Bénédicte Morin

1 H, 13C and 15N nuclear magnetic resonance analysis and chemical features of the two main radical oxidation products of 2′-deoxyguanosine: oxazolone and imidazolone nucleosides

The two primary one-electron oxidation and ˙OH-mediated decomposition products of 2′-deoxyguanosine 1 are 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-2,5-dihydrooxazol-5-one 3 (dZ) and its precursor, 2-amino-5-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one 2 (dIz). Here, we describe in detail the spectroscopic and chemical properties of both oxidative DNA lesions. The structures for dZ 3 and dIz 2 were determined by fast-atom bombardment mass spectrometry, UV spectroscopy, IR spectroscopy, together with 1H, 13C, 15N and 17O 1D and 2D NMR spectroscopy. In neutral aqueous solution dIz 2 is hydrolysed to dZ 3 (t½= 147 min at 37 °C) with the incorporation of one molecule of water. Hot alkali treatment (65 °C; pH 13) of dIz 2 and dZ 3 quantitatively results in the release of guanidine (t½= 3.3 and 3.1 min, respectively). The latter property allowed us to develop a specific and sensitive method for the detection of the two modified nucleosides 2 and 3. Methoxyamine reacts quantitatively with 3′,5′-di-O-acetyl-dIz 2a 120-times faster than with 3′,5′-di-O-acetyl-dZ 3a to form four 3,5-di-O-acetyl-2-deoxy-D-erythro-pentose–methoxyamine isomers.

Chain and conformation stability of solid-state DNA: implications for room temperature storage.

There is currently wide interest in room temperature storage of dehydrated DNA. However, there is insufficient knowledge about its chemical and structural stability. Here, we show that solid-state DNA degradation is greatly affected by atmospheric water and oxygen at room temperature. In these conditions DNA can even be lost by aggregation. These are major concerns since laboratory plastic ware is not airtight. Chain-breaking rates measured between 70°C and 140°C seemed to follow Arrhenius’ law. Extrapolation to 25°C gave a degradation rate of about 1–40 cuts/105 nucleotides/century. However, these figures are to be taken as very tentative since they depend on the validity of the extrapolation and the positive or negative effect of contaminants, buffers or additives. Regarding the secondary structure, denaturation experiments showed that DNA secondary structure could be preserved or fully restored upon rehydration, except possibly for small fragments. Indeed, below about 500 bp, DNA fragments underwent a very slow evolution (almost suppressed in the presence of trehalose) which could end in an irreversible denaturation. Thus, this work validates using room temperature for storage of DNA if completely protected from water and oxygen.

Embryotoxic and genotoxic effects of heavy metals and pesticides on early life stages of Pacific oyster (Crassostrea gigas)

This study evaluated embryotoxicity and genotoxicity of two dissolved metals copper and cadmium (Cu and Cd) and two pesticides (metolachlor and irgarol) occurring in Arcachon Bay (SW France) in Pacific oyster (Crassostrea gigas) larvae and investigated the relationship between those two endpoints. Embryotoxicity was measured by calculating the percentage of abnormal D-shaped larvae and genotoxicity was evaluated with DNA strand breaks using the comet assay. After 24h exposure, significant increases of the percentage of abnormal D-larvae and the DNA strand breaks were observed from 0.1 μg L⁻¹ for Cu, 10 μg L⁻¹ for Cd and 0.01 μg L⁻¹ for both irgarol and metolachlor in comparison with the controls. A strong positive relationship between embryotoxicity and genotoxicity was recorded for Cu, Cd and metolachlor. The current study suggests that copper, irgarol and metolachlor can induce larval abnormalities and DNA damage in a population of exposed oysters at environmentally relevant concentrations.

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.

A new spiked sediment assay using embryos of the Japanese medaka specifically designed for a reliable toxicity assessment of hydrophobic chemicals

Despite their low water solubility, hydrophobic pollutants are widespread in the aquatic environment and could represent a threat for living organisms. EU regulations on chemicals require accurate and reliable data on chemical toxicity. Current normalised fish toxicity assays, in particular those advocated by OECD guidelines, do not allow reliable toxicity assessment of hydrophobic compounds due to their low water solubility. In order to accurately evaluate the toxicity of this kind of compounds, a new spiked sediment assay using embryos of the Japanese medaka was developed. It consists of directly exposing fertilised eggs, during their entire embryonic development, onto the reference sediment spiked with the test compound. A large set of lethal or sublethal effects in embryos and newly hatched larvae, including non-invasive endpoints is analysed in order to maximise the sensitivity of the test. The approach was validated using four model pollutants with different modes of action: DMBA, PCB126, PCB153 and 4-nonylphenol (NP). All compounds, except PCB153, induced a dose-dependent increase in toxic effects. In fact, lethal effects only occurred at the highest tested concentration. In contrast, sub-lethal effects including skeletal deformations, cardiac activity modulation, body length reduction and hatching delay were observed at low to moderate concentrations of DMBA and PCB126. NP induced subtle effects in embryos, altering cardiac activity and hatching success but only at high concentrations. Although a few more improvements would make it a fully standardised assay, this spiked sediment assay using medaka embryos proves to be sensitive enough to measure hydrophobic chemical toxicity using an environmentally realistic mode of exposure.

Biliary PAH metabolites, EROD activity and DNA damage in dab (Limanda limanda) from Seine Estuary (France)

The Seine Estuary is well known to be widely contaminated by organic pollutants and especially by polycyclic aromatic hydrocarbons (PAHs). Fish are known to metabolize PAHs, leading to different toxic effects at both cellular and sub-cellular levels. In this work, we studied the relationships between the 7-ethoxyresorufin-O-deethylase (EROD) activity in the liver, the level of DNA strand breaks in blood cells and the concentration of PAH metabolites in the bile of the sentinel flatfish species Limanda limanda. Muscle and liver samples were analysed for parent PAH levels. Female and male dabs of two size classes (juveniles and adults) were collected by trawling in two sites with different degrees of pollution during March and September 2005 and 2006. Significant effects of sex, age, site and season were demonstrated on EROD activity and the level of strand breaks. Parent PAH concentrations in dabs did not allow discriminating of the two sampling sites. However, for PAH metabolites, significant differences were observed with sites and seasons. Dabs collected at the mouth of the estuary appeared to be the most impacted when looking at the results obtained with the three selected markers. The significant correlations observed between the level of PAH metabolites and the level of DNA lesions showed the importance of a combined analysis of chemical and biochemical markers to correctly assess the contribution of chemical contamination to the toxic effects measured in situ in fish.

Collaborative study for the detection of toxic compounds in shellfish extracts using cell-based assays. Part II: application to shellfish extracts spiked with lipophilic marine toxins

Successive unexplained shellfish toxicity events have been observed in Arcachon Bay (Atlantic coast, France) since 2005. The positive mouse bioassay (MBA) revealing atypical toxicity did not match the phytoplankton observations or the liquid chromatography−tandem mass spectrometry (LC−MS/MS) investigations used to detect some known lipophilic toxins in shellfish. The use of the three cell lines (Caco2, HepG2, and Neuro2a) allows detection of azaspiracid-1 (AZA1), okadaic acid (OA), or pectenotoxin-2 (PTX2). In this study, we proposed the cell-based assays (CBA) as complementary tools for collecting toxicity data about atypical positive MBA shellfish extracts and tracking their chromatographic fractionation in order to identify toxic compound(s). The present study was intended to investigate the responses of these cell lines to shellfish extracts, which were either control or spiked with AZA1, OA, or PTX2 used as positive controls. Digestive glands of control shellfish were extracted using the procedure of the standard MBA for lipophilic toxins and then tested for their cytotoxic effects in CBA. The same screening strategy previously used with pure lipophilic toxins was conducted for determining the intra- and inter-laboratory variabilities of the responses. Cytotoxicity was induced by control shellfish extracts whatever the cell line used and regardless of the geographical origin of the extracts. Even though the control shellfish extracts demonstrated some toxic effects on the selected cell lines, the extracts spiked with the selected lipophilic toxins were significantly more toxic than the control ones. This study is a crucial step for supporting that cell-based assays can contribute to the detection of the toxic compound(s) responsible for the atypical toxicity observed in Arcachon Bay, and which could also occur at other coastal areas.

Comparative responses of sperm cells and embryos of Pacific oyster (Crassostrea gigas) to exposure to metolachlor and its degradation products.

Metolachlor is one of the most intensively used chloroacetanilide herbicides in agriculture. Consequently, it has been frequently detected in coastal waters as well as its major degradation products, metolachlor ethane sulfonic acid (MESA) and metolachlor oxanilic acid (MOA) which are encountered at higher concentrations than metolachlor. Although a few studies of metolachlor toxicity have been conducted on marine organisms, little is known about the environmental toxicity of metolachlor degradation products. In this study, the deleterious effects of metolachlor and its degradation products on spermatozoa and embryos of Crassostrea gigas have been compared using biomarkers of developmental defects, DNA damage and gene transcription levels. After 24h exposure, significant increases in the percentage of abnormal D-larvae and DNA damage were observed from 0.01 μg L(-1) for S-metolachlor and 0.1 μg L(-1) for MESA and MOA. Results showed that S-metolachlor was more embryotoxic and genotoxic than its degradation products. Oyster sperm was also very sensitive to metolachlor exposure and followed the pattern: metolachlor (0.01 μg L(-1))>MOA (0.1 μg L(-1))>MESA (1 μg L(-1)). Metolachlor and MESA mainly triggered variations in the transcription level of genes encoding proteins involved in oxidative stress responses (mitochondrial superoxide dismutase and catalase). Overall, no significant variation in transcription levels could be detected in C. gigas embryos exposed to MOA. This study demonstrates that metolachlor and its main degradation products have the potential to impact several steps of oyster development and therefore recruitment in coastal areas exposed to chronic inputs of pesticides.

Environmental concentrations of benz[a]anthracene induce developmental defects and DNA damage and impair photomotor response in Japanese medaka larvae

Benz[a]anthracene (BaA) is a ubiquitous polycyclic aromatic hydrocarbon found in numerous aquatic ecosystems. However, ecotoxicological data in aquatic organisms are scarce. To remedy this lack of data, Japanese medaka (Oryzias latipes) embryos were exposed to BaA and toxic effects were investigated at multiple toxicological endpoints. Japanese medaka embryos were incubated onto BaA-spiked artificial sediment for 9 days at low or moderate environmental concentrations ranging from 0.9 to 12 µgg(-1) dw. BaA-exposed embryos exhibited significant tachycardia. BaA exposure was also shown to increase CYP1A activity in the hepato-biliary tissue as well as craniofacial deformities and DNA damage in pro-larvae. The photomotor response of BaA-exposed larvae was reduced in comparison to the control group. According to this set of tests, the lowest tested and observed effect concentration (LOEC) for Japanese medaka early life stages was equivalent to 0.92 µgg(-1) dw of BaA. This concentration fall into the range of concentrations frequently encountered in sediments of polluted aquatic ecosystems. Taking into consideration these results, BaA represents a threat for fish early life stages in particular those developing onto or into contaminated sediments.

Combined effects of temperature and copper and S-metolachlor on embryo-larval development of the Pacific oyster, Crassostrea gigas

This study evaluates the combined effects of two contaminants (copper and a herbicide S-metolachlor) and temperature on the early life stages of the Pacific oyster Crasssotrea gigas, which is native to Arcachon Bay (southwest France). The responses of D-larvae, obtained from wild and cultivated oysters, were investigated for one year during the oyster breeding period at different sampling sites and compared with the response of D-larvae from a commercial hatchery. Embryotoxicity was measured by considering the percentage of abnormal D-shaped larvae. Normal development of embryos occurred only from 22°C to 26°C. There were synergistic effects of copper at low and high temperatures. Native oysters appear particularly sensitive to an increase in water temperature, suggesting a future increase in the percentage of larval abnormalities as a result of global climate change. Hatchery oysters represent a good alternative model for studying the effects of both pollutants and climate change stressors.