Farida Akcha

Genotoxicity of diuron and glyphosate in oyster spermatozoa and embryos

We investigated the effects of genotoxicant exposure in gametes and embryos to find a possible link between genotoxicity and reproduction/developmental impairment, and explore the impact of chemical genotoxicity on population dynamics. Our study focused on the genotoxic effects of two herbicides on oyster gametes and embryos: glyphosate (both as an active substance and in the Roundup formulation) and diuron. France is Europes leading consumer of agrochemical substances and as such, contamination of Frances coastal waters by pesticides is a major concern. Glyphosate and diuron are among the most frequently detected herbicides in oyster production areas; as oyster is a specie with external reproduction, its gametes and embryos are in direct contact with the surrounding waters and are hence particularly exposed to these potentially dangerous substances. In the course of this study, differences in genotoxic and embryotoxic responses were observed in the various experiments, possibly due to differences in pollutant sensitivity between the tested genitor lots. Glyphosate and Roundup had no effect on oyster development at the concentrations tested, whereas diuron significantly affected embryo-larval development from the lowest tested concentration of 0.05 μg L⁻¹, i.e. an environmentally realistic concentration. Diuron may therefore have a significant impact on oyster recruitment rates in the natural environment. Our spermiotoxicity study revealed none of the tested herbicides to be cytotoxic for oyster spermatozoa. However, the alkaline comet assay showed diuron to have a significant genotoxic effect on oyster spermatozoa at concentrations of 0.05 μg L⁻¹ upwards. Conversely, no effects due to diuron exposure were observed on sperm mitochondrial function or acrosomal membrane integrity. Although our initial results showed no negative effect on sperm function, the possible impact on fertilization rate and the consequences of the transmission of damaged DNA for oyster development and physiological performances, requires further investigation. A likely hypothesis to explain the embryotoxic and genotoxic effects of diuron is that it may act via causing oxidative stress.

Relationship between PAH biotransformation as measured by biliary metabolites and EROD activity, and genotoxicity in juveniles of sole (Solea solea)

Polycylic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the marine environment. Their toxicity is mainly linked to the ability of marine species to biotransform them into reactive metabolites. PAHs are thus often detected at trace levels in animal tissues. For biomonitoring purposes, this findings have two main consequences, (i) the determination of the PAH tissue concentration is not suitable for the evaluation of individual exposure to PAHs (ii) it can explain sometimes the lack of correlations obtained with relevant markers of toxicity such as genotoxicity biomarkers. The aim of the present study was to better investigate the link between PAH exposure and genotoxicity in marine flatfish. During a laboratory experiment, juvenile soles were exposed for four weeks to a mixture of three PAHs, namely benzo[a]pyrene, fluoranthene and pyrene, followed by one week of depuration. Fish were exposed via the trophic route to a daily PAH concentration of 120 μg/g food. Fish were sampled at different time points. The bioavailability and the biotransformation of PAHs were assessed by the measurement of biliary metabolites using a sensitive UPLC MS/MS method. The 7-ethoxyresorufine-O-deethylase was also measured in liver subcellular fractions as a biomarker of phase I biotransformation activities. Genotoxicity was assessed in parallel by the measurement of DNA strand breaks in fish erythrocytes by the alkaline comet assay. During this study, the high amount of PAH metabolites produced in sole demonstrated the bioavailability of PAHs and their biotransformation by fish enzymes. A positive correlation was observed between the level of hydroxylated PAH metabolites and genotoxicity as measured by the alkaline comet assay.

Study of genetic damage in the japanese oyster induced by an environmentally-relevant exposure to diuron: evidence of vertical transmission of dna damage

Pesticides represent a major proportion of the chemical pollutants detected in French coastal waters and hence a significant environmental risk with regards to marine organisms. Commercially-raised bivalves are particularly exposed to pollutants, among them pesticides, as shellfish farming zones are subject to considerable pressure from agricultural activities on the mainland. The aims of this study were to determine (1) the genotoxic effects of diuron exposure on oyster genitors and (2) the possible transmission of damaged DNA to offspring and its repercussions on oyster fitness. To investigate these points, oysters were exposed to concentrations of diuron close to those detected in the Marennes-Oleron Basin (two 7-day exposure pulses at 0.4 and 0.6 μg L(-1)) during the gametogenesis period. Genomic abnormalities were characterized using two complementary approaches. The Comet assay was applied for the measurement of early and reversible primary DNA damage, whereas flow cytometry was used to assess the clastogenic and aneugenic effect of diuron exposure. Polar Organic Chemical Integrative Samplers (POCIS) were used in exposed and assay tanks to confirm the waterborne concentration of diuron reached during the experiment. The results obtained by the Comet assay clearly showed a higher level of DNA strand breaks in both the hemocytes and spermatozoa of diuron-exposed genitors. The transmission of damaged genetic material to gamete cells could be responsible for the genetic damage measured in offspring. Indeed, flow cytometry analyses showed the presence of DNA breakage and a significant decrease in DNA content in spat from diuron-exposed genitors. The transmission of DNA damage to the offspring could be involved in the negative effects observed on offspring development (decrease in hatching rate, higher level of larval abnormalities, delay in metamorphosis) and growth. In this study, the vertical transmission of DNA damage was so highlighted by subjecting oyster genitors to short exposures to diuron at medium environmental concentrations. The analysis of POCIS showed that oysters were exposed to integrated concentrations as low as 0.2 and 0.3 μg L(-1), emphasizing the relevance of the results obtained and the risk associated to chemical contamination for oyster recruitment and fitness.

Biliary PAH metabolites, EROD activity and DNA damage in dab (Limanda limanda) from Seine Estuary (France)

The Seine Estuary is well known to be widely contaminated by organic pollutants and especially by polycyclic aromatic hydrocarbons (PAHs). Fish are known to metabolize PAHs, leading to different toxic effects at both cellular and sub-cellular levels. In this work, we studied the relationships between the 7-ethoxyresorufin-O-deethylase (EROD) activity in the liver, the level of DNA strand breaks in blood cells and the concentration of PAH metabolites in the bile of the sentinel flatfish species Limanda limanda. Muscle and liver samples were analysed for parent PAH levels. Female and male dabs of two size classes (juveniles and adults) were collected by trawling in two sites with different degrees of pollution during March and September 2005 and 2006. Significant effects of sex, age, site and season were demonstrated on EROD activity and the level of strand breaks. Parent PAH concentrations in dabs did not allow discriminating of the two sampling sites. However, for PAH metabolites, significant differences were observed with sites and seasons. Dabs collected at the mouth of the estuary appeared to be the most impacted when looking at the results obtained with the three selected markers. The significant correlations observed between the level of PAH metabolites and the level of DNA lesions showed the importance of a combined analysis of chemical and biochemical markers to correctly assess the contribution of chemical contamination to the toxic effects measured in situ in fish.

Comet assay in phytoplankton as biomarker of genotoxic effects of environmental pollution

The alkaline comet assay was tested on different microalgae: the dinoflagellates, Karenia mikimotoi and Alexandrium minutum, and the diatom, Chaetoceros gracilis. The microalgae were exposed during their exponential growth to the model direct genotoxicant, hydrogen peroxide (1h, 5 and 100muM H2O2). Following H2O2 exposure, the comet assay was validated only for K. mikimotoi for which genotoxicity was observed from the lowest tested concentration of 5 microM with a concentration-dependent effect. C. gracilis was too small in size (4 microm) to be correctly analysed. For A. minutum, our lysis buffer was not strong enough to digest the cellulosic thecal plates. For K. mikimotoi, the comet assay was thus applied for the study of the genotoxic effects of different pesticides: epoxiconazole (as Opus formulation), chlorpyriphos-ethyl (as Dursban formulation) and endosulfan at 1, 10 and 100 microg of active substance/L for 24h. Exposure to epoxiconazole in formulation resulted in an increase in the extent of DNA strand breaks at the highest tested concentration icro/L. Endosulfan exposure resulted in DNA damage for K. mikimotoi nuclei. Genotoxicity was observed from 1 microg/L of endosulfan and was not concentration dependent.

Genotoxic and enzymatic effects of fluoranthene in microsomes and freshly isolated hepatocytes from sole (Solea solea).

The fluoranthene (Fluo) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) in human food and in marine compartments. However, the existing data on its genotoxicity is poor and controversial. The aim of this study was to assess in vitro the potential genotoxicity of Fluo in sole and its possible effect on CYP450 modulation. Freshly isolated hepatocytes were exposed for 24 h to a range of Fluo concentrations from 0.5 to 50 μM in both culture flasks and microplate wells. The ethoxyresorufin-O-deethylase (EROD) activity was measured as an indicator of the activity of the cytochrome P450 1A1 (CYP1A1). The genotoxic effects were evaluated by measuring both DNA strand breaks and DNA adducts by the alkaline comet assay and the postlabeling technique respectively. Calf thymus DNA was also exposed to Fluo in the presence of sole liver microsomes in order to check for Fluo DNA adduct formation. In sole hepatocytes, Fluo was shown to induce a decrease in the EROD activity in a concentration-dependent manner. A significant genotoxic effect was observed in terms of DNA strand breakage from an exposure concentration of 5 μM: despite a concentration-dependent effect was observed, it did not follow a linear dose-response. The response was similar whatever the way of exposure in flasks or in wells. One reproducible adduct was detected in the hepatocytes exposed to the highest concentrations of Fluo. The formation of Fluo adducts was confirmed by the detection of one reproducible adduct following in vitro exposure of calf thymus DNA to 100 and 200 μM of Fluo in the presence of sole microsomes. These results demonstrate the potential of sole hepatocytes to metabolize Fluo in 24 h into reactive species, able to induce genotoxicity by DNA strand breakage and DNA adduct formation. Moreover, a miniaturized cell exposure system was validated for further experiments using fewer amounts of hepatocytes and contaminants, and allowing exposure to PAH metabolites.

PAH metabolites in fish bile: From the Seine estuary to Iceland

Polycyclic aromatic hydrocarbons (PAH) are environmental contaminants that pose significant risk to health of fish. The International Workshop on Integrated Assessment of Contaminant Impacts on the North Sea (ICON) provided the framework to investigate biomarker responses as well as contaminant concentrations side by side in marine ecosystems. Concentrations of the main PAH metabolites 1-hydroxypyrene, 1-hydroxyphenanthren and 3-hydroxybenzo(a)pyrene were determined in bile by HPLC with fluorescence detection. Fish species under investigation were dab (Limanda limanda), flounder (Platichthys flesus) and haddock (Melanogrammus aeglefinus). A contamination gradient was demonstrated from the low contaminated waters of Iceland and off-shore regions of the North Sea towards higher concentrations in coastal areas. Concentrations of PAH metabolites differed primarily according to sampling region and secondarily to species.

Parental exposure to environmental concentrations of diuron leads to aneuploidy in embryos of the Pacific oyster, as evidenced by fluorescent in situ hybridization.

Changes in normal chromosome numbers (i.e. aneuploidy) due to abnormal chromosome segregation may arise either spontaneously or as a result of chemical/radiation exposure, particularly during cell division. Coastal ecosystems are continuously subjected to various contaminants originating from urban, industrial and agricultural activities. Genotoxicity is common to several families of major environmental pollutants, including pesticides, which therefore represent a potential important environmental hazard for marine organisms. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to short-term exposure to the herbicide diuron at environmental concentrations during gametogenesis. In this paper, Fluorescent in situ hybridization (FISH) was used to further characterize diuron-induced DNA damage at the chromosomal level. rDNA genes (5S and 18-5.8-28S), previously mapped onto Crassostrea gigas chromosomes 4, 5 and 10, were used as probes on the interphase nuclei of embryo preparations. Our results conclusively show higher aneuploidy (hypo- or hyperdiploidy) level in embryos from diuron-exposed genitors, with damage to the three studied chromosomal regions. This study suggests that sexually developing oysters are vulnerable to diuron exposure, incurring a negative impact on reproductive success and oyster recruitment.

Toxicity assessment of water-accommodated fractions from two different oils using a zebrafish (Danio rerio) embryo-larval bioassay with a multilevel approach

Petroleum compounds from chronic discharges and oil spills represent an important source of environmental pollution. To better understand the deleterious effects of these compounds, the toxicity of water-accommodated fractions (WAF) from two different oils (brut Arabian Light and Erika heavy fuel oils) were used in this study. Zebrafish embryos (Danio rerio) were exposed during 96h at three WAF concentrations (1, 10 and 100% for Arabian Light and 10, 50 and 100% for Erika) in order to cover a wide range of polycyclic aromatic hydrocarbon (PAH) concentrations, representative of the levels found after environmental oil spills. Several endpoints were recorded at different levels of biological organization, including lethal endpoints, morphological abnormalities, photomotor behavioral responses, cardiac activity, DNA damage and exposure level measurements (EROD activity, cyp1a and PAH metabolites). Neither morphological nor behavioral or physiological alterations were observed after exposure to Arabian Light fractions. In contrast, the Erika fractions led a high degree of toxicity in early life stages of zebrafish. Despite of defense mechanisms induced by oil, acute toxic effects have been recorded including mortality, delayed hatching, high rates of developmental abnormalities, disrupted locomotor activity and cardiac failures at the highest PAH concentrations (∑TPAHs=257,029±47,231ng·L(-1)). Such differences in toxicity are likely related to the oil composition. The use of developing zebrafish is a good tool to identify wide range of detrimental effects and elucidate their underlying foundations. Our work highlights once more, the cardiotoxic action (and potentially neurotoxic) of petroleum-related PAHs.

Parental trophic exposure to three aromatic fractions of polycyclic aromatic hydrocarbons in the zebrafish: Consequences for the offspring

In recent decades, PAH emissions due to extensive anthropogenic activities have risen sharply causing considerable pollution of aquatic ecosystems. This pollution represents a threat for organisms, among them are fish. Consequently, prenatal stress can have important repercussions, and may impact survival and population recruitment. To investigate this point, eggs were collected from zebrafish exposed during 6 months by trophic route to three aromatic fractions from two different origins, pyrolytic (PY) and petrogenic (light (BAL) and heavy (HFO) fractions) sources. Chronic dietary exposure of the parents was performed at environmentally relevant concentrations (0.3×, 1× and 3×; 1× represents an environmental concentration measured in French estuary). In order to explore the consequences of parental exposure for the next first generation, toxic responses were studied in both embryos and larvae using a multiscale approach. Toxic effects were assessed by looking at hatching success, developmental abnormalities, photomotor response and heartbeat. The level of PAH metabolites and EROD activity in fish larvae were measured to assess exposure to PAHs. Egg production of parents was significantly reduced compared to the Control; hence little information was available for BAL and HFO offspring. The size of larvae from PY parents was found to increase despite a reduced yolk sac compared to Control larvae. Furthermore, a high level of behavioral stress was observed in larvae originating from parents exposed to three-fold the environmental concentration. The cardiac activity was reduced in a concentration-dependent manner for the PY exposure group. No effect was however observed on biotransformation markers (cyp1a, EROD), nor on the level of DNA damage for all PY, BAL and HFO offspring. The absence of significant differences in metabolite levels may indicate a potential early depuration of transferred compounds or no PAH-transmission. The disruptions observed at the individual level in the next generation could impact on the longer-term, surviving population.