Evidence of a new dechlorinated ochratoxin A derivative formed in opossum kidney cell cultures after pretreatment by modulators of glutathione pathways: Correlation with DNA‐adduct formation

Abstract

Ochratoxin A (OTA), a nephrotoxic mycotoxin probably implicated in human Balkan endemic nephropathy and associated urothelial tumors, induces renal carcinomas in rodents and nephrotoxicity in pigs. OTA induces DNA-adduct formation, but the structure of the adducts and their role in nephrotoxicity and carcinogenicity have only partly been elucidated. In vivo, 2-mercaptoethane sulfonate (MESNA) protects rats against OTA-induced nephrotoxicity but not against carcinogenicity, indicating two different mechanisms leading to nephrotoxicity or carcinogenicity. To better understand how DNA-adduct could be generated, opossum kidney cells (OK) have been treated by OTA alone or in presence of several compounds such as MESNA or N-acetylcysteine (another agent that, like MESNA, reduces oxidative stress by increasing of free thiols in kidney), buthionine sulfoximine (BSO) (an inhibitor of glutathione-synthase), and alpha amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid (ACIVICIN) (an inhibitor of gamma glutamyl transpeptidase). Cytotoxicity of OTA on OK cells was evaluated by applying the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. None of the listed agents diminished OTA cytotoxicity significantly; ACIVICIN even increases OTA cytotoxicity. In contrast, analysis of the HPLC profiles of OTA metabolites produced during these incubations indicated that the pattern, the quantity of metabolites, and the nature of the derivatives were modulated by these agents. Ochratoxin B (OTB), open-ring ochratoxin A (OP-OA), 4 hydroxylated OTA, 10 hydroxylated OTA, OTA without phenylalanine, OTB without phenylalanine, and a dechlorinated OTA metabolite could be identified by nano-ESI-IT-MS.

Glutathione Conjugates of Ochratoxin a as Biomarkers of Exposure / Glutationski Konjugati Okratoksina A Kao Biomarkeri Izloženosti

“…To test the potential for conjugate formation by OTA, Dark Agouti rat liver and kidney samples were analysed for levels of OTA, OTα, OTB-GSH, OTHQ-GSH, OTB-NAC, and OTHQ-NAC, using HPLC with fl uorescence detection (34,35). The rat organ samples were available from a study in which 7-week-old Dark Agouti male and female rats were fed various concentrations of OTA in wheat for a period of 28 days; similar to the protocol previously reported (27).…”

“…The limit of detection (LOD) was 0.05 ng g -1 ; the limit of quantifi cation (LOQ) was 0.2 ng g -1 (34). The OTA metabolites were separated using a Prontosil C18 column (250 mm x 4 mm, particle size 3 μm) with solvent A: methanol:acetonitrile:6.5 mmol L -1 ammonium formate (200:200:600) adjusted to pH 3 with formic acid; and solvent B: methanol: acetonitrile:6.5 mmol L -1 ammonium formate (350:350:300) adjusted to pH 3 with formic acid, using gradient elution, as outlined previously (34,35). Detection was also performed using the fl uorimeter GTI spectrovision (λ ext =340 nm; λ em =465 nm) that permitted a LOD of ~ 0.05 ng g -1 for OTA.…”

Structure–activity relationships for the fluorescence of ochratoxin A: Insight for detection of ochratoxin A metabolites

“…In this case the LOD for OTA-GSH showed a 3-fold increase using OTHQ parameters (trace (1)) compared to the LOD using parameters for OTA detection (trace (2)). This increased sensitivity may facilitate the detection of OTA metabolites bearing a C5-OH group [28,30,38] in biological fluids and enhance our understanding of OTA-mediated toxicity.…”

Ochratoxin A and Aristolochic Acid Involvement in Nephropathies and Associated Urothelial Tract Tumours

“…(43). We have demonstrated that both oxidative stress and covalent binding are involved in OTA nephrotoxicity and carcinogenicity (90) while Faucet-Marquis et al (91) established a correlation between DNA adducts and OTA derivatives in an opossum kidney cell culture (91).When applied to renal tubular cells, OTA led to GSH depletion (92). Using primary human urothelial cells from several donors, Lebrun et al (93) pointed out the role of genetic GST expression and the extent of DNA damage induced by OTA.…”