Thomas H. Rushmore

Rapid lipid peroxidation in the nuclear fraction of rat liver induced by a diet deficient in choline and methionine

A diet deficient in choline and methionine, known to produce hepatocellular carcinoma in the absence of any added chemical carcinogen, induced lipid peroxidation in the nuclear fraction of the liver when fed to male Fischer 344 rats. This lipid peroxidation was detected within 1 day of feeding the diet by the appearance of diene conjugates and increased progressively up to 3 days. It was prevented completely by the addition of choline chloride to the diet. The close proximity of DNA may make it a possible target for attack by free radicals.

Initiation of carcinogenesis by a dietary deficiency of choline in the absence of added carcinogens

The feeding for 10 or 11 weeks of young male Fischer-344 rats, a diet devoid of choline and low in methionine, leads to the appearance of gamma-glutamyltransferase-positive foci of altered hepatocytes in the liver and to the induction of initiated resistant hepatocytes. The latter are known to contain the primary precursor cells for the ultimate development of hepatocellular carcinoma. This initiation of carcinogenesis with the choline-devoid diet is prevented by added choline. These observations indicate that a dietary deficiency may, by itself, without known contaminating or added carcinogens, initiate the carcinogenic process.

Identification of a characteristic cytosolic polypeptide of rat preneoplastic hepatocyte nodules as placental glutathione S-transferase

Evidence is presented that a distinctive type of glutathione-S-transferase (GSHTase-P), and a cytosolic polypeptide of Mr 52,000 (P-52), each appearing in greatly increased amounts in hepatocyte nodules during liver carcinogenesis in the rat, are so far indistinguishable. The probable identity of the two polypeptides was established with the use of SDS polyacrylamide gel electrophoresis and Western blot techniques with purified GSHTase-P and P-52 and their respective antibodies and by comparison of the sequence of the first 26 N-terminal amino acids. Since the enzyme and the polypeptide are each considered to be the best available early markers for hepatocyte nodules, as putative precancerous lesions, their probable identity makes them attractive cellular components for in depth studies on their transcriptional and translational regulation and their use in new approaches to the sequential analysis of liver carcinogenesis.

Mitogenic activity in platelet-poor plasma from rats with persistent liver nodules or liver cancer

Platelet-poor plasma (PPP) from F-344 rats with chemically-induced preneoplastic liver nodules or hepatocellular carcinoma stimulated S-phase DNA synthesis in monolayer cultures of normal rat hepatocytes. Similar mitogenic activity was detected in PPP 6 hrs to 1 week after partial hepatectomy (PH) or after necrotizing doses of CCl4 or diethylnitrosamine (DENA). Very little activity was found in PPP4 from control rats. The mitogenic activity in PPP from animals with nodules was non-dialyzable (greater than 14 kd) and bound to a heparin-sepharose affinity column. None of the mitogenic PPPs competed with [125I] epidermal growth factor (EGF) for binding sites on A431 cells or normal rat hepatocytes. These studies indicate that persistent proliferation of preneoplastic and neoplastic hepatocytes is associated with increased circulating levels of mitogenic hepatocyte growth factor.

Glutathione and enzymes related to free radical metabolism in liver of rats fed a choline-devoid low-methionine diet

Fischer F-344 male rats, fed a choline-devoid diet that leads to a highly reproducible sequence of biochemical and biological changes with an ultimate development of hepatocellular carcinoma, show elevated levels of glutathione in the liver at 3, 6 and 8 days. Several enzymes related to the metabolism of free radicals, including superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase and DT-diaphorase show neither increased nor decreased activity as measured between 12 h and 8 days on the diet. Thus, of several known cellular components related to the possible scavenger of free radicals in the liver, only glutathione responded to the feeding of the CD diet. It is tentatively concluded that a decrease in the levels of possible scavengers for free radicals is not a major basis for the nuclear and mitochondrial lipid peroxidation seen early in rats fed a choline-devoid diet.

Dietary Choline Deficiency as a New Model to Study the Possible Role of Free Radicals in Acute Cell Injury and in Carcinogenesis

Over the last few years, working with a diet that is devoid of choline and in methionine (CD) we have observed that when this diet is fed to rats, they develop not only fatty liver, but also hepatocellular necrosis and cancer. In the course of our investigation as to the mechanism of cancer development by a dietary deficiency without any added carcinogen, we found that early nuclear lipid peroxidation (1), DNA alteration (2) and cell proliferation are very common features in the liver. Free radical generation and DNA alteration in a proliferating organ has been proposed as the initiating event in the development of liver cancer (see Fig. 1). However, the nature of free radical generated and the nature of DNA alteration are not known.

Mechanism of Initiation of Liver Carcinogenesis by Dietary Imbalance

The role of diet in the modulation of carcinogenic process is known for several years. However, most of the emphasis was on how different macro and micro-constituents of diet can alter the biology of the system with respect to different carcinogens and modify the carcinogenic potential of the latter either by enhancing or diminishing the carcinogenicity to different organs. Recently, we and two other laboratories1–5 have shown that about 50% of rats develop hepatocellular carcinoma when fed a diet which is devoid of choline and low in methionine (CD) without any added carcinogen. The role of choline deficiency as a promoter in the carcinogenic process has been established6–7. The recent demonstration that CD alone without any added carcinogens caused liver cancer strongly suggests that CD both initiates and promotes, i.e. it is a carcinogenic regimen. This observation raised a puzzling question as to how the absence of something can cause initiation. A hypothesis has been proposed8 based on the following observations: (a) early hepatic nuclear lipid peroxidation which occurs in choline deficiency9 is followed closely by (b) DNA alteration10 and (c) liver cell proliferation11. These may represent the chain of events leading to initiation, promotion and eventually to cancer in this model where no known carcinogen is added.