Role of iron in gene expression and in the modulation of copper uptake in a freshwater alga: Insights on Cu and Fe assimilation pathways.

Metal uptake and toxicity can generally be related to its aqueous speciation and to the presence of competitive ions as described by the biotic ligand model. Beyond these simple chemical interactions at the surface of aquatic organisms, several internal biological feedback mechanisms can also modulate metal uptake. This is particularly important for essential elements for which specific transport systems were developed over the course of evolution. Based on the results of short-term Cu2+ uptake experiments and on the analysis of the expression of certain genes involved in Cu and Fe homeostasis, we studied the effects of Fe3+ on Cu2+ uptake by the freshwater green alga Chlamydomonas reinhardtii. We observed a significant increase in Cu2+ uptake rate in algal cells acclimated to a low Fe3+ medium up to 4.7 times greater compared to non-acclimated algal cells. The overexpression of the ferroxidase FOX1 and permease FTR1 genes suggests an activation of the high affinity Fe3+ assimilation system, which could constitute a plausible explanation for the increase in Cu2+ uptake rate in acclimatized algae. We show that Fe availability can have a significant impact on Cu uptake. Our observations reinforce the importance of considering physiological factors to better predict metal bioavailability.

Environmentally Relevant Mixture of Pesticides Affect Mobility and DNA Integrity of Early Life Stages of Rainbow Trout (Oncorhynchus mykiss).

The aim of this study was to analyze the impact of three concentrations of a pesticide mixture on the first development stages of rainbow trout (Oncorhynchus mykiss). The mixture was made up of three commonly used pesticides in viticulture: glyphosate (GLY), chlorpyrifos (CPF) and copper sulfate (Cu). Eyed stage embryos were exposed for 3 weeks to three concentrations of the pesticide mixture. Lethal and sub-lethal effects were assessed through a number of phenotypic and molecular endpoints including survival, hatching delay, hatching success, biometry, swimming activity, DNA damage (Comet assay), lipid peroxidation (TBARS), protein carbonyl content and gene expression. Ten target genes involved in antioxidant defenses, DNA repair, mitochondrial metabolism and apoptosis were analyzed using real-time RT-qPCR. No significant increase of mortality, half-hatch, growth defects, TBARS and protein carbonyl contents were observed whatever the pesticide mixture concentration. In contrast, DNA damage and swimming activity were significantly more elevated at the highest pesticide mixture concentration. Gene transcription was up-regulated for genes involved in detoxification (gst and mt1), DNA repair (ogg1), mitochondrial metabolism (cox1 and 12S), and cholinergic system (ache). This study highlighted the induction of adaptive molecular and behavioral responses of rainbow trout larvae when exposed to environmentally realistic concentrations of a mixture of pesticides.

Subchronic Exposure to Environmental Concentrations of Chlorpyrifos Affects Swimming Activity of Rainbow Trout Larvae.

Chlorpyrifos (CPF), an organophosphorous pesticide, can be found in aquatic ecosystems at concentrations of up to several hundred nanograms per liter because of water runoff from treated crops. While some studies have shown that low concentrations of CPF may have adverse effects on aquatic species, comparatively little is known about its effect on fish embryos and larvae. To investigate the developmental effects of CPF, rainbow trout (Oncorhynchus mykiss) eyed-stage embryos were exposed in semistatic conditions to 0.3 and 3 µg/L of CPF up to the end of the sac-fry stage, 3 weeks, at 12 °C. Several endpoints were analyzed including survival, hatching delay, hatching success, biometry, swimming activity, DNA damage, lipid peroxidation, protein carbonyl content, acetylcholinesterase (AChE) activity, and gene expression. At the end of the 3-week exposure, larvae exposed to the highest concentration of CPF were less mobile compared to the control and the lowest CPF conditions. No significant differences in AChE activity were observed in either set of CPF conditions compared to control, but it was significantly reduced for larvae exposed to 3 µg/L compared to those exposed to 0.3 µg/L of CPF. Expression of genes that encoded estrogen receptor beta was downregulated for larvae exposed to both CPF concentrations. Expression of cytochrome P450 family 19 subfamily A member 1 was also significantly repressed but only on larvae exposed to the highest concentration of CPF. Our results indicated that subchronic exposure to environmental concentrations of CPF could lead to sublethal effects on early-life stages of rainbow trout, especially effects on swimming activity that could affect foraging activity and escaping from predators. Environ Toxicol Chem 2021;40:3092-3102. © 2021 SETAC.

Sensitivity to cadmium of the endangered freshwater pearl mussel Margaritifera margaritifera from the Dronne River (France): experimental exposure.

Margaritifera margaritifera is a critically endangered species in Europe. Among the causes explaining its decline, metal pollution had never been deeply studied. Thus, an ecotoxicological investigation was developed on this species which comes from the Dronne River (South-West of France). Cadmium (Cd) exposure of mussels at 2 and 5 µg/L for 7 days was conducted to test their vulnerability to this metal, and also the potential endocrine disruption power of Cd. Morphometric analyses, gonad histological observations, metal bioaccumulation, metallothionein (MTs) production, measures of malondialdehyde (MDA), and finally quantitative relative expression analysis of genes involved in various metabolic functions were performed.The main results showed Cd accumulation increasing in a dose-dependent manner, especially in the gills. The same trend was observed for gene expression relative to oxidative stress. Histological analysis of the gonads highlighted a predominance of hermaphrodite individuals, but after 7 days of exposure to Cd, the percentage of female was largely increased compared with controls, from 17 to 33%. These results demonstrate the endocrine disruption effect of Cd on freshwater pearl mussels.The pearl mussel Margaritifera margaritifera is sensitive to cadmium since the metallothioneins are poorly induced, gene expression reveals oxidative stress, and gonads tend to be feminized.

Sub-lethal effects of waterborne copper in early developmental stages of rainbow trout (Oncorhynchus mykiss).

The aim of this work was to study the impact of copper during a sub-chronic exposure to environmental concentrations in the early life stages of rainbow trout (Oncorhynchus mykiss). Eyed-stage embryos of rainbow trout, at 265 °D, were exposed in semi-static conditions to sub-lethal concentrations of CuSO4 up to the larval stage (528 °D) under laboratory-controlled conditions. During 3 weeks, they were exposed to the environmentally-realistic concentration of 2 µg/L Cu and to a 10-fold higher concentration, 20 µg/L Cu. Several biological (survival, hatching success, malformation, growth) and behavioral (swimming activity) and molecular endpoints (genotoxicity and gene transcription) were studied. Exposure to 20 µg/L Cu had an inhibitory effect on hatching and increased half-hatched embryos (25%). At the end of the exposure, no significant differences were observed in growth of the larvae exposed to the highest Cu concentration. However, larvae exposed to 2 µg/L Cu exhibited increased growth in comparison with non-exposed larvae. The percentage of malformed larvae was significantly higher for both copper conditions, with skeletal malformations being the most observed. Expression of several genes was evaluated in whole larvae using quantitative real-time PCR. Genes involved in detoxification (gst, mt1 and mt2) and in cell cycle arrest (p53) were significantly repressed in both copper conditions when compared to control. In addition, potential genotoxic effects on larvae were investigated by the comet assay on blood cells, but this test did not demonstrate any significant DNA damage on larvae exposed to copper. This study confirms the adverse effects of copper on early life stages of rainbow trout even at the lowest environmentally relevant tested concentration.

Can pesticides, copper and seasonal water temperature explain the seagrass Zostera noltei decline in the Arcachon bay?

Dwarf eelgrasses (Zostera noltei) populations have decreased since 2005 in Arcachon Bay (southwest France). Various stressors have been pointed out, however the role of xenobiotics like pesticides or copper (Cu) and of parameters like water temperature warming have not yet been explored. To determine their impact, Z. noltei individuals were collected in a pollution-free site and transferred to the laboratory in seawater microcosms. This dwarf eelgrass was exposed to a pesticide cocktail and copper, alone or simultaneously, at temperatures (10°C, 20°C, 28°C) representative of different seasons. After a two-week contamination, leaf growth, leaf bioaccumulation of Cu, and differential expression of target genes were studied. Eelgrasses bioaccumulated Cu regardless of the temperature, with reduced efficiency in the presence of the Cu and pesticide cocktail at the two higher temperatures. High temperature also exacerbated the effect of contaminants, leading to growth inhibition and differential gene expression. Mitochondrial activity was strongly impacted and higher mortality rates occurred. Experimental results have been confirmed during field survey. This is the first report on the impacts on Z. noltei of pesticides and Cu associate to temperature.

Molecular and phenotypic responses of Japanese medaka (Oryzias latipes) early life stages to environmental concentrations of cadmium in sediment.

Japanese medaka embryos were exposed to environmental concentrations of cadmium (Cd) to investigate adverse and adaptive responses in fish early life stages. Embryos were exposed during their whole development by static sediment-contact to environmental Cd concentrations (2 and 20 µg/g dry weight). Cd bioaccumulation, developmental defects, biochemical and biomolecular (qRT-PCR) responses were analyzed in embryos and hatchlings. A dose-dependent increase of Cd bioaccumulation and developmental defects was observed at hatching. Cd had clear impacts on heartbeat and cardiac morphogenesis and also induced to spinal deformities. The profile and the level of gene transcription were differentially modulated according to the Cd concentration, the duration of exposure and/or the developmental stage of fish. Pro-apoptotic bax and DNA repair rad51 transcripts were significantly repressed in embryos exposed to the highest Cd concentration. Repression of these genes was correlated to the increase of heart rate in 6-day-old embryos. NADH-dehydrogenase nd5 gene transcription was inhibited in larvae at the lowest concentration suggesting mitochondrial respiratory chain impairment, in association with Cd-induced teratogenicity. Finally, wnt1 gene was overexpressed indicating putative deregulation of Wnt signaling pathway, and suggested to be implied in the occurrence of some spinal and cardiac deformities. Results of this study permitted to propose some promising markers at the transcriptional and phenotypical level, responding to environmental concentrations of Cd. The present work also highlights the usefulness of the modified version of the medaka embryo-larval assay with sediment-contact exposure (MELAc) to investigate the toxicity and the modes of action of sediment-bound pollutants.

How toxic is the depleted uranium to crayfish Procambarus clarkii compared with cadmium?

Due to a lack of information on the assessment of uranium's (U) toxicity, our work aimed to compare the effects of U on the crayfish Procambarus clarkii with those of the well documented metal: cadmium (Cd). Accumulation and impacts at different levels of biological organization were assessed after acute (40 µM Cd or U; 4-10 days) and chronic (0.1 µM Cd or U; 30-60 days) exposures. The survival rates demonstrated the high tolerance of this species toward both metals and showed that Cd had a greater effect on the sustainability of crayfish. The concentration levels of Cd and U accumulated in gills and hepatopancreas were compared between both conditions. Distinctions in the adsorption capacities and the mobility of the contaminants were suspected. Differences in the detoxification mechanisms of both metals using transmission electron microscopy equiped with an energy dispersive X-ray were also pointed out. In contrast, comparison between the histological structures of contaminated hepatopancreas showed similar symptoms. Principal component analyses revealed different impacts of each metal on the oxidative balance and mitochondria using enzymatic activities and gene expression levels as endpoints. The observation that U seemed to generate more oxidative stress than Cd in our conditions of exposure is discussed.

Single and mixture effects of pesticides and a degradation product on fluvial biofilms.

The Morcille River located in the Beaujolais vineyard area (Eastern France) is subjected to strong vine-growing pressure leading to the contamination by a range of herbicides and fungicides of the surrounding freshwater environment. Particularly high concentrations of norflurazon, desmethyl norflurazon and tebuconazole were recorded in spring 2010 at the downstream site of the river. Despite their occurrence in rivers, scarce toxicity data are available for these products, in particular in the case of desmethyl norflurazon (main norflurazon degradation product). Furthermore, the toxicity data are generally available only for single compounds and are issued from single species toxicity tests, leading to a lack of ecological relevance. Consequently, this study was undertaken to evaluate the toxic effects of norflurazon, desmethyl norflurazon and tebuconazole singly and in a ternary mixture on fluvial biofilm. Toxicity tests were performed in microplates for 48 h. Photosynthetic endpoints were measured using pulse amplitude-modulated fluorometry; diatom densities and taxonomic composition were determined. After 48 h of exposure, significant effects on optimal quantum yield (F v/F m) for desmethyl norflurazon and mixture were observed.

Effect of low-dose cadmium exposure on DNA methylation in the endangered European eel.

There is increasing evidence that epigenetics can play a key role in the etiology of diseases engendered by chronic pollutant exposure. Although epigenetics has received significant attention in the field of biomedicine during the last years, epigenetics research is surprisingly very limited in ecotoxicology. The aim of the present study was to investigate the possible effects of low-dose cadmium exposure on the DNA methylation profile in a critically endangered fish species, the European eel. Eels were exposed to environmentally realistic concentrations of cadmium (0.4 and 4 µg·L(-1)) during 45 days. The global CpG methylation status of eel liver was determined by means of a homemade ELISA assay. We then used a methylation-sensitive arbitrarily primed PCR method to identify genes that are differentially methylated between control and Cd-exposed eels. Our results show that cadmium exposure is associated with DNA hypermethylation and with a decrease in total RNA synthesis. Among hypermethylated sequences identified, several fragments presented high homologies with genes encoding for proteins involved in intracellular trafficking, lipid biosynthesis, and phosphatidic acid signaling pathway. In addition, few fragments presented high homologies with retrotransposon-like sequences. Our study illustrates how DNA methylation can be involved in the chronic stress response to Cd in fish.

Mitochondrial gene expression, antioxidant responses, and histopathology after cadmium exposure.

The present study investigates cadmium effects on the transcription of mitochondrial genes of Procambarus clarkii after acute (0.05, 0.5, and 5 mg Cd/L; 4-10 days) and chronic exposures (10 µg Cd/L; 30-60 days). Transcriptional responses of cox1, atp6, and 12S using quantitative real-time RT-PCR were assessed in gills and hepatopancreas. Additionally, the expression levels of genes involved in detoxification and/or oxidative stress responses [mt, sod(Mn)] and enzymatic activities of antioxidants (SOD, CAT, GPX, and GST) were analyzed. The histopathological effects in hepatopancreas of crayfish were evaluated by light microscopy. Relationships between endpoints at different levels of biological organization and Cd bioaccumulation were also examined. Cd induced high levels of bioaccumulation, which was followed by mitochondrial dysfunction and histological alterations in both experiments. Moreover, perturbations in the defence mechanisms against oxidative stress tended to increase with time. Results also showed that molecular responses can vary depending on the intensity and duration of the chemical stress applied to the organisms and that the study of mt gene expression levels seemed to be the best tool to assess Cd intoxication.

Effects of copper and cadmium spiked-sediments on embryonic development of Japanese medaka (Oryzias latipes).

Because of their high capacity to accumulate contaminants such as persistent organic pollutants and heavy metals, aquatic sediments are considered as a long-term source of contamination for aquatic organisms. In compliance with the increasing interest both for sediment quality evaluation and the use of fish early life stage (ELS) toxicity assays, we proposed an embryo-larval test to evaluate embryotoxicity and genotoxicity of sediment-bound contaminants. Pre-blastula stage medaka (Oryzias latipes) embryos were exposed by static sediment contact to two model heavy metals (cadmium and copper) at environmental concentrations during the whole 10-day embryonic development. Lethal and sub-lethal effects were recorded in both embryos and larvae for 20 days post fertilisation (dpf) using several global toxicity and phenotypic endpoints. The comet assay was also performed on medaka prolarvae to evaluate genotoxic effects of the tested chemicals. Environmental concentrations of cadmium (Cd) and copper (Cu) did not affect embryo and larval survival. However, both heavy metals significantly induced morphological abnormalities, particularly spinal and cardiovascular deformities. Cd but not Cu induced tachycardia. Both heavy metals induced a significant increase in DNA damage at all tested concentrations. Resulting LOEC values for Cd and Cu corresponded to 1.9 and 8.5 µg/g d.w. sediment, respectively. Although metal bioavailability is probably lower for naturally contaminated sediments, the relatively low toxicity thresholds for both Cd and Cu raise the question of possible risk for fish embryos developing in direct contact to sediments. This study demonstrates the applicability, sensitivity and relevance of the Japanese medaka embryo-larval assay (MELA) to evaluate sediment hazardous potency at environmental concentrations of heavy metals.

Cloning, characterization and gene expression of a metallothionein isoform in the edible cockle Cerastoderma edule after cadmium or mercury exposure.

Metallothionein (MT) genes encode crucial metal-binding proteins ubiquitously expressed in living organisms and which play important roles in homeostasis of essential metals and detoxification processes. Here, the molecular organization of the first metallothionein gene of the edible cockle Cerastoderma edule and its expression after cadmium (Cd) or mercury (Hg) exposures were determined. The resulting sequence (Cemt1) exhibits unusual features. The full length cDNA encodes a protein of 73 amino acids with nine classical Cys-X((1-3))-Cys motifs, but also one Cys-Cys not generally found in molluscan MT. Moreover, characterization of the molecular organization of the Cemt1 gene revealed two different alleles (A1 and A2) with length differences due to large deletion events in their intronic sequences involving direct Short Interspersed repeated Elements (SINE), while their exonic sequences were identical. To our knowledge, such large excision mechanisms have never before been reported in a bivalve gene sequence. After 10 days of Cd exposure at environmentally relevant doses, quantitative real-time PCR revealed a strong induction of Cemt1 in gills of C. edule. Surprisingly, neither induction of the Cemt1 gene nor of MT protein was shown after Hg exposure, despite the fact that this organism is able to bioaccumulate a high amount of this trace metal which is theoretically one of the most powerful inducers of MT biosynthesis.

Development of q-PCR approaches to assess water quality: effects of cadmium on gene expression of the diatom Eolimna minima.

This study was undertaken to develop molecular tools to assess water quality using diatoms as the biological model. Molecular approaches were designed following the development of a rapid and easy RNA extraction method suited to diatoms and the sequencing of genes involved in mitochondrial and photosystem metabolism. Secondly the impact of cadmium was evaluated at the genetic level by q-PCR on 9 genes of interest after exposure of Eolimna minima diatom populations cultured in suspension under controlled laboratory conditions. Their growth kinetics and Cd bioaccumulation were followed. Population growth rates revealed the high impact of Cd at 100 µg/L with total inhibition of growth. These results are linked to the high bioaccumulation values calculated after 14 days of exposure, 57.0±6.3 µg Cd/g dw and 734.1±70 µg Cd/g dw for exposures of 10 and 100 µg Cd/L respectively. Genetic responses revealed the impact of Cd on the mitochondrial metabolism and the chloroplast photosystem of E. minima exposed to 10 and 100 µg Cd/L with induction of cox1, 12S, d1 and psaA after 7 days of exposure for the concentration of 100 µg Cd/L and of nad5, d1 and psaA after 14 days of exposure for both conditions. This is the first reported use of q-PCR for the assessment of toxic pollution on benthic river diatoms. The results obtained presage interesting perspectives, but the techniques developed need to be optimized before the design of new water quality diagnosis tools for use on natural biofilms.

Effects of dietary cadmium contamination on bird Anas platyrhynchos--comparison with species Cairina moschata.

This study aimed to assess the effect of two dietary cadmium (Cd) levels (C1: 1 mgkg(-1); C10: 10 mgkg(-1)) on bird Anas platyrhynchos exposed for 10, 20 and 40 days (5 animals per experimental condition). Ducks were able to accumulate high amounts of Cd, especially in kidneys (after 40 days: C1 8.1 ± 1 mgkg(-1), C10 37.7 ± 4.3 mgkg(-1)). After 40 days, the lowest Cd level triggered oxidative stress and stimulated mitochondrial metabolism. At the same time, highest amounts of Cd (C10 group) only triggered repression of genes encoding for catalase and acetyl-CoA carboxylase, with repression factors of 1/50 and 1/5, respectively. High dose exposures were then associated with the repression of genes encoding for antioxidant, whereas low dose exposure triggered their induction. In contrast, the onset of MT gene expression appeared quickly for the C10 group even if a time delay was observed between gene expression and protein accumulation. Through the comparison of A. platyrhynchos and Cairina moschata, the response to Cd toxicity appeared species-dependent. Discrepancies between species could be explained by differential utilization of MT. This pathway of detoxification seemed sufficient to counter Cd toxicity.

Short-term metallothionein inductions in the edible cockle Cerastoderma edule after cadmium or mercury exposure: discrepancy between mRNA and protein responses.

Metallothioneins (MT) are essential metal binding proteins involved in metal homeostasis and detoxification in living organisms. Numerous studies have focused on MT response to metal exposure and showed an important variability according to species, metal, concentration and time of exposure. In this study, the expression of one isoform of MT gene (Cemt1) and associated MT protein synthesis were determined after 1, 3, 9, 24, 72 and 168h of cadmium (Cd) or mercury (Hg) exposures in gills of the cockle Cerastoderma edule. This experiment, carried out in laboratory conditions, revealed that in Cd-exposed cockles, induction of Cemt1 is time-dependent following a "pulse-scheme" with significant upregulation at 24h and 168h intersected by time point (72h) with significant downregulation. MT protein concentration increases with time in gills of exposed cockles in relation with the progressive accumulation of Cd in soluble fraction. On contrary, Hg exposure does not lead to any induction of Cemt1 mRNA expression or MT protein synthesis compared to control, despite a higher accumulation of this metal in gills of cockles compared to Cd. The localization of Hg (85-90%) is in insoluble fraction, whereas MT was located in the cytoplasm of cells. This gives us a first clue to understand the inability of Hg to activate MT synthesis. However, other biochemical processes probably occur in gills of C. edule since the remaining soluble fraction of Hg exceeds MT sequestration ability. Finally, since one of the first main targets of metal toxicity in cells was the mitochondria, some genes involved in mitochondria metabolism were also analyzed in order to assess potential differences in cellular damages between two metal exposures. Indeed, until T(168), no impact on mitochondrial genes was shown following Hg exposure, despite the complete lack of MT response. This result indicated the presence of other effective cellular ligands which sequester the cytosolic fraction of this metal and consequently inhibit metal reactivity. Such competition mechanisms with other cytosolic ligands more sensitive to Hg were particularly argued in the discussion.

Effect of dietary cadmium on lipid metabolism and storage of aquatic bird Cairina moschata.

In environment, birds often fast in connection with breeding, migration or drastic climatic conditions and need to mobilize lipid reserves during these periods. The impairment of lipid metabolism by cadmium (Cd; 1 mg kg(-1) added in diet) was investigated on palmiped Cairina moschata. Expression levels of genes involved in lipid metabolism, mitochondrial metabolism and detoxification were investigated in liver and muscle of ducks. Lipid content in muscle and liver were analysed and plasma triglycerides were quantified. After 20 days, ducks exposed to Cd displayed a lower body weight and lower lipid content in liver than controls. In muscle, the increase of lipid content was only significant for control ducks but not for exposed ducks. Exposed ducks appeared unable to sufficiently transport and store lipids into peripheral tissues. Cd impairs lipid metabolism by several ways. First, Cd triggered the down-regulation of fatty acids synthesis in liver even if the NADPH production and the mitochondrial metabolism are enhanced, suggesting a stronger energy needs. Secondly, the associated decrease of plasma triglycerides and lipoprotein lipase activity with Cd are consistent with impairment of lipids storage in peripheral tissues.

Biomarker responses in polar cod (Boreogadus saida) exposed to dietary crude oil.

Polar cod Boreogadus saida were exposed weekly to two doses of dietary crude oil for 4 weeks followed by 2 weeks of depuration. Administered doses corresponded on average to 4 and 9microgSigmaPAHsg(-1)fishweek(-1). Cytochrome P4501A1 (cyp1a1) and glutathione S-transferase (gst) mRNA expression, ethoxyresorufin O-deethylase (EROD) activity and metabolites in the bile showed strong and dose-dependent inductions at 2 and 4 weeks of exposure. Following 2 weeks depuration, mRNA expression of cyp1a1 and gst and PAH metabolites returned to basal levels while EROD activity and GST activity were still induced in the high oil treatment. The mRNA expressions of antioxidant defense genes (catalase, glutathione peroxidase and cytosolic and mitochondrial superoxide dismutase) did not change significantly during the experiment. Catalase activity was significantly depressed at week 2 in the high oil treatment. We conclude that the cyp1a1 mRNA expression, EROD activities and bile metabolites were the most reliable biomarkers of exposure while gst mRNA expression and GST activity were less sensitive and are considered only as complementary. Antioxidant defenses were poor biomarkers to assess effects of crude oil exposure in polar cod.

Biomarker responses in polar cod (Boreogadus saida) exposed to the water soluble fraction of crude oil.

In order to mimic the biological effects of an oil spill in Arctic waters, we examined several types of biomarkers (genes, enzymes, metabolites, and DNA damage) in polar cod Boreogadus saida experimentally exposed to the water soluble fractions of crude oil. During 4 weeks of exposure, induction of the studied biomarkers exceeded baseline levels. The mRNA expression of the cytochrome P4501A1 (cyp1a1) gene was the most promising biomarker, with glutathione S-transferase (gst) as a suitable complement. The delayed ethoxyresorufin O-deethylase (EROD) and GST activities and their persistence following 2 weeks of depuration may allow detection of previous exposures in field samples. The composition of PAH metabolites in the bile indicated the bioavailability of different PAH size-classes. Although mRNA expressions of antioxidant defense genes were induced at start of the exposure, with the strongest responses from catalase and cytosolic superoxide dismutase, they were poor for oil monitoring purposes due to their very short response times. Significant DNA damage demonstrated genotoxicity even at low PAH concentrations (<15microgL(-1)) and was correlated with benzo(a)pyrene and pyrene metabolites in the bile.

Cadmium-induced genotoxicity in zebrafish at environmentally relevant doses.

Genotoxic effects of cadmium on zebra fish Danio rerio have been assessed by random amplified polymorphic DNA and real time PCR, followed by a comparison of the melting temperature patterns between each amplification reaction. Fish were exposed to two concentrations of cadmium chloride dissolved in the medium (1.9+/-0.6 microg Cdl(-1), C(1); 9.6+/-2.9 microg Cdl(-1), C(2)) for 21 days. A discriminative RAPD probe, OPB11, was first selected producing differential band patterns between control and metal-exposed genomic DNAs. RAPD-PCR showed an increase in the relative hybridization efficiency of OPB11 on the genomic DNAs coming from fish exposed to both Cd concentrations as compared to the control condition. In addition, the RAPD-PCR melting temperature patterns showed that with the OPB11 probe, the frequency of PCR products whose fusion temperature belongs to the [86-87 degrees C] interval decreased with Cd contamination, whereas an increase of frequency for the [78-80 degrees C] and [85-86 degrees C] intervals was correlated with Cd exposure.