Document OEMeYDYe8ZqyzqrK92neNGz61

620 Light and Electron Microscope Study of Chlorobiphenyl Poisoning In Mouse and Monkey Liver Mannhirn Minhtzumi. MD. Ftihunha. Japan The affect* on mouse and monkey liver of long-term oral administration of chlorobiphenyl* (CBP), 1.5 mg/day or more, have been Investi gated at selected Intervals by light and electron . microscopy. Hepalocyies of mice contained large amounts of acidophilic materials In the cytoplasm, and tally vacuoles were observed later. Fine structural changes In the hepatocytes consisted of a marked Increase of smooth endoplasmic reticulum, a reduction of rough endoplasmic reticulum, "myelin figure" formation In the cylo- plasm, and Increase of microbodle* and tysoacmes. A marked Increase in lipid droplets was observed later. Results ol electron microscopy In monkey liver showed an Increase ol smooth en doplasmic reticulum In Ihe hepatocytes and swell ing of Kuptfer's calls, with an Increased number of lysosomss and vacuoles. Judging from the findings by electron microscopy, characteristic lesions ol livsr cells were produced by adminis tration of CBP. INGESTION of rice bran oil contaminated with chlorobiphenyls compound (Kanechlor 400) produced poisoning among the general population in western Japan from summer to fall of 1968,1 with a total of 600 patients to date. This event seems to be unique in the field of food poisoning. It was found that contamination of the oil occurred by leaking of chlorobiphenyls com pound into the rice bran oil through pinholes in a pipe used for heat exchange in the manufacturing process. Now, one year after the incident, many patients are anxious about the possible presence of latent lesions in internal organs, as well as persistent der matological signs. Various reports of poisoning by chlorobi phenyls (CBP)2-* since the first outbreak of acne-like lesions, due to high-boiling chlori nated compound in industry, was noted by Schwartz in 1936.* But all these cases were due to exposure to the fumes or dust of this compound. Several animal experiments have been car ried out to examine the toxic effects of CBP Submitted for publication Jen 27, 1970; accepted Mnrch IB. From the Department of Public Health, Faculty of Medicine, Kyushu University, Fukuoka. Japan. Rend in pert before the 42nd annuel meeting of the Japan Society of Induatrial Medicine, Fukuoka, Japan, March 29, 1969. Reprint requeate to Department of Public Health, Faculty of Medicine, Kyushu Univereity, Katekaeu 1270, Fukuoka. Japes (Dr. Niskir.umi). by the oral route* '' and have revealed lesions of skin and liver by light microscopy. How ever, the limited resolving power of light microscopy left unsolved problems of inter pretation. Moreover, these earlier reports were concerned primarily with the effects of high levels of exposure for a short period. This paper extends these observations to the levels of resolution provided by electron mi croscopy and more thoroughly explores the effects of relatively low levels of oral expo sure for a long period. Materials and Methods Experiment With Mice.--The ddN strain mice used were originally obtained from the National Institute of Genetics, Mishima. Japan, and raised in the mouse colony of this universi ty for 12 years. Sixty ddN female mice ten weeks old were separated into three groups: (1) mice receiving olive oil or nontoxic rice bran oil as a control group, (2) mice receiving 0.6% v/v CBP in olive oil, and (3) mice receiving toxic rice bran oil containing CBP at a concentration of 1,600 ppm. The CBP used was a mixture of chlorinated biphenyls contain ing 48% chlorine (equivalent to three to four atoms of chlorine per molecule), with a trace (0.01%) of naphthalenes. Each mouse was Riv en 0.2 ml of its respective oil by stomach tube every day. All animals were kept in metal cages, in groups of two to three per cage, and were given the commercial standard diet and water ad libitum. They were observed for toxic signs and weighed twice a week. Arch Environ Health--Vof 21, Nov 1970 \ DSW 028168 STLCOPCB4012159 CHL0R0B1 PHENYL POISONING--NISHIZUMI t -Ur-. * , ft V* < ` , IT* : ,y:, v^.'yVw 1 f?Y ' rvt I 'V ;i> * -, .V: v?'-Mv- 621 ,/'r-.;w,V.'y:.^'-T 'uti; ,< ''t-'V1-.'.y .* ' rig 1.--Hspstoeytss of control mouss that received olive oil for 26 wtekt. Smell vacuoles are seen In cytoplasm; 10% formaldehyde eolutlon fixation (hmitoxylln-oln, slightly reduced from X 560). Fig 2.--Hepatocytes from control mouse that re ceived nontoxic rtce bran oil for 26 weeks. No re markable abnormalities are present. Densely stained masses In ceils represent assoeleted mitochondria and ER; 4% glutaraldehyde fixation (toluldlne blue, slightly reduced from x 800). Seven or eight mice in each group were killed after four weeks, and two mice in each group at a time were killed at 13, 17, 22. and 26 weeks. All of them were investigated micro scopically for the presence of toxic alterations. They were killed by a sharp blow on the head, followed by decapitation, between 9 AM and 10 AM in nonfasted condition. Subsequent to exsanguination, the liver* were immediately ex cised and weighed. Samples from a constant site on the right lateral lobe were processed for light and electron microscopy. Tissues for con ventional light microscopy were fixed in neutral 10% formnldehyde solution, and paraffinembedded tissue was stained with hematoxylin-eosin. Tissues for electron microscopy were immediately placed in a drop of 4% glutaralde hyde in *-collidine buffer (pH 7.4) and cut into cubes of approximately 1 mm with a razor blade. Small blocks of tissue were fixed for two hours at room temperature in buffered 4% glut araldehyde and postfixed for one hour at 0 C in collidine-buffered 2% osmium tetroxide. Some of these tissues were also cut and fixed in 2% osmium tetroxide in s-collidine* or phosphate buffer" at pH 7.4 for 1V4 to 2 hours. The fixed and washed tissue slices were then dehydrated in a graded series of ethanol and in propylene oxide prior to embedding in epoxy resin (Epon).10 Thin and thick sections of the epoxy-embedded tissues were cut with glass knives on an ultramicrotome. Thick (0.5fi to 1.0m) sections were prepared and stained with toluidine blue11 for orientation. Thin sec tions of approximately 60 ms were stained with uranyl acetate12 and lead nitrate or Millonig's lead19 alone. Specimens were examined with an electron microscope operating at 50 kv. Experiment With Monkeys---Five cynomolgus monkeys, weighing about 1 kg (2.2 lb) each, and three squhrel monkeys, weighing about 500 gm (11.0 lb) each, were used for this experiment. The CBP was administered with fresh natural diet (potatoes, cabbages, carrots, bananas, oranges, apples, and a little quantity of of small, dried fishes). The total amount of CBP administered to a cynomolgus monkey ranged from 641 mg in 40 days (16 mg/day) to 346 mg in 239 days (1.4 mg/day). That admin istered to a squirrel monkey ranged from 320 mg in 46 days (7 mg/day) to 67 mg in 48 days (1.4 mg/day). They were observed for general skin alterations and behavior and weighed weekly. Histological and electron microscopical examination were carried out in the same way as in mice. Results General Observations.--Mice.--The three groups of mice were normal in appearance and behavior for the first three months ex cept for slight weight lo6s in two experimen tal groups. After three or four months, the group of mioe receiving CBP in olive oil and that receiving toxic rice bran oil revealed slight decrease in activity, and gross changes of the skin began to appear. The macroscopical changes seen of the Bkin during the Arch Environ Health--Vol 21, Nov 1970 \ DSW 028169 STLCOPCB4012160 622 CHLOROBIPHENYL POISONING--NISHIZVMI Fig 3.--Hapatocytse of imum given COP In olive oil for four Some of hapatocytsa Increase In sirs end contsln imall granular malarial I In cyto- plaam. NueM of hapatocytaa vary In alia; 10% for maldehyde solution fixation (hematoxytln-eoaln, slightly reduced from x 470). Fig 4.--Uvar section from mouse receiving CBP In olive oil for 13 weeks. Hyaline pranulaa and small vacuoles art seen In cytoplasm. Infiltration of smell round cells la noted at upper left; 10% formaldehyde solution fixation (hematoxylln-aoaln, slightly reduced from X 470). Fig 5.--Tissue from liver of mouse given CBP for 22 weeks. There Is marked variation In nuclear size and many small vecuolee seen In cytoplasm. Note lack of cell necrosis or alteration of other tIssue components; 4% glutaraldehyde fixation (toluldina blue, aDghtty reduced from x 470). Fig 6.--Hapatocytas of mouse receiving toxic rice bran oil for 13 weeks. Soma cells contain email vacu oles In cytoplasm and ravaal granular structure; 10% formaldehyde solution fixation (hemstoxytln-eosln, slightly reduced from X 470). experimental period were as follows: (1) eczematous changes of the skin around the eyelids, (2) erosion, ulceration, and perfora tion of earlaps, and (3) loss of hair, erosion, and ulceration at the skin around thn neck, forelegs, and sides of the chest. At the end of the experiment, the changes similar to those in experimental mice were seen in some control mice. In the former group, however, the changes were much more marked. Pathological examination at four weeks showed a marked difference in the size of the livers between the groups of mice receiving CBP in oil and the group receiving no CBP in oil (P<0.01). The mice receiving CBP in olive oil and those receiving toxic rice bran oil had a mean absolute liver weight of 2.9 0.4 gm and 2.1 0.3 gm, respectively, while the control mice had a value of 1.7 0.2 gm. Mean relative liver weights were 11.6 1.0 gm, 8.3 0.8 gm, and 6.8 0.9 gm/100 gm of body weight, respec tively. No gross tissue abnormalities wars noted other than marked liver enlargement Monkeyt.--All monkeys fed CBP gradual- Arch Environ Health--Vol 21, Nov 1970 DSW 026170 CHLORODIPHENYL POISONING--NlSHIZUMl 623 Fig 7.--Uvsr section from mourn given toxic rice bran oil for 26 weeks. Hepetocytes and Kupffer's cells are enlarged, and cytoplasm of some hepetocytes re veals reticular architecture containing brown pigment. Increased cell size Is due to both nuclear and cyto plasmic enlargement. Not all liver cells are equally altered; 10% formaldehyde solution fixation (hematoxytfn-eoeln, slightly reduced from X 470). Fig 8.--Hepstecytss of mouse given toxic rice bran oil for 26 weeks. Illustrating pratancs of light and dark calls. Phtnomenon of light tnd dsrk calls wss encountered more frequently In sxparlmsntal speci mens; 4% glutsrsldahyda fixation (toluldln* blue, lHghtiy reduced from x 470). Fig 10.--Hapatocyta from Control mouse given oil without CBP for four weeks. Various Intracsilulsr orgsneilet appeared but no Increase In lipid droplets. Glycogen srsa (01), lipid droplets (U; nucleus (N); glutsrsldahyda oxygen sulfate fixation (lead, reduced from x 7.360). Fig 9.--Tissue from liver of cynomolgus monkey given total of 346 mg of CBP In 229 days. Small fatty vacuoles are noted In cytoplasms of hepatocytas, and Kupffer's calls are enlarged; 4% glutsraldahyda fixation (tokitdtne blue, slightly reduced from x 800). ly loot weight One o! the squirrel monkeys who received a total of 20 mg of CBP in six day* revealed palpebral edema two days be fore death. Increased discharge from the eye with blepharitis and. kies of appetite were noted in a cynomolgus monkey who received 795 mg of CBP in seven weeks and were observed for three weeks after CBP was stopped. Although two cynomolgus monkeys who received 122 mg of CBP in six weeks revealed loss of appetite and weight loes during the six weeks, they gained weight after CBP was stopped. Pathological exami nation just before or after death demonstrat ed enlargement of the liver, but it was found that pneumonia or diarrhea was the main cause of death for all monkeys. Light Microscopic Findings.--Mice.--In the control mice receiving nonttndo rice bran oil, liven showed nearly normal histological appearance throughout the experiment ex cept for slight appearance of small cyto- Arch Environ Health--Vol 21. Nov 1970 DSW 0281 624 CHLORODIPHENYL POISONING--NISHIZVMl i IT' f * . vu r ' . IiTtt v u . . nr^i' is tqti s < ran Fig 11.--Hapatocyt* from contra) ntouM glvan nontoxic rico bran oil for four watka; RER ira abundant Lipid draplota (L), glycogan araa (01), nudaua (N), bila canallcufl (Be); oemlum fixa tion (load, raducad from X 37,600). . Fig 12.--Part of an hapatocyt* of control moua* at 17 waaka of experimental period, (Sowing, pretence of RER and abundant mitochondria (M); oamlum fixation (lead, raducad from X 30,400). . pinamin vacuoles in cel)* of the centrilobular regiona after 22 weeks or more (Fig 1 and 2). The hepatocytee of mice receiving CBP in olive oil for four weeks increased in size and contained small acidophilic granular materiala in the cytoplasm without changes of iobular structure (Fig 3). This suggested that the liver enlargement waa due to cytoplasmic hypertrophy since mitotic figures were not Arch Environ Health--Vol 21, Nov 1970 GSW 028172 STLCOPC Fig 13.--Portion of in hapatoeyta from mouH glvan CBP in ollva oil for four waaka. (flowing an Incraaaad amount of SER and lack of RER. Qlyeogan granulaa art praaant In dlaparalon. Nuclaua (N), mitochondria (M), microbodiaa (Mb): oamlum fixation (laad. raducad from x 24,300). Fig 14.--Portion of an hapatoeyta from mouta proparad at In Fig 13. Thara la "myalln flgura" (Mf) in which multfvaalcular body (Mv) It Inremortlnd with lipid W'rlv )>\ VtYWVWVm) FFh flrm. lamina lucliuuupJ41i niflluiiuW ara notad in araa aurroumimg "myalln flgura." Mlerobodlaa (Mb); oamlum fixation (load, raducad from x 30,400). Fig 15.--Part of an hepatocyta of a mouia glvtn CBP for 13 waaka, ahowlng anothar "myalln flgura" (Mf); SER la Incraaaad markadly, glutaraldehyda Oao, fixation (uranyl acatata and laad, raducad from x 16,000). Arch Environ Health--Vol SI, Nov 1970 DSW 028173 STLCOPCB4012164 626 CHLOROBIPHENYL POISONING--N1SHIZUMI M'" ' . *r*vi1 ub. I;# ,, Fig 16.--Artt of an hepatocytn prp*rd at In preceding flguraa, charactarlzed by unuaually abun dant mlcrebodlaa and prollftrttlon of vailcular SER. Mlerobodlat (Mb), mitochondria (M), lipid droptata (L). Oamlum fixation laad, raducad (ram x 29,600). likiUh Fig 17.--Part of an hapatocyta (ram mouaa glvan CBP for 22 waaka. Incraaaaa In lipid dreplata (L) and mlcrebodlaa (Mb) ara marked. Qlutaraldehyda Oao, fixa tion (uranyi acatata and laad, ra ducad from X 97,600). seen frequently. At 13 weeks after com mencement of this experiment, more eceinstained hyaline granules and small vacuoles in the cytoplasm began to be observed (Fig 4). After 17 weeks, the nuclei ot the hepatocytes revealed variation in size, and outlines were not clear in some cells. Stainability of cytoplasm generally decreased in sections stained with hematoxylin-eoein, and the cy toplasm was granular. Although lobular ar chitecture was well preserved and no necro sis was seen, there was remarkable variation in size of hepatocytes and their nuclei at 26 weeks. Some Kupffer's cells slightly inemtsed in size (Fig 5). The livers of mice receiving toxic rice bran oil showed the typical lobular structure, with hepatocytes arranged in cords radiating from the central vein. However, hepatocytes in centrilobular areas contained small vacuoles in the cytoplasm at 13 weeks (Fig 6). At 22 weeks, there was a marked increase in the number of small vacuoles, and most of the nuclei increased in size ununiformly. After 26 weeks, sinusoidal endothelial cells, Kupffer's cells, and some hepatocytes around the cen tral vein contained brown pigment in their enlarged cytoplasm. Hepatocytes and Kupffer'a cells increased in size and had a reticu lated architecture in their cytoplasm (Fig 7 and 8). Monkeys,--Histological findings in livers of monkeys fed CBP generally revealed the presence of enlarged Kupffer's cells, with vacuoles in the cytoplasm of hepatocytes. In a squirrel monkey that received a total of 51 mg of CBP per kilogram and died eight days from commencement of the experiment, Arch Environ Health--Vol 21, Nov 1970 DSW 0281?*t STLCOPCB4012165 CHLOROBIPHENYL POISO.XIXG--NISHIZUMI 627 r * ' i _ . * .; *V> - . : ' - .- * .'.y" ; . *` s N. * > kV;;.) ' ' ' v % ... ' v`, . V''.' \- " '* -i , . *", V .. '. * vV' . : ac,-; f7* ` 'i . ,--* i-A wl l| >. ' ' . :, * . . - . ' r / .i - */.- v v. . i- ..V?: of >c " - ' . 1*hi' * ,,* . . r,._ . i rv <- ' - \: ;- -j . ^ '*; .x*,.' - ^ ' *' ' ' ; V!i`,,v ' ' i'* i .A . * .* - . *; i . . *.*</ V* \'n*. a yA, ' .n * ,> >/ . ' '*j> '**). . . *>*/- . ;y-.t. ^ v.v v , . j * *p i` ,, * : 1 r ,4 " ^ *:;C. ~;;.v- 0;..\ v *. `V.v ~.0 i/; .! u _c; . -.It.'.-?.*4 '.rr;< >*( <! Ul' *\. i,.^h ii'.-dii Fig 18.--Part of an hapatocytn from mouta given toxic rice bran oil for four wseks; SER and microbodiee (Mb) are fairly abundant, but decrease in RER le not evident Nucleus (N); glutaraldehyde Oso, fixation (uranyf acetate and lead, rnduced from x 30,400). Fig 19.--Portion of an hepatocyte from mouse given toxic rice bran oil for 26 weeks. Illustrating ap pearance of lysosomes (Ly) and well-developed Golgi'a complexes (Q). Nucleus (N); osmium fixation (lead, reduced from x 28,400). the liver showed slight enlargement of Kupffer's oells. The liver of a squirrel mon key that received a total of 134 mg of CBP per kilogram in six weeks also showed en largement of Kupffer's cells containing erythrocytes and dilatation of sinusoidal spaces. In two cynomolgus monkeys that were fed about a total of 600 to 600 mg of CBP per kilogram in four to six weeks, notable changes included the appearance of largo vacuoles in the hepatocytes at the periphery of the lobules and enlargement of Kupffer's cells. Two other cynomolgus monkeys were Arch Environ Health--Vol 21, Nov 1970 DSW 028175 STLCOPCB4012166 028 CHI.OllOBIPHENYL POISONING--NISHIXII.VI . \ - , , ** *, * ... , B' %,\ \ A-4 \ -i: Of .. 4* . i //> V \'v, * , ' ' -/ . . ft $.* <" ' -v . J *. ' . *. . *. .,, ..# ,STP* ,,V '1 'j . . \ ,J > . / - *. . ! w' / \. , -'l * ' ' vl v ^ . ( ;> v .A.:. A;yA./'. 1 ,. k*>VV - aa a:-- <- \ ^ ' . , AA <" ,, < ' A. - W- A\. , [n., . .. Mi^.' * ,^i w'lA V .r \ 1 ; 'f' 4; , V' ` f v... a!,-.-V."* M' l, ' . >.,, *^ if V . ' ' ''? V -1 ?' .--.-'I \' . . \* 'V ,-,:,:.IVlb 1 '' vA'^ v ' - "u''f v ^ - -- '-w v`.r ,' 1 ' . .'>* :^* Fig 20.--Ares of tn hepstocyte from mouse prepared st In Fig 19. Mitochondria (M) vary In size. MlcroOodlet (Mb) are disperserI. Osmium fixation (lead, reduced from x 29.600). given a total ot about 300 mg of CBP per kilogram in 20 to 32 weeks. In theee cases, many vacuoles were seen in enlarged Kup(Tor's cells, and some hepatocytes contained granular materials and vacuoles (Fig 9). The hepatocytes of the monkey who died nfter 32 weeks revealed slight fatty degener ation in peripheral lobular areas. These mi croscopic alterations in the monkey livers resembled those in mice. Electron Microscopy.--Mice Given CBPFree Oil.--In electron microscopy, no ob vious differences were seen between the liv ers of mice given olive oil and those given nontoxic rice bran oil. After four weeks, slight increase in cytoplasmic lipid droplets were noted, and the abundant mitochondria varied in size and were spherical to ellipsoi dal in form. The nuclei were round in out line, and many contained nucleoli. Other cell organelles such as endoplasmic reticulum, Golgi's complexes, and microhodies were nor mally distributed. Stores of densely stained glycogen varied in amount and localization from ceil to cell (Fig 10 and 11). After 13 weeks, the livers showed slight increase in microbodies, as well as lipid droplets. Some of the lipid droplets were partially or com pletely surrounded by mitochondria. In 22 to 26 weeks, moderately increased lipid drop lets were noted in most of the hepatocytes. Golgi'B complexes and SER (smooth-surfaced membranes of the endoplasmic rcticulum) were more abundant and contained electron-opaque substances. Stacks of RER (rough-surfaced membranes of the endo plasmic reticulum) were fairly abundant for the duration of the experiment and, for the most part, were arranged in parallel (Fig 12). No novel observations were made with regard to lysosomes, peribiliary spaces, and nuclear structure. Mice Given CBP in Olive Oil.--Adminis tration of CBP led to widespread alterations of the ER (endoplasmic reticulum), which persisted for the duration of the experiment. Appearance of cytoplasmic Inclusions and increase in microbodies, lysosomes, and lipid droplets were also observed. Serial observa tions were as follows: 1. Four weeks after commencement of the experiment, proliferation of the SER was apparent, and individual cistemae contain ing electron-opaque materials were swollen and vesiculated (Fig 13). The RER were decreased with reorganization into rough surfaced sinuous cistemae. One to four myelin figures were present in about 10% of the hepatocytes (Fig 14 and 15). Slight increase in microbodies was observed at this period, but lipid droplets were not yet increased. Arch Environ Health--Vol 21, Nov 1970 : j j | i DSW 028176 ___________' STLCOPCB4012167 CHLOROBIPHENYL POISONING--N1SH1ZUM1 629 Fig 21.--Are* of n hplocyle from cynomolgui monky fed foul of 319 mg of CBP In 22 weekt, bowing IncreaMd vehicular SER. Nucleue (N); glutareldehyde 0o, fixation (lead, reduced from x 32, 800). Fig 22.--Enlarged Kupffer'a cell of monkey fad total of 348 mg of CBP In 34 weeks, allowing numerous vacuoles and nucleus of blood cell (Nbc) In cytoplasm. Nucleus of Kupffer'a cell. (Nk) glutareldehyde Oso, fixation (lead, reduced from x 12.000). Glycogen was dispersed among vesiculated or tubular SER. 2. After 13 weeks, the hepatocytes con tained an unusually large population of mi crobodies (Fig 16) and slightly increased lipid droplets. Mitochondria showed varia tion in size and form, and lipofuscin granules were obeerved in peribiliary areas. Fine gly cogen granules were dispersed among vesicu lar SER. 3. After 17 to 22 weeks, lipid droplets were increased considerably with groups of microbodies (Fig 17). Mitochondria showed a possible increased variation in size and shape, and. occasionally, intramitocbondrial inclusions were found. Golgi's region was well preserved and appeared active, ns judged by accumulation of electron-dense products within its membranes. 4. After 26 weeks, marked alterations of ER were sustained. An increase in lipid droplets or fat vacuoles was noted, and many microbodies were present close to lipid drop lets. Mice Given Toxic Rice Bran OU.--After four weeks, alterations of RER were not as remarkable as in mice given CBP in olive oil, but on increase in SER was clearly noted (Fig 18). The mitochondria varied in size. The hepatocytes contained abundant glyco gen stores, and lipid droplets were slightly increased. Arch Environ Health--Vol 21. Nov 1970 DSW 028177 STLCOPCB4012168 630 CHLOROBIPHENYL POISONING--NISHIZUMI After 13 to 17 weeks, increase in SER and decrease in RER were remarkable. Most mi tochondria were surrounded by parallel-ar ranged RER but were almost normal in size and shape. Glycogen deposits were preserved fairly well. At 26 weeks, increases in micro bodies and appearance of a well-developed Golgi's complex were revealed in addition to th alterations of ER (Fig 19 and 20). Gly cogen granules were dispersed sparsely. In conclusion, the hepatic cell alterations induced by administration of toxic rice bran oil were essentially very similar to those caused by administration of CBP in olive oil. It was considered that the slight difference in severity of alterations whb based upon the difference in the concentrations of CBP: about 1,600 ppm in the toxic rice bran oil and about 5,000 ppm in olive oil. Monkeys Fed CBP.--The hepatocytes of monkeys fed CBP revealed the increase in SER, with dilated vesicles (Fig 21). The mitochondria were generally decreased in the density of the internal matrix and varied in size. Some mitochrondria contained intramitochondrial myelin figures. Lipid drop lets and microbodies were of normal appear ance, but increase in lyaoeomes was slightly noted. Kupffer's cells showed moderate cyto plasmic swelling, with dilated ER. In the cytoplasm, there were an increased number of lysosomee and many vacuoles, some of which contained granular materials (Fig 22). In a few places, there were discontinu ities of the cell membrane, suggesting in creased fragility and subsequent rupture, possibly during processing. Comment The oral administration of CBP in oil resulted in enlargement of the liver and vacuolar or fatty degeneration of liver cells in light microscopy. These findings were in accordance with the observations of others.*-7-14 Bennett et al14 pointed out that chlorinated biphenyl resulted in liver changes markedly different from those caused by other well-known toxic agents such as carbon tetrachloride and chloroform poi soning and thought that this type of injury was persistent with slow recovery. In our study, the morphological alterations induced in hepatocytes by administration of CBP art generally slight when judged by conven tional light microscopy, but several marked alterations in cellular organelles were found by electron microscopy. An outstanding abnormality of liver cells caused by administration of CBP to mice and monkeys is an increase in the SER and a reduction in the RER throughout the ex periment. It is considered that proliferation of the SER in the hepatocytes corresponds to hyaline cytoplasmic inclusions (hyaline degeneration) recognized with the light mi croscope and is related to the liver cell hyper trophy. Proliferation of SER in the hepato cytes is generally seen after treatment with various toxic substances such as 3'-methyl-4dimethylaminoazobenzene,11 dimethylnitrosamine,1* thioacetamide,1T ethionine,1* o-na- phthyl isothianate,1* phenobarbital sodium,34 and chlorophenothane (DDT).*1 Clearly, proliferation of SER cannot be considered to be a specific response induced by CBP. Fouts33 has pointed out that the SER of hepatocytes may be an important site of action of several drugs and poisons. In fact, it has been observed that an increase of SER in liver cells is closely associated with an increase of drug-metabolizing enzyme in the microsomal portion of liver homogenates.33-3* A suggestion that these changes reflect adap tive phenomena for enhanced drug detoxi fication could also be made from CBP poisoning. Consequently, it is also fully con sidered that disturbances of lipid metabolism such as increase of triglyceride level in blood34 are induced as the result of the func tion of proliferated SER. TTiese changes of SER were accompanied by a disorganization of the polarized arrays of the RER and by loss of surface ribosomes from the structually intact paired membranes. Cytoplasmic inclusions, which are com posed of concentric whorls of closely packed onooth membranes, were seen in mice re ceiving CBP. These cytoplasmic inclusions have been generally referred to as "finger prints,"14-1* "nebenkem,"38 or "figures myeliniques"34 and have been observed after treat ment with various toxic chemicals.1*-31-37 38 Herdson et al** suggested that some enzyme functions reside in these cytoplasmic inclu sions. Noiback and Allen** considered that the membrane arrays would provide a great- Arch Environ Health--Vol 21, Nov 1970 DSM 028178 STLCOPCB4012169 CHLOnOBIPHENYL POISONING--NISHIZUMl G31 ly increased membrane surface area to en to the concentrations of administered CBP. sure maximal contact of the toxic agent with The microbodies in experimental mouse liv the detoxifying enzymes. Various suggestions er slightly increased in number early, but have been made as to the origins and func these organelles were markedly increased af tions of these inclusions on the basis of the ter 13 weeks. It has been reported that the morphological relationships of the formations microbodies take part in lipid metabolism to other organelles and the overall effects of and glyconeogenesis and that increase in the inducing agent on the cell. However, number accompanies changes of their struc many details still remain for later work. ture in liver injury.13'42 *In6 7this experiment, Diminution of glycogen stores is another an increase in microbodies might be recog interesting feature in the present experiment. nized as the morphological expression of This is partly due to the fact that the mice metabolic changes induced by CBP. receiving toxic oil consumed approximately In control mioe, fat was abundantly ob 80% of the amount of the diet consumed by served as cytoplasmic globules from an early the control mice. However, a hj'pothesis has period of the experiment whereas in experi been proposed to explain the reciprocal be mental mice, increase in lipid droplets was havior of the SER and glycogen stores.11 observed after 20 weeks. These findings sug Bruni suggested that when diminution or gest that altered metabolism induced by loss of glycogen and permeation of glycogen CBP prevents an accumulation of lipid in areas by the SER occur almost simultaneous the early period of the experiment and re ly, circumscribed acidophilic areas might be sults in an increase of neutral fat in the seen.34 As for our findings in light micro blood. In the monkey, an increase in lipid scopy, appearance of acidophilic granular ma bodies was noted in Kupffer's cells of the terials in the cytoplasm early and of vacuoles liver injured by CBP, but hepatocellular lip or hyaline cytoplasmic inclusions later might id droplets were not increased. This fact does be the results of hypertrophy of SER and not resolve the question of whether this dis diminution of glycogen stores. Four months parity is due to the difference in animals or after the administration of CBF in olive o3 other factors. For example, monkeys were was stopped, an increase of vesicular ER, fed CBP not dissolved in oils while mice with dilated sinuous cistemae, was still ob received it in oils. In our laboratory, other served, but in the same period, no alterations work is going on to clarify the differences of ER were noted in the hepatocytes of mice between these two cases. In the near future, given the toxic rice bran oil. It was suggested more refined combinations of pathological that these alterations of ER ore .adaptive and chemical analysis may elucidate its phenomena to CBP and persist in proportion . mechanism of action. References 1. Kurataune M, et al: An epidemiologic study on Yusho. Fukuoka Acta Mod 62(813-532, 1900. 2. Jonas JW, Aldan HR: An ama form dsnnetergoaia. Arch Derm Syph 33(1022-1034, 193a 3. Pucdmlll V: Dell'acn* Chloric*. Med Lavoro 45(131-145, 1954. 4. Msigs JK. Albom JJ, Kartin BL: Chloracn* from an imusual exposure to Aroclor. JAMA 154(1417-1418, 1964. & Schwarts L: Harmstitle from synthetic raaina and wax**. Am*r J Public Health 96(588-592.1938 6. Drinker CK, Warren MF, Bennett GA: 77i* problem of possible systemic effect* from certain chlorinated hydrocarbons. J' Induilr Hyg Toxic 1*1283-311, 1937. 7. Miller JW: Pathologic ehangm In animal* sxpossd to a oomoMictal chlorinated diphenyl. Pub lic Health Rep 59tl065-1093, 1944. & Bennett HS, Luft JH: f-OoUldlnt aa a basts for buffering fixatives. J Blophye Biochem Cylol 8ilI3114, 1969. 9. Millonlg G: Further observations on a phos phate buffer for osmium fixation, in Proceedingi o/ the Filth International Confrere on Electron Microecopy, Philadelphia, 1962. Nsw York. Academic Press Inc, 1962, p P-8. 10. Luft JH: Improvements in epoxy resin embedding method. J Blophye Biochtm Cytol 9(400-414. 1981. 11. Trump BF, Smuckier KA, Bendltt EP: A method for staining epoxy section* for light micros copy. J Vltraetruct Ree 5(343-348, 1961. 12. Wataoo ML: Staining of tissue sections for electron microscopy with heavy metals. J Blophye Biochem Cytol 4(475-478,1968. 18. MUlonig Q: A modified procedure for lead staining at thin sections. J Blophye Biochem Cytol 11(798-739, 1961. Arch Environ Health--Vol 21, Nov 1970 DSW 028179 STLCOPCB4012170 632 CHLOROBIPHENYL POISONING--NISHIZUMI M. Bennett GA, Drinker CK, Warren MF: Mor. pholofical changes in th livers of rata resulting from exposure to certain chlorinated hydrocarbons. J Induwtr Hyg Toxicol 20107-133, 1038. 15. Porter KJFt, Bruni C: An electron microscope study o( the early effects of T-Me-DAB on rat liver cells. Cancer Ree 101907 1009, 1969. 16. Gmmelot P, Benedetti EL: Changes in the fine structure of rat liver cells brought about by dimethylnltrosamine. J Biophye Biochem Cytol 71303-390, 1000. 17. Thooncs W, Bnnnauch P: Eleklronen- und lirhtmikmskopische Untersuchungen am Cytoplas ms dor Leberrellen nach skuter und chronltcher Thioacetamid-Vergiftung. Virchow Arch Path Anal 335:666-683, 1962. 18. Herman L, Eber L, Fitzgerald PJ: liver cell degeneration with ethicaline administration, in Proceedingt of the Fifth International Congreee on Electron Microtcopy, Philadelphia. 1962. New York, Academic Press Inc, 1962, p V-8. 19. Steiner JW, Bsglio CM: Electron microscopy of the cytoplasm of parenchymal liver cells in alpha-naphthylisothiocyanato-induced cirrhosis. Lab Inueet 12i768-790,1963. 20. Herdsoo PB, Garvin PJ, Jennings RB: Fine structural changes in rat liver induced by pheoobarbitaL Lab Inveet 13:1032-1037,1964. 21. Ortega P: Light and. Electron Microscopy of Dichlorodipbenyitrichlorethane (DDT) poisoning in the rat liver. Lab Invert 15*667-679, 1966. 22. Fonts JR: Interaction of drugs and hepatic microeomea. Fed Proc 2111107-1111, 1962. 23. Rammer H. Marker HJ: Drug-induced changes in the liver endoplasmic reticulum: Asso ciation with drug-metabolizing enzymes. Scisnce 142:1667-1668. 1963. 24. Uzawa H, et al: Hyperglyceridemia resulting from intake of rice oil contaminated with chlorinat ed biphenyla. Fukuoka Acta Med 60:449-464, 1969. 26. Emmelot P, Benedettl EL: Some obearvatlona on the effect of liver carcinogens on the Ana struc ture and function of the endoplasmic reticulum of rot liver cells, in Harris HJC fed): Protein Bioryntheeie. New York. Academic Press Inc, 1961, p 99. 26. Houiller C, Simon G: Contribution de la microscopic Slsctroniqus au progrSe da noe coonaisaances an cytologie at en histo-pathoiogie hfepetique. Rev Int Hepat 12:167-206, 1962. 27. Herdaon PB, Kaltenbach JP: Electron micro scope studies on enzyme activity end the isolation of thiohydantoin-induced myelin figures in rat liver. J Cell Biol 261486-493. 1966. 28. Hiuban Z, Swift H, Wisaler RW: Early changes in the fine structure of bepatocytas in beta-3-thianylalanine fed rata. Fed Proc 20:136, 1961. 29. Norback DH. Allan JR: Morphoganasia of toxic fat-inducad concentric membrane arrays in rat hepetocytee. Lab Invert 20i338-346, 1969. 30. Bruni C: Hyaline degeneration of rat Bvsr calls studied with tha electron microscope. Lab Invert 9t209-216, 1960. 31. Popper H, Sehaflner F: Fine structural change* of the liver. Ann Intern Med 59t674-681, 1963. ULTRA80NIC COMMT UNICATION Tlte upper frequency limit of human hearing is generally about 17-20 kHz in young people and, although for technical reasons audiometry is seldom continued above 12 kHz, it ia fairly easy to show that the limit tends to decline with age as an expression of presbycusis. It has been claimed, however (Pumphrey, 1950), that the limitation ia entirely due to conduction losa and that sounds presented at sufficient intensity by bone conduction are audible, without further sensation of pitch change, to more than 100 kHz. It appears that tha human cochlea ia capable of responding to high "ultrasonic" frequencies but does not normally experience them and is unable to perform frequency analysis upon them. These limitations of the human ear are not shared by many smaller mammals, for there is good evidence that many species not only hear and analyze ultrasound, but use these frequencies for communication. Ralls (1967) has shown that mice and Peromytcue show posterior oollicular responses to at least 100 kHz, with greatest sensitivity in the range 10-30 kHz, Noirot (1966) found that baby mice communicate with their mother at 60-90 kHz. Sewell (1967) has found similar behavior in ten other species of Myomorph rodents and shown that the adults of some species indicate aggressive and submissive intentions by ultrasonic signals. The hearing of bats is especially intereating, because the Microchiroptera <how a highly developed foran of ultrasonic echolocation.--Pye, J.D.: "Hearing in Bate " in de Reuck, A.V.S., and-Knight, J. (ods.): Hearing Mechanieme in Vertebratei, Boston: Little, Brown It Co., 1968, p 66. Arch Environ Health--Vof 21, Nov 1979 DSW 028180 STLCOPCB4012171