Postulated Carbon Tetrachloride Mode of Action: A Review

Abstract

Under the 2005 U.S. EPA Guidelines for Carcinogen Risk Assessment (1), evaluations of carcinogens rely on mode of action data to better inform dose response assessments. A reassessment of carbon tetrachloride, a model hepatotoxicant and carcinogen, provides an opportunity to incorporate into the assessment biologically relevant mode of action data on its carcinogenesis. Mechanistic studies provide evidence that metabolism of carbon tetrachloride via CYP2E1 to highly reactive free radical metabolites plays a critical role in the postulated mode of action. The primary metabolites, trichloromethyl and trichloromethyl peroxy free radicals, are highly reactive and are capable of covalently binding locally to cellular macromolecules, with preference for fatty acids from membrane phospholipids. The free radicals initiate lipid peroxidation by attacking polyunsaturated fatty acids in membranes, setting off a free radical chain reaction sequence. Lipid peroxidation is known to cause membrane disruption, resulting in the loss of membrane integrity and leakage of microsomal enzymes. By-products of lipid peroxidation include reactive aldehydes that can form protein and DNA adducts and may contribute to hepatotoxicity and carcinogenicity, respectively. Natural antioxidants, including glutathione, are capable of quenching the lipid peroxidation reaction. When glutathione and other antioxidants are depleted, however, opportunities for lipid peroxidation are enhanced. Weakened cellular membranes allow sufficient leakage of calcium into the cytosol to disrupt intracellular calcium homeostasis. High calcium levels in the cytosol activate calcium-dependent proteases and phospholipases that further increase the breakdown of the membranes. Similarly, the increase in intracellular calcium can activate endonucleases that can cause chromosomal damage and also contribute to cell death. Sustained cell regeneration and proliferation following cell death may increase the likelihood of unrepaired spontaneous, lipid peroxidation- or endonuclease-derived mutations that can lead to cancer. Based on this body of scientific evidence, doses that do not cause sustained cytotoxicity and regenerative cell proliferation would subsequently be protective of liver tumors if this is the primary mode of action. To fulfill the mode of action framework, additional research may be necessary to determine alternative mode(s) of action for liver tumors formed via carbon tetrachloride exposure.

Carbon tetrachloride-mediated lipid peroxidation induces early mitochondrial alterations in mouse liver

“…2 Indeed, several studies using radiolabeled CCl 4 reported a modest binding to hepatocyte DNA, [8][9][10] which was not confirmed by the identification of adducts by mass spectrometry. 2 As regards lipids, CCl 3 OO . radical initiates lipid peroxidation by pulling out a hydrogen atom in the vicinity of a polyunsaturated fatty acid double bond.…”

“…Both radicals are highly reactive species that may covalently bind to macromolecules to form nucleic acid, protein and lipid adducts. [1][2][3] However, the evidence for such interactions with liver DNA in vivo is limited. 2 Indeed, several studies using radiolabeled CCl 4 reported a modest binding to hepatocyte DNA, [8][9][10] which was not confirmed by the identification of adducts by mass spectrometry.…”

“…1 A consensus has emerged that CCl 4 toxicity is a mutifactorial process involving the generation of CCl 4 -derived free radicals, lipid peroxidation, covalent binding to macromolecules, loss of calcium homeostasis, nucleic acid hypomethylation and inflammatory cytokines. [1][2][3] Most of the studies related to the very early events of CCl 4 toxicity (ie minutes to few hours) have been performed in vitro on isolated hepatocytes, showing early findings such as swelling of hepatocytes, disorganization of endoplasmic reticulum, mitochondrial morphological injury and increased levels of free cytoplasmic calcium. [4][5][6] Consequently, little is known about the early effects of this organic solvent in vivo, particularly on mitochondrial function.…”

Sparassis crispaAttenuates Carbon Tetrachloride-Induced Hepatic Injury in Rats

“…Lipid peroxidation is accepted to be one of the principal causes of CCl 4 -induced liver injury and is mediated by the production of free radical derivatives of CCl 4 . Therefore, antioxidant activity and/or the inhibition of free radical generation are important in terms of protecting the liver from CCl 4 -induced damage [2]. In general, antioxidative enzymes such as SOD and CAT are easily inactivated by lipid peroxides or reactive oxygen species (ROS), which results in decreased activities of these enzymes in CCl 4 toxicity [1].…”

“…Liver injury induced by CCl 4 is a common model for screening the hepatoprotective activity of drugs because this chemical is a potent hepatotoxin and a single exposure can rapidly lead to severe hepatic necrosis and steatosis [2,14]. Increases in serum AST and ALT levels by CCl 4 have been attributed to hepatic structural damage because these enzymes are normally localized to the cytoplasm and released into the circulation after cellular damage has occurred [14].…”