Effects of pyrazole, 4-bromopyrazole and 4-methylpyrazole on mitochondrial function

Abstract

Pyrazole, an inhibitor of alcohol dehydrogenase, has been widely used in studies of ethanol metabolism. Since its specificity has recently been questioned, we studied the effects of pyrazole, methylpyrazole and bromopyrazole on mitochondrial function. These compounds inhibited oxidative phosphorylation, the ATP −32P exchange reaction, and energy dependent and independent calcium uptake. With α-ketoglutarate as substrate, state 3 (coupled) respiration was inhibited, whereas state 4 (resting) respiration was not affected. By contrast, state 4 respiration was stimulated when succinate or ascorbate served as the substrate, while state 3 respiration was slightly inhibited. Regardless of the substrate, the respiratory control ratio was depressed. The activities of succinic dehydrogenase and cytochrome oxidase were stimulated by pyrazole and its derivatives, which may explain the stimulation of succinate and ascorbate oxidation. The inhibitory effects of these compounds were reversed by washing the mitochondria, indicating that no permanent damage to mitochondria had occurred. This is supported by the lack of stimulation of latent ATPase activity and the unchanged barrier to the penetration of NADH. Pyrazole and its derivatives decreased the uptake of citrate and glutamate, but stimulated that of phosphate and malate. Methylpyrazole and bromopyrazole inhibited the transport of reducing equivalents into the mitochondria, as catalyzed by the malate-aspartate, fatty acid and α-glycerophosphate shuttles. The data mandate caution in advocating the therapeutic use of pyrazole or its derivatives in man, and suggest that the use of pyrazole to assess ethanol metabolism and its sequelae in vivo may have limitations.