Document gD4eXMz9pVQgEajEo9krwREJa

N . t and a IM'UEU p h a r m a c o l o g y 23, 5 6 3 -5 7 8 (19721 Comparative Toxicity Study of ^.S^'^'^'-Hexachlorobiphenyl and a Polychlorinated Biphenyl Mixture in Rabbits J. G. Vos and Erica ISotlnboovi-Rwi * Mr of Veterinary Pathology and Institute of Veterinary Pharmacology and Toxicology, University of Utrecht. BUtxtraat J72, Utrecht, The fvelherkokls Receded Decembei 6, IV7I Comparative Toxicity Study of 2.4,5,2',4',5'-Hexuchlorobiphenyl and a Polychlorinated Biphenyl Mixture in Rabbits. Vos, J. G. and NotenboowRam, Erica (1972). Toxicol. Ap;:l. 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'-hexachlorobiphenyl, 120 mg polychlorinated biphenyl (PCB) mixture (Aroclor 1260') and the solvent isopropanol, respectively. The dermal application resulted in early macroscopic skin lesions in the Aroclor group. The lesions in the 2.4.5.2',4',5'-he\achlorobiphcnyl group appeared Uteron and were levs severe. This difference was confirmed microscopically: nyperpiasia and hyperkeratosis of the follicular and epidermal epithelium ere 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 Icconsand elevated serum transaminase levels, was somewhat more severe in the hexachlorohphenyl group when compareJ with the Aroclor* group, though the mean liver content was ubout the same (respectively, 239 and 236 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 (he endoplasmic reticulum tSER), resulting in a displacement of rough surfaced membranes (RER) and mitochondria. The hyalinized cytoplasm was recognized as tightly packed tubules of proliferated SFR. that is considered as hypertrophic, hypoaciive SER. The contribution o f chlorinated dibenzolurans and puie PCB in the toxicity of crude PCB mixtures is discussed ,,.Morinated biphenyls (PCB) arc industrial chemicals with many applications, toxicity of these compounds lor industrial workers has been known for years, fspectally the occupational disease known as chloracne (Jones and Alden. 1936; jrtz, 1936; Meigs cl a l. 1954). Recently, the consumption of rice bran oil acci*Hv contaminated by PCB resulted in many cases of chloracne among Japanese jiitcrs (quoted by Crow. 1970); liver damage was also reported (quoted by i/umi, 1970). A critical report on the use. environmental contamination and the of PCB and other industrial halogcnaied hydrocarbons appeared recently ii 0 I W Academic Press. Inc. ..i rtw nduction in any lorm re in nJ. 563 Is /lC o l fy cS r, v- ^ 3 G r ? > ) VOS ANU NOTENBOOM-RAM (Zilko and Clioi, 1971). There is an additional danger for wildlife from the wide* environmental contamination with PCB through food chains, and this is, ulutialso a menace to human health. The purpose of the present study is to compare the toxicity of Arocior I2W)* v single isomer 2,4,5.2'.4'.5'-hexachlorobiphenyl. The commercial Arocior ? sample W2Sfound to be the least toxic in a comparative study using three PCB noriginatmg from different firms (Vos and Koeman. 1970). Chlorinated dibcn/**lconsidered to be responsible for the higher toxicity of two PCB mixtures, * rr found in the Arocior 1260 * sample (Vos et al.. 1970). To be more certain aho biological elfect attributable to PCB itself, a pure compound. 2.4.5.2.4'.5'-heu*i biphenyl, was used for comparison. This is also one step in the understanding c toxicity of the different components of PCB mixtures. Hepatic porphyria was also studied. Special attention was paid to PCB-ioalivcr damage, using light and electron microscopy for structural alterations ww chromatography for the determination of liver residues. METHODS The technical PCB mixture, a viscous fluid with an average of 60% chlorine. obtained from Monsanto in the United States (Arocior 1260s Lot No. Ai 2.4.5.2'.4',5'- Hexachlorobiphenyl. a crystalline compound, was syntheiucd core to the Ullman synthesis, using 2,4.5-trichloroiodobcnzenc and copper powder at (Tas and de Vos, 1971). Because of the chemical reaction, it is unlikeiyth.it thitp*^ lar isomer contains chlorinated dibcazofurans as impurity. The Arocior* uar was analyzed for the presence of chlorinated dibenzofuran and found to be ficrtii* of detection: I ppm). Adult female New Zealand rabbits, 3.5 mo old and weighing 2.5-2.9 kg. distributed at random into 3 groups of 4 animals. The performance of the vie* toxicity study was in general Ihe same as described earlier (Vos and Becmt. I*' Because of the lower solubility of the hexachlorobiphenyl isomer when compared ** the PCB mixture,20 mg of the PCBsfinsiead ofI20 mg in the prior study) weredw*** per milliliter of isopropanol. The PCB solutions were kept at body temperature * to keep the PCB dissolved. Six milliliters of PCB solution (120 mg) or of isuprepuj as a control was dropped daily, 5 times a week, on an area of 5 x 10 cm on thed** and shaved backs. After a test period of 4 wk (20 applications of 120 mg M . animals were killed. The coproporphyrin and protoporphyrin analyses of feces from the cccuav * macroscopical examination of tissues in Wood's light as parameters for eta** porphyria, and the hematological examination were done according to a forrw (Vos and Beems. 1971). At necropsy, body and organ weights were determined f* skin, liver and kidney were fixed in 10% buffered formalin. The spleen, ihxmuviufcv and mesenteric lymph node and appendix were fixed in Carnoy's fluid for 24 h/ m <* and stained with mcthylgrecn-pyronin according to Elias (1969). Paraplasi'cmbedfr sections were stained with hematoxylin and eosin. For detailed histology, wWv sections were stained with Peris' iron stain and with Best's carmine. Moreover. rryo*r sections of the livers were stained with Sudan black for the detection of lipxk tfr NPC00026558 753895 TOXICI1Y OF POLYCHLORINATED DIPHENYLS 565 Paraplast* embedded seciions and unstained cryostat sections of liver, kidney Ail) intestine were studied in a fluorescence microscope for the detection of i pigment and excess quantities of porpyrins, respectively. Jtrastructural examination, small blocks of liver tissue were fixed in 0.1 m tAte-HCI buffered 5% glularaldchyde (pH 7.0) for 5 hr. The liver tissue was Kd two times for 45 min in 0.1 m phosphate buffered 2,, osmium tetroxide TABLE 1 CopROPORPHYRtN AND PhOIOPORPHYRIN CON1'fcN'TS fug/g DRY WEIGHT) of Feces of R abbits T reated w ith PCB for F our W eeks' Coproporphyrin Proto|x>rpliyrin Hexachlorobiphenyl Aroclor* Hexach loro- Control biphenyl A roclor' Control 45.0 6.2 20.8 39.6 27.9 24.1 5.0 29.4 16.0 18.6 88.3 35.1 17.8 8.3 S.4 11.9 51.2 65.1 15.6 66.5 65.! 10.9 53.6 43.4 14.0 .smere the contents of fcccs, collected from the cccum of the individual animals, afnntly different from controls. p 0 025. TABLE 2 Absolute and R elative Liver W eights, M icroscopio L iver D amage. Liver R esidles and T ransaviinasc Values for R abbi i s T reated w iih PCB for Four W eeks- Liver weight 6/100 g 8 body weight hlnro 128 132 146 126 AIran: 133 4.6 4.2 4.8 5.1 4.7' or* 141 129 152 133 Mean: 139' Microscopic Liver damage +F - .i ill+ 11 M + 1-- ir - - T Liver Content (ppm) 249 140 261 304 239 SGPT It) 7l 56 70 92 72J SGOT (U) 16 34 30 21 131 110 91 83 Mean: 104 lues are given for the individual animal*. o -i -- 'a comparative estimate of the liver damage, nifcantly different from control*. / >0. 05. .-mhcantly different from controls, p c 0.025 NPC00026559 753896 Fig. I. Liver of a 2A5,2',4\5'-hexachlorobipfienyI treated rabbit. Subcapsular band g1 tissue (1) and area of hydropic degeneration (2). Hematoxylin and eosin. x 60. Fig. 2. Higher magnification of the subcapsular region of the liver seen in Fig. I. NotelfclN|Hk at 1, hydropic ceils at 2 and proliferation of mesenchymal cells at 3. Hematoxylin and ea tn W ktlW 753898 56S VOS A S H M )U .N R O m t-K A U Fig. 5, Liver section of an Aroclor1 treated rabbit. Toiuidir.e blue. ' 94(1. (a) Shmvmj povV- ami perinuclear displacement or cell organelles; note also (he foamy cvtopkiMn fanow). lb) lb * * cells showing vacuoles (I), that are scpaiaicd fr<rii-Hmogurieouv hvalicued cytoplasm(2| S*v perivacuolar localization of densely staining organelles. Fk.. n Severely damaged Itepafocyicv ofthe same 2,4.5.2'.4'..V-hexachiorobipiicnyl*tieairf m w as shown m rigs. 1 and 2 showing hydropic changes (I), lipid droplets ('). and focal p i t g w hyalin degu i-'fttu'ti f?l. Note ;i!m>the nuclear alterations such as iltc irregular outlines nf rfar% ami the prominent nucleoli. Toluidmc blue. - 940. 753899 TOXICITY OF POLYCHLORINATED BIPHENYLS & 72). Block staining was carried out with uranyl acetate. The tissue was then ' lied rapidly in ethanol and embedded in Dow epoxy resin (DERA) l Lockwood, lSections were cut with glass knives on a Reichert ultramierotome. Thin (0.5-1.0/i) for orientation and for light microscopic examination were stained with blue. Ultrathin sections poststained with lead citrate were examined by imicroscopy (Philips EM 100*) at 40 kV. iPCB concentrations in the iivers were determined by gas chromatography, as Icarrier (Vos. et a 1971V The significance of difference between treated and groups was determined on a one-tail significance level, using the Wilcoxon [fcrivso unrelated samples (van der Waerden, 1957). RESULTS 3 days of PCB treatment, the skin of rahbils in the Aroclor* group showed Mftdncss. After 6 days, definite redness and some thickening of the skin was noted <group. After 2 wk of treatment, hyperkeratosis and formation of transverse iwere seen. At this time, some redness and slight hyperkeratosis, together with jppaence of hexachlorobiphcnyl crystals on the skin, was seen in the Murobiphenvl treated group, although a slight hyperkeratosis was present in the a rfM lt 7.Cor.irol liver with hcpntocyta showing difTuutly veattered mitochondria and rework of the .ffifcflwiuc reticula of rough and smooth types. The c.'ecr areas may represent negative images of The inlcrceilulcr space between the bile cunaliculi may be an artifact. Nucleus (N); bile **SxScbIus(BC): space or Disic tD: endothelial cell (F>; nd simivOid IS). Uranvl ncetate and lead t. *4JOO. UPC00026563 f . i* W . />V 570 VOS K S n NOri:NBOOM-RAM control (sopropanol) group. Differences in ihc regrowih or hair between the i* groups were also noted. Regrowth was most reduced in the Aroclor* group a:in the hexachlorobiphenyl treated animals. During the course of the rxperimcr lesions in (he Aroclor group were clearly progressive, while the lesions in the clilorobiphcnyl group increased rather slowly. All animals, except 1 rabbit in ihe hexachlorobiphcnyl croup, showed a weigl during the experimental period of 4 wk. Mean weight gain in the hsxachlorotup animals was 142 g (ranging from - 215. to 365 g). in the Aroclor'5' group 325 g . 70 to 640) and in the control group 491 g (from 430 to 550). Fit*. 8. tkpatneytes ofthe.sameliver of an Arrvlnr"' treated rabbit asshown in Fi^s lain! foamy cytoplasm. The cytoplasm U mosli> occupied by smooth ciidoplasmtc reticulum (St ft* > perinuclear shift of mitochondria and RER can also be seen. N, nucleus. l*ranyl tvciair s.v citrate. .<4300. Coproporphyrin levels in ihc feces, collected at necropsy from the cecum of mdo.. animals, were significantly increased in the hexachlorobiphenyl aswcflasiniheAr.^^treated rabbits (Table Ij. Protoporphyrin excretion was also increased, though significantly. Macroscopically, fluorescence, was found in the bile of 2 rabbits pi texperimental groups. One animat of the Aroclor*' group showed also lluorisacrvi liver and bile. Examination of cryostat sections of liver, kidney, and small thiUi the fluorescence microscope showed fluorescence in the livers of 7 out oi 8 PCH it. animals. This fluorescence was mostly located in the bile duct and sometimes did*:* t NPC00026564 TOXICITY OF POLYCHLORINATEn BIPHFNYLS 571 tjeriportally located hcpalocyles. Some fluorescence was also noted in epithelial iHiheflhe small intestine in 4 PCB treated animals. Severe abdominal edema was Arcelor" treated rabbit that showed also the highest Jivcr content (317 ppm) iifwere liver damage {Table 2). f i t 9. Uijjhcr magnification of a part of Fig. The proliferation of SER comUts of vesicular amt w h r membranes. Clear areas of lire cytuplasin, awotiilcd vviih the SER. nmt likely reprewrt vgrmcimages of glycogen (arrimj. Note the dilataiumartd degrjiruilaluin of the rough endoplasmic Maftjm(RER). M. mitochondria; N. nucleus. Uranyl acetate and lead citrate. l.l.J00. Sosignificant ditferenccs were found in absolute and relative organ weights, except 1tlte liver, and in hematological findings, except for the scrum transaminase levels, fine values, together with the PCB concentrations in the livers and the comparative rule of liver damage are given in Tabic 2. The SGPT and SGOT values were i&tfcatuly increased in the hexachlorobiphenyl group. An increase was also noted t i e Arcelorx group. Mean liver content of PCB was the same in the experimental 5 7 : VOS AND .NOTL'NBOmf-RAM groups. The increase and severity ofliver damage in the Arocfor1*' group a-' the increase in liver residue. At examination of skin sections of the Aroclor* group, hyperplasia a. keratosis of the epidermal and follicular epithelium were found. The lesion* yet so pronounced as in the prior study (Vos and Bcems, 1971). Hexachlnfinduced skin damage, especially hyperplasia in the epidermal and follicular e; as well as follicular plugging, was clearly less when compared with the treated rabbits. A slight hyperkeratosis was found in some control animals. T be due lo the treatment with isopropanol (6 ml.'day); it was not found in t study (Vos and Beems. 1971) using a lower dose (1 ml;day). 753903 TOXICITY OF POLYCHLORINATED WPHFNYLS 573 rfhcpatocytes, enlarged nuclei and loss of glycogen. As shown in Table 2. liver damage jjbcxachlorobiphenyi was somewhat more severe when compared with the A roclor9j educed lesions. However, in the present study, areas of hydropic, degenerated and tcrctic cells dominated, often in the subcapsular region (Figs. I and 2). forming bauds /erotic and degenerated cells, accompanied sometimes by proliferation o f meseni Afoul cells. Necrosis was usually accompanied by hemorrhage. Loss of glycogen ^eiwi limited to the degenerated, hydropic or necrotic areas, but was also seen in foci ; ifewmgno alterations in ficmatoxylin-cosit sections. In the rest of the liver the glycogen `joBtent was also reduced when compared with the controls. The ecntro'obular damage - often zonal. This distribution can be explained hy the concept of structural and -Motional hepatic unit (Kappaporr rt a i%1954). The presence of Peris' positive material NPC00026567 km 574 VOS \N D NOTFXBOOM-RAM and ceroid pigment in Kuplfcr and parenchymal cells (characterized by a brownish fluorescence) was evidently less than in the preceding study and w-jv. to some necrotic areas. In both treated groups, numerous hcpatocytes were seen with light stainin' plasm. In these cells, the cell membrane and the .nuclear membrane were emp by the presence of basophilic material (Fig. 3). When plastic embedded liver to* examined by light microscopy, these alterations could be observed more (Figs. 4 and 5a). This was the general trend in the PCB treated animals. In c microscopy, the most striking feature of these ceils was :he proliferation of % hit;. 12. Severely damaged hcpaiocyte oi the Mine 2,415.2'.4'..V-hcxnchIorohiplicnvI inutcJ. as shown in Tig. A. Photograph show a cluster of luhu .ir smooth endoplasmic reticulum iniicruscopy visible as local hyaline degeneration), funning a dense, ctacly packed agjl<*r(SIIK). Note also :he lipid droplets fL>, and hydropic swelling, us shown by a great number and large vesicles (Vj. t.'ranyl acetate and ta d citrate, 31*00. surfaced membranes of the endoplasmic reticulum (SER). As a result of the (w- ' tion of SER, a perinuclear and peripheral displacement o f mitochondria and . surfaced membranes of the cndtiplasmic reticulum (RER) was apparent (Figv. ?iThe proliferation of SER could well explain the accentuated linings of cell and it,, in light microscopy and the increase in liver weight (Table 2) in the PCB treated p The RER showed some dilatation and degranulaimn (Fig. 9). In addition to the proliferative changes of the SER, degenerative changes war noted, Light microscopy showed the presence of hydropic cell* (Fig. 2). Examine plastic-embedded material under the light microscope showed some hydrops which consisted of large vacuoles separated from a homogeneous cytoplasm, - NPC00026568 TOXICITY (>[POLYCHLORINATED UIPHLSYLS 575 ci as focal cytoplasmic degeneration. At the rim oF the homogeneous cytoplasm rganrilcs were visible (Fig. 5b). The Focal hyalini/ed cytoplasm (Fig. 5b) was J byelectron microscopy to consist of tightly packed tubules of proliferated SER .* 10and 11). The condensed SF.R was separated from large vacuoles, sometimes . (locculent material in it. The mitochondriu were arranged around the vacuoles. Jes the hyalinized cytoplasm, lipid droplets and degenerative changes of the xus were Found in the more severely affected areas (Figs. 6 and 12). The nuclear ijes included irregular outlines of the nuclei and karyopycnosis. Myelin figures - found rarely in the cytoplasm. In the present study, no differences were noted in jpatotoxic action between the polychlorinated biphenyl mixture and 2.4,5.2\4\5'tuhlorc biphenyl. DISCUSSION ' Mtn the observed acnclike lesions, both From the PCB mixture and 2,4,5.2',4',5uchlorobiphcnyl, and assuming that the hexachlo rob phenyl is Free From contain inaiiih chlorinated dibenzofuran. it can be concluded thaL this particular compound Sc mixture PCB has a slight acnegenic action of itself. Considering the already tioncd dermal toxicity study, it is evident that the major acnegenic action of crude 0 mixtures comes From chlorinated dibenzofurans. In Tabic l it is shown that 1?2'4',5'-hexachJorobiphcnyI is even more porphyrogcnic than the PCB mixture, '.litis probable that hepatic porphyria comes only from PCB itself. hcontrast with the prior dermal toxicity study, only slight effects were noted from -Son the lymphatic tissue. This difference is explained by the longer experimental I in the Former study (38 days) which resulted in n less healthy condition of the ,Trimental animals. Stress (e.g.. release o f glucocorticoids) must be considered sponsible For the major effects on the lymphatic tissue. However, using the -ailive fluorescent antibody technique in tetanus toxoid stimulated guinea p, immunosuppression was Found (Vos and de Koij, 1972). liver damage was essentially the same after treatment with both the Aroclor* mure and 2.4,5.2',4',5'-hexachIorobiphenyL Liver damage caused by commercial m mixtures was due predominantly to the contaminants. This conclusion is based fthedifferences in liver toxicity between three PCB preparations (Vos and Kocman, r'fl; Vos and Bcems, 1971). The probable contribution of chlorinated dibenzofurans .id uncontaminated PCB in the toxicity of crude PCB mixtures is summarized in table 3. TABLE 3 Probable C ontribution ok C hlorinated D ibenzofuran and P urf. Polychlorinated Biphenyl in the T oxicity of T echnical PC B P reparations Chloracne Edema formation Liver damage Hepatic porphyria Tdojinaicd dibcn/oFuran 4..(. . . Vychlorinatcd biphenyl - t - +- 576 VOS ANO NO I'fcNHOOM-RAM Focal cytoplasmic hyalin degeneration, as well as ceroid pigment deposits m hepatic ceils, was more pronounced in the former dermal toxicity study (Vo* Bcems, 1971). probably due to the longer experimental period. Hyalin inclusions previously described after semichronic exposure lo PCB in rat ver (Bennett r>. 1938; Miller. 1944) and in mouse liver (Nishizumi. 1970). However, in the rabbit ' most important liver lesions described varied front fatty degeneration tu nut* hepatocytic degeneration and necrosis (von Wedel et a i. 1943: Miller. 1944). In i* study, the presence of cells with proliferated SER, resulting in a perinuclear amJ pr: pheral shift of RF.R and mitochondria, was more frequently seen (Fig. 81. Thu ;* liferation, also found by Nishizumi (1970) in mouse and monkey liver, must be cor dcred the structural indication for enhanced metabolism of foreign lipophilic * pounds. Thus, the activity of aniline hydroxylase and aminopynne .V-dcntcth>`.. (determined in vitro) was increased in the rabbit after administration of Aroclor* ; (Villeneuve ei a/., 1971). A decrease in sleeping time after treatment with hexobuiv* and enhanced in vitro rales of aniline hydroxylation and /Miitroanisolc demeritili'*were demonstrated hy Street ei a(.r (1969). They also found an increase of these ede-* with increasing chlorine content of different PCB preparations. Despite this incr** in drug enzyme activity caused by the higher chlorinated mixtures, the lower chloriasu compounds are very probably metabolized to a greater extent (Gram et oL l*V On the other hand, the presence of focal cytoplasmic hyalin degeneration wilful glycogen (Figs. Sband), in electron microscopy seen as densely packed agglomerai.*. ofSER. very probably represents hypertrophic, hypoaclive SER. Hullcrcr found reduced activity of microsomal oxidases (e.g. drug enzymes), while SER rcnvui^ hypertrophic in livers of rats treated with high doses of dieldriu. As seen under electron microscope, hypertrophic, hypoaclive SER was recognized as tight diwr o f tubular membranes, which could indicate a transition from adaptation toitv:r(Mutterer ct ai, 1968. 1969). Liver damage was also suggested by elevated trattami!* levels (Tabic 2). Myelin figures were seldom found in this study. Perhaps thisdegrs on the animal species, since Nishizumi (1970) described myelin figures in iuou<fcv. after PCB feeding, but not in monkey liver. The occurrence ofSER proliferai m. concentric membrane arrays is found in the livers of rats fed a chlorinated bipheni' : (Norback and Allen. 1970) and (oxic fat containing hexachlorodibcnzo-psiiot?.. , compound related to chlorodibsnzofuran (Norback and Allen, 1969). The resulti fra the former study suggested that the concentric arrays of agranular membranes nu> it structural inodiliealions of the endoplasmic reticulum having an enzymatic fcixv similar to that associated with ihc SER. ACKNOWLEDGMENTS We arc grateful to Dr. R. i. C. Kleipool and Mr. A. C. Tas or the Central institute for F**> and Nutrition Research. T.N.O., Zeist, for synthesis and supply of the 2.4.5,2\4'.5'-fcM chlorobiphenyl sample. Special thanks are due to Drv C. J. A. I !. V. van Vorstenbosch of the Institute ofVctctw Histology (Head: Professor Dr. W. A. de Voogd van der Stmaten) for use of the eta', .microscope, as well as for help and advice. NPC00026570 TOXICITY o r POLYCHLORINATED RIPHENYI.S 577 REFERENCES Iamtt, G. A., Drinker, C. K. and Warren, M. F. (1933). Morphological change* in the ben or mis mulling from exposure to certain chlorinated hydrocarbons. J. ind. Ihj.'. Tnicol. 20,97-123. *. K. D. (1970). Chlorucne. A critical review including u comparison or two series of as of acne from chluronaphthalcnc and pitch fumes. Trans. St. John'* Hasp. Dermatol. hr. 56,79-99. iu.J. M. (1969). Effects of temperature, poststaining rinses andethanol-butanol dehydrating phturcs on meihyigreen-pyronin staining. Stain Technol, 44, 201-204. 3usr, D, L.. Phillips. W. E. J. and Villekeijve, D. C. (1971). Metabolism of a polychlorincedbiphenyl (Aroclor 1254 *) mixture in the rat. Ball. Environ. Contom. Toxicol. 6,102 -112. ttmufcK, K,, Schaffnfk, F., K uos, F. M.and Popper, H. (1968). Hypertrophic, liypoactive poolh endoplasmic reticulum: A sensitive indicator of hepatotoxicity exemplified by iridrin. Science 161.1017-1019. artURER, F.. Klion, F. M., Wengraf, A., Schahner. F, and Popper, H. (1969). Hepare gular adaptation and injury. Structural and biochemical changes following dieldrin and nethyl butter yellow. Lab. Invest. 20, 455-464. ` h.J. W. and Aluen, H. S. (1936). An acneform dermatergosis. Arch. Dermatol. Sypbnid. J l . 1022--1034. lidwooD, W. R. (1964). A reliable and easily sectioned epoxy embedding medium. Anat. ftt. 150,129-140. ttiissj. W., Album, J. J. and Kartin, B. L. (19541. Chloracnc from an unusual exposure to Arucior.J. Amer. Med. Ass. 154,1417-1418. *iur, J. W. (1944). Pathologic changes in anir.uk exposed to a commercial chlorinated phenyl. U S. Pub. Health Rep. 59, 1085-1093. VodZUMt, M. (1970). Light and electron microscope study of chlorobiphertyl poisoning. trek. Environ. Health!1,620-632. Vuack. D. H. and Alijn, J. K. (1969). Morphogenesis of toxic fat-induced concentric siembr.ine arrays in rat heparocytcs. ImH. Incest. 20,338-346. VtBACK, D. H. and Allen, J. R. (1970). Enzymatic and morphologic alterations of hepatic rmloplasmic reticulum induced by a chlorinated aromatic hydrocarbon. Fed. Proc. Fed. Inr. Soc. Exp. Biol. 29.816,3220. tWAFOKT, A M., BoRnwv, Z. J., Lolghled, W. M. and Lotto, W. N. (1954). Subdivision rfhexagonal liver lobules into a structural and functional unit. Role in hepatic physiology md pathology. Anat. Rec. 119, II 34. inouxTZ, L. (19361. Dermatitis from synthetic resins and waxes. Anter. J. Pub. Health 26. 585-592. Liter, J. C., Urry. F. XL, Wagstai-f, D. J. and Blau, A. D. (1969), Comparative effects of polychlorinated biphenyls and organochlorine pesticides in induction of hepatic microsomal enzymes. Presented at AC'S meeting Div. Pesticides, Paper No. 17. September 8-12, New York. TaxA.C. and de Vos. R. H. (1971 j. Characterization of Tourmajor components in a technical polychlorinated biphenyl mixture. Environ. Set. Technol. 5, 1216-1218. its OCRWaerden, B. L. (1957). Mathcmatisti.\che Statistik, pp. 269-284. Springer-Verlag, Berlinand New York. '.iiXKEUVE, D. C.. G rant, D. L., Phillips, W. E. J.. C lark, M. L. and C legg, D. J. (1971). Effects of PCB administration on microsomal enzyme activity in pregnant rabbits. Ball. Environ. Contain. Toxicol. 6, (20 123. pwWeuel, H., Holla. W. A. and Denton, J. (1943j. Observations on the toxic effects mullingfrom exposure to chlorinated naphthalene and chlorinated phenyls with suggestions farprevention. Rubber Age (Hew York) 53,419-426. 1, j. G. and Seems, R. ft. (1971). Dermal toxicity studies o f technical polychlorinated riphenyls and fractions thereof in rahbits. Toxicol. AppL Pharmacol. 19,61? 633. 578 VOS AND NOTENBOOM'RAM Vos. J. G. iind oe Rou, T. (1972). Immunosuppressive activity of a polychlorinated bipl." preparation on the humoral immune response in guinea pigs. Toxicol. Appl. Phan*21, 549-555. Vos. J. Ci. and Koeuan, J. H. (1970). Comparative toxicologic study with polychlixm. biphenyls in chickens with special reference to porphyria, edema formation, liver neci.* and tissue residues. Toxicol. Appl. Pharmacol. 17, 656-66S. Vos. J. G., KotviAN. i. H,, van der Haas, H. L., ten*Noever df. Brauw, M. C..and w \ R. H. (1970). Identification and toxicological evaluation of chlorinated dibenrofuran t* chlorinated naphthalene in two commercial polychlorinated biphenyls. Food. Ci*** Toxicol. 8. 625-633. Vos, J. G., Strik. J. J. T. W. A., van Holstgun. C. W. M. and Pfnnings, J. H. (1971) P < chlorinated biphenyls as inducers of hepatic porphyria in Japanese quail, with speewt ercnce to 5-aminoIevclinic acid synthetase activity, fluorescence, and residues in thel..<Toxicol. Appl. Pharmacol. 20, 232-240. Zitko, V. and Choi, P. M. K. (1971). PCB and other industrial halogenated hydroc*.* in the environment. Fish. Res. Bd. Can. Techn. Rep. 272. NPC00026572