Document JNbkeEnz6R3qYm9d1e6nbJ8QK

No; 10 J" u./ tCbem. Pbrm. BuH.'l [,. . 11(10)2231-2236(1973),/ '' | !; M I.M <li,, ; ' ' ^ Metabolic Studies on Polychlorinated Biphenyls., II.1'2 3M4et*a6bolic ' Fate of 2,4,3',4'-Tetrachlorobiphenyl in Rats*1 ;1 .. . Hidetoshi Yoshimura, IIiro-aki Yamamoto, and Seitaro Saeki ' i'*nli / j* I '- I yi+*t \ i"hl'.V ; ,1; j.. : |,. I 1 Faculty of Pharmaceutical Sciences, Kyushu University*) 1 l,: '"'i11 (Received March 12, 1673)' " In order to understand the toxic nature of Kanechlor-400 (KC-ion.'a commercial 1.0 fl I )'Ji i!ii . :/< I -/.ift. )y \ preparation of polychlorinated biphenyls) and establish the treatment of the patients of this KCe400 intoxication (so-called Yuiho), metabolic fate of 2,4.3',-T-tetrachlorobipheny! (2,4,3',-T-TCB), a major component o/ KC-400, was investigated using rats. It was found ' that at least four metabolites having phenolic nature were excreted exclusively into the feces together with a large amount of unchanged 2,4,3',-T-TCB. Among these, a major metabolite (M-A,), mp 155--156, ami a minor metabolite (M-A,), mp 92 - 98*, were isolated from the feces and characterised to be monohydroxylated TCB by ultraviolet, infrared, nuclear magnetic resonance, and mass spectral analyses. '1 ' After 2,4,3',4'-TCB was orally administered at a single dole of 25 mg/body to the rat, a little less than one half of the dose was excreted as unchanged 2,4,3',4'-TCB during 12 duys, most of which were eliminated in the first day. The excretion of major metabolite reached maximum at the Bccond day, and total M-A, was accounted for about 10% of 1 dose during 12 days. Both 2,4,3',4'TCB and its major metabolite were still excreted in a small but significant amount on 12th day. ; . , iy ' hi..' rlifl.V- j , In addition to a widespread, contamination of environment . with polychlorinated bi phenyls (PCB), recent outbreaks of serious PCB intoxication in the southwest part of Japan prompted us tq investigate the metabolic fate of PCB, particularly of Kanechlor-400 (KC-400), a commercial PCB preparation of about 48% chlorine content. Because, this PCB intoxica tion was caused by ingestion of the specific lots of Kanemi Rice Oil which was accidentally contaminated with a large amount of KC-400 during the course of.manufacture of the rice bran oil.41 Therefore metabolic study of this KC-400 is very important to understand its toxic nature and also to establish the treatment of the intoxicated patients. . In our previous study11 on the elimination of KC-400 in rats using JH-Iabeled preparation, it was found that the urinary excretion of the radioactivity was very limited (only about 2% of the dose) while about 70% of the dose was excreted into the feces during 4 weeks after oral administration. The radioactivity excreted in the feces was further analyzed to be mostly composed of unchanged KC-400, but in part, of phenolic metabolites.81 More detailed informations on either qualitative or quantitative aspects of metabolites, however, seemed very difficult to obtain unless individual components were utilized for this investigation. Therefore, an attempt was made to isolate major components of KC-400., Four tetrachlorobiphenyls and one pentachlorobiphenyl were then isolated by a preparative gas chromato graphy and identified by ultraviolet (UV) and mass spectrometry and by chemical synthesis.81 Subsequently, a series of metabolic studies on individual isomers was initiated using firstly 1) Part I: II. Yoahimura and H. Yamamoto, Chem. Pharm. Bull. (Tokyo), 21, 1108 (1973), 2) This work was presented at the 4th Symposium on Drug Metabolism and Action, Sendai, Sept. l`J72; i Abstracts of papers, p. 63. . . . 3) j Location: Katakasu, Higashi-ku, Fukuoka. . . . /... . , . 4) H. Tsukamoto, el at., Fukuoka Ada Med,, 60, 403 (1900). ., . , j) H, Yoshimuru, H. Yamamoto, J. Nagai, Y. Yae, H. Uzawa, Y, Ito, A. Notomi, S. Minakami, A. Ito, ' * K. Kato, and H. Tiuji, Fukuoka Ada Med,, 62, 12 (1971). ' 6) S. Sack!, A. Tsutsui, K. Oguri, H. Yoshimura, and M. Hamana, I ukuoka Ada Med,, 62, 20 (1971). ~j-Vi ' \ ^$ OSH 025936 'iZW'L Voi. 21 (jyr3) .rh4,3',4'-tctrachIorobiphcn3'I (3,4,3',4'- TCB), one of the major components of KC-400, and tfie results were communicated shortly.11 The present paper will describe the another metabolic study on PCB isomers in rats using 2.4,3',4'-tetrachlorobiphcriyl (2,4,3',4'-TCB), a most abundant component of KC-400. Method Administration of Compound-------2,4,3',4'-TCB, which was prepared according to the method described in the previous paper,*' wns dissolved in soybean oil to make a 2.5% (w/v) solution. One ml of this solution, which contained 25 nig ol 2,4,.V,4'-TCB, was orally administered into adult male rat of Wistar King strain anesthetized with ether. For isolation of metabolites, repeated doses of 2,4,.V,4/-TCB (25 mg/body) were administered into 2d rats weighing 180--250 g every third day foi 0 days, while for the determination study a single dose was administered into 3 rats weighing about 120 g. Each animal was housed in an individual metabolic cage giving free access to food and water, and the urine and feces were separately collected for 12 days after the first administration. Extraction of Metabolites-------The urine samples described nbovc were adjusted to pH 2.0 with cone. 11C1 and extracted continuously with CHClj for 6 hr. The extract was dried over anhydrous NatSOj and the solvent was evaporated. A part ol the residue obtained was dissolved in MeOH for submitting to thinlayer chromatographic examination. Another part was dissolved in dry pyridine and trimcthylsilylatcd with N,0*bis(trimothy)s>lyl)Acctamido for gas chromatographic analysis. To the urine remained after CHCI, extraction was added cone. HC1 to make 10% HCl concentration, sind it was heated (or 1 hr on a boiling water bath. The hydrolyzed urine was extracted continuously with CilCl., and treated same as above. The feces, after dried in a dedicator (PjO,) and powdered with mortar, were extracted with CHCI, by Soxhlet extractor for W hr. The feces remained after CHCI, extraction was heated with 10% HCl for 1 hr on a boiling water bath and this hydrolyzed sample was continuously extracted with CHCI, lor 6 hr. ThinInver mui gas chromatographic samples of both extracts from unhydro/yzed and hydrolyzed feces were pre pared similarly as described in tire urinary extracts. Thin-Layer Chromatography (TLC)-------TLC was carried out using silica gel plates (Wakogcl B-5UA containing fluorescent indicators, 0.25 min thick, activated at 105 for 30 min). Solvent systems used were (A) hi'xnnc-CHCl, (1:2), (B) hcxane-CHC',-MeOH-2N% NH4OH (H: 28: 0:1) ami (C) hexanc-AcOF.tAcOll (40: 10: I). 2.4,3\4'-T('U and its metabolites were visualized ns fluorescent spots by ultraviolet lamp (Manasulu Light, short wavelength). Phenolic metabolite* were also revealed os blue or red spots by spraying with Folm-Ciocnltcu reagent or diazotized benzidine reagent, respectively* Gus-Llquld Chromatography (GLC)-------The instrument used was a Shimadzu OC-3AK gns chromato graph equipped with electron capture detector. The column wns a glass spiral tube (4 mm *2.5 in) and the velum.j packing whs 1,5% bL-30 on Chromosorb W (GO--mesh). The column temperature was main tained at 200. Nitrogen was used as a carrier gas with the flow rate of 00 tul/min (1.5 kg/ctn3}. Result Detection of Metabolites in the Urine The CHCI3 extracts of urine samples collected every day for 12 days after administration of 2,1,3',-V-TCB were submitted to TLC and GLC examinations according to the procedure described in Method, and the chromatograms were compared with those of control urine sample which was collected before administration of the compound. No difference, however, could be observed between chromatograms of test and control. In addition, no evidence was obtained for the excretion of conjugated metabolites by examinations on hydrolyzed urine samples. Detection of Metabolites in the Feces The CHCIg extracts of the feces collected every day for 12 days after administration of 2,l,3',4'-TCB were examined by TLC and GLC, comparing with those of control feces. The result indicated that these extracts contained at least 4 metabolites along with unchanged 2.4,'t',4'-TCB. A typical thin-layer chromatogram of the extract of the third day feces after the administration is shown in l;ig. 1. No. 10 As (- n only del. ' tc these, M v benzidine re CXpOMlI* 4 lTom f e 2,4,:ivi re: o,u C c<onrll' \ S e XUT.l ^ 2. 4. 4TU4 Fig. I- ' ill. Thu I'..:' C of it' I HIV in I i". 2.1,:;'. :'-T the | 1- jienl. ' M ' UK'llt. i:. bnllle- .0 '( liyell 1 , c Isokili m ; V :i',i'-t . volt],ill 1 : -i ' by C.i. rhi.it nii\l 1 coni,11. SV'-lc thin lnl' bolil. STLCOPCB4009892 No. 10 2233 As can be seen in Tig. 1, two spots having RJ values of 0.32 (M-a) and 0.60 (M-b) were only detected in the test extract with the solvent system of hexane-CHCl, (1:2). Among these, M-a was revealed as a blue or a red spot with Folin-Ciocalteu reagent or diazotized benzidine reagent, respectively, while M-b was detected as a red-violet fluorecent spot under exposure of ultraviolet lamp showing the same RJ value as that of unchanged compound. From these facts, M-a and M-b were assumed to be phenolic metabolites and unchanged 2,4,3',4'-TCB, respectively. . 0.0 5.0 111 extract CD S> ^ Mo-b 1 cunt ml extract 2. 4. 3'. 4 TCI) 1.017?/) Fig. 1. Thin-Layer Chromatogram of Extract of the Third Day Feces and 2,4,3',-T-TCI3 , *oivcnt iyi(cm: lrexim.-CHCJ, (1: 2} i; positive tu Polin-Ciocaiu-u rcap.-nt tad UV ltinp Q: poilllve to UV shoo 1 80 t 60 = * ZkJ 20 z oP <= 0 20 25 /u (min) Fig, 2-A. Gas Chromatogram o( Extract o( the Third Day Fccea --- : test ----: control -too tSv/i. 68.00. i. 40 20 az 00_____5__ __1_0_____15_____2_0____25 f i, (min) Fig. 2-B. Gas Chromatogram ol Triinetliylsilyiated Extract o( the Third Day I-cccs ------ : lest -- control Gas chromatograms of the extract of the third day feces, same as described above and of its trimethylsilylated product are shown in Fig. 2-A and 2-B, respectively. As indicated in Fig. 2-A, M-B was detected at tR 5.5 min, by which it was also identified to be unchanged 2,4,.i',4'-TCB, and M-A was revealed as a broad peak at t,, 11.0 min. As shown in Fig. 2-B, the peak of M-B remained unchanged after trimethylsilylation of the extract, while a broad ]K.ak of M-A were separated into 4 sharp peaks of M-A,, M-A,, M-A, and M-A, on this treat ment. From these results it was concluded that M-A should be a mixture of phenolic meta bolites, among of which M-A* was a major one, and M-B should be unchanged compound. Possible excretion of conjugated metabolites in the feces was negated by TI.C and GLC of hydrolyzed fecal samples. Isolation and Purification of Metabolites in the Feces The CHO, extract of 12 days feces of 20 rats administered with repeated doses of 2,4,3',4'-TCB was dissolved in hexane and chromatographed through a column packed with 10 volumes of silisic acid (Mallinckrodt, 100 niesli), using hexane and hexanc-CHCl, (1: 1 and ]: 2) as effluent solvents. Elution of unchanged compound and its metabolites were examined by GLC. As the result, M-B was isolated as colorless needles, nip 123--124", from the hexane cluate after recrystallization from MeOH and the metabolites (M-A) were obtained as oily mixture from the subsequent fractions with hexane-CHCl, (1: 1 and 1:2). The fractions containing metabolites were combined and further purified by preparative TLC using a solvent system of hcxane-CIICl, (1: 2). By this procedure, M-A, (RJ0AQ) was separated from other three metabolites (M-A,., M-A, and M-A4) showing Rf 0.32 and isolated as colorless needles, mp 32--08, after rccrystallization from MeOR-LLO (2:1). Separation of a major meta bolite, M-A,, irom M-A, and M-A, was finally conducted by preparative TLC using solvent DSW 025938 Vol, 21 (!!>7;i) system of hexane-CHCl,-MeOH-28% NH,OH (8:28:6: 1). M-A, located at Rf 0.60 was thus isolated as crystals which were recrystallized from Me0H-H,0 (2: 1) to colorless needles, nip 156--166. However, two minor metabolites, M-A, (Rf 0.32) and M-A, (Rf 0.38), were failed to obtain as crystalline forms because of their very limited amounts. Characterization of Metabolites M-B, mp 123--124, has the same melting point and also the same Rf and lR values in TLC and GLC as those of unchanged 2,4,:j',4'-TCB. The complete identy of M-B with 2,4,- 3',4'-TCB was further proved by comparison of various spectra of UV 263 mp.), IR (vg; cm-1: 1587,1400,1360,1022,890,808,834,806,778,726) and mass spectrum [mje: 290(M+), 292 (M + 2)]. In the mass spectrum, M + 2 peak showed approximately 130% of the intensity of the parent ion peak, and this is a characteristic of teri-addcro-compound.11 M-A,, nip 02--98, colored blue with Folin-Ciocalteu reagent being suggested to be a phenolic metabolite. The mass spectrum of M-A, showed M+ and M+2 ion peaks at mje 306 and 308, respectively, in which M+2 peak was about 130% of the intensity of M+ peak. These facts indicated that a single oxygen atom was introduced to the parent 2,4,3',4'-TCB molecule [mje: 290 (M+), 292 (M+2)] to form nionohydroxylated 2,4,3',4'-TCB. This as sumption was further supported by existence of a sharp band around 3500 cm-1 due to hydroxyl group in the infrared (IR) absorption spectrum (Fig. 3) and also by occurence of bathochromic shift with a change from neutral to basic condition in the UV absorption spectra (UV 7."?: 263, 290 m(i; M0H: 320 m(x). *' < i i- A major metabolite, M-A, nip 155--160, was also found to be a monohydroxy-metabolite same as M-A, by its spectral characteristic of mass [mje: 306 (M+), 308 (M + 2)], IR (vg;: 3500 cm-1, see Fig. 4) and UV : 255 (shouldeT), 300 mp; A^I*,0K: 325 mp). The] ocation ot hydroxyl group, however, could not be determined by these spectral analyses and decided by synthetic works which will be described in the following paper,1 Excretion Rate of 2,4,3',4-TCB and Its Major Metabolite, M-A, Excretion rate of unchanged compound and M-A, in the rat feces was determined by GLC during a period of 12 days after oral administration ot 2,413',4'-TCB at a single dose of 26 mg/body. The results are summarized in Table I. A little less than one half of the dose was excreted as unchanged compound during 12 days, most of which were eliminated in the first day. On the other hand, the excretion of M-A, in the feces readied maximum at the second day, and total M-A, was accounted for about 10% of the dose during 12 days after the treatment. It is rather surprising that a little 7) U.M. Silverstcin and G.C. Bassler, "Spectrometric Identification of Organic Compounds," 2nd ed., John Wiley and Sons, Inc., New York, JUG7, p. 29. 8) H. Yamamoto and H. Yoshimwa, Chcm. Phartn. Bull. (Tokyo), 21, 2237 (1973). DSW 025939 STLCOPCB4009894 .i i p,. iu v n c/> oo O O DO oU 0 CO 00 CO 01 No. 10 Tabt.e I. Excretion Rate of Unchanged 2,4,3',4'-TCB and M-A, in the Kat Feces Days after adminiKt ration Excretion rate (% of dose) Unchanged M-A, i 2 3 4 5--fi 7--8 9-12 Total 37.8 1.8 0.4 0.4 0.8 0.8 1.3 43.3 0.9 2.5 1.7 1.3 0.7 1.7 1.8 10.6 Value* in Tab!? represent mran of S rata. 2235 ,'i-3 but significant amount of both unchanged compound and its major metabolite, M-A,, still continued excreting on 12th day after a single oral administration. M Discussion Commercial PCB preparations such as KC-400 are complex mixture containing a large number of chlorobiphenyls and therefore unsuitable for metabolic study. In order to over came this difficulty to obtain general aspects of PCB metabolism by use of KC-400, we have started working on the metabolic fate of 3,4,3',4'-TCB followed by the present study on 2,4,,T,4'-TCB, since both compounds were representative components of KC-400.* By these studies, orally administered tetrachlorobiplienyls were found to be excreted together with their metabolites exclusively in the feces of rats. It was also elucidated that metabolites of these tetrachlorobiplienyls detectable in the feces of rats were all monohydroxylated deri vatives although metabolic rate were seemed different between 2,4,3',4'- and 3,4,3',4'-TCB, since excretion rate of the major metabolite of 2,4,3',4'-TCB was about 10% of the dose during a period of 12 days after the administration, while in 3,4,3',4'-TCB the major metabolite excreted during 14 days was only accounted for about 3.3% of the dose.11 Cl Cl Cl Cl Cl Cl Cl Cl 2,4,3',4'-TCH ' cKI>-0-cl OH monohydroxylated 2,4,3\4'-TCB ci_C^O^1 3,4,3',4'-TCB ci-0-<7>-ci k OH 5* or 2-hydroxy* 3,4,3\4'*TCBl> Fig. 5. Biotransformation of 2,4,3',4'- and 3,4,3',4'-TCB in the Rat These findings were well coincident with results obtained in the previous study using 'H-KC-400 that the radioactivity orally administered to rats was excreted mostly into the feces and only a little part was eliminated in the urine, and this excretion continued for a long period of time.51 The present investigation also provided strong support for the previous assumptions obtained by studies on elimination of PCB from the tissues of mice" and rats10' that all components were not metabolized at the same rate. In general, a major metabolic reaction of aromatic compound has been recognized to be i-W `il 11) H. Yoshimura ami M. Oshima, Fukuoka Ada Med., 62, 5 (1971). 10) D.L. Crant, W.lt.J. Phillips, and D.C. Villcncuve, Bull. F.nvir^.i. Contain. Toxicol., 6. 102 (1971). DSW 0259AQ STLCOPCB4009896 2236 Vol. 21 (1373) hydroxylation. In agreement with this, earlier studies on metabolism of biphenyl1" and 4-elilorobiphcnyl111 in various animals indicated that they were mainly hydroxylated at the para position and then conjugated with glucuronic acid being excreted into the urine. Using gas chromatographic and mass spectrometric techniques Hutzinger, el al.w reported quite recently that 2,5,2',5'-TCB injected iutraperitoncally into rats was largely excreted as un changed in the feres, and only a part was detected as a nronohydroxylated metalrolite in the urine. This result was essentially same as those obtained in the studies using isomeric 3,4, 3',4'- and 2,4,.V,4'-TCB. However, complete difference was observed in the elimination route of the metabolites. In the case of either 3,4,3',4'-or 2,4,3',4'-TCB, monohydroxylated meta bolites were exclusively excreted into rat feces, while a monol^ droxy-metabolitc of 2,5,2',5'- TCB was only eliminated in rat urine. The reason for this difference is uncertain at present. As can be seen in Table I, excretion of unchanged compound was highly concentrated in the first day feces, suggesting that they were probably those unabsorbed from the gastro intestinal tract rather than those excreted through biliary system. In other words, absor ption of 2,4,3',4'-TCB from the gastrointestinal tract was not so good in the dose level of 25 mg/lrody. On the contrary, M-A, was excreted very slowly in rat feces and assumed to lx1 excreted through the bile after metabolized in the liver, the major site of aromatic hydroxy- lation of foreign compounds. Concerning with this assumption Williams, ct reported that for extensil e biliary excretion in the rat the compound or its metabolite should have a molecular weight of 325 50 or more and a polar anionic group. It is, therefore, very probable to assume that the primary metabolites of 2,4,3',4'-TCB might be glucuronidcs which could be hydrolyzed by gut bacteria. However, this possibility was ruled out by careful examination of rat bile.161 . One more tiling to be discussed is that cither unchanged compound or the metabolite was excreted rather constantly from 3 or 4fh day to 12th day after the treatment. The reason for such a little but constant excretion for a long period of time is as yet not at all clear, however, similar results have also been reported on polychlorobcnzcnes by Farke and Williams.111 One of the possible explanations could be provided by our preliminary experi ment,111 in which a significant and constant excretion of 2,4,3',4'-TCB into tiie small intestine of rats from the blood stream could be observed for a long period of time after intraperitoneal or intravenous injection. Acknowledgement This work \v;is supported pnrtinlly by a Grant-inJAid for Scientific Rust-arch provided by the Ministry of Education and also by a research grant provided by the Ministry of Health and Welfare to which the authors are greatly indebted. JJ) H.n. West, J.K. Tawson, I.H, Miller, and G.It. Mathura, Arch. Piochnn, J)iof>hys., 60, 14 (JOjCI: P.J. Cronvcn, D,V. Rarkr, and R.T. Williams, Biochcm. J.t 96, 879 {196!)); P.J. Crcaven and D.V. I'arke, Hiochcm. Pharmacol.^ IS, 7 (1066). 12) W.I>. Block and H.H. Cornish, J. Dial. Chrm., 234, 3301 (1059). l.*|) O. 1 luczingcr, I).M. Nash, S. Safe, A.S.W. DcFrcitas, R.J. Norstrom, D.J. Wildish, and V. Zitko, Scirncc, 178, 312 (1972). 14) M.M. Abou-El-Mnknrcm, I\ MiJlburn, RX. Smith, and R.T. Williams, Diochcm. IQ5, 1280 (1967). lf) )!. Yoshimurs am! H. Yamamoto, unpublished data. 10) D.V. Rarkc and R.T. Williams, Biachem. J74, 5 (1960). . DSW 025941 No. ' fIrni.rm10),22 I (2.1,3 mono \u :*c w.is a vn tai by va h\ dre 'C C'.i' ini fo p. Ill f (W cn ' o) 1 (i.-; l._ Jo- STLCOPCB4009897