The inhibition by methionine and choline of liver carcinoma formation in male C3H mice dosed with diethylnitrosamine and fed phenobarbital

The ability of the dietary methyl donors methionine and choline to inhibit the carcinogenic and tumor-promoting effects of phenobarbital (PB) in the livers of male weanling C3H mice was examined. The mice were fed a commercial rodent diet with or without 0.05% PB. Thirty animals from each set received the diet with either: (1) no dietary supplementation, (2) an additional 1.0% choline chloride, (3) 1.5% DL-methionine or (4) both 1.5% DL-methionine and 1.0% choline chloride. Additional groups of 30 animals with the same eight dietary and PB-treatment regimens described above were given a single initiating dose of 150 mg diethylnitrosamine (DENA)/kg body wt dissolved in saline, or the saline solution only, 1 week prior to the start of PB feeding. The 16 treatment groups were fed their respective diets for 12 months. Statistical trend analysis showed that increasing levels of supplemental methyl donors gave highly significant protection in PB-treated mice (P less than 0.01). The incidence of liver carcinomas in the four dietary groups not receiving PB or DENA varied from 0 to 7%. The PB-treated animals not receiving an initiating dose of DENA developed hepatocellular carcinomas (HCCs) at incidences of 79% in group 1 animals, 74% in group 2 animals, 60% in group 3 animals, and 31% in group 2 animals respectively. Thus, incidence of HCCs in group 4 was significantly lower than in groups 1, 2 or 3 (P less than 0.01). However, the total incidence of liver tumors (adenomas plus carcinomas) was about the same in all DENA or PB-treated groups. Thus, dietary supplementation with methyl donors increased the proportion of animals bearing liver adenomas as their most advanced hepatic lesion in PB-treated mice. In DENA-treated mice fed PB, dietary supplementation with methionine and choline protected against the formation of liver carcinomas (P less than 0.02); however, methionine and choline had no significant effect on liver tumor formation in mice fed the PB-free diets. Methionine and choline supplementation gave significant protection against HCC metastases in the lungs of the tumor-bearing mice in groups initiated with DENA followed by PB promotion. These results support the hypothesis that PB exerts it tumorigenic activity in mice at least in part through a physiological insufficiency of labile methyl groups.     

Hepatocarcinogenesis in rats fed methyl-deficient, amino acid-defined diets

The ability of methyl-deficient, amino acid-defined diets toproduce liver tumors was studied in rats treated both with andwithout initiating doses of diethylnitrosamine (DENA). Male,weanling F344 rats were fed a complete, amino acid-defined dietfor one week. They were then injected once i.p. with one of3 doses of DENA (20, 70 or 200 mg/kg body weight) and fed thecomplete diet for an additional week. Thirty animals from eachdose group were then maintained for 76 weeks on the completediet (Diet 1) or one of 4 methyl-deficient diets: Diet 2, devoidof methionine and choline; Diet 3, devoid of methionine only;Diet 4, devoid of choline only and Diet 5, devoid of methionine,choline, folic acid and vitamin B₁₂. In Diets 2, 3 and 5 methioninewas replaced by equimolar amounts of its metabolic precursorDL-homocystine. Control rats were injected i.p. with the salinevehicle and maintained for the 76-week period on Diets 1 and2. Forty percent of the rats fed Diet 2, but receiving no DENA,developed hepato-cellular carcinomas or cholangiomas. A 90–100%incidence of hepatocellular carcinomas was seen in all groupsinitiated with DENA and fed Diet 2. No malignant liver tumorsdeveloped in Diet 1 rats that had received 0 or 20 mg/kg DENA;however, hepatocellular carcinomas were noted in one-half ofsuch animals receiving the 70 and 200 mg/kg doses. Liver metastasesgrew in the lungs of 60% of the tumor-bearing rats fed Diet2; none were seen in the Diet 1-fed rats. The singly deficientDiets 3 and 4 enhanced liver tumor formation to DENA-initiatedrats to a significantly lesser extent than did Diet 2. All DENA-initiatedrats fed the severely deficient Diet 5, died within 23 experimentalweeks with livers containing hepatocytes of atypical appearanceand, particularly at the 2 higher dosages, a cirrhotic pseudo-nodulararchitecture. No hepatocellular carcinomas or cholangiomas wereobserved in Diet 5-fed rats. None of the diets tested appearedto enhance tumor formation in extra-hepatic tissues. In fact,significant decreases were noted in the formation of spontaneoustesticular interstitial cell tumors in Diet 2-fed rats and ofpancreatic acinar tumors in rats fed Diets 2 and 3. Diet 2,devoid of both methionine and choline, also induced metaplasiaof pancreatic acinar cells to hepatocyte-like cells and wasasociated with moderate to severe hyperplasia of the transitionalepithelium lining the renal pelvis. Finally, DENA initiationgave weak but significant dose-dependent increases in the incidencesof kidney parenchymal tumors and lung bronchioalveolar celltumors that could not be directly correlated with the extentof methyl deprivation. The results indicate that dietary methyldeficiency markedly promotes liver carcinogenesis and exhibitscomplete carcinogenic activity in this organ in the rat.     

Profound postinitiation enhancement by short-term severe methionine, choline, vitamin B12, and folate deficiency of hepatocarcinogenesis in F344 rats given a single low-dose diethylnitrosamine injection

The potential promoting and/or complete carcinogenic activity of a methyl group-deficient (MD) diet lacking methionine, choline, vitamin B12, and folate on liver tumor induction in weanling male F344/NCr rats was examined. Each of 50 rats per group received one injection 20 mg diethylnitrosamine [(DENA) CAS: 55-18-5; N-nitrosodiethylamine]/kg body weight at 4 weeks of age, and then each was maintained on a methyl group-adequate (MA) diet for 52 weeks (groups 2 and 5) or on an MD diet for 15 weeks followed by the MA diet for 37 weeks (group 4). Controls received injections of saline and were maintained on the same two respective diet regimens (groups 1 and 3, respectively). Histologic results from sacrifices at 6, 10, 15, 22, 39, and 52 weeks revealed early development of foci of eosinophilic gamma-glutamyltransferase (GGT)-positive hepatocytes by week 6 in DENA-MD diet-treated rats, with subsequent development of a diffuse hyperplasia of hepatocytes, oval cell proliferation, cholangiofibrosis, nodular cirrhosis, and neoplastic nodule (NN) formation and, at 52 weeks, hepatocellular carcinomas (HCC) in 13 of 15 rats. Similar but significantly fewer lesions were observed at slightly later sacrifice times in the livers of saline-MD diet-treated rats, with development of NN in 5 of 12 rats and an HCC in 1 of 12 rats at 52 weeks. DENA-treated rats on MA diets developed relatively few GGT-positive foci, and none developed any neoplastic lesions. Except for basophilic foci, areas and foci of cellular alteration containing glycogen-rich hepatocytes frequently exhibited diminished uptake of injected iron and decreased glucose-6-phosphatase and ATPase contents focally or throughout. This study indicates that a relatively brief exposure of both untreated and DENA-treated weanling rats to a severely MD diet produces classical preneoplastic and neoplastic lesions in their livers.     

The effect of choline and methionine deficiencies on the number and volume percentage of altered hepatic foci in the presence or absence of diethylnitrosamine initiation in rat liver

The ability of methyl-deficient, amino-acid-defined diets to produce enzyme-altered foci was quantitatively determined in the livers of rats treated both with and without an initiating dose of diethylnitrosamine (DEN). Male weanling F-344 rats were fed a complete, amino-acid-defined diet for 1 week. They were then injected i.p. with a single dose of DEN (20 mg/kg body weight) and fed the complete diet for an additional week. Forty animals in each dose group were then maintained for 5-38 weeks on the complete diet (diet 1) or one of the three methyl-deficient diets customarily used in this laboratory: diet 2, devoid of methionine and choline; diet 3, devoid of methionine only; and diet 4, devoid of choline only. In diets 2 and 3, methionine was replaced by equimolar amounts of its metabolic precursor, DL-homocystine. Ten animals per group were killed 8, 12, 17, 24 and 41 weeks after DEN initiation. For 2 weeks prior to being killed, each group was maintained on the complete diet to minimize the histological abnormalities due to acute toxicity of the diets. Serial sections of the livers were obtained, stained sequentially for gamma-glutamyltranspeptidase, ATPase and glucose-6-phosphatase, and the quantitation of the focal lesions scored by these markers was carried out by quantitative stereology. The results indicated that, regardless of the enzyme marker(s) examined, there was a general correspondence between the volume and number of altered hepatic foci (AHF) formed and the previously described tumor-promoting activities of each diet. Thus, while all DEN-treated groups contained significant numbers of AHF 24 weeks after initiation, only the diet-2-fed animals displayed such foci at 8 weeks. Similarly, among the uninitiated rats, only those fed diet 2 exhibited the presence of AHF throughout the experimental period. Interestingly, the livers of uninitiated, choline-deficient rats showed a small number of AHF at 24 and 42 weeks; these foci were not observed at all in the corresponding DEN-untreated animals fed diet 3, deficient in methionine only. The results provide evidence that the carcinogenic effects of the methionine- and choline-deficient diet result more from its strongly promoting effect than from any initiating activity by the diet.     

On the role of compensatory mitogenesis in the hepatocarcinogencity of choline and multiple-lipotrope devoid diets

Female F-344 rats, in contrast to male rats of the same strain,are largely resistant to the hepatonecrogenic and hepatocarcinogenicactions of a diet devoid of choline and restricted in methionine(CD diet). A study was performed to determine whether the resistancewould be overcome by feeding a diet devoid not only of choline,but also of methionine, vitamin B₁₂ and folic acid (MGD diet).Three experiments were performed, to compare the degrees ofsteatosis and cell death and proliferation, and the onset ofpre-neoplastic and neoplastic lesions, in the liver of femaleF-344 rats fedeither the CD or the MGD diet Limited responseswere again observed in rats fed the CD diet On the other hand,feeding the MGD diet resulted in degrees of steatosis and ofcompensatory mitogenesis comparable to those previously foundto occurin male F-344 rats fed the CD diet It resulted alsoin the development of a marked cirrhosis, of neoplastic nodulesand of hepatocellular carcinomas. The results indicate thatin female F-344 rats overall availability of methyl groups maybe more critical than the dietary supply of choline in determiningthe severity and spectrum of hepatopathology. They emphasizealso the importance of compensatory mitogenesis in the inductionof neoplastic lesions by methyl- group deficient or devoid diets.     

Comparative tumor-promoting activities of phenobarbital, amobarbital, barbital sodium, and barbituric acid on livers and other organs of male F344/NCr rats following initiation with N-nitrosodiethylamine

Tumor-promoting abilities of four barbiturates, phenobarbital [(PB) CAS: 50-06-6], amobarbital [(AB) CAS: 57-43-2], barbital sodium [(BB) CAS: 144-02-5], and barbituric acid [(BA) CAS: 67-52-7], on the development of neoplasms in livers and other organs of rats following initiation with N-nitrosodiethylamine [(DENA) CAS: 55-18-5] were compared. Four-week-old F344/NCr male rats were given a single ip injection of 75 mg DENA/kg body weight. Beginning 2 weeks later, they were given either tap water (group 1) or drinking water containing 500 ppm of PB (group 2), the sodium salt of BB (group 3), AB (group 4), or BA (group 5) for the remaining experimental period. Control groups (groups 6-10) received an ip injection of saline alone and 2 weeks later were given either tap water or drinking water containing barbiturates as listed above. Animals were sacrificed at either 52 weeks or 78 weeks. None of the barbiturates altered the growth and survival of animals. PB and BB increased liver weights and significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 weeks and hepatocellular foci, hepatocellular adenomas, and trabecular carcinomas at 78 weeks in DENA-treated rats. No such enhancing effects were observed with AB or BA. PB or BB did not significantly enhance the incidence of nonhepatic tumors at 52 weeks. However, at 78 weeks BB significantly enhanced the development of renal tubular adenomas and carcinomas, while PB enhanced the development of thyroid follicular cell neoplasms in DENA-treated rats. These results clearly showed that barbiturates exhibited structure-promoting activity relationships and that their promoting abilities were not restricted to liver alone. Substitution of both hydrogen atoms at the C-5 position of the pyrimidine ring by alkyl or aryl groups appears to be essential but not sufficient for tumor-promoting activity of barbiturates.     

Hepatocarcinogenic and promoting action of a choline-devoid diet in the rat

Male Fisher 344 rats were solely fed a choline-supplemented diet for 65 to 105 weeks or a choline-devoid diet for 24 to 102 weeks. Hepatocellular carcinomas developed in the latter animals, beginning at 24 weeks. Other groups of rats were given a single dose of 20 mg diethylnitrosamine/kg, 18 h after a partial hepatectomy and were fed, 4 weeks thereafter, either a choline-supplemented, or a choline-devoid diet for up to 48 weeks. In rats fed the choline-supplemented diet, the only relevant lesion observed was a small transect number of foci of enzyme-altered hepatocytes. On the other hand, a significant number of foci, of preneoplastic nodules, and of hepatocellular tumors developed in rats fed the choline-devoid diet. The results obtained are consistent with those previously reported by others, indicating that diets devoid of choline, or of choline and methionine, are carcinogenic. The diets appear to act as complete carcinogens, since they are also efficient promoters of chemical hepatocarcinogenesis, as shown again, in the present study, by the results obtained in the diethylnitrosamine-pretreated rats.     

Tissue levels of S-adenosylmethionine and S-adenosylhomocysteine in rats fed methyl-deficient, amino acid-defined diets for one to five weeks

The levels of S-adenosylmethionine (AdoMet) and of S-adenosylhomocysteine(AdoHcy) as well as the ratio of AdoMet/AdoHcy were determinedin the liver, lungs, testes and kidneys of weanling male ratsfed a commercial chow diet or 5 different amino acid-defineddiets for 1– 5 weeks. The amino acid-defined diets usedwere as follows: diet 1, supplemented with methionine, choline,folic acid and vitamin B₁₂ diet 2, deficient in methionine andcholine; diet 3, deficient in methionine alone; diet 4, deficientin choline alone; diet 5, deficient in methionine, choline,folic acid and vitamin B₁₂ All methionine-deficient diets weresupplemented with an equimolar dose of its metabolic precursor,homocystine. The animals were sacrificed after 1, 3 and 5 weeksof treatment. In animals fed either the chow diet or diet 1,liver was the organ found to contain the highest levels of AdoMetand AdoHcy. Similarly, in animals fed diet 1 or chow, the testesand lungs contained the lowest level of AdoMet, while the lungscontained the lowest levels of AdoHcy. In general, the tissuelevels of AdoHcy and AdoMet in rats fed diet 1 were very similarto the corresponding values found in chow-fed rats. Diet 1 feeding,however, led to higher hepatic levels of AdoMet than did theadministration of the chow diet. The administration of the methyl-deficientdiets generally led to decreased hepatic AdoMet contents at3 and 5 weeks; the methyl-deficient diets also led to increasedAdoHcy contents and decreased AdoMet:AdoHcy ratios when comparedwith diet 1. Linear regression analysis show ed a significantdirect correlation between the observed hepatic AdoMet levelsand the methyl content of the diet as well as an inverse correlationbetween hepatic AdoHcy levels and dietary methyl contents. Unlikeliver, the lung and testes did not show any decrease in AdoMetcontent following feeding of the methyl-deficient diets. Thesetissues did show, however, early significant increases in AdoHcycontents and corresponding decreases in the ratios of AdoMet:AdoHcy.These changes were found to be proportional to the dietary methylcontent. The renal contents of AdoMet, AdoHcy and the ratioof AdoMet/AdoHcy were unaffected by any of the diets administeredexcept for diet 5. The administration of diet 5 to rats for5 weeks led to a significant increase in renal AdoHcy. Theseresults provide evidence indicating that dietary methyl insufficiencymay exert its role in carcinogenesis through a decreased availabilityof AdoMet in vivo.     

Comparison of lesions induced in the Syrian golden hamster by diethylnitrosamine, dimethylhydrazine, and dibutylnitrosamine: influence of subsequent butylated hydroxyanisole treatment

The target organ specificity of the carcinogens diethylnitrosamine [(DENA) CAS: 55-18-5], dimethylhydrazine [(DMH) CAS: 57-14-7], and dibutylnitrosamine [(DBN) CAS: 924-16-3] was examined in Syrian golden hamsters. Groups of male animals were given 8 weekly injections of one of these carcinogens and then were maintained on a basal diet or a diet supplemented with 1% butylated hydroxyanisole [(BHA) CAS: 25013-16-5], or they were given the respective carcinogens in the drinking water until they were sacrificed at week 34. While DENA specifically induced tracheal polyps and hepatocellular foci and nodules, DMH administration was associated with development of both hepatocellular and hemangiocellular liver lesions as well as forestomach papillomas and adenocarcinomas of the large intestine. DBN induced lesions in the urinary bladder, forestomach, and trachea, in addition to a few preneoplastic foci in the liver and lungs. In all organs studied, preneoplastic and neoplastic populations were essentially similar to those observed in other experimental animals, with colon and tracheal lesions demonstrating alteration in polysaccharide metabolism. While inhibiting the development of hepatocellular lesions, especially in the group initiated with DENA, and while itself inducing extensive papillomatous forestomach hyperplasia, BHA administration did not exert a significant modifying influence on tumorigenesis in other organs. The present results demonstrate the efficacy of Syrian golden hamster studies for investigation of comparative neoplasia. Of particular interest in this respect were differences in the degree of phenotypic instability demonstrated by glutathione S-transferase placental form-positive foci induced by the 3 carcinogens, which indicated a possible qualitative variation in "initiation."     

Effect of phenobarbital on the development of liver tumors in juvenile and adult mice

The effect of long-term exposure to phenobarbital (CAS: 50-06-6) subsequent to tumor initiation on the development of liver tumors in BALB/c and (C57BL/6 X C3H/Anf)F1 (B6C3F1) mice was determined. In male B6C3F1 mice that received either 15 or 45 ppm diethylnitrosamine [(DENA) CAS: 55-18-5] between 6 and 10 weeks of age, subsequent treatment with 500 ppm sodium phenobarbital in the drinking water resulted in the promotion of liver tumors. However, in male B6C3F1 mice initiated on day 15 of age with 25 mg DENA/kg, beginning long-term treatment of 500 ppm sodium phenobarbital at 4 weeks of age inhibited the development of liver tumors, whereas in male BALB/c mice initiated with 25 mg DENA/kg on day 15 of age, beginning the long-term treatment with 500 ppm sodium phenobarbital at 4 weeks of age promoted the development of liver tumors. Hence phenobarbital can either enhance or inhibit the formation of liver tumors, depending both on the mouse strain used and the animal's age at the start of exposure.     

Neoplastic response of F344 rats and B6C3F1 mice to the polymer and dyestuff intermediates 4,4'-methylenebis(N,N-dimethyl)-benzenamine, 4,4'-oxydianiline, and 4,4'-methylenedianiline

Feeding 4,4'-methylenebis(N,N-dimethyl)benzenamine [CAS: 101-61-1; 4,4'-methylenebis(N,N-dimethylaniline)] to inbred F344 rats increased the incidence of thyroid lesions (hyperplasia, adenomas, and carcinomas) in both sexes, especially in the female rats. 4,4'- Oxydianiline (CAS: 101-80-4) in the diet increased adenomas and carcinomas in the thyroid gland and neoplastic nodules and carcinomas in the liver of male and female F344 rats. In addition to increasing thyroid adenomas in females and hepatocellular adenomas or carcinomas in male and female B6C3F1 mice, 4,4'- oxydianiline increased adenomas of the harderian gland in male and female mice. 4,4'- Methylenedianiline (CAS: 101-77-9) in the drinking water increased neoplasms of the thyroid gland and liver in F344 rats and B6C3F1 mice.     

Development of hepatocellular adenomas and carcinomas associated with fibrosis in C57BL/6J male mice given a choline-deficient, L-amino acid-defined diet.

Development of hepatocellular carcinomas in rats caused by a choline-deficient, L-amino acid-defined (CDAA) diet, usually associated with fatty liver, fibrosis, cirrhosis and oxidative DNA damage, has been recognized as a useful model of hepatocarcinogenesis caused by endogenous factors. In the present study, in order to further explore involved factors and genes, we established an equivalent model in spontaneous liver tumor-resistant C57BL/6J mice. Six-week-old males and females were continuously fed the CDAA diet and histological liver lesions and oxidative DNA damage due to 8-hydroxydeoxyguanosine (8-OHdG) were examined after 22, 65 and 84 weeks. In male mice, fatty change and fibrosis were evident at 22 weeks, and preneoplastic foci of altered hepatocytes were seen at an incidence of 8/8 (100%) and a multiplicity of 6.6 +/- 4.0 per mouse at 65 weeks. Hepatocellular adenomas and carcinomas developed at incidences of 16/24 (66.7%) and 5/24 (20.8%), and multiplicities of 1.42 +/- 1.32 and 0.29 +/- 0.62, respectively, at 84 weeks. The female mice exhibited resistance to development of these lesions. The CDAA diet also increased 8-OHdG levels in male but not female mice. These results indicate that a CDAA diet causes hepatocellular preneoplastic foci, adenomas and carcinomas associated with fibrosis and oxidative DNA damage in mice, as in rats, providing a hepatocarcinogenesis model caused by endogenous factors in mice.     

Azaserine carcinogenesis: organ susceptibility change in rats fed a diet devoid of choline.

In rats, azaserine is primarily a pancreatic carcinogen and only induces hepatomas with a very low incidence. We have investigated the effects of feeding a choline-devoid (CD) diet upon the carcinogenicity of azaserine. Groups of male Wistar rats were fed a CD or a choline-supplemented (CS) diet. Azaserine (30 mg/kg in 2 ml of saline) was injected IP twice a week for the first 4 weeks, and once a week thereafter, for a total of 14 injections. Control groups were fed the CD or CS diet and were injected similarly with saline. Four to seven animals from each group were killed, 1, 4 and 6 months after the first azaserine injection, and the liver and pancreas were examined histologically. None of the control animals fed the CD or CS diet and given saline injections developed tumors of the liver or pancreas. No hepatomas were observed in 12 rats fed the CS diet and treated with azaserine. On the other hand, 3 of 5 and 5 of 7 rats killed after 4 and 6 months, respectively, of treatment with azaserine and the CD diet showed multiple hepatomas. Rats treated with azaserine developed multiple atypical acinar cell nodules (AACN) of the pancreas after 4 months irrespective of whether choline was present or absent in the diet. However, the number of AACN in rats fed the CD diet was significantly smaller than in rats fed the CS diet. It is concluded that a diet devoid of choline changes the organ susceptibility to the carcinogenic action of azaserine, enhancing hepatocarcinogenesis and reducing the action of the carcinogen on the pancreas.     

Ethynylestradiol protection against methyl insufficiency in castrated male Wistar/Furth rats fed a methionine-choline-deficient diet

The interactive effects of dietary methyl insufficiency andthe estrogenic compound ethynylestradiol (EE) on the levelsof S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH)were examined in the liver, lungs and pancreas of rats. In addition,such effects on the hepatic content of 5-methyl-deoxycytidine(5-MC) in nuclear DNA were determined. Castrated male Wistar/Furthrats were fed various levels of EE in either: (i) a complete,amino acid-defined diet (diet 1); (ii) the same diet lackingin choline and methionine and supplemented with 0.9% of DL-homocystine(equimolar to methionine) (diet 2); or (iii) diet 2 but onlywith 0.3% DL-homocystine (diet 2M). Methyl deficiency and EEeach independently produced decreased weight gains and increasedrelative liver weights (liver weight relative to total bodyweight) compared with control animals. Livers from rats feddiets 2 and 2M without EE had lower levels of SAM and lowerSAM: SAH ratios than did the livers from diet 1-fed rats nottreated with EE. Hepatic SAM: SAH ratios in diet 1-fed ratswere not altered by EE treatment. However, EE treatment increasedthe hepatic contents of SAM and restored the SAM: SAH levelsto normal in rats fed diet 2 or 2M. The levels of SAM + SAHin the livers of rats fed the low homocystine diet (diet 2M)were less than in those fed either diet 1 or diet 2. Thus, theaddition of EE at 10 p.p.m. gave protection against reducedlevels of SAM, and reduced SAM: SAH ratios in the liver, buthad little effect when added to the methyl-adequate diet. Nodifferences in hepatic 5-MC levels were observed in any of thegroups as a result of either methyl deficiency or EE treatment.Methyl deprivation alone caused no discernible difference inpancreatic SAM levels but did result in a significant rise inSAH levels and thus in decreased SAM: SAH ratios. EE had noconsistent effect on pancreatic SAM, SAH or SAM: SAH ratiosin any of the diet groups examined. Similarly, the chronic feedingof diet 2, diet 2M or of EE had no significant effect on theSAM contents of lungs, compared with the corresponding levelsin control rats. The protection conferred by EE against SAMinsufficiency in the livers of rats fed a methionine- and choline-deficientdiet is consistent with the relative insensitivity of femalerats to the hepato-toxicity of dietary methyl insufficiency.     

Effects of dietary selenium on bis(2-oxopropyl)nitrosamine-induced carcinogenesis in Syrian golden hamsters

In studies designed to determine the influence of dietary Se on pancreatic carcinogenesis, Syrian golden hamsters were fed unsupplemented torula yeast diet or diet supplemented with 0.1 or 5.0 ppm Se, from sodium selenite, starting at 4 weeks of age until the termination of the study. In separate groups, hamsters were given the diet supplemented with 0.1 ppm Se until 5 days after carcinogen treatment. Then they were fed either the unsupplemented diet or the diet supplemented with 5.0 ppm Se until the end of the experiment. N-Nitrosobis(2-oxopropyl)amine (BOP; CAS; 60599-38-4) treatment was given as a single sc injection of 20 mg/kg (body wt) at 8 weeks of age, and surviving hamsters were killed 50 weeks later. As a measure of Se status, glutathione peroxidase (GSHPX) activities were determined in plasma, erythrocytes, and liver. Values were elevated in animals fed higher levels of dietary Se. BOP treatment depressed plasma GSHPX at 24 hours and elevated erythrocyte and liver values at 4 weeks. Pancreatic ductular adenoma yields were inhibited with each elevation of dietary Se in female hamsters fed the diets, both before and after BOP administration, and were further inhibited in females that were fed diets containing 0.1 ppm Se before BOP administration and that were changed to the unsupplemented or 5.0-ppm-supplemented diets after BOP was given. Pancreatic ductular adenoma yields were highest in all male groups given diets of 0.1 ppm Se before BOP administration, irrespective of the Se level after BOP was fed. Adenoma yields in males were lowest in hamsters fed unsupplemented diet, both before and after BOP treatment. Pancreatic carcinoma yields were low and not influenced by dietary Se. The incidence of hepatic necrosis was elevated in BOP-treated hamsters fed the unsupplemented diet, and that of biliary cystic adenomas was highest in the group fed 0.1 ppm Se before and after BOP treatment.     

Altered tRNA methylation in rats and mice fed lipotrope-deficient diets

A diet that is deficient in methionine, choline, folic acidand vitamin B₁₂ has been found to induce alterations rapidlyin liver tRNA methylation in male Fischer rats. In vitro assaysindicated that activity of N2-guanine tRNA methyltransferaseII (NMG2) was increased to 150% of controls levels in 1 weekand 300% of control levels after 2 weeks or longer on this diet.Incompletely methylated tRNA was isolated from livers of thesesame animals, indicating that there was impairment of methylationin vivo. The effects on liver tRNA methylation of this methyl-deficientdiet were thus seen to mimic those of the liver carcinogen,ethionine, which also causes production of hypomethylated tRNAand increased activity of NMG2. The effect of the same dieton liver tRNA methyltransferase activity of C57BL/6J and C3H/HeJinbred mice were also studied. Intake of the lipotrope-deficientdiet Induced elevation in activity of liver N2-guanine tRNAmethyltransferase II activity in C57BL/6J mice, similar to thatseen in rats. In contrast, the methyl-deficient diet had verylittle effect on the same enzyme activity in C3H/HeJ animals.     

Carcinogenicity of betel quid. III. Enhancement of 4-nitroquinoline-1-oxide- and N-2-fluorenylacetamide-induced carcinogenesis in rats by subsequent administration of betel nut

The effect of betel nut on chemical carcinogenesis in the upper digestive tract and liver was examined in two different experimental models with ACI rats. The incidences of neoplasms and preneoplastic lesions of the tongue in animals given 5 ppm 4-nitroquinoline-1-oxide (4-NQO; CAS: 56-57-5) in the drinking water for 16 weeks and followed by 20% betel nut in the diet for 40 weeks were significantly higher than those in animals given 4-NQO alone. No enhancing effect from betel nut on the incidences of neoplastic and preneoplastic lesions in the upper digestive tract was found in animals administered 4-NQO for 12 weeks. The number of altered liver cell foci in rats given 200 ppm N-2-fluorenylacetamide (FAA; CAS: 53-96-3) in the diet for 8 weeks and followed by the betel nut diet for 16 weeks was significantly greater than that in animals fed the FAA diet alone. These results indicate enhancing effects of dietary administration of betel nut on oral carcinogenesis by 4-NQO and hepatocarcinogenesis initiated by FAA.     

Effect of 3-methylcholanthrene on the development of aortic lesions in mice

The effect of a carcinogen, 3-methylcholanthrene (3-MC), on the formation and growth of atherosclerotic lesions in mice was examined. Increasing doses of 3-MC from 15 to 1500 micrograms/kg increased the number and size of lipid-staining lesions in the aorta of AKXL-38a mice that were fed an atherogenic diet for 8 weeks. The number of lesions per mouse was 0.85 +/- 0.19 (SE) for animals treated with 3-MC (150 micrograms/kg) compared to 0.10 +/- 0.10 lesions/mouse for animals given solvent rather than 3-MC. The progression of lesions over time from 5 to 18 weeks showed that 3-MC-treated mice also differed from controls in the size of lesions. The total score per mouse at 18 weeks of atherogenic diet, based on the number of lesions and the size of each lesion, indicated by a score of 1 to 4, was 4.31 +/- 0.71 for 3-MC-treated animals and 2.67 +/- 0.74 for animals given solvent. The effect of 3-MC treatment could be observed at 18 weeks even though the entire dose of 3-MC was given during the first week on the atherogenic diet. These experiments do not distinguish whether 3-MC affects atherosclerotic lesions by acting as a mutagen or by some other mechanism. The composition of an atherogenic diet that produces lesions in mice without high mortality is given as well as a comparison of different methods of evaluating lesion formation.     

Effect of different levels of dietary corn oil and lard during the initiation phase of colon carcinogenesis in F344 rats

The effect of various levels of polyunsaturated fat (corn oil) and saturated fat (lard) fed during the initiation stage of colon carcinogenesis was studied in male F344 rats. The animals were fed the diets containing 5, 13.6, and 23.5% corn oil or lard 2 weeks before, during, and until 1 week after sc injection of 15 mg azoxymethane [(AOM) CAS: 25843-45-2]/kg body weight, once weekly for 2 weeks (designated as initiation). One week after AOM treatment, groups of animals were transferred to their respective 5% corn oil or lard diets. Additional groups consuming 5% corn oil or lard were transferred to 23.5% corn oil or lard, respectively (post-initiation stage). All animals were fed these diets until the termination of the experiment. Fecal bile acids and colonic mucosal ornithine decarboxylase activity were measured in vehicle-treated animals fed the experimental diets for 14 weeks. Body weights and intakes of total calories, protein, nonnutritive fiber, and micronutrients were comparable among the various dietary groups. The animals fed the 23.5% corn oil diet during the postinitiation stage had a higher incidence of colon tumors than did those fed the 5% corn oil diet, whereas feeding of 23.5 and 13.6% corn oil diets during the initiation stage had no effect. In contrast, animals fed the 23.5 and 13.6% lard diet during the initiation stage and 23.5% lard diet during the postinitiation stage developed more colon adenocarcinomas than did those fed the 5% lard diet. The excretion of fecal deoxycholic acid, lithocholic acid, and 12-ketolithocholic acid and the activity of colonic mucosal ornithine decarboxylase activity were higher in animals fed the 23.5% corn oil or lard diet during the postinitiation compared to the levels in animals fed the 5% corn oil or lard diet.     

Prolonged survival of female AKR mice fed diets supplemented with methionine and choline

Female mice of the AKR/J (AK) strain were fed a control diet (Purina Rodent Laboratory Chow) or a lipotrope-supplemented diet (Purina Rodent Chow plus 2% D,L-methionine and 1% choline chloride) beginning at 1 day after weaning. Food consumption and weight gain were found to be the same in both groups of animals. Mice of this inbred strain spontaneously develop thymic lymphoma, with close to 100% mortality expected by 12-13 months of age. Two separate experiments were carried out with 50 mice per group in one, and 40 mice per group in the other. The slopes of the survival curves for the animals in the control group and supplemented group of mice diverged after the animals reached 6.5 months of age. In both experiments, 20% of the mice receiving supplemented diet were still alive at 1 year, while 3% in one experiment and 8% in the other experiment survived in the control groups. Each experiment was terminated when the animals reached 13 months of age. At that time the survival rate of the controls was 2 and 4%, and survival in the groups of mice receiving supplemented diet was 14 and 18%. Necropsy revealed that the animals in both groups had advanced malignant lymphoma. Our results demonstrate that intake of a chow diet that is supplemented with moderate quantities of methionine and choline results in enhanced survival of spontaneously leukemic AK mice, in comparison with animals of this strain fed the same diet without supplements of choline and methionine.