Benito Lombardi

Isolation of a Golgi-rich fraction from rat liver

Abstract A fractiion rich is membranes of the Golgi apparatus was isolated from rat liver by discontinuous density gradient centrifugation. Electron microscopic analysis of the fraction revealed the presence of structures very similar to those of the Golgi apparatus in intact cells, namely stacked cisternae, secretory vesicles, and tubular elements. The Golgi-rich fraction contained over 90% of the UDP-galactose; N- acetylglucosamine galactosyltransferase, about 2% of the glucose-6-phosphatase and 12% of the AMP phosphohydrolase present in the post-nuclear supernatant of liver homogenates.

Detection of conjugated diene isomers of linoleic acid in liver lipids of rats fed a choline-devoid diet indicates that the diet does not cause lipoperoxidation

Abstract Male and female F-344 rats were fed for 1 week choline-devoid or control choline-supplemented diets containing either 5% corn oil and 10% partially hydrogenated vegetable oils (PHO diets) or 15% corn oil (CO diets). HPLC/second derivative UV spectrophotometric analyses, combined with on-line atmospheric pressure ionization mass spectrometry, were used to determine whether conjugated diene isomers of linoleic acid (CLA), present in the diets, were assimilated into liver and adipose tissue lipids. The CLA content in the PHO diets was an order of magnitude greater than that in the CO diets. CLA were detected in the adipose tissue of all rats but in the liver of only rats fed the PHO diets. In adipose tissue, the CLA levels clearly reflected those present in the diets, and no sex differences, or differences between rats fed the choline-devoid or control diets were noted. In addition to CLA, conjugated linolenic and eicosatrienoic acids, arising probably from desaturation and elongation of CLA, were detected in the liver. The results provide evidence that the conjugated dienes detected in liver lipids of rats fed a PHO-containing choline-devoid diet are of dietary origin and do not reflect lipid peroxidation.

Liquid chromatographic-mass spectrometric analysis of conjugated diene fatty acids in a partially hydrogenated fat

A commercially available partially hydrogenated fat was analyzed for fatty acids containing conjugated dienes. The fatty acids were isolated by high-performance liquid chromatography (HPLC), and analyzed with a photodiode array detector and an atmospheric-pressure ionization mass spectrometer. Conventional and second-derivative ultraviolet (UV) spectra of the peaks eluting from the HPLC were recorded with the photodiode array detector, and peaks displaying second-derivative UV spectra characteristic of the conjugated diene chromophore were analyzed by mass spectrometry. The UV and mass spectra of the fatty acids with conjugated dienes, present in the partially hydrogenated fat, were identical to those of reference preparations of linoleic acid isomers with conjugated dienes. The results obtained emphasize that care must be exercised in the interpretation of clinical and experimental data concerning the detection of conjugated dienes in tissues or body fluids of humans and experimental animals. The conjugated dienes may not reflect an ongoing process of lipid peroxidation, but may be of dietary origin.

Acute hemorrhagic pancreatic necrosis in mice. Alterations of pancreatic lipase activity, pancreas lipids, and serum lipoproteins.

Acute hemorrhagic pancreatic necrosis (AHPN) with fat necrosis was induced in female mice by feeding a choline-deficient diet containing 0.5% DL-ethionine. Pancreatic lipase increases significantly prior to the onset of AHPN and is not accompanied by gross alterations in the lipid composition of the organs. In the same animals significant activities of pancreatic lipase were detected in serum and peritoneal cavity after 3 days of dietary regimen. Analysis of serum lipids and lipoproteins indicated no hyperlipemia prior to or after onset of AHPN.

Expression of p53 mutant protein(s) in diethylnitrosamine-induced foci of enzyme-altered hepatocytes in male Fischer-344 rats.

Several types of human and animal tumors have been shown to carry mutations in the p53 gene. While the translation product of the wild type gene has tumor suppressor properties, mutant alleles of the gene produce proteins that can cooperate with other oncogene products in transforming cells. In this paper, evidence is presented indicating that a p53 gene mutation(s) occurs in foci of enzyme-altered hepatocytes induced by diethylnitrosamine in male Fisher-344 rats. The evidence was obtained by means of immunohistochemical and immunoblotting techniques, using antibodies directed against mutant forms of the p53 protein.

Free Radicals and Lipid Peroxidation in Liver of Rats Kept on a Diet Devoid of Choline

Rodents kept on a choline devoid (CD) diet up to 14 months develop hepatic lesions progressing through two broad stages. The first is characterized by severe steatosis and increase in cell turnover, the second by a gradual clearance of the deposited fat and fibrosis. Hepatocellular carcinomas eventually arise in rats fed for over 12 months, even though the animals aer not exposed to chemical carcinogens. It has been suggested that the diet may trigger generated thereby may be responsible for initiation of liver cancer and promotion. The radicals would lead to DNA damage, and the altered DNA in a proliferating liver would result in initiation of the carcinogenic process. In this communication we present evidence that the diet used in the above studies contained stable fatty acid isomers with conjugated dienes, which are absorbed and deposited in rat liver. This finding cast doubts on whether a CD diet does indeed cause a peroxidation of cellular membrane lipids. Electron spin resonance (ESR) spectroscopy was also used to investigate whether any abnormal pattern of free radicals exists in the liver of rats fed a CD diet. No significant differences were noted in ESR spectra of either transition metal-centered signals, or organic free radicals.

Tumorigenesis, protooncogene activation, and other gene abnormalities in methyl deficiency

The methyl deficiency model of hepatocarcinogenesis is notable because hepatocellular carcinomas (HCCs) arise in rodents chronically fed methyl-deficient diets, without intentional or adventitious exposure of the animals to chemical carcinogensJ -5 or genetic manipulations of the animals. 6-9 The tumorigenicity of these diets, therefore, appears to reside solely on effects they have on rodent liver. Unfortunately, a large number and variety of those effects are known, and have been known for some time, w-17 while newly discovered ones are being steadily reported in the literature. This situation obviously complicates the evaluation of and search for which effect(s) contribute(s) to the tumorigenicity of the diets, and, in particular, directly results in, or leads to, the genomic alterations that are responsible for cancer induction. Nonetheless, such an effort is ongoing in several laboratories, and several promising leads and working hypotheses are being pursued at the present time. In this paper we shall review briefly the results obtained by us in this effort, as well as the hypothesis that has been underlying our work. Our studies have been performed on rats fed semi-purified diets, either adequate in choline (CS diet), or essentially devoid of cho-

Nutritional model of hepatocarcinogenesis : rats fed choline-devoid diet

Rats fed a choline-devoid diet as the sole treatment develop hepatocellular carcinomas, the pathogenesis of which appears to reside exclusively in effects of the diet on the liver. Among the latter, most prominent is the induction of repeating cycles of liver cell injury, death, and regeneration. Two other models have been described recently in the literature, in which development of hepatic neoplastic lesions occurs after protracted periods of liver cell injury, death, and regeneration, without exposure of the animals to chemical carcinogens. The possibility is considered that an abnormal increase in cell turnover may result in all of the genomic alterations that are required for initiation, promotion, and neoplastic transformation of liver cells in these models of hepatocarcinogenesis. The possible involvement, in the same models, of endogenously initiated liver cells also is discussed briefly.

The Choline-Devoid Diet Model of Hepatocarcinogenesis in the Rat

The notion that a choline-devoid (CD) diet is hepatocarcinogenic in the rat has already gone through two historical phases, and is at the beginning of its third (for a detailed account, see ref. 1). In 1946, Copeland and Salmon published the first of a series of papers showing the development of hepatocellular carcinomas (HCCs) in rats chronically fed a CD diet2,3. The finding attracted much attention at the time, since the modalities of the experiments involved no addition to the diet of, or treatment of the animals with, chemical carcinogens. However, subsequent studies by Newberne et al.4,5 cast doubts on whether the diet, and the diet alone, was responsible for the genesis of the tumors, and attributed the latter, instead, to a likely contamination of the diet with aflatoxin B1, a newly discovered6 and most potent hepatocarcinogen in the rat7. In the last few years, the question was reopened by a repetition of the original findings of Copeland and Salmon in three different laboratories8–11. In these instances, both diets and rat’s environment were scrutinized for relevant contamination with chemical carcinogens, with negative results. At the present time, therefore, the conclusion seems unavoidable that CD diets are indeed hepatocarcinogenic, and that the genesis of the tumors resides in effects of these diets on rat liver. However, at least one primary issue awaits resolution, before the CD-diet model of hepatocarcinogenesis can be fully categorized; that is whether the diets act as complete carcinogens, able to initiate de novo liver cells, as well as to promote their evolution to cancer; or whether they merely promote the evolution to cancer of endogenous initiated cells.

Comparative Changes in the Liver of Female Fischer-344 Rats after Short-Term Feeding of a Semipurified or a Semisynthetic L-Amino Acid-Defined Choline-Deficient Diet

Groups of female Fischer-344 rats were fed a semipurified choline-deficient (CD) diet, or a semisynthetic L-amino acid-defined choline-deficient (CDAA) diet, for up to 12 wk and effects of the 2 diets on the liver were compared. Steatosis was diffuse and more severe throughout in rats fed the CDAA diet than in rats fed the CD diet. Greater elevations in serum aspartate and alanine aminotransferase activities were also present in the former rats, along with higher 2-bromodeoxyuridine labeling indices in the liver. Discrete amounts of 8-hydroxyguanine were detected in liver DNA, but were not significantly different in rats fed the 2 diets, or from those present in a group of control rats killed at 0 time. Glutathione S- transferase placental form-positive focal lesions were not observed in any of the rats. The results show that the CDAA diet causes more severe degrees of steatosis and liver cell death and proliferation than the CD diet, raising the possibility that it may, in contrast to the CD diet, result in the eventual induction of hepatocellular carcinomas in female Fischer-344 rats.