Diafenthiuron action: carbodiimide formation and ATPase inhibition

Dinfenthiuron (a thiourea insecticide/acaricide) is proposed by scientists at Ciba-Geigy to act in insects as a mitochondrial adenosine triphosphatase (ATPase) inhibitor following metabolic activation to the corresponding carbodiimide. The present research establishes methods for thin-layer chromatography and high-pressure liquid chromatography analyses of diafenthiuron and its carbodiimide and urea derivatives in enzyme preparations and tissues without artifacts from abiotic decomposition. The carbodiimide, but not the urea, is detected and quantitated as a diafenthiuron metabolite in mouse and housefly microsomal oxidase systems and in the liver, lung, and brain of intraperitoneally (ip)-treated mice. Carbodiimide formation by microsomes in vitro is dependent on the reduced form of nicotinamide adenine dinucleotide phosphate and inhibited by piperonyl butoxide (PB). Another microsomal oxidase metabolite of [3H]diafenthiuron cochromatographs with a major reaction product of hydrogen peroxide with diafenthiuron. This compound, proposed to be diafenthiuron sulfoxide, decomposes abiotically to the corresponding formamidine identified by X-ray crystallography. The carbodiimide, but not the thiourea itself, inhibits mouse liver mitochondrial oligomycin-sensitive ATPase activity in vitro with an IC50 of 0.18 micromolar. Diafenthiuron, its carbodiimide, and two classical ATPase inhibitors (dicyclohexylcarbodiimide and oligomycin A) at lethal ip doses strongly inhibit mouse liver mitochondrial oligomycin-sensitive ATPase in vivo at 1-6 hr after treatment. Although PB inhibits metabolism of the thiourea in vitro, it acts in vivo to increase the rate of poisoning and toxicity of diafenthiuron to mice by 2-fold consistent with the observed elevation in carbodiimide levels in liver, lung, and brain by 2- to 14-fold. Diafenthiuron and its carbodiimide act slowly in ip-treated mice with LD50 values of 15 and 0.3 mg/kg, respectively, and death 3-7 days after treatment; this delayed toxicity (also observed with dicyclohexylcarbodiimide but not oligomycin A) is not correlated with prolonged inhibition of liver ATPase activity.