Attenuation of Gossypol Cytotoxicity by Cyclic AMP in a Rat Liver Cell Line Richard W. Hutchinson,* Rola Barhoumi,* , ² Jared M. Miles,* and Robert C. Burghardt* , ² *Department of Veterinary Anatomy and Public Health, and ² Image Analysis Laboratory, Texas A & M University, College Station, Texas, 77843– 4458 Received December 15, 1997; accepted April 29, 1998 Attenuation of Gossypol Cytotoxicity by Cyclic AMP in a Rat Liver Cell Line. Hutchinson, R. W., Barhoumi, R., Miles, J. M., and Burghardt, R. C. (1998). Toxicol. Appl. Pharmacol. 151, 311–318. The effect of pretreatment of a rat liver cell line (Clone 9) with 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), an agent previously shown to increase gap junctional intercel- lular communication (GJIC), was used to determine if in- creased GJIC would attenuate the cytotoxic effects of gossypol acetic acid (GAA). Pretreatment with 8-Br-cAMP increased the inhibitory concentration 50%(IC 50 ) for gossypol from 4.1 to 6.1 M in Clone 9 cells. GJIC was suppressed by about 57%within 5 min when Clone 9 cells were treated with 1.0 M GAA alone, whereas cells treated with 3.0 or 10 M GAA were completely uncoupled within the same time frame. In contrast, GJIC was maintained near control levels in Clone 9 cells preincubated with 1.0 mM 8-Br-cAMP for 10 min prior to 1.0 M GAA exposure. 8-Br-cAMP partially restored GJIC in cells treated with 3.0 MGAA but was unable to protect cells exposed to 10 MGAA. The effects of GAA and 8-Br-cAMP pretreatment on connexin43 (Cx43) protein expression was analyzed with West- ern blots. GAA treatment at concentrations of 1 and 3 M caused a time- and dose-dependent increase in phosphorylation of Cx43 over a 20-min period, whereas 10 M GAA caused a time-dependent degradation of Cx43 over the same interval. Pretreatment of cells for 10 min with 8-Br-cAMP completely reversed the effect of 1 M GAA and partially blocked the effect of 3 M GAA on Cx43 phosphorylation and suppressed Cx43 degradation at 10 M. Additional fluorescence endpoints associated with cellular homeostasis mechanisms were also monitored to evaluate cytotoxicity and potential protective ef- fects of 8-Br-cAMP pretreatment in cells exposed to GAA, including generation of reactive oxygen species (ROS), cyto- plasmic acidification, glutathione (GSH) content, intracellular calcium levels ([Ca 2 ] i ), and mitochondrial and plasma mem- brane potential. The adverse effects of GAA on the production of ROS, cytoplasmic acidification, GSH content, and [Ca 2 ] i were also attenuated. The primary protective effect of 8-Br- cAMP was observed at orbelow the IC 50 of GAA, with greatest protective effects detected on early endpoints affected by GAA exposure. These studies suggest that the protective effect of 8-Br-cAMP in GAA-treated cells results from enhanced GJIC, which facilitates cellular homeostasis by providing cell–cell diffusion of essential metabolites, ions, and regulatory and informational molecules. © 1998 Academic Press Gossypol is a toxin produced in the seeds of the cotton plant (Berardi and Goldblatt, 1980). Cotton seeds are com- monly processed into oil and meal, which may contain high concentrations of the toxin, and further processing is nec- essary to reduce it to permissible levels. While gossypol poses a potential risk in human and animal foods, several pharmaceutical applications for gossypol have been inves- tigated, including use as an oral contraceptive for men (Csillag, 1996), as an antiviral agent (Bourinbaiar and Lee- Huang, 1994; Terry et al., 1992), and as an antineoplastic agent (Flack et al., 1993; Stein et al., 1992). Gossypol has inhibitory actions on many membrane-associ- ated enzymes (Stephens et al., 1983; Wu et al., 1991; Martinez et al., 1993; Cue ´llar and Ramı ´rez, 1993). In reproductive tissues, mitochondria are a primary target of gossypol action (Reyes et al., 1984; Reyes and Benos, 1988). Gossypol can bind covalently to proteins in membranes (Strøm-Hansen et al., 1989) as well as interact with lipid bilayers to increase proton and cation permeability (Reyes et al., 1984). Free radical generation by gossypol may also be a major factor causing cellular damage (Grankvist, 1989) by perturbing cellular redox status, which can lead to disruption of a number of cellular homeostasis mechanisms. Intercellular communication mediated by gap junctions (GJIC) is selectively blocked by gossypol in certain cell types but not others (Ye et al., 1990; Hutchinson et al., 1995; Barhoumi and Burghardt, 1996; Herve ´ et al., 1996), suggesting that this selectivity might contribute to the sensitivity of repro- ductive tissues, kidney, and cardiac cells to gossypol. We have recently employed noninvasive vital cellular im- aging methods with discrete functional endpoints to determine how gossypol affects the mechanisms and chronology of cel- lular injury in the rat liver cell line, Clone 9 (Hutchinson et al., 1995; Barhoumi and Burghardt, 1996). These studies revealed a very rapid and simultaneous production of reactive oxygen species (ROS) and suppression of GJIC that preceded cyto- plasmic acidification. This was followed by a simultaneous TOXICOLOGY AND APPLIED PHARMACOLOGY 151, 311–318 (1998) ARTICLE NO. TO988461 311 0041-008X/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.