Tolerated Doses in Zebrafish of Cytochalasins and Jasplakinolide for Comparison with Tolerated Doses in Mice in the Evaluation of Pre-clinical Activity of Microfilament-directed Agents in Tumor Model Systems In Vivo

摘要

Background/Aim: Chemotherapeutic approaches involving microtubule-directed agents such as the vinca alkaloids and taxanes are used extensively and effectively in clinical cancer therapy. There is abundant evidence of critical cytoskeletal differences involving microfilaments between normal and neoplastic cells, and a variety of natural products and semi-synthetic derivatives are available to exploit these differences in vitro. In spite of the availability of such potential anti-neoplastic agents, there has yet to be an effective microfilament-directed agent approved for clinical use. Cytochalasins are mycogenic toxins derived from a variety of fungal sources that have shown promising in vitro efficacy in disrupting microfilaments and producing remarkable cell enlargement and multi-nucleation in cancer cells without producing enlargement and multi-nucleation in normal blood cells. Jasplakinolide is a sponge toxin that stabilizes and rigidifies microfilaments. Insufficient in vivo data has been acquired to determine whether any of the microfilament-directed agents have valuable preferential anticancer activity in pre-clinical tumor model systems. This is partly because the limited availability of these agents precludes their initial use in large-scale mammalian preclinical studies. Therefore, the present study sought to determine the tolerated in vivo doses of cytochalasins and jasplakinolide in zebrafish (Danio rerio), a well-studied fish cancer model that is 1.5% the size of mice. We also determined the tolerated levels of a variety of clinically active anti-neoplastic agents in zebrafish for comparison with tolerated murine doses as a means to allow comparison of toxicities in zebrafish expressed as mu M concentrations with toxicities in mice expressed in mg/kg. Materials and Methods: Tolerated doses in zebrafish with various cytochalasins or jasplakinolide were determined by adding the solubilized test agent to water in which the fish were maintained for 24 h, then restored to their normal tanks and monitored for a total of 96 h. Results: Cytochalasin D at 0.2 mu M gave an approximate LD50 in zebrafish, while cytochalasin B was fully-tolerated at 5 mu M, and gave an LD50 of 10 mu M. 21,22-dihydrocytochalasin B was fully-tolerated at 10 mu M. Cytochalasin C was tolerated fully at 1 mu M, ten-fold higher than the level for cytochalasin D that was tolerated. Jasplakinolide at 0.5 mu M did not exhibit any apparent acute toxicity or affect fish behavior for four days, but delayed toxicity was evident at days 4 and 6 when the fish died. Further, the addition of 5 mu M glutathione (GSH) at the time of treatment substantially decreased the toxicity of 10 mu M cytochalasin B, a level of cytochalasin B that not otherwise tolerated in vivo. Such observations were likely due to GSH-mediated alkylation of C-20 in cytochalasin B, thereby reducing the rate of oxidation to the highly toxic congener, cytochalasin A, and reacting with any cytochalazin A formed. The protective effects of GSH are further supported by its ability to react with alpha, beta-unsaturated ketone moieties, as is found in cytochalasin A. GSH at 0.8 mu M was able to reduce the toxicity of 0.8 mu M cytochalasin D, but it took 20 mu M GSH to fully protect against the toxicity of 0.8 mu M cytochalasin D. Conclusion: Pre-clinical evaluation of rare natural products such as microfilamented-directed agents for efficacy in vivo in tumor-bearing zebrafish is a feasible prospect.