Ecotoxicoproteomic assessment of the functional alterations caused by chronic metallic exposures in gammarids.

Very few ecotoxicological studies have been performed on long-term exposure under controlled conditions, hence limiting the assessment of the impact of chronic and diffuse chemical pressures on the health of aquatic organisms. In this study, an ecotoxicoproteomic approach was used to assess the integrated response and possible acclimation mechanisms in Gammarus fossarum following chronic exposures to Cd, Cu or Pb, at environmentally realistic concentrations (i.e. 0.25, 1.5 and 5 µg/L respectively). After 10-week exposure, changes in protein expression were investigated in caeca of control and exposed males. Gel-free proteomic analyses allowed for the identification of 35 proteins involved in various biological functions, for which 23 were significantly deregulated by metal exposures. The protein deregulation profiles were specific to each metal, providing evidence for metal-specific action sites and responses of gammarids. Among the tested metals, Cu was the most toxic in terms of mortality, probably linked with persistent oxidative stress. Moulting and osmoregulation were the major biological functions affected by Cu in the long-term. In Pb-exposed gammarids, significant deregulations of proteins involved in immune response and cytoskeleton were observed. Reproduction appears to be strongly affected in gammarids chronically exposed to Cd or Pb. Besides, modified expressions of several proteins involved in energy transfer and metabolism highlighted important energetic reshuffling to cope with chronic metal exposures. These results support the fact that metallic pressures induce a functional and energetic cost for individuals of G. fossarum with potential repercussions on population dynamics. Furthermore, this ecotoxicoproteomic study offers promising lines of enquiry in the development of new biomarkers that could make evidence of long-term impacts of metals on the health of organisms.

Early exposure to Aroclor 1254 in vivo disrupts the functional synaptic development of newborn hippocampal granule cells.

Neurogenesis in the dentate gyrus is sensitive to endogenous and exogenous factors that influence hippocampal function. Ongoing neurogenesis and the integration of these new neurons throughout life thus may provide a sensitive indicator of environmental stress. We examined the effects of Aroclor 1254 (A1254), a mixture of polychlorinated biphenyls (PCBs), on the development and function of newly generated dentate granule cells. Early exposure to A1254 has been associated with learning impairment in children, suggesting potential impact on the development of hippocampus and/or cortical circuits. Oral A1254 (from the 6th day of gestation to postnatal day 21) produced the expected increase in PCB levels in brain at postnatal day 21, which persisted at lower levels into adulthood. A1254 did not affect the proliferation or survival of newborn neurons in immature animals nor did it cause overt changes in neuronal morphology. However, A1254 occluded the normal developmental increase in sEPSC frequency in the third post-mitotic week without altering the average sEPSC amplitude. Our results suggest that early exposure to PCBs can disrupt excitatory synaptic function during a period of active synaptogenesis, and thus could contribute to the cognitive effects noted in children exposed to PCBs.

Corrigendum to "Transcriptome of the freshwater amphipod Gammarus pulex hepatopancreas" [Genomics Data 8 (2016) 91-92].

[This corrects the article DOI: 10.1016/j.gdata.2016.04.002.].

Accumulation of neurotoxic organochlorines and trace elements in brain of female European eel (Anguilla anguilla).

Xenobiotics such as organochlorine compounds (OCs) and metals have been suggested to play a significant role in the collapse of European eel stocks in the last decades. Several of these pollutants could affect functioning of the nervous system. Still, no information is so far available on levels of potentially neurotoxic pollutants in eel brain. In present study, carried out on female eels caught in Belgian rivers and canals, we analyzed brain levels of potentially-neurotoxic trace elements (Ag, Al, As, Cd, Co, Cr, Cu, Fe, Hg, MeHg, Mn, Ni, Pb, Sn, Sb, Zn) and OCs (Polychlorinated biphenyls, PCBs; Hexachlorocyclohexanes, HCHs; Dichlorodiphenyltrichloroethane and its metabolites, DDTs). Data were compared to levels in liver and muscle tissues. Eel brain contained very high amounts of OCs, superior to those found in the two other tissues. Interestingly, the relative abundance of PCB congeners markedly differed between tissues. In brain, a predominance of low chlorinated PCBs was noted, whereas highly chlorinated congeners prevailed in muscle and liver. HCHs were particularly abundant in brain, which contains the highest amounts of ß-HCH and ϒ-HCH. p,p'-DDTs concentration was similar between brain and muscle (i.e., about twice that of liver). A higher proportion of p,p'-DDT was noticed in brain. Except for Cr and inorganic Hg, all potentially neurotoxic metals accumulated in brain to levels equal to or lower than hepatic levels. Altogether, results indicate that eel brain is an important target for organic and, to a lesser extent, for inorganic neurotoxic pollutants.

Transcriptome of the freshwater amphipod Gammarus pulex hepatopancreas.

So far, ecotoxicological studies used biomarkers of exposure or of effects in order to investigate the impacts of contaminated areas on biota (Peakall, 1994 [6]). However, although these results are important in the ecotoxicological risk assessment, biomarkers are very specific and only provide information on the biological processes or physiological pathways targeted by the biomarkers experimenters choose to test (Monsinjon and Knigge, 2007 [5]). In recent years, proteomics have become a major tool in ecotoxicology, as they provide a global insight into the mechanism of action of pollutants without the need of hypothesis testing or any preconception on the biological processes likely impacted (Gismondi et al., 2015; Trapp et al., 2015 [7]; Truebano, 2016 [8]). However, the analysis of proteomic results is often limited due to the lack of database, especially for non-model organisms, such as Gammarus sp, commonly used as biological model in ecotoxicology (Sornom et al., 2012 [11]; Vellinger et al., 2013 [9]; Gismondi and Thomé, 2014 [1]; Lebrun et al., 2014 [3]). Here, we performed Illumina HiSeq sequencing to total RNA isolated from the hepatopancreas (i.e. detoxification tissue) of Gammarus pulex males and females coming from uncontaminated river and contaminated river (e.g. PCB, benzo(a)pyrene). Approximately 290 M paired-end reads were assembled, filtered and sorted into 39,801 contigs whose 10.878 were similar of proteins available in databases. The assembled contigs could represent a reference hepatopancreas transcriptome for G. pulex, and constitute an important resource for future investigations on the impacts of pollutants on invertebrate biota, since it would improve the understanding of the mechanisms of action involved in toxicity. In addition, the hepatopancreas transcriptome will also allow the identification of new potential biomarkers for the ecotoxicological risk assessments. Assembled contigs were deposited in the European Nucleotide Archive under the BioProject number PRJEB13055, with accession numbers FJVI01000001-FJVI01039801.

Impact of three phthalate esters on the sexual reproduction of the Monogonont rotifer, Brachionus calyciflorus.

Phthalate esters are widespread contaminants that can cause endocrine disruption in vertebrates. Studies showed that molecules with hormonal activities in vertebrates and invertebrates can affect asexual and sexual reproduction in rotifers. We investigated the impact of di-hexylethyl phthalate (DEHP), di-butyl phthalate (DBP) and butylbenzyl phthalate (BBP), on the asexual and sexual reproduction of the freshwater monogonont rotifer Brachionus calyciflorus in order to determine a potential environmental risk for sexual reproduction. We observed that DEHP has no significant impact on both asexual and sexual reproduction up to 2 mg/L. DBP has a positive effect on asexual reproduction at concentrations from 0.05 to 1 mg/L, but depresses it at 2 mg/L. Sexual reproduction is only affected at 2 mg/L and the impact observed is negative. BBP displayed a negative impact on both asexual and sexual reproduction at 1 and 2 mg/L. However we showed that the impacts of BBP on mixis and fertilization rates observed are due to the decrease in population growth rates at these concentrations and not to a direct impact of BBP on the mixis and the fertilization processes. Our results show that sexual reproduction in B. calyciflorus is not more sensitive than asexual reproduction to any of the substances tested which indicates the mode of action of these molecules is related to general toxicity and not to an interference with potential endocrine regulation of sexual reproduction. Comparison of effect concentrations and surface water contamination by phthalate esters suggests these compounds do not constitute a risk for primary consumers in these environments.

Gender differences in responses in Gammarus pulex exposed to BDE-47: A gel-free proteomic approach.

Very few ecotoxicological studies have considered differences in toxic effects on male and female organisms. Here, we investigated protein expression differences in caeca of Gammarus pulex males and females under control conditions (unexposed) and after 96h exposure to BDE-47. Using gel-free proteomic analysis, we have identified 45 proteins, of which 25 were significantly differently expressed according to sex and/or BDE-47 exposure. These proteins were involved in several biological processes such as energy metabolism, chaperone proteins, or transcription/translation. In unexposed amphipods, 11 proteins were significantly over-expressed in females, and 6 proteins were over-expressed in males. Under BDE-47 stress, 7 proteins were differently impacted according to sex. For example, catalase was over-expressed in exposed females and under-expressed in exposed males, as compared to respective controls. Conversely, proteins involved in energy metabolism were up-regulated in males and down-regulated in females. Our proteomic study showed differences in responses of males and females to BDE-47 exposure, emphasizing that sex is a confounding factor in ecotoxicological assessment. However, due to the limited information existing in databases on Gammarids, it was difficult to define a BDE-47 mechanism of action. The gel-free proteomic seems to be a promising method to develop in future ecotoxicological studies and thus, to improve our understanding of the mechanism of action of xenobiotics.

Development of ecotoxicoproteomics on the freshwater amphipod Gammarus pulex: identification of PCB biomarkers in glycolysis and glutamate pathways.

PCBs are persistent organic pollutants largely distributed in the biosphere. Although their effects on vertebrates are well described, little is known about their action on freshwater invertebrate's metabolism. Gammarus pulex (Linné) was selected as an indicator model to develop a proteomic approach in order to characterize the effects of PCBs on the protein profile of this freshwater crustacean. Sublethal coplanar PCBs exposition and related 2D gel were performed. More than 560 spots were detected and a total of 21 proteins exhibiting significant expression differences in PCB exposed to G. pulex were identified by mass spectrometry. Database searches were conducted to relate the results to well-known metabolic pathways (pentose phosphate, cytoskeleton, energy, etc.). In particular, glyceraldehyde 3-phosphate dehydrogenase and arginine kinase were found to be sensitive to the PCB exposition of G. pulex. The aim of the present study was to assess the biochemical responses and the metabolic changes in G. pulex following intoxication to coplanar PCB congeners CB77 and CB169 by a proteomic approach. This approach allowed us, by the identification of key proteins, to highlight important biochemical mechanisms disturbed by the presence of these contaminants in G. pulex.

An unexpected parallelism between Vitamin A and PCBs in seal milk.

Bioaccumulating pollutants such as polychlorinated biphenyls (PCBs) induce a range of adverse effects in mammals. Vitamin A metabolism is prone to such pollutant disruption which may be particularly harmful for young animals. During lactation, maternal PCBs are transferred to the offspring through the milk. Seal milk is very fatty and consequently contains large amounts of these persistent lipophilic contaminants. In the present study, we investigated the relationships between PCBs and Vitamins A and E during lactation, in free-ranging grey seals, using longitudinal samples of milk and blubber. We discovered that, unlike Vitamin E or triglycerides, the dynamics of Vitamin A matches closely those of PCBs in milk throughout lactation. Levels of Vitamin A and PCBs remain constant during the first half of lactation and then increase at late lactation, indicating that pups ingesting higher levels of PCBs also ingest higher amounts of Vitamin A.

Effects of polychlorinated biphenyls on liver ultrastructure, hepatic monooxygenases, and reproductive success in the barbel.

Polychlorinated biphenyls (PCBs) are organochlorinated micropollutants ubiquitously distributed in the environment. They are known to be strong inducers of hepatic monooxygenases in fish. This can adversely affect reproduction by increasing steroid metabolism. In this work, adult barbels were contaminated with food containing Aroclor 1260, a commercial PCB mixture from Monsanto, at environmentally relevant concentrations. A significant increase in cytochrome P450 was observed, and two particularly sensitive enzymes, ethoxyresorufin o-deethylase (EROD) and ethoxycoumarin o-deethylase (ECOD), were strongly induced. Electron microscopy revealed alterations in liver ultrastructure in contaminated fish, principally an increase in the number of cisternae of the rough endoplasmic reticulum, drastic glycogen depletion, dissolution of mitochondrial contents, and appearance of myelin figures. Contamination was also studied in relation to reproductive success in a hatchery. Contaminated males displayed no alteration in milt quality, but PCBs did alter female reproductive parameters. Total mortality of eggs and larvae increased significantly with the level of PCBs in the eggs. The most highly contaminated fish did not even spawn. All the adverse effects recorded here tended to be reversible when the intoxication ended, sometimes after only a 1-year detoxication period.

Hierarchical cluster analysis of environmental pollutants through P450 induction in cultured hepatic cells.

Environmental pollutants are classically associated with increased drug metabolism. Cultures of rat hepatocytes, quail hepatocytes, and human hepatoma (Hep G2) cells were used to study the effects of pesticides on drug-metabolizing enzymes. Membrane integrity and mitochondrial activity were evaluated and induction of ethoxycoumarin-O-deethylase and ethoxyresorufin-O-deethylase activities were measured. Induced P450s were identified by immunoblotting. Pentachlorophenol and lindane appeared as the strongest inducers. On the immunoblots, specific antibodies revealed induced CYP1A1 in fetal rat hepatocytes, CYP2B in quail hepatocytes, and CYP3A7 in Hep G2 cells. Pesticide effects on these different activities in each type of cultured cells were compared by cluster analysis. Results obtained under similar conditions with reference inducers phenobarbital (PB) and benzo[a]anthracene and other environmental pollutants (polychlorobiphenyls) were added to previous data prior to multivariate analysis. The tested products fell into four major groups: a first group with pentachlorophenol, identified as a CYP3A inducer; a second group containing the methylcholanthrene-type inducers that increase CYP1A-related activities; a third class represented by dieldrin, a PB-type inducer; a fourth group including inert compounds or weak inducers. Lindane shares the criteria of the second and third groups and seems to induce both CYP1A and CYP2B activities. The current study results highlight the advantage of using several types of cultured hepatocytes to evaluate the short-term toxicity of environmental pollutants in vitro and constitute a useful model for predicting the potential toxicity of pesticides in humans (Hep G2 cells) and wildlife (fetal quail hepatocytes).

Ultrastructural modifications in cultured fetal quail hepatocytes exposed to pesticides and PCBs.

There is increasing interest in cultured hepatocytes as a tool for solving toxicological and pharmacological problems while reducing laboratory animal experimentation. In the present study, fetal hepatocytes from the Japanese quail (Coturnix coturnix japonica) were used as an in vitro alternative model for evaluating the effects of PCBs and various pesticide-type chemicals on cell ultrastructure. Major alterations were demonstrated. The most striking effects of toxicants were an increase in the number of cisternae of the rough endoplasmic reticulum (RER), various alterations of mitochondrial morphology, a decreased glycogen content, vacuolization of the cytoplasm, and the appearance of concentric membrane arrays (CMA's), also called myelin-like figures. Other changes were sometimes observed, such as altered cell junctions, an increased lipid content, deformations of the nuclei, or the appearance of crystalline structures. These ultrastructural modifications seem to be dose-dependent. The present in vitro findings are validated by similar observations previously made in vivo on Japanese quail. They confirm the effectiveness of this technique as a biomonitoring tool for the evaluation of environmental quality. Yet the multiplicity of possible toxic effects, even for xenobiotics of a same category, makes it necessary to screen additional indicators of toxicity, such as the detoxifying activity of monooxygenases.

P450 induction by Aroclor 1254 and 3,3',4,4'-tetrachlorobiphenyl in cultured hepatocytes from rat, quail and man: interspecies comparison.

Polychlorobiphenyls are potent inducers of hepatic cytochrome P450 in various species. Until now, no model based on cultured cells can be considered as a universal surrogate for in vivo metabolism. In this respect, cultured rat hepatocytes, quail hepatocytes, and human hepatoma (HepG2) cells were used to study the effects of 3,3',4,4'-tetrachlorobiphenyl (3,3',4,4'-TCB) and Aroclor 1254 on drug-metabolizing enzymes. The presence of dexamethasone in the culture medium allows the expression and the induction of several cytochrome P450 isoenzymes found in adult cells. Induction of ethoxycoumarin-(ECOD) and ethoxyresorufin-O-deethylase (EROD), activities were measured. Induced P450s were identified by immunoblotting and Northern blotting. Aroclor 1254 induced ECOD activity in all three cell types, but the effect was much stronger in fetal rat hepatocytes than in human or quail cells. Aroclor failed to induce EROD activity in quail cells, had a slight inducer effect in HepG2 cells, and a marked effect in rat hepatocytes. 3,3',4,4'-TCB had no effect in HepG2 cells but significantly increased EROD and ECOD activities, especially the latter, in rat and quail cells. On the immunoblots, specific antibodies revealed essentially CYP1A1 in fetal rat hepatocytes, CYP2B1/2 in quail hepatocytes and CYP3A1 in HepG2 cells. Analysis of Northern blots showed an hybridization with CYP1A1, 2B1 and 3A1 mRNA in fetal rat hepatocytes, CYP3A and 1A mRNA in HepG2 cells, and a form of CYP2 mRNA in fetal quail hepatocytes closely related to homolog rat CYP2E or CYP2C. In quail hepatocytes, induction did not increase proportionally with the concentration of inducer in the culture medium. Instead, the dose-response curves (for EROD activity especially) peaked sharply at 1 muM Aroclor 1254, an effect attributed to changes in membrane fluidity or lipid content. Our results highlight the advantage of using several types of cultured hepatocytes to investigate fundamental aspects of drug-metabolism-linked toxicity, the balance between xenobiotic bioactivation and detoxication being differently affected by PCBs in different animal species.

Effect of inducers and PCBs on the cytochrome P450 enzymes in cultured quail hepatocytes.

Hepatocytes isolated from fetal quail livers (Coturnix coturnix japonica) were cultured in vitro. Their capacity to metabolize drugs and xenobiotics was explored with typical cytochrome P450 substrates: ethoxycoumarin (known to be metabolized by several P450s), ethoxyresorufin (essentially dealkylated by P450IA1), and testosterone (specifically hydroxylated at several positions by several P450s). The cells could be kept metabolically active in culture for at least 4 days. Their drug-metabolizing activities were inducible by the usual P450 inducers, like phenobarbital and benzanthracene, but also by Aroclor 1254, a PCB mixture. The results obtained indicate that this experimental model could certainly be very helpful in ecotoxicological studies.

Cytotoxic effects of Aroclor 1254 on ultrastructure and biochemical parameters in cultured foetal rat hepatocytes.

The cytotoxicity of a commercial PCB mixture, Aroclor 1254, was assessed on cultured foetal rat hepatocytes. Under control conditions, dexamethasone stimulates immature hepatocytes to differentiate into both hepatocytes and biliary epithelial cells. Consequently, foetal rat hepatocytes maintain, in vitro, a liver-like organization with spaces corresponding to the lumen of biliary canalicules, many mitochondria, and a well-developed rough endoplasmic reticulum (RER). This in vivo-like organization of cultured rat hepatocytes remains unchanged in medium supplemented with Aroclor 1254 at concentrations below 25 microM. In the 25-125 microM concentration range, however, PCBs severely alter some cellular organelles, notably causing important development of the RER and the appearance of cytoplasmic lacunae containing laminated concentric membrane arrays. In addition, the number of lipid droplets increases, the glycogen islets disappear, and dramatic local alterations of the mitochondrial cristae occur. In exposed and unexposed cells, the following biochemical parameters were measured: the DNA content, protein synthesis, lipid peroxidation, and urea formation. The results show that Aroclor 1254 at concentrations exceeding 25 microM (but not at lower concentrations) causes irreversible damage to cultured hepatocytes. The observed ultrastructural modifications are in good agreement with several in vivo studies on rat liver. Thus, isolated foetal rat hepatocytes have considerable potential as an alternative to whole animals for use in (eco)toxicological studies.

Effects of PCBs (Aroclor 1254) on cytochrome-P450 expression and monooxygenase activities in cultured foetal rat hepatocytes.

Polychlorinated biphenyls (PCBs) are widespread residual micropollutants which accumulate in living organisms, probably as a consequence of their high lipophilicity. Cultured foetal rat hepatocytes used as target cells constitute an interesting in vitro model for studying the mechanisms of action of PCBs. In this paper, and the accompanying one (Toxicology 98 (1995) 83-94), we have used this model to investigate the effects of PCBs on several cellular parameters. The inducibility of CYPIA1 is the most sensitive parameter studied, as shown by the induction of ethoxycoumarin-O-deethylase and ethoxyresorufin-O-deethylase activities at PCB concentrations as low as 1 microM. Dexamethasone treatment of the cells potentiates this induction. PCB induction is reversible and occurs even in cells cultured for several days. CYP2B and CYP3A seem unaffected by PCBs in this experimental system. By inducing CYP1A1, PCBs can trigger the 'activation' of xenobiotics, such as polycyclic hydrocarbons, into mutagenic compounds.

Expression and induction of drug-metabolizing enzymes in cultured fetal rat hepatocytes.

An in vitro experimental model, fetal rat hepatocytes in culture, was metabolically characterized. Several enzymatic activities were expressed in these hepatocytes, namely, testosterone hydroxylations. Hepatocytes cultured up to 3 weeks in the presence of dexamethasone and phenobarbital still expressed some drug-metabolizing enzyme activities (e.g., ECOD). The enzymatic activities were measured both directly on monolayers during culture and on the corresponding harvested and homogenized cells. The results correlate perfectly with each other. The 'on cell' procedure allows us to repeat the assay or to measure several activities on the same cells at different time intervals. The presence of dexamethasone in the culture medium allows the expression and the induction of several cytochrome P450 isoenzymes, namely, those hydroxylating testosterone. This makes the model particularly attractive for induction experiments as well as for metabolic or toxicological studies needing longer treatments.

Use of cultured hepatocytes as an alternative method to study the effects of PCBs on living organisms.

In order to study the mechanism of action of polychlorinated biphenyls (PCBs) several parameters have been monitored in cultured foetal rat and quail hepatocytes. At low concentrations, the PCB mixture tested (Aroclor 1254) did not affect the biological and morphological parameters studied. Above 170 mug/ml, Aroclor induced cytotoxic effects and morphological damage, similar to those that have been observed in vivo, in both animal species (i.e. modification of the endoplasmic reticulum structure, appearance of cytoplasmic vacuoles, alteration of the mitochondrial cisternae). Concentrations as low as 10 mum (3 ppm) induced cytochrome P450IA1-dependent activities (ethoxyresorufin-O-deethylase, ethoxycoumarin-O-deethylase) in rat hepatocytes. In quail hepatocytes, a very significant induction was observed at concentrations as low as 1 mum. This induction was dependent on both dose and duration of exposure. Testosterone metabolism was not affected by the PCB treatment. These in vitro models are helpful alternatives to in vivo systems for the study of the mechanism of action of PCBs on hepatocytes.