CCl4

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CCl4
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Executive Summary Information

Compound Carbon tetrachloride
Toxicities Steatosis, cytotoxicity
Mechanisms Carbon tetrachloride is oxidized to the trichloromethyl free radical, from which ensues alkylation of proteins and DNA along with lipid peroxidation. Depletion of ATP precedes acute cytotoxicity. Carcinogenicity is observed on long-term, low dose exposure.
Comments This compound was selected to exemplify free radical-based chemical reactivity and has secondary utility as a standard for steatosis and fibrosis. Acute toxicity due to a solvent effect on cellular membranes can complicate toxicity profiling, especially at concentrations above 0.5 mM.
Feedback Contact Gold Compound Working Group (GCWG)
CCl4
Tetrachloromethane.png
Identifiers
Leadscope Id LS-1373
CAS 56-23-5
ChemSpider 5730
UNII CL2T97X0V0
ChEBI 27385
Pathway DBs
KEGG C07561
Assay DBs
PubChem CID 5943
ChEMBL 44814
Omics DBs
Open TG-Gate 00003
Properties
ToxCast Accepted yes
ToxBank Accepted yes
Target MOA standard for free radical generation
Toxicities Cytotoxicity,Steatosis


In Vivo Data ? Compound Assessment
Adverse Events ? Physiologic Effects

The immediate effect of acute CCl4 exposure by all routes is central nervous system (CNS) depression. If the patient survives this immediate effect, death is usually due to hepatic or renal injury. Adverse effects to other organs are likely to be secondary to CNS, liver, or kidney damage. CCl4 is classified as a potential human carcinogen based on results of studies that indicate ingested CCl4 increases the frequency of liver tumors in experimental animals. It has become a model for the study of agents that cause localized cellular injury via a free-radical mechanism.

Neurologic Effects
Acute exposure to CCl4 may lead to rapid CNS depression. CCl4 rapidly produces a narcotic effect on the brain. Immediate fatalities result either from respiratory depression (due to direct CNS effects) or from cardiac dysrhythmias. In severe cases, autopsy reveals permanent damage to nerve cells with focal areas of fatty degeneration and demyelination, Purkinje cell damage, and patchy pontine necrosis.

Hepatic Effects
Hepatic and renal toxicity are due to biotransformation of CCl4 to toxic metabolites. In acute lethal CCl4 exposures, autopsy reveals marked hepatic steatosis and centrilobar necrosis. The toxic metabolites of CCl4 block formation and release of low-density lipoproteins and deplete hepatic stores of glutathione. In addition, a dramatic increase in calcium concentration occurs in hepatic mitochondria, accompanied by alterations in electrolyte distribution with swelling of hepatic cells and depletion of liver glycogen.
Hepatic injury, which usually manifests after CNS effects have subsided, typically occurs 1 to 4 days after acute exposure. Jaundice develops in about 50% of poisoning cases and typically evolves rapidly. Recovery from acute exposure is usually complete, with no long-term sequelae. Chronic exposure may result in fibrosis or cirrhosis. A decrease in clotting factors (due to acute liver damage) may predispose the patient to hemorrhage.

Renal Effects
Exposure to CCl4 can result in nephritis, nephrosis, and renal failure. Within hours after manifestation of hepatic damage, renal failure may begin and typically reaches a peak in the second week after exposure. Oliguria or anuria may develop by the second to fourth day after exposure with concomitant edema, azotemia, proteinuria, hemoglobinuria, and glucosuria. Hypertension and acidosis may develop. Occasional moderate elevations in white cell counts occur, possibly in response to necrotic liver or kidney injury. Fluid overload can lead to pulmonary congestion and edema. CCl4 also may have direct toxic effects on the lungs. Changes in blood pressure or heart rate are probably secondary to renal effects on fluid and electrolyte retention or to CNS effects on the heart or blood vessels. Kidney failure is the main cause of death in many patients with acute CCl4 exposure.

References:

-Lora E.Fleming (1992)
-EPA IRIS
-Hoehme, S, Brulport, M, Bauer, A, et al. (2010) "Prediction and validation of cell alignment along microvessels as order principle to restore tissue architecture in liver regeneration." PNAS, 107: 10371-10376.

Carcinogenic Effects
Although data on the carcinogenic effects in humans are inconclusive, studies in experimental animals provide convincing evidence that ingestion of CCl4 increases the risk of liver cancer, in particular, hepatomas and hepatocellular carcinomas. Genotoxicity is difficult to demonstrate in vitro. Carcinogenicity in vivo is believed to be caused by regenerative cell proliferation secondary to necrotic tissue damage, at a time when the frequency of genetic damage is increasing and overwhelms DNA-repair mechanisms.

References:

-Mary K. Manibusan , Marc Odin, and David A. Eastmond, “Postulated Carbon Tetrachloride Mode of Action: A Review”, J Environ Sci Health, Part C, 25:3, 185-209 (2007).

Toxicity Mechanisms ? Molecular mechanisms of toxicity have been recently reviewed. Both covalent adduct formation and lipid peroxidation are observed.
-Steatosis and inhibition of protein synthesis require covalent adduct formation, but lipid oxidation is additionally required for cell death. Inhibition of protein and VLDL synthesis result from inhibition of methylation reactions.
- 170 uM CCl4 induces reversible changes in mitochondrial and ER calcium within 15 min.
- Longer exposures of 2 hr lead to depletion of ATP with loss of Ca homeostasis and can progress to cell death. It is the depletion of ATP rather than the influx of Ca that is the cause of cell death.
- TNFα, TGFβ, IL-1, IL-6, and IL-10 cytokines are implicated in activation of stellate cells and fibrosis in vivo.
- The details of toxicity are dependent on O2 concentration, with high O2 being protective.
References:
-Lutz WD Weber, Meinrad Boll, and Andreas Stampfl, "Hepatotoxicity and Mechanism of Action of Haloalkanes: Carbon Tetrachloride as a Toxicological Model", Critical Reviews in Toxicology 33:105-136 (2003).
- Boll, M., Weber, L. W., Becker, E., and Stampfl, A. "Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites", (2001) Z. Naturforsch. (C) 56, 111–121

CCl4 covalently modifies the MTP lipid transporter. This does not inhibit the transporter directly but induces its intracellular proteolysis.

References:

- Xiaoyue Pan, Farah N. Hussain, Jahangir Iqbal,Miriam H. Feuerman, and M. Mahmood Hussain, "Inhibiting Proteasomal Degradation of Microsomal Triglyceride Transfer Protein Prevents CCl4-induced Steatosis", J. Biol. Chem. 282:17078–17089 (2007).

CCl4 tiggers the Bax apoptosis gene. Bcl-2 overexpressing mice are resistant to CCl4-induced liver necrosis and fibrosis.

References:

- Claudia Mitchell, Marie-Anne Robin, Alicia Mayeuf, Meriem Mahrouf-Yorgov, Abdellah Mansouri, Marie Hamard, Dominique Couton, Bernard Fromenty, and He´ le`ne Gilgenkrantz, "Protection against Hepatocyte Mitochondrial Dysfunction Delays Fibrosis Progression in Mice, Am J Pathol (2009), 175:1929–1937.
-Waring, J. F., Ciurlionis, R., Jolly, R. A., Heindel, M., and Ulrich, R. G. (2001). Microarray analysis of hepatotoxins in vitro reveals a correlation between gene expression profiles and mechanisms of toxicity. Toxicol. Lett. 120, 359–368.

CCl4-induced hypermethylation of RNA disrupts protein synthesis.

References:

- Clawson GA. 1989. "Mechanisms of carbon tetrachloride hepatotoxicity", Pathol Immunopathol Res 8:104-112.

In rats, TGFβ activation of stellate cells via p38 and Smad3 pathways is involved in induction of fibrosis. TNFα is also implicated. However, the net effect of TNFα is not clear since it is implicated in fibrosis and inflammation but also in inducing repair in vivo.

References:

- Al-karim Khimji, Rong Shao, and Don C. Rockey, “Divergent Transforming Growth Factor- Signaling in Hepatic Stellate Cells after Liver Injury”, Am J Pathol 2008, 173:716–727.
-Petia P. Simeonova, Randle M. Gallucci, Tracy Hulderman, Robert Wilson, Choudari Kommineni, Murali Rao, and Michael I. Luster, “The Role of Tumor Necrosis Factor-a in Liver Toxicity, Inflammation, and Fibrosis Induced by Carbon Tetrachloride”, Tox Appl Pharm 177, 112–120 (2001).
-R.A. Roberts , N.H. James, S. Cosulich, S.C. Hasmall, G. Orphanides, “Role of cytokines in non-genotoxic hepatocarcinogenesis: cause or effect?”, Toxicology Letters 120 (2001) 301–306.

Therapeutic Target ?

PubMed references

The following resource link will perform a PubMed query for the terms "carbon tetrachloride" and "liver toxicity".
Carbon tetrachloride Search

PK-ADME ? Compound Assessment
PK parameters ? Carbon tetrachloride readily enters the body by inhalation, ingestion, and dermal absorption. Inhalation is the primary route of exposure, with pulmonary absorption in humans estimated to be 60%. The rate of absorption through the gastrointestinal tract is rapid and greatly affected by diet (e.g., fat or alcohol in the gut enhances CCl4 absorption). CCl4 also is absorbed through the skin, though less readily than by the lungs. Dermally, the liquid is more rapidly absorbed than the vapor, and prolonged skin contact with the liquid can result in systemic effects.

Few quantitative studies of CCl4 absorption and distribution in humans have been reported. In experimental animals, CCl4 is dispersed to all organs and tissues proportionate to blood perfusion and lipid content; the brain has the highest levels after inhalation or oral administration. The rate of inhalation absorption in humans decreases as the duration of exposure or dose increases, indicating a saturable metabolic pathway. In humans, approximately 50% to 80% of a dose absorbed by the lungs is exhaled unchanged. Some CCl4 temporarily enters fat tissue. After exposure ceases, CCl4 continues to emerge from fat and is removed by the lungs. About 4% of all metabolized CCl4 is converted directly to CO2 and is exhaled; the remainder forms adducts with proteins and other cellular molecules. The adducts are degraded (half-life about 24 hours) and their products excreted mainly in urine and feces.

References:

-Lora E.Fleming (1992)

PK parameters used to create PBPK models.

References:

- Mumtaz MM, Ray M, Crowell SR, Keys D, Fisher J, and Ruiz P., “Translational Research to Develop a Human PBPK Models Tool Kit-Volatile Organic Compounds (VOCs)”, J Toxicol Environ Health A. (2012) 75(1):6-24.
-Julian I. Delic, Patrick D. Lilly, Alex J. MacDonald, and George D. Loizou (2000) “The Utility of PBPK in the Safety Assessment of Chloroform and Carbon Tetrachloride”, Regulatory Toxicology and Pharmacology 32:144–155.

Therapeutic window ? The workroom standard for CCl4, mandated by the Occupational Safety and Health Administration (OSHA), is a time- weighted average (TWA) of 2 ppm. NIOSH recommends a 60-minute ceiling limit of 2 ppm. The air level considered by NIOSH to be immediately dangerous to life and health (IDLH) is 300 ppm. The EPA maximum contaminant level for CCl4 in drinking water is 5 parts per billion (60 nM).

References:

-Lora E.Fleming (1992)

Metabolically activated ? Bioactivation of CCl4 has become the model for chemical toxicity induced by free radicals—a mechanism of toxic injury similar to that associated with radiation and the aging process. The results of studies with experimental animals indicate that the first step in CCl4 metabolism involves the formation of a trichloromethyl free radical (•CCl3) via the cytochrome P-450 enzyme system . The •CCl3 radical is postulated to bind directly to microsomal lipids and other cellular macromolecules, contributing to the breakdown of membrane structure and disrupting cell energy processes and protein synthesis. Aerobically, the •CCl3 radical radical is trapped by oxygen to form the peroxy radical, •OOCCl3. This radical can induce lipid, protein, and DNA oxidation. The •CCl3 radical also may undergo anaerobic reactions, resulting in the formation of a variety of toxins including chloroform (CHCl3), hexachloroethane (Cl3CCCl3), and carbon monoxide (CO). In aerobic metabolism, the •CCl3 radical can yield trichloromethanol (Cl3COH), a precursor to phosgene (COCl2), which is a potent acylating agent, which is utimately hydrolyzed to form CO2.

References:

-Lora E.Fleming (1992)

Metabolic activation is via CYP2E1 with minor metabolism via CYP2B1, CYP2B2, and possibly CYP3A.

References:

- Lutz WD Weber, Meinrad Boll, and Andreas Stampfi, “Hepatotoxicity and Mechanism of Action of Haloalkanes: Carbon Tetrachloride as a Toxicological Model”, Critical Reviews in Toxicology 33:105-136 (2003).
-T Castillo, DR Koop, S Kamimura, G Triadafilopoulos, and H Tsukamoto, “Role of cytochrome P-450 2E1 in ethanol-, carbon tetrachloride- and iron-dependent microsomal lipid peroxidation.”, Hepatology (1992) 16:992-996.
- Plaa, G. L. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 42–65.
- Weber, L. W., Boll, M., and Stampfl, A. (2003) Crit. Rev. Toxicol. 33, 105–136.
-Frank J. Gonzalez, “The use of gene knockout mice to unravel the mechanisms of toxicity and chemical carcinogenesis”, Toxicology Letters 120 (2001) 199–208.

Omics and IC50 Data ? Compound Assessment
Gene expression profiles known. ? Gene expression for 20 uM CCl4 at 24 h clusters with methotrexate and monocrotaline in rat hepatocytes and in a separate study with allyl alcohol.

References:

- Jeffrey F. Waring, Rita Ciurlionis, Robert A. Jolly, Matthew Heindel, Roger G. Ulrich, “Microarray analysis of hepatotoxins in vitro reveals a correlation between gene expression profiles and mechanisms of toxicity”, Toxicology Letters 120 (2001) 359–368. ·
-Jeffrey F. Waring, Robert A. Jolly, Rita Ciurlionis, Pek Yee Lum, Jens T. Praestgaard, David C. Morfitt, Bruno Buratto, Chris Roberts, Eric Schadt, and Roger G. Ulrich, “Clustering of Hepatotoxins Based on Mechanism of Toxicity Using Gene Expression Profiles”, Toxicology and Applied Pharmacology 175, 28–42 (2001).

Differential gene expression in mouse liver after carbon tetrachloride and acetaminophen administration

References:

-EBI E-GEOD-4874

Gene expression in rats with custom glass microarrays at 3 days-, 14 days- and 28 days-repeated dose experiments

References:

-EBI E-GEOD-16394
-Jianping Huang, Weiwei Shi2, John Zhang3, Jeff W. Chou, Richard S. Paules, Kevin Gerrish5, Jianying Li, Jun Luo, Russell D. Wolfinger, Wenjun Bao, Tzu-Ming Chu, Yuri Nikolsky, Tatiana Nikolskaya, Damir Dosymbekov, Marina O. Tsyganova, Leming Shi8, Xiaohui Fan, J. Christopher Corton, Minjun Chen, Yiyu Cheng, Weida Tong, Hong Fang, and Pierre R. Bushel, “Genomic indicators in the blood predict drug-induced liver injury”, . Pharmacogenomics J. 2010 August ; 10(4): 267–277.
-Hidekuni Inadera, Shinjiro Tachibana, Aya Suzuki, Akiko Shimomura, “Carbon tetrachloride affects inflammation-related biochemical networks in the mouse liver as identified by a customized cDNA microarray system”, Environ Health Prev Med (2010) 15:105–114.
-Jiang Y, Liu J, Waalkes M, Kang YJ. “Changes in the gene expression associated with carbon tetrachloride-induced liver fibrosis persist after cessation of dosing mice.”, Toxicol Sci (2004) 79:404–10.
-Harries, H.; Fletcher, S.; Duggan, C.; Baker, V., “”, Toxicology in Vitro, 15:399-405 (2001).
-Bart A. Jessen, Jennifer S. Mullins, Ann de Peyster, and Gregory J. Stevens, “Assessment of Hepatocytes and Liver Slices as in Vitro Test Systems to Predict in Vivo Gene Expression”, Toxicological Sciences 75, 208–222 (2003).

Proteins involved in lipids and amino acids are downregulated (pyruvate dehydrogenase, senescence marker protein, 3-mercaptopyruvate sulfonotransferase, annexin V), while stress proteins were upregulated (catalase, uricase)

References:

- Michael Fountoulakis, Maria-Cristina de Vera, Flavio Crameri, Franziska Boess, Rodolfo Gasser, Silvio Albertini, and Laura Suter, “Modulation of Gene and Protein Expression by Carbon Tetrachloride in the Rat Liver”, Toxicology and Applied Pharmacology 183, 71–80 (2002).

10 studies from ArrayExpress:

References:

-Carbon tetrachloride and Acetaminophen hepatotoxicity study.
EBI E-GEOD-48744
-Rat liver. Control vs. Chemical treated, 28 days.
EBI E-GEOD-16394
-Rat liver. Control vs. Chemical treated: time course.
EBI E-GEOD-16752
-Rat liver. Control vs. Chemical treated, 14 days.
EBI E-GEOD-16743
-Rat liver. Control vs. Chemical treated, 3 days.
EBI E-GEOD-16340
-Foxf1 enchances pulmonary inflammation and mastocytosis (mouse).
EBI E-GEOD-11112
-Liver Pharmacology and Xenobiotic Response Repertoire (rat).
EBI E-GEOD-8858
-Transcription profiling of primary rat hepatocyte model toxicology.
EBI E-LGCL-7
- Transcription profiling of primary rat hepatocyte model toxicology.
EBI E-LGCL-6
-Non-genotoxic Hepatocarcinogens (rat).
EBI E-GEOD-8251
-Normal and injured liver sinusoidal endothelial cells (mouse).
EBI E-GEOD-1689

Proteomics profiles known. ? References:
-Rie Kikkawa, Masaaki Fujikawa, Toshinori Yamamoto, Yoshimasa Hamada, Hiroshi Yamada, and Ikuo Horii, “In Vivo Hepatotoxicity Study of Rats In Comparison with In Vitro Hepatotoxicity Screening System” J Toxicol Sci 31:23-34 (2006).
Metabonomics profiles known. ?
Fluxomics profiles known. ?
Epigenomics profiles known. ?
Observed IC50 for in vitro cellular efficacy. ?
Observed IC50 for in vitro cellular toxicity studies. ? Dose and time response for cell death in cultured mouse hepatocytes.

References:

- Randall J. Ruch, James E. Klaunig, Norman E. Schultz, Augusta B. Askari, David A. Lacher, Michael A. Pereira, and Peter J. Goldblatt, “Mechanisms of Chloroform and Carbon Tetrachioride Toxicity in Primary Cultured Mouse Hepatocytes” Environmental Health Perspectives Vol. 69, pp. 301-305, 1986.

Toxicity at CCl4 exposures above 0.5 mM in vitro should be considered with caution and are likely to represent non-specific solvent effects.

References:

- LM Berger, H Bhati, B Combes, and RW Estabrock (1986), “CCl4-induced toxicity in isolated hepatocytes: The importance of direct solvent injury”, Hepatology 6:36-45 (1986).
-Lutz WD Weber, Meinrad Boll, and Andreas Stampfi, “Hepatotoxicity and Mechanism of Action of Haloalkanes: Carbon Tetrachloride as a Toxicological Model”, Critical Reviews in Toxicology 33:105-136 (2003).
-David E Johnston and Christine Kroenig, “Mechanism of Early Carbon Tetrachloride Toxicity in Cultured Rat Hepatocytes”, Pharmacol Toxicol (1998) 83:231-239

Physical Properties ? Compound Assessment
Accepted and listed within the ToxCast/Tox21 program. ? No - Not included in ToxCast Phase I and II Chemicals List.
Substance stability. ? The chemical is non-flammable and fairly stable in the presence of air and light.

References:

- Environmental Health Criteria 208 CARBON TETRACHLORIDE World Health Organization Geneva, 1999

Soluble in buffer solution at 30 times the in vitro IC50 for toxicity. ? water (25°C): 0.785 mg/ml (5 mM)

References:

- Environmental Health Criteria 208 CARBON TETRACHLORIDE World Health Organization Geneva, 1999

estimated intrinsic solubility : 0.2877 mg/ml (1.9 mM) estimated solubility in pure water at pH 7: 0.2877 mg/ml estimated solubility in water at pH 7.4: 0.29 mg/ml (Calculations performed using ACD/PhysChem v 12.0)

Solubility in DMSO 100 times buffer solubility. ? DMSO is recommended for preparation of stock solutions.

References:

- Boll, M., Weber, L. W., Becker, E., and Stampfl, A. “Mechanism of carbon tetrachloride-induced hepatotoxicity. Hepatocellular damage by reactive carbon tetrachloride metabolites”, (2001) Z. Naturforsch. (C) 56, 111–121

Vessel binding properties. ?
Vapor pressure. (Non-volatile) ? Vapor pressure: 91 mmHg ( 20 °C)

References:

-Sigma Aldrich (02671) Product details

Estimated vapor pressure (25°C): 99.5 mmHg (Calculation performed using EPI Suite v4.1)


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David Bower, Egon Willighagen
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