Document zd7M2gN8OwEdEemxQjDpLXwV3

620 Light and Electron Microscope Study of Chlorobiphenyl Poisoning In Mouse and Monkey liv e r Maxahira N ishizum i. M l), Fuhtntka. Japan The effects on m ouse and monkey liver of long-term oral administration of ehlorobiphenyls (CBP). 1.5 m g/day or more, have been investi gated at selected intervals by light and electron microscopy. Hepatocytes of mice contained large am ounts of acidophilic m aterials in the cytoplasm, and fatly vacuoles were observed later. Fine structural changes in the hepatocytes consisted of a marked increase of sm ooth endoplasm ic reticulum, a reduction of rough endoplasm ic reticulum, "myelin figure" formation in the cyto- plasm, and Increase of m icrobodies and I some. A m arked increase in lipid droplets observed later. Results ot electron microscoc monkey liver show ed an Increase of smooth doplasm ic reticulum in the hepatocytes and si ing of Kupffer's cells, with an increased nur of fytosom es and vacuoles. Judging from findings by electron microscopy, characler lesions of liver cells were produced by adrr tration ol CBP. I n g e s t i o n of rice bran oil contaminated with ehlorobiphenyls comfxnmd i Kanechlor 400) produced poisoning among the general |*opul;tlion in western Japan from summer to fall of 1966.'- with a total of 600 patients to dale. This event seems to he unique in the field of food poisoning. It was found that contamination of the oil occurred by leaking of ehlorobiphenyls com pound into the rice hrnn oil through pinholes in a pipe used for heat exchange in the manufacturing process. Now, one year after the incident, many patients arc anxious about the possible presence of Inlent lesions in internal organs, as well as persistent der matological signs. Various rejwrts of poisoning by chlorobiphenyls fC B P )7 4 since the first outbreak of acne-like lesions, due to high-hoiling chlori nated compound in industry, was noted by Schwartz in 193G.ft Bui all these cases were due to exposure to the fumes or dust of this compound. Several animal experiments liave been car ried out to examine the toxic effects of CBP Subtnilleil for poblkaiion .Jnn 27. 1970. accepted March fa. Kmm the I lepnrtmem of I'nhlic Henllh, Faculty of Mcrlnioe. Kvurhu UnivciMlv, KukiNikn. -lapon. Head in pail liefore the 4Jnd annual meeting of the J.1JMI Society of IniluatnHl Medicine, KukiHikn. lxp.ui. March 21*. l`*Vf) Iteprim requests to Deportineu*. of I'nhlic Health. !^rtlltv of Medicine, Kvushu University KaUkosu 127h. Ktikuokx. Japan <Dr N ishi/unm by the oral route'11 and have revealed l*si of skin and liver by light microscopy. H ever, the limited resolving power of li microscopy left unsolved problems of in pretation. Moreover, these earlier rep< were concerned primarily with the effect.high levels of rxixisure for a short peri This paper extends these observations to levels of resolution provided by electron cmscopy and more thoroughly explores effects of relatively low levels of oral ex sure for a long peruxl. Material and Methods-- E x p e rim e n t W ith M ice.-- T h e rlilfsf str mice used w r e orientally obtained from National In-iitute of Genetics. M ishtm a. Jop and raised in the m ouse rolnnv of this uruvn tv for 12 v e ars S ix tv d d N fem ale mice week.- old were se|arali-l into th re e cruut (I) mice deceiving olive ml or nontnxic r b ra n oil as a -ontrol group. 12) m ice reochi 0.3% v /v CBT in olive oil. and (3) m receiving toxic rice hrau oil containing PBP a c o n c e n tra tio n of l.fiOO ppm . T h e C B P us w.is a m ix tu re of c h lo rm a le d h ip h e n v ls eor.ta: ing 48% c h lo rin e (cq m v n ien r to th ree to fn atom s of chlorine i*t m olecule), with a tn (0 01% ) of n a p h th a len e s. K a rh motive was c <*ii <>.? ml of its respective oil hv stom ach tu rverv dv All anim uN w ere kepi in m etal rag* in groups of two to fhn- p er cage, and given th e eoum ien-tal -ta n d a rd diet and wnl ad libitum T h e v w ere observed for toxic sip anrl weighed twa-c a week A rch F nrirun H ealth- -V oi '21. .Vor in/n NPC00026573 753910 CHLQROBl PHENYL POISONING--NISHIZVMI 621 Fig 1 -Hpatocytes of control mouse that received tve oil for 26 weeks Sma<l vacuoles are seen in loplasm: 10% formaldehyde solution fixation ematoxylin-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 s t;:nd masses in cells represent associated mitochond'ia and ER; 4% glutara.dehyde fixation (toluidine blue, slightly reduced from x 800). Seven o r eight m ire in each group were :lled a fte r four weeks, and two m ice in euch -uup at a tim e were killed a t 13, 17, 22. a n d 26 `dts. All of them were investigated m icroipically for the presence of toxic alterations. : l*ey wore killed by a sh a rp blow on th e head. ..Ilowed by decapitation, between 9 AM a n d 10 M in nonfasted condition. Subsequent to cxinguination, the bvers were immediately ex.I and weighed Sam ples from n c o n stan t :te on the right lateral lul*e were processed for ,-ht and electron m icroscopy. T issues for rnnviiuonal light microscopy were fixed in neutral form aldehyde solution, unrl |.traiTinmliedded tissue was stained with hem ntoxyu rosin. Tissues for electron microscopy were nmediately placed in a drop of 4*',', gluiuralrlcvHo in s-collidine buffer tp H 7.4) and cut info uhes of approxim ately 1 m m w ith a nixor 'xk. Small blocks of tissue were fixed for two ours at room tem p e ra tu re in buffered 4% glutratdehyde and |>ostfixed for one hour a t 0 C in '.llidme-bufiercd 2.', osmium tetroxidc. Som e of iese tissues were ulso cue and fixed in 2% mium tetroxide in s-collidine* or phosphate iiffeyj a t pH 7.4 for lVi to 2 hours T h e xed and washed (issue slice wore then hydrated in a graded senes of ethanol nil in propylene oxide p rio r to em bedding in .xixy resin (E p o n )." 1 T h in a n d thick sections f the epoxy-em bedded tissues were cut w ith lass knives on an ultram icrotom e. T hick (0.5u 1.0b) sections wore p rep ared and stain ed uth toluidine blue11 for orientation. T h in secnns of approxim ately 60 m^ were stainod with u ra n y l a ce ta te 1- ami lead n itra te or M illo n ig s lead*J alone. Specim ens were exam ined with an electron m icroscope o p e ratin g a t 50 kv. Experim ent W ith M onkeys.-- Five cynomolgus monkeys, weighing about 1 kg (2.2 lb) each, and three squirrel m onkeys, weighing a b o u t 500 gm (11.0 lb) each, wx-rc usi-d for this experiment. T he C B P was adm inistered with fresh natural diet (potatoes, cabbages, carrots, bananas, orange, apples, and a little quantity of of mall, dried fishes). T h o total a m o u n t of C B P administered to a cvnomolgus monkey ranged from 641 mg in 40 d a y s (16 m g /d n y ) to 3tH mg in 239 day (1.4 m g /d a y ). T h a t a d m in istered to a sq u irrel m onkey ranged from 320 mg in 4G days (7 m g/d:*y) to 67 mg in 48 day (1.4 m g/d n y ). T h ey were observed for general skin alterations and behavior and weighed weekly Histological and electron microscopical exam ination were carried out in the same way a in mice. Results General Observations.--Mice.--The three Rruujis of mice were normal in appearance and hehaviur 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 charges of the skin began to appear. The macroscpi ca! changes seen of tlie skin during the Arch Environ H ealth-- Vol 21, N ov 1970 NPC00026574 753911 622 C H L O liO B I P H E N Y L P O IS O N IN G -- S 'I S H I Z U M I ir toJ* . v i- _ _ _ _ 1 ^ c JL#' Pig 3.-- Hepatocytes of mouse given CBP in o!>ve oil for four weeks. Some of hepatocytes increase in s za 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). it Fig 4 -- Liver section from mouse receiving C8P * olive oil for 13 weeks. Hyaline granules and sma* vacuoles arc seen in cytoplasm. Infiltration of smiii round cells Is noted at upper left. 10% formaldehyde solution fixation (f-ematoxy.'in-eosin. s'ightly 'educed from x 470). ir m M Fig 5.--Tissue from l iw of mouse given CBP for 22 weeks. There is marked variation in nuclear size and many small vacuoies seen in cytoplasm. Note lack of cell necrosis or alteration of other tissue components. 4% glutaraldehyde fixation (toiuidme blue, siightiy reduced from x 470). Tig 6.-- Hepatocytes of mouse receiving toxic net bran orl for 13 weeks. Some ceils contain small vaev oles in cytoplasm and reveal granular structure: 10% formaldehyde solution fixation (hematoxylin eotui slightly reduced from x 470). , '. t experimental period were as follows: ( 1) eczematous changes of the skin around the eyelids, (2) erosion, ulceration, and perfora tion of earlaps, and f31 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 tliose 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 reeeivinc CBP in oil and the group receiving no CBP in oil (P c O .O l). The mice receiving CBP in olive oil and those receiving toxic ricr bran oil had a mean absolute liver weight of 2.9 0.4 gm and 2.1 0.3 gm, respectivrly. while the control mice had a value of 1.7 0.2 gin. Mean relative liver weight* were 11.5 1 .0 gm, 8.3 0.8 gm. nnd 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- 753912 lost weight. One of the squirrel monkeys no received a total of 20 mg of CBP in six vs revealed palpebral edema two days he re death. Increased discharge from the eye th blepharitis and loss of appetite were ted in a cynomolgus monkey wlw received 3 mg of CBP in seven weeks and were served for three weeks after CBP was typed. Although two cynomolgus monkeys to received 122 mg of CBP in six weeks .euled 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 denth fur all monkeys. Light Microscopic Findings.--Mice.-- In the control mice receiving nontoxic rice bran oil, livers showed nearly normal histological appearance tliroughout the experiment ex cept for slight appearance of small cyto- Ardi Endm n Health-- -Vol 21, Nov 1970 NPC00026576 CHLOnOBPHEN YL POtSOSlSG--S lS IU Z V M t x 753914 A rch F itr h on H ealth-- Vo! 21. <Vut< 1970 NPC00026578 753915 626 Cl lW H O M PHENYL POISONING -NISIIIZHMI Fig 16.-- Area of an hepatocyte prepared as in p'eceding figures, characterized by unusually abun dant microbodies and proliferation of vescuiar SER. Microbodies (Mb), mitochondria (M). lipid droplets (.). Osmium Fixation lead, reduced from x 29.600). Frg 17.-- Part of ar hepatocyte from mouse given CBP for 22 weeks Increases in lipid droplets (L) and microbodies (Mb) are marked. Glutara'dehyde Oso, fixa tion (uranyr 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 tl*e cytoplasm began to be observed (Fig 4). After 17 weeks, the nuclei of the hpato cytes revealed variation in size, and outlines were not clear in some cells. Stainability of cytoplasm generally decreased in sections 9tained 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 hcpatocytes and their nuclei at 26 weeks. Some Kupflfcr'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 vacuole* in the cytoplasm at 13 weeks fFig 6). At weeks, there was a marked increase in the number of small vacuoles, and most of tl nuclei increased in size ununifonmly. After? weeks, sinusoidal endothelial cells. KupfTrr'* cells, and some hepatocytps around the ern tra! vein contained brown pigment in thru enlarged cytoplasm. Hepatocytes and Kup ffer's cells increased in size and had a retioi later! architecture in their cytoplasm (Fig and 8). Monkeys. Histological findings in liver of monkeys fed CBP generally revealed hr presence of enlarged KupiTcr's cells, wi& vacuoles in the cytoplasm of hepatocytes. In a squirrel monkey that received a tnlil of J mg of CBP per kilogram and died rijhf days from commencement of the experiment. A rch E nviron H ea lth -- Vol 21, N o e 1970 NPC00026579 753916 CHLOHOBIPHENYL POISONING--NlSHMU&ti 627 Fg 18-- Part of on hpatocyte from mous* geven tonic nee bran oil for four weeks; 5ER and microbodies (Mb) are fairly abundant, but decrease in RER is not evident. Nucleus (N); glutaraldehyde 0so4 (ration (uranyl acetate and lead, reduced from x 30.4C0). Fig 19-- Portion of an hepatocyte from mouse given tosic rice bran oil lor 26 weeks, illustrating ap pearance of lysosomes (Ly) and wen-devcoped Gold's completes (G). Nucleus (N): osmium lirai-on (lead, reduced from x 28,400)- tie liver showed slight enlargement of \upffer's cdU. The liver of a squirrel mon ey that received a total of 134 mg of CUP i-r kilogram in six weeks also showed cnrsvment of Kupffcr's cells containing rythrocytcs and dibtation of sinusoidal ,iaces. In two cynomolgus monkeys that were fed about a total of 500 to 600 mg of CBP ir 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 Arrh Environ Health-- Val 'll, Nno 1970 753917 NPC00026580 r.2H ('HI.OHOIVPHE,VVt. POISQXINC- M S I l l / . i :MI j fig 20--- Area of n hepaiocyle from mouse prepared as in Fig 19. Mitochondria (M) vary in sire. Microbod'es (Mb) are dispersed. Osmium fixation (>ead. reduced from x 29 600) given n total of about 300 mg of CBl* j>er kilogram in 20 to 32 weeks. In these cases, many vacuoles were seen in enlarged Kup(Ter'< evils, and some hepatocytes contained granular materials and vacuoles i Fig 9 '. The hepatocytes of the monkey who died after 32 weeks revealed slight fatty degener ation in peripheral lobular arc;. 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 nonloxic 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 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 w*eks. the livers showed slight increase in microlwdies, as well as lipid droplets. Some of the lipid droplets were partially or com pletely surrounded by mitochondria. In 22 to 2fi weeks, moderately increased lipid drop let*. were noted in most of the hepilocytes. Golgi's complexes and SER i smooth-sur faced membranes of the endoplasmic reticu lum) were more abundant and contained electron-opaque suhstnnees. Stacks of RER <rough-surfaced membranes of the endo plasmic reticulum) were fairly abundant fur I Hie duration of the experiment and. for tlx* j most part, were arranged in parallel <Kig ' 12). No novel observations were made with j regard to lysosomes. peribiliury spaces, ynd * nuclear structure. 1 Mice Given CUP in Olive Oil Adminis tration of CBI* led to widespread alterations of the ER (endoplasmic reticulum ', which persisted for tlv duration of the exjjerimcnl Appearance of cytoplasmic inclusions and increase in microbodies, lysoson*1*, and lipid droplets were also observed. Serial observa tions were as follows: 1. Four weeks after commencement of thr experiment, proliferation of the SER was j apparent, and individual cisternae contain- I ing electron-opaque materials were swollen and vesiculated i Fig 13). The HER wrrr decreased with reorganization into rough- surfaced sinuous cisternae. One to four myelin i figures were present in about 10% of tlir hepatocytes (Fig 14 and 15). Slight mcrco* in microbodies was observed at this period, but lipid droplets were not yet increased ( Jkco tr tut- 2. / l.um-d inilifH lipid turn in wen* mgrn i.ir S E 3/ wen* l inicTol a |>osi Fh.l(K*. mcLusi Arch Envinui Health --Vol 21. Xov 1970 NPC00026581 753918 CHLOROBIPHENYL POISONING--N ISH IZ CMI 629 Fig 21.-- Area ot an hepatacyte from cynomotgus monkey tea total ol 319 mg of CBP in 22 weeks, showing increased vesieu'ar SER. N-cleus (N). glutaraldehyde Oso, fiiat.on (read, reduced from x 32.* 802). Fig 2 2 -- Enlarged Kupffer's ce'l of monkey fed total of 348 mg of CBP in 34 weeks, showing numerous vacuoles and nucleus cf blood ced (Nbc) in cytoplasm. Nucleus of Kuptfer's cell, (Nk) gluta.-aldehyde Oso, fixat:o~ (l.*jd, reduced from x 12.00C). vcngen was dispersed among vesiculatcd tubular S E R L After 13 weeks, tire hepatocytes con ned an unusually large population of miiudies (Fig 16) and slightly increased d droplets. Mitochondria showed variai in size and form, and lipofuscin granules e observed in peribiliary areas. Fine glyen granules were dispersed among vesicu* SER. i. After 17 to 22 weeks, lipid droplets v increased considerably with groups of .rcbodies (Fig 17). Mitochondria showed lossible increased variation in size and 1, and,. occasionally, mframitochondrial lusions 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 droplet; nr fat vacuoles was noted, and many microbodips were present close to lipid Uru|lets. M ice Given Toxic Rice Bran Oil.-- After four weeks, alterations of RER were not as remarkable as in mice given CUP in olivt oil, but an increase in SE R was dearly noted (Fig IB ). The mitochondria varied in size. The hepatocytes contained abundant glyco gen stores, and lipid droplets were slightly increased. Arch Environ 11rcllh-- Vo! 21. Nnv 1970 753919 630 KHLOROBIPitENYL PO!SOXL\'G--N lS n iy. UMI Attrr 13 to 17 weeks, increase in SER and decrease in R ER were remarkable. Must mi tochondria were surrounded by parallel-ar ranged REK but were almost nnrmal in size and shape. Glycogen deposits were preserved fairly well. At 26 weeks, increases in microbodies and appearance of a well-developed Golgi's complex were revealed in addition to the alterations of E R (Fig 19 and 201. 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 C BP 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 microbodics were of normal appear ance, but increase in lysosomes was slightly nnlrd. KupfTer's cells showed moderate cyto plasmic swelling, with dilated ER. In tlie 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 Tlie oral administration of CBP in oil resulted in enlargement o f the liver and vacuolar or fatty degeneration of liver cells in light microscopy. These findings were in accordance w ith the observations of others."-7-*4 Bennett rt a l14 pointed out that chlorinated biphenyl resulted in liver changes markedly different from those caused by other well-known toxic agents such os 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 hpatocytes by administration of CBP n generally slight when judged by conve tional light microscopy, but several marin alterations in cellular organelles were loui by electron microscopy. An outstanding abnormality of liver ce caused by administration of CBP to mi and monkeys is an increase in the SER at a reduction in the R ER throughout the i (riment. It is considered that prolifrtit of the S E R in the hepatocytes correspond to hyaline cytoplasmic inclusions (hyalii degeneration) recognized with the light in croscope and is related to the liver cd l hype trophy. Proliferation of S E R in the hepat< cytes is generally seen after treatment wit various toxic substances such as 3'-methyldimethylnminonzobcnzene,15 dimethylnitn sam ine.111 thioncetamidc.17 cthionine/* -re phlhyl isoth innate.10 phnobarbital sodtunv nnd chlomphennthane (D D T ).51 Clcarh* proliferation of S E R cannot be considered t be a specific response induced by CBI Fonts52 has pointed out that the SER f hepatocytes may be an important site < action of several drugs and poisons. Tn fat'! it has been observed that an increase of SEI in liver cells is closely associated with & Increase of drug-metabolizing enzyme in th microsomal portion of liver homogenates.5" A suggestion that these changes reflect adap tive phenomena for enhanced drug detox fication could also he made from CBI poisoning. Consequently, it is also fully con sidered that disturbances of lipid mvtabolim such as increase of triglyceride level ir blood-* are induced as the result of the func tion of proliferated S E R . These changea o S E R were accompanied by a disurpanizatinr of the polarized arrays of the R E R and h> loss o f surface, ribosomes from the stmctually intact paired membranes. Cytoplasmic -inclusions, which are com posed of concentric whorls of closely packed smooth membranes, were seen in mice (t ceiving CBP. These cytoplasmic inclusion.' have been generally referred to as "fingci prints/'1019 "nebenkem,"25 or "figures inyiliniques"90 and have been observed after treatment with various toxic chemicals.****57** Herdson et al20 suggested that some enzyme functions reside in these cytoplasmic inclu sions. Nnrhjck and A llen59 considered tha the membrane arrays would provide a gral- +P- NPC00026583 753920 C H I.O RO BIPH EN YL PO ISO N IN G -- N IS H IZ V M l G31 increased membrane surface area to en- to the concentrations of administered CBP. iie maxiiail contact of the toxic dgent with The microbodies in exj>eriinental mouse liv c detoxifying enzymes. Various suggestions er slightly increased in numlier early, but ive been made as to the origins and func these organelles were markedly increased af tus of these inclusions on the husls of the ter 13 weeks. It has lieen rejiortcd that the mphological relationships of the formations microbndies take part in lipid metabolism other organelles and the* overall effects of and glyconeogencsis and that increase in e inducing agent on the cell. However, number accompanies changes of their struc any details still remain for later work. ture in liver injury.11 In this experiment, Diminution of glycogen stores is another an increase in microbodies might be recog Cresting feature in the present experiment, nized as the morphological expression of his is partly due to the fact that the mice metabolic changes induced by CBP. reiving toxic oil consumed approximately In control mice, fat was abundantly ob of the amount of the diet consumed by served as cytoplasmic globules from an early * control mice. However, a hypothesis has period of the experiment whereas in exjeri- -en proposed to -xplain the reciprocal be mental mice, increase in lipid droplets was aver of the SER and glycogen stores.15 observed after 20 weeks. These findings sug mni suggested that when diminution or gest that altered metabolism induced by rj of glycogen and permeation of glycogen CBP prevents an accumulation of lipid in 'ms by the SER occur almost simultnneous- the early period of the experiment and re . circumscribed acidophilic areas might be suits in an increase of neutral fut in lire or50 As for our findings in light micro- blood. In the monkey, an increase in lipid ojjy, appearance of acidophilic granular ma- bodies was noled in Kupffer's cells of the rhls in the cytoplasm early and of vacuoles liver injured by CBP, but hepatocellular lip r hyaline cytoplasmic inclusions later might id droplets were not increased. This fact does r the results of hypertrophy of SER and not resolve the question of whether this dis jninution of glycogen stores. Four months parity is due to the difference in unimals or ler the administration of CBP in olive oil other factors. For example, monkeys were is stopped, an increase of vesicular ER, fed CBP not dissolved in oils while mice ilh dilated sinuous cUtemae, was still ob- received it in oils. In our laboratory, other med, but in the same period, no alterations work is going on to clarify the differences f ER were noted in the hepatocytes of mice between these two cases. In the near future, ven the toxic rice bran oil. It was suggested more refined combinations of pathological hat these alterations of ER arc adaptive and chemical analysis may elucidate its h-nomena to CBP and persist in proportion mechanism of action. References L Kuralsune M. at 1: An epidemiologic study on Aaho. Fukuoka Acta Med 62:513-532. 19G9. 1 Jones JW, Alden HS: An acne form derma terads. Arch Derm Syph 33:1022-1034, 1930. 3. Purcinelli V: Dell'am e Chlorica. Med Laix-ra 4:131-145, 1954. 4. Mens JK , Albom J J . Kartin BI.: Chlorarne .at an unusual exposure to Arorlor. JAM A 4:1417-1418, 1954. S Schwartz L: Dermatitis from svnihetic resin ad waxes. Amer J Public Health 26:586-592. 19.'k>. A Drinker CK, Warren MF. liennett CA- The oblam of possible systemic effects from certain hiorinated hydrocarbon.-, J Induttr Hyg Toxic *283-311, 1937. 7. Miller JW : Pathologic changes in animals rtposed to a commarcial chlorinated diphenyl. Pubtf Health Rep 59:1065-1093, 19-44. A Bennett HS, Iaift JH * Collidine as a basis tor bufTenng fixatives J Biophyx Biochem Cytnl 6:113- 114, 1959. 9 Millonig G: Further observations on a phos phate buffer for osmium fixation, in Proceedingi of the Fifth International Congrett on Electron M i croscopy. Philadelphia. 19>2 New York, Academic Prewt Inc, 1902. p P-8 10. T.uft JH : Impiovementa in epoxy resin embedding method. J Tlmphyt Biochem Cvtol 9:409-414, 19G1. 11 Trum p BF, Sm utkler FA, Benditt El*: A method for staining epoxy sections for light mun> copy J Ultrattruct He 5:343 348, 1901. 12. Watson ML: Stsininfr of tissue sections for electron microscopy with heavy metals. J Biophy* Biochem Cytnl 4:475-478. 1958. 13. Millonig G: A modified procedure for lead staining of thin sections J Biophyt Biochem Cvtol 11:736-739, 19*41. Arch Environ Health-- Vol 21. N ov 1970 NPC00026584 753921 6.12 CI/LOPOBIPHESYL POISONING--NISHIZUMI 14. Bennett GA, Drinker CK, ''Vorren Ml': Mor- 23. Hemmer H. Merker H J: Urug-indwnt jJmItntii-.il chances in the livers of rats resulting changes in the liver endoplasmic reticulum; Avo from exposure to certain chlorine led hydrocarbons. elation with drug-metabolizing enzymes. Reimt* J Induttr H yp Toxicol 20:97-123, 197. 142:1657-165, 1963. 15. t'orler K it, Dnini C: An electron microscope 24. Uzawa H, e t al; HypercKceridenda rasullint study of Ihe early effects of T-Me-DAB on rat liver cells. Cancer Rex 19:937-1009.1959. 15. Emmelot P, Henedetti RI.: Changes in the fine structure of rat liver cells brought about hy dimethylnitToaamine. J Biophys Biochem Cytol 7:393-396, I960. 17. Thoenes W, Rannagrli P: Elektronen- und Tirhlmikroskopisehe Untersuchungen tin Cytoplas ms der loiter/ellen nach akuter und chronischer Thkwcelomid-Vergiftung. Virchna Arch Path Anat 3351556-563.1962. 18. Hermsn L. Eber L, Fitzgerald PJ; Liver cell degeneration with ethionine administration, in Pro ceedings of the Fifth International Congress on Elrctmn Microscopy, Philadelphia, 1562. New York. Academic Press lor. 1962, p V-6. 19. Steiner JW, llaglio CM: Electron niicrosropy of the cytoplasm o parenchymal liver relLx in nlldia-naphthylbtotliiocyanate-indured cirrhosis. Lab Intent 12:765-799. .19!. from intake of rice oil contaminated with chlorinxt ed biphenyls. Fukuoka Acta Med 60:449-464,1909. 25. Fmmelot I1. Benedetti EL: Sc mo observai torn on tho effect or liver carrinoceng nn the fine strut lure and function of tho endoplasmic reticulum of \ rat liver cells, in Harris ItJC te d ': 1`rutetn iV>ethesis. New York. Arodemic Press Inc, 1961, p 99. Ra 26. Rouiller C, Simon G: Contribution de I * has microscopie iteotm nique mi prugri*s de nos con j amok naissances en rvtologie et en histo-pathologie hpa I quire tique. Reu Int HepaI 12:167-206. 1962. * aia ir 27. Herdson I'll, Kallenbach -IT; Electron mi,to. i in th scope studies nn enzyme activity and Ihe isolation ; 35 p ol thiohyrlmiloin-induced myelin figures in rat liter j The J Cell Biol 25:485-41X1, 1965. 28. Hrubnn 7-, S s il t H, W iw ler RW: Eariv rHanges in tho fine structure of hpatocytes n f pariti ptifiei hctn-3-thienylalanine fed mis. Fed Pror 20:1V - with 1961. 29. N orlnrk DH. Allen -TIL Morphogenesis of 20. Herdson PB. Garvin I\I. Jennings UB: Fine structural changes in rat liver induced by phenobaibital. Lob Invent 13:1032-11X17, 19&1. 21. Ortega P: Light nnd Electron Mirroccnpy of Dichloroiliphenyltrichlorethane (DDT) poisoning in the rat liver. Lab Invent 15:657-679, 1966. 22. Fouls JR: Inlerarlion of drugs and hepntir microsomeA. Fed Proc 21:1107-1111, 1962. toxic fat-induced concentric membrane n m y s in nl hpatocytes. fjuh Invest 20:338-3-11'. 1969. 30. Ilnini C: Hyaline degeneration of rat liver cells studied with the electron microscope, lab Invent 9:209-215,1960. 31. Popper H , Schn finer F: Fine structural changes of Ihe liver. Ann Intern M ed 59:674-6?!, 1963. : t ., th i uth The : lors > tutoli in vi' out i requi nf lo ULTRASONIC COMMUNICATION j lem." Tlie upper frequency limit of human hearing is generally about 17-20 kHz in young people and, although for technical reasons nudioinctry is seldom continued above 12 kHz. it is fairly easy to ehow that the limit tends tn decline with age an in expression of preibyruri*. It has been claimed, however (Pumphrey, 1950), that the limitation is entirely due to conduction loss and that sounds presented at sufficient intensity hy hone 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" ! direct I variai snintr f.'omf mal s in viti firlfl l frequencies but does not normally experience them and is unable to perform frequency munt 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 Pr.romyscus 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 Myomarph rodents and shown that the adults of some species indicate aggressive and submissive intentions by ultrasonic signals. r. Tho henring of bats is espediilly interesting, because the Micrichiruplcra show a highly developed form of ultrasonic echclocation.--Pye. "Hearing in Bats." in dr Houck. A.V.S.. and Knight. J. iedn.): Hearing Mechanisms in Vertebrates, Boston: Little. Brown & Co., 1968, p 66. OllM mque In bri* llietze til). a. mriscd Subir wl Mar From l'ppwl# flepri 1MI7, ] Arch Environ Health-- Voi 21, Sot' I97C NPC00026585 i