Differential effects of dihalogenated and trihalogenated acetates in the liver of B6C3F1 mice.

Haloacetates are produced in the chlorination of drinking water in the range 10--100 microg l(-1). As bromide concentrations increase, brominated haloacetates such as bromodichloroacetate (BDCA), bromochloroacetate (BCA) and dibromoacetate (DBA) appear at higher concentrations than the chlorinated haloacetates: dichloroacetate (DCA) or trichloroacetate (TCA). Both DCA and TCA differ in their hepatic effects; TCA produces peroxisome proliferation as measured by increases in cyanide-insensitive acyl CoA oxidase activity, whereas DCA increases glycogen concentrations. In order to determine whether the brominated haloacetates DBA, BCA and BDCA resemble DCA or TCA more closely, mice were administered DBA, BCA and BDCA in the drinking water at concentrations of 0.2--3 g l(-1). Both BCA and DBA caused liver glycogen accumulation to a similar degree as DCA (12 weeks). The accumulation of glycogen occurred in cells scattered throughout the acinus in a pattern very similar to that observed in control mice. In contrast, TCA and low concentrations of BDCA (0.3 g l(-1)) reduced liver glycogen content, especially in the central lobular region. The high concentration of BDCA (3 g l(-1)) produced a pattern of glycogen distribution similar to that in DCA-treated and control mice. This effect with a high concentration of BDCA may be attributable to the metabolism of BDCA to DCA. All dihaloacetates reduced serum insulin levels. Conversely, trihaloacetates had no significant effects on serum insulin levels. Dibromoacetate was the only brominated haloacetate that consistently increased acyl-CoA oxidase activity and rates of cell replication in the liver. These results further distinguish the effects of the dihaloacetates from those of peroxisome proliferators like TCA.

Dichloroacetate and trichloroacetate promote clonal expansion of anchorage-independent hepatocytes in vivo and in vitro.

Dichloroacetate (DCA) and trichloroacetate (TCA) are hepatocarcinogenic by-products of water chlorination and metabolites of several industrial solvents. To determine whether DCA and TCA promote the clonal expansion of anchorage-independent liver cells in vitro, a modification of the soft agar assay (over agar assay) was utilized to quantitate growth and analyze phenotype of anchorage-independent hepatocellular colonies. Hepatocytes from naïve male B6C3F1 mice were isolated and cultured with 0-2.0 mM DCA or TCA over agar for 10 days, at which time colonies of eight cells or more were scored. Both DCA and TCA promoted the formation of anchorage-independent colonies in a dose-dependent manner. Immunocytochemical analysis using a c-Jun antibody demonstrated that colonies promoted by DCA were primarily c-Jun+, whereas TCA-promoted colonies were primarily c-Jun-. This corresponds to the differences in c-Jun immunoreactivity reported in tumors induced by DCA and TCA. Neither DCA nor TCA induced c-Jun expression in hepatocyte monolayers, indicating that these haloacetates selectively affect subpopulations of anchorage-independent hepatocyts. The latency of colony formation was decreased by the concentration of DCA, although the same number of colonies appeared after 25 days in culture at all DCA concentrations used. The plating density of hepatocytes also affected colony formation. At lower cell densities, promotion of colony formation by DCA was significantly reduced. Pretreatment of male B6C3F1 mice with 0.5 g/liter DCA in drinking water resulted in a fourfold increase in in vitro colony formation above hepatocytes isolated from naïve mice, suggesting that DCA is promoting the clonal expansion of anchorage-independent hepatocytes in vivo. Results from this study indicate that DCA and TCA promote the survival and growth of initiated cells. Furthermore, results from over agar assays reflect observations made in vivo, indicating this assay provides a valid means to investigate the mechanism by which chemicals promote clonal expansion of initiated hepatocytes.

Differences in phenotype and cell replicative behavior of hepatic tumors induced by dichloroacetate (DCA) and trichloroacetate (TCA).

Dichloroacetate (DCA) and trichloroacetate (TCA) are two hepatocarcinogenic by-products of water chlorination. To compare the effects of DCA and TCA on cell replication in the nodules and tumors they induce, male B6C3F1 mice were administered 2.0 g/L DCA or TCA in their drinking water for 38 or 50 weeks, respectively. The pretreated mice were then given water containing 0, 0.02, 0.5, 1.0, or 2.0 g/L DCA or TCA for two additional weeks to determine whether cell proliferation in the normal liver or tumors that had been induced by DCA or TCA was dependent on continued treatment. Prior to sacrifice the mice were subcutaneously implanted with mini-osmotic pumps to label DNA in dividing cells with 5-bromo-2'-deoxyuridine (BrdU). Serial sections of nodules/tumors and normal liver were stained immunohistochemically for BrdU, the oncoproteins c-Jun and c-Fos, and hematoxylin and eosin (H & E); or with Periodic acid-Schiff (PAS) stain, BrdU, and H & E, respectively. DCA and TCA transiently stimulated the division of normal hepatocytes relative to rates observed in the livers of control mice. However, at 40 and 52 weeks of treatment, replication of normal hepatocytes was substantially inhibited by DCA and TCA, respectively. Cell division within DCA-induced lesions that were identified macroscopically was significantly higher with increasing dose of DCA administered in the last 2 weeks of the experiment. DCA-induced lesions were found to display immunoreactivity to anti-c-Jun and anti-c-Fos antibodies, were predominantly basophilic, and contained very little glycogen relative to surrounding hepatocytes. In contrast, rates of cell division within TCA-induced altered hepatic foci and tumors were very high and appeared to be independent of continued treatment. TCA-induced lesions did not display immunoreactivity to either c-Jun or c-Fos antibodies. Results from this study suggest that the mechanisms by which DCA and TCA induce hepatocarcinogenesis in the male B6C3F1 mouse differ.