Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.     

Effect of ethanol on early stages in nitrosamine carcinogenesis in rat liver

The effect of chronic alcohol consumption on the extent of adenosine triphosphatase(ATPase)-deficient preneoplastic lesions in rat liver induced by either diethylnitrosamine (DEN) (3 mg/kg, p.o.) or N-nitrosomorpholine (NNM) (40 ppm in the drinking water) was studied. Carcinogens were administered on 4 days in every week for 11 (DEN) and 15 (NNM) weeks, respectively. Ethanol was given at a concentration of 10% (w/v) in the drinking water either during carcinogen treatment or after withdrawal of carcinogen. An increase in both number and size of ATPase-deficient foci in liver was observed when the alcohol was given during the period of carcinogen administration. This increase may be associated with the known toxic action of ethanol which leads to single cell necrosis and liver regeneration. In contrast, when ethanol (10% in the drinking water for 16 weeks) was given after cessation of carcinogen treatment following a tumor-promotion feeding protocol, no such enhancement in preneoplastic response was obtained. Ethanol alone was ineffective in inducing ATPase-deficient foci. In liver, ethanol thus appears to possess, under certain conditions, co-carcinogenic but not tumor-promoting capacity.     

Immunological study of rat liver RNA in the early stages of hepatic carcinogenesis]

In tissues of non-lineal rats receiving dimethyl amino-azobenzene (DAAB) or its non-carcinogenic analog diethyl amino-azobenzene (DEAB) there was found, using rabbit serum against an artificial complex RNA + MBSA (methylated bovine serum albumin), the RNA-haptene in the reaction of counter-immunoelectrophoresis in the liver and serum of rats in definite terms since the start of DAAB administration. RSR reaction and immunoelectrophoresis have demonstrated the presence of circulating antibodies against hepatic cell RNA. The kinetic of antibodies against RNA is characterized by their increase after 15 DAAB injections, their decrease to the 60th day and again an increase by the time of the tumor appearance. It is suggested that the phenomena of sensibilization and desensitization to the antigens arising in the process of carcinogenesis play a definite role in "cancelling" the antitumor immunity.     

Multiple adaptive mechanisms to chronic liver disease revealed at early stages of liver carcinogenesis in the Mdr2-knockout mice

Molecular events preceding the development of hepatocellular carcinoma were studied in the Mdr2-knockout (Mdr2-KO) mice. These mice lack the liver-specific P-glycoprotein responsible for phosphatidylcholine transport across the canalicular membrane. Portal inflammation ensues at an early age followed by hepatocellular carcinoma development after the age of 1 year. Liver tissue samples of Mdr2-KO mice in the early and late precancerous stages of liver disease were subjected to histologic, biochemical, and gene expression profiling analysis. In an early stage, multiple protective mechanisms were found, including induction of many anti-inflammatory and antioxidant genes and increase of total antioxidant capacity of liver tissue. Despite stimulation of hepatocyte DNA replication, their mitotic activity was blocked at this stage. In the late stage of the disease, although the total antioxidant capacity of liver tissue of Mdr2-KO mice was normal, and inflammation was less prominent, many protective genes remained overexpressed. Increased mitotic activity of hepatocytes resulted in multiple dysplastic nodules, some of them being steatotic. Expression of many genes regulating lipid and phospholipid metabolism was distorted, including up-regulation of choline kinase A, a known oncogene. Many other oncogenes, including cyclin D1, Jun, and some Ras homologues, were up-regulated in Mdr2-KO mice at both stages of liver disease. However, we found no increase of Ras activation. Our data suggest that some of the adaptive mechanisms induced in the early stages of hepatic disease, which protect the liver from injury, could have an effect in hepatocarcinogenesis at later stages of the disease in this hepatocellular carcinoma model.     

Development of resistance during the early stages of experimental liver carcinogenesis.

The present study was designed to determine whether the resistant phenotype is acquired at the initiated cell stage itself or requires further exposure to a promoting regimen to express resistance. Male Fischer 344 rats were initiated with diethylnitrosamine (DENA) (200 mg/kg i.p.) and were subjected to either no further treatment or to the resistant hepatocyte (RH) model of liver tumor promotion. Six weeks later, the resistance of the focal lesions generated in these two groups to the mitoinhibitory effects of 2-acetylaminofluorene (2-AAF) was determined by subjecting the rats to two-thirds partial hepatectomy (PH) in the presence of a mitoinhibitory dose of 2-AAF (5 mg/kg i.p.) given at the time of PH. Labeling index was determined by administering multiple injections of [(3)H]thymidine. All rats were killed 48 h post-PH. While only a small percentage (23%) of the glutathione S-transferase-positive foci generated by DENA in the absence of an exogenous liver tumor promoting regimen were resistant to the mitoinhibitory effects of 2-AAF, a majority (85%) of the foci became resistant to 2-AAF following exposure to the RH model of liver tumor promotion. Further, initiated rats exposed to either 2-AAF or to CCl(4) alone, the two components of the RH model, resulted in 71% of the foci being resistant to the mitoinhibitory effects of 2-AAF. Similar patterns of results were obtained when the resistance of the foci to the mitoinhibitory effects of orotic acid, a liver tumor promoter and an inhibitor of DNA synthesis in normal hepatocytes, was monitored. These results suggest that the majority of initiated hepatocytes are not of resistant phenotype, however, they have acquired a unique ability to express resistance upon exposure to certain agents such as 2-AAF and CCl(4) or to a promoting regimen such as the RH model of liver tumor promotion.     

Ezymohistochemical study of the early stages of carcinogenesis in the rat peripheral nervous system

Early blastomatous changes in the peripheral nervous system induced by transplacental N-nitroso-N-ethylurea exposure were manifested in lemmoblasts proliferates, observed starting from the 14th day of the postnatal period in rats. The proliferates showed the increased activity of ortho-phosphoric and carbonic esters hydrolysis enzymes pentose cycle, ultimate glycolysis and the reduced citric acid cycle, tissue respiration, the synthesis of aminoacids, folic acid, nucleoproteids precursors, and a sharp suppression of oxidative desamination. Such profile of enzymic provision was retained in neurinomas developed 6 months following the postnatal period. Based on the data obtained, it is concluded that the process of glial cells malignant transformation is terminated long before the appearance of malignant neoplasms detected macroscopically.     

Pituitary grafts modify sex differences in liver tumor formation in the rat following initiation with diethylnitrosamine and different promotion regimens

We previously reported on sex differences in chemical hepatocarcinogenesisstudied in the resistant hepatocyte model and on the effectsof implantation of ectopic pituitary grafts into male rats onthe early stages of liver carcinogenesis. Marked sex differenceswere found in the area ratio of enzyme-altered foci (mm² foci/cm²liver section) in sexually mature male and female Wistar rats(>). Pituitary grafts implanted one week before 2-acetylaminofluoreneselection in male rats decreased the area ratio to a level nearthat of sham operated females. The present study was performedin order to investigate the relevance of the results in theshort-term experiment in terms of hepatoma formation. To studythe importance of 2-acetylaminofluorene selection as a determinantof the observed sex differences an experiment was also performedusing the Pitot-model, where male and female rats were initiatedwith diethylnitrosamine and promotion was performed with phenobarbital.The long-term experiments in the resistant hepatocyte modelshowed that male rats develop hepatomas earlier than femalerats and also showed a tendency towards a prolonged latencytime in male rats bearing pituitary grafts. A good correspondencewas thus achieved between the short-term and the long-term experiments. The rats treated according to the -Pitot-model did not develophepatomas within the experimental period. Of the rats killedat 13 months, three (out of five) male and five (out of five)female rats had hepatocyte nodules whereas, at 19 months two(out of six) male and five (out of six) female rats had developedsuch lesions in this model. We therefore con-dude that 2-acetylaminofluoreneis the factor responsible for the sex differences in the resistanthepatocyte model and that the hypothalamo-pituitary influenceduring 2-acetylaminofluorene selection is an important modifierof the carcino-genetic process.     

Effect of bombesin and caerulein on early stages of carcinogenesis induced by azaserine in the rat pancreas

This study was designed to analyze the effect of two pancreaticotrophic peptides on pancreatic carcinogenesis in the azaserine-rat model. The rats were treated with bombesin or caerulein for 16 weeks after initiation with azaserine. Two-week-old Lewis rats were given injections of a single dose of azaserine (30 mg/kg) and the control pups received an injection of saline. They were divided into ten groups for peptide treatment as follows: Group 1, azaserine-saline; Group 2, azaserine-bombesin, 10 micrograms/kg; Group 3, azaserine-bombesin, 30 micrograms/kg; Group 4, azaserine-caerulein, 5 micrograms/kg; Group 5, azaserine-caerulein, 15 micrograms/kg; Group 6, control-saline; Group 7, control-bombesin, 10 micrograms/kg; Group 8, control-bombesin, 30 micrograms/kg; Group 9, control-caerulein, 5 micrograms/kg; and Group 10, control-caerulein, 15 micrograms/kg. At 3 weeks of age, they were weaned. Peptides or saline were injected 3 consecutive days a week for 16 weeks. Rats were autopsied 4 months after the administration of azaserine. Pancreatic weight was increased by bombesin and decreased by caerulein treatment. Quantitative histological analysis of azaserine-induced atypical acinar cell nodules in the pancreas showed that the size and number of atypical acinar cell nodules were increased in both bombesin- and caerulein-treated groups. Thus, these peptides appear to stimulate the growth of preneoplastic acinar cell lesions.     

Irradiation and prolactin effects on rat mammary carcinogenesis: intrasplenic pituitary and estrone capsule implants

The implantation of silicone capsules that contained estrone and that were adjacent to grafts of anterior pituitary tissue in the spleens of adrenalectomized glucocorticoid-deficient inbred F344 rats resulted in high circulating prolactin (Prl) levels without the untoward effects of chronic hyperestrogenism or of grafts of Prl-secreting pituitary tumors. All peripheral serum estrone titers were below the titers in sera of proestrous untreated intact rats. Peripheral serum estrone and Prl levels were, however, a function of capsule surface area over the capsule sizes tested (12-74 mm2); the elevated Prl levels persisted for as long as 700 days. In adrenalectomized glucocorticoid-deficient female rats, both 5 Gy gamma-irradiation alone and intrasplenic pituitary-estrone implants alone induced mammary carcinomas; the combination of these treatments induced a greater incidence of first carcinomas and reduced first carcinoma latency. There were, however, no marked differences in tumor incidence or latency due to differences in estrone capsule size. Finally, ovariectomy reduced first carcinoma risk in irradiated, pituitary-estrone-implanted rats but did not change the time of maximum risk. Ovarian secretory activity thus persisted in such rats and ovarian hormones synergized with Prl in mammary carcinoma induction.     

Synthesis and accumulation of polyamines in rat liver during chemical carcinogenesis.

The activities of L-ornithine decarboxylase (EC 4.1.1.17) and S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) exhibited marked fluctuations in the livers of rats fed 4-dimethylaminoazobenzene. After 1 month of dye feeding the activity of ornithine decarboxylase rose to a level about six times higher than that of controls and the activity of adenosyl-methionine decarboxylase was more than twice the initial control level. These elevations were transient, and returned to near normal values within the next 2 months. Four months after the commencement of the azo-dye diet, ornithine and adenosylmethionine decarboxylase activities showed a second distinct stimulation and appeared to remain high until liver tumors appeared. In contrast, the activity of tyrosine aminotransferase (EC 2.6.1.5) initially decreased but increased after 3 to 6 months to levels slightly exceeding the control values. The biphasic response of the two decarboxylases to the carcinogen appeared to be specific for the liver since both enzyme activities showed a sharp decrease in the kidney during the first 3 months of dye feeding followed by a transient increase 1 month later. The concentration of putrescine in the liver roughly followed the fluctuations of ornithine decarboxylase activity, being about twice the control level in carcinogentreated rats. Spermidine and spermine concentrations were normal although the activities of spermidine synthase (EC 2.5.1.16) and spermine synthase appeared to be lower in animals fed the carcinogenic diet than in normal controls. The response of liver ornithine decarboxylase, adenosylmethionine decarboxylase and tyrosine aminotransferase to growth hormone, glucagon and hydrocortisone was measured several times during the course of the azo-dye diet. There were only small changes, if any, in the stimulation of tyrosine aminotransferase by growth hormone or hydrocortisone whereas the stimulation by glucagon was totally lost. The striking response of ornithine decarboxylase to porcine growth hormone was markedly reduced, and the moderate stimulation elicited by glucagon completely disappeared during the carcinogenic diet. The response of adenosylmethionine decarboxylase to growth hormone remained unchanged, while the marginal stimulation evoked by either glucagon or hydrocortisone in normal rats totally disappeared after 4 months of dye feeding. Elevated activities of ornithine decarboxylase and adenosylmethionine decarboxylase, together with a distinctly reduced response of these enzymes to hormonal stimulations, thus appear to be characteristics of rat liver undergoing chemical carcinogenesis.