Vet. Pathol. / 5: 753-762 (1978)
Effects of Anabolic Steroids on Liver Cell Ultrastructure in Sheep I. M . R EID. I. A. DONALDSO N and R. J. HEITZMAN
Agr icultural Researc h Council. Institute for Research on An imal Diseases. Compton. Newb ury . Berks hire . Eng land
Abstract. C astrated mal e shee p wer e sham impl a nted or impl a nted wit h estra dio l. tre n bolon e ace ta te or tre n bo lo ne ace ta te in combina tion with estrad iol. and sla ugh tere d afte r 56 days. Ultras truc tura l and mo rphome tric exam inatio n of the live rs of ste ro id-trea ted sheep showed a n in crease in the vo lume dens ity of the ce ll occupied by rough endoplasmic ret icu lum and micro bod ies. In four of the eight sheep treated wit h trenbolone acetate. e ithe r a lone or in com bina tion wit h estrad io l. enl arged mitoc hondria wit h cry stalli ne inclusions were seen . The increase in hepatic rough endoplasmic reticul um may be o f benefit to the shee p whereas the m itoch o nd ria l cha nges afte r anabolic ste roid use indicate a ce llular lesion of unkno wn significance for hea lth .
T renbolone ace ta te is a pot ent , synthetic anabolic steroid. In severa l co untries it is ad ministered as a subc uta neo us imp lant alone or in combination with estra dio l- 17,B and is cla ime d to grea tly increase the grow th rates of rum inant far m an ima ls [9, 10]. T he mode of action of anabolic steroids in promoting growth is not fully understood, but increased nitrogen retention is implicated and the liver almo st certai nly has a central role in the regulation of nitrogen retention [10]. T he possible hazard to the human po pulat ion of eati ng meat contai ning steroid residu es has received conside ra ble attention . T he potential hazard of impl anted anabo lic ste roids to the rec ipient far m a nima l has received less attention, despite the demon str at ed relati onsh ip between exoge no us steroid hormones (such as in ora l contracepti ves) and liver d isease in ma n [1-4] . Mate ria ls and Methods Sixteen castrated ma le sheep we re ra ndom ly assigned to four gr o ups and wit hi n each group assigned to one of four treatments. T hese treatments were I) contro l (sham implanted): 2) impl ant a tio n with 20 mi lligrams of 17{J-estrad iol: 3) implant at ion with 140 mi lligrams of tren bol on e acetate: and 4) impla ntati o n with a combi ned implant of 140 m illigra ms tren bo lo ne acetate an d 20 mi lligrams 17p-estrad iol. T he sheep were either Dorset Horn x Finn or Dorset Horn x Co b b Hyb rid a nd had a n average age of 7.25 mon ths (range 6 to 10 months) a t the sta rt of the ex pe rime nt. T hey were hou sed ind oor s in pen s 1.7x 1.9 mete rs a nd wer e fed dail y 0.4 kilograms of 22% crude prot ein concentrat e and 0.66 kilog ra m o f cho pped ha y. T he sheep were sla ug hte red 56 days a fter implantation . T hy m us. pituita ry. thyro id . hea rt a nd liver we re 753
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
754
Reid. Donaldson a nd He itzm a n
Table I. Body weight and organ weight of control, estradiol (OE2 ) , trenbolone acetate (T BA) and TBA+O E2 implanted sheep Slaughter weight, kg Liver, g Heart Thyroid Pituitary Thymus 1 2
Control
OE2
TBA
TBA+O E2
39.6 417.6 149.3 2.26 0.53 40.7
38.5 462.8 151.3 2.96 0.59 36.0
41.3 461.6 162.6 3.44 0.39 10.4 2
43.4 574.32 170.1 2.01 0.48 8.72
2.18 24.73 13.22 0.807 0.146 13 .1 8
Standard error of difference between means. Treatment mean significantly different from control ( P < 0.00I).
removed and weighed. Liver was fixed and processed for histological, electron microscopic and stereological examination by methods described [24, 25]. Results Liver weight a t sla ughte r was sign ifica ntly increased by 38% (P < 0.00 I) in shee p treated with trenbolon e acetate and estradiol together; th ymus weight was reduced significa ntly (P < 0.00 I) in sheep treated with trenbolo ne acetate alone or in combination with estradi ol (table I). Histology Examinatio n of paraffin sect ions of liver stained with haematoxylin and eosin (HE) a nd periodic acid -Schiff (PAS), and frozen sections stai ned with oil red 0 showed a norma l histological a ppea ra nce in all groups and no difference between contro l and treated sheep. Qualitative ultrastructure The ultrastructure of co ntro l sheep liver was in accord with previou s descriptions of cow [22] a nd sheep liver [23]. T he cyt oplasm contained large a mo unts of glycoge n arranged in rosettes a nd associated with areas of smoo th endoplasmic reticulum (fig . I). Parallel arrays of rough endopl asmic reticulum were freq uen tly seen surr ound ing or co ursing between mitochondria. The mitochondria ha d a fairly reg ular shape, being either oval or elongated with a length to d iameter ratio rarely greater than 5: I. The Golgi a ppa ratus a nd Iysosomes wer e mo st ofte n seen clo se to the bile canalicu li. In the sheep tre ated with trenbolone aceta te, either alone or in com bination with estradiol, changes in the hepatic mit och ondria of va rying severity wer e noted. In all four sheep treated with trenbolone aceta te alone a nd in three of the four shee p treated with the combined implant, enl ar ged mit ochondria were seen in ma ny of th e liver cells. In add ition, in two of the four sheep treated with trenbolone acetate and in two of the four sheep treated with the combined implan t large pleom orphi c mitoc hondria with crys talline incl usions were seen. T he size a nd shape of affected mitochondria were extremely va ria ble, some being enlarged with a roug hly spherical
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
Anabo lic Steroids and Hepatic Ultrastructure
755
Fig. I: Part of hepatocyte of con trol sheep showing mitochondria (M) , roug h endo plasm ic reticulu m (RER) and smoo th endoplasmic reticulum (S ER) . Bar = 1.0/lm. Fig. 2: Part of hepatocyte of sheep trea ted with trenbolone acetate and estradiol. Elongated biza rre mitocho ndria with crystalline incl usions. Bar = 1.0 iut» .
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
756
Reid . Dona ldson a nd He itzm a n
Fig. 3: Pa rt of hep atocyte of shee p trea ted with tren bolon e acetate. Enlarge d mitochondrion (M) with crysta lline inclu sions. Bar = 1.0 urn. Fig.4: Par t of hepat ocyt e of shee p treated with trenbolone ace tate . Enla rged mitocho nd ria. some with crys talline incl usio ns. Bar = 1.0 Il m .
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
757
Ana bo lic Ste roid s and Hep a tic Ultras truct ur e
Table II. Liver cell volume and volume den sity of fat droplets in liver of contro l and steroid treated sheep
Liver cell volu me, urn" Nu clear volume, l Volume den sity of fat dr oplets, flm :lj 100flm3 cell
5060
297 0.57
4159 251 1.07
4828 308 1.27
5149 258 0.53
475.2 39.4 0.540
Estrad iol. Trenbolone aceta te. :1 Standard error of di fference between mean s. I
2
shape (fig . 3,4), while o thers were elongated (fig . 2, 5) or bizarre in sha pe (fig . 6). In so me cases, the mitochondria l elongation was severe (fig. 5) with length to diameter ratios of 50: I a nd m itochondrial lengths of 18 micromete rs. Many of the m itochondria had fewer cri stae and numerous crysta lline inclu sion s (fig . 2-7). The inclusions con sisted of a regular, linear array of parallel stra nds with a centre-to-centre spa cing of around 100x 10- 7 mm (fig. 7). In th e e longated mit och ondria th e inclusions were parall el to th e lo ng axis of the mit och ond rion (fig. 2, 5) with peri odi c interruption s along th e length of th e crysta lline inclu sion mat er ial. In th e enla rged, sphe rica l mit och ondria, the inclus ions were see n d istributed in the matri x in a n a ppa re ntly random manner (fig . 3, 4). The per centage of cells affected by pleomorphic m itoch ond ria with crysta lline inclusion s va ried con siderably fro m block to block a nd sheep to she ep. In the most seve rely affected sheep, 20% of the liver ce lls exa mined contained mit och ondria with inclu sion s wh ereas in th e least seve re ly a ffected sheep about 5% of th e cells examined co nta ine d mit och ondria with inclusions. Mitochondri al inclusion s or pleomo rphic mit och ond ria were not see n in th e liver of control o r estradi ol-treat ed shee p. Steroid-related cha nges in o ther liver cell o rga nelles or in bile can ali culi were no t evide nt by inspect ion a ltho ug h spe cific cha nges in microbod ies a nd roug h endo plasm ic reti culum were reveal ed by stereolog ical method s as described in the foll owi ng sec tio n. Quantitative ultrastructure Live r cell vo lume, nuclear vo lume a nd volum e den sity of hepati c fat droplets, determ ined by point-counting toluidin e-blue stai ne d, I-m icr om eter sections of arald ire-embedded liver, wer e all un a ffect ed by steroid tr ea tment (table II). Implantation with all three ste ro id prep a rations, ho wever, resulted in a sta tistica lly significa nt 60% increase in hepati c ro ug h endo plas mic reti cu lum w hethe r ex pressed as vo lu me den sit y ([Lm 'f 100 um' cyto plas m) o r su rface den sit y ([Lm 2 f [Lm:1 cyto plas m) (table III ). In th e trenbolon e ace ta te gro up and in the es tradio l gro up, th ere was a sign ifica nt increase in th e volume den sit y of cyto plas m occ upied by microbodies (table III) . T he number and mean vo lume o f microbodi es a nd o f lysosom es, ca lcula ted by the for m ula o f Weibe l and Go mez [32] and a sha pe coe fficient of 1.45 (ta ble IV) were not significa ntly affec ted by ste ro id treatment. T he significa nt increase in vo lu me
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
75 8
Reid . Dona ldson and Heitzma n
6
7
5
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
Anabolic Steroids and Hepatic Ultrastructure
759
Table III. Volume den sity of cell organelle s and surface density of rough endoplasmic
reticu lum in liver of control and treated sheep
Mitochondria Microbodies Lysosomes Lipid droplets Golgi Smooth end oplasmic reticulum Rough endo plasmic reticulum
17.78 lAS 0.73
0.26 0.30 8.97 5.06
Volume dens ity fLm:1j 100fLm:1cytop lasm 18047 17.17 17.59 2.65[' 2.14 2.334 1.02 1.1 7 l.l 0 0.55 0.62 0.25 0.28 0.32 0.35 9.16 7.88 9.14 8.87['
8. 15"
8.9 1"
1.256 0.389 0.234 00462 0.112 1.700 0.921
Sur face density fLm1 jfLm :1 cytopla sm Ro ugh endoplasmic reticulum
0.83
1.32';
1.28['
1.26['
0.113
Estradi ol. Trenbolone acetate . 3 Stand ard error of difference between mean s. 4 Significantl y different from control P < 0.05. 5 Significantl y different from control P < 0.01. 6 Significantl y different from control P < 0.00 I. I
2
density of microbodies in the estradiol a nd in the trenbol one ac etate-trea ted grou ps is thus the product of non-significant in cr ea ses in both th e number a nd mean vo lumes of m icrobodies (table IV ). N o ne o f th e other ce ll o rg a ne lles sho wed any effect o f treatment. In view of th e m itoch ondrial enlargement seen in th e livers of sheep treated with trenbol one ac eta te, it wo u ld be o f interest to d et ermine th e number of mitochondria per unit vo lu m e of liv er cell and the ave rage m itoch ondrial vo lu me . This ca n be ac hie ved by a number of methods [17]; how e ver, a ll m eth ods m ake ce rta in assumptions a bo u t mitochondrial sha pe and size di stribution . It is cle ar that, in our case, such assumptions are not va lid becau se mitoch ondrial sha pe was a lte red by trenbolon e acetate treatment. The surface-to-vo lume ra tio o f mitochondria can be d eterm in ed , however, be cau se it is independent o f an y sha pe factors [15]. F ro m ta ble IV it can be see n that su rface/volu me ratio of h epatic mit och ondria was sign ifica ntly (P < 0.05 ) reduced in the tw o gr oups treat ed with tr enbol on e aceta te co m pa red wit h that in th e co n tro l g ro u p.
Fig. 5: Part of hepat ocyte of sheep treated with trenb olone acetate. Elongated mitochondria with blocks of crystalline material alon g long axis interrupted by gaps (a rrows). Bar = 0.8 fL m . Fig. 6: Part of hepat ocyte of sheep treated with trenbolone aceta te a nd estradiol. Bizarre shaped mitochond ria with inclusions. Bar = 1.0 iu v: Fig. 7: Mitochondri a from liver of sheep treated with trenbol one acetate. Periodicity of crysta lline material is IOOx 10- 7 mm. Bar = 004 iut» .
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
7 60
Reid. Do na ldson a nd Hei tzman
Table IV. Number and mean volume of microbodies and Iysosomes and surface:volume ratio in liver of control and treated sheep Number per 100 fl m" of cytoplasm Microbodies Lysosomes
2.08 2.26
3,44 2.62
3.89 2.24
3.83 2.97
0.924 0.574
0.747 0.382
0.2013 0.0752
Mean volume /lm:l Microbody Lysosome
0.682 0.322
0.620 0,462
0.765 0,457
Surface/Volume ratio flm 2 / urn" Mitochondria
5,46
4.864
5,40
4.97'
0.220
Estradiol. Trenbolone acetate. :1 Standard error of difference between means. 4 Significantly different from control P < 0.05. 1
2
Discussion Two significant cha nges occu rre d in hep a tocyte ultrastructu re afte r im plantatio n of sheep with the a na bo lic steroid, tren bolon e acetate. One, the inc rease in a mo unt of rough endoplasmic ret icu lum, is potentially of be nefit to the an imal: whereas the other, the occurrence of enlarged, pleomorphic mitochondria with cry sta lline incl usions , is potentially ha rm fu l to the animal. A major func tio n of th e liver is the partition of amino acids between utiliza tion for pro te in synt hesis and deam ination to yield substrates for energy production. In th e latter case , the amino group is incorporated into urea by liver enzymes. R ecent studies have shown that tre nbolone acetate reduced the urea en try ra te in cattle and shee p [6]. T his may resul t in relat ively more amino acids available for protein synthesis. Since a mino acid su pp ly is an im po rta nt regu la to r of protein syn thes is in liver [27], the increase in ro ugh endoplasmic retic ulum is consiste nt with increased hep ati c protein syn thes is after enha nced availa bi lity of a m ino aci ds . T he increased vo lume de nsity of micro bodies in liver of steroid treated shee p is of some in ter est. A variety of d rugs cau ses a n increase in hep ati c microbodies [29] including a number of hypolip ide mic age nts [13, 18]. It has been sugge sted th at the hyp olipidemi a is caused by an increased fatty acid ox ida tio n in hep a tic mic robod ies [13]. A number of ana boli c steroids are effective in lowering blood lipids in man [7] a nd we have so me pre limi nary ev idence that plasma cholesterol is lower ed in stee rs treated with tren bo lone acetate . There may be a rela tio nship between increased microbodies and hypo lipidemia in animals trea ted wit h anabolic steroids . C rysta lline inclusions in hepatic mitochondria have been described in a wide var iety of di sea ses of man [5, 8, 14,20,26,28] as well as in apparently no rmal livers o f man [33]. Giant mitochondria with crystalline inclusions a lso ha ve been described in the liver of healthy women on oral contrace ptives [12, 16, 19, 21], in patients given 17-alk ylated a nabolic steroids [ I I] and in ra ts treated with pregnenolone [30]. T he
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
A na bo lic Stero ids a nd Hepa tic U ltra stru ct ure
76 1
sig n ific a nce o f these c rys ta ll in e mitochondrial inclusi ons is co n t rove rs ia l; it is unclear w he the r th e in clusi ons indicate li ver injury o r are m ere exagge ra tio ns o f phy si ol o gical variation [5, 11 , 31]. T he occ u rre n ce , h o w e ve r, of mi toc ho n dr ia l in cl us io ns in the li ve r of wome n treated wit h co n trace p tive steroids a nd no w of shee p trea ted w it h anabo lic ste ro id s, a nd th e rela tio ns h ip between these ste ro id s a n d li ver tum ou rs [1-4] suggests tha t li ver d a m a g e s ho u ld be considered a po ten tia l h a zard o f a na bolic stero id imp la n ta tio n in fa r m a ni ma ls . F urt h er st udies are req uired to assess the seriousness of t he p ot ential h a zard .
Acknowledgements We th ank Me ssrs . R. A. C o llins a nd R. D. Harrison for techn ica l assis tance .
References I 2 3 4 5 6 7
8
9 10
II 12 13 14 15 16
17
ANONYMOUS: Live r tu mo u rs a nd ste roi d hormo nes. Lancet 2: 141\ L 1973 ANONYMO US: Live r tu mours a nd the pill. Br Me d 1 3:3-4, 1974 A NO NYM OUS: Oral contraceptives a nd live r nod ules. Lancet 1:843- 844, 1976 ANONYM OUS: Liver tu mo ur s an d the pill. Br Med 1 2:345, 1977 BHAGWAT, A.G .; Ross, R.e. : Hepa tic in tramitoc hondrial crysta lloid s. A rch Pa thoi 91:70- 77, 1971 DONALDSO N, I.A. : T he act ion of anabo lic steroids on nit rogen met ab olism a nd the endocrine system. Ph .D. the sis, U n iversity of Read ing, 1977 F ELTS, 1.M .: Clea ra nce of plasm a lipop ro tei ns: ro le o fl ipoprote in lip ase, lec ithin-c ho les tero l acyl-tra ns fera se, a nd th e effects of dru gs in Pharmaco logy of Lipid T ra nspo rt a nd At he rosclerotic Processes, ed . Ma so ro: pp . 375-402, Pergamo n Press, Oxford, 1975 GO MEZ- LAZA RO. e.: CARO- PATON, A.: PRIETO, 1.; COCA, M .e. : Intra mitocho ndria l pa racry stalline inclusions in hep at ocytes of patients with bili ary lithiasis. Lancet 2: 1417-1 418, 1972 HEITZMA N, R.J .: Ana bo lic ste roi ds: possible uses in meat a nd milk p roducti on . Ve t Rec 94:529 -532, 1974 HEITZM AN, R.J .: T he effective ness of a nabolic age n ts in increasing ra te o f growth in farm animals: re port on ex periments in cattle in Anabo lic Agents in An ima l Production . F AO/WHO Symposium , Rome, March 1975, ed . Lu an d Ren de l: pp. 89-98, Georg Thieme, St uttgart, 1976 KHAND EKAR, 1.0.: G ARG , B.D .: T UCHWEIIER, B.: KOVACS, K .: Effects of steroids u pon the liver ultrastruct u re . Rev Ca n Bio i 32: 121-135 , 1973 LARSSO N-COliN, U.: STENRA M, U.: Liv er ult rast ru cture and fu nction in icteric a nd nonicter ic wo men using o ra l co ntraceptive age nts. Ac ta Med Scand 181:257-264, 1967 LAZAROW, P.B.: Th ree hyp olip ide m ic drugs increase hepatic pa lmitoylcoe nzy me A oxi d ation in the rat. Science 197:560-5 81, 1977 LEEVY, e. M .: CHEN, T.: Z AKI, F.G .: Differential diagn osi s of ch olestasis. Adv Int ern Me d 17:323 -342, 197 1 LOUD, A.V. : BARANY, W .e.: PACK, B.A.: Qua nti ta tive eval ua tion of cy top las mic struc tu res in electro n m icrographs. Lab Invest 14:996-1 008, 1965 MART INEZ-MANAUTO U, 1.: AZNAR- RAMOS, R.: BAUTI STA-O'FAR RILL, 1.: GONZALEZ-ANGULO, A: T he ultrastructure of live r cells in wo men u nd e r ste roid the ra py. II. C ontrace ptive thera py . Acta Endoc rinol (Kb h) 65:207-22 L 1970 MAYH EW, T.M .: A com pa rison of several me thods fo r stereo logica l d eterminati o n of the n umbers o f orga nelles per u ni t vo lume o f cytoplasm. 1 Microsc 96:37 -44, 1972
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
76 2
Reid, Donaldson and Heitzman
18 MOODY, D.E.; R EDDY, 1.K .: Morphometric a na lysis o f the ultr astructural cha nge s in rat liver indu ced by the peroxisome p rolife rat or SaH 42-348. 1 Cell Bio i 7 1:768- 780. 1976 19 PER EZ. V.; G OROSDlSCH, S.; DE MARTIRE, 1.; NI CHOLSON, R.; Dl PAOLA, G .: Oral co ntraceptives: long term use prod uce s tine structu ra l cha nges in live r mit ochondria. Scienc e 165:805- 807, 1969 20 PETERSEN, P.: Abn orma l mit ochondria in hepa tocytes in hum an fatt y liver. Act a Pa thoi Microbiol Sca nd (A) 85:4 13- 420, 1977 2 1 PIHL, E.; R AISE, 0 .; Z EUCHNER, E.: F u nctiona l a nd mo rpho logical liver cha nges in wom en taking ora l contraceptives. Acta C hir Scand 134:639- 650, 1968 22 R EID, I.M .: An ul tra stru ctural and morphome tric study o f the liver o f the la cta ting cow in sta rva tion ketos is. Ex p Mo l Pat ho l 18:316- 330, 1973 23 R EID, I.M .; HALL, G .A.: A n ult ra structura l a nd biochemica l study o f hexachl o ro ph an eind uced fa tty liver in sheep. J Pathol 115:33- 43, 1975 24 R EID, I.M .; BAI RD, G .D .; H EITZMAN, R.J .: Effects o f fa stin g in th e non-lacta ti ng cow . A co rr ela ted biochemica l a nd stereo logical study o f fasting-induced fa tty live r. J Agric Sci 89:3 19-325, 1977 25 R EID, I.M. ; HARR ISON, R .D. : COLLINS, R .A. : Fa st ing a nd refeed ing in the la ctat ing dairy cow. 2. T he recovery o f liver cell stru cture a nd fun cti on followi ng a six-day fa st. 1 Co m p Pa th ol 87:253- 265, 1977 26 R UFFOLO, R. ; COVINGTO N, H.: Matrix inclu sion bod ies in the mit och ond ria o f th e hum an liver. Evidence o f hepatocellul ar inju ry. Am 1 Pa th o l 5 1: 101-11 6. 1967 27 SIDRANSKY, H.: N u tr itiona l di sturba nces o f protein meta boli sm in the liver. Am J Patho l 84:649- 667, 1976 28 STERNLI EB, I.: BERGER, 1.E.: O ptica l d iffra ctio n stud ies o f crysta lline structures in electron microgr aphs. II. C rys ta lline inclusion s in m itoch ond rial o f hum an hep at ocytes. 1 Cell Bioi 43:44 8-455, 1969 29 SVOBODA, D.; G RA DY, H .; AZAR NOFF, D.: Micr ob odies in ex perimen ta lly a ltered ce lls. J Cell Bioi 35: 127- 152, 1967 30 T UCHWEBER, B.; KOVACS, K .: K HANDEKAR, 1.0.; G AR G, B.D .: Intra mit ochon dri all a mell ar format ion s in du ced by pr egn en olon e-16-a -carbon itr ile in th e hep a tocyt es o f p regna nt rat s. 1 U ltrastruc t Res 39:456-464, 1972 3 1 VERIIEYEN, A.; BORGERS, M .: Effects o f levamiso le o n the ultr astructure o f mit och ondria in th e live r o f Beagle dogs. Vet Pa tho l 13: 131-1 37, 1976 32 W EIB EL, E.R.; G OM EZ, D.M .: A princip le for counting tissue stru ctu res o n rand om sections. 1 Appl Ph ysiol 17:343- 348, 1962 33 WILLS, E.J .: Crys ta lline stru ctur es in the mitoch ondria o f norma l h um an liver pa re nch ymal cells. 1 Cell Bioi 24:51 1- 5 14, 1965
Req ue st reprint s from Dr. 1. M . Reid , De part men t of C ellula r Pa th o logy, In stitute for Resear ch on Anima l Diseases, Co m pto n, N ewbu ry, Berk shire , R G 16 ONN , (U .K.).
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
Effects of Anabolic Steroids on Liver Cell Ultrastructure in Sheep I. M . R EID. I. A. DONALDSO N and R. J. HEITZMAN
Agr icultural Researc h Council. Institute for Research on An imal Diseases. Compton. Newb ury . Berks hire . Eng land
Abstract. C astrated mal e shee p wer e sham impl a nted or impl a nted wit h estra dio l. tre n bolon e ace ta te or tre n bo lo ne ace ta te in combina tion with estrad iol. and sla ugh tere d afte r 56 days. Ultras truc tura l and mo rphome tric exam inatio n of the live rs of ste ro id-trea ted sheep showed a n in crease in the vo lume dens ity of the ce ll occupied by rough endoplasmic ret icu lum and micro bod ies. In four of the eight sheep treated wit h trenbolone acetate. e ithe r a lone or in com bina tion wit h estrad io l. enl arged mitoc hondria wit h cry stalli ne inclusions were seen . The increase in hepatic rough endoplasmic reticul um may be o f benefit to the shee p whereas the m itoch o nd ria l cha nges afte r anabolic ste roid use indicate a ce llular lesion of unkno wn significance for hea lth .
T renbolone ace ta te is a pot ent , synthetic anabolic steroid. In severa l co untries it is ad ministered as a subc uta neo us imp lant alone or in combination with estra dio l- 17,B and is cla ime d to grea tly increase the grow th rates of rum inant far m an ima ls [9, 10]. T he mode of action of anabolic steroids in promoting growth is not fully understood, but increased nitrogen retention is implicated and the liver almo st certai nly has a central role in the regulation of nitrogen retention [10]. T he possible hazard to the human po pulat ion of eati ng meat contai ning steroid residu es has received conside ra ble attention . T he potential hazard of impl anted anabo lic ste roids to the rec ipient far m a nima l has received less attention, despite the demon str at ed relati onsh ip between exoge no us steroid hormones (such as in ora l contracepti ves) and liver d isease in ma n [1-4] . Mate ria ls and Methods Sixteen castrated ma le sheep we re ra ndom ly assigned to four gr o ups and wit hi n each group assigned to one of four treatments. T hese treatments were I) contro l (sham implanted): 2) impl ant a tio n with 20 mi lligrams of 17{J-estrad iol: 3) implant at ion with 140 mi lligrams of tren bol on e acetate: and 4) impla ntati o n with a combi ned implant of 140 m illigra ms tren bo lo ne acetate an d 20 mi lligrams 17p-estrad iol. T he sheep were either Dorset Horn x Finn or Dorset Horn x Co b b Hyb rid a nd had a n average age of 7.25 mon ths (range 6 to 10 months) a t the sta rt of the ex pe rime nt. T hey were hou sed ind oor s in pen s 1.7x 1.9 mete rs a nd wer e fed dail y 0.4 kilograms of 22% crude prot ein concentrat e and 0.66 kilog ra m o f cho pped ha y. T he sheep were sla ug hte red 56 days a fter implantation . T hy m us. pituita ry. thyro id . hea rt a nd liver we re 753
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
754
Reid. Donaldson a nd He itzm a n
Table I. Body weight and organ weight of control, estradiol (OE2 ) , trenbolone acetate (T BA) and TBA+O E2 implanted sheep Slaughter weight, kg Liver, g Heart Thyroid Pituitary Thymus 1 2
Control
OE2
TBA
TBA+O E2
39.6 417.6 149.3 2.26 0.53 40.7
38.5 462.8 151.3 2.96 0.59 36.0
41.3 461.6 162.6 3.44 0.39 10.4 2
43.4 574.32 170.1 2.01 0.48 8.72
2.18 24.73 13.22 0.807 0.146 13 .1 8
Standard error of difference between means. Treatment mean significantly different from control ( P < 0.00I).
removed and weighed. Liver was fixed and processed for histological, electron microscopic and stereological examination by methods described [24, 25]. Results Liver weight a t sla ughte r was sign ifica ntly increased by 38% (P < 0.00 I) in shee p treated with trenbolon e acetate and estradiol together; th ymus weight was reduced significa ntly (P < 0.00 I) in sheep treated with trenbolo ne acetate alone or in combination with estradi ol (table I). Histology Examinatio n of paraffin sect ions of liver stained with haematoxylin and eosin (HE) a nd periodic acid -Schiff (PAS), and frozen sections stai ned with oil red 0 showed a norma l histological a ppea ra nce in all groups and no difference between contro l and treated sheep. Qualitative ultrastructure The ultrastructure of co ntro l sheep liver was in accord with previou s descriptions of cow [22] a nd sheep liver [23]. T he cyt oplasm contained large a mo unts of glycoge n arranged in rosettes a nd associated with areas of smoo th endoplasmic reticulum (fig . I). Parallel arrays of rough endopl asmic reticulum were freq uen tly seen surr ound ing or co ursing between mitochondria. The mitochondria ha d a fairly reg ular shape, being either oval or elongated with a length to d iameter ratio rarely greater than 5: I. The Golgi a ppa ratus a nd Iysosomes wer e mo st ofte n seen clo se to the bile canalicu li. In the sheep tre ated with trenbolone aceta te, either alone or in com bination with estradiol, changes in the hepatic mit och ondria of va rying severity wer e noted. In all four sheep treated with trenbolone aceta te alone a nd in three of the four shee p treated with the combined implant, enl ar ged mit ochondria were seen in ma ny of th e liver cells. In add ition, in two of the four sheep treated with trenbolone acetate and in two of the four sheep treated with the combined implan t large pleom orphi c mitoc hondria with crys talline incl usions were seen. T he size a nd shape of affected mitochondria were extremely va ria ble, some being enlarged with a roug hly spherical
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
Anabo lic Steroids and Hepatic Ultrastructure
755
Fig. I: Part of hepatocyte of con trol sheep showing mitochondria (M) , roug h endo plasm ic reticulu m (RER) and smoo th endoplasmic reticulum (S ER) . Bar = 1.0/lm. Fig. 2: Part of hepatocyte of sheep trea ted with trenbolone acetate and estradiol. Elongated biza rre mitocho ndria with crystalline incl usions. Bar = 1.0 iut» .
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
756
Reid . Dona ldson a nd He itzm a n
Fig. 3: Pa rt of hep atocyte of shee p trea ted with tren bolon e acetate. Enlarge d mitochondrion (M) with crysta lline inclu sions. Bar = 1.0 urn. Fig.4: Par t of hepat ocyt e of shee p treated with trenbolone ace tate . Enla rged mitocho nd ria. some with crys talline incl usio ns. Bar = 1.0 Il m .
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
757
Ana bo lic Ste roid s and Hep a tic Ultras truct ur e
Table II. Liver cell volume and volume den sity of fat droplets in liver of contro l and steroid treated sheep
Liver cell volu me, urn" Nu clear volume, l Volume den sity of fat dr oplets, flm :lj 100flm3 cell
5060
297 0.57
4159 251 1.07
4828 308 1.27
5149 258 0.53
475.2 39.4 0.540
Estrad iol. Trenbolone aceta te. :1 Standard error of di fference between mean s. I
2
shape (fig . 3,4), while o thers were elongated (fig . 2, 5) or bizarre in sha pe (fig . 6). In so me cases, the mitochondria l elongation was severe (fig. 5) with length to diameter ratios of 50: I a nd m itochondrial lengths of 18 micromete rs. Many of the m itochondria had fewer cri stae and numerous crysta lline inclu sion s (fig . 2-7). The inclusions con sisted of a regular, linear array of parallel stra nds with a centre-to-centre spa cing of around 100x 10- 7 mm (fig. 7). In th e e longated mit och ondria th e inclusions were parall el to th e lo ng axis of the mit och ond rion (fig. 2, 5) with peri odi c interruption s along th e length of th e crysta lline inclu sion mat er ial. In th e enla rged, sphe rica l mit och ondria, the inclus ions were see n d istributed in the matri x in a n a ppa re ntly random manner (fig . 3, 4). The per centage of cells affected by pleomorphic m itoch ond ria with crysta lline inclusion s va ried con siderably fro m block to block a nd sheep to she ep. In the most seve rely affected sheep, 20% of the liver ce lls exa mined contained mit och ondria with inclu sion s wh ereas in th e least seve re ly a ffected sheep about 5% of th e cells examined co nta ine d mit och ondria with inclusions. Mitochondri al inclusion s or pleomo rphic mit och ond ria were not see n in th e liver of control o r estradi ol-treat ed shee p. Steroid-related cha nges in o ther liver cell o rga nelles or in bile can ali culi were no t evide nt by inspect ion a ltho ug h spe cific cha nges in microbod ies a nd roug h endo plasm ic reti culum were reveal ed by stereolog ical method s as described in the foll owi ng sec tio n. Quantitative ultrastructure Live r cell vo lume, nuclear vo lume a nd volum e den sity of hepati c fat droplets, determ ined by point-counting toluidin e-blue stai ne d, I-m icr om eter sections of arald ire-embedded liver, wer e all un a ffect ed by steroid tr ea tment (table II). Implantation with all three ste ro id prep a rations, ho wever, resulted in a sta tistica lly significa nt 60% increase in hepati c ro ug h endo plas mic reti cu lum w hethe r ex pressed as vo lu me den sit y ([Lm 'f 100 um' cyto plas m) o r su rface den sit y ([Lm 2 f [Lm:1 cyto plas m) (table III ). In th e trenbolon e ace ta te gro up and in the es tradio l gro up, th ere was a sign ifica nt increase in th e volume den sit y of cyto plas m occ upied by microbodies (table III) . T he number and mean vo lume o f microbodi es a nd o f lysosom es, ca lcula ted by the for m ula o f Weibe l and Go mez [32] and a sha pe coe fficient of 1.45 (ta ble IV) were not significa ntly affec ted by ste ro id treatment. T he significa nt increase in vo lu me
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
75 8
Reid . Dona ldson and Heitzma n
6
7
5
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
Anabolic Steroids and Hepatic Ultrastructure
759
Table III. Volume den sity of cell organelle s and surface density of rough endoplasmic
reticu lum in liver of control and treated sheep
Mitochondria Microbodies Lysosomes Lipid droplets Golgi Smooth end oplasmic reticulum Rough endo plasmic reticulum
17.78 lAS 0.73
0.26 0.30 8.97 5.06
Volume dens ity fLm:1j 100fLm:1cytop lasm 18047 17.17 17.59 2.65[' 2.14 2.334 1.02 1.1 7 l.l 0 0.55 0.62 0.25 0.28 0.32 0.35 9.16 7.88 9.14 8.87['
8. 15"
8.9 1"
1.256 0.389 0.234 00462 0.112 1.700 0.921
Sur face density fLm1 jfLm :1 cytopla sm Ro ugh endoplasmic reticulum
0.83
1.32';
1.28['
1.26['
0.113
Estradi ol. Trenbolone acetate . 3 Stand ard error of difference between mean s. 4 Significantl y different from control P < 0.05. 5 Significantl y different from control P < 0.01. 6 Significantl y different from control P < 0.00 I. I
2
density of microbodies in the estradiol a nd in the trenbol one ac etate-trea ted grou ps is thus the product of non-significant in cr ea ses in both th e number a nd mean vo lumes of m icrobodies (table IV ). N o ne o f th e other ce ll o rg a ne lles sho wed any effect o f treatment. In view of th e m itoch ondrial enlargement seen in th e livers of sheep treated with trenbol one ac eta te, it wo u ld be o f interest to d et ermine th e number of mitochondria per unit vo lu m e of liv er cell and the ave rage m itoch ondrial vo lu me . This ca n be ac hie ved by a number of methods [17]; how e ver, a ll m eth ods m ake ce rta in assumptions a bo u t mitochondrial sha pe and size di stribution . It is cle ar that, in our case, such assumptions are not va lid becau se mitoch ondrial sha pe was a lte red by trenbolon e acetate treatment. The surface-to-vo lume ra tio o f mitochondria can be d eterm in ed , however, be cau se it is independent o f an y sha pe factors [15]. F ro m ta ble IV it can be see n that su rface/volu me ratio of h epatic mit och ondria was sign ifica ntly (P < 0.05 ) reduced in the tw o gr oups treat ed with tr enbol on e aceta te co m pa red wit h that in th e co n tro l g ro u p.
Fig. 5: Part of hepat ocyte of sheep treated with trenb olone acetate. Elongated mitochondria with blocks of crystalline material alon g long axis interrupted by gaps (a rrows). Bar = 0.8 fL m . Fig. 6: Part of hepat ocyte of sheep treated with trenbolone aceta te a nd estradiol. Bizarre shaped mitochond ria with inclusions. Bar = 1.0 iu v: Fig. 7: Mitochondri a from liver of sheep treated with trenbol one acetate. Periodicity of crysta lline material is IOOx 10- 7 mm. Bar = 004 iut» .
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
7 60
Reid. Do na ldson a nd Hei tzman
Table IV. Number and mean volume of microbodies and Iysosomes and surface:volume ratio in liver of control and treated sheep Number per 100 fl m" of cytoplasm Microbodies Lysosomes
2.08 2.26
3,44 2.62
3.89 2.24
3.83 2.97
0.924 0.574
0.747 0.382
0.2013 0.0752
Mean volume /lm:l Microbody Lysosome
0.682 0.322
0.620 0,462
0.765 0,457
Surface/Volume ratio flm 2 / urn" Mitochondria
5,46
4.864
5,40
4.97'
0.220
Estradiol. Trenbolone acetate. :1 Standard error of difference between means. 4 Significantly different from control P < 0.05. 1
2
Discussion Two significant cha nges occu rre d in hep a tocyte ultrastructu re afte r im plantatio n of sheep with the a na bo lic steroid, tren bolon e acetate. One, the inc rease in a mo unt of rough endoplasmic ret icu lum, is potentially of be nefit to the an imal: whereas the other, the occurrence of enlarged, pleomorphic mitochondria with cry sta lline incl usions , is potentially ha rm fu l to the animal. A major func tio n of th e liver is the partition of amino acids between utiliza tion for pro te in synt hesis and deam ination to yield substrates for energy production. In th e latter case , the amino group is incorporated into urea by liver enzymes. R ecent studies have shown that tre nbolone acetate reduced the urea en try ra te in cattle and shee p [6]. T his may resul t in relat ively more amino acids available for protein synthesis. Since a mino acid su pp ly is an im po rta nt regu la to r of protein syn thes is in liver [27], the increase in ro ugh endoplasmic retic ulum is consiste nt with increased hep ati c protein syn thes is after enha nced availa bi lity of a m ino aci ds . T he increased vo lume de nsity of micro bodies in liver of steroid treated shee p is of some in ter est. A variety of d rugs cau ses a n increase in hep ati c microbodies [29] including a number of hypolip ide mic age nts [13, 18]. It has been sugge sted th at the hyp olipidemi a is caused by an increased fatty acid ox ida tio n in hep a tic mic robod ies [13]. A number of ana boli c steroids are effective in lowering blood lipids in man [7] a nd we have so me pre limi nary ev idence that plasma cholesterol is lower ed in stee rs treated with tren bo lone acetate . There may be a rela tio nship between increased microbodies and hypo lipidemia in animals trea ted wit h anabolic steroids . C rysta lline inclusions in hepatic mitochondria have been described in a wide var iety of di sea ses of man [5, 8, 14,20,26,28] as well as in apparently no rmal livers o f man [33]. Giant mitochondria with crystalline inclusions a lso ha ve been described in the liver of healthy women on oral contrace ptives [12, 16, 19, 21], in patients given 17-alk ylated a nabolic steroids [ I I] and in ra ts treated with pregnenolone [30]. T he
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
A na bo lic Stero ids a nd Hepa tic U ltra stru ct ure
76 1
sig n ific a nce o f these c rys ta ll in e mitochondrial inclusi ons is co n t rove rs ia l; it is unclear w he the r th e in clusi ons indicate li ver injury o r are m ere exagge ra tio ns o f phy si ol o gical variation [5, 11 , 31]. T he occ u rre n ce , h o w e ve r, of mi toc ho n dr ia l in cl us io ns in the li ve r of wome n treated wit h co n trace p tive steroids a nd no w of shee p trea ted w it h anabo lic ste ro id s, a nd th e rela tio ns h ip between these ste ro id s a n d li ver tum ou rs [1-4] suggests tha t li ver d a m a g e s ho u ld be considered a po ten tia l h a zard o f a na bolic stero id imp la n ta tio n in fa r m a ni ma ls . F urt h er st udies are req uired to assess the seriousness of t he p ot ential h a zard .
Acknowledgements We th ank Me ssrs . R. A. C o llins a nd R. D. Harrison for techn ica l assis tance .
References I 2 3 4 5 6 7
8
9 10
II 12 13 14 15 16
17
ANONYMOUS: Live r tu mo u rs a nd ste roi d hormo nes. Lancet 2: 141\ L 1973 ANONYMO US: Live r tu mours a nd the pill. Br Me d 1 3:3-4, 1974 A NO NYM OUS: Oral contraceptives a nd live r nod ules. Lancet 1:843- 844, 1976 ANONYM OUS: Liver tu mo ur s an d the pill. Br Med 1 2:345, 1977 BHAGWAT, A.G .; Ross, R.e. : Hepa tic in tramitoc hondrial crysta lloid s. A rch Pa thoi 91:70- 77, 1971 DONALDSO N, I.A. : T he act ion of anabo lic steroids on nit rogen met ab olism a nd the endocrine system. Ph .D. the sis, U n iversity of Read ing, 1977 F ELTS, 1.M .: Clea ra nce of plasm a lipop ro tei ns: ro le o fl ipoprote in lip ase, lec ithin-c ho les tero l acyl-tra ns fera se, a nd th e effects of dru gs in Pharmaco logy of Lipid T ra nspo rt a nd At he rosclerotic Processes, ed . Ma so ro: pp . 375-402, Pergamo n Press, Oxford, 1975 GO MEZ- LAZA RO. e.: CARO- PATON, A.: PRIETO, 1.; COCA, M .e. : Intra mitocho ndria l pa racry stalline inclusions in hep at ocytes of patients with bili ary lithiasis. Lancet 2: 1417-1 418, 1972 HEITZMA N, R.J .: Ana bo lic ste roi ds: possible uses in meat a nd milk p roducti on . Ve t Rec 94:529 -532, 1974 HEITZM AN, R.J .: T he effective ness of a nabolic age n ts in increasing ra te o f growth in farm animals: re port on ex periments in cattle in Anabo lic Agents in An ima l Production . F AO/WHO Symposium , Rome, March 1975, ed . Lu an d Ren de l: pp. 89-98, Georg Thieme, St uttgart, 1976 KHAND EKAR, 1.0.: G ARG , B.D .: T UCHWEIIER, B.: KOVACS, K .: Effects of steroids u pon the liver ultrastruct u re . Rev Ca n Bio i 32: 121-135 , 1973 LARSSO N-COliN, U.: STENRA M, U.: Liv er ult rast ru cture and fu nction in icteric a nd nonicter ic wo men using o ra l co ntraceptive age nts. Ac ta Med Scand 181:257-264, 1967 LAZAROW, P.B.: Th ree hyp olip ide m ic drugs increase hepatic pa lmitoylcoe nzy me A oxi d ation in the rat. Science 197:560-5 81, 1977 LEEVY, e. M .: CHEN, T.: Z AKI, F.G .: Differential diagn osi s of ch olestasis. Adv Int ern Me d 17:323 -342, 197 1 LOUD, A.V. : BARANY, W .e.: PACK, B.A.: Qua nti ta tive eval ua tion of cy top las mic struc tu res in electro n m icrographs. Lab Invest 14:996-1 008, 1965 MART INEZ-MANAUTO U, 1.: AZNAR- RAMOS, R.: BAUTI STA-O'FAR RILL, 1.: GONZALEZ-ANGULO, A: T he ultrastructure of live r cells in wo men u nd e r ste roid the ra py. II. C ontrace ptive thera py . Acta Endoc rinol (Kb h) 65:207-22 L 1970 MAYH EW, T.M .: A com pa rison of several me thods fo r stereo logica l d eterminati o n of the n umbers o f orga nelles per u ni t vo lume o f cytoplasm. 1 Microsc 96:37 -44, 1972
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
76 2
Reid, Donaldson and Heitzman
18 MOODY, D.E.; R EDDY, 1.K .: Morphometric a na lysis o f the ultr astructural cha nge s in rat liver indu ced by the peroxisome p rolife rat or SaH 42-348. 1 Cell Bio i 7 1:768- 780. 1976 19 PER EZ. V.; G OROSDlSCH, S.; DE MARTIRE, 1.; NI CHOLSON, R.; Dl PAOLA, G .: Oral co ntraceptives: long term use prod uce s tine structu ra l cha nges in live r mit ochondria. Scienc e 165:805- 807, 1969 20 PETERSEN, P.: Abn orma l mit ochondria in hepa tocytes in hum an fatt y liver. Act a Pa thoi Microbiol Sca nd (A) 85:4 13- 420, 1977 2 1 PIHL, E.; R AISE, 0 .; Z EUCHNER, E.: F u nctiona l a nd mo rpho logical liver cha nges in wom en taking ora l contraceptives. Acta C hir Scand 134:639- 650, 1968 22 R EID, I.M .: An ul tra stru ctural and morphome tric study o f the liver o f the la cta ting cow in sta rva tion ketos is. Ex p Mo l Pat ho l 18:316- 330, 1973 23 R EID, I.M .; HALL, G .A.: A n ult ra structura l a nd biochemica l study o f hexachl o ro ph an eind uced fa tty liver in sheep. J Pathol 115:33- 43, 1975 24 R EID, I.M .; BAI RD, G .D .; H EITZMAN, R.J .: Effects o f fa stin g in th e non-lacta ti ng cow . A co rr ela ted biochemica l a nd stereo logical study o f fasting-induced fa tty live r. J Agric Sci 89:3 19-325, 1977 25 R EID, I.M. ; HARR ISON, R .D. : COLLINS, R .A. : Fa st ing a nd refeed ing in the la ctat ing dairy cow. 2. T he recovery o f liver cell stru cture a nd fun cti on followi ng a six-day fa st. 1 Co m p Pa th ol 87:253- 265, 1977 26 R UFFOLO, R. ; COVINGTO N, H.: Matrix inclu sion bod ies in the mit och ond ria o f th e hum an liver. Evidence o f hepatocellul ar inju ry. Am 1 Pa th o l 5 1: 101-11 6. 1967 27 SIDRANSKY, H.: N u tr itiona l di sturba nces o f protein meta boli sm in the liver. Am J Patho l 84:649- 667, 1976 28 STERNLI EB, I.: BERGER, 1.E.: O ptica l d iffra ctio n stud ies o f crysta lline structures in electron microgr aphs. II. C rys ta lline inclusion s in m itoch ond rial o f hum an hep at ocytes. 1 Cell Bioi 43:44 8-455, 1969 29 SVOBODA, D.; G RA DY, H .; AZAR NOFF, D.: Micr ob odies in ex perimen ta lly a ltered ce lls. J Cell Bioi 35: 127- 152, 1967 30 T UCHWEBER, B.; KOVACS, K .: K HANDEKAR, 1.0.; G AR G, B.D .: Intra mit ochon dri all a mell ar format ion s in du ced by pr egn en olon e-16-a -carbon itr ile in th e hep a tocyt es o f p regna nt rat s. 1 U ltrastruc t Res 39:456-464, 1972 3 1 VERIIEYEN, A.; BORGERS, M .: Effects o f levamiso le o n the ultr astructure o f mit och ondria in th e live r o f Beagle dogs. Vet Pa tho l 13: 131-1 37, 1976 32 W EIB EL, E.R.; G OM EZ, D.M .: A princip le for counting tissue stru ctu res o n rand om sections. 1 Appl Ph ysiol 17:343- 348, 1962 33 WILLS, E.J .: Crys ta lline stru ctur es in the mitoch ondria o f norma l h um an liver pa re nch ymal cells. 1 Cell Bioi 24:51 1- 5 14, 1965
Req ue st reprint s from Dr. 1. M . Reid , De part men t of C ellula r Pa th o logy, In stitute for Resear ch on Anima l Diseases, Co m pto n, N ewbu ry, Berk shire , R G 16 ONN , (U .K.).
Downloaded from vet.sagepub.com at IOWA TESTING PROGRAMS on March 13, 2015
