Monitoring of pollution in the western Black Sea coast of Turkey by striped red mullet (Mullus surmuletus).

The striped red mullet (Mullus surmuletus) is an economically important demersal fish species. In this study, our aim was to monitor the pollution in the western Black Sea coast of Turkey using striped red mullet as a bioindicator species. Fish samples were caught from four different locations in the western Black Sea coast of Turkey in 2006, 2009-2011, and 2016. Highly elevated cytochrome P4501A (CYP1A)-related 7-ethoxyresorufin O-deethylase (EROD) activities were measured in striped red mullet caught from Zonguldak Harbor in all of the sampling years. The lowest EROD activities were measured in fish samples caught from Kefken. In addition to the EROD activity measurements, glutathione S-transferase (GST), glutathione reductase, and catalase activities were also measured in the striped red mullet samples. Higher GST and catalase activities were measured in the striped red mullet samples caught from Zonguldak Harbor than from Kefken in 2016. These results indicate that the striped red mullet is responsive to CYP1A inducer pollutants. This study covers intermittent measurements of the biomonitoring data from the striped red mullet caught around the western Black Sea coast of Turkey, over a 10-year period.

Mechanism of inhibition of CYP1A1 and glutathione S-transferase activities in fish liver by quercetin, resveratrol, naringenin, hesperidin, and rutin.

Quercetin, resveratrol, naringenin, hesperidin, and rutin are phenolic compounds/flavonoids that may have roles in the reduction of cancer susceptibility. In this study, in vitro modulatory effects of them were studied on liver CYP1A1 associated 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione S-transferase (GST) activity of leaping mullet (Liza saliens). All of the phenolic compounds/flavonoids used exerted an inhibitory effect on both EROD and GST activities of fish. Quercetin, resveratrol, hesperidin, and rutin were found to inhibit EROD activity in a competitive manner; on the other hand, naringenin was found to inhibit EROD activity in a noncompetitive manner. Ki values of quercetin, resveratrol, naringenin, hesperidin, and rutin were calculated from Dixon plots as 0.12 µM, 0.67 µM, 2.63 µM, 18 µM and 0.1 mM, respectively. Resveratrol, quercetin, and hesperidin were found to inhibit GST activity in a competitive manner; on the other hand, rutin and naringenin were found to inhibit GST activity in a mixed-type manner. Ki values of resveratrol, quercetin, hesperidin, naringenin, and rutin were calculated from Dixon plots as 3.2 µM, 12.5 µM, 45 µM, 128 µM, and 150 µM, respectively. The results suggest that quercetin and resveratrol containing foods are effective in the prevention and treatment of cancer.

Aldrin epoxidation in flathead mullet (Mugil cephalus): possible involvement of CYP1A and CYP3A.

The primary objective of this study was to determine specific cytochrome P450 isozyme(s) involved in the metabolism of aldrin to its toxic metabolite dieldrin in flathead mullet (Mugil cephalus) liver microsomes. To identify the cytochrome P450 isozyme responsible for the aldrin metabolism in mullet liver, the effects of mammalian-specific cytochrome P450 inhibitors and substrates were determined in the epoxidation reaction of aldrin. CYP3A-related inhibitors, ketoconazole, SKF-525A, and cimetidine, inhibited the metabolism of aldrin. The contribution of CYP1A to the aldrin metabolism was shown by the inhibition of 7-ethoxyresorufin-O-deethylase activity in the presence of aldrin. The results indicate that CY1A and CYP3A are the cytochrome P450s involved in aldrin epoxidase activity in mullet. In addition, the suitability of aldrin epoxidase activity for monitoring of environmental pollution was also assessed in the fish samples caught from four different locations of the West Black Sea coast of Turkey.

In vitro effects of myricetin, morin, apigenin, (+)-taxifolin, (+)-catechin, (-)-epicatechin, naringenin and naringin on cytochrome b5 reduction by purified NADH-cytochrome b5 reductase.

The microsomal NADH-dependent electron transport system consisting of cytochrome b5 reductase and cytochrome b5 participates in a number of physiologically important processes including lipid metabolism as well as is involved in the metabolism of various drug and xenobiotics. In the present study, we assessed the inhibitory effects of eight dietary flavonoids representing five distinct chemical classes on cytochrome b5 reduction by purified cytochrome b5 reductase. From the flavonoids tested, myricetin was the most potent in inhibiting cytochrome b5 reduction with an IC50 value of 0.35µM. Myricetin inhibited b5 reductase noncompetitively with a Ki of 0.21µM with respect to cofactor NADH, and exhibited a non-linear relationship indicating non-Michaelis-Menten kinetic binding with respect to cytochrome b5. In contrast to the potent inhibitory activity of myricetin, (+)-taxifolin was found to be a weak inhibitor (IC50=9.8µM). The remaining flavonoids were inactive within the concentration range tested (1-50µM). Analysis of structure-activity data suggested that simultaneous presence of three OH groups in ring B is a primary structural determinant for a potent enzyme inhibition. Our results suggest that inhibition of the activity of this system by myricetin or myricetin containing diets may influence the metabolism of therapeutic drugs as well as detoxification of xenobiotics.

Evaluation of bioreductive activation of anticancer drugs idarubicin and mitomycin C by NADH-cytochrome b5 reductase and cytochrome P450 2B4.

This study attempted to investigate the ability of microsomal NADH-cytochrome b5 reductase and cytochrome P450 2B4 to reductively activate idarubicin and mitomycin C. In vitro plasmid DNA damage experiments and assays using purified hepatic enzymes were employed to examine their respective roles in the metabolic activation of anticancer drugs. Mitomycin C was found to be not a good substrate for microsomal b5 reductase unlike P450 reductase. It produced low amounts of strand breaks in DNA when incubated with b5 reductase and its one-electron reduction by purified enzyme was found as negligible. Our findings revealed that P450 reductase-mediated metabolism of idarubicin resulted in a large increase in single-strand DNA breaks, whereas, b5 reductase neither catalyzed the reduction of idarubicin nor mediated the formation of DNA damage in the presence of idarubicin. The reconstitution studies, on the other hand, have identified rabbit liver CYP2B4 isozyme as being a potential candidate enzyme for reductive bioactivation of idarubicin and mitomycin C. Thus, the present novel findings strongly suggest that while b5 reductase could not play a key role in the cytotoxic and/or antitumor effects of idarubicin and mitomycin C, CYP2B4 could potentiate their activity in combination with P450 reductase.

Diverse action of acrylamide on cytochrome P450 and glutathione S-transferase isozyme activities, mRNA levels and protein levels in human hepatocarcinoma cells.

Humans are exposed to acrylamide in their diet and cigarette smoke. Acrylamide is metabolized into glycidamide by CYP2E1. However, very few studies regarding the effects of acrylamide on cytochrome P450 and Glutathione S-Transferase (GST) isozymes have been pursued. The aim of this study is to elucidate the effects of acrylamide on cytochrome P450 and GST isozymes in HepG2 cell line. Treatment with 1.25 and 2.5 mM acrylamide caused 9.5- and 3.7-fold increases and 4.0- and 3.3-fold increases in CYP1A-associated ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-demethylase (MROD) activities, respectively. These increases were consistent with increases in mRNA and protein levels of these isozymes. Similarly, CYP2E1-associated aniline 4-hydroxylase (ANH) activity, protein levels, and mRNA levels increased 2.1- and 2.6-fold, 2.4- and 3.2-fold, and 1.4- and 1.9-fold following 1.25 and 2.5 mM acrylamide treatments, respectively. In addition, GST-mu activity was increased 2.4- and 5.1-fold by acrylamide. Moreover, GST-mu mRNA and protein levels increased twofold as a result of acrylamide treatment. In contrast, GST-pi protein and mRNA levels decreased significantly. In conclusion, human cell exposure to acrylamide causes an increase in the levels of carcinogenicity and toxicity and a disturbance in drug metabolism, possibly due to complex effects on P450 and GST isozymes.

Association between polymorphisms of EPHX1 and XRCC1 genes and the risk of childhood acute lymphoblastic leukemia.

Microsomal epoxide hydrolase, EPHX1, plays a central role in the detoxification of potentially genotoxic epoxide intermediates. In this study, we firstly aimed to investigate the relationship between EPHX1 Tyr113His and His139Arg variants, and the risk of incidence of childhood acute lymphoblastic leukemia (ALL) in Turkish population, comprised of 190 healthy controls and 167 ALL patients. In exon 3 Tyr113His polymorphism, 113His/His homozygous mutant genotype with slow activity was 18.6% in ALL patients and 9% in controls, indicating 113His/His slow activity genotype was significantly associated with an increased risk of childhood ALL (OR: 2.3, 95% CI, 1.2-4.4, P = 0.01). No significant association was found between exon 4 His139Arg variant and the risk of ALL. When both exon 3 Tyr113His and exon 4 His139Arg polymorphisms were considered together, only the exon 3 113His/His, homozygous mutant, slow activity genotype with exon 4 wild-type genotype 139His/His was significantly increased the risk of ALL 2.4-fold (OR: 2.4, P = 0.02). We also evaluated whether haplotype analysis for EPHX1 Tyr113His polymorphism together with DNA protein XRCC1 Arg399Gln variant known for its deficient DNA repair capacity would represent more prominent risk factors for the development of childhood ALL. Accordingly, the co-presence of Tyr113His variant of EPHX1 and Arg399Gln variant of XRCC1 in the same individuals significantly increased the risk of childhood ALL up to 2.1-fold (OR = 2.1, P = 0.03). Moreover, homozygous mutant genotype for both genes significantly and considerably increased the risk of childhood ALL 8.5-fold (OR: 8.5, P = 0.03). In conclusion, individuals with EPHX1 113His/His slow activity genotype may not detoxify reactive carcinogenic epoxides efficiently, binding of reactive epoxides to DNA cause DNA damage. With the inadequate polymorphic DNA repair protein, XRCC1, this situation ultimately leads to significantly increased susceptibility for childhood ALL.

Evaluation of the protective effects of quercetin, rutin, naringenin, resveratrol and trolox against idarubicin-induced DNA damage.

PURPOSE: Idarubicin is a synthetic anthracycline anticancer drug widely used in the treatment of some hematological malignancies. The studies in our laboratory have clearly demonstrated that idarubicin can undergo reductive bioactivation by NADPH-cytochrome P450 reductase to free radicals with resulting formation of DNA strand breaks, which can potentially contribute to its genotoxic effects [Celik, H., Arinç, E., Bioreduction of idarubicin and formation of ROS responsible for DNA cleavage by NADPH-cytochrome P450 reductase and its potential role in the antitumor effect. J Pharm Pharm Sci, 11(4):68-82, 2008]. In the current study, our aim was to investigate the possible protective effects of several phenolic antioxidants, quercetin, rutin, naringenin, resveratrol and trolox, against the DNA-damaging effect of idarubicin originating from its P450 reductase-catalyzed bioactivation. METHODS: DNA damage was measured by detecting single-strand breaks in plasmid pBR322 DNA using a cell-free agarose gel method. RESULTS: Our results indicated that, among the compounds tested, quercetin was the most potent antioxidant in preventing DNA damage. Quercetin significantly decreased the extent of DNA strand breaks in a dose-dependent manner; 100 microM of quercetin almost completely inhibited the DNA strand breakage. Unlike quercetin, its glycosidated conjugate rutin, failed to provide any significant protection against idarubicin-induced DNA strand breaks except at the highest concentration tested (2 mM). The protective effects of other antioxidants were significantly less than that of quercetin even at high concentrations. Quercetin was found to be also an effective protector against DNA damage induced by mitomycin C. CONCLUSION: We conclude that quercetin, one of the most abundant flavonoids in the human diet, is highly effective in reducing the DNA damage caused by the antitumor agents, idarubicin and mitomycin C, following bioactivation by P450 reductase.

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 individual's 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.

Analysis of paraoxonase 1 (PON1) genetic polymorphisms and activities as risk factors for ischemic stroke in Turkish population.

BACKGROUND: Paraoxonase1 (PON1) is protective against the development of atherosclerosis, a risk factor for ischemic stroke. PON1 gene has one promoter region (-107T/C) and two coding region (192Q/R and 55L/M) polymorphisms that affect the levels and catalytic efficiency of the enzyme, respectively. In this study, we aimed to determine the importance of -107T/C, 192Q/R and 55L/M polymorphisms of PON1 gene and three PON1 activities (diazoxonase, paraoxonase, arylesterase) as risk factors for ischemic stroke. METHODS: Study population was comprised of 172 unrelated adult Caucasian patients with acute hemispheric ischemic stroke and 105 symptom-free controls. Genotypes were attained by PCR followed by restriction enzyme digestion and phenotypes were determined by spectrophotometric assays. RESULTS: This is the first study analyzing diazoxonase activity as a risk factor for ischemic stroke. Nevertheless, diazoxonase, paraoxonase and arylesterase activities were almost the same in stroke patients and controls. The -107TT genotype was associated with a 1.97 times increased risk for stroke in elderly (age > 59). Individuals with this genotype were found to have the lowest PON1 enzyme activities among the -107T/C genotypes. Triple combined haplotype QRLMTC was found to be 6.94- and 10.4-times protective against ischemic stroke in the overall and the elderly population, respectively. 55LL genotype was associated with a 1.78-fold increase in the risk of ischemic stroke. CONCLUSION: PON1 genotypes, but not activities, are related with the risk of stroke.

Assessment of pollution in the West Black Sea Coast of Turkey using biomarker responses in fish.

Aim of this study was to determine the extent of pollution in the West Black Sea Coast of Turkey by measuring CYP1A associated EROD activity, phase II enzyme, glutathione S-transferase and antioxidant enzymes, catalase and glutathione reductase activities and immunochemical detection of CYP1A protein level in the liver of mullet. The fish samples were caught from six locations having a varying degree of pollution in the West Black Sea Region of Turkey in August 2005, 2006 and 2007. Mullets caught from Zonguldak Harbour, Eregli Harbour and Gülüç Stream's Mouth displayed 6-9-fold higher EROD, 2-4-fold higher glutathione S-transferase and 2-3-fold higher catalase activities than the reference site, Amasra. Total polyaromatic hydrocarbon levels in mullets caught from these locations were also significantly higher (2-4-fold) than Amasra. The results of this study indicate that Zonguldak Harbour, Eregli Harbour and Gülüç Stream are highly polluted by polycyclic aromatic hydrocarbons and related contaminants.

Purification of CYP2B-like protein from feral leaping mullet (Liza saliens) liver microsomes and its biocatalytic, molecular, and immunological characterization.

In this study, CYP2B-immunoreactive protein was purified to electrophoretic homogeneity from the liver microsomes of leaping mullet. The purified cytochrome P450 (CYP) gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis having a M(r) of 49,300 Da. Absolute absorption spectrum of the purified CYP showed a maximum at 417 nm and CO-difference spectrum of dithionite-reduced cytochrome P450 gave a peak at 450 nm. The purified CYP was found to be active in N-demethylation of benzphetamine, erythromycin, and ethylmorphine, and O-dealkylation of pentoxyresorufin in the reconstituted system. However, it was unable to catalyze O-dealkylation of ethoxyresorufin, methoxyresorufin, benzyloxyresorufin, and hydroxylation of lauric acid and aniline. The purified CYP showed strong cross-reactivity with anti-sheep lung CYP2B, a homologue of CYP2B4. N-terminal amino acid sequence of the mullet P450 had the highest degree of homology with CYP2Bs among the known CYPs. Spectral, electrophoretic, immunochemical, N-terminal amino acid sequence, and biocatalytic properties of the purified CYP are most similar to those of mammalian cytochrome P4502B. All these data indicate that the purified CYP is certainly 2B-like. In this study, we not only purified biocatalytically active CYP2B-like protein from fish, but also demonstrated detailed functional properties of CYP2B-like protein for the first time.

Paraoxonase/arylesterase ratio, PON1 192Q/R polymorphism and PON1 status are associated with increased risk of ischemic stroke.

OBJECTIVES: In recent years, importance of enzyme activity measurements, in addition to genotyping, in epidemiological studies relating paraoxonase 1 (PON1) and vascular disease was emphasized. This is the first report evaluating paraoxonase and arylesterase activities as risk factors for ischemic stroke. In addition, PON1 192Gln(Q)/Arg(R) and 55Leu(L)/Met(M) polymorphisms were also analyzed. DESIGN AND METHODS: The study population was comprised of 108 ischemic stroke patients and 78 controls. Enzyme activities were determined by spectrophotometric assays and for genotyping, standard PCR protocols followed by restriction enzyme digestions were used. RESULTS: The prevalence of the PON1 192RR genotype was increased among stroke patients (16.7%) as compared to controls (9.0%, P=0.129). Paraoxonase and arylesterase activities and PON1 activity ratio (paraoxonase/arylesterase) were found to be lower in patients than in controls. Logistic regression analysis revealed PON1 activity ratio (odds ratio, OR=0.697, 95% CI, 0.541 to 0.898, P=0.005), PON1 192RR genotype (OR=3.434, 95% CI, 1.159 to 10.178, P=0.026) and PON1 status (PON1 activity ratio combined with PON1 192RR genotype; OR=1.406, 95% CI, 1.038 to 1.905, P=0.028) as significant predictors of stroke. CONCLUSIONS: This study identified PON1 activity ratio, PON1 192RR genotype and PON1 status as important risk factors for ischemic stroke.

Bioreduction of idarubicin and formation of ROS responsible for DNA cleavage by NADPH-cytochrome P450 reductase and its potential role in the antitumor effect.

PURPOSE: Idarubicin is a clinically effective synthetic anthracycline analog used in the treatment of several human neoplasms. Anthracyclines have the potential to undergo bioactivation by flavoenzymes to free radicals and thus exert their cytotoxic actions. In this study, our main objective was to investigate the possible involvement of NADPH-cytochrome P450 reductase in the bioreductive activation of idarubicin to DNA-damaging species. METHODS: A pBR322 plasmid DNA damage assay was used as a sensitive method for detecting strand breaks in DNA exposed to idarubicin in the presence of P450 reductase and cofactor NADPH under various incubation conditions. In addition, the rates of idarubicin reduction by P450 reductases purified from phenobarbital-treated rabbit liver, beef liver and sheep lung microsomes were determined by measuring NADPH oxidation at 340 nm. RESULTS: The plasmid DNA experiments demonstrated that idarubicin could undergo bioreduction by P450 reductase with the resulting formation of DNA strand breaks. The antioxidant enzymes SOD and catalase, and hydroxyl radical scavengers, DMSO and thiourea, afforded significant levels of protection against idarubicin-induced DNA strand breaks. These findings suggested that DNA damage by idarubicin occurs through a mechanism which involves its redox cycling with P450 reductase to generate reactive oxygen species (ROS). The extent of DNA damage by idarubicin was found to increase with increasing concentrations of drug or enzyme as well as with increasing incubation time. The capacity of idarubicin to induce DNA damage under above incubation conditions was compared with that of a model compound, mitomycin C. Finally, enzyme assays carried out with purified P450 reductases revealed that idarubicin exhibited about two-fold higher rate of reduction than mitomycin C. CONCLUSION: Our findings implicated bioreduction of idarubicin by P450 reductase and subsequent redox cycling under aerobic conditions as being one mode of idarubicin action potentially contributing to its antitumor effect.

Significance of genetic polymorphisms at multiple loci of CYP2E1 in the risk of development of childhood acute lymphoblastic leukemia.

BACKGROUND/AIMS: The molecular etiology of childhood acute lymphoblastic leukemia (ALL) is likely to involve interactions between environmental factors and genetic make up. Understanding these interactions between various predisposing genes for the risk of developing childhood leukemia is of considerable importance. CYP2E1 is a susceptible gene in this respect, especially for its capacity to bioactivate many procarcinogens including benzene and N-nitrosodimethylamine. The CYP2E1 gene possesses several polymorphisms in humans, and among them, CYP2E1*5B and *6 have been shown to be associated with increased risks of several chemical-induced diseases. There are limited and contradictory data on the association between the CYP2E1*5B variant allele and childhood ALL, and none on such associations of CYP2E1*6 and*7B variant alleles. The aim of this study was to investigate the possible association of CYP2E1*5B, *6 and *7B alleles, alone or in combination, with the risk of incidence of childhood ALL in a Turkish population. METHODS: The genotypes for both polymorphisms were determined by polymerase chain reaction/restriction fragment length polymorphism techniques on 207 healthy controls and 168 patients. RESULTS: Neither locus was associated with the occurrence of childhood ALL. On the other hand, when both CYP2E1*5B and *6 alleles were considered together, the risk of childhood ALL increased significantly (2.9-fold; OR = 2.9, 95% CI 1.0-8.5; p < 0.05). Moreover, the presence of at least 2 variant alleles of any combination increased the risk significantly 3.9 times, suggesting a combined effect (OR = 3.9, 95% CI 1.4-11.0). CONCLUSION: Individuals carrying combinations of CYP2E1*5B, *6 and *7B variants together are likely associated with the risk of developing childhood ALL.

The low frequency of defective TPMT alleles in Turkish population: a study on pediatric patients with acute lymphoblastic leukemia.

6-Mercaptopurine (6MP) is an essential anticancer drug used in the treatment of childhood acute lymphoblastic leukemia (ALL). Thiopurine methyltransferase (TPMT) polymorphisms are the major determinants of interindividual differences in the severe toxicity or efficacy of 6MP. Four variant alleles, TPMT*2, TPMT*3A, TPMT*3B, and TPMT*3C, are responsible over the 80% of low or undetectable enzyme activity. The frequencies of these variants were investigated among 106 children with ALL in Turkish population. TPMT*3A and TPMT*3C were the only deficiency alleles detected in Turkish population with an allele frequency of 0.9% for both. While *3C allele frequency in Turkish population was found to be very similar to Asian and other Caucasian populations, *3A allele frequency was significantly (P < 0.05) lower. So far, studies showed that the genetic polymorphisms of other drug metabolizing enzymes like CYP2E1, CYP1A1, GSTM1/ T1 in Turkish population were similar to Caucasian populations. However, we found that the distribution of TPMT polymorphisms in Turkish population was significantly lower than those in other Caucasians like British, French, and Italian whereas the distributions of TPMT variants were found to be very similar to Kazak population which is also Caucasian in ethnic origin. In this study, the clinical histories of the patients in the sample population were also examined, retrospectively. The patients with heterozygous or homozygous mutant genotypes had developed severe neutropenia and infection during 6MP therapy. The study provides the first data on the frequency of common TPMT variants in the Turkish population, based on analysis of pediatric patients with ALL.

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.

Effects of diabetes on rabbit kidney and lung CYP2E1 and CYP2B4 expression and drug metabolism and potentiation of carcinogenic activity of N-nitrosodimethylamine in kidney and lung.

There are limited number of studies regarding the influence of diabetes on the regulation of cytochrome P450s and associated drug metabolizing enzyme activities especially in extrahepatic tissues such as kidney. However, there is almost no such study in lung. Alloxan-induced diabetes did not change CYP2B4 expression as measured with immunoblot analysis and associated enzyme, benzphetamine N-demethylase, activity in rabbit kidney and lung. Induction of cytochrome P4502E1 by diabetes was identified by immunochemical detection on Western blots in the lung and kidney microsomes of rabbits. In parallel to CYP2E1 induction, aniline 4-hydroxylase and p-nitrophenol hydroxylase activities were markedly increased in diabetic rabbit lung and kidney. CYP2B4 and CYP2E1 dependent drug metabolism did not show any tissue variation in diabetic rabbit. These findings are in contrast to those of rats, mice and hamster. The results of the present work, in combination with those of the previous work [Arinç, E., Arslan, S., Adali, O., 2005. Differential effects of diabetes on CYP2E1 and CYP2B4 proteins and associated drug metabolizing enzyme activities in rabbit liver. Arch. Toxicol. 79, 427-433], indicate the existence of species-dependent response of CYP-dependent drug metabolizing enzymes to diabetes. A procarcinogen and food contaminant, N-nitrosodimethylamine (NDMA), is converted to its carcinogenic form after it is activated with NDMA N-demethylase. In the current study, a statistically significant increase of liver, kidney and lung NDMA N-demethylase activity associated with CYP2E1 was shown in diabetic rabbit. Thus, it is expected that, the risk of nitrosamine induced carcinogenesis will be greater in liver, kidney and lung of the diabetic subjects.

Effect of mercury, cadmium, nickel, chromium and zinc on kinetic properties of NADPH-cytochrome P450 reductase purified from leaping mullet (Liza saliens).

Information on the mechanism of metal ion inhibition of NADPH-cytochrome P450 reductase is limited. The purpose of the present paper was to elucidate in vitro effect of Hg(+2), Cd(+2), Ni(+2), Cr(+3) and Zn(+2) ions on the purified mullet NADPH-cytochrome P450 reductase. NADPH-cytochrome P450 reductase was purified from detergent-solubilized liver microsomes from leaping mullet (Liza saliens). All of the metal ions caused inhibition of the enzyme activity except Zn(+2). At 50 microM metal concentration, Hg(+2) inhibited the cytochrome P450 reductase activity completely (100%), while, at the same concentrations, Cd(+2), Cr(+3) and Ni(+2) caused 66%, 65% and 37% inhibition, respectively. At 50 microM metal concentration, Zn(+2) had no apparent effect on cytochrome P450 reductase activity. The IC(50) values of HgCl(2), CrCl(3), CdCl(2) and NiCl(2) were estimated to be 0.07 microM, 24 microM, 33 microM and 143 microM, respectively. Of the metal ions tested, Hg(+2) exhibited much higher inhibitory effect at lower concentrations, so it was evidently a more potent inhibitor than the others. All four metal ions displayed noncompetitive type of inhibition mechanism for the purified reductase as analyzed by Dixon plot. K(i) values of Hg(+2), Cr(+3), Cd(+2), and Ni(+2) were calculated from Dixon plots as 0.048 microM, 18 microM, 73 microM and 329 microM, respectively.

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.