Chromosome Damage Induced by Vinyl Acetate Through in Vitro Formation of Acetaldehyde in Human Lymphocytes and Chinese Hamster Ovary Cells1

[CANCER RESEARCH 45,4816-4821, October 1985]

Chromosome Damage Induced by Vinyl Acetate through in Vitro Formation of Acetaldehyde in Human Lymphocytes and Chinese Hamster Ovary Cells1

Hannu Norppa,2 Francesco Tursi,3 Pirkko PfÃ¤ffli,Jorma MÃ¤ki-Paakkanen, and Hilkka JÃ¤rventaus

Department ol Industrial Hygiene and Toxicology, Institute of Occupational Health, Haartmaninkatu 1, SF-00290 Helsinki, Finland [H. N., P. P., J. M-P., H. J.], and Istituto di Ricerche Farmacologiche "Mario Negri, " Via Eritrea 62,1-20157 Milan, Italy [F. T.]

ABSTRACT to the genotoxic effects of this compound. It was not mutagenic to Salmonella typhimurium (1,7-9), but workers exposed to vinyl A 48-h treatment with vinyl acetate (0.05-1 mM) induced a acetate were reported to have an increase in chromosome drastic increase in sister chromatid exchanges (SCEs) and (in aberrations in their blood lymphocytes (10). Inhalation of vinyl first division cells) structural chromosome aberrations in cultured acetate did not produce cancer in rats (11), while rats receiving human lymphocytes. The effects were more pronounced in cul vinyl acetate in their drinking water were reported to develop tures of isolated lymphocytes than in whole-blood cultures. A tumors in the uterus, thyroid, and liver (12). distinct dose-dependent induction of SCEs similarly occurred in In principle vinyl acetate could be epoxidized at the vinyl group, Chinese hamster ovary cells after a 24-h vinyl acetate treatment through microsomal or nonmicrosomal routes. However, in the (0.125-1 mÂ«).A pulse treatment of Chinese hamster ovary cells case of vinyl acetate, epoxidation seems to be of only theoretical for 4 h also yielded a clear increase in SCEs, but at higher importance. As early as 1959 Filov suggested that vinyl acetate concentrations (0.3-5 mw). The presence of rat liver S9 mix is rapidly hydrolyzed enzymatically to acetic acid and, via unsta enhanced the SCE-inducing effect of vinyl acetate in Chinese ble vinyl alcohol to acetaldehyde (13, 14). He detected acetal hamster ovary cells. Gas Chromatographie analysis of human dehyde in samples of human and rat plasma and whole blood 3 whole-blood lymphocyte cultures treated for 10 s-20 min with min after the addition of vinyl acetate, but not in heat-inactivated vinyl acetate (5.4 HIM) revealed a rapid degradation of vinyl plasma. Acetaldehyde is a well-known mutagen (15,16) and is acetate and formation of acetaldehyde. During the 20-min ob thus a probable reactive metabolite of vinyl acetate. servation period, no degradation of vinyl acetate or formation of In the present paper we have studied the cytogenetic effects acetaldehyde were observed in complete culture medium without of vinyl acetate in cultures of human lymphocytes and CHO cells. blood, which suggested that the reaction was enzymatic. Acet If microsomal epoxidation actually were important for the meta aldehyde induced SCEs in human whole-blood lymphocyte cul bolic activation of vinyl acetate, a positive result in the cytoge tures at concentrations (0.125-2 mw) comparable to those used netic tests would probably be expected only after the use of a for vinyl acetate. The results indicate that vinyl acetate induces metabolic activation system. If erythrocytes had the capacity to chromosome damage in cell cultures through enzyme-mediated activate vinyl acetate, more chromosome damage would be hydrolysis to acetaldehyde. observed in lymphocytes cultivated with whole blood than in isolated lymphocytes. On the other hand, if the hydrolysis of the INTRODUCTION ester bond were the critical reaction, an increase in chromosome aberrations and SCEs could probably be expected without ex The epoxidation of the vinyl moiety appears to be the most ogenous metabolic activation. In addition to cytogenetic analysis, important metabolic activation reaction involving vinyl com we utilized gas chromatography to measure the possible deg pounds (1). For example, two important plastic monomers, vinyl radation of vinyl acetate and formation of acetaldehyde in human chloride and styrÃ¨ne (vinyl benzene), both mutagenic in a number whole-blood lymphocyte cultures. of test systems, are converted by microsomal monooxygenases to corresponding epoxides (1-3). The oxidation of the vinylic MATERIALS AND METHODS double bond may in some cases occur through nonmicrosomal routes. StyrÃ¨ne is metabolized to styrene-7,8-oxide in human Test Chemicals. Vinyl acetate (>99% pure), containing 0.0015% of blood by the action of erythrocytes in a reaction probably cata hydroquinone as a stabilizer, was obtained from Fluka (Buchs, Switzer lyzed by oxyhemoglobin (4). Consequently the induction of land). For treatment of the cultures, vinyl acetate was dissolved in SCEs4 by styrÃ¨ne in human whole-blood lymphocyte cultures acetone; several solutions, from 0.5 to 0.05 ml/ml, were used. Acetal depends on the number of erythrocytes present (5, 6). dehyde (99.5%) was a product of Merck (Darmstadt, West Germany); Vinyl acetate (acetic acid vinyl ester), used, e.g., for the man two solutions, 0.1 and 0.01 ml/ml prepared in acetone, were used for the treatments. All solutions were made shortly before use. Because of ufacture of polyvinyl acetate and polyvinyl alcohol, is another the high volatility of acetaldehyde (boiling point, 20-21 Â°C),its solutions important vinyl monomer. In spite of the industrial importance of were prepared and kept in an ice bath. Cyclophosphamide was obtained vinyl acetate, only a limited amount of information is available as from LÃ¤Ã¤ke/Farmos-YhtymÃ¤Oy(Turku, Finland) and was dissolved (10 mg/ml) in equal volumes of 0.9% (w/v) NaCI solution and McCoy's 1Supported by Grant 83-0349 from the Swedish Work Environment Fund. 2To whom requests for reprints should be addressed, at the Department of Medium 5A (Grand Island Biological Co., Glasgow, United Kingdom). Industrial Hygiene and Toxicology, Institute of Occupational Health, Haartmaninkatu Studies with Human Lymphocytes. Lymphocytes were cultivated in 1, SF-00290 Helsinski, Finland. the dark at 37Â°C in air-tight glass injection bottles (20 ml) containing 6 3 Recipient of a grant from the European Science Foundation. ml of growth medium consisting of (in 100 ml) 15 ml of heat-inactivated 4 The abbreviations used are: CHO, Chinese hamster ovary; SCE, sister chro fetal calf serum (Grand Island Biological Co.), 1 ml of phytohemagglutinin matid exchange. Received 4/8/85; revised 6/10/85; accepted 6/24/85. (Wellcome, Beckenham, Kent, United Kingdom), 2 ml of 5-bromodeoxy-

CANCER RESEARCH VOL. 45 OCTOBER 1985 4816 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1985 American Association for Cancer Research. VINYL ACETATE-INDUCED CHROMOSOME DAMAGE uridine (Calbiochem, La Jolla, CA; 0.25 mg/ml in sterile water: final measurements were performed according to a head-space method concentration, 5 fig/ml), and 82 ml of RPMI Medium 1640 (with L- based on the procedure of Mendenhall ef a/. (23). The concentrations of glutamine; Grand Island Biological Co.). For whole-blood cultures, 0.27 vinyl acetate and acetaldehyde were determined, after the addition of ml of heparinized blood from a healthy male donor (age 30) was used. vinyl acetate (1 Â¡A;finalconcentration, 5.4 mM), from 2-ml aliquots (in air The final volume of these cultures was thus 6.27 ml. The number of tight 20-ml glass injection bottles) of RPMI Medium 1640 (Grand Island leukocytes and erythrocytes in the blood samples was evaluated by the Biological Co.) containing 0.15 ml of heat-inactivated fetal calf serum/ml Coulter ZM Counter (Luton, United Kingdom). (Grand Island Biological Co.), with or without 0.1 ml of heparinized whole Purified lymphocyte cultures were established from lymphocytes iso blood from a 35-year-old female donor. The reaction was stopped either lated with Ficoll-Paque (Pharmacia Fine Chemicals, Uppsala, Sweden) as immediately (after 10 s), or after various periods of incubation at 37Â°C described elsewhere (5). The numbers of leukocytes in the suspension (2, 5, 10, 15, and 20 min) by adding 1 ml of a mixture (1:3, w/v) of 40 of isolated cells were counted by the Coulter Counter and the proportions mM thiourea (Union Chemique, Brussels, Belgium) and 0.6 M perchloric of erythrocytes and different types of leukocytes were read on hemato- acid (Merck), and transferring the bottle to dry ice for 30 min. The sample logical slides. About 95% of the isolated cells were lymphocytes, 4% was then incubated at 80Â°Cfor 30 or 60 min, after which a volume of erythrocytes, and 1% monocytes. The number of leukocytes in the 500 /J was collected by an air-tight syringe from the gas phase of the purified cultures was adjusted according to their number in whole-blood bottle for immediate gas Chromatographie analysis of vinyl acetate and cultures. The volume of the isolated lymphocyte cultures was 6.5 ml. On acetaldehyde. The determination was performed with a Hewlett-Packard the average the cultures contained, per ml, 200,000 leukocytes and 200 Model 5740 A (Hewlett-Packard Co., Avondale, PA.) gas Chromatograph million (whole blood), or below 8,000 (isolated cultures) erythrocytes. equipped with a flame bnization detector and a Carbowax 20 M column The cells were treated 24 h after starting the cultures. The final (2 m, 10%, on Chromosorb W, acid washed, silanized). The camer gas concentrations of the test chemicals in the cultures were 0.05, 0.1, 0.2, was helium, column temperature was 80Â°C, injection temperature was 0.5, and 1 mw for vinyl acetate, and 0.063, 0.125, 0.25, 0.5, and 2 rriM 150Â°C, and detector temperature was 200Â°C. The concentrations of for acetaldehyde (whole-blood cultures only). Duplicate cultures were vinyl acetate and acetaldehyde were derived from the analysis of samples used for each treatment. Control cultures were treated with acetone with known amounts of vinyl acetate and acetaldehyde in serumless (9.1-19.3 rriM). The incubation was continued for an additional 48 h, RPMI Medium 1640. Retention time was 1.2 min for acetaldehyde and making a total of 72 h. Cell harvest, slide preparation, and staining were 2.8 min for vinyl acetate. performed as described earlier (17). Studies with CHO Cells. The cultures were set up as described by HytÃ¶nenef al. (18) and treated 24 h after initiation. In the first experiment RESULTS the duration of the treatment was 24 h. The vinyl acetate concentrations used were 0.125, 0.25, 0.5,1, and 2 mM. In the second experiment, the SCE Induction in Human Lymphocytes. In human whole- cells were first treated in serumless medium, caps of the flasks tightly blood lymphocyte cultures (with 200 million RBC/ml), there was closed, with different concentrations of vinyl acetate (0.31, 0.63, 1.25, a very clear dose-dependent increase in SCEs after a 48-h 2.5, and 5 mw) for 4 h, after which they were rinsed and allowed to treatment with vinyl acetate (Chart 1). The effect was statistically recover in complete medium for 24 h. The latter experiment was per significant (P < 0.001) at 0.1 mw and higher concentrations. At formed with and without S9 mix derived from the livers of Wistar rats 1 HIMthe number of SCEs/cell was almost 15 times the control induced with Aroclor 1254 (Analabs, Inc., North Haven, CT) as shown frequency. According to the proliferation index, a toxic effect of elsewhere (18,19). S9 mix was prepared using a method slightly modified vinyl acetate on cell division was apparent from concentrations from that of Nachtman and Wolff (20). The modifications were the use of MgSO4 (Merck) instead of MgCI, and the addition of 55 n\ of McCoys of 0.5 mw and up. At 1 mw only 13 cells could be analyzed for Medium 5A/ml (Grand Island Biological Co.). Duplicate cultures were SCEs. used for all treatments. Control cultures were treated with acetone (27 In purified lymphocyte cultures (with 8000 RBC/ml), the dose- mM). In the 4-h experiment, cyclophosphamide (10 ^M) was used as a dependent effect of vinyl acetate on SCEs was still more pro positive control agent. Cell harvest, slide preparation, and staining were nounced than in whole-blood cultures (Chart 1). A 2.4-fold in performed as described previously (17,18). crease in SCEs was already present at the lowest concentration Chromosome Analysis. All microscopic analyses were performed on tested (0.05 mM); and at 0.5 mw the mean number of SCEs per coded slides. One microscopist scored SCEs in human lymphocytes, cell was 10 times higher than in controls. The proliferation index another scored chromosome aberrations, and a third scored SCEs in decreased from concentrations of 0.2 mw and higher. In the CHO cells. For the analysis of SCEs, 25-50 harlequin-stained cells (30 for CHO controls, the frequency of SCEs was slightly, but nonetheless cells) were scored, whenever possible, from each of the duplicate cul significantly (P < 0.01), higher in isolated lymphocytes than in tures. Cell cycle delay was simultaneously evaluated by counting the lymphocytes cultivated with whole blood. number of first, second, and third (or further) division cells from 100 As with vinyl acetate, acetaldehyde also had a distinct dose- metaphases/culture. These data were transformed into cell proliferation dependent effect on SCEs in human whole-blood lymphocyte indexes ("replication indexes") as described by BÃ¶hlkeei a/. (16). This cultures (Chart 2). The increase in SCEs was statistically signifi index represents the average number of replications completed by the cant (P < 0.001), beginning with a concentration of 0.125 mw. cells during 5-bromodeoxyuridine supplementation. Concentrations from 0.5 mw and up clearly lowered the prolifer For the analysis of structural chromosome aberrations in first division ation index. cells of the human whole-blood lymphocyte cultures treated with vinyl Induction of Chromosome Aberrations in Human Lympho acetate, 100 cells/culture, as a rule, was inspected for the presence of cytes. Vinyl acetate induced a very clear dose-dependent in different categories of chromosome- and chromatid-type aberrations according to criteria suggested by Evans and O'Riordan (21). For each crease in chromosome aberrations in the first division lympho cytes of human whole-blood cultures treated for 48 h (Chart 3). treatment a total of 200 cells was analyzed. The statistical analysis of the SCE data was performed according to A statistically significant effect (P < 0.001) on the number of 1-tailed f test (22). The frequencies of cells with chromosome aberrations aberrant cells (gaps included or excluded), and the number of were compared using Fisher's exact probability test (22). cells carrying chromatid-type aberrations or chromatid-type ex Gas Chromatographie Measurements. The gas Chromatographie changes, was observed at 0.5 mw. By far the most frequent type

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-1 Concentration (mM) Chart 2. SCE induction ( ) and proliferation index ( ) in human whole- 0.5 1 Wood lymphocyte cultures treated with acetaldehyde for 48 h. Points, mean of 25- Concentration (mM) 50 cells (SCEs) or 200 cells (proliferation); oars, SE. Chart 1. SCE induction ( ) and proliferation index ( ) in cultured human lymphocytes treated with vinyl acetate for 48 h. Combined results from three The 4-h pulse treatment of CHO cells also led to a clear dose- experiments, each with duplicate cultures, are shown. Whole-blood cultures (â€¢) contained 200 million RBC/ml; purified lymphocyte cultures (O) contained less than dependent increase in SCEs, with or without S9 mix, but at 8000 RBC/ml. Points, mean of 13-200 cells (SCEs) or 200-600 cells (proliferation); higher concentrations than in the 24-h experiment (Chart 4). Both bars, SE. with and without S9 mix, a statistically significant (P < 0.001) response was present starting from the lowest concentration of aberration was the chromatid-type break. At 1 mw, 84% of tested (0.3 mM). The use of S9 mix clearly enhanced the effect the cells were aberrant (gaps included, 2% in controls), 38% of vinyl acetate. According to the proliferation index, vinyl acetate containing a chromatid-type exchange (none in controls). At this began to have an inhibiting effect on cell proliferation at 1.25 mM dose, chromosome-type breaks were also significantly (P < with S9 mix and at 5 mM without it. 0.001) increased (8% of cells, none in controls). Degradation of Vinyl Acetate and Formation of Acetalde In cultures of isolated lymphocytes, vinyl acetate significantly hyde. Within our observation period of 20 min, cultures contain (P < 0.001 ) increased the total number of aberrant cells (gaps ing blood showed a rapid disappearance of vinyl acetate and included or excluded), and the number of cells containing chro- formation of acetaldehyde (Chart 5). In solutions containing matid breaks or chromatid exchanges (P < 0.05) beginning with culture medium and heat-inactivated serum but no cells, there 0.2 ITIM(Chart 3). Thus, vinyl acetate was clastogenic in isolated was no degradation of vinyl acetate during this 20-min period. cultures at lower concentrations than in whole-blood cultures. From 0.5 mivi and higher chromosome-type breaks were also DISCUSSION increased (P < 0.001). At 1 mM, 97% of the cells included an aberration (gaps included, 5.5% in controls); 38% had a chro The results of this study show that vinyl acetate is a very matid-type exchange. The frequencies of aberrant cells appeared efficient inducer of both chromosome damage and SCEs in to be higher in the controls of isolated cultures than in those of cultured mammalian cells. This effect is probably mediated by whole-blood cultures, but the differences were not statistically the rapid hydrolysis of vinyl acetate. significant. In the experiments with human lymphocytes, vinyl acetate SCE Induction in CHO Cells. The effect of vinyl acetate on produced chromosome damage without exogenous metaboliz SCEs in CHO cells after a 24-h treatment was similar to the ing systems, which indicates that the compound, not expected effect obtained with the 48-h treatment in human whole-blood to be a direct mutagen, is activated in the in vitro system. The cultures (Chart 4). The increase in SCEs was already significant early studies of Filov (13) suggested that erythrocytes, as well (P < 0.001) at the lowest concentration tested (0.125 mw), and as plasma proteins, contribute to the hydrolysis of vinyl acetate. at 1 mivi it was 11.6 times the control frequency. The proliferation In our study, vinyl acetate induced more SCEs and chromosome index did not decrease below 1 mM. aberrations in isolated cultures than in whole blood. Conse-

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10 n. 01 2345 Concentration (mM) 572 0.5 Concentration (mM) Chart 4. SCEinduction( )and proliferationindex( ) in CHOcellstreated with vinyl acetate for 24 h (â€¢),or4 h with (A) or without (T) S9 mix. Cyclophos- Chart 3. Induction of chromosome aberrations in first division cells of human phamide (10 *im) with (â€¢)orwithout (O) S9 mix was used as a positive control in whole-blood lymphocyte cultures ( ) and cultures of isolated lymphocytes the 4-h treatment. Points, mean of 30-60 cells (SCEs) or 200 cells (proliferation); ( ) after a 48-h treatment with vinyl acetate. Points, mean of 100-200 cells; bars, SE. SCE data represent induced SCEs; i.e.. the mean number of SCEs in bars, SE. â€¢,aberrantcells including gaps; A, aberrant cells excluding gaps; â€¢ the corresponding control treatment has been subtracted from each value. cells with chromatid-type exchanges. quently, erythrocytes do not seem to play any important role in the activation of vinyl acetate. Because of their high number in a E 4 typical human whole-blood culture, erythrocytes may actually represent an important "inactivating" factor, offering additional Ja â€¢ targets for reactive molecules. Thus the lower SCE response in cultures of whole blood, as compared to those of purified lym phocytes, may represent a compromise between the hydrolysis ("activation") of vinyl acetate by plasma proteins, leukocytes, li and erythrocytes and, on the other hand, trapping ("inactivation") 10 15 20 of the reactive metabolite by erythrocytes. Incubation lime (min) The results of our experiments with CHO cells confirmed the Chart 5. Degradationof vinyl acetate (â€¢)andformation of acetaldehyde(â€¢)in findings of the lymphocyte assays. The high SCE induction, human whote-Woodlymphocyte cultures after treatment with vinyl acetate (5.4 which occurred with or without metabolic activation, again sug HIM).Points,mean of two determinations;bare, SE between two determinations. gested that vinyl acetate is activated by the cultured cells. In the 4-h pulse treatment, however, a clearly greater effect was re complete culture medium without blood. The rapid appearance ceived in the presence of S9 mix than in its absence. S9 mix of acetaldehyde in samples of blood, plasma, or erythrocytes obviously contains factors (probably esterases) capable of hy- treated with vinyl acetate was first shown by Filov Â¡n1959 (13). drolyzing vinyl acetate. The longer 24-h treatment yielded an He also reported that no formation of acetaldehyde could be increase in SCEs at lower concentrations than did the 4-h pulse detected in water, physiological saline, or heat-inactivated treatment, which showed that the SCE-inducing effect of vinyl plasma within 3 min after vinyl acetate treatment, which pointed acetate depended on exposure time. to an enzymatic reaction and to the unimportance of sponta In our experiments with whole-blood lymphocyte cultures, we neous hydrolysis during the short observation period. During found that practically all of the vinyl acetate administered had longer intervals, spontaneous hydrolysis of vinyl acetate may, on been transformed into acetaldehyde within 15 min. This process the other hand, occur. Lijinsky and Reuber (12) reported that in appeared to be enzyme dependent, as no hydrolysis occurred in water solution and at room temperature vinyl acetate decom-

poses, on the average, 8.5%/day. Sa/mone//a-microsome test, vinyl acetate gave no indication of It is supposed that vinyl acetate is attacked by a number of mutagenicity with or without S9 mix (1, 7-9). These negative esterases. It was hydrolyzed by porcine pancreatic lipase (24) results would appear to conflict with the clear clastogenicity of and was the best substrate, of a series of esters, for an acety- vinyl acetate found in the present paper. Our results suggest lesterase purified from Sclerotinia fungus (25). that S9 mix is able to activate vinyl acetate; thus it is probable It is very likely that the chromosome damaging effects of vinyl that acetaldehyde is also formed in bacterial assays in the acetate are due to acetaldehyde. At concentrations comparable presence of S9 mix. Acetaldehyde is itself, however, at best only to those used for vinyl acetate, acetaldehyde produced a clear a weak mutagen in the standard Salmonella plate test (38). The increase in SCEs in human whole-blood lymphocyte cultures. low mutagenicity of acetaldehyde may be related to the poor Vinyl acetate was a somewhat more efficient inducer of SCEs sensitivity of the conventional Salmonella strains to cross-linking than was acetaldehyde. This observation is probably explained agents, to the possible low capacity of acetaldehyde to induce by the high volatility and direct reactivity of acetaldehyde. Acet gene mutations, or, especially, to the high volatility of acetalde aldehyde has its boiling point at room temperature and is thus hyde. When the treatment was carried out in stoppered test much more critical to handle than is vinyl acetate, which boils at tubes, acetaldehyde clearly increased mutations in Escherichia 72Â°C.Consequently, the actual concentrations of the chemical coli (15). may have been lower than expected in cultures treated with Our unpublished results6 suggest that vinyl acetate induces acetaldehyde. In vinyl acetate treatment, at least part of the the formation of micronuclei in the polychromatic erythrocytes of acetaldehyde generated will be formed inside the target cells, C57BL/6 mice after a single i.p. injection. This effect is most which means fewer possibilities for binding to erythrocytes or probably mediated by acetaldehyde. Filov (13) suggested that growth medium components, and less distance to DNA than if vinyl acetate is converted into acetaldehyde very rapidly in vivo the chemical is added to the culture in a reactive form. Lambert in rats, possibly through the action of unspecific esterases in et al.5 showed recently that vinyl acetate and acetaldehyde blood (14). Acetaldehyde has been found to induce SCEs in the induce SCEs at equimolar concentrations in cultures of isolated bone marrow of mice and Chinese hamsters after i.p. exposure lymphocytes, which suggests that acetaldehyde binding to eryth (39, 40). The formation of acetaldehyde is also likely to take rocytes may be important. place in humans exposed to vinyl acetate. If this reaction oc Several earlier studies have shown that acetaldehyde is an curred mainly in blood, the acetaldehyde formed would be ex effective inducer of chromosome aberrations, sister chromatid pected to have an effect on leukocyte chromosomes. In fact, it exchanges, and micronuclei in cultured cells such as lympho has been reported that workers exposed to vinyl acetate have cytes, CHO cells, and rat skin fibroblasts (15,16, 26-32). Acet increased chromosome aberrations in peripheral lymphocytes aldehyde is also carcinogenic. Inhalation of acetaldehyde was (10). reported to induce laryngeal tumors in Syrian golden hamsters Acetaldehyde is the key metabolite of both vinyl acetate and and nose tumors in rats (33, 34). ethanol. This would suggest that ethanol is also a powerful All of the reactions of acetaldehyde with DNA have not yet clastogen. In vitro ethanol is clearly a less effective inducer of been determined. Acetaldehyde was found to make reversible chromosome damage than is acetaldehyde or vinyl acetate. It bonds, possibly Schiff bases, with exocyclic amino groups in the does, however, increase SCEs and chromosome aberrations in DNA bases adenine, cytosine, and guanine (35). After reduction cultured human lymphocytes without exogenous activation, and with sodium borohydrite, three stable products could be isolated. SCEs in CHO cells in the presence of S9 mix (26, 28, 30, 41, The main adduct was identified as /V2-ethylguanosine, and the 42). The difference in effectiveness between ethanol and its two minor ones were identified as A/2-(3-hydroxybutyl)- reactive metabolite is possibly due to the poor transformation of guanosines formed by aldol condensation of two acetaldehyde ethanol to acetaldehyde in cell cultures. For example in mitogen- molecules. Ristow and Obe (29) reported that acetaldehyde is stimulated mouse spleen cells, ethanol was not metabolized able to induce cross-links between the complementary strands during a 72-h observation period (43); and in cultures of CHO of DNA isolated from calf thymus or Bacillus brevis. Recently, cells, acetaldehyde production from ethanol by rat liver S9 mix Lambert et al.5 showed, using the alkaline elution technique, that was much slower than we observed for vinyl acetate in cultured acetaldehyde and vinyl acetate induce DNA cross-links in isolated lymphocytes (30). Vinyl acetate is a vinyl ester. As a group, such esters should human lymphocytes at equimolar doses. However, no direct all be hydrolyzed in a similar way, yielding acetaldehyde. Filov's strand breaking effect could be demonstrated for either of the compounds, which is in agreement with an earlier study which (13) results suggest that at least vinyl formate, vinyl propionate, obtained a negative result for acetaldehyde in the alkaline elution and vinyl butyrate are hydrolyzed to acetaldehyde, with vinyl assay with rat hepatocytes (36). It is probable that the clear formate hydrolysis occurring spontaneously in aqueous solution. clastogenic effects of acetaldehyde, and thus also of vinyl ace Several vinyl esters were also found to be substrates for porcine tate, result from the cross-linking ability of acetaldehyde; cross- pancreatic lipase (24). Thus the production of acetaldehyde may linking agents are usually very effective inducers of SCEs and be an important metabolic activation reaction for a number of chromosome aberrations. compounds. In spite of the well-documented mutagenicity of acetaldehyde, only one positive short-term assay, on enhancement of viral ACKNOWLEDGMENTS transformation in Syrian golden hamster cells (37), has been We are grateful to Professor Harri Vainio, who suggested cytogenetic tests on published on vinyl acetate. In four independent studies with the vinyl acetate. We would also like to thank Deborah Ruuskanen for linguistic editing and Leila Kuusela for typing the manuscript. 5 Unpublished studies of B. Lambert, Y. Chen, and S. He. 8 Unpublished studies of J. Maki-Paakkanen and H. Norppa.

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CANCER RESEARCH VOL. 45 OCTOBER 1985 4821

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1985 American Association for Cancer Research. Chromosome Damage Induced by Vinyl Acetate through in Vitro Formation of Acetaldehyde in Human Lymphocytes and Chinese Hamster Ovary Cells

Hannu Norppa, Francesco Tursi, Pirkko Pfäffli, et al.

Cancer Res 1985;45:4816-4821.

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