Time- and dose-dependent development of potassium bromate-induced tumors in male Fischer 344 rats.

Potassium bromate (KBrO3) is a rodent carcinogen and a nephro- and neurotoxicant in humans. KBrO3 is used in cosmetics and food products and is a by-product of water disinfection by ozonization. KBrO3 is carcinogenic in the rat kidney, thyroid, and mesothelium and is a renal carcinogen in the male mouse. The present study was designed to investigate the relationship of time and dose to bromate-induced tumors in male Fischer 344 (F344) rats and to provide some insight into the development of these tumors. KBrO3 was dissolved in drinking water at nominal concentrations of 0, 0.02, 0.1, 0.2, and 0.4 g/L and administered to male F344 rats as the sole water source for 12, 26, 52, 78, or 100 wk. Renal cell tumors were present after 52 wk of treatment only in the high-dose group. Mesotheliomas developed after 52 wk of treatment on the tunica vaginalis. Mesotheliomas were present at sites other than the testicle after 78 wk of treatment, indicating that their origin was the testicular tunic. Thyroid follicular tumors were present as early as 26 wk in 1 rat each from the 0.1- and 0.2-g/L groups. The present study can be used as a basis for the determination of dose-time relationships of tumor development for a better understanding of KBrO3-induced cancer.

The carcinogenicity of dichloroacetic acid in the male Fischer 344 rat.

The chlorinated acetic acids, in particular dichloroacetic acid (DCA), are found as chlorine disinfection by-products in finished drinking water supplies. DCA has previously been demonstrated to be a mouse liver carcinogen. Chronic studies are described in which male Fischer (F344) rats were exposed to DCA in their drinking water. In the first study, 28 day old rats were exposed to a regimen of 0.05, 0.5 and 5.0 g/l DCA. When animals in the high dose group began to exhibit peripheral hind leg neuropathy, the dose was lowered in stages to 1 g/l. These animals were sacrificed at 60 weeks due to the severe, irreversible neuropathy and were not included in this analysis. The remaining groups of animals were treated for 100 weeks. In the second study, rats were initially exposed to 2.5 g/l DCA which was lowered to 1 g/l after 18 weeks. The mean daily concentration (MDC) of 1.6 g/l was calculated over the 103 week exposure period. Time-weighted mean daily doses (MDD) based on measured water consumption were 3.6, 40.2 and 139 mg/kg bw/day for the 0.05, 0.5 and 1.6 g/l DCA respectively. Based upon the pathologic examination, DCA induced observable signs of toxicity in the nervous system, liver and myocardium. However, treatment related neoplastic lesions were observed only in the liver. A statistically significant increase of carcinogenicity (hepatocellular carcinoma) was noted at 1.6 g/l DCA. Exposure to 0.5 g/l DCA increased-hepatocellular neoplasia, (carcinoma and adenoma) at 100 weeks. These data demonstrate that DCA is an hepatocarcinogen to the male F344 rat. Calculation of the MDD at which 50% of the animals exhibited liver neoplasia indicated that the F344 male rat (approximately 10 mg/kg bw/day) is ten times more sensitive than the B6C3F1 male mouse (approximately 100 mg/kg bw/day). A "no observed effects level' (NOEL) of 0.05 g/l (3.6 mg/kg/day) was the same as for the mouse (3-8 mg/kg/day).

In vivo administration of dichloroacetic acid suppresses spontaneous apoptosis in murine hepatocytes.

Spontaneous apoptosis in hepatocytes of male B6C3F1 mice that received dichloroacetic acid (DCA) in their drinking water for 5-30 days (28-58 days of life) was examined as part of ongoing studies to determine the molecular basis of the hepatocarcinogenicity of this nongenotoxic water chlorination by-product. DCA at 0.5 and 5.0 g/liter, significantly reduced apoptosis relative to untreated controls in a dose-dependent fashion. Regression analysis indicated that apoptosis declined over the 30-day period in the livers of control, age-paired animals receiving no drug. Animals receiving low-dose DCA exhibited a similar, although quantitatively depressed, trend line, whereas animals receiving high-dose DCA showed maximal depression of apoptosis at 5 days, which was sustained throughout the course of the 30-day period. These studies suggest that DCA has the ability to down-regulate apoptosis in murine liver. When taken together with previous data demonstrating DCA-dependent decrease in labeling index in these same livers, these data further support the hypothesis that the carcinogenic mechanism of DCA may involve suppression of the ability of the liver to remove initiated cells by apoptosis rather than by induction of selective proliferation of initiated cells.

Species and strain sensitivity to the induction of peroxisome proliferation by chloroacetic acids.

B6C3F1 mice and Sprague-Dawley rats were provided drinking water containing 6-31 mM (1-5 g/liter) trichloroacetic acid (TCA), 8-39 mM (1-5 g/liter) dichloroacetic acid (DCA), or 11-32 mM (1-3 g/liter) monochloroacetic acid (MCA) for 14 days. TCA and DCA, but not MCA, increased the mouse relative liver weight in a dose-dependent manner. Rat liver weights were not altered by TCA or DCA treatment, but were depressed by MCA. Hepatic peroxisome proliferation was demonstrated by (1) increased palmitoyl-CoA oxidase and carnitine acetyl transferase activities, (2) appearance of a peroxisome proliferation-associated protein, and (3) morphometric analysis of electron micrographs. Mouse peroxisome proliferation was enhanced in a dose-dependent manner by both TCA and DCA, but only the high DCA concentration (39 mM) increased rat liver peroxisome proliferation. MCA was ineffective in both species. Three other mouse strains (Swiss-Webster, C3H, and C57BL/6) and two strains of rat (F344 and Osborne-Mendel) were examined for sensitivity to TCA. TCA (12 and 31 mM) effectively enhanced peroxisome proliferation in all mouse strains, especially the C57BL/6. A more modest enhancement in the Osborne-Mendel (288%) and F344 rat (167%) was seen. Dosing F344 rats with 200 mg/kg TCA in water or corn oil for 10 days increased peroxisome proliferation 179 and 278%, respectively, above the vehicle controls. These studies demonstrate that the mouse is more sensitive than the rat with respect to the enhancement of liver peroxisome proliferation by TCA and DCA and suggest that if peroxisome proliferation is critical for the induction of hepatic cancer by TCA and DCA, then the rat should be less sensitive or refractory to tumor induction.

Phosphorylation of specific rat plasma membrane proteins during promotion of gamma-glutamyl transpeptidase-positive hepatic foci and inhibition by di(2-ethylhexyl)phthalate.

The protein kinase activity of isolated plasma membranes from the livers of rats treated with three promoting regimens was examined using both exogenous proteins and endogenous plasma membrane proteins as substrates. Male rats first received either an initiating dose (30 mg/kg) of the hepatocarcinogen diethylnitrosamine or the 0.9% NaCl solution vehicle by i.p. injection at 18 h following partial hepatectomy. Ten days later, the three promoting regimens were begun. These consisted of 10 weeks of treatment with either (a) a choline-deficient (CD) diet, (b) a choline-supplemented (CS) diet containing 0.06% phenobarbital (PHB) (CS plus PHB), or (c) a CD diet containing 0.06% PHB (CD plus PHB). In addition, two other groups of rats received either (a) a CS diet containing 2% di(2-ethylhexyl)phthalate (DEHP) (CS plus DEHP) or (b) a CD diet containing 2% DEHP (CD plus DEHP). DEHP is a widely used plasticizer and environmental contaminant which we have shown previously inhibits the development of putative preneoplastic gamma-glutamyl transpeptidase (GGT) positive foci in rat liver. Total liver plasma membrane protein kinase activity using both protamine sulfate and histone was cyclic adenosine 3':5'-monophosphate independent and did not appear to be a marker of promotion. Its activity was increased by both DEHP which suppresses the development of GGT positive foci and a CD diet which promotes the appearance of GGT positive foci. The CD, CS plus PHB, and CD plus PHB dietary regimens, which promote the appearance of GGT positive foci, induced the phosphorylation of a Mr 40,000 plasma membrane protein in vitro by endogenous protein kinases. Plasma membranes from DEHP-treated rats did not demonstrate phosphorylation of this Mr 40,000 protein. DEHP dietary treatment also blocked the ability of epidermal growth factor to enhance the phosphorylation of its Mr 175,000 receptor protein in isolated liver plasma membranes. These results suggest that the phosphorylation of a Mr 40,000 plasma membrane protein may be important to the early promotional phase of liver carcinogenesis, and that one mechanism by which DEHP inhibits the emergence of GGT positive foci may be by blocking the response of initiated cells to stimulation by epidermal growth factor.

Concentration-dependent inhibition of development of GGT positive foci in rat liver by the environmental contaminant di(2-ethylhexyl) phthalate.

The ability of di(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer and environmental contaminant, to suppress development of putative preneoplastic lesions in rat liver was evaluated. gamma-Glutamyl transpeptidase-positive (GGT+) foci were initiated in the livers of Sprague-Dawley male rats with a single dose of diethylnitrosamine (DEN) following partial hepatectomy. Promotion of foci was commenced by feeding a choline-deficient diet (CD). A group of control rats was fed a choline-supplemented diet (CS). The ability of DEHP to suppress the emergence of GGT+ foci was evaluated by feeding additional groups of rats the CD diet containing either 0.1%, 0.5%, 1.0% or 2.0% DEHP. The CD diet promoted the number of GGT+ foci above levels in control livers. Inclusion of the plasticizer to the levels of 0.5%, 1.0% and 2.0% in the CD diet effectively inhibited the appearance of the foci. However, DEHP was unable to inhibit the promoting effect of the CD diet at a concentration of 0.1%. DEHP's ability to block development of GGT+ foci correlated with its ability to increase liver weight and to induce carnitine acetyltransferase (EC 2.3.1.7), a marker of peroxisome proliferation.

Inhibition of phenobarbital and dietary choline deficiency promoted preneoplastic lesions in rat liver by environmental contaminant di(2-ethylhexyl)phthalate.

The effect of di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer and environmental contaminant, on the emergence of gamma-glutamyltranspeptidase positive (GGT+) preneoplastic foci in the liver of rats fed promoting diets was studied. GGT+ foci were initiated in the liver of Sprague--Dawley male rats with a single dose of diethylnitrosamine (DEN) following partial hepatectomy. One series of control rats received saline vehicle alone. Promotion of foci was commenced by feeding: (1) a choline-deficient diet (CD); (2) a choline-supplemented diet (CS) containing 0.06% phenobarbital (CS + PHB); or (3) a CD diet containing 0.06% phenobarbital (CD + PHB). In the absence of initiation by DEN, dietary treatments did not increase the number of GGT+ foci. In rats receiving DEN, each promoting regimen effectively increased the number of GGT+ foci above levels in control rats fed only the choline-supplemented diet. Inclusion of the plasticizer at a level of 2% in each of the dietary promotion treatments, however, effectively inhibited the appearance of the foci.

Inhibition of development of preneoplastic lesions in the livers of rats fed a weakly carcinogenic environmental contaminant.

The effects of di(2-ethylhexyl)phthalate (DEHP), a widely used plasticizer and environmental contaminant, on the emergence of gamma-glutamyltrans-peptidase positive (GGT+) preneoplastic foci in the liver was investigated. Sprague--Dawley male rats initiated with diethylnitrosamine (DEN) following partial hepatectomy were placed on: (1) a choline supplemented diet (CS); (2) a CS diet containing 2% DEHP (CS + DEHP); (3) a choline deficient diet (CD); (4) a CD diet containing a 0.06% phenobarbital (CD + PHB); or (5) a CD diet containing 2% DEHP (CD + DEHP). Rats maintained on the CS + DEHP diet for 5 and 10 weeks showed no increase in GGT + foci. The plasticizer effectively inhibited the appearance of the preneoplastic foci when it was included with the CD diet.