Jean-Marc Porcher

Copper-induced oxidative stress in three-spined stickleback: relationship with hepatic metal levels

The aim of this study was to characterise biomarker responses in three-spined sticklebacks exposed to copper. For this purpose, adult sticklebacks were exposed for 3 weeks to copper sulphate at 0, 25, 100 and 200μgL(-1) as Cu. At days 4, 8, 12 and 21, several parameters were measured including liver, gonad and spleen somatic indexes, hepatic biomarkers (catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione (GSH), glutathione-S-transferase (GST) and 7-ethoxyresorufin-O-deethylase (EROD)) and hepatic copper and zinc concentrations. Copper induced a rapid and transient increase of antioxidant enzymes and a depletion of glutathione content during the first 8 days of exposure. Significant copper and zinc accumulation in fish liver were observed for the two higher exposure concentrations after 8 and 12 days, respectively. This study showed that copper induced an oxidative stress in fish liver before significant metal accumulation in the liver could be detected, suggesting the involvement of differential mechanisms in copper uptake and metabolism. Three-spined stickleback appears to be a sensitive model to study oxidative stress induced by metals.

Activation of the hsp70 promoter by environmental inorganic and organic chemicals: relationships with cytotoxicity and lipophilicity

Stress proteins (heat shock proteins, HSPs) have been proposed as general markers of cellular aggression and their use for environmental monitoring is often suggested. The aim of this work was to study the potency of various environmentally relevant organic and inorganic chemicals to induce the expression of the HSP70 marker. For this purpose, we used an established HeLa cell line containing the chloramphenicol acetyl transferase (CAT) gene under the control of the hsp70 promoter. The screening of three metallic and 15 organic chemicals revealed differences in their capacities to induce the hsp70 promoter. The three metals tested (cadmium, zinc and mercury) were able to induce a stress response. Some organochlorine compounds (chlorophenol derivatives, tetrachlorohydroquinone, 3, 4-dichloroaniline, ethyl parathion and 1-chloro-2,4-dinitrobenzene) induced a response, whereas other common halogenated pesticides or aromatic hydrocarbons (e.g. benzo(a)pyrene, 2, 4-dichlorophenoxyacetic acid, endosulfan, diuron, 4-nonylphenol) did not. The potency to induce hsp70 was significantly correlated to the octanol-water partition coefficient (log K(ow)) of the inducing chemicals, except for 1-chloro-2,4-dinitrobenzene and ethyl parathion. Cytotoxicity assays run in parallel to the induction measurements revealed that the three metals were effective at non cytotoxic doses whereas all organic compounds, except tetrachlorohydroquinone and 1-chloro-2,4-dinitrobenzene, induced the promoter at cytotoxic doses. These results suggest that hsp70 is induced by different mechanisms of toxicity. We propose that this model can be used in mechanistic studies for the detection of toxic effects of certain pollutants.

Wild gudgeons (Gobio gobio) from French rivers are contaminated by microplastics: preliminary study and first evidence.

Marine ecosystem contamination by microplastics is extensively documented. However few data is available on the contamination of continental water bodies and associated fauna. The aim of this study was to address the occurrence of microplastics in digestive tract of gudgeons (Gobio gobio) from French rivers. These investigations confirm that continental fish ingested microplastics while 12% of collected fish are contaminated by these small particles. Further works are needed to evaluate the occurence of this contamination.

Adverse effects in wild fish living downstream from pharmaceutical manufacture discharges.

A set of biochemical and histological responses was measured in wild gudgeon collected upstream and downstream of urban and pharmaceutical manufacture effluents. These individual end-points were associated to fish assemblage characterisation. Responses of biotransformation enzymes, neurotoxicity and endocrine disruption biomarkers revealed contamination of investigated stream by a mixture of pollutants. Fish from sampled sites downstream of the industrial effluent exhibited also strong signs of endocrine disruption including vitellogenin induction, intersex and male-biased sex-ratio. These individual effects were associated to a decrease of density and a lack of sensitive fish species. This evidence supports the hypothesis that pharmaceutical compounds discharged in stream are involved in recorded endocrine disruption effects and fish population disturbances and threaten disappearance of resident fish species. Overall, this study gives argument for the utilisation of an effect-based monitoring approach to assess impacts of pharmaceutical manufacture discharges on wild fish populations.

Bioanalytical characterisation of multiple endocrine- and dioxin-like activities in sediments from reference and impacted small rivers

A comprehensive evaluation of organic contamination was performed in sediments sampled in two reference and three impacted small streams where endocrine disruptive (ED) effects in fish have been evidenced. The approach combined quantitative chemical analyses of more than 50 ED chemicals (EDCs) and a battery of in vitro bioassays allowing the quantification of receptor-mediated activities, namely estrogen (ER), androgen (AR), dioxin (AhR) and pregnane X (PXR) receptors. At the most impacted sites, chemical analyses showed the presence of natural estrogens, organochlorine pesticides, parabens, polycyclic aromatic hydrocarbons (16 PAHs), bisphenol A and alkylphenols, while synthetic steroids, myco-estrogens and phyto-estrogens were not detected. Determination of toxic-equivalent amounts showed that 28-96% of estrogenic activities in bioassays (0.2-6.3 ng/g 17beta-estradiol equivalents) were explained by 17beta-estradiol and estrone. PAHs were major contributors (20-60%) to the total dioxin-like activities. Interestingly, high PXR and (anti)AR activities were detected; however, the targeted analysed compounds could not explain the measured biological activities. This study highlighted the presence of multiple organic EDCs in French river sediments subjected to mixed diffuse pollution, and argues for the need to further identify AR and PXR active compounds in the aquatic environment.

Monitoring of dioxin-like, estrogenic and anti-androgenic activities in sediments of the Bizerta lagoon (Tunisia) by means of in vitro cell-based bioassays: Contribution of low concentrations of polynuclear aromatic hydrocarbons (PAHs)

We used an array of in vitro cell-based bioassays to assess dioxin-like, estrogenic and (anti-)androgenic activities in organic extracts of sediments from the Bizerta lagoon, one of the largest Tunisian lagoons subjected to various anthropogenic and industrial pressures. The sediments were sampled both in winter and summer 2006 in 6 stations differently impacted and in one reference station located in the seawards entrance of Ghar el Melh lagoon. Chemical analyses of the 16 priority PAHs showed that the sediments were low to moderately contaminated (2-537 ng/g dry weight). By using the estrogen- (MELN) and androgen-responsive (MDA-kb2) reporter cell lines, significant estrogenic and anti-androgenic activities were detected only in the Menzel Bourguiba (MB) site, the most contaminated site, both in winter and summer. By using 7-ethoxyresorufin-O-deethylase (EROD) induction in the fish PLHC-1 cell line after both 4 and 24 h of cell exposure, dioxin-like activities were detected in all analysed samples. Dioxin-like activities were higher after 4 h exposure, and varied according to the sites and the sampling season. While highly significant correlation was observed between bioassay- and chemical analyses-derived toxic equivalents (TEQs), PAHs accounted for only a small part (up to 4%) of the detected biological activities, suggesting that other readily metabolised EROD-inducing compounds were present. This study argues for the use of short time exposure to assess biological TEQs in low contaminated samples and provides new induction equivalent factors (IEF(4h)) for 16 PAHs in the PLHC-1 cell line. Finally, our results stress the need to further characterise the nature of organic chemical contamination as well as its long-term impacts on aquatic wildlife in the Bizerta lagoon.

17alpha-ethinylestradiol disrupts the ontogeny of the forebrain GnRH system and the expression of brain aromatase during early development of zebrafish.

Until now, studies dedicated to the actions of endocrine disrupting chemicals (EDCs) on the reproductive axis have been concerned with their effects at the gonadal level leaving their actions on neuroendocrine circuits controlling reproduction virtually unexplored. In vertebrates, gonadotropin-releasing hormone (GnRH) is the key factor controlling the activity of the reproductive axis. The development and functioning of GnRH neurons are finely tuned by a series of factors, notably sex steroids, making these neurons potential targets of EDCs, notably in aquatic species. By means of immunohistochemistry, we examined the effects of low levels of ethinylestradiol (EE2 0.02 nM, 0.1 nM, 0.5 nM), a potent synthetic estrogen, on early development (at 5, 10, 20, 30 days post-fertilization) of the forebrain GnRH neurons in a model fish species, the zebrafish (Danio rerio). In parallel, the ER-regulated expression of cytochrome P450 aromatase B (AroB) protein, which is encoded by the cyp19a1b gene, was precisely mapped at the brain and pituitary levels in developing control and EE2-exposed zebrafish. We show that EE2 disrupts the ontogeny of GnRH system by inducing an increase in the number of GnRH-ir neurons and GnRH fibers based on their immunoreactivity as well as a decrease in the size of the GnRH-ir soma and a modification of the migration profile of GnRH-ir neurons. Furthermore, we report a spectacular dose and time-dependent induction of AroB expression in radial glial cells of the developing brain further illustrating the extreme sensitivity of AroB to xenoestrogen and the relevance of AroB as biomarker of xenoestrogen effects on the central nervous system. Collectively, these original and relevant observations highlight the sensitivity of GnRH and AroB to a synthetic estrogen during embryogenesis. These data reinforce the need to further study the mechanisms underlying EDC effects on key neuroendocrine circuits involved in reproduction and brain development of vertebrates.

Identification of synthetic steroids in river water downstream from pharmaceutical manufacture discharges based on a bioanalytical approach and passive sampling.

A bioanalytical approach was used to identify chemical contaminants at river sites located downstream from a pharmaceutical factory, where reproductive alterations in wild fish have been previously observed. By using polar organic compound integrative samplers (POCIS) at upstream and downstream sites, biological activity profiles based on in vitro bioassays revealed the occurrence of xenobiotic and steroid-like activities, including very high glucocorticoid, antimineralocorticoid, progestogenic and pregnane X receptor (PXR)-like activities (μg standard-EQ/g of sorbent range), and weak estrogenic activity (ng E2-EQ/g of sorbent range). Chemical analyses detected up to 60 out of 118 targeted steroid and pharmaceutical compounds in the extracts. In vitro profiling of occurring individual chemicals revealed the ability of several ones to act as agonist and/or antagonist of different steroids receptors. Mass balance calculation identified dexamethasone, spironolactone, and 6-alpha-methylprednisolone as major contributors to corticosteroid activities and levonorgestrel as the main contributor to progestogenic activities. Finally, RP-HPLC based fractionation of POCIS extracts and testing activity of fractions confirmed identified compounds and further revealed the presence of other unknown active chemicals. This study is one of the first to report environmental contamination by such chemicals; their possible contribution to in situ effects on fish at the same site is suggested.

Assessment of seasonal variability of biomarkers in three-spined stickleback (Gasterosteus aculeatus L.) from a low contaminated stream: Implication for environmental biomonitoring

In this study, wild three-spined sticklebacks were sampled every six weeks, between April and October, in a low contaminated stream. For all fish, physiological indexes, such as condition factor, hepato-, gonado- and nephro-somatic index were calculated to determine fish condition and reproductive status. Moreover, a set of biomarkers including biotransformation enzymes, oxidative stress parameters, neurotoxicity and endocrine disruption markers was measured. The results allowed to determine biomarker variability due to fish gender or sampling season. For example, 7-ethoxyresorufin-O-deethylase activity, glutathione peroxidase as well as vitellogenin and spiggin exhibited strong gender differences. Conversely, lipoperoxidation and acethylcholinesterase activity were characterised by a lack of gender and seasonal variation, and can be considered as more robust parameters for a field application. The present work allowed to establish practical guideline for biomarker measurements in wild sticklebacks and to define a reference system which can be used to analyze variations in future monitoring studies.

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.