Medroxyprogesterone Induced Changes in Rat Liver

Path. Res. Pract. 176, 115-124 (1983)

Department of Pathology, Umverslty of Oulu, Fmland

Medroxyprogesterone Induced Changes in Rat Liver A Light and Electron Microscopic Study A.

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Summary Medroxyprogesterone acetate (MPA) mduced morphological alteratIOns m the normal female rat liver and after carbon tetrachlonde exposure were assessed by light and electron microscopy and morphometry. AdministratIOn of MPA Increased the smooth endoplasmic reticulum (SER) m normal rat. Carbon tetrachlonde, Injected mtrapentoneally for four weeks, caused fatty accumulation, enlargement of the cell nucleus and decrease of the rough endoplasmic reticulum (RER) and Increase of the SER. After one week of recovery, Without treatment, the fatty infiltratIOn and the SER membranes decreased and the RER mcreased. MPA, follOWing inJury, Induced a decrease m the fatty accumulation and mltochondnal volume denSity and the hepatocyte nuclear and cytoplasm size normalized The SER membrane volume denSity mcreased and the surface denSity decreased, the RER membrane volume denSity Increased while the ~urface denSity did not alter slgmflCantiy. The volume densltles of the mltochondna and the peroxlsomes decreased m the MPA treated CCI 4 exposed group~ The results demonstrate that MPA mduce; a proliferatIOn of the SER In normal rat liver and after hepatic mJury the compound has a benefiCial effect on the regeneratIOn.

Introduction AdmInistratiOn of carbon tetrachlonde (CCI 4 ) m rats causes liver cell mJury detectable by hght (McCloskey and McGehee 1950, Stowell and Lee 1950), and electron microscopy (Oberhng and ROUiller 1956, Smuckler et aI., 1962), and histochemical (Recknagel and Littena 1960) methods. The mjury seen on light microscopy, consists of fatty accumulation, collagen increase and cytoplasmic and nuclear alterations (Stenger 1963). Ultrastructurally the changes are seen in the smooth and rough endoplasmIC membranes, mitochondria, lysosomes, perOXlsomes, Golgi apparatus and the cell membranes (Stenger 1963). Steroids have an mfluence on the liver cell organelles and modify the biochemical and morphologICal alteratiOns associated with liver injury (Hor-

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warth et aI., 1971, Schmuckler and Jones 1973). Medroxyprogesterone (MPA), Increases the drug metabolIzing enzyme activity In normal and damaged rat liver (Saarm et aI., 1980). MPA seems also to Improve the liver function In subjects with lIver parenchymal disease (Sotanieml et aI., 1978, RautIO et aI., 1980, Arranto et aI., 1982). Little is known how MPA influences the liver structure after hepatIC mjury. This study was undertaken to evaluate the effect of MPA on the injured hver by lIght and electron microscopy and morphometry.

Material and Methods Antmals Twenty-fIve, 2 months old female WI star rats weIghmg 160 ± 10 gr were used. They all came from the same farm. The anImals were mamtamed on a normal laboratory dIet, (HankkIJa Fodder MIxture, Central CooperatIve, FInland) and water ad lIbItum throughout the experIment. The anImals were dIVIded mto five groups, each conslstmg of fIve ammals. The rats were mamtamed on a controlled feedmg regImen at least 10 days pnor to use.

Treatment The fIrSt group of ammals received 100 mg MPA/kg of body weight, Injected Intrapentoneally for seven days. The rats m the second group were exposed to CCI 4 , dIssolved m corn od, 0.1 mlllOO gr of body weIght, Injected subcutaneously on the fIrSt and the thIrd day of the week for four weeks and sacnfICed at the end of the treatment. The thIrd group was exposed to CCI 4 as was group II, and saCrIfICed after one week WIthout treatment. The fourth group was exposed to CCI 4 , as was group II, and MPA was admmIstered Intrapentoneally dally for a week, 100 mg/lOO gr of body weIght, and then scanfIced. The fIfth group was used a control. The anImals were kIlled under lIght ether anestheSIa by cervIcal dIslocatIOn and exsangUInatIOn and the hver eXCIsed and weIghed. The organ was sectIOned and the samples taken for hght and electron mIcroscopy.

LIght and electron mIcroscopy For lIght mICroscopIc studIes lIver tIssue cubes, fIxed In 10% neutral phosphate buffered formalm, were embedded m paraffm wax, sectIOned senally 4-5 fUI1 thIck and stamed WIth haematoxylm and eosm, penodIc aCId-SchIff WIth and WIthout dIastase dIgestIOn, HerovIcI's collagen staIn and Gomon's sdver ImpregnatIOn techmque. For electron mICroscopIc analYSIS four tIssue blocks were randomly selected from ten tIssue cubes. For transmiSSIon electron mICroscopy the lIver tIssue samples were Immersed In 4% phosphate buffered glutaraldehyde and postfIxed m 1 % osmIUm tetroXIde In 3% calcIUm chlonde, dehydrated In alcohol and embedded In Epon®. SemIthIn, tolUIdIne staIned sectIOns, were exammed WIth a lIght mIcroscope for selectIOn of mIdzonal fIelds. Ultrathm, mterfererence colour Sliver to grey. uranyl acetate and lead CItrate contrasted sectIOns made on LKB Ultrotome III, were studIed In a Jeol 100 B electron mIcroscope.

Medroxyprogesterone Induced Changes . 117

Morphometry The morphometnc analysIs was performed accordmg to the pnnclples of Weibel et al. (1969), at several light (Arranto and Sotameml 1981) and electron ml(roscoplc magmficatlOn levels. The volume denSity of non-fatty and fatty parenchyma and fibrous trabeculae, with liver tissue as reference space, were assessed on Herovlcl's stamed slides. Five randomly selected areas were projected on a test pomt lattice, d 1.0 cm, at a magmfIcatlOn of 600 x. Pencellular collagen volume denSity m the pen portal, mldzonal and centnzonal areas of the hver lobule were assessed on Gomon's stamed slides. The randomly selected areas were photographed on Kodak Panatomlc film with a Zeiss Photomlcroscope II at a magmfIcation of 640 x. A test pomt lattice, d 0.5 cm, was supenmposed on the magmfIed prmts, 3.1 x, and the liver parenchyma was used as reference space. Semlthm tolUidme stamed slides were used to estimate the relative nuclear and cytoplasmic changes between the groups. The slides were projected at a magmflcatlOn of 2000 x on a test pomt lattice screen, d 0.5 cm, and twenty randomly selected representative hepatocytes were assessed for each ammal. The volume denSity of mltochondna, lysosomes and peroxlsomes was estimated on rwenty-fIve electron micrographs, pnmary magmfIcatlOn 10000, fmal 30000 x and test pomt lattice d 1.5 cm. The volume and surface denSities of the smooth and rough endoplasmiC reticulum were assessed on electron micrographs, pnmary magmfIcatlOn 20000 x, fmal 62000 x, usmg a multipurpose test screen, d 1.0 cm. The tissue and cell compartments were evaluated accordmg to the defmltlons of Riede and Reith (1980) and cm 3 of hver tissue as reference system.

StatIstIcal analysIs The mean and standard error were calculated and for group companson Student's t-test was employed.

Results The results of the morphometric assessments are given in tables 1 and 2. The lobular and trabecular formation of the hepatiC tissue and fine structure were normal in the control group. CCl4 induced liver injury (group 2). CCl4 mduced fatty accumulation of the centn-midzonallobular areas and an increase in hepatocyte nuclear and cytoplasmIC volume. The volume and surface densIty of SER vesicles increased and the volume fractIOn of RER decreased sIgnificantly. The mitochondna and peroxisome volume fractIOn remamed normal. MPA mduced changes (group 1). The histologiC architecture remained unaltered. Occasional eosinophilic cytoplasmic mclusions were seen m the hepatocytes. There was a sigmfIcant increase in the SER. The RER volume and surface density showed a tendency to decrease, however, not significant. The hepatocyte nuclear and cytoplasmiC volume fraction, perOXIsomes and mitochondria were normal.

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FIg. 1 and 2. LIght and electron mIcroscopIC changes Induced by medroxyprogesterone acetate In the normal rat liver and following carbon tetrachlonde inJury. p values for dIfferences between the means; x = companson to control group, y = companson between spontaneous and medroxyprogesterone Influenced recovery. x, y = p < 0.05, xx, yy = p < 0.01, xxx, yyy = p < 0.001.

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Fig. 5. ProliferatIOn of smooth endoplasmic reticulum lllduced by carbon tetrachlonde, 24000 x.

Spontaneous regeneration after CCl 4 mjury (group 3). The morphological alterations associated with recovery included a reductIOn of fatty inftltration and volume density of the liver cell nucleus and cytoplasm and an mcrease 10 the volume density of the mitochondna. The SER volume and surface densIty decreased. The volume density of the RER membranes decreased while the surface density mcreased. Effect of MPA after CCl 4 inJury (group 4). The effect of MPA on the repair process was charactenzed by a reduction of fat and mitochondnal volume densIty. The RER surface densIty ex ceded the normal range whtle the volume fraction was unaltered. The hepatocyte nuclear and cytoplasmIc size was normal. AlteratIOns 10 the lobular collagen fibre distnbution were not slgmftcant in the groups.

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Fig. 6 Whorl like prohferatlOn of rough endoplasmic reticulum around mltochondna Induced by medroxyprogesterone follOWing carbon tetrachlonde exposure, 6000 x.

Discussion MPA, a synthetic progesterone, has an induclOg effect on the liver. The microsomal enzyme activity is IOcreased (Saarm et aI., 1980), the protein and lipid synthesIs enhanced (Dahm et aI., 1978). Besides Improving the hepatocellular functIOn the compound seems also to be able to decrease the amount of pen cellular collagen 10 subjects with endstage CIrrhosis, thus Improving nutritIent contact between the hepatocytes and serum (Arranto et aI., 1982). The present study further demonstrates that the effects of MPA on intact and damaged lIver can be quantified histologIcally and morphometrically. In mtact ammals the amount of SER was increased and in the damaged lIver the drug effect was IOcrease m RER volume, decrease m mitochondrIal volume and a

Medroxyprogesterone Induced Changes . 123

reduction in fat content. Also normahzation in SER was visible. The changes indicate that MPA seems to improve the repair process after injury. The liver injury mduced by reactive metabolites of the CCI 4 , transformed by the endoplasmic reticulum, deplete the cytoprotectlVe substances and bmd to the macromolecules of the cytosol and membranes altering and deranging the function of the ER. The mjury is associated with detachment of nbosomes from the RER membranes, mcrease of the SER veSicles, mtralummal ER hpld droplets, decreased enzyme activity and protem syntheSIS, fatty change, collagen accumulation and parenchymal cell necrosIs (Meldoiesl et al., 1968). The RER of the hepatocytlc parenchymal cell IS the major site of the amino acid mcorporatlOn and protem synthesis (Keller et al., 1954, Smuckler et al., 1972). A decrease of the RER, frequently seen 10 injury and associated with a decreased protem syntheSIS, is related to the hepatic hpld accumulatIOn (Stenger 1963), also seen 10 thiS study. The compound had no mfluence on the RER membranes in the mtact ammals. Both light and ultrastructurally the recovery process was similar 10 control and injured rats, however, the changes, hpid decrease and RER mcrease, were more rapid 10 the MPA treated rats. Followmg CCl4 mJury MPA has a mitochondna volume reducing effect. Therapeutic agents and liver disease may cause a volume increase of the organelles, a phenomenon which was enhanced 10 the spontaneous repair process. The mechanisms mvolved are not yet known, however, syntheSIS blockade of the macromolecules have been suggested (Sternlieb 1979). Topographical relationship between mitochondria and the RER suggest mteraction between the organelles, also observed 10 thiS study. In the study MPA influenced mJury showed a decrease of the mltochondna volume denSity, suggestive of a protective mfluence of the compound on the mitochondria. ThIS study shows that the normahzatlOn of the ultrastructure and ltght microscopic changes were augmented by MPA after mjury and mdicate that the repair can be mfluenced by sterOid compounds like medroxyprogesterone.

References Arranto, A. J., and Sotameml, E. A.' MorphologIcal alteratIOns In patlent~ wIth alpha-methyldopa Induced hver Injury after short and long term exposure. Scand. J. Gastroenterol. 16, 853-863 (1981) Arranto, A. J., Rautio, A., and Sotameml, E. A.: Pencellular collagen In alcohohcs WIth hver CIrrhOSIS Res. Commun. Chern. Path. Pharmacol. 35, 121-136 (1982) Dahm, C H., Jelhnek, M., Mueller, E. J., RIckey, C, and Hertelendy, F.: Some effects of medroxyprogesterone acetate on IntermedIary metabolIsm In rat hver. LIfe SCI. 22, 165-169 (1978) Horvarth, E., Kovacs, K., Blascheck, J A, and SomogYI, A.: Ultrastructural changes Induced In the hver of rats by vanous sterOId compounds. Vlrchows Arch Abt B Zell Path. 7, 348-355 (1971)

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Keller, E. B., Zamcmk, P. c., and Loftfleld, R. B.: The role of mlcrosomes m the mcorporatlOn of ammo aCids mto protems. J. Hlstochem. Cytochem. 2, 378-386 (1954) McCloskey, J. F., and McGehee, E. Hoo Effect of subcutaneous and mtraoral admmlstratlOn of carbon tetrachlonde on the hver of the rat. Arch. Path. 49, 200-204 (1950) Meldolesl, J., Vmcenzl, L., Bassan, P., and Monm, M. T.: Effect of carbon tetrachlonde on the synthesIs of hver endoplasmic reticulum membranes Lab Invest. 19,315-323 (1968) Oberimg, c., and ROUiller, c.: Les effets de I'mtoxlcatlon algue au tetrachlorure de carbone sur Ie fOie du rat: etude au microscope electronnique. Ann. anat. path. 1,401-427 (1956) RautIO, A., Sotameml, E. A., Pelkonen, O. R., and Luoma, P. V.: Treatment of alcohohc CIrrhOSIS With enzyme mducers Chn. Pharmacol. Ther. 28, 629-637 (1980) Recknagel, R 0., and Llttena, M.: Biochemical changes m carbon tetrachlonde fatty hver. ConcentratIOns of carbon tetrachlonde m hver and blood. Amer. J. Path. 36, 521-531 (1960) Riede, U. N., and Reith, A. (Ed): Morphometry m pathology. Path. Res. Pract. 166, 143-404 (1980) Saarm, H, Ahokas, J T., Karkl, N. T., Pelkonen, O. R., and Sotameml, E. A.: Dose-dependant effects of medroxyprogesterone acetate on the hepatic drug-metabohzmg enzyme system m rats. BlOchem. Pharmacol. 29, 1155-1159 (1980) Schmuckler, D. L., and Jones, A. Loo Hepatic fme structure m young and agmg rats treated With oxandrolone: a morphologiC study. lipid Res. 16, 143-150 (1973) Smuckler, E. A, Isen, 0 A., and Benditt, E Poo An mtracellular defect m protem synthesIs mduced by carbon tetrachlonde. J. Exp. Med. 116,55-72 (1962) Sotameml, E. A., Hynynen, T., AhlqUist, J., Ahokas, J. T., Puoskan, U., and Pelkonen, O. R.: Effects of medroxyprogesterone on the hver functIOn and drug metabohsm of patients With pnmary blhary cmhosls and chromc active hepatitis. J. Med. 9, 117-128 (1978) Stenger, R.] HepatIC parenchymal cell alteration after long-term carbon tetrachlonde admmlstratlon A hght and electron microscoPIC study. Amer. J. Path. 43, 867-895 (1963) Sternheb, I.: Electron mICroscopy of mltochondna and peroxlsomes of human hepatocytes. In Progress m hver diseases. (Eds. Popper H and Schaffner, F.) vol IV, pp. 81-104, Grune and Stratton, New York, 1979 Stowell, R. E., and Lee, C. S.· HistochemICal study of mouse hver after smgle feedmg ot carbon tetrachlonde. Arch. Path. 50, 519-537 (1950) Weibel, E. R., Staub", W , Gnagl, HR., and Hess, E A.' Correlated morphometrIc and biochemICal studies on the hver cell. I Morphometnc model, stereologlcal methods and normal morphometnc data for rat hver. ]. Cell BIOI. 42, 68-91 (1969)

Received m reVised form August 25, 1982 Accepted August 25, 1982

Key words: L,ver inJury - Carbon tetrachloride - Medroxyprogesterone LIght and electron microscopy - Morphometry

Arno]. Arranto, Department of Pathology, Umverstty of Qulu, KaJaanmtie 52 D, SF-90220 Oulu 22, Fmland