The carcinogenicity of trichloroethylene and its metabolites, trichloroacetic acid and dichloroacetic acid, in mouse liver.

Trichloroethylene (TCE) has previously been shown to be carcinogenic in mouse liver when administered by daily gavage in corn oil. The metabolism of TCE results, in part, in the formation of trichloroacetic acid (TCA) as a major metabolite and dichloroacetic acid (DCA) as a minor metabolite. These chlorinated acetic acids have not been shown to be genotoxic, although they have been shown to induce peroxisome proliferation. Therefore, we determined the ability they have been shown to induce peroxisome proliferation. Therefore, we determined the ability of TCE, TCA, or DCA to act as tumor promoters in mouse liver. Male B6C3F1 mice were administered intraperitoneally 0, 2.5, or 10 micrograms/g body wt ethylnitrosourea (ENU) on Day 15 of age. At 28 days of age, the mice were placed on drinking water containing either TCE (3 or 40 mg/liter), TCA (2 or 5 g/liter), or DCA (2 or 5 g/liter). All drinking waters were neutralized with NaOH to a final pH of 6.5-7.5. The animals were killed after 61 weeks of exposure to the treated drinking water (65 weeks of age). Both DCA and TCA at a concentration of 5 g/liter were carcinogenic without prior initiation with ENU, resulting in hepatocellular carcinomas in 81 and 32% of the animals, respectively. DCA and TCA also increased the incidence of animals with adenomas and the number of adenomas/animal in those animals that were not initiated with ENU. While 2.5 micrograms/g body wt ENU followed by NaCl in the drinking water resulted in only 5% of the animals with hepatocellular carcinomas, 2.5 micrograms/g body wt ENU followed with 2 or 5 g/liter DCA resulted in a 66 or 78% incidence of carcinoma, respectively, or, followed with 2 or 5 g/liter TCA, resulted in a 48% incidence at either concentration. None of the untreated animals had hepatocellular carcinomas. Therefore our results demonstrate that DCA and TCA are complete hepatocarcinogens in B6C3F1 mice.

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)

Haloacetonitriles: metabolism, genotoxicity, and tumor-initiating activity.

Haloacetonitriles (HAN) are drinking water contaminants produced during chlorine disinfection. This paper evaluates metabolism, genotoxicity, and tumor-initiating activity of these chemicals. The alkylating potential of the HAN to react with the electrophile-trapping agent, 4-(p-nitrobenzyl)pyridine, followed the order dibromoacetonitrile (DBAN) greater than bromochloroacetonitrile (BCAN) greater than chloroacetonitrile (CAN) greater than dichloroacetonitrile (DCAN) greater than trichloroacetonitrile (TCAN). When administered orally to rats, the HAN were metabolized to cyanide and excreted in the urine as thiocyanate. The extent of thiocyanate excretion was CAN greater than BCAN greater than DCAN greater than DBAN much greater than TCAN. Haloacetonitriles inhibited in vitro microsomal dimethylnitrosamine demethylase (DMN-DM) activity. The most potent inhibitors were DBAN and BCAN, with Ki = 3-4 X 10(-5) M; the next potent were DCAN and TCAN, with Ki = 2 X 10(-4) M; and the least potent inhibitor was CAN, with Ki = 9 X 10(-2) M. When administered orally, TCAN, but not DBAN, inhibited hepatic DMN-DM activity. The HAN produced DNA strand breaks in cultured human lymphoblastic (CCRF-CEM) cells. TCAN was the most potent DNA strand breaker, and BCAN greater than DBAN greater than DCAN greater than CAN, which was only marginally active. DCAN reacted with polyadenylic acid and DNA to form adducts in a cell-free system; however, the oral administration of DBAN or DCAN to rats did not result in detectable adduct formation in liver DNA. None of the HAN initiated gamma-glutamyltranspeptidase (GGT) foci when assayed for tumor-initiating activity in rat liver foci bioassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-response relationship of phenobarbital promotion of diethylnitrosamine initiated tumors in rat liver.

The dose-response relationship was determined in rats for the enhancement by phenobarbital of diethylnitrosamine (DENA)-initiated neoplastic nodules and hepatocellular carcinomas. Male Sprague-Dawley rats received a single oral dose of either 80 mg/kg DENA or water. Seven days later, the animals were divided into groups that started to receive 0, 62.5, 125, 250, 500 or 1000 ppm sodium phenobarbital in the drinking water. Animals from each group were killed at 48 and 70 weeks after the DENA. No significant difference was observed in the low response of neoplastic nodules among the DENA-initiated groups. The incidence of DENA-initiated hepatocellular carcinoma was enhanced at 70 weeks by 250, 500 and 1000 ppm sodium phenobarbital but not by 62.5 or 125 ppm sodium phenobarbital. Equal enhancement of the incidence of hepatocellular carcinomas was obtained with 250, 500 and 1000 ppm sodium phenobarbital. In non-DENA-initiated rats, phenobarbital did not induce neoplastic nodules or hepatocellular carcinomas. Our results suggest that a daily dose of at least 250 ppm sodium phenobarbital is required in order for it to exert tumor promoting activity.

Effect of phenobarbital on the development of liver tumors in juvenile and adult mice.

The effect of long-term exposure to phenobarbital (CAS: 50-06-6) subsequent to tumor initiation on the development of liver tumors in BALB/c and (C57BL/6 X C3H/Anf)F1 (B6C3F1) mice was determined. In male B6C3F1 mice that received either 15 or 45 ppm diethylnitrosamine [(DENA) CAS: 55-18-5] between 6 and 10 weeks of age, subsequent treatment with 500 ppm sodium phenobarbital in the drinking water resulted in the promotion of liver tumors. However, in male B6C3F1 mice initiated on day 15 of age with 25 mg DENA/kg, beginning long-term treatment of 500 ppm sodium phenobarbital at 4 weeks of age inhibited the development of liver tumors, whereas in male BALB/c mice initiated with 25 mg DENA/kg on day 15 of age, beginning the long-term treatment with 500 ppm sodium phenobarbital at 4 weeks of age promoted the development of liver tumors. Hence phenobarbital can either enhance or inhibit the formation of liver tumors, depending both on the mouse strain used and the animal's age at the start of exposure.

The effect of single versus split doses of diethylnitrosamine on the induction of gamma-glutamyltranspeptidase-foci in the livers of adult and juvenile rats.

The induction of gamma-glutamyltranspeptidase (GGT)-foci by single and by split doses of diethylnitrosamine (DENA) was evaluated in the livers of juvenile and young adult male, Sprague-Dawley rats. A single dose of DENA was administered at either 32, 41, 46, 52 or 56 days of age and followed by a promoting regimen of sodium phenobarbital in the drinking water. In 32 days old rats, DENA resulted in a higher incidence of GGT-foci/cm3 than was observed when it was administered to 41 or 46 day old rats. The same dose of DENA was inactive in 52 or 60 day old rats. In rats initiated with DENA on day 32 of age, starting the phenobarbital promotion at 41 days of age resulted in a higher incidence of GGT-foci than when the phenobarbital treatment was begun at 60 days of age. When the dose of DENA was split into five daily doses of DENA starting on day 32 of age, the incidence of GGT-foci/cm3 was equivalent to the single dose given on day 32 of age. However, when the five daily doses were started on 48 days of age, the incidence of GGT-foci/cm3 was less than the five doses of DENA started on day 32 of age. Hence, juvenile rats were more sensitive to both single and split doses of DENA than adult rats. The dose-response relationship was also determined in juvenile rats for a single and for up to 64 daily doses of 3 mg/kg DENA started on day 32 of age. The dose-response relationship for a single dose of 3-96 mg/kg DENA was equivalent to the relationship of one to 32 daily 3 mg/kg doses. Thus, low daily doses of DENA were additive when started in juvenile rats. Neither a threshold nor a plateau was obtained for the dose-response relationship of DENA initiation of GGT-foci.

Effect of phenobarbital on the gamma-glutamyltranspeptidase activity and the remodeling of nodules induced by the initiation-selection model.

The effect of subsequent administration of phenobarbital on the gamma-glutamyltranspeptidase (GGT) activity and on the remodeling of nodules induced by the Solt-Farber procedure was examined in rats. GGT-nodules were initiated by diethylnitrosamine (DENA) followed by selection with 2-acetylaminofluorene (2AAF) in the diet and a partial hepatectomy (PH). Phenobarbital (500 ppm in the drinking water), administered to rats that were previously treated according to the Solt-Farber procedure, (1) increased the persistence of the GGT-nodules, (2) increased the percentage of the liver occupied by GGT positive cells, (3) increased the area of GGT activity per nodule and (4) increased the incidence of eosinophilic lesions. Subsequent treatment with phenobarbital did not alter the incidence of either GGT-nodules or hepatocellular carcinoma. Thus, phenobarbital increased the GGT activity of nodules induced by the Solt-Farber procedure and slowed both the loss of GGT activity by these nodules and their concurrent remodeling, but had no effect on the occurrence of cancer.

Effect of subsequent treatment of chloroform or phenobarbital on the incidence of liver and lung tumors initiated by ethylnitrosourea in 15 day old mice.

The effect of subsequent administration of chloroform or phenobarbital on the incidence of ethylnitrosourea (ENU) initiated liver and lung tumors was investigated. Fifteen day old Swiss mice were administered ENU, and at weaning they started to receive either 1800 p.p.m. chloroform or 500 p.p.m. sodium phenobarbital in their drinking water. The mice continued to receive either chloroform or phenobarbital until 51 weeks of age. They were sacrificed at 52 weeks of age. ENU at 5 and 20 mg/kg, caused a dose-dependent increase in liver and lung tumors. The male mice were more sensitive to the induction of liver tumors, while no sex preference was observed for the induction of lung tumors. In male mice chloroform inhibited, while in female and male mice phenobarbital promoted spontaneous and ENU-induced liver tumors. Subsequent treatment with either chloroform or phenobarbital did not affect the incidence of ENU-induced lung tumors. In conclusion, when administered in the drinking water, chloroform inhibited while phenobarbital promoted hepatocarcinogenesis in mice.

Effect of coadministration of phenobarbital sodium on N-nitrosodiethylamine-induced gamma-glutamyltransferase-positive foci and hepatocellular carcinoma in rats.

The effect of concurrent administration of phenobarbital on the hepatocarcinogenicity of N-nitrosodiethylamine (diethylnitrosamine; DENA) in rats was investigated by determination of the incidence of gamma-glutamyltransferase (gamma-glutamyltranspeptidase) (GGT)-positive foci and liver tumors. Male outbred Sprague-Dawley rats received either a weekly oral dose of DENA (0.08 mol/kg), phenobarbital sodium (500 ppm) in their drinking water, or DENA and phenobarbital sodium concurrently. After 16 weeks, only the animals treated concurrently with DENA and phenobarbital sodium had GGT-positive foci (3.65 foci/cm2). At 30 weeks, the group treated with DENA and phenobarbital sodium exhibited more foci (23.6 foci/cm2) compared to the group that received only DENA (3.08 foci/cm2). The average size of foci in both of the DENA-treated groups was the same. The tumors in the group that received DENA plus phenobarbital sodium showed a greater incidence of GGT activity compared to the tumors in the DENA group. Under the conditions of this study the incidence of GGT-positive foci did not predict the incidence of hepatocellular carcinomas.

Effects of strain, sex, route of administration and partial hepatectomy on the induction by chemical carcinogens of gamma-glutamyltranspeptidase foci in rat liver.

The incidence of gamma-glutamyltranspeptidase (GGT)-positive foci induced by 0.3 mmol/kg diethylnitrosamine (DENA) followed by promotion with 500 ppm sodium phenobarbital in drinking water and was the same in Fischer 344, Sprague-Dawley and Wistar-Lewis rats. There was no difference in the level of GGT-foci initiated by DENA, 7,12-dimethylbenz[a]-anthracene (DMBA), or 1,2-dimethylhydrazine (DMH) followed by promotion with phenobarbital with respect to sex or route of administration including gavage and intraperitoneal injection. Maximal stimulation by partial hepatectomy of DENA initiation of GGT-foci occurred when the DENA was administered 18 h after the operation. Our results indicate that the optimal protocol for the rat liver foci assay consists of using partial hepatectomized rats of 1 of the 3 strains and of either sex. The test substance should be administered by either gavage or intraperitoneal injection so that maximal DNA binding coincides with the maximal rate of DNA replication resulting from partial hepatectomy.