Document 7R88v28EQVDQyZQJaGOVaxpnE

VINYL CHLORfflE MUTAGENICITY AND CARCINOGENICITY VIA THE METABOLITES CHLOROOXIRANE AND CHLOROACETALDEHYOE MONOMER HYDRATE. Jim D. Elmore and John L. Wong* Department of Chemistry, University of Louisville Andrew D. Laumbach and Uldis N. Streips* Department of Microbiology, University of Louisville Louisville, Kentucky, USA 40208 . SUMMARY Mutagenicity tester strains of Bacillus and Salmonella were used to assay vinyl chloride in nutrient broth at a practical concentration level. Also screened without exogenous activation were seven potential metabolites of vinyl chloride in their pure forms as well as the related epichlorohydrin. Chlorooxirane, chloroacetaldehyde, chloroacetaldehyde monomer hydrate, chloroacetaldehyde dimer hydrate, chloroacetaldehyde trimer, and epichlorohydrin produced significant mutagenic activity in Salmonella typhimurium strains sensitive to base-pair mutation. A recombination repair deficient strain of Bacillus subtil is was inhibited in growth by these compounds, whereas excision repair deficient and wild type strains of Bacillus subtil is were relatively unaffected. On the basis of these assays a working hypothesis for the vinyl chloride carcinogenesis mechanism is proposed which involves chlorooxirane and chloroacetaldehyde monomer hydrate as the ultimate carcino genic metabolites of vinyl chloride. CMA 002747 2 INTRODUCTION The carcinogenic potential of vinyl chloride monomer 1 was initially established by Viola et al_. [1] and Maltoni et al_. [2] with inhalation experiments using laboratory animals. Detection of angiosarcoma in polyvinyl chloride workers suggested a causal relationship between industrial exposure to vinyl chloride and the development of pathological conditions in humans. This contention was supported by epidemiological data which revealed an association between exposure to 1 and the onset of hepatic abnormalities including angiosarcoma [3,4]. A report by Hefner et_ al_. [ 5] on the metabolic fate of 1 in rats indicated that 6735 of the vinyl chloride inhaled by the rats was metabolized and excreted in the urine. The metabolic products identified were N-acetyl-S(2-hydroxyethylJcysteine and thiodiglycolic acid [5,6] which are sulfhydryl conjugates of chloroethanol 2 and chloroacetic acid 3 respectively. Chloro-rA oxirane 4 and chloroacetaldehyde 5 were speculated to be the carcinogenic forms. V/e report herein the mutagenicity and carcinogenic potential of these t compounds in their pure forms. Several reports [7,8,9] have appeared recently using the Salmonella tester strains to test vinyl chloride and several of its supposed metabolic derivatives. Vinyl chloride and chloroethanol were found to be mutagenic after activation by liver homogenates [9]. Direct mutagenicity of vinyl chloride was also reported by McCann rt al_. [7] and Bartsch et_al_. [10] at 2035 v/v in air (200,000 ppm). Since the solubility of vinyl chloride in water at 25C and 1 atm has been determined to be 7.79 x 10"^ mole fraction [11] or 2,900 ppm, we have conducted further testing at this concentration level to secure a practical 002'748 3 dose-response comparison with its proximate metabolites. Regarding the latter, the exact chemical forms of the proximate metabolites previously tested are often questionable. Chloroacetaldehyde, like formaldehyde [12], dichloroacetaldehyde [13], and chloral [14], can exist in combinations of four forms depending on the history of sample preparations: the monomer 5, the monomer hydrate 6, the dimer hydrate 7, and the trimer 8. McCann et al. [7] used vacuum distilled chloroacetaldehyde without a follow-up analysis of its content. This distillate may have consisted of chloroacetaldehyde monomer 5 and its cyclic trimer 8 if water was totally absent, or it may have been a mixture of chloroacetaldehyde hydrates 6 and 7 in an aqueous medium. Bartsch et al_. tlOl tested a commercial aqueous chloroacetaldehyde solution which, according to our analysis reported herein, had an acidic pH and approximately equal concentrations of the two hydrates 6\> and ^7. This solution was also contaminated by ethanol to the extent of 10%. We have therefore conducted individual assays of pure compounds, or assays of a known mixture of the specific forms of chloroacetaldehyde. Furthermore, the mutagenicity observed for chlorooxirane 4 [9] may be attributed to a chloroacetaldehyde hydrate 'V rather than the chlorooxirane integrity. Under the 37C aqueous testing conditions reported, chlorooxirane decomposed with a half life of 1.6 min [10] to chloroacetaldehyde. For this reason we have also screened epichlorohydrin f9\j which is a stable chloro-epoxide homolog of r4\j as a comparative assay to interpret the observations of the activity of chlorooxirane. This investigation used the above-mentioned compounds 1^ - 9 in mutagen assays without exogenous enzyme activation. A preliminary screen was performed using DNA repair-deficient mutants of Bacillus subtil is. This was followed by quantitative testing of the compounds for mutagenicity cma 002749 4 with Salmonella typhimurium LT-2 strains ITS]. Thus, a combination of these two screening procedures have led to information on the mutagenicity and carcinogenic potential of these compounds as well as their chemical mode of action. MATERIALS AND METHODS The bacterial strains used in the bioassays are shown in Table I. The Salmonella tester strains were designed to detect chemical carcinogens as mutagens [15]. The recombination and DNA repair-deficient Bacillus subtilis strains were used in the repair assays as an indirect test for mutagenicity [16,17], Nutrient broth (Difco) and nutrient broth plus 0.5% NaCl was used for growth of stock cultures of Bacillus and Salmonel!a strains respectively. Nutrient agar (Difco) served as a solid medium for the growth of Bacillus strains in "repair-assays". The pour plates used with Salmonella strains consisted of molten (45C) soft agar which contained 0.6% agar, 0.6% NaCl, 0.5mM biotin, and 0.5 mM histidine. The minimal agar plate was composed of Vogel-Bonner E medium [18], 1.5% agar, and 2% glucose. Vinyl chloride gas was obtained from Matheson Scientific; aqueous chloroacetaldehyde (45% by wt.) from ICN Pharmaceuticals; epichlorohydrin from Matheson Coleman and Bell; and other chemicals from Aldrich Chemical Co. Mutagenicity Assays -S--a--l-m--o--n--e--l-la-- - Vinyl chloride 1 v/as tested by the method of Ames [7]. A mixture of 0.1 ml of 1 in broth and 2 ml of top agar was added to O.Tml of cell culture. The solutions were then mixed and poured immediately onto the surface of a minimal medium plate. After incubation for 48 hrs at 2?C, colonies were counted and recorded. For compounds 2 - 9, sample solutions of known concentration: cMA 002750 5 were prepared in dimethyl sulfoxide (DMSQ). A 0.1 ml aliquot of the sample solution was^admixed with 0.9 ml of the tester strain culture. Then, a 0.1 ml sample of this mixture was added to 2 ml of molten soft agar and applied to the surface of a minimal agar plate. Control plates for detection of the spontaneous reversion rates were prepared for each tester strain by omitting only the compounds tested. Pour plates were incubated for 48 hr at 37C before revertant colonies to prototrophy were counted. For positive mutagenesis control, plates containing the mutagen 4-nitroquinoline-N-oxide were used. Bacillus - The "repair-assay" procedure was a modification of the "recassay" procedure of Kada et al_. [19]. They were grown overnight in nutrient broth then diluted 10 fold in phosphate buffer (pH 7.0). Strains were streaked with pipettes onto nutrient agar plates. Filter paper discs (6 tun) saturated with test solution were placed upon the bacteria streaks. Following incubation for 24 hr at 37C, growing bacteria were visible except in the inhibition zone. The lethality and mutagenic potential of compounds were assessed by comparison of inhibition zones between the 168 wild type strain and the DNA repair-deficient strain and the DNA repair-deficient strains. The control used was 4-nitroquinolineN-oxide. Survival assays for 6 and 7 (cf.. Fig. 3.) were made in MY-1 broth [17] solutions. Cultures were grown in tryptose blood agar base for 16 hrs, inoculated into MY-1 broth, and viable cell counts were done on TBAB agar plates. Compound Synthesis and Purification Chlorooxirane 4 prepared by the method of Walling and Frederick [20] was in higher purity (95% pure) than that by molecular chlorination [21] (50% pure). Thus, t-butyl hypochlorite and ethylene oxide at -10C with 200 watt tungsten lamp irradiation yielded chlorooxirane 4; glpc (gas liquid phase chromatography) CMA 002751 6 c 1.2 mtrrat 50*C, infrared absorption ( vcm"^) 900, 1250, 1320, and 1710 as described previously [21], and PMR as shown in Table II. Derivatization of 4 with an excess of acidic 2,4-dinitrophenylhydrazine solution gave glyoxal-bis-dinitrophenylhydrazone, mp 317-318C (317C reported[21]). Chloroacetaldehyde monomer 5 was obtained in the purest form by crackinq a# the chloroactaldehyde trimer 8 at 95C and distilling it into dry DMS0; glpc tR * 2.25 min at 100"C and PMR as shown in Table II. Chloroaceta' ld~ehyde dimer hydrate 7 ^ - A solution containing 49.5 ml of 38% hydrochloric acid, 30 ml of the 45% aqueous chloroacetaldehyde solution, and 55.5 ml of water was distilled over a 9 inch Vigreaux fractionating column. The fraction (1/5 of the initial volume) collected from 87-100C was redistilled. This second distillate at 83-92"C crystallized after 3 days at -15C. Upon sublimation of SCC and 1 atm, white crystals of 7 were 'V obtained; mp 55C and PMR as shown in Table II. Chloroaceta' ld_ehyde trime""r 'V8 - Concentrated sulphuric acid (7.5 ml) was added to the 45% aqueous chloroacetaldehyde solution (5 ml) with vigorous stirring and external cooling (-5"C). The crystalline precipitate was filtered after standing overnight at -15C, washed with 5 ml of cold 20% aqueous methanol, and recrystallized 5 times from cold methanol; mp 87-88C, corresponding to that reported by Natterer[13], and pMR as shown in Table II. Quantitation of vinyl chloride in nutrient broth - The concentration of vinyl chloride 1 in the broth solution was determined by an extraction method in conjunction with glpc. This method involved (1) establishing the CMA 002752 7 linearity of the response of 1 on glpc., (2) constructing a standard curve of vinyl chloride weight vs_. peak area, and (3) extracting the broth with methylene chloride followed by glpc determination. (1) Linearity of response - A standard solution of 1 was prepared by condensing it (bp -13.4C) at -78C onto a known weight of methylene chloride in a 1 ml volumetric flask fitted with a serum cap. The condensed vinyl chloride was determined by weighing. A typical solution thus prepared was 0.2877 M and was subjected to glpc analysis by varying injection sizes from 1-9 ul. The correlation of vinyl chloride weight and peak area was made by a least square computer routine: slope * 0.877, with an index of correlation of 0.972 (ideal 1.00) up to 6 yl (0.1078 mg) of injection. (2) A standard linear curve using the above technique was established for 0.01-0.10 mg of vinyl chloride. (3) Extraction of broth Vinyl chloride was allowed to bubble through the nutrient broth for 30 min at 25C. A 1.0 ml broth sample was then extracted with 4 x 2 ml of methylene chloride, the extracts were combined and then made up to 10 ml in a volumetric flask with methylene chloride. Glpc analysis of this solution and application of the standard curve showed that there was 7.0 mg of 1 in the 10 ml solution, or the concentration of 'V1. in the broth was 0.0107 M. Repeated determination showed it to be 0.0105 M, High Pressure liquid Chromatographic (HPLC) Analysis of the Commercial 45% Aqueous Chloroacetaldehyde - Reverse phase HPLC on the commercial 45% chloroacetaldehyde solution (7 M, pH 2.6) resolved it into two components: t^l * 6.3 min and t^2 = 8.8 min in a ratio of 40:60. The ratios of the two peaks on the chromatogram changed as the solution pH was varied by addition of 1 N NaOH at room temperature: 43:57 (pH 3.7), 44:56 (pH 5.0), 48:53 (pH 7.6), CMA 002753 8 and 56:44 tpH 8.2). The first eluted component at t^l = 6.3 min increased while the second^ at t^2 * 8.8 min decreased at raised pH's. This indicated a retrograde aldol condensation type hydrolysis. The first peak was assigned to the monomer hydrate 6 and the second to the dimer hydrate 7. When the 45% solution was diluted to 0.44 M at pH 3.6, refluxed for 1 hr, cooled, and chromatographed, the ratio of t^l:t^2 became 33:67. This suggests an acidcatalyzed condensation of \6 to form the dimer hydrate '7Vi . When these two components were analyzed by glpc, both emerged at the same retention time (t_ = 2.8 min at 60C) as that of the monomer 5. Dehydration of 6 and 7 must have occurred under the glpc conditions thereby reverting them to the monomer form. As shown in Table II, the PMR spectra of 6 and 7 are resolved 'V \ from one another. Thus, the PMR spectrum of the 45% commercial solution also revealed the presence of both hydrates 6 and 7 with approximately the same integrals. Instrumentation High Pressure Liquid Chromatography (HPLC) - A Waters Associates model 600 pump combination (dual) with a model 440 differential 254 nm ultraviolet detector were used for the analysis of aqueous chloroacetaldehyde solutions. A Porasil Bondapak yC-jg 4 mm x 30 cm column was used with a 80:20 v/v 0.1 N NH4H2P04-Me0H isocratic eluant (pH 4.9) at 1 ml/min. These two components were also resolved on a Reeve Angel Parti si1 10 ODS 4.6 mm x 25 cm column using the same eluant. CMA 002754 9 Gas Ltqqtd Phase Chromatography (GLPC) - Analysis of vinyl chloride 1, chlorooxirane-4, and chloroacetaldehyde 5 were performed on a Carle model 9500 flame ionization gas chromatograph, A 10% SE-30 on Anakron 60/70 packed column was used at 30C for vinyl chloride determination, A 20% Carbowax on chromosorb W column at 50C and 40 ml/min of Helium was used for analysis of 4 and 5 unless specified otherwise. Both were 0,125 inch x 5 feet stainless steel columns. Proton Magnetic Resonance (PMR) - Spectra were obtained using a Varian A-60 A and a Perkin Elmer R-12 spectrometer. Solutions of D2O and DMS0-dg were used with 3-(trimethylsilyl)propanesulfonic acid sodium salt as internal reference, Tetramethylsilane was used as a reference in CDClg and CCl^, Probe temperature was 38 t. RESULTS AND DISCUSSION Mutagenicity assays with Bacillus and Salmonella for compounds 1-9 are A# summarized in Table III. They are grouped into three categories in subsequent discussion. Further testing data are included in Tables IV-VI and Figures 2-4. Nonmutagenicity of vinyl chloride, chloroethanol, and chloroacetic acid Only high concentrations of vinyl chloride (20% v/v in air) have produced mutagenic action in previous assays with the Salmonella tester strains [9,10]. We have found that tests with both the Salmonella and the Bacillus cultures were negative within the practical solubility range of vinyl chloride in the nutrient broth under ambient conditions. Figure ] shows the stability of a Cma 00275s 10 presaturated vinyl chloride broth solution at 25*C and 1 atm. The initial concentration of 0.022 M of vinyl chloride 1 rapidly decayed by 50% in 15 hr. Thereafter the-escape of 1 from the broth slowed considerably. In the next 45 hr there was a further decline of only 18%. Thus the bacteria strains (. subtil is MC-1 and Salmonella JA 100) were exposed to the stabilized solution of 0.0106 M (723 ppm) of vinyl chloride in the nutrient broth. The negative activity observed was not unexpected since vinyl chloride lacks the electrophilic character common to many mutagens [23). Although the direct mutagenicity of vinyl chloride at 200,000 ppm (20% v/v in air) observed previously [10] may be real, the chronic human exposure problem most likely requires metabolic activation of vinyl chloride to an electrophilic reactive form [8]. Also tested were chloroethanol 2 and chl'oroacetic acid 3 which probably are metabolic intermediates as shown in Scheme I. Both S-(2-hydroxyethyl)cysteine and SCHEME I: Vinyl Chloride Metabolites CH2- = CHC1 -1v cellular -C1CH,-CH,0H 2 --------- 2 2 ^ SH cellular C1-CH2-C02H 3 - -- _o2c-ch(nh3+)-ch2 H0-CH2-CH2-S S(CH2-C02H)2 thiodiglycolic acid in Scheme I have been identified [6] as the urinary metabolites of vinyl chloride. Neither chloroethanol 2 nor chloroacetic acid 3 exhibited any mutagenic effects at 1 mM concentration in our mutagenicity * assays (cf. Fig. 2 ). Although Bartsch et aJ^ClO] found considerable mutagenic cm 002756 n activity with chloroethanol 2 for TA 1530 strain in the absence of microsomal ----- <v activation, our observations corroborate with those of McCann et al_. [7] who showed that 2 was weakly mutagenic directly even at high concentrations for the sensitive TA 100 and that it showed only trace activity for TA 1535. The enhanced activity of 2 after microsomal activation observed by these groups suggests that chloroacetaldehyde derivatives were being formed from chloro ethanol. We have found potent mutagenicity and lethality with the various forms of chloroacetaldehyde as shown in Figures 2 - 4 and Tables IV-V. Mutagenesis of chloroacetaldehyde, the monomer hydrate, dimer hydrate, and the trimer - When chloroacetaldehyde 5 was distilled into distilled water, a mixture of the monomer hydrate 6 and the dimer hydrate 7 was formed instantly as determined by HPLC and PMR spectroscopy. Analysis of the commercial 45% aqueous chloroacetaldehyde solution with the same techniques showed a 50:50 mixture of the two hydrates. Upon standing under dry conditions, the monomer 5 cyclized to form trimer 8. The trimer was sparingly soluble in water, but >\# ^ was disproportionated upon heating in water to form the hydrates \6 and%7. Purified samples of chloroacetaldehyde 5, the commercial 45% chloroacetaldehyde solution containing a 50:50 mixture of the hydrates A6# andA#7, the dimer hydrate 7, and the trimer 8, in DMS0 solutions were tested for mutagenic potential. The data in Table IV summarizes the results of the repair assays with B. subtilis. Chloroacetaldehyde 5 and the monomer hydrate 6 specifically inhibited the growth of B_. subtilis MC-1, a mutant lacking recombination repair of DNA. These unrepaired DNA lesions then led to cell death. However, compounds 5 and 6 did not inhibit the wild type B. subtilis or those mutant CMA 002757 12 strains (Hcr-9, FB-13) having the capacity for recombination repair. In contrast, purified samples of the dimer hydrate 7 and trimer 8 inhibited all of the mutants as well as the wild type, although MC-1 again demonstrated the most sensitivity. It is unlikely that 7 and 8 caused DNA lesions that 'V are different from those by 5 and 6 because the excision repair mutants and the wild type were inhibited to an equivalent extent. Inhibition of these strains by 7 and 8 may result from a metabolic poisoning or cellular damage similar to that observed in Escherichia coli after exposure to vinyl chloride waste [24 J. Viability assays on B. subtil is strains during short term exposure to the chloroacetaldehyde 5 revealed that recombination repair was essential for recovery (Figure 2). The B. subtil is strains with recombination repair cap abilities displayed repair kinetics as evidenced by the shoulders in curves in Figure 3. The sensitive MC-1 strain lacking recombination repair yet having excision repair was rapidly killed following the exposure. We surmise that these DNA lesions caused by the chloroacetaldehydes v/ere repaired solely by the recombination repair mechanism. The direct mutagenic potential of the samples4-9 was determined by the mutations expressed in Salmonel!a strain TA 100. The dose response relation ships of these compounds (cf. Table V), and the dose-response curves (cf. Figure 4 ) were determined with strain TA 100. At high concentrations the dose response curves for all compounds became nonlinear because the toxicity of the chemicals reduced the number of potential revertants on the plates. The reduction of the cell population by high concentrations was confirmed by viable counts and observations of decreased background lawn on the test plates. CMA 002758 13 Data from the dose response curves (Figure 4 ) showed that chloroacetaldehyde 5 and the monomer hydrate 6 were more active than 7 and 8, % \ \ i\, The monomer 5 was the most mutagenic as predicted because its electrophilic *X# carbonyl group remains intact. The restoration of the carbonyl character to the monomer hydrate 6 can be projected in hydrophobic environment of the cell membrane by loss of water, hence its mutagenicity is also explicable. The substantial activity of the dimer hydrate 7 can be attributed to its 'X/ ready equilibrium with the monomer hydrate 6 in aqueous medium. The relationship of the activity of the cyclic trimer 8 to the chloroacetaldehyde action 'V cannot be deduced on the basis of the curves in Figure 4. Nevertheless, these mutations were the base-substitution type and were expressed in Salmonella strains TA 100 and TA 1535. The mutagenic response in TA 1535 was very weak as compared to TA 100. It should be noted that the latter contains an "R" factor that enhances mutation by an error-prone recombination repair mechanism following DNA damage [7]. Comparison of mutagenic response of chlorooxirane with its methylene homolog epichlorohydrin - The prevailing opinion [s] is that chlorooxirane 4 is the primary metabolite of vinyl chloride and is derived from the action of the microsomal mixed function oxidase. This compound, prepared independently from chlorination of ethylene oxide, was found to rearrange readily to chloro acetaldehyde in aqueous or DMSO solution at ambient temperatures. A kinetic study of a 0.15 M solution of 4 in a DgO-DMSO-dg (80:20) mixture at pD 7.1 and 4C by PMR technique showed that the rearrangement followed first order kinetics, k (sec"1) = 2.5 x 10`4. This translates to a half life of 46.2 min CMA 002759 14 at 4*C as compared to 1,6 min at 37"Cf9] . Such instability allows only limited testing in the cold as well as Interpretation of the results. On the other hand, epichlorohydrin 9, which can be considered the epoxidation metabolite of allyl chloride, is a methylene homolog of 4. Both 4 and 9 are structurally bis-alkylating agents, hence comparing their mutagenic activities will lend insight into the mode of action of chlorooxirane 25,26]. Table IV tabulates the mutagenic response of these two compounds in the Bacillus repair assay and Table VI the Salmonella TA 100 reversion. Due to the instability of chlorooxirane at 37C, the assays shown in Table VI were preincubated at 3*C in solution with the Salmonella TA 100 strain before plating to insure that chlorooxirane could reach the cells intact. Mutant strains of B_. subtil is were not inhibited when exposed to high concentrations of epichlorohydrin-9. v In contrast, chlorooxirane 4 selectively inhibited the rec` strain MC-1 in a manner similar to chloroacetaldehyde hydrate \6. Tests with S~a--lm- '--o--n--e--l-l-a-shov/ed that strain TA 100 was very susceptible to the mutagenic action of both epoxides 4 and 9. However 9 was less toxic to the tester strains than 4. The A* A. A, A low toxic effects of 9 indicate a different type of DMA lesion compared to that caused by chlorooxirane 4. It is possible that 9 may react with DNA by a a, a> mechanism which does not cause potential lethal strand-scissions. On the other hand, chlorooxirane 4 may act on the bacteria via a NIH shift [27,28] to a, form chloroacetaldehyde 5 or 6. Conceivably, chlorooxirane can also behave A, A. as a diradical intermediate rather than a conventional S^l or S^2 type alkyl ating agent in its reaction with DNA. Vinyl chloride carcinogenesis mechanism hypothesis - Among the comoounds tested in the metabolic Scheme II, chloroacetaldehyde 5 and chlorooxirane 4 CMA 002760 15 SCHEME II: _Ibe metabolic pathways of vinyl chloride [5], I. Cl-CH=CHc- ^1- Cl-CHj,-CH,,-0H 2- SCHEME I urine alcohol C1-CH2-CH0 5 dehydrogenase CUCH2-CH0 5- C1-CH2-C02H 3 % SCHEME I urine II. ci-ch2.ch2-oh h2o2 ase ci-ch2-ch2-ooh -C1-CH?-CH0 5 c III. oxidase C1-CH=CH2 1------------------- Cl-CH-CHo0 4 I____ h *\# C1-CH,,-CH0 5 were the most mutagenic with the lowest toxic side effects. Hence, they may qualify to be the active carcinogenic derivatives of vinyl chloride. Consider ing the aqueous milieu of the metabolic environment, however, the chloroacetal dehyde monomer hydrate 6 is a more realistic choice as an ultimate carcinogen than the monomer 5 which reacts immediately with water. Viability assays >\/ followino exposure of the B. subtil is mutants to 6 indicated that the compound induced recombination repair. The strain with the rec phenotype (MC-1) was immediately inactivated. All the rec+ strains showed survival ability and repair kinetics of similar nature. It has been shown [27] that mammalian cells have postreplication repair of DNA which has many similar features to the recombination repair in bacteria. In addition, a relationship between this mammalian postreplication repair process and'mutation as well as carcinogenesis CMA 002761 16 has been suggested 4]. Cells from patients with the skin disease xeroderma pigmentosum lack the ability to excise pyrimidine dimers and must rely on postreplication repair to remove these lesions [24]. It is believed that this error prone process of postreplication repair is responsible for the production of somatic mutations and cancer in patients with this disease. Since chlorcacetaldehyde monomer hydrate 6 induces recombination repair in bacteria responding to lesions, it may also be capable of activating the error prone postreplication repair in exposed mammalian cells. At the molecular level, chloroacetaldehyde is known to react with N 1 and N6 nitrogens of adenosine and N3 and N4 nitrogensof cytidine in single-stranded DNA [28,29]. It therefore appears that chloroacetaldehyde monomer hydrate 6 should merit our consideration as an ultimate carcinogenic metabolite of vinyl chloride. The lower mutagenic activity of chloroxirane 4 compared to 6 may reflect the unstable nature of chlorooxirane as an a-chloroether. While the carcino genic chloromethyl methyl ether is a bifunctional alkylating agent [3CJ, the mutagenic activity of chlorooxirane cannot be so categorized, especially when it is compared with epichlorohydrin 9. One mode of action of chloro oxirane is a rearrangement.to chloroacetaldehyde via the NIH shift [27,28]. Another is a homolytic ring cleavage to yield a stabilized diradical inter mediate C1CH-CH26. Both are capable of reacting with DNA, thereby accounting for the mutagenicity of 4. In mammalian cells, chlorooxirane.being a reactive epoxide, could be trapped by a glutathione epoxide transferase [31] at a faster rate than the detoxification of chloroacetaldehyde via the less active aldehyde dehydrogenase [32] . Such detoxification of chlorooxirane could CMA 002762 17 explain the-reported decrease in sulfhydryl level in liver cells during metabolism of short chain halo hydrocarbons including vinyl chloride [31]. Perhaps the lower mutagenicity of chlorooxirane 4 in these bacterial as^ ys, compared to the chloroacetaldehydes, is also attributable to its being detoxified faster. We therefore consider both chlorooxirane A4/ and the chloroacetaldehyde monomer hydrate 6 to be the ultimate carcinogenic metabolites of vinyl chloride. 'X/ Their reactions with DNA causing mutations in Bacillus and Salmonella suggest a causal relationship with vinyl chloride carcinogenesis in human and laboratory animals. Acknowledgements This work was supported by grants from the B. F. Goodrich Co. This grant program was initiated and administered by the Cancer Center of the University of Louisville. The Salmonella tester strains were kindly provided by Dr. B. N. Ames of the University of California, Berkeley. We also thank George D. Stratton, Jr. and S. E. Yen for their able assistance. CMA 002763 REFERENCES AND FOOTNOTES .18 *To whom correspondence should be addressed. 1 Viola, P. L., Bigotti, A. and Caputo A. (1971) Cancer Research 31, 516-522 2 Maltoni, C. and Lefemine, G. (1974) Environmental Research 7, 387-405 3 Creech, J. L. and Johnson, M. N (1974) Journal of Occupational Medicine 16, 150-151 4 Lee, F. I. and Harry, D. S. (1974) Lancet 1, 1316-1318 5 Hefner Jr., R. E., Watanabe, P. G. and Gehring, P. J. (1975) Annals of New York Academy of Science 246, 135-148 6 Watanabe, P. G., McGowan, G. R. and Gehring, P. J., Annals of New York Academy of Science, in the press 7 McCann, J., Simmon, V., Streitwieser, D. and Ames, B. N. (1975) Proceedings of National Academy of Science U.S.A. 72, 3190-3193 8 Rannug, U.,Johnsson, A., Ramel, C. and Wachtmeister, C. A. (1974) Ambio 3, 194-197 9 Malaveille, C., Bartsch, H., Barbin, A., Camus, A. M. and Montesano, R. (1975) Biochemistry Biophysics Research Cornnunication 63, 363-370 10 Bartsch, H., Malaveille, C. and Montesano, R. (1975) Internationl Journal of Cancer 15, 429-437 11 Hayduk, W. and Landle, H. (1974) Journal of Chemical Engineering Data 19, 253-257 12 Walker, G. (1953) Formaldehyde, p. 86, Reinhold, New York 13 Natterer, K- (1882) Monatsheffe fur Chemie 3, 443-464 CMA 002764 19 14 Harris-r G., ed (1965) Dictionary of Organic Chemistry, Vol. 2, pp, 589 and 956 15 McCann, J., Spingarn, N. E., Kobari, J. and Ames, B. N. (1975) Proceedings of National Academy of Science U.S.A. 72, 979-983 16 Okudo, S. and Romig, Vi. R. (1965) Journal of Molecular Biology 14, 130-142 17 La'umbach, A. D. and Felkner, I. C. (1972) Mutation Research 15, 233-245 18 Vogel, H. 0. and Bonner, 0. M. (1956) Journal of Biological Chemistry 218, 97-106 19 Kada, T. Tutikawa, K. and Sadaie, Y. (1972) Mutation Research 16, 165-174 20 Walling, C. and Fredericks, P. S. (1962) Journal of American Chemical Society 84, 3326-3331 21 Gross, V. H. and Freiberg, J. (1969) Journal fur Praktische Chemie 3.11, 506-510 22 Zief, M. and Schramm, C. H. (1964) Chemistry and Industry April 18, pp. 660-661 23 Kappus, H. Bolt, H. M., Buchter, A. and Bolt, W. (1975) Nature 257, 134- 135 24 Hagstrom, A. (1974) Ambio 3, 77-79 25 Voogd, C. E. (1973) Mutation Research 21, 52-53 26 Fjellstedt, T. A. Allen, R. H., Duncan, B. K. and Jakoby, W. B. (1973) Journal of Biological Chemistry 248, 3702-3707 27 Lehmann, A. (1974) Life Science 15, 2005-2016 28 Barbin, A., Bresil, H., Croisy, A., Jacquignon, P. Malaveille, C., Montesano, R. and Bartsch, H. (1975) Biochemical and Biophysical Research Communications 67,9 596-603 f CMA 002765 20 29 Kochetkov, N. K. Shibaev, V. N., and Kost, A. A. (1971) Tetrahedron Letters 22, 1993-1996 30 Leong, B. K., Macfarland, H. N. and Reese, W. H. (1971) Archives of Environmental Health 22, 663-666 31 Johnson, M. K. (1965) Biochemical Pharmacology 14, 1383-1385 32 Weiner, H., King, P., Hu, J. H. J. and Bensch, W. R. (1974) Alcohol and Aldehyde Metabolizing Systems (Thurman, R. G., ed.), pp. 101-113, Academic Press, New York ' CMA 002766 21 LEGENDS TO FIGURES Fig, 1. Stability of vinyl chloride in nutrient broth at 25C, 1 atm. Fig. 2. Survival of Bacillus subtil is MC-1 after incubation with: 1.0 mM chloroethanol 2 ( + -+); l.OmM chloroacetlc acid ^ (o --o); 5.76 mM (concentra tion based on UH2CCHO) chloroacetaldehyde (45% aqueous solution) 6 and ( t>--1>) 0.1 mM 4-nitroquinol ine-N-oxide (0"0); and untreated control cells (0--0), Mid-logarithmic cultures grown in MY-1 broth were incubated with compounds at 37C. Samples were plated at the times indicated from which viable cell counts were recorded. The mutagen 4-nitroquinoline-N-oxide served as a positive mutagenicity control. Fig. 3. Survival of Bacillus subtil is strains in the presence of 5.0 mM (concentration based on Ct^CCHO) chloroacetaldehyde (45% aqueous solution) Jd and 7. Cultures were grown to mid-logarithmic phase in MY-1 broth and then incubated with the chloroacetaldehyde solution at 37WC. Samples were plated at the times indicated from which the percent survival was determined. FB-13 uvr", rec+ (+--+);Hcr-9 her", rec+ (O-O); 168M wild type (0 -- 0); and MC-1 uvr+, rec" (c>--1>). Fig. 4. Dose response curves with Salmonella typhimurium TA100. Sample solutions of known concentrations prepared in DM50 were mixed with tester strain culture and soft agar. Plates were poured, incubated at 37C for 48 hrs, and then scorec for revertant colonies to prototrbphy. Chloroacetaldehyde monomer (0--0); chloroacetaldehyde (45% aqueous solution-concentration based on ClHjCCHO) 6 and 7 (0-0); chloroacetaldehyde dimer hydrate 0); chloroacetaldehyde trimer \ ( x--k); and epichlorohydrin jg (0--). 002T6T CONCENT RATI OK M IIL1 MOLAR Figure 1 Figure 2 OOK 4 -+ CMA 002769 60 Ml Hl)U$ 1 90 -J120 PERCENT SURVIVAL Figure 3 REVERT ANT COLONIES PER PLATE 26 TABLE I: Bacteria Tester Strains A. Salmonella typhimurium LT-2 Tester Strains3 aAll tester strains contain uvrB repair mutations which eliminate the excision repair system; mutations in the histidine operon; and rfa mutations which alter the cell wall by increasing permeability and eliminating pathogenicity. bThe resistance transfer factor,"R" factor,enhances the error-prone recombination repair system thus making the strains more susceptible to mutation [15], cThe strains susceptible to base-pair substitution contain mutations in the histidine G46 operon and those susceptible to frameshift mutation contain mutations in the histidine operon C 3076 (TA 1537) or D3052 (TA 1538, TA 98). Strains "R" factor5 Mutation detected0 TA 1535 TA 100 TA 1537 TA 1538 TA 98 base-pair substitution + base-pair substitution - frameshift - frameshift + frameshift B. Bacillus subtil is Tester Strains aTrp" denotes a requirement for tryptophan; Mit-S denotes sensitivity to mitomycin C. 5hcr+ denotes a host-cell reactivation ONA repair capacity; her- lacks a host-cell reactivation DNA repair capacity; rec+ denotes a recombin ation DNA repair capacity; rec~ lacks a recombination DNA repair capacity uvr* is sensitive to ultraviolet-induced DNA damage. Strains 168 M Hcr-9 FB-13 MC-1 Phenotype3 Prototroph (wild type) Trp" Trp" Trp~,Mit-S DNA Repair her , rec her-, rec uvr", rec her , rec 002772 27 TABLE II. PMR Spectra of Vinyl Chloride Derivatives Compounds C1HC| -CH2-0, %4 C1H2C-CH0 5 C1H2C-CH(0H)2 6 C1H-C-CH-0H c\ Q * C1H2C-CH-0H 7 ' Solvent cci4 cci4 DMSO-dg CDq0D-D20 1:8 3(p0 0.1) Spectra, 6TMS=0 (0=Hz) 2.75 (q, CH, J-1.5) 2.85 (q, CH, J=2.4) 4.90 (q, CH, J=2.4,l.5) 4.00 (d, CH, >2.2) 9.57 (t, CH?, 0-2.2) 3.50 (d, CH) 9.60 (t, CH2) 3.60 (d, CH2, >5) 4.60 (t, CHT J-5) dmso-d6 CD,0D-D,,0 1:8 3(pD 20.1) 3.55 (d, CH-, J=4.9) 5.05 (t, CHT 0=4.9) 3.60 (d, CHo, >4.5) 4.83 (t, CHT 0=4.5) CH2C1 S1 C^S'0'^SCH,C1 2c cci4 DMSO-dg 3.52 (d, CH2, 0=4.7) 5.08 (t, CH, J-4.7) 3.75 (d, CH2, 0=4.1) 5.45 (t, CHT 0=4.1) CMA 002773 28 IABLE III: Summary of Mutagen Activity in Microbial Systems NI No Inhibition of growth detected in Bacillus subtil is MC-1; NR * No increase of revertants in SalmoneTTa typhimurium TA 100 compared to control;+ ^active; ++ = very active. Acetaldehyde, a potential metabolite of and ally! chloride, the parent olefin of were negative in these two systems. Compounds Tested Control: 4-nitroquinoline-N-oxide 1 H9CCHC1 2 C1H2C-CH20H <3V( C1H2,C-C00H ~4 ClHCt -CH29-0| *5v C1H2-C-CH0 O6f C1H49C-CH(0H)9U -7V ClH49C-CH0H-0-CH0H-CH4,Cl 8 (ClH2C-CH0-)3 9 C1H-C-CH-CH-0 Bacillus subtil is Repair Assay ++ NI NI NI + ++ ++ NI Salmonella typhimuriui Reversion Assay ++ NR NR NR ++ ++ ++ ++ + ++ 29 -TABLE IV: Growth Inhibition of Bacillus subtil is Strains . Inhibition was measured in ran after 24 hr at 37C as described in text; NI denotes no inhibition Mutagen Molarjty C1HC/>C-CH0 r5u 0.100 Chloroacetaldehyde & \ (452 aqueous solution) 0.115 C1H,C-CH-0H 2i 0 7 ~ cih2c-ch-oh 0.097 I68M 2,0 NI 1.5 MC-1 27.7 22.5 9.5 Hcr-9 3.7 FB-13 2.7 NI NI 1.5 1.5 C,H2C>(^ i'CK2CI ch2ci 8 0.096 C1HC-CH-0 I Z1 4 * ClH,C-CH-CH-0 2 i Zj 9 ~ 0.260 0.113 4-Nitroquinoline-N-oxide 0.001 (control) 7.0 14.5 6.0 7.0 NI 10.0 NI NI NI NI NI NI 10.0 18.0 15.0 15.0 CMA 002775 4 30 TABLE-1L Relative Mutagenicity of the Four Forms of Chloroacetaldehyde with s.typhimurium TA 100 45% Aqueous Soln 6 : 7 50:50 Monomer 'X5# Dimer Hydrate 7 Trimer 8 'V Molarity 5.3x1 O'5 2.7x10" 1.4xl0"5 6.9xl0'6 3.4x10' 1.7x10" 8.6x1 O'7 4.3x1 O'7 Revertants Molarity 977 1.3x10" 723 6.7x1 O'6 512 3.3x1 O'6 194 1.7x1 O'6 120 8.4xl0"7 61 4.2xl0"7 36 2.1xl0'7 10 l.lxlO*7 Revertants 18 68 88 361 404 238 185 131 Molarity 4.8xl0"4 2.4x1 O'4 1.2x1 O'4 6.0xl0"5 3.0x10" 1.5x10" 7.5x10" 3.8x10" Revertants 311 259 193 107 88 30 23 11 Molarity 4.8xl0"4 2.4x10"4 1.2xl0"4 6.0x10' 3.0x10" 1.5x10" 7.4x10" 3.7x10" Revertants 144 159 101 39 27 18 12 -0 CMA 002776 C *> t < ( 31 TABLE VI: Reversion of $.typhimurium TA100 by Chlorooxirane 4 and Epichlorohydrin 9 *\i ^ Broth solutions of $ or % with TA100 were preincubated at 3C before plating. Duplicate plates were evaluated after 48 hrs at 370C." The average number of revertant colonies per plate minus the number of spontaneous reversions were recorded. Preincubation Time 0 hr 1 hr 2 hr 4 hr 6 hr Epichlorohydrin 9 (1.0 m) 186 204 297 202 154 Chlorooxirane 4 (0.26 mM) ^ 31 4 6 114 44 002777 CMA