Document rxDMvbVBGZ01J3Xmgry35jv7J

R&S 026406 K-5qu3-/oo)r 1965- f fK- ft5k*3 ~ K- in'i " PR--^12! - 5SC6 Mutagenicity of chloroacetaldehyde, a possible metabolic product of 1,2-dichloroethane (ethylene dichloride), chloroethanol (ethylene chlorohydrin), vinyl chloride, and cyclophosphamide (environmental carcinogcns/alkyl halides) Joyce McCann, Vincent Simmon,* David Streitwieser, and Bruce N. Ames Biochemistry Department, University of California, Berkeley, Calif, 9-1720; and Department of Toaieology, Stanford Reaearch Institute, Menlo Park California 91025 Contributed by Bruce N. Ames, June 2, 1975 ABSTRACT We have previously described a very sensi tive and efficient bacterial test designed to detect chemical carcinogens as mutagens. Chloroacetaldehyde is mutagenic in this system and is of interest because it is a possible me tabolite in mammals of the large volume industrial chemi cals 1,2-dichloroethane (ethylene dichloridc)(3.5 billion kg/ yr, U.S.) ana vinyl chloride (2.5 billion kg/yr, U.S.), and of the anlincoplastic agent cyclophosphamide. Chloroacetaldehyde reverts a new Salmonella bacterial tester strain (TA100). Chloroacetaldehyde is shown to be hundreds of times more effective in reversion of TA100 than is chloroethanol (ethyl ene chlorohydrin), a known metabolic precursor of chloroacctaldehyde and a possible metabolite of dichlorocthane and vinyl chloride, or than vinyl chloride, which is itself mu tagenic forTAlOO. Chloroethanol is shown to be activated by rat (or human) liver homogenates to a more highly mutagenic form with reversion properties similar to chloroacetal dehyde. Reversion properties of cyclophosphamide after in vitm metabolic ar-tiv-ntir,?. suggest that chlczcaeeizldchydc b not the active mutagenic form of this antineoplastic drug. We have previously described a rapid, sensitive bacterial test designed to detect chemical carcinogens as mutagens (1-7). The test utilizes a special set of histidine mutants of Salmo nella ttjphimurium for reversion, and a rat (or human) mi crosomal system {or metabolic conversion of carcinogens to their active forms. The standard bacterial tester strains have been described in detail (1, 3, 5) and contain histidine missense (TA1535) or frameshift (TA1537, TA153S) mutations. The strains also contain uurB and r/a (deep rough) muta tions which greatly increase their sensitivity to reversion by a variety of carcinogens: uorB causes loss of the excision re pair system and rja causes loss of the lipopolysaccharide permeability barrier. The compound to be tested, the bacte rial tester strain, and when required rat (or human) liver mi crosomal enzymes, arc combined on a petri dish and after incubation at 37" for 2 days histidine revertants arc scored. Hundreds of carcinogens and noncarcinogeus have been tested using tile .Sijlliiu/ieliu/niammulian-niicrosome bacte rial test system in this laboratory (1-13) and in many labora tories throughout the world. We (J M. and B.N.A.) are cur rently preparing a compilation of these results, and so far about Ho% of the known carcinogens tested have been de tected as mutagens in the test, and very few (<\Q%) noncar cinogens (many close relatives of carcinogens have been test ed) are positive. This and other evidence showing that a high percentage of carcinogens arc mutagens is most easily ex plained if carcinogens cause cancer by somatic mutation (4). We believe there is a high probability that chemicals found to be mutagens in the Salmonella test will turn out to be car cinogens. We have recently introduced two new tester strains (TA100 and TA98) which were constructed by transferring to our standard tester strains TA1S3S and TA1538 respec tively, a resistance transfer factor (R factor), pKMIOl (7). The new tester strains are greatly enhanced in sensitivity to reversion with a variety of potent carcinogens such as the fungal toxin aflatoxin, 7,12-dimethylbenz(a)anthracene, benzo(a)pyrene, and others, and also can be used to detect a variety of carcinogens and mutagens not detected by our standard tester strains, RESULTS AND DISCUSSION Chloroacetaldehyde. A compound of unusual interest that was positive on the new strain TA100 was chloroacetaldehvrie Eie 1 A shows that at the levels tested chlorccccta!dehyde is quite effective in reverting TA100, but does not revert TA1535. (We find at higher concentrations a slight activity of chloroacetaldehyde in reverting TA1535; weak activity has been found independently (14-17).) Chloroucetaldehyde is relatively specific for TA100, and does not sig nificantly revert the other R factor tester strain, TA9S (TA1538 containing the pKMICI plasmid), or our other standard tester strains. We originally tested chloroacetaldehyde because of its known unique reactions with nucleotides, which have been extensively studied in connection with its use as a fluorescent label (18-2J). It is known to react specifically with the N-i and NG nitrogens nf adenosine and the N--3 and N4 nitrogens of cytidine to form a highly fluorescent cyclic derivative. It has been used extensively for fluorescent labeling of a vari ety of adenine and cytosine containing compounds, includ ing single-stranded DNA. The mutagenic activity of chloro.tcetaldchydc reported Iutc strongly suggests it can also ie.u I with elirnu'.v-mal DW possibly at exposed singlesliandi-d regions Midi as the replication fork, or in regions of the DNA undergoing rccomhin-.itional repair Chloroacetaldehyde has also become of interest recently as a possible metuljolic product of the large volume indus trial chemical, and human carcinogen vinyl chloride (15-17, 22-25). It is also a possible active metabolite of several wide ly used chemicals and we discuss this in relation to its muta genicity. 1,2-Dichlorocth-iru- (ethylene dichlnride) is metabolized in mammals to chloioacctic acid, probably through clilorocthano! and chloroacetaldehyde (2R, 27), It is an extremely toxic chemical produced industrially in enormous quantities (about 3.5 X 10'1 kg in 197-1 in the U.S. (28) and about 20 X 3190 Gi-ncllcs: McCann at al. Fir.. ]. Reversion ofTAlOO and TA1535 with (A) eliloruacetsldehyde, (B) cliloroethanol. (G) vinyl chloride, anti (1)) cyclophos phamide. Procedure:! were as described previously (ref, and Table 1). S-9 (lfiO al per plalc) obtained Irotn phenohsirhitnl-induccd rats (-1) was added for vinyl chloride activation; S-9 ob tained from Aroclor-induccd rats (12) was added for rhlorocLhanul (150 >d S-H per plate) and cyclophosphamide (300 id 8-9 per plate) activation, D, TA1535; TA153K; O, TAJ00; , TA100 + S-!l; B, 'PA 1035 + S-9. 10 Ions worldwide (29)]. It is used primarily in the manu facture of vinyl chloride and other industrial chemicals, but is also used in huge amounts as a lead scavenging agent in gasoline (over 100 million kg in 1970), in various solvent ap plications, anti as a component of fumigants for gram, up holstery, and carpets (20) In Table 1 the reversion of TA100 with chloroacetaldeltydc is compared directly to reversion with two other metabolic pmdiicts of dichloroothane, i.o., chloroclhattol and chloroacelic arid. Although dicldoroethane and chloioellinnol are weakly mutagenic in bacteria without metabolic acti vation as previously shown (31-35)', on a molar basis oldoroaeetaldeliyde is hundreds of times more effective in re verting TA100, In similar assays chloroacelic acid was inact Dicliloroelhano lias also been rcjairtoil cpiiP- mutagenic in Droso phila (35. 37). l'wc. Nat. Acad. .So. USA 72 (1975) 3191 Table 1. Reversion of TA100 wiLli 1,2-didllornrlham:, vinyl chloride, and vvvrnl possible metabolites RcverUnl colonics DieliloroeUi.-iiie* Clilomacelnldehyde ChlorooLh.iiuil GHtomaertic arid Vinvl chloride vu 1.3 X 10* 30 2.1 X 10* 1.0 x 10' 7.-1 x 10' per plate 25 285 159 0 128 per /Jtrtnl 0.19 7d G 0.G -- 1.0 ]{ -ulK are lrun linear <! <'`-re'-ponse rurvr s ltor subl rncl 'mil 'p'wo l.'im , ,ir inn I :;:nui.'i [i wilaul-. I .ippri.y iinai idi 130 pi r pl.ilrl 1; i * \ c: !-,n v.,r (Ii 11 i in:i,r i! iv' jpi 11; pi.pi- lit nu hi p.nal in;; in,;' ,c*i n and I i.uli-i i,i 11 in i 11 \ j -1 . 1 . o , - | -, i \ a- | ,ri -, i- m I- i'- i d (3), i'-i n in, ,'ini u .a, n ,.(! 1-.a \ in; 1 , l;l u nl,-. I A MnI \ti - p" iti nil pel ri pi.'It'-- 'n 11 r -111 ,1111 r - lui tailun- time-. In a t ,i,\ 1 chloride at inii-.pliere i20Vi \ /vl 1 laj, Kctcrianl loliilllei. were stored oiler Hit nil.u lull ol Ihe [)em pi,lies loi -IS hr al 37. All tllemuaU welt- ol 1 he hi;; Ileal pul lit at ,i da I lie; nntl ehloride 0)9.97, pure I ttilt from Gnion (`arlntle, taeiuim disldletl t ldiirt>aeetaldeliyde was a ^ill ol Nelson l.eunartl. 1,2-rlit hluroet hone ami chloroelliaiioi tvt-re from Aldi ich Dicldornelliane is an oytremelt tve.ih mutagen. ami reproducible dose-response curves ttere nnl ohl,lined, resnlis repurletl are averages limn seieial experiment1-, live. The metabolites were also tested on the complete Set of tester strains (TA 15.35, TA1537. TA1.53S, TA98) and except for the expected weak reversion of TA 1.535 with chloioethanol and eliloriijcet.ildehyde, the other strains were not re verted. Attempts to activate dicldoroethane to a more muta genic foin. using the standard rat liver S-9 Mix (.), 12) for activation were unsuccessful. This could be related to the relatively inefficient conversion of cidoroethanol to cliloro;i t-r-in Ini-i i y rj rj in inr in ruro m sicni frig, t 11), or iri utuei un determined factors. We are currently optimizing our rat liven activation system for the metabolic conversion of this and a variety of other alkyl halides such as carbon tetrachlo ride and the results of these experiments will Ire published elsewhere. Chloroothaiiol, itself an industrial chemical, is a likely metabolic product of dicldoroethane and precursor of chloroacetaldehsde. It is known to be metabolized in vivo (3S) and m vitro to clilornacctaldchvde by lat (3S) or human (39) liver alcohol dehydrogenase We have examined the rever sion of TA 100 and TA1.33.5 with cliloroethanol, ami K,g. IB shows that in (he presence of r;u live! microsorncs, chlorocthnnol is activated to a form which shows clear activity on TA100 and a very slight activity for TA1535F Similar resuits were obtained for TA100 using human liver extracts. Fig JH also shows that cliloroethanol. at higher concentra tions, is weakly mutagemc directly (without mierosomes) for TA100 and shows a trace of activity for TA1533, as pre vious))' repoi ted (31, 33, 3-1). In contrast to the microsomal activation, we have not been able to show any clear muta genic effect on incubating these amounts of cliloroethanol with horse liver alcohol dehydrogenase and NAD, and other engines in our S-9 homogenate could be involved (.10--12). Exogenously added NADP, normally present in om S-!J Mix, was not neccssaiy lor the S*9 activation. The enhanced re version of TA100 relative to TAI535 after metabolic activa tion could Miggei.t that chlortucoUUlehydr. which has simi- t Malavodle. c( al (Hi) have independently shown the weak {rvjypi ovimatelv 2*h>ld) enlunerntent in irversinn of TA13-'() (mie of our SuhntMU'Ihi lesier sir,mis siuuLu It* TA 1333) with ehlnovtlij' no! after in I'ttro nu*uU>lic :u.Uv,uiuu R&S 026407 R&S 026408 1 sm 3192 Gcnclics: McCann et at. lar sjiocificily in reversion of TA100 (Fig. 1A), is 1 lies active mctalxditc. Vinyl Chloride, a major industrial elieinieal and a carcin ogen in humans and rodents (13-15), has lieen recently rcporlcd mutagenic in yeast (46). and Salmonella (15-17, 22), and chromosome breaks have Ix-en observed in humans exposed to high doses of vinyl chloride (-17, 43). Vinyl chloride is metatmli/ed in rats, and it appears likely that a metabolite is (lie active mutagenic or carcinogenic form (15-17, 22-25). Two of the metabolites of dichloroethanc (ehloroacctaldehyde and cldoroethanol) are also suggested metabolites of vinyl chloride (15, Hi, 22-25), Another proposed active me tabolite of vinyl chloride, chloroethylene oxide (22, 23), also has been recently shown to be unite mutagenic in the Sal monella test (Hi, 17) and in a recent study both chloroacetaldehyde and chloroethylene oxide have been implicated as likely in vitro metabolites of vinyl chloride (23). Vinyl chloride itself has been shown to be mutagenic in the presence of the S-9 microsonntl fraction by Raumig at at. (22) and later by bartsch at at. (15). bartsch at at, also re ported consideiable mutagenic activity in the absence of microsoincs. Wc find that most of the mutagenic activity of vinyl chloride (Table 1, Fig. 1C) is direct activity, not re quiring activation with rat livei homogenates {S-9 Mix)5, So far the cause of the reported differences in direct and microSomally activated mutagenesis with vinyl chloride is not clear. It is also not clear whether the mutagenic activity of vinyl chloride in the absence of microsomes is due to a possi ble bacterial activation One argument against cldoroacetaldehyde being involved in the mutagenic activity of vinyl chloride is the equal activity of vinyl chloride against tester strains TA100 and TA1535 (compare Fig 1C with Fig. 1A), On the other hand wc sec so little metabolic activation of vinyl chloride with the S-9 Mix that this argument may not be pertinent. We arc also uncertain about the involvement of chloroacetaldehyde as an active mutagenic form of vinyl chloride because of the relatively inefficient metabolic acti vation of chloroethauol (Fig. lb) in our system. An alterna tive metabolic intermediate between vinyl chloride and chloroacetaldehyde is chloroethylene oxide, which is known to rearrange spontaneously to chloroacetaldehyde (49). The mutagenicity of both chloioelliylene oxide and chlnroacetaldchyde suggest that one or both of these forms may be the true active carcinogenic form of vinyl chloride. Cyclophosphamide (also called Fndoxan, or Cytoxan) is used extensively as an antineoplastic agent, and is also a known carcinogen (50); its mutagenic activity (51-53) re quires metabolic conversion to an active form. Several meta bolic products of cyclophosphamide arc known, hut the exact nature of the active form is still uncertain. It has re cently lieen suggested, largely on liypothetieal grounds, that olilotoaool.tIdi'iivile i mild he the active product (54) We has c emu pared directly tile rc s ci simi ui TA 100 and TA 1555 s We have used liver iiuetosoiues fuun hulk pltenobarbilal- and Aroelor-iiulueed rats, AlU-mpls to obtain more significant activa tion of vinyl chloride as a mntagen in liquid suspensions in sealed vials using the standard S-9 Mix, purified rat liver inicrosoines, or using S-!) Mix from which small molecules (i c , glutathione) were reitinvcd by passage through a Sephadex G-50 column svrro nil unsuccessful, as were attempts to add liver alcohol dehydrogenase and NAD to die standard S-9 Mix. Nnle Added in Proof. In more recent otperimrnls on the mutagen icity of vinyl chloride sve have hern aide to obtain a 2-fold increase over the direct activity adding -10 id uf S-9 (Aroclor-indueed rat liver) to tin1 0.5 ml of S-9 Mix Tins microsomal dependent activity is ahoul the same for lioth TA 1335 and TAI00 and thus does not ap|x'uv to lx' due to ctilnroaoctaldehyde. Proc. Sal. Acad. Sal. USA 72 {1975) with cyclophosphamide before and after activation with rat liver S-9 Mix and the results are slinsvn in Fig. ID, Cyclo phosphamide is clearly mutagenic for Uith tester strains after metalxdic conversion, hut there is no preference for TA100. Wc conclude that the primary mutagenic metabo lite of cyclophosphamide in vitro is not chloroacetaldehyde. Other possibilities arc the known metabolite, aldophnspliamidc (55), or nitrogen mustard itself. Wc base previously shown that a close relative of nitrogen mustard, bis-2-chlororlhylaniine, ieverts TA1535 and TA100 to a similar de gree (7), and thus has reversion properties similar to activat ed cyclophosphamide. In view of the accumulating evidence of the correlation between mutagenicity and carcinogenicity, wc believe that the mutagenic activity of chloroacetaldehyde indicates it has a high probability of being a carcinogen. Wc arc not aware of any studies on the carcinogenicity of chloroacetaldehyde. However, in an extensive study dealing with its toxicity, Lawrence at at. (53) report changes of respiratory epitheli um in lungs of rats exposed to cldoruacetaldohyde that are suggestive of a pic-malignant condition. The likely involvement of chloroacetaldehyde in the me tabolism of dichloroethaue and chloroethauol indicates the carcinogenicity of these industrial chemicals should be thor oughly examined. Though dichloroethanc is pioduced in bil lions of kg/yr it is not unique among large industrial chemi cals in never having been thoroughly tested for carcinoge nicity'. Carcinogenicity studies with chloroethauol (59, 60) are somewhat conflicting and are by no means definitive. Angiosarcoma, the rare form of liver cancer associated with human exposure to vinyl chloride might also he found in workers exposed to dichloroethanc or chloroethanol if chloroacetaldehyde is actually the aetive enreinneenie form of all three chemicals. For information prior to publication and criticisms we thank: C. Brunei, II Barisch, Y J Gehring, F. Weisburger, N. Leonard, N. Lopneoo, F. Mukai. and II .5 nosenkr.ui'4 This wurk was support ed lax 14it DA Contract A 1(01-3) 5-1 P. A 156 j McCann was sup ported by a post-doctoral fellowship from die California Division of the American Cancer Society. 1 Ames, B N (1971) in Chemical Mutagens: Principles and Methods for Their Detection, oil. 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