A.M. Schumann

The pharmacokinetics and macromolecular interactions of perchloroethylene in mice and rats as related to oncogenicity

Abstract The pharmacokinetic and macromolecular interactions of perchloroethylene were evaluated in B 6 C 3 F 1 mice and Sprague-Dawley rats in an attempt to explain, mechanistically, the sensitivity of the mouse and the resistance of the rat to perchloroethylene-induced hepatocellular carcinoma. When compared to rats, mice were found to metabolize 8.5 and 1.6 times more perchloroethylene per kilogram of body weight following inhalation of 10 ppm or a single oral dose of 500 mg/kg perchloro[ 14 C]ethylene, respectively. Since the initial metabolism of perchloroethylene is an activation process, the increased extent of metabolism in the mouse resulted in a greater extent of irreversible binding of radioactivity in hepatic macromolecules of the mouse compared to that in the rat after inhalation of 10 or 600 ppm or a single oral dose of 500 mg/kg perchloro[ 14 C]ethylene. Repeated oral administration of perchloroethylene for 11 days resulted in histopathological changes in the liver of mice at doses as low as 100 mg/kg/day, while minimal treatment-related effects were observed in the liver of rats only at the 1000 mg/kg/day level. Approximately a twofold increase in hepatic DNA synthesis, indicative of hepatic regeneration, was observed in mice but not in rats after repeated oral administration of perchloroethylene at dose levels which are tumorigenic to mice in lifetime studies. The absence of any pronouced direct interaction of perchloroethylene with hepatic DNA in mice at times of peak hepatic macromolecular binding suggests that hepatic tumors are induced in B 6 C 3 F 1 mice by recurrent cytotoxicity which enhances the spontaneous incidence of liver tumors in this highly susceptible strain of mouse. The implication of these results for hazard assessment is that recurrent tissue damage is necessary for tumors to be induced. Thus, levels of perchloroethylene which do not induce organ toxicity are not likely to pose a carcinogenic risk to man.

Genetic and nongenetic events in neoplasia

It has become increasingly evident that all chemical carcinogens do not act via the same mechanism of tumorigenicity. Based upon the extent of a chemicals interaction with DNA, a general classification scheme of various mutational and nonmutational theories of chemical carcinogenesis is presented. Compounds that directly interact with DNA are classified as genotoxic whereas those that do not interact directly with DNA are classified as epigenetic carcinogens. Under each general heading, several mutational and nonmutational mechanisms of carcinogenesis are believed to be possible. Data are also presented to support the existence of one such mechanism, an epigenetic-mutational theory of chemical carcinogenesis based upon recurrent cytotoxicity. In this case, increased regenerative DNA synthesis in response to tissue injury is believed to result in an enhancement of the normal spontaneous mutation rate, conceivably leading to a cellular transformation. The carcinogenic risk posed by such epigenetic carcinogens appears to differ greatly from that posed by genotoxic carcinogens. Thus, consideration of data concerning the possible mechanism of carcinogenicity of a chemical, along with pharmacokinetic data, will allow a better understanding of bioassay results and a more accurate assessment of carcinogenic risk.

[14C]Methyl chloroform (1,1,1-trichloroethane): pharmacokinetics in rats and mice following inhalation exposure.

Studies on the pharmacokinetics of [14C]methyl chloroform (1,1,1-trichloroethane) in male Fischer 344 rats and B6C3F1 mice were undertaken to characterize the disposition of the inhaled chemical over a wide range of exposure concentrations. The animals were exposed to 150 or 1500 ppm of [14C]methyl chloroform vapor for 6 hr and the elimination of 14C activity was followed for 72 hr. Following exposure to either concentration of methyl chloroform, both species excreted >96% of the total recovered radioactivity during the first 24 hr. The major route of elimination of methyl chloroform was via exhalation of unchanged chemical in the expired air which constituted approximately 94–98% of the total recovered radioactivity in rats and 87–97% in mice at 150 and 1500 ppm, respectively. Mice were found to eliminate methyl chloroform in the expired air more rapidly than did rats. The remaining radioactivity (approximately, 2–13%) was detected as metabolized methyl chloroform in the expired air (14CO2) and as nonvolatile radioactivity in the urine, feces, carcass, and cage wash. Although mice were found to metabolize two to three times more methyl chloroform on a body weight basis, the biotransformation of methyl chloroform was shown to be a saturable, dose-dependent process in both species. Since the biotransformation of methyl chloroform occurred to such a limited extent, saturation of its metabolism did not impact markedly on the distribution or elimination of the parent chemical. The body burden, end-exposure blood level, and tissue concentration of methyl chloroform were found overall to increase in direct proportion with the exposure concentration. [14C]Methyl chloroform was more concentrated in the fat of both species than in the liver or kidneys immediately after exposure. However it was rapidly cleared from the fat so that by 24 hr <2% of the initial radioactivity remained. Thus, methyl chloroform shows little potential for significant bioaccumulation in rodents.

Activation of a Cellular Proto-Oncogene in Spontaneous Liver Tumor Tissue of the B6C3F1 Mouse

The controversy surrounding the interpretation of observed increases in the high spontaneous liver tumor incidence of the B6C3F1 mouse after administration of certain chemical agents necessitates a mechanistic understanding into the nature of tumor development in this particular strain of mouse. Recently, cancer genes (oncogenes) have been detected in the DNA from a variety of human tumors and tumor cell lines. These genes have been implicated to play a role in the transformation of normal cells into cancerous ones. To investigate the role that cellular oncogenes might play in the development of spontaneous liver tumors in the B6C3F1 mouse, DNA was isolated from spontaneously occurring liver tumors and transfected into NIH 3T3 fibroblasts. DNA from this tumor tissue was capable of transforming NIH 3T3 cells from 82% of the animals examined strongly suggesting the presence of an active cellular oncogene. In contrast, DNA isolated from surrounding non-tumorous liver tissue and liver tissue from non-tumor bearing mice did not cause any transformation in the NIH 3T3 assay. These data demonstrate that the active cellular oncogene is not present in the hepatic tissue via a germ-line transmission but is activated only in those cells of the tumor tissue. Experiments using Southern blot hybridization analysis have identified this active cellular oncogene to be a member of the ras oncogene family. Identification of this cellular oncogene will now allow the evaluation of factors which might modify its expression. These future studies will lead to an increased understanding of potential mechanisms by which hepatic tumors are enhanced and should provide more informed estimates of risk for man based on bioassay data generated in this strain of mouse.

Metabolism and disposition of ethylene carbonate in male fischer 344 rats

Ethylene carbonate (EC) has a toxicity profile which resembles that of ethylene glycol (EG). To determine whether the toxicity of EC could be explained on the basis of its metabolism to EG, male Fischer 344 rats were given 200 mg/kg of uniformly labeled [14C]EC in water by gavage and the disposition of the radiolabel was then followed for 72 hr. EC was rapidly metabolized, with approximately 57 and 27% of the administered dose eliminated in the expired air as 14CO2 and in the urine, respectively; the remainder was found in the carcass. Separation of the urinary metabolites using liquid chromatography revealed a single radioactive peak. This metabolite was unequivocally identified as ethylene glycol via gas chromatography-mass spectrometry with the aid of 13C enrichment of the EC dose. Measurement of whole blood levels of EC and EG in rats given 200 mg/kg of EC by gavage revealed blood levels of EG approximately 100-fold higher than the levels of EC in these same animals, with a half-life of EG in blood of 2 hr, indicating rapid conversion of EC to EG. In a separate group of animals administered an equimolar dose of [14C]EG (141 mg/kg), approximately 37% of the dose was expired as 14CO2 and 42% was excreted in the urine as parent compound. When expressed on the basis of the ethanediol moiety, the disposition of EC was identical to that of EG. In view of the rapid and extensive biotransformation of EC to EG and the similarity of the existing (though limited) toxicity data base of EC compared to EG, utilization of the extensive EG systemic toxicity data base for assessing the safety of EC appears justified.

Genetic vs. Nongenetic Chemical Carcinogenesis and Risk Assessment

The fundamental goal of toxicological research is to provide a rational basis for recommending acceptably safe levels of human exposure to potentially harmful agents. Chemically induced cancer is a toxic response that has received primary attention in recent years. The potential lethality of cancer, its generally irreversible nature, and its long latent period have placed carcinogenesis in the forefront of public concern.