Clinical Science ( 1992) 82, I8 I - I84 (Printed in Great Britain)
Effects of methotrexate on rat liver regeneration afker partial hepatectomy lkuyo TSUKAMOTO* and Shosuke KOJOJr *Department of Food Science and Nutrition, Nara Women’s University, Nara, Japan, and ?Department of Life and Health Sciences, Hyogo University of Teacher Education, Yashiro, Hyogo, lapan (Received 20 March127 August 199 I; accepted 9 September 199 I) 1. Methotrexate was administered immediately after
partial (70%) hepatectomy, resulting in complete inhibition of dihydrofolate reductase in 24 h-regenerating liver. 2. At 48 h and 72 h after partial hepatectomy, thymidylate synthase activity was increased, whereas thymidine kinase was inhibited, by the injection of methotrexate. The DNA and RNA contents and the liver weight were also reduced in methotrexate-treated rats. 3. The immunoblotting assay showed that methotrexate stimulated the synthesis of thymidylate synthase protein in 48 h-regenerating liver. At the same time, thymidylate synthase activity was directly inhibited by methotrexate. The mechanisms of inhibition of these enzymes by methotrexate appeared to be different.
INTRODUCTION Methotrexate (MTX), one of the most widely used and effective anti-neoplastic agents, is believed to exert its anti-tumour effects by virtue of its tight-binding inhibition of dihydrofolate reductase (EC 1.5.1.3). The depletion of cellular pools of tetrahydrofolates after exposure to MTX may disturb the biosynthesis de novo of thymidylate and/ or purines depending on the cell type. The growth inhibitory effects of MTX and its protection by thymidine or purine have been extensively studied in various cell lines [ 1-51. There have been a few studies on the effect of MTX on liver regeneration after partial hepatectomy (PH). Brown et al. [GI and Ngu et al. [7] reported that MTX had no significant growth-repressing effect on regenerating liver after PH. Labow et al. [S] argued that the injection of MTX inhibited the enzymic activities related to DNA synthesis, with the exception of thymidylate synthase (EC 2.1.1.45), in regenerating liver. The action of MTX on the regenerative response in the liver after PH has not yet been characterized. As a part of our systematic work on the regulation network of liver regeneration [9-201, we have investigated the effect of MTX on liver regeneration. The regenerative response in the present experiment was evaluated by
measuring the activities of thymidylate synthase and thymidine kinase (EC 2.7.1.21), which are the ratedetermining enzymes of DNA synthesis in regenerating rat liver [15], and by determining DNA and RNA contents and liver weight. In this paper, we report the first evidence that MTX stimulates the synthesis of thymidylate synthase protein in regenerating liver as well as inhibiting the induction of thymidine kinase.
MATERIALS AND METHODS Materials L-Tetrahydrofolate and dihydrofolate were prepared by catalytic hydrogenation of folic acid [21] and by reduction of folic acid with sodium hydrosulphite [22], respectively. [5-3H]Deoxyuridine monophosphate ( 10.9 Ci/mmol) and [rnethyl-3H]thymidine (65 Ci/mmol) were purchased from Amersham International and E N Radio-Chemicals, respectively. MTX was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). All other reagents were of analytical grade.
Animals and treatments Male Wistar rats weighing 180-200 g (5-1 1 animals in each group) were used in this study. The animals were permitted free access to food ( M E Oriental Yeast Co., Osaka, Japan) and water at all times. PH (70%) was performed by the procedure of Higgins & Anderson [23]. MTX (5, 25 and 50 mg/kg body weight), dissolved in 0.1 mol/l NaHCO,, was injected intraperitoneally immediately after PH. In the experiments with 48 h-regenerating liver, the injection of MTX was repeated 24 h after PH. For the 72 h-regenerating liver, MTX was administered at 0, 24 and 48 h after PH. Control rats received the same quantity of 0.1 mol/l NaHCO, as did the experimental animals.
Analytical methods The rats were killed under ether anaesthesia at 24, 48 or 72 h after PH. The liver was perfused in situ with 0.9%
Key words: dihydrofolate reductase, liver regeneration, methotrexate, thymidine kinase, thymidylate synthase. Abbreviations: MTX, methotrexate; PH, partial hepactectomy. Correspondence: D r lkuyo Tsukamoto, Department of Food Science and Nutrition, Nara Women’s University, Nara 630, Japan.
I, Tsukamoto and S. Kojo
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(w/v) NaCl and homogenized in 50 mmol/l Tris-HCI buffer, pH 7.5, containing 0.25 mol/l sucrose, 10 mmol/l /3-mercaptoethanol, 1 mmol/l phenylmethanesulphonyl fluoride and 1 mmol/l EDTA. The DNA and RNA contents of liver homogenate were measured by the diphenylamine [24] and orcin [25] reactions, respectively, after extraction with trichloroacetic acid by the procedure of Schneider [25]. Protein was determined by the method of Lowry et 01. [26]using BSA as standard.
and then reacted with goat anti-(rabbit 1gG)-horseradish peroxidase conjugate. After the colour development, the reflectance of the stained band was determined. The thymidylate synthase content was expressed as ng/40 pg of protein of the liver supernatant fraction.
Statistical analysis This was performed with Student's t-test.
Enzyme assays A 20% (w/v) homogenate was centrifuged at 36 000 g for 30 min at 4"C, and the supernatant fraction was used as the enzyme preparation. Dihydrofolate reductase activity was assayed spectrophotometrically by determining the decrease in absorbance at 340 nm (30°C) as described by Gunderson et at. [27]. The assay mixture contained 50 mmol/l Tris-HCI buffer, pH 7.5,O.l mmol/l NADPH, 0.06 mmol/l dihydrofolate and enzyme preparation (150-400 pg of protein). The activities of thymidylate synthase and thymidine kinase were determined as described previously [9].
Determination of thymidylate synthase content The thymidylate synthase content was determined by an immunoblotting assay developed by us utilizing the 36 000 g supernatant fraction of the 20% (w/v) liver homogenate [ 161. Briefly, the supernatant fractions (10-40 pug of protein) were submitted to SDS/gel electrophoresis on 1 0% polyacrylamide-slab gels. Proteins separated on the gel were electrophoretically transferred to Durapore membrane filters. The filters were incubated with anti-(rat liver thymidylate synthase)-rabbit serum
RESULTS Effect of MTX on the enzymic activities, the contents of DNA, RNA and protein and the liver weight The activities of dihydrofolate reductase in the liver were 5.2k0.4, 7.8k0.7, 7.4fO.G and 8.1f0.6 nmol of dihydrofolate reduced min- mg- of protein ( m e a n s * s ~ ~at) 0 h, 24 h, 48 h and 72 h after PH, respectively. The injection of MTX completely inhibited the dihydrofolate reductase activity of 24 h-, 48 h- and 72 h-regenerating liver at doses of 5, 25 and 50 mg/kg body weight. The activities of thymidylate synthase and thymidine kinase after PH are shown in Table 1. At 24 h after PH the thymidylate synthase activity was increased by about six times compared with normal (resting in Go state; just after PH). The injection of MTX immediately after PH at a dose of 5 mg/kg body weight produced a small reduction in thymidylate synthase activity in 24 hregenerating liver compared with the control (PH only), but this decrease was not significant. When the dose of MTX was increased to 25 or 50 mg/kg body weight, no significant effect was observed on the thymidylate
'
Table I. Effect of M T X on liver regeneration. MTX (5, 25 o r 50 mgikg body weight), dissolved in 0.1 moll1 NaHCO,, was injected intraperitoneally just after PH and repeated at every 24 h. A t 24, 48 o r 72 h after PH, the activities of thymidylate synthase (TS) and thymidine kinase (TK), the total contents of DNA, RNA and protein in liver and the liver weight were determined as described in the Materials and methods section. Values are expressed as meanstsm. Statistical just after PH). significance: * P (0.05 compared with the corresponding control group; t P (0.05 compared with normal (Go, Treatment
Time after PH (h)
No. of rats
Total liver content
Enzyme activity (pmol min-l mg-l o f protein) TS
Liver weight (8)
(mg)
TK
DNA
RNA
Protein
Control
24 48 72
10 II 6
41.77f4.08t 38.70 f3.06 36.66 t4.78
3 14.90 f26.22t 196.23 f9.47 153.20 f 16.36
6.68fO.31t 10.66i.0.67 15.02f 1.21
35.5 f I .ot 45.8 f2.9 60.7 t I .4
649.2 f 37.4t 806.6 f5 I .8 1024. I t52.0
3.43 f 0 . 0 5 t 4.91 f0.13 6.03 i.0.22
MTX (5 mgikg body wt.)
24 48 72
6 8
5
32.47 t 3.421. 107.07 i 8 . 6 6 * 101.90 i.22.96*
329.47 f 36.821. 109. I 6 f 22.57* 33.30 i.I I.84*
6.82f0.33t 8.42*0.55* I1.05f0.34*
32.0 f2.3t 33.7 f3.3* 41.5 f 3 . 9 *
573.9 f 28.5 695.3 540.2 872.3 i.75.5
3.40 kO.09t 4.55 i.0. I I 5.54 t0.21
MTX (25 mglkg body wt.)
24 48 72
6 6 6
36.10k i.34t 121.975 10.13* 122.62f 11.29'
292.45 i.33.68t I 10.0 I f20.70* 28.9 I +4.05*
6.34 t 0 . 0 8 t 8.47 t0.25* 10.62 f0.66*
32.0 f I 4 35.8 f I .3* 43.5 f2.8*
584.9 i.37.0 750.5 f 18. I 806.9 f 18.5'
3.40f0.14t 4.49 f0.09 4.98f0.24*
MTX (50 mgikg body wt.)
24 48 72
6 6
5
29.0 I f4.441 105.85 t 8.74* 102.32t9.72*
235.80f39.65t 137.16+20.38* 28.85 i.7.94*
6.68i.0.22t 8.6 I +0.38* IO.I2f0.51*
31.1 f 1.4t 34.0 f I .6* 37. I f I .9*
586.0 f 4 5 . l 641.9i.31. I * 818.6f45.l*
3.33 i.0.08t 4.16 +0.19* 5.08 f0. I2*
8
6.74 f0.6 I
25.34f3.34
5.46f0.31
23.7 t I .2
488.0+ 37.3
2.95 i.0.09
Normal (Go,just after PH)
Effect of methotrexate on liver regeneration
synthase activity of 24 h-regenerating liver. In a similar manner, thymidine kinase activity was not affected at 24 h after PH by the injection of MTX at the doses employed. At 48 h after PH, the thymidylate synthase activity was similar to that at 24 h after PH in the control group. In the MTX ( 5 mg/kg body weight)-treated group, the thymidylate synthase activity at 48 h after PH was increased by 3.3 times compared with 24 h-regenerating liver. This activity was significantly higher than that of the corresponding control. The injection of MTX (25 or 50 mg/kg body weight) also markedly increased the thymidylate synthase activity, as shown in Table 1. On the other hand, thymidine kinase activity in the MTX (5 mg/ kg)-injected group was decreased to 55% of the corresponding control value at 48 h after PH. When MTX was administered at doses of 25 and 50 mg/kg, the extent of the reduction in thymidine kinase activity was similar to that of the group given MTX at a dose of 5 mg/kg body weight. At 72 h after PH, the thymidylate synthase activity in the MTX-treated group (5 mg/kg body weight) was increased by 2.8 times compared with the control. The injections of 25 and 50 mg of MTX/kg body weight produced increases of 3.3 and 2.8 times compared with control, respectively. However, the thymidine kinase activity in 72 h-regenerating liver was decreased to the normal (Gostate, just after PH) level, which was about 20% of the control, on treatment with 5, 25 or 50 mg of MTX/kg body weight. Treatment with MTX had no effect on the DNA content in 24 h-regenerating liver, as shown in Table 1. The DNA contents of MTX-treated 48 h- and 72 hregenerating liver were approximately 80% and 70% of the corresponding control values, respectively. These results showed that the DNA content was significantly decreased by injection of MTX in 48 h- and 72 hregenerating liver. In analogy with the DNA content, the RNA content was also affected at 48 h and 72 h after PH in all MTX-treated groups. Gross parameters, such as protein content and liver weight, were not influenced significantly by the injection of 5 mg of MTX/kg body weight. When MTX was administered at a dose of 25 mg/ Table 2. Effect of MTX on the thymidylate synthase in 48 hregenerating liver. MTX (5, 25 o r 50 mglkg body weight) was administered as described in the Materials and methods section. A t 48 h after PH, the liver was homogenized and centrifuged at 36 000 g for 30 min. The supernatant fraction was used t o determine the activity and the content of thymidylate synthase (TS) as described in the Materials and methods section. Values are expressed as meansksm o f four t o six experiments. Treatment
Control MTX (5 mglkg body wt.) M T X (25 mglkg body wt.) MTX (50 mglkg body wt.)
TS activity (pmol min-' mg-' of protein)
TS content (ng140 pg of protein in the supernatant)
53k1.5
4.0 k 0.98
I30 t 5.7
16.5 k I .29
l 3 8 k 3.7 98k4.9
19.6k1.18 25.0 k 2.57
I83
kg body weight significant decreases in protein content and liver weight appeared at 72 h after PH. With an increase in the dose to 50 mg/kg body weight, the reductions in protein content and liver weight were significant at 48 h and 72 h after PH. These results indicated that MTX inhibited the regeneration of the liver after PH, but that the effects of MTX on liver regeneration were not observed at 24 h after PH, except for the inhibition of dihydrofolate reductase. Differential responses of the two thymidylate-synthesizing enzymes to MTX were demonstrated. Thymidylate synthase activity was remarkably increased, whereas thymidine kinase activity was suppressed to normal. Thymidine kinase was not directly inhibited by MTX at 1 mmol/l in our assay system in vitro.
Effect of MTX on the thymidylate synthase content To understand the mechanism of stimulation of thymidylate synthase, the enzyme protein content was determined by immunoblotting as previously reported [16].The results are shown in Table 2. In previous studies [16, 18, 201, the activity of thymidylate synthase correlated very closely with the enzyme content. When thymidylate synthase activity was increased to 2.45 times the control level by injection of MTX (5 mg/kg body weight), the enzyme protein content was found to be 4.13 times that of control. The administration of MTX at 25 mg/kg and 50 mg/kg body weight raised the thymidylate synthase content to 4.9 and 6.3 times that of control, respectively, without any increment in the activity. These results clearly demonstrate that MTX stimulates the synthesis of thymidylate synthase protein in a dosedependent manner and at the same time MTX inhibits the enzyme, resulting in the discrepancies between the enzyme content and the activity of the enzyme. MTX is an inhibitor of thymidylate synthase, as we reported previously [28]. In our assay system, MTX inhibited thymidylate synthase activity by 40%, 60% and 90% at concentrations of 0.01, 0.1 and 1 mmol/l, respectively. This result suggests that the decrease in thymidylate synthase activity in MTX-treated rats could be explained by direct inhibition of the enzyme by MTX contained in the liver.
DISCUSSION The results of this study clearly demonstrate that MTX stimulates the synthesis of thymidylate synthase protein in regenerating liver in a dose-dependent manner, as shown in Table 2. This is the first evidence of the increase in the synthesis of thymidylate synthase protein as well as the inhibition of induction of thymidine kinase by injection of MTX, although Labow et al. [8] have reported the stimulation of thymidylate synthase activity at 48 h after PH. It was found that MTX completely inhibits dihydrofolate reductase activity within 24 h of injection even at a dose of 5 mg/kg body weight. However, the effects of MTX on the rate-determining enzymes of DNA synthesis
I. Tsukamoto and S. Kojo
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(thymidylate synthase and thymidine kinase), the contents of DNA, RNA and protein, and liver weight were not seen at 24 h. This result indicates that the reservoir of tetrahydrofolate derivatives in the liver remained sufficient, at least for 24 h after PH, to support the regenerative responses. At 48 h after PH, thymidine kinase activity was depressed to about 60% of control and the DNA content was also decreased. This suggests that thymidylate deprivation caused by the inhibition of both thymidine kinase and thymidylate synthase was responsible for the depression of the increase in DNA content. Although the activity of thymidylate synthase measured itz vitro increased after 48 h of PH in MTX-treated animals, the enzyme may not be functional in the liver, where the storage of the cofactors, tetrahydrofolates, was depleted. The depletion of tetrahydrofolate cofactors may result in the suppression of purine nucleotide synthesis and a subsequent decrease in RNA synthesis, as shown in Table 1.The depression of RNA synthesis appears to cause the decrease in thymidine kinase activity, since the induction of thymidine kinase was inhibited by actinomycin D [20]. On the other hand, the induction of thymidylate synthase was enhanced because it is insensitive to the inhibition of RNA synthesis, as previously reported [20]. In contrast with the report by Mdler et al. [29], thymidine kinase was suppressed to the normal level in 72 h-generating liver of MTX-injected rats. It has been reported that DNA synthesis in nonparenchymal cells such as endothelial, Kupffer and bile ductule cells, occurred 1 day later than in hepatocytes during liver regeneration [30]. Therefore, it may be argued that the delayed effect of MTX is caused partly by the influence of the drug on non-parenchymal cells.
REFERENCES
regenerating rat liver and the effect of methotrexate administration. Cancer Res. 1964; 24,989-93. 8. Labow, R., Maley, G.F. & Maley, F. The effect o f methotrexate on enzymes induced following partial hepatectomy. Cancer Res. 1969; 29, 366-72. 9. Nakata, R., Tsukamoto, I., Miyoshi, M. & Kojo, S. Liver regeneration after carbon tetrachloride intoxication in the rat. Biochem. Pharmacol. 1985; 34,
586-8. 10. Nakata, R., Tsukamoto, I., Nanme, M., Makino, S., Miyoshi, M. & Kojo, S. a-Adrenergic regulation of the activity of thymidylate synthetase and thymidine kinase during liver regeneration after partial hepatectomy. Eur. J. Pharrnacol. 1985; 114, 355-60. I I. Nakata, R., Tsukamoto, I., Miyoshi, M. & Kojo, S. Liver regeneration in streptozotocin-diabetic rats. Biochem. Pharmacol. 1986; 35,865-7. 12. Nakata, R., Tsukamoto, I., Miyoshi, M. & Kojo, S. Effect of thyroparathyroidectomy on the activities of thymidylate synthetase and thymidine kinase during liver regeneration after partial hepatectomy. Clin. Sci. 1987;
72,455-6 I. 13. Tsukamoto. I. & Kojo, 5. Effect o f phorbol ester on rat liver regeneration. Biochem. Pharmacol. 1987; 36, 2871 -2. 14. Tsukamoto, I. & Kojo, S. Effect of calcium channel blockers and trifluoperazine on rat liver regeneration. Eur. J. Pharmacol. 1987; 144, 159-62. 15. Tsukamoto, I. & Kojo, S. One evidence supporting that thymidylate synthetase and thymidine kinase are the rate-determining enzymes o f D N A synthesis in regenerating rat liver. Chem. Lett. 1987; 23 13-6. 16. Tsukamoto, I.,Nakata. R., Miyoshi, M.. Taketani, S. & Kojo, S. A new immunublotting assay for thymidylate synthetase and its application t o the regulation o f enzyme activity in regenerating rat liver. Biochim. Biophys. Acta 1988; 964, 254-9. 17. Tsukamoto, I. & Kojo, S. Effect of glucocorticoid on liver regeneration after partial hepatectomy in the rat. Gut 1989; 30, 387-90. 18. Tsukamoto, I. & Kojo, S. Effect of colchicine and vincristine on D N A synthesis in regenerating rat liver. Biochim. Biophys. Acta 1989; 1009,
191-3. 19. Tsukamoto, I. & Kojo, S. The sex difference in the regulation of liver regeneration after partial hepatectomy in the rat. Biochim. Biophys. Acta
1990; 1033, 287-90. 20. Tsukamoto, I. & Kojo, S. Effect o f cycloheximide and actinomycin D on thymidylate synthase and thymidine kinase in regenerating rat liver after partial hepatectomy. J. Nutr. Sci. Vitaminol. 1990; 36, 357-63. 21. Huennekkens, F.M., Mathews, C.K. & Scrimgeour, K.G. Preparation and properties o f tetrahydrofolic acid. Methods Enzymol. 1966; 6, 802-5. 22. Futterman, S. Preparation and properties of dihydrofolic acid. Methods Enzymol. 1966; 6,801-2. 23. Higgins, G.M. & Anderson, R.M. Experimental pathology o f the liver: restoration o f the liver o f white rat following partial surgical removal. Arch. Pathol.
1931; 12, 186-202. 24. Burton, K. A study of the conditions and mechanism o f the diphenylamine
I. Hryniuk, W.M., Fisher, G.A. & Bertino, J.R. S-phase cells o f rapidly growing
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and resting populations: differences in response t o methotrexate. Mol. Pharmacol. 1969; 5, 557-64. 2. Johnson, L.F., Fuhrman, C.L. & Abelson, H.T. Resistance o f resting 3T6 mouse fibroblasts t o methotrexate cytotoxicity. Cancer Res. 1978; 38,
J. 1956; 62,315-22.
2408-1 2. 3. Hakala, M.T. & Taylor E. The ability of purine and thymine derivatives and 4.
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o f glycine t o support the growth of mammalian cells in culture. J. Biol. Chem. 1959; 234, 126-8. Pinedo, H.M., Zaharko, D.S., Bull, J.M. & Chabner, B.A. The reversal o f methotrexate cytotoxicity t o mouse bone marrow cells by leucovorin and nucleosides. Cancer Res. 1976; 36,441 8-24. Balk, S.D., Mitchell, R.S., Lestourgeon, D. & Hoon, B.S. Thymidine and hypoxanthine requirements for the proliferation of normal and Rous Sarcoma virus-infected chicken fibroblasts in the presence of methotrexate. Cancer Res. 1979; 39, 1854-6. Brown, S.S.,Neal, G.E. & Williams, D.C. Lack o f effect of methotrexate on hepatic regeneration. Nature (London) 1965; 206, 1007-9. Ngu, V.A., Roberts, D. & Hall, T.C. Studies o f folic reductase. I. Levels in
25. Schneider, W.C. Determination o f nucleic acids in tissues by pentose analysis. Methods Enzymol. 1957; 3, 680-4. 26. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 1951; 193, 265-75. 27. Gunderson, L.E., Dunlap, R.B., Harding, N.G.L., Freisheim, J.H., Otting, F. & Huennekens, F.M. Dihydrofolate reductase from amethopterin-resistant lactobacillus casei. Biochemistry 1972; I I , 1018-22. 28. Nakata, R., Tsukamoto, I., Miyoshi, M. & Kojo, S. Purification and characterization of thymidylate synthetase from rat regenerating liver. Biochim. Biophys. Acta 1987; 924, 297-302. 29. Maller, U.,Keiding, N. & Larsen, J.K. Effect o f methotrexate on cells in a keratinized epithelium with an active thymidine salvage pathway assessed by autoradiography. Cell Tissue Kinet. 1984; 17,497-507. 30. Grisham, J.W. A morphologic study o f deoxyribonucleic acid synthesis and cell proliferation in regenerating rat liver; autoradiography with thymidine'H. Cancer Res. 1962; 22,842-9.
Effects of methotrexate on rat liver regeneration afker partial hepatectomy lkuyo TSUKAMOTO* and Shosuke KOJOJr *Department of Food Science and Nutrition, Nara Women’s University, Nara, Japan, and ?Department of Life and Health Sciences, Hyogo University of Teacher Education, Yashiro, Hyogo, lapan (Received 20 March127 August 199 I; accepted 9 September 199 I) 1. Methotrexate was administered immediately after
partial (70%) hepatectomy, resulting in complete inhibition of dihydrofolate reductase in 24 h-regenerating liver. 2. At 48 h and 72 h after partial hepatectomy, thymidylate synthase activity was increased, whereas thymidine kinase was inhibited, by the injection of methotrexate. The DNA and RNA contents and the liver weight were also reduced in methotrexate-treated rats. 3. The immunoblotting assay showed that methotrexate stimulated the synthesis of thymidylate synthase protein in 48 h-regenerating liver. At the same time, thymidylate synthase activity was directly inhibited by methotrexate. The mechanisms of inhibition of these enzymes by methotrexate appeared to be different.
INTRODUCTION Methotrexate (MTX), one of the most widely used and effective anti-neoplastic agents, is believed to exert its anti-tumour effects by virtue of its tight-binding inhibition of dihydrofolate reductase (EC 1.5.1.3). The depletion of cellular pools of tetrahydrofolates after exposure to MTX may disturb the biosynthesis de novo of thymidylate and/ or purines depending on the cell type. The growth inhibitory effects of MTX and its protection by thymidine or purine have been extensively studied in various cell lines [ 1-51. There have been a few studies on the effect of MTX on liver regeneration after partial hepatectomy (PH). Brown et al. [GI and Ngu et al. [7] reported that MTX had no significant growth-repressing effect on regenerating liver after PH. Labow et al. [S] argued that the injection of MTX inhibited the enzymic activities related to DNA synthesis, with the exception of thymidylate synthase (EC 2.1.1.45), in regenerating liver. The action of MTX on the regenerative response in the liver after PH has not yet been characterized. As a part of our systematic work on the regulation network of liver regeneration [9-201, we have investigated the effect of MTX on liver regeneration. The regenerative response in the present experiment was evaluated by
measuring the activities of thymidylate synthase and thymidine kinase (EC 2.7.1.21), which are the ratedetermining enzymes of DNA synthesis in regenerating rat liver [15], and by determining DNA and RNA contents and liver weight. In this paper, we report the first evidence that MTX stimulates the synthesis of thymidylate synthase protein in regenerating liver as well as inhibiting the induction of thymidine kinase.
MATERIALS AND METHODS Materials L-Tetrahydrofolate and dihydrofolate were prepared by catalytic hydrogenation of folic acid [21] and by reduction of folic acid with sodium hydrosulphite [22], respectively. [5-3H]Deoxyuridine monophosphate ( 10.9 Ci/mmol) and [rnethyl-3H]thymidine (65 Ci/mmol) were purchased from Amersham International and E N Radio-Chemicals, respectively. MTX was obtained from Nacalai Tesque, Inc. (Kyoto, Japan). All other reagents were of analytical grade.
Animals and treatments Male Wistar rats weighing 180-200 g (5-1 1 animals in each group) were used in this study. The animals were permitted free access to food ( M E Oriental Yeast Co., Osaka, Japan) and water at all times. PH (70%) was performed by the procedure of Higgins & Anderson [23]. MTX (5, 25 and 50 mg/kg body weight), dissolved in 0.1 mol/l NaHCO,, was injected intraperitoneally immediately after PH. In the experiments with 48 h-regenerating liver, the injection of MTX was repeated 24 h after PH. For the 72 h-regenerating liver, MTX was administered at 0, 24 and 48 h after PH. Control rats received the same quantity of 0.1 mol/l NaHCO, as did the experimental animals.
Analytical methods The rats were killed under ether anaesthesia at 24, 48 or 72 h after PH. The liver was perfused in situ with 0.9%
Key words: dihydrofolate reductase, liver regeneration, methotrexate, thymidine kinase, thymidylate synthase. Abbreviations: MTX, methotrexate; PH, partial hepactectomy. Correspondence: D r lkuyo Tsukamoto, Department of Food Science and Nutrition, Nara Women’s University, Nara 630, Japan.
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(w/v) NaCl and homogenized in 50 mmol/l Tris-HCI buffer, pH 7.5, containing 0.25 mol/l sucrose, 10 mmol/l /3-mercaptoethanol, 1 mmol/l phenylmethanesulphonyl fluoride and 1 mmol/l EDTA. The DNA and RNA contents of liver homogenate were measured by the diphenylamine [24] and orcin [25] reactions, respectively, after extraction with trichloroacetic acid by the procedure of Schneider [25]. Protein was determined by the method of Lowry et 01. [26]using BSA as standard.
and then reacted with goat anti-(rabbit 1gG)-horseradish peroxidase conjugate. After the colour development, the reflectance of the stained band was determined. The thymidylate synthase content was expressed as ng/40 pg of protein of the liver supernatant fraction.
Statistical analysis This was performed with Student's t-test.
Enzyme assays A 20% (w/v) homogenate was centrifuged at 36 000 g for 30 min at 4"C, and the supernatant fraction was used as the enzyme preparation. Dihydrofolate reductase activity was assayed spectrophotometrically by determining the decrease in absorbance at 340 nm (30°C) as described by Gunderson et at. [27]. The assay mixture contained 50 mmol/l Tris-HCI buffer, pH 7.5,O.l mmol/l NADPH, 0.06 mmol/l dihydrofolate and enzyme preparation (150-400 pg of protein). The activities of thymidylate synthase and thymidine kinase were determined as described previously [9].
Determination of thymidylate synthase content The thymidylate synthase content was determined by an immunoblotting assay developed by us utilizing the 36 000 g supernatant fraction of the 20% (w/v) liver homogenate [ 161. Briefly, the supernatant fractions (10-40 pug of protein) were submitted to SDS/gel electrophoresis on 1 0% polyacrylamide-slab gels. Proteins separated on the gel were electrophoretically transferred to Durapore membrane filters. The filters were incubated with anti-(rat liver thymidylate synthase)-rabbit serum
RESULTS Effect of MTX on the enzymic activities, the contents of DNA, RNA and protein and the liver weight The activities of dihydrofolate reductase in the liver were 5.2k0.4, 7.8k0.7, 7.4fO.G and 8.1f0.6 nmol of dihydrofolate reduced min- mg- of protein ( m e a n s * s ~ ~at) 0 h, 24 h, 48 h and 72 h after PH, respectively. The injection of MTX completely inhibited the dihydrofolate reductase activity of 24 h-, 48 h- and 72 h-regenerating liver at doses of 5, 25 and 50 mg/kg body weight. The activities of thymidylate synthase and thymidine kinase after PH are shown in Table 1. At 24 h after PH the thymidylate synthase activity was increased by about six times compared with normal (resting in Go state; just after PH). The injection of MTX immediately after PH at a dose of 5 mg/kg body weight produced a small reduction in thymidylate synthase activity in 24 hregenerating liver compared with the control (PH only), but this decrease was not significant. When the dose of MTX was increased to 25 or 50 mg/kg body weight, no significant effect was observed on the thymidylate
'
Table I. Effect of M T X on liver regeneration. MTX (5, 25 o r 50 mgikg body weight), dissolved in 0.1 moll1 NaHCO,, was injected intraperitoneally just after PH and repeated at every 24 h. A t 24, 48 o r 72 h after PH, the activities of thymidylate synthase (TS) and thymidine kinase (TK), the total contents of DNA, RNA and protein in liver and the liver weight were determined as described in the Materials and methods section. Values are expressed as meanstsm. Statistical just after PH). significance: * P (0.05 compared with the corresponding control group; t P (0.05 compared with normal (Go, Treatment
Time after PH (h)
No. of rats
Total liver content
Enzyme activity (pmol min-l mg-l o f protein) TS
Liver weight (8)
(mg)
TK
DNA
RNA
Protein
Control
24 48 72
10 II 6
41.77f4.08t 38.70 f3.06 36.66 t4.78
3 14.90 f26.22t 196.23 f9.47 153.20 f 16.36
6.68fO.31t 10.66i.0.67 15.02f 1.21
35.5 f I .ot 45.8 f2.9 60.7 t I .4
649.2 f 37.4t 806.6 f5 I .8 1024. I t52.0
3.43 f 0 . 0 5 t 4.91 f0.13 6.03 i.0.22
MTX (5 mgikg body wt.)
24 48 72
6 8
5
32.47 t 3.421. 107.07 i 8 . 6 6 * 101.90 i.22.96*
329.47 f 36.821. 109. I 6 f 22.57* 33.30 i.I I.84*
6.82f0.33t 8.42*0.55* I1.05f0.34*
32.0 f2.3t 33.7 f3.3* 41.5 f 3 . 9 *
573.9 f 28.5 695.3 540.2 872.3 i.75.5
3.40 kO.09t 4.55 i.0. I I 5.54 t0.21
MTX (25 mglkg body wt.)
24 48 72
6 6 6
36.10k i.34t 121.975 10.13* 122.62f 11.29'
292.45 i.33.68t I 10.0 I f20.70* 28.9 I +4.05*
6.34 t 0 . 0 8 t 8.47 t0.25* 10.62 f0.66*
32.0 f I 4 35.8 f I .3* 43.5 f2.8*
584.9 i.37.0 750.5 f 18. I 806.9 f 18.5'
3.40f0.14t 4.49 f0.09 4.98f0.24*
MTX (50 mgikg body wt.)
24 48 72
6 6
5
29.0 I f4.441 105.85 t 8.74* 102.32t9.72*
235.80f39.65t 137.16+20.38* 28.85 i.7.94*
6.68i.0.22t 8.6 I +0.38* IO.I2f0.51*
31.1 f 1.4t 34.0 f I .6* 37. I f I .9*
586.0 f 4 5 . l 641.9i.31. I * 818.6f45.l*
3.33 i.0.08t 4.16 +0.19* 5.08 f0. I2*
8
6.74 f0.6 I
25.34f3.34
5.46f0.31
23.7 t I .2
488.0+ 37.3
2.95 i.0.09
Normal (Go,just after PH)
Effect of methotrexate on liver regeneration
synthase activity of 24 h-regenerating liver. In a similar manner, thymidine kinase activity was not affected at 24 h after PH by the injection of MTX at the doses employed. At 48 h after PH, the thymidylate synthase activity was similar to that at 24 h after PH in the control group. In the MTX ( 5 mg/kg body weight)-treated group, the thymidylate synthase activity at 48 h after PH was increased by 3.3 times compared with 24 h-regenerating liver. This activity was significantly higher than that of the corresponding control. The injection of MTX (25 or 50 mg/kg body weight) also markedly increased the thymidylate synthase activity, as shown in Table 1. On the other hand, thymidine kinase activity in the MTX (5 mg/ kg)-injected group was decreased to 55% of the corresponding control value at 48 h after PH. When MTX was administered at doses of 25 and 50 mg/kg, the extent of the reduction in thymidine kinase activity was similar to that of the group given MTX at a dose of 5 mg/kg body weight. At 72 h after PH, the thymidylate synthase activity in the MTX-treated group (5 mg/kg body weight) was increased by 2.8 times compared with the control. The injections of 25 and 50 mg of MTX/kg body weight produced increases of 3.3 and 2.8 times compared with control, respectively. However, the thymidine kinase activity in 72 h-regenerating liver was decreased to the normal (Gostate, just after PH) level, which was about 20% of the control, on treatment with 5, 25 or 50 mg of MTX/kg body weight. Treatment with MTX had no effect on the DNA content in 24 h-regenerating liver, as shown in Table 1. The DNA contents of MTX-treated 48 h- and 72 hregenerating liver were approximately 80% and 70% of the corresponding control values, respectively. These results showed that the DNA content was significantly decreased by injection of MTX in 48 h- and 72 hregenerating liver. In analogy with the DNA content, the RNA content was also affected at 48 h and 72 h after PH in all MTX-treated groups. Gross parameters, such as protein content and liver weight, were not influenced significantly by the injection of 5 mg of MTX/kg body weight. When MTX was administered at a dose of 25 mg/ Table 2. Effect of MTX on the thymidylate synthase in 48 hregenerating liver. MTX (5, 25 o r 50 mglkg body weight) was administered as described in the Materials and methods section. A t 48 h after PH, the liver was homogenized and centrifuged at 36 000 g for 30 min. The supernatant fraction was used t o determine the activity and the content of thymidylate synthase (TS) as described in the Materials and methods section. Values are expressed as meansksm o f four t o six experiments. Treatment
Control MTX (5 mglkg body wt.) M T X (25 mglkg body wt.) MTX (50 mglkg body wt.)
TS activity (pmol min-' mg-' of protein)
TS content (ng140 pg of protein in the supernatant)
53k1.5
4.0 k 0.98
I30 t 5.7
16.5 k I .29
l 3 8 k 3.7 98k4.9
19.6k1.18 25.0 k 2.57
I83
kg body weight significant decreases in protein content and liver weight appeared at 72 h after PH. With an increase in the dose to 50 mg/kg body weight, the reductions in protein content and liver weight were significant at 48 h and 72 h after PH. These results indicated that MTX inhibited the regeneration of the liver after PH, but that the effects of MTX on liver regeneration were not observed at 24 h after PH, except for the inhibition of dihydrofolate reductase. Differential responses of the two thymidylate-synthesizing enzymes to MTX were demonstrated. Thymidylate synthase activity was remarkably increased, whereas thymidine kinase activity was suppressed to normal. Thymidine kinase was not directly inhibited by MTX at 1 mmol/l in our assay system in vitro.
Effect of MTX on the thymidylate synthase content To understand the mechanism of stimulation of thymidylate synthase, the enzyme protein content was determined by immunoblotting as previously reported [16].The results are shown in Table 2. In previous studies [16, 18, 201, the activity of thymidylate synthase correlated very closely with the enzyme content. When thymidylate synthase activity was increased to 2.45 times the control level by injection of MTX (5 mg/kg body weight), the enzyme protein content was found to be 4.13 times that of control. The administration of MTX at 25 mg/kg and 50 mg/kg body weight raised the thymidylate synthase content to 4.9 and 6.3 times that of control, respectively, without any increment in the activity. These results clearly demonstrate that MTX stimulates the synthesis of thymidylate synthase protein in a dosedependent manner and at the same time MTX inhibits the enzyme, resulting in the discrepancies between the enzyme content and the activity of the enzyme. MTX is an inhibitor of thymidylate synthase, as we reported previously [28]. In our assay system, MTX inhibited thymidylate synthase activity by 40%, 60% and 90% at concentrations of 0.01, 0.1 and 1 mmol/l, respectively. This result suggests that the decrease in thymidylate synthase activity in MTX-treated rats could be explained by direct inhibition of the enzyme by MTX contained in the liver.
DISCUSSION The results of this study clearly demonstrate that MTX stimulates the synthesis of thymidylate synthase protein in regenerating liver in a dose-dependent manner, as shown in Table 2. This is the first evidence of the increase in the synthesis of thymidylate synthase protein as well as the inhibition of induction of thymidine kinase by injection of MTX, although Labow et al. [8] have reported the stimulation of thymidylate synthase activity at 48 h after PH. It was found that MTX completely inhibits dihydrofolate reductase activity within 24 h of injection even at a dose of 5 mg/kg body weight. However, the effects of MTX on the rate-determining enzymes of DNA synthesis
I. Tsukamoto and S. Kojo
I84
(thymidylate synthase and thymidine kinase), the contents of DNA, RNA and protein, and liver weight were not seen at 24 h. This result indicates that the reservoir of tetrahydrofolate derivatives in the liver remained sufficient, at least for 24 h after PH, to support the regenerative responses. At 48 h after PH, thymidine kinase activity was depressed to about 60% of control and the DNA content was also decreased. This suggests that thymidylate deprivation caused by the inhibition of both thymidine kinase and thymidylate synthase was responsible for the depression of the increase in DNA content. Although the activity of thymidylate synthase measured itz vitro increased after 48 h of PH in MTX-treated animals, the enzyme may not be functional in the liver, where the storage of the cofactors, tetrahydrofolates, was depleted. The depletion of tetrahydrofolate cofactors may result in the suppression of purine nucleotide synthesis and a subsequent decrease in RNA synthesis, as shown in Table 1.The depression of RNA synthesis appears to cause the decrease in thymidine kinase activity, since the induction of thymidine kinase was inhibited by actinomycin D [20]. On the other hand, the induction of thymidylate synthase was enhanced because it is insensitive to the inhibition of RNA synthesis, as previously reported [20]. In contrast with the report by Mdler et al. [29], thymidine kinase was suppressed to the normal level in 72 h-generating liver of MTX-injected rats. It has been reported that DNA synthesis in nonparenchymal cells such as endothelial, Kupffer and bile ductule cells, occurred 1 day later than in hepatocytes during liver regeneration [30]. Therefore, it may be argued that the delayed effect of MTX is caused partly by the influence of the drug on non-parenchymal cells.
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