Amiya K. Ghoshal

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.

Effects of peroxidative stress and age on the concentration of a deoxyguanosine-malondialdehyde adduct in rat DNA.

The effect of age and peroxidative stress on the concentration of a deoxyguanosine malondialdehyde adduct (dG-MDA) in rat tissues was investigated. Vitamin E deficiency had not effect on the dG-MDA content of liver DNA in rats fed a diet containing 10% corn oil. When 2% cod liver oil was added to this diet, the dG-MDA content of liver DNA doubled in the positive controls fed a high level of vitamin E (100 ppm dl-α-tocopherol), and there was a further increase when vitamin E was deleted. Neither iron nitrilotriacetate administration nor choline deficiency had any effect on the dG-MDA content of liver DNA. Carbon tetrachloride had a lowering effect. The failure of iron or carbon tetrachloride administration and of vitamin E deficiency to increase liver dG-MDA is consistent with their failure in previous experiments to affect the urinary excretion of dG-MDA. In contrast, these forms of peroxidative stress produce large increments in the urinary excretion of MDA adducts with lysine, reflecting increased formation and degradation of MDA-modified proteins. DNA appears to be protected from modification by MDA produced at extranuclear sites. The frequency of dG-MDA in different tissues of 4-month-old rats varied markedly: brain ≫ liver > kidneys and testes. Higher concentrations of dG-MDA were found in the liver and kidneys, but not the testes, of 25-month-old rats. The determinants of the concentration of dG-MDA in DNA merit further investigation.

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.

New Insight into the Biochemical Pathology of Liver in Choline Deficiency

A diet deficient in choline can cause liver cancer in rats. The previous work since 1932 emphasized the fat-removing ability of choline from the liver. There are other dietary factors, including methionine, which, like choline, can remove fat from the liver. These factors were termed as lipotropes. Since then, choline deficiency and lipotrope deficiency are used synonoumously. Recent work since 1980 has clearly demonstrated that choline deficiency (CD) and lipotrope deficiency (LD) are not the same. Generation of free radicals, DNA alterations, liver cell death, and liver cancer that occur due to CD are not generated by LD. Generation of free radicals due to CD diet and some of the agents that counteract free radical action also prevent CD effects except for lipid accumulation in the liver. Despite the recent observations on the role of phospholipase A2 (PLA2) as the protector of the membranes, it has been found that by preventing the rise of PLA2 in the liver, cell death can be prevented. These new findings give choline a distinct role in liver cell death and cancer rather than the role of lipotrope. A new hypothesis linking dietary choline deficiency and liver cancer has been discussed.

Ethionine in the Analysis of the Possible Separate Roles of Methionine and Choline Deficiencies in Carcinogenesis

The importance of ethionine, the ethyl analogue of methionine, as a metabolic probe to study the possible roles of methionine and choline in liver carcinogenesis has been briefly reviewed. Ethionine-induced liver carcinogenesis is similar in many aspects, including initiation, promotion, and progression, to carcinogenesis with other agents. However, the special role of methionine in preventing virtually all metabolic and pathologic effects of ethionine, including liver cancer, places ethionine in a special position. On the basis of these observations and our current knowledge about choline deficiency in the genesis of liver cancer, we proposed that choline and methionine play separate but overlapping roles in the initiation and promotion of liver carcinogenesis.

Changes in fatty acid composition of phospholipids from liver microsomes and nuclei in rats fed a choline-free diet

Male F-344 rats were fed a choline-free (CF) diet, and changes in phospholipid content, phospholipid fatty acids and phospholipase A2 activity in liver nuclei and microsomes were examined during the first 72 hr. Both nuclei and microsomes showed a decrease in phosphatidylcholine (PC) content. Microsomes showed an increase in PC arachidonate while nuclei showed a decrease. Also, microsomes showed increased activity of phospholipase A2 (PLA2) while nuclei did not. These observations are consistent with the hypothesis that the absence of diene conjugates in liver microsomes in the rats on the CF diet may reflect the increased rate of removal of peroxidized fatty acids by phospholipase A2.

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.