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.     

Histochemical study on rat liver glycogen during DAB carcinogenesis

The distribution of glycogen in livers of DAB-fed rats has been examined by the PAS reaction. At early stages of DAB feeding, a loss of glycogen was observed in cells of centrolobular areas undergoing a degenerative process. Intense glycogen staining was shown, on the other hand, by the hyperplastic nodules which develop from cells of periportal areas. At later stages, the formation of hyperbasophilic foci was found to be accompanied by a loss of glycogen, and the hepatomas, which apparently arise from such foci, were PAS-negative. It thus seems that a radical change in glycogen metabolism is associated with the neoplastic transformation. Alterations inglycogen metabolism being considered irrelevant to neoplasia per se, these results suggest that hyperbasophilic foci are the sites not only of modifications in the regulation of the cell cycle that initiate neoplastic growth but also of parallel changes responsible for secondary features of tumors.     

DNA polymerases in replication and repair of DNA during carcinogenesis induced by feeding N-acetylaminofluorene

To study the roles of DNA polymerases and ß duringreplication and repair of damaged DNA, use was made of the factthat during chronic treatment with carcinogens, replicationand repair do not necessarily follow the same time sequence.Early cell damage and restorative hyperplasia cause a transientwave of DNA synthesis, while repair replication might be expectedto continue throughout the period of treatment with the carcinogen.N-acetylaminoflluorene (AAF) was fed in the diet for periodsof up to 35 weeks, and at intervals during the feeding periodmeasurements were made of DNA synthesis in vivo, and off DNApolymerases a and ß as assayed in vitro after fractionation.The activity of polyararise increased and decreased with thetransient early wave of DNA synthesis. Polymerase ßshowed an initial rapid increase in activity which peaked beforethe increase in DNA synthesis, and then decreased. The decreasein activity may be due to the fact that, although AAF continuesto be fed in the diet, the foci and nodules which develop nolonger metabolise the carcinogen to a form which damages DNA.Thus replication occurs in the nodules while DNA damage andrepair occur in the surrounding non-neoplastic liver. With therapid growth of nodules there is overall an increase in neoplastictissue, a relative decrease in nonneoplastic tissue, and thusa relative decrease in DNA damage, repair, and induction ofpolymerase ß. Histo-logical examination showed thatby 35 weeks the conversion to neopflasia was virtually complete.These results support the concept that polymerase functionsin de novo replication of DNA, and is induced during cell replication,while polymerase it functions in repair replication, and increasesin activity during chronic damage to DNA. Whether it is inducedby treatment with carcinogens depends on the duration of treatment,and on other processes (e.g. metabolism of the carcinogen) whichtake place during the development of malignancy.     

Sequential analysis of DNA damage and repair during the development of carcinogen-induced rat liver hyperplastic lesions

The level of DNA fragmentation, as evaluated by alkaline elution, and of unscheduled DNA synthesis (UDS), as measured by autoradiography, was determined in the parenchymal cells from the entire liver during the development of hyperplastic lesions induced in the rat by the following treatment: diethylnitrosamine (DEN) (200 mg/kg i.p.) on Day 0; CCl4 (2 ml/kg intragastrically) on Day 21; dietary administration of 0.02% 2-acetylaminofluorene during the third and the fourth wk; and of 0.05% phenobarbital from the sixth wk. Both DNA fragmentation and UDS were constantly detected, concomitantly with the presence of gamma-glutamyltransferase (gamma-GT)-positive hepatocytes, in the primary cultures derived from the liver of rats of this experimental group sacrificed at 4, 5, 6, and 7 wk after DEN injection, their amount being approximately the same at the fourth and at seventh wk. Moreover, evidence of DNA alterations was still present, albeit diminished, 22 wk after the beginning of treatment. In contrast, DNA fragmentation and UDS did not persist past the fifth wk, and gamma-GT-positive hepatocytes were very few or totally absent in hepatocyte primary cultures from control rats treated with DEN alone, 2-acetylaminofluorene alone, or 2-acetylaminofluorene:CCl4. CCl4 alone, and phenobarbital alone caused only a modest, albeit statistically significant, increase in DNA elution rate and UDS, respectively. In a comparison performed on hepatocyte primary cultures obtained from rats of the experimental group sacrificed at the fifth wk after the injection of DEN, the level of UDS was higher in gamma-GT-positive than in gamma-GT-negative hepatocytes. These results indicate that the regimen used to induce the selective proliferation of initiated hepatocytes actually produces extensive DNA lesions which can give rise to additional carcinogenic initiations.     

The galactosyltransferase activity of hepatic nodules during rat liver carcinogenesis

Galactosyltransferase has been isolated from putative preneoplastic hepatocyte nodules generated in the resistant hepatocyte model by the procedure of Solt et al. (Am. J. Pathol., 88 (1977) 595-609). The following observations have resulted from these studies: (a) the specific activity of galactosyltransferase isolated from hepatocyte nodules by affinity chromatography was reduced to about 1/3 that of the enzyme in control and in liver tissue surrounding the nodules; (b) the galactosyltransferase activity from normal rat serum eluted from the alpha-lactalbumin affinity column as a single peak (spec. act. = 1.57 nmol/min per mg) while that from the serum of nodule-bearing rats eluted in two distinct peaks (spec. act. = 2.49 and 0.49 nmol/min per mg protein); (c) the elution profile of the enzyme from hepatocyte nodules was broad compared to that from normal liver, surrounding liver or serum; (d) the Km for N-acetyl-D-glucosamine (GlcNAc) was lower in all four independent batches of nodules compared to the Km for GlcNAc from control and surrounding liver; (e) the Km for uridine diphosphogalactose (UDP-Gal) was higher for the enzyme from nodules compared to that from control tissue. These data suggest that the hepatocyte nodule produces several glycoforms of galactosyltransferase the kinetic properties of which differ from those of the enzyme from control liver.     

Alterations of ornithine decarboxylase gene during the progression of rat liver carcinogenesis

Liver nodules and carcinomas, developing in F344 rats initiated.with diethylnitrosamine, exhibit high ornithine decarboxylase(ODC) activity and DNA synthesis. ODC-related RNAs of 1.8, 2.1and 2.6 kb are produced by normal rat liver. Early preneoplasticnodules, developing 10 weeks after initiation, showed overproductionof 1.8 and 2.1 kb RNAs, while the 2.6 kb RNA was barely detectable.Rises in the 1.8, 2.1 and 2.6 kb RNAs occur in late nodules(30 weeks after initiation) and in carcinomas. The comparisonof different tissues for relative increase in ODC activity,RNA levels and DNA synthesis showed that these parameters behavedin the same way: highest increases occurred in early nodulesand carcinomas. These observations suggest that overexpressionof ODC gene and alterations in regulatory mechanisms of ODCgene expression may be implicated in the progression of preneoplasticlesions to malignancy. Southern blot analysis of PstI DNA digestsrevealed the presence of ODC gene rearrangement in two carcinomasand in one late nodule. However, the role of this phenomenonin the progression of preneoplastic lesions is unclear, dueto the possibility that ODC pseudogenes are involved insteadof or in addition to ODC gene.     

Orotic acid, a promoter of liver carcinogenesis induces DNA damage in rat liver

Orotic acid, a precursor of pyrimidine nucleotide biosynthesisand a promoter for liver carcinogenesis, when fed at 1% levelin a diet for 5 weeks resulted in liver DNA damage. The damagecan be monitored as alkali-labile lesions using alkaline sucrosegradients as well as alkaline elution technique. Furthermore,the induced DNA damage persists for up to three weeks afterwithdrawal of the orotic acid diet. The fad that several skin-tumourpromoters also induce DNA damage raises the question whetherDNA damage is a component in tumour promotion.     

Morphometric study of the rat duodenal epithelium during the initial six months of 1,2-dimethylhydrazine carcinogenesis

Male Wistar rats weighing 100 g received 1,2-dimethylhydrazine (25 mg/kg) s.c.) twice a week for 2 months and once a week thereafter for an additional 4 months. Groups of four to six rats were sacrificed monthly. Paraffin sections of duodenum were prepared and stained with periodic acid-Schiff and hematoxylin for cell counts and with toluidine blue for measuring nucleolar area. As an index of villus size and crypt size, the mean number of epithelial cells per representative sections of villi and crypts were used. Mitotic activity was assessed by counting the mean number of mitotic figures per representative crypt section. Nucleolar area was assessed from the analysis of drawn (camera lucida) images of nucleoli of columnar cells at six levels of the epithelium: lower, mid, and upper parts of the crypts and villi. Villus size increased progressively during the 6-month treatment, from 272 +/- 2 (S.E.) to 349 +/- 8. Crypt size increased from 118 +/- 2 in a wavy fashion, showing maximum (139 +/- 5, 143 +/- 2) at 3 and 6 months and minimum (123 +/- 3, 127 +/- 1) at 1 and 4 months. Mitotic number displayed a similar pattern of increase so that the percentage of mitotic figures in the crypts (mitotic index) remained constant (about 5%) in control and experimental animals. Nucleolar area in the controls decreased with age from 4.2 +/- 0.08 sq micron in lower crypt at 1 month to 2.8 +/- 0.04 sq micron at 6 months. During 1,2-dimethylhydrazine treatment, lower crypt nucleoli increased to 4.5 +/- 0.12 sq micron after 3 months and decreased slightly thereafter, reaching 4.0 +/- 0.14 sq micron by the sixth month. The nucleoli furthermore decreased gradually along epithelium (nucleolar compaction) by an average of 0.23% per cell position in control as well as treated animals. It appeared, then, that the main effect of 1,2-dimethylhydrazine was the enlarging of the four parameters measured. This effect seemed to relate in some manner to tumor formation as all the enlargements were attenuated in intestinal tissue adjacent to tumors.     

Dietary modulation of rat liver carcinogenesis

Rat liver carcinogenesis was induced according to the resistanthepatocyte model of Solt and Farber. One week after the endof the procedure for the rapid growth of altered hepatocytes,one group of rats was submitted to a high fat (20%) regimenup to the end of the experiment. The incidence of histologicallyconfirmed malignant hepatocarcinomas was compared with thatobserved in a group that remained on a normal diet. The modulating(promoting) effect of the high fat regimen was evident sincenine out of 10 animals in this group bore macroscopically detectabletumors and eight out of 10 presented histologically confirmedhepatocellular carcinomas as early as 24 weeks after the beginningof the experiment. At that time, no malignant tumors were detectedin the group submitted to the normal fat regimen. These resultsare similar to those resulting from a porto-caval shunt or thechronic administration of liver tumor promoters. This suggeststhat at this stage of the carcinogenic process, any treatmentinducing chronically metabolic adaptation in a tissue containingpreneoplastic nodules modulates positively the progression ofthese lesions as demonstrated by the dramatic reduction of thelag period for their malignant transformation.     

Glucose-6-phosphatase activity in rat liver parenchyma during azo-dye carcinogenesis

The glucose-6-phosphatase (C-6-Pase) activity was investigated by the Wachstein and Meisel method in preneoplastic rat liver parenchyma, as well as in normal liver and hepatomas induced by 4-dimethylaminoazobenzene (DAB). Normal liver shows a high G-6-Pase activity restricted to the cytoplasm of hepatocytes. At early stages of DAB feeding, the centrolobular degenerating zones give a coarser reaction while portal areas retain a normal activity. In cirrhotic liver, the intensity of the reaction varies greatly from one nodule of regeneration to another but is relatively uniform within each nodule. Hyperbasophilic foci are found in nodules with either high or low G-6-Pase activity. The foci developing from highly active nodules show a loss of G-6-Pase activity and the tumors are negative. It appears from these studies that a) the variations in G-6-Pase activity observed in regenerating nodules play no role in the development of hyperbasophilic foci and hepatomas, and b) the loss of activity occurring in hyperbasophilic foci and tumors probably represents a secondary feature of the neoplastic transformation.     

Nuclear DNA content of altered hepatic foci in a rat liver carcinogenesis model

In individual altered hepatic foci (AHF), aneuploidy occurs before malignant changes can be diagnosed histologically (O. Sudilovsky and T. K. Hei. Fed. Proc., 42:2225, 1983). In the current experiments Sprague-Dawley rats of both sexes were given i.p. injections of diethylnitrosamine (50 mg/kg body weight) 18 h after partial hepatectomy and were given a choline-sufficient diet (CS) for 1 wk. Four treatment groups were then formed and fed CS, CS containing 0.05% phenobarbital (PHB), choline-deficient diet (CD), and CD with 0.05% PHB. An extra female group received infusions of saline after the hepatectomy and fared CD. Control animals were partially hepatectomized, inoculated i.p. with saline, and placed on CS. The rats were sacrificed 16 wk later, liver sections were stained with a combined Feulgen-gamma-glutamyl transpeptidase stain, and the DNA content of gamma-glutamyl transpeptidase-positive foci was measured cytospectrophotometrically. There were no AHF in the control animals. Hepatocytes from control livers and cells adjacent to foci in treated livers had peaks corresponding to the 2C, 4C, and 8C range. In AHF the ploidy, however, was predominantly diploid, tetraploid, or heterogeneous. The ratio of diploid to tetraploid cells in foci of rats provided with CS + PHB was 5.5 and in those supplied with CD + PHB was 0.09. This suggested that dietary manipulations change the nuclear DNA distribution of AHF. Aneuploidy was also present, as expected, in 4 of 33 AHF in the animals placed on CD + PHB. It was observed as well in 2 of 26 AHF of rats given CD but in none of the 20 AHF fed CS + PHB. These data indicate that CD (which acts as both initiator and promoter) may be responsible for the appearance of aneuploidy. A general model, based on these results and the clonality of each individual focus, is proposed for the development of cells through the preneoplastic stage.