Cytotoxicity, DNA integrity and methylation in mammary and kidney epithelial cell lines exposed to Ochratoxin A

Ochratoxin A (OTA) is a secondary metabolite of moulds that may contaminate food and feedstuffs. OTA is recognized as a nephrotoxic, hepatotoxic and teratogenic substance in different animal species. The kidney is the target organ of OTA, whereas lower OTA concentrations could be detected in other tissues, such as adipose tissue, liver and muscles. In addition, OTA transfer to milk has been demonstrated in several species, such as humans, rabbits, rats and ruminants, identifying the mammary gland as one of the potential target of this mycotoxin. This study aimed to investigate the in vitro damage induced by OTA using two well established epithelial cell lines. MDCK cells have been used as a model of the kidney epithelium, while BME-UV1 have been employed as a model of the mammary gland epithelium. The effect of OTA on cultured cell lines, with subsequent evaluation of cell viability (MTT test) and membrane stability (LDH test), was assessed. In all experiments performed, control consisted of MDCK and BME-UV1 cells exposed to basal medium alone. After 24h of OTA treatment (concentration range 0.15 up to 10µg/mL), MDCK and BME-UV1 cell viability was strongly reduced in a dose-dependent way and LC50 has been calculated. LC50 for MDCK cells was 1 μg/mL while, for BME-UV1 cells, LC50 was 0.8 μg/mL. In light of LC50, the range of concentrations for further experiments was determined (0.3 up to 1.25µg/mL). The percentage of LDH released by MDCK and BME-UV1 cells increased in a dose-dependent way. In particular, 1.25 μg/mL of OTA determined a 35% of LDH released in MDCK cells and a 46% of LDH released in BME-UV1 cell line. Subsequently, the effect of the addition of OTA to MDCK and BME-UV1 cells has been evaluated on DNA integrity, which was detected by gel electrophoresis, by the analysis of DNA oxidation biomarker 8-OHdG (8-OHdG adduct) and the global DNA methylation status (5-mC). The level of 8-OHdG adduct was significantly (P<0.05) increased in BME-UV1 cells treated with 1.25 μg/ml of OTA. The analysis of 5-mC revealed that in MDCK and BME-UV1 cells, OTA has not induced alterations in the global DNA methylation status. The results obtained herein could represent the basis for the development of future studies investigating the in vitro relationship between DNA damage and the global DNA methylation status, promoting new strategies to control OTA cytotoxicity at different tissue level.