Chlorpromazine protection against Ca(2+)-dependent and oxidative cell injury. Limitations due to depressed mitochondrial function.

Chlorpromazine (CPZ), a phenothiazine, demonstrated both cytoprotective and toxic effects on cardiomyocytes. CPZ markedly reduced cytotoxicity caused by two toxic challenges, each with a distinct cytotoxic mechanism. Lethal cell injury was induced in cultured neonatal cardiomyocytes by either: (1) ionomycin, a Ca2+ ionophore that caused Ca(2+)-dependent cell injury; or (2) ethacrynic acid (EA), a glutathione (GSH) depletor that killed cells primarily via peroxidative damage. Pretreatment with 50 microM CPZ reduced the extent of ionomycin-induced cell death, as measured by lactate dehydrogenase (LDH) leakage, but enhanced the loss of intracellular ATP and collapsed the mitochondrial transmembrane potential (delta psi). In EA-treated cultures, 50 microM CPZ also lowered LDH leakage and diminished the peroxidative damage responsible for the cytotoxicity, but again enhanced the loss of intracellular ATP and collapsed the delta psi. CPZ protection was incomplete and limited to a narrow concentration range that was essentially identical for both toxic challenges. Maximum protection was observed with 50 microM CPZ, yet the amount of residual damage was similar to the degree of injury caused by a mitochondrial uncoupler, carbonylcyanide-m-chlorophenylhydrazone alone. In the absence of either challenge, 50 microM CPZ did not affect cellular energy status or kill the cells, but a higher concentration of CPZ (150 microM) did deenergize unchallenged cardiomyocytes. These data demonstrate that CPZ can reduce cytotoxicity caused by either Ca(2+)-dependent events or oxidative stress. However, even at an optimally protective level, CPZ in combination with either ionomycin or EA deenergized the cells, although neither toxic challenge nor 50 microM CPZ alone seriously affected delta psi. It would appear that intracellular perturbations induced by either challenge promote a depression of mitochondrial function by CPZ, which limits the protective action of the drug. Since both of the challenges used contain toxicologic features exhibited by a wide variety of toxic insults, results of this study have both mechanistic and clinical implications.

Comparative toxicity and mutagenicity of N-hydroxy-2-acetylaminofluorene and 7-acetyl-N-hydroxy-2-acetylaminofluorene in human lymphoblasts.

Exponentially growing TK6 human lymphoblasts were exposed to either 0-50 microM N-hydroxy-2-acetylaminofluorene (N-OH-AAF) or 0-10 microM 7-acetyl-N-hydroxy-2-acetylaminofluorene (7-acetyl-N-OH-AAF) in both the absence and presence of a partially purified preparation of hamster-liver N-arylhydroxamic acid N,O-acyltransferase (AHAT). Neither N-arylhydroxamic acid was toxic to the lymphoblasts, nor mutagenic at the thymidine kinase (tk) locus, in the absence of AHAT over the concentration range examined. In the presence of AHAT, an enzyme that activates N-arylhydroxamic acids to electrophilic N-acetoxyarylamine intermediates, both compounds caused toxicity and mutagenicity in TK6 cells. The 7-acetyl-N-OH-AAF was approximately 10-fold more toxic and mutagenic than the unsubstituted N-OH-AAF. These data demonstrate that metabolism of these N-arylhydroxamic acids, presumably to N-acetoxyarylamine intermediates by AHAT, is a key event in the biological activity of these agents. In addition, the presence of electron-withdrawing 7-acetyl substituent that is thought to stabilize N-acetoxy intermediates, appears to enhance the biological activity of the unsubstituted N-OH-AAF.