Acute toxicity of a commercial glyphosate formulation on European sea bass juveniles (Dicentrarchus labrax L.): gene expressions of heme oxygenase-1 (ho-1), acetylcholinesterase (AChE) and aromatases (cyp19a and cyp19b).

Acute toxicity of Roundup, a commercial glyphosate--based herbicide, was evaluated in a teleost marine fish, the European sea bass, after 96 h of exposure. The LC50 96-h value of Roundup was 529 mg/L. Juveniles (Dicentrarchus labrax L.) were exposed to a sublethal concentration (35% of the LC50, i.e. 193 mg/L) of Roundup for 96-h. The study of heme oxygenase-1 (ho-1) gene expression was performed in four tissues (liver, gills, brain and gonads) and highlighted the disruption of antioxidant defence system. Results showed that ho-1 mRNA levels in liver and gills significantly decreased (p<0.001 and p<0.01 respectively) in fish exposed to 193 mg/L of Roundup, whereas in brain and gonads, ho-1 mRNA level was not altered. The analysis of acetylcholinesterase expression was used to evaluate the overall neurotoxicity of the herbicide and aromatase genes to assess the alteration of the endocrine system. Results showed that AChE and cyp19b gene transcriptions significantly increased (p<0.01) in brain of sea bass, whereas aromatase gene expression (cyp19a) in gonads was not significantly altered. Our results showed complex tissue-specific transcriptional responses after 96 h of exposure to a sublethal concentration. All these disruptions confirmed the deleterious effects of this glyphosate-based herbicide in a marine species.

Effect of water temperature increase on HO-1 expression in European sea bass (Dicentrarchus labrax L.) tissues.

One of the most pertinent environmental factors influencing the marine organism life is temperature. It has been demonstrated that an increase of temperature is able to induce the synthesis of heat shock proteins (HSP). In this study we investigated the expression of HO-1 mRNA, also referred to as HSP32, in different tissues of European sea bass (Dicentrarchus labrax, L.) at several time points after increased temperature exposure (from 12degC to 30degC). Our results showed that HO-1 was not expressed in gills, heart, muscle and brain while it was expressed at a basal level in intestine. In liver, spleen and kidneys, HO-1 expression was influenced by temperature increases. In the spleen, we found a significant decrease of the HO-1 expression at the end of 4 weeks. In kidneys a very fast collapse of HO-1 expression level was recorded reaching null value as soon as one hour after exposure to 30degC. In liver, HO-1 expression increased from one hour of exposure to 30degC confirming HO-1 involvement to heat shock response in this organ. This increasing trend reached a 4.5-fold higher value than the initial level after 4 weeks.

Effects of water temperature increase and heavy metals contamination on WAP65 gene expression in sea bass (Dicentrarchus labrax) liver.

It has been previously demonstrated that "Warm temperature Acclimation-related 65 kD Protein" (WAP65) is involved in temperature acclimation, response to intoxication and infection, as well as in development. The expression of wap65-1 was investigated in the liver of European sea bass (Dicentrarchus labrax) during exposure to the increased temperature (from 12 deg C to 30 deg C) and during intoxication with four heavy metals: lead, cadmium, copper and zinc. Post temperature increase wap65 expression was highest after one hour at 30 deg C. After 1 to 4 weeks at 30 deg C wap65 transcript levels did not differ from the 12 deg C control group, similar to observations regarding the heat shock protein, hsp70. Upregulation of wap65 was detected after treatment (intoxication) with cadmium (0.5 µg/l). In contrast, a slight, but significant down regulation of wap65 was seen after copper (5 µg/l) intoxication. These data indicate that functional analyses of WAP65 are needed to understand the differential regulation of this gene by metals. The role of WAP65 may be similar to that of HSP70, which has generalized functions in responding to certain stressors and maintaining normal cell physiology.

Cloning of Wap65 in sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) and expression in sea bass tissues.

The complementary DNA encoding WAP65 protein was cloned from the liver of two fish species sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata). Full-length cDNA sequences were obtained from reverse transcribed total RNA, followed by 5' and 3' rapid amplification of cDNA end (RACE) experiments. The full-length cDNA sequence of D. labrax is 1709bp and the coding sequence is flanked by a 67bp 5'-UTR and a 358bp 3'-UTR. The full-length cDNA sequence of S. aurata is 1599bp, and the coding sequence is flanked by a 48bp 5'-UTR and a 273bp 3'-UTR. The deduced amino acid putative primary sequences are composed of 427 and 425 amino acid residues for D. labrax and S. aurata, respectively. They display high homologies with previously described fish WAP65 and other hemopexin-like proteins from rabbit (Oryctolagus cuniculus). Expression of Wap65 has proved to be a natural physiological adaptive answer of teleost fish to warm temperature acclimation. In all fish species studied to date, Wap65 was found expressed mainly by the liver, although other tissues seem able to express Wap65 in response to a warm temperature acclimation, in a specie specific manner. Here, we investigate the tissue specific expression of Wap65 in D. labrax and S. aurata in response to a warm temperature acclimation, by RT-PCR analysis.

cDNA sequencing and expression analysis of Dicentrarchus labrax heme oxygenase-1.

The liver cDNA encoding heme oxygenase--1 (HO-1) was sequenced from European sea bass (Dicentrarchus labrax) (accession number no. EF139130). The HO-1 cDNA was 1250 bp in nucleotide length and the open reading frame encoded 277 amino acid residues. The deduced amino acid sequence of the European sea bass had 75% and 50% identity with the amino acid sequences of tetraodontiformes (Tetraodon nigroviridis and Takifugu rubripes) and human HO-1 proteins, respectively. A short hydrophobic transmembrane domain at the C--terminal region was found, and four histidine residues were highly conserved, including human his25 that is essential for HO catalytic activity. RT-PCR of mRNA from eight different European sea bass tissues revealed that, in a homeostatis state, the heme oxygenase--1 was abundant in the spleen and liver but not in the brain.