Prema M. Rao

Orotic Acid, a New Promoter for Experimental Liver Carcinogenesis

Male Fischer 344 rats initiated with 1,2-dimethylhydrazine 2HCl (100 mg/kg) given 18 hr after partial hepatectomy and exposed to a diet containing 1% orotic acid for 13 months developed a 100% incidence of hepatocellular carcinoma. The creation of nucleotide pool imbalances by dietary orotic acid, for e.g., an increase in uridine nucleotides and a decrease in adenine nucleotides, was considered as a possible mechanism for the promotional effect of orotic acid on liver carcinogenesis. The significance of this hypothesis is that altered nucleotide pools affect both genomic as well as membrane organization. Consistent with this hypothesis is our finding that feeding rats with a diet containing 1% orotic acid for 10 weeks resulted in a liver DNA damage as monitored by its slower sedimentation in alkaline sucrose gradients compared to the corresponding controls. To assess the general applicability of this hypothesis, nucleotide pool imbalances were created by using methods other than feeding orotic acid and their effect on the incidence of γ-glutamyltransferase positive foci in carcinogen initiated rats was determined. The results obtained indicated that rats initiated with 1,2-dimethylhydrazine.2HCl (100 mg/kg) given 18 hr after partial hepatectomy and exposed to diet deficient in arginine, a regimen that causes an increased synthesis and excretion of orotic acid, or were fed diets containing 1% thymidine or 1% thymine developed greater number of γ-glutamyltransferase positive foci compared to the corresponding controls fed the basal diets. These results were interpreted to indicate that orotic acid exerts its promotional effect probably by creating an imbalance in nucleotide pools. One of the mechanisms by which an imbalance of nucleotide pools influences the pathogenesis of the carcinogenic process may be by inducing perturbations in the DNA.

Effect of β-carotene on the expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat liver

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), is a rate-limiting enzyme in the biosynthesis of not only cholesterol but also a variety of non-sterol isoprenoids. It is subjected to multivalent feedback suppression by transcriptional and post-transcriptional control mechanisms mediated by sterols and non-sterol substances. In the present study, the effect of a plant isoprenoid, beta-carotene, on the expression of HMG-CoA reductase in rat liver was investigated. In control rats the hepatic levels of mRNA transcripts of HMG-CoA reductase increased following 2/3 partial hepatectomy with two peaks, one at 8 h and the other at 24 h. Administration of the carotenoid (70 mg/kg, given every alternate day for 3 consecutive weeks) partially inhibited the increase in the transcript level with a 50% reduction at 8 h and 30% reduction at 24 h post partial hepatectomy. Nuclear run-off assays with nuclei isolated from the resting liver and from livers of control rats and rats exposed to beta-carotene for 3 consecutive weeks and killed 8 h after partial hepatectomy indicated that beta-carotene did not inhibit the rate of transcription of HMG-CoA reductase gene. These observations suggest that beta-carotene regulates the expression of HMG-CoA reductase by some post-transcriptional mechanisms.

Modulation of carcinogen chromatin-DNA interaction by polyamines.

Abstract The methylation of rat liver chromatin DNA has been studied in vitro by the direct-acting carcinogen N-methyl N-nitrosourea. It is shown that spermine inhibits the methylation of chromatin DNA at the N 7 and O 6 positions of guanine and the N 3 position of adenine. However, spermine does not inhibit the methylation of 2-deoxy-5′-guanilic acid included as an internal control in the reaction. Under the experimental conditions, spermine exerts no influence on the degradation of N-methyl N-nitrosourea. The study has revealed that compounds like spermine or spermidine which bind tightly to DNA can modulate carcinogen-DNA interaction either by altering the net charge and/or the conformation of DNA.

Chronic mitoinhibition during promotion of hepatocarcinogenesis.

We have reported previously that orotic acid (OA), a precursor for pyrimidine nucleotide biosynthesis, is able to promote carcinogenic process in both liver and duodenum of rats. The present study investigates the possible role of mitoinhibitory effects of OA as being responsible for its promotional effects. Male Fischer 344 rats were given a semisynthetic basal diet (BD) or a diet containing 1% OA for four weeks coupled with 2/3 partial hepatectomy (PH), and all animals were then continued on BD for an additional four weeks. This protocol is known to exert a promoting effect on the initiated rat liver. Livers were perfused, and the labeling index (LI) of isolated cultured hepatocytes was monitored. Hepatocytes isolated from livers of rats fed a BD or 1% OA exhibitedin vitro an LI of 39±2 and 24±1%, respectively. The loweredin vitro LI was seen even upon exposure to epidermal growth factor (EGF) (67±2% in OA-treated livers compared to 91±2% in hepatocytes from control rat liver). A similar four-week exposure to OA coupled with PH decreased hepatic DNA synthesis induced by a choline-deficient dietin vivo by about 50%. These results indicate that OA is able to decrease the response of normal hepatocytes to growth factors and suggest a possible mechanism of chronic differential mitoinhibition as a basis for promotion induced by OA.

Dietary and Metabolic Manipulations of the Carcinogenic Process: Role of Nucleotide Pool Imbalances in Carcinogenesis"

Perturbations in DNA and/or membranes are considered to be important for the carcinogenic process. A search for nutritional and metabolic means of disturbing the homeostasis of DNA and membranes revealed that nucleotide pools of fer an exciting possibility. An imbalance in nucleotide pools can exert a two-pronged attack on both DNA and membranes. When given to rats, orotic acid, a precursor of pyrimidine nucleotides, results in an imbalance in nucleotide pools (an increase in uridine nucleotides and a decrease in inosine/adenine nucleotides), alterations in both DNA and membranes, and promotion of carcinogenesis in the liver initiated by chemical carcinogens. Agents such as adenine and allopurinol, which inhibit the metabolism of orotic acid and thereby decrease the formation of uridine nucleotides, and galactosamine, which traps uridine nucleotides, inhibited the promotional effects of orotic acid in the liver. These results suggested that orotic acid needs to be metabolized to uridine nucleotides and the creation of a subsequent imbalance in nucleotide pools is important for the promotional effects of orotic acid. To determine whether the creation of a nucleotide pool imbalance is a more general mechanism of tumor-promotion, two lines of approach were investigated. One was to determine the effect of orotic acid on promotion of carcinogenesis in other organs, and the second approach was to determine how to induce nucleotide pool imbalances by means other than orotic acid administration. It is interesting to note that orotic acid promotes carcinogenesis in duodenum initiated by azoxymethane. Regarding the second approach, it became apparent that several metabolic disturbances result in increased orotic acid synthesis and alterations in nucleotide pools. For example, increased administration of amino acids, ammonia, certain disturbances in urea cycle enzymes and/or metabolites, and certain types of liver dysfunction result in increased synthesis of orotic acid. Similarly, folic acid deficiency also results in increased levels of deoxyuridine nucleotide levels.

Effect of Glycine on the Induction of Orotic Aciduria and Urinary Bladder Tumorigenesis in the Rat

The mechanism by which amino acids increase the cellular levels of orotic acid (OA) was investigated. Administration of glycine (2.5 mmoles/100 g) to rats resulted in a 100-fold increase in urinary OA excretion, which was inhibited by pretreatment with cycloheximide or actinomycin D. The induction of OA synthesis from NH4Cl but not from carbamoylaspartate (CA) was inhibited by cycloheximide, indicating that the cycloheximide sensitive step was after the formation of ammonia and before the formation of CA. The glycine-stimulated OA synthesis was not inhibited by acivicin, a potent inhibitor of the cytosolic carbamoylphosphate (CP) synthetase, implicating the mitochondrial CP synthetase in supplying the CP for OA synthesis. Preliminary results indicated that cycloheximide did not inhibit glycine-induced urea synthesis to any significant extent. The results thus suggest that (i) the increased OA synthesis induced by glycine requires a transcription-translation dependent step and (ii) the regulatory step may be the transport of mitochondrial CP to cytosol and/or the synthesis of cytosolic CA. Attempts to determine whether increased exposure of urinary bladder to high concentrations of OA will influence bladder tumorigenesis revealed that chronic administration of glycine (2.5 mmoles/100 g, ip, daily, 5 days a week for 20 weeks) resulted in a 44% increased incidence of hyperplastic, preneoplastic, and neoplastic lesions. Some of these rats also exhibited stones in urinary bladders. The mechanism by which glycine induces tumorigenesis in the urinary bladder is currently being explored.

Ribonucleotide reductase: a possible target for orotic acid induced mitoinhibition in normal hepatocytes in primary culture.

The present study was designed to determine the mechanism by which orotic acid, a rat liver tumor promoter, inhibits DNA synthesis in normal hepatocytes in primary culture. Our results indicate that orotic acid inhibited the epidermal growth factor induced expression (mRNA) of both M1 and M2 subunits of ribonucleotide reductase while the expression of c-fos, c-myc, c-Ha-ras and beta-actin was not inhibited to any significant extent. These studies suggest that ribonucleotide reductase may be one target for orotic acid-induced mitoinhibition.

Sequential histopathological analysis of hepatocarcinogenesis in rats during promotion with orotic acid

In the present study, sequential histopathological changes during hepatocarcinogenesis promoted by orotic acid were examined. Male Fischer 344 rats were given 1,2-dimethylhydrazine.2HCl (100 mg/kg, i.p.) 18 h after 2/3 partial hepatectomy. After 1 week of recovery, they were divided into 2 groups; group 1 was continued on a semisynthetic basal diet while the group 2 received the basal diet containing 1% orotic acid. Rats were sacrificed after 5, 10, 20, 29, 40 and 53 weeks of promotion. Histopathological analysis indicated that emergence of hepatocellular carcinomas was preceded first by foci of morphologically and histochemically altered hepatocytes and then by the appearance of hepatocyte nodules. Clear cell foci, eosinophilic ground glass foci and gamma-glutamyltransferase positive foci were detectable after 5 weeks in initiated rats fed orotic acid. Hepatocyte nodules developed in 56% of the rats after 20 weeks of promotion, while the first hepatocellular carcinoma was observed in one rat sacrificed after 29 weeks of orotic acid promotion. Cancer incidence steadily increased with the duration of the orotic acid treatment and 59% developed hepatocellular carcinomas with 30% metastasis to lungs by 53 weeks of promotion. A relevant feature of this model is that during exposure to orotic acid no liver hyperplasia, nor bile duct or oval cell proliferation were seen and the liver architecture in the tissue surrounding focal lesions was well preserved throughout the sequence.

Transient inhibition by orotic acid does not abolish the in vivo response of rat hepatocytes to a direct mitogen, lead nitrate

BACKGROUND Orotic acid (OA) is able to inhibit hepatocyte proliferation in vivo induced by 2/3 partial hepatectomy. The present studies were aimed at establishing: (i) whether OA also inhibits hepatocyte proliferation induced by a direct mitogen and, if so (ii) whether the stimulus provided by the mitogen is still expressed following transient inhibition by OA. METHODS/RESULTS In the first experiment male Wistar rats were injected with either lead nitrate (100 mumol/kg, i.v.) or saline and 20 h later some animals receiving the mitogen were also implanted with a 400-mg OA tablet (as OA-methyl ester. i.p.). Multiple injections of 3H-thymidine were given to each rat (50 microCi each, 6 h apart, i.p.) until 2 h before killing. All groups were killed 3 days after the initial treatment. Results indicated that OA almost completely inhibited hepatocyte DNA synthesis and labelling induced by lead nitrate (e.g. labelling index was 1.9 +/- 0.5% in the saline-treated group, 44.7 +/- 4.0% in the lead nitrate group and 1.4 +/- 0.3% in the group receiving lead nitrate + OA). Based on the above results, in a second experiment rats were given a similar dose of lead nitrate and a subset of animals was implanted 20 h later with a 400-mg OA tablet, as previously described. Multiple doses of 3H-thymidine were again given to each rat (20 microCi each, 6 h apart) until 2 h before killing. Animals from both groups were killed at 3, 6 or 8 days after lead nitrate. Results indicated that, while at day 3 lead nitrate-induced DNA synthesis was effectively inhibited by OA, at day 6 the proliferative response was resumed in the group receiving OA. Cumulative labelling index over 6 days was 30.3 +/- 1.4 in rats given the mitogen alone and 52.1 +/- 2.2 in the group exposed to lead nitrate + OA. CONCLUSIONS These data indicate that: (i) OA is also able to inhibit hepatocyte proliferation induced by a direct mitogen such as lead nitrate; this, in turn, suggests that its inhibitory effect is not unique to the stimulus elicited by partial hepatectomy. (ii) The proliferative response triggered by the mitogen is not abolished by the transient (3-4 days) inhibitory phase imposed by OA. Possible mechanisms underlying these effects are considered in the discussion.

Studies on Metabolic Perturbations and Tumor Promotion: Glycine Induces the Synthesis of Orotic Acid, a Tumor Promoter and the Expression of Certain Cell Cycle Dependent Genes

Recently we have demonstrated that orotic acid (OA), a normal cellular constituent is an efficient promoter of liver carcinogenesis in the rat1–4. Being a precursor of pyrimidine nucleotides, feeding OA results in an increased synthesis of uridine nucleotides in the liver. This increase in uridine nucleotides is associated with a decrease in adenine nucleotides thereby creating an imbalance in the nucleotide pool. It was hypothesized that creation of such an imbalance in the nucleotide pool is an important factor in the mechanism by which OA exerts its promoting effect4,5. One of the significant aspects of this hypothesis is that since each cell has its own nucleotide pool pattern geared to its needs, disruption of this pattern could be one mechanism to achieve promotion in a variety of organs. Indeed OA has been shown to promote carcinogenesis in the duodenum initiated by azoxymethane6.