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The differential effects of three forms of interferon alfa on hepatic regeneration after partial hepatectomy in the rat
The Differential Effects of Three Forms of Interferon Alfa on Hepatic Regeneration After Partial Hepatectomy in the Rat S T E P H E N W O N G , TONY GAUTHIER, KELLY D. E . KAITA, AND GERALD Y. M I N U K
tive activity, few are considered essential. One such class of a g e n t s is t h e polyamines, which include putrescine a n d its metabolites s p e r m i d i n e a n d spermine. 2 In t h e absence of t h e s e p o l y v a l e n t cations h e p a t o c y t e reg e n e r a t i o n will not occur. 3 P o l y a m i n e s y n t h e s i s in t h e liver is r e g u l a t e d b y a r a t e - l i m i t i n g enzyme, o r n i t h i n e decarboxylase (ODC), which also serves as a biological m a r k e r of h e p a t i c r e g e n e r a t i v e activity. 4 Recently, it h a s b e e n r e p o r t e d t h a t I n t e r f e r o n (IFN) inhibits ODC activity in t h e liver. 5'6 This effect raises concerns t h a t I F N m i g h t i m p a i r or d e l a y h e p a t i c recovery from viral-induced injury. Indeed, I F N t h e r a p y is r a r e l y advocated in p a t i e n t s w i t h viral h e p a t i t i s a n d a d v a n c e d liver disease w h e r e h e p a t i c r e g e n e r a t i o n is vital to the m a i n t e n a n c e of h e p a t i c function. 7 A l t e r n a tively, I F N inhibition of ODC activity could theoretically be beneficial as i n c r e a s e d ODC activity h a s b e e n described in h e p a t o c e l l u l a r c a r c i n o m a tissue, s P r e s e n t l y , t h e r e are t h r e e f o r m u l a t i o n s of I F N alfa (IFN a) t h a t are e i t h e r commercially available or undergoing p h a s e III trials, two r e c o m b i n a n t forms (IFN a - 2 a a n d I F N a-2b) a n d one l y m p h o b l a s t o i d form (IFN a-n1). T h e p u r p o s e of the p r e s e n t s t u d y was to determ i n e w h e t h e r each of t h e s e a g e n t s i n t e r f e r e s w i t h hepatic r e g e n e r a t i o n to the same e x t e n t a n d w h e t h e r the i n h i b i t o r y effects of I F N can be p r e v e n t e d by the coadm i n i s t r a t i o n of exogenous putrescine.
The p u r p o s e o f this s t u d y w a s to d e t e r m i n e w h e t h e r all c o m m e r c i a l l y available f o r m s o f i n t e r f e r o n alfa (IFN ~) h a v e t h e s a m e i n h i b i t o r y effect o n h e p a t i c regeneration a n d w h e t h e r this i n h i b i t o r y effect c a n be p r e v e n t e d by putrescine, a h e p a t i c g r o w t h p r o m o t o r . Adult m a l e S p r a g u e - D a w l e y rats (n = 92) r e c e i v e d either IFN ~-2a, 2b, n l , or saline, 0 a n d 8 h o u r s after partial h e p a t e c t o m y (PHx) or 16 h o u r s before PHx. A s u b g r o u p o f 29 rats b e i n g t r e a t e d w i t h I F N ~-2a or saline also r e c e i v e d put r e s c i n e (5, 50, or 500 mg/kg) 16 h o u r s before PHx. Hepatic r e g e n e r a t i o n w a s d o c u m e n t e d by d e t e r m i n i n g [SH]-thymidine i n c o r p o r a t i o n into h e p a t i c D N A (DNA synthesis), h e p a t i c p u t r e s c i n e levels, and, in s e l e c t e d cases, o r n i t h i n e d e c a r b o x y l a s e (ODC) activity at 24 h o u r s after PHx. The results o f t h e s t u d y s h o w e d that h e p a t i c r e g e n e r a t i o n w a s u n a f f e c t e d w h e n IFN ~ w a s a d m i n i s t e r e d 0 a n d 8 h o u r s after PHx. When administered at - 1 6 hours, o n l y IFN a-2a significantly inhibited D N A s y n t h e s i s a n d w a s a s s o c i a t e d w i t h d e c r e a s e d hepatic p u t r e s c i n e levels. I n h i b i t i o n w a s d o s e - d e p e n d e n t in that a 10-fold i n c r e a s e in IFN ~-2a c a u s e d a further d e c r e a s e in b o t h D N A s y n t h e s i s a n d h e p a t i c p u t r e s c i n e levels. At t h e h i g h e r dose, IFN ~-2b a n d n l also inhibited D N A s y n t h e s i s a n d l o w e r e d h e p a t i c p u t r e s c i n e levels a n d ODC activity. E x o g e n o u s p u t r e s c i n e (5 a n d 50 mg/ kg) r e s t o r e d h e p a t i c r e g e n e r a t i v e activity to n o r m a l but w a s toxic at h i g h c o n c e n t r a t i o n s (500 mg/kg). T h e s e data i n d i c a t e that n o t all c o m m e r c i a l l y available f o r m s o f IFN inhibit h e p a t i c r e g e n e r a t i o n in t h e rat to the s a m e extent. (HEPATOLOGY1995;21:883-886.)
MATERIALS A N D M E T H O D S
H e p a t i c r e g e n e r a t i o n is a n i m p o r t a n t c o m p o n e n t of the recovery process t h a t occurs a f t e r various forms of h e p a t i c i n j u r y including viral hepatitis. 1 O f t h e different a g e n t s t h a t are k n o w n to p r o m o t e h e p a t i c r e g e n e r a -
Abbreviations: ODC, ornithine decarboxylase; IFN-a, interferon alfa; PHx, partial hepatectomy; EDTA, ethylenediaminetetraacetic acid. From the Liver Diseases Unit, Depa~ments of Medicine and Pharmacology, University of Manitoba, and Health Sciences Clinical Research Centre, Winnipeg, Manitoba, Canada. Received November 17, 1994; accepted April 14, 1995. Supported by the Medical Research Council of Canada and Roche Canada. Dr Kaita is the recipient of a Roche Canada/University of Manitoba Hepatology Fellowship Award. Address reprint requests to: G. Y. Minuk, MD, FRCP(C), Liver Diseases Unit, Health Sciences Centre, 820 Sherbrook St, Winnipeg, Manitoba, RSA 1R9, Canada. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2203-002753.00/0
Adult male Sprague-Dawley rats weighing 250 to 300 g were maintained on standard rat chow and water ad libitum until the day before surgery. The surgery consisted of either a 70% partial hepatectomy (PHx) as described by Higgins and Anderson 9 or a sham operation in which the liver was exteriorized and manipulated to the same extent as for a PHx before being returned into the abdominal cavity. Treatments with physiological saline, IFN, and/or putrescine were by intraperitoneal injection at the times specified. Unless otherwise stated, each administered dose of IFN was 600 IU per gram of body weight, which approximates the rat equivalent dosage of 5 MU in a 60-kg human. 1° Four series of experiments were performed. In the first series, rats received either saline (n = 11), IFN a-2a (n = 7), or IFN a-n1 (n = 7) at arbitrarily selected times 0 and 8 hours after PHx. In series 2, rats received either saline (n = 5), IFN a-2a (n = 5), IFN a-2b (n = 5), or IFN a-n1 (n = 6) at - 1 6 hours before PHx. In series 3 experiments, 10 times the dose of IFN used in series 1 and 2 (6,000 IU per gram of body weight) was used in all study groups (n = 4 to
883
884
WONG ET AL
HEPATOLOGYSeptember 1995
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FIG. 1. DNA synthesis rates at 24 hours after PHx in rats treated at -16 hours with 3 forms of IFN a (600 IU/g). *Comparisons with saline, IFN ~-2b- and IFN a-hi-treated groups.
8). The schedule of drug administration was identical to that of series 2. Series 4 experiments included rats treated at - 1 6 hours before PHx (as in series 2 and 3) with either physiological saline (n = 5), IFN a-2a (600 IU per gram of body weight) + saline (n = 6), or IFN a-2a (600 IU per gram of body weight) + putrescine, at doses of 5 mg/kg (n = 6), 50 mg/kg (n = 6), or 500 mg/kg (n = 6). Hepatic Regeneration. Hepatic regenerative activity was documented by determining the rates of DNA synthesis, hepatic putrescine levels, and, in selected cases, ODC activity, at 24 hours after PHx. DNA Synthesis. Briefly, [3H]-thymidine was administered by intraperitonea] injection i hour before killing by cardiac puncture. Immediately after killing, the livers were excised, weighed, and homogenized in 2.5 mmol/L of ethylenediaminetetraacetic acid (EDTA) buffer, pH 7.4. [3H]-thymidine radioactivity was determined in pellets of homogenates that were precipitated with 5% trichloroacetic acid and read in a Beckman LS 9800 liquid scintillation counter (Beckman Instruments, Palo Alto, CA). DNA content was measured by reaction with 3,5-diaminobenzoic acid at 37°C. ~ Putrescine Determinations. At the time of killing, livers were excised and homogenized 1:9 (wt/vol) in a 100 mmol/L sodium phosphate buffer containing 5 mmol/L of dithiothreitol. 2 Homogenates were deproteinized with the addition of sulfasalicyclic acid in the presence of an internal standard (1,6-hexanediamine) and centrifuged at 3,000g for 15 minutes. The supernatants were adjusted to a pH of 2.2 with 0.4 mol/L of NaOH before analysis. Levels of putrescine and its metabolites spermidine and spermine were obtained with an Alpha Plus Amino Acid analyzer (LKB Biochrome Ltd., Cambridge, England) equipped with an 80 mm × 40 mm stainless steel column packed with Ultropac 8 (8 _+ 1 urn) cation resin and an LKB 4460 fluorescence detector, with orthophthalaldehyde as the fluorescence reagent. Amino acid elution was performed first with buffer i (1.2 mol/L of Na+ citrate, pH 6.45) for 6 minutes at 58°C, which was followed by buffer 2 (Na+/K+ citrate mixture containing 0.56 mol/L of Na+ and 1.6 mol/L of K+, pH 5.60) for 12 minutes at 58°C. Elution with buffer 2 was continued for another 11 minutes, and the temperature was raised to 90°C. Regeneration and equilibration of the column were accomplished by elution with buffer 3 (0.40 mol/L of NaOH) for 3 minutes at 90°C, followed by elution with buffer 1 for 4 minutes. The temperature was
lowered to 58°C, and elution with buffer I was performed for 15 minutes. Buffer flow was 35 mL/hr; reagent flow was 17 mL/hr. Automatic integration was performed with a Nelson 900 series integrator (Nelson Analytical, Inc., Cupertino, CA). In previous studies we documented that hepatic putrescine levels correlate with hepatic regenerative activity. 2 ODC Aetivity. To further support DNA synthesis and hepatic putrescine data and to confirm that decreases in hepatic putrescine levels result from inhibition of putrescine synthesis, ODC activity was measured in the liver homogenates of series 3 and 4 rats by quantitation of 14CO2liberated from 14Clabeled ornithine, as described by Luk. 3 The reaction mixture contained 0.2 mL of liver homogenate and 50 #L of pyridoxal phosphate (final concentration 0.8 retool/L) in a total volume of 0.2 mL of buffer containing 5 mmol/L of Na2HPQ, 5 mmol/ L of HaH2PO4, 0.1 mmol/L of EDTA, and 2 mmo]/L of dithiothreitol (pH 7.4). The reaction was started with the addition of 10 #L of ornithine solution (2.5 mmol/L of cold ornithine and 25 uCi of [14C]-ornithine [Du Pont-New England Nuclear, Boston, MA]). After a 2-hour incubation at 37°C, the reaction was stopped with 0.5 mL of 50% trichloroacetic acid. The ~4CO2 liberated by the decarboxylation of ornithine was trapped on glass microfilters (Whatman International Ltd., Maidstone, England) impregnated with 200 ttl of hyamine hydroxide, which was suspended in a center well above the reaction mixture. The 14CO2 trapped in the filter paper was measured in a liquid scintillation counter. Protein contents of liver homogenates were assayed according to the method of Hartree 11 with bovine serum albumine (BSA) as the standard. Statistics. Two-way ANOVAs, Student's t-tests, and Wilcoxon's rank sum tests for unpaired data were used in statistical calculations. P values -< .05 were considered significant. The results provided represent the mean _+ SD. RESULTS W h e n I F N w a s a d m i n i s t e r e d at 0 a n d 8 h o u r s after P H x (series 1 e x p e r i m e n t s ) D N A s y n t h e s i s r a t e s were similar in saline (27.9 _+ 11.8 d i s i n t e g r a t i o n s per minute [DPM]/#g D N A ) - , I F N a - 2 a (33.9 +_ 17.0 DPM/ # g D N A ) - , a n d I F N a - n l (22.4 _+ 9.7 D P M / # g D N A ) t r e a t e d rats. As s h o w n in Fig. 1, w h e n I F N was a d m i n i s t e r e d 16
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FIG. 2. Hepatic putrescine levels at 24 hours after PHx in rats treated at - 16 hours with 3 forms of IFN a (600 IU/g). Comparisons with saline, IFN a-2b- and IFN a-nl-treated groups.
HEPATOLOGY Vol. 22, No. 3, 1995
WONG ET AL
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FIG. 3. DNA synthesis rates at 24 hours after PHx in rats treated at - 1 6 hours with 3 forms of IFN a (6,000 IU/g).
STUDY
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FIG. 5. Hepatic ODC activity at 24 hours after PHx in rats treated at - 1 6 hours with 3 forms of IFN a (6,000 IU/g).
hours before PHx, DNA synthesis rates when compared with saline-treated rats were similar in IFN a - 2 b - and IFN ~ - n l - t r e a t e d rats but significantly decreased in the IFN a - 2 a - t r e a t e d group (P < .05). Similar results were obtained with hepatic putrescine levels (Fig. 2). A 10-fold increase in the dose of IFN resulted in more consistent decreases in DNA synthesis rates in IFN a2 a - t r e a t e d rats compared with saline-treated controls (P < .005; Fig. 3). DNA synthesis rates were now also decreased (P < .05) in rats treated with the higher dose of IFN ~-2b and IFN a-n1 (Fig. 3). Hepatic putrescine levels and ODC activity were significantly lower in all IFN-treated groups compared with saline-treated controls (Figs. 4 and 5). The results of putrescine supplementation in IFN a2 a - t r e a t e d rats are shown in Figs. 6 and 7. At both 5 and 50 mg/kg of exogenous putrescine, DNA synthesis rates (Fig. 6) and hepatic putrescine levels (Fig. 7) in IFN a - 2 a - t r e a t e d rats were restored to saline-treated control values. Because four of six rats treated with 500 mg/kg of putrescine died before killing, the results of this group were not included in the analyses.
Total hepatic DNA was similar in all study groups throughout series i to 4 experiments (data not shown). DISCUSSION
The results of this study show that not all forms of IFN a inhibit hepatic regenerative activity to the same extent. IFN a-2a appears to be more potent in that regard, whereas IFN a-2b and IFN a-n1 only manifest an inhibitory effect at dose equivalents that are higher than what is generally recommended for patients with chronic viral hepatitis. ~2 The reason why IFN a-2a inhibits hepatic regeneration more so than the other forms of IFN a tested, particularly IFN a-2b, is unclear. All three forms of IFN consist of 166 amino acids in the mature protein. Of these 166 amino acids, IFN a-2a and IFN a-2b differ from each other by only 1 amino acid with IFN a-2a having a lysine molecule and IFN a-2b having an arginine molecule at position 23 instead of glycine, the amino acid in the natural sequence. How one amino
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FIG. 4. Hepatic putrescine levels at 24 hours after PHx in rats treated at - 1 6 hours with 3 forms of IFN a (6,000 IU/g).
FIG. 6. DNA synthesis rates at 24 hours after PHx in rats treated at - 1 6 hours with either saline or IFN a-2a (600 IU/g) and 5 or 50 mg/kg of putrescine. *Comparison with saline-treated control group.
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WONG ET AL
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hepatic regeneration to the same extent in this animal model of regeneration.
Acknowledgment: We thank D. Byron for her prompt and accurate typing of the manuscript. REFERENCES
Saline * p < 0.05
IFN A-2a+Sal
IFN A-2a+Put:5 IFN A-2a+Put:50
STUDY GROUPS
FIG. 7. Hepatic putrescine levels at 24 hours after PHx in rats treated at -16 hours with either saline or IFN a-2a (600 IU/g) and 5 or 50 mg/kg of putrescine. *Comparisons with saline-, putrescine 5 mg/kg-, and putrescine 50 mg/kg-treated groups.
acid substitution might alter the rate of hepatic regeneration has yet to be determined. However, it is apparent that this substitution is physiologically relevant in that the incidence of neutralizing antibodies developing to IFN a-2a is three times more common than with IFN a-2b. 13 Moreover, Lok et a114 have reported that less myelosuppression occurs in IFN a-2a-treated patients with high titers of these antibodies than in those without such antibodies. That the inhibitory effect of IFN a-2a was associated with low hepatic putrescine levels, had decreased ODC activity, and could be reversed by exogenous putrescine, suggest that IFN-induced suppression of hepatic ODC, the enzyme responsible for putrescine synthesis, may be the mechanism involved. Similar findings have been reported by other groups. 6'15 The reason why IFN a-2a treatment at times 0 and 8 hours after PHx had no effect on hepatic regeneration while treatment at - 1 6 hours significantly inhibited regenerative activity was not delineated. However, previous studies have documented that the absorption of compounds from the peritoneal cavity in the presence of hepatic dysfunction can be significantly delayed. 1~ Our findings may reflect that phenomenon. The fact that signs of hepatic regenerative activity appear within minutes to hours after PHx further supports the possibility that delayed absorption kinetics from the peritoneal cavity may be relevant. 17 In conclusion, the results of this study show that not all commercially available forms of IFN a inhibit
1. Bucher NLR. Regeneration of mammalian liver. In: Bourne GH, Danielli JF, eds. International review of cytology. New York: Academic, 1963:245-300. 2. Minuk GY, Gauthier T, Benarroch A. Changes in serum and hepatic polyamine concentrations after 30%, 70% and 90% partial hepatectomy in rats. HEPATOLOGY1990; 12:542-546. 3. Luk GD. Essential role of polyamine metabolism in hepatic regeneration. Gastroenterology 1986;90:1262-1267. 4. Francavilla A, Panella C, Polimeno L, Giangaspero A, Mazzaferro V, Pan C, Van Thiel DH, et al. Hormonal and enzymatic parameters of hepatic regeneration in patients undergoing major liver resections. HEPATOLOGY1990; 12:1134-1138. 5. Nishiguchi S, Otani S, Matsui-Yuasa I, Morisawa S, Monna T, Kuroki T, Kobayashi K. Inhibition of ornithine decarboxylase induction of interferon (alpha + beta) and its reversal by didutyryl adenosine 3,5-monophosphate. Eur J Biochem 1988;172: 287-292. 6. Marth C, Kirchebner P, Daxenbichler G. The role of polyamines in interferon and retinoic acid mediated synergistic antiproliferative action. Cancer Letters 1989;44:55-59. 7. Shindo M, di Bisceglie AM, Hoofnagle JH. Acute exacerbation of liver disease during interferon alpha therapy for chronic hepatitis C. Gastroenterology 1992; 102:1406-1408. 8. Janne J, Poso H, Raina A. Polyamines in rapid growth in cancer. Biol Chimica et Biol Physica Acta 1978;473:241-293. 9. Higgins GM, Anderson RM. Experimental pathology of the liver of the white rat following partial surgical removal. Arch Pathol 1931; 12:186-202. 10. Cancer drug development: cancer. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Principles and practice of oncology. Philadelphia, PA: Lippincott; 1987:270-300. 11. Hartree EF. Determination of protein content: a modification of the Lowry method that gives a linear photometric response. Anal Biochem 1972;48:422-427. 12. Peters M. Mechanisms of action ofinterferons, Semin Liver Dis 1989;9:235-239. 13. Antonelli G, Currenti M, Turriziani O, Dianzani F. Neutralizing antibodies to interferon alpha: relative frequency in patients treated with different interferon preparations. J Infect Dis 1991; 163:882-885. 14. Lok ASF, Lai CL, Leung EKY. Interferon antibodies may negate the antiviral effects of recombinant a-interferon treatment in patients with chronic hepatitis B virus infection. HEPATOLOGY 1990; 12:1266-1270. 15. Takeda T, Nishiguchi S, Nakajima S, Shiomi S, Kuroki T, Kobayashi K, Otani S. Growth inhibition by interferon on a human hepatoma cell line mediated by inhibitionof putrescine synthesis [Abstract]. HEPATOLOGY1992; 15:A897. 16. Scharl A, Kullander S, Beckmann MW, Spicer JA, Baranczuk RJ, Holt JA. Prolonged clearance of intraperitoneal 16 alpha (125I)iodo-17 beta-estradiol in presence of ascites. Am J Obstet Gynecol 1991;165:1847-1853. 17. Michalopoulos GK. Liver regeneration: molecular mechanisms of growth control. FASEB J 1990;4:176-187.
tive activity, few are considered essential. One such class of a g e n t s is t h e polyamines, which include putrescine a n d its metabolites s p e r m i d i n e a n d spermine. 2 In t h e absence of t h e s e p o l y v a l e n t cations h e p a t o c y t e reg e n e r a t i o n will not occur. 3 P o l y a m i n e s y n t h e s i s in t h e liver is r e g u l a t e d b y a r a t e - l i m i t i n g enzyme, o r n i t h i n e decarboxylase (ODC), which also serves as a biological m a r k e r of h e p a t i c r e g e n e r a t i v e activity. 4 Recently, it h a s b e e n r e p o r t e d t h a t I n t e r f e r o n (IFN) inhibits ODC activity in t h e liver. 5'6 This effect raises concerns t h a t I F N m i g h t i m p a i r or d e l a y h e p a t i c recovery from viral-induced injury. Indeed, I F N t h e r a p y is r a r e l y advocated in p a t i e n t s w i t h viral h e p a t i t i s a n d a d v a n c e d liver disease w h e r e h e p a t i c r e g e n e r a t i o n is vital to the m a i n t e n a n c e of h e p a t i c function. 7 A l t e r n a tively, I F N inhibition of ODC activity could theoretically be beneficial as i n c r e a s e d ODC activity h a s b e e n described in h e p a t o c e l l u l a r c a r c i n o m a tissue, s P r e s e n t l y , t h e r e are t h r e e f o r m u l a t i o n s of I F N alfa (IFN a) t h a t are e i t h e r commercially available or undergoing p h a s e III trials, two r e c o m b i n a n t forms (IFN a - 2 a a n d I F N a-2b) a n d one l y m p h o b l a s t o i d form (IFN a-n1). T h e p u r p o s e of the p r e s e n t s t u d y was to determ i n e w h e t h e r each of t h e s e a g e n t s i n t e r f e r e s w i t h hepatic r e g e n e r a t i o n to the same e x t e n t a n d w h e t h e r the i n h i b i t o r y effects of I F N can be p r e v e n t e d by the coadm i n i s t r a t i o n of exogenous putrescine.
The p u r p o s e o f this s t u d y w a s to d e t e r m i n e w h e t h e r all c o m m e r c i a l l y available f o r m s o f i n t e r f e r o n alfa (IFN ~) h a v e t h e s a m e i n h i b i t o r y effect o n h e p a t i c regeneration a n d w h e t h e r this i n h i b i t o r y effect c a n be p r e v e n t e d by putrescine, a h e p a t i c g r o w t h p r o m o t o r . Adult m a l e S p r a g u e - D a w l e y rats (n = 92) r e c e i v e d either IFN ~-2a, 2b, n l , or saline, 0 a n d 8 h o u r s after partial h e p a t e c t o m y (PHx) or 16 h o u r s before PHx. A s u b g r o u p o f 29 rats b e i n g t r e a t e d w i t h I F N ~-2a or saline also r e c e i v e d put r e s c i n e (5, 50, or 500 mg/kg) 16 h o u r s before PHx. Hepatic r e g e n e r a t i o n w a s d o c u m e n t e d by d e t e r m i n i n g [SH]-thymidine i n c o r p o r a t i o n into h e p a t i c D N A (DNA synthesis), h e p a t i c p u t r e s c i n e levels, and, in s e l e c t e d cases, o r n i t h i n e d e c a r b o x y l a s e (ODC) activity at 24 h o u r s after PHx. The results o f t h e s t u d y s h o w e d that h e p a t i c r e g e n e r a t i o n w a s u n a f f e c t e d w h e n IFN ~ w a s a d m i n i s t e r e d 0 a n d 8 h o u r s after PHx. When administered at - 1 6 hours, o n l y IFN a-2a significantly inhibited D N A s y n t h e s i s a n d w a s a s s o c i a t e d w i t h d e c r e a s e d hepatic p u t r e s c i n e levels. I n h i b i t i o n w a s d o s e - d e p e n d e n t in that a 10-fold i n c r e a s e in IFN ~-2a c a u s e d a further d e c r e a s e in b o t h D N A s y n t h e s i s a n d h e p a t i c p u t r e s c i n e levels. At t h e h i g h e r dose, IFN ~-2b a n d n l also inhibited D N A s y n t h e s i s a n d l o w e r e d h e p a t i c p u t r e s c i n e levels a n d ODC activity. E x o g e n o u s p u t r e s c i n e (5 a n d 50 mg/ kg) r e s t o r e d h e p a t i c r e g e n e r a t i v e activity to n o r m a l but w a s toxic at h i g h c o n c e n t r a t i o n s (500 mg/kg). T h e s e data i n d i c a t e that n o t all c o m m e r c i a l l y available f o r m s o f IFN inhibit h e p a t i c r e g e n e r a t i o n in t h e rat to the s a m e extent. (HEPATOLOGY1995;21:883-886.)
MATERIALS A N D M E T H O D S
H e p a t i c r e g e n e r a t i o n is a n i m p o r t a n t c o m p o n e n t of the recovery process t h a t occurs a f t e r various forms of h e p a t i c i n j u r y including viral hepatitis. 1 O f t h e different a g e n t s t h a t are k n o w n to p r o m o t e h e p a t i c r e g e n e r a -
Abbreviations: ODC, ornithine decarboxylase; IFN-a, interferon alfa; PHx, partial hepatectomy; EDTA, ethylenediaminetetraacetic acid. From the Liver Diseases Unit, Depa~ments of Medicine and Pharmacology, University of Manitoba, and Health Sciences Clinical Research Centre, Winnipeg, Manitoba, Canada. Received November 17, 1994; accepted April 14, 1995. Supported by the Medical Research Council of Canada and Roche Canada. Dr Kaita is the recipient of a Roche Canada/University of Manitoba Hepatology Fellowship Award. Address reprint requests to: G. Y. Minuk, MD, FRCP(C), Liver Diseases Unit, Health Sciences Centre, 820 Sherbrook St, Winnipeg, Manitoba, RSA 1R9, Canada. Copyright © 1995 by the American Association for the Study of Liver Diseases. 0270-9139/95/2203-002753.00/0
Adult male Sprague-Dawley rats weighing 250 to 300 g were maintained on standard rat chow and water ad libitum until the day before surgery. The surgery consisted of either a 70% partial hepatectomy (PHx) as described by Higgins and Anderson 9 or a sham operation in which the liver was exteriorized and manipulated to the same extent as for a PHx before being returned into the abdominal cavity. Treatments with physiological saline, IFN, and/or putrescine were by intraperitoneal injection at the times specified. Unless otherwise stated, each administered dose of IFN was 600 IU per gram of body weight, which approximates the rat equivalent dosage of 5 MU in a 60-kg human. 1° Four series of experiments were performed. In the first series, rats received either saline (n = 11), IFN a-2a (n = 7), or IFN a-n1 (n = 7) at arbitrarily selected times 0 and 8 hours after PHx. In series 2, rats received either saline (n = 5), IFN a-2a (n = 5), IFN a-2b (n = 5), or IFN a-n1 (n = 6) at - 1 6 hours before PHx. In series 3 experiments, 10 times the dose of IFN used in series 1 and 2 (6,000 IU per gram of body weight) was used in all study groups (n = 4 to
883
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WONG ET AL
HEPATOLOGYSeptember 1995
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FIG. 1. DNA synthesis rates at 24 hours after PHx in rats treated at -16 hours with 3 forms of IFN a (600 IU/g). *Comparisons with saline, IFN ~-2b- and IFN a-hi-treated groups.
8). The schedule of drug administration was identical to that of series 2. Series 4 experiments included rats treated at - 1 6 hours before PHx (as in series 2 and 3) with either physiological saline (n = 5), IFN a-2a (600 IU per gram of body weight) + saline (n = 6), or IFN a-2a (600 IU per gram of body weight) + putrescine, at doses of 5 mg/kg (n = 6), 50 mg/kg (n = 6), or 500 mg/kg (n = 6). Hepatic Regeneration. Hepatic regenerative activity was documented by determining the rates of DNA synthesis, hepatic putrescine levels, and, in selected cases, ODC activity, at 24 hours after PHx. DNA Synthesis. Briefly, [3H]-thymidine was administered by intraperitonea] injection i hour before killing by cardiac puncture. Immediately after killing, the livers were excised, weighed, and homogenized in 2.5 mmol/L of ethylenediaminetetraacetic acid (EDTA) buffer, pH 7.4. [3H]-thymidine radioactivity was determined in pellets of homogenates that were precipitated with 5% trichloroacetic acid and read in a Beckman LS 9800 liquid scintillation counter (Beckman Instruments, Palo Alto, CA). DNA content was measured by reaction with 3,5-diaminobenzoic acid at 37°C. ~ Putrescine Determinations. At the time of killing, livers were excised and homogenized 1:9 (wt/vol) in a 100 mmol/L sodium phosphate buffer containing 5 mmol/L of dithiothreitol. 2 Homogenates were deproteinized with the addition of sulfasalicyclic acid in the presence of an internal standard (1,6-hexanediamine) and centrifuged at 3,000g for 15 minutes. The supernatants were adjusted to a pH of 2.2 with 0.4 mol/L of NaOH before analysis. Levels of putrescine and its metabolites spermidine and spermine were obtained with an Alpha Plus Amino Acid analyzer (LKB Biochrome Ltd., Cambridge, England) equipped with an 80 mm × 40 mm stainless steel column packed with Ultropac 8 (8 _+ 1 urn) cation resin and an LKB 4460 fluorescence detector, with orthophthalaldehyde as the fluorescence reagent. Amino acid elution was performed first with buffer i (1.2 mol/L of Na+ citrate, pH 6.45) for 6 minutes at 58°C, which was followed by buffer 2 (Na+/K+ citrate mixture containing 0.56 mol/L of Na+ and 1.6 mol/L of K+, pH 5.60) for 12 minutes at 58°C. Elution with buffer 2 was continued for another 11 minutes, and the temperature was raised to 90°C. Regeneration and equilibration of the column were accomplished by elution with buffer 3 (0.40 mol/L of NaOH) for 3 minutes at 90°C, followed by elution with buffer 1 for 4 minutes. The temperature was
lowered to 58°C, and elution with buffer I was performed for 15 minutes. Buffer flow was 35 mL/hr; reagent flow was 17 mL/hr. Automatic integration was performed with a Nelson 900 series integrator (Nelson Analytical, Inc., Cupertino, CA). In previous studies we documented that hepatic putrescine levels correlate with hepatic regenerative activity. 2 ODC Aetivity. To further support DNA synthesis and hepatic putrescine data and to confirm that decreases in hepatic putrescine levels result from inhibition of putrescine synthesis, ODC activity was measured in the liver homogenates of series 3 and 4 rats by quantitation of 14CO2liberated from 14Clabeled ornithine, as described by Luk. 3 The reaction mixture contained 0.2 mL of liver homogenate and 50 #L of pyridoxal phosphate (final concentration 0.8 retool/L) in a total volume of 0.2 mL of buffer containing 5 mmol/L of Na2HPQ, 5 mmol/ L of HaH2PO4, 0.1 mmol/L of EDTA, and 2 mmo]/L of dithiothreitol (pH 7.4). The reaction was started with the addition of 10 #L of ornithine solution (2.5 mmol/L of cold ornithine and 25 uCi of [14C]-ornithine [Du Pont-New England Nuclear, Boston, MA]). After a 2-hour incubation at 37°C, the reaction was stopped with 0.5 mL of 50% trichloroacetic acid. The ~4CO2 liberated by the decarboxylation of ornithine was trapped on glass microfilters (Whatman International Ltd., Maidstone, England) impregnated with 200 ttl of hyamine hydroxide, which was suspended in a center well above the reaction mixture. The 14CO2 trapped in the filter paper was measured in a liquid scintillation counter. Protein contents of liver homogenates were assayed according to the method of Hartree 11 with bovine serum albumine (BSA) as the standard. Statistics. Two-way ANOVAs, Student's t-tests, and Wilcoxon's rank sum tests for unpaired data were used in statistical calculations. P values -< .05 were considered significant. The results provided represent the mean _+ SD. RESULTS W h e n I F N w a s a d m i n i s t e r e d at 0 a n d 8 h o u r s after P H x (series 1 e x p e r i m e n t s ) D N A s y n t h e s i s r a t e s were similar in saline (27.9 _+ 11.8 d i s i n t e g r a t i o n s per minute [DPM]/#g D N A ) - , I F N a - 2 a (33.9 +_ 17.0 DPM/ # g D N A ) - , a n d I F N a - n l (22.4 _+ 9.7 D P M / # g D N A ) t r e a t e d rats. As s h o w n in Fig. 1, w h e n I F N was a d m i n i s t e r e d 16
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FIG. 2. Hepatic putrescine levels at 24 hours after PHx in rats treated at - 16 hours with 3 forms of IFN a (600 IU/g). Comparisons with saline, IFN a-2b- and IFN a-nl-treated groups.
HEPATOLOGY Vol. 22, No. 3, 1995
WONG ET AL
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STUDY
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FIG. 5. Hepatic ODC activity at 24 hours after PHx in rats treated at - 1 6 hours with 3 forms of IFN a (6,000 IU/g).
hours before PHx, DNA synthesis rates when compared with saline-treated rats were similar in IFN a - 2 b - and IFN ~ - n l - t r e a t e d rats but significantly decreased in the IFN a - 2 a - t r e a t e d group (P < .05). Similar results were obtained with hepatic putrescine levels (Fig. 2). A 10-fold increase in the dose of IFN resulted in more consistent decreases in DNA synthesis rates in IFN a2 a - t r e a t e d rats compared with saline-treated controls (P < .005; Fig. 3). DNA synthesis rates were now also decreased (P < .05) in rats treated with the higher dose of IFN ~-2b and IFN a-n1 (Fig. 3). Hepatic putrescine levels and ODC activity were significantly lower in all IFN-treated groups compared with saline-treated controls (Figs. 4 and 5). The results of putrescine supplementation in IFN a2 a - t r e a t e d rats are shown in Figs. 6 and 7. At both 5 and 50 mg/kg of exogenous putrescine, DNA synthesis rates (Fig. 6) and hepatic putrescine levels (Fig. 7) in IFN a - 2 a - t r e a t e d rats were restored to saline-treated control values. Because four of six rats treated with 500 mg/kg of putrescine died before killing, the results of this group were not included in the analyses.
Total hepatic DNA was similar in all study groups throughout series i to 4 experiments (data not shown). DISCUSSION
The results of this study show that not all forms of IFN a inhibit hepatic regenerative activity to the same extent. IFN a-2a appears to be more potent in that regard, whereas IFN a-2b and IFN a-n1 only manifest an inhibitory effect at dose equivalents that are higher than what is generally recommended for patients with chronic viral hepatitis. ~2 The reason why IFN a-2a inhibits hepatic regeneration more so than the other forms of IFN a tested, particularly IFN a-2b, is unclear. All three forms of IFN consist of 166 amino acids in the mature protein. Of these 166 amino acids, IFN a-2a and IFN a-2b differ from each other by only 1 amino acid with IFN a-2a having a lysine molecule and IFN a-2b having an arginine molecule at position 23 instead of glycine, the amino acid in the natural sequence. How one amino
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FIG. 4. Hepatic putrescine levels at 24 hours after PHx in rats treated at - 1 6 hours with 3 forms of IFN a (6,000 IU/g).
FIG. 6. DNA synthesis rates at 24 hours after PHx in rats treated at - 1 6 hours with either saline or IFN a-2a (600 IU/g) and 5 or 50 mg/kg of putrescine. *Comparison with saline-treated control group.
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WONG ET AL
HEPATOLOGYSeptember 1995
hepatic regeneration to the same extent in this animal model of regeneration.
Acknowledgment: We thank D. Byron for her prompt and accurate typing of the manuscript. REFERENCES
Saline * p < 0.05
IFN A-2a+Sal
IFN A-2a+Put:5 IFN A-2a+Put:50
STUDY GROUPS
FIG. 7. Hepatic putrescine levels at 24 hours after PHx in rats treated at -16 hours with either saline or IFN a-2a (600 IU/g) and 5 or 50 mg/kg of putrescine. *Comparisons with saline-, putrescine 5 mg/kg-, and putrescine 50 mg/kg-treated groups.
acid substitution might alter the rate of hepatic regeneration has yet to be determined. However, it is apparent that this substitution is physiologically relevant in that the incidence of neutralizing antibodies developing to IFN a-2a is three times more common than with IFN a-2b. 13 Moreover, Lok et a114 have reported that less myelosuppression occurs in IFN a-2a-treated patients with high titers of these antibodies than in those without such antibodies. That the inhibitory effect of IFN a-2a was associated with low hepatic putrescine levels, had decreased ODC activity, and could be reversed by exogenous putrescine, suggest that IFN-induced suppression of hepatic ODC, the enzyme responsible for putrescine synthesis, may be the mechanism involved. Similar findings have been reported by other groups. 6'15 The reason why IFN a-2a treatment at times 0 and 8 hours after PHx had no effect on hepatic regeneration while treatment at - 1 6 hours significantly inhibited regenerative activity was not delineated. However, previous studies have documented that the absorption of compounds from the peritoneal cavity in the presence of hepatic dysfunction can be significantly delayed. 1~ Our findings may reflect that phenomenon. The fact that signs of hepatic regenerative activity appear within minutes to hours after PHx further supports the possibility that delayed absorption kinetics from the peritoneal cavity may be relevant. 17 In conclusion, the results of this study show that not all commercially available forms of IFN a inhibit
1. Bucher NLR. Regeneration of mammalian liver. In: Bourne GH, Danielli JF, eds. International review of cytology. New York: Academic, 1963:245-300. 2. Minuk GY, Gauthier T, Benarroch A. Changes in serum and hepatic polyamine concentrations after 30%, 70% and 90% partial hepatectomy in rats. HEPATOLOGY1990; 12:542-546. 3. Luk GD. Essential role of polyamine metabolism in hepatic regeneration. Gastroenterology 1986;90:1262-1267. 4. Francavilla A, Panella C, Polimeno L, Giangaspero A, Mazzaferro V, Pan C, Van Thiel DH, et al. Hormonal and enzymatic parameters of hepatic regeneration in patients undergoing major liver resections. HEPATOLOGY1990; 12:1134-1138. 5. Nishiguchi S, Otani S, Matsui-Yuasa I, Morisawa S, Monna T, Kuroki T, Kobayashi K. Inhibition of ornithine decarboxylase induction of interferon (alpha + beta) and its reversal by didutyryl adenosine 3,5-monophosphate. Eur J Biochem 1988;172: 287-292. 6. Marth C, Kirchebner P, Daxenbichler G. The role of polyamines in interferon and retinoic acid mediated synergistic antiproliferative action. Cancer Letters 1989;44:55-59. 7. Shindo M, di Bisceglie AM, Hoofnagle JH. Acute exacerbation of liver disease during interferon alpha therapy for chronic hepatitis C. Gastroenterology 1992; 102:1406-1408. 8. Janne J, Poso H, Raina A. Polyamines in rapid growth in cancer. Biol Chimica et Biol Physica Acta 1978;473:241-293. 9. Higgins GM, Anderson RM. Experimental pathology of the liver of the white rat following partial surgical removal. Arch Pathol 1931; 12:186-202. 10. Cancer drug development: cancer. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Principles and practice of oncology. Philadelphia, PA: Lippincott; 1987:270-300. 11. Hartree EF. Determination of protein content: a modification of the Lowry method that gives a linear photometric response. Anal Biochem 1972;48:422-427. 12. Peters M. Mechanisms of action ofinterferons, Semin Liver Dis 1989;9:235-239. 13. Antonelli G, Currenti M, Turriziani O, Dianzani F. Neutralizing antibodies to interferon alpha: relative frequency in patients treated with different interferon preparations. J Infect Dis 1991; 163:882-885. 14. Lok ASF, Lai CL, Leung EKY. Interferon antibodies may negate the antiviral effects of recombinant a-interferon treatment in patients with chronic hepatitis B virus infection. HEPATOLOGY 1990; 12:1266-1270. 15. Takeda T, Nishiguchi S, Nakajima S, Shiomi S, Kuroki T, Kobayashi K, Otani S. Growth inhibition by interferon on a human hepatoma cell line mediated by inhibitionof putrescine synthesis [Abstract]. HEPATOLOGY1992; 15:A897. 16. Scharl A, Kullander S, Beckmann MW, Spicer JA, Baranczuk RJ, Holt JA. Prolonged clearance of intraperitoneal 16 alpha (125I)iodo-17 beta-estradiol in presence of ascites. Am J Obstet Gynecol 1991;165:1847-1853. 17. Michalopoulos GK. Liver regeneration: molecular mechanisms of growth control. FASEB J 1990;4:176-187.