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620 Light and Electron Microscope Study of Chlorobiphenyl Poisoning In Mouse and Monkey Liver Masahiro Nishizumi. Mil, Fukuoka. Japan \ The effects on mouse and monkey liver of long-term oral administration of chlorobiphenyls (CBP), 1.5 mg/day or more, have been investi gated at selected intervals by light and electron . microscopy. Hepatocytes of mice contained large amounts of acidophilic materials in the cytoplasm, and fatty 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 cyto- plasm, and increase of microbodies and lysosomes. A marked increase in lipid droplets was observed later. Results of electron microscopy in monkey liver showed an increase of smooth en doplasmic reticulum in the hepatocytes and swell ing of Kupffer's cells, with an increased number of lysosomes and vacuoles. Judging from the findings by electron microscopy, characteristic lesions of liver 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)2-4 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 Jan 27, 1970; accepted March 18. From the Department of Public Health, Faculty of Medicine, Kyushu University, Fukuoka, Japan. Head in part before the 42nd annual meeting of the Japan Society of Industrial Medicine, Fukuoka, Japan, March 29, 1969. Reprint requests to Department of Public Health, Faculty of Medicine, Kyushu University, Katakasu 1276. Fukuoka. Japan (Dr. Nishizumi). by the oral route6'7 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 0.5% 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 giv 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--Vol 21, Nov 1970 OSVM 255222 K STLCOPCB4060913 CHLOROBlPHENYL POISONING--NISHIZUMI rr&'&& * - , ,'f;1..*?<} ^ ?&*;.>'','. > ,f ' :^\0'T .yj^-.V^Vr / ]ffjf /V). * s' v-./M ^^ *v* .'i^r ^ * ; ^ V '<0'4^, y.;. V .*. 4 k ._*, . .v#*; -T/" 621 Ar'. ' iv. ;.t' V- ^ s!^: C^CV^t-.i Fig 1.--Hepatocytes of control mouse that received olive oil for 26 weeks. Small vacuoles are seen in cytoplasm; 10% formaldehyde solution fixation (hematoxylin-eosin, slightly reduced from X 560). Fig 2.--Hepatocytes from control mouse that re ceived nontoxic rice bran oil for 26 weeks. No re markable abnormalities are present. Densely stained masses in cells represent associated mitochondria and ER; 4% glutaraldehyde fixation (toluidine 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 livers 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% formaldehyde solution, and paraffinembedded tissue was stained with hematoxy lin-eosin. Tissues for electron microscopy were immediately placed in a drop of 4% glutaraldehyde in s-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% glutaraldehyde 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-collidine8 or phosphate buffer0 at pH 7.4 for iy2 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. to 1.0/0 sections were prepared and stained with toluidine blue11 for orientation. Thin sec tions of approximately 60 mg were stained with uranyl acetate12 and lead nitrate or Millonig's lead13 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 squirrel 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 348 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 loss in two experimen tal groups. After three or four months, the group of mice 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 skin dining the Arch Environ Health--Vol 21, Nov 1970 osvJ255223 622 CHLOROBIPHENYL POISONING--N1SHIZUMI .ScviV -V '*3?*t vr:-?-.::< Vv^ V l * j* J Iti'"''*'**'* - ' ' *.-'! ! 4 . -r-1 * v '.' .. ' '.-tv;;,'/ft ::; -V'-/"' r . V/w*i l*.t 0* >. J-. ^ . -1 Fig 3.--Hepatocytes of moose given CBP in olive oil for four weeks. Some of hepatocytes increase In size and contain small granular materials in cyto plasm. Nuclei of hepatocytes vary in size; 10% for maldehyde solution fixation (hematoxylin-eosin, slightly reduced from x 470). Fig 4.--Liver section from mouse receiving CBP in olive oil for 13 weeks. Hyaline granules and small vacuoles are seen in cytoplasm. Infiltration of small round cells is noted at upper left; 10% formaldehyde solution fixation (hematoxylin-eosin, 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 vacuoles seen in cytoplasm. Note lack of cell necrosis or alteration of other tissue components; 4% glutaraldehyde fixation (toluidine blue, slightly reduced from x 470). . Fig 6.--Hepatocytes of mouse receiving toxic rice bran oil for 13 weeks. Some cells contain small vacu oles in cytoplasm and reveal granular structure; 10% formaldehyde solution fixation (hematoxylin-eosin, 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 the 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 were noted other than marked liver enlargement. Monkeys.--All monkeys fed CBP gradual- Arch Environ Health--Vol 21, Nov 1970 DSV\I 255224 STLCOPCB4060915 CHLOROBIPHENYL POISONING--NISHIZUMI 623 Fig 7.--Liver section from mouse given toxic rice bran oil for 26 weeks. Hepatocytes and Kupffer's cells are enlarged, and cytoplasm of some hepatocytes 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 (hematoxylin-eosin, slightly reduced from x 470). Fig 8.--Hepatocytes of mouse given toxic rice bran oil for 26 weeks, illustrating presence of light and dark cells. Phenomenon of light and dark cells was encountered more frequently in experimental speci mens; 4% glutaraldehyde fixation (toluidine blue, slightly reduced from X 470). Fig 10.--Hepatocyte from control mouse given oil without' CBP for four weeks. Various intracellular organelles appeared but no increase in lipid droplets. Glycogen area (Gl), lipid droplets (L); nucleus (N); glutaraldehyde oxygen sulfate fixation (lead, reduced from x 7,360). Fig 9.--Tissue from liver of cynomolgus monkey given total of 348 mg of CBP in 229 days. Small fatty vacuoles are noted in cytoplasms of hepatocytes, and Kupffer's cells are enlarged; 4% glutaraldehyde fixation (toluidine blue, slightly reduced from x 800). ly lost weight. One of the squirrel monkeys who received a total of 20 mg of CBP in six days revealed palpebral edema two days be fore death. Increased discharge from the eye with blepharitis and loss 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 loss 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 nontoxic rice bran oil, livers showed nearly normal histological appearance throughout the experiment ex cept for slight appearance of small cyto- Arch Environ Health--Vol 21, Nov 1970 OS* 255225 STLCOPCB4060916 624 CHLOROBIPHENYL POISONING--NISHIZUMI , Jv - f,m m-:- . .-V.-'Y ' a a- ; '..'V' .wHV` ?* zr-v''fcwjsfc***>'5* ' v* ji r;-vi; . - -a i..*.; rs A af " , sv* .* V-W i-v-VT*-. yV- .' v , \.: ; vv . . v .. . v '. v; - ' . . : / -V *' - 'Sf *'.'< ' .< Fig 11.--Hepatocyte from control mouse given nontoxic rice bran oil for four weeks; RER are abundant. Lipid droplets (L), glycogen area (Gl), nucleus (N), bile canaliculi (Be); osmium fixa tion (lead, reduced from x 37,600). Fig 12.--Part of an hepatocyte of control mouse at 17 weeks of experimental period, showing, presence of RER and abundant mitochondria (M); osmium fixation (lead, reduced from X 30,400). , plasmic vacuoles in cells of the centrilobular regions after 22 weeks or more (Fig 1 and 2). The hepatocytes of mice receiving CBP in olive oil for four weeks increased in size and contained small acidophilic granular materials in the cytoplasm without changes of lobular structure (Fig 3). This suggested that the liver enlargement was due to cytoplasmic hypertrophy since mitotic figures were not Arch Environ Health--Vol 21, Nov 1970 DSW 255226 STLCOPCB4060917 Fig 13.--Portion of an hepatocyte from mouse given CBP in olive oil for four weeks, showing an increased amount of SER and lack of RER. Glycogen granules are present in dispersion. Nucleus (N), mitochondria (M), microbod ies (Mb); osmium fixation (lead, reduced from x 24,300). Fig 14.--Portion of an hepato- cyte from mouse prepared as in Fig 13. There is "myelin figure" (Mf) In which multivesicular body (Mv) Is incorporated with lipid hortt/ \\\ o.eis'. taming electing opaque uieteiiab are noted in aroa surrounding "myelin figure." Microbodies (Mb); osmium fixation (lead, reduced from x 30,400). Fig 15.--Part of an hepatocyte of a mouse given CBP for 13 weeks, showing another "myelin figure" (Mf); SER Is increased markodly; glutaraldohyde Oso, fixation (uranyl acetate and lead, reduced from x 16,000). Arch Environ Health--Vol 21, Nov 1970 DSW 255227 STLCOPCB4060918 626 CHLOROB1PHENYL POISONING--NISHIZUMI Fig 16.--Area of an hepatocyte prepared as in preceding figures, characterized by unusually abun dant microbodies and proliferation of vesicular SER. Microbodies (Mb), . mitochondria (M), lipid droplets (L). Osmium fixation lead, reduced from x 29,600). *b i. : (: L ti'iil *''*>!UL-'.'i.W.i V>1 ' Fig 17.--Part of an hepatocyte from mouse given CBP for 22 weeks. Increases in lipid droplets (L) and microbodies (Mb) are marked. Glutaraldehyde Oso, fixa tion (uranyl acetate and lead, re duced from x 37,600). seen frequently. At 13 weeks after com mencement of this experiment, more eosinstained hyaline granules and small vacuoles in the cytoplasm began to be observed (Fig 4). After 17 weeks, the nuclei of the hepatocytes revealed variation in size, and outlines were not clear in some cells. Stainability of cytoplasm generally decreased in sections stained with hematoxylin-eosin, 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 in creased 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 Kup ffer's 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 OS\N 255228 STLCOPCB4060919 CHLOROBIPHENYL POISONING--NISHIZUMI 627 Fig 18.--Part of an hepatocyte from mouse given toxic rice bran oil for four weeks; SER and micro bodies (Mb) are fairly abundant, but decrease in RER is not evident. Nucleus (N); glutaraldehyde Oso, fixation (uranyl acetate and lead, reduced 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's complexes (G). Nucleus (N); osmium fixation (lead, reduced from X 28,400). the liver showed slight enlargement of Kupffer's cells. 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 500 to 600 mg of CBP per kilogram in four to six weeks, notable changes included the appearance of large 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 dS\N 255229 STLCOPCB4060920 628 CHl.OROBlPHENYL POISONING--NISHH/.UMl Fig 20__ Area of an hepatocyte from mouse prepared as in Fig 19. Mitochondria (M) vary in size. Microbodies (Mb) are dispersed. Osmium fixation (lead, reduced from X 29,600). given a total of about 300 mg of CBP per Golgi's complexes and SER (smooth-sur kilogram in 20 to 32 weeks. In these cases, faced membranes of the endoplasmic reticu many vacuoles were seen in enlarged Kup- lum) were more abundant and contained fTer's cells, and some hepatocytes contained electron-opaque substances. Stacks of RER granular materials and vacuoles (Fig 9). (rough-surfaced membranes of the endo The hepatocytes of the monkey who died plasmic reticulum) were fairly abundant for after 32 weeks revealed slight fatty degener the duration of the experiment and, for the ation in peripheral lobular areas. These mi most part, were arranged in parallel (Fig croscopic alterations in the monkey livers 12). No novel observations were made with resembled those in mice. regard to lysosomes, peribiliary spaces, and Electron Microscopy.--Mice Given CBP- nuclear structure. Free Oil.--In electron microscopy, no ob Mice Given CBP in Olive Oil.--Adminis vious differences were seen between the liv tration of CBP led to widespread alterations ers of mice given olive oil and those given- of the ER (endoplasmic reticulum), which nontoxic rice bran oil. After four weeks, persisted for the duration of the experiment. 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 microbodies were nor mally distributed. Stores of densely stained glycogen varied in amount and localization from cell to cell (Fig 10 and 11). After 13 weeks, the livers showed slight increase in microbodies, as well as lipid droplets. Some 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 of the lipid droplets were partially or com figures were present in about 10% of the pletely surrounded by mitochondria. In 22 to hepatocytes (Fig 14 and 15). Slight increase 26 weeks, moderately increased lipid drop in microbodies was observed at this period, lets were noted in most of the hepatocytes. but lipid droplets were not yet increased. Arch Environ Health--Vol 21, Nov 1970 DSW 255230 STLCOPCB4060921 CHLOROBlPHENYL POISONING--NISHIZUMI IV " >//?>;_ '/*'*'?* `'^V<= ' ; ."' ' V'.7..'*# ^T-C-:- ' V--.V .-WV -. ; -.VV'-vi t- v:-.V'!5 ; ;; c v* 629 h W,*./->-.-r' /V' :.-! fe>v-:: " ^ _.;^v ,/ * /lit.. I'lh wVl. 1 VV W?; rV`$, * .'tf* Fig 21.--Area of an hepatocyte from cynomolgus monkey fed total of 319 mg of CBP in 22 weeks, showing increased vesicular SER. Nucleus (N); glutaraldehyde Oso, fixation (lead, reduced from X 32, 800). *' jjjJ Fig 22.--Enlarged Kupffer's cell of monkey fed total of 348 mg of CBP in 34 weeks, showing numerous vacuoles and nucleus of blood cell (Nbc) in cytoplasm. Nucleus of Kupffer's cell, (Nk) glutaraldehyde 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 observed 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, intramitochondrial inclusions were found. Golgi's region was well preserved and appeared active, as 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 Oil.--After four weeks, alterations of RER were not as remarkable as in mice given CBP in olive oil, but an 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 255231 STLCOPCB4060922 630 CHLOROB1PHENYL POISONING--NISHIZUMl 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 th6 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 was 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 lysosomes was slightly noted. Kupffer's cells showed moderate cyto plasmic swelling, with dilated ER. In the cytoplasm, there were an increased number of lysosomes 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.9'7-1* 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,18 dimethylnitrosamine,10 thioacetamide,17 ethionine,18 -naphthyl isothianate,19 phenobarbital sodium,20 and chlorophenothane (DDT) .21 Clearly, proliferation of SER cannot be considered to be a specific response induced by CBP. Fouts22 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.22-23 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 blood24 are induced as the result of the func tion of proliferated SER. These 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 smooth membranes, were seen in mice re ceiving CBP. These cytoplasmic inclusions have been generally referred to as "finger prints,"16-10 "nebenkem,"28 or "figures myeliniques"29 and have been observed after treat ment with various toxic chemicals.16-21-27-28 Herdson et al20 suggested that some enzyme functions reside in these cytoplasmic inclu sions. Norback and Allen29 considered that the membrane arrays would provide a great Arch Environ Health--Vol 21, Nov 1970 DSW 255232 STLCOPCB4060923 CHLOROBIPHENYL POISONING--NISHIZUMI 631 ly increased membrane surface area to en sure maximal contact of the toxic agent with the detoxifying enzymes. Various suggestions have been made as to the origins and func tions of these inclusions on the basis of the morphological relationships of the formations to other organelles and the overall effects of the inducing agent on the cell. However, many details still remain for later work. Diminution of glycogen stores is another interesting feature in the present experiment. This is partly due to the fact that the mice receiving toxic oil consumed approximately 80% of the amount of the diet consumed by the control mice. However, a hypothesis has been proposed to explain the reciprocal be havior of the SER and glycogen stores.15 Bruni suggested that when diminution or loss of glycogen and permeation of glycogen areas by the SER occur almost simultaneous ly, circumscribed acidophilic areas might be seen.30 As for our findings in light micro scopy, appearance of acidophilic granular ma terials in the cytoplasm early and of vacuoles or hyaline cytoplasmic inclusions later might be the results of hypertrophy of SER and diminution of glycogen stores. Four months after the administration of CBP in olive oil was stopped, an increase of vesicular ER, with dilated sinuous cistemae, was still ob served, but in the same period, no alterations of ER were noted in the hepatocytes of mice given the toxic rice bran oil. It was suggested that these alterations of ER are adaptive phenomena to CBP and persist in proportion to the concentrations of administered CBP. The microbodies in experimental mouse liv er slightly increased in number early, but these organelles were markedly increased af ter 13 weeks. It has been reported that the microbodies take part in lipid metabolism and glyconeogenesis and that increase in number accompanies changes of their struc ture in liver injury.31 In this experiment, an increase in microbodies might be recog nized as the morphological expression of metabolic changes induced by CBP. In control mice, fat was abundantly ob served as cytoplasmic globules from an early period of the experiment whereas in experi mental mice, increase in lipid droplets was observed after 20 weeks. These findings sug gest that altered metabolism induced by CBP prevents an accumulation of lipid in the early period of the experiment and re sults in an increase of neutral fat in the blood. In the monkey, an increase in lipid bodies was noted in Kupffer's cells of the liver injured by CBP, but hepatocellular lip id droplets were not increased. This fact does not resolve the question of whether this dis parity is due to the difference in animals or other factors. For example, monkeys were fed CBP not dissolved in oils while mice received it in oils. In our laboratory, other work is going on to clarify the differences between these two cases. In the near future, more refined combinations of pathological and chemical analysis may elucidate its mechanism of action. References 1. Kuratsune M, et al: An epidemiologic study on Yusho. Fukuoka Acta Med 62:513-532, 1969. 2. Jones JW, Alden HS: An acne form dermatergosis. Arch Derm Syph 33:1022-1034, 1936. 3. Puccinelli V: Dell'acne Chlorica. Med Lavoro 45:131-145, 1954. 4. Meigs JK, Albom JJ, Kartin BL: Chloracne from an unusual exposure to Aroclor. JAMA 154:1417-1418, 1954. 5. Schwartz L: Dermatitis from synthetic resins and waxes. Amer J Public Health 26:586-592, 1936. 6. Drinker CK, Warren MF, Bennett GA: The problem of possible systemic effects from certain chlorinated hydrocarbons. J ' Industr Hyg Toxic 19:283-311, 1937. 7. Miller JW: Pathologic changes in animals exposed to a commercial chlorinated diphenyL Pub lic Health Rep 59:1085-1093, 1944. 8. Bennett HS, Luft JH: a-Collidine as a basis for buffering fixatives. J Biophys Biochem Cytol 6:113 114, 1959. 9. Millonig G: Further observations on a phos phate buffer for osmium fixation, in Proceedings of the Fifth International Congress on Electron Mi croscopy, Philadelphia, 1962. New York, Academic Press Inc, 1962, p P-8. 10. Luft JH: Improvements in epoxy resin embedding method. J Biophys Biochem Cytol 9:409-414, 1961. 1L Trump BF, Smuckler EA, Benditt EP: A method for staining epoxy sections for light micros copy. J Vltrastruct Res 5:343-348, 1961. 12. Watson ML: Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475-478,1958. 13. Millonig G: A modified procedure for lead staining of thin sections. J Biophys Biochem Cytol 11:736-739, 1961. Arch Environ Health--Vol 21, Nov 1970 DSW 255233 STLCOPCB4060924 632 CHLOROBIPHENYL POISONING--NISHIZUMI 14. Bennett GA, Drinker CK, Warren MF: Mor phological changes in the livers of rats resulting from exposure to certain chlorinated hydrocarbons. J Industr Hyg Toxicol 20:97-123, 1938. 15. Porter KR, Bruni C: An electron microscope study of the early effects of 3'-Me-DAB on rat liver cells. Cancer Res 19:997-1009, 1959. 16. Emmelot P, Benedetti EL: Changes in the fine structure of rat liver cells brought about by dimethylnitrosamine. J Biophys Biochem Cytol 7:393-396, I960. 17. Thoenes W, Bnnnasch P: Elektronen- und Ijchtmikroskopische Untereuchungen am Cytoplas ms der Leberzellen nach akuter und chronischer Thioacetamid-Vergiftung. Virchow Arch Path Anat 335:556-583, 1962. 18. Herman L, Eber L, Fitzgerald PJ: Liver cell degeneration with ethionine administration, in Pro ceedings of the Fifth International Congress on Electron Microscopy, Philadelphia, 1962. New York, Academic Press Inc, 1962, p V-6. 19. Steiner JW, Baglio CM: Electron microscopy of the cytoplasm of parenchymal liver cells in alpha-naphthylisothiocyanate-induced cirrhosis. Lab Invest 12:765-790, 1963. 20. Herdson PB, Garvin PJ, Jennings RB: Fine structural changes in rat liver induced by phenobarbital. Lab Invest 13:1032-1037,1964. 21. Ortega P: Light and Electron Microscopy of Dichlorodiphenyltrichlorethane (DDT) poisoning in the rat liver. Lab Invest 15:657-679, 1966. 22. Fouts JR: Interaction of drugs and hepatic microsomes. Fed Proc 21:1107-1111, 1962. 23. Remmer H, Merker HJ: Drug-induced changes in the liver endoplasmic reticulum: Asso ciation with drug-metabolizing enzymes. Science 142:1657-1658, 1963. 24. Uzawa H, et al: Hyperglyceridemia resulting from intake of rice oil contaminated with chlorinat ed biphenyls. Fukuoka Acta Med 60:449-454, 1969. 25. Emmelot P, Benedetti EL: Some observations on the effect of liver carcinogens on the fine struc ture and function of the endoplasmic reticulum of rat liver cells, in Harris RJC (ed): Protein Biosyn thesis. New York, Academic Press Inc, 1961, p 99. 26. Kouiller C, Simon G: Contribution do la microscopie 61ectronique au progrfes do nos connaissances en cytologie et en histo-pathologie hepatique. Rev Int Hepat 12:167-206, 1962. 27. Herdson PB, Kaltenbach JP: Electron micro scope studies on enzyme activity and the isolation of thiohydantoin-induced myelin figures in rat liver. J Cell Biol 25:485-493, 1965. 28. Hruban Z, Swift H, Wissler RW: Early changes in the fine structure of hepatocytes in beta-3-thienylalanine fed rats. Fed Proc 20:136, 1961. . 29. Norback DH, Allen JR: Morphogenesis of toxic fat-induced concentric membrane arrays in rat hepatocytes. Lab Invest 20:338-346, 1969. 30. Bruni C: Hyaline degeneration of rat liver cells studied with the electron microscope. Lab Invest 9:209-216, 1960. 31. Popper H, SchafEner F: Fine structural changes of the liver. Ann Intern Med 59:674-691, 1963. ULTRASONIC COMMf UNICATION Tlie 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 is 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 is entirely due to conduction loss 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 the human cochlea is 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 Peromyscus show posterior collicular 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 interesting, because the Microchiroptera -how a highly developed form of ultrasonic echolocation.--Pye, J.D.: "Hearing in Bats." in de Reuck, A.V.S., and Knight, J. (eds.): Hearing Mechanisms in Vertebrates, Boston: Little, Brown & Co., 1968, p 66. Arch Environ Health--Vol 21, Nov 1970 DSW 255234 STLCOPCB4060925