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1,,iii.0L00' Am lfl1 i,haAm*(.olocv 13. i63-i7 (IV72) Comparative Toxicity Study of 2,4,5,2',4',5'-Hexachlorobiphn)rl and a Polychlorinated Biphenyl Mixture in Rabbits J. C. Vos and Erica Notimoom-Ram /minute of Veterinary Pulholofy anti Institute of I 'etermary Pharmacology and ToxfcaJagp, University of Utrecht, Billuraat 172, Utrecht, The Netherlands Received December 6, 1971 Comparative Toxicity Study of 2,4I5,2',4'.5'-Hex*chlorobiphcnyl and a Polyehiohnaied Biphenyl Mixture in Rabbits. Vos, J. G. and Notcn*oom~ Rah, Erica (1972). Toxicol. Appt. Pharmacol. 23, 563-371. Three groups of rabbits were treated daily (5 limes per week for 2S days) with 120 mg 2,4,5,2\4*,5'-hex*chlorobiphenyi, 120 mg polychlorinated biphenyl (PCB) mixture (Aroclor 1260*) and the solvent isopropanol, respectively. The dermal application resulted in early macroscopic skin lesions in the Arodor group. The lesions in the 2,4,5,2'.4',5'>hexachlorobiphenyl group appeared later on and were less severe. This difference was confirmed mtcroscoplcafly : hyperplasia and hyperkeratosis of the follicular and epidermal epithelium were more severe in the Aroclor* group. Fecal coproporphyrin levels were significantly increased in the experimental groups. Enhanced liver weights were found in both test groups. Liver injury, as judged by light microscopic icMoni and elevaied serum transaminase levels, was somewhat more severe ia the hexachlorobiphenyt group when compared with the Aroclor* group, though the mean liver content was about the same (respectively, 239 and 23f> ppm). Light microscopic findings included subcapsular necrosis, zonal necrosis, hydropic degeneration, as well as a peripheral and perinuclear shift of cell organelles, and focal cytoplasmic hyalin degeneration. In electron microscopy the shift was found to be due to a proliferation of smooth surfaced membranes of the endoplasmic reticulum (SER), resulting in s displacement of rough surfaced membranes (RER) and mitochondria. The hyatinized cytoplasm was recognised as tightly packed tubules of proliferated SER, that is considered as hypertrophic, hypoective SER. The contribution of chlorinated dihenzofurans and pure PCB in the toxicity of crude PCB mixtures is discussed. Polychlorinated biphenyls (PCB) ate industrial chemicals with many applications. The toxicity of tiiese compounds for industrial workers has been known for years, and especially the occupational disease known as chloracne (Jones and AWen, 1936; Schwartz, 195b; Meigs rt at., 1954). Recently, the consumption of rice bran oil acci dentally contaminated by PCB resulted in many cases of chloracne among Japanese consumers (quoted by Craw, 1970): liver damage was alto reported (quoted by Nithtzumi, 1970). A critical report on the use, environmental contamination and the toxicity of PCB and other industrial halogenatcd hydrocarbons appeared recently Corn C IUTZ hr AtaamUx Am. It. Alt tights of rwrroducuon * tny (to* rwmi U1 * i*- :' ;-s - : . ,.f `J : T ;p 4%.T-r. .-, i V?fi:r>.v4^?4 *brt *" fepH' P*mWSm4 mwu JW-rifc-" ,41**.'.-, >A fefl pH teil ^:.v'V`V'Xw . f .j v'-JB''. v-J : <-S >4Sr*l* 564 VOS ANI> NOTkh'nOOM'KAM (Zitko and Choi, 1971) There is an additional danger for wildlife from (he widespread environmental contamination with PCB through food chains, and this is, ultimately, also a menace to human health The purpose of the present study is to compare the toxicity of Aroclor 12G0 ' wnh a single isomer 2.4,5,2',4`,5'-hexachlorobphenyl. The commercial Aroclor 1260' sample was found to he the least toxic in a comparative study using three PCB mixtures originating from different firms (Vos and Koeman, 1970). Chlorinated dibenzofurans, considered to be responsible for the higher toxicity ftCllpgi PCB mixtures, were not found in the Aroclor 1260* sample (Vos el a/., 1970). XBh|p more certain about the biological effect attributable to PCB itself, a pure rotnpOlllBfi.V I.......hi m biphenyl, was used for comparison. This is also .one step JgtJhe understanding of the toxicity of the different components of PCB mixtures. Hepatic porphyria was also studied. Special attention was paid to PCB-induced liver damage, using light and electron microscopy for structural alterations and gas chromatography for the determination of liver residues. METHODS The lechnica! PCB mixture, a viscous fluid with an average of 60% chlorine, was obtained from Monsanto in the United States (Aroclor 1260* Lot No, AK-3). 2.4.5,2',4',5'- Hexachlorobiphenyl, a crystalline compound, was synlhelizcd according to the Ullman synthesis, using 2,4,5-lrichloroiodobetuene and copper powder at 220'C (Tasand de Vos. 1971). Because of the chemical reaction,]! is unlikely that this particu lar isomer contains chlorinated dibenzofurans as impurity. The Aroclor* sample was analyzed for the presence of chlorinated dibenzofuran and found to be free (limit of detection: 1 ppm). Adult female New Zealand rabbits, 3.5 mo old and weighing 2.S-2.9 kg, were distributed at random into 3 groups of 4 animals. The performance of the dermal loxicity study was in general the same as described earlier (Vos and Beems, 1971). Because of the lower solubility of the hexachlorobiphenyl isomer when compared with the PCB mixture, 20 mg ofthe PCBS (instead of 120 mg in the prior study) were dissoh ed per milliliter of isopropanol. The PCB solutions were kept >1 body temperature in order to keep the PCB dissolved. Six milliliters of PCB solution (120 mg) or of isopropanol as a control was dropped daily, 5 times a week, on an area of 5 x 10 cm on the clipped and shaved backs. After a test period of 4 wk (20 applications of 120 mg PCB) the animals were killed. The coproporphyrin and protoporphyrin analyses of feces from the cecum, the microscopical examination of tissues in Wood's light as parameters for chemical porphyria, and the hematological examination were done according to a former study (Vos and Beems, 1971). At necropsy, body and organ weights were determined. The skin, liver and kidney were fixed in 10% buffered formalin. The spleen, thymus,axillary and mesenteric lymph node and appendix were fixed in Carnoy's fluid for 24 hr at 4 C and stained with melhylgreen-pyronin according to Elias (1969). Paraplast`embedded sections were stained with hematoxylin and eosin. For detailed histology, selected sections were stained with Peris'iron stain and with Best's carmine. Moreover, cryostat sections of the livers were stained with Sudan black for the detection of lipids. Un> ' .V- * '* ' ' ' i - -fj- .aa . a. . * pA .-f. 'f.~ '-~ ,;T i1 > VjV^'sr'- - \ .' }' ' Ii-`^iriln " if-j* ti"'-',-'(i |H>iinfy s t TOXICITY OF POLYCHLORINATED BIPHENYLS 565 stained Paraplast1* embedded sections and unstained cryostat sections ofliver, kidney and small intestine were studied in a fluorescence microscope for the detection of ceroid pigment and excess quantities of porpyrins. respectively. For ultrastructural examination, small blocks of liver tissue were fixed In 01 m cacod>laie-HCl buffered S% glutaraldehyde (pH 7.0) for 5 hr. The liter (issue was postfixed two times for 45 min in 0.1 m phosphate buffered 2% osmium tetroxide TABLE I COPROPORPHVRIN AN'D PROTOPORPHYRIN CONTENTS f^a/fl DRY WEIGHT) of Feces of Rabbits Treated with PCB for Fou* Weeks* Coproporphyrin Protoporphyrin Hexachloro- biphenyl Arodor* Hexachlortw Control biphenyl Aroclor* iControl Mean: 45.0 6.2 20.8 39.6 27.9* 24.1 5.0 29.4 16.0 18.6* 4.5 3.7 3.6 3.5 3.8 88.3 8.3 51.2 665 53.6 35.1 8.4 65.1 65.1 43.4 17.8 11.9 15.6 10.9 14,0 Figures are the contents of feces, collected from the cecum of the individual animals. * Sifnifiesntly different from comroJi,^ < 0*025. TABLE 2 Absolute and Relative Liver Weights, Microcoopic Ltvca Damage, Liver residues and Transaminase Values for Rabbits Treated with PCB for Four Weeks* Liver weight g/J0O 8 body weight Microscopic Liver damage* Liver Content (ppm) SOFT (U) SGOT (U) HtxaciiloroHiphenyl Mean: 128 132 146 126 133 Aroclur * Mean: 14] 129 152 133 139* Control 131 110 91 83 Mean: 104 4.6 4.2 4.8 5.1 4.7* 5.0 4.0 4.8 4.2 4.5 4J 3.5 19 2.8 3.4 -H-+ ++++ +++ ++ f++ ++++ + -- -v - 249 140 261 304 239 198 248 317 182 236 0.8 0.6 0.6 0.7 0.7 71 16 56 34 70 30 92 21 72* 25* 64 29 32 to S3 13 34 12 46 16 43 9 22 10 20 11 31 11 29 10 * Values are liven for the individual animats. * in - -r-* j - a comparative eaiimai* of the liver damaae. ' Significant!/ different from controls, p < 0.05. ' significantly differeni from controls. P < 0.025. 20 * .;' - i ; - x Tf. -r, - ite.Vt.gj/. mtm - V"'-' * jss`; fr?r0?.; -mM 'ft* Ifr-gjfV ; HOMS *-00663 ' 1.. Flo. 1. Liver oF 2.a,J,2',4.J'-he*echlorobiphenyl treated rabbit. Subcapiular bend of necrotic tiiiut (I) and area of hydropic defeneration (2). Hematoxylin and eoain. >. 60. Fic. 2. Higher magnification of the subcapuilar rtgron of the liver teen in Fig. I. Note the nrcrovi al I, hydropic oelli at 2 and proliferation of mcacnchymal cclli al 3. Hematoxylin .nd coin - liO. < <BJ nmvm**!* *n in" iS. V 'l MOHS 200664 irrr $ f-'ic. 4 Hepaiocjie* of 4 control animal, Clear areas (arrow) represent negative images of glycoten. Derive muienel representing cell organelles is tcaiierrd in iKe cytoplasm. Tolujdine blue, * 940, MOWS 200445 Fig, 5. Liver section of in Aroctor* treated rabbit. Toiitidine blue. . 940. (a) Showing peripharal and perinudear displacement of eeN organelles: note also the foamy cytoplasm (arrow) (b) Hydropic cells ibowing vacuoles (I), that are separated from homogeneous hyalinirad cytoplasm (2|. Mon (he perivacuolar localization of densely staining organelles. *.. . * ' V * ' Fki. 6. Severely damaged hepaiocyles of the same l,4.J,2'.4',5''he*achlorobiphfnyl-treiied .inims as shown in Figs. I and 2 showing hydropic changes (I), NpM droplets (2). and focal cytoplasm* hyalin degeneration (3). Note also the nuclear alterations such as the irregular outlines of the nui:It and the prominent nucleoli. Toluidine blue. v 940 MOMS 200666 V1 ;- ^ *' ;V-'/r ' V ^ -V ,y.`. n723^*?f'r'*- * -.j'-.f. : . f H V\< v  TOXICtTY Oh POLYCHLORINATED BIPHENYLS 569 ipH 7 2) Block staining was carried out with uranyt acetate. The tissue was then dehydrated rapidly in ethanol. nd embedded in Dow epoxy resin (DER*) {Lockwood, i U6-t) Sections were cut with glass knives on a Reichert ultramicrotome. Thin (0.5-1.Oft) sections for orientation and for light microscopic examination were stained with to.'uidinc blue. Ullralhin sections poststained with lead citrate were examined by electron microscopy {Philips EM 100*) at 40 kV. The PCS concentrations in the livers were determined by gas chromatography, as described earlier (Vos. era!., 1971). The significance of difference between treated and control groups was determined on a one-tail significance level, using the WiJooxon test for to unrelated samples {van der Waerden, 1957). RESULTS Afier 3 days of PCB treatment, the skin of rabbits in the Aroclor* group showed some redness. After 6 days, definite redness and some thickening of the skin was noted in this group. After 2 wk of treatment, hyperkeratosis and formation of transverse wrinkles were seen. At this time, some redness and slight hyperkeratosis, together with the presence of hexachlorobiphenyl crystals on the skin, was seen in the hexachlorobiphenyl treated group, although a slight hyperkeratosis waa present in the l ie. 7. Control liter whh hepatocytcs showing diffusely scanered mitochondria and network of the endoplasmic reticula or rough and smooth types. Ttie clear areal may represent negative images of Lyenscn. The intercellular space be I ween [he bile canalicuH may be an artifact. Nucleus (N); bite canaliculus rC:. space of Disse ID]; endothelial cell (E); and sinusoid (Sj. Uranyt acetate and lead curaie 4J00. HONS tOO**7 570 VOS AND NOT EN ROOM-HAM control (isopropanoi) group, Differences in the regrowth ofhair between ,c two groups were also noted. Regrowth was most reduced in the Arodor*- p*.*nd ii-^ in the hexachlorobiphenyl treated animals During the course of the e\p-rr.Tient, thg lesions in the Aroclor group were clearly progressive, while the lesionsthe h*,.,. chlorobiphenyl group increased rather slowly. ` All animals, except 1 rabbit in the hexachlorobiphenyl group, showed a -eight during the experimental period of 4 wk. Mean weight gain in the hexachlc robiptenM animals was 142 g (ranging from -215 to 365 g), in the Aroclor* group ''5 g^from 70 to 640) and in the control group 491 g (from 430 to 550). Fig. 4. Hcpetocyies of the tome Hw ofan Aroclor* treated rabbit as shown in Fips. 1 and Ja, showing foamy cytoplasm. The cytoplasm is mostly occupied by smooth endoplasmic reticulum iSE A) Some perinuclear shift of mitochondria and REA eaa also be saon. N. nucleus Uranyl scneis end lead citrate. * 4100. Coproporphyrin levels in the feces, collected at necropsy from the cecum of individual animals, were significantly increased in the hexachlorobiphenyl at weliasinthe Aroclor* treated rabbits (Table I). Protoporphyrin excretion was also increased, ihough not significantly. Macroscopically. fluorescence was found in the bile of 2 rabbits of both experimental groups. One animal of the Aroclor * group showed also fluorescence of liver and bile. Examination or cryostat sections of liver, kidney, and small intestine in the fluorescence microscope showed fluorescence in the livers of 7 out ol 5 PCR tree led animals. This fluorescence was mostly located in the bile duct and sometimes diffusely HONS 200*46 .v `'r>' TOXICITY OF POLYCHLORINATED BIPHENYLS 571 m penportally located hepatocyies. Sonia fluorescence was also noted in epithelial cells of the small intestine in 4 PCB treated animals. Severe abdominal edema was seen in an Aroclor * treaied rabbi) lhai showed also the highest liver content (317 ppm) and se\ ere liver damage (Table 2), Fic 9. Higher maysMredan of a pan at PI*. I. The proliferation of SER consists of vesicular and tubular membranes. Clear araaa of the cytoplasm, associated with the SER, most likely represent nee*nue jmafes of *lyco*m (arrow). Note the dilatation and dc|ranulnion of the rough endoplasmic reticulum (RER). M. optochondria: N. nucleus. Uranyl acetate and lead citrate. * 13,300, No significant differences were found in absolute and relative organ weights, except for (he liver, and in hematological findings, except for (he serum transaminase levels, These values, together with the PCB concentralions in the liters and the comparative estimate of liver damage'are given in Table 2. The SGPT and SGOT values were significantly increased in the hexachtorobiphenyl group. An increase was also noted in the Aroclor* group. Mean liver content of PCB was the same in the experimental HOMS 290669 VOS AMD NOTCNBOOM-RAM groups. The increase and severity of liver damage in the Aroclor' group agreed with the increase in liver residue. At examination of skin sections of the Aroclor* group, hyperplasia and hyper keratosis of the epidermal and follicular epithelium were found. The lesions were not yet so pronounced as in the prior study (Vo* and Haems, If7l). Hexaehlorobiphenyl induced skin damage, especially hyperplasia in the apkteniR}ind follicular epithelium as well as follicular plugging, was clearly less wtara OMfegwed with the Aroclor* treated rabbits. A slight hyperkeratosis was found in soma diatrol animals. This could be due to the treatment with isopropano) (6 ml/day); it was not found in the prior study (Vos and Beems, 1971) using a lower dose (1 ml/day). Fk>. 10. Hepalocytes of an Aroclor* treated animal. Similar hydropic cell* as teen in Fig. 3b. showing large eonfluent vacuole* wHh Bocouknt material within them fV). The hyalimxed cytoplasm of hydropic hepalocytes shown in Fig. Jb consists of hypertrophied, densely packed SER. Note the arrangement of mitochondria around the vacuoles, farrow). Uranyi acetate and lead ciirsle. 5300. When the lymphoid system was examined, no outstanding differences were seen in spleen, appendix and lymph nodes between control and treated groups Some atrophy of the cortex, together with less pyroninophilic cells in the marrow, were found in the thymuses of rabbits in the PCB treated groups. The atrophy agrees with the lower absolute and relative (about IS%) thymus weights in these groups. No kidney damage was found, except for the presence of polyploid tubular epithelial cells in a hexachlorobiphenyl treated rabbit. Pathologic changes in the livers of the treated animals were, in general, the same as described in a previous study (Vos and Beems, 1971). These findings included cenlrolobular degeneration and liver cell atrophy, focal hyalin degeneration of the cytoplasm 200670 . -I v- _ .,, . 4 *> - ' TOXICITY OF POLYCHLOatNATEO IIPHENYLS 573 of hepatocytes. enlarged nuclei end loss of glycogen. As shown inTsble 2, liver damage by he.\achlorobipheyl was somewhat more severe when compared wilh the Aroclor induced lesions. However, in the present study, areas of hydropic, degenerated and necrot ic cells d ominucd, often in the subcapsular region (Figs. I and 2), forming bands W*r.V*' . ) *-*"** - - ... , * . .;<* s ''tr , > . 1- I 'J *' * l :i;* : R.I aktw* 'll wT.*' . i Fig. It. Higher mafniAeeiioA of a pan of Fig. 10 reveals the tubular structure of (he densely hyper trophied smooth endopJasai* retteufcan. Umnyt aealate and Had citrate, a 13,300. of necrotic and degenerated cells, accompanied sometimes by proliferation of mesen chymal cells. Necrosis was usually accompanied by hemorrhage. Lots of glycogen was not limited to the degenerated, hydropic or necrotic areas, but was also seen in foci showing no alterations in hemtioxylm-eosin sections. In the rest of the liver the glycogen content was also reduced when compared with the controls. The cenirolobular damage was often rortal. This distribution can be explained by the concept of structural and functional hepatic unit (Rappaport eta!., 1954). The presence of Peris* positive material nous a ` ' . ..&. ' /. fci*- . .' v. - ft- i.",''"i ^'aVv.-*;-v;..'*4i--vl 'Ay-* -'.; :^'. -*3 ;`.i .V - '.-. * .... ' .; ,v1.-? * ,, iI C v>-v *rt ' *. % tv *'' . \ s-' ~ W ViK -..-vr i'v^r.! i'al Aj ic'- ` ;-.s ,'S''%} ?:o -Av-W.A 574 VOS AND NOTENSOOM-MAM and ceroid pigment in KupflTer and parenchyma] cell* (characterised by a yellowbrownish fluorescence) was evidently less than in the preceding study and was limited to some necrotic areas. In both treated groups, numerous hepatocytes till |jjgt S lilt light staining cyto plasm. In these cells, the cell membrane and the nurtwr jBpttbrane were emphasized by the presence of basophilic material (Fig. 3), When pfclftieambedded liver tissue was examined by light microscopy, these alterations chM lie observed more clearly (Figs. 4 and 5a). This was the general trend In the PCI treated animals. In electron microscopy, the most striking feature of these cells was the proliferation of smooth Fic 13. Smnly dsmaf+d hepalocyre of the same 3,4,J,2',4`,$') achlorobiphenyl (rriicd animal as shown in Fit- 6. Photograph shows a duster of tubular smooth endoplasmic reticulum (in light microscopy visible at focal hyaline deganeration), formini a dense, closely packed spatomeralioo (SER). Nose also the lipid droplets (L), aod hydropic swelling, as shown by a treat number of small and larte vesicles (V). Uranyl acetate and lead citrate. * MOO. surfaced membranes of the endoplasmic reticulum (SER). As a result of the prolifera tion of SER, a perinuclear and peripheral displacement of mitochondria and rough surfaced membranes of the endoplasmic reticulum (RER) was apparent (Figs. 7 and B). The proliferation of SER could well explain the accentuated linings of cell and nucleus in light microscopy and the increase in liver weight (Table Z) in the PCB treated groups. The RER showed some dilatation and degranulation (Fig. 9). In addition to the proliferative changes of the SER, degenerative changes were also noted. Light microscopy showed the presence ofhydropic cells (Fig. 2). Examination of plastic-embedded material under the light microscope showed some hydropic cells which consisted of large vacuoles separated from a homogeneous cytoplasm, inier- L-rS- . MOMS 200672 TW,-" -/M* maiaaaiwij g TOXICITY OF POLYCHLORINATFO BIPHENYLS 575 prcied as focal cytoplasmic degeneration. At the rim of the homogeneous cytoplasm cell organdies were visible (Fig, 5b). The focal hyaiinized cytoplasm (Fig. 5b) was found by electron microscopy to consist of tightly packed tubules of proliferated SER (Figs 10 and [ I). The condensed SER was separated from large vacuoles, sometimes with floccuient material in it. The mitochondria were arranged around (he vacuoles. Besides the iiyatinixed cytoplasm, lipid droplets and degenerative changes or the nucleus were found in the more severely afTected areas (Figs. 6 and 12). The nuclear changes included irregular outlines of (he nuclei and karyopycnosis. Myelin figures were found rarely in the cytoplasm. In the present study, no differences were noted in the hepatotoxic action between the polychlorinated biphenyl mixture and 2,4,5,2`,4',5*licxachlorobiphenyl, DISCUSSION From the observed acnelike1 lesions, both from the PCB mixture and 2,4,3,2',4`,3`he.xachlorobiphenyl, and assuniingthat the hexachlorobiphenyl is free from contamina tion with chlorinated dibenrofuran, it can be concluded that this particular compound of the mixture PCB has a slight acnegenic action of itself Considering the already mentioned dermal toxicity study, it is evident that the major acnegenic action of crude PCB mixtures comes from chlorinated dibenzofurvns. fn Table I it is shown that 2,4,5,2',4 ,5-hexachlorobiphenyl is even more porphyrogenic than the PCB mixture. Thus it is probable (hat hepatic porphyria comes only from PCB itself. In contrast with the prior dermal toxicity study, only slight effects were noted from PCB or the lymphatic tissue. This difference is explained by the longer experimental period in the former study (38 days) which resulted in a less healthy condition of the experimental animals. Stress (e.g., release of glucocorticoids) must be considered responsible for the major cfliecis on the lymphatic tissue. However, using the sensitive fluorescent antibody technique in tetanus toxoid stimulated guinea pigs, immunosuppression was found (Vos and de Roij, 1972). Liver damage was essentially the same after treatment with both the Aroclor* mixture and 2,4,5.2',4',5'-hexachIorobiphenyl. Liver damage caused by commercial PCB mixtures was due predominantly to the contaminants. Thia conclusion is based oitlbe differences in liver toxicity between three PCB preparations (Vos and Koeman, 1970; Vos and Betms, 1971). The probable contribution ofchlorinated dibenaofumns and uncontaminated PCB in the toxicity of crude PCB mixtures is summarized in Table 3. TABLE 3 Pnoaxau ComtaxmoM or Chuuunatid Dimnzowiun and Puaa Polychuminatid hrrwirvT- in Tin ToncstY or Tschnical PCB Px{taxations CMoracne Edema formaiioo Liver damage Hepatic porphyria Chlorinated dibenxofuran +4- ++ ++ - Polychlorinated biphenyl + + ++ ... . 'X r rr* ` b - -rv;* vx * * f*1- ,, p-.'A**- -5; w LW"' '* \ - - **Kj-u**>!- V'i'-"- `C .. . PSfcV'.?.: I '.:V 'M'SV:7.;." , - ;^LT<>*rv: ( ty; vh *' A . -\ v.-1; > . *' * : - : v ' t ./ ^.VV-rV;' .1 'yV'3 .w'gL -St* M; 1-0 f0mi *V 576 VOS AND NOTEKtCJOM-XAM Focal cytoplasmic hyalin degeneration, at well at ceroid pigment deposits in the hepatic cellt, wat more pronounced in the former dermal toxicity study (Vos and Becmi, 1971), probably due to the longer experimental period. Hyalin indutiont were previously described alter temichronic exposure to PCS ill eat liver (Bennett ei a!., 1938; Miller, 1944) and in mouse liver (Nithtzvmi, I3%^wtver, in the rsbbit, the most important liver lesions described varied from ftidy degeneration to marked hepatocytic degeneration and necrosis (von Wade! era!., 1983; Miller, 1944). In this study, the presence of cells with proliferated SER, resulting in a perinuclear and peri pheral shift of RER and mitochondria, was more frequently seen (Fig. 8). This pro liferation , also found by Nishizumi (1970) in mouse and monkey liver, must be consi dered the structural indication for enhanced metabolism of foreign lipophilic com pounds. Thus, the activity of aniline hydroxylase and aminopyrine Mdemethylase (determined in vitro) was increased in the rabbit after administration of Arodor* 1254 (Villeneuve *t at, 1971). A decrease in sleeping time after treatment with hexobarbiul and enhanced in vitro rates of aniline hydroxylation and p-nitroanisoie demetfayiation were demonstrated by Street at at., (1969). They also found an increase of these effects with increasing chlorine content of different PCB preparations. Despite this increase in drug enzyme activity caused by the higher chlorinated mixtures, the lower chlorinated compounds are very probably metabolized to a greater extent (Grant el al1971). On the other hand, the presence of focal cytoplasmic hyalin degeneration without glycogen (Figs. 5b and 6). in electron microscopy seen as densely packed agglomerations of SER, very probably represents hypertrophic, hypoactive SER. Hutterer et at. (1968) found reduced activity ofmicrosomal oxidaaas (a.g. drug enzymes), while SER remained hypertrophic in liven of rats treated with high dotes of dieldrin. As seen under the electron microscope, hypertrophic, hypoactive SER was recognized as tight clusters of tubular membranes, which could indicate a transition from adaptation to injury (Hutterer ere/., 1968,1969). Liver damage was also suggested by elevated transaminase keek (Table 2). Myelin figures a*re seldom found in this study. Perhaps this depends on the animal species, since Nifhizumi (1970) described myelin figures in mouse Nver after PCB feeding, but not in monkey liver. The occurrence of SER proliferation and concentric membrane arrays is found in the liven of rats fed a chlorinated biphenyl diet (Norback and Allen, 1970) and toxic fat containing hexachtorodibenzo-p-dioxin, a compound related to chlorodibenzofurmn (Norback and Allen, 1969). The results from the former study suggested that the concentric arrays of agranular membranes may be structural modifications of the endoplasmic reticulum having an enzymatic function similar to that associated with the SER. acknowledgments We are grateful to Dr. R. J. C. Kleipool and Mr. A. C. Tas of the Centra] Institute for Food and Nutrition Research, T.N.O., Zeiss, for synthesis and supply of the 2,4,5,2'.4,5'-he*a. chiorobiphcnyl sample. Special thanks are due to Drt. C. J. A. H. V. van Vontenbosch of the Institute of Veterinary Histology (Head: Professor Dr, W. A. de Voogd van der Straaten) Tor use or the electron microscope, m well ts for help and advice. MOMS 200674 m'e i 'nwtmi .n-min1 4W w." [.w,3 ;. &S&V *'fc$ - ;5S% r ; *nimiiiaii.t. v *-- u*^--L-ia..`-, ^irrHlit' - ----t- TOXICITY Ol- POLYCHLORINATED BIPHENYLS 577 REFERENCES Bennett, G. A.. Drinker, C. K, and Warren, M. F. (1931). Morphological changes in [ha liven of rats resulting from exposure to certain chlorinated hydrocarbons. J, bid. Hyg. Toxicol. 20,97-123. Crow, K. D (1970), Chloracne. A critical review including a comparison of two aeries of cases of acne from chloronaphthalene and pitch fumet. Tram. Si. John's Hang, Dermatol. Soc. 56, 79-99, Elias, J. M (I969). Effects oftemperature, pottsta in i ng rinses and eihanol-butanol dehydrating mix turn on meihylgreen-pyrorrin staining. Slain Techno!. 44, 201-204. Grant, D. L , Phillips, W, E. J. and Villiniuve, D. C. (1971). Metabolism of a polychlorin- aitd biphenyl (Aroclor 1254*) mixture in (he rat. Bull. Environ. Contain, Toxicol, t, 103-112, Kuitehf.r, F., Schatfner, F,, Klion, F. M. and Popper, H. (1961). Hypertrophic, hypoocthte smooih endoplasmic reticulum: A sensitive indicator oT hepaloioxicfly exemplified by dieidrin. Science 161,1017-1019. HurttRER. F., Klion, F. M., Wenorap, A,, Schaffner, F. and Popper, H, (1969). Hepato cellular adaptation and injury. Structural and biochemical changes following dieidrin and meihyl butler yellow. Lab. Invest. 20, 455-464. Jones, J. W. and Alden, H. S, (1956). An acneform dermatergotis. Arch. Dermatol. SyphttoL 33. 1022-1034. Lockwood, W. R. (1964). A reliable and easily sectioned epoxy embedding medium. Altai. Rcc. 150. 129-140. Meigs. J. W., Alcon, J. J, and KxaTiH, B. L. (1954), Chioracna from an unusual expoaun (o Aroclor. J. Amor. Med. Ass. 154, 1417-1411. Miller, J. W. (1944). Pathologic changes in animals expoeed to a commercial chlorinated diphenyl. U.S. Pub. Health Rep. 99.1015-1093. Nuhizi'mi, M. (1970). Light and electron microscope study of chlorobiphenyi poisoning. Arch, Environ. Htalth 21,620--632. Norbacx, D. H. and Allin, J. R. (1969). Morphogenesis of toxic fat-induced concentric membrane arrays in rat hepaiocytes. Lab. Invest. 20,331-346. Norsack, D. H. and Allen, J. R. (1970). Enzymatic and morphologic alterations of hepatic endoplasmic reticulum induced by a chlorinated aromatic hydrocarbon. Fed. Five. Fed. Aincr. Soc. Exp Biol. 29,116, 3220. Rappaport, a. M., Borowy, Z. J., Louohezd, w. M. and Lotto, W. N. (1954). Subdivision . or hexagonal liver lobules Into a structural and functional unit. Role in hepatic physiology end pathology. Anat. Fee. 119,11-34. Schwartz, L. (1936). Dermatitis from synthetic retina end waxes. Amer. J, Fob. Health 26, SB6-592. Street, J. C.. Urry, F. M., WaCstarf, D. J. and Blau, A. D. (1969). Comparative Wbcti of polychlorinated biphenyls and otganochloriac pesticides in induction ofhepatic microtome! enzymes. Presented it ACS meeting Div. Pesticides, Paper No. 17. September t-12. New York. Tas. A.C. andDf Vos, R.H. (1971). Characterization of four major components ina technical polychlorinated biphenyl mixture, ftadrea. Set. Tiebmol, 5,1216-1211. van der WaEroln, B. L. (1957). Mothematistixhe StallitIk, pp. 269-264. Springer-Varteg. Berlin end New York, Villeneuve, D. C.. Grant, D. l_, Pwlups, W. E. J,, Clak, M. L. andCuoo. D. J. (1971). Efleci* of PCB atknMMatioo on mtcmomaJ enzyme activity In pregnant rabbits. Bull. Environ. Contem. 7mr*nL6, 120-121. von Wedel, H., Hncu, W, A. and DtNTOw, J. (1943). ObservatioRS on the toxic effects resulting fromexposta* to chlorinated naphthaleneand chlorinated phenyls with suggestion* for prevention. Bobber Age (New Yerh) S3,419-426. Vos. J. G. and Beems, R. B. (1971). Dermal toxicity studies of technical polychlorinated biphenyls end fractions thereof in rabbits, Toxicol. Appt. Pharmacol. 19,617-633. , '#r- 9' , MOMS'-J0M7S .hit. 578 vos and notenkx>m-jum Vot, J. C *nd m Rou, T. (1972). ImmuMwppiSMitt teiMiy of a polychlorinated biphenyl preparation on the humoral immune response in guinea pips. Toxicol. Appl Pharmacol. 21, J49-3J3. **; ' Vos, J. G. and Kocman, J. H. (1970). Comparelive tealnotopie ejpdy with polychlorinaied biphenyls in chickens with special reference to porphyri% siwlji thr motion, liver necrosis, and tissue residues. Toxicol. Appl. Pharmacol. 17, (56*4K> . U&i, Vos, J. G., Kocman, J. H,, van du Haas, W. L,sin Nodvqa nuu, M, C., and dc Vos, R. H. (1970). IdentiflcatloR and tosicokipical evaluation of chlorinated dibenzofuran and chlorinaied naphthalene in two commercial polychlorinated biphenyls. FcoA. Cosmrt. Toxicol, t, 625-0J3. ' Vos, J. G., Stsik, J. J. T. W, A., van Houium, C. W. M. and Rinnisos, J. H. (1971). Poly, chlorinaied biphenyls as inducers of hepatic porphyria In Japanese quail, with special ref erence to (aminolevulinic add synthetase activity, fluorescence, end residues in the liver. Toxicol. Appl. Pharmacol. M, 232-240. Zjtko, V. and Choi, P. M. K. (1971). KB and other industrial halotenated hydrocarbons in the environment. FUh. Pei. BA. Cam. Trchn. Pep. 271, WMS 200^7*