The Neurodevelopmental Toxicity Of Monocrotophos On The Embryos Of Sea Urchin Hemicentrotus Pulcherrimus

Recently, more and more attention has been paid on the neurodevelopmental toxicity of organophosphorous pesticides. Monocrotophos is a kind of highly toxic organophosphorous pesticide. Due to its water-solubility, monocrotophos is apt to contaminate the environment with residue and even threaten the health of human's. More and more researches demonstrated that the influence of organophosphorous on reproduction and differentiation of neurocyte, genesis of axon and dendrite and function of dendrite can cause defected behavior. Sea urchin embryo is an idealistic model for studying the toxicity of pesticide to embryo and nervous system. Therefore, in this study we use embryos and larvae of H. pulcherrimus as model to study the effects of monocrotophos on the formation and function of serotonergic and pre-dopaminergic nervous systems. As both serotonergic and pre-dopaminergic nervous systems have the function of regulate embryo and larvae's swimming behavior. The morphology and swimming behavior of larvae were also observed here. The neurogenesis of serotonergic nervous system was illustrated by whole-mount immunohistochemistry and immunoblotting. Since the axon growth and serotonin receptor network formation were inhibited by monocrotophos. We also further studied spatio-temporal expression of HpNetrin in the sea urchin H. pulcherrimus during serotonergic axon extension. The mainly results are as follow:(1) Morphologically, MCP inhibited the body and arm growth of pluteus larvae; besides, larvae lost the ability of upright swimming, and this effect is dose dependant.(2) MCP delayed the early neurodevelopmental process of sea urchin. The results demonstrated that serotonergic cell decreased in number, and the axon growth of serotonin apical ganglion was significantly inhibited. The malformation of neuron morphogenesis also included the disorder of serotonin receptor network, especially the lost of oltort, which is one of the most important nerve fibers.(3) The spatio-temporal expression of HpNetrin was disordered from the concentration of 10μg/ml MCP. The whole-mount immunohistochemistry result showed that there is no increase intensity of HpNetrin protein near the animal pole of prism larvae, and very weak signal of HpNetrin along the ciliary band of 2-arm pluteus. The immunoblotting and RT-PCR results indicated that the amount of protein expression pattern was also disorded by MCP.(4) Dissociated cells lost the ability to re-aggregate under the exposure of 0.1μg/ml MCP. Single cells shaked weakly, but still alive; Epith II immuno stain and Trypan Blue stain results showed that the cell membrane were destroyed by MCP; The neurogenesis of serotonergic neurons were also affect in re-aggregated cells, which is correspondence to the results obtain by study on intact organism. But the effect of MCP on the formation of serotonin receptor network formation can't be detected in re-aggregated cells. Thus, although re-aggregated cell is more sensitive than larvae, it still has limitations in the screening of neuro-toxicants.(5) The positive signal of dopamine receptor D1 (DRD1) can be detected since the swimming blastula stage of sea urchin. Our results illustrated that the DRD1 granules decrease obviously with the incubation of 10μg/ml MCP. We proved that reproduction of DRD1 along the ciliary band of 48-hpf larvae reduced greatly in number by BrdU incorporation experiment.