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Citrinin (CIT) is a mycotoxin produced as a secondary product by the genera Aspergillus, Penicillium, Monascus, and other strains. CIT has the potential for contaminating animal feed and human food such as maize, wheat, rye, barley, oats, rice, cheese, and sake. Although CIT is primarily known as a nephrotoxic mycotoxin, it also affects other organs, including the liver and bone marrow, and its mechanisms of toxicity have not been clearly elucidated. There is a further lack of studies investigating the potential for CIT-induced neurotoxicity and its mechanisms. In the current study, SH-SY5Y human neuroblastoma cell line was treated with CIT for 24 h to evaluate various toxicological endpoints, such as reactive oxygen species (ROS) production and apoptosis induction. Results indicate that CIT has an IC50 value of 250.90 lM and cell proliferation decreased significantly at 50 and 100 lM CIT concentrations. These same concentrations also caused elevated ROS production (34.76%), apoptosis (9.43-fold) and calcium ion mobilization (36.52%) in the cells. Results show a significant decrease in the mitochondrial membrane potential (86.8%). We also found that CIT significantly upregulated the expression of some genes related to oxidative stress and apoptosis, while downregulating others. These results suggest that apoptosis and oxidative stress may be involved in the mechanisms underlying CIT-induced neurotoxicity

Glyphosate (N-phosphonomethyl glycine) is a non-selective, organophosphate herbicide widely used in agriculture and forestry. We investigated the possible toxic effects of the glyphosate active compound and its commercial formulation (Roundup Star®) in the human hepatocellular carcinoma (HepG2) cell line, including their effects on the cytotoxicity, cell proliferation, reactive oxygen species (ROS) levels, and expression of oxidative stress-related genes such as HO-1, Hsp70 Nrf2, L-FABP, and Keap1. MTT and NRU tests indicated that the IC50 values of Roundup Star® were 219 and 140 μM, respectively, and because glyphosate failed to induce cell death at the studied concentrations, an IC50 value could not be determined for this cell line. Roundup Star at concentrations of 50 and 100 μM significantly increased (39.58% and 52%, respectively) cell proliferation, which 200 μM of glyphosate increased by 35.38%. ROS levels increased by 27.97% and 44.77% for 25 and 100 μM of Roundup Star and 32.74% and 38.63% for 100 and 200 μM of glyphosate exposure. In conclusion, Roundup Star and glyphosate significantly increased expression levels of selected genes related to the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway. This suggests that ROS production and the MAPK/ERK signaling pathway may be key molecular mechanisms in the toxicity of glyphosate in liver cells.

Perfluorooctanoic acid (PFOA) is environmentally persistent and has been classified by The International Cancer Research Agency (IARC) as a possible human pancreatic carcinogen. In this study, the epigenetic alteration, the changes in the expression levels of endoplasmic reticulum stress-related and metabolism-related genes, as well as DNA methyltransferase expression were investigated using RT-PCR and ELISA assays. PFOA induced a significant increase in the methylation ratio (5-mC%), impacted DNA methylation maintenance gene expression and decreased lipid metabolism-related genes except for PPARγ (≥ 13-fold increase). While PFOA induced the expression of ATF4 (≥ 5.41-folds), CHOP (≥ 5.41-folds) genes, it inhibited the expression of ATF6 (≥ 67.2%), GRP78 (≥ 64.3%), Elf2α (≥ 95.8%), IRE1 (≥ 95.5%), and PERK (≥ 91.7%) genes. It is thought that epigenetic mechanisms together with disruption in the glucose-lipid metabolism and changes in endoplasmic reticulum stress-related genes may play a key role in PFOA-induced pancreatic toxicity.

Background and Aims: Prochloraz (PCZ) is an imidazole fungicide which is used in agriculture and gardening. PCZ, with endo crine disrupting effect, disrupts reproductive and developmental functions. Previously, the effects of PCZ on the estrogen and androgen receptors have been shown in vitro and in vivo. Because of endocrine disrupting effects, PCZ could influence nuclear receptors which acted as ER and AR antagonists. Besides, PCZ has been thought to have no genotoxic effects. Therefore, we aimed to investigate possible effects of PCZ on nuclear receptor genes and epigenetic mechanisms in human prostate carci noma (PC-3) cells. Methods: In the present study, MTT and LDH tests were applied to evaluate the cell viability. Expression levels of nuclear receptor genes such as AhR, PXR, PPARα, PPARγ were studied on real-time quantitative PCR. For global DNA methylation analysis, the levels of 5-methylcytosine (5-mC%) were measured by elisa kit. Results: According to MTT and LDH test results, IC50 value of PCZ has been determined as 144.19 and 116.65 μM, respectively. There were significant changes for the expression levels of AhR, PPARα and PPARγ genes after 5-50 μM of PCZ treat ments. 5 and 50 μM of PCZ decreased the levels of 5-mC% in the rates of 22.6% and 26.9%, respectively. Conclusion: It has been suggested that PCZ may cause alterations on the expressions of nuclear receptor genes which could be related to endocrine disrupting effects and may have implications on global DNA methylation.

Diosgenin is an important precursor of steroidal drugs and a number of biotechnology techniques, including gene transfer and elicitation studies, are used to produce it in higher volumes. With diosgenin levels of 0.2–0.9%, fenugreek is a useful source of this important substance. This study investigated the effect of yeast extract elicitation on diosgenin levels in suspension culture of fenugreek and determined the expression levels of important genes involved in the diosgenin pathway. Callus cultures from plants grown in vitro were used to establish cell suspension cultures. Yeast extract was applied at a concentration of 0.01 g/mL for 12 h and 24 h to elicit 13-day-old cultures, leading to 1.66- and 1.40-fold increase in diosgenin levels, respectively. Furthermore, we examined the expression of CYP86A2, an enzyme recently proposed to participate in C26 oxidation in sterol chain during diosgenin formation, and the enzyme genes of β-glucosidase and cycloartenol synthase, which are key enzymes in diosgenin formation. β-glucosidase and cycloartenol synthase gene expression decreased but CYP86A2 expression increased in cell suspension culture of fenugreek. With this study, we have evaluated the potential of yeast extract to increase diosgenin production in fenugreek cell suspension cultures. As a result, we have concluded that yeast extract is a promising potential elicitor for increasing diosgenin levels in fenugreek plants. Yeast extract increased the amount of diosgenin content in fenugreek cell suspension cultures max 1.66 fold. 24-hour yeast extract treatment increased CYP86A2 gene expression and diosgenin content also.

Objectives:Bisphenol A (BPA) is a synthetic monomer used in the production of polycarbonate and an environmental contaminant with endocrine disrupting properties. BPA release from plastic carriers is thought to cause high amounts of exposure, which result in high risk to human and environment health.Materials and Methods:The study examined the possible changes in global DNA methylation, CpG promoter DNA methylation, and gene expressions of Rassf1a and c-myc after BPA exposure in rat kidney epithelial cells (NRK-52E).Results:The IC50 values of BPA in NRK-52E cells were 133.42 and 101.74 μM in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red uptake tests, respectively. The cells were treated with BPA at 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM concentrations for 24 h and at 100 nM concentration for 24, 48, 72, 96 h, and 6 days. Decreased global 5-methylcytosine levels were observed after 48, 72, 96 h, and 6 days at the concentration of 100 nM BPA. Changes in CpG promoter DNA methylation were detected in the genes of Rassf1a and c-myc in BPA-treated NRK-52E cells. Expression levels of Rassf1a and c-myc changed in response to BPA at the high concentrations after 24 h treatment, whereas 100 nM exposure to BPA altered gene expression after 48, 72, and 96 h.Conclusion:These results indicate that changes in global and gene-specific DNA methylation may play an important role in the mechanism of BPA toxicity in kidney cells.

Zearalenone, produced by various Fusarium species, is a non-steroidal estrogenic mycotoxin that contaminates cereals, resulting in adverse effects on human health. We investigated the effects of zearalenone and its metabolite alpha zearalenol on epigenetic modifications and its relationship with metabolic pathways in human hepatocellular carcinoma cells following 24 h of exposure. Zearalenone and alpha zearalenol at the concentrations of 1, 10 and 50 μM significantly increased global levels of DNA methylation and global histone modifications (H3K27me3, H3K9me3, H3K9ac). Expression levels of the chromatin modifying enzymes EHMT2, ESCO1, HAT1, KAT2B, PRMT6 and SETD8 were upregulated by 50 μM of zearalenone exposure using PCR arrays, consistent with the results of global histone modifications. Zearalenone and alpha zearalenol also changed expression levels of the AhR, LXRα, PPARα, PPARɣ, L-fabp, LDLR, Glut2, Akt1 and HK2 genes, which are related to nuclear receptors and metabolic pathways. PPARɣ, a key regulator of lipid metabolism, was selected from among these genes for further analysis. The PPARɣ promoter reduced methylation significantly following zearalenone exposure. Taken together, the epigenetic mechanisms of DNA methylation and histone modifications may be key mechanisms in zearalenone toxicity. Furthermore, effects of zearalenone in metabolic pathways could be mediated by epigenetic modifications.

Neonicotinoid insecticides, known for their selectivity and low mammalian toxicity, have been widely used in recent years as alternatives to organophosphate insecticides. Although neonicotinoids are generally considered to be safe, data show that they can cause harmful effects on human and environmental health. Due to the lack of information on their mechanism of toxicity, the effects of imidacloprid and thiamethoxam on DNA methylation as the most used marker for epigenetic effects were investigated in human neuroblastoma (SH-SY5Y) cells. The cells were exposed to imidacloprid and thiamethoxam in concentrations of 100, 200, and 500 μM for 24 hours, then global DNA methylation and expression of genes involved in global DNA methylation ( DNMT1, DNMT3a and DNMT3b) were investigated. Global DNA methylation significantly increased after imidacloprid exposure at 100 μM, and thiamethoxam exposures at 200 µM and 500 μM (>1.5-fold). Imidacloprid significantly decreased the expression of DNMT1 and DNMT3a, whereas thiamethoxam did not cause any significant changes in the expression of DNMT genes. Our findings suggested that alteration in global DNA methylation may be involved in the toxic mechanisms of imidacloprid and thiametoxam.

Synthetic cannabinoids have become a significant public health problem in the world because of inexpensive, easy to obtain, difficult to detect by many screening methods, constantly new compounds are produced and marketed, and so on. Despite their effect on ∆9-tetrahydrocannabinol (THC) like properties on cannabinoid receptors, they exert a very strong effect as opposed to THC and could cause serious side effects to death. Studies in the literature on causative epigenetic mechanisms of long-term usage of drugs like cocaine, cannabis, and heroin are available however, few studies have been conducted on synthetic cannabinoids, which are very common in use. Also, recently the endocrine disrupting effects of such substances have come into prominence. We aimed to examine potential effects of AKB48, one of the important synthetic cannabinoids, on cytotoxicity, global methylation and CpG island promoter methylation of cannabinoid receptor genes (CB1 and CB2) and gene expressions of DNA methyltransferase enzymes (DNMT1 and MGMT), CB1 and CB2 genes in prostate adenocarcinoma (PC-3) cells following 24 h exposure. In MTT assay, IC50 value was calculated as 343.8 µM for AKB48 in PC-3 cells. Percentage of 5-methylcytosine (5-mC%) levels were significantly decreased by 27% at 100 μM concentration. No significant alteration was shown in the promoter methylation of CB1 gene compared to the control. These results will contribute to literature for determination of potential effects of synthetic cannabinoids on epigenetic modifications in the tissues of endocrine system.

Background and Aims: Occurence of the mycotoxin citrinin could increase during Monascus purpureus fermentation in red yeast rice resulting in contamination of meat products when the red yeast rice is used as food additive. The aim of this study was to investigate extraction of citrinin from meat products by different extraction methods including LLE, QuEChERS and IAC methods. Methods: Because of high sensitivity IAC method was selected for the extraction of citrinin in 33 meat products (salami, sausage and minced meat), then citrinin was analysed by HPLC- FLD. Results: IAC method was linear in the range of 0.25-100 ng/g (r2 ≥0.999) in meat products. Citrinin was found at 0.28 to 1.79 ng/g in 6 of the 19 samples (36.84%); and in one sausage sample contained citrinin at LOD level. However, citrinin was not detected in the salami and minced meat samples. Conclusion: Our results indicated that citrinin in meat products could be an important health concern. Since no limits were set in food products, regulations should be developed concerning the presence of citrinin in other products as well as meat products, in Turkey.

Objectives The aim of this study was to determine the levels of Pb, Cd, and OTA in frequently used medicinal plants. Materials and methods Twenty-one samples of linden, chamomile, and sage were obtained during the spring and summer period of 2016 from local markets and traditional bazaars in Istanbul, Turkey. Microwave-assisted digestion was applied for the preparation of the samples and the ICP-OES was used for the determination of Pb and Cd. Determination of OTA was performed using HPLC-FLD after immunoaffinity column clean-up. Results OTA was detected in only one chamomile sample with a low concentration level of 0.034 µg/kg. According to the results of ICP-OES analysis, Pb in the concentration range of 4.125-6.487 mg/kg, 3.123-5.769 mg/kg and 3.229-5.985 mg/kg and Cd in the concentration range of 0.324-0.524 mg/kg, 0.365-0.51 mg/kg and 0.321-0.474 mg/kg was found in linden, chamomile, and sage teas, respectively. Conclusion We indicated that levels of Pb and OTA were found below the maximum permissible level whereas high levels of Cd were observed in medicinal plants, which may not pose a health risk for consumers according to the exposure assessment. However, it is suggested that other mycotoxins and heavy metal contents should be carefully considered in medicinal plants.

In nature, plants are frequently exposed to various stress. Since plants are sessile organisms, their ability to cope with stress is crucial. It is known that epigenetic mechanisms such as cytosine methylation play significant role in plant stress response. In this study, we evaluated the morphological, physiological and molecular responses of Arabidopsis thaliana plant and calli after NaCl and abscisic acid (ABA) treatment in vitro. While plants were morphologically affected, on the contrary there was no morphological change in calli. It is known that phenotypic changes under stress conditions are also dependent on epigenetic modifications such as DNA methylation, chromatin modifications and siRNA mediated mechanisms in addition to DNA sequence variants. In this context, we analysed global DNA methylation patterns and their influence on transcription in four methylation related genes. We found that while NaCl application caused hypomethylation in plants, ABA application did not make a change in genomic methylation. According to the qPCR results, the most obvious difference was the increase in expression of Pol IV after NaCl application in both samples and for the DRM2 gene is the increase in plant samples after ABA application. In conclusion, although we found evidence for epigenetic regulation after NaCl treatment, but not related to ABA treatment. These results demonstrate the mutual interaction between morphophysiology, DNA methylation and the associated gene expression, and provide insight into the abiotic stress response of Arabidopsis.

Acetamiprid, which is a neonicotinoid insecticide, is used to control leafy vegetables, fruiting vegetables, fir seeds, citrus fruits,pome fruits, grapes, cotton and ornamental plants and absorbent insects on flowers. The present study aim to evaluate globalDNA methylation and gene expression of DNA methylation related enzymes in liver and brain tissues of male Sprague-Dawleyrats after a 90-day subchronic exposure to acetamiprid at low doses of 12.5, 25 and 35 mg/kg body weight (b.w.). Global DNAmethylation resulted in a significant decrease in the levels of 5-methylcytosine (5-mC%) at the doses of 25 and 35 mg/kg b.w.in the liver and 35 mg/kg b.w. in the brain compared to the vehicle control group. Consistently, expression of DNA methyltransferase enzymes decreased at doses of 12.5, 25 and 35 mg/kg b.w. in liver and 35 mg/kg b.w. in brain. It has been suggested thatnon-genotoxic (epigenetic) mechanisms may be involved in the toxicity of acetamiprid and further investigations are neededto elucidate the epigenetic effects of neonicotinoid insecticides.Cite this article as: Arıcan YE, Karaman EF, Özden S (2019). The subcronic effects of acetamipride on the global DNA methylation levels in Sprague-Dawley rat brain and liver. Istanbul J Pharm 49 (3): 167-172.

Background Cardiovascular effects of omega-3 polyunsaturated fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been widely reported. However, there are limited studies concerning their effects on human blood vessels. Therefore, the aim of this study was to investigate the direct vascular effects of EPA and DHA on the human saphenous vein (SV) precontracted with either prostaglandin F2α (PGF2α), or thromboxane A2 analogue (U46619), or norepinephrine (NE). Moreover, we aimed to investigate the protein expression of free fatty acid receptor 4 (FFAR4) in human SV. Methods Pretreatment of human SV rings with EPA and DHA (100 μM, 30 min) was tested on vascular reactivity induced by PGF2α (10 nM to 5 μM), NE (10 nM to 100 μM), and U46619 (1 nM to 100 nM). In addition, direct relaxant effects of EPA/DHA (1–100 μM) were tested in human SV rings precontracted by PGF2α, NE, and U46619. Furthermore, the involvement of potassium channels on their vascular effects was investigated in the presence of the nonselective K+ channel inhibitor tetraethylammonium chloride. Results Pretreatment with EPA and DHA resulted in a significant decrease in vascular reactivity induced by U46619 and PGF2α compared to NE. In the presence of TEA, the relaxant effects of EPA and DHA were significantly decreased in SV preparations precontracted by U46619 and PGF2α for DHA. Furthermore, FFAR-4 protein was expressed in tissue extracts of human SV. Conclusions Our study demonstrates that both EPA and DHA reduce the increased vascular tone elicited by contractile agents on the human SV and that the direct vasorelaxant effect is likely to involve potassium channels.

[4‐(Adamantane‐1‐carboxamido)‐3‐oxo‐1‐thia‐4‐azaspiro[4.4]nonan‐2‐yl]acetic acid (4a) and [4‐(adamantane‐1‐carboxamido)‐8‐nonsubstituted/substituted‐3‐oxo‐1‐thia‐4‐azas‐ piro[4.5]decane‐2‐yl]acetic acid (4b–g) derivatives were synthesized; their structures were verified by elemental analysis, infrared spectroscopy, 1 H nuclear magnetic resonance (NMR), 13C NMR, and mass spectroscopy data; and their in vitro cytotoxicity activities were investigated against human hepatocellular carcinoma, human prostate adenocarcinoma, and human lung carcinoma cell lines (HepG2, PC‐3, and A549, respectively), and a mouse fibroblast cell line (NIH/3T3). All compounds, except compound 4e, were found as cytotoxic, especially on A549 cells as compared with the other cells (selectivity index = 2.01–11.6). As a further step, the effects of compounds 4a–c on apoptosis induction were tested and the expression of selected apoptosis genes was analyzed. Among the selected compounds, compound 4a induced apoptosis remarkably. Moreover, computational calculations of the binding of compounds 4a–c to the BIR3 domain of the human inhibitor of apoptosis protein revealed ligand–protein interactions at the atomistic level and emphasized the importance of a hydrophobic moiety on the ligands for better binding.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by Fusarium fungi. ZEN has endocrine disruptor effects and could impair the hormonal balance. Here, we aimed at investigating possible effects of ZEN on metabolism-related pathways and its relation to epigenetic mechanisms in breast adenocarcinoma (MCF7) and breast epithelial (MCF10F) cells. Using the MTT and neutral red uptake (NRU) cell viability tests, IC50 values of ZEN after 24h were found to be 191 mu mol/L and 92.6 mu mol/L in MCF7 cells and 67.4 mu mol/L and 79.5 mu mol/L in MCF10F cells. A significant increase on global levels of 5-methylcytosine (5-mC%) was observed for MCF7 cells, correlating with the increased expression of DNA methyltransferases. No alterations were observed on levels of 5-mC% and expression of DNA methyltransferases for MCF10F cells. Further, at least threefold upregulation compared to control was observed for several genes related to nuclear receptors and metabolism in MCF7 cells, while some of these genes were downregulated in MCF10F cells. The most notably altered genes were IGF1, HK2, PXR, and PPAR gamma. We suggested that ZEN could alter levels of global DNA methylation and impair metabolism-related pathways.

Bisphenol A (BPA), as synthetic monomer used in the production of polycarbonate plastic and epoxy resins, has endocrine disruptor properties and high risk on human health. Epigenetic alterations could act an important role in BPA-induced toxicity, but its mechanism has not been fully understood. We investigated the effects of BPA on gene expression of chromatin modifying enzymes, promoter methylation of tumor suppressor genes and histone modifications in human prostate carcinoma cells (PC-3). IC50 value of BPA was determined as 217 and 190 mu M in PC-3 cells by WIT and NRU tests, respectively. We revealed an increase in global levels of 5-methylcytocine and 5-hydroxymethylcytocine at 10 mu M of BPA for 96 h. We observed a significant increase on promoter DNA methylation and decrease on gene expression of p16 gene while no change was observed for Cyclin D2 and Rassf1. Significant changes were observed in global histone modifications (H3K9ac, H3K9me3, H3K27me3, and H4K20me3) in PC-3 cells. According to these results, we investigated wide-range epigenetic modifications using PCR arrays. After 96 h BPA exposure, chromatin modifying enzymes including KDM5B and NSD1 were significantly downregulated. Also, promoter methylation of tumor suppressor genes including BCR, GSTP1, LOX, MGMT, NEUROG1, PDLIM4, PTGS2, PYCARD, TIMP3, TSC2 and ZMYDN10 altered significantly. ChIP results showed that H3K9ac, H3K9me3 and H3K27me3 modifications on p16 gene showed significant increases after 1 and 10 mu M of BPA exposure. In conclusion, epigenetic signatures such as DNA methylation and histone modifications could be proposed as molecular biomarkers of BPA-induced prostate cancer progression. (C) 2019 Elsevier Ltd. All rights reserved.

Neonicotinoids are a relatively new type of insecticide to control a variety of pests. Although they are generally considered to be safe, they can lead to harmful effects on human and environmental health. We aimed to investigate possible effects of common neonicotinoid insecticides (acetamiprid, clothianidin, imidacloprid, thiacloprid, and thiamethoxam) on cytotoxicity and DNA damage in human neuroblastoma (SH-SY5Y) and human hepatocellular carcinoma (HepG2) cells. Our results indicated that 50% of inhibitory concentration values of neonicotinoids are in the range of 0.96 to >4 mM in SH-SY5Y cells and 0.53 to >4 mM in HepG2 cells by the methyl tetrazolium and neutral red uptake tests after 24 and 48 h exposure. We observed significant DNA damage at 500 µM of five neonicotinoids in SHSY-5Y cells, while only imidacloprid, thiametoxam, and thiacloprid showed some alterations in HepG2 cells after 24 h exposure using the alkaline comet assay. In conclusion, neonicotinoid insecticides may induce cytotoxicity and DNA damage in cell cultures; therefore, further studies are needed to better understand the toxicity of neonicotinoids.

Bisphenol A (BPA), an estrogenic endocrine disruptor, is widely used in the production of polycarbonate plastic and epoxy resins, resulting in high risk on human health. In present study we aimed to investigate the effects of BPA on global and gene specific DNA methylation, global histone modifications and regulation of chromatin modifiying enzymes in human neuroblastoma cells (SH-SY5Y). Cells were treated with BPA at 0.1, 1 and 10 mu M concentrations for 48 and 96h. IC50 value of BPA was determined as 183 and 129 mu M in SH-SY5Y cells after 24h by MTT and NRU tests, respectively. We observed significant alterations on the 5-mC% levels (1.3 fold) and 5-hmC% levels (1.67 fold) after 10 mu M of BPA for 96h. Significant decrease was identified in H3K9me3 and H3K9ac after 10 mu M of BPA for 96h while decrease was observed in H3K4me3 at 10 mu M of BPA for 48h. Alterations were observed in chromatin modifiying genes including G9a, EZH2, SETD8, SETD1A, HAT1, SIRT1, DNMT1, RIZ1 and Suv39h1 after 96h of BPA exposure. Taken together, this study suggests that BPA might modulate the epigenetic regulators which would be key molecular events in the toxicity of endocrine disrupting chemicals.

3-Monochloropropane-1,2-diol (3-MCPD) is a well-known food processing contaminant that has been regarded as a rat carcinogen, which is known to induce Leydig-cell and mammary gland tumors in males, as well as kidney tumors in both genders. 3-MCPD is highly suspected to be a non-genotoxic carcinogen. 2,3-Epoxy-1-propanol (glycidol) can be formed via dehalogenation from 3-MCPD. We aimed to investigate the cytotoxic effects of 3-MCPD and glycidol, then to demonstrate the possible epigenetic mechanisms with global and gene-specific DNA methylation in rat kidney epithelial cells (NRK-52E). IC50 value of 3-MCPD was determined as 48mM and 41.39 mM, whereas IC50 value of glycidol was 1.67mM and 1.13mM by MTT and NRU test, respectively. Decreased global DNA methylation at the concentrations of 100 mM and 1000 mu M for 3-MCPD and 100 mu M and 500 mu M for glycidol were observed after 48 h exposure by using 5-methylcytosine (5-mC) ELISA kit. Methylation changes were detected in promoter regions of c-myc and Rassf1a in 3-MCPD and glycidol treated NRK-52E cells by using methylation-specific PCR (MSP), whereas changes on gene expression of c-myc and Rassf1a were observed by using real-time PCR. However, e-cadherin, p16, VHL and p15 genes were unmethylated in their CpG promoter regions in response to treatment with 3-MCPD and glycidol. Alterations in DNA methylation might be key events in the toxicity of 3-MCPD and glycidol. Alterations in DNA methylation might be key events in the toxicity of 3-MCPD and glycidol.

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.

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.

Ochratoxin A (OTA) is a mycotoxin that can be found in several food commodities including cereals, wine, coffee, cacao, spices or dried fruits, resulting in chronic human exposure. The aim of our study was to investigate the presence of OTA in widely consumed cereal-derived products commercialized in Turkey. A total of 142 samples were collected from different supermarkets and traditional bazaars in Istanbul during 2008–2010 years. The analytical methods used in our study involved the liquid/liquid extraction of OTA, immunoaffinity clean-up and high performance liquid chromatography determination with fluorescence detection. The frequencies of OTA contamination were 21.62%, 19.05% and 55.95% and the mean concentrations of positive samples were 0.32 μg/kg, 0.14 μg/kg, 0.41 μg/kg for breakfast cereals, cereal-based baby foods and tarhana samples, respectively. Our findings show that contamination levels of OTA in all cereal-derived products were lower than the permitted level by European Commission Regulation.

Scope: Furan, a food contaminant formed during heat processing, induces hepatocellular tumors in rodents and high incidences of cholangiocarcinomas in rats even at the lowest dose (2 mg/kg b.w.) administered. Initial estimates suggested that human intake of furan may be as high as 3.5 μg/kg b.w./day, indicating a relatively narrow margin of exposure. The aim of this study was to establish dose–response data for cytotoxicity, regenerative cell proliferation and secondary oxidative DNA damage in livers of male F344 rats treated with furan at doses ≤2 mg/kg b.w. for 28 days. Methods and results: No significant signs of hepatotoxicity other than a mild, dose-dependent increase in serum cholesterol and unconjugated bile acids, and no evidence of oxidative DNA damage were seen. Histopathological alterations and proliferative changes were restricted to subcapsular areas of the left and caudate liver lobes. Conclusion: Although statistically significant effects were only seen at the 2 mg/kg b.w. dose during the course of our study, a ∼two and ∼threefold increase in 5-bromo-2′-deoxyuridine labeling index was observed at 0.1 and 0.5 mg/kg b.w., respectively, suggesting that chronic exposure to doses even below 2 mg/kg b.w. may cause proliferative changes in rat liver and highlighting the need to assess furan carcinogenicity at lower doses.

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.

The contribution of DNA adduct formation in the carcinogenic action of the mycotoxin ochratoxin A (OTA) has been subject to much debate. Recently, a carbon-bonded ochratoxin A-2'-deoxyguanosine adduct (dGuoOTA) formed by photochemical reaction in vitro has been shown by 32 P-postlabeling/ TLC to comigrate with a spot detected in DNA isolated from rat and pig kidney following exposure to OTA. Considering the large body of evidence arguing against covalent DNA binding of OTA and the poor resolution and specificity of postlabeling analysis, we developed a stable isotope dilution LC-MS/MS method to analyze dGuoOTA in kidney DNA isolated from rats treated with OTA. dGuoOTA and nitrogen-15-labeled dGuoOTA ( 15 N 5 -dGuoOTA) were prepared by photoirradiation of OTA in the presence of dGuo or nitrogen-15-labeled dGuo. Conditions for DNA hydrolysis were optimized using a synthetic oligonucleotide containing dGuoOTA to ensure complete release of dGuoOTA. The LOD of the method (S/N > 3) was 10 fmol dGuoOTA on-column. However, dGuoOTA was not detected in DNA samples isolated from male F344 rats treated with OTA for up to 90 days at doses known to cause renal tumor formation. Detection limits, calculated for each individual sample based on the absolute LOD and the amount of DNA injected, were as low as 3.5 dGuoOTA/10 9 nucleotides. These data are consistent with previous results showing lack of DNA adduct formation by OTA and demonstrate that dGuoOTA is not formed in biologically relevant amounts under physiological conditions in vivo.

Organophosphorus insecticides may induce oxidative stress leading to generation of free radicals and alteration in antioxidant system. The aim of this study was to examine the potency of trichlorfon, an organophosphate insecticide, to induce oxidative stress response in human erythrocytes in vitro. For this purpose trichlorfon solutions in different concentrations and erythrocyte solutions were incubated at 37 degrees C for 60 min. At the end of the incubation time, malondialdehyde (MDA), an end product of lipid peroxidation, total glutathione, reduced glutathione (GSH) levels, activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) enzymes were determined by spectrophotometric methods. Trichlorfon increased MDA formation depended on the concentration. On the other hand, decreases in the GSH-Px activity, GSH levels and increases in the total glutathione levels, SOD and CAT activities were seen in the studied concentrations. The present findings indicate that the in vitro toxicity of trichlorfon may be associated with oxidative stress.

The aim of this study was to investigate the presence of Ochratoxin A (OTA) in red pepper flakes commercialised in Turkey. A total of 75 samples were collected from different supermarkets and traditional bazaars in Istanbul during 2012-2013. OTA analysis was performed by high-performance liquid chromatography equipped with fluorescence detection after immunoaffinity column clean-up. The method was linear in the range 0.05-40 μg kg(-1) (r(2) = 0.9997). Twenty-seven out of 31 (87.1%) packed red pepper flake samples contained OTA at concentrations ranging from 0.6 to 1.0 μg kg(-1), whereas 100% of the unpacked red pepper flake samples contained OTA, in the range 1.1-31.7 μg( )kg(-1). Overall, only 4 unpacked samples (5.3%) contained OTA, with a mean value of 23.4 μg kg(-1), which is higher than the European Union limit. It is suggested that OTA content should be carefully considered in red pepper flake samples especially marketed in unpacked form.

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.

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., Deutscher Akademischer Austauschdienst Firat University Scientific Research Projects Management Unit: UDP-30102, NP-2919, UDP-17621, YADOP-20728 SBAG-109S187

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.

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.

Prochloraz is a broad-spectrum contact imidazol fungicide used against several diseases in wheat, barley and oleaginous plants but also for treatment of flower production. Although prochloraz has endocrine disrupting and hepatocarcinogenic effects, there is lack of data on toxic effects of prochloraz. Therefore, we aimed to investigate the DNA damage effects of prochloraz in NRK-52E cells by using Ames and Comet assay. By using a standard alkaline Comet assay procedure, there was no DNA damage observed after 24 h prochloraz exposure. It also showed that prochloraz caused neither base-pair substitution nor frame shift mutations by using TA98, TA100 strains, respectively, with/without metabolic activation in Ames assay. Both Comet and Ames assays, the exposure concentrations were 12.5, 25, 50 and 100 µM. IC50 value of prochloraz was determined as 110.76 µM in NRK-52E cells by MTT cytotoxicity test. Also, we evaluated possible effects of prochloraz on lipid peroxidation, reduced glutathione (GSH), oxidized glutathione (GSSG) and antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) in NRK-52E cells at 1-50 µM concentrations. Prochloraz induced lipid peroxidation and altered glutathione contents and antioxidant enzyme activities in NRK-52E cells. Our results indicated that prochloraz showed no evidence of mutagenicity and DNA damage; however, some alterations were observed on lipid peroxidation and antioxidant systems in prochloraz treatment.

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.

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.

Ochratoxin A (OTA) is nephrotoxic and a potent renal carcinogen. Male rats are most susceptible to OTA toxicity, and chronic administration of OTA (70 and 210 microg/kg bw) for 2 years has been shown to induce high incidences of adenomas and carcinomas arising from the straight segment of the proximal tubule epithelium. In contrast, treatment with a lower dose of 21 microg/kg bw did not result in increased tumor rates, suggesting a nonlinear dose response for renal tumor formation by OTA. Since the mechanism of OTA carcinogenicity is still largely unknown, this study was conducted to investigate early functional and pathological effects of OTA and to determine if sustained stimulation of renal cell proliferation plays a role. Male F344/N rats were treated with OTA for up to 13 weeks under conditions of the National Toxicology Program (NTP) bioassay. Cell proliferation in the renal cortex and outer stripe of the outer medulla (OSOM) was determined using bromodeoxyuridine incorporation and immunohistochemistry. Histopathological examination showed renal alterations in mid- and high-dose-treated animals involving single-cell death and prominent nuclear enlargement within the straight proximal tubules. Treatment with OTA at doses of 70 and 210 microg/kg bw led to a marked dose- and time-dependent increase in renal cell proliferation, extending from the medullary rays into the OSOM. No effects were evident in kidneys of low-dose-treated animals or in the liver, which is not a target for OTA carcinogenicity. A no observed effect level in this study was established at 21 microg/kg bw, correlating with the dose in the NTP 2-year bioassay that did not produce renal tumors. The apparent correlation between enhanced cell turnover and tumor formation induced by OTA indicates that stimulation of cell proliferation may play an important role in OTA carcinogenicity and provides further evidence for an epigenetic, thresholded mechanism.

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 (C(18)) Empore extraction disks, octadecyl (C(18)), 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 (C(18) and CN) to confirm identification. Analytical separation was performed simultaneously on C(18) 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 C(18) disks, from 75.1% to 100.0% for C(18) cartridges, and from 54.0% to 98.0% for SDB cartridges over concentrations at 0.025--0.4 microg L(-1). The limits of detection were 0.012--0.035 microg L(-1).

The unquestionable therapeutic success of the anticancer drug cis-platin and its secondand third-generation analogues has triggered, during the past fifty years, the development of several metal-based potential chemotherapeutic drugs. Among all, gold complexes of the type [AuX2(dtc)] (X=Cl, Br; dtc = various dithiocarbamates), have been gaining increasing attention due to their capability to strongly inhibit tumor cells growth by exploiting mechanisms of action different from those recognized for the clinically established platinum drugs. As known, survival of tumor cells is favored by mitochondrial changes that make death induction harder in a variety of stress conditions. These changes likely involve mitochondria, and include the inhibition of permeability transition pore (PTP) opening through a variety of kinase signaling pathways. Here, we present new biological findings for gold(III)-containing molecules showing a potent anticancer activity coupled with a peculiar selectivity toward cancer cells. The gold(III)-complexes are able to induceoxidative stress and tumorcell death favoring thePTP opening. Therefore, they target a specific hallmark of cancer cells, such as the resistance to apoptosis. These findings pave the way for a novel strategy allowing to hit crucial mechanisms that shield the neoplasms from the toxicity ofmany anti-tumor strategies, and identify the gold(III)-dithiocarbamates as a promising chemotherapeutic drugs.