Modulation of Caco-2 Colon Cancer Cell Viability and CYP2W1 Gene Expression by Hesperidin-treated Lacticaseibacillus rhamnosus GG (LGG) Cell-free Supernatants.

BACKGROUND AND OBJECTIVE: Ensuring colon homeostasis is of significant influence on colon cancer and delicate balance is maintained by a healthy human gut microbiota. Probiotics can modulate the diversity of the gut microbiome and prevent colon cancer. Metabolites/byproducts generated by microbial metabolism significantly impact the healthy colonic environment. Hesperidin is a polyphenolic plant compound well known for its anticancer properties. However, low bioavailability of hesperidin after digestion impedes its effectiveness. CYP2W1 is a newly discovered oncofetal gene with an unknown function. CYP2W1 gene expression peaks during embryonic development and is suddenly silenced immediately after birth. Only in the case of some types of cancer, particularly colorectal and hepatocellular carcinomas, this gene is reactivated and its expression is correlated with the severity of the disease. This study aimed to investigate the effects of hesperidin-treated Lacticaseibacillus rhamnosus GG (LGG) cell-free supernatants on CaCo2 colon cancer cell viability and CYP2W1 gene expression. METHODS: Alamar Blue cell viability assay was used to investigate the cytotoxic effect of cell-free supernatant of LGG grown in the presence of hesperidin on CaCo2 cells. To observe the effect of cell-free supernatants of LGG on the expression of CYP2W1 gene, qRT-PCR was performed. RESULTS: Five times diluted hesperidin treated cell-free supernatant (CFS) concentration considerably reduced CaCo2 colon cancer cell viability. Furthermore, CYP2W1 gene expression was similarly reduced following CFS treatments and nearly silenced under probiotic bacteria CFS treatment. CONCLUSION: The CYP2W1 gene expression was strongly reduced by cell-free supernatants derived from LGG culture, with or without hesperidin. This suggests that the suppression may be due to bacterial byproducts rather than hesperidin. Therefore, the CYP2W1 gene in the case of deregulation of these metabolites may cause CYP2W1-related colon cancer cell proliferation.

Xenogenic Neural Stem Cell-Derived Extracellular Nanovesicles Modulate Human Mesenchymal Stem Cell Fate and Reconstruct Metabolomic Structure.

Extracellular nanovesicles, particularly exosomes, can deliver their diverse bioactive biomolecular content, including miRNAs, proteins, and lipids, thus providing a context for investigating the capability of exosomes to induce stem cells toward lineage-specific cells and tissue regeneration. In this study, it is demonstrated that rat subventricular zone neural stem cell-derived exosomes (rSVZ-NSCExo) can control neural-lineage specification of human mesenchymal stem cells (hMSCs). Microarray analysis shows that the miRNA content of rSVZ-NSCExo is a faithful representation of rSVZ tissue. Through immunocytochemistry, gene expression, and multi-omics analyses, the capability to use rSVZ-NSCExo to induce hMSCs into a neuroglial or neural stem cell phenotype and genotype in a temporal and dose-dependent manner via multiple signaling pathways is demonstrated. The current study presents a new and innovative strategy to modulate hMSCs fate by harnessing the molecular content of exosomes, thus suggesting future opportunities for rSVZ-NSCExo in nerve tissue regeneration.

Association of cholesterol 7α-hydroxylase (CYP7A1) promoter polymorphism (rs3808607) and cholesterol 24S-hydroxylase (CYP46A1) intron 2 polymorphism (rs754203) with serum lipids, vitamin D levels, and multiple sclerosis risk in the Turkish population.

BACKGROUND: Patients with multiple sclerosis (MS) have significantly lower vitamin D levels. Cholesterol is known to be the precursor for vitamin D synthesis, and cholesterol removal is regulated by cholesterol 7α-hydroxylase (CYP7A1) in the liver and cholesterol 24S-hydroxylase (CYP46A1) in the brain. In this study, single nucleotide polymorphisms (SNPs) within the genes CYP7A1 (rs3808607) and CYP46A1 (rs754203) were investigated for their effects on serum lipid profiles, vitamin D levels, and the risk of developing MS. METHODS: Patients with MS (n = 191) and controls (n = 100) were tested using the PCR-RFLP method to determine their genotypes for rs3808607 and rs754203 SNPs. RESULTS: The minor (C) allele frequency for CYP7A1 rs3808607 variation was 0.380 in patients with MS and 0.305 in control subjects (P = .074). For CYP46A1 rs754203, the frequencies of the minor (C) allele were 0.272 and 0.250 in patients and control subjects, respectively (P = .563). Serum vitamin D (25(OH)D3) concentrations were significantly lower in patients than in control subjects (P = .002). The CYP46A1 rs754203 SNP was associated with total cholesterol levels in patients, whereas the CYP7A1 rs3808607 variant was not associated with serum lipid parameters or vitamin D levels in patients or control subjects. CONCLUSION: CYP7A1 rs3808607 and CYP46A1 rs754203 variations are not likely to confer an independent risk for MS development in the Turkish population. To the best of our knowledge, this is the first study to investigate the association between CYP46A1 rs754203 and MS risk.

Missense Genetic Polymorphisms of Microsomal (EPHX1) and Soluble Epoxide Hydrolase (EPHX2) and Their Relation to the Risk of Large Artery Atherosclerotic Ischemic Stroke in a Turkish Population.

PURPOSE: Soluble epoxide hydrolase (sEH) and microsomal epoxide hydrolase (mEH) both catalyze the metabolism of epoxyeicosatrienoic acids (EETs), lipid signaling molecules that are protective against ischemic brain injury owing to their participation in the regulation of vascular tone and cerebral blood flow. In addition, mEH metabolizes polycyclic aromatic hydrocarbons, one of the causative factors of atherosclerotic lesion development. In this study, we aimed to investigate the association of enzyme activity-modifying missense single nucleotide polymorphisms (SNPs) of the sEH gene (EPHX2) and mEH gene (EPHX1) and ischemic stroke risk in a Turkish population. PATIENTS AND METHODS: Genomic DNA of patients with large artery atherosclerotic ischemic stroke (n=237) and controls (n=120) was isolated from blood samples, and genotypes for Tyr113His (rs1051740) and His139Arg (rs2234922) SNPs of EPHX1 and Arg287Gln (rs751141) SNP of EPHX2 were attained by the PCR/RFLP method. RESULTS: Minor allele frequency and genotype distributions for Arg287Gln, Tyr113His and His139Arg SNPs did not differ significantly between stroke patients and controls. However, hypertension- and diabetes-associated ischemic stroke risk was decreased by EPHX1 and increased by EPHX2 variants in stratification analyses. CONCLUSION: This study has shown for the first time that the polymorphic alleles of EPHX1 were unlikely to be associated with large artery atherosclerotic ischemic stroke susceptibility; however, protective effects were evident within subgroups of hypertension and diabetes. In addition, EPHX2 Arg287Gln polymorphism, which has been studied for the first time in a Turkish population, was not significantly related to ischemic stroke, but increased the stroke risk in subgroup analysis.

Association analysis of Glutathione S-transferase omega-1 and omega-2 genetic polymorphisms and ischemic stroke risk in a Turkish population.

OBJECTIVES: Oxidative stress is a known risk factor for the pathogenesis of atherosclerosis, the main cause of ischemic stroke. Glutathione S-transferase (GST) omega-1 and omega-2, members of phase II enzymes, play a role in the antioxidant system. The single nucleotide polymorphisms (SNPs), C419A and A424G in GST omega genes can cause a decrease in enzyme activity. The aim of this study was to investigate the possible association between these polymorphisms and ischemic stroke risk in a Turkish population. METHODS: The genotypes and allele frequencies for 239 patients and 130 controls were determined by the PCR/RFLP method. No significant differences were found between patients and controls in terms of genotype and allele frequencies. RESULTS: The frequency of the polymorphic 'A' allele was 0.358 in patients and 0.342 in controls for the C419A polymorphism in the GSTO1 gene. The frequency of the polymorphic 'G' allele for GSTO2 A424G SNP was 0.370 in patients and 0.404 in controls. The combined homozygous wild type genotype 'CCAG' was significantly higher in control group than in the patients. CONCLUSION: No significant difference was observed between the stroke patients and controls in terms of genotypes and allele distributions. Double combine haplotype CCAA was found to be protective against ischemic stroke when compare to other haplotypes. However, different genotypes of GSTO1 and GSTO2 were observed to have effects on stroke risk in subgroups of diabetics and smokers. In conclusion, the current study is the first to report this finding.

Genetic polymorphisms of vitamin D3 metabolizing CYP24A1 and CYP2R1 enzymes in Turkish patients with ischemic stroke.

Objective Vitamin D deficiency is known as an important risk factor in pathogenesis of atherosclerosis, which contributes to stroke development. Genetic variations including single nucleotide polymorphisms (SNPs) in enzymes involved in vitamin D metabolism can affect susceptibility to the development of stroke. Therefore, the objective of this study was to investigate the association between polymorphisms of vitamin D metabolizing enzymes (rs927650 SNP in CYP24A1, and rs10741657 SNP in CYP2R1 genes,) and ischemic stroke risk in Turkish population. Materials and methods To test this hypothesis, we designed a case-control study which consisted of 256 ischemic stroke patients and 132 controls. Genotypes were determined by PCR-RFLP technique. Results No significant differences were found between patients and controls in terms of CYP24A1 rs927650 and CYP2R1 rs10741657 genotype frequencies. Polymorphic allele frequencies of CYP24A1 rs927650 and CYP2R1 rs10741657 were 0.414 and 0.660 in stroke patients, respectively. Conclusion This is the first study conducted regarding the association of CYP24A1 rs927650 and CYP2R1 rs10741657 genetic polymorphisms and ischemic stroke risk. The polymorphic genotypes of these polymorphisms, together with hypertension, diabetes, smoking, and obesity, were found as significant risk factors for ischemic stroke.

Relationship between the hippocampal expression of selected cytochrome P450 isoforms and the animal performance in the hippocampus-dependent learning task.

Despite very extensive studies on the molecular mechanisms of memory formation, relatively little is known about the molecular correlates of individual variation in the learning skills within a random population of young normal subjects. The role of cytochrome P450 (CYP) enzymes in the brain also remains poorly understood. On the other hand, these enzymes are known to be related to the metabolism of substances important for neural functions including steroids, fatty acids, and retinoic acid. In the present study, we examined the potential correlation between the animals' performance in a place learning task and the levels of selected CYP isoforms (CYP2E1, CYP2D1 and CYP7A1) in the rat hippocampus. According to their performance, rats were classified as "good" learners (percent error/number of trials to criterion ≤ group mean - 3SEM) or "poor" learners (percent error/number of trials to criterion ≥ group mean + 3SEM). The CYP enzyme levels were determined by Western Blot at the early, intermediary and advanced stages of the task acquisition (day 4, day 8 and after reaching a performance criterion of 83% correct responses). In this study, as expected, CYP2E1 and CYP2D1 isoforms have been found in the rat hippocampus. However, a putative CYP7A1 isoform was also visualized. Hippocampal expression of these enzymes was shown to be dependent on the stage of learning and animals' cognitive status. In "good" learners compared to "poor" learners, significantly higher levels of CYP2E1 were found at the early stage of training, significantly higher levels of CYP2D1 were found at the intermediate stage of training, and significantly higher levels of CYP7A1-like protein were found after reaching the acquisition criterion. These findings suggest that the differential expression of some CYP isoforms in the hippocampus may have impact on individual learning skills and that different CYP isoforms may play different roles during the learning process.

Association of cytochrome P4502E1 and NAD(P)H:quinone oxidoreductase 1 genetic polymorphisms with susceptibility to large artery atherosclerotic ischemic stroke: a case-control study in the Turkish population.

Stroke, a major cause of death and disability, is described as interruption or severe reduction of blood flow in cerebral arteries. Oxidative stress plays an important role in the pathogenesis of atherosclerosis and carotid atherosclerosis is a risk factor for stroke. Combination of multiple environmental and genetic risk factors is thought to increase stroke. Therefore, investigation of the polymorphisms of enzymes is of crucial importance to determine the molecular etiology of the disease. To test this hypothesis, we performed a case-control study in which we compared the distribution of CYP2E1 and NQO1 genotypes between 245 large artery atherosclerotic ischemic stroke patients and 145 controls, using PCR-RFLP. A significant difference was observed between stroke patients and controls with respect to the CYP2E1*5B genotype (odds ratio; OR 8.069, P = 0.011) and allele (OR 7.876, P = 0.011) distribution. However, this polymorphism was not a significant predictor of disease status in logistic regression analysis. NQO1*2 polymorphism genotype distribution was significantly different between patients and controls (P = 0.027) and heterozygote *1*2 genotype was found to be a protective factor against large artery atherosclerotic ischemic stroke in logistic regression analysis (OR 0.562, P = 0.018). This is the first study conducted regarding the association of CYP2E1 and NQO1 genetic polymorphisms and large artery atherosclerotic ischemic stroke risk in Turkish population.

Tannic Acid Inhibits Proliferation, Migration, Invasion of Prostate Cancer and Modulates Drug Metabolizing and Antioxidant Enzymes.

The aim of this study was to investigate the effects of plant phenolic compound tannic acid (TA) on proliferative, metastatic, invasive properties of prostate cancer (PCa) cells; PC-3 and LNCaP, as well as drug metabolizing and antioxidant enzymes. Characterization of TA was done by using FT-IR and NMR. TA dose dependently inhibited the proliferation of PC-3 and LNCaP cells with IC50 values 35.3 µM and 29.1 µM, respectively. Wound healing assay showed that TA significantly inhibited (92.7%) migration of PCa cells (p<0.0001). In addition, TA was found to have anti-invasive potential on PC-3 cells and it inhibited (80.9%, p<0.0001) invasion of PC-3 cells into matrigel. Only 17.8% of PC-3 cells can form colony in the 0.7% agarose after treatment of cells with TA at the IC50 value concentration. Furthermore, flow cytometry analyses with Annexin V-APC and 7-AAD staining demonstrated that TA increases early apoptosis rate of PC-3 cells by 25.8% and LNCaP cells by 20.9%. Besides, Western blot and qRT-PCR analyses also demonstrated that TA regulates protein and mRNA expressions of CYP17A1, CYP3A4, CYP2B6, NQO1, GSTM1 and GSTP1 enzymes. The results obtained from this study show that TA might be a good candidate for combinational therapy and highly effective strategic molecule for reducing the occurrence of PCa.

Inhibitory action of Epilobium hirsutum extract and its constituent ellagic acid on drug-metabolizing enzymes.

Epilobium hirsutum (EH) is a medicinal plant for treating various diseases. Despite its wide usage, there is no available information about its potential influences on drug metabolism. The present study was undertaken to determine the in vivo effects of EH on hepatic CYP2B, CYP2C, CYP2D, and CYP3A enzymes that are primarily involved in drug metabolism. Male Wistar rats were injected intraperitoneally with EH water extract (EHWE) and ellagic acid (EA) at a daily dose of 37.5 and 20 mg/kg, respectively, for 9 days and hepatic drug-metabolizing enzymes were assessed at activity, protein and mRNA levels. Erythromycin N-demethylase activity was inhibited by 53 and 21 % in EHWE- and EA-treated rats, respectively. Benzphetamine N-demethylase and 7-benzyloxyresorufin-O-debenzylase activities were decreased by 53 and 43 %, and 57 and 57 % in EHWE-and EA-treated rats, respectively. Moreover, protein levels of CYP2B1, CYP2C6, CYP2D2, and CYP3A1 also decreased by 55, 15, 33, and 82 % as a result of EHWE treatment of rats, respectively. Similarly, CYP2B1, CYP2C6, CYP2D2, and CYP3A1 protein levels decreased by 62, 63, 49, and 37 % with EA treatment, respectively. qRT-PCR analyses also showed that mRNA levels of these enzymes were significantly inhibited with bothEHWE and EA treatments. In conclusion, inhibition of drug clearances leading to drug toxicity because of the lowered activity and expression of drug-metabolizing enzymes might be observed in the people who used EH as complementary herbal remedy that might be contributed by its EA content.

Contribution of ellagic acid on the antioxidant potential of medicinal plant Epilobium hirsutum.

In the present study, the possible role of ellagic acid (EA) on antioxidant potential of Epilobium hirsutum (EH) in rat liver was investigated. Wistar rats were intraperitoneally treated with 37.5 mg/kg of EH and 10 mg/kg of EA for 9 days. Effects of EH and EA on antioxidant [glutathione peroxidase (GPx) and superoxide dismutases (SOD)] and Phase II [NADPH quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTs)] enzyme activities, as well as protein and mRNA expressions of those, were investigated. Polyphenolic content of EH was determined by LC-MS/MS analysis. EH and EA injection to rats resulted in a significant increase of NQO1 (3.6-fold and 4.7-fold), GPx (1.45-fold), and SOD (1.34-fold and 1.27-fold) enzyme activities, whereas total GST (46% and 57%) and its isoforms,and GST mu (57% and 72%), and GST theta (60% and 68%) activities were significantly decreased. Western-blot and qRT-PCR analysis showed that NQO1 and GPx protein and mRNA expressions were increased significantly (P < 0.0001), whereas GST mu and GST theta were significantly decreased (P < 0.0001).

Importance of NOS3 genetic polymorphisms in the risk of development of ischemic stroke in the Turkish population.

In the present study, we aimed to investigate the relationship between endothelial nitric oxide synthase 3 (NOS3) G894T, T-786C, and intron 4 variable number of tandem repeat (VNTR) variants, alone or in combination, and the risk of incidence of ischemic stroke in the Turkish population. The genotypes for all polymorphisms were determined by polymerase chain reaction/restriction fragment length polymorphism techniques on 245 ischemic stroke patients and 145 controls. In the case-control analysis, no significant difference was observed between stroke patients and controls with respect to NOS3 G894T, T-786C, and intron 4 VNTR polymorphisms genotype and allele frequency distribution. However, the copresence of G894T and intron 4 VNTR risk-elevating genotypes in the same individual increased the risk of stroke seven times (odds ratio=7.083, 95% confidence interval=0.866-57.963, p=0.029).

A comparative study for the evaluation of two doses of ellagic acid on hepatic drug metabolizing and antioxidant enzymes in the rat.

The present study was designed to evaluate different doses of ellagic acid (EA) in vivo in rats for its potential to modulate hepatic phases I, II, and antioxidant enzymes. EA (10 or 30 mg/kg/day, intragastrically) was administered for 14 consecutive days, and activity, protein, and mRNA levels were determined. Although the cytochrome P450 (CYP) 2B and CYP2E enzyme activities were decreased significantly, the activities of all other enzymes were unchanged with the 10 mg/kg/day EA. In addition, western-blot and qRT-PCR results clearly corroborated the above enzyme expressions. On the other hand, while the NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities were increased significantly, CYP1A, 2B, 2C, 2E, and 19 enzyme activities were reduced significantly with 30 mg/kg/day EA. In addition, CYP2B, 2C6, 2E1, and 19 protein and mRNA levels were substantially decreased by the 30 mg/kg/day dose of EA, but the CYP1A protein, and mRNA levels were not changed. CYP3A enzyme activity, protein and mRNA levels were not altered by neither 10 nor 30 mg/kg/day ellagic acid. These results indicate that EA exerts a dose-dependent impact on the metabolism of chemical carcinogens and drugs by affecting the enzymes involved in xenobiotics activation/detoxification and antioxidant pathways.

Hippocampal levels of ChAT, PKA, phospho-PKA and phospho-CaMKIIα but not CaMKIIα positively correlate with spatial learning skills in rats.

Despite very extensive investigations on molecular processes underlying memory formation, there are very few studies examining potential differences in the brain biochemistry between "good" and "poor" learners belonging to a random population of young animals. In the present study, an attempt was made to correlate individual variation in spatial learning in young-adult Long-Evans rats with hippocampal levels of protein kinase A (PKA), calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα), and choline acetyltransferase (ChAT). Additionally, in order to indirectly estimate the activity of CaMKIIα and PKA, hippocampal levels of their phosphorylated forms (pCaMKIIα and pPKA) were assessed using Western blot technique. Rats were classified as "good" and "poor" learners on the basis of their performance in a partially baited 12-arm radial maze. The biochemical assays did not reveal a significant difference in the basal hippocampal levels of the CaMKIIα, however, the level of pCaMKIIα, was significantly higher in "good" learners. Also, hippocampal levels of both PKA and pPKA, as well as that of ChAT, were significantly higher in "good" as compared to "poor" learners. Our results suggest that the differences in the expression level of PKA and ChAT (but not of CaMKIIα), as well as the differences in the activation of both PKA and CaMKIIα, may contribute to the individual variation in learning skills and episodic-like memory in a random population of young-adult subjects.

Epilobium hirsutum alters xenobiotic metabolizing CYP1A1, CYP2E1, NQO1 and GPx activities, mRNA and protein levels in rats.

CONTEXT: Natural products have attracted increasing interests due to their use in flavoring, nutrition, cosmetics, pharmacy and medicine. Epilobium hirsutum L. (Onagraceae) is known for its analgesic, antimicrobial, and antiproliferative activity. CYP1A1 and CYP2E1, xenobiotic metabolizing enzymes, serve as a metabolic activation route yielding reactive metabolites that are eliminated by the action of NQO1 and glutathione peroxidase (GPx) enzymes. OBJECTIVE: This study investigated in vivo effects of Epilobium hirsutum (EH) on CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA expressions in liver. MATERIALS AND METHODS: Male Wistar Albino rats were injected with EH at a dose of 37.5 mg/kg i.p. daily for 9 d. CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA levels were determined by enzyme assays, Western blotting and qPCR, respectively. RESULTS: CYP1A1 associated ethoxyresorufin-O-deethylase activity of control and EH-treated animals were found as 6.54 ± 1.21 and 4.48 ± 1.67 nmol/min/mg, respectively. CYP2E1 associated aniline 4-hydroxylase of control and EH group were 0.537 ± 0.011 and 0.109 ± 0.01 nmol/min/mg, respectively. However, EH treatment increased the GPx and NQO1 activities from 0.069 ± 0.015 to 0.107 ± 0.026 nmol/min/mg and from 163.34 ± 92 to 588.3 ± 14 nmol/min/mg, respectively. Furthermore, protein and mRNA expression analysis revealed that CYP1A1 and CYP2E1 levels were decreased while those of NQO1 and GPx increased after EH treatment. DISCUSSION AND CONCLUSION: Our current data suggest that the metabolism of xenobiotics, including drugs, may be altered due to changes in the expression and activity of these proteins by EH.

Flavin containing monooxygenase 3 genetic polymorphisms Glu158Lys and Glu308Gly and their relation to ischemic stroke.

Ischemic stroke is a multifactorial disease leading to severe long-term disability and it is the third leading cause of death in developed countries. Although many studies have been reported to elucidate etiological and pathological mechanisms of stroke, the genetic and molecular basis of disease remains poorly understood. Recent studies have shown that reactive oxygen species causing oxidative stress play a pivotal role in the pathogenesis of atherosclerosis that is the main cause of a group of cardiovascular diseases including ischemic stroke. In this study, we aimed to investigate the relationship between FMO3 Glu158Lys and Glu308Gly variants, and the risk of incidence of ischemic stroke in Turkish population. Two single nucleotide polymorphisms (SNPs) within the FMO3 gene were genotyped by using PCR-RFLP technique in a sample set of 245 cases and 145 controls. In the case-control analysis, no significant difference was observed between stroke patients and controls with respect to FMO3 Glu158Lys and Glu308Gly polymorphisms' genotype and allele frequency distribution. However, heterozygote 158Glu/Lys (OR=6.110, P<0.001) and 308Glu/Gly (OR=6.000, P=0.006) genotypes increase the risk of stroke 6 times in hypertensive subjects. On the other hand, the wild type genotypes 158Glu/Glu and 308Glu/Glu had 6.2-fold and 4.8-fold higher risk of ischemic stroke in obese subgroup, respectively. Our results clearly showed that the risk of hypertension-related ischemic stroke was higher in the heterozygote genotype carriers. This is the first study conducted regarding the association of FMO3 Glu158Lys and Glu308Gly genetic polymorphisms and ischemic stroke risk in Turkish population.

Apolipoprotein A5 polymorphisms in Turkish population: association with serum lipid profile and risk of ischemic stroke.

Atherosclerosis, a major cause of ischemic stroke, may be associated with variability of triglyceride (TG) levels. Apolipoprotein A5 (APOA5) genetic polymorphisms are associated with altered TG levels. The objective of this study was to investigate the coding region polymorphisms S19W (rs3135506) and G185C (rs2075291) and the promoter region polymorphism -1131T>C (rs662799) of the APOA5 gene as risk factors for ischemic stroke in Turkish population. Study group consisted of 272 ischemic stroke patients and 123 controls. Genotypes were determined by real-time polymerase chain reaction (PCR) for S19W and PCR-restriction fragment length polymorphism analysis (PCR-RFLP) for -1131T>C and G185C. 19W allele frequency was 0.090 in stroke patients and 0.062 in controls (P = 0.191). Minor allele frequencies of -1131T>C and G185C in patients were 0.106 and 0.004, respectively, and were nearly the same in controls. Total cholesterol and LDL-cholesterol levels were significantly higher for stroke patients having at least one 19W allele compared to non-carriers. A significant difference was also found for LDL-cholesterol levels of stroke patients; higher in -1131C allele carriers compared to wild type patients. There was a trend for higher frequency of ischemic stroke among -1131C allele carrier hypertensive, diabetic or obese subjects compared to non-carriers. However, APOA5 genotypes were not associated with the risk of ischemic stroke by logistic regression analysis. The present study demonstrated that carrying rare alleles of APOA5 S19W, -1131T>C and G185C alone do not constitute a risk for ischemic stroke in the studied Turkish subjects.

Correlation between hippocampal levels of neural, epithelial and inducible NOS and spatial learning skills in rats.

In the present study, to better understand the role of different nitric oxide synthase (NOS) isoforms in hippocampus-dependent forms of learning, we examined the expression of neural, endothelial, and inducible NOS in the hippocampus of young-adult rats classified as "poor" and "good" learners on the basis of their performance in the partially baited 12-arm radial maze. Taking into consideration strain-dependent differences in learning skills and NOS expression, experiments were performed on two different lines of laboratory rats: the inbred Wistar (W) and the outcrossed Wistar/Spraque-Dawley (W/S) line. The hippocampal levels of NOS proteins were assessed by Western Blotting. In the present study, genetically more homogenous W rats showed a slower rate of learning compared to the genetically less homogenous outcrossed W/S rats. The deficient performance in the W rat group compared to outcrossed W/S rats, and in "poor" learners of both groups compared to "good" learners was due to a higher percentage of reference memory errors. The overall NOS levels were significantly higher in W group compared to outcrossed W/S rats. In both rat lines, the rate of learning positively correlated with hippocampal levels of nNOS and negatively correlated with iNOS levels. Hippocampal eNOS levels correlated negatively with animals' performance but only in the W rats. These results suggest that all 3 NOS isoforms are implemented but play different roles in neural signaling.

Association analysis of GSTT1, GSTM1 genotype polymorphisms and serum total GST activity with ischemic stroke risk.

Oxidative stress plays a major role in pathogenesis of atherosclerosis which is responsible for stroke. Glutathione S-transferases (GSTs) detoxify metabolites produced by oxidative stress within the cell and protect the cells against injury. This study aimed to investigate the association of polymorphisms in GSTT1, GSTM1 genes and GST activity with ischemic stroke risk. Patients had almost the same GST activity as that of controls. No significant differences were found between patients and controls in terms of GSTT1 null, GSTM1 null and GSTT1/GSTM1 double null genotype frequencies. Besides, both patients and controls with double GSTT1/GSTM1 null genotypes had the lowest serum GST activities. Compared to the present genotypes, GSTT1 null (OR = 4.888; P = 0.006) and GSTM1 null (OR = 2.383; P = 0.011) genotype groups contained relatively more hypertensive stroke patients. This study showed that GSTT1 and GSTM1 null genotypes, together with hypertension, may play a significant role in the pathogenesis of ischemic stroke.

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.