Document 6w3Xv5VeZn79bBoZgw5Qyv36R

!CANGER PSSEAR;h 26 2419-2422, July 1976] Covalent Interaction of Metabolites of the Carcinog Trichloroethylene in Rat Hepatic Microsomes1 Benjamin L. Van Duuren and Sipra Banerjee LAW "lTL: V U.S. CODE) Laboratory o! Organic Chemistry and Carcinogenesis, institute of environmental Medicine. New York University Medical Center, New York. New York 10076 SUMMARY Trichloroethylene epoxide has not been characterized as a metabolite of TCE, but the synthetic compound is known Trichloroethylene (TCE). a structural analog of vinyl chlo (B. L. Van Duuren and S. Kline, unpublished data). ride, is known to induce hepatocellular carcinoma and The present study was undertaken in order to obtain evi other tumors in C57BL/6 x C3H/He F, (hereafter known as dence for the covalent interaction of TCE metabolites with B6C3F,) hybrid mice. TCE epoxide, a possible metabolite, is rat liver microsomes in vitro. expected to be highly reactive toward cellular nucleophiles, e.g., proteins and nucleic acids. Hence, the microsomal metabolism of TCE and its covalent binding to microsomal MATERIALS AND METHODS protein were examined. Rat liver microsomes were incu bated in vitro with ['*C]TCE. The results showed that TCE Chemicals. [1,2-,4C]TCE (specific activity, 1 mCi/mmole) binds covalently to microsomal protein since extensive or was custom-labeled by New England Nuclear, Boston, ganic extractions and Pronase digestion do not dissociate Mass. PB was obtained from Elkins-Sinn, Inc., Cherry Hill, the TCE-protein complex. The binding was decreased by N. J. 7,8-BF was purchased from Aldrich Chemical Co., 7,8-benzoflavone. blocked by SKF-525A, and enhanced by Milwaukee. Wis. SKF-525A and TCPO were provided by Dr. i.p, administration of phenobarbital. The possibility that A. Lu of Hoffmann-La Roche, Nutley, N. J. G-6-P, G-6-P TCE epoxide, once formed, could be converted to water- dehydrogenase, GSH, mercaptoethanol, urea, and Pronase soluble products through enzymatic hydrolysis by epoxide were all purchased from Sigma Chemical Co., St. Louis, hydrase was also investigated. Addition of 3.3,3-tnchloro- Mo. propene oxide, a potent inhibitor of epoxide hydrase. to the cubation system markedly enhanced the binding of TCE. fese observations support the view that, in order to bind to Preparation of Microsomes. Male Sprague-Dawley rats (A. R. Schmidt. Madison, Wis.), 5 to 9 weeks of age, were maintained on a commercial diet and water ad libitum. Liver otein, it is necessary for TCE to be metabolized to its epoxide, a reactive intermediate that is most likely involved m TCE carcinogenesis and toxicity. microsomes were prepared according to the method of Levin ef a/. (12). A 25% liver homogenate was prepared in O. 05 m Tris-HCI buffer, pH 7.5, containing 1.15% KCI. It was then centrifuged at 12,000 x g for 15 min at 4. The microso mal pellet was collected from the supernatant obtained INTRODUCTION from the above centrifugation by spinning at 105,000 x g for 60 min. The microsomal pellet was washed twice with 1.15% TCE* has been used for many years as an anesthetic (5). It KCI containing 10 him EDTA. The washed pellet was sus is also widely used as a degreasing agent for metals and as pended in 0 05 m potassium phosphate buffer, pH 7.5, at a an extractant in the foodstuff industry (18). Several metabo protein concentration of 8 to 9 mg/ml. The microsomal lites of TCE in humans and animals have been characterized pellet was at times stoi ed at -20s under nitrogen and a layer (4, 10, 11) and it has been suggested that TCE is metabol of 0.25 m sucrose. Such samples retained enzymatic activity ized via an epoxide (16). On the basis of its known metabolic oroducts and the possibility of an epoxide intermediate, it has also been predicted that TCE is probably carcinogenic, unchanged up to 2 weeks. Induction of Mixed-Function Oxidases. Animals weigh ing 40 to 45 g were given PB i.p. in 0.9% NaCI solution at a particularly to the liver (20). This suggestion was made, in dose of 100 mg/kg of body weight, once daily, for 3 days. Part, because of its structural similarity to vinyl chloride, a The animals were fasted for the last 18 hr and killed 24 mown liver carcinogen in animats and man (6, 20). Re hr after the last injection. The control animals received cently, TCE was shown to be carcinogenic by feeding to injections of 0.9% aqueous NaCI solution. Microsomal 36C3F, hybrid mice, but not in Osborne-Mendel rats (19). preparations were then made as described above. Binding of TCE to Microsomes. Microsome preparations were incubated with [14C]TCE in the presence of a NADPH- ' Supported by USPHS Grants ES-01150, ES-00260, and CA-13343. t The abbreviations used are TCE, 1,2,2-tricfiloroethylene; PB. sodium ohenobarbital; 7.8-BF, 7,8-benzoflavone; SKF-525A. /J-diethylammoethyl diobenylpropyl acetate; TCPO. 1.2-epoxy-3,3.3-tnchloropropane; G-6-P, glu-ose 6-phosphate, GSH reduced glutathione; TCA, trichloroacetic acid. Received February 13, 1976; accepted April 9, 1976. regenerating system in a closed vial with shaking at 37 for 60 min. The complete incubation mixture consisted of a microsomal preparation containing 500 of microsomal protein, 10 0 /zmoles of MgCL, 2.5 ^moles of G-6-P, 1.3 units of G-6-P dehydrogenase, 1.0 ^mole of NADP, 0.83 1976 032827 SL 2419 8. L. Van Duuren and S. Baner/ee Mmole of TCE (specific activity, 1 MCi/Vmole) in 1% acetone and 0.38 ml of 0.05 M potassium phosphate buffer, pH 7 4, in a total volume of 1,0 ml. The reaction was terminated by the addition of TCA to a final concentration of 5%. The mixture was then centrifuged at low speed for 15 min and the supernatant was discarded The protein pellet was heated at 90' for 15 min in the presence of 5 ml of 5% TCA for hydrolysis of RNA. The protein residue obtained by lowspeed centrifugation was suspended in 5 ml of methanoLether (3:1) and heated at 60' for 15 mm. After the super natant was discarded, the protein was extracted 10 times with the same soivent mixture (17) and, finally, washed once with ether. The residue was air dried and dissolved in 0.2 ml' of 0.5 n NaOH. Aliquots were then taken for liquid scintilla tion counting, using Aquasol (New England Nuclear, Bos ton, Mass.) as counting solution, and also for the determi nation of protein content The protein was assayed by the method of Lowry et at. (13). with crystalline bovine serum albumin as standard. For determination of the 0-hr value of the binding, microsomes were denatured with TCA prior to the substrate addition and incubation. In order to determine whether TCE itself binds covalently with microsomal protein, the washed protein residue ob tained as described above was suspended in buffer and then digested with 100 m9 of Pronase at 37 for 4 hr. This digest was then extracted with 4 volumes of ether to remove any TCE that was not covalently bound. Radioactivities were determined before and after hydrolysis RESULTS Binding of TCE to Microsomal Protein. From measure ments of the amount of TCE bound per mg of protein with time, it was determined that maximum binding is reached within 60 min and then remains at a plateau. The binding between 0 and 60 min was linear. In subsequent expen- ments. the microsomes were incubated for 60 min only. The purified microsomal protein was hydrolyzed with Pro nase. The hydrolysate, after extraction with ether, was found to retain all of its radioactivity. This result indicated that the binding of TCE occurs with the amino acid residues of microsomal protein. Effect of SKF-525A and 7,8-BF on the Binding of TCE to Microsomal Protein. Table 1 shows the effect of 2 inhibitors of mixed-function oxidase on the binding of TCE to micro somal protein. When microsomes were incubated with [,4CjTCE in the presence of either SKF-525A or 7.8-BF, a diminution in the amount of TCE bound to protein was observed. At a concentration of 2.5 x 10 3 m, SKF-525A ''C1 S;-! 4fff^ 25'0 CCrT'T^O'^ '.Mlb tr*1 . con Mrjbor. wa.j m'.reaped to 3 76 '.0 3 ,v SKF-525A almost totally blocked TCE from binding'lo pro tein (95%). 7,8-BF had a similar effect on TCE binding to microsomal protein, as shown in Table 1. Acceleration of the Binding of TCE to Microsomes by in Vivo Treatment With PB. Table 2 shows the effect of PB on the TCE binding to protein when administered in vivo. PB at a dose of 100 mg/kg of body weight administered for 3 days increased the binding of TCE to microsomal protein by 64%. Table The effect of SKF-525A and 7,8-BF on the binding of !'*CJTCE : hepatic microsomal protein Microsomes were incubated witn [,4C]TCF for 60 min, as Cscribed in Materials and Methods Addition to the incubation sys tem (M) ['4C|TCE bindmg to protein --------- ------ --. -- nmolesdpm/mg protein mg protein % change over the con trol None SKF-525A 2.5 x 10 3 3.75 x 10 3 7.8-BF 1.25 x 10 3 2.5 x 10 3 5726 r 113 4289 ? 243 291 - 67 4188 - 128 4028 ? 85 2 59 1 92 0 13 1 89 1 81 -25 - 95 -27 -30 " Results are the average Of 4 analyses m each group mean S.O. Table 2 The effect of in vivo treatment of PB on ['4CITCE-protein bmdin: The animals were treated either with 0.9% NaCI solution or Pg , vivo Hepatic microsomes were isolated and incubated wit ['*C]TCE in vitro as described in "Materials and Methods." Result are the average of 4 animals in each group. ['`CJTCE binding to protein In vivo treatment 0.9% NaCI solu'ton PB " Mean - S D. dpm/mg protein 4348 r 631" 7121 - 388 nmoles/ mg pro- tern 1.96 3 21 % change over the control +64 compared with control animals, when microsomes wen incubated for 60 min. Effect of Various Agents on the Binding of TCE to Micro somal Protein. In order to obtain further information con cernmg the nature of the binding, several agents were incu bated individually with microsomes (Table 3). When micro somes were incubated with different concentrations o urea, GSH, 1-methy-2-mercaptoimidazole, and mercapto ethanol, they all decreased the extent of bindmg of TCE tc microsomal protein Effect of TCPO on TCE-Protein Binding. Table 4 demon strates the enhancing effect of TCPO, a potent inhibitor o: epoxide hydrase (15), on the TCE binding to microsoma protein. TCPO stimulates TCE binding, which is concentra tion dependent At a concentration of 1.25 x 10 2 m or more, it caused an augmentation of the binding of 76 tc 91% DISCUSSION A n-,.m.;-r pi a -'p+nmentai s;u cs.-jj rave orc video e-.: j = n', - that some indirect-acting carcinogens are metabolized tc their activated carcinogenic intermediates by means of a cytochrome P-450-dependent mixed-function oxidase (2, 14), The intermediate epoxides from such indirect-acting carcinogens may then interact covalently at nucleophilic sites in nucleic acids and proteins and one or more of these processes are probably responsible for their carcinogenic activity. 2420 SL 032828 CANCER RESEARCH VOL 36 Interaction of ~ncnicroethy'ene with f.'.zroscries It hus been suggested that an epoxiae 'ntc-rrneb'ate Chart l, Structure 1 := rpc- activ'tec carcnccecir intermediate of vinyl ch'O'ide i2u) ~h.s suggestion was cases on its similar ity in structce to eeox za carcinogens such ?= giycidaldehyde. Structure 2 and i-ch'oro-ether carcinogens such as fcs(chloron'iatnyl;einer. Structure 3. Chloroethyiene cxide, Structure 1. contains both the epoxide and u-chioroether moieties of Structures 2 and 3. In a recent report it was shown that rat liver microsomes catalyze the covalent bind ing of [,4Cjvinyl chloride to macromolecules (7), In another study (1), evidence; was obtained for the 'crmgtion of epox- Table 4 Tne effect of rCPO cn ['*CjTCE bmd:ng to protein Hepatic rmccsomes were isolated from amma's wt'5"i"-g ao 45 g. as in Table 2 ar.rf as desentori in `Materials ana Methods Addition to the incubat.cn system |M) (`C'FCE o.ncbng to protein nmches / dprn/ng protein mg protein 'a change ever f-e cci trei None TCPO 2 5- 10 * 5.0 - 10 1 4400 .-. 3601 5056 = 427 5706 - 937 20 2,28 2,37 - 13 . 'O m, cr-3ones ~' "c i;y; a--:- ctm-item ith me pro posal (20) that an epoxide intermediate is involved in the metabolism of vinyl chloride. On the basis of these considerations, it is likely that TCE, Structure 4, is also metabolized via an epoxide Structure 5 (20). This was suggested earlier by Powell (16) on the basis of metabolic studies. SKF-525A is a known inhibitor of the metabolism of var ious substrates of cytochrome P-450 (3). This agent is shown in the present study to inhibit the covalent binding of TCE to rat liver microsomal proteins. 7,8-BF, an inhibitor of 7,l2-dmnethylbenz(a)anthracenemduced mouse skin carcinogenesis (8). is known to de crease the covalent binding of 7,12-dimethyibenz(a)anthracene to DNA, RNA and protein in mouse skin (9) In line with these earlier findings, the present work showed that 7.8-BF inhibited the covalent binding of TCE to rat liver microsomal proteins. The experiments with TCPO, a potent inhibitor of epoxide hydrase (15). showed that this agent causes an enhance ment of TCE binding to microsomal protein. This otserva- is also consistent with the formation of an epoxide from when then binds to microsomal protein. PB injected 2p . * w 2 5 10 ; 7-v3 _ 8518 _ 162 3 ;3 3.84 -91 Results are the average of 4 analyses in each group, mean = SD lli ; i h2cJ-Vc:/u ;-'CM-- C ClO20!>2Cl c\ / c=c \ C\r.~ c /Cl ci7 ''o' N Chari 1 /1 chloroethyiene oxide 2, glycidaldehyde: 3, bis(chloromethyl) ether, 4, trichloroethylene; 5, trichloroethylene epoxide into the animals prior to the examination of TCE binding also enhanced the binding of TCE to microsomal proteins. The other agents, listed in Table 3. all decreased the bind ing of TCE to hepatic microsomal proteins as expected. The present report provides evidence for the covalent binding of TCE to rat liver microsomal proteins from in vitro experiments. These results suggest that the binding is via an'epoxide or other related electrophilic species. Table 3 The effect of various agents on the binding of ['"CjTCE to hepatic nhcrosomal protein Microsomes were incubated with (MCjTCC fcr GO min as de scribed in ' Materials and Methods. ' [,JC]TCE binding to pro tein Addition to the incuba dpm, mg pro tion system (m) tein nmoles/ ing pro tein % change over the control None GSH 5 v 10 5 5 >. 10 4 1~Methyl-2-mercap- touwdazole 5 '< ID ` 5 x 10 4 Mercaptoethanol 5 '< 10 5 Urea 0 05 01 0.5 5756 - 113" 3119 _ 102 1954 - 67 2.o9 1.4 0.88 2510 u. 299 990 z. 28 4533 z 174 5566 - 248 4434 - 141 3317 :. 124 1.13 0.45 2.04 2 51 2.0 1.49 -46 -66 -56 -83 -21 -3 - 23 -42 " Results arc the average: of 4 analyses in each croup mean = SD REFERENCES 1 Sarbrn, A . Bresil, H Croisy. A . Jacquignon. P , MaiaveJle. C , Montesano R , and Bartsch, H Luer-Microsome-Meaiated Formation o( Alkylanng Agents from Vinv' Bromine and Vinyl Chloride. Eiocnem, B.ophys F?es. Common , 67 596-503, 1375. 2 Conney. A H. 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