Azzarone, A

Francavilla, A

Starzl, TE

Zeng, QH

English

PubMed

Name not available

Hepatic growth effects of methylprednisolone azathioprine, mycophenolic acid, and mizoribine

D-Scholarship@Pitt

>. i 1993 BRIEF COMMUNICATIONS 13. Wiekre CG. Norman DJ. Bennison A. Barry JM. Bennett WM. Postrenal transplant erythrocytOSis: a review of 53 patients. Kidney Int 1983: 23: 73l. 14. Friman S. Nyberg G. Blohme l. Erythrocytosis after renal trans-plant: treatment by removal of the native kidneys. Nephrol Dial Transplant 1990: 5: 969. 15. Bakris GL. Sauter ER. Hus8ey JL. Fisher JW. Gaber AO. Winsett R. Effects of theophylline on erythropoietin production in normal subjects and in patients with erythrocytosi8 after renal trans-plantation. N Engl J Med 1990: 323: 86. 16. Gould AB. Goodman SA. Green D. An in vivo effect of renin on erythropoietin formation. Lab Invest 1973; 28: 719. 17. Fried W. BI angiotensi Lab Clin l 18. Bauer C. Kl erythropoi 19. Curtis JJ. L of remove plantatioz Received 27 May 1992. Accepted 17 September 1992. HEPATIC GROWTH EFFECTS OF METHYLPREDNISOLONE. AZATHIOPRINE. MYCOPHENOLIC ACID, AND MIZORIBINE I Cyclosporine I eSA) and FK506 augment liver regeneration after partial hepatectomy in rats (1. 2), an effect that is not the consequence of immune modulation (3). In addition. both drugs increase the hepatocyte proliferative response in dogs that is characteristic after Eck fistula. and they also prevent the hepatocyte atrophy and organelle disruptIon that is caused by this operation (4). Substances causing these changes have been termed hepatotrophic. Rapamycin (RPM)! a third potent immunosuppressant. has opposite 1 antihepatotrophic) actions without evidence of inherent hepatocyte toxicity 15). A hypothesis that these growth control alterations are caused by altered signal transduction of the hepatocytes independent of immunologic pathways (4) has been supported by in vivo and in vitro experimentation (3), However. there is a paucity of information about the influence of other immunosuppressive drugs on growth and regeneration. Using in vivo (partial hep-atectomy) and in vitro (tissue culture) rat models described elsewhere (2, 3. 5. 6), we have investigated the effect upon hepatocyte proliferation of methylprednisolone (MP). myco-phenolic acid (YtPA). mizoribine (MZ). and azathioprine IAZA). In vivo experiments were performed with adult male inbred Fisher 344 rats weighing 180-200 g (Hilltop Lab Animals. Scottsdale. PAl. The rats were given standard rat laboratory diet and water ad libitum, and kept in a temperature and light-controlled room \light 0730-19301. Immunosuppressive drugs were given preoperatively on days -3. -2. and -1. and as a tinal dose just after 70% hepatectomy. The operations were performed between 0900 and 1030 under light ether anesthesia (2, 5). Immediate resumption of food and drink made it unnec-essary to provide parenteral fluid and electrolyte support. At 24 hr after the hepatectomy when the rat liver regeneration response is at its peak. 185x104 Bq JH-thymidine was admin-istered by intraperitoneal injection. followed 2 hr later by guillotine killing. Hepatic DNA was extracted and purified as previously described (2. 5). DNA content was measured with a standard of calf thymus DNA (Sigma. St. Louis). Mitotic indices were obtained in 3 animals in the groups 1. 3. 5, i and : This work was supponed by Research Grants from the Veterans Admimstratlon and Project Grant OK 29961 from the National Institutes 01 Health. Bethesda. MD. This study has also been sup-poned bv ConSiglio Nazionale delle Ricerche. ACRP ProgT8m. Grant 92.02187.PF39. • Abbreviations: AZA. azathioprine: MP. methylprednisolone: MP A. mycophenolic acui; MZ. mizonbine: RPM. rapamycin. 9. and in 6 animals in the groups 2. 4. 6, 8 and 10 (Table 1) that were sacrificed at 33 hr. The liver specimens from these animals were formalin-fixed. stained with H&E. and examined for the percentage of mitoses. The in vivo experimental groups are summarized in Table 1. After partial hepatectomy, the contemporaneous vehicle con-trols showed considerable variability from group to group (Fig. 1). In control experiments. the highest DNA synthesis and mitotic rates were in control animals injected with saline (Table 1. groups 1 and 3) whereas the lowest were in animals with drug vehicle containing CMC solvent alone (group 9) and CMC mixed with Tween and alcohol (group 5). This suggested a potential inhibitory effect of the CMC carrier and emphasized the need for concurrent controls for each test group. The only drug that significantly augmented regeneration relative to the controls was MP. P<O.Ol (Fig. 1). The only drug that significantly inhibited regeneration was MZ (P<O.Ol). The MZ inhibition was profound. reducing DNA synthesis >85% and the mitosis rate >70% (Fig. 1). MPA at a standard dose and AZA at low (1 mg/kg orall and high (6 mg/kg i.m.) doses were without effect on regeneration in the whole animal (Fig. U. In vitro experiments were performed with hepatocyte culture preparations made from the livers of the same strain of unal-Group 1 (n-S) 2 (na 13l 3 (n=8) 4 (n=13) 5 (n=8) 6 (n==13) -; (n=8) TABLE 1. Conditions of in vivo experiments" Dose used Drup Img/kg/ Route Vehicle daHl MP MZ 20 MPA 15 1M 1M 1M 1M PO PO Saline Saline Saline Saline 0.5% CMC: 0.4% Tween 0.9 alcohol in saline 0.5% CMC; 0.4% tween 0.9% alcohol in saline PO Olive oil 8 (n=13) AZA PO Olive oil 9 (n=8) 1M 1 % CMC in saline 10 (n-13I AZA 6 1M 1 % CMC in saline • Methylprednisolone IMP) and mizoribine (MZl were disaolved in saline: mycophenolic acid (MP A 1 was dissolved in 0.5 % carboxymethvl-cellulose ICMCl. 0.4% Tween and 0.9% alcohol in saline; azathiopnne (AZAl was mixed with olive oil or dissolved with 1% CMC in saline . Drug solutlOn8 were made daily for each dOll8 of each drug. 220 TRANSPLANTATION VoL 56, No.1 45 .. iii 30 2 :i c B cf 15 C MP C MPA C MZ C AZA C AZA 1 fTI9III4I 6 "'9*9 FIGURE 1. JH·thymidine incorporation and mitoses/lOOO hepato· cytes in 70% hepatectomized rats treated or not treated with methyl· prednisolone (MP), mycophenolic acid (MPA), mizoribine (MZ), and azathioprine (AZA). Drug doses are as shown in Table 1. Twenty·one animals were used for each drug: 8 rats were used as control and 13 were treated with the drugs. Three rats of each control group and six of each experimental group were used for determinations of the number of mitoses. Bars are mean :t: SD. (*)P<O.OI (drug versus control). tered male rats (180 and 200 g). Hepatocytes were isolated with a previously standardized in situ two· step collagenase perfusion technique (3. 6). The hepatocytes were dispersed and washed twice with cold CA"''''·free perfusion buffer and resuspended in basal medium (MEM) supplemented with pyruvate (1 mM). proline to.26 mM), insulin (10-7 Ml. and 5% FCS. Cell number· w"as determined with a hepatocytometer. Viability was deter· mined by trypan blue exclusion. For drug testing, only prepa· rations with >95% viability were used. DNA content of the harvest cells was determined by a microfluorometric method (2, 5), and DNA synthesis was measured by the method of Michelopoulos et al. (7) and expressed as cpm/Ilg DNA. The hepatocytes were plated at a cell density of 2xI06/well in a Corning 35-mm tissue culture dish containing 1.5 ml medium and maintained at 37°C in a 5% CO2 atmosphere. In prelimi· nary viability studies in which the basal medium was enriched with insulin but not with EGF, the viability of these resting hepatocytes over the 48 hr following a 3-hr attachment was diminished only by azathioprine (Table 2). After a 3-hr attachment period in the definitive in vitro experiments, the medium was replaced with 1.5 ml basal me-dium tMEMl, to which EGF no ng/ml) and insulin (10-7 M) were added. MPA was dissolved in DMSO. and the other 3 immunosuppressive drugs tested were dissolved in alcohol and added in the concentrations shown in Figure 2. The minute quantities of DMSO or alcohol added to the medium (1 Ill/mi) were shown not to affect hepatocyte proliferation (data not TABLE 2. Viability, DNA concentration. and tranumina .. levels of hepatocytel in media enriched with immunOluppreuive drugs in the abNnce of EGF" 24 br 48 br Viability DNA Viability DNA ALT percentap (",/diabl percentage (",/diab) tU/mll Control 95%' 3.2%0.2 96%3 3.3%0.2 5. 1::t 1 MPA 10 "g/ml 94%3 3.3%0.3 96%' 3.3%0.2 4.1%0.6 AZA 1 "g/ml 56%' 2.1%0.3 35±5 1. 7%0.3 16%3 MZ l00ng/ml 95%6 3.1±0.2 95%9 3.3%0.2 4.5%0.7 MP 10 "g/ml 9'%3 3.3%0.3 97±3 3.3%0.2 5.1%0.8 • MPA: mycophenolic acid: AZA: azathioprine; MZ: mizoribine; MP: methylprednisolone. c ~ III 30 i C _liZ O .. C ~ " a 20 -~~ Ql II· ~:O E :i .. 10 ,.. .. e .:Wa. 7 ... ~ 0 PI .... C = Me.llylDrednilolone J e. MycoDllenolic acid lIa/m' o 0.8 1.2 2.4 5 10 . ~. .... . • ~ ... .Y h---tl ;; I"V ~~ .. !-- i II i +-=:1 : . ~q-~~~~-1~-D~~ 038 12 25 50 lOa A. Mlzoribine (na/m" FIGURE 2. Doses versus DNA synthesis response with MP, MPA. and MZ of cultured hepatocyte. (n=9 for all data points (3 experiments with triplicate determinations,. Mean :t: SD; (*)P<O.05; (")P<O.Ol. shown). During the last half of the 48-hr culture period. 3 Ilei lH·thymidine was added to the wells containing hepatocytes and MP. MPA. or MZ. MZ and MPA inhibited the DNA synthesis of the cultured hepatocytes while the MP caused a near doubling of thymidine incorporation (Fig. 2). These results and the previously reported ones with cyclo· sporine. FK506. and RPM provide a basis for further inquiry. They suggest a fundamental difference between the growth effects of cytotoxic agents (AZA, MPA. and MZ) versus previ· ously studied drugs like CsA. FK506. and RPM that are thought to act by altering signal transduction (8). The cytotoxic agents act by selectively inhibiting the synthesis of purine nucleotides (adenine or guanine), thereby reducing DNA synthesis of a variety of immunologic and other specialized cells. including hepatocytes. However. in the intact animal. the hepatocyte proliferative response of regeneration was not demonstrably affected by AZA or MPA. although it was drastically reduced by MZ. In contrast. the hepatocytes in vitro' were killed (AZA) or rendered nonproliferative (MPA and MZ). Whether the striking effect of MZ in both testing conditions was a high· dose phenomenon or has a more specific explanation could not be determined from our experiments. The in vivo results that we obtained with a 4-day course before hepatectomy of either 1 or 6 mg/kg/day of azathioprine were remarkedly different than those reported by Kim et a1. (9) who observed striking augmentation of DNA synthesis in essentially the same experiments. They interpreted this as evidence that hepatic regeneration is immune-modulated. Be-... i July 1993 BRIEF COMMUNICATIONS 221 cause of the possibility that Kim (9) might have interposed a delay between the last drug dose and the partial hepatectomy, thereby allowing the beginning of regeneration in response to a chemical injury, we performed more experiments in which the last azathioprine dose was not given at the time of opera-tion. Although the animals with the premature cessation of AZA treatment tended to have more vigorous regeneration \ Table 3), this difference was not statistically significant. How-ever. the possibility of a "timing" artefact has not been ruled out and will require further study. Unlike AZA and the other cytotoxic drugs (MPA and MZ), ~m caused a striking increase of proliferation of hepatocytes in culture and it augmented liver regeneration in the intact animal. Such steroid-stimulating effects on hepatocyte division have not been noted before-and. in fact, Richman et a1. (10) reported inhibition by dexamethasone. which in our model also was stimulatory (unpublished observations l. MP is known to inhibit the synthesis and expression of multiple cytokines including ILl, IL./, and migration inhibitor factor \ 11. 12l. Although ILl and ILl are thought from reported in vitro exper-iments to be growth suppressors \ 13) this could not be dem-onstrated by us in a sensitive in vivo test system in which the recombinant cytokines in question were infused directly into the tied-off portal vein of the Eck tistula liver 114). Thus. the proliferative response to MP reported herein both in vivo and in vitro cannot be explained by what is currently known about steroid actions. In contrast. the inhibition of hepatocyte proliferation by cytotoxic drugs is consistent with previous reports about AZA (15). A seemingly obvious explanation could be that AZA. MPA. and MZ selectively inhibit synthesis of purine nucleo-tides (adenine with AZA and guanine with MPA and MZ) that are required for DNA synthesis. The molecular basis of growth alterations by immunosup-pressants is best understood with FK506. esA. and RPM-drugs that alter signal transduction with pleiotropic effects not limited to Immune cells (3. 8). These agents. which are not inherently cytotoxic. are inactive "prodrugs" until they bind with ubiquitous cytosolic immunophilins and form complexes that act upon a common target\s) (8l. Although these molecular mechamsms have been determined principally with the lym-phocyte and mast cell, the hepatocyte may be the best to investigate growth control (6). .\s the search continues for immunosuppressive drugs, it is self-evident that growth control qualities should be systemati-cally determined. Conversely, drugs under study primarily for their hepatocyte growth control activity (14) should also be screened for possible immunosuppressive activity. TABLE :3. Effects on hepatocyte proliferation ot' AZA administration ( ~roup n Operation .... ZA adminIstration Route cpmlmg DNA ,) Sham 1.5 mgl kg for 3 davs PO 4.47i±4:20 ., K~ 70% PH 1.5 mg/kg for 3 days PO 87.574±18.315 '; -;'0% PH 1.5 mgl kg for 4 days PO 55.318±15.422 . Croups 1 and :2 were treated for 3 days but did not receive AZA on the dav of the operation as In our experiments shown In Figure 1 (group :l in this table). A. AwwAolkb~·P A. FRANCAVILLA2• J Q. H. ZENGJ T. E. STARZL' The Department of Surgery University Health Center of Pittsburgh Pittsburgh, Pennsylvania The Department of Gastroenterology University of Bari Bari, Italy 2 University Health Center of Pittsburgh. J University of Bari. • Address requests for reprints to Thomas E. Starz!. M.D .• Ph.D .• Department of Surgery, 3601 Fifth Ave .. University of Pittsburgh. Pittsburgh. PA, 15213. REFERENCES 1. Makowka L. Svanas G. Esquivel CO. et al. The effect of cyclospor-ine on hepatic regeneration. Surg Forum 1986: 37: 353. 2. Francavilla A. Barone M. Todo S. Zeng Q. Porter KA. Stsnl TE: Augmentation of rat liver regeneration by FK 506 compared with cyciosporme. Lancet 1989: 2: 1248. J. Francavilla A. Starzl TE. Barone M. et al. Studies on mechanisms of augmentation of liver regeneratIOn by cyclosporme and FK 506. Hepatology 1991: 14: 140. -I. Starzl TE. Porter KA. Mazzaferro V. Todo S. Fung J. FrancaVilla A. Hepatotrophic effects of FK 506 in dogs. Transplantation 1991; 51: 67. 5. Francavilla A. Starzl TE. Scotti C. et al. Rapamycin inhibits liver. kidney, and intestine regeneration. Transplantation (in pressl. 6. Francavilla A. Carr B. Stanl TE. Azzarone A. Carrieri G. Zeng QH. Effects of rapamycin (RPM) on cultured hepatocyte prolif· eration and gene expression. Hepatology (in press). 7. Michalopoulos G. Cianciulli HD. Novotny R. KUgerman AD. Strom SC. Jirtle RL. Liver regeneration studies with rat hepatocytes in primary culture. Cancer Res 1982: 42: 4673. 8. Schreiber SL. Liu J. Albers MW. et a1. Immunophilin·ligand com· plexes as probes of intracellular signaling pathways. Transplant Proc 1991: 23: 2839. 9. Kim Y1. Kawano K. Iwao Y. et al. Stimulation of liver regeneration by pretreatment with azathioprine a8 well as cyciosporine and FK506. Transplantation 1992: 53: 949. 10. Richman RA. Claus TH. Pilkis SJ. Friedman DL. Hormonal sum· ulation of DNA synthesis in primary cultures of adult rat hepa-tocytes. Proc Nat! Acad Sci USA 1976: 73: 3589. 11. Kern JA. Lamb R.I. Reed JC. Daniele RP. Nowell PC. Dexameth· asone inhibition of interleukin 1 beta production by human monocytes. J Clin Invest 1988: 81: :237. 12. Horst HJ. Flad HD. Corticosterold-interleukin 2 interaCtions: in· hibition of binding of interleukin 2 to interleukin :2 receptors. Exp Immunol 1987; 68: 156. 13. Nakamura T. Arakaki R. Ichihara A. Interleukin-1B is a potent growth inhibitor of adult rat hepatocytes in primary culture. Exp Cell Res 1988; 179: 488. 14. Francavilla A. Stanl TE. Porter K. et a1. Screenmg for candidate hepatic growth factors by selective portal infusion after camne Eck fistula. Hepatology 1991: 14: 665. 15. Gonzalez EM. Krejczy K. Malt R. Modification of nucleic aCid synthesis in regenerating liver by azathioprine. Surgery 1970: 68: 254 . Received 19 December 1991. Accepted 11 September 1992. 

Hepatic growth effects of methylprednisolone, azathioprine, mycophenolic acid, and mizoribine

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Hepatic growth effects of methylprednisolone, azathioprine, mycophenolic acid, and mizoribine

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