Mohan R. Nemali

Almost total conversion of pancreas to liver in the adult rat: A reliable model to study transdifferentiation

Study of transdifferentiation provides an excellent opportunity to investigate various factors and mechanisms involved in repression of activated genes and derepression of inactivated genes. Here we describe a highly reproducible in vivo model, in which hepatocytes are induced in the pancreas of adult rats that were maintained on copper-deficient diet containing a relatively non-toxic copper-chelating agent, triethylenetetramine tetrahydrochloride (0.6% w/w) for 7-9 weeks and then returned to normal rat chow. This dietary manipulation resulted in almost complete loss of pancreatic acinar cells at the end of copper-depletion regimen, and in the development of multiple foci of hepatocytes during recovery phase. In some animals, liver cells occupied more than 60% of pancreatic volume within 6-8 weeks of recovery. Northern blot analysis of total RNA obtained from the pancreas of these rats revealed the expression of albumin mRNA. Albumin was demonstrated in these pancreatic hepatocytes by immunofluorescence. The advantages of this model over the previously described models are: a) low mortality (10%), b) depletion of acinar cells, and c) development of multiple foci of hepatocytes in 100% of rats.

Differential induction and regulation of peroxisomal enzymes: Predictive value of peroxisome proliferation in identifying certain nonmutagenic carcinogens☆

Hypolipidemic drugs and certain plasticizers markedly increase the number of peroxisomes in liver parenchymal cells. Continued exposure to peroxisome proliferators has been shown to produce essentially similar pleiotropic responses leading eventually to the development of liver tumors in rats and mice. These agents are not mutagenic in short-term test systems and do not appear to interact with or damage DNA. Accordingly, the events leading to or associated with the induction of peroxisome proliferation have been postulated to play a role in the development of liver tumors. Recent evidence indicates that persistent peroxisome proliferation leads to the formation of 8-hydroxyguanosine in rat liver DNA, which supports the role for oxidative stress. The mRNAs of the three peroxisomal beta-oxidation genes are induced over 20-fold in the livers of rats treated with nafenopin, Wy-14643, BR-931, and other structurally diverse peroxisome proliferators. This increase in beta-oxidation mRNAs is evident within 30 min to 1 hr and was maximal 8 to 16 hr after the administration of a single dose of these agents by gavage. The peroxisomal catalase and urate oxidase mRNAs increase about 2-fold in the livers of rats treated chronically with peroxisome proliferators. These results indicate that peroxisome proliferators differentially regulate different peroxisomal enzymes. The tissue specificity of peroxisomal beta-oxidation gene regulation by xenobiotics supports the contention that the development of liver tumors following exposure to peroxisome proliferators correlates well with the inducibility of peroxisome proliferation and the beta-oxidation genes. Although these agents are known to exert mitogenic response in liver, it is unlikely that stimulation of DNA synthesis alone is responsible for tumor development. Cell proliferation may, however, play a secondary role. The morphological phenomenon of peroxisome proliferation should serve as a simple, sensitive, and valuable biological indicator for the identification of nongenotoxic or nonmutagenic chemicals that may be carcinogenic. An understanding of the cellular and molecular basis of peroxisome proliferation is a prerequisite for the evaluation of toxicological implications of this phenomenon.

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.

Induction of Hepatic Peroxisome Proliferation by Xenobiotics

Significant increase in the number of peroxisomes in liver parenchymal cells and in the activity of H2O2 generating peroxisomal fatty acid β-oxidation enzyme system, accompanied by an increase in certain other hepatic enzymes, are produced by the administration of several structurally dissimilar hypolipidemic agents and some other xenobiotics. Continued feeding of these non-mutagenic peroxisome proliferators for extended periods of time results in the development of hepatocellular carcinomas in rats and mice. Available evidence indicates that maximal peroxisome prolfieration is a tissue-specific phenomenon restricted largely to the hepatocyte. The mechanism by which structurally diverse peroxisome proliferators produce a similar pleiotropic response is not known, but the tissue specific biological response and rapid rate of transcription of peroxisomal fatty acid β-oxidation enzyme genes support the hypothesis that these agents act by binding to a specific recognition molecule(s). Identification and molecular characterization of peroxisome proliferator-specific receptor(s) will be necessary to understand the tissue-specific and species-sensitive differences in the induction of peroxisome proliferation. Since hepatocarcinogenicity by peroxisome proliferators is not attributable to their direct effect on DNA, it is postulated that oxidative stress emanating from sustained induction of peroxisome proliferation plays a role in the initiation and/or promotion of carcinogenesis. Whether the oxidative stress or the continued peroxisome proliferator-receptor interactions lead to amplification or rearrangement of the peroxisomal β-oxidation genes or oncogenes remains to be elucidated.