Quantitative Analysis of Smooth Endoplasmic Reticulum Proliferation in Hepatocytes of Early Postnatal and Adult Mice Treated With Phenobarbital

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1 GASTROENTEROLOGY 1984;87; Quanttatve Analyss of Smooth Endoplasmc Retculum Prolferaton n Hepatocytes of Early Postnatal and Adult Mce Treated Wth Phenobarbtal KAZUO KANAI, SHINSUKE KANAMURA, MARl ASADA-KUBOTA, JUN WATANABE, and MOTOKO OKA Department of Anatomy, Kansa Medcal Unversty, Fumzonocho 1, Morguch, Osaka, Japan The smooth endoplasmc retculum n hepatocytes of early postnatal and adult ddy male mce njected wth phenobarbtal was analyzed stereologcally, The anmals receved daly ntrapertoneal njectons of phenobarbtal and were klled about 24 h after the last njecton. In early postnatal anmals (njected wth 35 mg/kg of the drug at days 1 and 2 after brth, or njected wth 35 mg/kg at day 2 and wth 5 mg/kg at days 3 and 4 after brth), the smooth endoplasmc retculum prolferated both n pervenular and perportal hepatocytes; there was no sgnfcant dfference n amount of smooth endoplasmc rectculum between the cells of the two zones. In adult anmals, however, prolferaton occurred only n per venular cells after the admnstraton of 35 mg/kg of phenobarbtal for 2 days, and predomnantly n per venular cells even after the admnstraton of 15 mg/kg of the drug for 7 days. These results suggest that the conspcuous prolferaton of the smooth endoplasmc retculum n pervenular hepatocytes n response to phenobarbtal admnstraton, whch s characterstc of adult lver, does not occur n early postnatal mce but becomes manfest durng postnatal development. Hepatocytes of the adult mouse, rat, rabbt, dog, monkey, and human reveal functonal and morphologc dfferences dependng on ther postons wthn the lver acnus (1). For example, perportal and Receved November 29, Accepted May 11, Address requests for reprnts to; Dr. Shnsuke Kanamura, Department of Anatomy, Kansa Medcal Unversty, Fumzonocho 1, Morguch, Osaka, 57 Japan. Ths work was supported, n part, by a grant (No. 5571) from the Japanese Mnstry of Educaton. The authors thank Dr. Tbor Barka, Mount Sna School of Medcne of the Cty Unversty of New York, for hs crtcal readng and correcton of the manuscrpt by the Amercan Gastroenterologcal Assocaton /84/$3. pervenular (around the termnal hepatc venule) hep&tocytes not only dffer ultrastructurally (2,3) but also n enzyme actvtes (4), n fastng glycogen content (5-7), n the rate of proten synthess (8), and n response to cell njury after carbon tetrachlorde treatment (9). Phenobarbtal (PB) admnstraton to the anmals nduces a promnent prolferaton of the smooth endoplasmc retculum (SER) n pervenular hepatocytes that progressvely nvolves mdacnar cells (1-17). However, n the frst few days after brth, the actvtes of glucose-6-phosphatase (18,19), ornthne carbamoyltransferase, succnate dehydrogenase, the reduced form of ncotnamde adenne dnucleotde dehydrogenase (2), and ble canalcular alkalne phosphatase (21) are evenly dstrbuted throughout the acnus. A smlar unform dstrbuton of hepatocytes that ncorporated [3Hlthymdne has been descrbed n the acn n the lver of newborn rats (22). The zonal dstrbuton of varous enzyme actvtes and of cells syntheszng deoxyrbonuclec acd (DNA), whch s characterstc of adult lver, appears gradually durng postnatal development. Further, t has been shown recently that the ultrastructural heterogenety among hepatocytes, evdent n adult anmals, s not present n newborn anmals but arses durng postnatal development (23,24). Therefore, t s of nterest to know whether the predomnance of SER prolferaton n pervenular hepatocytes s seen n early postnatal anmals treated wth PB or only appears durng postnatal development. Therefore, we have quanttated stereologcally the amount of the SER n perportal and pervenular hepatocytes of early postnatal and adult mce njected wth PE. Abbrevatons used n ths paper: PB, phenobarbtal; SER. smooth endoplasmc retculum.

2 1132 KANA! ET AL. GASTROENTEROLOGY Vol. 87, No.5 Fgure 1. Electron mcrograph of a porton of pervenular hepatocyte from a postnatal mouse njected wth PB (35 mg/kg) at days 1 and 2 after brth and klled 3 days after brth. Lver peces were fxed by mmerson n buffered 2.5% glutaraldehyde and then n buffered 1% OS4' Thn sectons were cut from surface portons of the fxed peces. Preservaton of fn e structure s satsfactory. Prolferaton of the smooth endoplasmc retculum s seen. x13,. Materals and Methods Male ddy mce, some 1-5 days old and others -3 mo old, were used n the experment. The lactatng and nonlactatng anmals were fed Orental laboratory chow (Orental Yeast Company, Tokyo, Japan) and water ad lbtum. The anmals receved daly ntrapertoneal njectons of PB (sodum phenobarbtal) dssolved n.9% salne n the mornng. Early postnatal and adult anmals were each dvded nto two groups. Early postnatal anmals n one group were njected wth 35 mg/kg of PB at days 1 and 2 after brth; mce n the other group were njected wth 35 mg/kg of PB at day 2, and wth 5 mg/kg at days 3 and 4 after brth. The average body weght of the postnatal mce njected wth PB ncreased only slghtly durng the expermental perod. One group of adult anmals was njected wth 35 mg/kg of PB for 2 days. The other group of adult anmals was njected wth 15 or 2 mg/kg of PB for 7 days n order to ascertan whether the predomnance of SER prolferaton n per venular hepatocytes occurs even after admnstraton of hgh doses of PB; morphometry was not carred out n ths group. Control anmals were njected wth salne. There were 3 treated and 3 control anmals n each group except the last, whch conssted of 24 treated anmals. The anmals were klled by cervcal dslocaton about 24 h after the last njecton of the drug. Peces (smaller than 1 mm 3 ) from the left lobe of lver were fxed by mmerson n 2.5% glutaraldehyde n.1 M sodum cacodylate, ph 7.3, at 4 C for 3 h and rnsed n.1 M sodum cacodylate contanng 8% sucrose at 4 C for 3 mn. Lvers of adult anmals njected wth 15 mg/kg of PB for 7 days were perfused va the portal ven wth the fxatve soluton for 15 mn, washed n the buffer for 3 mn, and then cut nto peces. These peces were postfxed n buffered 1% osmum tetroxde for 3 h, dehydrated n ethanol, and embedded n Spurr medum. Four peces, two ncludng the termnal hepatc venule and two contanng the portal area, were selected randomly from each anmal. One thn secton of slver nterference color was cut from the surface porton of each pece wth a glass knfe on an LKB Ultrotome (LKB Instruments, Inc., Rockvlle, Md.), staned wth uranyl acetate and lead ctrate, and examned n a Htach H-5 electron mcroscope (Htach Ltd., Tokyo, Japan). Electron mcrographs (5.1 x 7.3 cm for early postnatal anmals and 9.2 x 7.3 cm for adult anmals) of hepatocytes and portons of hepatocytes that were wthn a three-cell radus of the portal area or of the termnal hepatc venule and that were located n the upper or lower rght corner of the supportng copper grd (2 mesh) were taken at a magnfcaton of 25 from each thn secton. From these, sx mcrographs (fve plus a spare) were randomly chosen. For early postnatal anmals, each electron mcrograph was dvded nto four equal

3 November 1984 SER PROLIFERATION BY PHENOBARBITAL 1133 Table 1. Surface Densty of the Endoplasmc Retculum n Hepatocytes From Early Postnatal and Adult Mce Treated Wth Phenobarbtal SER RER TER Perportal Pervenular Perportal Per venular Perportal Per venular Early postnatal 1 C 2.72 ± ± ± ± ± ±.135 E 4.66 ±.438" 4.25 ±.483" 3.85 ±.282" 4.28 ± ± ±.97 Early postnatal 2 C 2.78 ± ± ± ± ± :+:.281 E 6.62 ±.12b 6.39 ± ± ± ± ±.9" Adult C 2.18 ± ± ± ± ± ±.691 E 2.55 ± ±.766" 4.34 ± ± ± ±.826" Values represent mean ± SE. Surface densty: J-L2/ J-L3 hepatocyte cytoplasm. Early postnatal 1: mce njected wth 35 mg/kg at days 1 and 2 after brth and klled at 3 days after brth. Early postnatal 2: mce njected wth 35 mg/kg at day 2 and 5 mg/kg at days 3 and 4 after brth. and klled at 5 days after brth. Adult: adult mce njected wth 35 mg/kg for 2 days. C. control; E, experment; RER, rough endoplasmc retculum; SER, smooth endoplasmc retculum; TER, total endoplasmc retculum. "p <.5, h P <.1: sgnfcantly dfferent from control value (Student's t-test). parts, and four photographs enlarged to a fnal magnfcaton of 3, were prepared. For adult anmals, each electron mcrograph was dvded nto nne equal parts, and four photographs at a magnfcaton of 3, (upper left and rght, and lower left and rght of the nne parts) were prepared. However, photographs n whch the cytoplasm occuped less than about one-half of the area were dscarded and sutable photographs were suppled from spare electron mcrographs. Thus, the surface densty (square mcrometer per cubc mcrometer of hepatocyte cytoplasm) of the endoplasmc retculum was estmated usng a multpurpose test system (25,26)' 24 x 24 cm, ::E S2 -.J.. >- () 1 5 u.. "'::::; Fgure 2. V p V p V 3 DAYS 5 DAYS A Surface denstes of the smooth endoplasmc retculum n hepatocytes from early postnatal and adult mce treated wth phenobarbtal. Three days: anmals njected wth 35 mg/kg at days 1 and 2 after brth and klled 3 days after brth. Fve days: anmals njected wth 35 mg/kg at day 2 and 5 mg/kg at days 3 and 4 after brth, and klled 5 days after brth. A: adult anmals njected wth 35 mg/kg for 2 days. P: perportal hepatocytes. V: pervenular hepatocytes. Open hstogram: control. Dotted hstogram: expermental. *p <.5, ***p <.1: sgnfcantly dfferent from control value (Student's t test). whch contaned 168 test ponts spaced at 2. cm, on 4 photographs per anmal. The data were subjected to statstcal analyss usng Student's t-test for dfferences between perportal and pervenular cells, and control and expermental anmals. Results Immerson fxaton of lver peces <1 mm:) and cuttng of thn sectons from surface portons of the peces resulted n satsfactory preservaton of hepatocyte ultrastructure (Fgure 1). Quanttatve Results The values are summarzed n Table 1. Smooth Endoplasmc Retculum In control postnatal anmals njected at days 1 and 2 after brth, or at days 2, 3, and 4 after brth, the surface densty was not sgnfcantly dfferent between the perportal and per venular hepatocytes (Fgure 2, Table 1). The value was greater n pervenular cells than n perportal cells n control adult anmals njected wth salne only. After PB admnstraton, the values ncreased both n the perportal cells (71 %, p <.5) and pervenular cells (82%, p <.5) n anmals njected at days 1 and 2 after brth (35 mg/kg), and also both n perportal cells (138%, p <.1) and pervenular cells (122%, p <.5) n anmals njected at 2 days (35 mg/kg) and 3 and 4 days (5 mg/kg) after brth. There were no sgnfcant dfferences between these ncreases n perportal and pervenular cells. In adult anmals njected for 2 days wth PB (35 mg/kg), however, the value ncreased (66%, p <.5) only n per venular cells.

4 1134 KANA! ET AL. GASTROENTEROLOGY Vol. 87. No.5 5 t :::E, { ; (/), «....J I j : [l. am ( 'j. :.,;,.:. \ &; fk 1 rt '. ' D ;g. ' : ; ;. j. : ; : ' '. u. '" :... j '.... ; j. ; ls., 1 ;/I. ",) '1 ";; -","!.. ;,:" - I l " ; I " \.1r p v p v p V 3 DAYS 5 DAYS A Fgure 3. Surface denstes of the rough endoplasmc retculum n hepatocytes from early neonatal and adult mce treated wth phenobarbtal. See legend of Fgure 2 for explanaton. Rough Endoplasmc Retculum Sgnfcant decrease was seen only n perportal cells of anmals njected at days 1 and 2 after brth (35 mg/kg) (Fgure 3, Table 1). In other groups, the values dd not change after PB admnstraton. Total Endoplasmc Retculum (Smooth Endoplasmc Retculum Plus Rough Endoplasmc Retculum) The values ncreased sgnfcantly n perportal and pervenular cells of anmals njected at 2 (35 mg/kg) and at 3 and 4 days (5 mg/kg) after brth, and n per venular cells of adult anmals njected for 2 days (Fgure 4, Table 1). Dscusson Morphology of Hepatocytes From Anmals Treated Wth 15 mg/kg of Phenobarbtal for Seven Days All 12 anmals ded wthn a few days after the admnstraton of 2 mg/kg of PB; 8 of 12 anmals survved after the admnstraton of 15 mg/ kg of PB for 7 days. Prolferaton of the SER was marked n pervenular cells (Fgure 5), but not n perportal cells n the lvers of survvng anmals (Fgure 6). Thus, the predomnance of SER prolferaton n pervenular cells perssted even when such hgh doses of PB were gven for 1 wk. These data ndcate that durng the early postnatal perod, PB admnstraton causes SER prolferaton both n pervenular and perportal hepatocytes. In adult mce, admnstraton of PB results n prolferaton of the SER only (35 mg/kg for 2 days) or predomnantly (15 mg/kg for 7 days) n the pervenular hepatocytes. * 'f; ;;J, * -. * * * In the adult mouse and rat, the response of hepatocytes to PB admnstraton dffers accordng to the locaton of the cell wthn the lver acnus. The prolferaton of the SER occurs n per venular hepatocytes and progressvely nvolves mdacnar cells after PB admnstraton (1-17). As revealed n the present study, however, n early postnatal mce admnstraton of PB leads to a smlar prolferaton of the SER n the pervenular and perportal hepatocytes. Generally, the so-called "functonal and structural heterogenety among hepatocytes" s not present n newborn anmals but arses durng postnatal development. Actvtes of several enzymes (18-21) and the cells that synthesze DNA (22) are evenly dstrbuted throughout the acnus n the frst few days after brth. The zonal dstrbuton of these enzyme actvtes and cells, whch s characterstc of adult lver, appears gradually durng postnatal development. Smlarly, dfferences n the ultrastructure between perportal and pervenular hepatocytes are not present n newborn anmals but arse durng postnatal development (23,24). The present results ndcate that the predomnance of the SER prolferaton n pervenular hepatocytes after PB admnstraton, evdent n adult lver, also appears durng postnatal development. It s lkely that ths s related to the gradual establshment of adult lobulaton that occurs durng the postnatal perod. LeBouton and Marchand (22) and LeBouton (27) suggested that the postnatal changes observed n the subacnar dstrbuton of labeled nucle after [3H]thymdne ncorporaton are caused by the dras- 1 : t )\:. ; : \* f '-l..- :::E 1:: J (/) :f. :., «...J ff' [l.,=, I >. -;:.. r I- - ' 1 >-.; r. ";'.';- u 5.?..': 7 -. ;. ;,:; 1 U. r. t. ;,;;.l:." } ;: - ' 1,. f t ; :., r., -,!. t I p V P V P V 3 DAYS 5 DAYS A Fgure 4. Surface denstes of the total endoplasmc retculum n hepatocytes from early neonatal and adult mce treated wth phenobarbtal. See legend of Fgure 2 for explanaton.

5 November 1984 SER PROLIFERATION BY PHENOBARBITAL 1135 Fgure 5. Electron mcrograph of portons of pervenular hepato cytes from adult mce treated w t h phe nobarbtal [15 mg/kg) for 7 days. Lvers were fxed by perfuson wth buffered 2.5% gluta rald ehyde and then mmersed n buffered 1% OS4. X8. Marked prolferaton of the smoot h endoplasmc ret cu lum s seen. Intercellula r bls ters on both sd es of the ble ca nal c ulu s a re probably artfact. tc alteraton, at brth, n the amount and composton of blood crculatng through the lver. The fetal lver receves a relatvely large volume of welloxygenated blood rch n nutr ents va the umblcal ven. In ths stuaton, the mcroenvronment along the snusods mght be constant throughout the acnus and the hepatocytes of three zones mght be able to synthesze DNA equally. After brth, the bulk of the blood crculatng through the lver derves from the portal ven, and ths blood s lower n amount, pressure, oxygen saturaton, and nutrent content (28,29). Such a change n blood supply concevably alters the mcroenvronment along the snusods and produces a dfference n hepatocyte functon dependng on the poston of the cell wthn the acnus. The dfference n the subacnar dstrbuton of hepatocytes responsve to PB between early postnatal and adult mce, observed n ths study, may also be related to ths change of hepatc blood supply at brth. On the other hand, Gumuco and Mller (3) suggested that hepatocyte heterogenety may not be entrely related to the zonal mcroenvronment along the snusods but may be the result of ntrnsc cellular dfferences n each zone. Such ntrnsc cellular dfferences may develop durng the postnatal perod. Therefore, the dfference n the subacnar dstrbuton of PB-responsve cells between growng and adult anmals may be the result of the postnata l development of ntrnsc dfferences n hepato cytes of each zone. Loud (2) descrbed consderable quanttat ve ultrastructural dfferences b etw een hepatocytes wthn a three-cell radus of the portal area or the termnal hepatc venule and those n the remander of the acnus. In the present study, hepatocytes wthn a three-cell radus of the portal area and the termnal hepatc venule were consdered as perportal and pervenular hepatocytes. It s possble that the restrcted cell populatons descrbed by Loud may reveal alteratons consderably more (or less) severe than the rest of the perportal or per venular hepatocytes after PB admnstraton. It has been shown repeatedly that the admnstraton of PB nduces a marked prolferaton of the SER n hepatocytes of vertebrates. Most nvestgatons were concerned, however, wth the lver of adult anmals. To our knowledge, there s only one report n the lterature (17) descrbng the prolferaton of SER n pervenular cells (wthout morphometrc

6 GASTROENTEROLOGY Vol. 87, No KANA! ET AL. Fgure 6. Electron mcrograph of portons of perportal hepatocytes from adult mce treated wth phenobarbtal (15 mg/kg) for 7 days. Lvers were fxed as ndcated n legend for Fgure 5. The prolferaton of the smooth endoplasmc retculum s not so marked as n Fgure 5. analyss) n the lvers of early postnatal anmals. The present results ndcate that hepatocytes of early postnatal mce and fully dfferentated hepatocytes of adult mce respond dfferently to the admnstraton of PB References 1. Rappaport AM. Acnar unts and th e pathophysology of the lver. In: Rouller C, ed. The lver. Morphology, bochemstry, physology, Vol. 1. New York: Academc, 1963: Loud VA. A quanttatve stereo logcal descrpton of ultrastructure of normal rat lver parenchymal cells. I Cell Bo 1968;37: Schmucker DL, Mooney JS, Jones AL. Stereologcal analyss on hepatc fne structure n the Fscher 344 rat. Influence of sublobular locaton and anmal age. J Cell Bo 1978;78: Novkoff AB. Cell heterogenety wthn the hepatc lo bule of the rat (stanng reactons). I Hstochem Cytochem 1959; 7: Corrn B, Aterman K. The pattern of glycogen dstrbuton n the lver. Am J Anat 1968;122: Babcock MB, Cardell RR. Hepatc glycogen patterns n fasted and fed rats. Am J Anat 1974;14: Kanamura S, Asad a-kubota M, Kana K. The pattern of glycogen wthdrawal wthn the lver acnus durng fastng. Anat EmbryoI198;16: LeBouton AV. Heterogenety of proten metabolsm between lver cells as studed by radoautography. CUff Mol Bo 1968; 2: Wlson ER, Wllams WL. Responses of fatty lver of mce to carbon tetrachlorde. Anat Rec 1969;165: Jones AL, Fawcett DW. Hypertrophy of the agranular endoplasmc retculum n hamster lver nduced by phenobarbtal. J Hstochem Cytochem 1966;14: Burger PG, Herdson PB. Phenobarbtal-nduced fne structural changes n rat lver. Am I Pathol 1966;48: Becker FF, Lane BP. Regeneraton of the mammalan lver. VI. Retenton of phenobarbtal nduced cytoplasm c alteratons n dvdng hepatocytes. Am J PathoI1968;52: Bolender RP, Webel ER. A morphometrc study of the removal of phenobarbtal-nduced membranes from hepatocytes after cessaton of treatment. I Cell Bo 1973;56: Menard D, Berteloot A, Hugon JS. Acton of phenobarbta l on the ultrastructure and the enzymatc actvty of the mouse ntestne and the mouse lver. Hstocheme 1974a;38: Menard D, Penasse W, Drochmans p, Hugon JS. Glucose-6phosphatase heterogenety wthn the hepatc lobule of the phenobarbtal-treated rat. Hstocheme 1974b;38: Wanson IC, Drochmans P, May C, Penasse W, Popowsk A. Isolaton of centro lobular and per lobular hepatocytes after phenobarbtal treatment. J Cell Bo 1975;66: Kanamura S, Ogawa K. Cytochemcal localzaton of glucose6-phosphatase actvty n the lver of growng mce after phenobarbtal treatment. Acta Hstochem Cytochem 1977; 1: Kanamura S. Postnatal changes n the localzaton of glucose6-phosphatase actvty wthn the lver lobule of the mouse. Anat Rec 1975 ;181 :635-4.

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