Blind and naive classification of toxicity by fish chromatophores

Cellular and molecular pathways involved in the ability of animals to change color have been studied previously as biosensors and cytosensors of active and toxic agents, but such studies generally have been limited to just a few standardized agents. Here we describe the performance of cultured chromatophore pigment cells from the fin tissue of Siamese fighting fish as sensors of toxic agents under blind sampling conditions at the September 2002 EILATox-Oregon Workshop. Detection was accomplished by monitoring motor protein-mediated movements of cellular pigment in chromatophores at both the gross population level as well as in singly imaged cells. Pigment responses were recorded both during the exposure of chromatophores to each blind sample as well as afterwards when the cells were examined for after-effects by challenging them with clonidine, an adrenergic drug that induces standardized pigment movements. After recording all results and upon breaking the key to reveal the identities of the toxic agents, it was found that all of the toxic samples in the study had been distinguished accurately from non-toxic controls that were included among the blind samples. Furthermore, it was revealed that most of the toxic agents detected had never before been tested or calibrated against chromatophores, demonstrating that detection can be achieved under naive conditions that have not been optimized for the analysis of any particular toxic agent. Finally, by organizing the results into categories of pigment responses, a binary classification tree was generated that distinguished each toxic agent as having a distinct response pattern from the others. Thus, chromatophore-based cytosensors can discover toxicity in the absence of prior knowledge of the agent in question, and the categories of responses of the cells can be used to distinguish one toxic agent from another.