Potential impacts of bisphenols on prostate cells: An overview of cytotoxicity, proliferation, oxidative stress, apoptosis, and ER-stress response activation.

This study aimed to evaluate the toxic effects of Bisphenol A (BPA), Bisphenol F (BPF) and Bisphenol S (BPS) on PNT1A and PC-3 cells, focusing on their effects on endoplasmic reticulum (ER) stress and related pathways. PNT1A and PC-3 were treated with BPA, BPF and BPS at concentrations of 0.1, 1 and 10 µM for 48 h cytotoxicity, BrdU cell proliferation, ROS generation, apoptosis detection, gene expression analysis and Western blot analysis were performed. BPA induced proliferation and late apoptosis in PNT1A cells, whereas it induced both late apoptosis and early apoptosis in PC-3 cells. BPF and BPS induced late apoptosis in PC-3 cells. Increased ROS levels were observed in PNT1A cells exposed to 1-10 µM BPA. BPA, BPF and BPS increased the expression levels of ER stress-related genes in PNT1A cells. Furthermore, exposure to BPA increased the expression of ER stress-related CHOP/DDIT3 protein in PNT1A cells. These findings highlight the potential health risks associated with BPA, BPF and BPS exposure and emphasize the importance of investigating the underlying mechanisms by which these chemicals may affect human health. Further research is required to comprehensively understand the role of ER stress pathways in cellular responses to these substances.

The role of chromatin-modifying enzymes and histone modifications in the modulation of p16 gene in fumonisin B1-induced toxicity in human kidney cells.

Fumonisin B1 (FB1) poses a risk to animal and human health. Although the effects of FB1 on sphingolipid metabolism are well documented, there are limited studies covering the epigenetic modifications and early molecular alterations associated with carcinogenesis pathways caused by FB1 nephrotoxicity. The present study investigates the effects of FB1 on global DNA methylation, chromatin-modifying enzymes, and histone modification levels of the p16 gene in human kidney cells (HK-2) after 24 h exposure. An increase (2.23-fold) in the levels of 5-methylcytosine (5-mC) at 100 µmol/L was observed, a change independent from the decrease in gene expression levels of DNA methyltransferase 1 (DNMT1) at 50 and 100 µmol/L; however, DNMT3a and DNMT3b were significantly upregulated at 100 µmol/L of FB1. Dose-dependent downregulation of chromatin-modifying genes was observed after FB1 exposure. In addition, chromatin immunoprecipitation results showed that 10 µmol/L of FB1 induced a significant decrease in H3K9ac, H3K9me3 and H3K27me3 modifications of p16, while 100 µmol/L of FB1 caused a significant increase in H3K27me3 levels of p16. Taken together, the results suggest that epigenetic mechanisms might play a role in FB1 carcinogenesis through DNA methylation, and histone and chromatin modifications.

Effects of perfluorooctanoic acid on endoplasmic reticulum stress and lipid metabolism-related genes in human pancreatic 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.

Assessment of Global DNA Methylation in SH-SY5Y Cells Exposed to the Neonicotinoid Insecticides Imidacloprid and Thiamethoxam.

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.

Alterations in cell viability, reactive oxygen species production, and modulation of gene expression involved in mitogen-activated protein kinase/extracellular regulating kinase signaling pathway by glyphosate and its commercial formulation in hepatocellular carcinoma cells.

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.

Mechanisms underlying citrinin-induced toxicity via oxidative stress and apoptosis-mediated by mitochondrial-dependent pathway in SH-SY5Y cells.

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 µM and cell proliferation decreased significantly at 50 and 100 µM 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.

Zoledronic acid-induced oxidative damage and endoplasmic reticulum stress-mediated apoptosis in human embryonic kidney (HEK-293) cells.

Zoledronic acid, a nitrogen-containing bisphosphonate drug, is used for the treatment of osteoporosis, Paget's disease of bone, and tumor-induced osteolysis. Zoledronic acid has also gained a place in cancer treatment due to its cytotoxic and antiproliferative effects in many cancer cells. Although zoledronic acid is considered safe, kidney damage is still one of the concerns in therapeutic doses. In the study, the aim was to assess the nephrotoxic profiles of zoledronic acid in the human embryonic kidney (HEK-293) cells. Cytotoxicity evaluation was performed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) and neutral red uptake tests, while oxidative stress was performed by reactive oxygen species (ROS) production via flow cytometry, and the incomprehensible evaluation of ROS-related genes by RT-PCR and apoptosis was performed with Annexin-PI analysis in flow cytometry. The obtained result showed that zoledronic acid inhibited cell viability (IC50 values were determined as 273.16  by MTT) and cell proliferation in a concentration-dependent manner, induced ROS production, caused glutathione depletion, and increased oxidative stress index and endoplasmic reticulum (ER) stress, indicating severe cellular stress. The expression levels of oxidative damage (L-fabp, α-GST, Nrf2, and HMOX1), ER stress (CASP4, IRE1-α, GADD153, and GRP78), and apoptosis (Bcl-2, Bax, Cyt-c, p53, CASP9, CASP3, NF-κB, TNF-α, and JNK) related genes were altered as well as IRE1-α protein levels. Herein, we were the first to show that increased oxidative stress and ER stress resulting in apoptosis are the key molecular pathways in zoledronic acid-induced nephrotoxicity equivalent to clinically administered concentrations.

Effects of local anaesthetics (neural therapy) on pain and hand functions in patients with De Quervain tenosynovitis: A prospective randomised controlled study.

OBJECTIVE: De Quervain tenosynovitis is the most common cause of lateral wrist pain. The diagnosis can be made with the Finkelstein test when pain is provoked with wrist ulnar deviation. Conservative treatment including rest, non-steroidal anti-inflammatory medication and physical therapy is applied first, then there may be a need for corticosteroid injections, and in resistant cases, surgery. The aim of this study was to evaluate the effectiveness of neural therapy (NT) on pain and hand functions in patients with De Quervain tenosynovitis. METHODS: A total of 36 patients admitted between May 2019 and March 2020 were randomly assigned to neural therapy (NT) and control groups. Hand rest and thumb spica splint were applied to all the patients, and NT interventions to the NT group only. A visual analogue scale (VAS) and the Duruöz Hand index (DHI) were used to measure pain and functionality at baseline, then at 1 and 12 months after the end of the treatment. RESULTS: The NT and control groups both showed improvements in VAS and DHI scores at 1 and 12 months compared with baseline (P < .001) according to within group comparisons. The VAS scores were significantly lower at both 1 and 12 months compared with baseline in the NT group (P < .001, P = .002 respectively). The DHI scores were lower in the NT group at 1 month (P = .009), and at 12 months there was no significant difference between the two groups (P = .252). No adverse effects were seen in any patient. CONCLUSION: NT seems to be effective in reducing pain and improving hand functions in patients with De Quervain tenosynovitis.

Synthesis, in vitro cytotoxic and apoptotic effects, and molecular docking study of novel adamantane derivatives.

[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), 13 C 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.

Global DNA Hypomethylation and Rassf1a and c-myc Promoter Hypermethylation in Rat Kidney Cells after Bisphenol A Exposure.

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.

Evaluation of the epigenetic alterations and gene expression levels of HepG2 cells exposed to zearalenone and α-zearalenol.

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, PPARÉ£, L-fabp, LDLR, Glut2, Akt1 and HK2 genes, which are related to nuclear receptors and metabolic pathways. PPARÉ£, a key regulator of lipid metabolism, was selected from among these genes for further analysis. The PPARÉ£ 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.

In Vitro Effects of Eicosapentaenoic and Docosahexaenoic Acid on the Vascular Tone of a Human Saphenous Vein: Influence of Precontractile Agents.

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.

Global and region-specific post-transcriptional and post-translational modifications of bisphenol A in human prostate cancer cells.

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 µM in PC-3 cells by MTT and NRU tests, respectively. We revealed an increase in global levels of 5-methylcytocine and 5-hydroxymethylcytocine at 10 µM 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 µM 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.

Alterations in global DNA methylation and metabolism-related genes caused by zearalenone in MCF7 and MCF10F cells.

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 24 h were found to be 191 µmol/L and 92.6 µmol/L in MCF7 cells and 67.4 µmol/L and 79.5 µmol/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 PPARγ. We suggested that ZEN could alter levels of global DNA methylation and impair metabolism-related pathways.

Investigation of the genotoxic and cytotoxic effects of widely used neonicotinoid insecticides in HepG2 and SH-SY5Y cells.

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.

Levels of Heavy Metals and Ochratoxin A in Medicinal Plants Commercialized 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.

Effects of BPA on global DNA methylation and global histone 3 lysine modifications in SH-SY5Y cells: An epigenetic mechanism linking the regulation of chromatin modifiying genes.

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 10µM concentrations for 48 and 96h. IC50 value of BPA was determined as 183 and 129µM 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 10µM of BPA for 96h. Significant decrease was identified in H3K9me3 and H3K9ac after 10µM of BPA for 96h while decrease was observed in H3K4me3 at 10µM 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.

DNA methylation analysis in rat kidney epithelial cells exposed to 3-MCPD and glycidol.

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 48 mM and 41.39 mM, whereas IC50 value of glycidol was 1.67 mM and 1.13 mM by MTT and NRU test, respectively. Decreased global DNA methylation at the concentrations of 100 µM and 1000 µM for 3-MCPD and 100 µM and 500 µM 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.

Ochratoxin A in red pepper flakes commercialised in Turkey.

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.

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.