Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone-induced toxicities in rats: comparative study with other mitochondrial uncouplers (2,4-dinitrophenol, OPC-163493 and tolcapone)

Abstract

FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) is known to inhibit oxidative phosphorylation as a protonophore, dissipating the proton gradient across the inner mitochondrial membrane. To understand the toxicity of FCCP, 3-day, 2- and 4-week repeated oral dose studies were performed in male rats. In the 3-day and 2-week repeated dose toxicity studies, observations included salivation, increased body temperature, and dead and moribund animals. Increased liver weight was observed in conjunction with hydropic degeneration and centrilobular necrosis of hepatocytes. In addition, pathological changes were observed in the pancreas, testis, epididymal duct, stomach and parotid gland. Electron microscopic examination revealed mitochondrial pleomorphism in the hepatocytes. Swelling of mitochondria was observed in the alpha cells and beta cells of the pancreas. Dilatation of rough endoplasmic reticulum, Golgi bodies and loss of secretory granules were also noted in the beta cells of the pancreas. FCCP was also compared with three other mUncouplers (DNP, OPC-163493 and tolcapone) with regard to in vitro mitochondrial uncoupling (mUncoupling) activities. FCCP produced the peak ΔOCR (oxygen consumption rate) at the lowest concentration (0.4 μM), followed by OPC-163493, tolcapone, and DNP, based on peak values in ascending order of concentration (2.5, 10, and 50 μM, respectively). Considering the relationship between the mUncoupling activity and toxicity profile of the four mUncouplers, there is no parallel relationship between the in vitro mUncoupling activity and the degree of in vivo toxicity. These findings may contribute to the efficient development of new mitochondrial uncoupler candidates.

Supplementary information: The online version contains supplementary material available at 10.1007/s43188-023-00189-x.

Keywords: Blood toxicity; FCCP; Liver toxicity; Mitochondrial uncoupler; Pancreas toxicity.

Fig. 1

Oxygen consumption rate (OCR) measurement in cultured human liver carcinoma HepG2 cells by an extracellular flux analyzer. ΔOCR evoked by mUncouplers was defined as its mUncoupling activity (inset). ΔOCR are plotted against mUncouplers’ concentrations. Data represent mean ± SD (n = 3–6) *p < 0.05, **p < 0.01, OPC, FCCP, tolcapone, or DNP vs. DMSO-treated group. One-way ANOVA followed by Dunnett’s test was performed. *p < 0.05, **p < 0.01

Fig. 2

Treatment-related changes were observed in the liver for the rats treated with FCCP in the 2-week toxicity study. Control (a), FCCP 40 mg/kg (b). a No abnormalities detected. b Focal necrosis of hepatocytes (N) are seen

Fig. 3

Treatment-related changes were observed in the pancreatic for the rats treated with FCCP in the 2-week toxicity study. Control (a), FCCP 20 mg/kg (b). a No abnormalities detected. b Apoptotic islet cells (arrows) are seen

Fig. 4

Treatment-related changes were observed in the skeletal muscle for the rats treated with FCCP in the 2-week toxicity study. Control (a), FCCP 40 mg/kg (b). a No abnormalities detected. b Degeneration/necrosis of muscle fibers (arrows) are seen

Fig. 5

Treatment-related changes were observed in the testis for the rats treated with FCCP in the 2-week toxicity study. Control (a), FCCP 40 mg/kg (b), in the epididymis, control (c), FCCP 40 mg/kg (d). a No abnormalities detected. b Degeneration/necrosis of germ cells (arrows) are seen. c No abnormalities detected. d Decrease in sperms and exfoliated germinal epithelial cells in the epididymal ducts

Fig. 6

Treatment-related changes were observed in the liver for the rats treated with FCCP in the 2-week toxicity study. Electron micrograph of the hepatocyte. Control (a), FCCP 40 mg/kg (b). a Normal shaped mitochondria are seen. b Abnormal shaped mitochondria (arrows) are seen. (Original magnification × 5000)

Fig. 7

Treatment-related changes were observed in the pancreas for the rats treated with FCCP in the 2-week toxicity study. Electron micrograph of the alpha cell in the islet. Control (a), FCCP 20 mg/kg (b). a No abnormalities detected. b The alpha cell shows degeneration with swelling mitochondria (arrows). Decrease in normal secretory granules are also observed in the alpha cell. (Original magnification × 10,000)

Fig. 8

Treatment-related changes were observed in the pancreas for the rats treated with FCCP in the 2-week toxicity study. Electron micrograph of the beta cell in the islet. Control (a), FCCP 20 mg/kg (b). a No abnormalities detected. b The beta cell shows degeneration with swelling mitochondria (arrows), dilatation of rough endoplasmic reticulum and Golgi bodies. Loss of secretory granules are also observed in the beta cell. (Original magnification × 10,000)