V. Subbarao

Induction of hepatocytes in the pancreas of copper-depleted rats following copper repletion

Pancreatic hepatocytes are induced in rats maintained on copper-deficient diet containing 0.6% D-penicillamine for 8-10 weeks, followed by copper repletion. These induced hepatocytes are morphologically and functionally very similar to parenchymal cells of the liver. Immunofluorescence stains demonstrated the presence of albumin and catalase in these cells. Stains for pancreatic enzymes and hormones were negative. As expected, the hypolipidemic compound, ciprofibrate, induced peroxisome proliferation in these cells. These results indicate that a simple depletion and repletion of copper can trigger transdifferentiation in the pancreas of adult rats.

Evaluation of liver cell proliferation during ciprofibrate-induced hepatocarcinogenesis☆

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 ethoxyquin 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 combination 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 proliferation induced by peroxisome proliferators may be less important in carcinogenesis than peroxisome proliferation induced by these compounds.

Chemical Carcinogens Without Mutagenic Activity: Peroxisome Proliferators As A Prototype*

Chemical carcinogens can be classified into two categories (i.e. mutagenic and non-mutagenic) on the basis of positive or negative evidence of DNA damage, mutagenicity or chromosomal aberrations in short-term test systems. Evidence indicates that carcinogenic peroxisome proliferators are negative in short-term test systems. This paper outlines approaches which may be useful in identifying a chemical carcinogen without mutagenic activity. It is conceivable that an alteration in DNA, if essential for initiation of neoplasia, may be mediated indirectly by the biological effects of nonmutagenic carcinogens.

Quantitative analysis of hepatocellular lesions induced by di(2‐ethylhexyl)phthalate in F‐344 rats

Di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, has been shown to be a weak hepatocarcinogen in rats and mice. However, in previous studies no quantitative analysis of tumors was carried out. In the present study, F-344 male rats were given a diet containing 2% DEHP ad libitum for 108 wk. At necropsy livers were quantitatively analyzed for total tumor incidence and the number of lesions per liver after slicing the entire organ at 1- to 2-mm intervals. Neoplastic nodules and/or hepatocellular carcinomas were observed in 11 of 14 rats (78.5%). When evaluated according to the size, 57, 16, and 36% rats contained nodules ranging from 1 to 3, 3 to 5, and greater than 5 mm in size, respectively. The number of nodules per liver ranged from zero to four. These results indicate that DEHP induces tumors in a large number of animals at 2% dose levels. It is clear from this study that when a weak peroxisome proliferator is evaluated for carcinogenic effects, a complete and thorough gross examination of the liver is essential to obtain accurate tumor incidence.

Induction of peroxisome proliferation and hepatic tumours in C57BL/6N mice by ciprofibrate, a hypolipidaemic compound.

The hepatic effects of ciprofibrate, a potent peroxisome proliferator, were evaluated in male C57BL/6N mice, a mouse strain with very low incidence of spontaneous liver tumour development. Dietary feeding of ciprofibrate (0.0125% or 0.025% w/w) for 2 weeks resulted in a marked proliferation of peroxisomes (9-fold increase) and several-fold increase (8- to 10-fold) in the activity of peroxisomal beta-oxidation enzymes. Feeding ciprofibrate at 0.025% concentration for 15 months followed by a 0.0125% for 6 months led to the development of hepatic adenomas in 8/14 (57%) and hepatocellular carcinomas (HCC) in 3/14 (21%) mice. In mice given 0.0125% ciprofibrate for 18 months 5 of 8 (62%) and 3 of 8 (37%) developed adenomas and HCC respectively. Similar to the findings observed in rats, both the adenomas and HCC were negative for gamma-glutamyltranspeptidase. These results in C57BL/6N mice of hepatocarcinogenic effect of ciprofibrate, a non-genotoxic chemical, indicate that peroxisome proliferation can be used as a reliable parameter to evaluate the carcinogenicity of hypolipidaemic compounds.

Inhibition of spontaneous testicular Leydig cell tumor development in F-344 rats by dehydroepiandrosterone

The incidence of spontaneous Leydig cell tumors of testis is very high in old F-344 rats. We have examined the effect of dehydroepiandrosterone (DHEA), a steroid hormone with antimitotic and anticarcinogenic properties, on spontaneous Leydig cell tumorigenesis. Fifteen-week-old male F-344 rats were fed a diet containing DHEA (0.45% w/w) for 84 weeks. At the termination of experiment none of the 13 rats had Leydig cell hyperplasia or Leydig cell tumors. All the eight control rats of comparable age had Leydig cell tumors. These findings suggest that DHEA is a potent inhibitor of spontaneous Leydig cell tumors of testis in aged rats.

Osteopontin contributes to hepatocyte growth factor-induced tumor growth and metastasis formation.

The cytokine hepatocyte growth factor (HGF)/scatter factor-1 and its cognate receptor, Met, are involved in the etiology and progression of many types of cancer. Despite recent advances in understanding the signal transduction pathways activated by HGF, the mechanism by which HGF exerts its tumorigenic effect is not well understood. To identify proteins that may be involved in mediating HGF-induced cell motility, invasiveness, and tumorigenesis, we used two separate differential display screening methods to identify changes in gene expression that are initiated by HGF in an epithelial cell culture system. Among several known and unknown genes whose expression was modified, osteopontin (OPN), a protein previously associated with tumorigenesis, was found to be upregulated within 6 h following HGF stimulation. OPN expression was dependent on activation of the PI-3 kinase pathway. Autocrine secretion of HGF resulted in sustained expression of OPN. Downregulation of opn expression by stable antisense transfection attenuated OPN expression and repressed HGF-induced invasiveness in vitro and decreased HGF-mediated tumor growth and metastasis formation in vivo. Constitutive expression of OPN in itself exerted partial invasiveness in vitro, but its expression itself was not sufficient to initiate tumor growth or metastasis formation in vivo. Thus, together with other molecules, OPN activity contributes to HGF-induced tumor growth and invasiveness.

Comparison of the peroxisome proliferator-induced pleiotropic response in the liver of nine strains of mice.

We have investigated the hepatic effect of ciprofibrate, a potent peroxisomal proliferator, in 9 strains of mice to ascertain whether all strains show similar peroxisome proliferation or if there are any that are resistant to the induction of peroxisome proliferation. Dietary feeding of ciprofibrate at 2 concentrations (0.0125% or 0.025% w/w) for 2 weeks resulted in a significant increase in liver weight (170 to 200%) and a 7- to 11-fold increase in volume density of peroxisomes. Catalase and peroxisomal β-oxidation enzymes increased by 1.7- to 2.7- and 1.9- to 9.3-fold, respectively, over the controls. SDS-polyacrylamide slab gel electrophoresis of post-nuclear fractions of livers showed a marked increase in 80,000-mol. wt. polypeptide. Immunocytochemical studies, as expected, revealed higher levels of PBE. Ciprofibrate treatment also induced hepatic DNA synthesis in all strains as determined by [3H]thymidine incorporation and autoradiography. Dot blot analysis of total RNA from livers of ciprofibrate-treated mice (5 strains) showed a significant increase in peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE) mRNA. When the 9 strains were ranked for each parameter, CBA/Ca was the least responsive mouse strain and the B6C3F1 was the most responsive. However, the results of this study indicate that there is no significant interstrain difference in rankings across strains to ciprofibrate-induced hepatic pleiotropic response.

DEHYDROEPIANDROSTERONE-INDUCED PEROXISOME PROLIFERATION IN THE RAT : EVALUATION OF SEX DIFFERENCES

Abstract Dehydroepiandrosterone (DHEA) is a newly identified peroxisome proliferator that causes hepatomegaly, peroxisome proliferation, and induction of peroxisome-associated enzymes in rats and mice, and hepatocellular carcinomas in rats. In the present study, we have systematically analyzed sex differences and the effect of castration on DHEA-induced peroxisome proliferation in male and female rats, since no Information is available on this subject. DHEA was fed in diet at a concentration of 0.45% for 2 weeks and livers were analyzed for hepatomegaly, peroxisome volume density, peroxisome proliferator associated Mr 80,000 polypeptide (PPA-80), and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (PBE) mRNA. Both intact and castrated rats showed similar response to DHEA characterized by increased peroxisome volume density, PBE mRNA, and PPA-80. Significant difference was observed in the liver weights between castrated and intact animals in both the sexes. Castrated rats that received DHEA had 20%–30% more liver weight than DHEA-administered intact rats. These results clearly indicate that peroxisome proliferative effect of DHEA is not influenced by sex hormones and it is equally potent in both males and females.

Phenotypic Properties of Liver Tumors Induced by Dehydroepiandrosterone in F‐344 Rats

Dehydroepiandrosterone (DHEA), a C19 adrenal steroid hormone, induces peroxisome proliferation in liver cells and is hepatocarcinogenic in the rat The present study deals with the phenotypic properties of DHEA‐induced liver lesions. A majority of the altered areas (80–87%), neoplastic nodules (>94%) and hepatocellular carcinomas (HCC, 80–100%) lacked the marker enzymes 7‐glutamyltranspeptidase and placental form of glutathione S‐transferase (GSTP). Northern blot analysis of HCC from 4 rats revealed no detectable GSTP mRNA. These HCC, however, showed a marked decrease in the staining of glucose‐6‐phosphatase and adenosine triphosphatase. These results indicate that the phenotypic properties of liver tumors induced by DHEA and amphipathic carboxylate peroxisome proliferators are similar.