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Adenofibrosis in the Rat Liver With Persistence of Polychlorinated Biphenyls in Adipose Tissue Rrnatr D. Kimbrough, MD, Ralph E. Linder; Virlyn W. Buret; Ralph W. Jennings, Chambltt, Ga Fifty mils Shtrmsn strain rati wars fad 500 ppm of a polychlorinated biphenyl (PCI) (Aroelor 1254) for six months. Fivo each wart kill'd tiro, ont, two. throo. fonr, six, siiht, and tan months aftor exposure to Arofifor had caasad. Tho Uvars of thosa rats woro oxaminod by light and oioctron mi croscopy. Uvor lotions porsistod although oiposurs to PCI* ciastd. Tan months aftor oxposuro coasod, 1,192 ppm PCIs woro still prosont in tho rats' adipate tissue and 22.55 ppm in tho rat (Ivors. Aroelor pottoms found in tho tissues by etoctron capture gas chromatography dif fered from patterns of dietary Arociors. Maos spectral analysis of liver and adipose tissue rovoolod throo major Aroelor compoAonit with mosses of 324, 359, and 392. Those contained isotopic clusters indica tive at tho pretence of Cl,, Cl,, and C1,, roapeetiveiy. n a previously reported study1 I Sherman strain rata were fed poly chlorinated biphenyls (PCBs) (Aroelor 1204 and Aroelor 1260). Tho rats developed characteristic mor phological changes in the liver. These changes consisted of hypertrophy of the liver cells, a brown pigment in Kupffer cells, lipid accumulation in the cytoplasm of hepatocyteg and, at the higher dietary levels, adenofi brosis. Because of the wide distribu tion of PCBs in the environment* and Submitted Tor publication March & 1973; ac cepted April IP From Ih# Environmental Protection Agency, Bio-Effect* Brunch, CHamblee, Ga. Reprint requesta to 16S Avery Dr NE, Atlanta 30309 (Dr KimbroufhV their persistence, the hepatic lesioa was studied further. The present study was undertaken to see whether, once exposure to PCB6 was stopped, the morphological changes produoad in the liver might disappear. Materials and Methods A total of 50 male weanling, specificpathogen-free, random-bred Shermaa strain rata were tagged individually and group-caged, ten rats per cage. They wet* given Aroelor 1254 in their diet for at months. The dose of 500 ppm, which it equivalent to an average daily intake af 36.4 mg/kg body weight.' was chosen b* cause in the previous study ail ten exposed male rats developed adenoftbrosia within a period of eight months. The Aroelor was mixed into ground laboratory chow aa pre viously described.' After six months cf Fig 3,-Seci-wrt Insgnrfication 5 | Frg 4. - Port-"', Icitrata-uraiHi -:i Fig 1.-Area of adenofibrosis in llvar section consisting primarily of duels surrounded by (laltonad epithelium. Oucis are surrounded by little tlbrosts (hamatoxylin-eosin, original magnification x 150). Fig 2.-Rat liver section showing cluster of small columnar cells mem bling pancreatic tissue adjacent to blood vessel. Hepatocytes surround ing lesion are vacuolated (hsmatoxylln-eosin, original magmticatisa x 1751. 390 Arch Environ Heslth/Vol 27, Dec 1973 !f Adenofibrosis/Kimdrough et a! lArcti Environ HONS 081765 hepnlic lesion Tin- presont to sue whether, 4 wilt. Mopped, nge* produced (fig 3.-SCtion of hepatic tissue in latter part of recovery phase Illustrating numerous small lipid vacuoles (LV) (lead citrate-uranyl acetate, original 'pear. Lgniflcationx 51,300). Method* imling. Npociflc* hied Sherman jtdivnlunlly and They were fir dirt Tor ail 1 ppm. which ia daily minke of won Imaen be* veil fen exposed iihro.i.< within e ' ho AMtclor wea iryi l. i\v oa pre> six months of inner i Mis resem- loeyli vtirrounOnei m.'Kinlicetion imbrough it el Itrch Environ Health/Vot 27, Dec 1973 Adenofibrosis/Kimbrough et al 381 MUMS 081766 -M .L o *1 in z o a VI Tig 5.- Border of are* of edenotibrosia in liver. Note round clutters of dark granules intermixed with web-like clusters of vacuoles. These formations iduitrate appearance of brown pigment (BP). C represents collagen; 1C, liver cell (lead citrate-uranyl acetate, original magnification x 20,235) rucplving the experimental diet, the rats received no more Aroclor and were fed plain Inlminlmy cl>w At thin lime five rntx were killed. Additionnl killings of five randomly soluclvd rule were made one. two. three, four, nix, eight, and ten months alter ox|Mure to PCI) had been dixcontinlied. At autopsy the livers were removed, woighed, and the tiaaue fixed in phoa* plmle-buflured 5% giuUraldehyde for three hours for elect non microscopic exam ination, post-fixed in 1% osmium tclroxide. vmliedded in mornplass, sectioned with o glass knife, and stained with lead citrate and urnnyl acetate. Tissue for the light microscope was fixed in 4% buffered hiininldehydo solution and stained with hematoxylin and eonm. Frown tissue of selecti*d formalin-fixed he|Mtic tissue waa cut on l he cryostat and eloined with oil red (I to demonstrate lipids. At the time of the lost Hocriflcr, ten months after exposure to ft'D-oontnining diota had boon discontin ued, adipose tiaaue and liver from aeven previously exposed rota ond the adipose tissue nnrl liver from two rata of the some age that hud been fed only plain chow were nniil.vzed for the presence of PClis by electron enpture <BC> gns chromatography according to methods described by Curley et al.* In order to determine which Aroclor constituents were of such chemical stabili ty thnt they remuined at a mensurable residual level ten months after diolery in take had ceased, adipose and hepatic com posites were analyzed by gas chromatography-maae spectrometry The conditions for moss spectral analysis were as follows: the mass spectrometer was interfaced with a gas chromatograph and equipped with n moss mnrkcr accurate within 0.3 mass units. The gas chromatographic column temperature was 220 C; flash healer, 235 C; glass-coiled column. 3.05 mxQ.64 cm-. 1.5% OV-17/1.95% QF-1 on 60/80 mesh chromosorb "W" (high performance, acid washed, dimethyldichlorosilane treated), helium pressure: 0.84 kg/sq cm; flow rate (rotameter setting 3), 45 ml/min; separa tor temperature, 225 C; source energy, 70 ev; accelerating voltage 3.5 kv; trap cur rent 60Mamp; box current SOjiamp, and leak current Spamp. Results During the course of the experi ment three rats died. The livers of the rats that were killed at the time expo sure to Aroclor 1254 was discontin ued were enlarged, with average weights of 6.32% of the body weight. When the experimental diets had been discontinued for four months, the liver made up only 3.6% of the body weight, and after eight months recovery the average weight amounted to 3.34% of the average body weight. The fraction of the body weight for the liver in control adult male rats ranges from 2.44% la 2.59%.' Forty livers were studied micros copically. The hepatocyte.i were en larged in 39 livers and all 40 liven showed either vacuolated or foamy cytoplasm because of increased lipid accumulation. Cytoplasmic inclu sions similar to those described previously4 were observed in the liv ers of 24 rata. Adenofibrosis. which in many instances was very extensive, was present in 36 of the 40 livers, and a brown pigment was noted in the KupfTer cells and other macrophage? in 25 livers. Of the four rats that did not develop adenofibrosis, one each was killed two and six months after the poisoned diets were discontinued The other two rats without adenofi brosis were killed ten months after onset of exposure. The adenofibrosis ranged in size from grayish-white le sions measuring 0.5 to 1 cm to areas of 5 cm with a pitted surface. Quite often the lesion was multicentric The adenofibrosis1 is illustrated in Fig 1 and 2. In this study the appearance of tr Qi Fig 6 -rj , tract ol > 1 m the k-sn-i external \ lesions rglunduhii cells ih > roundi-i larger 1 and con duels ' lithciiul 1 392 Arch Environ Health/Vol 27, Dec 1973 Adenotibrosis/Kimbrough et el Arch En SONS 081767 It*-- `mmattom .st H overage ii ,>i i hi* body cmilrol ndull m J. 4401. to uli. il micros* lex wvvo on* utl m liven ud pi* foamy ictvnxud Upid Hitiic indu- v described d in flu* Itv* win. which in i\v extensive, | i() liwrx, and luted in the i nncmphages riitH that did iH, one each mint)ix after f i| 6. -Gil ctiromstegrsphic traces ot Aroctor 1254 standard (A), hepatic tissue extract (B). and Iattract of adipose tissue (Cl Response axprassad as percentages. with 100%-1.0 mv. (iacontimied. the lesion, particularly whan it was niut rnlonoA* extensive, varied a great deal. Small ninths niter lesions oonaistod predominantly of ilunnfiliroiii (Itndular pale-staining epithelial sh-\\ liitc le- tells that formed ducts and were sur cm to areas rounded by very little fibrosis. The 'face Quite larger lotions had extensive fibrosis centric. The ind contained collagen, and often the 'd in Fig 1 ducts which were formed by the epi <|N*nrm)ccof thelial palc-stnining cells were markedly dilated. The dilated ducts contained mucus or necrotic debris, and at times formed small cysts mea suring up to 0.5 cm in diameter. Small dilated ducts were lined by co lumnar epithelium, and larger ones by flattened epithelium (Fig 1). In areas of extensive fibrosis, and also adjacent to blood vessels in areas of normal hupntie parenchyma tFig2), clusters of srmill glandular cells were seen in 15 of the livers which resembled the epithelium observed normally in the pancreas. These small clusters of pancreatic-like tissue have not previously been described in the livers of' rats or of other species Special stoins for esterase, esterase with sodium taurocholntc. and tryp tophan showed a positive reaction indicative of salivary gland tissue. The significance of these colls is pres ently not understood. Normally tis sue of a pancreatic type is not ob served in the livers of our rats Clus ters of small columnar epithelial cells resembling pancreatic tissue also occurred in portal triads without i>eing surrounded by typical adcnofihrosis In the present study, one to two rats at each killing showed these small columnar cells with highly aci dophilic cytoplasm and a small, round nucleus. In the older animals, the fibrosis was quite extensive in some instances and the glandular proliferation was present mainly at the periphery of the lesion. Signs of regression of the lesion were not noted, except for the disappearance of epithelial cells from the center of the lesion. Furthermore the lipid accumulation and the hyper trophy of the hepatic cells remained constant throughout the study. Ex tensive areas of necrosis were ob served in three of the 40 livers. In addition to the change described previously,' electron microscopic examinations of sections of liver tis sue showed numerous small lipid vacuoles in the cytoplasm of many hepatocytes, particularly in the latter part of the recovery phase (Fig 3). The smooth and rough endoplasmic retic ulum was arranged in n disorderly fashion (Fig 4). Many of the mito chondria were atypical and surround ed by double membranes. The electron microscopic appear ance of the brown pigment present in the macrophages and KupfTer cells is illustrated in Fig 5. Large clusters of dark granular material, intermixed with lipid vacuoles, were observed, as well us round clusters of granular material about the size of mitochon dria. In our previous studies it was found that nt hast some of Ihis pig- ibrout'.h et *1 I Arch Environ Hesllh/Vol 27, Dec 1973 Adenofibrosis/Kimbrough et si 393 SONS 081768 1 ment contained iron' but in addition high concentrations of uroporphyrin <75% 8-cnrboxyprophyrins and 25% 7-carboxyprophyrins) were found in the livers,1* which could account for some of tho pigment. In addition to these findings, oil red 0 stain demon strated granules of red-staining lipid material in the area of the brown pigment. Since the morphological changes in the liver did not disappear, the re maining seven rats were killed after a ten-month recovery phase and their adipose tissue and livers analyzed for Aroclor. In adipose tissue, the concen trations of PCBs ranged from 924 ppm to 1,688 ppm, with an arithmetic moan of 1,192 ppm. The concentra tions in the liver ranged from 17.30 ppm to 26.24 ppm, with an arithmetic mean of22.65 ppm. The PCB levels in control adipose and liver tissues were <1.0 ppm. The gas chromatogram obtained from tho EC analysis of adipose tissue did not differ from that of liver, but was significantly different from standard Aroclor 1254. An average of seven peaks was observed as a resi due in the tissues. Under these gas chromatographic conditions, at least 18 peak* have been observed in the standard material (Fig 6). The differ ence is gas chromatographic patterns between stored and ingested Aroclor hns been observed before in this labo ratory and bv other researchers in the field."-' The total ion current (TIC) chro matogram for the liver tissue com posite is shown in Fig 7. Mass spec tral analysis revealed the presence of only three major Aroclor components with masse* of 324, 358, and 392. These components contained isotopic clusters indicative of the presence of Clv Cl,,, And ClT, respectively. Adi;>osv tissue analysis by mass spec trometry revealed the same patterns. Although only three different mo lecular components were present, examination of the EC and TIC chromatograms coupled with mea sured intensities from multiple scan ning of eluting peaks indicates that stereoisomers of these three mole cules nru obviously present. Peaks 1 to 3 (Fig 7) did not contain any chlorinated moieties, while other peaks observed in the liver TIC con tained the following molecular ions: peak 4 (324); peaks 5 to*7 (392 and 358); and peaks 8 to 9 (392). Comment The adenofibroais of the liver pro duced in this study and the one pre viously reported1 is the result of Aro clor consumption. Whether the Aro clor itself, or a contaminant such as a chlorinated dibenzofUran, is responsi ble still needs to be resolved. A study of the lesion shows that ppm eight th;i' S\lv Tli r a|V\ sai< PI'"' MV *1 it pii'dt Stir l* a-1- n> ).. ol . '' h (I.'IV Mi i>: . HIV* SU IV i! ".I Inpll In li linn lv I U'l i <!-. lv*: i VI ul epithelial proliferation occurs in the periphery, while the central portion of the lesions contains primarily fibrous tissue. The present study does not completely resolve the question of reversibility of this lesion since the storage levels of PCBs in adipose tis sue and liver may have been suffi ciently high to stimulate continued growth of the altered hepatic tissue. The levels in the liver suggest that a constant low-level turnover of PCBs takes place in the rata. In a previous study, Curley et aF found levels of 10,000 ppm Aroclor 1254 in adipose tissue of rats that had been fed 500 I* pvu* \\ lv lbi-hli r ti ll..b M II lb 394 Arch Environ Heslth/Vot 27, Dec 1973 Adenotibrosis/Kimbrough et *1 MQNS 081769 wm of the mixture in their diet for light months. This finding indicates Jhat a great deal of the Aroclors had Ln mobilized from the adipose tie* also observed in the earlier study.1 Electron microscopic examination of the pigment shows that particularly the large clusters of it resemble very we and excreted in the present study, closely illustrations of ceroid pig p* storage levels found ten months ment.19 Experimentally ceroid pig ifter onset of recovery are in the Ume range of those of rats fed 100 ment can be produced by hemorrhage into fatty tissues rich in unsaturated Km PCBb in their diet for eight nths.* The dietary level of 100 ppm produces a much lower incidence of fatty acids, particularly in the ab sence of on antioxidant (vitamin E deficiency). Ceroid pigment is com idenofibrosis,1 and it is possible that monly referred to aa lipofuscin. These idenofibrosis represents a permanent lipid pigments are at times associated Itsion, particularly since the percent with hemosiderin and may contain i body weight of the livers had de iron-positive granules as in the pres based. I The Aroclors found in the tissues ent case.' Ceroid granules were also described by Edwards and White in Uflered from those fed in the diet. the livers of rats fed butter yellow. iMolaboKsm of lower chlorinated bi- Bennett et al also observed a brown phenyls is considered as a possible mplanation for the diflerences ob- pigment in rats fed e mixture of chlorinated naphthalenes and chlori nrved analytically between con- nated biphenyls. These authors turned and stored PCB. Earlier work claimed that it failed to stain in a ifBlock and Cornish11 has shown that manner characteristic of either hemo biphenyls and chlorobiphenyls can be siderin or hemofuscin. The porphy kydroxylated and excreted in the rins demonstrated in the livers of our urine of rabbits. A more recent study rats fed Aroclor may also contribute by Hulxingor et al* indicates that the to the overall pigment deposits. Cer rat is capable of hydroxylating mono-, oid pigment increases in people with ii-, and tetrechlorobiphenyls, but not hexachiorobiphenyls. Since Aroclor age and has therefore been called "A bnutzungspigmtnt" by the Ger 1264 averages five chlorines per mol- mans. Attempts have been made to kuIo, it is conceivable that the heav* relate it to senility. It has been associ itr chlorinated moietiea could remain ated with neuronal ceroid-lipofusci u a residue. nosis (Batten disease) in man. In Pdimcthylaminoazobenzene also minks, a disease called yellow fat produces adenofibrosis. Edwards and disease14 is associated with the accu White1* and Bennett et alu described mulation of excessive amounts of cer the lesion in rets when a mixture of oid in adipose tissue. The accumula chlorinated naphthalenes and chlori tion of ceroid in mink can be prevent * >r*maa n nated diphenyls were fed. Unfortu nately, the finding was not well de ed by addition of vitamin E to the diet It occurs when the food rations scribed in Bennett et al's report, but contain high percentages of fish occurh in the Edwards end White mentioned Ben scrap. However, Gorham et ai suggest Dtrol portion nett et el's finding as another exam that other factors may also be in us primarily ple of the induction of adenofibrosia volved, since high dietary percent nl nudy does by chemicals in an excellent descrip ages of fish scrap did not promote > i leslionof tion of the lesion. A critical review of ;tm since the the histopathogenesis of this lesion u iulipose Hi- | was made by Stewart and Snell1*. i* U*en suffl- 1 The presence of the brown pigment tc Kiitinuod in Kupffer cells and macrophages is of growth as well as the stock ration, even when vitamin E was added to the diet These observations raise the question whether high doses of vita min E, and possibly the addition of pji'ir tissue, interest and was to a lesser extent choline to the diet, could prevent or i.'xcsl that s ivfi of PCBs 0 n previous :ncl levels of 1 in adipose n fed 500 minimize the observed effects of PCBs on the liver of our rats. Richard 1. Moor* assisted wilh (hr animal (cull Linda W Anderson assisted with preparation and interpretation of t}w clt'lrom>rrogriipJi* Annie H. Alford performed ihc tissue prepara tion* H L Stewart, MD, and the (iiu<- laboratory of the National Cancer InMitiilc, JI<Hlic*ln, Md, performed evuluotitm* of liver slide*. References 1. Kimbrough RR. Under HE, Coin** TIL MorphnktgiLiil changes in liver of ml* led puivchlorinated biphenyls. AnA Kmnron Health 21 354-304.1012 2. Risehrnugh RW, et al. Polychlorinated hiphenyla in !> global econyaic-m Nature 220 1078-1102, IMS. 3. Curley A, ot al: Polychlorinated biphenyls: Distribution and storage in Imdy fluid* and tistfutm of ahermnn rats. Snviron Ret 4:481-496. 1971 4. Kimbrough HD.Caine*Til, Under RE The uUmetruclurc of liver* of rnt* fed IU)T and cheldrin. Arth Environ Health 22 4HU-4U7. JS7J. 5 Goldstein JA, Hickman P, Joe I) Hepatic porphyria induced hy polychlorinated biphenyls (PCII'el: Relationship between porphyria. 4aminoievuhmc acid synthetase and cytochrome P-460, iiiwiriicted. Fed /'me 32:702, IW7II 6. Grant OL. Phillipa Will, VilWneuve DC Metabuliam of a polychlorinated biphenyl (Aroclor 1214) mixture in the rat Hall Kirnmn Contain 7iiiico/0 1O2l 12.1971 7. Vos JC, Kouman Jll: Comparative tu.til ngic study wilh polychlorinated biphenyls in chickens with special roloivnce to pnrphyria, edom* !brmatvon. liver necrosis and tissue resi dues. To&itol Appl Pharminvl 17 U.'WI O.'VM. 1970 8 lliork Wl), Cornish lilt Metitlmlisin of In phenyl nnd 4-chlorobiphenyl in the i iildot J Mini Cheni 234:3:101-3302. I!#H V. HotringerU.et at: Hulychtormaied hiphtmyla: ntetobolic behavior of pine isomer* m |ngeons. rats, and brook trout. Srirnrr I7H :tl2 313,1972. 10. Edward* JE, White JFathnlogic chunyes with special reference lu pigmentation and ciaasificution of he|mlic tumors m ruts list pJimHh yliiminnasobensene ibutlei- yellow). J Nut/ Cower Inti 2157183. 1941. 11. Bennett CA, Drinker CK. Wnrren ME MoiphohigHiil chnnges m the livers of ruts re sulting front CKtHMorr In rerluin i hhinniilid hy droenrttons. J hull/yn 7'u.nro/ 20 U7-12U, IthIH 12 -Stcwurl ML,Snell KC. The liiHtopnthology of experimental tumor* of the liver of tin- rot: A critical review or the hitlopathngCHfsi*. Aria Umo int Contra Cancrum 13 770-Mfi;i. |f,7 13. Harlroft WS: Observation and interpreta tion of lipid pigments t Lipofusctnt) in tin- pathol ogy of lobornlory aniiniils. Cat Hit Tiaicol 1:370 411, 11(72. 14. Gorham JR, Baker GA. line N Ohst-rv.ilions on the etiology of yellow Int tliscnse in mink: Preliminurv report. Vrt Mrtl 4G 1(11)11)2 1961. '.itmueii at al Arch Environ Health/Vol 27, Oec 1973 Adenofibrosis/Kimbrough et ai 39$ f MONS 081770