Sibel Ozden

Methiocarb-induced oxidative damage following subacute exposure and the protective effects of vitamin E and taurine in rats

Methiocarb, is used worldwide in agriculture and health programs. Besides its advantages in the agriculture, it causes several toxic effects. In this study, we aimed to investigate subacute effects of methiocarb on lipid peroxidation, reduced glutathione (GSH), antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) and histopathological changes in rat tissues. Moreover, we examined the possible protective effects of vitamin E and taurine on methiocarb-induced oxidative damage in rat tissues. Rats were randomly divided into six groups as follows; I-control group; II-methiocarb group; III-vitamin E group; IV-vitamin E+methiocarb group; V-taurine group and VI-taurine+methiocarb group. Methiocarb significantly increased lipid peroxidation in liver and kidney when compared to control groups. Levels of GSH and activities of SOD, CAT and GSH-Px were found to be decreased, while GSH-Rd remained unchanged in rat liver and kidney treated with methiocarb. Pretreatment of vitamin E and taurine resulted in a significant decrease on lipid peroxidation, alleviating effects on GSH and antioxidant enzymes. The degenerative histological changes were less in liver than kidney of rats treated with methiocarb. Pretreatment of vitamin E and taurine showed a protective effect on the histological changes in kidney comparing to the liver of rats treated with methiocarb.

Low Doses of the Carcinogen Furan Alter Cell Cycle and Apoptosis Gene Expression in Rat Liver Independent of DNA Methylation

Background Evidence of potent rodent carcinogenicity via an unclear mechanism suggests that furan in various foods [leading to an intake of up to 3.5 μg/kg body weight (bw)/day] may present a potential risk to human health. Objectives We tested the hypothesis that altered expression of genes related to cell cycle control, apoptosis, and DNA damage may contribute to the carcinogenicity of furan in rodents. In addition, we investigated the reversibility of such changes and the potential role of epigenetic mechanisms in response to furan doses that approach the maximum estimated dietary intake in humans. Methods The mRNA expression profiles of genes related to cell cycle, apoptosis, and DNA damage in rat liver treated with furan concentrations of 0.1 and 2 mg/kg bw were measured by quantitative polymerase chain reaction (PCR) arrays. We assessed epigenetic changes by analysis of global and gene-specific DNA methylation [methylation-specific PCR, combined bisulfite restriction analysis (COBRA), and methylated DNA immunoprecipitation chip] and microRNA (miRNA) analyses. Results The expression profiles of apoptosis-related and cell-cycle–related genes were unchanged after 5 days of treatment, although we observed a statistically significant change in the expression of genes related to cell cycle control and apoptosis, but not DNA damage, after 4 weeks of treatment. These changes were reversed after an off-dose period of 2 weeks. None of the gene expression changes was associated with a change in DNA methylation, although we detected minor changes in the miRNA expression profile (5 miRNA alterations out of 349 measured) that may have contributed to modification of gene expression in some cases. Conclusion Nongenotoxic changes in gene expression may contribute to the carcinogenicity of furan in rodents. These findings highlight the need for a more comprehensive risk assessment of furan exposure in humans.

Determination of Commonly Used Herbicides in Surface Water Using Solid-Phase Extraction and Dual-Column HPLC-DAD

The present study describes the application of different solid-phase extraction techniques for the extraction, separation, and quantitative determination of 10 commonly used herbicides with different chemical structures (chlorsulfuron, diuron, bentazone, linuron, chlorpropham, fenoxoprop-ethyl, MCPA, diclofop-methyl, fluazifop-butyl, trifluraline) in water. Octadecyl (C18) Empore extraction disks, octadecyl (C18), and stryene divinylbenzene (SDB) Bond Elut Env cartridges were compared for solid-phase extraction efficiency. Herbicides were separated and quantified by reversed-phase high performance liquid chromatography with diode-array detection (HPLC-DAD) with simultaneous separation on two columns of differing polarity (C18 and CN) to confirm identification. Analytical separation was performed simultaneously on C18 and CN columns. Reanalysis of the sample extracts on a (cyano) CN column were used to confirm the identity of these compounds. Method optimization and validation parameters were presented in this work. Recoveries varied from 76.0% to 99.0% for C18 disks, from 75.1% to 100.0% for C18 cartridges, and from 54.0% to 98.0% for SDB cartridges over concentrations at 0.025–0.4 μg L−1. The limits of detection were 0.012–0.035 μg L−1.

Effects of methiocarb on lipid peroxidation and glutathione level in rat tissues

Methiocarb is an N-methylcarbamate insecticide used worldwide in agriculture and health programs. The aim of this study was to investigate the possible effects of methiocarb to induce lipid peroxidation (LPO) in tissues of male Wistar rats following single and repeated oral exposures. Animals were divided into six different groups, and methiocarb was administered by orally at doses 25, 10, and 2 mg/kg body weight for 1, 5, and 28 days, respectively. Liver, kidney, brain, and testis tissues were taken from the rats for the biochemical examinations. LPO and reduced glutathione (GSH) levels were determined in the tissues. LPO was significantly increased in liver, kidney, brain, and testis after 1-, 5-, and 28-day treatments of methiocarb. GSH levels were significantly increased in the 1-day period and significantly decreased in the 5- and 28-day periods in all tissues after methiocarb administration. It is concluded that methiocarb may induce LPO and produce disturbances on the GSH levels in liver, kidney, testis, and brain of rats. This suggests that methiocarb-induced toxicity may be associated with oxidative stress to cellular membranes. Further studies are required to better understand the role of oxidative stress on methiocarb-induced toxicity.

Acute effects of methiocarb on oxidative damage and the protective effects of vitamin E and taurine in the liver and kidney of Wistar rats

Methiocarb (MC) is a widely used carbamate pesticide in agriculture and health programs. Although the main molecular mechanism of carbamate toxicity involves acetylcholinesterase inhibition, studies have also implicated the induction of oxidative stress. Therefore, the present study was aimed to evaluate the effect of acute MC exposure on lipid peroxidation, antioxidant defense systems, histological changes in Wistar rats and the protective effect of pretreatment with vitamin E and taurine. A total of 48 rats were randomly divided into six groups. Rats in group I were given corn oil, while those in group III were dosed with vitamin E (100 mg/kg body weight (b.w.)) and in group V were dosed with taurine (50 mg/kg b.w.). Rats in group II were administered with MC only (25 mg/kg b.w., 1/4 of median lethal dose (LD50)), while those in groups IV and VI were pretreated with vitamin E (100 mg/kg b.w.) and taurine (50 mg/kg b.w.) for 20 days, respectively, and then exposed to MC (25 mg/kg b.w.). The rats administered with MC showed significant increase in the levels of malondialdehyde in the liver and kidney as an index of lipid peroxidation. Levels of glutathione and activities of superoxide dismutase, catalase and glutathione peroxidase were significantly increased, while activity of glutathione reductase remained unchanged in both the tissues after MC treatment. Mild degenerative histological changes were observed in liver tissue, while the changes in kidney tissue were more severe then liver after MC treatment. Pretreatment with vitamin E and taurine resulted in a significant decrease in the lipid peroxidation and alleviating effects on antioxidant defense systems in both the tissues, while protective effects on the histological changes were shown only in kidney when compared with liver. In conclusion, the study has demonstrated that the acute MC exposure in Wistar rats caused oxidative damage on liver and kidney, which were partly ameliorated by the pretreatment of vitamin E and taurine.

Assessment of global and gene-specific DNA methylation in rat liver and kidney in response to non-genotoxic carcinogen exposure.

Altered expression of tumor suppressor genes and oncogenes, which is regulated in part at the level of DNA methylation, is an important event involved in non-genotoxic carcinogenesis. This may serve as a marker for early detection of non-genotoxic carcinogens. Therefore, we evaluated the effects of non-genotoxic hepatocarcinogens, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), hexachlorobenzene (HCB), methapyrilene (MPY) and male rat kidney carcinogens, d-limonene, p-dichlorobenzene (DCB), chloroform and ochratoxin A (OTA) on global and CpG island promoter methylation in their respective target tissues in rats. No significant dose-related effects on global DNA hypomethylation were observed in tissues of rats compared to vehicle controls using LC-MS/MS in response to short-term non-genotoxic carcinogen exposure. Initial experiments investigating gene-specific methylation using methylation-specific PCR and bisulfite sequencing, revealed partial methylation of p16 in the liver of rats treated with HCB and TCDD. However, no treatment related effects on the methylation status of Cx32, e-cadherin, VHL, c-myc, Igfbp2, and p15 were observed. We therefore applied genome-wide DNA methylation analysis using methylated DNA immunoprecipitation combined with microarrays to identify alterations in gene-specific methylation. Under the conditions of our study, some genes were differentially methylated in response to MPY and TCDD, whereas d-limonene, DCB and chloroform did not induce any methylation changes. 90-day OTA treatment revealed enrichment of several categories of genes important in protein kinase activity and mTOR cell signaling process which are related to OTA nephrocarcinogenicity.

Ochratoxin A in dried grapes and grape-derived products in Turkey

Ochratoxin A (OTA) is a naturally occurring mycotoxin and widespread food contaminant which results in a probable human exposure. A total of 85 samples (50 dried grapes, 10 grape juices and 25 pekmez (boiled and concentrated grape juices) were collected from different supermarkets and traditional bazaars in Istanbul during 2008–2009. An analytical method based on immuno-affinity column for clean-up and high-performance liquid chromatography equipped with fluorescence detection was used to determine the OTA. Contamination frequencies were 8%, 20% and 88% with mean concentrations of positive samples of 1.15, 1.40 and 2.04 µg/kg for dried grapes, grape juices and pekmez samples, respectively. These levels were lower than the maximum levels as set by the European Commission (EC). However, 12 of 25 pekmez samples had higher levels than permitted by the European Union (EU) for safe consumption.

Global histone modifications in Fumonisin B1 exposure in rat kidney epithelial cells

Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in maize-based food and feed. Although the effects of FB1 on sphingolipid metabolism are clear, little is known about early molecular changes associated with FB1 carcinogenicity. It has been shown that FB1 disrupts DNA methylation and chromatin modifications in HepG2 cells. We investigated dose- and time-dependent effects of FB1 in global histone modifications such as histone H3 lysine 9 di-, trimethylation (H3K9me2/me3), histone H3 lysine 4 trimethylation (H3K4me3), histone H4 lysine 20 trimethylation (H4K20me3), histone H3 lysine 9 acetylation (H3K9ac) and the enzymes involved in these mechanisms in rat kidney epithelial cells (NRK-52E). The increased levels of global H3K9me2/me3 were observed in FB1 treated cells, while the global levels of H4K20me3 and H3K9ac were decreased. FB1 caused some changes on the activities of H3K9 histone methyltransferase (HMT) and histone acetyltransferase (HAT) at high concentrations in NRK-52E cells. Further, the effects of trichostatin A (TSA), a histone deacetylase inhibitor, were investigated in NRK-52E cells. TSA was found to cause an increase on H3K9ac levels as expected. In this study we suggest that FB1 may disrupt epigenetic events by altering global histone modifications, introducing a novel aspect on the potential mechanism of FB1 carcinogenesis.

Role of fumonisin B1 on DNA methylation changes in rat kidney and liver cells

Abstract Context: Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium verticillioides (Sacc.) Nirenberg (Nectriaceae) mold that contaminates maize and other agricultural products. Although the effects of FB1 on sphingolipid metabolism are clear, little is known about early molecular changes associated with FB1 carcinogenicity. Objective: Alteration on DNA methylation, as an early event in non-genotoxic carcinogenesis, may play an important role in the mechanism of FB1 toxiciy. Materials and methods: Dose-related effects of FB1 (1–50 µM for 24 h) on global DNA methylation by using high-performance liquid chromatography with UV-diode array detection (HPLC-UV/DAD) and CpG promoter methylation by methylation-specific PCR (MSP) were performed in rat liver (Clone 9) and rat kidney (NRK-52E) epithelial cells. Results: Cell viability reduction is 39% and 34% by the XTT test and LDH release in the growth medium is 32% and 26% at 200 µM of FB1 treatment in Clone 9 and NRK-52E cells, respectively. No significant dose-related effects of FB1 on global DNA methylation which ranged from 4 to 5% were observed in both cells compared with controls. Promoter regions of c-myc gene were methylated (>33%) at 10 and 50 µM of FB1 treatment in Clone 9 cells while it was unmethylated in NRK-52E cells. Promoter regions of p15 gene were unmethylated while VHL gene were found to be methylated (>33%) at 10, 25, and 50 µM and 10 and 50 µM of FB1 treatment in Clone 9 and NRK-52E cells, respectively. Discussion and conclusion: Alteration in DNA methylation might play an important role in the toxicity of FB1 in risk assessment process.

Effects of bentazone on lipid peroxidation and antioxidant systems in human erythrocytesin vitro

Abstract Bentazone, a benzothiadiazole herbicide, is widely used for a variety of crops including cereals, maize, peas, rice and soy beans. The concern for human health is stil very high because bentazone is continuously monitored in environment and several studies to evaluate its potential carcinogenic effects when chronic and high doses were administered to animals. We aimed to investigate the possible effects of bentazone on lipid peroxidation, levels of glutathione and activities of antioxidant enzymes in human erythrocytes in vitro. For that, erythrocyte were incubated with bentazone in different concentrations (0–50 nM) at 37 °C for 1 hr. Bentazone showed significant increase in the levels of malondialdehyde (MDA) at the highest concentration in erythrocytes as an index of lipid peroxidation. Besides, alterations in the levels of reduced glutathione (GSH) and activities of glutathione peroxidase (GSH-Px) were observed while the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GSH-Rd) were unchanged. In conclusion, findings from this study indicate that in vitro toxicity of bentazone may be associated with oxidative stress and this work warrants further in vivo investigations.