KINETIC-ANALYSIS OF THE CHRONOLOGY OF PATULIN-INDUCED AND GOSSYPOL-INDUCED CYTOTOXICITY IN-VITRO

Kinetic analyses of the mechanisms of patulin- and gossypol-induced ce llular toxicity in an immortalized rat hepatocyte cell line were exami ned using a battery of vital fluorescence bioassays. Intracellular glu tathione (GSH) content and intracellular Ca2+ ([Ca2+](i)) were monitor ed simultaneously using fluorescent probes requiring uv excitation (35 1-363 nm); reactive oxygen species (ROS) production, mitochondrial and plasma membrane potential, and intracellular pH were monitored simult aneously with visible wavelength probes (488 nm). Changes in gap junct ion-mediated intercellular communication (GJIC) were monitored using t he gap FRAP technique. Cells were exposed to different concentrations of patulin (0, 1.0, 10, 100, and 1000 mu M) or gossypol (0, 1.0, 3.0, and 10 mu M). All parameters were monitored directly after addition of toxin for 20 min. The analyses provided the following chronology of c ellular injury caused by patulin: simultaneous suppression of GJIC and GSH depletion --> ROS generation --> mitochondrial membrane depolariz ation --> simultaneous increase in [Ca2+](i) and cytoplasmic acidifica tion --> depolarization of plasma membrane, A distinct chronology of g ossypol-induced cellular injury was also identified: simultaneous supp ression of GJIC and generation of ROS --> cytoplasmic acidification -- > simultaneous elevation of [Ca2+](i) and partial depletion of GSH --> mitochondrial membrane depolarization --> depolarization of plasma me mbrane. This report indicates the utility of these vital assays as imp roved mechanistically based methods for toxicity testing in vitro. (C) 1996 Society of Toxicology