Kinetic Analysis of the Chronology of Patulin- and Gossypol-Induced Cytotoxicity in Vitro
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Kinetic analyses of the mechanisms of patulin- and gossypol-nduced cellular toxicity in an immortalized rat hepatocyte cell line were examined using a battery of vital fluorescence bioassays. Intracellular glutathione (GSH) content and intracellular Ca2+ ([Ca2+](i)) were monitored simultaneously using fluorescent probes requiring uv excitation (351-363 nm); reactive oxygen species (ROS) production, mitochondrial and plasma membrane potential, and intracellular pH were monitored simultaneously with visible wavelength probes (488 nm). Changes in gap junction-mediated intercellular communication (GJIC) were monitored using the gap FRAP technique, Cells were exposed to different concentrations of patulin (0, 1.0, 10, 100, and 1000 M) or gossypol (0, 1.0, 3.0, and 10 M). All parameters were monitored directly after addition of toxin for 20 min. The analyses provided the following chronology of cellular injury caused by patulin: simultaneous suppression of GJIC and GSH depletion ROS generation; mitochondrial membrane depolarization simultaneous increase in [Ca2+](i) and cytoplasmic acidification depolarization of plasma membrane. A distinct chronology of gossypol-induced cellular injury was also identified: simultaneous suppression of GJIC and generation of ROS cytoplasmic acidification simultaneous elevation of [Ca2+](i) and partial depletion of GSH mitochondrial membrane depolarization depolarization of plasma membrane. This report indicates the utility of these vital assays as improved mechanistically based methods for toxicity testing in vitro.