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.

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.

Five caffeine metabolite ratios to measure tobacco-induced CYP1A2 activity and their relationships with urinary mutagenicity and urine flow.

To choose a sensitive protocol to discriminate populations exposed and not exposed to inducers, five urinary metabolite ratios (MRs) [MR1 (17X + 17U)/137X, MR2 (5-acetylamino-6-formylamino-3-methyluracil [AFMU] + 1X + 1U)/17U, MR3 (17X/137X), MR4 (AFMU + 1X + 1U + 17X + 17U)/137X, and MR5 (AFMU + 1X + 1U)/17X] were calculated in 4-5 h and 0-24 h urine samples after caffeine intake. One hundred twenty-five healthy volunteers (59 nonsmokers and 66 smokers) were included in the study. All ratios showed a log-normal distribution. MR2 in the two time intervals was the only ratio nondependent on the urine flow. Differences between nonsmokers and smokers could be detected with all ratios at 4-5 h. However, only MR2 and, to a lesser extent, MR5 allowed the discrimination of higher cytochrome P450 1A2 (CYP1A2) activity in smokers in the 0-24 h sample. Although smokers had increased urinary mutagenicity in relation to nonsmokers, a significant association between MRs and urine mutagenicity was observed only with MR2 in the 4-5 h interval; this ratio/time schedule being that of higher association with tobacco consumption. The most flow-dependent ratios, MR1, MR3, and MR4, were closely correlated with each other at the two intervals. The flow dependency profile of each ratio may explain their different power to indicate both tobacco exposure and tobacco-derived mutagenicity. In conclusion, MR2 in the period of 4-5 h after caffeine intake seems preferable, especially at high urine flow rates.

Antioxidant intracellular activity of genistein and equol.

Antioxidant effects of isoflavonoids have recently been described. To learn whether the isoflavonoids genistein and equol have actions on the intracellular free radicals, human neutrophils and J774 monocyte-macrophage cell line were used to measure the intracellular production of O(2) (superoxide anion) and H(2)O(2) (hydrogen peroxide) by flow cytometry. The results shown significatives decrease in O(2) and H(2)O(2) production after 1 hour of incubation with equol and genistein. The phagcytic oxidant production decreased owing to the effects of both isoflavonoids in a concentration-dependent manner.

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.

Apoprotein B in subretinal fluid.

We studied the presence of apolipoprotein B (apo B) by immunoelectrophoresis in subretinal fluid (SRF) and serum from 15 rhegmatogenous retinal detachments and 5 retinal detachments with proliferative vitreoretinopathy (PVR). Apo B concentration +/- standard deviation was 1.49 +/- 1.06 mg/dl in SRF and 108.41 +/- 40.22 mg/dl in serum. Only in four cases was apo B not detected in SRF. We found no significant correlation between apo B concentrations in SRF and apo B levels in serum. We did not find a positive correlation between apo B concentrations in SRF and the duration and size of the detachments. There was no statistical relationship between the presence of PVR and apo B levels. This phenomenon suggests the preservation of the outer blood retinal barrier during rhegmatogenous retinal detachment.

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.

Thioether excretion, urinary mutagenicity, and metabolic phenotype in smokers.

In 81 healthy individuals (51 smokers and 30 nonsmokers) biological indicators of internal exposure to electrophiles derived from tobacco smoke through metabolism were evaluated. Subgroups of smokers have been established in relation to the amount and type of tobacco smoked. Acetylator and hydroxylator phenotypes have been used as biomarkers of genetically determined susceptibility to cancer development. Urinary concentrations of thioethers (UT) and mutagenicity, with S9 mix for microsomal activation (MI-S9), were higher in smokers in relation to the level of tobacco consumption, but not to the type of tobacco. The "Slow acetylators-rapid oxidizers" category was not significant from the "rapid acetylators-rapid oxidizers" for values of UT and MI-S9. Data suggest that the biomarkers of exposure used in this study lack the necessary specificity to ascertain genetically determined susceptibility to cancer induced by tobacco smoking.

Apoprotein A and A-I profiles in subretinal fluid.

Previous reports have described the presence of apoproteins A in subretinal fluid (SRF). We studied the presence of total apoprotein A (apo A) and apoprotein A-I (apo A-I), using the method described by Laurel in SRF and its levels in serum in 20 patients with retinal detachment. By this method we can quantify the concentrations of apoproteins in SRF. All cases showed the presence of these apoproteins in SRF. The mean +/- standard deviation obtained was 78.7 +/- 26.94 mg/dl and 173.35 +/- 30.08 mg/dl for total apo A in SRF and serum respectively. For apo A-I these values were 32.62 +/- 14.36 mg/dl in SRF and 123.4 +/- 24.11 mg/dl in serum. We found no correlation between levels of total apo A and apo A-I in SRF and its levels in serum. Statistical differences were found between apo A-I content in SRF from detachments with the size of 3 quadrants and that of 1 and 2 quadrants but not between 3 and 4 quadrants. When the detachment affected 1 and 2 quadrants the concentrations of apo A-I were statistically lower. No statistical differences were found between concentrations of total apo A and apo A-I in SRF and the duration or presence of PVR in the detachments. These findings suggest that the outer blood retinal barrier is preserved during rhegmatogenous retinal detachment.

Levels of vancomycin in aqueous humor after topical eye drops administration.

Vancomycin is an antibiotic which is especially active against Gram positive bacteria. At present, numerous infections of the anterior segment of the eye are caused by the Staphilococcus aureus and epidermis. Strains which are resistant to methicilin are more and more frequent and for this reason Vancomycin is becoming the antibiotic of choice to combat these infections since no resistance of the Staphilococcus to it has been demonstrated. The authors evaluated the levels of Vancomycin in aqueous humor after the administration of topical eye-drops. For the first two hours after the last administration, levels of 0.52 micrograms/ml were detected. These inhibit the growth of the majority of bacteria sensitive to Vancomycin. Between two and four hours, the levels decreased to 0.15 micrograms/ml; these are therapeutic levels for a large number of Gram positive bacteria. From four hours after the last administration, these levels are undetectable. Due to the success of its penetration, topical administration of Vancomycin should be considered as a therapeutic modality against infections by gram positive bacteria of the anterior segment. The ideal guideline for administration would be one drop every two hours, especially if the infection is severe. In this way, subconjunctival injection could be avoided.

Cytogenetic study in peripheral blood lymphocytes from asthmatic patients receiving continued therapy with theophylline.

Possible cytogenetic effects of theophylline have been investigated in asthmatic patients undergoing continuous therapy with this drug. Sister-chromatid exchanges (SCE), chromosome aberrations (CA) and proliferating rate indices (PRI) were evaluated in cultured peripheral blood lymphocytes from patients receiving theophylline alone, theophylline plus inhaled beta 2 adrenergic drugs or theophylline in combination with beta 2 adrenergic agents and corticoids. Two samples from each individual were obtained in order to perform a prospective study: before the theophylline medication (sample A) and at a time after the beginning of treatment (sample B). After treatment (66.3 +/- 37.8 days), an increase in SCE was observed without modifications either in PRI or in CA. Patients receiving beta 2 adrenergics or beta 2 adrenergics plus glucocorticoids before and during theophylline treatment, did not respond differently than those on theophylline alone.

Urinary mutagenicity and N-acetylation phenotype in textile industry workers exposed to arylamines.

Primary aromatic amines have been identified epidemiologically as human carcinogens. It has been suggested that the target organ affected by aromatic amines is dependent on the rate of metabolic activation. Epidemiological studies have shown an association between low acetyl transferase activity and bladder cancer risk. On this basis, our working hypothesis was that the slow acetylators could follow in a higher extent the metabolic pathway independent of N-acetylation, leading to the excretion of conjugates of electrophyles with glucuronic acid. The instability of these glucuronides could be responsible for the association between arylamine-induced bladder cancer and slow acetylator phenotype. A total of 153 individuals were included in this study: 70 exposed to arylamines (working in textile industry) and 83 nonexposed. The following parameters were determined in urine: mutagenic index in the absence of metabolic activation, S9; mutagenic index in the presence of S9; and the mutagenic index after incubation of the urine with beta-glucuronidase. All individuals were phenotyped according to their capacity of N-acetylation by using isoniazid as drug test. The results show that the mutagenic index after incubation of the urine with beta-glucuronidase is statistically higher in exposed subjects when compared with nonexposed individuals (P less than 0.001), this parameter being statistically higher among exposed subjects who were slow acetylators than among rapid metabolizers, independent of the fact that they were smokers or nonsmokers. There were no significant differences between groups for the mutagenicity in urine not incubated with beta-glucuronidase.

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.

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.

[Erythrocyte glutathione and urinary thioethers in smokers]. / Glutatión eritrocitario y tioéteres urinarios en fumadores.

The carcinogenic mechanism of smoking is related with the production of electrophilic reactants and their possible covalent binding with DNA. On the other hand, there are detoxifying mechanisms such as glutathione-S-transferase, which results in mercaptopuric derivatives that are excreted in the urine. The integrity of the erythrocyte membrane is maintained by reduced glutathione among other factors. In the present study, the concentrations of erythrocyte reduced glutathione (GSH) and urinary thioethers (UT) were measured in a sample of 81 subjects divided in two groups. Group I: 30 nonsmokers; group II: 51 smokers, subdivided in group IIA (26 individuals smoking 10-20 cigarettes/day) and group IIB (25 individuals smoking more than 20 cigarettes/day). In the present study, the usefulness of GSH and UT as markers of collective internal contamination and of individual risk regarding tobacco exposure were evaluated. A higher concentration of GSH was found in smokers than in nonsmokers (F = 6.84, p less than 0.02). Regarding VT elimination, a significant increase in these parameters was found in association with the grade of smoking (p less than 0.05). They were higher in the subjects from the subgroup IIB than in the subgroup IIA (moderate smokers).

Cytogenic effects of diatrizoate and ioxaglate on patients undergoing excretory urography.

Possible cytogenic alterations due to radiologic contrast medium in patients undergoing a common radiologic examination is studied. Two groups of 20 patients each were used. Group I consisted of patients undergoing excretory urography, using sodium and meglumine diatrizoate as contrast. A different agent, sodium and meglumine ioxaglate, was used with group II. Three blood samples were taken from each patient before urography, immediately after urography, and 1 week later. The frequency of sister chromatid exchanges (SCE) and chromosomal aberrations (CA) were found to increase significantly in the B samples from both groups, that of group I being higher (P less than .01 compared with P less than .05). Furthermore, these alterations were found to persist in the C samples from group I. No modification of the Proliferating Rate Index (PRI) was found. The osmolarity or other components of the contrast media studied could be involved in the process. The results indicate that ioxaglate produces less cytogenic damage than diatrizoate.

Chromosome aberrations and urinary thioethers in smokers.

Chromosome aberrations, classified as chromosome- and chromatid-type aberrations, were evaluated in 94 individuals. The concentration of thioethers in the urine was also determined. The sample consisted of 41 non-smokers (control group) and 53 smokers, 25 smoked 10-20 cigarettes/day (subgroup IIA) and 28 smoked more than 20 cigarettes/day (subgroup IIB). Our aim was to perform internal dosimetry on smokers using a cytogenetic test, and a test for urinary excretion of thioethers, in order to determine the relation between these 2 methods of dosimetry. Our results show a higher frequency of chromosome aberrations (p less than 0.0001) and a higher excretion of urinary thioethers (p less than 0.0001) in smokers as compared to non-smokers. However, linear regression between these parameters was not statistically significant. In view of the variation between different individuals with regard to the amount of urinary thioethers, it seems more accurate to perform the biomonitoring of smoking by analyzing chromosome aberrations.