Document b5eEZGgY59zJrYZDb7EVY5YRy

! t PCB (igjfc tf y x. ~ jv) K 't 6 ng m so ^ 10 n fa fj fa m W.fz E A W^ fK ^f The Fifth Reports of the Study on "Yusho" and PCB (Polychlorinated Biphenyls) October, 1975 Study Group for the Therapy of Yusho Faculty of Medicine, Kyushu University Fukuoka 812, Japan DSW 032563 t \ \ The Fifth Reports of the Study on "Yusho" and PCB (FUKUOKA ACTA MEDICA Vol. 66, No. 10, 1975) CONTENTS 1. Foreword....................................................................................................... T. Omac---( 1) 2. Metabolic studies on PCBs VI. Contribution of the hydroxylatcd metabolites to the acute toxicity of 2,4, 3',4'-tctrachloroblphenyl ............................................... H. Yoshlmura, H. Yamatoto and H. Kinoshila---( 3) 3. Metabolic studies on PCBs VII. Metabolic fate of 2, 5, 2', S'-tctrachlorobiphcnyl in rats.......... H. Yoshimura, H. Yamamoto and K. Yonczawa---( 9) 4. EtTecl of PCB on rat liver microsomal phospholipids ......................... .............................................................. Y. Maeda, K. Takahashi and K. Kato--.(17) 5. Effect of PCB on HcLa cells-..................................................................... -- R. Mori, K. Fujita, M. Yamaguchi, H. Yoshimura, and H. Yamamoto---(22) 6. Hemolytic and "hcmagglulinating" activity of PCB ............................. K. Fujita, R. Mori, M. Yamaguchi, H. Yoshimura and H. Yamamolo---(28) 7. PCBs toxicity and nutrition IV. PCBs toxicity and vitamin A (3) .............. ........ S. Innami, A. Nakamura, K. Kato, M. Miyazaki, ............... ... S. Nagayama and E. Nishidc--(33) 8. Influences of some agents on the enzyme-inducing activity of PCB-- ............................................................... Y. Araki, T. Hidaka and K. Tanaka-- (39) 9. Influences of PCB on the brain catecholamine levels In rats.............. .................................... N. Sucnaga, K. Yamada, T, Hidaka and T. Fukuda (43) 10. Chlorinated dibenzofurans in Kancchlors and rice oils used by patients with Yusho ............................. J. Nagayama, Y. Masuda and M. Kuratsunc---(47) 11. Comparison of hyperkeratosis induced by PCBs, PCDF and PCDD application.............................M, Nishizumi, M. Kuratsune and Y. Masuda---(54) 12. PCB residues in plasma of Yusho children born to mothers who had consumed oil contaminated by PCB---S. Abe, Y. Inouc and M. Takamatsu --(59) 13. PCB, DDT and BHC levels in human plasma as a measurement of tissue residue-..............Y. Inoue, S. Abe, M. Takamatsu, N. Aokl, S. Miki and K. Fujiwara---(64) 14. Abnormality of scrum enzyme in PCB poisoning patients with special .. reference to ribonuclcase.........................M. Yamanaka, JC. Akagi, . N. Hirao and K. Murai --(71) 15. Consecutive, six year follow-up study on scrum triglyceride levels in ' patients with PCB poisoning ............................................................... ' ............... ......... M. Okumura, M. Yamanaka, S. Nakamuta and H. Uzawa---(74) 16. Relation between PCB level in the blood and clinical symptoms of Yusho patients........................................................... H. Koda and Y. Masuda---(78) 17. Alteration in skin severity grading of Yusho In 1973 and 1974, and presentation of a new standard for the skin severity of Yusho by point count system............................. M. Asahi, H. KOda and S. Toshitani---(83) 18. Anterior pituitary function of Yusho paiicnts........................................ ......................... -...........................M. KusucKs, Y. Nagata and M. Nakamura---(89) DSW 032564 STLCOPCB4016526 19. Further ophthalmic studies on patients of chronic PCB poisoning ................................. Y. Ohnishi, H. Ikui, S. Kurimoto and K. Knwashima...(94) 20. Ocular findings of PCB intoxication and histological changes of the . palpebral conjunctiva in rats fed with Kaneclor 500..................... A. Aoki-->(96) 21. A follow-up study of fasting therapy of Yusho patients..............M. Imamura*-*(100) DSW 032565 l STLCOPCB4016527 43REI?. 66 (10): 547--548, 1975 # -g ifcfiEfttfifiJFftJiCK, AfttA&S|!&g m &i m m Foreward Teruo Omae Chief of the Study Group for the Therapy of " Yusho", Professor of Medicine, Second Department of Internal Medicine, Faculty of Medicine, Kyushu University, Fukuoka, Japan . In succession lo the 4th report (Fukuoka Acta Mcdica, Vol. 65, No. ], January, 1974), 'he outline of the recent studies made by the Study Group for the Therapy of "Yusho" was described. The total number of the patients reached 1291 on April, 30th, 1975, among which 29 died. Elevation of blood PCB concentration and abnormality in gas chromatographic pattern of blood PCB, acnciform eruptions, pigmentation (skin, conjunctiva, gingiva or nail) and hypertrophy or hypersecretion of Mcibonf glands have been considered as most important for the diagnosis of "Yusho". Some changes in clinical profiles were noticed as the time elapsed. Studies concerning the pathogenesis or pathophysiology of "Yusho" were also described. . R1-. JdUfiifc PCB KMI-SflfftWS-Ji. (W&imf60 #6*3. B3?n44Jp; 62$H3-. B3fn46^; 63$10-!\ BSfn47^1 65 $ 1-5J, ft 49 #) B3fn50 4p6 E Wfli&!8SffSfe8Ki. fn50*MEJSfc, fBgm Elftm A* K 1. CW3fn 50 4p 5 ^ 15 El IS?3E> AA E m . ' 0 m 0 H 0 0 0 .0 0 0 0 0 0 w Si J ft *> A ? to 8K *1 RW& 0 &WAI4 P & 'h % w 0 ft fi aii tk g // 0 Wttg Jft E JSb&g > 0 Bit Sc ti g W 1)5 to iii U1L ik) lb ^ Si 6ff ft A ii E & &_ m a to th P IE ft $ B3 5k 2 ., IS HI 51 2 tt TS -- 6K *B to 0 0 %A0 A A ft * * US P3 A E 0 0 A*AE 0 0 0 0 1*1 Sl rc ? ft W\ to *1 *i H R* ft ^ flt-rtH RH g fis-rtW <h R 11 to *1 R#8r& 0 9) Hr q/j 0 i g 0 ft fllrttg g 0 0 0 0 0 // fill Gg S? & AH W HI n g 15 If m J3 * t} 3S (A 1m IS &AW ft <a ifi AM 1 Vl *'J 8 to ft J life Cl Si ft H) to Or T A tt # PJ A K 1* k n id A H) & A EH 8i a ( -l ) OSW 032566 STLCOPCB4016528 1 I0IHH4) ._**___ fillSlElt 66 CIO): 549--554, 1975 PCB (g!6$B) 2, 4, 3', 4'-^ h =5*7 n o fcf7x-;K7)RKji 549 . * 1 1 1 1 , --------- Il'tWrtJX Cl'frl'. Djfl&K-O 1 . ft Uj * & W *T ` Metabolic Studies on Polychlorinated Biphenyls. VI. Contribution of the Hydroxylafed Metabolites to the Acute Toxicity of 1,4,3', 4'-Tetrachlorobipbenyl Hidetoshi Yoshimura, Hiro-aki Yamamoto and I-Iaruki KinosiiitA Department of Hygienic and Forensic Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University , In our previous study, the acute toxicity of 5-hydroxy-2,4, 3', 4'-lctrachlorobiphcnyl (TCB), the major metabolite of 2,4, 3'. 4'-TCB, was found to be about 5 times as high as that of 2,4, 3', 4'-TCB in mice. In order to learn the contribution of this metabolite to the toxicity of the parent compound, it was studied whether 2,4, 3\4'-TCB would exert more toxic effect or not after pretreatment of mice with phcnobarbital (PB) or 3-mclhylcholanthrene (3-MC), the typical inducers of liver microsomal drug metaboliz' ing enzymes system. Effects of the same pretreatment of rats on the biliary excretion ra(0 0f 5-hydroxy-2,4, 3', 4'-TCB after iv injection of 2,4, 3', 4'-TCB and on the in vitro metabolic rate of 2, 4, 3\4'-TCB using liver 9000 g supernatant fraction were also inves tigated. By these studies the following findings were obtained. 1. Pretreatment with 3-MC increased the acute toxicity of 2,4,3',4'-TCB, but on the contrary PB pretreatment decreased that toxicity in mice. 2. Liver 9000 g supernatant fraction obtained from the rat pretreated with 3-MC showed much higher activity to hydroxylate 2,4,3', 4'-TCB than that of the control rat, but PB pretreatment did not increase the activity at all. 3. By pretreatment with PB or 3-MC, rats increased their biliary excretion of 5hydroxy-metabolite about 2 times or more than 10 times, respectively, comparing with that of untreated rats, during 24 hr after iv injection of 2,4,3', 4'-TCB. It was suggested that the increase of excretion of 5-hydroxy-metabolite in 3-MC pretreated rats should be due to marked etevation of the metabolic rate, whereas that in PB pretreatment could be explained by increase of biliary flow rate. 4. From these results, it was concluded that at least a part of the acute toxicity of 2,4.3', 4'-TCB was attributable to the more toxic metabolite, 5-hydroxy-2,4,3', 4'TCB, which was produced in the body. t o -A'-MO ic& 3- a tV * * a u -*-400 tp 2. 4, 3'. 4'-x 1'< 1-5* a Of 7*-A (2.4,3', 4'- . *5,fctf3, 4,3', 4'-x t*7i-M3,4,3',4'-TCB)*>`> <3) DSW 032567 STLCOPCB4016529 lochtm. 1. Biochcm. J. PJiar- ntlh, R. L. nacol, 19: fflRJKlt- 56 (0): 555--562, 1975 pcb oi-tmicmt mm) 2,5,2' 5'-t b 7^ O Bt:7i = A7? h fCfrtf 655 . . & *j 3i Si iJi * 5A W # iR spa fyj. ' Metabolic Studies on Polychlorinated Biphenyls. VII. Metabolic Fate of 2, 5, 2', S'-Tetrachlorobiplienyl in Rats Hidetoshi Yoshimura, Hiro-aki Yamamoto ` and Kazuaki Yonezawa ' Department of Hygienic and Forensic Chemistry, Faculty of Pharmaceutical Sciences, Kyushi University . In the previous studies from this laboratory, it was found that both of 2, 4,3', 4'and 3,4. 3\4'-tctrachloTobiphenyl (TCB) were metabolized to the monohydroxylated derivatives in rats and the metabolites were excreted almost exclusively into their fcccs. Hutzingcr et al., on the other hand, reported that when 2,5,2', 5'-TCB was injected inlraperitoneaily into rats, large quantities of the unchanged material could be extracted from the feces and only a monohydroxy derivative was identified in the urine. In order to confirm this discrepancy for excretion route of the metabolite, wc re-investigated the metabolic fate of 2, 5, 2', 5'-TCB orally given to rats and obtained the following results. 1. In the case of 2, 5,2', 5'-TCB, the major metabolite was excreted mostly in the feces. By GLC analysis, this metabolite excreted during 7 days accounted for about 45 % of the dose, whereas that excreted in the urine during the same days accounted for only 0.4% of the dose. This metabolic rate was much higher than those of 2,4,3', 4'and 3,4,3', 4'-TCB (about 10% and 3.3% of the dose, respectively, during 12 days). The structure of this major metabolite was elucidated to be 3-hydroxy-2,5,2', 5'-TCB by various spectrometries and finally by comparison with the authentic sample. A trace amount of 4-hydroxy-metabolite was also identified in the urine. 2. In above experiments, about 7 % of the dose was excreted unchanged into 7 days feces, most of which was done in the first day. A trace amount of unchanged material could be detected in the urine. This result also suggested that the absorption rate of 2,5,2\5'-TCB from the gastrointestinal tract was quite higher than those of 2,4, 3',4'-and 3,4, 3'. 4'-TCB (the absorption rate of the latter two TCB was calculated to be about 60% and 40%, respectively). 3. 2, 5,2'. 5'-TCB was injected inlraperitoneaily into rats with cannulatcd bile duct and excretion of the metabolite in the bile was examined by GLC. The result indica ted clearly the excretion of 3-hydroxy-2, 5,2', 5'-TCB as the major metabolite and also of 4-hydroxy-mciabolilc in a trace amount, but no unchanged material could be detected in Ihc bile. Since 4-hydroxy-metabolite was not excreted in the faces, above result suggested that a part of the metabolites excreted through the biliary system to the small intestine might be rc-absorbcd from the intestine. '6 (9> DSW 032568 STLCOPCB4016530 . and Saoki, 2231, 1973. Mira, 11.: 37, 1973. Tiolo, It.: 68, 1973. 55: 5, 1974. G.: Nature ., Safe, S., I., Wildish, J2, 1972. fcitko, V.: 209, 1971. ter, G. C., Janie CornSons, Inc., ,: J. Arocr. Roach, i. 'i Biophys. Olburn, 1'., llochcm. J. : ft 1 IWA pi] K It 66 (10): 563--567, 1975 563 77 (PCB) (D&m Ift m Bit lia B ffi & - Effect of Biphenyl Chloride on Rat Liver Microsomal Phospholipids Yasuo Maeda, Keikichi Taicahashi and Keitaro Kato Faculty of Pharmaceutical Sciences, Kyushu University Fukuoka, Japan The proportion of different phospholipids and fatty acid component of phosphatidyl choline, phosphatidylethanoJamine and phosphatidylinosilol were studied in rat liver microsomal membranes, after a long period administration (60days; total dose, 100mg) of Kanechlor-400 (KC-400). The proportion of different phospholipids did not change, but KC-400 caused the proportion of stearic acid in phosphatidylcholine to increase to J25? of the control and the proportion of palmitic acid to decrease to 70 96. The effect of a long period administration of KC-400 on drug metabolizing .emzymes was also studied, and the relationship between elevation of the emzymes and change of phospholipids was discussed. -, m n IBtoKite?,??fbli, Phenobarbital UiWC- PCB 61, fflSZibmwtSififICkV $3l0<iTk'o,,,>. Phenobarbital li Cytochrome P-450 (Cyt. P-450) i>>, Methyl- cholanthrene Cyt. P-448 PCB Cyt. P-450 t Cyt. P-448 If? i'o7-Afcfai)PTiti*>Jj]i)nTC` JIM). Phenobarbital t Mctylcholanthrene "Pfi, $ b )5. If? 6-o7-A65^18fil<,UTt,'S'; vJSKtctei (if Hate 131 $ nx V'*. L i\b &fe utzt. t fnslcICli&lbinfib ?I'i1, Phosphatidylcholine t Phosphalidylcthanol8mine T13, fuJftlifrteEfsitS iaifb, ttttr'Mmtttftiimv&iwmczet c1 6'*C) ^.Phosphatidylcholine Phenobarbital -p Metylcholanthrene (Ci--<)5 t, PCB Bf 5^o >/- 0 VBUJ3ItRiJTE3W C'Ot,'X5)l?tli'l?fj:^"'. 5 V PCB *>*, tSltBT? 9p7-Aglets,'iii2<nst;&>(.7S*. 5i", nc pcb zmm-'jb Bf ? i> p 7- AB51C ') 5R^KR6l^'X(>S!l^f7V\ BT?^o'/-iK It&t ?.SS-li5ftsy57J:^ftliPtS!S 9 ^J5rZ7l!fffiS5 Bfi &&lii!;t>3K-p l'T %% illA t:. *5 K * ifc i. #*a BKlt&li Sprcaue Dawley y y htj (ill 300g) % BUULf;. Kanechlor-400 (KC-400) * Corn oil c in DSW 032569 STLCOPCB4016531 563 tJISJEjfc 66(10) ; 568--573, J975 (PCS) i*| HeLa }ajS|CJU5-rg 3 /,, jj & & -m m m ~ ill d ; | *f & fit U4 * & W Effect of Polychlorinated Biphenyls (PCB) on HeLa Cells. 41 <\ 4 A Ryoichi Mori, Koichi Fujita, and Midori Yamaguchi Department of Microbiology, Faculty of Medicine, Kyushu University, Fukuoka 4t Jl H Hidetoshi Yoshimura and Iliroaki Yamamoto Department of Hygienic and Forensic Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka Suspension of Kanechlor 400 (KC 400), a commercial brand of polychlorinated biphenyls (I'CB), was found to have cytotoxic and cytolytic activity against HeLa cells. Tho minimum effective dose was 3~6pg/ml in media without serum protein. Time- lapse cinematography revealed that HeLa cells treated with KC 400 25 A'g/ml undergo retraction of processes, bleb formations, balloon formations followed by burst of the cells, - yUHbfc'7x--A (T PCB B3t) flilS g,0--9T&> Z> * * 7 D -to 400 (&T KC 400 BS t) U, &JWITJ 7 V Hcta;il-r5i)IFlt:HjgH;SEtb%3-r *> c t. 0l!fto>'3iief5frftT*j *>, % pH 7.2 CPBS) *f;f Eagle minimum essential medium (MEM) IClm^T (0.SS^iaiS:lBiRtLft. /fjis^njt,'. {fmiutj lo&iin MEM %JSl(S EXllItt& ftxl> i HeLa *6IKa S 3 fcfDWc. Ki8i^(t'jaii(fcK)?fStLTti-/7 7. 9 ? 9 l Zo-fUd b (Falcon 3040) ft35X10 mm !) r r y t'j (Falcon 3001) r ! r>: i;( I \ ) 5>l t l i! :A ii i i i b-7-(Sfl3* MbWert'A* HeLa*ajja/!H\ tftlc&IJ-f KC 400#glcoiyT&!j&Oof;fflT<flft*-i. *t 14 t is jfe : Wd3fctJJ5fc-r *!:>& bsksm. fianas'-aKK^fl o. 5 % b y /o^a-- KtliaiTjlniJJtfifSlcXft. Jfc&SftSfTOfcyEi&il lf L yta: pcb ii3-: it kc 400 *{j;iijGf:. kc 400 K (DMSO) 1C 10 mg/ ml5Ci;icifi^f:{>,iiB'il/, WrP m C, 15J>*-h^u^^icj: eft ttBilCiSteJiC Falcon 3001 IclfiftUc HeLa *{0K4 MEM V5&-otC0h, '354ffi KC 400 fcStr MEM 2ml *inA., t*--*?- SSfciM), 20(3 DLL fcol). (22) DSW 032570 i STLCOPCB4016532 574 tSWKJfc 06(10); 571-578, 1975 l (pcb) m m $ -$ &- lii ?> t' K> . ihm&mmtmMmm % n & t& ai * & bj] ' Hemolytic and "Hcmagglulinaling" Activity of Polychlorinated Biphenyls (PCB). Koichi Fujita, Ryoichi Mori and Midori Yamaguchi Deportment of Microbiology, Faculty of Medicine, Kyushu University, Fukuoka Hidetoshi Yoshimura and Hiroakt Yamamoto Department of Hygienic and Forensic Chemistry, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka Kanechlor 400 (KC 400) dissolved in dimelhylsulfoxidc and emulsified in PBS was found to have "hemagglunating" activity at 4"C and hemolytic acitivity at 37'C. The '`hcmagglutinating" phenomenon was not due to the dimer formation or aggregate for /nation. The phenomenon was blocked by serum protein and cardiolipin. Most of the tsomers of tctrachlorobiphenyls, chief components of KC 400, were shown to have hemolytic activity at the same concentration as KC 400. fi&fll1' v#<)iuikd7 crcB) ml tciStf Utc tjti>C>(5f-!S;tfi9aScWi^tt!G ~^>V * * *** o-* 400 (KC 400) *5, tgJJ U. Hcha C (4-* ' *Hi A>C.taifre%<D{t,i/:X 1/ p otr7x--') ti, rnfnTCt*Tt>--T71- strasiti/, pcb tfc^&H.a&JWAU 7 x ----A'(4.4' tjm* t asK-5v-xsaj . w*. --S?7 P P k'7x-- -- 7V) 7i>7'/--'J *5f20jtl3aiifiil?.i?i4I!iSli PCB G?igS2L A^o'J K^CltST-S&HBGT^^Gfct,^. *ts4 4*cicimt&1, &ifitif.&ij&iAtails as^ifct:* 7 x5 7 x(Analabs 'fiW&rtt-'sletZCt&umu. 37`Ct?ti UW li;f5*K^fmr&U JRlfitElC*** t?.& $ tile X- tafif 5. Uis KC 400 fc&fflCf i:831HafiiflF-ffl5fefitKii. fit* fc i fihUSU.'SJRjC -fi&IC-ot, tT'IC 7 7 -7Wt 5 mg/ml lti8A$-ttfc&, ^SIK of&m^frsuLte. h t jet a (2) jfiUSiStffiiKtt /UX^-, tA-t? h, 7t>7, -- 7 t!) i !?t? (DttJflSito . jfiiG. AlseverSctcfSfr-U^lfu^^ttKtSffiMfiffi PCB bTttlvT KC 400 *2!/ta,fc. KC * (PBS) T-3iRt:ttLT;S. ftifi;*> 1500 400 K (DMSO) tclOnig/ rpm, 10#fn' PB0S &ftil,'T0.4% (v/v) ifol* ( ) DSVi 032571 1 T 7f' \ STLCOPCB4016533 (PCI!) l 66: 568-573 l~: l'CB (H! Kit!. ii% 9 a-tu oi'T. WPJK JSWBffc 66 (10): 579-584, 1975 579 Polychlorinated Biphenyls0 Toxicity and Nutrition IV PCBs Toxicity and Vitamin A (3) Satoshi Innami, Atsuko Nakamura, Katsushige Kato and Motoyoshi Miyazaki The National Institute of Nutrition, Tokyo 162, Japan Sumiko Nagayama Teikyo College, Tokyo 151, Japan Eiichi Nishide Department of Fisheries, College of Agriculture and Veterinary Medicine, Nihon University, Tokyo 154, Japan The investigations have been made to elucidate the interaction between PCBs toxicity and vitamin A. The authors (Innami, Tojo el al.) have already reported that rats fed for 6 weeks a 0.1 #? PCBs diet supplemented <,.'ith 3,400 1U of vitamin A showed belter growth than those fed a 0.1#! PCBs diet without supplementation. Ho wever, rats given a vitamin A deficient diet with 0.1 % PCBs showed more significant growth retardation than those given a 0.1# PCBs diet without supplementation (Innami, Nakamura et al.). All rats which were fed a vitamin A deficient diet with PCBs had a bad coat of fur and closed their eyes 50#! or completely at the 3rd week of feeding, and some of them died in the last period of the experiment. But such symptoms were not at all seen in the rats given a 0.1#! PCBs diet without supplementation ora vitamin A deficient diet. It was also observed that the vitamin A content in the liver decreased significantly with 0.1#? PCBs administration (Innami, Nakamura et al.). This result was not inconsistent with that of Villcneuvo et. al. who demonstrated the decrease of vitamin A content in the livers of rabbits and fetuses receiving Aroclor 1221 and 1254 for 28 days. Therefore, it seemed that the nature of the symptoms which appeared in the rats fed a vitamin A deficient diet with 0.1# PCBs was strongly possible to be of a vitamin A deficiency itself. However, Tanaka et al. previously described that almost similar symptoms appeared when rats fed a slock diet were dosed 0.1 g of PCBs per kg per day orally for 4 weeks. For such reason, the authors have mentioned in the previous paper (Innami, Nakamura et a!,) that further investigations would be required to clarify the cause of this phenomenon. In the present study, the experiments have been conducted to determine whether these symtoms would be a characteristic phenomenon of PCBs poisoning or based on vitamin A deficiency. . Methods Weanling male rats of the Sprague Dawley strain were housed individually in wire Cages maintained in a laboratory with 12 hours of light and 12 hours of darkness. The temperature in the animal laboratory was controlled near to 22C. The composition of the basal diet was (#?): milk casein 20.0, sucrose 63.0, soybean oil 9.0, mineral mixture 1) The following abbreviation is used: PCBs, poiychlorinated^biphcnyls. C 33 ) DSW 032572 STLCOPCB4016534 680 lnnaml et al. (Harper, A. E.) 4.0, cellulose flour 2.0, vitamin mixture (Harper, A. E.) 0.85, cholinc- HC1 0.15 and vitamin oil 1.0 (one gram of this oil contains 300 1U of vitamin A-acetate, 30 IU of vitamin D, and 10 mg of a-tocophcryl E-acetatc), In the vitamin A deficient group, vitamin A was removed from vitamin oil and vitamin free casein (Wako Pure Chemical Inc.) was directly used without further purification. One hundred mg of PCBs was added to 100 g of the basal diet in substitut ing for an equal part of sucrose, PCBs used in this experiment were purchased from Wako Pure Chemical Jnc., and were a mixture of the isomers, of which tetrachloride was dominant. ... The diets were prepared every two weeks and stored in the refrigerator except feeding time. Six to seven rats of each group were'fed the experimental diets. Food and water were given ad libitum throughout the experiment. Food consumption and body weight were recorded 3 times per week. In the first experiment, rats were fed on the PCBs diets with and without vitamin A for 8 weeks. On the way of feeding, half of the rats receiving a vitamin A deficient diet with PCBs was switched to the diet containing 3,000 1U of vitamin A-acetate per 100z of the PCBs diet after the characteristic symptoms appeared. In the second experiment, after the characteristic symptoms appeared each 100 JU of vitamin A-acetato and 0-carotenc was directly ingested every two days into the stomach of the rats fed a vitamin A deficient diet with PCBs by using a syringe with a round-top needle (9.5 cm In length). The purity of crystal ^-carotene purchased from Merck was found to be 80% by the alumina chromatographic analysis (Tabata et al.). Vitamin A-acetate and /J-caroteno were dissolved in the soybean oil (the Japanese Pharmacopoeia) and the potency was determined every two days. At the ond of experiment the food cups were removed at 7 : 00 a.m. After fasting for 7~8 hours, the rats were sacrificed by decapitation. The liver was quickly removed, weighed and stored in a freezer maintained under --20*C. As early as possible after sacrifice, the liver vitamin A was determined by a colorimetric method using a mixture ofantlmony trichloride and glycerol-1, 3-dichlorohydrfn following saponification in KOH (Fujita and Kimura). . Results and Discussion The first experiment was carried out to further clarify whether the characteristic symptoms which appeared in the rats given a vitamin A deficient diet with PCBs in a comparatively early period of the experiment, as previously reported, would be based upon a vitamin A deficiency or not. Rats wero fed on the PCBs diets with and without Vitamin A for 8 wcoks. As shown In Fig. 1, rats fed a vitamin A deficient diet with PCBs showed more prominent growth retardation than the PCBs group at the 4th week after feeding started. At this time, all rats of this group had a bad coat of fur and eye mucus, and closed their eyes completely as shown in Fig. 2 (A). The results were completely the same as previously reported (Innami, Nakamura et al.). . On the other hand, the growth of the vitamin A deficient group was almost equal to that of the control group until the 4th week after feeding started, and since then the growth of this group tended to slow down slightly as compared with the control group. However, not any eye symptoms in this group have been delected by the end of experiment. Even in the PCBs-fed group, the same was true, but the only difference from the vitamin A deficient group was that two out of 6 rats died at the last week of the experiment. They liad a bad coat of fur and a part of hair was lost. In the group on the vitamin A deficient diet with PCBs one out of 7 rats died at the 4lh C ) . DSW 032573 STLCOPCB4016535 PCBs toxicity and vitamin A set week of feeding, and after (hat the rema ining rats of this group died successively until (he 6th week of feeding. While, it was found that the rats which were switched from a vitamin A deficient diet with PCBs to the diet con taining 3,000 1U of vitamin A-acelate per 100 g of the PCBs diet at the 26th day after feeding started, ns shown in Fig. 1, recovered from their body weight loss rapidly and gained significantly over the PCUs group at the end of experiment. They regained a fine coat of fur, and remarkably, their closed eyes opened and the eye mucus disappeared, as seen in Fig. 2(B). It was also observed in this experiment that the vitamin A content in the liver of rats fed a PCBs diet or a vitamin A deficient diet with PCBs decreased signi ficantly, as shown in Table 1, The vitamin A content in the liver of rats fed 3,000 1U of vitamin A~acetate with 0.1# PCBs was significantly higher titan that of rats fed a vitamin A deficient diet with PCBs. However, the liver vitamin A content of these rats, despite that a large amount of vitamin A was administered, was much Pig. 1. ElTect of vitamin A supplementation on recovery of growth retardation in rats fed a vitamin A deficient diet with PCBs A : Control, B : Vitamin A deficient, C: 0. \% PCBs. D: V.A dcf.40. \ % PCBs, E: 3,000 LU of V.A-acetalc was added to JOOg of a 0.1 % PCBs diet. less than that in the previous experiment (Innami, Nakamura et at.). The reason may be due to the difference of experimental conditions between the present experiment and the previous one. At any rate, this means that if animals receiving PCBs would once Tig. 2. Appearance of characteristic symptoms tn rats received a vitamin A deficient diet with 0. \% PCBs and recovery of these symptoms by vitamin A administration. (A) A rat received a vitamin A deficient diet with 0. \% PCBs for A su-eks. (B) A rat received a diet containing 3,000 IU of vitamin A per 100 g of a 0. PCBs diet for 3 weeks after the characteristic symptoms appeared. ( 35 ) ,. V DSW 032574 STLCOPCB4016536 ) j ) 562 Innami ct al. 'table J. Effects of vitamin A and ^ carotene on weight gain and vitamin A content in liver of rats fed a vitamin A deficient diet n 'n' DO"- Experiment number Group PCBs (7) Body weight gain ..... e____ psiis' Liver weight E 9&" 14.39+1.66 9.47 + 0. 61 Vitamin A content lU/g liver 2. 5710. 51 Exp. 1 V.A def. + PCBs (7) 17 4 8. 10+0. 38 10.510. 26 1.67 + 0.22 V.A def. 1 PCBs--> V.A suppl." H PCBs (7) 137 + 21 17.80 + 2. 10 9.03 + 0. 53 12. 352. 70 V.A def. + PCBs (6) 24+ 3 8. 0310.61 9. 6010. 57 1. 1710. 31 Exp. 2 V.A def. -1- PCBs--* V.A dose" + PCBs (6) 75+20 12.42 + 1.67 9.35 + 0. 48 " ................ 5. 5512. 09 V.A def. -f PCBs-->^-carotene dose" +PCBs (6) 91 23 14. 1411.88 10. 32+1.75 0 Mean + S.F.. 1) (Liver \vcight)+-(Body wcighOXlOO 2) 3,000 IU of vitamin A-acctate was supplemented to 100 g of a 0. 1# PCBs diet on and after the 26th day of feeding. 3) Each 100 1U of vitamin A-acetate and ^-carotene was ingested every two days into tlie stomach of rats fed a vitamin A deficient diet with 0. 1 % PCBs on and after the 33 id day of feeding. Figures in parentheses arc the number of rats used. become deficient in vitamin A, much time may be needed to restore vitamin A in lire liver even though a large amount of vitamin A is supplied, Jn the second experiment, the effectiveness of ^-carotene on the PCBs toxicity in the vitamin A deficient animals was compared with that of vitamin A. Each 100 1U of vitamin A-acctatc and /7-carotene was directly ingested every two days into the stomach of rats fed a vitamin A deficient diet with PCBs after the characteristic symptoms appeared. The results are shown in Fig. 3 and Table 1. All rats fed a vitamin A deficient diet with PCRs died until the 6th week of feeding. However, in the groups which ingested either vitamin A or ^-carotene orally since the 33 rd day of feeding, only one out of 6 rats in each group died within 10 days after the vitamin administration. Thcro were no differences between vitamin A and /?-carotcne in the recovery of reduced body weight and the characteristic symptoms caused by a vitamin A deficient diet with PCBs, However, the recovery of growth by vitamin A or /7-carolenc admin istration was not remarkable as compared with the first experiment. The reasons for this or for the appearance of one dead rat in each ease of this experiment might be due to the delay of time for the ingestion of vitamins and the low level of vitamin administration as compared with the first experiment. Vitamin A content in the liver of rats ingested 100 IU of vitamin A every two days was not so high, but higher than that of rats fed a vitamin A deficient diet with PCBs. On the other hand, vitamin A was not detectable in the liver of rats ingested ^-carotene every two days. This seems to indicate that, as far as the present experimental condtlions concern, the ingestion of 100 IU /?-carotenc every two days is inadequate to store vitamin A in tho liver of rats fed a vitamin A deficient diet with PCBs for few weeks. (3*3 DSW 032575 STLCOPCB4016537 PCBs toxicity and vitamin A At any rate, the above results mean that a large part of the symptoms which appeared in the rats fed a vitamin A deficient diet with PCBs is based on a vitamin A deficiency. In other words, it means that PCBs act to accelerate vitamin A deficiency in the animals or make animals deficient in vitamin A. Thus, the possible explanation on the PCBs toxicity is that the toxic effect, at least a part of it, appears through the occurrence of vitamin A deficiency. However, it is not yet clear whether the symptoms described by Tanaka et al. would also be based upon a vitamin A deficiency caused by subacute toxicity of PCBs or not. Further investigations will be needed about it. 583 Summary The experiment!: have been conducted to determine whether the symptoms which appeared in the rets fed a vitamin A deficient diet with PCBs at an earlier time than expected would be a characteristic phenomenon of PCEs poisoning or based on a vitamin A deficiency. ingestion on recovery of growth retardation in rats fed a vitamin A deficient diet with PCBs A: Control.B: Vitamin A deficient, C : 0. i% PCBs, D : V.A dcf.-l 0. \% PCBs, E: 100 IU of V. A was Ingested, F : 100 IU of ^-carotene was Ingested. Rats fed a vitamin A deficient diet with PCBs showed the characteristic symptoms until the 4 lh week ifter feeding started, and all rats in this group died until the 6th week of the experiment. While, the rats whoso diet was switched from a vitamin A deficient diet with FCBs to the diet containing 3,000 IU of vitamin A-acclate per 100 g of the PCBs diet at the 26 th day of feeding after the characteristic symptoms appeared, restored their body weight rapidly and gained significantly over the PCBs diet group without supplementation at the end of experiment. They regained a fine coat of fur and remarkably, their closed-eyes opened clearly and the eye mucus disappeared, It was also four.d from another experiment that there were no differences between vitamin A and /9-carotenc In the recovery of reduced body weight and the characteristic symptoms caused by a vitamin A deficient diet with PCBs. However, a significant difference was seen in the vitamin A content of the liver between the rats dosed 100 JU of vitamin A and the rats dosed 100 IU of ^-carotene. These results indicate that a large part of the symptoms which appeared in the rats fed a vitamin A deficient diet with PCBs is based on a vitamin A deficiency. Thus, it Is concluded that PCBs act to accelerate vitamin A deficiency in the animals or make animals deficient in vitamin A. Therefore, animais received PCBs require more vita min A than usual. ' References 1) Fujita, A. and Klmura, K.: An Improved mlcromethod for fractional determination of vitamin A alcohol and color and tho changes In both types of. vitamin A in blood plasma, '( 37 > DSW 032576 STLCOPCB4016538 564 lnnaml et at. milk and liver after loading with both types of the vitamin A and R-carotcnc. J. Vitaminol. 6: 6-15, I960. 2) Harper, A. E.: Amino acid balance and imbalance 1. Dietary level of protein and amino acid imbalance. J. Nutr, 68: 405-4IB, 1959. 3) lnnaml, S,, Nakamura, A,, and Nngayama, S.: Polychlorobiphcnyl toxicity and nutrition 11, PC toxicity and vitamin A (2). J. Nutr. Sci. Vitaminol., 20: 363-370, 1974, 4) lnnaml, S., Tojo, Id,, Utsugl, Y., Nakamura,'A. and Nngayama, S.: ECU toxicity and nutrition I. PCB toxicity and vitamin A (I). Jap. J. Nutr. 32: 58-66, 1974 (in Japanese), 5) Tabflta, R.( Baba, H,, Fukuba, H., Takal, Y., and Iwao, H,: The determination methods of vitamins in foods (2) The determination method of ^-carotene. Jap. J. Nutr. 21 : 54-56, 1956 (In Japanese). 6) Tanaka, K., Fujila, S,, Komatsu, F. and Tamura, N.: Experimental subacute poisoning of cblorobiphcnyls, particularly the Influence on the scrum lipids In rats. Fukuoka Acta Med. 60: 544-547, 1969 (in Japanese). 7) Villeneuve, D. C., Grant, D. L,, Phillips, W, E. J., Clark, M, L., and Clegg, D, J.: Effects of PCB administration on microsomal enzyme activity in pregnant rabbits. Bull, Envir. Contam. Toxicol. 6: 120-128, 1971. ' pcb a (3) I Ep Aft ili tt & 7- Iw j& B & A 3S 17 & ill x i 7' B-wmm n m 3S -- . V>p ** ? fc PCB fcatrtf fi l VA (&T V. A) WtiV, CO&ikV V. A'X5.lt h {,<!>' $>ii4 pcb Sprague DawIcy ?f;CDift4)j->P ;?-X i 16 0.1 % pcb mmtmu&w satr v.A*Mffl*i*4f*. Uft*. *6SAainjMill4r'lcA.'?Mih *&NI&j&* X $Vi t l'^fc1$#ftM>'mw.:. * CV&miklt BBIcUlf>!iiUK& SW:** t 4>2lfflt5>U, Z 01 BtOkffl Sr 0.1 9i PCB 3F| lOOg 3.0001U V.A it, ia-usasifiifSTj JfcttJiWi* itt &&'> t: -1J, V. A V ^6'fliimret4 tC. 0.1 % PCB HTTIi7~8i3rjl6^,tJT 6 HUM12 EkEAlibi. C 2 < (rofcWClK teia;vii c &l, Cn^?i=fJtW^*I^S> ft-t>aT>W'l $IJMP- V. A * ix t Mt. U r:. t t. V. 0.1 % PCB DTli 3 ~4 aBKlJm'tmt D, 613 Q * Tit: 01: Lj6>DCj6-'0, M 7? 33 0016 V. A t -*o?-Vfc'cn?n 1001U -f'P2B^cnica:ADf:l-ei;t, 3n I H32^Wi6idT, & Ilf DIM V. A in/li'S'iiH %.U:tCi, PCBfff, V.A^aPCBr.fJlilc V.A V.Ai>fK-liV.A ; Rfms&tbhim. bi'L, p-hvr'sWj-nn-li. smic V.A *Jg: 0.154? PCB S-Afr V.A9KWM tfT-faff D t: ffl fi-K 5dn i M 14, V. A Xi2.!t jLZLW&Zct&iayjt:. T(tDf,, PCB llifo fel6*dDTV.A'r<2tflafi-t2'Z>:&', *514 V.A X5.&iiiic icmwi 11ti&s nsx, PCB tzijpmvu v. a -to'irJ^-p 61 \'Ki. 56 ' A , DSW 032577 STLCOPCB4016539 66(10): 585-588, 1975 585 5E . * m fo* 0 Z5 H m t|> u Influences of Some Agents on the Enzyme-inducing Activity of PCB Yasunori Araki, Tadaslii Hidaka and ' Kiyoshi Tanaka . Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka, Japan The enzyme-inducing activity of PCB was measured pharmacologically with the method of testing hexobarbital sleeping time in young female mice (Araki & Tanaka: Fukuoka Acta Med. 65: 47, 1974). Combination of PCB with a small dose of methyl mercury or clofibrate enhanced the inducing activity, whereas alkyibcnzcnc sulfonate counteracted the activity or PCB. Cholestyramine strongly Inhibited the shortening of hexobarbital sleeping time induced by a mixture of PCB, DDT and BHC, indicating that this anion exchange resin suppressed the intestinal absorption of the mixture. pen it sir * /fctt &TE tuxm im <t> u, myMtRmmwm-vbi). trj:c miBClZt * MCUKXiiftWyZZ nts (Araki & Tanaka). LWMXmZ'c, PCB Id DDT &f;ld bhc tttjju, /Hii-rocDOf/n-p^u^iit; 'irfttlWlftT'.'i (M* m4>). 9iftd 5 <f?AWB5it:iciJt,'>Ttd, pcb to *3w&T,<b{kommvint m ft mu t tiwer $> z. ufitarrn t ipttBtfl] t \ ^ vmjwmn * lithTlf, PCB fctur:. &fcarattirsmi--o\tm? ft MJtsaa clofibrate (>>k 3pri) t PCB fcwWJUfc&W:. - PCB JSJftRttfe WWf Z Hit DT cholestyramine it?6H,$ftt:it (Tanaka & Araki), C. ft in PCB+DDT+BHC 3 iift(fWlkmwux {,imtemt'HiKte zfrt} IE R # & Araki & Tanaka #f7;ICfil:<i\ tflft J5--20 g JSC-fit CF 1 -e 101 f(f t DT, hexobarbital-Na 100 mg/kg REWfrlH ted fcffSSWffflfcaU&Df:. PCB t VXiit/X 0 a -A' 400 CMC TvkttGMtetef'P 9 , fl'J >r Xti PfftlCfoRlLK. 2 H, 7 B, 14 0 ($2l<bft|d`21 Blot) ffitCtfSfit&KifcDT, *t fiWiajic^t zfmmtv* o nk tfl^ldBittifc ?*$$?iL'itSucfiMt'-f;. W-Mi . wiass cs.e.) *> *> 2 nf i,uvm&* &Tit 1,r. DTIddiib^ rptl-'Ct ftjil/TId sodium dodecylbcnzenesulfonate (ABS) zkiSHlc, clofibrate, DDT, BHC $, fric fitlS l Id CMC U ft fc 0 '/srvt&PiZXVK. mm: ( 39 ) <- OSW 032578 STLCOPCB4016540 & sobarbltal-Na |4rKt5i. It PCB i liPJjfi'j IWV>7s3-4!, !;nii rcn oMWf ibralc t PCB 0Ot !>. HMIfflttJ: iipjb/HcMjhtolcfli M ,ka, K.: A simple no-inducing agents 47-57, 1974. B i DDT, ltllC t 68, 1974. W.a|/7,f d-;i'5S liipa ja, K. Rnd Fujlwara, mJ with simultaneous sulfonic acid salt. Hi. jd Salminen, K.: yl mercury on tbc s. Acta phnrmacol. :aVI, Y.: Inhibitory on the intestinal t 65: 53-57, 1974. . (ABS) ftlM /M 1975. It|tr. BftiHJi BRfSrt 66(10): 589--592, 1975 B9 PCB * f- }l7 > [C72i[%tW am- Bwemts) 5k & m T- ill ra bs a. ss. 55 se*ta m * Influences of PCB on the Brain Catecholamine Levels in I?**is Noriko Suenaga, Katsushi Yamada, Tadashi Hidaka and Takeo Fukuda Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka, Japan Norepinephrine (NE) levels in the cerebral cortex, brain stem and cerebellum, and dopamine (DA) level in the brain stem were determined fluoromclrically in rats treated with PCB (Kancclor 400). When J00mg/kg/day of PCB was given to rats for 7 days, NE In the cortex increased and NE in the cerebellum decreased. Methyl mercury chloride in the daily dose of 2~5 mg/kg did not change the NE levels. Combination of PCB with methyl mercury did not potentiate the NE decrease in the cerebellum. In the subacute experiment, in which 20~50mg/kj* of PCB was given twice a week for 7 weeks, a definite and dose-dependent decrease in the cerebellar NE and a slight decrease in the cortical NE were evidenced. PCB OrfWftJUOTMlz-zl'Tlt. tbUi'O (*&). t im "id. ii5as?m 'nAgtfi we fib (JiHM(>). fl4fttt0Waics*&HE5B <fcn5ffij<a`K*e>ft* Osto). uh'Utmm*. t- t HaitEEibiitt <. rswij I'j^osa t nb u t'b oshhm. isn-ko h-viat^Mbb^t,' (# n>. ^Inlli7 y He PCB 3/1/7 5 > b, W KMsffif'MJ fiftt t BTigfttH 'itA. 'vO&Bfc&bT. PCB Ffimf'6BltjJS^t!ia:-r 5f?iU'mm\H>!;bf:<.'iB5. Hcftm PCB b * *DMci00f/Hi!f, $ m(>*(>), 3-I51Sl!l8X{,*to3t)f WVK^ibtlcbf:. kaa E0$u Wistar King A JR-?* f-0$t!fflb/;. PCB Id** ? p -jv400 fc CMC -ejfct&ESffJiftlc. fl=b. ? y-A'TkfJJti methyl mercury chloride |c tS*-* cysteine jbPA.XTiersHX t b, t'-TtHW pen 7 *3ira-^J-A. 31i&&:*Kiia2ig]7i3!Hl Oil 14 |gl) a^bf:. bTId.(-(inclined screen test)fcfjo/:. CttldCEO -j h0-tf, ty, %. tiTi'BfilH'ttA (decapitation convulsion) (i, [Pi L-oxvmt iTi&atLoia ntktn'nAX, ^CDrtir.SiIdti:y 05) 4-Bf51ilbf:. . &fStKXIEl ^{tiJjSJitfSitKifj t.'BKBi, l5aSL5lii5K-littE^J-0 2 QfjtltfDiM 8t6&&f5k'3 BtfticiKzfibf:. ti/iEgftS^tid^ tttfBb. *S*&n. d''43Si'.5>lC'P& nor epinephrine (NE) >e, {SJt-Vli dopamine (DA) irt, Shellenberger f,0 TSlfcttJ: bflii&bfc. < 43 ) 0 DSW 032579 STLCOPCB4016541 pcBr;r& WCtt, 66: WKKMRK 13: 378- 381, PCBOBM 65: 69- P *S V 1C J. z> 3: 622-623, 1CD8ER, N.: imlnc on llic Iroshock find 14 : 579-583, ., Oliver, A. the pathway illar I'urkinje 19, 1973. Y. and Line, , hydrocarbon 31-88, 1975. PEil'l* p k - fcfllCfcl) 62: 157-158, enlc amines: xposure. Life 3 Cordon, J. re for sirnnldopamine and ie brain areas. 71. ' 53MBit 66(1): 593-599, 1975 593 Chlorinated Dibervzofurans in Kanechlors and Rice Oils Used by Patients with Yusho . Junya Nagayama", Yoshito Masuda'5 and Masanori Kuratsune1' Department of Public Health, Faculty of Medicine, Kyushu University, ' Fukuoka, Japan" and Daiichi College of Pharmaceutical Sciences, Fukuoka, Japan1' 11 was reported (hat the causal agent of the epidemic of Yusho which occurred in the Western Japan in 1968 was polychlorinated biphenyls (PCBs)". According to the animal experiments by Vos and his associates marked differences in toxicity were seen among foreign commercial brands of PCBs". They reported that the more toxic brands made in France and West Germany contained 20 and 5 ppm of polychlorinated dibenxofurans (PCDF), respectively, while the less toxic one made in the United States no such contaminants". It seems quite possible that the toxicity of commercial PCBs is greatly affected by their relatively small amount of contaminants such as PCDF""". On the other hand, it has been well known (hat "chick edema factor-', the causal agent of an extensive poisoning of chickens which killed more than a million chicks in the United States in 1957, is polychlorinated dibenzo-p-dioxins (PCDD)"", and that these compounds arc extremely toxic and acnegenic to man, too"". All these facts raise a question whether or not PCDF or PCDD are involved in Yusho in any sense. Thus, it is without doubt needed to investigate the possible contamination by these compounds of Kanechlor-400, a commercial brand of PCBs of Japanese make which is known to have caused Yusho epidemic. In view of the fact that very few studies have been made in this respect, we analyzed various types of Kanechlor and samples of the toxic rice oil used by patients with Yusho for PCDD and PCDF. This paper describes the main results of such analysis. Materials and Methods 1, Materials analyzed 1) Kancchlor-300. 400, 500, and 600: Kanechlor-300 and -500 were those donated Kanegafucbi Chemical Industry Co., Ltd. in 1968 and Kancchlor-400 and -600 were supplied by the Ministry of Health and Welfare in 1972. 2) Rice oil used by patients with Yusho ("Yusho oil"): 3 samples of rice oil used by 3 independent families with Yusho in 1968. These samples had been kept in glass bottles with glass stoppers at room temperature in our laboratory until 1974 when the analysis was made. 2. Chemicals 1) n-Hcxanc, ethanol, water, potassium hydroxide, anhydrous sodium sulphate, and silica gel: Purified as described previously1". 2) Carbon tetrachloride and methylen chloride: Reagent grade, distilled with a fractional distillation column. 3) Activated alumina: Alumina for column-chromatography (about 3C0 mesh), Wako Pure Chemical Industry, Ltd., activated by healing at 150`C for 12 hours before every use. 4) PCDF and PCDD: Synthesized by chlorination of dibenzofurans or by condcn- ( 47 ) * DSW 032580 STLCOPCB4016542 694 Nagayama, Mnsnda & Kuralsunc sation of 2, 4. 5-trichlorophcnoF". PCDF consisted mainly of telra- and pcnta-chloro- dibenzofurans, while PCDD mainly of 2, 3, 7, 8~tctrachlorodibcnzo-p-dioxin. 5) Octachlorodibenzofuran and octachlorodibenzo-p-dioxin: Purchased from Analabs Jnc., U.S.A. ` 3. Apparatus 1) Gas chromatograph (ECD-GC): Beckman GC 72-5 with a detector of electron capture type, using a glass column (4 mm X2 m) containing chromosorb W AW DMCS (100-120 mesh) coated with 2 % SE-30. Column temperature was 200'C and helium was used ns carrier gas, flow rate 30ml/min, 2) Gas chromatograph-mass spectrometer (GC-MS): Type D-100, GC-MS, Japan Electron Optic Laboratory. Column used was similar to the one used for gas chro matography. Ionization energy was 25eV. ( 4, Analytical methods | The alkaline decomposition method described in the official standard analytical j jmethods for PCBs established by the Ministry of Health and Welfare was applied for the isolation of PCBs fraction from samples of Yusho oil (5-I0g). Isolation of PCDF . jand PCDD from the PCBs fraction mentioned above or from Kanechlor samples (0.1- 0.5 g) was made in the following ways. Each material was placed with 20 ml of j n-hexanc onto a column (1X7,5cm) of activated alumina and then eluted with 120ml t of n-hexanc containing 20 % carbon tetrachloride, 10 ml of n-hexane, and then with j 30 ml of n-hexane containing 20 % mcthylcnchloride. PCDF and PCDD were expected to be contained in the final cluatc. If their separation from PCBs was not adequate, ' the above chromatographic fractionation was repeated or another similar column chro matography was tried using a shorter column Of activated alumina (1X2.5 cm) and a half volume of each of the eluents mentioned above. The elualc consisting of n-hcxnnc containing 20 % mcthylcn chloride thus collected was evaporated by a rotary evaporator, then dissolyed in n-hexanc and subjected to ECD-GC and GC-MS analysis. Determination of PCDF and PCDD was made by two methods, namely by mcasur- . ing the gas chromatographic peak heights and by measuring the gas chromatographic ' peak area after perchlorination of these compounds. The former consists of comparison of each of the peaks of gas chromatogram confirmed as those of PCDF or PCDD by GC-MS with the corresponding one of the synthesized PCDF or PCDD used as standard, , assuming that all the PCDF or PCDD have the same peak height sensitivity regardless , of the number of chlorine and the site of substitution by chlorine. The latter consisted | of chlorination by the BMC reagent1" of the evaporation residue of the fraction contain- j ing PCDF and PCDD to yield octachlorodibenzofuran and octachlorodibenzo-p-dioxin, } followed by the gas chromatographic determination of such octachioro-dcrivativcs. For determination of PCBs, the peak height calculation method and the pcrchlor- : Ination method, described in the official analytical methods for PCBs set by the Ministry of Health and Welfare, were used. \ . . Results i> The gas chromatograms of PCDF fractions separated from Kancchlor-400 and -500 arc shown in Fig. 1, while the gas chromatogram of the corresponding fraction from Yusho oil in Fig. 2. Fig. 3 and 4 show the mass-spectra or tetra- and pcnta-chlorodibcnzofurans isolated from these PCDF fractions, together with those of the standard PCDF. Table 1 summerizes tire results of analysis by ECD-GC and GC-MS, indicating that from dichloro to hoptachlorodibenzofurans were contained in the samples analyzed. The Kanechlors designated larger numbers, starting from 300 to 600, contained the DSW 03Z581 STLCOPCB4016543 Cl-dibenzofuran in Yusho oil S95 Retention time (min) Fig. 1. Gas chromatogram of PCDF fraction A. Kanechior-400, B. Kancchlor-500. C. Authentic specimen Fig. 2. Retention time (min) Gas chromatogram of PCDF frac tion. A. Rice oil ("Yusho oit'D, B. Authentic specimen Table 1. Concentration of chlorinated dibenzofurans in Kancchlors and "Yusho oil1' a: calculated from peak heights, b: calculated by perchlorination method. higher chlorinated isomers of dibenzofuran. The concentrations of PCDF in Kancchlor300. -400, -500, and -600 were 1, 18, 4, and 5 ppm by the peak -height calculation method and 1.5, 17, 2.5, and 3 ppm by the pcrchlorinatic^p method, respectively. Thus, C).' _______ . . .............................................................- - ....................... DSW 032582 STLCOPCB4016544 596 Kuratsunc 'A - 275 338 s, i I, ILjjuL___ LiflLi _u_LlL fii 200 250 300 % FJg. 3. GC-MS of tctrachlorodibcnzofuran. A. Kancchlor-400, B. Kicc oil ("Yusho oil"), C. Authentic specimen Fig. 4, GC-MS of pcntnchlorodibcnzofuran. A. Kanechlor-400, B. Rice oil ("Yusho oil"), C. Authentic specimen Table 2. Concentration of PCBs in "Yusho oil" "Yusho oil" Concentration (ppm) ab A 830 870 B 900 920 C 1.030 980 a: calculated from peak heights b: calculated by pcrchlorinatiou method two methods gave similar results, demonstrating that Kancchlor-400 contains a consider ably higher concentration of PCDF than the other types. Three samples of Yusho oil showed similar gas chromatographic patterns, indicat ing that the PCDF contained in the oil consisted mainly of tetra- and penta-chlorodibenzofurans together with a small amount of tri- and hexa-chlorodibenzofurans. Both the peak height calculation method and the pcrchlorinatiou method gave the same figure, 5 ppm of PCDF. Contrary to the above findings in regard to PCDF, PCDD was not detected in any of the materials examined. As shown in Table 2, the samples of Yusho oil contained 800-1,000 ppm of PCBs, and both calculations gave very close figures. In additional experiments, 10 g of a vegetable oil containing 10 mg of Kancchlor-400 was subjected to o series of analytical procedures, the same as those employed in the above analysis, including the alkaline decomposition process maintained for 6 hours. ( 60 ) DSW 032583 STLCOPCB4016545 Cl-dibenzofuran In Yusho oil 597 PCDF found from the oil thus treated was about 0.2^e. just as much as expected from (he PCDF content of 10mg of Kancchlor-400. Similar blank experiments were repented 3 times, yielding practically the same results. Therefore, any possible transformation of PCBs to PCDF during the analytical procedures could be denied. The recovery ratio of 1 fig of PCDF added to 10 g of n vegetable oil was determined to be approximately 90 Discussion Analysis of a trace amount of PCDF and PCDD is generally known to be fairly difficult. .As mentioned, it is not conceivable that the I'CDF found in our analysis were the artificial products due to the-analytical procedures employed. In addition, the recovery ratio was also fairly high. Therefore, we can say with confidence that Kancchlors and "Yusho oil" contain PCDF. It scorns to be noteworthy that the concentrations of PCDF in Kanccldor-400 and in Yusho oil, as determined by the present analysis, are considerably h'ghcr than the figures so far reported. Roach cl ah, found about 1 ppm of PCDF in Kanechlor-dOO'1'. According to Vos, Howes, Space Science Laboratory, University of California, could de monstrate the presence of PCDF in Kancchlor samples and in a sample of "Yusho oil". The figures for the formers have not yet been supplied but the figure for the latter, ap proximately 1 ppm, have been provided usM>. Thus, the informations so far accumu lated do not very precisely coincide with each other, but they agree at least in that both Kancchlors and "Yusho oil" contain PCDF. Since "Yusho oil" which is known to have been contaminated with Kancchlor-400 contained 5 ppni of PCDF and 1,000 ppm of PCBs, the ratio of the concentration of PCDF to that of PC3s is 5: 1,000, much larger than the expected ratio 0 018 : 1,000 which can be calcurated from our figure concerning PCDF concentration in Kanechlor- 400. The. reasons fer this great discrepancy are not well understood, but it shculd be noted that the sample of Kancchlor-400 we analyzed was an "unused" one, while Hie Knncchlor-400 present in "Yusho oil" must be "used" one as heat transfer medium. Very few studios have been made in which the toxicity of PCDF was directly compared with that of PCBs. Araki observed that PCDF was about 170 times slrongcr than Kancchlor-400 in the activity of hepatic enzyme induction151, Nithizumi and Masuda, however, could not observe any marked difference in acncgenicity between PCBs and PCDF when applied to the skin of the ears of rabbits. The ratio of the concentration of PC.3s needed to exert a certain degree of acncgenicity to the corres ponding concentration of PCDF needed to exert the same degree of acncgenicity was shown to be 1: 0.3"'. There are several studies, however, in which "Yusho oil" and Kancchlor-400 were compared in toxicity, lkeda reported that "Yusho oil" was twice or more toxic, when fed to broilers, than expected by the concentration of Kancchlor-400 contained in the 0iji>,T>^ jn feeding experiment with CF | 1 mice, it was shown that both a diet added "Yusho oil" to attain about 110 ppm of PCBs in the diet and a diet containing 500 ppm of Kancchlor-400 exerted an approximately similar toxicity. The former diet, however, seemed to affect nto::c the skin and liver than the latter and the authors suggested a possibility that "Yusho oil" might contain some chlorinated compounds which were more toxic than Kancchlor-4001".' In a feeding experiment with chicken, Goto ei al, observed that a diet containing 5?<?.of "Yusho oil" showed simllar'toxic effects to those induced by feeding a diet containing 400 ppm of Kancchlor-400. if the concentration of PCBs in the "Yusho oil" used could be assumed to be about 2,000 ppm, the concen tration of PCBs in the diet added "Yusho oil" would be about ]00ppm, considerably <. C 51 > . DSW 032584 STLCOPCB4016546 598 Nagayama, Masuda &. Kuratsune lower than that of the diet added Kancchlor-4001*1. Furthermore, there seems to be a great difference in acute toxicity between PCDF and Kanechlors. LD,, of Kancchlor-400, when fed to mice and rats, was 1.1-1.9 g/kg and that of KanechIor-300 0.6g/kg when fed to rabbits1''. PCDF, on the contrary, killed rabbits due to severe liver necrosis after a single oral administration of as little as about 1 mg/kg" Toxicity of PCBs and possibly of PCDF must greatly vary from isomer to isomer. Rational comparison of toxicities of PCBs and PCDF docs not seem to be easy. However, in view of all these past findings described above, PCDF seems to be con siderably more toxic than PCBs. The possible role played by PCDF in Yusho should not be dismissed. Summary In order to answer the question whether or not polychlorinated dibenzofurans (PCDF) and polychlorinated dibenzo-p-dioxins (PCDD) which arc more toxic than PCBs are involved in causation of Yusho, various types of Kanechlor, PCBs of Japanese make, and 3 samples of "Yusho oi!" used by Yusho families were analyzed for these compounds. Kanechlor-300, -400, -500, and -600 were shown to contain 1, 18, 4, and 5 ppm of PCDF, respectively, as calculated by gas chromatographic peak heights and 1.5, 17, 2.5, and 3 ppm of PCDF, as calculated by the perclilorination method. Thus, Kanechlor -400 was most contaminated with PCDF. "Yusho oil" samples also contained about 5 ppm of PCDF, much higher than expected. PCDD, however, was not detected in any materials analyzed. The possible role of PCDF in causation of Yusho was suggested. References J) Tsukamolo, H. et at,: The chemical studies on detection of toxic compounds in the rice bran oils used by the patients of Yusho. Fukuoka acla Med. 60: 496-512, 1969 (In Japanese). 2) Vos, J. G, and Kocnian, J. H.: Comparative toxicologic study with PCBs in chickens with special reference to porphyria, edema formation, liver necrosis and tissue residues. Toxicol. Appl, Pharmacol. 17: 6S6-66B, 1970. . 3) Vos, J. G., Kocman, J. H., Van der Mass, H. L., Ten Noevcr de Brauw, M. C, and dc Vos, R. H.: Identification and toxicological evaluation of chlorinated dibenzofuran and chlor inated naphthalene in two commercial PCBs. Food cosmct. Toxicol, 8: 625-635, 1970. 4) Vos, J. G. and Beems, R. B,: Dermal toxicity studies of technical PCBs and fractions thereof in rabbits. Toxicol. Appl. Pharmacol. 19: 617-633, 1971. 5) Vos. J. G. and Nolcnboom-Ram, E.: Comparative toxlcily sludy of 2,415,2',4',5'-hcxachloroblplicnyl and PCB mixture in rabbits. Toxicol. Appl, Pharmacol. 23; 563-578, 1972. 6) Cantrell, J. S,, Webb, N. C. and Mabis, A. J.: Identification and crystal structure of a hydropcricardiuni producing factor. Chem. Eng, News. 45: 10, 1967. 7) Firestone, D., Flick, D. F.p Rcss, J. and Higginbotham, G. R.: Distribution of chick edema factor in chick tissues. J. Ass. ofiic. Anal. Chcm, 54 : 1293-1298, 1971. 8) Bauer. H., Schulz, K. H. und Spiegclborg, U.: Bcruflichc Vergiflungcn bci der Hcrstcllung von Chlorphenol-Verbindungcn. Arch. Gewcrbcpath. Gcwerbchyg. 18: 538-555, 1961. P) Oliver, R. M.: Toxic efTccts of 2.3.7,8 telrachlorodibcnzo 1.4 dioxin in laboratory workers. Br. J. lnd. Med. 32: 49-53, 1975. 10) Roach, J. A. G. and Pomcrantz, I. H.: The finding of chlorinated dibenzofurans in a Japanese PCB sample. Bull. Environ. Contain. Toxicol. 12: 338-342, 1974. 11) Masuda, Y,, Kagawa, R. and Kuratsunc, M. : Polychlorinated biphenyls in Yusho patients and ordinary persons. Fukuoka Acta Med. 65: 17-24, 1974 (In Japanese). 12) Nishizuml, M., Masuda, Y. and Kuratsunc. M.: Comparison of hyperkeratosis induced by PCBs, PCDF and PCDD application. Fukuoka Acta Med. 66: 600-604, 1965 (In Japanese). ( 52 ) DSW 032585 Viml STLCOPCB4016547 Cl-dibcnzofuran In Yusho oil 599 13) Hulzingcr, O., Safe, S. and Zitko, Y.: Analysis of chlorinated aromatic hydrocarbons by exhaustive chlorination: Qualitative and structural aspects of the pcrchlorodcrivativcs of biphenyl, naphthalene, tcrphcnyl, dibenzofuran, dibenzodioxin and DDK, Intern. J. Environ. Anal. Chem. 2: 95-106, 1972. 14) Vos, J. Q.: From private communication, 1974. 15) Arakl, Y.: Comparison of enzymc-induc.ing activities of chlorinated dibc'zofuran and chlorinated dibenzodioxin. Fukuoka Acta Med. 6J: 61-64, 1974 (In Japanese). 16) National Hygienic Laboratory, Ministry of Health and Welfare: Studies on toxic substances In rice-bran oil, with special reference to organochlorinc compounds. (5) Studies on toxicity of noxious substances. Special studies on prevention, diagnosis and treatment of Yusho.: 74-124, 1969 (In Japanese). 17) Ikeda, Y,: Toxicity of PCU. J. Food llyg, Soc. Japan. 13: 359 367, 1972 (In Japanese). 18) Livestock Hygiene Experiment Station, Ministry of Agriculture and Forestry: Studies on toxic substances in rice-bran oil, with special reference to organochlorinc compounds. (5) Studies on toxicity of noxious substances, Special studies on prevention, diagnosis and treatment of Yusho.: 74-124, 1969 (In Japanese). 19) Goto, M., Sakaguchi, K. and Ogawa, K.: Hydropericardium assay of rice oil which caused Yusho and of Kanechlor 400 in chickens. Fukuoka Acta Med. 60: 533-538, 1969 (In Japanese). 20) Hofmann, H, Th.: Neuere Erfahrungen mil hochtoxischen Chlorkohlenwasserstoflen Arch, exp. Pathol. Pharmakol, 232: 228-230, 1958. t> * y n ~ )\> fe J; U V A * * < JV VOiU.it V W 7 v V . & 0j gs i0 ra m a" ta m i> 7)9 (PCDF) hi (PCDD) K K i K >, *4.^0- J; Ckillifit'flJl^fiiJii 5 -f x f) 4 4 4-H PCDF *3j;Ck PCDD <0})U, r>tz. t **$>o-* 300, 400, 500 teitk 600 * ft'Uift: J: ) , x 1 , 18, Ail XV 5 ppm , ifClfcJ'oMfcHilCi *). 1.5, 17, 2.5 ti IV 3ppm PCDF 4-fcfcmi'f:. ***0-* 400 PCDF mb5>4HrtA-7>f:. ifcfcft&te&t&te/ll? 4 5 ppm PCDF iu'lill! 3 ft /:. Dj&>D PCDD lit,->-f<lt):H:*.f> t(J;tl|$nfii>o tc. 5 4^44MP PCB Bt&ti 800~ 1,000 ppmT'A> I) , 000--3.000 ppm |c< ti'tt bOU'lWOS-Jtz. PCDF Hi&??li,lio|L t, ftltfiK.lA^iClI/IJ 5 d x* -f *rp PCB > * 4- > o -*400 t> sm^t,> t t, x) h ,c PCDF JbJftiJiEl c* >2^ IQLJ u -C t,' ^ T.J fjl?(i; * ; hi. C 53 ) 4. DSW 032586 > STLCOPCB4016548 600 larao;:;: 66(10): coo-toe 197 Polychlorinated Biphenyls, Polychlorinated Dibenzofuran Polychlorinated Dibenzodioxin 00 rn tl- ^ 15 & is E & 1ft m & a Comparison of Hyperkeratosis Induced by PCBs, PCDF and PCDD Application ' Masahho NismzuMi and Masanori Kuratsune Department of Public Health, Faculty of Medicine, . Kyushu University, Fukuoka Yoshito Masuda ' Daiichi College of Pharmaceutical Sciences, Fukuoka Polychlorinated biphenyls (Kancchlor 400, a commercial preparation), polychlorinated dibenzofuran (a mixture of tclra- and penfa-chloro-dibcnzofurnn) or polychlorinated dibenzodioxin (2, 3,7, 8-telrachloro-dibenzodioxin) was dissolved into acetone in different concentrations, and was applied on the inside or depilated rabbit's ear for consecutive 3 days. The redness on the local skin was followed by thickening of the skin within 1 to 2 weeks. Judging from the extent or hyperkeratosis induced by these three compounds, PCDD Ir approximately 1,000 times stronger than PCBs or PCDF as an acncgcn. Polychlorinated Dibenzofuran (PCDF)fcJ;7/ Polychlorinated Dibenzodioxin (PCDD) (DgUl} ftftffZmf.t bT, IU3J litT'IC 10***". ctu?d> Polychlorinated Biphenyls (PCBs) irM.L-tfc1). PCBs iT-tit t DT PCDF TydSHSKSUiftiH***'" ct &t>. & pcbs b M3 DTO t, fgjs* 5a t> b l. Uti. M)fffi0&5-U: Kanechlor <00 d'lciif&fi PCDF tr'ftlifiiSSStiXV'V' *f, pcdd ?/&*&&? PCDF fct >' PCDD 3/ifcU PCBs i&ictne ka>ik *- s ftmi&i-tttt U tft&^WU:tfl&tfHa;tt588Kfija UlKZlltJttZ 1 ftZtztb, 14 t is ( I ) PCDF &/$ Dibcnrofurao 5g, juifc'Sl 2 (t). 5g 3 t>5fl0. 5g isio' Chloroform 100nil <DiTdttfc? Site. KHUmHiVv'slfymt, ') > a j; t)5Sa-;^ &$&ttz. nmuta^ a-clswiBi, jktSVIimmvb 5 n)|(ijf5-o/:. k J&fiT&f-^'C^^ Chloroform It J; t R&fcfflii-fttfiig tb-otz. u&u wxm 36$-tttzi&, l5 Florisil column V n-llcxane fc-icx Benzene fc/UOTf.'iWt SCtICJ: bf^fe tiUMWbtif: (mP 180--195*). C &, t' OC- MS (05; PMU-6MG) XW&tlt. Fie 1 () 6 Kabt No. 1 2 3 4 5 6 7 8 9 10 It i D6f D (2)2. 2. 4. 5-'l '.<) 5g -T f'dWMvf 'CIU') I 111 Vt<t)i'Jl > I- V o : osw 032587 l l l STLCOPCB4016549 tftr&XrT HWilWt (3;6W)tt4bi0'5 dlbenzodioxin (juft J&U *!.> >+' ijftlfffli 1'CKs (Ka/: 'inmiVUvW naff>c>. i'cbs ic 'uran li |J IfRiR, 1/1000 fflTiaiJR) tt d dibcnzoftiran &J; n touxmmm. ns 1. H, u. Splcgclbcrg, i bri dcr Hcrstcllung tern. Arch. Gower5S-555, 1961. Izek, H.: A technic :y in rabbils, applied , 3. 7.8-tclrnchIorodiJermat. 39: 511-517, : TctrachlorobiRtt. 1131% IS Millilfti: Kancchlors Hftjlt 30: 126, I Pomeranlz, I. H.: dlbcnjofurans in a phenyl sample. Bull. 12: 338-342, 1974. n, J. H., Van Dcr Dr Brau vv, M. C ficatfon and toxico1 nail'd dibenzofuran e In two commercial Fd. Cosnict. Toxicol. mt, R. B.: Dermal ical polychlorinated eof In rabbits. Toxic. 13, J97I. tcnboonvRam, R.: y of 2,4.5,2',4',yI PCB mixture In nacol. 23: 563-J78, If!|59Kit 66(10): 605--609, 1975 pcb PC15 (COOT 60S H 5 I'll . A' & J: *4 A S Polychlorinated Biphenyl Residues in Plasma of Yusho Children Born to Mothers Who Had Consumed Oil Contaminated by PCB Surniko Abe, Yoshito Inoue and Makoto Takamatsu Department of Environmental Health, Ktirumc University School of Medicine, Kuritme, Japan Medical examination and analysis of PCB in plasma were performed on 30 children who were born in the period of February 1967 to December 1973. and on 18 mothers who had taken oil contaminated by PCB in Tamahoura-cho of Nagasaki-kcn. Plasma samples were also obtained from 11 ordinary children in Miiraku-cho. The average PCB concentrations in plasma of Yusho children, mothers and ordinary children were 6.0 ppb, 10.8 ppb and 3.7 ppb, respectively. The average PCB concen tration in plasma of Yusho children who had been fed on mother's milk in infancy was higher than that of children fed on none of mother's milk. Gaschromatograms of PCB in plasma of Yusho children and mothers had a peculiar pattern in most cases. Though the skin symptoms of the Yusho children had subsided, they still complained of atypical symptoms such as easily getting cold, diarrhea, physical dullness and abdo minal pain. - liBSfn 47 ^ 5 B It &*3j5iifilirii{ilim Xffifcvit 4 j&yt u&ih, h *1 Knife i3 n 108 tintftf **(,. PCB itfSJftDfc** < MlfcMItUc 22 Tffi ol->TrrDf-'ifnSJ PCBWttjiiWcDT, gfWJFU JS&4* Z>ttt> fcGfttTMi'i VI: essLWtffiWi*s)i/3a?irtiiuiyait5>5rt$nict. * v>. BJfifeffc*> 4 /J'JfcltOk'Ttt tl it. ^m#Mait mm'M&v satniii'-p-ci'i. . B) & Jr tk s ti!iffifli?fit-3i'Tii, tmicm 3ififlRNii * M ? * 4(3 WT *ifcK&*> !>. tfc${lUti:'IXWIWIAD bft Tl' ZCDV. Wits 14^ ztX-{!i&rj.M-GK&r} < f5i)i-C $ Tk' *. C COgftlC Jt oT ivtfu 49 tr 1 i {.iffiftft. 7 A It * A-1 iili&VUfttt SOO fcf,y$$.i't 4 Hi1, bvtctKU'Mz. ^^f,ta(u49 Mktr2{It4 5UTfM' PCB M4-4)?rtT4 tV C A-Kd> 3 PilltD/: t> Hs^mPtflttl: . BUK137J 4* T, ftliffim#f/ilK&3!t Z'a 6 11 t, It. M di PCB &tz. 'C DTC^f3lt Jt-pT PCB iciOUaUf: i>%- \ till 4'm2 bf: (3 *> b * i n t:Hi 4'S O' f: V, -c 6>Z itti-atritZCtfiiV&t:. 8lE ^jH3 bt R^ W4'Eli ^ t- UitliJiHrj H'- {* ( 59 ) DSW 032588 STLCOPCB4016550 . 610 tStfJEtt 66C10): 610--616, 1975 lUStth PCB fee.y'IC DDT, BHC tZftbCO ffi ft 3S S BS ft i A B $ *& -T- ft & t& fi ft: in m H ft: rn ft m k>: n m PCB, DDT and BHC Levels in Human Plasma as a Measurement of Tissue Residue Yoshito Inoue, Sumiko Abe and Makoto Takamatsu Department oj Environmental Health, School of Medicine, Kurume University, Kurume Nobuo Aokj and Shosaku Miki Horikawa Hospital, Kyoto Kunisato Fujiwara Kyoto City Institute of Public Health, Kyoto Polychlorinated biphenyls (PCB) in plasma has been used for diagnosis of Yusho (PCB poisoning) since 1974. But it was not obvious that PCB in plasma reflected the total residue in human body. This study was performed to this relation. Pair samples of plasma and adipose tissue were obtained from 33 hospital patients at the time of operation. And the other samples--blood samples (21), subcutaneous fat tissue (2), liver tissue (1). and kidney tissue (1) were obtained from 21 persons. The PCB concentrations in plasma and adipose tissue, and the patterns of gaschroma- tograms were examined. At the same time /5-BHC, pp'-DDE, pp'-DDT were measured. (1) It was observed that PCB, /9-BHC, pp'-DDE in plasma reflected the residual condition of these compounds in adipose tissue respectively. (2) The concetrations of these compounds of the emaciated group were higher than those of non-cmaclated group. . (3) The PCB gaschromatograms patterns varied with the type of PCB exposure. (4) It was found that two cases had very high PCB concentrations in plasma (230 ppb, 92 ppb). . jfod! PCB iGEJSlft It ftWi SUSt fc fc * t ES6t> tx x, 49 tv 3 n i nit t> 1/ e, n, b*>bifo'h PCBicJt SfciMfi, gilMi.Kvl.'XOVlgl&tlXl'iOV&'y X, (U&flK&fflbTOi PCB omit* 35*.* -TT-icfttatiiSjnaaiiiiot'Tii. jfarbE&taiia t Mxiittira** a * 11 *>&> b nxc* * x, MHatcioT, pen MS/jPp (M) DSW 032589 t-r *". rv,,*t'rr7' A i- fid is, '> Uhl (Stc-v t: tb 1. hii JtL'.?;c *33 ft. t 18 ft'JC a c* w*. iitb bft. bft. C* itMi. PCB Oft. (2 tc A! &*$! ft. 2. TkKii f&. i. ffi. X, thru. K-Vsv fi-. ax 296 otj rnl/i du STLCOPCB4016551 : }lliSi:.'ll imKIL TST3/.T-: riW No. : Ji{fcC7 x A<+si?m C.: Chlo ric in liuniRn !0: 432-457, 112 A>: t f nn, W. B., nan pesticide > burden and . Pharmacol. K., Crabtree, gnnochlorhic human blood Jlcallli. 13; ft i h py u [j&ft'i' Pen ftffl&liJfc. 329- . r-: Jfhffi't' PCB n, 1974. , J., Baldwin, Dijk, M. C.: .ontent of huilth. 17; 759, Afi|'|> PCB 31 CWKIt 66(10); 617-619, 1975 617 RNA 5)gISi5rp:i>IC aa ^ e?- eu - an 7n& (3=.tt mmmm) lU iE m ft * & 1<3 m sf as ** # 5E-SR Abnormality of Serum Enzyme in PCB Poisoning Patients ' with Special Reference to Ribonuclease Masayoshi Yamanaka, Kimiliiro Akagi, Nobuyuki Hirao and Koichiro Murai Second Deportment of Internal Medicine, Faculty of Medicine, Kyushu University Fukuoka 8J2, Japan Serological enzyme analysis was performed on 101 sera of patients with PCI! poisoning, including RNase, GOT, LDH and alkaline phosphatase. The means+SD of RNase activity in young (under 40 yr) and old (above 41 yr) patients were 194 + 51 and 220+50, whereas those in each healthy control groun were 142+26 and I6239, re spectively. The enzyme levels in both groups of PCB poisoning were highly significant statistically compared with those of normal subjects (p<0.005). The respective abnor mal elcvaiion of GOT, LDH and alkaline phosphatase was observed in 6.4, 7.3 and 9.0>o of the cases examined. The mean values of abnormal GOT, LDH and alkaline phos phatase activities were 64.8 + 41.3 (mU/ml), 251+41.5 (mU/ml) and 169 + 27.9 (mU/ml), respectively. Electrophoretic analysis of alkaline phosphatase on agar gel indicated a single active band of hepatic origin. Niffismifoiflwsht, got, $$ 6, M RNase *Wfcbt: 101 ftllfl LDH. 7 ju>j 9 & 7 4 x 7 7 2 -M(DJrMWWlit 3 ft TO *5) K : 61 A. : 17 A. : 11 A. 9 A. fBJIlMiK: 3 A) Xbh. fcJJKli j?i-mjEiimraic.tffr{a)fct!ai!M LT, Rjtafcfj-p t&m&Ti. ttKlltt&m^MxTxvjtt,-.". jMih tz. it">. MiffifllffiiBUfKAJSaffiT t MCOFM *B!R Ml RNase fljf&i, Anfinsen f," &*& L. l LT itlfieH- bTt' ALTffifflL/;. SfULTd-xf RNA (Sigma 25. * L-V eittfi8'"1"'. tt) . 50mM b <) xaiiiStSSiSi (pH 8.5) T-3 0 BlBkl&^TSWU O.S&'iTIfKiLTfflWc. RlGli RNA (t RNA RUfSt 1 ml ICtfaJiV 50/d i|)A, 37`C, 30 5) T RNase) ii'KPJ](E:Wt ZWLXT.&ri*, iilifiUU !HH b blzbt. O^mm*?-**?* frjy&u $ wmm got, ldh tSbt/TjUij * 77 7 tf 3&8S4'D.2nil fcftljLTEltibltbtz. 7k tfdlMC. 10 5)(!{JiiKLTiti(,. 2,OOOXgT5 5)Pi)S-i> MfcinA.*:. &, 4Aifi 0.2ml 3 ml LdHAT+ti/JU ?3 3t-fe JUT' 260 nm BDfcffi biz. C -MITT jfaJH RNase |, A#,,,, id 0.45 T'EK-Sfrjlt. RNA M2MNW <9 if i n k .(71 ) i. . DSW 032590 STLCOPCB4016552 620 IGraEtt 66(10): 620-623, 1975 i* * (PCB 4> g) JH # (C & tt 3 tin if h U ^ y is 7 (iff Ml flrttlS) Jfe (fa #Lffl^2ga-rtHaa cff mmrmm lii t{J JE S r|i#rfl 8! ?- (iff iiKeftee) m h & >al ' . Consecutive Six Year Follow-up Study on Serum Triglyceride Levels in Patients with PCB Poisoning Makoto Okumura , First Department of Internal Medicine, Fukuoka University Medical School Masayoshi Yamanaka, Sumiko Nakamuta Second Depatlment of Internal Medicine, Faculty of Medicine, Kyushu University Haruo Uzawa Department of Geriatrics, Institute of Constitutional Medicine, . Kumamoto University, Japan. Scrum triglyceride (TG) concentrations of 40 subjects (14 males and 26 females) with PCil poisoning were determined at least once a year for 6 years successively from the onset of poisoning in 1969. The determination of TG was carried out under the supervision of cooperative triglyceride standardization program, Georgia, U. S. A. Total number of determinations of scrum TG was 219 in 1969, 166 in 1970, 206 in 1972,. 75 in 1973, and 46 in 1974, respectively. ' . In male subjects, the mean values and standard deviations of the results(mg/100 ml) were 15957 in 1969, 16655 in 1970, 169 60 in 1971, 17469 in 1972, 164+68 in 1973, and 160+118 in 1974. No significant change was obesrved in these values for 6 year follow-up study. Contrarily, in female subjects, values obtained In these 6 years were 155+75, 161 + 70. 15580, 15363. 129 + 50, and 1U56, respectively. The differences of mean vaiues between 1973 or 1974 and those of 1969 to 1972 were found tt> be statistically significant. It was suggested that serum TG levels of female subjects with PCB poisoning are decreasing gradually, though those of males still remain in elevated levels through out the term of this study. The mechanism underlying these sex differences is yet to be elucidated. (i&ffct'? ijmijfc-r-etc7ii^i ovwimt t (, tr PCB ijijg) IF&fi* 1'. RSmflT'liSW/)*fi1fPai',,J 4 P 3*F-iraK!RTtitJ iA.lfTlnI Vfi-hixtt'-'t l zwm&ut:". it DSW 032591 624 ft! |SJE It 66(10): 624--628, 19?5 % J'I'I -X ^ PM IM )fi 1$ >& e ^ H # 0 ifn 143 pcb t m r m.t o 4$ Cfeff: &WfemW) ^ ft 3A i$ ffi t A Relation between PCB Level in the Blood and Clinical Symptoms of Yuslio Patients Hiromu Koda ' Department of Dermatology (Director: Prof. H. Urate}, Faculty of Medicine, Kyushu University, Fukuoka, Japan Yoshio Masuda Daiichi College of Pharmaceutical Sciences, Fukuoka Blood PCB levels and clinical symptoms were studied in 72 patients with Yusho who had visited at the outpatient department of Kyushu University during April 1973 1o March 1974. Blood PCB was analysed with gas chromatography. Patterns of PCB on gas chromatogram were classified in three types, type A and B which were peculiar to Yusho patients and type C which showed similar pattern to controls. The mean value of blood PCB levels of 72 patients was 5.94-5 S. D. ppb. The mean + S. D. of PCB levels ,of type A group composed of 43 patients (59.7 %) was 7.24.9 ppb, that of type B group composed of 26 patients (36.1 %) was 4.3 + 3.1 ppb, whereas that of type C group composed of 3 patients (4.2%) was 1.7 + 02 ppb. Clinically, dermatological symptoms were mostly observed in type A group. Type C group hardly showed such symptoms. General signs such as fatigue and headache were noticed on patients with any types of PCB pattern. Same type of PCB pattern in al) of the family members was observed in 15 families out of 21 families. rasi 48 tii 4 a + 9 -jiMimnfciim flJEMffOtfa'h PCB0M*fro-U'**:. ** JlblfeW/iEfll-S-lco^T, 1(11PCB t --itat&zct iiX'tllVVWfti. Kb* PCB assis: cow ft H3fl! 48 ip 4 fi X !> 1 if-ram 9W* &nutz fi!iS?.Et)itU7f72. ttfcitii &*&RUsXim,i,t-9Z (^Wifiiffis) ic-oi'Ti. Mifcbf:'?, 4fK'HH6W3B-r*. (2) 11 PCB ic Efci3Uf:#5x!!iBiiJ!Bfo-J: (3) immh 5><fr7*jtf;Cr>3r<atC-^>t>X(AJtlfI7 ^ TIC XU60-C, *Wtn9>r!). CtTI-Afi5fiB32-f* IttFtbZ. lOgte 10ml <D*-p J -A'isJutfc tgztmik, j Ktim 10ml T-3EHlitiU/, tSffi'PJiSkStft, is ') f>A> 1 g A 7 a AH* #5 6UTS1&U (mr/-*S]3E?ifej&Ili S8M) V%ritz. *5A|i5#SE-30*U!/U:. PCB 0 --A- 500+600 (i:i) tU, ^c'-J'Wofiitci DJECtCf:. C 78 ) OSW 032592 P3fctt; 660 0): 629- 631, 1975 629 Hgfn48^jf, 49*J $Lmnm.&wmwu (fiE: fifiPfelP&S) M iE -- 3 m & (iff: ?'JSB3te&&) 5F0 PH & Alteration in Skin Severity Grading of Yudho in the General Examination in 1973 and 1D74, and Presentation of a New Standard for the Skin Severity of Yusho by Point Count System Masakazu Asahi, Hiromu Koda Department of Dermatology (Director; Prof. H. Urabe) Faculty of Medicine, Kyushu University, Fukuoka, Japan Shoji ToshitanI Department of Dermatology (Director: Prof, S. Toshitam") Faculty of Medicine, Fukuoka University, Fukuoka, Japan , Alteration in skin severity grading index of Yusho was investigated by analysing Ihe results of the general examination of Yusho in 1973 and 1974. In the 1973 exami nation, almost no change was found in the skin severity index and in the 1974 exami nation, this time about half number or the whole patient showed improvement more or less. Some problems in the standard for skin severity grading of . Yusho was discussed and the new tentative standard for the skin severity was presented. This new standard was characterized by its quantitative nature (point count system). MTri^hbT-fT-ie7tmmutz. c jig S0Khlifi&J5&{'fc 5 KfSifibT & tzfrlt^i'X li. *i"t'ic4 Inlitiaf: Mas!t$a&bT&re6>*"~", ^liiilit 48 tfffi, 49 stftcuftsjfc it t v>, . T*ifcC, ffifviKiffiCS(3)73^ fr t iz-an 5 Utz& U vHWi liu-ftttfiiiffiiBPWsyi ic^x, * MsiV-ttti 9 tt < i'&*>&z ox, c fti? ym-yuY^'Cswccatt, &i ?ic. m IE5SH-Kl*a 43 t, fc'ii h. if l 0MS Vtxtc 6 ESf jfcfetvX l' * *>\ 47ff-KtP?ftifi $*1 IPcfjofcMe&fcfcoTi' *. C <DtzVjWf&mtt13e4dr-ffiic 1 <i:fPi>'-C c fPiiRiii;?5!2i3lti utzmw&i'l: 9 U1-1ZVS"', !RT>fit VAU Tfe^T'8B&4:5 t BWT-DfJKis''ftf;l<z>xbzid". *UiCitt'd' -roi 5^Si&5Uffi5icliW e.**. JffA:2SiSiX;td ffiTGfct t. 07?-iiic^SJ!et d-:b->*>'mr&fztzVj, HUZbZXb itt^u&&J47ifffi-tT&;j5lt M'f'UX s-tz. 48. 49if&.'i t'iy-'lCljUBT l tfifaiz b **', CGJlb (CJK ( 83 ) U OSW 032593 STLCOPCB4016555 y: -103, li ffi. Jttfc 1 ISWKtfi 6600): 635-639, 197S 63t> ca- ts-wafa)' ffii tu m && m ft n . . tt> ft e I Anterior Pituitary Function of "YUSIIO" Patients (Polychrolinatcd Biphenyls Poisoning) Masahiko Kusuda, Yukihiro Nagata and Masahiko Nakamura Department of Obstetrics and Gynecology (Director: Prof, 1. Tnki') Faculty of Medicine, Kyushu University, Fukuoka 812, Japan Twelve patients, consisting of 9 women and 3 men who had been suffering from "YUSHO" (polychlorinated biphenyls poisoning), were subjected to this study. Anterior pituitary function was examined with regard to basal levels of gonadotro pins and its response to synthetic LH-Relcasing Hormone in total number of 16 times. One hundred meg. of LH-RTI was administered intravc -.ously, blood was drawn just before the injection and 15, 30, 60, and 120 minutes after lhat. Scrum specimens were kept at -- 20"C until assayed. All samples from the same patient were assayed with the same radioimmunoassay system. Criterion on pituitary response to LH-RH was settled as indicated in Table 1. Results obtained were listed in Table 2-B and Table 3-B and these were compared with the control groups (Table 2-A, 3-A). In 10 cases out of 11 patients normal basal levels of LH and FSH were noted, and pituitary response to LH-RH also revealed "good response" In each phases of menstrual cycles, postmenopausal women, a castrated woman, and men, respectively. One patient in which the test was performed in pre ovulatory phase showed rather high basal level and strong response. However, in midfollicular phase it showed normal "good response". Although pituitary response of "YUSHO" patients seems high in normal range, any decisions could not be drawn, because criteria on "abnormally strong response" have not been established as yet. From these results it can be mentiond: anlerior pituitary lobe function concerning with gonadotroph is not deteriorated at least. Consequently, the cause of abnormality of sexual function might be pursued in hypothalamus or peripheral steroid metabolism. jHMKUgut, k, fs&wua 11. tiz-wtar Jdt'TliEdUfitwK- R t, fSfltiHX (.iUfttMb, Mild'-WAT-liffilb t *$. Mi SI.113 T&(MifSni;d F Kfnt'>&>H fi605*` b mit * T1, c ft 4 X- <ffiW 4,tU?;di. t> b U> tb A-7-9&, WT-2&ICOI' T, ^16Mft^ LH-RH (LRF 3Lf:|i Gn- RH) HHijit jt ? THeIt r!)iftifil ft?A'^ fe Fit a L, t Uli. fi fo* Y- 2 (fill We t&MA. 1 3 tgt3K5Ax- i> *. 6amarjiltimiaffcin W&'eVt&ls, m^LTl'^ftilA (B9) DSW 032594 STLCOPCB4016556 640 (SHEUi 66(10): 640--641, 1975 & e a t bb ii vt css 3 m x m ft isi*dfc *r- ?& m* S- Jil . & K - a$ Further Ophthalmic Studies on Patients of Chronic Chlorobiplienyls Poisoning ("Yusho") Yosliilaha Ohnishi, Hiroshi Ikui, Shinzi Kukimoto and Kenichiro Kawashima Department of Ophthalmology, Faculty of Medicine, Kyushu University, Fukuoka, Japan Since 1968 many patients have been registered as chronic chlorobiphenyls poisoning in the western part of Japan. One hundred and forty-seven registered patients (male 67 and female 80) of the disease were re-examined for follow-up study on January, 1975. Hypersecretion of the Meibomian gland and abnormal pigmentation of the con junctiva, which are main ocular signs of the disease, have been improved. Kifn 43 if. 6 Ji mo. $&b/;/ii]tm Kifn504):i /j 27 EJ*r> s nr.IJic^/j 9. K It i h Kift 49 -YS&M OeBlaW m *. > * -vn^tc. *roa tfU:SV?J175 147fcit-p i. n t L-fc I47M5;il&#li-:^fc,ktftt fitlflflifi# 1 ItrjilyfcJS t '0* *. 2. &&0m. sjssitfy]4?Kb. OKffi^sax tii, fcgf&|?dC**i!*.|I3S. ClK!ltttfi3 miztv&fz. L< . 0UldlW5S'Wlt1?:'PT**,!:fc2J; ptttr *. JH8S#`lt rBiWJiS<<f5J, rB5-4iP!*-'til6j Ut'<DJ&mi&l>''3t:. t IzmitfiK Mi, TK^lt BH(c&fe3Kof;,fc ?Ittcb, lt<<//SJ tV'5ITfA.T-*of:. <r <t K fc it 0~I0 t 4 12 * 1. ^i| t8mvS*!t 11--20 2I~30 31--40 41~50 8 7 7 19 10 12 14 19 J8 19 21 38 . 51---60 10 19 61~ 8 2 29 < JO ft 67 80 147 (94) DSW 032595 2 (2 M W. -7 "U t' t'l i- 3. g fife lit i)ma flVtttj! 6h. s< tr3). O) fli'ff <0'A',Zf. lilWK era:-! t>nt ftt5'.:tHt men tit. ~z>k. r. m imsfri STLCOPCB4016557 Vh 642 SSMKIt 660 0): 642--645, 1975 $ Se m # 0 83 fjfr H. fe <fc PCB a ^ (id : ft * PS g Ocular Findings of Chlorobiphenyls Intoxication and Histological Changes of the Palpebral Conjunctiva in Rats Fed with Kaneclor 500 Akihiko Aoki Department of Ophthalmology, Kurume University School of Medicine . Light and electron microscopic observations of the palpebral conjunctiva including the fornix in rats, fed with Kaneclor 500 in the dose of 50 mg/kg 4 times a week, were carried out. Light microscopically, marked lymphocytic and plasmacytic infiltrations were recognized in the submucosal area, and slightly in the Meibomian gland which contained condensed secretion. Electron microscopically, no abnormal findings could be seen in the pigment epithelium and In me visual cells ol the retina the palpebral conjunctiva, an increase of mclanin-Iikc pigment was recognized. LtiMifV.li, -j 4 * * 4 rWDSiffiililcttiffctr? x - - /maxit: X b !iis L-Tin b, V&X 7 4FK&0 OTt'J. iffiWM'fcfiB *- naisfi'-iattc.ooTt^si b, ZCoumWlSi'-tt C). SiidE'dl ( + ). i>tR* (). Kb (-) Umbt:. ttttoztitiittczh:. Ksm i OO ClTfldJ MhWlvm%me>&mW(C{>t-3<<'T, HRifiSLK ot'TflVrfcfcBJ&fiof;. it:--A. 1C ft^irv A* 400 J; I; I'fcftTt-'S * * > p 500 V. KfU 1>T. JtaiJj:o;aaitS'f5of:-CCir i. 4titS2f8tt nafil 49 tp 8 n isX 50 if. l tiit:, , S2&m#tt;&f5'of:K A# NJdiKKfctl:** iWiil&'/tlU&T, Rifii 50tpi fi\t 25 SlC-^TW/rtaffi-*- *. 25 5&At}(i, jlJUfc, * T. g^li, Ctx > 58fVX'bX>t:. mximsitLxu, BUKiti'iisiciffittSf, ?* ^ McfctJ^gSS tk85~105g ?y h7E?tC, #*?p-.i6 500 * 50 mg/kg &J&-C 1 Jld'lt 4 [BJ, KtUKUCKr-liS; i}hmmnx4 sfiTOff&Kisiffi i, t:. pj^ fiSfc&frllMiKfetmU 3tm:if4HV/L HB Jfcfefcfiot;. ifclSfti Luft 1kK$i-z>X'.WM?A tffcftWSU B*5Grf T7SffiT'Si&Lfc. ftlSWilieu. amifAK^,ri'4T(tCf <) we t 11 iMd&mwmt&h A2>. <:&{V -{*- AJBIt {, fits !> *ia*zg, i ftiaatmtc & mei-# ig*t: (HI. 2). WAiei *fol&Tli. mmitiW'lifcTitZtXojy 5 = >ri!jtWasaHtr'lc0fi/l,tt{ri??aLTi'f:. f:ir<fttfelt;iad2~3 nEK-XfpiWS. dense body ) 1 DSW 032596 EHSS c Win. 4). Iii t; t fcfcrh *. Kw< 11: - 'XL- < EKi i li. J Y/bx mm ZA... J; o T : A-6. *L4o. ICJl 6 5 *3' HiWt'1 x, Wir b-bbmm STLCOPCB4016558 CiO teMKife 66(10): 616--648, 1976 jim<r*w#r,i; (ii!s?sa) h-m^mjMi . 'i- &m A Follow-up Study on Fasting Therapy of Yuslio Patients Motoo Imamura Tsushi Clinic, Goshikicho, Tsunagun, Hyogoken Thirty three patients with Yusho, who had sumitted to a fasting therapy more than once, were followed up. Beneficial effects of the therapy were still confirmed in 79 percent of the patients 0.3~4.6 years after leaving hospital. The mechanism responsible for the symptomatic relief resulted from this this therapy was discussed. 1970 <p IO/]$.f, 1975 tr.7;J iTi: 34 J|!| zvimutx-it 2 m WKCW'*', t0W6TOf>hx t972ty-10j|(c tdWihft HiiKiL'WjtftfttStJ tciBCti/lliNU:. AMU 1975 (p 2JJ iT'lcitiKlyr: 33 f.icoi't, iUFAlfSmMtitlE.itft't h/]~4 i\- 8 I'rMf.iabWlr AUUJii *im'i-ftWMfil,, r<)<]Xbimcx`com; 4-U:. 1. W ft l o) au#*. mm. niiti |^ 16-67} fJ:Kll m i< #. `Mi: 19# tQtfeK SWTO 25# KW.l 3 # iKflW 3 # . Mill?;* 2# (2) sAtm 8U 1# 10U 16# I2[) 10# 14U 6# (3) 1M. .24# 2M 8# 3 fcl 1# I ) fi'.lttlttj fciftfl'l 1-211 fi'ijti ii a ft. 2) ftsfiiwo&ti untoTpjfc23 H net KT'fll li%!WX-WI>Ji4-U6> 4. 3) l&ftlll|WWX2HfHJ(d*/:'U 1 11 fit! 500-2000 ml -t-J**. 4 ) KiftUflw 3 UI Id 1; y a - x (5&f5.'W' i ItlfeOWnl-) 4 200 ml -jf-p'rit'l, 'Fffi: 2 ("I 'j A. (IKOs). 5) <4 4), ttifJ, Of?dlu'.^ ^ VStiiL, x5 ~ 6 II B d !> Hitt'l'-rifrW. ^ 200 ml -f-i'SA. * aim. 6) Km20 #, tsm 13 7) fft Wltc'J 44^=63-, *#"3-. filftt 2ClCtlC^OTl><. tffUdlfcl hU'J 1800--2000Cat 8) ^WJ4'a fj. 4flfi) fcMfid-tt*. 9 ) Klfi# 0) 80 % < D t Md, l&ftftjnffl&ii. C 2 OBtc6d7TlWJj'i5, f?r>nt*, 3-4 1BtcttntdSWmWt/j:2>. io) f,ii!itty#id, fcfe3. 4 nc^&T-u&.viMuji *&, ms.tn tdt'i&Jiiiaiffif7id^"t'^-^r hotr>, A$ffll,iSfi>J&MS-f:r-?fc (33#d'3#). 2. . 3. tfcB$!g 1) Srftlftf (&&&!&, SifiiS, wire. *&! (too) OSW 032597 Mi, 3 e 4, 5 It Id (3 I'KiC. 6 iff l'. 2) jfi. ; tty;*' l'<. hri'. 4. . AM - h JtP,b7 A. CO B. 1 CD 1 CD 6i in, tmety. x-um 33 # tUtX-ce ffli&ii' 5J6:( STLCOPCB4016559