Alain Devaux

Alkaline comet assay in rainbow trout hepatocytes

The alkaline comet assay was performed to measure DNA integrity in fish hepatocytes. Primary cultures of rainbow trout hepatocytes were exposed to two known genotoxic compounds, hydrogen peroxide (H(2)O(2)) and benzo[a]pyrene (B[a]P), and to organic extracts of river sediments. The DNA damage in the form of single-strand breaks was monitored following the formation of DNA comets after alkaline electrophoresis. Exposure of the hepatocytes to H(2)O(2) for 2 hr increased strand breaks in a dose-related manner at the concentration range reported previously in studies with mammalian hepatocytes. B[a]P treatment led to a significant increase in strand breaks at the concentrations ranging from 0.1 to 10 muM after 4 hr of exposure. After 48 hr of exposure to B[a]P, the level of DNA strand breaks was lower than that of the control. The organic extracts obtained from river sediments significantly increased DNA strand breaks in trout hepatocytes, indicating the presence of genotoxic compounds in the sediment. The results show that the alkaline comet assay is a promising method by which to study the genotoxic potential of xenobiotics found in the aquatic environment.

Monitoring of the chemical pollution of the river Rhône through measurement of DNA damage and cytochrome P4501A induction in chub (Leuciscus cephalus)

Abstract The in vivo response of freshwater fish exposed to pollutants was assessed using two biomarkers, 7-ethoxyresorufin- O -deethylase (EROD) induction and DNA single strand breaks. Chub ( Leuciscus cephalus ) were caught in spring and in fall at various locations in the river Rhone watershed. EROD activity was measured in the liver while DNA damage was evaluated in chub erythrocytes using the recently developed Comet assay. Chemical contamination was evaluated both in fish muscle (PCBs) and in sediment (PCBs, PAHs, heavy metals) collected at each sampling station. Sex of individuals was shown to influence the level of EROD activity but not the level of DNA damage. The EROD activity as well as the DNA damage were found to be higher in the mostly contaminated stations compared to the reference one. This study shows that multibiomarker-based approach provides complementary informations about early effects in feral fish exposed to complex chemical pollution and highlights the interest of the Comet assay in genotoxicity assessment.

Bioaccumulation, oxidative stress, and neurotoxicity in Danio rerio exposed to different isotopic compositions of uranium.

Experiments were carried out on adult male zebrafish (Danio rerio) to assess early changes induced by waterborne exposure to different isotopic compositions of uranium (depleted uranium associated or not with 233U). Oxidative stress and neurotoxicity were selected as effect endpoints to characterize uranium chemo- and radiotoxicity. Catalase, glutathione peroxidase, and superoxide dismutase activities and total glutathione content of hepatic extracts, as well as brain acetylcholinesterase activity and uranium bioaccumulation, were measured. Oxidative stress induced by uranium exposure led to decreases in superoxide dismutase and catalase activity levels as well as total glutathione content in liver extracts. These perturbations were significantly more marked in 233U-exposed fish. Furthermore, significant increase in acetylcholinesterase activity was observed in brain extracts at the same level, whatever the isotopic composition of uranium.

Reproduction impairment following paternal genotoxin exposure in brown trout (Salmo trutta) and Arctic charr (Salvelinus alpinus)

This work describes some consequences of paternal germ cell DNA damage on the reproduction success in two fish species. Male brown trout (n=31) and male Arctic charr (n=28) were exposed to the model genotoxicant MMS at the end of spermatogenesis to generate a significant DNA damage level in mature spermatozoa (28% and 25% tail DNA in trout and charr sperm, respectively, evaluated through the comet assay). Sperm from each MMS exposed and control fish was then used to fertilize in vitro an aliquot of a single pool of eggs collected from 4 unexposed females for each species. Each batch of fertilized eggs was monitored individually in the hatchery to follow embryonic and larval abnormalities during the fry development. Paternal exposure did not influence fertilization rate or survival rate at hatching in either species. However, MMS paternal treatment resulted in a large array of morphological abnormalities during embryonic and larval development. At the eyed stage, malformations exhibited a 8 fold increase in trout and a 2 fold increase in charr for larvae stemming from MMS treated males as compared with controls. At the end of yolk sac resorption, an increase in the gross morphological abnormality incidence was found in trout larvae originating from MMS exposed males (2.10% vs. 0.93% in control, p<0.05). When looking more in detail at bony structures after Alizarin red S staining, a 20% incidence of skeletal defects was recorded at the swimming stage. A positive correlation was found between the paternal sperm DNA damage level and the skeletal abnormality incidence of its progeny. During the next 2 months of development, mortality in trout originating from DNA damaged sperm was 3 times higher than in control. After one year, no effect of paternal treatment was found on growth traits (length and weight) but the gross morphological abnormality incidence was still very high in the treated group (27% malformation incidence vs. 0.5% in control). These results demonstrate ecologically relevant consequences of fish spermatozoa DNA damage and stress the value of using this parameter as a biomarker signaling potential long term effects of environmental genotoxins in aquatic systems.

Genotoxicity assessment in the amphipod Gammarus fossarum by use of the alkaline Comet assay.

Many xenobiotics and newly developed substances released in the aquatic environment have been found genotoxic for living organisms. There is interest in developing biomarkers of genotoxicity in different phyla and the need to increase our understanding of the impact of genotoxic insult on invertebrates, particularly on crustaceans. Freshwater invertebrates and particularly amphipods are highly relevant species ecologically. However, genotoxic responses of such species are rarely studied, whereas understanding these responses is becoming an urgent concern. The aim of this study was to develop and optimize the Comet assay in the freshwater invertebrate Gammarus fossarum by use of different cell-types: haemocytes, oocytes and spermatozoa. In a first step, the Comet assay was performed on these three cell types after exposure to the model genotoxicant methyl methanesulfonate (MMS) in vitro and in vivo. Results showed a clear dose-response relationship for all tissues, a low variability and a high sensitivity of the response, demonstrating the effectiveness of the Comet assay to detect genotoxic insult in amphipods. In a second step, to explore the potential of this technique for use in ecotoxicological studies with amphipods, these organisms were exposed to five known or suspected genotoxic compounds. The results demonstrated the possibility to use the freshwater amphipod G. fossarum in environmental genotoxicity studies with the Comet assay.

Uranium bioaccumulation and biological disorders induced in zebrafish (Danio rerio) after a depleted uranium waterborne exposure

Because of its toxicity and its ubiquity within aquatic compartments, uranium (U) represents a significant hazard to aquatic species such as fish. In a previous study, we investigated some biological responses in zebrafish either exposed to depleted or to enriched U (i.e., to different radiological activities). However, results required further experiments to better understand biological responses. Moreover, we failed to clearly demonstrate a significant relationship between biological effects and U radiological activity. We therefore chose to herein examine U bioaccumulation and induced effects in zebrafish according to a chemical dose-response approach. Results showed that U is highly bioconcentrated in fish, according to a time- and concentration-dependent model. Additionally, hepatic antioxidant defenses, red blood cells DNA integrity and brain acetylcholinesterase activity were found to be significantly altered. Generally, the higher the U concentration, the sooner and/or the greater the effect, suggesting a close relationship between accumulation and effect.

Genotoxic pressure of vineyard pesticides in fish: Field and mesocosm surveys

The present study deals with the genotoxicity assessment of vineyard pesticides in fish exposed in the field or in mesocosm conditions. Primary DNA damage was quantified as strand breaks using the single cell gel electrophoresis assay (Comet assay) applied to fish erythrocytes. In a first experiment, a significant genotoxic effect was observed following an upstream-downstream gradient in early life stages of brown trout (Salmo trutta fario) exposed in the Morcille River contaminated by a mixture of vineyard pesticides during three consecutive years. The pronounced response in terms of DNA damage reported in the present study could argue for a high sensitivity of fish early life stage and/or a high level of exposure to genotoxic compounds in the Morcille River. This stresses the interest in using trout larvae incubated in sediment bed to assess genotoxic compounds in the field. In a second experiment, adult European topminnow (Phoxinus phoxinus) were exposed in water running through artificial channels to a mixture of diuron and azoxystrobin, two of the main pesticides detected in the Morcille watershed. As compared with the unexposed channel, a 3-5-fold increase in the DNA damage was observed in fish exposed to chronic environmental pesticide concentrations (1-2 microg L(-1) for diuron and 0.5-1 microg L(-1) for axoxystrobin). A single 6h pulse of pesticide (14 microg L(-1) of diuron and 7 microg L(-1) of azoxystrobin) was applied to simulate transiently elevated chemical concentrations in the river following storm conditions. It did not increase genotoxicity. After a 1-month recovery period, DNA damage in exposed fish erythrocytes recovered to unexposed level, suggesting possible involvement of both repair mechanisms and cellular turnover in this transient response. This work highlights that vineyard treatment by pesticides and in particular diuron and azoxystrobin can represent a genotoxic threat to fish from contaminated watershed rivers.

DNA damage in caged Gammarus fossarum amphipods: a tool for freshwater genotoxicity assessment.

The aim of this study was to propose a tool for freshwater environmental genotoxicity assessment using Gammarus fossarum, a high ecologically relevant species. In a first part, gammarids were caged upstream and downstream wastewater treatment plant effluent output. The sensitivity of genotoxic responses of haemocytes, oocytes and spermatozoa was compared using the Comet assay. Spermatozoa appeared to be the most sensitive, suitable and relevant cell type for genotoxicity risk assessment. In a second part, a watershed-scale study was conducted over 2 years to evaluate the applicability of our caging procedure. The genotoxic impact of a contamination was followed, taking into account seasonal variability. DNA damage in spermatozoa exhibited low basal level and low variability in control upstream sites, providing a reliable discrimination of polluted sites. Finally, DNA damage in caged G. fossarum has been proved to be a sensitive and reproducible tool for freshwater genotoxicity assessment.

DNA repair activity in fish and interest in ecotoxicology: A review

The knowledge of DNA repair in a target species is of first importance as it is the primary line of defense against genotoxicants, and a better knowledge of DNA repair capacity in fish could help to interpret genotoxicity data and/or assist in the choice of target species, developmental stage and tissues to focus on, both for environmental biomonitoring studies and DNA repair testing. This review focuses in a first part on what is presently known on a mechanistic basis, about the various DNA repair systems in fish, in vivo and in established cell lines. Data on base excision repair (BER), direct reversal with O⁶-alkylguanine transferase and double strand breaks repair, although rather scarce, are being reviewed, as well as nucleotide excision repair (NER) and photoreactivation repair (PER), which are by far the most studied repair mechanisms in fish. Most of these repair mechanisms seem to be strongly species and tissue dependent; they also depend on the developmental stage of the organisms. BER is efficient in vivo, although no data has been found on in vitro models. NER activity is quite low or even inexistent depending on the studies; however this lack is partly compensated by a strong PER activity, especially in early developmental stage. In a second part, a survey of the ecotoxicological studies integrating DNA repair as a parameter responding to single or mixture of contaminant is realized. Three main approaches are being used: the measurement of DNA repair gene expression after exposure, although it has not yet been clearly established whether gene expression is indicative of repair capacity; the monitoring of DNA damage removal by following DNA repair kinetics; and the modulation of DNA repair activity following exposure in situ, in order to assess the impact of exposure history on DNA repair capacity. Since all DNA repair processes are possible targets for environmental pollutants, we can also wonder at which extent such a modulation of repair capacities in fish could be the base for the development of new biomarkers of genotoxicity. Knowing the importance of the germ cell DNA integrity in the reproductive success of aquatic organisms, the DNA repair capacity of such cells deserve to be more studied, as well as DNA repair capacities of established fish cell lines. The limited amount of available data, which shows low/slow DNA repair capacities of fish cell lines compared with mammalian cell lines, concerned mainly the NER system; thus this point merits to be explored more deeply. Additionally, since some of the DNA repair systems appear more efficient in embryo larval stages, it would be of interest to consider embryonic cell lineages more closely.

Linking genotoxic responses in Gammarus fossarum germ cells with reproduction impairment, using the Comet assay

Germ cells perform a unique and critical biological function: they pass down DNA that will be used for the development of the next generation. Thus there is an increasing need to understand how the adult exposure to genotoxicants could show negative impact on the offspring of aquatic organisms. Hence this work addresses the question of the consequences of germ cell DNA damage resulting from parental exposure on reproduction quality in the freshwater crustacean Gammarus fossarum, a high ecologically relevant species. Initially, the sensitivity response of mature oocytes and spermatozoa to two model genotoxicants, MMS and K(2)Cr(2)O(7) was compared by implementing the Comet assay after the exposure of these gammarids in the laboratory and after the exposure of caged organisms in the field. Spermatozoa appeared significantly more susceptible than the oocytes to genotoxicants whatever were the exposure conditions. Secondly, a significant correlation between the level of damage to the sperm DNA of exposed parents and the abnormality rate in embryos that had developed in non-contaminated water were demonstrated. Interestingly, this relationship bridges the biomarker response measured in germ cells at molecular level and its consequences at individual level for the subsequent generation. Moreover, reproduction defects were observed for a level of DNA damage exceeding a minimal threshold, which could have significant consequences for the population dynamics of this high ecologically relevant species.