Male rats fed a diet deficient in choline and methionine develop hepatocellular carcinomas without the administration of carcinogenic agents (Ghoshal and Farber 1984, Mikol et al. 1983). In these rats, fat accumulates and there is cell necrosis and increased proliferation of hepatocytes, followed by the development of -glutamyl transferase-positive foci and cirrhosis leading to hepatocellular carcinoma as a final event (Ghoshal and Farber 1993). The diet most frequently used in these long-term studies is the Lombardi diet (Shinozuka et al. 1978), which contains 9% peanut meal and 8% soy protein isolate as major sources of protein and is therefore low in methionine. Additionally, choline is not provided as a supplement. Male rats fed this diet deficient in choline and methionine have a reduced content of hepatic choline (<50% of control), betaine (30% of control), methionine (80% of control), S-adenosylmethionine (SAM)⁴ (60% of control) and folate (69% of control) (Selhub et al. 1991). These findings indicate that this diet causes a stress on the methyl pool and on the folate-dependent remethylation of homocysteine to methionine. Another diet commonly used for this methyl-deficient rat model is an amino acid-defined diet without choline and low in methionine. Nakae et al. (1992) compared this amino acid-defined, choline-deficient, low-methionine diet to the Lombardi diet and showed a higher incidence of hepatocellular carcinomas with the amino acid-defined diet. The investigators proposed that lack of oligopeptides in the amino acid-defined diet causes less transintestinal absorption of methyl donor amino acids and therefore results in a more severe methyl donor deficiency (Nakae et al. 1992).

Because folate is an essential cofactor in the endogenous formation of methionine from homocysteine, it seems likely that a combined folate-choline-methionine deficiency will cause a more severe methyl donor deficiency than a diet deficient in only choline and methionine. There are a few studies supporting this view (Henning et al. 1989a, Tuma et al. 1975). Feeding rats a diet low in folate, choline and methionine resulted in greater fat accumulation and a lower ratio of SAM to S-adenosylhomocysteine (SAH) in the liver compared with rats fed a diet deficient in only choline and methionine (Henning et al. 1989a). Tuma et al. (1975) also observed an increase in hepatic triglyceride accumulation when the folate antagonist methotrexate was administered to rats in addition to a choline-methionine-deficient diet adequate in vitamin B-12.

In previous experiments (James et al. 1989, Zhang et al. 1993a), we observed that in rats fed a methyl-folate-deficient diet, concentrations of NAD in liver, muscle and spleen were decreased. The activity of poly(ADP-ribose) polymerase (PARP) (EC 2.4.2.3.0), an enzyme requiring NAD as its substrate, was increased in rats fed the methyl-folate-deficient diet at 3 wk (Henning et al. 1989b). Poly(ADP-ribose) polymerase is a nuclear enzyme associated with DNA repair, cell replication and differentiation (Lautier et al. 1993). Poly(ADP-ribosylation) has been linked to tumorigenesis because of its function in the alteration of chromatin structure and regulation of protein activity involved in the metabolism of DNA strand breaks (Boulikas 1991). Experiments with cells in culture and animals showed that inhibitors of PARP activity alter the transformation of cells in vitro and tumorigenicity in vivo (Boulikas 1991). It also was shown that PARP activity was increased in rat liver during 2-acetylaminofluorene-induced hepatocarcinogenesis (Kiehlbauch et al. 1993).

The two experiments described in this article were performed to optimize the carcinogenic effect of methyl-choline-deficient diets. We hypothesize that the carcinogenic effect of a methionine-choline-deficient diet can be enhanced by additional folic acid and niacin deficiencies.

MATERIALS AND METHODS

Table 1. Diet composition [View Table]

For the first 6 mo, rats were weighed and food consumption was determined three times per week, and from 6 to 17 mo these procedures were continued twice per week. After 2 and 6 mo, three or four rats of each group were killed by exsanguination under ether anesthesia, and liver tissue was removed for histologic examination. At 12, 15 and 16 mo, one rat per group was killed; the remaining rats were killed at 17 mo. In addition to the histologic examination, total lipid and PARP activity were determined in liver and the NAD concentration in several tissues.

In Experiment 2, 52 rats were randomly assigned to either a folate-deficient, 6% casein, 6% gelatin, 15% soybean oil diet (MCFD-B) or to the control-B diet, which was a similar diet with both folate and niacin added. Fat concentration in the diets in Experiment 2 was increased to 15 g/100 g and cysteine concentration was halved compared with that of the MCFND-A diet in Experiment 1 to make the diet more similar to the Lombardi diet (Shinozuka et al. 1978). After 2 mo, the MCFD-B-fed group was subdivided, and rats were fed either the MCFD-B (n = 21) or MCFND-B diet (n = 21). The methionine and cysteine concentrations of the diets were calculated to be 7.7, 3.7, 2.5, 2.3, 2.3 and 2.3 g methionine/kg for the control-A, 12% MCFD, 8% MCFD, MCFND-A, control-B and MCFND-B diets, respectively, and 0.46, 0.46, 0.3, 4.3, 2.3 and 2.3 g of cysteine/kg for the control-A, 12% MCFD, 8% MCFD, MCFND-A, control-B and MCFND-B diets, respectively. After 2 and 6 mo, three rats from each group were killed; the remaining rats were killed after 12 or 15 mo. At the end of each time period, blood was collected from the abdominal vein into a heparinized syringe and immediately cooled on ice. Liver, kidney, lung and a piece of skeletal muscle (rectus femoris from the right rear leg) were removed and frozen in small aliquots in liquid nitrogen and stored at 70°C or immediately extracted for NAD determinations. At 12 and 15 mo the number and size of hepatic tumors were evaluated. The procedures for the care and treatment of the rats received prior institutional approval and followed the Guide for the Care and Use of Laboratory Animals (NRC 1985).

RESULTS

Table 2. Weight gain and food consumption in Fischer 344 rats fed methyl-folate-deficient diets with or without niacin1 [View Table]

Table 3. Concentrations of NAD in tissues of Fischer 344 rats fed methyl-folate-deficient diets with or without niacin for 6 and 17 mo (Exp. 1)1 [View Table]

In Experiment 2, NAD levels were significantly lower in whole blood and liver at all time intervals in rats fed the methyl-deficient diets compared with control rats (Table 4). Muscle NAD was significantly lower only after 15 mo. Liver NADP concentrations were also lower in rats fed the MCFD-B and MCFND-B diets compared with those fed the control diet. Whole-blood NADP, however, was not different (Table 4).

Table 4. Total liver lipid and NAD concentrations in tissues of Fischer 344 rats fed methyl-folate-deficient diets with or without niacin for 2 to 15 mo (Exp. 2)1 [View Table]

Table 5. Liver concentrations of total lipid and activity of poly(ADP-ribose) polymerase (PARP) in Fischer 344 rats fed methyl-folate-deficient diets with or without niacin for 2 to 17 mo (Exp. 1)1 [View Table]

Table 6. Number and size of tumors developed in Fischer 344 rats fed methyl-folate-deficient diets with or without niacin1 [View Table]

In Experiment 2, 100% of rats fed the MCFD-B and MCFND-B diets developed tumors. Some tumors were first detected at 12 mo (Table 6). The number and size of tumors was increased at 15 mo. There were no significant differences in the number or size of tumors that developed in rats fed the MCFD-B and MCFND-B diets.

DISCUSSION

The lesions in the methyl-folate-deficient rats fall into all three of the above categories. Most importantly, the largest nodules exhibit complete loss of plate architecture with focal pseudoglandular change, absent portal tracts, and marked nuclear pleomorphism with prominent mitosis. By both present and older criteria these nodules represent hepatocellular carcinoma of moderate differentiation. The certain criterion for malignancy is metastasis, which was not encountered in our animals, possibly because they were not allowed to live long enough. There were no spontaneous deaths. A test of malignancy by growing the tumor cells in culture is planned with the next experiment.

All rats fed the methyl-deficient diets in both experiments showed typical effects of a methyl donor deficiency, such as fatty livers, decreased hepatic SAM concentration and either increased or unaltered hepatic SAH levels (data not shown). In Experiment 1, decreasing the methionine in the diet by decreasing the casein content increased the severity of the effects. In both experiments, however, hepatocellular carcinomas developed only in rats fed the 6% gelatin, 6% casein diet with or without niacin. Rats fed either the 12 or 8% casein diet did not develop carcinomas, although the methionine contents of the 8% casein (2.5 g/kg diet) and 6% casein and gelatin diet (2.3 g/kg diet) were similar. One striking difference in the amino acid composition of casein and gelatin is that gelatin contains more glycine than does casein (MCFD-B, 14.9 g/kg diet; 8% MCFD, 1.6 g/kg diet). In an experiment by Krumdieck et al. (1992), a severe shift in the folylpolyglutamate distribution towards shorter forms was observed in livers of rats receiving supplemental glycine (25 g/kg diet). More than a 10-fold increase in monoglutamate and a reduction of hexa- and pentaglutamates were observed. Although these rats had a normal folic acid intake, the total liver folates in these rats dropped to about one third of control levels. It is possible that also in the case of the MCFD-B diet the high glycine content caused a further decrease in liver folate levels and therefore rendered rats more methyl deficient.

In Experiment 2, 100% of rats fed the casein-gelatin diets (MCFD-B and MCFND-B) developed tumors, whereas in the first experiment only one out of four rats fed the MCFND-A diet showed hepatocarcinomas. The MCFD-B and MCFND-B diets contained 2.3 g of methionine, no choline, no folate and 10 µg of vitamin B-12 per kilogram of diet. In experiments in which Fischer 344 rats were fed the Lombardi diet, 20-50% of the rats were reported to develop hepatocellular carcinoma after 11-24 mo (Ghoshal and Farber 1984, Mikol et al. 1983, Nakae et al. 1992). The Lombardi diet contained 2 g of methionine, traces of choline, 10 µg of vitamin B-12 and 2 mg of folate per kilogram of diet. The diet composition in regard to the methionine, choline, cysteine and oil content of the Lombardi diet is very similar to the diet used in our laboratory except for the absence of folate and increase in glycine. We hypothesize that the folate deficiency, enhanced by the increased glycine content, leads to a more severe methyl donor deficiency and an increased carcinogenic effect. This is not surprising because the endogenous formation of methionine from homocysteine depends on folate as a cofactor (Krumdieck 1990). If the folic acid supply is compromised, less methionine is formed and less S-adenosylmethionine is available for methylation. In Experiment 2, weight gain was significantly decreased in methyl-folate-deficient rats (fed the MCFD-B and MCFND-B diets) compared with controls although the food intake was increased per kilogram of body weight. Comparing the effect of our diet on weight gain with that of the choline-deficient Lombardi diet, rats fed the Lombardi diet did not show this reduction in weight gain compared with the choline-sufficient controls (Chandar and Lombardi 1988). The pattern of weight reduction in Experiment 2 again reinforces the hypothesis that the additional folate deficiency leads to a more severe methyl donor deficiency, which is reflected in the reduced weight gain compared with that of rats fed the Lombardi diet.

No differences were found in either number and size of tumors in rats fed the MCFD-B or MCFND-B diet. In rats fed the MCFND-B diet, tissue and plasma NAD levels were not significantly different from those of rats fed the diet containing niacin (MCFD-B). Rats apparently had enough NAD through the conversion of tryptophan to maintain niacin levels as in those fed MCFD-B. To produce a more severe niacin deficiency, it may be necessary to reduce the amount of tryptophan in the diet.

In previous publications it has been shown that the PARP activity was increased in livers of methyl-deficient rats after 3 wk (Henning et al. 1989b). In the present study, the hepatic PARP activity was enhanced during a mild reduction in hepatic NAD concentrations (about 80% of controls), and the activity was lower than that of controls following a further reduction in NAD concentration (<60% of control) (Table 3). Because DNA strand breaks have been reported in the choline-folate-deficient rat model (James et al. 1989), we hypothesize that PARP activity was stimulated by DNA strand breaks to facilitate DNA repair. Stimulation of PARP activity may lead to a reduction of NAD below 60% of the control value leading to an inhibition of PARP activity, as observed in livers of rats fed the MCFND-A diet. The NAD concentrations were decreased in liver and blood at 6 mo, and at 17 mo other tissues showed alterations in NAD levels, suggesting a general change in niacin metabolism, possibly secondary to alterations in PARP activity. These alterations in PARP activity and NAD may slow DNA repair processes and increase DNA damage.

Only rats fed the MCFND-A, MCFD-B and MCFND-B diets developed hepatocarcinomas after 12-17 mo. Because all rats fed the MCFD-B diet developed tumors, the effect of a concomitant niacin-deficient diet could not be ascertained. In summary, this study provides preliminary results that methyl-deficient diets devoid of folic acid enhance tumor development. Additional studies are required to resolve the question of whether tumor development will be influenced by a more severe niacin deficiency.