Genotoxicity and activation of cellular defenses in transplanted zebra mussels Dreissena polymorpha along the Seine river.

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.

DNA adduct formation and induction of detoxification mechanisms in Dreissena polymorpha exposed to nitro-PAHs.

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.

Development of an in vitro method for the prediction of mycotoxin binding on yeast-based products: case of aflatoxin B1, zearalenone and ochratoxin A.

To date, no official method is available to accurately define the binding capacity of binders. The goal is to define general in vitro parameters (equilibrium time, pH, mycotoxin/binder ratio) for the determination of binding efficacy, which can be used to calculate the relevant equilibrium adsorption constants. For this purpose, aflatoxin B1 (AFB1), zearalenone (ZEA) or ochratoxin A (OTA) were incubated with one yeast cell wall in pH 3, pH 5 or pH 7 buffers. The percentage of adsorption was recorded by quantitation of remaining mycotoxins in the supernatant and amount of mycotoxin adsorbed on the residue. The incubation of yeast cell wall in the presence of mycotoxins solved in buffer, lead to unexpected high adsorption percentage when the analysis was based only on remaining mycotoxins in the supernatant. The decrease of mycotoxins in the supernatant was not correlated to the amount of mycotoxins found in the residue. For this reason we modified the conditions of incubation. Yeast cell wall (5 mg) was pre-incubated in buffer (990 µl) at 37 °C during 5 min and then 10 µl of an alcoholic solution of mycotoxin (concentration 100 times higher than the final concentration required in the test tube) were added. After incubation, the solution was centrifuged, and the amount of mycotoxins were analysed both in the supernatant and in the residue. A plateau of binding was reached after 15 min of incubation whatever the mycotoxins and the concentrations tested. The adsorption of ZEA was better at pH 5 (75 %), versus 60 % at pH 3 and 7. OTA was only significantly adsorbed at pH 3 (50 %). Depending on the pH, the adsorptions of OTA or ZEA were increased or decreased when they were together, indicative of a cooperative effect.

Impact of pH on the stability and the cross-reactivity of ochratoxin A and citrinin.

Mycotoxins are secondary metabolites produced by several fungi contaminating crops. In several countries, the maximum permitted levels of mycotoxins are found in foodstuffs and feedstuffs. The common strategy of mycotoxin analysis involves extraction, clean-up and quantification by chromatography. In this paper, we analyzed the reasons of underestimation of ochratoxin A (OTA) content in wine, and overestimation of OTA in wheat, depending on the pH of the clean-up step and the simultaneous presence of citrinin (CIT). We demonstrated that the increase of pH by adding polyethylene glycol (PEG) to wine led to an underestimation of OTA by conversion of OTA into open ring ochratoxin A OP-OA. In comparing three methods of extraction and clean-up for the determination of OTA and CIT in wheat--(i) an inter-laboratory validated method for OTA in cereals using immunoaffinity column clean-up (IAC) and extraction by acetonitrile/water; (ii) a validated method using IAC and extraction with 1% bicarbonate Na; and (iii) an in-house validated method based on acid liquid/liquid extraction--we observed an overestimation of OTA after immunoaffinity clean-up when CIT is also present in the sample, whereas an underestimation was observed when OTA was alone. Under neutral and alkaline conditions, CIT was partially recognized by OTA antibodies.

Genotoxicity assessment and detoxification induction in Dreissena polymorpha exposed to benzo[a]pyrene.

The use of DNA adduct analysis has previously focused on the use of marine organisms for biomonitoring, whereas similar investigations in freshwater organisms are sparse. In that context, we have investigated the relevance of DNA adducts as biomarkers of genotoxicity in the freshwater mussels Dreissena polymorpha. The objective of the present study is to determine the stability of DNA adducts induced by benzo[a]pyrene (B[a]P) in zebra mussels. Mussels were exposed to dissolved B[a]P (10-100 µg/l) for 4 days. Afterwards, mussels were kept in clean water for 28 days and DNA adduct levels were subsequently measured in two different organs, the digestive glands and the gills, using the (32)P-postlabelling technique. In parallel, the expression of genes involved in the detoxification system was assessed by qPCR (catalase, superoxide dismutase, glutathione S transferase, HSP70, aryl hydrocarbon receptor, P glycoprotein). We observed a higher level of DNA adducts in the digestive glands compared to the gills. Moreover, in gills, the level of DNA adduct was dependent on the B[a]P concentration. The levels of adducts tended to decrease in both organs after 28 days in clean water. In addition, an early induction of HSP70, PgP, AHR and SOD mRNA levels was noticed in the gills compared to the digestive glands. CAT and GST gene expression increased from 12h exposure in both organs. A higher gene expression level of those genes was observed in the gills, except for AHR and CAT genes. Data converge towards the fact that DNA adducts hence represent a very promising biomarker of B[a]P contamination and potentially of exposure to polycyclic aromatic hydrocarbons. In addition, for the first time in this study, B[a]P detoxification system was characterised in D. polymorpha.

Cytotoxicity micropollutant removal in a crossflow membrane bioreactor.

The application of membrane bioreactor (MBR) technology was investigated with the aim of evaluating its potential for cytostatic drug and cytotoxicity bioremoval. The toxicity removal was assessed from biomarker test. CP removal of up to 80% was achieved under the operating conditions studied (HRT of 48 h and a SRT of 50 days). The increase of TMP was associated with an increase of supernatant toxicity as if fouling led to retention of the toxicity. Peaks of supernatant cytotoxicity were correlated with peaks in supernatant humic acid contents. It may suggest that molecules with a toxic effect may be adsorbed or entrapped in humic acids substances. Our study then points out that advances in wastewater treatment using an MBR can provide a suitable process for lowering CP concentrations before discharge into the aqueous environment. However, a tertiary treatment is necessary if complete elimination of toxicity is targeted.

Structures of covalent adducts between DNA and ochratoxin a: a new factor in debate about genotoxicity and human risk assessment.

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.

Genotoxicity of the hydroquinone metabolite of ochratoxin A: structure-activity relationships for covalent DNA adduction.

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.

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.

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.