Effect of Ischemia on Nucleosides and Bases in Rat Liver and Hepatoma 3924A1

Home , Deoxycytidine

[CANCER RESEARCH 47, 3092-3096, June 15. 1987] Effect of Ischemia on Nucleosides and Bases in Rat Liver and Hepatoma 3924A1

Konrad Pillwein, Hiremagalur N. Jayaram, and George Weber2

laboratory for Experimental Oncology, Indiana University School of Medicine, Indianapolis, IN 46223

ABSTRACT MATERIALS AND METHODS

A high-pressure liquid Chromatographie method was developed which Materials. Nucleobases and nucleosides were obtained from Sigma achieved a separation and quantitation of 20 biologically important Chemical Co., St. Louis, MO. Ammonium acetate, ammonium phos nucleosides and bases. The concentrations of pyrimidine nucleosides and phate, sodium acetate, and acetonitrile (HPLC grade) were purchased bases, namely deoxycytidine, cytosine, cytidine, uracil, and uridine (22.6, from Fisher Scientific Co., Springfield, NJ. Radial Pak, Resolve Ct8 5 10.1, 5.2, 2.9, and 2.4 nmol/ml, respectively) were high in plasma, Mm(8.0 mm x 10 cm), Partisil 10-SAX (8.0 mm x 10 cm) columns, whereas purine nucleosides and bases were present in concentrations less and HPLC apparatus consisting of 720 System Controller, 730 Data than 2.5 nmol/ml. In erythrocytes, the pools of xanthine, hypoxanthine, Module, 71OB-WISP auto injector, 510 pumps, and 440 dual channel and xanthosine were 32-, 27-, and 22-fold larger, respectively, whereas absorbance detector were obtained from Waters Co., Milford, MA. cytidine, uridine, and deoxycytidine were only 21, 12, and 5% of plasma Animals and Tumors. Inbred male rats of the ACI/N strain weighing concentrations. The results suggest a compartmental system for transport 180-200 g (HarÃanIndustries, Cumberland, IN) were housed in indi of some of the purine and pyrimidine nucleosides and bases in the whole vidual cages and fed laboratory chow; water was available ad libitum. blood. The rats were inoculated s.c. on both flanks with 2 x IO7cells of the Studies on the effect of ischemia on nucleoside and base pools in rat transplantable hepatoma 3924A. The biological and biochemical prop liver indicated marked increases within 30 s in the concentrations of erties of this tumor and the advantages of this biological model system adenine, adenosine, inosine, hypoxanthine, uridine, and xanthine, whereas were outlined elsewhere (12, 13). in hepatoma the effects were less pronounced. By 2 and 5 min ischemia Sample Preparation. Two weeks after inoculation, the tumor-bearing these perturbations were most marked in both liver and hepatoma. These rats were lightly anesthetized with ether and the hepatoma was exposed results indicate a need for rapid freeze-ciamp preparation of tissue without severing the blood supply. A portion was rapidly excised and samples to obtain precise and repeatable results in the determination of freeze-clamped within 1 s as described earlier (12, 14). The remaining tissue nucleoside and nucleobase concentrations. part of the tumor was excised and kept at 23Â°C(room temperature) for periods up to 5 min. This provided the ischemia samples. Livers from INTRODUCTION normal rats were obtained under similar conditions. The freeze-clamped tissues were ground under liquid nitrogen, then homogenized and An imbalance in purine and pyrimidine metabolism has been extracted with 10% TCA (1:4 w/v). The acid soluble extracts were reported in inborn errors of metabolism ( 1), immune disease immediately neutralized with 0.5 M tri-n-octylamine in freon (1:2 v/v) (2), and in cancer cells (3-5). Quantitative measurement of the (15, 16). metabolites of purine and pyrimidine biosynthesis and degra Preparation of Plasma Samples. Rats were anesthetized with ether dation is important for diagnosis and to monitor therapeutic and blood was drawn from the inferior vena cava into a heparinized syringe and kept on ice. The blood sample was centrifuged for 2 min effects of drugs (6-8). Furthermore, knowledge of substrate at 4"( ' at 1000 x g. Plasma was separated and extracted with cold 10% concentrations for various enzyme systems is essential in as TCA (1:2 v/v) and centrifuged at 12,000 x g for 0.2 min in an Eppendorf sessing the importance of biosynthetic and catabolic pathways centrifuge. Acid extract was neutralized as detailed above. for DNA and RNA metabolism. Preparation of Erythrocyte Extract. Erythrocytes were separated from A significant advance in the determination of nucleobases plasma as described above, buffy coats were quickly removed, and then and nucleosides was achieved by the HPLC3 technique and red blood cells were washed twice with cold phosphate buffered saline. several methods for the analysis of nucleobases and nucleosides After counting in a Coulter counter (Coulter Electronics, Luton, Beds, in plasma and urine were described in the literature (9-11). England) cells were frozen at â€”80Â°C.Frozencells were homogenized HPLC has provided flexibility in the analysis of nucleosides in cold 10% TCA (3.5 ml TCA/g of packed cells) and acid extract was and bases in biological fluids, but ribonucleotides, deoxyribo- neutralized as described earlier. Determination of Nucleobases and Nucleosides. Neutralized acid sol nucleotides, and cyclic nucleotides present in tissue extracts uble plasma extracts were directly analyzed on a Radial-Pak, Resolve exhibit absorption at 254-280 nm and cause problems in nu CIBcolumn for nucleobases and nucleosides. Samples from liver, hep cleoside and base analysis by broadening the peaks and thus atoma 3924A, and erythrocytes were processed to separate interfering decreasing the accuracy in their determinations. nucleotides. Aliquots of neutralized acid extract were loaded onto a In the present communication, we report the concentrations column of Partisil 10-SAX preequilibrated with 1 HIM ammonium of nucleosides and bases in rat liver and rat hepatoma 3924A. acetate, pH 4.7; the nucleosides and bases were eluted isocratically for We have examined the effect of ischemia on the nucleoside and 7 min with the same buffer at a flow rate of 2 ml/min and collected. nucleobase pools in tissues by measuring the concentrations of The fractions were immediately pooled, frozen, and lyophilized. The nucleosides and bases in freeze-clamped rat liver and hepatoma column was then washed with 2 M ammonium acetate, pH 6.1, at a 3924A, obtained immediately (within 1 s) and after periods of flow rate of 4 ml/min for 10 min followed by reequilibration for 10 min with 1 IHMammonium acetate, pH 4.7. ischemia (up to 5 min). The lyophilized fraction containing nucleobases and nucleosides was Received 1/14/87; revised 3/11/87; accepted 3/13/87. dissolved in water and neutralized with tri-n-octylamine in frÃ©onas The costs of publication of this article were defrayed in part by the payment detailed earlier. Aliquots were then analyzed on a Radial Pak, Resolve of page charges. This article must therefore be hereby marked advertisement in Cig column which was preequilibrated with buffer A (1 HIMsodium accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by USPHS, National Cancer Institute, Grant CA-13526, and by acetate buffer, pH 3.9) and eluted isocratically with the same buffer for an Outstanding Investigator Grant CA-42510 to G. W. and by USPHS Grant 5- 4 min at a flow rate of 1 ml/min and then a 0-6% linear gradient was S07RR5371 to H. N. J. applied with acetonitrile for 16 min. The buffer system was then 2To whom requests for reprints should be addressed, at Laboratory for switched to 5 HIMsodium acetate, pH 5.4 (buffer B) over a period of 1 Experimental Oncology, 702 Barnhill Drive, Indiana University School of Med min. A 0-10% linear gradient was applied with buffer B and acetonitrile icine, Indianapolis, IN 46223. 3The abbreviations used are: HPLC, high-pressure liquid chromatography; over a period of 19 min and then maintained at 10% level for 5 min. TCA, trichloroacetic acid. The column was regenerated with buffer A and 40% acetonitrile for 5 3092 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1987 American Association for Cancer Research. NUCLEOSIDES AND BASES IN TISSUES AND BIOLOGICAL FLUIDS

min at a flow rate of 2 ml/min. The column was equilibrated for 5 min Only cytosine and cytidine coeluted with this method, but were with buffer A before the application of the next sample. separable with a different gradient elution we developed for Quantitation of Cytosine and Cytidine. To separate cytosine and that purpose. cytidine, the above method was slightly modified by altering gradient Reproducibility. The reproducibility of the elution profile conditions, maintaining the same flow rate and buffers. After loading from one analysis to another was examined by injecting a the sample onto a preequilibrated column with buffer A, linear gradient (0 to 2%) was applied with acetonitrile for 13 min. Buffer A was mixture containing 5 nmol each of 20 different standard nu replaced with buffer B over a period of 0.5 min and then the concentra cleosides and bases consecutively 10 times. The average varia tions of buffer B (98%) and acetonitrile (2%) were kept constant for tion of retention time was 0.4 Â±0.04% (SE), and day-to-day 8.5 min. During the next 0.5 min, the concentration of buffer B was variability (10 days) was 0.8 Â±0.07%. Column reproducibility increased to 100% and maintained for 2.5 min, followed by a 0-15% (three columns from the same batch) was 1.2 Â±0.04% and linear gradient with acetonitrile for 15 min. The column was then batch reproducibility (three columns from different batches) regenerated with buffer A and 40% acetonitrile for 5 min. The analysis was 2.6 Â±0.2% with maximum variations for guanine (4.4%) of the next sample was performed after equilibrating the column for 10 and xanthine (5.8%). However, the sequence of elution of min with buffer A. Under the conditions of analysis, the order of elution compounds remained identical on all columns. of the compounds was, in min: uracil, 8.2; uric acid, 12.4; hypoxanthine, Linearity. Linearity was established with 11 different concen 14.9; uridine, 15.6; xanthine, 16.3; thymine, 18.2; deoxyuridine and trations (0.005-20 nmol) of hypoxanthine, guanosine, uracil, guanine coelute, 20.9; cytosine, 23.5; cytidine, 24.6; inosine, 25.2; deoxycytidine, 28.1; guanosine, 29.3; deoxyinosine, 31.3; xanthosine, and thymidine as representatives of purine and pyrimidine 35.0; deoxyguanosine, 36.0; thymidine, 37.9; adenine, 40.5; adenosine, nucleosides and bases (data not shown). 42.4; and deoxyadenosine, 43.2. Accuracy of Integration. Ten different nucleoside and base Statistical Evaluation. Results were subjected to statistical analysis standards (adenine, adenosine, cytidine, guanine, guanosine, by Student's t test (17). Differences between means yielding a probabil hypoxanthine, inosine, thymidine, uracil, and uridine) were ity of less than 5% were considered as statistically significant. analyzed consecutively 10 times and their integrated areas were compared. Standard errors were in the range of 0.006-0.7% of the amount injected. RESULTS Recovery. Studies with 12 different purine and pyrimidine Removal of ribonucleotides, deoxyribonucleotides, and cyclic nucleosides and bases (adenine, adenosine, guanine, guanosine, nucleotides from nucleobases and nucleosides by ion-exchange inosine, hypoxanthine, xanthine, cytidine, thymine, thymidine, chromatography should facilitate clear separation and removal uracil, and uridine) were performed with a mixture containing of interfering broad peaks in the UV detecting region of 254 each component at final concentrations of 10, 50, or 75 nmol/ and 280 nm. Nucleosides and bases were not retained and g, employing the entire extraction and analysis procedures. eluted close to the void volume, whereas nucleotides were Nucleobases and nucleosides were added to the freeze-clamped absorbed onto the Partisil 10-SAX column. powder of liver or hepatoma 3924A, immediately homogenized Chromatographie Separation of Nucleobases and Nucleosides. with cold 10% TCA and carried through the whole procedure. The Chromatographie elution profile of the biologically impor Triplicate analyses were performed for each concentration and tant nucleosides and bases is illustrated in Fig. 1. As the recovery values were statistically evaluated. Recoveries averaged chromatogram indicates, 18 deoxynucleosides, ribonucleosides, 95% with a standard deviation of 0.6%. and nucleobases were separated by this analytical technique. Nucleobases and Nucleosides in Rat Plasma. To account for

z Ã› I ~ o

Fig. l. Chromatographie elution profile of Standard nucleobases and nucleosides on Radial-Pak Resolve Cu S (.m column. Numbers, elution time (min) of the compounds. 3093

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1987 American Association for Cancer Research. NUCLEOSIDES AND BASES IN TISSUES AND BIOLOGICAL FLUIDS the variability in biological samples, retention times for the were not detectable in the hepatoma (<5 pmol/g). nucleobases and nucleosides were determined and confirmed by Effect of Ischemia on Concentrations of Nucleobases and cochromatography of standards of interest and by determining Nucleosides in Rat Liver and Hepatoma 3924A. Earlier studies peak-height ratio at 254 and 280 nm. Table 1 lists the concen demonstrated a significant alteration in the nucleotide pools trations of nucleobases and nucleosides in rat plasma, which following ischemia (12, 14). Since our preliminary studies in was rich in circulating deoxycytidine, cytosine, and cytidine. dicated marked alterations in concentrations of nucleosides and The concentrations of deoxyadenosine, deoxyguanosine, deox- bases in rat liver and hepatoma 3924A after 10 min ischemia yinosine, xanthine, xanthosine, and deoxyuridine were not de (data not shown), we undertook a detailed time course study at tectable in the plasma (detection limit, 5 pmol/ml). shorter intervals (0-300 s) (Table 3). Ischemia caused a signif Nucleobases and Nucleosides in Erythrocytes. The total pools icant decrease in the concentration of adenine in hepatoma, of nucleobases and nucleosides in the rat erythrocytes were 14% and in liver a significant increase. The effect of ischemia on the of those in rat plasma (6.9 and 50.3 nmol/ml, respectively) pools of other purine nucleosides and bases was similar in liver (Table 2). The concentrations of hypoxanthine, xanthine, and and hepatoma, but the perturbations were more pronounced in xanthosine in erythrocytes were 20- to 30-fold higher than in the liver. Among the pyrimidine nucleosides and bases, is plasma. Adenosine, deoxyadenosine, guanine, guanosine, de chemia resulted in increases in thymine and uracil pools only oxyguanosine, deoxyinosine, cytosine, thymine, thymidine, and in the hepatoma and the perturbations in the pools of other deoxyuridine were not detectable in erythrocytes (detection pyrimidine nucleosides and bases were similar in liver and limit, 5 pmol/109 cells). hepatoma, although more marked in the liver. Nucleobase and Nucleoside Concentrations in Rat Liver and Hepatoma 3924A. In normal rat liver, except for deoxycytidine, DISCUSSION adenine, cytidine, and uracil, the concentrations of nucleobases and nucleosides were lower than 5 n\t (Table 3). Deoxyguano The results demonstrate that the HPLC method is well suited sine, deoxyinosine, and deoxyuridine were not measurable in for the determination of the concentrations of nucleobases and the rat liver (detection limit, 5 pmol/g). Deoxyadenosine pools nucleosides in plasma, liver and hepatoma. Quantitative recov in the liver could not be estimated accurately due to coelution eries of nucleosides and bases were obtained by extraction with with an as yet unidentified compound. TCA and the results reported here are in agreement with the In hepatoma 3924A as in normal rat liver, adenine, uracil, finding of Kin m (15). cytidine, and deoxycytidine concentrations were higher than In rat plasma the values for deoxycytidine and cytidine were those of other nucleobases and nucleosides (Table 3). Deoxy in general agreement with those published (9, 18). The concen adenosine, deoxyguanosine, deoxyinosine, and deoxyuridine tration of cytosine in the present study was 2- to 3-fold lower than that reported earlier (9). The concentrations of thymine, Table I Concentrations of nucleobases and nucleosides in rat plasma thymidine, uracil, and uridine obtained in the present study Values are means Â±SE of plasma samples obtained from 4 or more rats. were at variance with those in the literature (9, 18). Among the Blood was drawn from the inferior vena cava of ether anesthetized rats; plasma was separated, extracted with TCA and analyzed on HPLC as detailed in "Ma purine nucleosides, inosine concentration was 41-fold lower, terials and Methods." whereas adenosine and adenine values were 4-fold lower than Nucleobase or values that reported (9, 18). Variations in the concentrations of some nucleosideAdenineAdenosineGuanineGuanosineInosineHypoxanthineCytosineCytidineDeoxycytidineThymineThymidineUracilUridineConcentration(nmol/ml)2.0 (nmol/ml)8.28.120.524.23.3, of the nucleosides and bases from the published values might Â±0.22.4 relate to the age, sex, and strain of the rats used in these studies Â±0.50.4 (female Charles River CD Sprague-Dawley rats were used in Â±0.070.3 Â±0.060.5 Ref. 8; male Sprague-Dawley rats weighing 300 g, in Ref. 18; Â±0.70.1 and male ACI/N rats weighing 180-200 g, in the present study). Â±0.0110.1 Â±0.75.2 Unlike the figures available in the literature for only a few Â±0.822.6 9.817.64.712.87.81.0Reference99999,18999918compounds, the present study provides the concentrations in Â±3.60.1 the plasma for the major important nucleobases, ribonucleo- Â±0.011.3 Â±0.32.9 sides, and deoxyribonucleosides. Â±0.62.4 Concentrations for some of the nucleosides and bases were Â±0.6Literature published for rat heart (19), human placenta (20), cardiac muscle (21), African green monkey kidney cells (22), human Table 2 Nucleobases and nucleosides in rat erythrocytes skin fibroblasts (23), and human lymphocytes (24); values for Values are means Â±SE and are also expressed in percentages of plasma rat erythrocytes and liver were not available. concentration. Erythrocyte samples were obtained from seven or more rats. For comparing values, I â€¢10"cells were considered equivalent to 1 ml of plasma. The 20- to 30-fold higher concentrations of purine nucleo Blood was obtained from the inferior vena cava of ether-anesthetized rats. sides and bases found in erythrocytes have relevance because of Erythrocytes after separation from plasma and buffy coat were washed twice with the postulated role of erythrocytes in transporting purines into cold phosphate buffered saline, homogenized in TCA, and analyzed on HPLC according to the procedure given in "Materials and Methods." bone marrow of normal and leukemic patients (10, 25). Total of pyrimidine nucleobase and nucleoside concentrations were Nucleobase or plasma con higher in plasma (44.6 nmol/ml) than in erythrocytes (2.57 centration"2670 nucleosideHypoxanthine (pmol/10'cells)2670 nmol/g), suggesting a compartmental system for some purines Â±980 and pyrimidines in the whole blood. Xanthine 650 Â±230 >3250 Xanthosine 450 Â±90 >2250 The pools of adenine were significantly lower in hepatoma Adenine 390 Â±100 19 3924A than in liver which is in accord with the high adenine Inosine 170 Â±70 34 phosphoribosyltransferase activity observed in this tumor (26). Deoxycytidine 1220Â± 230 5 Cytidine 1070 Â±260 21 Adenosine concentrations were lower in hepatoma than in liver UridineConcentration 280 Â±40Percentage 12 which might reflect high adenosine deaminase activity in the " All values are significantly different from the plasma values (/' < 0.05). tumor (27). Hypoxanthine and xanthine pools were elevated to 3094 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1987 American Association for Cancer Research. NUCLEOSIDES AND BASES IN TISSUES AND BIOLOGICAL FLUIDS

Table 3 Effect of ischemia on concentrations of nucleobases and nucleosides in rat liver and hepatoma 3924A The techniques used for the determinations are detailed in "Materials and Methods." Values are means Â±SE of four or more samples.

ischemiaNucleobases Period of

and nucleosidesAdenineAdenosineInosineGuanineGuanosineHypoxanthineXan Â±1.73.7 Â±1.80.6 Â±0..6Â±0..4Â±0..0Â±0..2Â±0..5Â±0.11.9 Â±0.11.6 Â±0.20.4 Â±0.12.3 Â±0.23.8 thineXanthosineCytosineCytidineDeoxycytidineThymineThymidineUracilUndineOsÂ«Liver22.7Â±0.22.9 Â±0.24.3 Â±0.13.0 Â±0.73.4 Â±0.317.0 Â±0.310.5 Â±1.523.3 Â±2.549.0 Â±1.70.1 Â±3.70.6 Â±0.050.3 +0.10.2 Â±0.0317.5 Â±0.116.7 Â±0.62.9 Â±4.32.8 Â±0.1Hepatoma13.4 Â±0.2Liver164'70S'425'79100367'147'83979677Â«14090103286'30sÂ»Hepatoma76ISC'94100112182'1131129110376'1008098114'120s*Liver240e1162'5362'

192 and 253% in the hepatoma, which might be due to the nucleosides and nucleobases were different in liver and hepa markedly lower xanthine oxidase activity in the tumor com toma from those in the plasma; thus plasma values do not pared to liver (13). Cytidine levels were lower in hepatoma provide a reliable indication of the availability of substrates for which might reflect the elevated cytidine kinase activity in that salvage enzymes but this information is to be found in the tissue (13). Increased thymine (to 600%) and decreased thymi- tissues. From the tissue concentrations reported in Table 3 for dine (to 33%) concentrations in the hepatoma, compared to the nucleoside and nucleobase substrates and for other ligands liver, might be due to the markedly decreased dihydrothymine (ATP, PRPP) determined in the same tissues, reported else dehydrogenase and the high thymidine kinase activities in the where (12, 13), we calculated that in the hepatoma 3924A the tumor (13). activities of the purine and pyrimidine salvage enzymes were The novel results mainly concern the behavior of the concen orders of magnitude higher than those of the de novo synthetic trations of the precursors and breakdown products of the mi pathways (26, 28). Therefore, for a successful anticancer che cleotides. In ischemia, the concentrations of deoxycytidine and motherapy inhibition of strategic enzymes of the de novo path cytosine progressively decreased; that of uracil was unaltered, ways must be coupled with blocking salvage enzyme activities whereas those of cytidine, and particularly of uridine, increased or salvage transport as shown in combinations such as using as a result of degradation of the uridylates and cytidylates in acivicin as an inhibitor of de novo synthesis and dipyridamole rat liver and hepatoma (14). The decline in thymidine concen as the transport inhibitor (13). Measurement of the concentra tration suggests its degradation through thymine into catabolic tions of strategic nucleosides and nucleobases along with key products. As a consequence of ischemia, the pools of guanine ribonucleosides and deoxyribonucleotides should provide a bio sharply decreased and were depleted within 2 min. By contrast, chemical monitor of drug action and a powerful assistance in the pools of inosine, hypoxanthine, xanthine, and adenosine the design of treatment protocols for maximized chemothera- markedly increased in rat liver and hepatoma. The rapid de peutic targeting with the lowest host toxicity (13, 29). crease in guanine concentration resulted from high guanase activity. The increase in the concentrations of inosine reflects ACKNOWLEDGMENTS the elevation in IMP pools and catabolic breakdown of some of the nucleotides (12, 14). The increase in hypoxanthine and The technical assistance of Cicero CalderÃ³n and Leopoldo Soliven xanthine concentrations reflects the functioning of IMP nucleo- is greatly appreciated. tidase and xanthine oxidase which have high activities in rat liver. These results taken together illustrate the importance of REFERENCES rapid freeze-clamping of the tissues for the accurate determi nations. A similar requirement for measurement of nucleotide 1. Dillon, M. 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Konrad Pillwein, Hiremagalur N. Jayaram and George Weber

Cancer Res 1987;47:3092-3096.

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