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Role of Polychlorinated Dibenzofuran in Yusho (PCB Poisoning) / TAKASHI KASHIMOTO, Ph D. MOEAKt MIYATA, Ph.D. SHINjI KUNITA, Ph.D. Oab Prefectund Institute of Public Health 1-3-49, Nakamtehi, Htpuhineri-ku O--ka. japan TA-CHENG TUNG, M.D., PtuD. SHU-TAO HSU, M.D., Ph.D. KING-jEN CHANG, M.D. SHU-YING TANG, M.D. Department of Biochemistry National Taiwan University Caiepa of Medicine 1 jen-Ai Road Taipei, Taiwan GEN OHI, M.D., Ph.D. |UNICHI NAKAGAWA SHUN-ICHI YAMAMOTO, M.D., Ph.D. Department of Hygiene and Preventive Modtebie Faculty ef Medicine University of Tokyo 7-3-1, Honpo, Bunkyo ku Tokyo 113, Japan ABSTRACT. In the Mood of 15 patients with Yusho or "polychlorinated biphenyl pollerring** that occurred in 1979 In Taiwan, wee found polychlorinated cSberuofurara (14 of 15) and polycMorinoad (folw^nnyb (15 of 15). ao ad a palydilarinalad biphwndt (15 of 15). Tha moon concontntion ratio of diaaa sufatancai ma ajmroximotoly 1:140: 500. hoi on Ho followln, orldonco, w propmo d>a< polyddotlnoaad qutirp6.nyta wow major poihofmic wtioncn In tho dadi,nin of Ymho: (1) aimed monihdadora and count of Ymho padoMl an dhproporttoniioty itwara and panlawnt far dw abwnwd Wood lorofa of polycMorlnotad biphanyli. whilo potfonM who wt occupad anally txpoaod to pun paiycMorinalad biphanyb taka diaracwltdcally mM and bonlpi clinical cooratdaaplM porycmonnaceo ospnenyi leven nan muen ntpitf nan those noted In Yusho petients; (2) Polychlorinated debenzofuram show a markod tendency to accurmriate In the liver, which m&t explain frequent preeenre of jaundtee and other abdominal symptoms in Yusho, which are, apfai, net ebserved In these with occupetional potyefriurinated biphenyl poisoning; (3) Toxicity of polychlorinated <fben- tkMtolbmik. 1MI |Vol. 34 (No. 4)| zofarans is a hundred to ten thousand times greater than that of polychlorinated biphonyIs and palychlorinated quaterphenyls in animal experiments. "YUSHO" is a poisoning characterized clkwcaHy by acnetike dermal lesions and a variety of constitutional tymgtomz. In 1968, an outbreak occurred In waatam parts of Jipamit Archipelago and as a result, more than a thousand persons suffered. The outbreak was initially thou^it to result from polychlorinated biphenyl (PCB) poisoning1'* caused by the consumption of rice oil contaminated with KanecMor 400^ a heat transfer medium.* Subsequently, the "toxic" rice oil was also found to contain elevated levels of potycMovodh benzofurans (PCDFs)*; highly toxic luntawrinirm of PCBas; and polychlorinatad-quaterphenyls (PCQs)* soxkologk ally ill-defined, but substances perhaps rtpiaiy toxic aa PCB*.7** Both PCDFs and PCQs can be gmarated when PCBs are heated.* Tha ratio of PCDFs or PCQs to PCtocan become high during the dcodorization process of rice o9 under high temperature and reduced pressure.1* Taxkoiog- HONS 010623 331 kil contribution of PCDFi In Yusho was further implicated by discovering that the Hnr of Yusho patients who died from other causes after the outbreak of the poisoning con* taifwd fCOFt one thousand times higher than that found m normal populations.11*aA recent analysis of the blood of Yusho patients conducted 11 yr afar the Yusho outbreak, detected PCQs but not PCDFs, suggnting the role of PCQs as a suitable marker for the past episode of exposure to toxic oils.1* During the spring and summer of 1979, cases of peculiar skin disease characterized mainly by acne; cheese-Jike dis charge from the Meibomian gland; pigmentation of the skin, gingiva and the nail beds; and abdominal pains were report ed in two prefectures in the mickle part of Taiwan, Repub lic of China.1* Samples of suspected "toxic" rice oil deliv ered to us in the autumn were found to contain PC0s. Gas chromatographic patterns of these samples were similar to those found in Japanese "toxic" rice oils. All blood sam ples from the patients likewise contained PCBs ranging from 54 to 136 ppb (unpublished data). The number of patients exceeded 1,800 at the end of 1980; a detailed epidemiolog ical and clinical report will be made elsewhere. Of the 15 patients in the present study, PCDFs were found in the blood of aN except for one who had mild clin ical manifestations. In this study strong evidence suggesting a major role of PCDFs in developing clinical manifestations and prognosis which characterize Yusho (not mere "pure PCS" poisoning) Is presented. MITHOOS AND MATERIALS Three samples of "toxic" oil, and 10-mi blood samples were coHetted from 15 patients with various clinical sever ities, who were pupils living in the dormitories on the school campus. Every meal was provided by the school. The pupils were estimated to turn consumed the contaminated oil In the school chow served during a 4-month period, up to 6 months prior to the time of blood sampling. Analytical methods for PCBs, PCQs, end PCDFs. Ten milKilters of Mood were saponified with 20 mi of 2 N KOH ethanol solution for 1 hr under refluxing. After adtfition of 20 ml water, the saponified sample was extracted with 30 ml of m hexane. The *-hexane layer was then washed with 50 ml of water, dried over anhydrous sodhrm sulfate and concentrated to approximately 5 ml in a K.D. concentrator. The n-hexane extract was put on a Florisll column (20 g of 80-100 mesh Florisii, activation at 130*C overnight; 1.8 cm ID) and eluted successively with 130 ml of e-hexane, 50 ml of 2% dtethyi ether In e-hexane, 100 ml of 591 dtethyi ether in e-hexane, 250 ml of 50% dtethyi ether in e-hexane, and 230 ml of acetone at a rate of approximately 2 ml/min. The first and second ehiates were combined, concentrated, and analyzed for PCBs by gas chromatography (GC) on a Varian Aerograph 2100 machine equipped with **Ni-ECO. as described previously.1 Polychlorinated quaterphenyh eluted In the third frac tion ware chlorinated exhaustively with antimony-pent* chloride, cleaned up on an alumina column to remove impurities, and determined by a Shimadzu GC-6A gas chromatograph fitted with NI-ECDl Polychlorinated dibenzofurans were eluted in the fourth and fifth fractions from a Florisii column, and after carefrd evaporation to dryness were subjected to a final dean-up on an aluntina column (20 g alumina, Merck Ca, LtiL, Art Na 1077; 1.5 cm ID). To achieve more efficient removal of impurities, 200 ml of 291 methylene chloride in n hexane was used as the first duate. Polychlorinated dibenzofurana were recovered with 200 ml of 2098 methylene chloride in -hexane. The ehmte was evaporated to drynew and the residue was dissolved inn-hexane and analyzed by GC using a Varian Aerograph 2100 ps chromatograph machine with **Ni-ECD. Gas chromatograph conditions were as follows: column, 2 m X 2 mm glass column pcJted with 291 OV-17 on Gas Chrom Q (100/120 mesh); injection, detection, and column temperatures, 255,320, and 250*C, respectively; carrier gas, Na (30 ml/min). Polychlorinated dibenzofurans were estimated by comparing the peak hei^its of the gas chromatogram with those of FCDF standard, assuming tint PCDF isomers with the same number of chlorine atom aN had the same sensitivity, regartfless of the sites of chlorine substitution {Fig 1). Polychlorinated dibenzofurans In the samples were iden tified (Fig 2) by gaschromotnmasspectrographic (GC-MS) methods, using a j EOL-20KP gas chromaiopaph-)MS-300 mass spectrometer with a computer system. The conditions were as follows: column, 2 m X 2 mm glass column packed with 3% OV-17 on Gas Chrom Q (80/100 mesh); inlet and column temperatures, 300 and 300*C, respectively; ionizing energy, 70eV; acceleration, 3.0 KV, carrier ps, He (Z0 kg /cm*). GOMS Chromatograms at m/z (M4) and (M4 - 63) of PCDF in "toxic" oil in Taiwan (Fig 2)L The PCDF frac tion of the oil was injected into a GC-MS using a JEOL20KP gas chromatograph-JMS-300 mass spectrometer with a computer system. The GC-MS conditions were as follows: column, 2 mX 2 mm glass column packed with 398 OV-17 on Gas Chrom Q (80/100 mesh); inlet and column temper atures, 300 and 300*C, respectively; ionizing energy, 70 eV; acceleration, 3.0 KV; carrier gas, He (Z0 kg/cm*). RESULTS The pi chromatographic patterns of PCDFs (Fig 1) extracted and purified from toxic oil samples in both coun tries show fair resemblance in peeks 10 through 18, mdtal big similarity of constituent isomers between the two. A GC-MS confirmation was also done on each PCDF peak for toxic oil and Taiwanese blood samples. A similar relation ship was likewise present between the blood of e Yusho patient (No. 11) in Taiwan and the liver of a Japanese patient. A GC-MS chromatogaphic analysis of the toxic oil from TaiwdrT indicates that peaks 5 through 14 represent 4-6 chlorinated dibenzofurans with molecular km (M4) and fragmental ion (M4 - 63) resulting from the deletion of COO (Fig 2).* The blood concentrations of PCBs, PCQs, and PCDFs found in patients from Taiwan and Japan, Japanese workers occupationally exposed to PCBs, and control Japanese sub jects are shown in Table 1. Polychlorinated dibenzofurans were detected In ail the Taiwanese patients (6 months after termination of toxic oil ingestion), except for one who had 972 Archives of Environmental Health HONS 010624 n*. 1. Gaa thwwNpwi fCOF (rtrtaa: (A) Mud af THw "Yu*. " padaat (No 111; ()a<l-P-- ~YJ**r. pMtoM; (C| "taala" ad to Taiwan; (D) "wide aM to lao Campaimd a toch part ily to |M III l **; 'J"; w,_* Mtowf br COKISaatoyda; paak No.3, uUacMaraMpfcairl; aaafc Haa. 3-7, tawicMaraatoamaftwao. paafc Waa. . II.and 11 aanlaatoaraOaiatofaa: paa> Maa 11.1.md 16. IwaarlllaraJbaolQftwan. Novantoar/Dacaiwaaf 1911 [Vol. 34 (No. 6)1 HONS 010625 133 m/z (241) m/x(M*) * 12 `` 345 67 8 (min) Fig. 7. GC-MS cbreeMtepsmi a mj* (M4) and (M4 -3)ef PCOF In "toxfc" #U In Tin*. Each flgwe Mmwm ht Mm Iimt mm drumfyin we* tqwl te Mm fmk mnebw In Mm pi ihriwUi pMlfFIpil. the mildest clinical manifestation! and corresponding tow levels of PCBs and PCQi DISCUSSION Although attempts to identify PCDFs in the Wood of Japanese Yusho patients 11 yr after the outbreak failed, PCDF concentrations exceeding those found In the preen* cases, in relation to PCB levels, would have been found shortly after the outbreak. Japanese toxic oils have a greater FCDF: PCB ratio, and roughly 10 times higher contamin ation with both substances than Taiwanasa toxic oils (Table 1). However, a direct comparison between the two out breaks with regard to the intake of toxic substances and severity of poitoning is impossible became only some "toxkM lot samples from the Taiwanese incident are avail able for anatysbt What do the PCDF levels noted In our study siptify? We believe that the following evidence strongly suggests that FCDFs wart mainly responsible In the pathogenesis of Yusho. (1) Those Htdhriduals occupationally exposed to pure PCBs art usually symptom-free; sometimes mild dermal man ifestations such as chforacne accompanied by mild consti tutional symptoms occur.14"14 This occurs despite that Wood concentrations of PCBs In individuals 7 yr after leav ing their workplace are equal to or peatsr by one digit than concentrations observed among Taiwanese patients in the current study (TaWe 1). When occupational exposure to PCBs was terminated, their dermal lesions quickly improved14 in contrast to persistent cfinical signs and symp toms among Yusho patients that have angered more then 10 yr.19 It should be apin emphasised that most Japwmse Yusho patients now have Wood PCB levels that barely exceed those of the gsneral population.19 (2) PCDFs show strong hepatotropfsm,17 as do their analogue*1*--polychlorinated dfoenaodfoxines. Whtit the PCDF to PCB ratfoln the Mood ofTaiwanese patients It approximately 1 : 500, which is similar to that found in Taiwanese toxic oils (l.a., 1: 30(9, the ratio found in til* liver of Japanese patients, who succumbed 1 to 9 yr after the incident, Is 1 : 5--a value far above the ratios noted in the Japanese toxfc oils (1 :100 In average (TaWe l)|.Cfo tally, no abdominal pains nor jaundfce have been dascribad among the three series of workers occupationally expoaed to PCBs,1*** while 45 to 73%of J*mese Yusho patients experienced abdominal palm3* and 11% had jaundice accompanied by other abdominal symptoms." Presence of hepafiotropic and hepatotoxic9 PCDFs In Yiuho can account for this symptomatic discrepancy. (3) Althou^i toxicity of PCDFs varies dapandbig on the Wotopcal parameters (e.g^ chick embryo,9 fiver necro sis,9 thymic atrophy,9*9 animal speciey* etc.) and on the structural difference of isomen,9'9*9 most of these 324 HONS 010626 Archive! of Egtrimwal HssUh ttin-Trtrw--i mmimttmmmH< wi"yii>r 9409*1 VWwfftfnvtoTtfM* 1 VMIptMl*NN NM4W MB 4lapMM*hw) M IS M II 17 4 Kllwl <** 4} 94 49 99 $4 49 M 4 M 4 M9 H 7* ST Hilll Ul 4.4 njtai zsrztjsf t 1 14944 1792 m *--4**--fc|M-- s 5 3 HwCOi ... 1M--" Tdl a4 4>T4tm 4 > > , 7 Uil.l au 114.4 49.4 1397 71* nJ 114.4 49,999 4 12.499 UMNtWM rcgtMi WM l*J 34J 344 17J M 1IJ I1J 9.9 14 1U 49.9 1M 34.4 IU I9.J * IMl 34*2.9 1U>. -- H9.I 7J 41.7 27.9 99.7 4 444 - ITJNtlJM 09299 147,009 7CK NnMfty hnw, m h. Ti4a KB* (*** 9071 9.14 9991 911 BMC 919 917 HA.* 9999 9949 927 9991 9099 99S7 91449971 MAt - - - 997 92 91 2S.7 J7.9 141 * 37 SJS942J1B KS:K9:IW 199 s >9 1914 149:44 > 92* 199:94:921 !: >9:912 1: I:9I2 149 i M 19*9 149:91:917 M9.*39:<9>2 199:14: 914 199:24:911 199: S4: 914 199:79:927 199:91:911 N9: 49:919 199:39:914 199:34: <917 199: < 999: -- 199: < 1 : -- 199: M: 29 199 : 49 > S 199: |9:9 199:99:29 MB: -- : 992 199:39:902 MB: 147:929 | i tMNkAMtMMt W99944r/P*C4iw4f >9tl fvf. 36 {No. 6)] HONS 010627 32S tosiroio^nl comporiw win mode using the PCDF and FCB MWgtet wftfch dbuctfy yrivod "froth" and "pure" frammmCKtwn UtffiiiHg PCDFs end FCBs that were either reproducerf widar tin ori^nat conditions (ij^ temper aturo and coexisting Mr) at tin rice ofl production plant, or purified from the "tosk oR"~the causative agent of Japan ese Yuiho--we observed that the toxicity of PCDFs was hundreds to ten thousand dims pater than PCB toxicity in monkey/ mouse,7* and rat7 In addhfon to hepatotropism of PCDFs, the Mood PCOF: PCB ratio In the current study alerts one to the toxic contribution of PCDFs in Yusho (4) Aithou0i the toxfcofo^c role of PCQs has been barely defined in Yusho, the present study shows that the toxicity of PCQs Is almost equal to that of PCBs.7** There fore, the Mood levels of PCQs observed among Taiwanese Yusho patients suggest that these substances were relatively insignificant in toxicological contribution. Perhaps PCQs are more important in enabling one to differentiate whether the patient has had a past exposure to "Yusho-type" chlor inated hydrocarbons (ij., PCBc PCQs, and PCDFs) or to only PCBs.1* Submitted for publication Augwt a, 1991; accepted for peM- cadon Sspte-bw 13, 1991. ReRumte hr reprints dioeH be smt te: Gen ON, M.D., Mt.D., - --------af - mJ nmiih. lltadM. SdMal at MadU-. University of Tokyo, 7-9-1, Kongo, Benkye ke, Tokyo 113, japan. REFERENCES !. TsukamoSo, H.; Mafctowmi, S4 Hbose, R; Keg--a, T.; Fvkumeto, li; Fakumato, 1C; Kerutrant, R; Nlshtou--I, kt; 5hibita, Mj Nejpd, I4 Yaa, Y4 Seweda, 1C; V--ihl--, Rj Tatse--1,1C; OgvH, 1C; Shlieena, R; llano, 1C: KohayasM, R; Yano, T.; lea, A4 Okada, T.; Irageed, 1C; Kop, T4 Te--Ha, Y4 Kop, T4 Yamadi, Y4 MtyaaecM, M.; Sepne, kL; Heri, 1C; Takaddna, 1C; Manake, 1C; Wakimen. Y^and Shim--rl, R1999. The chamlcd Undies on desecdon of tonic compounds te the rteo bran oHe used ty the pedena of Yueho. Ftdmodo Actt X-- (In jepee see) Mt 499-511 2. Tanaha, R 1970. Ad hnc study on prevention, dapwaais and tinta-- of Yueho, Study Report Worn japenaae Apeney of Tschnalepy (1970), in jepeneee 3. Rerelwme,kL; Yo>hlmura, Mateunka, and Ye--p--hi, A. u19p7a1.nrapoid*Men-eieilosqnlFcailias--tudnyaonnaYAi aud--te,.isae-p-e-eah-ae- ne i.ce..--i.e.ua-rr-an- en-du ob- v*y poiyeklerlraMd Mpheoyte. fakes AbeHB mr^ecr 1:119-2B. . lee^ywra,--asooe, r^ ano xumwune, --. it/s. lrwmhm dlteeiRuawi In Kanoekler and Has od uead by patients with 5. Vue, ).C{ Keemen, HJi; Van dar Mem, H.L; ten Mssur da onyis. Food Cisneer Toxkot B: 925-33. 9. Mfyoti, R; Kedil--ale, T.; and Kunita, R 197B. Detection of In Kane--I rice ode which caused "Kanend Ywbe". / Food 70. Sot fo^m 19t394- 7. Hocl, s.; Obana, H.; Kadilmoee, nriiida.T 1979. MW tribune* and bMo0cal effect erPCO,PCT and rCQeofeUk Prot /w Conprser Food Hy (In Jtpeiw) November, 1979: 31 . Hori.S. 19<0. BWeglai effects efPCIs and rehaedpelycMew Inatetf compounds on awo Monkeys. Proe 7nt Jy--pi HknmSoc /ep (In Japanese), 99-99. 9. Mlyata, R; Murakami, Y.; and Kwhiewte, T. 1971. Detorodnm tfon of polychlorinated quaterphenyl (FCQ) In Keneini rise oR that riwad "Yusho** and tnmUptfton on PCQ tor--flow. / Feed MygSoc/tpm 19: 417-24. 10. Mlyata, H., end Kashi--oto, T. 1979. Invesdpdee ee erpne- chlorinated compounds fanwsd in Kanand rtea oN thacaauaad -Yusho". / Food Hyf Soc /qp 20:1-9. 11. Mlyata. H.: KasMmoto, T.;and KuaUa.R 1977. OatecUon and datermhndon of pplythleruRkaniutor-- In nanral henan dw was and Kamnd Yusho./FoodNpgSec/qp It: 29*45. 12. keehlmoto, T.; Mlyata, R; and Xunite, R The peemrae of poSy- cMorlrated quaterphenyls In the desuas of Yeehe lirtteu. Food Cosifwt Toxtcoi (In pram). 13. Chen, F.R; Gaw, JJd.; Wong, C.K.; and Chen, C.J. 19*0. Lewie and Geechramatopaphto pettsrmof pelyrMiilnMiJ Mphewyto In the Mood of patterns of pslyrhtoriiien9 Mphsayh tod- Mood of patients after PCB poisoning In Taiwan, td teidee Conran Toxicol 25: 325-29. 14. HJrayama, R 1979. A study of PO ~~rmlrtlltn enwog tod-- trtai workers. Konttm Mod A--oc (tn |apaneae)41:1-14. 15. Hara, 1.; Hbata. M.; Witenaha. I.; VokwMg, T4 Tikab--I, IR; and NMiManf, N. 1979. Haallh states of Ute wethers oaeupedute- illy expo--d to PCB at a peint --fact-tog fertery.Ano (MW Mtepd fnt Ad MmM (le lapeen) 17:11-31. IS. Ouw, RIC; Stmpeon, F.IC;and Slyad, OiS. 197C Uaaaidhotoh effects of Arodor 1231, a poiychiorinated MphenyS, he m stow 17. Napya--e,).; Maeuria, Y.; and Kmatnene, M. 1977. Oetesedew tlon of polychlorinMed dlbonaofUrane in Uensaaof padente with '*Yueho". Food Cotmot Toxin/ 15:19S-9BL IS. Firestone, O.; FIMe, D.F.; Rees,).; and HMpnbodtem, G.R. 1971. DHtrlhudon of chkfc ode--a factors In chick Umax / AOAC 54:1393-99. - 19. A--hi. 5.; Koda, R; Urabe, R; and ToWtent, s. 1979. Permute toped symptoms of Ywho aReraltoee In this decada. Fatosto Ado ttedko (In japanaw) 70:172-90. 20. YoeMtnura, T.; Mori, R;and Kumteuns,M. 1970. Omi nip 1 im raladondilp batwaan tesdc oR intaka and sy--pte--a in Y--bo padente: Short report./ap/ Nrt (to japeraea) 32: 111. 21. Keratoma, M.; YeeMmura. T.; Mai--aka, |.; and Va--pHL A. 1971. Ywho, a poiooninccaused by rfca oil eonteedneeadwRh pniyrhtorlratid Mphonyb. H5MHA Med Rep 09c 1003-91. 22. Vos, J.G^ and Koeman, j.R 1970. C--per-- todeatep^ study with poiythiorlnated liphsnyb In chtoitera uHll qp--kd rof--nca lo porphyria, ado--a tenwetlaa. Rear aa--usteand UW sue misuse. Toxko! Ap* Fhsr-ecof 17:950-9BL 23. OMsM, S.; Marto, M.; and Fukuto, R197R Ca-peraths tesddty of polychlortaated Mphenyto a Toxko! dppf Phoemocot 43:12-21 24. Moore, jJC; Gupta, %Jt; and Voa, j.G. 1990. TtalrRy of 2,3,7, S tetrachlorodlberaoft Metfenef CootPCM (CPA) 77-90. 25. Moore, JJC; MeTeuell, E.E.; Oelprd, and Mwrto, hLW. 1979. Co--perath* textofty of their hatoprraead dtoenooftwMn in plsu pip, mice and rhesus monkeys. A--mbMKAesdJOl W. Nkhotoon and I. A. Mourn, cdx. pp, 151-193. 29k Saefd, S.; Ozawa, N.; and Yedii-ara, R 1977. f; misill of 2- chtoroand 1,4, B-trirhtorodfteniofVmn and Utelr elhtt on dm growth of --ice and on the Rear --toreee--wi drag --ataboRring enzyme systems of rets. Fukuoka Acte Mcdke (ht japantee) m 99-101 MOMS 0X0626