In vitro methods for measuring cytotoxic activity of cylindrospermopsin as alternatives to animal testing

Cylindrospermopsin (CYN) is a cyanotoxin produced by cyanobacterial blooms found in brackish and fresh water. CYN is implicated in toxicity to fish, cattle, and humans, by contaminating water, affecting millions of people worldwide. Limitations of current detection methods of CYN, like chromatography and ELISA, are that they cannot differentiate between the deactivated harmless, and active cytotoxic harmful form of toxin. The only current test to detect biologically active CYN is a mouse bioassay. We show here that CYN inhibits in a dose-dependent manner cell metabolic activities and transduced viral replication and protein expression. We used these findings to develop novel in vitro activity assays to identify, quantify, and differentiate the harmful active CYN and the harmless inactive one. These new in vitro cell-based activity assays have similar sensitivities to that of the currently used in vivo LD50 mouse bioassay. Utilizing these in vitro methods, we also demonstrate that CYN is heat-resistant but can be inactivated by pretreatment with H2O2 at a neutral pH after exposure to direct sunlight. Our results suggest that these in vitro assays can be used as an alternative to animal use, to evaluate the presence of biologically active cytotoxic CYN, to ensure water safety.