Document jgM0xg25Z6vXY2jNopENk4RgN

Gwunc VOLUME 45 NO. 1 CNREA 8 PP 1 -480 January 1985 \ JAN. 3 1985 065757 4 (CANCER RESEARCH 45,188-193. J*nu4fy 19851 Formation of ie Conjugates by Reactive Metabolites of Vinylidene Chloride in Micf les and Isolated Hepatocytes*1 2 Daniel C. Uebler,* Michael J. Meredith, and F. Peter Guengerich3 Departments at Pharmacology [0. C. L] ana Btocfimiatry [U. J. U, F P. <3.1 ana Camas m UotaoXm Toxicology ID. C. L. V. J. U, F. P. a/, VandarbuSchool ol IVaaiana. NasheOa. Tammsaa 37233 r5* 30 ' dTERtALS ABSTRACT Oxidation of the vinyl halide carcinogen and hepatotoxin vi nylidene chloride (VDC) by microsomal cytochrome P-450 yields 2,2-dichloroacetaWehyde, 2-chloroacetyi chloride, 2-chioroacetlc add, and 1,1-cfichloroethylene oxide. The rotes of these metab olites in covalent modification of proteins and reduced glutathione (GSH) were examined. 2-Chloroacetyt chloride reacted with model thiols at least 1 CP-fold faster than did 1,1-dichloroethytene oxide and at least 1 CP-fold faster than did 2,2-dichioroacetaMehyde or 2-cNoroacetic add. Microsomal covalent binding of ("*C}VDC was inhibited by GSH but not by lysine, suggesting that protein thiols, rather than amino groups, are major targets. Liver microsomes catalyzed the formation of three GSH:VDC metabolite conjugates, identified as S-(2,2-dichioro-1 -hy droxyjethytghjtathione, 2-<S-glutathionyi)acetate, and S-(2-gtutathionyi)acetytgiutathione, a novel conjugate containing both sta ble (thtoettiw) and labile (thioester) linkages. The latter two conjugates also were formed in isolated rat hepatocytes and measurable amounts of 2-(S-glutathionyf)acetate were released into the incubation medium. Both 2-(S-gtutathionyt)ac8tate and S-(2-$utathionyl)acetylglutathione wore formed with ["SJGSH added to the hepatic medium, indicating that reactive VDC metabolites are capable of crossing the plasma membrane to react with extracellular targets. Unlabeled S-(2-glutathionyi)acetytglutathione underwent carbonyl substitution with added ["S)GSH, suggesting that this conjugate may partidpate in modification of protein thiols. This conjugate also underwent hydrolysis with a half-life of approximately 3 hr. GSH:VDC me tabolite conjugates may serve as accessible models for labile covalent adducts formed between VDC metabolites and protein thiols. INTRODUCTION Vinyl halide monomers hive afforded investigators an oppor tunity to study the conaMpances of procardnogen bioactivation using relatively sxnpte wripounds. Early studies demonstrated that vinyl chloride is ntdifttiart to chioroethylene oxide by micro somal cytochrome P-480 (1, 2, 8). and it was inferred that concomitant production of 2-chloroacetaidehyde was the result of epoxide rearrangement (8,11). These and other observations contributed to the widely held assumption that epoxides are the ' This wo* was supported by USFHS Owns ES 03206 and ES 00267 and by Vanderbilt UrWerety Research Com) Orem 361024. 1 Recipient oi a Ptwnaceubcal Manufacturer's Aaaooadon Foundation AdvancadPiedoctotWFelowanip, Present adfreesOepirtment of Biocfiatnieby and Biophysics, Oregon State University. CorvHf*. OR 97331. 1 Burroughs WeHcome Scholar > Toxicology (1963 to 1988). To whom request* lor reprints should be addressed, at Department at Biochemistry, VjnderMl University School of Median*. NashvMe. TN 37232. Received May. 21.1984-. accepted September 27.1964. principal products of microsomal vinyl halide oxidation, which Owd '-I as such, occupied a position of central importance in covalent modification of cettular macromolecules and production of more i vdK * stable metabolites (3). Recent mechanistic studies in this labo mtfqi is-J ratory have chaflenged this view, Epoxides are not obdgaie intermediates in vinyl halide biotransformation but are formed, together with carbonyl products (hatoacetaktahydes and- 'oacyi halides) via partitioning of a common catalytic intermed =>(i7 t9f****K ; *a | mP 4d 20). *>.,, t. Selectivity of irxfividuaf virM ctton with particular cellular tarmxWtwn oMW*ved.Stij0ko with vinyl chloride indicated tht sli^othylane oxtie is primary ; tfiCadM mam*** Mtmt Mi responsible for modBfcatiorvof DNMv*d and In Wire, whde 2 chloroacetaldehyde btads prknarty ID protein (TO. 37V The cresomai oxidation ef VOC* yia|M VDC oxide, 2-didroace chloride, 2-chtoroacaflc add. awe) 2,2-dchtoroacataidehyCe 17). which may react wfttl A of AdhAir nuttaophites .Tip variable reactivity of ttae^ mt||io8ree may,J%*!il|Wc*9M:,fc influence their target! are bound to DNA ittoMjsdfjipNta Twr i -4S6* dc : rdiw j fycaf wnm yi) sM Ms- et at. (28) suggested z re sponse via interaction wlfticefciar componlMwWM-- DNA Alternative target* ppidbli V* mechanism may include c^^auMtfMf theWlqdK|ilNA trar scription. chttxnatlitAl^fW^^pRaelulir ^pjHjESatus (191 Information concefTtiflgllMdfcMftjta of wpfmreiltire wood therefore be required (dfiMajURH^ieir i4NR ftflM Ifmation ut carcinogenic or toxic lesions. Several reports have suggested that ceflutar thiMk r-<>rt ,i protective influence in VDC intoxication and serve todet can* VDC metabolites (13,16, 27). AcconSngly, deptetionof' oat* GSH by starvation or pretreatment with dwthytondsats er noxi VdMuMMI Him ah horn Hn wtdwto "V oonoanai ll' Ml -lSw i inc -'Smar jjm bid The ce VDC hepatotoxidty in rets (13, 27). Anknsis adtnir .tereo ['XJVDC excrete radiolabel in the urine as IW-acetytcareoxyme- thytaysteine, thtogtycoMe add, thfodiglycoio add, and uniderefied sulfur-containing metdaoUss (16, 1ft). The importance ot reHlk...lra MflWClor Cellular GSH as a detoxicating nudeopMs for VDC metabolites implies protda thiols may themadves bemajor targets w covalent modification. This suggestion has been verified for other WC..HC- compounds which interact with GSH in a simflar manner (22,31 32). Because urinary adducts reflect significant rend as ^ 4 as lid maf hepatic btotransformsMonythafr iility at Mcators of intra metabolts disposition d fcnHdt In tNb$qj/k, VDC meta. Ates were compared on the basis of relative reactivities towafds sutihydryi compouids, relieve contrtxjtigneto protein covalent MIMPr,, SH ** l binding in vitro, andafiity tO'form GSH oonjugates in hepato* `The ebwMw UMO me VDC. WiyfcMn* Oieartd* (1,1-<fcoroe<hylW VDC cnuOe, 1.1-4cMoraMftyMneakte; OSH, reducsdgWsiritoo*: GSSQ. ***?~ glutsthions: HPLC, higrt-pertanienoe Iqucf chnxnMpgraphy: FDNB. 1-fXxxo-Z-* ijmvobuinns; OHP. 2,4^HbaHwny4 *" 30 tor! I of CANCER RESEARCH VOL 45 JANUARY 1985 186 SL 065758 VDC ADDUCTS ^ and microsomal systems. The formation of extracellular conjugates was used as an index of the ability of VDC dtes to cross hepatocyte membranes. The data indicate multiple VOC metabolites participate in covalent modification yjrolens and GSH. ittlEBIALS AND METHODS 00Dials. [l/-,4CJVDC was synthesized from 1.1.2-trichloro-{U-'`C] (Anersham. Arlington Heights. IL: 10 mCi/mmol). The labeled 'jgtaf material was diluted with unlabeled 1.1,2-tnchloroethane to a i4 strife activity of approximately 0.2 mCi/mmol and treated with T(TthiryrkVS.4.0lLindec-7-ene for 1 min at 60. A stream of nitrogen through the mixture to sweep VDC into a tube immersed n iSyiaKisopropyi aicohoi bath. The (U-''C]VDC thus collected was of yHfcfwjcchemical purity as assessed by gas chromatography (Tenax. 'Hf\{"SJMethtonme {1170 Ci/mmol) and [BS]GSH (69 Ci/mmol) were mi n*w England Nuclear (Boston. MA). voC end thiophenol were purchased from Aldnch (Milwaukee, WIV. roc ms tfctJled before use. Chloroacetyl chloride was from Eastman Pattster. NY) and was also distilled before use. GSH. 2-(S-cystei-ygatate. and glutatfiione-S-transferase were purchased from Sigma & iffii*. MO). This commercial glutathione-S-transferase preparation i conposed largely of isozyme B and lesser amounts of other isozymes, -wit her aicohoi dehydrogenase was purchased from Boehringermth&n (Indianapolis. IN). Petal calf serum was from Grand Island ndogcalCo. (Grand island. NY). Okrtoroacetaldehyde and VDC oxide were synthesized as described neuter* (17). AM other chemicals were of the highest punty commer- able. Hepetccyte and Microsomal Incubations. Rat liver and human liver -ooeomes were prepared as descnbed previously (9. 36). Rat heoarqm were prepared by collagenase perfusion (29). and viability was ansed by trypan blue exclusion. Fischer's medium, supplemented an 15% fetal calf serum but deficient in sulfur amino acids, was die rdxm used for all experiments with nepatocytes. Ntpwocytes (2 to 3 x io* ceils in a volume of 1 ml) were incubated riMltd 15-mi glass scintillation vials containing Fischer s medium. VDC vw added to the suspension from a i m stock solution in acetone to a concentration of 5 him. ["SJGSH was added to cell suspens-cns nn a 0.4 u stock solution prepared immediately before use. Hepato ma* were incubated for 3 hr in Fischer s medium supplemented with 15 mi [``Sjmethionine at a specific activity of 2 Ci/mmol to label surfur it**. The tens were then washed twice and resuspended in fresh containing 0.5 m unlabeied methionine. The specific activity of tstwpatoceMular ("SJGSH produced was 89 mCi/mmol and was deterWNd tiom fractions collected from HPLC analyses (23). After incubatxxi ^YOC tor 60 min at 37, the ceMs were rapKfly separated from the by centrifugation at 1000 x g tor 30 sec. and the medium was "frwd tar analysis of GSH adducts. The pellet was resuspended n 1 *df10%HC10 and recentnfuged. The resulting supernatant contained >,*t*A4arGSH and GSH;VDC metaboliteconjugates and was analyzed ^`Exchange HPLC. itebsomal incubations contained 100 mM potassium phosphate. pH T.in NADPH generating system consisting of 0.35 IU glucose-6T'C'Chite dehydrogenase per ml, 10 mm glucose 6-phosphate, and 0.5 'NHAOP*, and microsomal protein at a final concentration of 5 to 10 ,IW' SSH was added from a 40 miu aqueous stock solution: this yMhowas prepared immediately before use. VDC was added from a JMWi sokition in acetone or methanol. Incubations were terminated *50 mn at 37" by addition of ZnSO* to a final concentration of 1% W*r HOO* to a final concentration of 3.5% (w/v) and centrifuged at tor 5 min. The supernatants were then neutralized with KHCOj of GSH conjugates. Depletion of GSH during aqueous incubations with VDC metabolites was monitored using 5,5 -dittiiobis-2.2'-dinitrobenzoic acid (23). VDC metabolites were added directly to solutions containing 0.5 mM GSH in 200 mM potassium phosphate, pH 8.0, at 37*. with an initial metabolite concentration of 50 mw. Aliquots were then removed at various times for assay of residual thiol content. 2-Chloroacetic acid was added as its sodium salt to minimize pH changes. Less than 5% of the GSH was oxidized to GSSG during these incubations. Depletion of thiophenol in CHClj by VDC metabolites was monitored using 5,5'dithiotxs-2,2-dinitrobenzoic add in aqueous acetone. Thirty /imd of each VDC metabolite were added to 30 >imol thiophenol in 6 ml CHCI3 at 25". Twenty-iii aliquots were then transferred to tubes containing 1 ml of acetone plus 200 mI of 10 mM 5,5'-dithiobis-2.2'-dinitrobenzoic acid in 85% acetone: 15% HsO. Twenty m> of tnethylamme were added, and the absorbance was immediately recorded versus a blank at 490 nm. Sec ond-order rate constants for the reaction ot thiophenol with VDC metab olites were determined from plots of reciprocal absorbance versus time. Covalent binding of [,4C]VDC radiolabel to microsomal proteins was assayed by the method of Wallin et a/. (35). incubations contained 3 mg microsomal protein (from untreated rats) per ml and 10 mM ('*C]VDC (0.03 iiCi) and were for 30 mm at 37*. Proteins were precipitated on 2.4cm glass fiber filters (Fisher G6). Following 3 washes each with ethanol, methanol, and acetone, the filters were counted using ACS scintillation cocktail (Amersham). Separation and Characterization of Glutathione Conjugates of VDC. Glutathione and cysteine coniugates were analyzed using ionexchange HPLC as described by Reed er at. (23). Aminopropyi siMca was prepared from 5 nm Sphensorb silica (Ramin. Woburn, MA). and 0.5- x 30-an columns were slurry packed as described previously (24). Super natants from ZnSOi- or HCKVquenched microsomal incubations were neutralized with KHCO? and treated with ethanoMc FDNS tor 30 to 60 mm before injection. Elution Solvent A contained 80% methanol and 20% H:0 (v/v), and Solvent B contained 3 m sodium acetate, pH 4.5. in 64% methanol. The column was loaded isocraticaMy at 95% Solvent A for 10 min. Conjugates were eluted during a 30-min linear gradient from 5 to 99% Solvent B at a flow rate of 1 mi/min. Fractions (0.5 ml) were collected and counted using ACS scmtBation cocktail, wMs elution of DNP derivatives was monitored at 360 nm. For preparative and some analytical chromatography, ammonium acetate was substituted tor so dium acetate in Solvent B. Effluent tractions containing 3 GSH conjugates (Conjugates A. 8. and C) were collected, and several collections were pooled. Methanol was removed from the pooled fractions m vacuo, and the fractions were then lyophiiized. The residue corresponding to Conjugate A was treated with 2 n HC1 tor 12 hr at 25". The solution was then extracted with 3 portions of ether, the extracts were concentrated under a stream of nitrogen and then analyzed for 2,2-dichloroacetalCtehyde by gas chromatography (17). A portion of the residue corresponding to conjugate B was dissolved m M HO and heated at 110" for 30 min. A second portion was dissolved in 6 s HCI and heated under nitrogen at 110* for 30 hr. Both samples were then neutralized with KHCOj and gently gassed with oxygen overnight to oxidize liberated thiols to disulfides. The mixtures were then analyzed by ion-exchange HPLC as described above. Residue containing Conju gate C was hydrolyzed with 6 n HO at 110" tor 24 hr. The hydrolysate was reanalyzed for 2-(S-cysteinyl)aeetate by ion-exchange HPLC. RESULTS Reaction of VDC Metabolites with Thiols. Thiols reacted with major VDC metabolites at measurable rates in aqueous and nonaqueous solutions (Table 1). In aqueous buffer at pH 8, GSH reacted with 2,2-dichtoroacetaldehyde and 2-chloroacetate at moderate rates. Despite rapid hydrolysis. 2-chloroacetyt chloride reacted with GSH at least 4 orders of magnitude faster than did 2-chioroacetic acid or 2,2-dichtoroacetaldehyde. The rate of re action of GSH with 2-chloroacetyi chloride was calculated using CANCER RESEARCH VOL 45 JANUARY 1985 187 SL 065759 VDC ADDUCTS RmconatvttMltl witfi VDC mMboWM Reactions were started by I matacoltaa to Mkrttans of tfw thtato. Aliquots wara than andyaad fc,. and Methods.' MM* VDC iiMUflalle.ilof ntolir raOoe were t:i n me CHCt, system end 100:1 n the aqueous system. Psaudo-Vstoroar rata constants tor the GSH teecdon were dMernsned tram plots of the logwtthm of absorbance versus tsne. Second-order rate constants for the thiophanal reaction were detemtoed from plots of redprocst absorbance versus dm* System MataboMe VOC oxide z-uvoroacvcyi cnonov 2.2-OtchMreKataldanyde 2-CNoroaPattc add * Mean S.D. from 3 individual * --, not detsmsned. TNophanocCHCt*. 25*. k (m" mm "I 90 20* >7x 10* <8 <6 GSH:200 mu potasskan phos phate, pH 8 0.37*. k* (min") >3.6 x 10* 0.024 0.002 0.006 0.000 Table 2 Mlcrtaan* contort binding at f*C/VDC ! Complete incubation mixtwes contained 100 inMpotasaasn phosphate pH 7 - [ 3 mg rat or human tver meroaomn protein per ml, 0.35 IU yeest gii.toi^i I phosphate dehydrogenase par ml. 10 mu glucose S-phospnate. 0.5 m- saop- ; and 10 mm f'*C|VDC (0.03*0). Rat War microsomas wars from umreate rm+. t Covalent binding of [,4C]VDC rarSdabel to microsomal protein was a ,wj described in `Matenals and Methods.' I System Complete -NADPH 5 mM GSH 4-Alcohd denytkoganase (0.5 mg/mfc 0.5 raM NADH +Alcohol denytkogenata (0.5 mg/ml) (da)ad):0 5 mM NADH 4-0.5 mM NADH 4-5 mM lysine Human War 31 Human War 80 nmol bound/mg/30 mm 22*3* 2 1 12 1 9 1 % of L -pfcr. syv-im i-O 9 55 41 23 3 28 3 18 1 11.10* 20.22* 105 127 -2 * Mean S.D. Horn 3 individual exptfdnents. Results of dupdcUe expenments. i u. m a hydrolysis rate constant of 4 s~' for the acyl chloride. This value was estimated from earlier studies comparing the hy drolyses of acetyl chloride and 2-chloroacetyl chloride in aqueous acetone (33) and from the reported hydrolysis rate constant for acetyl chloride in water at 0s (7). The assumptions made were than the ratios of hydrolysis rate constants of the 2 acyl chlorides were similar in water and aqueous acetone, and that the hydrol ysis rate constant doubled with each 10s increase in tempera ture. It also was assumed that the phosphate buffer would not significantly affect the solvolysis rate. VDC oxide was not studied in the aqueous system, because it can be produced only as a dilute solution in chloroform. Addition of the required volume of epoxide solution thus produced a 2-phase mixture unsuitable for the experiment, and attempts to concentrate the epoxide solu tion were unsuccessful. To compare the relative reactivities of VDC oxide and 2- chioroacetyl chloride with thiols in the absence of competing hydrolysis, the reaction of each metabolite with an equimolar concentration of thiopheno! in chloroform was studied. The acyl chloride reacted with thiophenol at least 800-fbkj faster than did the epoxide, white neither 2,2-dfchtoroacetakjehyde nor 2-chioroacetic acid depleted the thiol at a measurable rate in this system (Table 1). The slow reaction of 2-chfcxoacetic acid relative to 2-chloroacetyl chloride suggests that acylation, rather than alkylation, is the major reaction between 2-chloroacetyl chloride and the thiol. The rate constants presented describe bimolecular reactions, although pewfe-flrst-order conditions were selected for the aqueous sysMi^gp^ concentrations of VDC metabolites were used in order to owe rates of GSH conjugation signifi cantly in excess of thatOf GSH oxidation. Covalent Binding of [,4CJVDC Radiolabel in Microsomes. Rat and human liver microsomes catalyzed covalent binding of ('*C]VDC radiolabel to microsomal proteins (Table 2). No radio activity was bound when NADPH was omitted from the incuba tion mixtures. Inclusion of 5 rriM GSH in the incubation mixtures inhibited roughly half of the binding, but 5 mM lysine inhibited the binding only slightly. These results suggest that protein thiols, rather than protein amino groups, are major targets for covalent modification by VDC metabolites. Inhibition of covalent binding by GSH was not increased when glutathione-S-transferase (0.3 mg/mi) was added to the incubations (data not shown). Covalent binding of [14C]VDC radtoiabQl also wqs decreased in rracrr somes supplemented with 0.3 mg alcohol dehydrogenase per t plus 0.5 mM NADH. Substitution of heat-denatured dehydroge'' ase returned binding to control levelB. while addition of 0.5 ir* NADH alone increased bindng by 25%. Microsomes prepare from 2 human Ever samples catalyzed covalent binding at toe* comparable to rat microsomes. Production of GSHrVDC Metabolite Conjugates ir **cr* somes. Rat Ever microsomes supplemented with G H an' f*C]VDC produced 3 GSH conjugates which were ana J' N-ONP derivatives using ion-exchange HPLC. NADPH and GSr were required for formation of al 3 conjugates. Represent8^ chromatograms of both rat and human Ever microsomal prot**are shown in Charts 1 and 2. Three GSHVDC metabolite co jugates were designated A. B, and C, in order of elution. Coopgate A (23 min) was present in higher levels in the 2 hum* samples than in the rat samples, while the relative amounts Conjugates B (36 min) and C (39 min) were similar in bo#*,J lH **SHcoi totiiatote *mm ,ot VTtf WbnofOJ ** ion ***" Wf aSt *lh.*jn *WC,but 3 9 Hugaie: kaanjpy CANCER RESEARCH VOL. 45 JANUARY 1985 188 SL 065760 VOC ADDUCTS B were not present in measurable amounts. The presence of measurable levels of a GSH-.VDC metabolite conjugate in the hepatocyte incubation medium indicates that conjugates may be released from the hepatocyte. Alternatively, the extracellular conjugates may be formed outside the cells by VDC metabolites which cross cell membranes to react with extracellular GSH. In order to determine if one or both of these processes occur, unlabeled rat hepatocytes were incubated with 5 mM VDC in medium containing 10 him [3SS]GSH. Cells incubated in this manner accumulated 0.25 nmol [MS]GSH per 10 cells during the incubations, a level which corresponds to approximately 0.5% of the total cellular GSH content and which may represent GSH leakage into a small fraction of nonviable cells. Thus, labeled conjugates would be formed only by VDC metabolites which cross the plasma membrane to react with extracellular GSH. The major conjugate formed extracellulariy was Conjugate C (Table 3). although lesser amounts of Conjugate B were also detected. Conjugate A was not formed outside the ceils at detectable levels. Conjugate B was formed in the medium at approximately 10% of Conjugate C levels, but the amount of each conjugate formed increased with increasing medium GSH content (Table .7 Table 3 fymttkm of <3SH:VDC matabclita con/ugate* by isolated tiepetoeyfes - SWM m hepatocyte* were incubated at a concentration of 2 to 3 x 10* cads/ lafMw1s medurn containing 5 him VOC. After 1 hr of incubation, cells were X&f Moereted from the medium by centrifugation, and GSH:V0C metabolite vwgMMi cdk and medium were analyzed separately as described in 'Materials a Mods' _____ 3). Chemical Characterization of GSH:VDC Metabolite Conju gates. Chemical characterization of GSH-.VDC metabolite con jugates was based on chromatographic properties and chemical degradation of the conjugates. All 3 conjugates required derivatization with FDNB to elute as shown in Chart 1. Conjugate A, which was produced in microsomal preparations nmol conjugate/10* calls/60 min Cells* Medium* Medkjm: Medium; 5 mu GSH 10 mu GSH A* <0.4* <0.4 <0.4 8 4.8 0.8 <0.3 0.6 C 12.5 3.0 1.5 *1.1 11.1 <0.4* 3.9* 45.7* 'lepatocytas were premcubated with ["sjmetbionine to label hepatocyte GSH ' netoead in `Materials and Methods.* I Unmm hepatocytes were incubated m medium containing 5 or 10 mu TCSH (2J mO/mmol). I , w ( S.D.) of 3 individual experiments. 1 Vikm from a single experiment, ft Hen of dlxAcat# experiments. but not in isolated hepatocytes, disappeared from FDNB-treated samples after standing 48 hr at pH 8. Conjugate A was collected from ion-exchange HPLC and subjected to hydrolysis with 2 n HO at 25s for 12 hr. Gas chromatography (electron capture) (17) of ether extracts of the hydrolysate indicated that 2.2-dfchloroacetaldehyde was released during hydrolysis. Further, addition of 2.2-dichloroacetaldehyde to neutral aqueous solutions of GSH, followed by S-carboxymethylation of unreacted GSH with iodoacetate and treatment with FDNB. produced (in addition to the expected GSH and GSSG derivatives) a product with a retention time identical to Conjugate A. Both GSH and N-acetytcysteine reacted at a similar rate with 2.2-dichloroacetaldehyde, *4 fwnan samples. Microsomes from Human Liver 80, which suggesting that a thiohemiacetal rather than a Schiff base was greater levels of covalent protein binding than micro- the reaction product. 'H nuclear magnetic resonance of the N- ro )*Mi from Human Liver 31 (Table 2), also produced more of acetytcysteine adduct in DjO was also consistent with this fit KM3SH conjugate than microsomes from Human Liver 31. interpretation (5.33 4, muitiplet. 1H (CljCti--]; 4.42 4. triplet, 1H tatte total amount of conjugates formed and the relative [--CfcftNHCOCHaJCOiH]; 3.32 4. muitiplet, 1H [--Cb(OH)S--J; Tie ^Portions of individual conjugates were similar in several in- 3.12 4, doublet, 2H [--SCt!r""]i 2.03 4, singlet, 3H ted tattUB-with rat liver microsomes and were unaffected by the [--NHCOCtfeD- els "tasnof 0.3 mg of purified gfcjtathione-S-transferase per ml to Conjugate C coeluted with authentic N-DNP:2-(S-gluta- leinadJeOon mixtures. thiony!)acetate. Add hydrolysis of Conjugate C. prepared using ;rtr Jtatcdon of GSH:VDC Metabolite Conjugates in Isolated microsomes, GSH, and ['*C]VDC, followed by analysis of the ird JjJjtaytes. Isolated rat hepatocytes preincubated with hydrolysate by HPLC, indicated that Conjugate C was deaved I* ptaWonine to label cellular GSH (24) produced [MS]GSH at to 2-(S-cysteinyi)(14C]acetate (Chart 3). The carboxymethylated ISH ta-Hte activity sufficiently high (89 mCi/mmol) to permit de- thiol is an expected VDC metabolite and may be formed by non jd? .-dd | mv [ tiah sif i 7TM GSHtVDC metabolite conjugates both within the cells jtattie surrounding medium. Detectable levels of Conjugates but not of Conjugate A, were found in "S-prelabeled "bv/k*8 <^oniu9a'e C, the major intracellular conjugate, at levels approximately 3-fokJ higher than Conjugate >,, ytagpts C also was found in the incubation medium at conjugation of GSH with VDC oxide, 2-chtoroacetyl chloride, or 2-chloroacetate. This conjugate was stable for at least 48 hr at pH 8. Conjugate B, the major adduct formed in microsomal incuba tions, was only moderately stable at pH 8 and had disappeared completely from derivatized samples within 48 hr. Virtually all of t M' l qtaately 10% of intracellular levels, but Conjugates A and the radioactivity initially appearing as Conjugate B eluted as . -T>, .'7 CANCER RESEARCH VOL 45 JANUARY 1985 189 SL 065761 VDC ADDUCTS n TIm, wiW CMrt3. kxt-Mching* HPLC of 2-fS-cysteinyfH,<C]acMata released Oy add hydrolysis of ,4C-ConjugaM C. '*C-Conjug>ta C was produced usng microsomes, OSH. and ('*CjVOC and cdectad tong ion-exchanga HPLC. following derivataa- ticn with FDNB. The ccscted. darivatlzed '*C-Con|ugate C was subjected to add hydrolysis (S a HO. 110*. 24 hr), and the products ware analyzed using cnexchwigs HPLC. Arrows 1, 2. 3, and 4 mark the retention times of N-(2,4dinitrophenyl) danvatlves of cystine. 2-(S<ystainyl)acetate. 2-(S-glutathio- nyoeostate. and gMathfone dbuiiide, respectively. Chart 4. Substitution of [*S]GSH into unlabaled Conjugate 3 jugate S was prepared by incubating rat liver microsomes with VDv , ... OSH. Alter precipitation of microsomal protein and neutraazau o (*S)QSH (5 uCi) was added. Alter incubatxxi for 30 mm. the - t, denvatized with FONB and analyzed by ion-exchange HPLC a3 , . Materials and Methods.' Arrows mark the retention times of Conjugal = ,. DISCUSSION Conjugate C [W-ONP:2-(S-glutathiony0acetate] in samples treated with FDNB and left standing at pH 8 for 48 hr (Chart 1). Lyophiiized samples of Conjugate B collected from ion-exchange HPLC were subjected to add hydrolysis and oxidative work-up to convert liberated thiols to disulfides. Treatment of Conjugate B with n HO at 110 for 1 hr produced 2-<S-glutathionyl)acetate and GSSG in a 2:1 molar ratio. Hydrolysis of a second sample in 6 n HCI at 110 for 24 hr cleaved Conjugate B to 2-(Sglutathionyljacetate, cystine, and glutamate. Glycine was not retained on the ion-exchange HPLC column used for these analyses. These results suggest that Conjugate B contains 2 GSH molecules bridged by a stable (thioether) linkage and a labile (thioester) linkage. Such abisglutathionyl conjugate would be expected to undergo both transesterification and hydrolysis reactions. To determine if Conjugate B would transacylate ex ogenous thiols, unlabeled Conjugate 8 was generated in microsomes incubated with VDC and unlabeled GSH. After precipita tion of microsomal protein and neutralization to pH 7.5 with KHCOptKOH, ["SjGSH was added, and the mixture was allowed to stand 15 min. The mixture was then derivatized with FDNB and analyzed by HPLC. Although some radiolabeled Conjugate C was detected, moet of the radiolabel appearing in this region of the chromatogram ohited as Conjugate B (Chart 4). The appearance of radioiabei in Conjugate C may be attributed to Scarboxymethylation of ["SjGSH by chioroacetate present in the neutralized supernatant. The presence of radiolabel in Conjugate B indicates that the (^SjGSH substituted for unlabeled GSH in the thioester. Further, when unlabeled GSH was omitted from the original microsomal incubation, [*S]GSH radiolabel did not elute as Conjugate B. In a separate experiment, "S-labeted Conjugate B was derivatized with FDNB, the pH was adjusted to 7.5, and aliquots of the mixture were analyzed periodically by HPLC. Conjugate B underwent a pseudo-first-order decay with a half-life of approximately 3 hr (data not shown). Previous investigators assigned a central role to VDC wMlScfiffft the formation of covalent adducts with cellular nuclec >Ves, \ Min uec 16, 25). The data in Table 1 show that 2-chtoroao- :hn . - MNft of was considerably more reactive toward thiols th ,a> epoxide or other metabolites. While the epoxide was su. - in aqueous buffer, it is most likely that it would also ^c:.. - GSH. These data indicate that both the epoxide and ac.1 cnr. / > Mg -su may modify thiols despite rapid hydrolysis. 2-Chlorozi:e!ii: and 2,2-dichloroacetaldehyde are considerably more sub Ccr***'1 aqueous solution, and while they react more slowly wtm ;r -.. their stability may enhance their overall contribution to cov; f*dL. modification of target macromolecules in vivo. i qsmpriM The importance of protein thiols as major targets fc :o. modification by VDC metabolites is suggested by th: ita ; tWSO 1 sented in Table 2. Inhibition of binding by GSH and r v Vwould suggest that protein thiols are major targe; '-d ' <Mtaj| *W&cn j protein amino groups are minor ones. The possible to otom significance of modifying even a small population of ammo j mm I cannot be discounted. The comparative chemical property Amir thiol and amine nucleophiles may account for the predominant i of thiol adducts. Protonation of amines at physiological pH wd-r j largely restrict the population of available nitrogen nucleopn*" to those lodged in hydrophobic regions of proteins or mem branes. These targets may themselves be exposed to tow* concentrations of VDC metabolites. The selectivity of' DC m*. tabolites for thiols over other nucleophiles may reflect 1 ov*p preference of VDC metabolites for `soft" nucleophiles hre>* the VDC metabolites, 2-chloroacetic acid, 2-chioroacei -fiton* (a-carbon), and VDC oxide, would most likely alkylate v a an S'- mechanism and would be expected to prefer the softer nix*v philes (thiols) to the somewhat harder amines. The data in Table 2 support a role for 2,2-dichloroacetaiden^ in microsomal covalent binding. Approximately 60% of the *Acys ing was inhibited by added alcohol dehydrogenase plus naD'1 indiating that the aldehyde and not the corresponding aico^' CANCER RESEARCH VOL 45 JANUARY 1985 190 SI* 065762 VOC AOOUCTS (,* .*1 'r?M| ' ^ ,wrff xiOe f'S|1? iOf !* tS ttw :udHxi :t witr ;kx*lr C dlX .ole ' itno*1. jaiet)'. i/ait.'"' 3 (XI* lysir* J IflHt ogcd argcij ties o* 'nanc* wouW >pfuk manx low*1 C aveta* iree0* -ilond* inSJ lode- letiyd* j Cur** vIADH I ilcoW ; These observations also suggest that cytosolic i1ffm,,MTal dehydrogenases capable of reducing or oxi2^2^#chloroacetaldehyde may afford partial protection StfeeKuiar damage due to aldehyde production. TjSajctfvity of VDC metabolites toward thiols is indicated by J&Ltha influence of GSH on covalent binding but also the Zfrdi VDC metabolites to form 3 different GSH conjugates. fjjjL, yyorkers have shown that the hepatotoxicity and co- of VDC are potentiated in vivo when hepatic GSH **3 reduced by fasting or diethyimaleate pretreatment (13. Ottw studies have shown that (UCJVDC radiolabel is exjjuJj j(j (he urine as sulfur-containing metabolites apparently iiewd from GSH conjugates (16, 18). The urinary metabolites 3*101 secondary modifications due to renal biotransformation jrtentsrctepatic recirculation which may mask the identities of ftftfidiY formed products. The use of microsomes and isolated lipocytes to study the formation of GSH conjugates was effpfM to minimize such secondary modifications. Conjugate cat be synthesized by adding 2,2-dichloroacetaldehyde trat aqueous solutions of GSH, exhibits properties exrf s-(2.2-dichloro-1 -hvdroxvlethvlQlutathione. a thiohem- jgqflof the aldehyde and GSH. The assignment of a thiohemxttf stnicture, rather than a Schiff base (with the a-amino jNup of the 7-glutamyl residue of GSH) is based on several federations. Both GSH and A/-acety<cysteine (which cannot bjmiSchiff base) react with 2,2-dichloroacetaidehyde at similar iiwin aqueous buffer (data not shown). 'H nuclear magnetic aonsice of the /V-acetytcysteine conjugate also supported a inhemiacetai structure. Mild add hydrolysis of Conjugate A itoed 22-dichloroacetaldehyde. While aldehydes do not gen* Mflyfomi stable thiohemiacetals, introduction of electron-withA#ing u-substituents stabilizes thiohemiacetals formed with ittiydesand thiohemiketals formed with ketones (6). Conjugate B is identified as S-(2-glutathionyl)acetyiglutathione, tibgkitathionyi conjugate formally derived from one molecule <* 2-chlcrDacety) chloride and 2 of GSH. This conjugate is *BPropriatety labile for a thioester and undergoes carbonyl subsWon with added ["S]GSH at neutral pH (Chart 4). The sweteristic reactions of the acyt moiety of Conjugate B are **>*>flaly the same as those for 2-chloroacetyi chloride, i.e., *DWon and hydrolysis. However, the conjugate hydrolyzes 'Wt more slowly than the highly reactive acyl chloride and is teavatable to a greater number of targets for a greater period this case, conjugation serves to prolong the life of a Sf*s VDC metabolite, albeit as a metabolite of somewhat ^Wf raactiVTty. While Conjugate B formally derives from 2gSKfciityl chloride, attack of GSH on the methylene carbon of Wtofofc followed by acylation of a second thiol by the Intermediate, would also yield Conjugate B. However, fW** studfes of VDC oxidation demonstrated that VDC oxide much smaller amounts than 2-chloroacetyi chloride tead to the conclusion that the epoxide is only a minor 3**to the production of Conjugate B. 'dwkfentlty of Conjugate C with 2-(S-glutathionyl)acetate was *x*kirnmediately evident This product is an expected metab- 2S*,,wGd, upon renal biotransformation, give rise to several ^Ported urinary metabolites, including 2-{S-(A/-ace- jpftfofrjyljacetate, thiodiglycollic acid, dithioglycollic acid, affl601*6 acid, and possibly W-acetyHS-(2-hydroxy)- US. 18). Conjugate C is also the most stable of the 3 conjugates, due most likely to the stable thioether linkage between the VDC-derived carbon and cysteine sulfur. Conjugate C may be formed by the direct carboxymethylation of GSH by 2-chloroacetate, or by alkylation of GSH by 2-chloroacetyl chlo ride or VDC oxide, followed by hydrolysis of the intermediate product Conjugate B hydrolysis also may provide a significant source of Conjugate C. The relative amounts of GSH:VDC metabolite conjugates dif fered in microsomal and hepatocyte incubations. The production of conjugates in the microsomal system is probably limited only by the amounts of metabolites generated, their stability, and the concentration of GSH. In contrast to the microsomal system, the formation of GSH:VDC metabolite conjugates in isolated hepatocytes is subject to additional factors present in whole cells. Conjugate C was the major GSH conjugate found in the hepa tocytes, and Conjugate B was found at approximately one-fourth the level of Conjugate C (Table 3). Conjugate A was not found in the hepatocytes. an indication that 2,2-dichloroacetaldehyde produced in the cells is so efficiently oxidized or reduced by cellular dehydrogenases that only negligible amounts are conju gated. The relative ratio of Conjugate B to Conjugate C in hepatocytes is nearly the opposite of that seen in microsomes (Chart 1; Table 3). The reduced levels of Conjugate B in hepa tocytes may be due to reduced availability of 2-chloroacetyl chloride or VDC oxide. A more attractive alternative explanation for the inversion of Conjugate B:C ratios in microsomes versus hepatocytes is a possible contribution of enzymatic hydrolysis of Conjugate B in the hepatocytes. However, the ability of Conju gate B to serve as a substrate for hepatocyte esterases is entirely speculator^. Hepatocytes that were incubated with VDC released measur able amounts of Conjugate C but not Conjugates A or B into the medium. The extracellular conjugate in "S-preiabeied cells may represent conjugate efflux from the cells or conjugate formed outside the cells by VDC metabolites that cross the plasma membrane to react with labeled GSH released into the medium. To determine the extent to which the latter process occurred, unlabeled cells and VDC were incubated in medium containing ["CjGSH. Because hepatocytes cannot accumulate GSH from the medium, only extracefluterty formed conjugates contain ra diolabel. Labeled Conjugates B and C were recovered from the medium (Table 3), demonstrating extracellular formation of both conjugates. Formation of extracellular conjugates increased with increasing medium GSH content Celts that were incubated without added GSH would release approximately 2.5 nmol/hr/ 10* ceils into the medium (5). Assuming all glutathione efflux as GSH, the maximum amount of GSH present outside the cells during a 1-hr incubation would be 5 to 10 n**. In view of the fact that the levels of extraceflulariy formed conjugates with 5 imi medium [*S]GSH were approaching the limits of detection, it is likely that levels of extraceflulariy formed conjugates in *Sprelabeted cells were substantially below detection limits. The conjugates observed in the medium of the "S-prelabeled cells therefore represented efflux of Conjugate C from the ceils. Extracellular formation of Conjugate B demonstrates that 2chloroacetyl chloride and VDC oxide are stable enough to mi grate a considerable distance through ceils and perhaps within a tissue. This phenomenon has been demonstrated previously for metabolites of benzo(a)pyrene (30), dimethyinitrosamine (34), vinyl chloride (10), and trichloroethylene (21). The phenomenon CANCER RESEARCH VOL 45 JANUARY 1985 191 SL 065763 VDC ADDUCTS txt tor discussion. of metabolite ceB-to-ceH migration has been offered as an expla nation for formation of DNA:vinyl chloride metabolite adducts in hepatic nonparenchymal cells, which do not efficiently metabolize vinyl chloride (10). The covalent modification of macromotecules within target cells which do not produce VDC metabolites may be a consequence of metabolite migration. An integrated scheme for the major oxidative and conjugative pathways of VDC metabolism is presented in Chart 5. The pathways presented represent major reactions related to cova lent modification of GSH (and protein thiols). Secondary oxidative and reductive pathways for 2,2-dich<oroacetaldehyde have been omitted as have possible reactions of VDC metabolites with DNA, which will be a subject of further study in this laboratory. The data presented here argue that multiple VDC metabolites participate in the covalent modification of cellular proteins and that protein thiols are major targets. Formation of GSH:VDC metabolite conjugates offers a model for VDC metabolite-protein thiol interaction that presents advantages over the isolation of amino acid adducts via protein hydrolysis. The GSH conjugates may be isolated and quantitated rapidly, and individual conju gates contain characteristic metabolite:thio! linkages. Stability of these conjugates may reasonably approximate the stability of protein thioi:metabo!ite adducts. Interestingly, Jaeger et a/. (14) observed that hepatic [,4C]VDC covalent binding, measured as trichloroacetic acid-insoluble radioactivity, decayed with a halflife of 2 to 3 hr. This figure closely approximates the half-life of Conjugate B. In contrast, hepatic covalent binding of acetami nophen, which apparently forms stable phenyfthioether adducts with protein thiols (32), decays with a half-life of approximately 12 hr (15). These data suggest that a significant fraction of the covalent binding of VDC metabolites consists of labile adducts which are subsequently lost to hydrolysis or dissociation. The consequences of formation of such adducts have not been addressed, but it may be hypothesized that such metastable adducts play a significant role in the initiation of tumohgenic and toxic processes. REFERENCES 1. Bamin. A., 6<ni. H.. Crony. A.. Jacqugnon. p., Maiaveiae. C,, Montesano, a, and Bartsch. H. Liver microaome mediated formation of alkylating agents from vinyl cdonde and vinyl bromide. Biochem. Biophys. Res. Commun., 67: 596-603.1975. 2. Bartsch. H.. MataveMe. C.. Bartn, A., and Ptanche, G. 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F, Carrey. 0.. and Guengench. F P. Purification and characterization of six cytochrome P-450 isozymes from human liver mtcrosomes. Biochemistry, 22 5375-5383.1983. 37. Zaideia. F.. Crtxsy. A.. Bartxn, A., MaiaveiUe, C.. Tomatis, L. and Bartscn. H. Carcmogemcrty of chtoroethytene oxide, an ultimate reactive metabolite of vmyl chtohde. and txs<chtoromethyf)etner after subcutaneous administration and in initiation-promotion experiments m mice. Cancer Res., 40:352-356.1980. `.AtM-il UjM'pi ''.W* ' .im .a , w * -W" >7 if it"1 diffi*" '9 m V* ' xiCOi W i D*/*' ' mcrf X* ..hJ mJH* , rteffl 'ft1* ``'ii: * -I... .'i-i'`Av* , 'r'v, :iS'-;k:- ... .`rti 'VZ'f' CANCER RESEARCH VOL. 45 JANUARY 1985 193 <>57 65