Document EdzRGVkLaJxE8drMVbK7aqen4

I, "** <**.! iinirtr**"" * fHmi 4 T11 V`llUl.y',, i * *1 L. killmr i 0:Mn ^ v*lior| <tJt <>l I V*,, ,, , " Caitfr, I I I Induction of Adenofibrosis and Hepatomas of the Liver In BALB/cJ Mice by Polychlorinated Biphenyls (Aroclor 1254} 1 Renate D. Kimbrough, M D.,5 5 and Ralph E. Linder 14 * SUMMARY--Two groups of 50 BALB/cJ Inbred male mice were fed 300 ppm of a polychlorinated biphenyl, Aroclor 1254, In the diet for 11 and 6 months, respectively. The 5 months' feeding was followed by 5 months' recovery. Two additional groups of 50 mice each were fed plain chow. Alt 22 surviving mica fed Aroclor 1254 for 11 months bad greatly enlarged livers representing 25% of their body weight, whereas those fed the experimental diet for 6 months only had slightly, but significant!y, enlarged livers. Adenofibrosis was observed in all 22 livers of mice fed Aroclor 1254 for 11 months but not in the other groups. Of the 22 mice fed 300 ppm Aroclor 1254 for 11 months, 9 had 10 hepatomas measuring 0.1-1.5 cm In diameter. One of 24 surviving mice fed Aroclor 1254 for only 6 months, followed by a control diet for 5 months, had a hepatoma 0.3 cm In diameter. No controls had hepatomes.--J Natl Cancer Inst 53: 547-552, 1974. ether and then mixed with cornstarch. The ether was allowed to evaporate, and the Aroclor 1254-corn starch mixture was added to increasing amounts of ground chow, Early in the experiment, some mice were lost be cause of fighting. All mice, including controls, were caged in pairs 2 months after onset of the experiment. Since most mice that died did so early in the study and often showed a great deal of aulolvsis, they were not included in the final evaluation of the liver pa thology. Organs of all mice that were killed were in spected grossly for lesions, the livers were removed, weighed, fixed, and then carefully inspected grosslv for hepatomas. Any abnormal-appearing tissue was also examined microscopically. The tissues were fixed in 4% buffered formaldehyde, stained with hemntoxvlin and eosin, and examined with the light microscope. IN PREVIOUS STUDIES with Sherman rats, prolonged feeding of polychlorinated biphenyls (PCB's) Aroclor 1254 particularly, but also Aroclor 1260, induced a lesion, classified as adenofibrosis, in the liver (], 2). Edwards and White (3) and Stewart and Snell (4) described this lesion in detail. On gross inspection adenofibrosis, also called cholangiofibrosis, is firm and grayish white and has a pitted or granuiar suriace. Microscopically, it con sists of fibrosis and proliferated, atypical-appearing glandular epithelium that forms cysts and ducts. To ascertain whether this lesion could also be produced in mice, we conducted the present study. MATERIALS AND METHODS Two hundred 5- to 6-wcek-old BALB/cJ inbred male mice were purchased from The Jackson Labor atory, Bar Harbor, Maine. According to information from the Jackson Laboratory (5-7), this strain has a very low incidence of spontaneous tumors, particularly hepatomas. The mice were distributed randomly into 4 groups of 50. They were tagged individually and caged in groups of 5. The mice were weighed every 2 weeks for the first 6 weeks and thereafter every 4 weeks. Two groups of 50 mice each were fed 300 ppm Aroclor 1254 in ground Purina Laboratory Chow. After 6 months of exposure, 1 group was given plain ground chow, while the other group consumed the experi mental diet for 11 months. The 2 additional groups were controls and were fed plain ground laboratory chow throughout the 11-month study. Food con sumption was measured during the 2d and 6th week of exposure and thereafter every 8 weeks in the group exposed to the experimental diet for 11 months and in 1 control group. The Aroclor 1254 (Lot AK-38, from Monsanto Chemical Co., St. Louis, Mo.) was first dissolved in RESULTS In the control group, the food consumption ranged from 167 to 198 g/kg body weight/day. In the group fed Aroclor 1254 for 11 months, it ranged from 159 to 1 71 g/kg body weight/day, which resulted in a mean dosage level of Aroclor 1254 of 49.8 mg,.kg day. During the first 4 months, 23 mice died in the 2 control groups and 20 in the 2 experimental groups. Table i gives the total survivors in th~ different groups, body and liver weights, and iv.cidencc of hepatomas. A slight, but statistically significant, increase in body weight was observed in mice receiving the experimental diet for 11 months. Compared to controls, these mice had large livers, representing 25'; of their body weight. The surface of the liver of 9 of these mice contained nodules of varying sizes up to 1.5 cm in diameter. All livers of the mice that were killed in this group showed orange fluorescence under ultraviolet light, indicative of eievaud por phyrin levels. The body weights of the mire fed Aroclor 1254 for only 6 months were comparable to those of the control groups at the lime they were killed; but, when the treated diet was discontinued, they weighed an average of 3 g more than the controls. Upon microscopic examination of the livers of the 34 and 24 surviving controls in the 2 groups, 45 livers were normal. A few times a liver cell showed a large, hyperchromatic nucleus or binuclcation. The re- 1 Received March 1, 1974; accepted April 18, 1174. Formerly F.nvironmcntal Protection Agency, Chamblre Toxicology Laboratory, 4770 Buford Highway, Ch.nnblrr, Ga. 30341. ,. Presenl address: Center for Disease Control. "I oxirnlosiy Section, 1000 Clifton Road, N.K., Atlanta, Ca. 30333. 4 Present address: Environmental Protection Agency, Research Triangle Park, N.C. 27711. 1 Dr. M. L. Stewart kindly reviewed the slides and Dr. M. Dcringer supplied the information on the spontaneous incidence of hepatomas. JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 53, NO. 2, AUOUST 1974 547 1 iWlf '*"> jjeiipyUMenjjwm, ^ DSW 031425 STLCOPCB4015387 i ,i`- ..... aob 548 JUMBROUOH AND UNDER o Bg> % St 4 "13 K.S.S I! ift. BS " "*! g1". ?s7 *-* 0 r-t d o^r CO CO Sa u ro o' maining 13 livers had focal, round-cell infiltrates and occasionally small areas of necrosis and fibrosis. All mice with round-cell infiltrates in the liver had small skin abscesses, usually near the groin, which appeared to be abscesses of the preputial gland. Microscopic examination of the livers of the 22 survivors in the group receiving the experimental diet for 11 months (fig. 1) showed markedly enlarged liver cells. The nuclei of the hepatocytes were enlarged, hyperchrornatic, and atypical. The cytoplasm was I liver i i the seen t that sho\ a sn live diff l son 31 * J5 o E aO 1/3 either smooth or vacuolated. Macrophages and Kupffer's cells contained a brown pigment. Slight, diffuse interstitial fibrosis and numerous, large extra fint DIS |c to I-' * lQ. 8^ o 13 15 cL8 4 s g "S CO *-t CIO N >e 33 23 .*?.g. Os i x>oSE 00 XI3'Etsi e*5 2e "0J*5 cellular hyaline bodies were also seen. Some livers had extensive areas of coagulation necrosis and fibrosis (fig. 2). A general pleomorphism was noted in a number of livers. Degeneration, necrosis, and atrophy of individual liver cells were also observed. A few nuclei showed karyorrhexis. Some smaller cells and proliferation of the focal nodular type were also noted. The irregular outer surface of the liver with areas of retraction indicated that proliferation, atrophy, and regression had occurred. Occasional calcareous deposits = 5-8*i in diameter were present. In each of the livers, several areas were seen where the parenchyma was replaced by glandular formations of proliferated epithelial cells which formed ducts and often produced mucus. These epithelial cells were surrounded by either sparse (fig. 3) or abundant connective tissue. In addition to the above findings for all 22 livers of mice treated for 11 months (table 1), 10 hepatomas were found (fig. 4); of these, 3 measured 0.1-0.4 cni in diameter, 3 measured 0.5 cm, and 4 measured StU ad no Pr ho CO Il P! of iw i a< V lc o iS is 1. c Xa 1-1.5 cm. The tumors developed in a total of 9 mice. On gross inspection, they appeared as tan nodules. On microscopic examination, they consisted of well- 31 differentiated hepatocytes that were relatively uniform Ho >b and usually smaller than the surrounding liver cells. The tumors were well circumscribed and surrounded i by compressed hepatic parenchyma or strands of 3S oc oto to o*o HE fibrous tissue. The larger tumors contained areas where the sinusoids were dilated and filled with a pinkstaining amorphous material. On gross inspection of the organs at autopsy, metastascs were not seen. However, detailed screening for metastases including i serial sectioning of lungs was not undertaken. I ifs ?3*> Most livers of the 24 mice surviving 6 months of dietary exposure to PCB's followed by a 5-month 3' .t* dl recovery had enlarged cells; often these liver cells had I enlarged, atypical, hyperchrornatic nuclei, although l the livers at autopsy weighed only slightly more than those of the controls. Necrosis of individual liver cells \ was observed and some nuclei showed karyorrhexis. 0$ S" fefl E o> ^ TVJ O fc The macrophages and Kupffer's cells usually con tained a brown pigment. Many liver cells, particularly those in the periphery of the lobules, had vacuolated cytoplasm. The livers of about two-thirds of these mice showed slight-to-moderate diffuse, interstitial fibrosis. The outer surface of the livers still showed small i i i I'm S' indentations but not as pronounced as those in the l Iia Q -' group receiving PCB's for 11 months. Numerous extracellular hyaline bodies were observed in some t W*vfVUl .... . HWWWJl " u DSW 031426 STLCOPCB4015388 trte and brosis. All bad imall Ppeared af the 22 *ntal diet rgcd Jiver enlarged, iasm waj 'g and t. Slight, ^ extrs* iver* had 1 fibrosis ted in a trophy smaller *pe were ver with feration, casional present, here the itions of cts and 1* Were undam ivers of atomas 0.4 cm tasured i mice, xlules. f wellnifonn ' cells, unded ids of where pinkion of seen, tiding ihs of ion tii ( had ough than cells :is. conlarly ated nice osis. nail the T>US ime ADENOFIBROSIS AND hepatomas in mice fed fcb's 549 livers. In addition, many calcareous deposits, light in the center and darker in the periphery, were also seen (fig. 5). They were more frequent in this group than in the one fed PCB's for 11 months. A few livers showed bile duct proliferation. Only 1 liver contained a small hepatoma measuring 0.3 cm in diameter. The liver cells comprising the hepatoma were welldifferentiated hepatocytes. Except for the subcutaneous abscess formations in some mice and 1 sweat gland adenoma, no other findings of note were made in the mice of any groups. DISCUSSION Two significant findings were made in the present study: adenofibrosis of the liver and hepatomas. Thus adenofibrosis can also be induced in mice and does not occur solely in rats where it has been described reviously (/, 3). A similar lesion may occur in amsters (8). In the mouse lesion, the epithelial component was much better differentiated and less pleomorphic than in the rat lesion (/). The pattern of the lesions and the interrelationship with fibrosis were the same. Ducts and cysts formed in areas of adenofibrosis in both the mouse and rat livers. Whether adenofibrosis is a precursor of a malignant lesion or occurs concomitantly with malignant lesions of the liver is still debated. In an excellent review, Stewart and Snell (4) concluded that the lesion showed no convincing evidence of a precancerous lesion. Reuber (8) postulated that cholangiocarcinonias would develop from adenofibrosis in rats as well as hamsters. The mou:c strain used in this study only rarely develops hepatomas spontaneously. Peters et al. (5) observed 6 hepatomas in a group of 2000 BALB/cCr mice, a subline from the Andcrvont subline at the Na tional Cancer Institute; one would expect incidences similar to those observed in the BALB/cJ subline. Madison et al. (6) found 13 (0.62%) hepatomas in 2088 BALB/cCr mice 18 months of age. In the pres ent study none of the controls, 1 of 24 mice fed PCB's for 6 months, and 9 of 22 mice fed PCB's for 11 months developed hepatomas. Although the hepatomas were well differentiated, according to Andervont and Dunn (9), they represent potentially malignant lesions, a certain percentage of which can metastasize and be transplanted. From the appearance of the lesion, it is impossible to predict which tumor will be malignant. The size of the liver tumors cannot be used as a criterion. Quite possibly the tumors would have been larger and would have occurred more often had the mice been allowed to complete their lifespans. A Japanese PCB, Kanechlor 400, was recently re ported to have induced hepatomas in female Donryu rat* (10), and Kanechlor 500, when fed at a dietary level of 500 ppm for 32 weeks, produced hepatomas in male dd mice (//). The dietary levels of PCB's in our study, as well as those of the Japanese studies, were high compared to levels that could be found in the normal human diet. Presently, temporary tolerance levels, ranging from 0.2 ppm in infant food to 5 ppm in fish and poultry, have been set by the U.S. Food and Drug Administration (FDA) under the authority of Section 406 of the Federal Food, Drug, and Cos metic Act. The temporary tolerance level in poultry was set on a fat basis. The FDA concluded that the total daily food intake of PCB's should not exceed 1.4 mg/adult (12). The mice in the present study consumed daily =50 ing PCB's/kg body weight, which is 2500 times as much as the theoretical daily human consumption of 0.02 mg/kg body weight calculated by the FDA. The present study docs not indicate whether lower levels would also induce hepatomas or whether a "no-effect level" for hepa tomas could be established in rodents. We have pot previously observed calcareous de posits in livers of experimental rodents. The deposits we presently observed could represent calcification of small foci of tissue necrosis, particularly in areas where extracellular hyaline bodies were present. REFERENCES (/) Kimbrough RD, Linder RE, Gmnes TB: Morphological changes in livers of rats fed polychlorinated biphenyls. Arch Environ Health 20:354-364, 1972 (2) Kimbrough RD: Pancreatic type tissue in livers of rats fed polychlorinated biphenyls. J Nall Cancer Inst 51:679-681, 1973 (3) Edwards JE, White J: Pathologic changes with special reference to pigmentation and classification of hepatic tumors in rats fed p-dimethylaminoazobenzene (butter yellow). I Nad Cancer Inst 2:157-183, 1941 (f) Stewart llL, Snell KC: The histopathoic,,, af experi mental tumors of the liver of the rat. A critical review of the histopathogenesis. Acta Un Int Contr Cancr 13:770-803, 1957 (5) Peters RL, Rabstein LS, Spahn GJ. et al: Incidence of spontaneous neoplasms in breeding and retired breeder BALB/cCr mice throughout the natural life span. Int J Cancer 10:273-282, 1972 (6) Madison RM, Rabstein LS, Bryan WR: Mortality rate and spontaneous lesions found in 2928 untreated BALB/cCr mice. J Natl Cancer Inst 40:683-685, 1968 (7) Derinoer MK: Occurrence of mammary tumors, reti cular neoplasms, and pulmonary tumors in strain BALB/cAnDe breeding female mice. J Natl Cancer Inst 35:1047-1052, 1965 (8) Reuber MD: Histogenesis of cholangioftbrosis and well differentiated cholangiocarcinoma in Syrian hamsters given 2-acetoamidofluorene or 2-diacetoamidofluorene. Gann 59-239-246, 1968 (9) Andervont HB, Dunn TB: Transplantation of spon taneous and induced hepatomas in inbred mice, j Natl Cancer Inst 13:455-503, 1952 (10) Kimura NT, Baba T: Neoplastic changes in the rat liver induced by polychlorinated biphenyl. Gann 64:105 108, 1973 (//) Ito N, NacasaxI H, Arai M, et al: Histopathologic studies on liver tumnrigrnrsis induced in mice by tech nical polychlorinated biphenyls and its promoting effect on liver tumors induced by benzene hcxachloride. J Natl Cancer Inst 51 :I637-I64b, 1973 (12) Food and Dkuc Administration: Final environmental impact statement rule making on polychlorinated biphenyls. Dec. 18 1972 i to-sto o - n - It OSW 031427 W|IUIl^!i>,**^lUltl||If^^|LWH41WP.Pt| Willi STLCOPCB4015389 jrtbj2-t-t#vvo'vj' hi .?-=>{*&*:f-v--rt/iZ*^* >?: v> :4*;<-?uifr";v-->:1.'| yu-v.,v ;**,**. 1>c'y'.!> t.^-.w-s' 21 r? v* 1> * ;'* 7^: *r; * r V . * ->&:' Vi X^ /T.. (fA - -"** *-' . ^C, % *' ,. ' ; - . v * - .'j* -; ?_ ;>* *. . -1.1 ' * . * . Vv^ ; y .yXV=-,^ 2 :* * ; , : :t. ? ' * . .* . V + . C JV;-:;*{ Fiovre 1.--Liver of mouif fed 300 ppm Aroclor 1234 for 11 months. Note enlarged pleomorphic hepatocytei and interstitial fibrosis in tome areas. Hematoxylin and coin (H & E). X 100 Fiouar. 2.--Liver of mouse fed 300 ppm Aroclor 1254 for 11 months. Note extensive coagulation necrosis and fibrosis. H & E. X 100 550 w DSW 031428 KIMBROUGH AND LINDER u; iMi.jmff1-* Iu>'^jmhhwJffji11J,r-' STLCOPCB4015390 t Tkvkt. 3.--Liver of mouse fed 300 ppm Aroclor 1254 for 11 months. Not' epithelial cells forming duels and surrounded by connective tissue. H & E. X 125 fibrosis X 100 4DLR KIMBROUGH AND LINDF.R mtiPMi DSW 031429 551 STLCOPCB4015391 STLCOPCB4015392