[CANCER RESEARCH (SUPPL.) 52. 2078s-2081s. April 1. 1992] , -Pool Imbalance, and Liver-Tumor Promotion: A Possible Mechanism for the Mitoinhibitory Effects of Orotic Acid in Isolated Rat Hepatocytes1

Sharmila Manjeshwar, Aisha Sheikh, Giuseppina Pichiri-Coni, Pierpaolo Coni, Prema M. Rao, Srinivasan Rajalakshmi, Peter Pediaditakis, George Michalopoulos, and Dittakavi S. R. Sarma2

Department of Pathology, university of Toronto, Toronto, Ontario, Canada A/55 IAS fS. M., A. S., G. P-C., P. C., P. M. R., S. R., D. S. R. S.J, and Department of Pathology, Duke University, Durham, North Carolina 27710 fP. P., G. M.j

Abstract (4) and membranes, the two targets often implicated in carcinogenesis. This study was designed to determine the possible mechanism by Feeding orotic acid, a precursor of nucleotide which orotic acid exerts its mitoinhibitory effect on rat hepatocytes in primary culture. Orotic acid inhibited, dose-dependently DNA synthesis , to rats results in an imbalance in nucleotide pools in hepatocytes induced by epidermal growth factor, transforming growth characterized by an increase in hepatic and factor a, hepatocyte growth factor, acidic fibroblast growth factor, or a decrease in adenosine nucleotides (5-8). It also induces alter plasma from rats exposed to various liver cell-proliferative stimuli, such ations in DNA (9) and membrane glycosylation (10) and pro as two-thirds partial hepatectomy, lead nitrate, cyproterone acetate, motes liver carcinogenesis in initiated rats (11-15). It was ethylene dibromide, or a diet deficient in choline. Further, orotic acid postulated that creation of such an imbalance in nucleotide inhibited DNA synthesis even when added 24 h after the hepatocytes pools is crucial for the tumor-promoting effect of orotic acid were primed with transforming growth factor a. Taken together, these (16). Consistent with this postulation is the observation that results suggested that the target site may not be at the level of the growth- inhibition of the of orotic acid to uridine nucleo factor receptor and receptor-mediated early events. In a preliminary tides by agents such as adenine resulted in a decrease in the experiment, orotic acid inhibited the expression of the ribonucleoside tumor-promoting potential of orotic acid (17). diphosphate reducíasegene. Exposure to orotic acid results in an imbal ance in nucleotide pools characterized by an increase in uridine nucleo- The present study reflects our continued efforts to compre tides and a decrease in adenosine nucleotides. It is hypothesized that this hend the mechanism by which orotic acid and the resulting imbalance in nucleotide pools inhibits the expression of the ribonucleoside imbalance in the nucleotide pools promote liver carcinogenesis. diphosphate reducíasegene and, therefore, is a likely target for the Operationally, promotion results in focal proliferation of initi mitoinhibitory effect of orotic acid. ated hepatocytes to form foci of enzyme-altered hepatocytes and hepatic nodules (18, 19). Therefore, any mechanism pro Introduction posed for promotion should be able to explain how focal pro liferation is achieved. Accordingly, a promoter can act as a Nucleotides, in addition to being precursors of template- mitogen selectively on initiated hepatocytes or can mitoinhibit directed nucleic acid synthesis, play a key role in major biolog the noninitiated hepatocytes differentially while permitting the ical processes. For example, after conversion to nucleotide initiated hepatocytes to respond to growth stimuli and form sugars, nucleotides participate in glycosylation reactions of foci and/or nodules. Orotic acid is not a mitogen to hepatocytes proteins and lipids, including those of the membranes. Further, (12), but orotic acid is a mitoinhibitor (20, 21). We provide uridine nucleotides participate in the synthesis of polysaccha- evidence from in vitro experiments to suggest that one of the rides, cytidine nucleotides in the synthesis of lipids, and aden target sites at which orotic acid exerts its mitoinhibitory effect osine and guanosine nucleotides in the synthesis of proteins. is beyond the growth-factor receptor and the growth factor- Although the ATP-ADP system is involved in the mainstream mediated early events. of energy transfers, 5'-di- and triphosphates of other ribo- and deoxyribonucleosides also participate in cellular energy trans Materials and Methods fer. As second messengers, nucleotides also influence signal- transduction mechanisms. In addition, nucleotides also regulate Experiments using Defined Growth Factors. Hepatocytes were iso the activity of enzymes such as RNR1 (1,2) and galactosyltrans- lated, by a collagenase-perfusion technique (22), from 180-200-g male Fischer 344 rats (Charles River Breeding Laboratories, St. Constant, ferase (3). Because nucleotides participate in several cellular Quebec, Canada) maintained on a semisynthetic basal diet (diet 101 functions, an imbalance in the levels of nucleotides may have from Dyets Inc., Bethlehem, PA). Low centrifugal speeds (50 x g) were profound effects in the pathogenesis of many disease processes. used during cell washing to minimize the contamination of hepatocytes One of our approaches to understanding the carcinogenic with smaller nonparenchymal cells. Cell viability, estimated by the process is identifying those metabolic events that, when per trypan blue exclusion method, was > 90%. Viable hepatocytes (2 x turbed, influence pathogenesis. Nucleotide pools appear to be IO5)were cultured on 35-mm dishes coated with collagen (Vitrogen, 60 Mg/dish), in modified William's E medium containing fetal bovine an important cellular influence on carcinogenesis. An imbalance in nucleotide pools can disturb the homeostasis of both DNA serum (10%, v/v), insulin (20 units/liter), L-glutamine (2 m\i), 4-(2- hydroxyethyl)-l-piperazineethanesulfonic acid (10 HIM),penicillin (100 ' Presented at "Nutrition and Cancer," the first conference of the International units/ml), and streptomycin (100 Mg/ml). After an attachment period Conference Series on Nutrition and Health Promotion, April 17-19, 1991, of 3 h at 37'C in air-CO2 (95:5), the medium and nonattached cells Atlanta, GA. The study was supported in part by USPHS Grants CA37077 were removed. The medium was changed to serum-free, modified (D. S. R. S.) and CA43632 (G. M.), from the National Cancer Institute, and by William's E medium supplemented with L-proline (2 mivi)and sodium grants from the National Cancer Institute, Canada (D. S. R. S.). *To whom requests for reprints should be addressed. pyruvate (10 mM). Dishes contained different growth factors, TGF-a 'The abbreviations used are: RNR, ribonucleoside diphosphate reducíase; (40 ng/ml), HGF (100 ng/ml), or acidic FGF (20 ng/ml). Control EGF, epidermal growth factor; HGF, hepatopoietin-A or hepatocyte growth factor; TGF-a, transforming growth factor a; FGF. fibroblast growth factor; 2/3 dishes contained only the basal medium. Orotic acid was added as PH, two-thirds partial hepatectomy; PRPP, 5-phosphoribosyl-l-pyrophosphate; orotic acid methyl ester (Sigma Chemical Co., St. Louis, MO). All PPP, platelet-poor plasma; OTC, ornithine transcarbomylase. dishes also contained ['Hjthymidine (5 //('I dish). The dishes were 2078s

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. MITOINHIBITION BY OROTIC ACID IN RAT HEPATOCYTES incubated for 48 h. At the end of the experiment, cells were processed for measuring DNA synthesis either by determining labeling index or by determining acid-precipitable radioactivity. For determining labeling index, the cells were washed in cold phosphate-buffered saline, fixed in 10% buffered formalin, and processed for autoradiography (20, 21). For determining the acid-precipitable radioactivity, the cells were washed in cold phosphate-buffered saline and subsequently dissolved in 1.5 ml of 0.33 N NaOH. A sample was taken for DNA precipitation with 1 ml of an ice-cold solution of 40% trichloroacetic acid and 1.2 N HCI. The precipitate was then filtered onto 0.45-Mm Millipore filters and washed with ice-cold 5% trichloroacetic acid. The air-dried filters BM TGF -a OA OA OA OA OA were dissolved in 1 ml of Cellosolve (Fisher Scientific, Canada) and (40ng/ml)(24|jM) (48uM) (72uM) (96nM) (120u,M) counted with 10 ml of Aquasol (NEN, Canada) in a scintillation counter. The data are expressed as mean cpm/dish ±SD of three Fig. 1. Inhibition of TGF-n-induced DNA synthesis by orotic acid (OA) in hepatocytes in primary culture. Isolated hepatocytes were incubated with basal dishes. In experiments with HGF, the conditions of the experiment and medium (BM) or basal medium containing TGF-a and different concentrations media were slightly different from those described above and were of orotic acid. All dishes contained | 'I I]iln midinr. After 48 h of incubation, the detailed earlier (23, 24). cells were processed for measurement of acid-precipitable radioactivity. Other Experiments using Platelet-poor Plasma. PPP was prepared (25) from details are given in the text. Values are the mean ±SD of three dishes. The control sham-hepatectomized rats or rats subjected to 2/3 PH, treated experiment was repeated 3 times with a similar pattern of results. with lead nitrate (10 umol/100 g i.v.), ethylene dibromide (10 mg/kg intragastrically), or cyproterone acetate (10 mg/100 g intragastrically) 24 h before being killed, or from rats exposed to a choline-deficient diet for 5 days. After the hepatocytes were allowed to attach for 3 h, PPP was added to the medium (20% final concentration). Some dishes also contained orotic acid. The effect of orotic acid on DNA synthesis in hepatocytes stimulated by PPP was monitored by using the meth odology described above. Addition of Orotic Acid 24 h after Priming the Hepatocytes with Growth Factors. In some experiments, after the 3 h for attachment the T medium was changed to a serum-free medium and the growth factor was added to the medium in appropriate dishes. Twenty-four h later, the medium was washed off, fresh serum-free medium containing n n n different concentrations of orotic acid and ['HJthymidine was added, OA OA OA OA OA OA 20uM 28uM 40uM and the dishes were incubated for another 24 h. At the end of the experiment, cells were processed for measuring DNA synthesis as Fig. 2. Inhibition of HGF-induced DNA synthesis by orotic acid (OA) in described above. Routinely, the effect of EGF (20 ng/ml) on the hepatocytes in primary culture. Experimental conditions were identical to those induction of DNA synthesis and the effect of orotic acid (120 MM)on given in the legend to Fig. 1. The basal medium (BM) used in these experiments EGF-induced DNA synthesis in hepatocytes were determined as posi was different from that used in the experiments in Fig. 1, and its composition tive controls. All experiments were repeated at least two or three times was described earlier (23, 24). Values are the mean ±SD of three dishes. The experiment was repeated 2 or 3 times. and gave similar patterns of results.

Table 1 Effect of orotic acid (OA) on DNA synthesis in hepatocytes induced by Results and Discussion plasma from rats subjected to several liver-cell-proliferative stimuli Platelet-poor plasma was prepared from control rats or rats subjected to various The first series of experiments was designed to determine liver-cell-proliferative stimuli, as indicated. Different preparations of PPP have 57-59 »ig'inIprotein. A 120 JJMconcentration of orotic acid was used in this whether orotic acid would inhibit the growth response of he study. Other details of the experiment are given under "Materials and Methods." patocytes, in primary culture, to several growth factors that The values are the mean ±SD of three dishes. The experiment was repeated 2 or presumably use different receptors. The rationale was that, if 3 times. this occurs, it is likely that at least one site of action is not at Labeling index (%) the level of growth-factor receptor. The results presented in Proliferative stimuli used to PPPControlprepare 20% OA20 OA2.0 Figs. 1 and 2 indicate that orotic acid dose dependently inhib ited the growth response of hepatocytes to both TGF-a and ±0.8 2/3 PH 39 2.5 1.5 ±0.6 HGF. A comment should be made on the differences in dose Lead nitrate 37 3.1 1.0 ±0.2 response of orotic acid towards TGF-a and HGF. Experiments Ethylene dibromide 41 3.4 0.5 ±0.1 Cyproterone acetate 40 2.9 0.5 ±0.1 dealing with HGF were done by Pediaditakis and Michalopou- Choline-deficient dietWithout 65:2.0b3.8With 2.0 ±0.4 los (23, 24) at Duke University, with a slightly different me dium. Currently we are exploring the possibility of whether the supersensitivity of hepatocytes to orotic acid when HGF was jected to various liver cell-proliferative stimuli. These include used was because of the differences in the composition of the 2/3 PH, liver mitogens such as lead nitrate, cyproterone acetate, basal medium. and ethylene dibromide (26), and a choline-deficient diet (27). The effect of orotic acid was also examined by using acidic The results presented in Table 1 indicate that orotic acid FGF as the inducer of DNA synthesis. Orotic acid again inhib inhibited DNA synthesis in hepatocytes stimulated by plasma ited the response of hepatocytes to acidic FGF. However, the from rats subjected to the different types of liver-cell prolifera- stimulation of DNA synthesis by acidic FGF, compared with tive stimuli. These data, together with our earlier observation the controls, was not consistently statistically significant be that orotic acid inhibited DNA synthesis in hepatocytes induced tween different experiments. Because of these variations the by EGF (20, 21), suggest that one of the target sites for the data are not presented. In addition to these defined growth mitoinhibitory effect of orotic acid may be beyond events related factors, we also examined the effect of orotic acid on the growth to growth factor receptors. This statement may be valid, because response of hepatocytes stimulated by plasma from rats sub- EGF and TGF-a, HGF, and acidic FGF appear to have different 2079s

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. MITOINHIBITION BY OROTIC ACID IN RAT HEPATOCVTES receptors (28, 29). Even though we do not know the nature of 2.00-1 the plasma growth factors induced by liver-cell-proliferative stimuli, the in vivo response of the liver to 2/3 PH and different liver mitogens, in terms of induced expression of c-fos and c- myc, is different (30, 31). 1.00- The next series of experiments was designed to determine whether orotic acid inhibits DNA synthesis by interfering with the growth factor-mediated early events. The protocol in these experiments was to prime the hepatocytes with a growth factor for 24 h, wash the cells free of the growth factor, and study the 0.00 effect of orotic acid. The rationale was that, if orotic acid still BM TGF-n OA OA OA OA OA (40ng/ml)(24uM) (48uM) (72uM) (96nM) (120uM) inhibits DNA synthesis, then the target site may not be growth factor-mediated early events. Fig. 3. Effect of orolic acid (OA) on TGF-a-induced DNA synthesis in primary hepatocyte cultures, when added 24 h after the growth factor. Experimental The results indicated that orotic acid can inhibit TGF-a- details were identical to those given in the legend to Fig. 1, except that hepatocytes induced DNA synthesis in hepatocytes irrespective of whether were incubated with TGF-n for 24 h. The cells were then washed and the incubation was continued with fresh medium for another 24 h with different orotic acid is added at the beginning of the experiment (i.e., concentrations of orotic acid and ['H|thymidine. The cells were processed for together with the growth factor) (Fig. 1) or 24 h after the measurement of acid-precipitable radioactivity. Other details are given in the text. hepatocytes are primed with the growth factor (Fig. 3). Similar Values are the mean ±SD of three dishes. The experiment was repeated 2 or 3 patterns of results were obtained when EGF (21) and acidic times with a similar pattern of results. FGF (data not shown) were used instead of TGF-a. Taken together, these results suggest that one of the target ders may pose increased risk of tumor promotion. For example, sites for the inhibition by orotic acid of DNA synthesis in feeding a diet deficient in arginine, a amino acid, hepatocytes is at some point beyond growth factor receptor- results in increased levels of orotic acid (35-37) and an imbal and growth factor-mediated early events. We, therefore, hy ance in hepatic nucleotide pools similar to that caused by pothesized that the target site could be some step related to the feeding orotic acid (37). In addition, an arginine-deficient diet cell cycle. also promotes liver carcinogenesis in both rats and mice (37). Earlier work suggested that for orotic acid to induce its Certain urea cycle disorders, such as deficiency in OTC (38, biological effects it needs to create an imbalance in nucleotide 39), arginosuccinate sythetase (40), arginosuccinate lyase (41), pools, characterized by an increase in uridine nucleotides and a or arginase (42) or a decreased transport of ornithine to mito decrease in adenosine nucleotides (5-8). Consistent with this chondria (43), are associated with increased levels of orotic postulation are the observations that adenine, which inhibits acid. If the excess orotic acid is utilized for the synthesis of the metabolism of orotic acid by competing for PRPP, also uridine nucleotides, this could result in an imbalance in nucleo inhibits the efficacy of orotic acid to induce fatty liver (5-8), tide pools similar to that found in rat liver after administration liver-tumor promotion (16), and inhibition of EGF-induced of orotic acid. Interestingly, livers of OTC-deficient patients DNA synthesis (21). This argument favors the suggestion that exhibited a higher ratio of uridine to adenosine nucleotides, the target sites for mitoinhibitory effects mediated by orotic compared with that in the non-OTC-deficient patients (44). acid could be enzymes whose expression and/or activity are Although no extensive epidemiológica! data are available about regulated by the levels of nucleotide pools. It is tempting to these disorders and cancer incidence, there have been a few speculate that one of the target sites for orotic acid to induce reports about hepatocellular carcinoma in patients with orotic its mitoinhibition could be RNR. RNR is a key rate-limiting aciduria (45, 46). enzyme in DNA synthesis, and its activity is regulated by is also seen under conditions of hepatic nucleotides. ATP is one of the positive effector molecules for insufficiency resulting from partial hepatectomy (47), CCU RNR (1, 2). In addition, this enzyme is sensitive to the levels toxicity (47), portacaval shunt (48), hepatic cirrhosis, and al of deoxynucleotides (1, 2). In rat liver exposed to orotic acid, coholic hepatitis in rats (49) and humans (47). Interestingly, there is not only a decrease in the levels of ATP but also an these altered conditions are associated with an increased risk increase in uridine nucleotides (5-8). of liver-cancer development. However, implicating orotic acid In a preliminary study, we observed that orotic acid inhibited as the risk factor under these conditions needs further study. EGF-induced expression of RNR in hepatocytes and that such There are also genetic and metabolic disorders that result in an inhibition was counteracted by adenine (32). It is very likely increased levels of uridine nucleotides without a concomitant that an imbalance in nucleotide pools inhibits DNA synthesis increase in orotic acid. Folie acid deficiency results in up to a by regulating the expression of the gene for RNR and perhaps 10-fold increase in dUMP because of inadequate levels of folie other genes sensitive to the levels of nucleotides and acid needed in the conversion of dUMP to TMP (50). Interest deoxynucleotides. ingly, folie acid deficiency is known to enhance cell transfor One of the significant features of the orotic acid model of mation (50). In addition, Lesch-Nyhan syndrome is attributed tumor promotion is that orotic acid, being a normal cellular to a deficiency of guanine phosphoribosyl trans- constituent and a precursor of pyrimidine nucleotides, may ferase, the enzyme that catalyzes the conversion of hypoxan have the potential to be a multiorgan tumor promoter. Inter thine and guanine to and guanylic acid in the estingly, orotic acid appears to promote azoxymethane-induced presence of PRPP. Deficiency of this enzyme makes the PRPP carcinogenesis in rat duodenum (33) and dimethyl- available for orotic acid to form orotidylic acid and results in a benzo(a)anthracene-induced mammary carcinogenesis in rats 6-fold increase in uridine nucleotides (51). One wonders (34). whether such an imbalance in nucleotide pools has any role in Another interesting feature of the orotic acid model of tumor the pathogenesis of this disease process. promotion is that there are metabolic and genetic disorders that In view of the important roles nucleotides play in various are associated with increased levels of orotic acid. These disor- cellular functions, an imbalance in their levels not only may 2080s

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1992 American Association for Cancer Research. Orotic Acid, Nucleotide-Pool Imbalance, and Liver-Tumor Promotion: A Possible Mechanism for the Mitoinhibitory Effects of Orotic Acid in Isolated Rat Hepatocytes

Sharmila Manjeshwar, Aisha Sheikh, Giuseppina Pichiri-Coni, et al.

Cancer Res 1992;52:2078s-2081s.

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