Emel Arinç

Genotype and allele frequencies of polymorphic CYP2E1 in the Turkish population

Cytochrome P4502E1 (CYP2E1) gene shows genetic polymorphisms that vary markedly in frequency among different ethnic and racial groups. We studied the genotype distributions and allele frequencies of three CYP2E1 polymorphisms: CYP2E1*5B (RsaI/PstI RFLP, C-1053T/G-1293C SNP, rs2031920 /rs3813867), CYP2E1*6 (DraI RFLP, T7632A SNP, rs6413432), and CYP2E1*7B (DdeI RFLP, G-71T SNP, rs6413420) by PCR/RFLP technique in a sample of 206 healthy subjects representing Turkish population. CYP2E1*5B polymorphism analysis yielded the genotype distribution as 96.12% for *1A/*1A (c1/c1), and 3.88% for *1A/*5B (c1/c2). The genotype frequencies for CYP2E1*6 polymorphism were found as 83.98% for *1A/*1A (T/T), 15.53% for *1A/*6 (T/A) and 0.49% for *6/*6 (A/A). For CYP2E1*7B (G-71T) polymorphism, the genotype frequencies were determined to be 86.89% for *1A/*1A (G/G), 12.62% for *1A/*7B (G/T) and 0.49% for *7B/*7B (T/T). Accordingly, the allele frequencies for *5B, *6 and *7B were 1.94, 8.25, and 6.80%, respectively. The genotype distributions of CYP2E1*5B and *6 in Turkish population were similar to those in other Caucasian populations, while differed significantly from East Asian populations. Recently, a novel and functionally important CYP2E1*7B polymorphism was identified in the promoter region. There have been few studies and limited data on CYP2E1*7B polymorphism frequency in the world and, so far, no information has been available for Turkish population. The genotype frequencies of CYP2E1*7B in Turkish population were found to be similar to those of other Caucasian populations. Population studies like this could be useful in assessing the susceptibility of different populations to chemical-induced diseases, including several types of cancer.

Hepatic cytochrome P4501A and 7-ethoxyresorufin O-deethylase induction in mullet and common sole as an indicator of toxic organic pollution in Izmir Bay, Turkey

Abstract In this study, the degree of induction of cytochrome P4501A-associated 7-ethoxyresorufin O-deethylase (EROD) activity and immunochemical detection of cytochrome P4501A in leaping mullet (Liza saliens) and common sole (Solea vulgaris) were used as biomarker for assessment of polycyclic aromatic hydrocarbon (PAH) or/and polychlorinated biphenyl (PCB) type organic pollutants along the Izmir Bay on the Aegean Sea coast, Turkey. Fish were captured in May 1995 and February and June 1996. Mullet caught from Pasaport, a highly urbanized and industrial section of the Bay, had approximately 62 times more EROD activity than the feral fish sampled from an uncontaminated site in the Outer Bay. Mullet caught along the pollutant gradient at the three other sites in the Bay exhibited less but highly significant induced EROD activity. An inverse relationship was found between the EROD activity in the fish and the distance between the catch point and the discharge region of polluted rivers and of industrial and domestic wastes into the Harbour. Studies using the polyclonal antibodies produced against mullet cytochrome P4501A showed a similar trend. In addition, EROD activities of benthic fish, common sole, captured from three different sites of the Bay also confirmed and extended the results obtained with those of mullet. Except that, common sole caught from site 5, the edge of Gediz river, exhibited higher EROD activity than that of fish caught from site 4, Tuzla. Although, detailed qualitative and quantitative analyses of organic chemicals in the waters and sediments of Izmir Bay are not available, the results of this study indicate that Inner and Middle Bays of Izmir Bay are highly contaminated with PAH and/or PCB type organic pollutants.

DNA repair XRCC1 Arg399Gln polymorphism alone, and in combination with CYP2E1 polymorphisms significantly contribute to the risk of development of childhood acute lymphoblastic leukemia

It is now well established that genetic polymorphisms impairing the DNA repair capacity can disrupt the genomic integrity and potentially modulate individuals susceptibility to various cancers. In this study, we investigated the possible association of X-ray repair cross-complimenting group 1 (XRCC1) Arg399Gln and Arg194Trp variants with the risk of incidence of childhood acute lymphoblastic leukemia (ALL) in Turkish population comprised of 190 healthy controls and 167 ALL patients. For Arg399Gln polymorphism, the heterozygous (Arg/Gln) and homozygous mutant (Gln/Gln) genotypes were significantly more common in the ALL patients than the controls (OR: 1.6, p=0.04). Particularly, the Gln399Gln genotype significantly increased the risk of disease up to 2.0-fold (OR: 2.0, p=0.04). Besides, Gln399Gln genotype has been found to be associated with considerably increased risk of ALL among females (OR=2.9, p=0.03). In case of codon 194 polymorphism, no significant associations have been found with risk of childhood ALL. In addition, none of the combinations of XRCC1 codon 194 and 399 polymorphisms have been found to be significantly associated with childhood ALL risk. In the scope of this study, we have also showed that the co-presence of XRCC1 codon 399 and CYP2E1*5B and *6 polymorphisms (data for CYP2E1 polymorphisms drawn from previously published study conducted in our lab) in the same individuals considerably increased the risk for childhood ALL to 3.7-fold with borderline significance (p=0.049). The observed combined effect was considerably more prominent among females (OR=17.4, p=0.001) and need to further investigation. This is the first study showing combined associations of XRCC1 399Gln, CYP2E1*5B and *6 alleles with the risk of development of childhood ALL.

Comparative studies of sheep liver and lung microsomal aniline 4-hydroxylase

1. The specific activity of the aniline 4-hydroxylase which catalyses hydroxylation of aniline to p-aminophenol was found to be 0.65 (N = 10) and 0.15 (N = 13) nmol p-aminophenol formed/mg protein/min, in sheep liver and lung microsomes, respectively. 2. The effects of aniline concentration, pH, cofactors, amount of enzyme and incubation period, on enzyme activity were studied, and the optimum conditions for maximum activity of liver and lung microsomes were determined. 3. Liver and lung microsomal aniline 4-hydroxylase activity was found to be completely dependent on the presence of cofactor NADPH. 4. The Lineweaver Burk and Eadie Hofstee plots of the liver enzyme were found to be curvilinear, suggesting that the enzyme did not follow the Michaelis Menten kinetics. From these graphs, two different Km values were calculated for the liver enzyme as 3.21 and 0.072 mM aniline. Km of the lung enzyme was calculated to be 1.43 mM aniline from its Lineweaver Burk graph. 5. The effects of magnesium, nickel and cadmium ions on the liver and lung aniline 4-hydroxylase activity were examined. Magnesium ion was found to have stimulatory effect, whereas nickel and cadmium ions inhibited the activity of the both liver and lung enzyme.

Effects of in vivo benzo(a)pyrene treatment on liver microsomal mixed-function oxidase activities of gilthead seabream (Sparus aurata)☆

Benzo(a)pyrene [B(a)P] treatment of gilthead seabream, 25 mg/kg, i.p. for 5 consecutive days, did not cause any significant changes in ethylmorphine N-demethylase and aniline 4-hydroxylase activities of liver microsomes. The same treatment did not alter the liver microsomal cytochrome b5 content, NADH-cytochrome b5 reductase and NADPH-cytochrome P450 reductase activities. However, benzo(a)pyrene treatment caused a 2-3-fold increase in 7-ethoxyresorufin O-deethylase (7-EROD) activity of gilthead seabream liver microsomes. Although, upon treatment, total cytochrome P450 content of liver microsomes increased about 1.7-fold in 1990 fall, no such increase was observed in spring 1991. However, a new cytochrome P450 with an apparent M(r) of 58,000 was observed on SDS-PAGE of liver microsomes obtained from benzo(a)pyrene treated gilthead seabream. Besides, in vitro addition of 0.2 x 10(-6) M benzo(a)pyrene to the incubation mixture inhibited 7-ethoxyresorufin O-deethylase activity by 93%. Gilthead seabream liver microsomal 4-ethoxyresorufin O-deethylase activity was characterized with respect to substrate concentration, amount of enzyme, type of buffer used, incubation period and temperature.

Preparation of highly purified cytochrome P4501A1 from leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular and immunochemical properties.

Cytochrome P4501A1 was purified to electrophoretic homogeneity from the liver microsomes of feral fish leaping mullet (Liza saliens) collected in Izmir Bay, Aegean coast of Turkey. Purification of cytochrome P4501A1 involved anion exchange chromatography of Emulgen 913-cholate solubilized microsomes on first- and second-DEAE-cellulose columns, hydrophobic interaction chromatographies of the partially purified cytochrome P4501A1 on Porapak Q and phenyl-Sepharose CL-4B and further purification on adsorption chromatography on the hydroxylapatite column. Finally, it is further concentrated and purified on the third DEAE-cellulose column. The purified cytochrome P4501A1 was characterized with respect to spectral, electrophoretic, immunochemical and biocatalytic properties. Cytochrome P4501A1, purified 32-fold with a specific content of 15-17 nmoles P450 (mg protein)-1, produced a single band on SDS-polyacrylamide gel electrophoresis having monomer molecular weight of 58,000 +/- 500. Absolute absorption spectrum of the purified cytochrome P4501A1 fractions showed maximal absorption at 417.5 nm and CO-difference spectrum of dithionite-reduced cytochrome P4501A1 gave a peak at 448 nm. Purified P4501A1 was found to be active in the O-deethylation of 7-ethoxyresorufin in the reconstituted system containing purified fish liver cytochrome P450 reductase and synthetic lipid. However, it was unable to catalyze the oxidation of the other monooxygenase substrates such as benzphetamine and aniline known to be specific for the other isozymes. Purified L. saliens liver microsomal cytochrome P4501A1 showed strong cross-reactivity with the antibodies directed against the cytochrome P4501A1 homologues purified from other teleost species such as rainbow trout and scup. Spectral, electrophoretic, immunochemical and biocatalytic properties of the purified cytochrome P4501A1 strongly suggested that it is the CYP1A1 in the L. saliens liver.

In vivo effects of 3-methylcholanthrene, phenobarbital, pyrethrum and 2,4,5-t isooctylester on liver, lung and kidney microsomal mixed-function oxidase system of guinea-pig: A comparative study

The optimum conditions (pH, microsomal protein amount and substrate concentration) of guinea-pig liver, lung and kidney microsomal aniline 4-hydroxylase, ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities were determined. Male guinea-pigs weighing 500-700 g were administered 3-methylcholanthrene (25 mg/kg, i.p. 3 days), phenobarbital (75 mg/kg, i.p. 3 days), pyrethrum (120 mg/kg, i.p. 2 days) and 2,4,5-T isooctylester (200 mg/kg, i.p. 3 days). 3-Methylcholanthrene treatment caused significant increases in liver microsomal benzo[a]pyrene hydroxylase and kidney microsomal aniline 4-hydroxylase activities. However, with phenobarbital treatment the only significant increase was observed in liver microsomal ethylmorphine N-demethylase activity. Pyrethrum treatment decreased kidney microsomal ethylmorphine N-demethylase activity significantly. 2,4,5-T isooctylester treatment increased liver microsomal aniline 4-hydroxylase and lung microsomal ethylmorphine N-demethylase activities significantly. Liver microsomal NADPH-cytochrome c reductase activity was increased significantly by phenobarbital and pyrethrum treatment. The other treatments did not cause any significant changes in microsomal NADPH-cytochrome c reductase activities of liver, lung and kidney. Cytochrome P-450 content of guinea-pig liver microsomes were increased significantly about 2.5-fold and 2-fold by treatment with 3-methylcholanthrene and phenobarbital, respectively. 3-Methylcholanthrene also caused 1 nm spectral shift in the absorption maxima of CO difference spectrum of the dithionite-reduced liver microsomal cytochrome P-450, forming P-449.

Characterization of cytochrome P450 dependent mixed-function oxidase system of gilthead seabream (sparus aurata; sparidae) liver☆

Abstract 1. 1. The average cytochrome P450 content of gilthead seabeam liver microsomes was found to be 0.078 ± 0.012 (n = 4) nmol/mg protein, which was about 50%, 24% and 13% of that measured in trout, sturgeon and scup liver microsomes, respectively. 2. 2. Gilthead seabream liver microsomes contained a relatively higher amount of cytochrome b5, as 0.32 ± 0.05 (n = 4) nmol/mg protein. 3. 3. Effects of microsomal protein amount and temperature ranging from 15 to 30°C on aniline 4-hydroxylation and ethylmorphine N-demethylation activities were examined. No significant differences were observed between the enzyme activities at these temperatures. 4. 4. The liver microsomal aniline 4-hydroxylase activity of gilthead seabream in the 1990 fall (0.016–0.021 nmolp-aminophenol/min/mg protein) was 4.5–6 times higher than that in the 1991 spring (0.003–0.004 nmolp-aminophenol/min/mg protein). 5. 5. Liver microsomal ethylmorphine N-demethylase activity measured in 1991 spring (0/371) nm ol formaldehyde/min/mig protein) was 41% higher than that in 1990 fall (0.263 nmol formaldehyde/min/mg protein).

Electrophoretic, spectral, catalytic and immunochemical properties of highly purified cytochrome P-450 from sheep lung

1. Cytochrome P-450LgM2 was purified from sheep lung microsomes in the presence of detergents, Emulgen 913 and cholate. 2. The purification procedure involved the chromatography of the detergent solubilized microsomes on DEAE-cellulose and hydroxylapatite. 3. Cytochrome P-450LgM2 was further purified on second DEAE-cellulose and hydroxylapatite columns. 4. The specific content of the highly purified P-450LgM2 was 16-18 nmol P-450/mg protein and purified 164-fold. 5. The yield was 16% of the initial content in microsomes. 6. The SDS-polyacrylamide slab gel electrophoresis (PAGE) of the purified lung cytochrome P-450LgM2 showed one protein band having the monomer molecular weight of 49,500. 7. The absolute CO-difference spectrum of dithionate-reduced P-450LgM2 gave a peak at 451 nm. 8. When sheep lung cytochrome P-450LgM2 and P-450LM2 purified from liver of phenobarbital (PB)-induced rabbit were subjected to Western Blotting and visualized immunochemically with anti-P-450LM2, they showed identical mobilities. 9. P-450LgM2 was found to be very active in N-demethylation of benzphetamine in a reconstituted system containing purified sheep lung reductase and synthetic lipid. 10. Turnover numbers (min-1) for benzphetamine, aniline, ethylmorphine and p-nitrophenol were determined to be 273, 1.2, 15.5 and 1.05, respectively, in a reconstituted microsomal lung monooxygenase system. 11. Spectral, electrophoretic, biocatalytic and immunochemical properties of sheep lung P-450LgM2 were found to be similar to those of P-450 isozyme 2, purified from PB-treated rabbit liver and of rabbit lung microsomes.

Differential effects of diabetes on CYP2E1 and CYP2B4 proteins and associated drug metabolizing enzyme activities in rabbit liver

The effects of diabetes on cytochrome P450 (CYP)-dependent drug metabolizing enzymes are yet to be clarified. The most widely used animals in these studies have been rats, and information on the effects of diabetes on rabbit liver drug metabolizing enzymes have been unavailable until now. In this study, for the first time, a significant induction of liver CYP2E1 is demonstrated via immunoblot analysis in alloxan-induced rabbits. The CYP2E1 content of diabetic microsomes was highly correlated with the activities of liver aniline 4-hydroxylase (r=0.82, p<0.05), and p-nitrophenol hydroxylase (r=0.86, p<0.01), and diabetes increased the activities of the enzymes associated with CYP2E1. The activities of aniline 4-hydroxylase and p-nitrophenol hydroxylase were significantly increased by 1.7 and 1.8-fold, respectively compared to those of control rabbits. In marked contrast, diabetes had no effect on the protein levels of CYP2B4 as determined by immunoblotting and on benzphetamine N-demethylase activity, which is known to be specifically metabolized by CYP2B4 in rabbit liver. The present study demonstrates that diabetes increases the activities of CYP2E1 and associated enzymes but does not change the activity levels of CYP2B4 and associated enzymes in diabetic rabbits. These findings are in contrast to those of mice, hamsters and rats, and that suggest the presence of species-dependent responses of CYP-dependent drug metabolizing enzymes to diabetes.