Sea urchin embryos as an in vivo model for the assessment of manganese toxicity: developmental and stress response effects.

Manganese (Mn), one of the most abundant metals in nature present in rocks, soil and water, is also found in soft bottom sediments of the oceans. It represents a trace element that is accumulated and utilized by all forms of life and plays multiple roles ranging from bone mineralization to cellular protection. Although Mn is an essential nutrient, exposure of cells/organisms to high levels of Mn cause toxicity. In the marine environment, increased concentrations of bio-available Mn often result from anthropogenic activities, and consequently, Mn represents a new important factor in environmental contamination. Emission of Mn into the marine environment occurs from metallurgic and chemical industries including municipal wastewater discharges, sewage sludge, mining and mineral processing, emissions from alloy, steel, and iron production. Thus, a sharp Mn solubility gradient exists along the oxic/anoxic interfaces in such water columns. During hypoxia, that in many costal areas occurs as a result of eutrophication, Mn is released in a divalent ionic form (Mn2+) and reaches high concentrations in bottom waters. In this study, we investigated the effects of manganese chloride (MnCl2) on embryos of the sea urchin Paracentrotus lividus used as a model of Mn-induced toxicity. Embryos were continuously exposed to different MnCl2 concentrations from fertilization to the pluteus stage and examined after 6,12, 20, 24, 48, 72 hours. We found a severe dose-dependent inhibition of embryonic development, with consequent specific malformations. The EC50 value was determined in 7,7 mg/l, defined as the concentration causing 50% abnormal embryo development. These effects were rapidly reversed upon Mn washout, except after 40 hours of treatment. Major developmental defects consisted in the absence or reduced elongation of skeletal rods (spicules), suggesting a key role for Mn in embryonic skeleton development. Mn accumulation was determined in exposed embryos by AAS analysis and compared to physiological calcium (Ca) concentration found in the same specimens. We found that the Mn is accumulated into embryos in a time and dose-dependent manner, with a drastic increase 24 hrs post-fertilization. In contrast, Ca concentration is reduced in an opposite proportional way, consistent with its poor detection in primary mesenchyme cells, observed by in vivo labelling with cell permeable fluorescent calcein. A direct correlation has been observed between malformations, accumulation of Mn ions and the regulation of key stress proteins that provide the major protection against stressors. We found that Mn exposure caused an increase in HSC70 and HSC60 levels, but not in HSC90 levels and did not induce synthesis of the HSPs inducible forms. Analysis by 2D gel electrophoresis showed different patterns of protein spots in control and exposed embryos highlighting qualitative protein expression differences in response to Mn exposure. No increase in apoptosis in comparison to controls was measured by the TUNEL assay, as well as no reactive oxygen species (ROS) production was found by a fluorescent detection assay on live embryos. To conclude, developmental and stress response effects observed could be explained as a Mn toxicity linked to the accumulation and bio-concentration of Mn into the embryos tissues and a possible competition between Mn and Ca for membrane transport sites or other specific molecular targets