Document 5D1qMo86djJ5K7nVnO0eXqby5

Hanscll, H. H. and D. J. Ecobichon. 1974, Effects of chemically pure chlorobiphenyla on the morphology of rat liver. Toxicology and Applied Pharmacology 28(4): 418-427. HONS COOMO vmi o>t f*oy As-t Af im ini pharmacouajV 2B, 4J8-427 (1974) Effects of Chemically Pure Chlorobiphenyls on the Morphology of Rat Liver* M. M. Hansell ano D. J. Ecobichow Deportments of Anatomy nmi Phormacobxy. Dnlbousie Unkeriity, Haltfas, Voi n Scotia, Canada Recettetl September 4, 1973; accepted December 17, 1973 Effects of Chemically Pure Chlorobiphenyls on (he Morphology of Ral Liver. Hammll. M. M. ami Ecobichon. D. J (1974). Toxicol. Appl, Hhafm* oral. 28, 418-427. Pure biphenyl and a scries of isontericatly pure mono-, di-. terra-, he*A- and octa-chlorobiphenyls of known chemical composition were injected ip (SO Mtg/kg) into young male Wisiar rals for 2 consecutive days and the rals were killed 4 days after the last injection. In a second experi ment. rats nere injected ip with 4-mono, 4.4'-di* and 2,5,2',5'-telrchlofobiphenyl (100 mg/kg) for 7 days. Tire effects of Ihe pure PCBs were com pared with those caused by o.p- and p.p'-UDT and Aroclor 1254 and 1260. Samples ofliver 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. Diphenyl and 2,2'-dkhlorohphenyl produced few morpho logic effects whereas 4,4'-di-, 2.5,2,5,- and J.4,J'.4'-leirochlorobipl*enyl caused more changes. The highly chlorinated hexa- and ociachtorobiphenyls produced morphological changes that were comparable to those produced by DDT, and Aroclors 1154 and 1260. The degree of ultrastructural altera tion appeared lo be related directly to the number of chlorine atoms and the presence of chlorine in Ihe 4- or 4'-position, The structure of the liver of several species of animals irealed with polychlorinated biphenyls (PCBs) has been examined using light and electron microscopy. With on; exception (Vos and Notenboom-Ram, 1972), all these studies have employed com mercially available mixtures such as Aroclors. Phenctclors, and Kancclors (Bennett el of.. 1938; von Wedel el of., 1943; Miller, 1944; Nishizumi, 1970; Kimbrough ei at,, 1972; Norback and Allen, 1972; Keller and Zinkl, 1973; Bruckner er at., 1973). Some of these commercial mixtures were reported to he contaminated with impurities such ns chlorinated dihenzofurans and dibenzo-p-dioxins (Vos ct al.. 1970; Sissons and Welti. 1971; Zilko and Choi, 1971; Greig, 1972; Hutzinger et at., 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 sublethal effects w. . directly correlated with chlorine content, while lethal effects were inversely correlated. 1 Presented in part ml the Sixteenth Annual Meeting of the Canadian Federation of Biological Societies. Saskatoon, Saskatchewan, June 26-29, 1973. CoCrritkl ft IV74 hy Academic Inc. All iiftH* of rcin wjr fom icvcrvcd. Pitftittf in CrcM flnui* .. 4t& \ nms toMai PCK AND LIVMt MORPHOLOGY 419 ' o clnmnoic [lie effects of possible contaminant* in commercial mixture* and lo determine [lie rein non ship between position and degree of chlorination, and effects on the li\or, a senes of isomcrically pure chlorobiphenyl compound* of known position find decree 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 arc reported elsewhere (Johnstone ct at,, 1974). METHODS The methods for the preparation of (he pure chlorobiphenyl compounds and the doses administered were described in detail by Hutzinger er at. (1971) and Johnstone ct at, (1974) The compounds* used arc listed in Table I. A group of 7 young male Wistar rats (Woodlyn strain) weighing 50-65 g was treated ip with each agent dissolved ill pi.ii tint oil, at a dosage ofSOmg/kgdaily for J days. The rats were killed within overdo** of CHClj 4 days after the final injection following the determination of pentobarbital sleeping time. Control rats were treated with equivalent volumes of peanut oil and were killed by CHClj overdose after pentobarbital sleeping time was performed, In a second c\pr!hnent, 3 compounds (4-mono-, 4,4'-di- and 2,5,2 ,5 -telrachlorobiphcnyl) 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 CHClj. After euthanasia, the abdomen of the rals was quickly opened and samples of the liver were removed for fixation 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. Foreleclron microscopy, 1-mm cubes of liver were fixed in 1 % osmium ted oxide in sodium cacodylalc buffer at 0CC for 2 hr, dehydrated in ti scries of graded alcohol solutions and propylene oxide and embedded in Spurr low viscosity embedding medium. Ultralhin sections were cut on 3 Porter-Blum MT2B ultiumicrotome stained wilh uranyl acetate and lead citrate and examined with a Philips 200 electron microscope. Thick plastic sections were cut from the same block and stained with methylene blue and azure A for light microscopy (Pease, 1964). RESULTS The results of ultrastruclural evaluation and the hepatic weights for the rats trealed wiih the agents at 50 mg/kg for J days are presented in Table 1. Hepatic weights were significantly increased following treatment withpjs'-DDT, Aroclor 1254, Aroclor 1260 and J^.S^'^S'-hcsachlorobiphenyl. These compounds also induced major altera tions in hcpatocylc ultrastructure. Hcpatocytcs of treated rats were compared wilh hepatocyles from control, vehicletreated animals for the presence of these alterations. The grading system for alterations in amounts ol smooth endoplasmic reticulum (SER), and numbers of lipid droplets and ' Abbreviations used: e,p*-DDT, l,t,t-tricMoro-2-(0-clilorophenyl)-2>(p-chlorophenyl)e1hane; />,/>-i)DT, l.l.t-nrcliluro-Z^-MaCp-ohlorophcnyOrihane. The nomenclature of the polychlorinated bipSicnyl tl'CH) compounda fcWows the accepted font, the position of chlorines on one phenyl bcinj indented hy numbers from 2 to A white chlorine position* on l he second rinf arc designated by a num> licr wall n superscript prime, i.c.. 2,2'-dichlorohiphenyl. ih f ?' l.' y. v .. ' tvr '* V .<!' -3. t'.'-'V,. W, V v -r , v i; j v t-- < *r -V \ * * *. L-vv * %'/S . . * i w-vr I-.,-: * i" * ' 1 ' ` J* * ' I f. no *, , , ' , ' t v\utr-r y . ! 1 ,s 1. V si --1 .. ,. .`s-; : :.: ...*,. v . a.-: -..,`S i. ,*tjX >*. >f>'.\ F/\ r^''rf'' #v*,.'V-e i* -,.* !^^i.-V-;T'j*.-.:>V'.-i`u'v.' -l - - -: 1 ...T%*''*'"-''A, ... :r\'"-i -A + , . V: '?.;:'':* ''::;?;*.>.& v -' :Xi ..v-* '-' . . , . .--* ;>."y- 'a' ^.VxV.vi .i4 4 `1 .| 1 j t `j <* 1 , 4 Nj 5.*i !;<Vv*^*$&S* ^iM' 420 iiansli.l ano rcotiicmiM microbodics is based on a comparison of (he amounts present in any cell with the mount present in a control hcpatocylc(value ^ +) mid a hcpatocylc having the ma\imum amount of change (value = 4 +4 1 +). The grading system reflects only relative numbers since morphometric techniques were not used for quantitation. The major ultra structural alterations induced by the various purechlorobiphenyls were prolifera tion of SER, alteration of the rough endoplasmic reiicwtum (RER) and increased num bers of lipid droplets and tnicrobodics (Table 1). yjstf* TABLE 1 j. Cuanoes in Livtk Weight, Hepatic Smootii Smartasmic Ri ncut um. Lli-ln DaOI'Lhis AND MtCftoaODIE*, FOLLOWING TH* AOMtNlSTRAIION or DOT. Aroclor I2S4 and 1260 and Isomenically Pure Chiokobiphenyisio Rais* Compound Liver weight* (g) Smooth Lipid ER* droplets M tcrobodies Vehicle-Treated otp-DDT p.p'-DDT Aroclor 1254 Aroclor 1260 Biphenyl (BP) 4-Chloro BP 2,2'-Dichloro BP 2,4`-DichloTO BP , 4,4 -Dichloro BP 2.5.2,.5,-TetTachtoro BP 2.4.2',4'-Tctrachloro BP 3.4.3,4-lctrachloro BP 2,4,5,2',4',5'-Hcxachloro BP 2,4,6.2'.4',6'-Hexachloro BP 2,J,5,2',3,,5'-HcxaditoTO BP 2.3.4.5.2',3',4',5'Octachloro BP 4.77 1.14 4.91 0.46 5.59*0.89* 6.23 + 0.30* 6.15 + 0.49* 4.87 0.11 4.76 0.69 4.8$ 0.96 4.69 0.34 4.21 0.44 4.68 0.69 5.03 0.48 4.73 0.47 4.18 0.91 3.821 1.29 $.80 0.78* 4.53 1.46 + ++++ ++++ + 4+4 + +++ + ++ + +++ 44 + +H 4 4-4 +4 + +4H 4+ + 4 ++++ +4+4 4 +4 4 +4-4 +4 4 4 4 4+ +4 ++4 + +4 44 4 41 44 1 44 44 44 44 4 4H1 4444 44 !4 4 1+4 4t 44 + 4 4 +4 44 4 4 4+ 14 +1f 444 44 1 1 4 i44 1 * Animals were Injected ip with a peanut oil solution al a dose of SO mg/Vg for J days and killed 4 days after the last injection. ' * Values presented are mean weights t SD of 14 (vehicle-!ruled) and 7 (egeru-lrcalcd) animals per group. Values arc based on wet I issue weight. . ' Values are statistically significant from control weights at a probability level p < 005. `See text (Reeulta) for grading system. Isomeric-ally pure hexa- and oein-chlorobtphenyls, administered at a concentration of SO mg/kg Tor 3 consecutive days, caused alterations in hepatic morphology that were visible with the light microscope (Fig. 1). The changes included large numbers ofhcpnto- cytes with vacuolated cytoplasm and small foci of necrosis involving S or 6 cells. The necrolic foci were particularly numerous at the periphery or the lobule. Treatment with 4-mono-, 4, 4'-di and 2,5,2',5'-lclrachlorobiphenyt at 100 mg/kg Tor 7 days resulted in larger numbers of necrotic Toct, centrolobular necrosis and proliferation of biliary ductules (Fig. 1). The lesions were most severe in rats given 4,4`-dichlorobiphenyl for 7 days, Hepalocyles or rats treated with or pj'-DDT (Fig. 2A) had increased amounts of SER in the form of vesicles (S) which tended to accumulate at the periphery of the r*fm HCO AND LIVER MORPHOLOGY 421 cell. Then' nas a reduction in the amount orRER (R) which often displayed expanded or sinuous cisternac. The number of lipid droplets and microbodies (M) was increased. Hepatocytes fiont rats given the commercial Aroclors were similar to those from rata treated with DDT. Aroclor 1254 treatment (Fig. 2 Ft) caused more pronounced altera tions titan Aroclor 1260 treatment. Hepatocytes of treated animals contained very abundant SLR, reduced RLR, increased numbers or microbodies (M) and lipid droplcts(L). Hepatocytes from rats treated with pure PCB isomers for 3 days were compared with hepatocytes from control rats and rals treated w'ith the commercial Aroclors and DDT ' jf , ' t > ' jV i uaFs^***#***r t I'm. I. Liver from a on treated with SO mf/Lg/day o14-chtorohiphenyl for 7 days showing hcptloc; 'c. iiii vacmilatcdcyluplasin (V) and a small area of nscrosis (N). Hematoxylin and eosin. x2IJ. at the same dosage. The amount of hepatic smooth endoplasmic reticulum in rats treated with biphenyl and 2,2*-diclilorobiphenyl was comparable to that in control rats; but in rats treated with 4-mono-, 4,4*'di-, 2,5,2*,5*- and J^.S'^'-tetra-chlorubiphenyl, the hepatocytes contained'increased amounts of SER. All other compounds tested (2,-r-di, 2.4,2 .4 -tetra-, 2.4,5,2',4',5'., 2,4,6,2',4',C'- and 2,3.5,2\3'.5'-hexa- and 2,3,`J,5,21,3',4',5'-ocla-chlorobiphcnyI) caused increases in the amounts of SER that were equivalent to the increases observed in hepatocytes of rals treated with DDT or Arodur 12(i0(l"ig. 3A). The RER often had sittuouscisiernae(Fig. 38) and was reduced in n-nount in hcpatocylcs of rats treated with hexa- or octachlorobiphenyl (Fig. 3C). 1 he 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 (F ip. 3C). I.csscr effects were observed following treatment with biphenyl, 2,2*-di- and *: *Ls-y-' M'-V t ; . . . ' ' - S r' HONS 200*64 ,1* ~ NT -. * '>S'.t , 1 ^ > .-. i"- > f- . > * 1 . ' - y <t . 1 -V '. S.'; I, t i > . V-: * - , Wj .W.w:; > '* - VJ' ' it'\v : x. r' r.t/ -;V. W i V-S* 422 IIANSF.LL AND LUHHCltON ' 1 /! Fkj. 2. Hcpitocytci from rati treated with JO mj'kjtMay of p,/>'-DDT (A) and clor 1254 (B) for 3 day* allowing proliferation of imooth endoplasmic reticulum (S), cipimttu' . ;rm.ir of rouih endoplasmic reticulum (R) and microbodiei (M) and lipid dropleti (1-). A, x 12.000; B. x 14,000. fJ!^w..'.' W'l.jMimn>wvi*mvrw. HONS 200665 \.J\* * ' .fir; i 1 ' v>*. >'/- ^i.- ' -V: . , ^ ~-fc * -d.thlu.rk `. *. ---i-*- * - *. - 'a r* *'**1 1 ; te.-;: PCII AND L1VLK MOKfHULOCY 'K(i.f..,J' -t-Jt.ff.-'j.f.'>*f:'J ;:" I ii; l Hc|wioryi from deferent rail 1retied wilh isomerjcilfy pure rhlorobiphenyb at conccntra<io!i> or so mg/kg/day for 3 days. (A) Treated with ^a^'^'-ieinchlorobiphetiyl, showing abundani i.r;oo<h endoplaimic reticulum (S), glycogen (C) and a microbody (M| *25,673. (B) Treated with i,4,.V,4'-iciracliloiol>iplienyl, showing timious cistemte of rough endoplasmic nelieulum (R) sur rounding a mimehondrion (M). *23,500- (C) Treated wiili 2,3,5,2\V,4'.heichlnrobiphenyl, showing prolirci ,nio)i (>f j-inoo;h endoplasmic-reticulum (S), roicrobodies (M) and lipid droplclt (L), *14,100. , .> .: . NOONN* 200606 r...... - * ' - V. - - O'..*; - 1. > A. a - * v.' <U ' v*1 I, - K,h - ;-r;..--i ?:>?>?sjnr v-x^v rr~ ^ .. ' ` .*' ' ,j" tV ' : . . 1 . .. ; ' ' . ... /. .i, 1X" * . -fL'. ^ tV 1 ;<* ' ---`zi ?/. \ i , -r . \ ` X' \Tf- ' <,i"i--*.4,i f- -.s'j;,f*--* . .*^* ,T -pl,` i' .; v; , .. ... .-v., vf- -.>> \ 4-*:? - v .."i-. ? v ' : .. - ry'-'.'. h .t "'{'s.V-'r* a' -' . * sw 424 tlANSUL AND rCOBICMON 2,5,2'l5'-te(rMchlorobiphcnyl. The number of lipid inclusion! in hcpalocyics wav nevei s pre1 as thal observed following treatment with DDT and the Arodors The atlminisiration of 4-mono-, 4,4`'di' and 2(5,2`>5i-letra-chlorobiphenyl al a dos;ipe 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'-dldMorohiphcnyl caused more drusiit Tr-*;,'.. 'a is.-1' .v *>+.> VfT . VCUX'+i fKi. 4. ilejulocytes from rats treated with 100 mg.'k(/day of4,4'-dichlorobiphenyt for 7 dayt sh<>'si abundant smooth cndoplatmic reticulum (S), mictobadiw (M> and lipid droplets (t) A. *H 10 B, >IHD, . \ij- i MOMS 200687 i '>>* irr*' a *a lamrw Un'*^4f.HTwi tom' PCii AND LIVER MORPHOLOGY alterations rhan 4-i)ionocltlorobiphcnyl or 2,5,2',5'-teirachlorobiphet)yl, the effects of i Jjc latter two agents being approximately (he 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 Ihe 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). Jn all previous studies, administration of PCBs has resulted in increased amounts ofSER in licputocyics (Nishizumi, 1970; Kimbrough el at., 1972; Vos and Notenboom-Ram, 1972; Nnrback and Alien, 1972; Roller and Ztnkl, 1973). In this study, the proliferation of SH< was closely related to the structural characteristics of the compound used. Biphenyl arid 2,2'-<Jichloiobiphcnyl appeared not to induce extensive proliferation of M lt, Inn with the other compounds, (he proliferation appeared to be related to the degree ofchlarimition and particularly to the presence ofa chlorineitom in tlse4 and/or 4' position finblc 1). The morphological alterations correlated well with the activities of drug-metabolizing enzymes within ihe same rats (Johnstone et at., 1974), When commercial mixtures were employed, mixtures having a greater chlorine content (Arorlors 1254, 1260) luid greater enzyme-inducing effects and caused more marked morphological changes than those having lower chlorine content (Villcneuvc et of., 1971; Pickers et at., 1972; Roller and Zinkl, 1973), The more highly chlorinated com pounds nitty have greater effect because metabolic studies show that low chlorine analogs ate eliminated from tissues more rapidly than the more highly chlorinated compounds (Grain ct at., 1971; Hatley and Bunyan, 1972; Bickers et at,, 1972; Rehfeld et af.t 1972). In this study and the report of Kimbrough et at. (1972), administration of Aroclor 1254 resulted in more severe morphological alterations than Aroclor 1260 or any of the pure compounds used. It was also a more powerful inducer of drug-metabolizing enzymes Ilian Aroclor 1260 (Johnstone et of., 1974). Aroclor 1254 was not Ibe most highly chlorinated compound used, and Ihe response to its administration was incon sistent with the view that increased chlorine content caused greater alterations. The fnilurc of Aroclor 1254 lo follow the rule of increased tissue response with increased chlorination might be due lo the particular combination of chlorobipbenyls present in the mixture or possibly lo the presence of contaminants. Chlorinated dibenzofurans iukI dihcnzo-p-dioxans have bsMefound in some commercial PCB mixtures and these compounds caused alteratfeM fn hepatocyle morphology tbat were similar to those caused by PCIIs (Vosera/T. lWBrZitkoand Choi, 1971 ;Greig, 1972; Hutringere/ at., 1972; Wchh and McCall, 1972; Norback and Allen, 1972). Such contaminants are not reported to be present in Arodor 1260 (Vos flat., 1970). Kimbrough etat. (1972) staled Unit u preliminary analysis of the lot of Aroclor 1254 used in their study suggested pos sible con trim million with a chlorinated dibenzofuran. It is likely, therefore, that increas ing chlorination of ihe biphenyl nucleus does lead to proliferation of SER and lo inctcuMil activities of drug-meiabolizing enzymes. The enhanced efleet observed with Aroclor 1254 can be due to some other factor than chlorination. Treatment of rats with 4-cli]cro-,4,4'-dichloro- and 2,5,2',5'-tetrachloro-biphenyl at a dosage of 100 mg kg day for 7 days resulted in marked proliferation of SER. The if >'. ; -a' HOMS 200MS 'I ' A-. * .%*""/ r . . V\;.; v *:"' "v-rsr'&n' /.v>*Vv i- ..*.-. f.. . < . , - , " v p* I .* * ^ ' 4. . J ,, . '*'- V; 'l?- ** \ \t :, s C>.'. 'i ! . *> 4 . < .. , . * i* i l* ^ * V' , -J .*1 ''- -V, ; `\V,*** `^i)?**, *fl V, .'. ` ` \ * _.' ' Jj V-r: , 426 HANSTLL AND tCORICHON dosages, modes of administration and duration of exposure have varied greatly in pub lished reports of the morphological alterations produced by PCBs. With the exception of the study by Kimbrough el al, (1972), most studies have used much higher concen trations than (he ones used in this study. Most authors have noted increa&cJ hepatic weight and hepatic cell size. In our study, the increurift hepatic weight was signifies :u only after treatment with pjt'-DDT, Aroclors 1254 tad 1260, and 2,3,5,2',3'.5'-hexachlorobiphenyl. There was no significant Jnerea*riif4^i7c. This result may be due to the short exposure time and relatively low doses Mill. Concentric membrane arrays or whorls within the cytoplasm of hcpatocytcs have been noted in several studies (Nishizumi et ai, 1970; Norback and Allen, 1972; Roller and Zinkl, 1973). Their formation may be species-related as such structures were found 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-ietrachlorobipheny). Many of (he 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) 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 maximize exposure to degradative enzymes in the membranes. Vr- '}/, r>v Z si-osTOMt fe :f? 1 ACKNOWLEDGMENTS This study was supported by funds from the Medical Research Council or Canada (Crums MA-4807 and MA-361)). Samples of Aroclor 1254 and 1260 from the Monsanto Chemical Company and Of the pure Chlorobiphenyls from Dr. O- Hulzinger, National KcscarcliCouncil, are gratefully acknowledged. The assistance of Dr. T. Ghost, Department of Pathology, Dilhousie University in the histological eva hiatton oftissue sections was grea tty appreciated. REFERENCES Bailey, S. and Buntan, P. J. (1972), Interpretation of persistence and effects of polychlorin ted biphenyl* in birds. Natme (London) 236, 34-36. Bennett, G. A., Drinker, C. K., and Warren, M. F. (1938), Morphological changes in ttw livers of rats resulting from exposure to certain chlorinated hydrocarbons. J. hut Hyg Toxicol, 20, 97-123. Bickers, TV R- Harrer, L. C., Kawas, A. and Aivarf.s. A. P,(1972). 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