The induction of resistant hepatocytes during initiation of liver carcinogenesis with chemicals in rats fed a choline deficient methiomne low diet

Feeding a choline deficient methiomne low (CLD) diet for twoweeks can substitute for partial hepatectomy in the genesiswith benzo[a]pyrene (B[a]P) or 1,2-dimethylhydrazine (1,2-DMH)of resistant hepatocytes that can be selected with dietary 2-acetylaminofluoreneplus partial hepatectomy. With single doses of B[a]P or 1,2-DMHduring the feeding of the CLD diet for 3 weeks, the number offoci of hepatocytes positive for -glutamyl transferase is approximatelythe same as with the same dose of each carcinogen given afterpartial hepatectomy. DL-ethionine was now found to be positivewhen given as a single dose to animals on the CLD diet. Feedingethionine for 6 or 12 weeks in a similar choline lipotrope deficientdiet also induced a significant increase in foci of enzyme alteredhepatocytes. The results indicate that dietary imbalance canhave an important influence on initiation of chemical carcinogenesis.     

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.     

Enhancement of 2-acetylaminofluorene liver carcinogenesis in rats fed a choline-devoid diet.

The effects of feeding a choline-devoid (CD) or a choline-supplemented (CS) diet on the induction of liver tumors in rats by 2-acetylaminofluorene (AAF) were investigated. Male Sprague-Dawley rats were fed either a CD or a CS diet containing 0.0075% AAF. Three to eight animals were killed 1, 3, 4 and 6 months thereafter. Well to moderately well differentiated hepatocellular carcinomas developed in 50% and 75% of rats fed the CD + AAF diet for 4 and 6 months, respectively. Cholangiocarcinomas developed, after 6 months, in 38% of the animals. No tumor developed in rats fed the CS + AAF diet. The results extend to AAF those previously obtained with DL-ethionine and azaserine showing that feeding a CD diet greatly potentiates the induction in rats of liver tumors by chemical carcinogens.     

Effect of methionine and choline on liver tumor promotion by phenobarbital and DDT in diethylnitrosamine-initiated rats

The effec t of the chronic feeding of methionine or cholineon liver tumor promotion by phenobarbital (PhB) or 1, 1 bis(p-chlorophenyl)-2,2, 2-trichloroethane (DDT) was studied in rats receiving aninitiating dose of diethylnitrosamine (DEN). Male weanling ratswere injected i.p. with DEN (200 mg/kg body wt). Control ratswere injected with saline. Five days after the injection, therats were placed on different diets containing 0.05% PhB or0.05% DDT with or without added 1.5% DL-methionine or 1.0% cholinechloride. Each diet was administered for 72 weeks, when theanimals were placed on the unsupplemented chow diet for an additional30 weeks. Rats treated with DEN and then fed PhB or DDT developedan 85–100% incidence of hepatocellular carcinomas (HCCs).Single injection of DEN alone produced a 60% incidence of HCCs.Dietary feeding of methionine and choline either alone or incombination with PhB or DDT did not have any significant effecton the incidence of HCCs. Liver tumor formation was negligiblein uninitiated rats. Lung metastases developed in 42% and 46%of the DEN + PhB-and DEN + DDT-treated groups, respectively.Supplementation of methionine in the diet lowered the incidenceof lung metastases to 14% in the DEN + PhB-treated rats andto 19% in DEN + DDT-treated rats. Choline was not effectivein inhibiting the development of lung metastases in either case.The injection of DEN alone produced a 54% incidence of lungtumors. PhB and DDT feeding lowered the DEN-induced lung tumorincidence to 23% and 14% respectively. Further, when the datafrom different diet groups were combined it was found that singleinjection of DEN also doubled the incidence of leukemia normallyseen in F344 rats. The present report constitutes the firstevidence that a single injection of DEN induces lung tumorsand enhances the incidence of leukemia in rats.     

Enhancement of DL-ethionine-induced liver carcinogenesis in rats fed a choline-devoid diet.

The effects of a choline-devoid (CD) or a choline-supplemented (CS) diet on the induction of liver tumors in rats by DL-ethionine were investigated. Groups of male outbred Sprague-Dawley rats were fed a plain CD or a plain CS diet, or the same diets containing 0.05% DL-ethionine. Hepatocellular carcinomas developed in 50% of the rats fed the CD+ethionine diet for 14 weeks and in about 80% of the rats fed the same diet for 22-30 weeks. No hepatocellular carcinomas developed in rats fed the CS+ethionine diet, the plain CD diet, or the plain CS diet up to 30 weeks. The findings suggest that a CD diet alters the response of rat liver to DL-ethionine and leads to an early and enhanced induction of hepatocellular carcinoma.     

Hyperplastic nodules of the liver induced in rats fed bracken diet

Hyperplastic nodules (HN) of the liver were induced in Charles River Sprague-Dawley rats (CD rats) and ACI rats fed a diet containing 30% bracken for 260 and 180 days, respectively. HN were also induced in high incidence in CD rats fed a diet containing the carcinogenic fractions of bracken extract.     

High incidence of hepatocellular carcinomas induced by a choline deficient L-amino acid defined diet in rats.

The carcinogenicities of a choline deficient L-amino acid defined (CDAA) diet and a semipurified choline deficient diet were comparatively examined. A total of 60 male Fischer 344 rats, 6 weeks old, were divided into 5 experimental groups each consisting of 12 rats. Group 1 received the CDAA diet chronically to the end of the 52-week experiment while Group 2 was given the same diet for the first 24 weeks and then a basal diet for the following 28 weeks. Groups 3, 4, and 5 received a choline supplemented L-amino acid defined diet, the semipurified choline deficient diet, and a semipurified choline supplemented diet, respectively, throughout the experimental period. All surviving rats were subjected to complete macroscopic examination at Week 52. Histopathologically diagnosed hepatocellular carcinomas were induced in Group 1 at an incidence of 100%; multiple metastatic nodules were seen in the lungs of one of the animals. Hepatocellular carcinomas were also induced in Group 4 rats at a significantly lower incidence of 20%. No hepatocellular carcinomas were observed in rats in Groups 2, 3, and 4. The results indicate that the CDAA diet exerts more potent carcinogenicity for the livers of rats than does the semipurified choline deficient diet. However, limited exposure for 24 weeks may have not been sufficient for hepatocellular carcinoma induction by the CDAA diet at Week 52 although a high incidence of hyperplastic nodules and slight cirrhosis were evidence of persistent lesions.     

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.     

Reduced accumulation of I-compounds in liver DNA of rats fed a choline-devoid diet

Groups of rats were fed for 1, 4 or 7 months choline-devoidor choline-supplemented diets, that provided 50% of the methionine,and 0.15% or 150% of the choline requirements of young, growingrats. liver DNA was isolated and analyzed by the nuclease Pl-enhancedversion of the ³²P-postlabeling assay, which detects aromatk/hydrophobicDNA adducts and I-compounds (adduct-like DNA modifications shownto accumulate in tissues of aging rats). DNA adducts and qualitativedifferences in the patterns of I-compounds were not observedin rats fed the two diets. However, in rats fed the choline-devoiddiet there was a drastic reduction in the accumulation of I-compounds,compared with that in rats fed the control diet. These resultsextend previous evidence of a lack of relevant DNA adducts inthe liver of rats fed the choline-devoid diet, and suggest thepossibility of a role of I-compounds in the carcinogenicityof this diet.     

32P-Postlabeling analysis of liver DNA adducts in rats chronically fed a choline-devoid diet

Liver DNA, obtained at various time intervals from rats chronicallyfed a choline-devoid diet, was analysed for the presence ofaromatic or alkyl adducts by the ³²P-postlabeling assay. Alkyladducts were not detected. Aromatic DNA adduct lesions wererevealed, but only at levels (1 adduct per 0.5–3x10⁹ nucleotides)which are at the limits of the extremely high sensitivity ofthe method used, levels which remained constant throughout theperiod of feeding. Thus, contamination of the total environmentof the animals with chemical carcinogens does not appear tobe responsible for the genesis of the hepatocellular carcinomasthat develop in rats chronically fed a choline-devoid diet.The diet, therefore, either acts as a complete carcinogen, orpromotes the evolution to cancer of endogenous, ‘spontaneously’initiated liver cells.     

Elevated levels of prostaglandin E2 in Yoshida hepatoma and the inhibition of tumour growth by non-steroidal anti-inflammatory drugs

Prostaglandin (PG) E2 biosynthesis in Yoshida hepatoma (AH 130) was evaluated by radioimmunoassay. When hepatoma cells were incubated in vitro, the levels of PGE2 in the medium were similar to those found in hepatocytes for the first 2 h; this was followed by a rapid increase in PGE2 formation, and the 6h incubation levels were 4-fold higher than in hepatocytes. Addition of sodium arachidonate markedly and dose-dependently stimulated PGE2 synthesis; the increase was largely prevented by the addition of indomethacin (1 microM) or L 8027, a prostaglandin synthetase inhibitor. Experiments in vivo indicated that indomethacin treatment of tumour-bearing rats significantly reduced the tumour mass. When rats were injected with PGE2 after receiving the drug, the number of tumour cells was very similar to that of untreated animals. This, as well as the inhibition of tumour growth by acetylsalicylic acid, strongly suggests that the inhibition of PG biosynthesis by anti-inflammatory drugs and the inhibition of tumour proliferation may be closely associated events. It was also found that injections of indomethacin very significantly prolonged survival of hepatoma-bearing rats. Since PGE2 does not appear to affect the cyclic AMP levels of hepatoma cells, it is possible that hepatoma may use PGE2 to subvert the immune system. This could help to explain the effectiveness of anti-inflammatory drugs in the control of tumour growth.     

Autoradiography of "oval cells" appearing rapidly in the livers of rats fed N-2-fluorenylacetamide in a choline devoid diet

Autoradiographic analysis of liver sections from rats fed thehepatocarcinogen N-2-fluorenylacetamide (FAA) in a choline devoid(CD) diet suggests that proliferating small "oval" cells arisefrom a few small portally-situated cells, and spread rapidlyacross the entire liver lobule. Small cells with detectablegrains are first located where liver plates meet the portalareas. This cell type gradually increases in number over a 10–12day period, then proliferates rapidly. After 28 days, microscopicnodules consisting of heavily labeled large eosinophilic cellsappear, whereas residual hepatocytes are not labeled. Combinedimmunofluorescent and autoradiographic labeling studies revealthat many of the small cells contain AFP; approximately halfof the a-fetoprotein-containing cells are labeled with [³H]thymidine(dT). Feeding CD-FAA diets to rats with hepatocytes prelabeledwith [³H]dT after 70% hepa-tectomy 7 weeks earlier providesdata which suggest that small "oval" cells do not arise fromprelabeled hepatocytes but, instead, infiltrate the prelabeledhepatocytes during the diet induced proliferative phase. Weconclude that "oval" cells arise from a small number of portalcells, not from hepatocytes. Exact identification of the ovalcell precursor is not possible, but it could be a "stem" cell.Although hepatocyte-like properties are found in small cells(e.g., albumin staining), there is no evidence that they differentiateinto normally functioning hepatocytes.     

Cyclooxygenase-2 inhibitor celecoxib inhibits promotion of mammary tumorigenesis in rats fed a high fat diet rich in n-6 polyunsaturated fatty acids

Fifteen pentacyclic triterpene diols and triols, consisting of: six taraxastanes, faradiol (1), heliantriol B0 (2), heliantriol C (3), 22alpha-methoxyfaradiol (4), arnidiol (5), and faradiol alpha-epoxide (6); five oleananes, maniladiol (7), erythrodiol (8), longispinogenin (9), coflodiol (10), and heliantriol A(1) (11); two ursanes, brein (12) and uvaol (13); and two lupanes, calenduladiol (14) and heliantriol B2 (15), isolated from the non-saponifiable lipid fraction of the edible flower extract of chrysanthemum (Chrysanthemum morifolium) were evaluated for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, in Raji cells as a primary screening test for anti-tumor-promoters. All of the compounds tested showed inhibitory effects against EBV-EA activation with potencies either comparable with or stronger than that of glycyrrhetic acid, a known natural anti-tumor-promoter. Evaluation of the cytotoxic activity of six compounds, 1-3 and 5-7, against human cancer cell lines revealed that compound 5 possesses a wide range of cytotoxicity, with GI50 values (concentration that yields 50% growth) of mostly less than 6 microM.     

Early histological and functional alterations of ethionine liver carcinogenesis in rats fed a choline-deficient diet.

The effects of feeding a choline-deficient (CD) or a choline-supplemented diet upon the early stages of DL-ethionine carcinogenesis in rat liver were investigated. Low levels of DL-ethionine (0.05 and 0.10%) when fed with a CD diet were found to induce within 4 weeks a massive proliferation of oval cells without significant cell necrosis or presence of inflammatory cell infiltrates. The same levels of ethionine when fed with a choline-supplemented diet caused no significant histological alteration of the liver. In rats fed the CD plus ethionine diets concomitant with the proliferation of oval cells, there was a marked elevation in the content of alpha1-fetoprotein in both liver and plasma. After specific immunofluorescence staining, oval cells stained intensely for albumin and alpha1-fetoprotein. Hepatocytes stained only for albumin, and bile duct cells stained for neither albumin nor alpha1-fetoprotein. These results indicate that a diet deficient in choline markedly alters the response of rat liver to carcinogenetic doses of ethionine. Thus, ethionine hepatocarcinogenesis in rats fed a CD diet may be a useful model for the exploration of the mechanism(s) whereby a dietary factor influences hepatocarcinogenesis.     

Selenium and difluoromethylornithine additively inhibit DMH-induced distal colon tumor formation in rats fed a fiber-free diet

We investigated the effects of difluoromethylornithine, an inhibitorof ornithine decarboxylase (ODC) and selenium supplementationon tumor formation induced by the carcinogen 1, 2-dimethylhydrazine(DMH) in Sprague-Dawley rats. A biochemical link between polyaminebiosynthesis and selenium metabolism to its cancer preventativeform has been suggested by the common requirement of S-adenosylmethio-nine.One-hundred and twenty male Sprague-Dawley rats were dividedinto experimental (n = 80) and control (n = 40) groups. Experimentalanimals received DMH 20 mg/kg s.c. for 20 weeks. Animals werefed either a regular diet (selenium content 0.2 p.p.m.) or ahigh selenium diet (5 p.p.m.) with or without 0.2% DFMO in thedrinking water. At death, week 30, animal weights within experimentalor control groups were not different between the four diet treatmentgroups. Tumor number and incidence in the proximal colon wasnot affected by DFMO treatment, selenium supplementation orthe combined treatment. In contrast, in the distal colon, 19tumors developed in the DFMO treated group, 22 tumors in thehigh selenium group and only 12 tumors in the combined highselenium/DFMO treatment group compared to 32 tumors in the regulardiet group. Similarly, tumor incidence was decreased by DFMOand selenium supplementation and their effects were additive.In control animals, ODC activity was decreased by DFMO treatmentand selenium supplementation in the distal colon and liver,but not the proximal colon. ODC activity of tumor tissue wasgreater than normal colon tissue from diet paired animals forproximal and distal colon, except for distal colonic tumorsin the high selenium/DFMO treatment group. Polyamine content,however, did not correlate with ODC activity in normal or neoplastictissue. In general, S-adenosylmethionine levels from normalcolon and liver tissue were unaffected by diet treatment. Seleniumsupplementation in combination with DFMO treatment selectivelyinhibited distal colon tumor formation in rats fed a fiber-freediet.     

Role of the prostaglandin E2 receptor in mammary tumor metastasis.

Both clinical and experimental breast tumors often synthesize high levels of prostaglandins, most notably prostaglandin E2 (PGE2). We have reported previously that metastatic murine mammary tumor cells also express a high-affinity PGE2 receptor. We have now shown that the receptor plays a functional role in the metastasis of two mammary tumor cell subpopulations, lines 66 and 4526. We showed that three agents, LEO101 (LEO Pharmaceuticals), SC19220 (Searle Co.), and AH6809 (Glaxo Co.), antagonize [3H]PGE2 binding to these cells and block PGE2-mediated elevation of intracellular cyclic AMP. Pretreatment of line 66 cells with nontoxic concentrations of any of the three receptor antagonists prior to i.v. injection results in more experimental lung colonies. As shown previously, and confirmed here, pretreatment of these cells with indomethacin (which inhibits endogenous PGE synthesis and therefore increases detectable PGE receptor) inhibits metastasis. Thus, the tumor cell PGE2 receptor contributes to the ability of murine mammary tumor cells to metastasize.     

Epoprostenol sodium, a prostaglandin I2, lacks tumor promoting effects in a medium-term liver carcinogenesis bioassay in rats

Potential modifying effects of epoprostenol sodium administration on liver carcinogenesis were investigated in male F344/DuCrj rats initially treated with N-nitrosodiethylamine (DEN). Two weeks after a single dose of DEN (200 mg/kg, intraperitoneally), rats daily received subcutaneously epoprostenol sodium at doses of 0, 1, 10 and 100 microg/kg, or were fed phenobarbital sodium (PB) at a dietary level of 500 parts per million (ppm) as positive control for 6 weeks. All animals were subjected to partial hepatectomy at week 3, and were killed at week 8. Prominent flushing of extremis and signs of behavioural depression occurred after injection and lasted for 1 h in rats given 100 microg/kg epoprostenol sodium. Such clinical signs were slight in rats treated with 10 microg/kg, but not observed with 1 microg/kg. Marked decrease in body weight gain was noted in rats given 100 microg/kg. Statistically significant changes in relative liver weights were not found in any group given the test chemical. Epoprostenol sodium did not significantly increase the quantitative values for glutathione S-transferase placental form (GST-P) positive liver cell foci observed after DEN initiation, in clear contrast to the positive control. The results thus demonstrate that epoprostenol sodium lacks modifying potential for liver carcinogenesis in our medium-term bioassay system.     

Hot-spot mutations in the p53 gene of liver nodules induced in rats fed DL-ethionine with a methyl-deficient diet.

Male F-344 rats were fed for 15 weeks a methyl-deficient L-amino acid defined diet containing 0.05% DL-ethionine. Nodules protruding from the surface of the liver were dissected free of surrounding tissue, and polyadenylated RNA isolated from the nodules was reverse transcribed. The region of the p53 gene comprising codons 120-290 was amplified by the polymerase chain reaction, and cDNAs were sequenced. Mutations were detected in nodules obtained from 7 of 12 rats. In all seven cases, the same two point mutations were present. The first was at the first base of codon 246 and consisted of a C-->T transition (C:G-->T:A, Arg-->Cys), while the second was at the second base of codon 247 and consisted of a G-->T transversion (G:C-->T:A, Arg-->Leu). It is concluded that the hepatocarcinogen ethionine induces specific hot-spot p53 gene mutations; this is in contrast to the mutations at various sites previously observed to occur in rats fed a hepatocarcinogenic methyl-deficient diet alone. The results also provide the first evidence that ethionine is mutagenic in the rat.