Document 4J9y7djdBjwOvm4kD7KLqLawx

i>xi<mouv am> AI`1'1 Ifi) phaamacologv 23, 563-578 (IV72) Comparative Toxicity Study of 2,4,5,2',4',5'-Hexachlorobiphenyl and a Polychlorinated Biphenyl Mixture in Rabbits J. G. Vos and Erica Notevboom-Ram /mttiuic of Veterinary Pathology and Institute of Veterinary Pharmacology and Toxicology, University of Utrecht, Biltstroat 172, Utrecht, The Netherlands Received December 6, 1971 Comparative Toxicity Study of 2,4,5,2',4',5'-Hexachlorobiphenyl and a Polychlorinated Biphenyl Mixture in Rabbits. Vos, J. G. and Notenboom- Ram, Exica (1972). Toxicol. Appl. Pharmacol. 23, 563-578. Three groups of rabbits were treated daily (5 times per week for 28 days) with 120 mg 2,4,5,2',4',5'-hexach!orobipheny!> 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 microscopically: 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 lesions and elevated serum transaminase levels, was somewhat more severe in the hexachlorobiphenyl group when compared with the Aroclor* group, though the mean liver content was about the same (respectively, 239 and 236 ppm). Light microscopic findings included subcapsuiar 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 hyalinized cytoplasm was recognized as tightly packed tubules of proliferated SER, that it considered as hypertrophic, hypoactive SER, The contribution of chlorinated dibenzofurans and pure PCB in the toxicity of crude PCB mixtures is discussed. Polychlorinated biphenyls (PCB) aTe industrial chemicals with many applications. 1 lie toxicity of these compounds for industrial workers has been known for years, and especially the occupational disease known as chloracne (Jones and Alden, 1936; Schwartz, 19J6; Meigs et a!., I9S4). Recently, the consumption of rice bran oil acci dentally contaminated by PCB resulted in many cases of chloracne among Japanese consumers (quoted by Crow. 1970); liver damage was also reported (quoted by Nishizumi, 1970). A critical report on the use, environmental contamination and the toxicity of PCB and other industrial halogenated hydrocarbons appeared recently Conjxiski p 1972 by Acodcmfc In*. All ii|hu or reproduction In any fbim marred. 333 ' 4m 564 VOS AND NOTfcN BOOM-RAM (Zitko and Choi, 1971). There is an addilional danger for wildlife from ihe 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 1260' with a single isomer 2,4,5,2',4',5`-hexachlorobiphenyl. The commercial Aroclor I260` 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 of two PCB mixtures, were not found in the Aroclor 1260* sample (Vos et a/., 1970). To be more certain about the biological effect attributable to PCB itself, a pure compound, 2,4,5,2',4',5'-hexachlorobiphenyl, was used for comparison. This is also one step in the 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 technical 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',S'- Hexachlorobiphenyl, a crystalline compound, was synthetized according to the Uilman synthesis, using 2,4,5-trichloroiodobenzene and copper powder at 220 C (Tasandde Vos, 1971). Because of the chemical reaction, it 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: I ppm). Adult female New Zealand rabbits, 3.5 mo old and weighing 2.5-29 kg. were distributed at random into 3 groups of 4 animals. The performance of the dermal toxicity study was in genera! the same as described earlier (Vos and Betms, 1971). Because of the lower solubility of the hexachlorobiphenyl isomer when compared with the PCB mixture, 20 mg of the PCBs (instead of 120 mg in the prior study) were dissolved per milliliter of isopropanol. The PCB solutions were kept at 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 or 120 mg PCB) the animals were killed. The coproporphyrin and protoporphyrin analyses of feces from the cecum, the macroscopical 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 methylgreen-pyronin according to Elias (1969). Puraplast* 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. Lin- HQtiS 062700 y.'iv'V- ^V '' ' V -. At.-.It.'3. ...v.*r*rny iif r" i'ii irC'iWiUiirin TOXICITY OF POLYCHLORINATED BIPHENYLS 565 stained Paraplast* embedded sections and unstained cryostat sections of liver, 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 0.1 m cacodylate-HCl bulfered 5% glutaraldehyde (pH 7.0) for 5 hr. The liver tissue was postfixed two limes for 45 min in 0.1 m phosphate buffered 2% osmium tetroxide TABLE I COPHOPORPHYR IN AND PROTOPORPHYRIN CONTENTS (/ig/g DRY WEIGHT) of Feces of Rashits TatArto with PCB for Four Weeks* Coproporphyrin Protoporphyrin Mean: Hexachloro* biphenyl 45.0 6.2 20.8 39.6 27.9* Arodor* 24.1 5.0 29.4 16.0 18,6* Control 4.5 3.7 3.6 3.5 3.8 Hexachloro* biphenyl 88.3 8.3 31.2 66.5 53.6 Arodor* 35.1 8.4 65.1 65.1 43.4 Control 17.8 11.9 15.6 10.9 14.0 * Figures are (he contents of feces, collected from the oeeum of the individual animals. * Significantly different from controls, p < 0.025. TABLE 2 Absolute and Relative Liver Weights, Microscopic Liver Damage, Liver Residues ano Transaminase Values for Rabbits Treated with PCB ro* Four Weeks* Liver weight g/lOOg g body weight Microscopic Liver damage* Liver Content (ppm) SGPT (U) SGOT (U) Hexachloro* 12S biphenyl 132 146 126 Mean: 133 Arodor* Mean: 141 129 152 133 139c Control 131 no 91 83 Mean: 104 4.6 4.2 4.3 5.1 4.V 5.0 4.0 4.8 4.2 4.5 4.3 3.5 2.9 2.8 3.4 ++ +++ -<-+++ +++ +4+++ +++-F + - 249 71 16 140 56 34 261 70 30 304 92 21 239 72* 25* 198 64 29 248 32 10 317 53 13 182 34 12 236 46 16 0.8 43 0.6 22 0.6 20 0.7 31 0.7 29 9 10 11 11 to * Value* are given for the individual animals. * * to i +-M- ft comparative estimate of the liver damage. * Significantly different from controls, p < 0.05. * Significantly different from controls, p < 0.025. 20 1 . i- ^ 'i'- 2 . vV,;. HONS 062709 ps. v'S-J.-A- !. '?--"C-* -- m*urj,*'9`^'^^-^'.V j'. -j, ~ 'i'?t*-si 566 VOS AND NOTENBOOM-RAM Fig. I. Liver of a 2,4,3,2',4,5-hexachlorobiphenyl treated rabbit. Subcaptular band of necrotic tissue (I) and area of hydropic degeneration (2). Hematoxylin andeosin. >. 60. HONS 062110 >>4: ' I,. .'. y *.,!., " ,.''*`-jJ^ *"' '^ '"i,' " r **\. ' * * . **.- , TOXICITY OF POLYCHLORINATED BIPHENYLS 7 *. ilj- . i '- mm'*- r fcyo-'`Xi-p- Ji *> rCtr-.v Fig. X Ctnirolobulkr area of an Arcelor* treated rabbit liver. A marked peripheral and perinuclear displacement of basophilic organelles can be seen. Hematoxylin and eosin. x 375. It^v'-' i I&&* llW&stfS 5 Fig. 4. Hepitoc) tes of a control animal. Clear areas (arrow) represent negative images of glycogen. Dents material icpieseniing cell organelles is scattered in the cytoplasm. Toluidine blue. * 940. ^yywi^i> \m yay^iwuF i jii, ."?/. ,y* - * -r - Z,~'A \ v ; .i - *" 'ffiw.i J ' * * r * I<*,r ' V. , -V*. - VVtC ^v^"v"":^^'-i.'>T iKii*-afi./f'.T-1V% ts'/na.** V - f * ' " >;;.; > ii " K>" ' HONS QbZlll 568 VOS AND NOTENBOOM-RAM Fig. 5. Liver section of an Aroclor* treated rabbit. Toluidine blue. * 940. (a) Showing peripheral and perinuclear displacement of cell organelles; note also the foamy cytoplasm (arrow) (b) Hydropic ceils showing vacuoles (I), that are separated from homogeneous hyalinized cytoplasm (2). Note the perivacuolar localization of densely staining organelles. Fig. 6. Severely damaged hepatocytes of the same 2.4l5,2'.4\5'-he*achlorobiphenyl-treated animn as shown in Figs. I and 2 showing hydropic changes (1), lipid droplets (2). and focal cytoplasmn hyalin degeneration (3). Note also the nuciear alterations such as the irregular outlines of the nude and the prominent nucleoli. Toluidine blue. * 940 MOMS 062712 TOXICITY OH POLYCHLORINATED BIPHENYLS 569 (pH 7.2). Block staining was carried out with uranyl acetate. The tissue was then dehydrated rapidly in ethanol; ud embedded in Dow epoxy resin (DER*) (Lockwood, 1964). Sections were cut with glass knives on a Reichert ultramicrotome. Thin (0.5-1 .On) sections for orientation and for light microscopic examination were stained with toluidme blue. Uitrathin sections poststained with lead citrate were examined by electron microscopy (Philips EM 100*) at 40 kV. The PCB concentrations in the livers were determined by gas chromatography, as described earlier (Vos, et a!., 1971). The significance of difference between treated and control groups was determined on a one-tail significance level, using the Wilcoxon test for two unrelated samples (van der Waerden, 1957). RESULTS After 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 2t4,5t2'A',hexachlorobiphenyi treated group, although a slight hyperkeratosis was present in the Tie. 7. Control liver with hepatocytei showing diffusely scattered mitochondria and network of the endoplasmic reticula of rough and smooth types. The clear areas may represent negative images of glycogen. The intercellular space between the bile cunaliculi may be an artifact. Nucleus (N); bite canaliculus (BC); space of Dissc <D); endothelial cell (E); and sinusoid IS). Uranyl acetate and lead citrate. < 4300. HONS 062713 ' *. > V'. .. ' 570 VOS AND notenboom-ram control (isopropanol) group. Differences in the rearowth of hair between _-e two groups were also noted. Regrowlh was most reduced in the Aroclor8 cr. .p and in the hexachlorobiphenyl treated animals. During the course of the e\;erment. ihc lesions in the Aroclor group were clearly progressive, while the lesionsthe hexichlorobiphenyl group increased rather slowly. All animals, except I rabbit in the hexachlorobiphenyl group, showed a --eight cam during the experimental period of 4 wk. Mean weight gain in the hexachk \->biphen\| animals was 142 g (ranging from -215 to 365 g), in the Aroclor^ group ::_S g (from 70 to 640) and in the control group 491 g (from 430 to 550). Fig. 8. Hepaiocyleiof the same liver ofan Aroclor' treated rabbit as shown in Figs. 3 and 5a, showing foamy cytoplasm. The cytoplasm is mostly occupied by smooth endoplasmic reticulum (SER). Some perinuclear shift of mitochondria and HER can also be seen. N, nucleus. Uranyl acetate and lead citrate. x 4300. Coproporphyria levels in the feces, collected at necropsy from the cecum of individual animals, were significantly increased in the hexachlorobiphenyl as well as in the Aroclor' treated rabbits (Table I). Protoporphyrin excretion was also increased, though not significantly. Macroscopically, fluorescence was found in the bile of 2 rabbits of both experimental groups. One animal of the Aroclor8 group showed also fluorescence of liver and bile. Examination of cryostat sections of liver, kidney, and small intestine in the fluorescence microscope showed fluorescence in the livers of 7 out ot R PCB treated animals. This fluorescence was mostly located in the bile duct and sometimes diffusely w-;..z&ii&v-yw&t `1 'J .'t -* MONS 062714 TOXICITY OF POLYCHLORINATED BIPHENYLS in pcriportaily located hepatocytes. Some fluorescence -aas also noted in epithelial cells of the small intestine in 4 PCB treated animals. Severe abdominal edema was teen in an Aroclor treated rabbit that showed also the highest liver content (317 ppm) and seNere liver damage (Table 2). c i* fit t* Fin. 9. Higher magnification of a part of Fig. 8. The proliferation of SER consists of vesicular and tubular membranes. Clear areas of the cytoplasm, associated with ihe SER, most likely represent negative images of glycogen (arrow). Note the dilatation and degranulation of the rough endoplasmic reticulum (RER). M, mitochondria; N, nucleus. Uranyl acetate and lead citrate, * 13,300. No significant differences were found in absolute and relative organ weights, except for the liver, and in hematological findings, except for the serum transaminase levels. These values, together with the PCB concentrations in the livers and the comparative estimate of liver damage are given in Table 2. The SGPT and SCOT values were signiHcanily increased in the hexachlorobiphetiyl group. An increase was also noted in Ihe Aroclor* group. Mean liver content of PCB was the same in the experimental W (ip-sS* ' >. n! \ `. *-- * <: -s \p: ' ' MCNS 062715 ?Vl :;: ..'. A ,V ; .* ,,V. *' ` /'-^ ,? fetew 572 VOS AND NOTLNBOOM-RAM groups. The increase and severily of liver damage in the Arodor' 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 (Vos and Beems, 1971). Hexachlorobipheny! induced skin damage, especially hyperplasia in the epidermal and follicular epithelium as well as follicular plugging, was clearly less when compared with the Aroclor* treated rabbits. A slight hyperkeratosis was found in some control animals. This could be due to the treatment with isopropanol (6 ml/day); it was not found in the prior study (Vos and Beems, 1971) using a lower dose (1 ml/day). ' Sfcii Jill fegp j55/ f^Vi/jr. ns .ISgi Hit Fig. 10. HepatocytesofanAroclor treated animal. Similar hydropic cell* as seen in Fig. 5b. showing large confluent vacuole* with flocculcnt material within them (V). The hyaiinized cytoplasm of hydropic hepatocytes shown in Fig. 5b consists of hypertrophied, densely packed SER. Note the arrangement of mitochondria around the vacuole*, (arrow). Uranyl acatate and lead citrate. * 5500. 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, wete found in the thymuses of rabbits in the PCB treated groups. The atrophy agrees with the lower absolute and relative (about 18%) 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 centrolobular degeneration and Jiver cel! atrophy, focal hyalin degeneration of the cytoplasm 7*" mf ,"i HONS 062716 TOXICITY OF POLYCHLORINATED BIPHENYLS 573 of hepatocytes, enlarged nuclei and loss of glycogen. As shown in Table 2, liver damage by hexachlorobiphenyl was somewhat more severe when compared with (he Avoclor* induced lesions. However, in the present study, areas of hydropic, degenerated and necrotic cells dominated, often in thcsubcapsular region (Figs. I and 2), forming bands Fig. 11. Higher magnification of a part of Fig. 10 reveals the tubular structure of the densely hyper trophied smooth endoplasmic reticulum. Urenyl acetate end lead citrate, x 13,300. of necrotic and degenerated cells, accompanied sometimes by proliferation of mesen chymal cells. Necrosis was usually accompanied by hemorrhage. Loss of glycogen was not limited (o the degenerated, hydropic or necrotic areas, but was also seen in foci showing no alterations in hemaloxylin-eosin sections. In the rest ofthe liver the glycogen content was also reduced when compared with the controls. The centrolobular damage was often ronul. This distribution can be explained by the concept of structural and functionul hepatic unit (Rappaport e/a/., 1954). The presence of Peris' positive material V*S7SF &^ 574 VOS AND NOTENBOOM-HAM and ceroid pigment in KupfTer and parenchymal cells (characterised by a yellow, brownish fluorescence) was evidently less than in the preceding study and was limited to some necrotic areas. In both treated groups, numerous hepalocytcs were seen with light staining cyto> plasm. In these cells, the cel! membrane and the nuclear membrane were emphasized by the presence of basophilic material (Fig. 3). When plastic embedded liver tissue was examined by light microscopy, these alterations could be observed more clearly (Figs. 4 and 5a). This was the general trend in the PCB treated animals. In electron microscopy, the most striking feature of these cells was the proliferation of smooth Fio. 12. Severely damaged hepaiocyte of the same 2,4,5,2',4',5'-hexchlorobiphenyl treated animal as shown in Fig. 6. Photograph shows a duster of tubular smooth endoplasmic reticulum (in light microscopy visible as focal hyaline degeneration), forming a dense, closely pecked agglomeration (SER). Note also the lipid droplets (L), and hydropic swelling, as shown by a great number of small and large vesicles (V). Uranyl acetate and lead citrate, x 3000. surfaced membranes of the endoplasmic reticulum (SER). As a result of the proliferalion of SER, a perinuclear and peripheral displacement of mitochondria and rough surfaced membranes of the endoplasmic reticulum (RER) was apparent (Figs. 7 and Si). The proliferation ofSER could well explain the accentuated linings of cell and nucleus in fight microscopy and the increase in liver weight (Table 2) 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 of hydropic 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, inter- HONS 062718 TOXICITY OF POLYCHLORINATED BIPHENYLS 575 prcted as focal cytoplasmic degeneration. At the rim of the homogeneous cytoplasm cell organelles were visible (Fig. Sb). The focal hyalinized cytoplasm (Fig. 5b) was found by electron microscopy to consist of tightly packed tubules of proliferated SER (Figs. 10 and 11). The condensed SER was separated from large vacuoles, sometimes with flocculenl material in it. The mitochondria were arranged around the vacuoles. Besides the hyalinized cytoplasm, lipid droplets and degenerative changes of the nucleus were found in the more severely affected areas (Figs. 6 and 12). The nuclear changes included irregular outlines of the nuclei and karyopycnosis. Myelin figures were found rarely in the cytoplasm, fn the present study, no differences were noted in the hepatotoxic action between the polychlorinated biphenyl mixture and 2,4,5,2',4',5'hexachlorobiphenyl. DISCUSSION From the observed acnelike'lesions, both from the PCB mixture and 2,4,5,2',4',5'hcxachlorobiphenyl, and assuming that the hexachlorobiphenyl is free from contamina tion with chlorinated dibenzofuran, 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 dibenzofurans. In Table I it is shown that 2,4,5,2',4',3'-hexach)orobiphenyl 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 on 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 (c.g., release of glucocorticoids) must be considered responsible for the major effects 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 Arodor mixture and 2.4,5,2',4',5'*hexachlorobiphenyl. Liver damage caused by commercial PCB mixtures was due predominantly to the contaminants. This conclusion is based on the differences in Jiver toxicity between three PCB preparations (Vos and Koeman, 1970; Vos and Beems, 1971). The probable contribution ofchlorinated dibenzofurans and uncontaminated PCB in the toxicity of crude PCB mixtures is summarized in Table 3. TABLE 3 Probable Contribution of Chlorinated Dibenzofuran and Purs Polychlorinated Biphenyl in the Toxicity of Technical PCB Preparations Chloracne Edema formation Liver damage Hepatic porphyria Chlorinated dibenzofuran ++ ++ -M- _ Polychlorinated biphenyl + - ++ jfie tmm;y-'-gyuiiv'>11 1..'.ggHT'iff"wryag ..... *' 576 VOS AND notenboom-ram Focal cytoplasmic hyalin degeneration, as well as ceroid pigment deposits in the hepatic cells, was more pronounced in the former dermal toxicity study (Vos and Beems, 1971), probably due to the longer experimental period. Hyalin inclusions were previously described after semichronic exposure to PCB in rat liver (Bennett et al., 1938; Miller, 1944) and in mouse liver (Nishizumi, 1970). However, in the rabbit, the most important liver lesions described varied from fatty degeneration to marked hepatocytic degeneration and necrosis (von Wedel et al, 1943; 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 N-demethylasc (determined in vitro) was increased in the rabbit after administration of Aroclor* 1254 (Villeneuve et al, 1971). A decrease in sleeping time after treatment with hexobarbital and enhanced in vitro rates of aniline hydroxylation and p-nitroanisole demethyiation were demonstrated by Street et a/., (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 et al., 1971). 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 al. (1968) found reduced activity ofmicrosomal oxidases (eg. drug enzymes), while SER remained hypertrophic in livers of rais treated with high doses of dieldrin. As seen under the electron microscope, hypertrophic, hypoactive SER was recognized as tight dusters of tubular membranes, which could indicate a transition from adaptation to injury (Hutterer el al., 1968,1969). Liver damage was also suggested by elevated transaminase levels (Table 2). Myelin figures were seldom found in this study. Perhaps this depends on the animal species, since Nishizumi (1970) described myelin figures in mouse liver after PCB feeding, but not in monkey liver. The occurrence of SER proliferation and concentric membrane arrays is found in the livers of rats fed a chlorinated biphenyl diet (Norback and Allen, 1970) and toxic fat containing hexachlorodibenzo-/-dioxin, a compound related to chlorodibenzofuran (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 Central Institute for Food and Nutrition Research, T.N.O., Zeist, for synthesis and supply of the 2,4,5,2\4',5'-he*achiorobiphenyl sample. Special thanks are due to Drs. C. J. A. H. V. van Vorstenbosch of the Institute of Veterinary Histology (Head: Professor Dr. W. A. de Voogd van der Straaten) for use of the electron microscope, as well as for help and advice. HONS 062720 TOXICITY Q* POLYCHLORINATED BIPHENYLS 577 REFERENCES Bennett, G. A., Drinker, C. K. and Warren, M. F. (1938). Morphological changes in (ha livers of mis resulting from exposure to certain chlorinated hydrocarbons. J. hid. Hyg. Toxicol. JO. 97-123. Crow, K. D. (1970). Chlomcne. A critical review including a comparison of two series of cases of acne from chloronaphthalene and pitch fumes. Trans. St. John's Hasp. Dermatol. Soc. 36, 79-99. Elias, J. M.(1969). Effecti oftempera lure, poststaining rinses and eihanoMmtanol dehydrating mixtures on methylgreempyronin staining. Stain Technol. 44, 201-204. Grant, D. L, Phillips. W. E. J. and Villeneuve, D. C. (1971). Metabolism of a polychlorinaied biphenyl (Aroclor 1254*) mixture in the rat. Bull. Environ. Contam. Toxicol. 6,102-112. Huttekkr, F., Schaffner, F., Klion, F. M. and Popper, H. (1968). Hypertrophic, hypoactive smooth endoplasmic reticulum: A sensitive indicator of hepatotoxidty exemplified by dieldrin. Science 161,1017-1019. Huttlrer, F., Klion, F. M., Wenorap, A., Schaffner, F. and Popper, H. (1969). Hepato cellular adaptation and injury. Structural and biochemical changes following dieldrin and methyl butter yellow. Lab. Inwest. 20, 455-464. Jones, J. W. and Aloen, H. S. (1936). An acneform dermatergosis. Arch. Dermatol. Syphilol. 33, 1022-1034. Lockwood, W. R. (1964). A reliable and easily sectioned epoxy embedding medium. Anar. Rer. 150. 129-140. Meios, J. W., Alsom, i. 3. and Kartin, B. L. (1954). Chloracne from an unusual exposure to Aroclor. J. Amer. Med. Ass. 134, 1417-14)8. Miller, J. W. (1944). Pathologic changes in animals exposed to a commercial chlorinated diphenyl. U.S. Pub. Health Rep. $9. 1083-1093. Nishlzi'MI, M. (1970). Light and electron microscope study of chlorobiphcnyl poisoning. Arch. Environ. Health 21, 620-632. Norback, D. H. and Allen, J. R. (1969). Morphogenesis of toxic fat-induced concentric membrane arrays in rat hepatocytcs. Lab. Invest. 20, 338-346. Nor back, D. H. and Allen, J. R. (1970). Enzymatic and morphologic alterations of hepatic endoplasmic reticulum induced by a chlorinated aromatic hydrocarbon. Fed. Proc. Fed. Amer. Soc. Exp. Biol. 29, 816, 3220. Rappaport, A. M.. Borowy, Z. J., Louoheed, W. M. and Lotto, W. N. (1934). Subdivision of hexagonal liver lobules into a structural and functional unit. Role in hepatic physiology and pathology. Anat. Rec. 119,11-34. Schwartz, L. (1936). Dermatitis from synthetic resins end waxes. Amer. J. Pub. Health 26, 586-592. Street, J. C., Urry, F. M., Wagstafp, D. J. and Blau, A. D. (1969). Comparative effects of polychlorinated biphenyls and organochiorine pesticides in induction ofhepatic microsomal enzymes. Presented at ACS meeting Div, Pesticides, Paper No. 17. September 8-12, New YoTk. Tas. A. C. and de Vos, R. H. (1971). Characterization of four major components in a technical polychlorinated biphenyl mixture. Environ. Sci. Techno!. 3,1216-1218. van der Wairdln, B. L. (1957). Mathematlstische Stotistik, pp. 269-284. Springer-Verleg, Berlin end New York. Villeneuve. D. C, Grant, D. L., Phillips, W. E. J., Clark, M. L. andCLEOo, D. J. (1971). Effects of PCB administration on microsomal enzyme activity in pregnant rabbits. Bull. Environ, Contam. Toxicol. 6,120-128. von Weoel, H., Holla, W. A. and Dbnton, J. (1943). Observations on the toxic effects resulting from exposuretochlorinatcd naphthaleneand chlorinated phenyls with suggestions for prevention. Rubber Age (New York) 53,419*426. Vos, J. G. and Been*, R. B. (1971), Dermal toxicity studies of technical polychlorinated biphenyls and fractions thereof in rabbits. Toxicol. Appl. Pharmacol. 19, 617-633. fUJlli I MON 5 VOS AND NOTEN BOOM-RAM Vos, J. G. and de Rou, T. (1972). Immunosuppressive activity of a polychlorinated biphenyl preparation on the humoral immune response in guinea pigs. Toxicol. Appl. Pharmacol 21,549-555. Vos, J. G. and Koeman, J. H. (1970). Comparative toxicologic study with polychlorinated biphenyl* in chickens with special reference to porphyria, edema formation, liver necrosis, and tissue residues. Toxicol. Appl. Pharmacol. 17, 656-668. Vos, J. G., Koeman, J. H., van dch Haas, H. L., ten Noevex de Brauw, M. C.. and os Vos, R. H. (1970). Identification and toxicological evaluation of chlorinated dibenzofuran and chlorinated naphthalene in two commercial polychlorinated biphenyls, Feed. Cosmrt. Toxicol. 8, 625-633. Vos, J. G., Stair, J. J. T. W. A., van Holsteijn, C. W. M. and Pinnings, J. H. (1971). Poly* chlorinated biphenyls as inducers of hepatic porphyria in Japanese quail, with special ref* erence to 5-aminolevulinic acid synthetase activity, fluorescence, and residues in the liver. Toxicol. Appl. Pharmacol. 29, 232-240. Zitko, V. and Choi, P. M. K. (1971). PCB and other industrial halogenated hydrocarbons in the environment. Fish. Res. Bd. Can. Ttchn. Rep. 272. H0Ns 06212*