In Vitro Hepatotoxicity of the Cyanobacterial Alkaloid Cylindrospermopsin and Related Synthetic Analogues

Abstract

Cylindrospermopsin (CY), a sulfate ester of a tricyclic guanidine substituted with a hydroxymethyluracil, is a cyanobacterial toxin of increasing environmental import as it frequently occurs in drinking water reservoirs. As a toxin, CY mainly targets the liver but also involves other organs. In hepatocytes CY inhibits the synthesis of protein and of glutathione, leading to cell death. The total chemical synthesis of CY has recently been reported (Xie et al., 2000, J. Am. Chem. Soc. 22, 5017-5024). The synthesis has provided analogues of CY to study aspects of the relationship between chemical structure and activity that contribute to toxicity. Protein synthesis inhibition was measured in vitro using a rabbit reticulocyte system. Primary cultures of rat hepatocytes were used to determine the biological activity of CY and analogues in intact cells. Protein synthesis and cell glutathione levels were measured. We could distinguish between CY transport and biological activity by comparing the results in vitro to those in intact cells. The role of the sulfate group in CY toxicity was examined by comparing biological effects of CY with that of CY-DIOL (synthetic CY lacking the sulfate group). The sulfate group was found not to play a role in CY activity or in its uptake into cells, since there was no significant difference in biological activity in vitro or in cells between natural CY and CY-DIOL. The orientation of the hydroxyl group at C7 also had no impact on biological activity or transport of CY, since the C7 epimer of CY (EPI-CY) and the corresponding diol (EPI-DIOL) had activity similar to RAC-CY in vitro and in intact cells. AB-MODEL, the analogue lacking an intact C ring, and the methyl and hydroxyl groups of ring A could inhibit protein synthesis (but at concentrations 500-1000-fold higher than natural CY). Other structurally simpler synthetic analogues lacked biological activity.

Characterization of the Gene Cluster Responsible for Cylindrospermopsin Biosynthesis

“…Besides posing a threat to human health, cylindrospermopsin also causes significant economic losses for farmers due to the poisoning of livestock with cylindrospermopsin-contaminated drinking water (38). Cylindrospermopsin has hepatotoxic, general cytotoxic (33)(34)(35), and neurotoxic (19) effects and is a potential carcinogen (17). Its toxicity is due to the inhibition of glutathione and protein synthesis as well as the inhibition of cytochrome P450 (13,(33)(34)(35).…”

Experimental immunology First report of cylindrospermopsin effect on human peripheral blood lymphocytes proliferation in vitro

“…Such properties has been observed in rodent model experiments and studies involving human cell lines [11][12][13]. There has been also two confirmed epidemic cases of CYN poisoning (Palm Island, Australia, 1979 andCaruaru, Brazil, 1996) resulting among many in: painful hepatomegaly, bloody diarrhea, vomiting, anorexia and dehydration.…”

Cyanobacterial Toxins of the Laurentian Great Lakes, their Toxicological Effects, and Numerical Limits in Drinking Water

“…While the exact mechanism of protein synthesis inhibition is not completely understood it appears to be the primary mode of toxicity at lower doses of cylindrospermopsin, while at high doses cytochrome P450 metabolism of cylindrospermopsin producing ROS induces cell death [22,245].…”