ATP Depletion Is Associated with Cytotoxicity of a Novel Lipid Regulator in Guinea Pig Adrenocortical Cells

Abstract

A novel lipid regulator (PD132301-2) produces degeneration and necrosis of adrenal fasciculata in guinea pigs. Primary adrenocortical cell cultures from male Hartley guinea pigs were utilized to investigate potential mechanisms of this toxicity. Concentration-dependent loss of viability, measured by neutral red (NR) accumulation or MIT reduction, was observed within 6 hr at concentrations of 0.01 to 10 μM PD132301-2. At 10 μM, NR and MIT indices were 50% of those of control after 6 hr exposure. Maximal decreases in NR and MTT indices to 20% of control values occurred by 24 hr at ≥ l μM PD132301-2. Adenine nucleotide analysis after PD132301-2 challenge indicated that ATP depletion preceded loss of viability. At 10 μM PD132301-2, ATP levels were 80% of those of control after 30 min and 25% of those of control after 6 hr. Supplementation of glucose-free buffer with 20 mM fructose protected adrenocortical cells from PDI32301-2-induced toxicity. Fructose protection was blocked by inhibiting glycolysis with 1 mM sodium fluoride. Pretreatment of cultures with 100 μM metyrapone, an inhibitor of cytochrome P-450, did not block cytotoxicity induced by 10 μM PD132301-2, but did block cytotoxicity of 100 μM o,p′-DDD. In adrenocortical mitochondrial preparations, inhibition of respiration by PD132301-2 was site II-specific. Both state 3 and state 4 respiration were inhibited 50-75% at 1-30 μM PD132301-2. Thus, ATP depletion resulting from direct inhibition of mitochondrial respiration is a critical early event in adrenocortical cytotoxicity of PD132301-2.