Search

Studies on hepatocyte primary cultures have suggested that loss of expression of the placental form of glutathione S-transferase in peroxisome proliferator (PP)-induced hepatocarcinogenesis is due to inhibition of glutathione S-transferase P (GSTP) transcription by the PPs. In the present study, we have analyzed the effect of a PP, ciprofibrate, and of another ligand of nuclear receptors, 3,3', 5-triiodo-L-thyronine (T3), on GSTP mRNA and protein levels in an in vivo model where GSTP expression was induced in Wistar rats by pre-treatment with a single dose of lead nitrate. Results indicate that administration of ciprofibrate or T3, immediately after lead nitrate treatment, did not exert any inhibitory effect on GSTP mRNA and protein levels, as revealed by both Western and immunohistochemical analysis. The results indicate that PPs do not inhibit hepatocyte GSTP expression induced in vivo by lead nitrate and suggest that inhibition of GSTP expression by PPs may not necessarily be the cause for the rapid disappearance of GSTP-positive preneoplastic lesions observed after a short term exposure to these agents.

In this study we examined the ability of peroxisome proliferators to induce oxidative DNA damage in the form of 8-hydroxydeoxyguanosine (OHdG). We studied the hypolipidemic drug ciprofibrate, which is among the most potent and efficacious of the peroxisome proliferators, and perfluorodecanoic acid (PFDA), which is an inhibitor of peroxisomal beta-oxidation. Rats were fed 0.01% ciprofibrate in the diet, or were injected with PFDA at doses of 3 or 10 mg/kg every 14 days (controls and ciprofibrate-fed rats were given equivalent doses of corn oil). Rats were maintained for 10 days, 24 days, 6 weeks, 26 weeks, or 54 weeks. DNA was isolated from the liver at these times and hydrolysed to nucleosides, and the levels of OHdG as well as normal nucleosides were analysed by high-performance liquid chromatography with electrochemical detection. Ciprofibrate increased OHdG concentrations at all times except for the initial 10-day timepoint. Both doses of PFDA increased OHdG levels at all times except the last timepoint, at which only the higher dose produced a significant increase. This study shows that both ciprofibrate and PFDA induce oxidative DNA damage in the form of OHdG. Furthermore, the inhibition of peroxisomal beta-oxidation by PFDA does not affect the development of OHdG.

This study was conducted to determine if the peroxisome proliferator perfluorodecanoic acid (PFDA) has promoting activity in two-stage hepatocarcinogenesis. Because PFDA is a non-competitive inhibitor of the peroxisomal bifunctional enzyme and thus inhibits the peroxisomal β pathway, we hypothesized that PFDA may not have promoting activity as do other peroxisome proliferators, because hydrogen peroxide production is inhibited. Twenty-four hours after partial hepatectomy, female Sprague-Dawley rats were given an initiating dose of 10 mg/kg diethylnitrosamine by gavage. The rats were divided into five groups that received monthly i.p. injections of 0.0, 0.05, 0.50 or 5.0 mg/kg PFDA in corn oil or were placed on diets that contained either 0.01% ciprofibrate or 0.05% phenobarbital for 9 or 18 months. Both ciprofibrate and the highest dose of PFDA increased the activity of the peroxisomal enzyme fatty acyl CoA oxidase. PFDA treatment did not increase the tumor incidence or the number of altered hepatic foci at 9 or 18 months, although the mean volume of foci was increased at 9 months. Ciprofibrate increased the incidence of hepatocellular carcinomas at 18 months but did not increase the number or volume of altered hepatic foci at 9 or 18 months. Phenobarbital increased the number and volume of foci but did not influence the tumor incidence. The results of this investigation indicate that PFDA is not a promoter of hepatocarcinogenesis.

The effect of two members of the hypolipidemic medicine class of hepatocarcinogenic peroxisome proliferators on proliferation of hepatocytes in primary mixed cultures with liver epithelial cells was studied. Rat hepatocytes present in primary mixed cultures with rat liver epithelial cells were maintained for 3 months retaining their differentiated characteristics and proliferative potential. Hepatocyte clusters in mixed cultures stained positive for albumin, indicating that they retained some metabolic functions. Furthermore, in mixed cultures exposed to 0.2 mM clofibric acid or ciprofibrate for 3 months, hepatocytes were engaged in proliferation as shown by the expression of proliferating cell nuclear antigen (PCNA) and the presence of mitotic figures. This in vitro system could be useful to obtain more information about responses of liver cells during prolonged exposure to peroxisome proliferators.

It is hypothesized that hepatic tumors in rats induced by peroxisome proliferators is dependent on peroxisome proliferative effect of these compounds and the resulting oxidative stress. However, it is argued that since these compounds also induce tumors in pancreas and testes, the two organs in which there is no proliferation of peroxisomes, the carcinogenic effect is unlikely to be related to oxidative stress. To clarify this controversy we have systematically analyzed the incidence of pancreatic acinar cell foci and nodules, and testicular Leydig cell tumors in ciprofibrate treated and control rats. In animals treated with 0.025% ciprofibrate for 22 months the incidence of Leydig cell tumors and acinar cell lesions was 100% and 66%, respectively. In age-matched controls the incidence of testicular and pancreatic lesions was 93% and 66%, respectively. These findings clearly demonstrate that the Leydig cell tumors and pancreatic lesions develop spontaneously and are not induced by ciprofibrate.

To determine if the carcinogenic potential of peroxisome proliferators is dependent upon their ability to induce cell proliferation, we have investigated the extent of cell proliferation in the livers of rats fed ciprofibrate, a peroxisome proliferator. Male rats were maintained on a diet containing ciprofibrate (0.025% w/w) and killed at selected intervals following 1 week of continuous [3H]thymidine labeling. Evaluation of labeling indices demonstrated a significant increase in cell proliferation during the first week but not in rats killed at the end of 5 and 20 weeks of treatment. Increases in hepatocyte nuclear labeling were found at 40 and 70 weeks of ciprofibrate administration which coincided with the appearance in livers of putative preneoplastic and neoplastic lesions. In a short-term feeding study, ciprofibrate and ethoxquin were fed to rats at a dietary concentration of 0.025% and 0.5%, respectively, either alone or in combination for 7 days. Ciprofibrate and ethoxyquin either alone or in com- bination produced marked hepatomegaly and a significant increase in DNA synthesis as demonstrated by [3H]thymidine incorporation and autoradiographic studies. DNA synthesis in the group receiving ciprofibrate and ethoxyquin simultaneously, was slightly more than in animals that received either compound alone, suggesting a synergistic effect, although chronic feeding of these agents together resulted in inhibition of liver carcinogenesis (Rao, M.S. et al. (1984) Cancer Res., 44, 1072–1076). The results of this study further suggest that cell pro-liferation induced by peroxisome proli-ferators may be less important in carcinogenesis than peroxisome proliferation induced by these compounds.

The peroxisome proliferators Wy-14,643, BR-931, nafenopin and ciprofibrate were tested in the primary hepatocyte culture-unscheduled DNA synthesis assay and in the Ames Salmonella microsome mutagenicity assay. The amount of unscheduled DNA synthesis (UDS) in hepatocytes was determined by quantifying the amount of [ 3 H] thymidine incorporated into DNA in the presence of hydroxyurea after isolation of nuclei from hepatocytes treated with the test agent. Wy-14,643 and BR-931 induced unscheduled DNA synthesis in rat hepatocytes, whereas nafenopin and ciprofibrate had no effect. All of the peroxisome proliferators were negative in the Ames Salmonella assay.

The incidence of lung metastasis in rats with hepatocellular carcinomas (HCC) induced by ciprofibrate, a peroxisome proliferator and the expression of gammaglutamyl transeptidase (GGT) in the metastatic lesions was studied. HCC were induced in 75 male F-334 rats by chronic dietary administration of ciprofibrate (0.025% w/w) for 16–22 months. The incidence of lung metastasis was 25% in rats killed between 14 and 16 months which increased to 56% in rats killed between 20 and 22 months. The lung metastases were multifocal and present in both the blood vessels and parenchyma. All the metastatic foci examined for the expression of GGT by histochemical stain were devoid of this enzyme. The results of this study clearly demonstrate the malignant behavior of ciprofibrate-induced liver tumors and the absence of GGT expression in metastatic lesions a phenotypic property that is peculiar to the primary HCC induced by several peroxisome proliferators.

Altered areas (AA), neoplastic nodules (NN) and hepatocellular carcinomas (HCC) induced by chronic administration of ciprofibrate and Wy-14,643 were examined for iron accumulation after systemic iron overload. Eighty percent of AA, 90% NN and 100% HCC showed increased resistance to iron accumulation. However, marked heterogeneity was observed in the amount of iron from area to area within the same lesion with some cells containing no iron, while others showing blue reaction to iron. These findings indicate, that putative preneoplastic and neoplastic hepatic lesions induced by peroxisome proliferators exclude iron in a fashion similar to that of hepatic nodules and carcinomas induced by DNA damaging carcinogens.

The peroxisome proliferator ciprofibrate was examined for its ability to initiate hepatocarcinogenesis in rats. Ciprofibrate was fed in the diet at levels of 0%, 0.01% and 0.025% for 2 weeks in order to induce steady-state peroxisome proliferation. Rats were then subjected to partial hepatectomy and then maintained for 6 months on a basal diet or one containing 0.05% phenobarbital. Ciprofibrate treatment did not increase the number or volume of altered hepatic foci (putative preneoplastic lesions). However, ciprofibrate treatment increased liver weights in a dose-dependent manner in rats which did not receive phenobarbital. It is concluded that ciprofibrate-induced peroxisome proliferation is not sufficient to induce initiation, but that a permanent change is produced which results in an increased liver weight.