Date Published: April 27, 2017
Publisher: Public Library of Science
Author(s): Hiroki Yoshioka, Haruki Usuda, Nobuhiko Miura, Nobuyuki Fukuishi, Tsunemasa Nonogaki, Satomi Onosaka, Matias A. Avila.
The aim of this study was to determine whether calcium potentiates acute carbon tetrachloride (CCl4) -induced toxicity. Elevated calcium levels were induced in mice by pre-treatment with cholecalciferol (vitamin D3; V.D3), a compound that has previously been shown to induce hypercalcemia in human and animal models. As seen previously, mice injected with CCl4 exhibited increased plasma levels of alanine aminotransferase, aspartate aminotransferase, and creatinine; transient body weight loss; and increased lipid peroxidation along with decreased total antioxidant power, glutathione, ATP, and NADPH. Pre-treatment of these animals with V.D3 caused further elevation of the values of these liver functional markers without altering kidney functional markers; continued weight loss; a lower lethal threshold dose of CCl4; and enhanced effects on lipid peroxidation and total antioxidant power. In contrast, exposure to V.D3 alone had no effect on plasma markers of liver or kidney damage or on total antioxidant power or lipid peroxidation. The potentiating effect of V.D3 was positively correlated with elevation of hepatic calcium levels. Furthermore, direct injection of CaCl2 also enhanced CCl4-induced hepatic injury. Since CaCl2 induced hypercalcemia transiently (within 3 h of injection), our results suggest that calcium enhances the CCl4-induced hepatotoxicity at an early stage via potentiation of oxidative stress.
Carbon tetrachloride (CCl4) is widely used in experimental animal models of liver failure that mimic human hepatic toxicity. The mechanism of CCl4 hepatotoxicity has been thoroughly studied since 1967, including the use of in vivo models of acute and chronic CCl4 poisoning, ex vivo perfusion of livers, and the use of isolated or cultured hepatocytes [1, 2]. CCl4-induced toxicity is a multifactorial process involving the generation of CCl4-derived free radicals [2–5]. The first step is metabolic activation of CCl4 by CYP2E1, whereby CCl4 is converted to free radicals (trichloromethyl and trichloromethyl peroxy radicals). The second step is binding of these radicals to antioxidant enzymes, including the sulfhydryl (protein thiol) groups of glutathione (GSH). In the third step, these overproduced free radicals increase membrane lipid peroxidation, bind covalently to macromolecules, deplete ATP, and interfere with calcium homeostasis [6–8]. Since sulfhydryl groups are essential elements of the molecular arrangements responsible for the Ca2+ transport across cellular membranes, loss of function of these proteins is expected to impair the capacity of microsomes and mitochondria to regulate cellular calcium levels.
The present study demonstrated that pre-treatment with V.D3 potentiated CCl4-induced hepatotoxicity and enhanced mouse mortality, without increasing renal toxicity and generation of liver fibrosis. Our previous investigation demonstrated that single i.p. injection of mice with a fatal dose of CCl4 (4 g/kg) induced severe hepatotoxicity and moderate renal toxicity [20, 22, 24]; however, the critical target organ that led to mouse death following CCl4 injection was not defined. In the current study, V.D3 potentiation of toxicity was observed only in the liver, as indicated by plasma levels of ALT and AST, biochemical markers of hepatic damage. Although pre-treatment with V.D3 significantly increased renal calcium levels compared to those in animals pre-treated with olive oil, renal calcium content did not differ significantly between mice treated with olive oil + CCl4 and those treated with V.D3 + CCl4 (data not shown). Together, these data suggest that the liver is the primary target organ of acute CCl4 toxicity.