Immunohistochemical localization and differential expression of cytochrome P450 3A27 in the gastrointestinal tract of rainbow trout.

In mammals the cytochrome P450 3A (CYP3A) subfamily isoforms are primarily expressed in liver and intestines with lesser amounts found in other tissues. The aim of this study was to examine the cellular localization and the expression pattern of CYP3A27 in the gastrointestinal tract (GI tract) of a freshwater teleost species, the rainbow trout (Oncorhynchus mykiss), a fish model used extensively for toxicological and carcinogenesis research. Using an avidin biotinylated enzyme complex and 3,3'-diaminobenzidine staining, strong cytoplasmic immunohistochemical staining was observed for CYP3A27 protein in hepatocytes and in enterocytes of the intestinal ceca and the proximal descending intestine when probed with a polyclonal antibody raised against rainbow trout P450 LMC5, a CYP3A protein. The intensity of epithelial staining decreased distally along the GI tract with faint staining observed in the epithelial cells examined near the vent. Western blot analysis was supportive of the immunohistochemistry results. Northern blot analysis also demonstrated that CYP3A27 mRNA was expressed along the entire GI tract. The major area of CYP3A27 mRNA expression was in the intestinal ceca, followed by the proximal descending intestine, at levels that were about three- to five-fold and two- to four-fold, respectively, greater than seen in the liver of the fish studied. Monooxygenase activities of intestinal ceca microsomes against testosterone and progesterone confirmed the presence of active CYP3A enzyme in this tissue. These results suggest that the intestine of rainbow trout may possesses substantial capacity for first-pass metabolism of xenobiotics by CYP3A27, which makes it an excellent model in which to study the consequence of such metabolism.

Determination of coumarin metabolism in Turkish population.

Cytochrome P450 2A6 is an important human hepatic P450 which activates precarcinogens and oxidizes some drug constituents such as coumarin, halothane, and the major nicotine C-oxidase. Genetic polymorphism exists in the CYP2A6 gene. CYP2A6*1 (wild type) is responsible for the 7-hydroxylation of coumarin. The point mutation (T to A) in codon 160 leads to a single amino acid substitution (Leu to His) and the resulting protein, CYP2A*2 is unable to 7-hydroxylate coumarin. Gene conversion in exons 3, 6, and 8 between the CYP2A6 and the CYP2A7 genes creates another variant, CYP2A6*3. In this study, healthy male and female Turkish volunteers (n = 50) were administered 2 mg coumarin, and urine samples were analyzed for their content of the coumarin metabolite, 7-hydroxycoumarin (7OHC), by high-performance liquid chromatography (HPLC). Genetic polymorphism for CYP2A6 was detected by using two-step polymerase chain reaction (PCR) to identify CYP2A6*1, CYP2A6*2, and CYP2A6*3 in 13 of these subjects. The percentage of the dose excreted of total 7OHC in relation to CYP2A6 genotype and excretion of nicotine/cotinine was also evaluated to demonstrate the role of CYP2A6 in nicotine metabolism. The majority of Turkish subjects (68%) excreted less than 60% of the 2-mg dose as coumarin metabolite. The allelic frequencies were detected as 0.88 for CYP2A6*1 allele; 0.12 for CYP2A6*3 allele in 13 individuals. No heterozygous and homozygous individuals were identified for the CYP2A6*2 allelic variant. Phenotyping and genotyping for drug metabolizing enzymes are of great importance in studies correlating precarcinogen activation or drug metabolism to the CYP2A6 genotype in smoking behavior when populations are investigated.

Urinary cotinine levels of smokeless tobacco (Maras powder) users.

Smokeless tobacco is used in various forms in some countries of the world. "Maras otu" is a kind of smokeless tobacco usage in the Southeastern region of Turkey. The use of smokeless tobacco causes nicotine addiction and dependence. Moreover this type of smokeless tobacco usage is one of the risk factors for oral cancers and genotoxic damages for users. Cotinine is widely used as a biomarker of tobacco consumption and intake of nicotine. Therefore, urine samples were collected from people who are using Maras powder and smoking cigarettes, and passive smokers, and the levels of cotinine investigated. The purpose of this study is to determine the cotinine levels of Maras powder users and to compare the results with cigarette smokers and passive smokers. Urinary cotinine levels of subjects were determined by using capillary gas chromatography with FID detection. The mean (+/- SD) urinary cotinines have been determined as 6467.35+/-3198 microg/g creatinine for 26 Maras powder users, 1943.92+/-1443 microg/g creatinine for 26 cigarette smokers and 198.62+/-420.82 microg/g creatinine for 26 passive smokers. A significant difference has been found between cotinine levels of Maras powder users and cigarette smokers, which is three times higher in Maras powder users (p<0.001). The present study suggests that smokeless tobacco poses a threat to public health and it should not be viewed as a safe alternative to cigarettes.

Cloning, sequencing, and tissue expression of CYP3A27, a new member of the CYP3A subfamily from embryonic and adult rainbow trout livers.

Screening of lambdagt11 and lambdagt22A cDNA libraries of livers from adult females and embryos of rainbow trout (Oncorhynchus mykiss), respectively, using rabbit anti-rainbow trout cytochrome P450 LMC5 polyclonal antibodies showed that there were identical cDNAs of 1802-bp nucleotides with open reading frames coding for proteins containing 518 amino acids (59,206 Da, pI = 6.39). The cDNA was assigned CYP3A27 by the P450 Nomenclature Committee to represent the first CYP3A subfamily member reported for aquatic species. The deduced N-terminal sequence of CYP3A27 was in agreement with 8 of the first 12 confirmed amino acid residues from Edman degradation of LMC5, a P450 previously isolated from juvenile trout liver. In similarity comparisons between species by positional alignment, the deduced amino acid sequence of rainbow trout CYP3A27 was 56.4% identical with dog CYP3A12, 56.0% with monkey CYP3A8, 54.9% with human CYP3A4, 54.7% with rat CYP3A9, and 54.2% with sheep CYP3A24. Marked differences in sex, age, and tissue expression of CYP3A27 in rainbow trout were observed at the mRNA level as shown by Northern blots. The major extrahepatic expression site for CYP3A27 was upper small intestine. Females expressed considerably more CYP3A27 mRNA than male in the fish examined. Southern blot analysis of restriction enzyme-digested rainbow trout genomic DNA demonstrated that multiple CYP3A27-related genes exist in rainbow trout.

Expression of CYP2M1, CYP2K1, and CYP3A27 in brain, blood, small intestine, and other tissues of rainbow trout.

Expression of five constitutive forms of cytochrome P450 [(LMC1 (CYP2M1), LMC2 (CYP2K1), LMC3, LMC4, and LMC5 (CYP3A27)] in selected tissues from sexually immature 2-year old female and male rainbow trout (Oncorhynchus mykiss) were examined at the translational level by Western blot using polyclonal antibodies raised in rabbits against those purified trout hepatic P450s. Tissues examined were from brain, liver, muscle, blood, head kidney, trunk kidney, upper intestine, stomach, heart, and gonad (ovary or testis). The results showed that the liver was the major organ for expression of all the trout P450s studied. Trunk kidney was the secondary expression site except for LMC5. Selective translational expression of these P450 isoforms or similar proteins was observed for LCM1 and LMC5 in brain; for LMC2 and LMC5 in female upper intestine; and for LMC2 in blood plasma of the fish studied under the experimental and sampling conditions.

N-acetylation phenotyping with sulfamethazine in an Iranian population.

The acetylation polymorphism is one of the most common inherited variations in the biotransformation of drugs and chemicals. Its association with drug toxicity and increased risk of developing certain diseases and certain types of chemically induced cancers has made it one of the oldest and best studied examples of pharmacogenetic conditions. N-Acetylation of sulfamethazine was studied in 74 unrelated healthy Iranian volunteers. The frequency of slow acetylators, determined by using free and total plasma sulfamethazine concentrations, was 78.4%. The mean acetylation ratio was 19.48% for slow acetylators, and the frequency of the recessive allele controlling slow acetylation was found to be 0.88. This percentage is similar to that observed in various Arab countries and higher than that observed in Europeans.

N-Acetyltransferase phenotype of patients with bladder cancer.

N-Acetyltransferase phenotype has been determined in 109 control subjects and in 23 patients with bladder cancer. Sixty-two per cent of control, and 39% of patients with bladder cancer were phenotypically slow acetylators. This difference was not significant. N-Acetyltransferase phenotype is unlikely to be a major determinant in the development of bladder cancer in the Turkish population.