MORPHOMETRIC ANALYSIS OF THE ULTRASTRUCTURAL CHANGES IN RAT LIVER INDUCED BY THE PEROXISOME PROLIFERATOR Sail 42-348

The changes occurring in hepatocytes of F-344 male rats during a 3-wk treatment with a hypolipidemic agent, 1-methyl-4-piperidyl-b/s[p-chlorophenoxy]acetate (Sail 42-348), have been evaluated by morphometric and biochemical methods. The twofold increase in liver weight resulted from a significant increase in hepatocyte cytoplasm as well as a moderate increase in the number of liver cells. The peroxisome population and SER played an overwhelming part in the hypertrophy of hepatocytic cytoplasm. The relative volume and the surface density of peroxisomes increased ninefold and sevenfold, respectively. The increase in the collective peroxisome volume resulted from an increase in both the number and the average volume of peroxisomes. The SER also demonstrated a substantial increase in these values. The relative volume and surface density of mitochondria were not significantly altered in comparison to controls, while these values for R E R decreased onefold. Studies on the lobular distribution of cytoplasmic organeUes before and during treatment revealed that the relative volume and surface density of peroxisomes and SER increased from periportal to centrilobular cells of the hepatic lobule, whereas mitochondrial values decreased from periportal to centrilobular cells. The RER values were fairly constant in different parts of the hepatic lobule. The increase in peroxisome and SER volume and surface area was first evident within the first 3 days of Sail 42-348 treatment and these values continued to increase, reaching a steady state within 2 wk. The time course of increase in catalase and carnitine acetyltransferase activities correlated with the morphometric data on the peroxisomes. After cessation of Sail 42-348 treatment, the peroxisome values decreased rapidly within the first 3 days and reached control levels within 1 wk. Moderate reduction in SER values occurred after withdrawal of the drug, but these values remained higher than controls even after 2 wk, suggesting that the reduction in the amount of circulating peroxisome proteins may result in empty SER channels. On the 4th day of drug withdrawal a significant increase in the relative volume and surface density of lysosomes was 768 TI-IE JOURNAL OF CELL BIOLOGY 9 VOLUME 71, 1976 " pages 768-780 on O cber 9, 2017 jcb.rress.org D ow nladed fom observed, suggesting that these organelles may play some part in the removal of cellular membranes. However, the rapid reduction in peroxisome values after Sail 42-348 withdrawal appears to be due to cessation of enhanced peroxisome protein synthesis. The function of mammalian peroxisomes (microbodies, microperoxisomes) has yet to be elucidated. Rhodin first described these organelles in the renal proximal tubule as a single-membranelimited cytoplasmic constituent with a fine granular matrix (39). Further studies have since demonstrated peroxisomes in several animal tissues, protozoans, and plants. The content of mammalian peroxisomes was shown by de Duve and co-workers to include catalase and several oxidative enzymes such as urate oxidase, D-amino acid oxidase, L-a-hydroxy acid oxidase, and isocitrate dehydrogenase (21). More recently, the activities of carnitine acetyltransferase, NAD+:a-glycerol phosphate dehydrogenase, and NADH-cytochrome c reductase have also been reported in peroxisome fractions (10, 15, 26). One area of study which may help to elucidate the function of peroxisomes and their contents is the use of certain hypolipidemic drugs which have been shown to induce proliferation of hepatic peroxisomes. In 1964, Duncan and co-workers reported that the hypolipidemic drug, clofibrate, caused a marked hepatomegaly in rats (11). The following year, Hess and co-workers found that the hepatomegaly arose from a striking proliferation of peroxisomes in the liver cells (17). A proliferation of the smooth endoplasmic reticulum (SER) also contributed to the hepatomegaly, but to a lesser extent. Methyl clofenapate, nafenopin, and Sai l 42-348 (1-methyl-4-piperidyl b/s[p-chlorophenoxy]acetate), which are hypolipidemic analogues of clofibrate, have also been found to induce hepatomegaly with a proliferation of peroxisomes (34, 35, 37). In addition, a few other compounds which are structurally unrelated to clofibrate have been shown to induce both hypolipidemia and peroxisome proliferation (18, 20, 36). On the basis of this relationship, it has been suggested that peroxisomes may be related to lipid metabolism (36). The increase in peroxisome proliferation after treatment with these drugs was shown to be accompanied by an increase in catalase activity (27, 37), the marker enzyme for peroxisomes (21). An increase in the activities of camitine acetyltransferase (CAT) and NAD+:ct-glycerol phosphate dehydrogenase also occurs upon treatment with these compounds (19, 27, 37). The purpose of this study is to describe quantitatively the structural events which lead to hepatomegaly, on the basis of a morphometric analysis (47, 48) of hepatocytes in rats treated with Sai l 42-348. These structural alterations in liver cells are correlated with the changes in hepatic catalase and carnitine acetyltransferase activities. Also included in this report are studies on the effect of withdrawal of Sa i l 42-348 on the reversal of peroxisome proliferation, in order to assess the role of the lysosomal system in the removal of peroxisomes. Preliminary results of this study have been presented elsewhere (28). MATERIALS AND METHODS