Document qa82g2M7KMzq5LXVLoQ2X9enq

;<-\l 1f_0oo) L;:i 1UJ R&S 134660 /1 . Archives of TOXICOLOGY Archiv fur TOXIKOLOGIE Edited tor the Deutscho Pharmnkologische Gesollschaft end ii'io Deutsche Gesollschalt fur Rechlsmcdizin l,';:iiusgegcbon von der Deutschen Pharma.' elngi.-.u-eri Guse'i- h ift Fditr.nal Beard W. D. Erdmann Gottingen (Editor-in-Chicf) W. N. Aldridge Carshalton P. Baum M unz H, Edery Ness Ziona, Israel H, Frohberg Darmstadt L. Golberg Research Triangle Park. N. C. D. Henschler B. Holmstedt Stockholm W. Koransky Marburg a d l.at.n G.A.Neuhaus Berlin H. Remmer Tubingen F. Sakai Tm-m G. Schmidt Heidelberg W. Schwerd Wurzhurn H. Uehieke !!> J. H. Weisburyer Valhalla. N.Y. G. Zbinden Zurich Advisory Board F. Bar .Berlin J, Baumler Basel M. v. Clarmann Munc*:er< Wm, B. Deichmann Coral Gables. Honda R. Gfcdeke Fruibjry i Br G. Heeht Brodttn R. Hess Basel G. M achat a Wu.n W. Masshoff t Grnunrt W. Meior-Ruge Basol H, Oattal LudAiO'-'i.ih-i O. Pribilla Lubeck L. Schlosser. Lubcck Vol. 35 No. 3 1976 Springer-Verlag Berlin Heidelberg New Aren Tot'CGl. -- ATXKAA 30(*J) 1VJ--2J4 I I Arch. Toucoi, 33, 133- 162 (1976) TOXICOLOGY C by Springer VrU 1,7* Original Investigations ii h '0 Disposition of [1,2-I4C] Vinyl Chloride In the Rat H. M. Boit, H. Kappus, A. Buchtcr and W. Bolt Imtitwt (Or Tonikoktpe der Univcnitlt, Tubincen and ImtHut und Potiklirtik fur Arbeit** und $ozi*imcdizin dcr L'mveniUt, tCdln, Federal Republic ofGermany Abstract. Rats were exposed to |1,2-MC| vinyl chloride in a closed system at initial concentrations below 100 ppm. When the system was occupied by 3 rati, a half-life of vinyl chloride in the system's atmosphere of 1.13 0.12 h was observed. The volume of the system was 10.3 L Calculation of the clearance of vinyl chloride from the system revealed that about 40% of inspired vinyl chloride is absorbed by lung. Therefore, changes in respiration did not influence uptake of vinyl chloride. Uptake of vinyl chloride by the rats was completely blocked by acute pretreatment with potent inhibitors of cytochrome-P-450-dependent microsomal dmg metabolism (be., by 35 mg/kg 3-brotnophenyM(5)-imidazolc or $0 mg/kg 6-nitro-l,2J-bcnzothiadiazole in 0.6 ml/kg DMSO). A weaker inhibition was observed after dosing SKF 525 A or 5,6-dimethyl-1.2.3-bemothiadtatole (50 mgAg in 0.6 ml/kg DMSO). Metyraponc did not cause inhibition. Uptake of vinyl chloride was increased by pretreatment with DDT and. to a lesser extent, with clotrimazoL No significant stimulation of uptake was ob served after pretreatment with phenobarbetal, 3-methylcholamhrene, rifampiria, or chronic ethanol treatment Immediately after exposure, highest radioactivity levels were observed In liver and kidney. The radioactive metabolites of ```C-vinyl chloride were rapidly excreted, largely by the kidneys. Excretion of radioactivity in the urine was 69.4 2.6% within 24 h. Key words: VinylchJoride -- 3-Bromophcnyl-4(5}-imidazoie -- 6-Nitro-1.2.3benzothiadiazole -- Inhibitor of vinyl chloride metabolism - Induction of vinyl chloride metabolism. Znsannnenfassung. In einem geschlossenen System wurden Ratten inltlakn Konzcntrationen an |1,2-I4C| Vinylchlorid von unter 100 ppm ausgesetst Bel eincr Besetzung des Systems dutch 3 Ratten wurde in der AtmospMre rine To h'horn offprint requests should be sent: Priv.-Doi. Dr. Dr. Hctmm M. lot. Iiiuu IV Tosikotope der Univeraiti!, Wilhdmttr. 56. D- 7-400 Tdbutten. Federal RepuMw at Oueoumy 0 % t * / 154 H. M. Hud cl *L Halbwcrtszeit des gasformigcn Vinylchlorid von 1,13 0.12 Std gcmessen. Bci eincm Volumen dcs Systems von 10,3 1 ergab die Bercchnung tier Vinylchlorid Clearance, dafl nur ca. 40% des von den Ratten eingeatmeten Vinylchlorid re sorbiert wurde. Aus dicsem Grunde fuhrten Andcrungen der Atcmtltigkeit nicht zu Andcrungen der Aufnahmegeschwindigkai von Vinylchlorid. Durch schr wirksame Inhibitoren von Cytochrom-P-450-abhangigcn mi krosomalen Oxidationcn (3-Bromphcnyl-4(5)-imkiazol und 6-Nitro- l.2.3ben*o thiadiazol) konnte die Aufnahme von Vinylchlorid vollstindig verhindert wer den. SKF 525 A und 5,6-Dimethyl-I,2J-bcnzothiadiazol waren in dieter Min sicht weit weniger wirksam. Durch Vorbehandlung der Ratten mit DDT und. zum gcringeren Made, mit Clotrimazol wurde die Aufnahme von Vinylchlorid gesteigert. Kcine tignifikante Steigerung trat auf nach Vorbehandlung mit Phenobarbetal, 3 Methylehoi anthren. Rifampicin und nach chronischcr Aikoholgabc. Unmitteibar nach Beendigung der Exposition wurden die hdchsten Radioak tivitatswerte in Leber und Niere festgcstcllt. Die Metabolite von **C Vinylchlorid wurden schr schneil ausgeschiedcn. Bercits nach 24 Std wurden im Urin 69,4 * 2,6% der inkorporierten Radioaktivitat gemessen. SeM6*a*hbrtert Vinylchlorid 3-Bromphcnyl-4(5)-imidazoi ~ 6Nitro 1.2.3 benzothiadiazoi -- Induktion dcs Vinylchlorid-Stoffwechscls - Hcmmung det Vinylchiorid-StolTwechsels, Recent reports of angiosarcoma of the liver due to occupational esposure to vinyl cluoride (Creech and Johnson, 1974: Lee and Harry. 1974; Buchtcr and Bolt. 1974; Thomas cl ah, 1975) had Focused attention on metabolism and disposition of thu compound, particularly since it has been suggested (Jaeger ct al.. 1974; Bartsch et aL, 1975) that hcpatotoxicity and carcinogenicity of vinyl chloride may be attributed not to the compound itself, but to a hitherto unknown reactive metabolite. However, due to the gaseous nature of vinyl chloride, metabolic studies require some special techniques. Recently, Hefner ct al. (1975) exposed rats to vinyl chloride in a dosed system and monitored concentration in the atmosphere by infrared absorption. The ob served decline could be described by a logarithmic function. Uptake of vinyl chloride from air by the rats was inhibited by acute high doses of ethanol or pyrazoie. This indicated that uptake was dependent on metabolism; apparently the organism takes up vinyl chloride only to that extent which is metabolized. At concentrations of rinyl chloride above 200 ppm the metabolizing system was saturated and metabolism therefore was much slower than at low atmospheric concentrations of vinyl chloride. In these studies, Hefner et al. (1975) were faced with cooskierable difficulties of minimizing losses of vinyl chloride from their exposure system, apparently due to the solubility of vinyl chloride in rubber and diffusion through rubber membranes. The present report aims at further extending knowledge of (he fate of vinyl chloride in vivo and deals with the inhibition of uptake of vinyl chloride by ipwafk and potent inhibitors of cytochrome-P-450-dependcnt mixed function oxidations. Furthermore, the effect of agents known to induce the cytochrome P 4M) system has been examined. The use of an all-glass exposure system and avoidance of rubber ,kf-*>> ,4 \m, materiel vhhwafc was use metahidnes ta IS cn/ywwv m ttm pkd MUr ^ 33 1 IgUlM K it KH U*i eg 1 CO 4* O) o> N> 1 es k hki 4 1 ** . * i H+ i a* ^S --i Adi 0 rv II i ft v I. i I( 4 I It* h * i4 (NI kg a Ait tv *sJt sd 1 )i *4 <*ti eg* *t akt Ua I n4t I ts-d* 1 V left ** Hi l i Disposition of Vinyl Chloride l.<3 material prevented losses of vinyl chloride from the exposure system. |MC| Vinyl chloride was used which also permitted determination of distribution of radioactive metabolites in the rat organism. In order to avoid saturation of the metabolizing enzymes, in these experiments, concentrations below 100 ppm have been ap plied. Materials aad Methods Materials. I U '*CI Vinyl chloride, specific radioactivity 10.7 mCi/mmoL was synthesized hy the Radiochemical Department of Farbwcrkc Hocchst. Frtflkfurt/Main. Synthesis started from 112 "Cl acetylene. The purity of the product was 94.9%, as checked by gasb<|uid chromatograph) (tiaiaata/) phase: Tenant: carrier fas: He. 70-73 ml/min: temperature 70-130 C: FID). For stora<c. the prrpe ration u diluted with helium (3 pta.) to prevent polymerisation. A sample, also diluted wlh helium, which was stored for 2 months at room temperature, com.lined about 8% oi s-.iyl chlortd: iWignmers. The rest was unchanged vinyl chloride monomer (Farbwerkc Hocchst. personal comtnutMciiainl. In our laboratory, the radioactive preparation was stored in the dark at C and used within I momIt after synthesis. 3-Brontophcnyl-4l3Fimidaeole. 6-nitro-U.3-benzothiadiaaolc. and V6 Jimethyl 1.7.3 htn/iahu diaaolc were kindly donated by Dr. C. F, Wilkinson. Cornell L-niacriity. Ithaca. N.Y. SKF 323 A <2-diethylaminocthyl-2J-diphcnylvaJeratc HClt was obtained from Smith. Klust. A French, Philadelphia. Pa, and metyrapone was obtained from Ciba. Basei.Swtt/erland. Pyrarok was a commercial product of Fluka. Buchs, Switzerland, Pretreatment of Animals. Male Wistar rats (200-230 g) from Ivanmas. Ktttlcgg. AHgau were used for the experiments. Rats were prttreaied with phenoharbitaj by a sinfle i.p. injection of (W mgkg. foltowrd by 3 days cf 0.1Si phcnobarbital as a constituent of the drinkinf water. 3-McthylchuUnthrcne was given as a sinfle Lp. dose of 40 mg/kg in olive oil. 4 days prior to (he experiment. Di (pchlorophmyll.i tn chloroetnane (DDT) was fiven 1 week before the experiment by a single i.p. dose of 200 irf kf in olitr oiL Clotrimazol (Bay b 3097: Cuneaten* of Bayer. I.ocrkuacnl was administered at a dad) up. dm* of 20 mf/kf in olive oil foe 3 days, followed by 2 days without treatment. Rifampscsa (20 mg. kgl was given orally for 3 days; ran were used I day after cevvatum of treatment. Chrome tkam<i treatment of rats was achieved by adding 30% (v/v) ethanol uml 3<rv, i si sucrose to the dnnkusg water for 3 weeks. Treatment ceased I day before the experiment. Exposure of Rais to "C-F/rt.r/ Chloride. Rats were exposed to '(' wntl chloride in a timed all fjau system as shown in Figure I. A desiccator was used as the exposure cage. It contained a tenter R&S 134663 Fig. I. All-glass exposure sys tem. (A) inlet for uC-vinyl i chloride: (B) septum (rubber, j coated with teflon). For deter- I minxlion of concentration of | ,4C-vinyl chloride in system's c ! atmosphere, tempi's of air (2 I mi) were withdrawn by means | of syringe, containing toluene i scintillator (see text). (C) inlet i for oxygen: connected with j spirometer, (D) CO, absor bent. Three rats were placed | in chamber. Volume of system was 10.3 I 136 It. M. Ik* rt J (D) fitted with the carbon dioxide abtorbem (~lndikator-Aientkaik', Drifer. Liibeck). A tfuromc ter. filled with oxjrgcn. w connected with the oxygen inlet tCX. The total volume of the vyvtctn wav 10.3 L The vpirometer uxed foe theie experiments was of type 336 (A. Dazgitx. Hamburg). and aUtiwnl mechanical recording of the consumed fas. When starting an experiment. 3 rats wtre placed in the desiccator and oxygen consumption wav monitored by the iqwromerer. ,4C-Vtnyl chloride was mixed with atmospheric air and the mixture wax injected with a 20*mi syringe into the rat exposure cage via inlet A. Concentration of radioactive vinyl chloride in the air of the system was measured ever) .Ul min. Measurement of RadtoactMty. Because of the solubility of vinyl chloride in toluene, a totume scintillation mixture (0.4% PPQ plus 0.01% POPOP in toluene) could be used for determination i4 **C vinyl chloride. A J-ml syringe was filled with 3 ml of this mixture. Air of the exposure system 11 mil was drawn by this syringe with a long needle through the rubber septum IB). This septum was coaled with teflon to avoid diffusion of vinyl chloride through the rubber. After withdrawal of the in, :Nr contents of the syringe, scintillation mixture and uC-vinyl chloride contaim *g air. were shaken fie 2 mm to dissolve the vinyl chloride, and the scinlillaror wax poured into a scintillation vial with an additional 7 ml of scintillator. The scintillation mixture was counted for radioactivity in a Bertholu III $000 liquid scintillation spectrometer. Determination of RadtoactMty In Tissues and Urine, For determination ot radioactive met*o litei of vinyl chloride in tissues, organs of rats exposed to MC-vinyl chloride were homogenized h 4 vd of water by means of an Ultra-Turrax tissue homogenucr. Losses of nonmciaNiiucd vinyl chtorvdr due to diffusion from the tissue into air could be minimized by immediately sacrificing the rats a"wr withdrawal from the exposure chamber, and by immediate removal of the organs. The Untrihutn* of nonmetabolixcd vinyl chloride in rat (issues after exposure will he reported separately. After homogenization. '/, ml of homogenate, corresponding to 0.1 g of wet tissue, was digevast with 0.$ ml 1 M hydroxide of hyaminc. After achieving solution. 0.3 ml uf preside mixture tsupema ant of 12.5 g commercUl benxoylpcroxide plus JI.3 g CaCl) plus 100 ml toluene) was added, stsvesl overnight at 37. and counted for radioactivity in 10 ml of Bray's solution (Bray. IvMI) For determination of excretion of radioactivity in urine of rats exposed to "C smyt ridoredr. the rats were placed in a metabolism cage after exposure. Urine was counted for radwacusuy m Bray's solution (Bray. I960). Rcxoiti Uptake of >4C-Vinyl Chloride. Before starting the animal studio, control npen menu with the unoccupied chamber showed that no measurable radioactivity was lost from the exposure system (Fig. 2, curve A). When the chamber was occupied by 3 rats, a logarithmic decline of radioactivity in the air of the exposure system was observed (Fig. 2, curve B) with a half-life of 1.13 + 0.12 h (6 determinations, x 1 ,L This agrees with the data of Hefner ct al. (1975) who measured a similar decline by means of infrared spectroscopy. Changes in the uptake of oxygen from the spirometer (Fig. 2. curve C) do not coincide with changes of uptake of vinyl chloride by the animals from the system (Fig. 2, curve B). Inhibilon of Uptake of ,4C~ Vinyl Chiot ide. Hefner et al. (1975) had shown that inhibition of metabolism of vinyl chloride. e.g., by acute high doses of pyrajote or ethanol, results in a corresponding inhibition of uptake of vinyl chloride from air. However, they failed to show inhibition by specific inhibitors of microsomal mixed function oxidases. In their experiments SKF 525 A did not inhibit the uptake of vinyl chloride to a significant extent. Much more potent inhibitors of microsomal oxidative drug metabolism than SKF 525 A have been synthesized recently by Wilkinson and W coworiers (WU IKJWM4WM V-t.ll * lift. L IWshm >4 t . * (k ttAtf |||4 a **I 'll fc (< kizvwwi IV? IgWTxl 4x w* |,\ t hrxiiti Mswda/xiii (1 DO Mt (/> vwyl xMvewlx nwv*4 dt the m w*t) tii semw The x*iN CO 4* _* CD ay ww , 4* it* revre vihxVr ( wl CxmsecvM v 4e. _..*v Keif Mr i*t *")* hLawit m U'*x4 At U pro** DMHOtUtv *A#t by tp Ihr uptake td regyyt <Mtnah (curve Hi Twemy h** h*w x-Mxwsde *w (he vkwwl vy * rxfwrevmem wwh |)MVI Bw hv* Wi mux* Is veer itgwt t Deposition of Vinyl Chloride rtf. 2. Deckn* of ,4C*virty| chloride in closed system due to uptake by 3 ran. (A) control experiment with unoccu pied chamber. (B) chamber occupied by rats. Half-life of vinyl chloride - 1.1 h. (C) oxygen consumption (ml/min) of rau during experiment B. Change* in respiration do not influence uptake of vinyl chloride. \ V *-- a. 137 \ \ \ 1A oy9*n consumption 4n kinson. 1971; Wilkinson and Bruttsten, 1972). These inhibitors were originally tie signed as insecticide synergists. In the present study two benzothiadiazotes (6-nitro 1.2.3-benzothiadiazole; 5,6-dimethyl- 1,2,3-beruothiadiazolc) and one substituted imidazole (3-bromophenyl-4(5)-imidazole) have been studied as to their inhibition of vinyl chloride metabolism. Because these compounds turned out to be insoluble in most of the solvents commonly used for application to animals in vivo it was twees sary to administer litem in DMSO solution. The inhibition of uptake of vinyl chloride by 3-bromophcnyl'4(3MniidazoJe and its reversibility is shown in Figure 3. These experiments have been carried out on consecutive days, always using the same animals (3 male Wistar rats of 230 gk The half-life of vinyl chloride in the closed system was 1.1 h when the rats were untreated (curve A). In contrast, pretreatmem with 33 mg/kg 3-bromophcnyl-4{3Hmidazote in DMSO (0.6 ml/kg) by i.p. injection, 30 min prior to exposure, completely blocked the uptake of vinyl chloride for 5 h. After this period inhibition became incomplete (curve B). Twenty-four hours after application of the inhibitor, the half-life of vinyl chloride in the closed system turned to the normal value (curve C). The control experiment with DMSO showed that this solvent inhibited upukc of vinyl chloride, but to n much lesser degree than was observed in presence of the imidazole: initially. R&S 134665 15K 10 44 II. M pa^-STSSSE c 0*1SO K CflflilS u. conttsl \ 24 h **' " iraelrnsrit <i t * \ 2 Ah 2 v *8 h- 4b h FI*. 3. Reverjibic inhibiii.m of uptake of *'tC-vinyl chloride. Experiments performed Hh *an* 3 (at consecutive days. (Atcoolro) experiment. /,. of vinyl chloride in *yiem 1.1 h. <B| total inh-Mmn iW uptake of vinyl chloride by acute dosage of 35 mg/kg 3 bromoptvenyI MSt inwda/otr in On ml kg DMSO (Section It. Inhibition becomev incomplete after J h (Section lit. 1C) control experiment. 24 h after dosage of 3 hromophenyl -H.'Firmdarole. /,. 0.U2 h. (C) solvent control. O.ft ml kg (IMS!) *t min prior to experiment. Initial (Section I) .1.3 h. lasting {& 4 h (Seeton lit the half-life of vinyl chloride in the system was 3.3 h (curve D, Section 0. but Jhi* initial inhibition lasted only for 3.5 h (curve D, Section IU. In order to compare the inhibitory potency of some further compounds (SKF 525 A, metyrapone. pyrazole) with th;tt of the benzothiadiazoies and of bromophe 1-imidazole, all compounds in a similar manner were administered in DMSO volu lion. The results are given in Table 1. Complete inhibition of uptake of vinyl chloodc was achieved by 3-bromophenyl*4(5)-imidazole (35 mg/kg) and by 6-tuiro ben/o thiadiazole (50 mg/kg), 5,6-Dimethyl-bcnzothiadiazole (50 mg/kg) showed a lesser effect, comparable to that ofSKF 525 A (50 mg/kg). Metyrapone did not inhibit. Tafcta 1* Inhibition of uptake of ,4C-vinyl chloride by scute dosage of potential wihUvittev. the wn pounds were administered 30 min prior to the experiment by Lp, injection m DMSO suhuaiw. Halt Ur of vinyl chloride in the closed system (/,,) and tl.e corresponding rate convtam of ckmmaoms from the system are shown. System wax occupied by 3 rats /11 k % (h) (It DMSO control (see Fig. 3) 35 mg/kg r-oroin|iiKTi)n-n,r.....--.... . 50 mg 6-Nitro-l.2.3-beiuothiadi*ioie in DMSO 50 mg/kg 5,6-Dimethyl-1 JJ-beozothiadiaaote in DMSiO 50 mg/kg SKF 525 A in DMSO 50 mg/kg Metyrapone in DMSO 50 mg/kg Pyrazole in DMSO 100 mg/kg Pyraaote in DMSO 3.3 a.. 13.7 140 3.2 s. ... 0.21 0 0 0.051 0.040 0.22 0071 0 KA too X X *4 10Q \*l Iht and a rtmtp.'tundi IW w# k|l Husna .nospkuty hkneked sat titan (hut nrpnrwd by (he *g OMW) tah dim. Vs hen given m t cMnneit tests M 7 lx fuJtv*fc*t i*. i/srrsn nvdw.e dsug mgtakuthea cwnpam ih* had Me v It tv vseyeivng that ptsg arnh ihrxsutssrhv' vl tt a**ly alter the rentmet So MgndWant mh 3 nvrlhy h-tudautWarv* h qfcvWiaw v reduct awe nh etheawd. uhsvi usds |7?| shaxtaed any Vvx increave dunMayUtuvs cnansg-d uptake of vinyl t twatmgnt usth IH>T 1) lexeesg ndwiun even ut Use fats wav tmfcantnd /IttfrdvfM uad / see to fbstaf wei ghs4<aev has hi the tsadated prrfuaed tel My weasureJ m Ibsen aft wetnhtNNet father than the of haghevt v'.enent d tadut Ina j. i n*. i ,t pv fer **0 M# w4 i an* I tkiwnA m i &4U pMH i ( * lu Kfan htnpi j i Mn we* < NUweipwwt 1 tstaa^e tee t Imiiaii l not * tbw isSw ID* Bum M K+ I Dwpouti.m of Vinyl Chloride l!W and a comparatively weak inhibition was observed after administration of pyraroic (50 mg/kg). However, increase of the dose of pyrazole u. !00 .ng/kg in OMSO completely blocked uptake of vinyl chloride. This inhibition by pyra/oic is greater than that reported by Hefner et al. (1975). but it has to be taken into account that the solvent DMSO which has been used in our study, exerts a synergistic inhibitory effect. When given in aqueous solution, 100 mg/kg pyrazole blocked uptake of vinyl chloride oi.ly by 71% (data not shown in the table). Induction Experiments. Rats were pretreated with agents which are known to induce drug metabolizing enzymes in the endoplasmic reticulum of the liver. Table 2 compares the half-life of vinyl chloride in our system before and after pretreatment. It is surprising that phcnobarbital does not shorten the half life. After pretrvatmem with phcnobarbital it was of no influence whether the rats were examined immcdi atdy after the treatment period or I day later. No significant enhancement of uptake was observed after application of 3-mcthylcholanthrene. Neither rifampicin, which induces the microsomal NAOPIt cytochromc-c-reductase in the rat (Otani and Remmcr. 1975), nor chronic treatment with ethanol, which induces a specific species of cytochrome P 450 (Ullrich et al.. 1975) showed any effect. Pretreatment with clotrimazol which has been found to increase demethylatior. of mestranol in rats (Bolt and Kappus, 1973) slightly in creased uptake of vinyl chloride, but a marked effect was observed only after pre treatment with DDT. The well-known persistence of DDT in fat cfTects a lonjt lasting induction: even when assessed I month later, the uptake of vinyl chloride b, the rats was enhanced. Distribution and Excretion of Radioactivity. Rapid conversion of vinyl chloride to polar metabolites has been reported in vivo, using rats (Hefner et al.. 1975k and in the isolated perfused rat liver (Radwan and Hcnschlcr. 1975). Therefore, radioactiv ity measured in tissues after exposure to ,4C-vinyl chloride should mostly represent metabolites rather than the parent compound. As demonstrated in Table 3. organs of highest content of radioactivity arc liver and kidney. Radioactivity in fat and in Table 2. Effect of pretreatmevu of rati itti inducing aicnli on uptake of 'V tiny! t blonde Data | the half-lire of vinyl chloride in the doted tystem when occupied hy 3 male ran I2UU 2*41 ft gats pretreated with Half life (/, ,1 of tiny 1 chkindr ihi Phcnobarbital: 1st experiment 2nd experiment*1 3 McthylchoUmhrcTjc Rifampidn Ethanol (chronic treatment for 3 weeks: 1 day ecssab-n: Ootrimaroi DDT Before pretrtatment After pretfcatmmt I.I 1.2 1.2 1 h I.I 1.(1 I.I 1 1 1.1 1 0 I.I USX 1.0 u nr ` This value was taken 1 week after a tingle i.p. dose of 200 mg k| DDT One m,wtth later, ihr rati showed a iln of 0.80 h * For the second phcnobarbital experiment rats of 175 g were used .<aaBe~rnii,i.jcnTn. * *4 *0** I*' A a 160 H. M. tuM ti / TaUe 3. Radioactivity in ofgani of nu after exposure to **C vinyl chloride (initial concentration 30 ppm, 3 h exposure). Mate Wijtir rats. 200 f. Mean value* + standard deviation are ihown: n = 3 TaMe 4. hiCTdjon taifekauWs ity m urine of ran after 3 h n> ure to ,4t` vwyl cMnrsk nndial concentration 50 ppmi % of incorporated vinyl chloride radioactivity/g (issue - Immediately after exposure 48 h after beginning of exposure Brum Liret Spleen Kidney Adipose liisue Muick (M. psoas) 0.171 + 0.008 1.86 0.30 0.73 0.03 2.13 + 0.43 0.22 0.06 0.32 0.07 0.038 0.004 0.32 002 0.37 + 0.01 0.21 * 0.03 0.044 t 0.004 0.03 + 0.01 Day after exposure % exiTrtvn of incorisval*1 raduscln*) tx * a ti lu 6V 4 2 a 2nd 1.74 (14a 3rd 0.4V UUa 4th 0.42 0 0.1 brain, organs which may be expected to be of affinity for the very lipid soluble vinyl chloride, is comparably low. Metabolites of vinyl chloride are largely excreted during the first 24 h after exposure, predominantly in the urine (Table 4). Fourty-eight h after exposure, a comparatively small part of incorporated radioactivity remains in the organism (Table 3). Discussion The results suggest that respiration is not a r.ne-limiting factor for toe uptake of vinyl chloride by lung. This is also reflected by calculation of the clearance of vinyl chloride in the closed system. For k = 0.61 h*' (i.e.. * 1,13 h) and the volume of the system of 10.3 I the clearance of vinyl chloride for three ratv each of 230 g. amounts to k v -- 6.3 1/h. On the other hand, the mean value of oxygen consump lion has been determined to be 13 ml/min or 0.78 1/h (Fig. 2k If we assume a difference of oxygen content between inspired and expired air of about J*V total respiration of the 3 rats averages 13.6 1/h. A comparison with the above clearance of vinyl chloride shows that only about 40% of inspired vinyl chloride is taken up by the organism. This means that normally respiration should not limit the rate of et*ry of vinyl chloride into the organism. The dependence of uptake upon metabolism is clearly expressed by its total, and reversible, inhibition after dosing potent inhibitors of oxidative microsomal drug metabolism. It is currently thought that substituted imidazoles occupy the type It binding ate at the 6th ligand of heme iron in cytochrome P-430 (Wilkinson t ak. 1974), whereas benzothiadiazoles may trap a tractive oxygen ipecies invoriud at drug oxidation (Marshall and Wilkinson. 1973k The much lesser affect of 5>dl methyl-benzothiadiazolc compared to 6-ftitrobeniothiadsaxole which Is found in the present study, lias also been observed in ttudici of microsomal drug metabolism m vitro. Using rat liver microsomes, the oxidation of aniline, ethylmorphme. and anw nopyrine was markedly inhibited by k-nilrobcnxothiediazole. but not by 3.ft <h I 4 kMfl --rtt~)friniiiglusi Mtresgans a* mi Kassaf |U7*> h . spevws vv*k tnetakdisni d ssa basis tu simsmUiss The task id Manuk missmg inManm he sgwvdV sgwtw 1 wthet esgWM **enr Im> res eeks kdai rate id nsetah vales, ahhiiagh hshad rasuftst f his . vhhatdv m f-*--- t-t atatUM id nsatagaM pratraaMd --* -n** uhsersata "shkii ** , that smu go y subs Cft lh* f* se rapaJH M. . d aksJM > O) g espaaiaM v gj (, fhvnavat. s M Ihii WarnId ia ns'wfial amm S| i hsnd ti< -tvsyivi^ia>Mi the bmssmMhs i4 and a* Msnrdnagi, any IVH| *,** t SI. IM *Wi HSfW. W s linn ** H M S sni M I Mi* Mi** a*, i ju ,m'ii *" M M taaw H a> **) Disposition of Vinyl Chloride |n| mcthylbenzothiadixzolc, whereas demethylation ofp-nitroanisolc and hydro*ylatton of estrogens was inhibited by both compounds to nearly the same extent (Boh and Kassel, 1976). It may be considered that vinyl chloride is metabolized by a distinct species of cytochrome P-450. This may also explain the lack of stimulation of metabolism of vinyl chloride by phenobarbital and methylcholanthrcnc which con trasts to stimulation by the chlorinated lipophilic compounds DOT and ciotrimazol. The tack of stimulation by phenobarbetal in the present experiments coincides with a missing inhibitory effect by metyrapone. Metyrapone is currently thought to inhibit the specific species of cytochrome P-450 which is induced by phenobarbital. Further experiments in vitro on metabolism of vinyl chloride by rat liver micro somes also revealed no stimulation by plienobarbitai pretreatment, neither of the total rate of metabolism, nor of covalent binding of vinyl chloride to macromole cukes, although cytochrome P-450 was enhanced under these conditions (unpub lishcd results). This is in contrast to recent reports on enhanced liver tonicity of vinyl chloride in phcnobarbitaJ-pretrcated rats (Jaeger et al.. 1974). and on increased foe (nation of mutagenic metabolites from vinyl chloride by liver microxomes from rats pretreated with phenobarbital (Baruch ct al.. 1975). However, our results agree with observations of Gearing (personal communication) who also did not find increased metabolism of vinyl chloride in rats after pretreatment with phenobarbital. We think that most probably strain differences may be responsible foi these diverging re suits. The present experiments further prove that vinyl chloride is metabolized very rapidly. Most of the metabolites leave the body via the kidneys within 24 h. The rest of about 30% which is not excreted in urine is partly excreted via frees and via expiration of ,4COj (Green and Hathway. 1975: Gchring. personal communication). However, some metabolites remain in tissues even 48 h after esposure (Table .0. This should be viewed along with the previous findings that vinyl chloride can be converted in vitro by rat liver microsomes to reactive metabolites which covalently bind to macromolecular structures (Bolt ct al.. 1975: Kappuv ct al.. IV7J). Hence, the possibility of long-lasting covalent binding to cellular macrumolecuks in vivo and its toxicologic implications will be a matter of a further survey (Bolt et al. 1976). AcknawMgemenit. The authors wish to express their thanks to the iladiuchettucaJ tkpanmv*: .a Fartowerke Hocchit. Frankfurt, for the .ymhesii of I1.2 ,4CI vinyl chiunde, am) to the l>yiwmu Nobel AG. Troisdorf, for valuable support. A (cnerous gift of insecticide synergists by Dr. C. F. Wilkinson Ithaca. NY. n giatetwht acknowledged. The study was financially supported by the Deutsche FortchungsgemeincchaA. B*mn MaJ (mU,, berg (grant No. Bo 349/Ik References Baruch. H- Malaveillc. C,, Montciano. R.: Human, rat and mouse liver mediated awuenwet >4 vinyl chloride w S. Typhimurium strains. Int. J. Cancer IS. 429 437 <|97'| Bolt. H. M- K-^pus, H.: Effect of ckrtrimuol treatment on the demeth) latent of mnaramd tt>em Mctab. Res. S. 234 (19731 BolL H. M, Kappus. H., Buchter. A,, Bolt. W.: Metabolism if tvt)l chluode I .antes 1931 t, 1425 162 M M * <t A Boh, H. M.. Kappus. H., Kaufmann, R.. Appel, K. E_ Bitchier. A- Btih. W,: MeiaN*srw. I 'V nail chloride in vitro and in vivo. Symposium: Environmental pollution and cancerttgevtc n*t*. L)urv. Nov. 3-5. 1975. INSERM. Pari* 1976. In press Bolt, H,, Kassel. H.: Effect of insecticide syner|iit* on microwimal mulatto* id nestratfed and cthynyl-ocstradiol and on microsomal drug metabolism. Xeitohtutici 4. ,1.1 .1* i!v?M Bray. G. A.: A simple efficient liquid scintillator for counting aqueous sotuteons tn a ttqual vaswtiAataw counter. Anal, Biochem, I. 279--283 (I960) Bitchier. A- Bolt. W.: Arbeitsmedizinische Aspckte der Vinykhlorid Krankhcn. 14. Jahrestagung <acr Ocutschen Gcsdlschafl fur Arbeiumedixin, Hamburg 1974. pp. 307 312. Stuttgart. A. W. (#**t ner 1974 Creech, J. L. Johnson. M. N.: Angiosarcoma of liver in the manufacture of puiysmyl cbVeaJ* I Occupational Med. 16, 150-131 (1974) Gchring. P. J.: Personal communication (1975) Green. T,, Hathway. D. E.: The biological fate of vinyl chlondc in relation u> at uncogermitv Chcm-biol Interactions II. 545-562 (1975) Hefner. R. E.. Wannabe. P. G.. Gchring. P. J.: Preliminary studies of the fate of lahalvd untl iWvtb monomer in rats. ,>nn. N.Y. Acad. Sci. 246. 133-148 (1975) Jaeger. R, J- Reynolds, E. S.. Conolly, R. B.. Moslen. M. T- Szahu. 5.. Murph). S 0 Acute hvpat*. injury by vinyl chloride in rats pretreated with phcnobarbitaJ. Nature (Lund t 232. '-'S ''a (1974) Kappus, H, Boh. H. M.. Buchtcr. A. Bolt. W.: Rat liver microtomes catal) te cosaktit (undine i4 'X vinyl chloride to macromoiecuiei. Nature (LondL) 237. 154 - 135 (1975) Lee. K J,, Harry. D. S.: Angiosarcoma of the liver in a vinyl chloride worker Lantvt 1974 L 1316-1318 Marshall,-R. S_ Wilkinson. C. F.: The interaction of insecticide synergists with nonmr) matte nuuM osidation systems. Pesticide Biochem. PhyiioL 2, 423-436 f|973i Otani, G- Rcmmer, H.: Participation of microsomal NAOPII cytochrome c rvdwciat* hi ihr mrtJi* ohsm cf rifampicin. Naunyn-Schmicdcberg's Arch. PharmacuL, Suppl 287. R 7a (|v7<i Radwan. Z.. Henschler. D.: Uptake and metabolism of vinyl chloride hi the isolated pcrlustd mi l.in preparation. NaunynSchmjedeberg's Arch. Pharmacol. Suppl. 287. R |<U(|9l<t Thomas. L, B- Popper. H., Berk. P, D.. Schkoff. I,, Falk. H.: Vinyl chloride induced hire dtteete New EngL J. Med. 292. 17-22 (1975) Ullrich. V.. Weber. P., Wollenberg. P.: Tctrshydrofurane: An mhthtWir for rthamd induced kin cm CTosomai cytochrome P-450. Biochem. biophys. Res. Commuo. 64. KN g 13 llvtJi Wilkinson. C. F.: Insecticide synergists and their mode of action. In; Ptsucnle chenuury lal 4 \ TahoeiK pp. 117-159. Proc, 2nd Internal, Congr. Pest. Chem, New York: Gurdtm A B*v*,h 1971 Wilkinson, C. F,, Brattsten. L. B,: Microsomal drug metabolizing enzymes in trisects- ITrvtg Menbokrtm Rev. 1. 133-228 (1972) Wilkinson. C. F- Hetnarski, K.. Hicks. L J.: Substituted imtda/utcs at udnhities id auctiwitmct oxidation and insecticide syncrgiui. Pesticide Biochem. PhyuoL 4. 299 312 1 |v74i Xrrrfrrd Stpttmbrr 10, 1975 1 11 4 mms* JJ (/) 03 4* O) *4 O Vt4 II | Paftmkfc AU- 15 RaraNrhsii atal IwUUaW it 1 | t |t as Itsrmtsgs id ywt, I* lots (/was* rsUm iW pmlnusz hvrvusml to* g I tfAt tint** MsedptwoMsmt <d d ku 4 4*18 |A lhsri> hiwen lunusvd* y *"* Mi M Ml i AmOvI g hgjA6y M, Ami pre*Wtw*> a <W 4m6sn d phemtvrnuaae ( l*A<*f**- slWfsi *n n**, hfuditsri f tags* met I wag yg^, Nug* 1 IWt. ItesaAwrg | 'V m . 41(8 Agin llnm ( |g film m i Hi l m dig 1