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H)NI< Ot.OOY AND API-l IEI> PHARMACOLOGY 28, 418-427 (1974) Effects of Chemically Pure Chlorobiphenyls on the Morphology of Rat Liver1 M. M. Hansell and D. J, Ecobichon Departments of Anatomy and Pharmacology, Dalhoitste Unirersity, Halifax, S'ocn Scotia, Canada Receded September 4, 1973; accepted December 17, 1973 Effects of Chemically Pure Chlorobiphenyls on the Morphology of Rat Liver. Hansell, M. M. and Ecobichon, D. J, (1974). Toxicol. Appl. Pharm acol. 28, 418-427. Pure biphenyl and a scries of isomerically pure mono-, di-, (etra-, hexa- and octa-chlorobiphcnyls of known chemical composition were injected ip (50 mg/kg) into young male Wislar rats for 3 consecutive days and the rats were killed 4 days after the last injection. In a second experi ment, rats were injected ip with 4-mono, 4,4'-di- and 2,5,2',5'-lclrachlorobiphenyl (100 mg/kg) for 7 days. The effects of (he pure PCBs were com pared with those caused by o.p'- and /j,//-DDT and Aroclor 1254 and 1260. Samples of liver were examined by light and electron microscopy. The major alterations observed within the hepatocyles were marked proliferation of smooth endoplasmic reticulum and increased numbers of microbodies and lipid droplets. Biphenyl and 2,2'-dichlorohiphenyl produced few morpho logic effects whereas 4,4'-di-, 2,5,2',5'- and 3,4,3',4'-tetrachlorobipheny! caused more changes. The highly chlorinated hexa-and oclachlorobiphenyls produced morphological changes that were comparable to those produced by DDT, and Aroclors 1254 and 1260. The degree of ultrastructural altera tion appeared to be related directly to the number of chlorine atoms and (he presence of chlorine in the 4- or 4'-posilion. The structure of ihe liver of several species of animals treated with polychlorinated biphenyls (PCBs) has been examined using light and electron microscopy. With one exception (Vos and Notenboom-Ram, 1972), all these studies have employed com mercially available mixtures such as Aroclors, Phenoclors, and Kaneclors (Bennett el al., 1938; von Wedel cl a!., 1943; Miller, 1944; Nishizumi, 1970; Kimbrough el til., 1972; Not back and Allen, 1972; Koller and Zinkl, 1973; Bruckner et al., 1973). Some of these commercial mixtures were reported to be contaminated with impurities such as chlorinated dibenzofurans and dibenzo-p-dioxins (Vos ct at., 1970; Sissons and Welti, 1971; Zitko and Choi, 197); Greig, 1972; Hutzinger et al., 1972; Webb and McCall, 1972). Hammond (1972), in summarizing the relationship between toxicity and degree of chlorination of PCBs based on studies using commercial mixtures, found no consis tent relationship valid for different species and routes of exposure. Tucker and Crabtree (1970) suggested that sublelhal effects wv.e directly correlated with chlorine content, while lethal effects were inversely correlated. 1 Presented in part at the Sixteenth Annual Meeting of (lie Canadian Federation of Biological Societies, Saskatoon, Saskatchewan, June 26-29, 1973. Copyright (f) 1974 by Academic Press. Inc. AH lights or reproduction in any form reserved. Primed in CJrcnt Britain 418 OSW 025744 STLCOPCB4009699 PCI* AND LIVLR MORPHOLOGY 419 To eliminate (lie effects of possible contaminants in commercial mixtures and to determine the relationship between position and degree of chlorination, and effects on the liver, a series of isomerically pure chlorobiphenyl compounds of known position and degree of chlorination was administered ip to rats. The influence of these com pounds on functionally diverse hepatic drug-metabolizing enzymes and hepatic mor phology were studied. The effects on various drug-metabolizing enzymes are reported elsewhere (Johnstone cl al., 1974). METHODS The methods for the preparation of the pure chlorobiphenyl compounds and the doses administered were described in detail by Hutzinger ef al. (1971) and Johnstone et al. (1974). The compounds2 used are listed in Table 1. A group of 7 young male Wistar rats (Woodlyn strain) weighing 50-65 g was treated ip with each agent dissolved in peanut oil, at a dosage of 50 mg/kg daily for 3 days. The rats were killed with an overdose of CHClj 4 days after Ihe final injection following the determination of pentobarbital sleeping, lime. Control rats were treated with equivalent volumes of peanut oil and were killed by CHCI3 overdose after pentobarbital sleeping time was performed. In a second experiment, 3 compounds (4-mono-, 4,4'-di- and 2,5,2',5'-telrachlorobiphenyl) were injected ip at a dosage of 100 mg/kg/day for 7 days, the rats being killed 24 hr after the last injection by an overdose of CHCI,. After euthanasia, the abdomen of the rats was quickly opened and samples of the liver were removed for fixalion for light and electron microscopy. For light microscopy, samples were fixed in 10% buffered formalin and embedded in paraffin; sections were stained with hematoxylin and eosin. For electron microscopy, I-mm cubes of liver were fixed in 1 % osmium tetioxide in sodium cacodylate buffer at 0CC for 2 hr, dehydrated in a series of graded alcohol solutions and propylene oxide and embedded in Spurr low viscosity embedding medium. Ultratbin sections were cut on a Porter-Bium MT2B ultramici otome stained with uranyl acetate and lead citrate and examined with a Philips 200 electron microscope. Thick plastic sections were cut from the same block and stained wilh methylene blue and azure A for light microscopy (Pease, 1964). . RESULTS The results of ultrastructural evaluation and the hepatic weights for the rats treated with the agents at 50 mg/kg for 3 days are presented in Table 1. Hepatic weights were significantly increased following treatment withp,p'-DDT, Aroclor 1254, Aroclor 1260 and 2,3,5,2',3',5'-hexachlorobiphenyl, These compounds also induced major altera tions in hcpatocytc ullrastructurc. Hcpatocytes of treated rats were compared wilh hepatocytes from control, vehicletreated animals for the presence of these alterations. The grading system for alterations in amounts ol smooth endoplasmic reticulum (SHR), and numbers of lipid droplets and * Abbreviations used: o,//-DDT, l,f,l-lricli!oro-2-(-cliloroplienyl)-2-(/>-clilorophcny!)ethanc; 1,1,1 -ti ichloro-2,2-bis(/>-ch!orophenylX'lbanc. The nomenclature of the polychlorinated biphenyl (PCH) compounds follows the accepted form, the position of chlorines on one phenyl being indicated by numbers from 2 to 6, while chlorine positions on (he second ring arc designated by a num ber with o superscript prime, i.e., 2,2'-diclilorobiphcny). , f 3X A>.j ^ ,,* ... ' * f> DSW 025745 -. '1 y. ,;r : ....................... STLCOPCB40097 420 MANSELL AND ECOIHCHON microbodics is based on a comparison of the amounts present in any cell with the amount present in a control hcpalocyte (value -- 4) and a hcpatocyte having the maxi mum amount of change (value =The grading system reflects only relative numbers since morphometric techniques were not used for quantitation. The major ultrastructural alterations induced by the various pure chlorobiphenyls were prolifera tion of SER, alteration of the rough endoplasmic reticulum (RER) and increased num bers of lipid droplets and microbodics (Table 1). TABLE 1 Changes in Liver Weight, Hepat ic Smooth Endoplasmic Reticulum, Lipjd Droplets and Microbodies, Following the Administration ot DD T, Aroclor 1254 and 1260 and Numerically Pure Chlorobiphenyls to Rats" Compound Liver weight6 (8) Smooth ER" Lipid droplets M icrobodies Vehicle-Treated o,p'-DDT ap'-ddt Aroclor 1254 Aroclor 1260 Biphenyl (BP) 4-Chloro BP 2,2'-Dichloro BP 2,4'-Dichloro BP ; 4,4'-Dich!oro BP 2,5,2',5'-TctrachIoio BP 2,4,2',4'-Tctrachloro BP 3,4,3',4'-Tctrachloro BP 2,4,5,2',4',5'-lTcxachloro BP 2,4,6,2',4',6'-Hexachloro BP 2,3,5,2',3',5'-Hcxachloro BP 2,3>4,5,2',3',4',5'-Oclach!oro BP 4.77 1.14 4.91 0.46 5.59 0.89c 6.23 0.30c 6.15 0.49c 4.87 0.11 4.76 + 0.69 4.85 0.96 4.69 0.34 4.21 0.44 4.68 4 0.69 5.03 0.48 4.73 4 0.47 4.18 0.91 3.82 4 1.29 5.80 4. 0.78c 4.53 1.46 4 ++F4 +4+4 +4+4-4- 4-4+ + +4 + +44 4++- +4 444 +4 +4-4-4-I-++4 ++++ +4-++ 4+4 4 +4-1 +4+4 44 4 +4 +4-1 + +1 4+ 444 4-4-144 4+ +4 4+ 4 4 +44 44 4 4 4 4 4+ 4 t 4+ 4+ 44+ 4 4+4 444 4-4+ 4-4 4 4 44 44 44 4 44 1 4 4-1 * Animals were injected ip with a peanut oil solution at a dose of 50 mg/kg for 3 days and killed 4 days after (lie last injection. * Values presented are mean weights SD of 14 (vehicle-treated) and 7 (agent-treated) animals per group. Values are based on wet tissue weight. c Values are statistically significant from control weights at a probability level p < 0.05. * Sec text (Results) for grading system. Isomerically pure hexa- and octn-chlorobiphenyls, administered at a concentration of 50 mg/kg for 3 consecutive days, caused alterations in hepatic morphology that were visible with the light microscope (Fig. 1). The changes included large numbers ofhepatocytes with vacuolated cytoplasm and small foci of necrosis involving 5 or 6 cells. The necrotic foci were particularly numerous at the periphery of the lobule. Treatment with 4-mono-,4, 4-di and 2,5,2',5'-tctrachlorobiphenyl at 100 mg/kg for7 days resulted in larger numbers of necrotic foci, ccntrolobular necrosis and proliferation of biliary ductules (Fig. I). The lesions were most severe in rats given 4,4'-dichlorobiphenyl for 7 days. Hepatocyles of rats treated with o,p'-t orp,p'~DDT (Fig. 2A) had increased amounts of SER in the form of vesicles (S) which tended to accumulate at the periphery of the OSW 025746 STLCOPCB4009701 -MV-".-' v V. !' {';'?- I'CB AND UVER MORPHOLOGY 421 cell. There was a reduction in the amount of RER (R) which often displayed expanded or sinuous cisternac. The number of lipid droplets and microbodies (M) was increased. Hcpalocytes from rats given the commercial Aroclors were similar to those from rats treated with DDT. Aroclor 1254 treatment (Fig. 2B) caused more pronounced altera tions than Aroclor 1260 treatment. Hepatocytes of treated animals contained very abundant SER, reduced RER, increased numbers of microbodies (M) and lipid drop lets (L). Hepatocytes from rats treated with pure PCB isomers for 3 days were compared with hcpalocytes from control rats and rats treated with the commercial Aroclors and DDT '- A ,v Vv t* % v* i -1" l ie. ). Liver from a ra( treated with 50 mg/kg/day of 4-chloi obiphenyl for 7 days showing hepato cytes wills saettohued cytoplasm (V) and a small area of necrosis (N). Hematoxylin and eosin. x285. at the same dosage. The amount of hepatic smooth endoplasmic reticulum in rats ircaicd with biphenyl and 2,2'-diclilorobipheny! was comparable to that in control rats; but in rats treated with 4-mono-, 4,4'-di-, 2,5,2',5'- and 3,4,3',4'-tetra-chlorobiphenyl, the hepatocytes contained increased amounts of SER. Ail other compounds tested (2,4'-di, 2,4,2',4'-tctra-., 2,4,5,2',4',5'-( 2,4,6,2',4',6'- and 2,3,5,2',3',5'-hexa- and 2,3,4,5,2`,3',4',5'-octa-chlorobiphcnyl) caused increases in the amounts of SER that were equivalent to the increases observed in hepatocytes of rats treated with DDT or Aroclor 1260 (Fig. 3A). The RER often had sinuous cisternae (Fig. 3B) and was reduced in amount in hepatocytes of rats treated with hexa- or octachlorobiphenyl (Fig. 3C). The highly chlorinated chemicals and those possessing a chlorine in the 4 and/or 4' position caused the greatest increases in the numbers of microbodies and lipid inclusions (Fig. 3C). Lesser effects were observed following treatment with biphenyl, 2,2'-di- and * u....... DSW 025747 V-t STLCOPCB4009702 '.' . '''.'/'kv: /. f ' v, v ' j:.", ,v.V: ,v' .1 V. ':: V:,";*v>; ^r;... ,`v*//^;l.' .^S?;^;`.(,'-?v,>>.-.^1**; :*V*'?*><'-i:' `A ` vty'';rv'`` '? *'*!.*? <Jr4,s*T*-.V'-* Aw*'. Vr''1'*'> >'.. 'Y- v' jy.:: . Nv . v :i" l4.'' '; l's^vVt-i\/'r ri4*f^vv" > l.^.'0: ' , V '' ' '* /:;;- ;. ;v ./ :^;?;::.v-^.'mw v."i 422 MANSELL AND I.COU1CMON Fio. 2.1lepatocyles from rats treated with 50 mg'kg/day of /iji'-DDI (A) and . .dor 125-1 (1!) for 3 days showing proliferation of smooth endoplasmic reticulum (S), expanded -.iei-nae oT rough endoplasmic reticulum (R) and microbodies (M) and lipid droplets (L). A, x!2,000; B, xld.OOO. ttlilllll^^ OUSdW 0257#4-rw8 V':. * .rv\ .:v-; 'a:-i / .'; .`j,sv '',W J '*v '*j * * V' ^- ;* r>/';' >|.-v; ?<Aj ;v2 ;*WV:'V*'-vv-J;d .* v-s ..!'' ;v. .>v-,Ym`v .. . '. '.> v... . .' i STLCOPCB4009703 ................... . #'_ J ) . ^ . I . f ) 1 b, .' ... .'.b.j.. '. I y i li)lll aka S*a`.al *>jf**' it i4<|l`..^:iil/l a&U^tl.fi..,< fi^li^jft..*: A-'^`*'^ al'i 1^, ,1 t. u . */ V` ' * ' ,f *'.." 1 1 *. 1 jo. 3. Hcpalocytcs from different ra(s treated with isomerically pure chlorobipbenyls at concentra tions or 50 mg/kg/day for 3 days. (A) Treated with 2,4,2',4'-ie(rachlorobiphcayl, showing abundant smooth endoplasmic reticulum (S), glycogen (G) and a microbody (M) x25,675. (B) Treated with 3,4,.V,4'-tetrachloiobiphcnyl) showing sinuous cislernac of rough endoplasmic reticulum (R) sur rounding a mitochondrion (M). x23,SOO. (C) Treated with 2.3,5,2'13'I4'-hexachlorobipbcnyl, showing prolifeunion of smootli endoplasmic reticulum (S), microbodies (M) and lipid droplets (L), xl4,100. \';' :'$.'V'/k' '-! ' fcr>, : /&v\'v & V r.=: rv .:;; '> >.. V -- ;>;.. DSW 025749 it ..*. v .-* ; / v'5:i, ;*./ w-.x v.-u. 'x.,-.vV' ..... sy<;\.' * - ' SCTT;LCrOPCB4009704 ^r *:!.v 7 *; *,*',Vr ..,^< 't..;, N 'V ^ V1 *T a)\Vi* ' - Uiw.VW.WJ 424 HANSKLL AND ECOBICIION 2,5,2',5'-tetrachlorobiphenyl. The number oflipid inclusions in hepatocytes was never as great as that observed following treatment with DDT and the Aroclors. The administration of 4-mono-, 4,4'-di- and 2,5,2', 5'-letra-chlorobiphenyl at a dosage of 100 mg/kg/day for 7 days caused more pronounced alterations than those observed after treatment for 3 days (Fig. 4). The 4,4'-dichlorobiphenyl caused more drastic Fig. 4. Hepatocytes from rats treated with 100 mg/kg/day of4,4'-diclilorobiphenyl for 7 days showi' abundant smooth endoplasmic reticulum (S), microbodies (M) and lipid droplets (I.). A. xl4,10 B, X8580. DSU 025750 STLCOPCB4009705 J*Cfl AND LIVL'R MORPHOLOGY 425 alterations than 4-moochlorobiphenyl or 2,5,2',5'-tetrachlorobiplicnyl, the effects of the latter two agents being approximately the same. DISCUSSION Proliferation of SER is a response of the hepatocyle to a wide variety of drugs and poisons and is considered to be a structural reflection of the enhanced metabolism of foreign lipophilic compounds. The increase in SER can be related to increased amounts of drug metabolizing enzymes within the liver (Vos and Notenboom-Ram, 1972). In all previous studies, administration of PCBs has resulted in increased amounts of SER in hcpatocytes (Nishizumi, 1970; Kimbrough et af,, 1972; Vos and Notenboom-Ram, 1972; Norback and Allen, 1972; Koiler and Zinkl, 1973). In this study, the proliferation of SER was closely related to the structural characteristics of the compound used. Biphenyl and 2,2'-dichlorobiphcnyl appeared not to induce extensive proliferation of SER, but with the other compounds, the proliferation appeared to be related to the degree of chlorination and particularly to the presence of a chlorine atom in the 4 and/or 4' position {Tabic 1). The morphological alterations correlated well with the activities of drug-metabolizing enzymes within the same rats (Johnstone et al., 1974). When commercial mixtures were employed, mixtures having a greater chlorine content (Aroclors 1254, 1260) had greater enzyme-inducing effects and caused more marked morphological changes than those having lower chlorine content (Villcneuvc et al., 1971; Bickers et al1972; Koller and Zinkl, 1973). The more highly chlorinated com pounds may have greater effect because metabolic studies show that low'chlorine analogs arc eliminated from tissues more rapidly than the more highly chlorinated compounds (Grant ct al., 1971; Bailey and Bunyan, 1972; Bickers el al., 1972; Rehfeld el al., 1972). In this study and the report of Kimbrough et al. (1972), administration of Aroclor 1254 resulted in more severe morphological alterations than Aroclor 1260 or any of the pure compounds used. Jt was also a more powerful inducer of drug-metabolizing enzymes than Aroclor 1260 (Johnstone et al., 1974). Aroclor 1254 was not the most highly chlorinaied compound used, and the response to its administration was incon sistent with the view that increased chlorine content caused greater alterations. The failure of Aroclor 1254 to follow the rule of increased tissue response with increased chlorination might be due to the particular combination of chlorobiphenyls present in the mixture or possibly to the presence of contaminants. Chlorinated dibenzofurans and dibcnzo-/;-d;oxans have been found in some commercial PCB mixtures and these compounds caused alterations in hepatocyte morphology that were similar to those caused by PCBs (Vos et al., 1970; Zitkoand Choi, 1971; Greig, 1972; Hutzingeref al., 1972; Webb and McCall, 1972; Norback and Allen, 1972). Such contaminants are not reported to be present in Aroclor 1260 (Vos et al., 1970). Kimbrough el al. (1972) stated that a preliminary analysis of the lot of Aroclor 1254 used in their study suggested pos sible contamination wiih a chlorinated dibenzofuran. It is likely, therefore, that increas ing chlorination of the biphenyl nucleus does lead to proliferation of SER and to in creased activities of drug-metabolizing enzymes. The enhanced effect observed with Aroclor 1254 can be due to some other factor than chlorination. Treatment of rats with 4-chloro-,4,4'-dichIoro- and 2,5,2',5'-tetrach!oro-biphenyl at a dosage of 100 mg/kg'day for 7 days resulted in marked proliferation of SER. The pfTit**v**v# . 426 HANSIZLL AND I.COBICHON dosages, modes of administration and duration of exposure have varied greatly in pub a lished reports of tire morphological alterations produced by PCBs. With the exception -v of the study by Kimbrough el al. (1972), most studies have used much higher concen trations than the ones used in this study. Most authors have noted increased hepatic weight and hepatic cell size. In our study, the increase in hepatic weight was significant only after treatment with p,p'-DDT, Aroclors 1254 and 1260, and 2,3,5,2',3\5'-hexachlorobiphenyl. There was no significant increase in cell size. This result may be due to the short exposure lime and relatively low doses used. Concentric membrane arrays or whorls within the cytoplasm of hcpatocytcs have been noted in several studies (Nishizumi elal., 1970; Norback and Allen, 1972; Roller and Zinkl, 1973). Their formation may be species-related as such structures were found 3 in rats and mice, rarely in rabbits but not in monkeys (Norback and Allen, 1972; Nishizumi, 1970; Vos and Notenboom-Ram, 1972). One such array was observed in a hepatocyte of a rat treated with 2,4,2',4'-telrachlorobiphenyl. Many of the pure chlorobiphenyls as well as DDT and the Aroclors resulted in a rearrangement of the rough endoplasmic reticulum into sinuous arrays which Norback and Allen (1972) --j believe to be the first stage of whorl production. Hepatocytes from treated rats contain increased numbers of lipid droplets, many of which were large. Norback and Allen (1972) noted that such lipid droplets were often incorporated within the membrane arrays. They suggested that the membrane arrays provided sites for storage of lipid droplets that contain lipophilic exogenous, toxic compounds, such segregation would 3 maximize exposure to degradative enzymes in the membranes. ACKNOWLEDGMENTS This study was supported by funds from the Medical Research Council of Canada (Grants MA-4807 and MA-3611). Samples of Aroclor 1254 and 1260 from the Monsanto Chemical Company and of the pure chlorobiphenyls from Dr. O. Hutzingcr, National Research Council, are gratefully acknowledged. The assistance of Dr. T. Ghose, Department of Pathology, Dalhousie University in the histological evaluation of tissue sections was greatly appreciated. .;,vt REFERENCES ) Bailey, S. and Bunyan, P. J. (1972). Interpretation of persistence and effects of polychlorin .'"i ated biphenyls in birds. Nature (Low/on) 236, 34-36. A Bennett, G. A., Drinker, C. K. and Warren, M. F. (193S). 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Dept, of Interior, Bureau of Sport Fisheries and Wildlife. Pub. 84. Viu.enu.ive, D. C., Grant, D. L., Phillips, W. E. J., Clark, M. L. and Clegg, D. J. (1971). Effects of PCB administration on microsomal enzyme activity in pregnant rabbits. Bid!. Environ. Contain. Toxicol. 6, 120 128, Vos, J. G. and Koiman, J. H. (1970). Comparative toxicologic study with polychlorinated biphenyls in chickens with special reference to porphyria, edema formation, liver necrosis, and lissue residues. Toxicol. Appl. Pharmacol. 17, 656-668. Vos, J. G. and Notinboom-Ram, E. (1972). Comparative toxicity study of 2,4,5,2',4',5'- hexacliloiobiphenyl and a polychlorinated biphenyl mixture in rabbils. Toxicol. Appl. Pharmacol. 23, 563-577. Vos, J. G,, Koi man, J. 11., Van Der Mass, H. L., ienNoever de Brauw, M. G. andde Vos, R. 11, (1970). Identification and toxicological evaluation of chlorinated dibenzofuran and chlorinated naphthalene in two commercial polychlorinated biphenvls. Food Cosmet. Toxicol. 8, 625-655. ' Wi hu, R. G. and McCall, A. C. (1972). Identities of polychlorinated biphenyl isomers in Aroclors. J. Ass. Ojfie. Anal. Chan. 55, 746-752. von Wi.di l, H., Holla, W. A. and Denton, J. (1943). Observations on Iheloxic effects result ing from exposure to chlorinated naphthalene and chlorinated phenyls with suggestions for prevention. Rubber Age 53, 419-425. Ziiko, V. and Choi, P. M. K. (1971). PCB and other industrial halogenated hydrocarbons in (lie environment. Fish. Res. Bd. Can. Tech. Rep. Til, 1-64. -r ` V V';:V; > , )' V iy-: v; pym-y iv-*. ; 0 yy , Hv-i. hfte J h\i ,, > * LVciv. 0SW 025753 c,*?V , yfyry V*A:-7- V .3 ; 's1ii y'fr,1^ .. * a*'1 * * . . .* i * s, * y-r- f ^ ,>y^rp.;yyy,iy"A/;^ ^'y-yyyfyv> 7. y fyyyyydc-./>>: t y--r 'Ail ,;'c A" -; >: ,\y;: v-A -l-m - STLCOPCB4009708