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Morphological Changes in Livers of Rats Fed Polychlorinated Biphenyls Light Microscopy and UUrastructure linnale D. Kimbrough, MD; Ralph E. Linder; and Thomas B. Gaines, Chamblee, Ga Male and femala Sharman attain rata war* fad polychlorinated biphenyl* Aroclor 1260 and Aroclor 1264 at 0. 20, 100, 600 and 1,000 ppm In their diet. Rate received the dietary levels for eight months. Light microscopic changes con sisted of hypertrophy of the liver cells, inclusions in the cytoplasm, brown pigment in Kupller cells, lipid accumulation, and, at the higher dietary levels, adenoflbrosls. Ultrastructural changes of the livers of exposed anlmets consisted 0| : Increese in smooth endoplasmic reticulum r atyptcet mitochondria. Lipid vacuoles were o< ceslonally surrounded by concentric membrane The epllheliel component of adenoflbrosls to ileted of goblet cells and cells that restmbit the epithelium which lines the bile ducts, in g,r erat, the effect of Aroctor 1254 on the liver wi more pronounced than that nf Aroclor 1260. Polychlorinated biphenyls (PCBs) are widely distributed in the environment.1 They are marketed in the United States under the trade name Aroclor followed by a four digit number with the last two digits indicating the percent of chlorine. They were first marketed in the 1930s. Jones and Alden* reported a case of chloracne in a worker who had been heavily exposed to a chlorinated biphenyl. Miller4 reported pathological changes in animals ex posed to a commercial chlorinated biphenyl. He studied rats, rabbits, and guinea pigs, and found liver damage in all series of ex periments, as well as skin changes in the animals receiving subcutaneous injections or applications of the material to the skin. Liver damage was most pronounced in the guinea pig. The rat livers had intercellular hyaline bodies in addition to the fatty de generation that was observed in all three Bpecies. Bennett and his co-workers4 also reported that Aroclors affected the livers of rats. Von Oettingen5 discusses these earlier findings in his book "The Halogenaled Hy drocarbons: Toxicity and Potential Dan gers." After the Second World War, the PCBs were increasingly used for moisture Submitted for publication Jan IB. 1972; accepted April 28. From the Chamblee Toxicology Laboratory, Envi ronmental Protection Agency, Chamblee, Ga. Rend in part before the 12th annual meeting of the Society of Toxicology, Williamsburg, Va, March 6-9,1972. ' Reprint requests to Chamblee Toxicology Labora tory, Environmental Protection Agency, 4770 Bu- foid Hwy, Chamblee, Ga 30341 <Dr. Kimbrough). proofing, sealing, impregnation, and vnj> suppression in prolonging the residual life c insecticides and as fire retardants. They c\: be found in synthetic resins, synthetic an. natural rubber, cellulose resins, paint, var nish, wax, asphalt, and allyl starch. The; have also been used in printing inks, textitdyes, and as heat transfer fluid. In 196f an outbreak of poisoning that involved :i least GOO people occurred in western Jajn; where rice bran oil was contaminated witl Kanechlor 400, a PCB. The contaminate of the oil occurred because PCBs used a heat exchangers in the manufacturing proess leaked into the oil through pinholes it the pipes. This incident prompted Nishizu miT to study the effect of PCBs on the liven of mice and monkeys. He found a larct amount of acidophilic material in the cyto plasm of hepatocytes and fatty vacuoles Using electron microscopy, he observed "my elin figures" in the cytoplasm, and an in crease in smooth endoplasmic reticulum am! lipid droplets. More recently, PCBs leaked into fishmeal from a heat exchanger of a processing plnr.; in North Carolina. The contaminated fish meal was fed to adult chickens and breeders. The contamination was discovered when the eggs from the breeders did not hatch. Some of the chickens also exhibited symptoms of chick edema.4 The fact that PCBs can pro duce chick edema has been described repeatedly.4-11 Contaminants may be at least partially responsible for some of these toxic effects. Arch Environ i--Vo! 25, Nov 1972 STLCOPCB4009535 *.*;.* V-t? iw'i+jSlafroerfaa " Trwthnk^ttif^ii BIPHENYLS--KIMBROUGH ET AL Chlorinated dibenzofuran and chlorinated naphthalenes have been found as contami nants in two European commercial PCBs.12 jt js possible that other PCBs are also coninniinated with these or other highly toxic substances.111 The effect of two PCBs, Aroclor 1254 and Aroclor 1260, on Sherman itroin rats, was studied in our laboratory to jain better insight into the toxicity of these commercial products. The Aroclor 1260 was from the same lot as that studied by Vos and Koeman.11 Materials and Methods Sherman strain rate were used in these ex periments. The animals were caged individually nd weighed weekly. To determine tho single dote oral LDS,,, Aroclor 1254 and 1260 were given in peanut oil to groups of ten rata/doaage level by stomach tube as described by Gaines.14 Survivors were observed for a minimum of 15 days after treatment. The method of Lichtfleld and Wilcoxon18 was used to calculate the LD, values. When the Aroclors were fed in tho diet they were dissolved In ether and mixed with corn starch. The ether was allowed to evaporate and the Aroclor-cornstarch mixture was mixed Into increasing amounts of ground chow. In the feeding studies, 3 to 4-week-old male and fe male rats were distributed in groups of ten rats of each sex according to a table of random numbers. Their food consumption was mea sured during the second, fifth, 11th, 22nd, and 30th week of exposure to the dietary levels of 0, 100, 500, and 1,000 ppm, and at similar inter vals in the groups fed 20 ppm and their con trols. The rats fed 20 ppm were set up at a later date than the other dietary levels. For Tabm 1.--f/ean Body Weight Gain and Liver Weights of Rats Fed Aroclor 1260* Relative Liver Weights Fraction of Dietary Level Dosage Level Watgfct Gain Liver Welghte Body Weight Difference From Six (ppm) (mg/kg/dey) (gms) (gms) (*> Controla M 00 451 14.56 2.59 see M 100 6.5 463 18.87 3.23 P<.001 M 500 32.8 398 22.01 4.25 P<eOOl i< M 1,000 71.4 351 22.46 4.91 P<.001 F 00 256 11.23 3.12 ... F 100 7.2 253 12.20 3.40 P>.10 F 900 38.2 185 12.67 4.52 Pc.OOlt F 1,000+ 72.4 148 . 15.85 6.35 ... M 00 435 12.77 2.39 e.e M 20 1.4 455 16.63 3.02 PC.001 F 00 226 11.94 3.74 ... F 20 1.6 233 12.68 3.86 P>.50 * In diet for eight months; ten rats/group. t Only twg animate survived. t For the actual liver weight P>,05. Table 2,--Mean Body Weight Gain and Liver Weights of Rats Fed Aroclor 1254* Relative Liver Welghte Fraction of Dietary Level Dosage Level Weight Qaln Liver Weights Body Weight Dlfferenco From tea (ppm) (mg/kg/dey) (gme) (gms) <%) Controla M 0 O 484 14.14 2.44 ,,, M 100 6.8 459 16.01 2.92 P<.025 M 500 36.4 315 24.48 5.95 PC.001 F0 F 100 O 244 10.59 3.19 eea 7.5 235 11.86 3.62 P>.05t F 500 37.6 158 18.98 7.47 PC.001 M0 0 453 13.9B 2.53 M .F _F 20 0 20 1.4 439 15.38 2.90 P<.025 0 238 11.21 3.34 e# 1.6 249 12.49 3.72 PC.025 * In dlst for tig ht months; ten rats/group. 1 Cquel numb* re of rsts ltd 100 ppm and 20 ppm, respectively, lor same length of time did not show significant ncrease In actual liver weight (P<. 1 and >.05); however, the Increase In relative liver weight was significant P<.01. Arch Environ Health--Pol 25, Nov 1072 > t C .rir.; ;.rp, c *x _ . STLCOPCB4009536 356 DIPHENYLS--KIMBROUGH F.T AL this reason, a second group of controls for each sox was added to the experiments (Tables 1 and 2). AU animals that -were fed PCB as well as their controls were used in reproduction studies. The results of the reproduction studies will be reported separately. At autopsy, the tissues were fixed in a buffered 4% solution of formaldehyde (formalin). They were stained with hematoxylin-eosin for light microscopic study. Selected liver sections were cut on the cryostat and stained with Oil red O, Peris' stain for iron and a PAS stain were also performed on a few selected livers. Tissue from the livers of two male and two female rat3 at each dietary level except 20 ppm were minced and fixed in chilled 6% buffered glutaraldehyde for two hours, postfixed for two hours in 1% osmic acid (oeraium tetroxide), dehydrated through changes of alcohol and propylene ox ide, and embedded in resin. Sections were cut with a glass knife, stained with lead citrate and uranyl acetate, and examined with the electron microscope. In a preliminary experiment, Aroclor 1254 at the dietary level of 1,000 ppm for three months killed most of the rats. Tissue from this preliminary experiment was examined only under the light microscope. Results There was considerable variation, in both sexes, in the acute, oral, toxic effects of Aroclor 1260 and 1264 at various dosage levels. It was impossible to calculate LD-U values. The data indicated that the LDr.,, values were about 4,000 to 10,000 mg/kg for both mixtures. Autopsies of rats killed by either mixture revealed hemorrhage into the lung, stomach, and pancreas. Foci of ulcera tion surrounded by a severe inflammatory reaction were observed primarily in the cicodenum and occasionally in the glandular part of the stomach. When the material was fed in the diet over a period of time, porphyria was observed in a number of animals poisoned with either mix ture. The liver fluoresced most consistently under the UV light. Occasionally, fluores cence of bones, serum, and urine was ob served. A more detailed study of this phe nomenon will be reported later. When Aroclor 1260 was fed duily to groups of ten male and ten female rats each Table 3.--Light Microscopic Findings in the Liver of Rets Fed Aroclor 1260 Dietary Level (ppm) 0 20 100 500 1,000 0 20 100 500f l.ooot Enlarged Inclusion* Hepato- In Adno- lncr**d Foamy Sex eytes Cytoplasm fibrosl* Upld Cytoplasm Pigment M ... ... ... ... M JO 9 ... 3 M 10 9 ... 10 M 10 4 10 7 1 M 10 6 2 10 9 2 r .1. ... ... ... ... F3 ... ... F 72 1 10 ... i F 95 1 9B 6 F6 4 74 2 * In their diet (or eight month*; tn rat*/group. t Only nine anlmali were studied. (Only *even animal* were itudied, Table 4.--Light Microscopic Findings in the Liver of Rats Fed Aroclor 1254* Dietary Level (ppm) Enlarged Inclusions Hapato- In Adno SK cyit* Cytoplasm fibrosis 0 M ... ... ... 20 M 7 1 ... 100 M 20 7 1 500 M 10 2 10 0F 20 F ... ... ... ... a.. 100 F 10 7 500t F 92 7 9 * In their diet for eight month*; ten rt/group. t Only nine teted *ince one rat that died showed too much autolyala. Increased Upld ... to 10 ... ... 10 9 Foamy Cytoplasm i 10 4 ... 9 2 Plpmsnt ... 3 2 ... 1 10 7 Arch Environ Health--Vol 25, Nov 1972 DSW 025582 STLCOPCB4009537 ** ' '" j-v BIPHENYLS--KIMBROUGH ET At 357 . i ., <Vt'l *1 o cn , '>> ' ' *' ^ Stf?'# K:u, - -*o* V> .,c . J. * ' >,X\fA VAi.v'-f*c V: l'# 'fc'c`' *?'v . ` w.ai. V v . - ,, * ,! , * s, y V '.':v /<. . . a #*'- - ' * . 4 < Fig 1,--Liver teetfon of mile rat fed 500 ppm Flo 2.--Section of liver from male rat fed 3,000 ppm !i Vlor 1260 for eight month*. Noto foamy cytople*m Aroclor 1254 for eight month* Illustrating adenoflbro- m!: o( many of hepatocyte* (hemetoxylln'Soiln x 125). *1*. Noto area of fibrosis which surround* ductule* of glandular cells. Cellular debris, polymorphonuclear leukocytes, or mucus can be observed In lumen of ductule* (hematoxylin eosin X125). at dietary levels of 0, 20, 100, 500, and 1,000 ppra for eight months, none of the control rats and none of the male rats fed the mixturo died. One female fed 100 ppm died (,^8 ^d^ooppmTicl^^M for eight LnZ filter SIX months xposuro And two females Ara w* xtm!n*d under electron microscope (Ftg 8* fed 500 ppm died after one and two months 1Q) (toiuidin* blue x30Q). exposure. Eight females fed 1,000 ppm died within two to six months after experiment. The rats fed 600 and Aroclor 1260 in their diet gained Ilian the controls. At autopsy, oil male rats fed Aroclor 1260 K\l nificantly more than the livers trols (Table 1). The livers of the here larger also but a significant weight difference was not observed, except that at 500 ppm the percent of body weight of the livers was increased as a result of the re* duced weight gain of these animals (Table 1). Grossly, the livers of the exposed animals were soft and often yellowish brown or dark dive. Gmyish-white, firm, glistening areas, measuring up to 0.5 cm in diameter, were seen on cross section in a number of livers. The dark livers showed pink-orange fluoresc ence under the UV light. Arch Environ Health--Vol 25, A'ou 1972 J. V . .. if. V >' <1: rr>"U c. *. AMi lAEi:V-59fc^ t0mm K- .w fHtff;V..V.-.V: 'si DSW 025583 STLCOPCB4009538 358 BIPHENYLS--KIMBROUGH ET AL The incidence of the various light micro scopic findings of the livers are given in Table 3, They consisted of hypertrophy of the individual liver cells. Often the nuclei were hyperchromatic and showed pleomorphism, and occasional mitotic figures were observed. The cytoplasm had either large vacuoles or appeared foamy (Fig 1). Eosinophil inclusions identical to those de scribed in the liver of rats that have been exposed to DDT,10 mlrex,,T or dieldrin18 were also observed. The Kupffer cells and perivascular macrophages of some livers con tained a brown pigment. Frozen sections of livers containing the brown pigment flu oresced pink under UV light which is consis tent with the presence of porphyrin. Some of the pigment showed a positive staining reac tion for iron with Peris' stain as well. In addition to these findings, some livers also exhibited focal areas of fibrosis which con tained glandular structures (Fig 2 and 3) that either formed strands or dilated cystic ducts that were filled with cellular debris (polymorphonuclear leukocytes) or a mucus like PAS-positive substance. The glandular cells had a pale, scanty, baBophilic cyto plasm and usually a hypocbromatlc nucleus, The areas of adenofibrosis corresponded to the grayish-white, glistening areas described grossly. The glandular epithelium appeared stratified in some areas. Electron microscop ic study revealed (Fig 4 to 10) an increase in smooth endoplasmic reticulum (Fig 5) in the hepatocytes of all but the control rats (Fig 4). Many large and small lipid vac uoles and occasional atypical mitochondria were also Been. At the dietary level of 100 ppm, dark and light cells, dilated bile ducts, and granular cytoplasmic inclusions wen* additional findings (Fig 6). At the dietary level of 600 and 1,000 ppm, concentrically arranged membranes which surrounded lipid vacuoles were also observed. They liave been previously described in livers of rats fed DDT and dieldrin.18 The cytoplasm of many cells had a loosened-up, "moth eaten" appearance (Fig 7), with occasional vac uoles containing a few strands of electrondense material. These cells corresponded tn the hepatocytes with foamy cytoplasm ob served under the light microscope. Widenin'* of intercellulur spaces, occasional phagocytized red blood cells, and an increase in collagen were observed. The nodular, gray ish-white areas (Fig 8) that represented Flo 4.--Section from normel ml ret liver. N, nucleus: M, mltochondrle; RER, rough endopleimic reticulum; SER, smooth endoplesmlc reticulum (teed cltrete, urenyl ecetete x 13,490). DSW 025584 Arch Environ Health--Vol 25, Nov 1972 **' *'** yy*\' - '&* "' t ;*.V * -.'v, V ?.. .I;.' Fig 5,--Section from mala rat llvar fed 100 ppm Aroclor 1260 for eight month*. Note large amount of imooth endoplaimlc reticulum. SER, smooth endoplasmic reticulum; M, mitochondria; N, nucleus (lead citrate, uranyl acetate x34,200). Fla 6.--Section from llvar of female rat fed SOO ppm Aroclor 1260 for eight month*. Not* small and largt vacuole* within cytoplasm, soma filled with lipid and others with strand* of dark granular material (arrows). V. vacuoles; M, mitochondria; CB, cell border; BC, bile canaliculus; RER, rough endoplasmic retlculum; SER, smooth endoplasmic reticulum (lead citrate, uranyl acetate x 13,490). ffigMyai 'ft/-.. ^ So** *: Arch Environ Health--Vol 25, Nov 1972 STLCOPCB4009540 360 BIPHENYLS--KIMBROUGH ET AL Fig 7.--Section from llvar of mile rat fed 1,000 ppm Aroelor 1260 for eight month*. Not* "molheiten" appearance of the cytople*m (lead cltnte, urmyl acetate x 13,490). Fig 8.--Section from liver of femile rat fed Aroelor 1284 for eight month*. Not* large amount of collagen, flbroblaitt, end eplthallal cell* lining a ductule. C. collagen; FB, flbroblatb N. nueleu*; EC, epithelial celt (arrow); DL, lumen of ductule (lead citrate, urartyl acetate XS.320). ill A| dii li DSW 0 2 5 5 8 6 STLCOPCB4009541 tifYi^i^IlLi^l';^1^1'~-~.jTI""' .:'" i* '!.'* p?'Vi.^/ft*' l* ,'..* t> '* .* . Si Wi;3U*tf ,*. '.`,y ~:'* ;; BIPHENYLS--KIMBROUGH F.T AL 361 extensive foci of adenoflbrosis consisted of fibroblasts and a great deal of collagen that fin-rounded rosettes of epithelial cells. These ceils wero cuboldal or columnar in shape. Tlie free surface was lined by microvilli, and cells contained large, pale nuclei. The cyto plasm was granular, with many ribosomes and a few endoplasmic membranes. Some of these liver duct cells contained a great deal of mucus and resembled goblet cells similar to those observed in the intestinal tract (Fig 9) . The cytoplasm contained tonofliaments and, particularly near the duct lumen, the boundaries between adjacent cells were marked by prominent terminal bars (Fig 10). In a preliminary study in which 1,000 ppm Aroclor 1264 was fed in the diet to ten male and ten female rats, five of the males and eight of the females died. At autopsy of die exposed animals, the livers were pale and enlarged and contained firm, grayishwhite, glistening areas. One male rat that died after 49 days exposure showed only slight enlargement of the liver cells. The hepatocytcs of all the other exposed rats were enlarged, vacuolated often with hyperchromatic--sometimes enlarged and some times pyknotio--nuclei which occasionally displayed mitotic figures. Varying degrees of fibrosis around the bile ducts were observed. Hyperplasia and proliferation of glandular cells simulating bile ducts were also seen in these areas. In some areas, the sinusoids were dilated and contained cellular debris, lined by FM II "compressed" hepatocytes. A brown pi;;' t was seen within the macro phages. A v livere stained with Oil red O showed a large amount of red staining (lip id) material. The animals that died not only had liver lesions but also suffered from se vere pulmonary congestion and, in a few instances, from massive hemorrhages into the soft tissue or the peritoneal cavity. When 600, 100, 20, and 0 ppm Aroclor 1264 were fed to the rats for eight months, two males and one female rat died at 600 ppm, but none died at the lower dietary levels. The rats fed 600 ppm Aroclor 1254 gained less weight than the controls. The Fig 9.--Section from lima liver 11 Fig 8. Note goblet call containing mucus and degenerated cells within lumen of ductule. C, collagen; EC, epithelial cell; N, nucleus; CB, cell border (arrow); DL, lumen of ductule (arrow); MU, mucus goblet cell (lead citrate, uranyl acetate x 11,400). Arch Environ Health--Vol 25, Nov 1S72 STLCOPCB4009542 Fl0 10.--Higher mQnl(ictloo of two epithelial cells from Flo 9 showing T. tonofilements1 D d mosomet; N, nucleus; M, mitochondria; CB, cell border (leed citrate, urenyl acetate X34.200).' livers of the exposed groups were larger than those of the controls (Table 2), This was significant in all but the female rats fed 100 ppm. At autopsy, the livers of the exposed animals, particularly at the higher dietary levels, were soft, yellowish-brown, or dark olive. Firm, grayish-white, glistening areas were seen, especially in those fed higher PCB levels. Occasionally, the liver surface was nodular. These livers also fluoresced when exposed to UV light. The light micro scopic findings are given in Table 4. The observed changes in the livers of exposed rats were quite similar to those described for Aroclor 1260, except that all male rats at 600 ppm and one male rat at 100 ppm had areas of adenofibrosis in their liver, while none of the males demonstrated this at the respective dietary levels of Aroclor 1260. In the females, the incidence of adenofibrosis was much higher at the same dietary levels of Aroclor 1254 than in those fed Aroclor 1260. The electron microscopic examination of the different liver changes summarized in Table 4 corresponded to those already de scribed for Aroclor 1260. The main difference between the two com pounds was the much higher incidence of adenofibrosis at a lower dietary level of Aro clor 1254. When fed over a period of time, Aroclor 1260 Seemed to he more toxic to female rats. Such a sex difference could not be established for Aroclor 1254. Comment Aroclor 1260 and 1264 have a definite effect on the liver. This effect is more pro nounced with Aroclor 1264 when all mor phologic changes of equivalent dietary levels of Aroclor 1254 and 1260 are compared. All dietary levels tested so far showed an in crease in the size of the individual liver cells and often an increase in the liver weights. Accumulation of fat was observed at 100 ppm, and it was very pronounced at the higher dietary levels. The incidence of cyto plasmic inclusions was higher at the lower dietary levels. This is probably due to the fact that at the higher dietary levels most of the cytoplasmic components have been re placed by lipids. The pigment observed in the Kupffer cells showed a positive pruwian blue reaction and represented, at least in part, hemosiderin. The fluorescence of the livers under UV light was caused by the presence of porphyrin, which resulted in the dark discoloration of the liver. An additional finding in the livers was the presence of grayish-white, glistening areas which microscopically corresponded to aden ofibrosis, This lesion consisted of glandular, often mucus-producing, epithelial cells form ing ductules. They were surrounded by fi brosis. Synonyms for tin's lesion are cholunglofibrosis, bile duct proliferation, bile duct adenomatosis, and fibroadenoma. Stewart DSW 025588 Arch Environ Health--Vo/ 25, Nov JOT2 STLCOPCB4009543 BIPHENYLS--KIMBBOVGH ET AL 363 * * Snell'" give an excellent description of joenotlbiosis of the liver of rats. The authors concluded that "adenofibrosis is a contract ing lesion built up by the incorporation and transformation of normal (hepatic cords) and pathologic (pseudotubules) components of the surrounding liver. Thus far there is no convincing evidence that adenofibrosis is a precancerous lesion.'* Adenofibrosis, for inctftnce, has been described in livers of rats fed butter 'yellow (p-dimethylaminoazoben1 renel.-'0 Recently, Reuber21 fed hamsters , 2-aeelamidofluorene or 2,-diacetamidofluorene &nd produced adenocarcinomas. He thought (hat some of these carcinomas originated from bile ducts. The author interpreted ade nofibrosis as cholangiofibrosis, suggesting that , adenofibrosis in the rat was a precancerous lesion and was identical to the lesions he ob- served in hamsters which, in his opinion, also represented a precancerous lesion. He postu lated that the observed cholangiocarcinomas developed from the adenofibrosis. Glaser and his colleagues22 produced a highly malignant transplantable tumor, derived from the liver of a hamster fed 2-acetyIaminofluorene, that did not appear to be malignant in the donor but showed changes similar to the adenoflbrwis described in tills paper. It is possible that the malignant lesion was not simply de rived from the adenofibrosis-type lesion but from cells that already manifested irrevers ible preneoplasiic changes that were not mi croscopically delectable. Miller* found fatty livers, liver necrosis, and inclusions in the cytoplasm of the hepatocytes in rats that had been exposed to a biphenyl with 42% chlorination. He did not mention adenofibrosis in his report. How ever, he studied a different biphenyl and his animals were exposed to the chemical for much shorter periods of time. Nishizumi1 studied the effect of a biphenyl with 48% chlorination in monkeys and mice. Tire long est exposure time was 26 weeks. He de scribed enlargement of the liver, fatty :hanges, granular cytoplasm of the hepato:ytes, increase in size of Kupffer cells and lepatocytes, and a brown pigment in some >f these cells, but did not observe adenofirrosis. At some stages of exposure he ob served, with the electron microscope, an in:rease in the Bmooth endoplasmic reticulum, ariation in tho appearance of mitochondria, and an increase in the number of microbod ies, as well as `'myelin figures" which seem to be similar to the concentrically arranged membranes that have been described in rats after exposure to chlorinated hydrocarbons.18 The increase in liver weights and the obser vation of inclusions in the cytoplasm has been reported for several persistent insecti cides.1018 To our knowledge adenofibrosis has not been produced with chemicals such as DDT, dieldrin, pyrethrum, piperonyl butoxide, and mirex. Unlike Nishizumi's find ings,1 an increase in the number of micro bodies was not observed in the rats fed PCBs in this study. The increase in smooth endoplasmic retic ulum has also been observed with other chemicals, and it is not surprising that chlor inated biphenyls induce microsomal enzymes.28-2 Study of the adenofibrosis under the elec tron microscope shows the epithelial compo nent of the lesion consisting of two types of cells^-goblet cells and epithelial cells with granular cytoplasm, tonofilaments, and desmosomes. The latter cells resemble the epi thelial cells lining the bile ducts rather than hepatocytes. The epithelial cells are reminis cent of "oval" cells which are thought to represent ductular cells. However, the oval cells are smaller and have a hyperchromatic nucleus.4" Whether thoy develop originally from bile duct epithelium or from other cells in the liver needs further investigation. The fact that adenofibrosis has not been observed in previous feeding studies with PCBs may have a variety of reasons such as the length of exposure, the animal species or rat strain, and the composition of PCB given to the rats. Contamination of PCBs with traces of other chemicals such as chlorinated dibenzodioxins, chlorinated dibenzofurans, or chlori nated naphthalenes may also be responsible. Vos and Koeman11 checked Aroclor 1260 of the same lot and also did not demonstrate any contaminants. However, preliminary chemical analyses of this lot of Aroclor 1254 suggest possible contamination with a chlori nated dibenzofuran. This is still under Investigation.21 The significance of adenofibrosis will be further investigated in order to establish whether it is an irreversible lesion and whether it is transplantable. From experi- Arch Environ Health--Vol 25. Nov 1972 m |iiinMI*U ' inII.1 364 BIPHENYLS--KIMBROUGH ET A1 ence with DDT and other chlorinated hy _ 1L Vos JG, Koeman JH: Comparative toxicolog drocarbons, it can be deduced that the liver ic .study with polychlorinated biphenyls in chickens enlargement, as expressed by hypertrophy of with special reference* to porphyria, edema forma tion, liver necrosis and tissue residues. Toxic Appl the liver cells and the observation of inclu sions in the cytoplasm, is reversible after cessation of exposure. The observed porphyria, which was also noted by Vos and Koeman,11 and the effect of PCBs on the liver, particularly the adeno- Pharmacol 17:656-668, 1970. 12. Voe JG,_ Koeman JH, van der Mass HL, et al: Identification and toxicological evaluation ot chlorinated dibenzofuran and chlorinated naphthal ene in two commercial polychlorinated biphenyls Food Coemet Toxic 8:626-666,1970. ' 13. Kimbrough RD: Toxicity of chlorinated hy drocarbons and related compounds including chlori fibrosis, make it necessary to reevaluate our present concept of the toxicity of PCBs. Additional information is also needed on the nated dlbenzodioxins and chlorinated dibenzofurana; A review. Arch Environ Health 25:125-131, 1972. 14. Gaines TB: The acute toxicity of pesticides to rata. Toxic Appl Pharmacol 2:88-99, 1960. i , interaction of PCB3 with substances such as 15. Ldchtfleld JJ, Wilcoxon F: A simplified alcohol, chlorinated hydrocarbons, sex hor method of evaluating dose-effect experiments. J Pharmacol Exp Ther 96:99-133,1949. mones, lead, and, of course, the various con 18 Kimbrough RD, Gaines TB, Hayes WJ: f taminants that may or may not be present Combined effect of DDT, pyretbeum, and piperonyl butoxide on rat liver. Arch Environ Health 16:333 In the PCBs. The quality of the diet may 341,1968, have an impact on the effect of PCBs. The 17. Gaines TB, Kimbrough RD: Oral toxicity ot findings described in this paper support the mirex in adult and suckling rats: With notes on the ultrastructure of liver changes. Arch Environ Health t conclusion of Lichtenstein et al28--that the 21:7-14,1070. toxicity of the PCBs decreases as the level 18. Kimbrough RD, Gaines TB, Linder RE: The t of chlorination increases. uUrastructiire of livers of rata fed DDT and dieldrin. Arch Environ Health 22:460-467,1971, Richard L. Moore assisted with the animal teats. 19. Stewart HL, Snell KC: The hlstopathology of Estelle C. Gray did the statistical analysis. Linda experimental tumors of the liver ot the rat: A W, Anderson and Annie R. Alford prepared the critical review of the histopetbogenesU. Acta Unio tissues. Internatlonalis Contra Cancrum 13:770-803, 1957. Arcelor 1264 (lot No. AK-38) and Arodor 1260 20. Edwards JE, White J: Pathologic changes (lot No. AK-8) were provided gratia by Monsanto with special reference to pigmentation and classifi- - Chemical CO, St. Louis, Mo. cation of hepatic tumors in rata fed p-dlmethytami- noazobenzene (butter yellow). J Nat Cancer Inrt References 2:167-183, 1941. 1. Rlaebrough RW, Rieche P, Peakall DB, et al; 21. Reuber MD: Histogenesis of cholangiofibrosin end well differentiated cholangiocarcinoma in Syrian tk Polychlorinated biphenyls in the global ecosystem. Nature 220:1,078-1,102, 1088. 2. Jones JW, Aldan HS: An acnefonn dermatergosls. Arch Dtrm Syph 33:1,022-1,034,1038. 8. Miller JW: Pathologic changes in animals exposed to n commercial chlorinated diphenyl. Publie Health Rep 50:1,088-1,003, 1044. 4. Bennett GA, Drinker CK, Warren MF: Mor phological changes in the livers of rate resulting from exposure to certain chlorinated hydrocarbons. J Induit Hyg Toxicol 20:97-123,1938. 6. 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