Effect of sulfhydryl compounds and glutathione depletion on rat heart myocyte toxicity induced by 4-hydroperoxycyclophosphamide and acrolein in vitro

Neonatal rat heart myocytes were used to study the acute cardiotoxic effects of cyclophosphamide (CTX) metabolites. The myocytes were equi-sensitive to 4-hydroperoxycyclophosphamide (4HPC) and acrolein in vitro with 50% inhibitory concentrations (IC₅₀) of 123 μM and 152 μM, respectively. The parent compound, CTX, was inactive and the myocytes were much less sensitive to the bischloroethylamine alkylator, mechlorethamine (IC₅₀ = 1161 μM). Cellular glutathione (GSH) levels were initially reduced and then transiently elevated following exposure to 4HPC. With acrolein, GSH levels were reduced to unmeasurable levels at all time points. With the addition of 0.5 mM l-buthionine sulfoximine (BSO), myocyte GSH levels were reduced from control values of 120 nmol/mg protein to < 10 nmol/mg protein without causing direct cytotoxicity. However, pretreatment with BSO significantly enhanced the cardiotoxic effects of 4HPC. Finally, two exogenous sulfhydryl-containing compounds were shown to block the myotoxic effects of both 4HPC and acrolein. Mesna and N-acetylcysteine increased at concentrations of 0.01 μg/ml and 0.1 μg/ml roughly increased the IC₅₀ of 4HPC by 2-3-fold. These results suggest that acrolein and 4HPC are equipotent cytotoxins and that a transient depletion in GSH accompanies this toxic effect in cardiac myocytes.