Abstract
Ochratoxin-A (OTA), is toxic secondary metabolite and is found to be a source of vast range of toxic effects like hepatotoxicity, nephrotoxicity. However, the information available currently regarding neurotoxic effects exerted by OTA is scanty. Hence, the present study was aimed to evaluate the neurotoxic effects of OTA and the possible mechanisms of toxicity as well as the role of cytotoxic oxidative stress on neuronal (Neuro-2a) cell line was evaluated in vitro. Results of the MTT and LDH assay showed that, OTA induced dose-dependent cell death in Neuro-2a cells and EC50 value was determined as 500 nM. OTA induced high levels of reactive oxygen species (ROS) and elevated levels of malondialdehyde, also loss of mitochondrial membrane potential was observed in a dose depended manner. Effects of OTA on ROS induced chromosomal DNA damage was assessed by Comet assay and plasmid DNA damage assay in which increase in DNA damage was observed in Neuro-2a cells by increasing the OTA concentration. Further western blotting analysis of OTA treated Neuro-2a cells indicated elevated expression levels of c-Jun, JNK3 and cleaved caspase-3 leading to apoptotic cell death. Other hand realtime-Q-PCR analysis clearly indicates the suppressed expression of neuronal biomarker genes including AChE, BDNF, TH and NOS2. Further N-acetylcysteine (NAC) pretreatment to Neuro-2a cells followed by OTA treatment clearly evidenced that, the significant reversal of toxic effects exerted by OTA on Neuro-2a cells. In the present study, results illustrate that ROS a principle event in oxidative stress was elevated by OTA toxicity in Neuro-2a cells. However, further in vivo, animal studies are in need to conclude the present study reports and the use of NAC as a remedy for OTA induced neuronal stress.
Highlights
Ochratoxin, is produced by filamentous fungi species Aspergillus and Penicillium as secondary metabolite (Pitt, 1987)
Total cellular protein was quantified by Lowry et al (1951) method and 100 μg of protein from each sample was loaded onto 10% gel separated on sodium dodecyl sulfate (SDS)-PAGE and nitrocellulose membrane was used to transfer proteins using electro blotting apparatus (Cleaver Scientific Ltd, UK) according to previous method (Anand et al, 2012)
The membranes were probed with β-actin, JNK3 (55A8), phospho-SAPK/jun amino-terminal-kinase (JNK) (Thr183/Tyr185; 98F2), c-jun, phospho c-jun at 1: 1000 dilutions whereas active caspase 3 antibody at 1:400 dilution and incubated at 37◦C for 3 h after transfer
Summary
Ochratoxin, is produced by filamentous fungi species Aspergillus and Penicillium as secondary metabolite (Pitt, 1987). It was first discovered in 1965 as a fungal toxin in animals and first isolated from South African A. ochraceus isolate (van der Merwe et al, 1965). It occurs in nature in 3 different isoforms as Ochratoxin A, B, and C, among these Ochratoxin A (OTA) is the most potent. OTA is proven to be carcinogen in animals, and has been classified as a class 2B, possible. Kidney has been considered as the key target organ of OTA toxicity in most of the mammalian species (Patharajan et al, 2011; Jilani et al, 2012)
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