The phytoalexin, resveratrol ameliorates ochratoxin A genotoxicity in human embryonic kidney (HEK293) cells.
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Date
2014
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Abstract
Background: Ochratoxin A (OTA) is a mycotoxin produced by fungal species of Aspergillus and Penicillium. OTA is nephrotoxic and carcinogenic in several animal models; it frequently contaminates human and animal food products. Chronic exposure is associated with progressive renal fibrosis in humans (Balkan endemic nephropathy). Resveratrol is a phytoalexin that possesses both anti-cancer and antioxidant properties. We investigated the mechanism of cellular oxidative stress induced by OTA in the human embryonic kidney (HEK293) cell line.
Methods: An IC50 value of 1.5μM was determined from a dose-dependent cell viability curve using the methylthiazol tetrazolium (MTT) assay on HEK293 cells treated with a range of OTA concentrations (0.25μM–50μM) for 24hrs. Glutathione levels were quantified by luminometry and gene expression of Nrf2, OGG1, CAT, SOD and GPx was determined by qPCR. Protein expression of Nrf2 and phosphorylated SIRT1 (pSIRT1) was assessed by western blot, DNA damage was determined using the comet assay, and flow cytometry was employed for intracellular ROS detection.
Results: Resveratrol decreased mRNA expression of OGG1 (p<0.05) and OTA significantly increased OGG1 expression (p<0.05). The comet assay proved that while OTA induced DNA damage, resveratrol protected the DNA against strand breaks. Both resveratrol and OTA significantly increased antioxidant defence gene expression (Nrf2, CAT, GPx and SOD) (p<0.05). OTA decreased intracellular ROS, while resveratrol-treated cells exhibited the lowest percentage of intracellular ROS. Luminometry analysis showed the OTA+Resveratrol co-treatment to have a synergistic effect on the concentration of GSH and GSSG. Western blot analysis of protein showed that resveratrol significantly increased the levels of pSIRT1 while
concomitantly decreasing the protein levels of Nrf2 (p<0.05) and OTA significantly decreased pSIRT1 protein levels.
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M. Med. Sc. University of KwaZulu-Natal, Durban 2014.
Keywords
Ochratoxins., Balkan nephropathy--Etiology., Resveratrol--Health aspects., Clinical biochemistry., Theses--Medical biochemistry.