Document wDrXmdyDL4k45X1Z1G66MpowQ

Vol. 76, No. 2, 1977 ' BIOCHEMICAL AND DIOPHYSICAL RESC/'KCH COMMUNICATIONS ALKYLATION OF DNA AND PRO fr. INS III :;ICL EXPOSED TO VINYL CHLORIDE S. Osterman-Golkar, D. Ilultmork, 0. f^qci'bdcl'-, C.J. Calleman, R. Giithe, L. Ehrenbcrg ai:<i C. A. Wachtmeister Wallenberg Laboratory, University of Stockholm, Lilia Frescati, S-104 05 StockheUi Sr, Sweden Received March 22/1977 SUMMARY: Experiments with mice show that the pre-carcinogen vinyl chloride i: mctaSoTically converted to a short-lived aiiy.ating intermediate which introduces the 2-oxoethyl group onto nucleophilic sites in DHA and proteins. The absolute and relative amounts of alkylated product; support the hypothesis that the main reactive metabolite is chlorooLhylene oxide. INTRODUCTION ; The degree of alkylation of specific cmino acids in Hb* or erythrocytes may be used as-a measure of the hi vivo dose be1:; of electrcphilic agents and of cotr.pounds which are metabolically convert1-.! To electrophilic agents (1). The present study on vinyl chloride is the basis .if work in progress at the Wallenberg Laboratory on a) factors modifying the fmm'f.on in vivo of electrophilic inter mediates from vinyl chloride and b) the de-vicement of a gas-chromatographic method to alkylated amino acids in Hb fro;.: ran occupationally exposed to vinyl chloride. Exposure of a model nucleophile, 3 ,*T 'diahlorobcnaenelhiol to vinyl chloride in a microsomal system in vitro yields an Alkylated product, identified as 3,A-dichlorophenylmercaptoacetaldehyde (2). This could result irom chloroethylene oxide or chloroacotaldohydc as reactive mcUbolius of vinyl chloride. In their react ions with nucleophiles, both these compeuu-h yield alkylated products containing the 2-oxoethyl group. Reduction of the 2-oxoethyl groups i t.h sodium borohydride gives the possi bility of determining the degree of alkylation of macromolecules from the amounts of S-(2-hydroxycthyl)cysteir.c and (N-l-ond `l-3-)hydroxyethylhistidine or N-7- 'Abbreviations: Hb, globin precipitate of haemoglobin; HOCtCys, S-(2-hydroxyethyl jcysteinc; HOEtHis, (N-l-and t:-3-)hyilroxyethylhistidine; HOEtGua, N-7- -hydroxyethylguanine. Copyright 1977 by Academic Prat, hu\ All rights of reproduction in eiuy form reserved. 259 ISSN 0006*29IX ' vl `V'X.Vjy ,v'(".k * y!'". R & S 027QQQ Voi. 76, No. 2, 1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS -hydroxy; tkyluuanir.c in hydrolysates from proteins and nucleic acids, respectively. The present investigation comprises quantitative measurement of these alkylated products in IIP, tcsLcs proteins and liver DJIA after exposure of mice to '^C-label led vinyl cnloride. Tne -act that vinyl chloride binds covalently to proteins and RNA in the presence f fortified microsomal preparations has been demonstrated earlier (3). lio charjc.or'zation of the mode of binding to macronolecules in vivo has been published, MATERMETHODS A n i 11 s . ; mice frcn three strains. CBA, 3AL3 and ATL, were used. The mice `..ere "c- ' - ths old (average weight 20 g), They were f d on a standard pellet diet and ,ii- wi theut restriction. Chermcal t. inyl * 11 ,2-^C | chloride, specific activity 0.43 aiCi/mmol , radio- C;'4Tr.Ci'i i fy 09'., -(,55 purchased from Hew England iTuclear, Boston, Mass. The vinyl chi . ` was dissolved in m-xylene to obtain 0.1 ciCi/ml xylene. Chloro- ethyl ere c : d.-> and ciiloroacetaldehyde were prepared as described earlier (4). Hydroxys: cysteine was synthesized according to ,!ilkha and Rappoport (5) and the tv.C 1.; crin hydroxyetiiylhistiaines were synthesized by reacting ethylene oxide v.'N'. -acotylhiscidine methyl ester in m.etanol-HC1 , followed by hydrolysis of tr.n seed "t (6), ryicoxyetny 1 au-mine was synthesized from guanosine and ethyl ere ; ,e in nc: r.ic acid (7, 8). 1:-so.-uv.- . ;`-A -ini,.". Is to vinyl chloride. Ten to twelve mice at a time were "v/nVd'"vTiyi chloride ui an 11 1 all-glass system. The vinyl chloride c frcii t:.e solvent at O^C into an evacuated 150 ml vessel and was : "j to the treatment chamber by flushing with air (600 ml), in the chamber was circulated by a propeller and the expired COj sn an Accarite layer. The pressure was kept constant by passing a U SI f 0Z through a capillary inlet. The atmosphere was analyzed for `iis by taking 2 ml sam.oles by a gas syringe and rapidly injecting i. ;h a small membrane on tho cover of a scintillation flask, pre- filled .. : .nl of scintillation liquid (5 g PP0 and 0.C5 g P0RQP/1 toluene), !n . i frol e.-perii'ient the 0? concentration in Nip chamber was measured as follows: :ow stream of 00? was Ted tlirounh 1 ml of water in a plastic cell ('opt ' .) with a Clark-typo oxygen electrode connected with a recorder. Air Stmnli'S i rapidly injected into the CO2 stream. The O2 level was constant over a ; - i .'! of 11 hours with 10 mice in the chamber. Ter !-. .-Ives tie layout of the experiments with regard to the number of aniR.il; w "i-il, duration of exposure, doses expressed in ppm-h and time from And of , r ...re to tilling. Table 1 also gives the half-life to the radio- ictiv'l r. : ' /n.' aii' :nd the total radioactiv1fy in Hb. Isoiati.- 1 ,J1. of!;Ar tissue proteins and DJIA. After anesthetizing the animals th.. i o*., ' . re MS was collected and suspended in 0.9,, JinCl solution con- ; 1 r* :,n, ' i`.e -rythrocytcs were gently spun down and thoroughly washed with 0.'- The cells were lyzcd with water and cell membranes and debris were ;! i 000 g for 10 min. The supernatant was dialyzed over night O J fl 1 1 i *1 C ! .to buffer pll 7 ind was then brought to pH 4 by addition of Ifltt j" ` 1 *i;cr 3 .1 the pH was cautiously brought to pll 6 arid half of the , Uijj" : d> '`IhCn t.iih sodium bor.ihydrido (50 mj/10 ml). The glcbin was pre- .'f a.i li cion of 10 volumes of 1 tiCl in acetone (9) and was then ively with IIC1-acetone, 5'. aqueous TCA, ethanol and ethyl ether. Table Exot. A ) ' ' (_ phenol ; the ae, buffer ethai e 1 liv.iroiT.yj-~ -'vacua ' d i sso1 2-chloi pi 0 to in' aeded a' (SO x 1 er fleer 170 ml respect corresp (0.9 x f011 ewe 4 3 and acid; v; S.. After ., B-awex E hvdro.v. i:mO*l SUT-? Radicac scinti I and com ion-exC The cou r-VacHC tor me with Hu. The dat -'iy. 'll ed ) ide V Vol. 76, No. 2, 1977 BIOCHEMICAL AMO BIOPHYSICAL RESEARCH COMMUNICATIONS Table I. Details of the layout of the experiments A, C and C. Expt. Number of mice and Strain Duration of exposure (h) Dose Half-1ife (ppm-h) of act. in the air <h) Time from end of exposure to killing (h) Tot. act. in Hh (dpn/mg) A 11 CCA 10 160 3.3 B 4 CBA 10a> 302 1 .2 4 BALE 4 ATI C 3 CBA 2.33a) 98 0.9 7 BAIB 12 12 1 1 3 25 - 16 38 14 7.7 14.2 14.0 15.6 ^^C-labelled vinyl chloride was added at intervals to maintain a sufficently high vinyl chloride concentration ir, the treatment chamber (in expt. 3 every 2 h, in expt. C every 50 nun). Proteins and nucleic acids from livers and testes were separatee! by the phenol m-cresol -hydroxyqui no! i ne net hod (10). The DNA was precipitated from the aqueous phase by 2 volures of 2~elho/,',-ethanol , dissolved in 0.1 M^fhcsphate buffer pH 7, reduced wi tii sod 1 uni borohydrid; and reprec i p i ta ted with 2-othoxy- ethanol. Ilydrolysis and ion-excha_nne_chromatoqraohy of proteins anti QUA. Samples of 10'0~300~T of protein were nynrolyzea with 6 .*< hcT liupupu r (1 mg / 0.1 r;l) in evacuated tubes at 120C for 15 ii. After evaporation to dryness the product was dissolved m 2 - 5 ml of 0.1 11 HC1 and incubated at 37C for 1 h to hydrolyze 2-chioroethylcysteine which forms from part of the hydroxyethylcystei ne during protein hydrolysis. Hydroxyethylcysteme and the two hydroxyo thyl hi s ti di nes wore added as carriers. The amino acids were then separated on a Dowex 5C..'-.\4 column (80 x 1 cm) which was eluted with 400 mi of 1 II KC1 followed by 2 II KC1 . The effluent was collected in 4.5 - 5 ml fractions. Hydroxyethylcysteme eluted after 170 ml 1 H HC1 , and the hydroxyethylhisfiriines after 1G0 ml a no 190 ml 2 II HCi , respectively. To achieve a better separation of the histidine products, the correspondin'] fractions i.'S-ru re-chrunatograpncd on an Aminex A-5 column (0.9 x 20 ). The- column v.v eluted with 160 ml 0.12 M citrate buffer pH 4.26, followed by 0.12 1-1 buffer pH 5.30. The two hydroxyethyl hi stidines eluted after 43 and 53 ml, respectively, of the last-mentioned buffer. The presence of amino acids was detected by thin layer chromatonraphy (1). 0 Samples of about 20 ng DNA were Iv/drolyzed in 1 II HCI for 1 h at 100 C. After addition of hydroxyethylguaninc, the hydrolysates were chromatographed on a Dowex SOW-'XI2 column (18 x 1.1 cm) with 1 II HCI. The fractions containing hydroxycthy 1quanine (eluted after ca. 700 ml) were evaporated to dryness and measured for radioactivity. Radioactivity determinations were made using an Intertechnique SI 30 liquid "scTTuTlTaViun "spec"tron'etcrT~Rrotein samples were dissolved in 1 ml 0.01 II NaQH and counted after addition of if) ml of Instaoel (Pacl.ard). Tractions from the ion-exchange columns were counted after addition of equal amounts or Instagel. The counting efficiency was determined using automatic external standardization. Reaction Sureties of conceivable vinyl chloride refnlipl i tes. The rate constants Tor tnV^-caelTciis o7 chlorocThy'Iene o'-V.Te', chToroa'ceta'Iuc'hy'de and chloroethanol with nucleophiles in water were determined by methods described eailier (10, 12). The data arc partly published in another context ( 1 3). 2G1 L O lZ 0 C.:.:.-^1 i~Vi;,< j w?;.--,y-.Si :,Vf-Yitr *;..& -cry Vol. 76, No. 2, 1977 EIOCHL/.MCAL AND BIOPHYSICAL RH5LARCH COMMUNICATIONS Vol. 76, ,20 10 , R&S 027011 Fin. la, Part of the ion-exchange separation of acidic amino acids from a hydro lysate o* lib without j`-rccc: d i n f] reduction of Hb, experiment A. Tito po sitions of "'C.v: ` nown u.'iino .icids arc indicated 1) serine 7) aspartic acid 3) rjl/cine 'i) *'. Siiine 5) liOLtCys. Hi. I ho sate fr'.rt.ioiis as iii la but after reduction of the lib (333 mg) jr. Part of Voe ion-exchange separation on Aminex A-S of basic amino acids from a hydro I,"at* of reduced lib (333 raj), experiment A. The positions of the two isomeric !iO'dthis, b and 7, are indicated. PL'suus and ir.:r:jon The concc-n r.U.iun of vinyl chloride in the inhalation chamber decreased ex ponentially with ti.ve. From Table I it is clear that an increased proportion of EAL8 2(12 T-Vc v f > , \ -f ^ "`.i'kz.'izt*J?/"?* *! `Xtf-tTr *'! Vol.76, Wj. 2. 1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COM/AUHICATIONS R&S 027012 -d exof I'All! :ig. ton-e/.clnnge separation of reduced liver DMA, experiment B. 24 mg Dr;.', from GALB nice, 2b. 8.7 mg QUA from C3A nice. mice in the treatment led to a faster clearance of vinyl chloride from the system. The total incorporation of radioactivity found in Hb from BALE mice is about twice as hir:,. at that Of the strains C13A and ATI. These observations are consistent with the rii.ding that the rate of uptake of vinyl chloride reflects the rate of meta bolic conversion of 'Hnyl chloride {14, 15). Tlx* elution patterns Of chromatographed hydrolysates Of Hb and liver DMA are Shown in Pigs. 1 and 2, respectively. Pig. la shows part of the chromatogram after hydrolysis of I'b without preceding borohydride reduction, and Pig. lb shows the same fractions after such reduction. Only in the latter case is a radioactive peak corresponding to hydroxyethylcysteine obtained. The basic fractions, Fig. lc, show the appearance of the two isomeric hydroxycthylhistidims in the hydrolysate of reduced lib. The hydrolysate of unreduced material contained no radioactivity in the corresponding fractions. The hydroxycthylguanine recovered after hydrolysis of reduced DMA presents a peak which is well separated from that of guanine {Fig. 2a, b). The labelling of the latter has to be ascribed to metabolic incorporation of ^C. These results show that vinyl chloride through metabolic activation gives 203 ' '" 1 :'?l'- ** ^ ' ^.......... - ...................................... Vcl. 76, No. 2, 1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Table II. Rate constants for reactions of chloroethylcne oxide, chloroacetaldehyde, chloroetlianol and, for comparison, ethylene oxide (cf. 17) with diffe rent nucleophiles in water; ku n in h , other constants in l-molo'1*!!" . "H 20 Alkylating agent Nucleophile k (20C) k (37C) ,Ja) Chloroethylene oxide Chloroacetaldehyde Z-Chloroethanol Ethylene oxide h2o aniline W H,0 S2U3 sb20u3 HjO <o O l LJ 6.9 820 13-103 52 2*10"6 13 . 4*10-2 0.8 1.3 0.96 a^Ths s-value describes the sensitivity of an alkylating agent to changes in the nucleophilic strength, n, of a nucleopnile. The s-value is calculated from the Swain-Scott equation (cf. e.g. refs. 11 and 17). R&S 027013 rise to an alkylating agent which introduces the 2-oxoethyl group onto nucleo philic sites. It may also be concluded that alkylation by chloroetlianol is negligible. The degree of alkylation, [ry] / [Y] , of a nucleophilic group Y in vivo is proportior.a 1 to the rate constant, k^, for the reaction of the alkylating agent RX i/ith Y, to the initial concentration (or the total amount formed) of RX, , and to its mean-life, 1/A, in the immediate environment of Y, according to the equation Qtfl/M = ky - [RXJo l/A [1] where >. is the rate constant for the disappearance of RX (1, 8, 17). The rate constants for the reactions of chloroacetaldehydc and chloro ethylcne oxide `with amino acids in lib and 'with guanine-ft-7 in DilA are not known, but may be estimated from known rate constants for reactions with simple nucleo philes (Table II) and by comparison with corresponding values for ethylene oxide (1, 3, 17). Estimated values of the rate constants for the reaction of histidine in lib with chloroacetaldehyde and chloroethylcne oxide at 37C are 3-10 and 0.1 l-(g lib) '-h respectively. 26-1 Vol. 76, No. 2, 1977 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Table III. Degree of alkylation of cysteine and histidine of haemoglobin and guaninc-H-7 in liver 0?IA. Expt. A B Strain HOEtCys (nmole/g Hb) CBA CBA BALB 0.17 1.5 3.4 The two iso meric llOEttlis (nmole/g Hb) 0.21 0.07 1.9 O.B 4.2 1.3 HOEtGua (nmole/g CHA) 0.60.4 4.3 2.6 Insertion Of this rate constant of chloroacetaldchyde into eqn lj shows that, in order to account for the found degree of alkylation of Hb, it has to be assumed that practically all vinyl chloride contained in the alveolar venti lation (8) is converted to chloroacetaldehyde, and that this compound has a mean retention time (1/x) of several hours. These are very unlikely assumptions. On the contrary, if vinyl chloride is converted to chloroethylcne oxide, the r.oan-life of this compound could bo estimated to he in the order of seconds, which seems very reasonable. Considering the s-values of Lise proposed intermediates (Table II) and the fact that cysteine and histidine residues differ by about 2 units in the Swain-Scott nucleophilicity scale (cf. 17), the ratio of cysteine co histidine alkyl ation in Hb is expected to be 2 times lower for chloroethylcne oxide and about S times higher for chloroacetaldehyde than for ethylene oxide. The experiments showed this ratio to be 0.56 (cf. Table III) compared to 0.75 for ethylene oxide (17). This further strengthens the conclusion that chloroethylcne oxide is the main reactive metabolite. The degree of alkylation of guanine-tl-7 in DIIA is close to that expected on the basis of Hb data, assuming the dose to be equal in the liver and the blood (cf. 1, 8, 17). BALQ mice show a higher ratio of Hb to DiTA alkylation (Table III), and a higher ratio of metabolic ineorporat ion to alkylation of guanine in Dllft (fig. 2) than CCA mice. Tim indicates that there are Strain differences at several levels. Small amounts of the two hydroxyethylhistidines were found in proteins from 2C5 R&S 027014 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS pooled testes (12 and 6 dpm, respect)vely, in 134 mg- of testis proteins from BAL8 mice, expts. B and C). Thus the male gonads are exposed to the alkylating intermediate and a risk of heritable damage, in addition to the cancer risk must be expected. Further studies on the recovery of alkylation products by this method, and studies on the relative role of alternative pathways, e.g. formation of imidazo derivatives with adenosine and cytidine (18), are required for meaningful risk evaluations. ACKNOWLEDGEMENT This work 'was financially supported by the Swedish Board of Occuoational Safety and Health, KenaNord AB, Stockholm, Sweden, Norsk Hydro A/S, Oslo, Norway, and Pekema Oy, Helsinki, Finland. REFERENCES Ustlrman-Golkar, S. , Ehrenberg, L., Segerback, D. and Hallstrom, (1976) Mu tat. Res., 34, 1-10. Gothe, R., Calleman,"C.J., Ehrenberg, L. and Wachtmeister, C. A. (1974) Ambio. 3, 234-236. Bolt, H. M. , Kappus, H., Buchtcr, A. and Bolt, W. ( 1 975) Lancet, June 28, p. 1425. Rannug, U., Gotne, R. and Wachtmeister, C. A. (1976) Cnem.-Biol. Interact. 12, 251-263. Zilkha, A. and Rappoport, S. (1963) J. Org. Chem., 23, .105-1107. Calleman, C, J., to be published. 3rooke^f P. and Lawley, P. 0,(1961) J. Chem * Soc., 39t3'394S. Ehrenberg, L., Hiesche, K. 0., 0sterman-Golkar, S. and Wennberg, ( 1974) Mutat. Res., 24, 83-103. Anson, M. L. and MirsTy, A. E- (1930) J. Gen. Physiol,. 13. 469-476. ..10. Kirby, K. S., Pox-Carter, E. and Guest, M. (1967) Biochem. J. 204, 2 S8"Z 6 2 11. Ostermaii-Golkar, S., Ehrenberg, L. and Wachtmeister, C.A. (1370) Radiat. Got., 10, 303-327. 12. Ehrenberg, L.,"5sterman-Golkar, S., Singh, D. and Lundqvist, U. (1974) Radiat. Got., 15, 185-194. 13. Hussain, S. and 0sterman-Golkar, S. (1976) Chem. Biol. Interact., 12. 265-267. , s, U. Kit, H, 11., Kappus, H., Buchter, A. and Bolt, VI. ( 1976) Arch. Toxicol., 35, 153-162. ,,v 15. Hefner, R.~T. , Watanabe, P. G. and Gehnng, P, J- ( 1975) Ann. N. Y. Acad. Sci., 246, 135-148. 16. Bartsch, H. and Montcsano, R. (1975) Mutat. Res., 31., 93-114. 17. Osterman-Golkar, S. Doctoral thesis, Stockholm 1975 and unpublished 18. Barrio, J. R., Secrist, J. A. andleonard, N. J. ( 1972) Biochem. Biophys. Res. Commun., 46, 597-604. CD Rea; Si.'MM t..e AM At' 'iMm' - I