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One of the proposed mechanisms by which orotic acid (OA) promotes liver carcinogenesis is by differentially mito-inhibiting the normal hepatocytes while permitting the initiated ones to respond to growth stimuli to form foci/nodules. In an attempt to examine this hypothesis, the present study was designed to determine (i) whether OA inhibits DNA synthesis in normal hepatocytes in vivo, and (ii) whether hepatocytes from hepatic foci/nodules are relatively resistant to the mito-inhibitory effects of OA. The results of this study indicate that OA given i.p. as a tablet of 300 mg at the time of partial hepatectomy (PH) almost completely inhibited liver DNA synthesis. Three days later--a time period by which the implanted tablet disappeared--the hepatocytes resumed DNA synthesis. Exposure to OA results in an accumulation of uridine nucleotides and a decrease in adenosine nucleotides. Creation of such an imbalance in nucleotide pools appears to be important for OA to inhibit DNA synthesis. Adenine (a tablet of 300 mg), an agent that inhibits the metabolism of OA to uridine nucleotides, counteracted the mito-inhibitory effects of OA. To determine whether the hepatocytes in foci/nodules are resistant to the mito-inhibitory effects of OA, rats were initiated with diethylnitrosamine (DENA; 150 mg/kg) and promoted by the resistant-hepatocyte model. Fourteen weeks after the administration of DENA, the rats were subjected to PH in the presence of absence of OA (300 mg tablet). The results indicated that, in contrast to hepatocytes in normal or surrounding non-nodular liver, a subpopulation of hepatocyte foci/nodules appear to be relatively resistant to the mito-inhibitory effects of OA. These findings support the hypothesis that differential mito-inhibition is a possible component in the promoting effect of OA. However, whether this is the mechanism by which OA promotes liver carcinogenesis needs to be further investigated.

This study was designed to determine the possible mechanism by which orotic acid exerts its mitoinhibitory effect on rat hepatocytes in primary culture. Orotic acid inhibited, dose-dependently DNA synthesis in hepatocytes induced by epidermal growth factor, transforming growth factor alpha, hepatocyte growth factor, acidic fibroblast growth factor, or plasma from rats exposed to various liver cell-proliferative stimuli, such as two-thirds partial hepatectomy, lead nitrate, cyproterone acetate, ethylene dibromide, or a diet deficient in choline. Further, orotic acid inhibited DNA synthesis even when added 24 h after the hepatocytes were primed with transforming growth factor alpha. Taken together, these results suggested that the target site may not be at the level of the growth-factor receptor and receptor-mediated early events. In a preliminary experiment, orotic acid inhibited the expression of the ribonucleoside diphosphate reductase gene. Exposure to orotic acid results in an imbalance in nucleotide pools characterized by an increase in uridine nucleotides and a decrease in adenosine nucleotides. It is hypothesized that this imbalance in nucleotide pools inhibits the expression of the ribonucleoside diphosphate reductase gene and, therefore, is a likely target for the mitoinhibitory effect of orotic acid.

The present study was undertaken to determine the effect of two different types of liver cell proliferative stimuli, namely compensatory regeneration and direct hyperplasia on DNA synthesis of normal and preneoplastic isolated hepatocytes. Platelet-poor plasma (PPP) isolated from male Wistar rats treated with three different hepato-mitogens, lead nitrate (LN), cyproterone acetate (CPA) and ethylene dibromide (EDB), or subjected to surgical partial hepatectomy (PH), was tested for its ability to stimulate DNA synthesis in normal and preneoplastic hepatocytes in primary cultures. Induction of DNA synthesis was detected as early as 30 min after CPA, EDB and PH administration and persisted up to 5 days after the LN administration. In addition, hepatocytes isolated from preneoplastic liver nodules were also able to respond in culture to the DNA synthesis stimulus induced by these factors.

One percent orotic acid supplemented diet is a promoting treatment in the rat model of liver carcinogenesis. After treatment with this type of diet, DNA alterations were observed using alkaline sucrose gradients and alkaline elution methods. In this work we have utilized two unwinding methods for the detection of DNA fragmentation. One method is a viscosimetric method in which the rate of increase in DNA viscosity with time is related to the rate of alkaline DNA unwinding. The second method measures fluorimetrically the amount of renatured and denatured DNA after different times allowed for alkaline DNA unwinding. These two methods are very sensitive in detecting DNA breaks induced by typical alkylating agents, X-rays and H2O2. The two unwinding methods were clearly negative for the orotic acid supplemented diet. We suggest that the DNA alterations detected with alkaline sucrose gradients and alkaline elution methods, after promoting treatment with orotic acid, are probably different from the DNA breaks induced by typical alkylating agents, X-rays and H2O2.

The present study was undertaken to determine the kinetics of DNA synthesis and expression of cell cycle dependent proto-oncogenes in response to two types of cell proliferative stimuli in male Wistar rat liver. The peak of DNA synthesis was approximately 24 h after a compensatory cell proliferative stimulus induced by 2/3 partial hepatectomy and approximately 36 h following a mitogenic stimulus obtained with a single dose of lead nitrate (10 micromol/100 g body wt, through femoral vein). Even though both proliferative stimuli induced the expression of c-fos, c-myc and c-Ha-ras, the extent of the increase in c-fos expression was 4- to 5-fold less in mitogen-induced cell proliferation. In addition, while the expression of c-myc, following partial hepatectomy returned to basal level by 4 h, the induced expression of c-myc persisted for up to 40 h during the lead nitrate-induced liver cell proliferation.

The degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.

Experiments were designed to determine whether DNA conformation and sequence play any role in its methylation by N-methyl-N-nitrosourea, methylmethanesulfonate, and dimethylsulfate, agents that are known to methylate DNA by different mechanisms but yield 7-methylguanine as the major product. The approach taken was to bind ligands to DNA that interact with it stereospecifically and to study their effect on the formation of 7-methylguanine by the three methylating agents. The results indicate that both distamycin A and spermine shielded the formation of 7-methylguanine in vitro in rat liver DNA by N-methyl-N-nitrosourea but not by methylmethanesulfonate or dimethylsulfate; they did not, however, protect 2-deoxyguanylic acid against methylation by N-methyl-N-nitrosourea. Based on the mechanism by which the methylating agents and the ligands react with DNA, these results are interpreted to suggest that 1) guanines methylatable at the N-7 position by N-methyl-N-nitrosourea are located at, or close to, the binding sites of the ligands, probably the A-T-rich regions, and those methylatable by methylmethanesulfonate and dimethylsulfate are distal to these regions, and/or 2) the conformation DNA assumes in the presence of distamycin A or spermine permits methylation by methylmethanesulfonate and dimethylsulfate but not by N-methyl-N-nitrosourea. The study implicates DNA structure and/or its sequences in carcinogen-DNA interaction.

This investigation was designed to study whether methylation of liver chromatin DNA by dimethylnitrosamine (DMN) and the subsequent in vivo removal of DNA-bound methylated products are random. Liver chromatin DNA was fractionated into nuclease-digestible and nondigestible material 4 hr following the administration of [3H]DMN (0.5 mg/250 muCi/100 g body weight). Digestion of such methylated liver chromatin with pancreatic DNase I or micrococcal nuclease and analysis of nuclease-digested acid-soluble products revealed a discrepancy between the radioactivity released (72%) and the nucleotides released (50%) as measured by the absorbance at 260 nm. This discrepancy disappeared, and the rate and extent of release of both the radioactivity and the absorbance at 260 nm were identical when the total purified DNA isolated from methylated chromatin was used as the substrate instead of chromatin DNA in the nuclease reaction. These results, together with the fact that guanine contents of the DNA of the two fractions of the chromatin isolated by nuclease digestion were identical, suggest that methylation of the nuclease-accessible region of hepatic chromatin DNA is relatively greater than that of the inaccessible region. The study of the removal of methylated products in the accessible region of the chromatin DNA further reveals that, of the methylated products present at 4 hr, 62% is lost by 3 days, 87% is lost by 1 week and 94% is lost by 2 weeks. However, loss from the nuclease-inaccessible region of chromatin DNA is only 27% by 3 days, 49% by 1 week, and 86% by 2 weeks, thereby suggesting that the removal of methylated products from this region of chromatin DNA is relatively slower compared with that from the nuclease-accessible region of chromatin-DNA. The results of this study thus indicated (a) an increased methylation and faster rate of removal of DMN-induced methylated products in nuclease-accessible regions of chromatin DNA and (b) decreased methylation and slower rate of removal from the nuclease-inaccessible regions of chromatin DNA. It is concluded that the distribution and removal of DMN-induced methylated products in liver chromatin DNA is nonrandom as measured by this technique.

Orotic acid, a precursor of pyrimidine nucleotide biosynthesis and a promoter for liver carcinogenesis, when fed at 1% level in a diet for 5 weeks resulted in liver DNA damage. The damage can be monitored as alkali-labile lesions using alkaline sucrose gradients as well as alkaline elution technique. Furthermore, the induced DNA damage persists for up to three weeks after withdrawal of the orotic acid diet. The fact that several skin-tumour promoters also induce DNA damage raises the question whether DNA damage is a component in tumour promotion.