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

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, first step at 2.5 W·kg with increments of 0.5 W·kg 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 Student's 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.

Genetic polymorphisms of CYP2C8, CYP2C9 and CYP2C19 in Ecuadorian Mestizo and Spaniard populations: a comparative study.

This study was designed to investigate the potential differences between Spaniards and Ecuadorian Mestizo people regarding CYP2C8, CYP2C9, and CYP2C19 genetic polymorphisms. DNA from 282 Spaniard and 297 Ecuadorian subjects were analyzed by either a previously reported pyrosequencing method (CY2C8*3, CYP2C9*2, CYP2C9*3, CYP2C19*2 and CYP2C19*3) or a nested PCR technique (CYP2C19*17). Whereas CYP2C19*17 allele distribution was higher in Ecuadorians than in Spaniards (P < 0.001) and the frequency of CYP2C19*3 was similar in these two populations (P > 0.05), the other allelic variants were detected at significantly lower frequencies in Ecuadorians than in Spaniards (P < 0.05). According to the diplotype distributions, the prevalence of the presumed CYP2C9 and CYP2C8 extensive metabolizers was higher in Ecuadorians than in Spaniards (P < 0.05). Individuals genotyped CYP2C19*1/*17 and *17/*17 who were considered as ultrarapid metabolizers were overrepresented in Ecuadorians in relation to Spaniards (P < 0.001). By contrast, among Ecuadorians no poor metabolizers (PMs) of either CYP2C8 or CYP2C9 were found and only two individuals were CYP2C19 PMs. These data are compatible with a higher CYP2C8, CYP2C9, and CYP2C19 activity in Mestizo Ecuadorians as opposed to Spaniards, which could imply differences in dosage requirements for drugs metabolized by these cytochromes and should also be considered in allele-disease association studies.

CYP3A5 3, CYP3A4 1B and MDR1 C3435T genotype distributions in Ecuadorians.

Polymorphisms in CYP3A genes, such as CYP3A5} and CYP3A4, as well as in the MDR1 gene, which encodes for P-glycoprotein, have been implicated as genetic markers in several disorders. Differences in the frequency distribution of the allelic variants CYP3A5 3, CYP3A4 1B, and MDR1 3435T have been demonstrated between distinct ethnic groups. In this study we examined the frequency of these allelic variants in 317 healthy Mestizo individuals from Ecuador and made comparisons with results reported in the literature. The genotypes were determined by PCR-RFLP. Allele and genotype differences were studied by chi-square test. The MDR1 T allele frequency was similar to that of Spaniard or Asian populations, which is consistent with the ethnic origin of Ecuadorian Mestizo individuals (Amerindian and Spaniard Caucasians). By contrast, the CYP3A5 3 allele frequency was significantly lower in Ecuadorians than in Spaniards and other white populations and higher than in Central Americans, Asians and blacks. CYP3A4 1B was more common in Ecuadorians than in Caucasian or Asian populations but less present than in blacks. The differences in the polymorphism found in this work should be considered in allele-disease association studies.

Characterization of a novel CYP2A7/CYP2A6 hybrid allele (CYP2A6*12) that causes reduced CYP2A6 activity.

The human CYP2A6 enzyme metabolizes certain drugs and pre-carcinogens and appears to be the most important enzyme for nicotine metabolism. At present, more than 10 different allelic variants are known that cause abolished or decreased enzyme activity. Genetic polymorphism in this gene might be of particular importance for an individual's need for nicotine and for susceptibility to lung and/or liver cancer. We have identified a new CYP2A6 allele (CYP2A6*12) which carries an unequal crossover between the CYP2A6 and CYP2A7 genes in intron 2. This results in a hybrid allele where the 5' regulatory region and exons 1-2 are of CYP2A7 origin and exons 3-9 are of CYP2A6 origin, resulting in 10 amino acid substitutions compared to the CYP2A6(*)1 allele. Phenotyping with the CYP2A6 substrate coumarin indicates that it causes reduced CYP2A6 activity in'vivo. Furthermore, when expressed in mammalian COS-1 cells, the enzyme variant catalyzed 7-hydroxylation of coumarin at a rate approximately 60% of that of the wild-type enzyme. The CYP2A6(*)12 allele was present at an allele frequency of 2.2% among Spaniards, but was absent in Chinese.

Urinary mutagenicity, CYP1A2 and NAT2 activity in textile industry workers.

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 beta-glucuronidase (MIbeta). 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 MIbeta, 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.

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 the CYP2C19*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 micronuclei 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.

Toward a clinical practice guide in pharmacogenomics testing for functional polymorphisms of drug-metabolizing enzymes. Gene/drug pairs and barriers perceived in Spain.

The development of clinical practice recommendations or guidelines for the clinical use of biomarkers is an issue of great importance with regard to adverse drug reactions. The potential of pharmacogenomic biomarkers has been extensively investigated in recent years. However, several barriers to implementing the use of pharmacogenomics testing exist. We conducted a survey among members of the Spanish Societies of Pharmacology and Clinical Pharmacology to obtain information about the perception of such barriers and to compare the perceptions of participants about the relative importance of major gene/drug pairs. Of 11 potential barriers, the highest importance was attributed to lack of institutional support for pharmacogenomics testing, and to the issues related to the lack of guidelines. Of the proposed gene/drug pairs the highest importance was assigned to HLA-B/abacavir, UGT1A1/irinotecan, and CYP2D6/tamoxifen. In this perspective article, we compare the relative importance of 29 gene/drug pairs in the Spanish study with that of the same pairs in the American Society for Clinical Pharmacology and Therapeutics study, and we provide suggestions and areas of focus to develop a guide for clinical practice in pharmacogenomics testing.

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.

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.

Polymorphism C3435T of the MDR1 gene in Central Americans and Spaniards.

The human multidrug resistance gene (MDR1) encodes for P-glycoprotein (P-gp) which is a transmembrane transporter protein that acts as an efflux pump for a number of lypophilic compounds. It plays a protective role for cells against DNA damage. The wobble C3435T polymorphism at exon 26 has been associated with different expression levels and activity. Differences in allele frequency of the C3435T polymorphism have been demonstrated between distinct ethnic groups. In our study we examined these polymorphisms in 433 healthy individuals. From these, 229 were Central American mestizos from Nicaragua (n = 117) and El Salvador (n = 112) to be compared with a group of 204 North Spaniards, with the aim of detecting potential genotypic differences between these populations. The genotypes were determined by PCR-RFLP. The frequencies of the C allele were very similar among Central Americans (0.53) and Spaniards (0.52), which is consistent with the ethnic origin of Central American individuals (Amerindians and European Caucasians). In comparison to other previously studied populations, the C allele frequency in Central Americans was significantly lower than that found in African populations and higher than that observed in the Indian and Southwest Asian populations. These data may be relevant for dose recommendation of P-gp substrate drugs and also for studies of allele disease association in the Central American population.

Growth hormone does not alter CYP2A6 activity in growth hormone-deficient children.

A large number of metabolic alterations are increasingly being treated with growth hormone. Despite the fact that growth hormone is known to be the main regulator of several hepatic drug metabolizing enzymes in rodents, few studies deal with the effect of growth hormone on hepatic enzyme activities in human beings. The aim of this study was to determine the effects of growth hormone replacement therapy for 4 weeks on CYP2A6 activity in children, because changes in this enzyme activity may have important therapeutic and toxic consequences. A total of 31 growth hormone-deficient children (age range 4.1-13.1 years; mean age 9.88 +/- 2.89 years) participated. The genotypes of CYP2A6 gene, CYP2A6*1A, CYP2A6*1B, CYP2A6*4, CYP2A6*1x2 and CYP2A6*9, were determined by polymerase chain reaction. To assess the enzyme activity, we used caffeine as a probe drug at two points in time: before starting growth hormone therapy (Day 0) and after 4 weeks of growth hormone therapy (Day A). Caffeine and metabolite concentrations in urine were assayed by high-pressure liquid chromatography. The metabolite ratio 1,7-dimethilxanthine to 1,7-dimethylurate (17U/17X) served to indicate CYP2A6 activity. Median value and 95% confidence interval at baseline was 1.08 (0.98-1.24). The value after treatment was 1.08 (0.86-1.21). Data comparison between periods showed lack of statistically significant differences (P > 0.05). The relative change, measured by the ratio of medians and 90% confidence interval, was 1.02 (0.84-1.19). There were no significant differences when the ratio between genotype groups were compared. These results indicate that growth hormone replacement therapy of growth hormone-deficient children for 4 weeks does not modify the CYP2A6 activity and hence the efficacy or toxicity of the CYP2A6 substrate compounds.

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.

Lack of effect of growth hormone replacement therapy on CYP1A2 and xanthine oxidase activities in growth hormone-deficient children.

BACKGROUND AND OBJECTIVES: The recombinant human growth hormone (rhGH) is being increasingly used for a number of metabolic alterations. GH is the main regulator of several hepatic drug metabolizing enzymes in rodents. In addition, GH could play a major role in defining the interface between pharmacogenetics and development. However, little is known about the effect of GH on the activity of hepatic enzymes in children. The aim of this study was to determine the effect of rhGH replacement therapy for 4 weeks on CYP1A2 and xanthine oxidase (XO) activities in children. METHODS: We used caffeine as a probe drug to assess the enzyme activities at two points in time: before starting GH treatment (day 0) and after 4 weeks on rhGH therapy (day A). A total of 31 GH-deficient children (age range: 4.1-13.1 years, mean age: 9.88+/-2.89 years) participated. Urinary concentrations of caffeine and metabolites were determined by high-performance liquid chromatography (HPLC) to calculate the metabolite ratios: (AFMU+1X+1U)/17U for CYP1A2 and 1U/(1X+1U) for XO. RESULTS: Four weeks of GH substitution did not importantly alter the markers of the enzyme activities measured in this study. Median values and 95% confidence intervals (CI) at baseline were 5.17 (3.87-5.59) for the CYP1A2 ratio and 0.62 (0.56-0.65) for the XO ratio. These values, after treatment, were 4.57 (3.90-5.97) for the CYP1A2 marker and 0.62 (0.59-0.67) for the XO ratio. Data comparison between periods showed lack of statistically significant differences (P>0.05). The relative changes measured by the ratios of medians and 90% CI were 1.14 (0.90-1.31) and 0.99 (0.94-1.06) for CYP1A2 and XO, respectively. CONCLUSIONS: The absence of significant changes in the markers of enzyme activities CYP1A2 and XO suggests that rhGH replacement therapy of GH-deficient children for 4 weeks could not noticeably modify the efficacy or toxicity of substrates of these metabolic enzymes.

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 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.

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.

Effects of growth hormone deficiency and rhGH replacement therapy on the 6beta-hydroxycortisol/free cortisol ratio, a marker of CYP3A activity, in growth hormone-deficient children.

OBJECTIVE: To determine the effect of both growth hormone deficiency (GHD) and rhGH replacement therapy on CYP3A activity as well as the potential influence of gender. METHODS: The sample consisted of 35 GHD children (16 males and 19 females), aged 2.9-13.1 years, and a control group of 35 healthy children matched for age and sex. The urinary ratio 6beta-hydroxycortisol/free cortisol was used as a marker of CYP3A activity. In patients, urine samples were collected at two times, prior to starting rhGH replacement treatment and 30 days after beginning therapy. RESULTS: A significantly higher metabolic activity in GHD children was observed in relation to controls ( P=0.0001) without sex differences. Paired comparisons demonstrated a sexually dimorphic effect of rhGH therapy on the CYP3A activity. While boys displayed a significant decrease ( P=0.003), girls showed no significantly different values of CYP3A marker ( P>0.05). Unpaired comparison between controls and GHD children after therapy demonstrated absence of significant differences in boys ( P>0.05) and a strikingly higher activity in girls ( P=0.0001). CONCLUSIONS: The data suggests that: (a) GHD in children increases CYP3A activity in a non-sex-dependent manner, (b) rhGH treatment for 30 days to GHD children results in a sexually dimorphic effect on CYP3A activity, with a significant decrease in males toward normalization in relation to controls and non-significant changes in females. The results of this study may have important clinical implications for GHD children, since changes in CYP3A activity importantly affect the metabolism of both steroid hormones and CYP3A substrate drugs.