Julien Rouxel

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.

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.

Multigenerational exposure of the microalga Tetraselmis suecica to diuron leads to spontaneous long-term strain adaptation

To investigate the ability of microalgae to develop stable, long-term resistance to herbicides, the marine microalga Tetraselmis suecica was exposed to the herbicide diuron (5 μg/L) for a 43-generation exposure period followed by a 12-generation depuration phase. During the first 25 generations, diuron-exposed cultures showed doubling times ranging from 1.95 to 2.6 days, which was 2 to 2.5-fold longer than control cultures. Between generations 25 and 38, during diuron exposure, two out of the three exposed cultures exhibited a spontaneous drop in doubling time. These results provided evidence of culture adaptation to diuron. To assess persistence of the diuron adaptation observed on growth performance, one of the adapted cultures (D3) was maintained for 12 months in unexposed conditions and then tested by a second, short-term exposure to diuron 5 μg/L, in parallel with a control culture (C1) for six generations. Flow cytometry analyses were used to monitor cell density, viability, morphology, relative chlorophyll content and intracellular reactive oxygen species (ROS) level. Under these conditions, diuron induced a strong increase of doubling time in exposed-C1 cultures (2.5-fold longer than unexposed-C1 cultures), but no significant increase occurred in exposed D3-cultures compared with unexposed D3- and unexposed C1-cultures, showing the persistence of adaptation in the previously-exposed strain D3. Intracellular ROS level showed the same trend. Significant differences were observed between these strains, with weaker effects of diuron on strain D3 compared with strain C1: forward scatter (FSC), representing relative cell size, decreased in exposed cultures (67.8% and 95% of the controls for C1 and D3, respectively), whereas FL3 as relative chlorophyll content increased in exposed cultures (115.6% and 108.6% of the controls for C1 and D3, respectively). Results of second exposure to diuron revealed that the adaptation of strain D3 had persisted after 12 months of depuration, as no growth impairment was observed. This study demonstrates the possible appearance of stable diuron resistance in microalgae in cases of strong, multigenerational chronic exposure to this herbicide in polluted environments.

Effects of an environmentally relevant concentration of diuron on oyster genitors during gametogenesis: responses of early molecular and cellular markers and physiological impacts

Genitors of the Pacific oyster Crassostrea gigas were submitted during gametogenesis to a short pulse exposure to the herbicide diuron at a realistic environmental concentration. Histological analysis showed no effect of diuron on gametogenesis course, sex ratio and reproductive effort. A non-significant increase in testosterone and progesterone levels was observed in genitors exposed to the herbicide. At cell level, diuron exposure was shown to modulate the phagocytic activity of circulating hemocytes. The results of a transcriptional analysis showed that diuron affected the expression of genes belonging to functions known to play a major role during oyster gametogenesis such as gene transcription regulation, DNA replication and repair, DNA methylation and cytokinesis. Taking into account the results we previously obtained on the same genitors, this study showed a negative effect of diuron on oyster reproduction by inducing both structural and functional modifications of the DNA.

Parental diuron-exposure alters offspring transcriptome and fitness in Pacific oyster Crassostrea gigas

One of the primary challenges in ecotoxicology is to contribute to the assessment of the ecological status of ecosystems. In this study, we used Pacific oyster Crassostrea gigas to explore the effects of a parental exposure to diuron, a herbicide frequently detected in marine coastal environments. The present toxicogenomic study provides evidence that exposure of oyster genitors to diuron during gametogenesis results in changes in offspring, namely, transcriptomic profile alterations, increased global DNA methylation levels and reduced growth and survival within the first year of life. Importantly, we highlighted the limitations to identify particular genes or gene expression signatures that could serve as biomarkers for parental herbicide-exposure and further for multigenerational and transgenerational effects of specific chemical stressors. By analyzing samples from two independent experiments, we demonstrated that, due to complex confounding effects with both tested solvent vehicles, diuron non-specifically affected the offspring transcriptome. These original results question the potential development of predictive genomic tools for detecting specific indirect impacts of contaminants in environmental risk assessments. However, our results indicate that chronic environmental exposure to diuron over several generations may have significant long term impacts on oyster populations with adverse health outcomes.

Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas

Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis.

Development of a comet-FISH assay for the detection of DNA damage in hemocytes of Crassostrea gigas.

In this work, the DNA-damaging effect of hydrogen peroxide on the structural integrity of nucleolar organizer regions (NORs) was studied for the first time by comet-FISH in the Pacific oyster Crassostrea gigas. Global DNA damage was assessed in hemocytes using an alkaline version of the comet assay. Next, NOR sensitivity was analyzed by mapping major rDNA repeat unit by fluorescence in situ hybridization (FISH) on the same comet slides. Exposure of hemocytes to 100 μM of hydrogen peroxide induced a significant increase in both DNA damage and number of FISH-signals of major ribosomal genes versus the control. Moreover, a significant positive correlation was shown between DNA damage as measured by the comet assay (percentage of DNA in comet tail) and the number of signals present in comet tails. This study demonstrates the potential value of the comet-FISH assay for the study of DNA damage induced by genotoxicant exposure of target genes. It offers a perspective for better understanding the impact of genotoxicity on animal physiology and fitness.