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The aim of the present study was to confirm the relevance of studying DNA adduct formation in a field study. In that context, freshwater mussels Dreissena polymorpha, collected in a reference station, were transplanted in different sites with a pollution gradient. After one and two months, mussels were collected and DNA adduct formation was analyzed using the (32)P post labelling technique on both gills and digestive glands. In addition, the expression of genes involved in the detoxification system (catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), HSP70, aryl hydrocarbon receptor (AHR), P glycoprotein (PgP), metallothionein (MT)) was assessed by RT-PCR. DNA adducts were observed at amount comparable to data from literature. Increase of DNA adducts after two months of transplantation could be correlated with strong modulation of gene expression implicated in detoxification processes. Indeed, PgP and HSP70 gene expressions were similarly induced in gills and digestive glands while SOD and CAT expressions were down regulated in both tissues. AHR, GST and MT genes were differently regulated depending upon the tissue studied and the level of contamination in the different sites. We demonstrated that mussels transplanted in the different stations with pollution gradient were able to biotransform PAHs, assessed by DNA adduct formation and the high decrease of detoxification genes. Specific DNA adducts pattern obtained after one and two month mussel transplantations demonstrated the relevance of DNA adduct as biomarker of environmental pollution., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Derived polycyclic aromatic hydrocarbons (PAHs) such as nitro-PAHs are present in the environment and are known to be much more toxic than PAHs compounds. However, very few studies have analysed their effects on the aquatic environment and none have investigated the freshwater environment. In the present study, we determined whether 1-nitropyrene (1-NP), a model of nitro-PAHs, can induce DNA adducts in gills and digestive glands of the freshwater mussel Dreissena polymorpha. Two concentrations of 1-NP (50 and 500 μM) were tested. In addition, in order to understand the metabolic pathways involved in 1-NP genotoxicity, mRNA expression of genes implicated in biotransformation mechanisms was assessed by quantitative reverse transcription-PCR. Results showed the presence of DNA adducts in both gills and digestive glands, with highest levels obtained after 5 days of exposure to 500 μM. Metallothionein mRNA levels were enhanced in digestive glands exposed to 50 μM. Surprisingly, at the higher concentration (500 μM), aryl hydrocarbon receptor and HSP70 genes were only up-regulated in digestive glands while PgP mRNA levels were increased in both tissues. Results suggested a cytotoxic and genotoxic effect of 1-NP. Mussels seemed to be able to partially detoxify this compound, in view of the low amount of DNA adducts observed after 5 days exposure to 50 μM. For the first time, 1-NP biotransformation and detoxification systems have been characterised in D. polymorpha., (© The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The potent renal carcinogenicity of ochratoxin A (OTA) in rats, principally in the male, raises questions about mechanism. Chromatographic evidence of DNA adducts after (32)P-postlabeling analysis contrasts with experimental attempts to demonstrate the absence of OTA in such adducts. Proffered schemes for alternative epigenetic mechanisms in OTA carcinogenicity remain unsatisfying, while structural data substantiating DNA-OTA adducts has also been lacking. We report refined (32)P-postlabeling methodology revealing one principal adduct isolated in small amounts from the kidneys of all five Fischer and five Dark Agouti rats to which OTA had been given on four consecutive days. We also describe structural data for the principal adduct from OTA/DNA interaction in vitro and its subsequent preparative isolation by the postlabeling methodology (as C-C8 OTA 3'dGMP), essentially creating an ochratoxin B-guanine adduct. Reasoning for the unsuitability of experimental protocols in published evidence claiming nongenotoxicity of OTA is given. In vivo exposure of renal DNA to cycles of adduction with OTA, necessarily protracted for carcinogenesis to occur, can reasonably explain an occasional focal neoplasm from which metastasizing carcinoma could develop.

Ochratoxin A (OTA) is a mycotoxin that shows potent nephrotoxicity and renal carcinogenicity in rodents. One hypothesis for OTA-induced tumor formation is based on its genotoxic properties that are promoted by oxidative metabolism. Like other chlorinated phenols, OTA undergoes an oxidative dechlorination process to generate a quinone (OTQ)/hydroquinone (OTHQ) redox couple that may play a role in OTA-mediated genotoxicity. To determine whether the OTQ/OTHQ redox couple of OTA contributes to genotoxicity, the DNA adduction properties, as evidenced by the (32)P-postlabeling technique, of the hydroquinone analogue (OTHQ) have been compared to OTA in the absence and presence of metabolic activation (pig kidney microsomes) and within human bronchial epithelial (WI26) and human kidney (HK2) cells. Our experiments show that OTHQ generates DNA adduct spots in the absence of metabolic activation. These adducts are ascribed to covalent DNA adduction by OTQ generated through autoxidation of the hydroquinone precursor, OTHQ. Although OTA does not interact with DNA in the absence of metabolism, the OTQ-mediated DNA adduct spots noted with OTHQ are also observed with OTA following treatment with pig kidney microsomes and NADPH, suggesting that OTA undergoes oxidative activation to OTQ by cytochrome P450 or enzymes with peroxidase activity. Comparison of DNA adduction by OTHQ and OTA in human cell lines shows that OTQ-mediated adduct spots form in a dose- and time-dependent manner. The adduct spots form at a faster rate with OTHQ, which is consistent with more facile generation of OTQ from its hydroquinone precursor. These results establish structure-activity relationships for OTA-mediated DNA adduction and provide new evidence for the potential role of the OTQ/OTHQ redox couple in OTA-induced genotoxicity.

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.