Paule Vasseur

Antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or field exposure in the gills and the digestive gland of the freshwater bivalve Unio tumidus

The aim of this work was to evaluate the potential utility of antioxidant parameters as indicators of exposure to toxicants and of toxic effects in the freshwater mussel Unio tumidus. Antioxidant enzymes (glutathione peroxidase (EC 1.11.1.9), glutathione reductase (EC 1.6.4.2), Superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6)), redox status of glutathione and lipid peroxidation were measured at first in the gills and the digestive glands of mussels after exposure to copper (30 μg l−1) or/and thiram (100 μg l−1) for 3 days. The effects of a complex industrial effluent on the antioxidant system were investigated afterwards in a field study: encaged mussels were placed in a river upstream and downstream from a pollution source consisting of the effluent of a cokery for 1 week. These studies demonstrated that in both experimental exposures, the most sensitive parameters were selenium-dependent glutathione peroxidase activity (SeGPx), reduced glutathione levels (GSH) and especially glutathione reductase activity (GRd) which significantly decreased. Decreases in SeGPX and GRd activities were more pronounced in the gills under coexposure to copper and thiram, with 74% and 78% of reduced activity, respectively (P < 0.01); reduction of GSH levels was 50% in this case. In the field study, the activities could be reduced by 35% and 72% in the gills for SeGPx and GRd, respectively; reduction of GSH levels could reach 45%. In contrast, superoxide dismutase and catalase activities remained generally constant in all the treatment groups compared with controls. Lipid peroxidation, as expressed by malonaldehyde content measured by HPLC, was slightly enhanced in experiment with copper+thiram and this increase (from 1.8 to 2.8 fold) was concomitant with a depletion of antioxidant defences. The responses of antioxidant parameters were overall greater in the gills than in the digestive glands of exposed mussels. These results suggest that among antioxidant defence systems, SeGPx, GSH levels and particularly GRd, could represent biomarkers of interest for the estimation of the effects of environmental contamination on freshwater invertebrates.

Biomarkers and community indices as complementary tools for environmental safety.

Research on biomarkers as early bioindicators of perturbation in populations and individuals has been gaining ground over the last decade. This ecotoxicological approach relies on the fact that changes occur at low levels of organization before the community is affected and thus they can be monitored to assess environmental safety. Changes may concern behavior, physiology, biochemistry, or genomic structure and functioning, and may impair population dynamics in the long-term. Ecotoxicity studies based on biomarkers allow us to measure the impact of environmental stressors and to easily follow the evolution of the systems towards degradation or restoration. Over and above their use as simple indices of exposure to specific pollutants, biomarkers can give an insight into ecosystem health. The results of our experience in field studies involving ecotoxicologists and ecologists will be presented in order to illustrate the relevance of such an integrating strategy for environmental quality assessment.

Nongenotoxic carcinogens: development of detection methods based on mechanisms: a European project

While the accumulation of genetic changes in a somatic cell is considered essential for the genesis of a cancer, it has become clear that not all carcinogens are genotoxic, suggesting that some carcinogens indirectly participate in the generation of genetic changes during carcinogenesis. A European project funded by the European Community was thus conceived to study mechanisms of nongenotoxic aspects of carcinogenesis. Two main strategical approaches were adapted: (i) to study whether and how Syrian hamster embryo (SHE), Syrian hamster dermal (SHD) and BALB/c 3T3 cell transformation systems simulate in vivo carcinogenesis, and to examine whether they can detect nongenotoxic carcinogens; (ii) to study, refine and validate mechanisms-based end-points for detection of nongenotoxic carcinogens. For mechanisms-based research, the proposed end-points included gap junctional intercellular communication (GJIC) inhibition, altered expression of critical genes, immortalization and aberrant cell proliferation. We also selected model compounds commonly usable for various endpoints. Our major results can be summarized as follows: (1) SHE and BALB/c 3T3 transformation systems reflect both genotoxic and nongenotoxic carcinogenic events; they detect not only genotoxic but also many although not all, nongenotoxic carcinogens. This is further supported by the fact that both genotoxic and nongenotoxic carcinogens were able to immortalize SHD cells. (2) Many nongenotoxic carcinogens, although not all, inhibit GJIC in vitro as well as in vivo. Mechanistic studies suggest an important role of blocked GJIC in carcinogenesis and that different mechanisms are involved in inhibition of the communication by different agents used. However, inhibition of GJIC is not a prerequisite for the enhancement (or induction) of transformation of SHE or BALB/c 3T3 cells. (3) Among compounds examined, there was a good correlation between induction of micronuclei and cell transformation in SHE cells while no such correlation was found between the induction of cell transformation and ornithine decarboxylase activity. (4) Two transgenic mouse mutation assays (lacI and lacZ) were established and validated. The genotoxin dimethylnitrosamine was shown to be mutagenic to the liver in both assays. Ortho-anisidine, a bladder-specific carcinogen that was inactive in standard rodent genetic toxicity assays was uniquely mutagenic to the bladder of the transgenic mice. The peroxisome proliferator methyl clofenipate was established as nonmutagenic to the liver of both transgenic mice. That eliminated DNA damage as a cause of the liver tumours produced by this chemical and weakened the idea that induced cell division leads to mutation induction. (5) With an in vitro DNA replication model, it was found that DNA damage induced by genotoxic agents can be responsible for inhibition of DNA replication, while certain nongenotoxic agents such as phorbol esters increase DNA replication. (6) An attempt to use structure-activity relationship for subfamilies of nongenotoxic carcinogens, e.g., receptor-mediated carcinogens, has been initiated with some promising results. Our results support the idea that there are multiple nongenotoxic mechanisms in carcinogenesis, and that working hypothesis-oriented approaches are encouraged rather than simple screening of chemicals in developing test systems for the detection of nongenotoxic carcinogens.

Comparison of two types of sensors using eukaryotic algae to monitor pollution of aquatic systems

Abstract Two types of amperometric environmental sensor incorporating eukaryotic algae were investigated for use in monitoring pollution of aquatic systems. Both sensors allowed the monitoring of photosynthetic events, one by measuring the rate of reduction of a redox mediator by the illuminated biocatalyst, the other by monitoring its photosynthetic oxygen production using a semi-protected oxygen electrode. Comparison of sensor response obtained prior to challenge with pollutants with that following challenge allows rapid detection of disturbance in photosynthetic activity. The oxygen electrode-based biosensor gave good sensitivity with long operational life, and proved to be a better approach than mediator system to monitor algal biocatalysts for detection of aquatic pollutants displaying toxicity on photosynthetic organisms.

In Situ Assessment of Phytotechnologies for Multicontaminated Soil Management

Due to human activities, large volumes of soils are contaminated with organic pollutants such as polycyclic aromatic hydrocarbons, and very often by metallic pollutants as well. Multipolluted soils are therefore a key concern for remediation. This work presents a long-term evaluation of the fate and environmental impact of the organic and metallic contaminants of an industrially polluted soil under natural and plant-assisted conditions. A field trial was followed for four years according to six treatments in four replicates: unplanted, planted with alfalfa with or without mycorrhizal inoculation, planted with Noccaea caerulescens, naturally colonized by indigenous plants, and thermally treated soil planted with alfalfa. Leaching water volumes and composition, PAH concentrations in soil and solutions, soil fauna and microbial diversity, soil and solution toxicity using standardized bioassays, plant biomass, mycorrhizal colonization, were monitored. Results showed that plant cover alone did not affect total contaminant concentrations in soil. However, it was most efficient in improving the contamination impact on the environment and in increasing the biological diversity. Leaching water quality remained an issue because of its high toxicity shown by micro-algae testing. In this matter, prior treatment of the soil by thermal desorption proved to be the only effective treatment.

Use of in vitro (Ames and Mutatox tests) and in vivo (Amphibian Micronucleus test) assays to assess the genotoxicity of leachates from a contaminated soil

Two in vitro bacterial assays (Ames test and Mutatox test) and one in vivo Micronucleus test on amphibians were used to assess the genotoxicity of aqueous leachates from a soil sample contaminated with polycyclic aromatic hydrocarbons (PAHs) and heavy metals. Positive results were obtained with the three tests, suggesting that this contaminated soil may be a threat to the aquatic environment. The test sensitivity was shown to be influenced by the experimental conditions. The response was higher with the nonfiltered leachate than with 0.45 μm filtered one, which was the standard protocol (Norm X31-210). This standard procedure must be reconsidered in order to account for the pollutants adsorbed on solid particles which were biologically active. Incubation of bacteria in a liquid medium enhanced the sensitivity of the Ames test, by increasing the bioavailability of pollutants in comparison with the agar plate method. Biological tests appeared to be a useful complement to physico-chemical analysis, since they account for bioavailability and bioaccumulation of chemicals, and interactions between pollutants.

Algal Microplate Toxicity Test

The test method described below can be employed on its own, or as part of a battery of tests approach, to estimate the phytotoxicity potential of diverse types of liquid samples. Because it is conducted in a 96-well microplate format, the technique is simple and offers other advantages which include cost-effectiveness and spacesaving features. It is theoretically applicable to any liquid. Testing can be undertaken, for example, with samples representing the following media: (1) domestic and industrial wastewaters, treated or untreated; (2) surface, groundwater or leachates; (3) sediment interstitial waters; (4) any chemical that is soluble in water; (5) any water-insoluble chemical that can be rendered soluble by means of an organic solvent or other techniques (e.g., sonication or emulsification).

In vitro effects of Thiram on liver antioxidant enzyme activities of rainbow trout (Oncorhynchus mykiss)

The effects of the dithiocarbamate Thiram on the activities of Superoxide dismutase, catalase and glutathione peroxidase in young rainbow trout livers were studied in vitro. Catalase and glutathione peroxidase activities were evaluated with standardized methods. Superoxide dismulase was assayed by a method based on the inhibiting action of the enzyme on the rate of oxidation of NADH. The activities of superoxide dismutase, catalase and glutathione peroxidase were found to be 11.3 μg/min/mg, 33.1 μmol/min/mg and 12.2 nmol/min/mg protein respectively, in control animals. Incubation of liver supernatants with 10−6 M, 10−5 M and 10−4 M Thiram for 1.5 h resulted in a total loss of Superoxide dismutase activity at 10−4 M, whereas catalase activity showed no significant reaction to the same range of toxic concentrations. The activity of glulathione peroxidase decreased at 10−6 M of Thiram but was not further reduced at higher concentrations of dithiocarbamate.

The genotoxicity of iron and chromium in electroplating effluents

Electroplating effluents were tested for their genotoxicity with the micronucleus test on newt larvae. The metallic content of the tested samples was responsible for the induction of micronuclei in red blood cells (RBC). Then, iron (Fe3+), chromium (Cr3+, Cr6+) and zinc (Zn2+) which were identified in these samples, were tested either separately or combined, at their concentrations in the electroplating effluents. Fe3+ induced a high level of micronuclei at 12.5 and 25 mg/l (nominal concentrations). Both soluble and non-soluble forms of iron were responsible for these genotoxic effects. At lower concentrations (0.6 and 4.5 mg/l) Fe3+ was not systematically genotoxic. Zinc could not be considered genotoxic on newt. Cr3+ gave negative responses, but exposure to Cr6+ (1 mg/l) could result in a significant number of micronucleated RBC in some cases. The most dramatic genotoxic effects were registered when Fe3+ and Cr6+ were combined. This study demonstrates that interactions between pollutants and the effects of non-soluble chemicals on aquatic vertebrates and invertebrates can no longer be neglected.

OECD Detailed Review Paper (DRP) number 31 on "Cell Transformation Assays for Detection of Chemical Carcinogens": main results and conclusions.

The Detailed Review Paper (DRP) number 31 of the Organisation for Economic Co-operation and Development (OECD) analyses the performance of the three models used in Cell Transformation Assays (CTAs) to screen the carcinogenic potential of chemicals: the Syrian hamster embryo (SHE) cells, and the mouse cell lines BALB/c 3T3 and C3H10T1/2. The CTA results have been compared to results from recent genotoxicity tests using mammalian and non-mammalian cell systems. The performance of the CTAs in predicting carcinogenic potential has been established on several hundreds of chemicals, comprising organic and inorganic substances. The results have been analysed and the chemicals classified as rodent and/or human carcinogens. Based on this comparison and on their performance - concordance, sensitivity, specificity, positive and negative predictive capacity, and evidence for intra- and inter-laboratory reproducibility - OECD recommended that the CTAs using the SHE cells (carried out at physiological or acidic pH) and the BALB/c 3T3 cell line should be developed into OECD test guidelines. The CTA using the C3H10T1/2 cell line was considered to be useful to elucidate molecular mechanisms of cell transformation at the genomic and transcriptomic level. However, due to the limited data available on reproducibility, a test guideline was not recommended at that time.