Gilles Monod

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.

Effects of 4-Nonylphenol on Sex Differentiation and Puberty in Mosquitofish (Gambusia holbrooki)

Three days post-parturition mosquitofish were exposed to different concentrations of 4-NP following a semi-static protocol. Exposure lasted up to the development of male anal fin in male individuals of the control group. Exposure to 50 μg/L 4-NP resulted in 100% females considering secondary sexual characters, while external sex-ratio did not statistically differed from unity in control group. In group exposed to 0.5 and 5.0 μg/L sex-ratio did not differ from unity but incompletely developed gonopodium was observed in several individuals. Individuals exposed to 50 μg/L 4-NP exhibited female or undeveloped gonads, while gonadal sex-ratio did not statistically differ from unity in control group. Percentage of undeveloped gonads increased with 4-NP concentration. Additional observations demonstrated hepatic histopathology in fish exposed to the highest concentration and growth reduction dependent on 4-NP concentration. In a complementary experiment, extensive metabolism of [3H]4-n-NP was characterized following in vivo exposure of juvenile mosquitofish suggesting that metabolism could modulate 4-NP toxicity. This study suggests susceptibility of early life stages of mosquitofish to endocrine modulators with regard to development of reproductive capabilities.

Main neuro-endocrine, endocrine and paracrine regulations of fish reproduction, and vulnerability to xenobiotics.

The reproductive function of fish, which is very sensitive to the variations of environmental factors, appears also to be particularly vulnerable to the presence of xenobiotics in the aquatic medium. Many physiological processes can be impaired, from sexual differentiation to female and male gametogenesis, due to disruptions among complex neuro-endrocrine, endocrine or paracrine regulations. This paper describes the main regulation steps that are known or can be suspected to be disrupted by xenobiotics and gives some examples. The large interspecific diversity of reproductive strategies and the complexity of underlying mechanisms are particularly highlighted to draw attention to possible confusions between real endocrine disruptions and natural physiological variations.

DNA adduct formation and 7-ethoxyresorufin O-deethylase induction in primary culture of rainbow trout hepatocytes exposed to benzo[a]pyrene

The formation of DNA adducts, using the (32)P-postlabelling assay, and induction of 7-ethoxyresorufin O-deethylase (EROD) were investigated in a primary culture of rainbow trout hepatocytes exposed to benzo[a]pyrene (B[a]P). Concentrations of 0.1 and 1 mum-B[a]P were shown to induce EROD whereas 10 mum was an inhibitory concentration. DNA adducts were detected for 12 hr to 72 hr after exposure to 1 mum-B[a]P whereas EROD activity was increased 36 hr after treatment. The pattern of adducts was shown to be identical to that obtained after B[a]P treatment of rainbow trout in vivo, as demonstrated by co-chromatography of the adducts. Pre-exposure of hepatocytes for 48 hr to beta-naphthoflavone (betaNF) and subsequent 24-hr exposure to 1 mum-B[a]P did not lead to increased DNA adduct formation although betaNF treatment led to a 3.4-fold induction of EROD activity at the time of B[a]P addition. This study suggests that primary culture of rainbow trout hepatocytes provides a suitable method for studying DNA adduct formation and its modulating factors in vitro.

Variability of physicochemical and biological parameters between replicated outdoor freshwater lentic mesocosms.

Micro- and mesocosms are frequently required in regulatory procedures of aquatic risk assessment for pesticides. However, many questions are still a matter of debate with regard to the use of these systems for environmental risk assessment, especially considering the inter-system variability of the measured parameters and its consequences on experimental design and data analysis. In this paper, variability of physico-chemical and biological parameters measured during two long-term experiments (8 to 9 months) in uncontaminated outdoor freshwater lentic mesocosms (8 m3) is analysed. Consequences on the design of ecotoxicity tests in mesocosms and on data analysis are also addressed. Water temperature, pH, dissolved oxygen concentration and concentration of suspended solids exhibited a very low variability whereas nutrient concentrations displayed elevated levels of variability. Among biological parameters, those measured at the individual level were less variable than those measured at the community level. Functional descriptors frequently exhibited a lower inter-mesocosm variability than structural descriptors. Aggregation of data proved to significantly reduce inter-mesocosm variability. The results indicate that univariate statistical methods may be used for physico-chemical or species-level (e.g. biometric parameters) data which exhibit a moderate inter-mesocosm variability. The use of multivariate techniques is suggested for other levels of investigation. Nevertheless, variability is not sufficient to identify useful parameters. The sensitivity towards chemicals and ecological relevance of descriptors within the experimental context must also be considered.

Maintenance of cytochrome P450 content and phase I and phase II enzyme activities in trout hepatocytes cultured as spheroidal aggregates

We investigated for 1 month the capacity of aggregated trout hepatocytes to metabolize xenobiotics and steroids. As a first approach, the cytochrome P450 content and markers of phase I and II metabolic reactions were examined in subcellular fractions prepared from cultured liver cells. Ethoxyresorufin-O-deethylase (EROD) was chosen as a marker of CYP1A1. The induction of this enzyme was studied using β-naphthoflavone as inducer. For phase II reactions, glutathione-S-transferase (GST) activity was measured using 1-chloro-2,4-dinitrobenzene as substrate. A control of monooxygenase and transferase activities in intact cells was obtained by measuring selective hydroxylation of testosterone, and glucuronidation of 4-nitrophenol and testosterone. All the results were compared with those obtained for freshly isolated hepatocytes or conventional primary culture. The cytochrome P450 content decreased gradually until day 5 and then maintained on the same level (ca 50% of the initial content) for 1 month in aggregate culture. EROD and GST activities were constant or even increasing during 1 month. After 1 month culture, 2 days exposure of cells to β-naphthoflavone led to a 10-fold increase. After 30 days, testosterone hydroxylase activities were about 30% of the activities found on freshly isolated hepatocytes. A similar decrease appeared for testosterone glucuronidation, whereas a stable UDPGT activity was observed during the same period when 4-nitrophenol was used as substrate.

Use of the fish cytochrome P-450-dependent 7-ethylresorufin O-deethylase activity as a biochemical indicator of water pollution. Study of the liver and the kidney of male and female nase (chondrostoma nasus) from the River Rhône

7-Ethylresorufin O-deethylase (EROD) activity and the cytochrome P-450 content of liver and kidney microsomes were measured in male and female nase (Chondrostoma nasus) from the River Rhône (France) caught downstream and upstream of a PCB incineration plant. Concurrently, PCB concentrations in the flesh of nase were also measured. The results showed that sex affected hepatic, but not renal, EROD activity during gonadal maturation. The male nase demonstrated higher activity than the female. Upstream/downstream comparisons clearly revealed a more elevated hepatic EROD activity in fish contaminated by PCB pollution than in fish captured in the reference area throughout the reproductive cycle, demonstrating the reliability of this enzymatic activity as a biochemical indicator. Renal EROD activity did not seem to be sensitive to PCB pollution, since neither male nor female nase showed significant upstream/downstream differences.

Prochloraz-induced Oocyte Maturation in Rainbow Trout (Oncorhynchus mykiss), a Molecular and Functional Analysis

In the present study, we aimed at characterizing the effect of prochloraz, an imidazole fungicide, on the oocyte meiotic maturation process in a freshwater teleost species, the rainbow trout (Oncorhynchus mykiss). Full-grown post-vitellogenic ovarian follicles were incubated in vitro with prochloraz, Luteinizing Hormone (LH), or a combination of prochloraz and LH. The occurrence of oocyte maturation was assessed by monitoring germinal vesicle breakdown (GVBD) after 62-h in vitro incubation. Experiments were repeated in presence of actinomycin D, cycloheximide, or trilostane. The effect of prochloraz on the production of 17,20β-dihydroxy-4-pregnen-3-one (17,20βP), the natural maturation-inducing steroid, was quantified by radioimmunoassay. In addition, the effect of prochloraz on ovarian expression of 12 genes was monitored by real-time PCR. Prochloraz (10(-5)M) administered alone was able to induce 100% GVBD in the most responsive females. The occurrence of GVBD observed after prochloraz stimulation of follicles originating from various females was similar and highly correlated with the occurrence of GVBD observed after stimulation with low LH concentration. In addition, oocyte maturation induced by LH or prochloraz was totally inhibited by actinomycin D, cycloheximide, and trilostane. Similarly to LH, prochloraz was able to trigger 17,20βP production by the ovarian follicle. Finally, prochloraz induced the overexpression of genes participating in 17,20βP production, intercellular communication, and paracrine control of preovulatory follicular differentiation such as igf, igf2, connexin 43, and 20β hydroxysteroid dehydrogenase (hsbd20). Together, our results demonstrate that prochloraz administered alone is able to trigger oocyte maturation through the induction of specific genes, some of them being also triggered by LH. Finally, our results clearly indicate that the effects of prochloraz and LH on oocyte maturation are synergistic.

Xenobiotic metabolizing enzyme activities in aggregate culture of rainbow trout hepatocytes

Abstract Rainbow trout hepatocytes were isolated by a two step perfusion technique and maintained either in monolayer culture for 5 days or in aggregate culture for 30 days. Cytochrome P450 content decreased from day 0 to day 5 in both culture systems, and then was preserved at the same level after one month in aggregated cells. 7-Ethoxyresorufin O-deethylase (EROD) and UDP-glucuronosyl transferase were not significantly different in freshly isolated cells and in 30-day aggregated hepatocytes, whereas a substantial increase in glutathione S-transferase was observed. Two-day exposure of cells to β-naphthoflavone led to a significant increase in EROD activity in both culture systems, especially after one month of aggregation (10-fold increase). According to these results, aggregate culture of rainbow trout hepatocytes seems to be a promising in vitro model to investigate the biotransformation pathways in fish and their regulation by endogenous and exogenous compounds.