Choline deficient diet enhances the initiating and promoting effects of methapyrilene hydrochloride in rat liver as assayed by the induction of gamma-glutamyltranspeptidase-positive hepatocyte foci.

Earlier we demonstrated that short-term feeding of methapyrilene hydrochloride (MPH) and of a choline deficient (CD) diet to rats induced peroxidative damage of microsomal membrane lipids of liver cells. In the present study, we investigated whether a CD diet modifies the extent of MPH-induced lipid peroxidation and whether the modifications lead to changes in the initiating and promoting action of these agents using assays of the induction of gamma-glutamyltranspeptidase (GGT)-positive hepatocyte foci. Addition of 0.1% MPH to a CD diet enhanced the extent of microsomal lipid peroxidation induced by a CD diet alone. Feeding a choline supplemented (CS) or a CD diet containing 0.1% MPH for 2 weeks followed by 7 weeks promotion by a CD diet plus phenobarbital was ineffective in inducing GGT-positive foci. Feeding MPH in a CS or a CD diet for 4 weeks, however, resulted in the development of substantial numbers of GGT-positive foci. There was a 3 fold increase in the number of foci in rats initiated with a CD + MPH diet over that in rats initiated with a CS + MPH diet. 0.1% MPH in a CS diet or a CD diet exerted significant promotional effects on the induction of GGT-positive foci in rats initiated with a single injection of diethylnitrosamine. Addition of MPH to a CD diet was additive in inducing GGT-positive foci. The results suggest that lipid peroxidation of the liver may be involved in the carcinogenic and/or promoting effects of MPH and a CD diet.

Free radical injury and liver tumor promotion.

One of the underlying mechanisms of tumor promotion both in the skin and liver involves free radical mediated injury to informational macromolecules of target cells. A choline-deficient (CD) diet, which is an efficient liver tumor promoter, induces peroxidative damage of liver cell membrane lipids. By modifying components of a CD diet, we have shown that the efficacy of the promotion is correlated with the extent of lipid peroxidation. The substitution of fats in a CD diet with predominantly polyunsaturated fat and the addition of methapyrilene to a CD diet enhances membrane lipid peroxidation and the promoting effects. An antioxidant (BHT) and hypolipidemic peroxisome proliferators (BR931 and DEHP) suppress both of these effects. Contrary to these findings, phenobarbital did not induce membrane lipid peroxidation, and its addition to a CD diet inhibited the diet-induced lipid peroxidation, though such a combination exerted a stronger promoting action. Thus, a CD diet and phenobarbital exert their promoting actions through different mechanisms. The consequence of membrane lipid peroxidation in the liver cells induced by a CD diet may be multiple. Our recent study of surface membrane insulin receptors of liver cells of rats fed a CD diet showed a decrease in number and an enhanced binding affinity leading to altered responsiveness of liver cells to insulin mediated glycogen synthesis. It is suggested that CD diet-induced lipid peroxidation leads to functional alterations of membrane receptors involved in cell growth control and may thereby exert its promoting action.

Enhancement of induction of intestinal adenocarcinomas by cyclosporine in rats given a single dose of N-methyl N-nitrosourea.

Dietary administration of cyclosporine (CsA) to rats induces lymphoproliferative disorders involving the gut lymphoid plaques with frequent mucosal ulceration. In this study, we investigated whether administration of a known chemical carcinogen prior to CsA treatment modifies the development of the lesions induced by CsA. Male Sprague-Dawley rats were given either a single i.p. injection of N-methyl N-nitrosourea (MNU) (25 mg/kg) or the solvent--and one week thereafter they were fed a diet containing 0.011% CsA. Control rats were fed a basal diet with or without prior MNU treatment. Of the rats that received MNU and CsA, 60% developed adenocarcinomas of the small and large intestine arising in the regions of the intestinal lymphoid plaques. Only 1 of 12 rats given MNU followed by a basal diet developed adenocarcinoma. No tumors developed in rats treated with CsA alone, but there was atypical proliferation of the intestinal mucosa in conjunction with lymphoid hyperplasia of the gut. The mucosal damage secondary to CsA-induced lymphoid lesions probably acted as a promoting stimulus for the development of intestinal carcinomas.

Alterations in hepatocyte insulin receptors in rats fed a choline-deficient diet.

Specific insulin binding and glycogen synthesis were studied in control hepatocytes, hepatocytes from rats fed a choline-deficient (CD) diet for 7 to 14 days, and hepatoma cells induced with a CD diet and DL-ethionine in culture. Both the binding affinity and the number of receptors were affected in hepatocytes by the CD diet. The number of receptor sites was 26,000/cell and the dissociation constant (Kd) for the high affinity binding site was 2.6 nM at 30 degrees C, in contrast to the control values of 205,000 sites/cell and 23.2 nM, respectively. In the hepatoma cells, receptor cell number and Kd were further diminished to 6,400 sites/cell and Kd = 1.1 nM. The basal level of glycogen synthesis in control hepatocytes and in CD hepatocytes was similar; however, the basal rate of glycogen synthesis in hepatoma cells was only 16% of that in the control cells. The glycogen synthesis in hepatoma cells was stimulated by insulin, but at a 3-log higher concentration compared to the control cells. This loss of sensitivity to insulin is consistent with the marked decrease in insulin receptors. CD hepatocytes had a decrease in insulin receptors with a concurrent decrease in Kd (increase in binding affinity), such that, sensitivity to insulin did not differ significantly from that of control hepatocytes. However, the maximal stimulation of glycogen synthesis was only 27% that of the control cells. The changes in receptor number and Kd of hepatocytes from rats fed a CD diet may be due to alterations in cell membrane lipid composition and this alteration may be responsible for the enhanced sensitivity of hepatocytes to chemical carcinogens and for the tumor promoting effect of the diet.

Suppression of choline-deficient diet-induced hepatocyte membrane lipid peroxidation in rats by the peroxisome proliferators 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)- acetamide and di(2-ethylhexyl)phthalate.

Many hypolipidemic peroxisome proliferators have been shown to induce liver tumors in rats after long-term feeding. In short-term assays, however, some of them prevent the development of gamma-glutamyl transpeptidase-positive foci, the putative preneoplastic lesions, in the liver of carcinogen-initiated rats and inhibit the promoting effect of a choline-deficient (CD) diet on these lesions. The CD diet-induced lipid peroxidation in the liver has been implicated as one of the underlying mechanisms of the promoting effect. In the present study, the effects of 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio(N-beta-hydroxyethyl)acetamid e (BR931) and di(2-ethylhexyl)phthalate (DEHP) on CD diet-induced liver membrane lipid peroxidation were investigated by determining the extents of conjugated diene formation. No evidence of lipid peroxidation was detected in the microsomal lipids of the liver after administration of BR931 or DEHP at concentrations of 0.16% and 1%, respectively, for 1, 2, and 4 wk. When added to a CD diet, both BR931 and DEHP effectively protected against the diet-induced lipid peroxidation. There was an increase in cellular glutathione levels after 4 wk and an increase in catalase activity after 2 wk in the liver of rats fed BR931 or DEHP. The levels of activity of the glutathione peroxidases and glutathione-s-transferase were significantly reduced. The results suggest that, in the acute stage, hypolipidemic peroxisome proliferator-induced effects of excess production of H2O2 and potential lipid peroxidation are balanced by stimulation of some cellular detoxifying systems. The inhibition of lipid peroxidation by hypolipidemic peroxisome proliferators may account for their inhibitory effects on the CD diet-induced promotion of preneoplastic foci.

Enhancement of the induction of murine thymic lymphomas by cyclosporine.

The effects of cyclosporine (CsA) on the induction of thymic lymphoma in male Swiss Webster mice were investigated using the classic model of two-stage carcinogenesis with N-methyl N-nitrosourea (MNU) as an initiator and CsA as a promoter. The mice treated with a single dose of MNU followed by chronic feeding of 0.015% CsA developed an eight-fold higher incidence of thymic lymphomas than the mice treated with a single dose of MNU followed by a basal diet. No mice treated with CsA alone or mice kept on a basal diet developed tumors. The results suggest that CsA enhances the induction of thymic lymphomas by its promoting effect and that the disturbance of thymic microenvironment induced by CsA may be one of the underlying mechanisms of the promoting action by CsA.

Hereditary dysplastic nevus syndrome: lymphoid cell ultraviolet hypermutability in association with increased melanoma susceptibility.

The hereditary dysplastic nevus syndrome (DNS) is a well-characterized disorder in which affected individuals have increased numbers of premalignant (dysplastic) nevi and a markedly increased risk of developing cutaneous melanoma. Seeking evidence of a systemic disorder in DNS, we examined the effect of ultraviolet radiation on cultured lymphoid cells. Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with hereditary DNS had similar survival values following treatment with 2.3 to 9.0 J of 254-nm ultraviolet radiation per m2 as did lines from control individuals. Mutagenesis at the hypoxanthineguanine phosphoribosyltransferase locus was assessed by measuring the induction of resistance to thioguanine using a microtiter well assay. Three lymphoblastoid cell lines from patients with hereditary DNS and melanoma had a 2- to 3-fold greater frequency of induced mutants per clonable cell than three normal lines following exposure to 4.5 to 9.0 J of ultraviolet radiation per m2. Expanded clones of mutated DNS lymphoblastoid cell lines had less than 6% of normal hypoxanthine-guanine phosphoribosyltransferase activity. Inhibition and recovery of DNA synthesis following ultraviolet exposure were similar in 2 DNS and 2 normal lines. Repair by DNS lines of ultraviolet-induced DNA damage was in the normal range as measured by alkaline elution. Thus, hereditary DNS exhibits in vitro hypermutability which may reflect increased susceptibility to ultraviolet-induced somatic mutations in vivo. This abnormality may be related to the increased melanoma susceptibility of patients with hereditary DNS.

Choline deficiency and chemical carcinogenesis.

We have reviewed the current status of our knowledge concerning the biologic effects of dietary choline (lipotrope) deficiency in modifying chemical carcinogenesis in experimental animals and discussed its possible mechanisms. Choline deficiency produces various pathologic lesions, involving virtually every organ of the body, as a result of a decrease in phospholipid and acetylcholine synthesis and in the supply of labile methyl groups. The liver is the only organ in which a relationship has been consistently demonstrated between choline deficiency and chemically induced tumors. The deficient diet enhances the initiating potency of several carcinogens and acts as a strong cocarcinogen. Diet also exerts a strong promoting effect, though the possibility that it is a complete carcinogen cannot be ruled out. Phase I enzymes of the carcinogen metabolizing system are uniformly depressed by choline deficiency, but very little information is available regarding the effects of diet on Phase II enzymes that detoxify carcinogen metabolites. Possible modifications of carcinogen-induced DNA damage and their repair processes have not been adequately scrutinized. Solid evidence suggests that feeding a choline-deficient diet leads to enhanced liver cell proliferation, an inadequate supply of methyl groups for transmethylation reactions, and membrane lipid peroxidation. Induced cell proliferation and hypomethylation of DNA may alter the state of gene expression, including that of specific cellular oncogenes. Lipid peroxidation may alter the structure and function of membrane receptors related to liver cell growth or may directly damage cellular DNA. Thus these alterations, individually or in combination, could play a critical role in the diet-induced modification of chemical carcinogenesis.

Lipid peroxidation of liver microsome membranes induced by choline-deficient diets and its relationship to the diet-induced promotion of the induction of gamma-glutamyltranspeptidase-positive foci.

The effects of varying the type of dietary fat in the choline-deficient (CD) diet on the development of gamma-glutamyltranspeptidase (GGT)-positive foci in the liver of carcinogen-treated rats were investigated, and the results were correlated with the extent of membrane lipid peroxidation induced by the diets. Male Sprague Dawley rats were initiated with a single dose of diethylnitrosamine. Thereafter, groups of rats were fed choline-supplemented or CD diets in which the amount of saturated fat was varied by using hydrogenated vegetable oil (Primex) and corn oil (CO), either alone or in combination. The number and size of GGT-positive foci induced by the CD diet with CO as the sole source of fat were larger than those induced by the diet containing mixtures of Primex and CO. The CD diet with Primex alone was the least effective in inducing GGT-positive foci. Peroxidation of liver microsomal membrane lipids in rats fed regular CD or CD:CO diets was examined by determining the formation of conjugated dienes. The generation of diene conjugate in rats fed a CD:CO diet was evident after 2 days of the diet feeding, and the levels increased at 1 and 2 weeks. No significant diene conjugate was demonstrated in rats fed a regular CD diet for 2 days. However, after 1 and 2 weeks, there was generation of diene conjugate, the levels of which were lower in rats fed the CD diet than those on a CD:CO diet. Addition of an antioxidant, 0.25% butylated hydroxytoluene, to both CD and CD:CO diets abolished the generation of diene conjugate in rat liver microsomal membranes and markedly inhibited the promotion of GGT-positive foci in the liver of diethylnitrosamine-initiated rats. The results suggest that membrane lipid peroxidation in the liver may be related to the promotion of the induction of GGT-positive foci by a CD diet. The enhanced promotion by the inclusion of a higher level of polyunsaturated fat in the diet may be, in part, due to its greater susceptibility to peroxidation.

Methapyrilene induces membrane lipid peroxidation of rat liver cells.

Male Sprague Dawley rats were fed either a basal diet or the same diet containing 0.1% methapyrilene hydrochloride for 1, 2 and 3 days and 1 and 2 weeks. Peroxidative degradation of membrane lipids from liver cell mitochondria and microsomes was evaluated by determining the extent of diene conjugate (u.v. absorption 233 nm) formation. Feeding methapyrilene for 1, 2 and 3 days resulted in lipid peroxidation of both mitochondrial and microsomal membranes. After 1 and 2 weeks feeding, peroxidation was no longer evident in lipids isolated from mitochondria, while the microsomal membrane lipids showed persistent conjugated dienes. The results suggest that membrane lipid peroxidation of the liver may play a role in the promoting and/or carcinogenic effects of methapyrilene hydrochloride.

Accelerated regression of carcinogen-induced preneoplastic hepatocyte foci by peroxisome proliferators, BR931, 4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio (N-B-hydroxyethyl)acetamide, and di-(2-ethylhexyl)phthalate.

Enzyme altered preneoplastic hepatocyte foci were induced in male Sprague Dawley rats by a single injection of diethylnitrosamine (DEN) followed by dietary promotion of a choline-deficient (CD) diet for 4 weeks or by feeding a diet containing 0.02% 2-acetylaminofluorene for 4 weeks. BR931, a hypolipidemic agent, administered to rats bearing preneoplastic foci caused a rapid regression of the foci within 4-6 weeks. Di(2-ethylhexyl)phthalate (DEHP), a plasticiser, exerted the similar effect when tested on rats bearing preneoplastic foci induced by DEN + CD diet. Since BR931 and DEHP have been shown to be carcinogenic after long term feeding, the results suggest that the short-term effects of the class of agents known as hypolipidemic peroxisome proliferators are different from the chronic effects. Alternatively, the precursor lesions of hepatocellular carcinomas induced by hypolipidemic, peroxisome proliferators may differ from enzyme altered hepatocytic foci.