The Establishment Of Zebrafish Toxicity Microplate Model And Its Application In Secreening And Evaluation Of Marine Natural Products

In marine environment, marine organisms have developed variety of species diversity and chemical deversity. Marine animals, especially sponges, corals and other invertebrates, have developed efficient chemical defensive mechanisms to face the tough competitive situations by producing various secondary metabolites. These metabolites were found to serve as defensive agents with ichthyootoxicity, allelopathy, antifouling, algal inhibition, insecticidal, antipredatory, and antimicrobial activities and other chemical ecology effects, and were also valuable natural resources for discovery of marine drugs. During the progress of searching for drug lead compounds from marine medicinal bioresources, one of the key procedure is safety assessment of compounds. It is hard currently to use common mammilary models in large-scale screening during short time with low consumption. Zebrafish, however, possessing many features, such as relatively small entity, external development, high fertility and transparent embryos, should be used as a highly useful micro-scale model system in the field of drug development.In this paper, zebrafish toxicity microplate model was established in order to supply a dual model for drug discovery and for the research of chemical ecology. The secondary metabolites isolated from sponges, corals and their symbiotic microbes were screened and evaluated for their toxicity and chemical ecological activity with the established model. And the feasibility of this model for drug safety assessment and chemoecological effect evaluation was discussed.In the zebrafish toxicity microplate model, zebrafish Danio rerio were utilized to take fertilized embryos, which were cultured in 24-cell plates to investigate the toxicity and activity of test substances. The indexs of abnormal development of embryos, including coagulated eggs, death, no spontaneous movements, abnormal heartbeat, delayed hatch, delayed development, tail malformation, notochord malformation, and heat malformation, were obeserved by using microscrope. The repeatability, stability, accuracy, and sensitivity of the establish model were also investigated.By using the established model, the toxicity and activity of secondary metabolites (134 compounds) isolated from sponges, corals and symbiotic microbes were sreened and evaluated. And based on the data from the assay, the structure-activity relationships of the tested compounds were analyzed and discussed. It was found that, in steroids, 24-ketal steroids and pregnane steroids have more powerful impact than other type steroids on embryo development. In sesquiterpenes, most of azulene sesquiterpenes and part of bisabolane sesquiterpenes exhibited obvious toxicity. In diterpenes, the toxicity of brianane-type diterpenes were low commonly, however, diterpenes with furan ring revealed more obvious toxicity and activity. In sesterterpene, most of scalarane-type sesterterpenes had no effects on the development of zebrafish embryos. Most of biphenylene oxides from marine organisms showed diverse toxicity toward embryo development leading to lethal, sublethal and teratogentic effects. And partial polyketides exhibited certain toxicity, suggesting that the polyketides wth paraquinone moiety had potent toxicity and activity. These results further confirmed the feasibility and practical ability of zebrafish toxicity microplate model for the screening and evaluating marine natural products.In conclusion, zebrafish toxicity microplate model was established, and was applied to evaluated the toxicity and activity of marine natural products for the first time. This model supply a dual model for drug discovery and chemical ecology, which is suitable both for assessing chemical toxicity in drug discovery and for evaluation chemical ecological effects in study of marine chemical ecology.