Carcinogenicity of by-products of disinfection in mouse and rat liver.

By-products of disinfection were tested for initiating and/or promoting activity in rat liver by using the rat liver foci bioassay. The assay uses an increased incidence of gamma-glutamyltranspeptidase-positive foci (GGT foci) as an indicator of carcinogenicity. The by-products of disinfection, including chloramine, halogenated humic acids, halogenated ethanes, halogenated acetonitriles, halogenated methanes, halogenated ethylenes, and N-Cl-piperidine, did not initiate GGT foci, which would indicate that they are not capable of initiating carcinogenesis. Chloroform and halogenated benzenes were tested in this assay for their ability to promote the occurrence of GGT foci and tumors initiated by diethylnitrosamine (DENA). Chloroform (1800 ppm in the drinking water) either had no effect or inhibited the occurrence of GGT foci when administered subsequent to a single dose of DENA. However, when the chloroform was administered in drinking water concurrently with weekly doses of DENA, it enhanced the formation of liver tumors. Of 20 halogenated benzenes tested, only 1,2,4,5-tetrachlorobenzene and hexachlorobenzene promoted the occurrence of DENA-initiated GGT foci. Thus in rat liver, the tested by-products of drinking water disinfection did not demonstrate tumor-initiating activity, although a few appeared to possess tumor-promoting activity. Chloroform was also tested for tumor-promoting activity in 15-day-old Swiss mice initiated with ethylnitrosourea (ENU). At weaning they started to receive either 1800 ppm chloroform or 500 ppm sodium phenobarbital (the positive control for tumor promotion) in their drinking water. The mice continued to receive either chloroform or phenobarbital until 51 weeks of age and were sacrificed at 52 weeks of age.(ABSTRACT TRUNCATED AT 250 WORDS)


Introduction
Experimental chemical carcinogenesis is generally considered to be a multistage process consisting of at least the stages of initiation and promotion. Initiationpromotion was first observed in mouse skin, where an increased yield of tumors resulted from repeated applications of croton oil to skin that had been previously exposed to a single subcarcinogenic dose of benzo(a)pyrene (1)(2)(3). Initiation-promotion has been described in tissues other than skin, including liver, bladder, and stomach (4)(5)(6)(7)(8)(9). Initiation occurs when a carcinogen binds to the DNA followed by fixation of the alteration during cellular replication (10,11). The altered genotype can be observed as an altered phenotype. Promotion is believed to result in clonal expansion of the initiated cell (12). This clonal expansion can occur through direct stimulation of cellular replication of the initiated cells or through indirect stimulation of initiated cells as the result of the inhibition of cellular replication in the surrounding noninvolved cells. Clonal expansion results in a focus of cells possessing the altered phenotype.
The administration of ethylnitrosourea (ENU) to 15day-old mice has been shown to be an effective procedure for initiating liver tumors (30,31), and the admin- istration of phenobarbital has been shown to be an effective procedure for promoting mouse hepatocarcinogenesis (31)(32)(33)(34). The administration of chloroform in corn oil by gavage induced liver tumors in B6C3F1 mice (35). Chloroform has been shown to lack or at most possess minimum genotoxic activity (31,(36)(37)(38)(39)(40)(41)(42) and has also been shown to not bind significantly to liver or kidney DNA in rats (43)(44)(45). These findings have led to the proposal that the hepatocarcinogenicity of chloroform in mice results from a nongenotoxic mechanism such as tumor promotion (45,46). We therefore attempted to demonstrate the hepatic tumor-promoting activity of chloroform in mice.

Experimental Design
Rat Liver Foci Bioassay. Unless otherwise stated, these studies followed the protocol outlined in Figure   1. The rats were treated with the test compound 18 or 24 hr after a % partial hepatectomy. The positive control for initiation was DENA. Seven days after initiation, promotion by 500 ppm phenobarbital in the drinking water was begun. After 10 weeks of exposure to phenobarbital, the rats were removed from the exposure to the promoter for 1 week and then sacrificed. At sacrifice, 10 x 10 x 2 mm blocks of liver tissue were removed, frozen in optimum cutting temperature compound, cut into 8-,um sections, and stained for the presence of GGT activity according to the procedure of Rutenburg et al. (47). The sections were analyzed for the incidence of GGT foci and other lesions.
When the ability of a chemical to promote hepatocarcinogenesis was evaluated, DENA was used as the initiator, and phenobarbital was used as the positive control for promotion. Starting 1 week after the administration of the DENA, the test chemical was given over a period of at least 8 to 10 weeks. In one study, promotion was continued until 32 weeks, at which time tumors were observed. As with studies in which the initiating activity of drinking water disinfection byproducts were tested, the incidence of GGT foci and other lesions was determined.
Tumor Promotion Assay of Chloroform in Mouse Liver. The protocol for this experiment has been previously published (31). Briefly, 15-day-old mice were administered by intraperitoneal (IP) injection either 20, 5, or 0 pug ENU/g body weight. After weaning (5 weeks of age), the pups were divided according to sex and were randomly assigned to one of the following groups: group 1, 20 ,ug ENU/g; group 2, 5 jig ENU/g; group 3, 0 ,ug ENU/g; group 4, 20 jig ENU/g followed by 1800 ppm chloroform; group 5, 5 ,ug ENU/g followed by 1800 ppm chloroform; group 6, 0 ,ug ENU/g followed by 1800 ppm chloroform; group 7, 20 ,ug ENU/g followed by 500 ppm sodium phenobarbital; group 8, 5 ,ug ENU/g followed by 500 ppm sodium phenobarbital; and group 9, 0 ,ug ENU/g followed by 500 ppm sodium phenobarbital. The sodium phenobarbital and the chloroform were administered from weaning until the mice were 51 weeks old. The mice were sacrificed at 52 weeks of age, and a complete necropsy was conducted on each mouse.

Results and Discussion
The rat liver foci bioassay has been proposed for use in determining whether a chemical or a complex mixture possesses tumor-initiating activity (16,48). The assay has detected the tumor-initiating activity of 69% of the  (10) aThe protocol for the rat liver bioassay used to determine tumor initiating activity is presented in Fig. 1. bThe time the animals are administered the test substance following a 2/3 partial hepatectomy (PH).
c The results are expressed as the mean ± standard error of the mean. d The number of animals.
carcinogens tested, whereas none of the tested noncarcinogens were active in the assay (49). The only carcinogens that were not detected in the assay for tumorinitiating activity were either nongenotoxic or were direct-acting carcinogens (7,49). Because initiating activity in the rat liver foci bioassay depends on the genotoxic activity of the test substance, the lack of activity by nongenotoxic chemicals in the assay was not unexpected. Table 1 contains a list of the hepatic and nonhepatic carcinogens that have been tested for tumorinitiating activity in our laboratory using the protocol of the experiments described in this report. All these carcinogens exhibited tumor-inititating activity in the rat liver foci bioassay. It is recognized that the rela-tionship between carcinogenesis and GGT foci and other altered foci used as endpoints in the assay has yet to be proved. Even so, the ability of the rat liver foci bioassay to distinguish carcinogens from noncarcinogens and to detect the initiating activity of hepatic and nonhepatic carcinogens makes it suitable for determining whether a chemical or a complex mixture possesses tumor-initiating activity.
The results from the rat liver foci bioassay for the initiating activity of drinking water disinfection byproducts are presented in Table 2. Of all the chemicals found in drinking water, including disinfection by-products that were tested for initiating activity, only 1,2dibromoethane when given 24 hr after a partial hepa- 0.43 ± 0.14 (8) PB 1.38 ± 0.26 (9) 0.05 ± 0.05 (9) aThe protocol for the rat liver foci bioassay used is presented in Fig. 1. The animals received a 2 partial hepatectomy followed 1 day later by 0.3 mmole/kg DENA. Seven days later, they started to receive either 1800 ppm chloroform or 500 ppm phenobarbital in drinking water for a total of 8 weeks. The animals were sacrificed at the termination of the exposure to the promoters.
bThe results are expressed as the mean ± standard error of the mean. c The number of animals.
tectomy exhibited initiating activity. The initiating activity of 1,2-dibromoethane was minimal in comparison to the activity observed with the DENA (the positive control). Milks et al. (50) observed in a similar rat liver foci bioassay that 1,2-dibromoethane did not initiate GGT foci. Our in vivo initiation-promotion bioassay results also conflict with a previous study by Hatch et al. (51) in which chlorinated methanes and ethanes enhanced viral tramsformation of Syrian hamster embryo cells. Some of the chemicals tested were chloroform, 1,2-dichloroethane, and 1,1-dichloroethane. Although the in vitro data indicate that some of the compounds may enhance cell transformation and therefore possibly carcinogenesis, the in vivo data indicate that they do not initiate carcinogenesis. In conclusion, disinfection by-products, with the possible exception of 1,2-dibromoethane, did not induce GGT foci, which indicates that they do not possess significant ability to initiate carcinogenesis. Chloroform administered in corn oil has been shown to induce hepatocellular carcinomas in mice and epithelial tumors in the kidney of male rats (35). The inability of chloroform to bind significantly to liver and kidney DNA (44)(45)(46) and its lack or very low level of genotoxicity (31,(36)(37)(38)(39)(40)(41)(42)(43) has led to the proposal that the carcinogenicity of chloroform results from a nongenotoxic mechanism such as tumor promotion (45,46). Because the mechanism of carcinogenicity (i.e., genotoxic or non-genotoxic) of a substance has major implications in interspecies and low-dose extrapolation, we attempted to demonstrate the nongenotoxic and tumor-promoting mechanism for the carcinogenic activity of chloroform.
The ability of chloroform (CHCl3) to promote the appearance of GGT foci was evaluated in male Fischer 344 rats ( Table 3). The rats received a % partial hepatectomy 1 day before being treated with DENA (0.3 mmole/kg body weight [bw]); 7 days later these rats were started on chloroform (1800 ppm) in drinking water. Phenobarbital (PB) (500 ppm) in drinking water was used as a positive control. Exposure to the promoters was continued for 8 weeks, and the animals were then sacrificed. Phenobarbital enhanced the incidence of GGT foci, whereas chloroform did not alter the incidence of GGT foci/cm2. Chloroform was then tested in male Sprague-Dawley rats for its ability to act as a cocarcinogen (Table 4). Chloroform (1800 ppm in the drinking water) was given concurrently with weekly doses of DENA (8.2 mg/kg body weight). Concurrent administration of phenobarbital (500 ppm) with DENA was used as the positive control. Treatment was continued for either 16 or 32 weeks, at which time the rats were sacrificed. At neither time point did co-administration of chloroform increase the incidence of GGT foci over the incidence observed in animals that received only DENA or over the level in animals that received only the water vehicle control (Table 4). Phenobarbital did increase the incidence of GGT foci when co-administered with DENA. The concurrent administration of either chloroform or phenobarbital with DENA did not affect the incidence of animals with tumors or the number of tumors per animal. Hence, phenobarbital greatly increased the incidence of GGT foci without altering the yield of tumors induced by DENA. This finding indicates that a quantitative relationship between the incidence of GGT foci and the incidence of tumors does not exist in all experimental protocols. Thus, a substance under certain experimental situations can increase the incidence of GGT foci without altering the incidence of tumors.
c Not done.
Treatment with chloroform (1800 ppm) subsequent to the ENU administration decreased the incidence ofliver tumors by approximately one-half. In mice not initiated with ENU, chloroform did not induce altered foci/areas and tumors. Our results demonstrate that instead of promoting the development of liver tumors initiated by ENU administered to neonatal mice, chloroform inhibited the formation of the tumors. These results are in contrast to the NCI-NTP-sponsored bioassay in which liver tumors were found following the administration of chloroform in corn oil by stomach gavage (35). The observed differences between the two studies might be the result of the different strains of mice used or the result of vehicle differences (corn oil versus drinking water) (31). Thus the mechanism by which chloroform administered in corn oil can induce hepatocarcinogenesis in mice and by which it can inhibit hepatocarcinogenesis in mice when administered in drinking water is poorly understood. It is the conclusion of our studies that the proposed ability of chloroform to act as a tumor promoter has still not been proven. Hexachlorobenzene has been shown to be carcinogenic in rodent liver (52)(53)(54). Similar to that of chloroform, its carcinogenic activity has been proposed to result from the nongenotoxic mechanism of tumor promotion. Therefore, a series of halogenated benzenes, including hexachlorobenzene, were tested for their ability to enhance the incidence of DENA-initiated GGT foci in male and female Sprague-Dawley rats ( Table 6). The rats were administered DENA by gavage (0.5 mmole/kg body weight). Groups of ten rats each were then administered one of the various halogenated benzenes by IP injection at 1 and 5 weeks after the administration of DENA. Two weeks after the final dose of the halogenated benzene was administered, the rats were sacrificed. Of the various halogenated benzenes tested, only 1,2,4,5-tetrachlorobenzene and hexachlorobenzene enhanced the occurrence of GGT foci in male rats and only hexachlorobenzene enhanced the occurrence of GGT foci in female rats. These results would indicate that 1,2,4,5-tetrachlorobenzene and hexachlorobenzene can promote tumor development in rats and that the other halogenated benzenes either are not tumor promoters in rat liver, are tumor promoters by virtue of a mechanism independent of GGT foci, or are too weak to be detected by the protocol used. Validation of the tumor-promoting activity of 1,2,4,5-tetrachlorobenzene and hexachlorobenzene still requires that their enhancement of the incidence of tumors initiated by DENA be demonstrated. This paper is being presented in honor of the retirement of Mr. Leland J. McCabe from a long and very successful government service. His guidance and support as Division Director was instrumental in the successful completion of the research described in this paper.
Some of these studies were supported in part by U.S. Environmental Protection Agency contract 68-03-3072 with the Department of Laboratory Animal Medicine at the University of Cincinnati and contract 68-03-3215 with Pathology Associates, Inc. This paper has been subject to the Agency's review and has been approved for publication as an EPA document. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.