, Spain—The two major causes of bladder cancer have been recognised to be cigarette smoke and occupational exposure to arylamines. These compounds are present both in tobacco smoke and in the dyes used in textile production. Aromatic amines suffer oxidative metabolism via P450 cytochrome CYP1A2, and detoxification by the polymorphic NAT2. The aim of the present work was to assess the association between occupational‐derived exposure to mutagens and CYP1A2 or NAT2 activity. This cross‐sectional study included 117 textile workers exposed to dyes and 117 healthy controls. The urinary mutagenicity was determined in 24 h urine using TA98 Salmonella typhimurium strain with microsomal activation S9 (MIS9) or incubation with β‐glucuronidase (MIβ). Urinary caffeine metabolite ratios: AFMU+1X+1U/17U, and AFMU/AFMU+1X+1U were calculated to assess CYP1A2 and NAT2 activities, respectively. The results show that workers present a strikingly higher urine mutagenicity than controls (p<0.0001), despite the implementation of the new restrictive norms forbidding the industrial use of the most carcinogenic arylamines. Neither NAT2 nor CYP1A2 activity had any effect on the markers of internal exposure to mutagens, since no significant differences were observed when the urinary mutagenicity of slow and fast acetylators (p>0.05) was compared, and the urinary mutagenicity was not significantly associated with the CYP1A2 activity marker (r=0.04 and r=–0.01 for MIS9 and MIβ, respectively). This study clearly indicates the need for further protective policies to minimise exposure to the lowest feasible limit in order to avoid unnecessary risks.

Differences between Spaniards and Ecuadorians in CYP2A6 allele frequencies: comparison with other populations

This study was designed to investigate the potential differences between Spaniards and Ecuadorian Mestizo people regarding CYP2A6*1A, CYP2A6*1B1, CYP2A6*1x2A, CYP2A6*9A, and CYP2A6*4A variant alleles at the CYP2A6 gene and also to compare the observed frequencies with those previously reported in different ethnic groups. DNA from 234 Spaniard and 300 Ecuadorian subjects were analyzed by either PCR or PCR‐restriction fragment length polymorphism. Differences between Spaniards and Mestizo Ecuadorians were detected in relation to the frequencies of the alleles linked to either absent enzyme activity, CYP2A6*4A (4 and 7.1%, respectively), or reduced CYP2A6 enzyme activity, CYP2A6*9A (6.4 and 10.3%, respectively). CYP2A6*4A and CYP2A6*9A frequencies in Ecuadorians were higher than those in Africans or Caucasian groups and lower than those in Asians. This study provides, for the first time, the result of the analysis of CYP2A6 allele frequency in a South American population and demonstrates the presence of ethnic differences in CYP2A6 genetic variants between Spaniards and Mestizo Ecuadorians, which should be considered in allele–disease association studies and, in particular, in those involving CYP2A6 genetic polymorphisms and tobacco‐related cancer.

Omeprazole treatment: genotoxicity biomarkers, and potential to induce CYP1A2 activity in humans.

Omeprazole is one of the most used acid-suppressing medications. This fact emphasizes the questions concerning the safety of this compound. Healthy volunteers (n-33) were included in this prospective study. All study subjects were analysed for their CYP2C19 genotype. Of the 33 individuals, 24 were homozygous for the wild type CYP2C19*1 allele, 7 were heterozygous for theCYP2C19*2 variant allele, and 2 were homozygous for the CYP2C19*2 variant allele. Before and after 14 days of omeprazole treatment at a daily dose of 20 mg, one blood sample was taken from each individual to determine five cytogenetic biomarkers of genotoxicity: chromosome aberrations, micronuclei, proliferating rate index, sister chromatid exchanges, and mitotic index. The only significant change was that of a weak increase in micro nuclei count after treatment in relation to baseline values (day 0) (P-0.026). To assess the potential of omeprazole to induce P450 CYP1A2, the urinary ratio AFMU-1X-1U/17U in the interval of 4 / 5 hours after caffeine intake was calculated twice (days 0 and 15), using the caffeine test in 27 of the 33 individuals. This result suggests that omeprazole does not increase CYP1A2 activity after 14 days of treatment.

Variants of the Solute Carrier SLC16A1 Gene (MCT1) Associated With Metabolic Responses During a Long-Graded Test in Road Cyclists

Abstract González-Haro, C, Soria, M, Vicente, J, Fanlo, AJ, Sinués, B, and Escanero, JF. Variants of the solute carrier SLC16A1 gene (MCT1) associated with metabolic responses during a long-graded test in road cyclists. J Strength Cond Res 29(12): 3494–3505, 2015—Variants of the solute carrier SLC16A1 gene have been associated with alterations in MCT1 expression, because of a lactate (La−) transport deficiency across the cell membrane and a blood La− accumulation. The aim of this study was to associate the allelic and genotypic frequencies of 1470T>A, 2917(1414) C>T, and IVS3-17A>C variants relative to the blood La− kinetics and metabolic responses to a progressive effort until exhaustion. Twenty-five well-trained road cyclists performed a long-graded laboratory test: 10 minutes at 2.0 W·kg−1, first step at 2.5 W·kg−1 with increments of 0.5 W·kg−1 every 10 minutes until exhaustion. Blood La−, nonesterified fatty acids (NEFAS), and glucose levels were measured; fat and carbohydrate oxidation rates were estimated through stoichiometric equations. Three variants of SLC16A1 gene were determined for each subject, which were divided in two groups: wt (wild type)/mt (mutated type) and mt/mt genotype group versus wt/wt genotype group. Metabolic responses were compared between both groups with an unpaired Students t-test; Friedman and Wilcoxon tests were performed for nonparametric data. The statistical significance was set at p ⩽ 0.05. For 1470TA polymorphism, no significant blood La− differences were found between groups. 2197(1414)C>T allele carriers and IVS3-17A>C carriers showed significantly higher blood La− levels, lower blood NEFAS, and glucose levels at submaximal intensities. These findings open a new perspective to investigate SLC16A1 variants (1470TA and IVS3-17A>C) on La− deficiency transport and its regulation/interaction with other metabolic pathways. Future studies would be needed to clarify whether 1470T>A, 2917(1414)C>T, and IVS3-17A>C allelic/genotypic distribution benefit performance in endurance athletes.