An alternative mechanism has been suggested to underlie the carcinogenicity of methylene chloride. Casanova and colleagues (1997) proposed that formaldehyde, a metabolite of methylene chloride, causes the formation of DNA-protein cross-links. When hepatocytes from humans and from several other species were incubated with methylene chloride, DNA-protein crosslinks were found only in the mouse samples. That suggests that mice are more susceptible than other species to the carcinogenic effects of methylene chloride.

Chloroform

Chloroform was introduced in 1847 as an inhalation anesthetic (Bruckner and Warren, 2001). It is no longer used as an anesthetic in humans, but it is used in some endodontic procedures and in the administration of drugs for the treatment of some diseases. It is used in the production of chemicals, as an extraction solvent, as a heat-transfer medium in fire extinguishers, and as an intermediate in the preparation of dyes and pesticides.

The metabolism of chloroform (Figure 4.2) is important to its toxicity, because its toxicity is mediated by reactive metabolites that bind to macromolecules (lipids and proteins) of the endoplasmic reticulum. Most chloroform metabolism occurs by oxidation.

A primary target of chloroform is the liver, and liver necrosis is one of the major toxic effects that has been observed in humans and animals after inhalation and oral exposure to high doses of chloroform (Bruckner and Warren, 2001). Leakage of cytoplasmic liver enzymes into the bloodstream, increases in liver triglycerides, and decreases in liver reduced-glutathione levels have been seen in rodents after exposure to chloroform. Other effects seen in rats and mice are centrilobular hepatocyte necrosis, vascular degeneration in midzonal and periportal portions of the liver lobule, and a decrease in the eosinophilia of the centrilobular and midzonal hepatocyte cytoplasm, acute hepatitis, increased liver weights, and diffuse centrilobular swelling.

The kidneys are another major target of chloroform after both inhalation and oral exposure (Bruckner and Warren, 2001). Tubular necrosis, increased kidney weight, increased cell proliferation, and epithelial cell lesions in the proximal convoluted tubules have been seen in mice after inhalation exposure to chloroform. Tubular necrosis, tubular swelling, and increased kidney weight have occurred in rats after oral exposure.

Liver and kidney tumors have also been seen in animals after exposure to chloroform; the tumors depend on the species, strain, and sex of the animal and on the dosage of chloroform (Bruckner and Warren, 2001). Inhalation exposure to chloroform in BDF1 male mice resulted in renal tubular tumors (Nagano et al., 1998). Exposure to chloroform in drinking water resulted in increased hepatic neoplastic nodules in female Wistar rats and adenofibromas in male and female Wistar rats (Tumasonis et al., 1987). Oral exposure studies indicate a dose-related increase in hepatocellular carcinomas in both male and female mice receiving chloroform in corn oil by gavage (NCI, 1976). An increase in renal tumors was seen in male rats receiving chloroform by gavage (tubular adenoma and carcinoma) (NCI, 1976) and in drinking water (renal tubular adenomas and adenocarcinomas) (Jorgenson et al., 1985). Male mice, but not female mice or male and female rats, demonstrated an increase in renal tumors following exposure to chloroform in a toothpaste base (Palmer et al., 1979; Roe et al., 1979). Those carcinogenic effects, however, occur only at high doses. The nonlinearity of the relationship between chloroform dose and tumor formation is consistent with the evidence that chloroform is not genotoxic. The



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