Document n8x39Zkn2EBxqb2dGkzOyw5a
Ini. J Cancer: 16. 6.19-644 (I975j
Rrpnnlrd from the lnlrrn.tlional Journal of Canter
(^) In-crruiional Union Agaimi Cancer
Printed in SilJrlan<)
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MUTATION INDUCTION IN CHINESE HAMSTER V79 CELLS
BY TWO VINYL CHLORIDE METABOLITES.
CHLOROETHYLENF OXIDE AND 2-CHLOROACETALDEHYDE
by
I.Inver 1 li.'iii kman. Helmut ll,M<nni 1 .mil l.eo Samis
Department at Cenelii s. Ueizniinoi Institute o) Sclent e, Rehosoth. Israel, and ' International Ayem r /or Research an Canter, Unit a! Chemical Carcinogenesis.
Ctt (ours Alhet t I homos. CAHIS l.volts. hranee
( Idoroelliylene n\nlc and 2-cliloroaci`tahleln de, two possibly t ttrcinnttcnie metahalites a/ vinvl cll/nritlc in mammals, caused a tlnsc-depcntlcnl induction of R-azattiiimineanil tmaliain-rc.sislani mutants in Chinese hamster l'7d cells m vitro. Cp t" onc-hinuhedfold higher concentrations id 2-chioroelhtinol m mullnc/doi tmce/ic aeid. a nnntirs vinsl chloride metabolite in rots anti man. seere inactive.
I he various aiKerse hiological elVeels of s ins I et td.. 1974) 2-Chlo;oacetaldchyde (111) and
chloride (I, Fig I), Us toxicity (Pally el a!., 1963; chloioethy lene oxide (II) have been identified
Tribukh el al,, 1949) anil carcinogenicity in as vinyl chloride metabolites by in sitro tech
experimental animals and in man (Creech and niques (Bartsch and Montesano, 1975; Clothe
Johnson. 1974; IARC. 1974m 1975; Fee and et td. J974); monochloroacetic acid (IV) is a
Harry, 1974; Maltoni and l.efemme, 1974; urinary metabolite in rats and man exposed to
Viola el a!.. 1971 ). iis mutagenic action m micro vinyl chloride (Cirigorescu and Toba. 1966;
bial systems (Bartsch et til.. 1975; Bartsch and Helner et id.. 1975). Because the relationship
Montesano. 1975; Malavedle et at.. 1975; between carcinogenicity and mutagenicity is
Foprieno et nl.. 1975; Karmug et id.. 1974) and becoming more formally established (Miller and
perhaps its chromosomal breaking activity in Miller, 1971), we have studied the mutagenic
human cells (Ducatman et til. 1975; F'uncs- ell'ect of these compounds in mammalian cells.
Cravioto et til.. 1975) appear to be largely We have used Chinese hamster V79 cells, a
dependent upon its metabolic conversion into mammalian cell system that has already been
chemically reactive metabolites. In vitro studies successfully used for the detection of other
have shown that microsomal hepatic mixed- chemical carcinogens and/or their metabolites
funciion oxidases of rats, mice and humans are as mutagens (Huhermen et id.. 1971; Huherman
equally effective m metabolizing vinyl chloride et td.. 1972; Huherman and Sachs, 1974).
into reactive metabolites (Bartsch et id.. 1975;
Bartsch and Montesano. 1975).
2-Chloroethanol has been suggested as a possible intermediate in vinyl chloride meta
('hemietd.s
MAII l< 1AI AM) Millions
bolism in rats (Hefner et id.. 1975) and exhibits
C'hloroethylene oxide (b.p., 65-67 C) was
a weak mutagenic activity in hacteria (Bartsch prepared by chlorination of ethylene oxide in
et at., 1975; Malavcille et ah. 1975; Rosenkran/* 1 the gas phase (Cross and Freiberg. 1969) by
Received: May 20. 1975.
1 Correspondence for this author should he addressed lo the Inlernaiional Ageiics lor Research on Cancer.
1
111 (IIIUIW I I \ |
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Hu>-1r:insl<lrnKLl(iin <>l vinvl lh11<11111,- 111 in thi**rn.-lli>lone ovule (111. wills'll rearranges In 2-chIt>ri'.n.t-1.iKlt.11^tic (III) I lie killer is luithci uvuli/ed In tnunochloruaceiic acid (1V|. .i urin.irv inctuboluc in Imnuih and int-. lollnu mg their cvposurc In vinyl chlunde
Drs. A Cioisy and l` .lacquignon. (NRS, Clil'-sur-'i'\cue, Irancc I he compound. which contained traces ol' 2-chlornacotaldehyde I I and eihylene ovide ( 5",,). was stored at he low
M) Undei these conditions no change in the NMR spe< trum (in CIX'I,) was noted after 12 days. 2-C'hlorottcetaldehyde (50",, aqueous solution) and monochlomaeeiie acid (m.p,, hi -63 C) were obtained from Rluka Co., Buchs. Switzerland and 2-chloroethanoI (h.p.. 120130 C) from Merek-Sehuehardt, Darmstadt, l-ederal Republic of Ciermany. K-A/aguamne and ouabain weie purchased from Sigma Chemical Co., St. Louis, Mo., USA
Cell cull ine.',
The V79 cells denied from a clone isolated from the male Chinese hamster cell line V7V-4. kindly supplied by Dr, L. M. Y Clui, Universits of Michigan, Ann Arbor, Mich. USA. Cells weie grown m Ragle's medium containing a four-fold concentration of amino acids and vitamins and 10",, foetal calf serum ; 5 ml medium were used per 50 mm plastic Rein dish (Nunclnn. Nunc-A/S. Roshilde, Denmatk), and the cultuics were incubated at 37 0 5 C m a humidified meubatot supplied with a constant llow ol lt)",, CO, m air. No detectable mvcoplusmic con tamination was found when the cultures were tested on mycoplasma agar (Chanock cl a!.. I%2|. Stock solutions of 2(K)-fold conccntiatioiis of S-a/aguamne in dimethylsulphoside (DMSOi were piepated and stored for up to 4 weeks at
20 ( I he Imal concentration ol DMM) m the medium a..is 0.5it did not ailed the cloniue cllieicncv of V7V tells
( l Inin Ill'll l II will
Dishes weie seeded with 2(X) sells pci dish Alter I (>-1N h. various concentrations ol the compouiKls to be tested were added lot 3 h. alter which Ircsli medium was added Alter incubation lor n-Is days, the ilishes were lived and tlie colonies counted l.acli point of cloning ellicteney is based on an average value taken from 5-h dishes Ol 200 cells plated, about W , weie still single cells IN hours later.
\ /nluci'iiit 111 imi/i
Selection of N-a/uguanine- or ouabain-resistant mutants was pet formed in a sinnlui manner to that described above1 16-IN h after seeding. 2 It)1 sells dish for N-a/aguaninc resistance and I 10' ccllsAlish for ouabain ie..istance, the cells were treated for 3 h with dillerent coikciitiations of the compounds to be tested, I resit medium was then added and the cells weie incubated for an additional 4N h. 1 or selection ol N-u/ugunnine-rcsisiani colonies, the cultures weie re-led cveiy 2 days with medium containing 0 2 mst N-a/aguanmc. usually for 10-12 days, lor selection of ouabain-resistant colonies, the cultures were tic.tied once with I mw ouabain lor 14-16 days At the end of treatment, the colonies weie lived and stained: the numhei
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R&S 026401
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of cells was determined from 10-12 dishes pei value for X-u/aguaninc resistance and from 32-40 dishes per value for ouabain resistance. The heritability of this resistance was shown from experiments with nine isolated ouahainrcsistant colonies (Huberman and Sachs, un published) and 17 isolated X-a/aguaninc-rcxixtant colonics (Huberman cr al., 1971. Huberman and Sachs, 1974), all of which remained resistant to the drug used for selection after growth for I month in the absence of X-a/aguanine. Mutation frequency was calculated for 10'' or 1 ()*' survivors, taking into account the number of cells seeded and the cytotoxic cllcct of the mutagen.
KKSUI.'JS
The mutagenic activity of chloroethylene oxide and its rearrangement product 2-chloroacctaldchyde (Gross and (-"reiberg, 1969; Zief and Schramm, 1964) was assayed in Chinese hamster V79 cells. Mutation frequency was scored with two dilTcrcm genetic markers either by resistance to X-a/aguanine (Chu and Mailing, I96X) or by resistance to the steroidal compound ouabain (Hakcr cl al., 1974). The mutation frequency has been expressed as the number of X-a/aguanine- or ouabain-resistant mutants per 10'' and 10*' survivors, respectively.
The mutagenic response increased as a function of the concentration of chloroethylene oxide and 2-chloroaeeialdehyde in the medium (Table I). Al a concentration of 6 //m, chlotoetliylene oxide or 2-chloroacclnldchyde mutation frequency of X-a/aguanine resistance reached 4 or 13 times that of the control. Al the same concentration, the number of ouabain-resistant mutants per 10" survivors was 10 limes higher than in the control with chloroethylene oxide hut only twice as high as in the control with 2-chloi oacetaldehyde. Al a concentration of 13 ,/im chloroethylene oxide, the mutation fre quency. determined with X-a/aguanine or ouabain resistance, reached X or 23 limes, respectively, the spontaneous mutation frequency, while the cloning ethciency was 45",,. 2-C`hloroacetaldcliyde, however, at 13 ,m, had strong cytotoxici ty, and cloning elliciency was less than 0.1 thus, no mutagenic effect was detectable. Helow a concentration of 2.5 mst, 2-cbloroeihanol or monochloroacetic acid had no activity in inducing X-a/aguanine- and ouabain-resistant mutants.
discussion
The data, with mammalian cells, confirm previous icsulls concerning the mutagenicity of 2-chloroethylene oxide and 2-chloroacetal-
R&S 026402
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delivde m .9. typhiinnriion strum I A 1 5.10 (Ikn tsch i'i til.. 1975; Malavcillc cr til.. 1975). As is evident from Table I. diloroethylcnc oxide caused a mutagenic response with a much lower toxicity as eomptited to that of 2-chloroaceialdchyde tit equimolar concentration: the same result was obtained with microbial indicatots (Malavcillc ct til., 1975). Thus, 2-chloroelhylene oxide acts as a mutagen per sc and not exclusively vio its spontaneous rearrangement product 2-chloroaceluldchydc (Gross and l-'tciberg. 1909; /Jell' and Schramm, 1964). The strong mutagenicity of these two compounds, as compared to that of 2-chloroethanol and monochloroacetic acid, points to a possible role for chloroeihyiene oxide and 2-chloroacetaldehyde in vinyl chloride carcinogenesis, since it is now recognized that the majority of chemical carcinogens, some of which cause cancer in man, have been found to be mutagens when tested as their ultimate reactive forms or when the parent compounds were combined with an in vitro or in vivo metabolic activation system (Miller and Miller. 1971; Huberman and Sachs, 1974).
The genetic activity of chloroeihyiene oxide can be ascribed to its strong alkylating potency which can be shown in assays with 4-(/r-nurobenzyl) pyridine (Bartsch and Montesano, 1975, Malavcillc ct a!., 1975). This properly, as well as its short half-life in aqueous solution at neutrality (ill 1.6 min), links this compound to the group of highly reactive u-chlormated aliphatic ethers, among which his (chloromcthyl) ether (if;- 2 min (Van Duuren ct ol.. 1972]) is carcinogenic m animals and in man (IARC. 19741>).
2-Chloroacctaldehydc is also a chemically reactive compound, although its rate of hydrolysis in aqueous solution dill'ers from that of chlorocthylenc oxide by a factor of I ,(XX) (i'j 24 h [Van Duuren ct til., 1972]), and under conditions whereby chloroeihyiene oxide reacts with 4-ipnitrobenzyl) pyridine, 2-chloroacetaldehyde shows no detectable rate of reaction (Bartsch and Montesano, 1975; Malavcillc ct til., 1975). At acidic pit, it reacts slowly with adenosine or cylidine to give cyclic fluorescent derivatives which have been characterized as 2 /f-D-ribofuranosyl-imida/o-(2,l-i)purine or 5,5-dihydro-5oxo-5-/>-D-ribofuranosyl-imida/o (1,2-c) pyrimi dine (Barrio ct a!.. 1972). Alterations of this kind in DNA activity may explain the ellecl of 2-
chloroacetaldehydc as a mutagen in bacterial (Malavcillc cl ol., 1975) and mammalian cells Gable I). I he role of 2-ehloroacelaldchydc m carcinogenesis still needs to be studied.
Although we have obtained, evidence that chloroeihyiene oxide is formed as a primary metaholile of vinyl chloride by microsomal enzymes in vitro (Bartsch and Montesano. 1975). the significance of this compound in vinyl chloride carcinogenesis can be indicated only by circumstantial evidence: chloroeihyiene oxide is a strong mutagenic and alkylating agent, mid epoxides are now recognized as obligatory inter mediates in the metabolism of many olefins by hepatic microsomal mixed-function oxidases (Daly ct a/.. 1972). Compounds structurally related to chloroeihyiene oxide, such as 1,2epoxybutcne-.l or epichlorhydrinc, are known to be carcinogenic, while 2-chloroaceialdehvde (assayed as diethylacctal) and monochloroacetic acid have been reported to induce no turnouts (Van Duuren cl til.. 1972. 1974).
Alternative pathways of metabolic activation of vynil chloride to ultimate carcinogenic forms cannot be extended, i.c. tin enzymic dediloi ill ation (Ciandolli and Van Dyk, 1973) of wnyl chloiide to produce ethylene which may he further oxidized to ethylene oxide, a mutagenic compound (Khrenberg and Ciustafsson. 1957) Studies on the nature of the binding products of vinyl chloride and/or Us metabolites to cellular macromolecules are in progress.
Mill AUDIO IN I'KI.SS
Since completion of this work, McCann cr ol. (1975) have teported a pronounced mutagenic effect of vinyl chloride and 2-chloroacetaldehydc m S. lyp/ninnrinni TAI(X). while the concept of involvement of chloroeihyiene oxitle m the covalent binding of ''C-labclled vinyl chloride to cellular macromolccules mediated by a rat liver microsomal system has heen strongly supported by the results of Kappus ct ol. (1975).
AC KNOW1.I.IX.II.MI n is
The authors acknowledge the excellent tech nical assistance of Miss Sara Etkin and Mr. Arie Goldcnberg, and the help of Mrs. Elizabeth Ward in preparing this manuscript.
This study was supported by the National Cancer Institute. USA, Contracts NOI-CP-5563C) and NOI-CP-0221 7.
642
iataiifia i r &s 026403
i
Ml/rAliF.MCI r Y Of | WO VINYL CIU.OKim MIIAHOIIUS
MUTAGl-NICITf- Dll DLIJX MI-TAtiOLH'LS DU C'HLORURH DU VINVLU. LU CHLOROLTHYLFNL OX YD H UT LA 2-CHLOROACLTALDHYDL, DANS 15US CLLLULLS V79
DE HAMSTURS C'HlNOiS
Lev cellules L79 lie hamster cliiniiis penvent etre miners cf/icaccment cl devenir resisttnues a la R-azaynnninc an la onahalnc, en Junction ilc la dose nrilixec, par le chlorocthylenc iixyile el la 2-chloracict'tnlilchyde, deux metabolites da cldorare tie vinyle ehez lex mammi/eres. Acer ties concentrations cent Joix pins lanes, le 2-chloroelhantd an / acide monochloroacelit/ue, an metabolite nrinaire tin chlnrnre tie via vie die: le rat ct Vhamme, stmt inacti/s.
Kl.l I'KI Nf`f s
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