ATP depletion is associated with cytotoxicity of a novel lipid regulator in guinea pig adrenocortical cells.
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 MTT reduction, was observed within 6 hr at concentrations of 0.01 to 10 microM PD132301-2. At 10 microM, NR and MTT 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 > or = 1 microM PD132301-2. Adenine nucleotide analysis after PD132301-2 challenge indicated that ATP depletion preceded loss of viability. At 10 microM 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 PD132301-2-induced toxicity. Fructose protection was blocked by inhibiting glycolysis with 1 mM sodium fluoride. Pretreatment of cultures with 100 microM metyrapone, an inhibitor of cytochrome P-450, did not block cytotoxicity induced by 10 microM PD132301-2, but did block cytotoxicity of 100 microM 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 microM PD132301-2. Thus, ATP depletion resulting from direct inhibition of mitochondrial respiration is a critical early event in adrenocortical cytotoxicity of PD132301-2.
Department of Pathology and Experimental Toxicology, Parke-Davis Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105.
L A
LA
Vernetti
J R
JR
MacDonald
G H
GH
Wolfgang
M A
MA
Dominick
D G
DG
Pegg
D000244:Adenosine Diphosphate / Q000032:analysis; D000255:Adenosine Triphosphate / Q000032:analysis* / Q000096:biosynthesis; D000302:Adrenal Cortex / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D005632:Fructose / Q000494:pharmacology; D006168:Guinea Pigs; D008297:Male; D008797:Metyrapone / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D010101:Oxygen Consumption / Q000187:drug effects; D010671:Phenylurea Compounds / Q000633:toxicity*
D010671:Phenylurea Compounds; D005632:Fructose; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000324:Adrenocorticotropic Hormone; C079534:N-(2,6-bis(1-methylethyl)phenyl)-N'-((1-(4-(dimethylamino)phenyl)cyclopentyl)methyl)urea hydrochloride; D008797:Metyrapone