Mutagenicity of Pesticides Containing I ,3-Dichloropropen&

[CANCER RESEARCH 37, 1915-1917, June 1977] Mutagenicity of Pesticides Containing I ,3-Dichloropropen&

Francesco Dc Lorenzo, Sandro Degl'lnnocentl, Aldo Ruocco, Lorenzo Silengo, and Riccardo Cortese I and II Cattedradi ChimicaBiologica,II FacoltÃdiMedicinae Chirurgia,Universityof Naples,ViaSergioPansini5, 80131Naples,Italy

SUMMARY cial chemicals present in the environment, e.g. , pesticides, food additives, dyes (2), and others. In a systematic study of the mutagenic effect of chemical This bacterial tester system allows the detection of muta compounds used as pesticides, we found that D. D. soil genic events even when their probability is low, since it is fumigant and Tebone are mutagenic. The test was per performed on a population of 100,000,000 independent ge formed using the bacterial tester strains following the pro nomes. The diffusion of artificial chemicals in our environ cedure developed by Ames. The active principle of D. D. soil mont is such that millions of individuals are continuously fumigant and Tebone is a mixture of the cis and trans exposed to them. We have used the bacterial tester strains isomers of 1,3-dichloropnopene. Both isomers are muta to screen the potential mutagenic property of pesticides genic in Salmonella strains TA 1535 and TA 100. 2,3-Di used in Italy that were obtained from Ministero della SanitÃ , chboro-i-pnopene, a minor component (5%) of the common Rome, Italy. In this paper we describe the mutagenic prop cial preparation Tebone, was also found to be mutagenic in erties of 2 widely used pesticides, D. 0. soil fumigant and strains TA 1535 and TA 100. Mutagenesis of these tester Tebone, the active principle of which is a mixture of cis and strains is an indication of a base-pain substitution event trans isomers of I ,3-dichboropropene. causing a missense mutation. 1,3-Dichboropnopene is widely used in agriculture all oven the world. In Italy 2,187,100 kg were produced in 1972. In MATERIALS AND METHODS California over 1,000,000 kg of 1,3-dichboropnopene-con taming pesticides were used in 1971. The bacterial strains and the experimental procedure are those previously described (1, 7). Pesticides were diluted in dimethyl sulfoxide (Carlo Erba Chemical Company, Milan, INTRODUCTION Italy) as specified under â€œResults.â€•Allyl chloride and 1,3- dichboro-2-butene were purchased from Aldrich Chemical The papers of Ames (1) and McCann et a!. (7) have do Company, Milwaukee, Wis. cis and trans isomers of 1,3- scnibed a simple and sensitive bacterial test for the detec dichboropropene, 1,2-dichboropropane, and 2,3-dichbono-i - tion of mutagens. This test measures the number of back propene, as well as the pesticides in their commercial for mutations induced by mutagens in strains of Salmonella mubas, were given to us by Ministeno della SanitÃ .The data typhimurium auxotrophic for histidine. The compound to be reported in Tables 1 and 2 are obtained from an experiment tested , the bacterial tester strain , and , when required , a that was repeated twice and that gave similar results. liven microsomal extract are combined directly on a histi dine-free agamplate. The number of bacteria reverted back to prototnophy are counted after a 48-hr incubation. RESULTS The tester strains have been selected for their sensitivity and specificity in being reverted to prototmophy by a variety Table 1 shows the results of a standard mutagenicity test of mutagens. Strain TA 1535 detects mutagens causing of the commercial preparation of D. D. soil fumigant and missense mutations, while TA 1537 and TA 1538 detect Tebone. The mutagenicity in strains TA 1535 and TA 100 frame-shift mutagens. All these strains back the DNA-exci is probably a result of a covalent interaction with DNA sion repair system, and this lack greatly increases their (1, 7). Mutagenicity in strain TA 1978 increased significantly sensitivity to mutagens. Strain TA 1978 has the same muta following treatment with liven microsomal fraction. This tion as does TA 1538, but it has a normal DNA-excision shows that probably a metabolite with new mutagenic prop repair system. More recently (7), McCann et al. have con erties was formed following treatment with liver microsomal structed 2 more tester strains, TA 100 and TA 98, which fraction. show increased sensitivity to reversion with a variety of D. D. soil fumigant is composed of 40% 1,3-dichboropro potent carcinogens such as the fungal toxin aflatoxin, 7,12- pene, 40% 1,2-dichbonopropane, and 20% other unknown dimethylbenzanthnacene, and benzopyrene and which can chemicals. Tebone is composed of 30% cis-i ,3-dichboropro also be used to detect a variety of carcinogens and muta pene, 30% trans-i ,3-dichboropropene, 20% 1,2-dichboropro gens not detected by the other tester strains (7). This muta pane, 5% 2,3-dichbono-1-propene, 2% allyl chloride, and genic bacterial test system can be used for screening artifi about 15% unknown compound. We tested the mutagenic ity of these compounds separately. The results are shown in Table 2. cis and trans isomers of 1,3-dichboropropene were I Supported by Ministero della SanitÃ , Rome, Italy. Received August 9, 1976; accepted January 27, 1977. strongly mutagenic in strains TA 1535 and TA 100, therefore

JUNE 1977 1915

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1977 American Association for Cancer Research. F. Do Lorenzo et a!. Table 1 Mutagenicityof Teloneand 0. 0. soil fumigant with and without liver microsomal fraction D. D. soil fumigant on Tebone were diluted with dimethybwereNo. sulfoxide, and aliquots containing the different amount of the compoun addedto the plates. For the experimental procedure and amount of liver microsomal fraction usedsee Refs. 1 and 6.ds strainsTA of mutant colonies/plate with Salmonella 98Com 1978 TA 1535 TA 100 TA 1537 TA

WithoutWithTebonepound Amount/plate Without With Without With Without With Without With 22221000 8 8 17 17 71 71 15 15 [email protected] 24 115 12 15 178 151 25 23 35311mg@g 36 225 48 59 225 191 31 27 27362.5mg 45 249 75 90 263 242 18 19 33395mg 53 270 115 135 425 385 34 32 243010mg 61 365 150 220 282 500 27 25 3141D.D.soil 15 150 78 61 192 212 12 35

2222fumi- 0 8 8 17 17 71 71 15 15 3330gant 500 @g 11 123 35 42 125 112 28 33 2833155mg 38 181 45 61 198 250 23 26 362325mg 80 300 151 151 350 450 32 24 mg 75 446 145 150 470 512 19 31 2527

Table 2 Mutagenicity of dichloropropene and dichloropropane with and without liver microsomal fraction For experimental1.CompoundAmount/plateNo. pnocedure see Table strainsTAof mutant colonies/plate with Salmonella 100WithoutWithWithout1535TA 1978TA

WithWithoutWithNocompound192125 288789(control)cis-1

21594731propene50,3-Dichbono 20 @g2437719 @g 71 1551trans-i 100kg680 1210490 99090 119 1311800 17502100 31362650propene50,3-Dichlono 20 @g23510927 @g 75 15502,3-Dichbono-1-20 100 @g430 925381 82868 115 911750 18202200 51531450propene50 @g19021231 @g 97 13551 100 j@g650 1080451 87585 98 811520 19001091

,2-Dichlonopno38220185pane20 10 mg758127 mg 21 50 mg210 41 1185 31238 48 15480 850450 920 causing base-pair substitutions (missense mutations), and widely used pesticides. In 1972 in Italy 2,187,100 kg were only weakly mutagenic in strain TA 1978. 2,3-Dichboro-i- produced (Annuanio Italiano di Statistica); in the Nether propene showed similar behavior. 1,2-Dichboropnopane was lands it has been used on more than 300 sq km/year to mutagenic in strains TA 1535 and TA 100 but only at con combat potato cyst nematodes (11). The California State centrations at least 500-fold higher than those of dichboro Department of Agriculture reported (State Pesticide Use propene. We tested allyl chloride and 1,3-dichbonopropene Report 1971) that in 1971 approximately 1,284,84i kg of 2-butene, which showed structural similarity with 1,3-dich pesticides containing 1,3-dichboropropene were used in the boropropene, but they did not show any mutagenic effect state and that it could be estimated that 8% (72,576 kg) was (see Chart 1). The mutagenic properties ofavailable purified lost in the atmosphere (8). The residues of 1,3-dichboropro components do not fully account for the mutagenicity of the pene in crops are below the limit of determination (10). 1,3- commercial preparations of Tebone and D. D. soil fumigant; Dichboropropene disappears from the soil at the rate of 2 to the difference might be due to the presence of the 20% 20% per day depending on the composition of the soil (5). unknown compounds in the batter. It is difficult to evaluate quantitatively the danger of the contamination of the environment resulting from the use of DISCUSSION 1,3-dichboropropene. We must emphasize, however, that due to the nature of the danger (mutagenic effect) even 1,3-Dichboropmopene is the active principle of many trace amounts in the environment must be considered

1916 CANCER RESEARCH VOL. 37

H CH2CI on the carcinogenicity of dichboropropene are now laboratory.CISTRANS 1,3 DICHLOROPROPENE MUTAGENies being carried out in this CI H

HH & 1,3 DICHLOROPROPENE MUTAGENACKNOWLEDGMENTSWe @I&17c1 thank Professor Bruce N. Ames, University of California, Berkeley, â€˜iiCl of2,3 Calif., for providing strains of S. typhimurium and for his helpful criticism work.REFERENCESALLYL C=C DICHLORO-1PROPENEthis H CH@Cl

l@IH

s@=? CHLORIDE NOT MUTAGEN1 . Ames, B. N. The Dectection of Chemical Mutagens with Enteric Bacteria. H CH@Cl In: A. Hoblaender (ed.), Chemical Mutagens, Vol. 1, pp. 267-282. New York: Plenum Press, 1971. @H.H Mutagenic:1.3DIcHLOROPROPENE-2BUTEt* 2. Ames, B. N., Kammen, H. 0., and Yamasaki, E. Hair-dyesAre i!: itt!: NOT MUTAGENIdentification of a Variety of Mutagenic Ingredients. Proc. NatI. Acad. ciCH2CI Sci. U. 5., 72: 2423-2427, 1975. 3. Ames, B. N., Lee, F. D., and Durston, W. E. An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcino HH 1973.VINYL gens. Proc. NatI., Aced. Sci, U. S., 70: 782-786, CHLORIDE MUTAGEN4. Bartsch, H., and Montesano, R. Mutagenic and Carcinogenic Effects of Cl H Vinyl Chloride. Mutation Res., 32: 93-114, 1975. 5. Castro, C. E., and Belser, N. 0. Hydrolysis ofCis and Trans 1,3-Dichboro HHH in Wet Soil. J. Agr. Food Chem., 14: 69-70, 1966. I II H._? â€”Câ€”liâ€”H1,2-DICHLOROPROPANE MUTAGEN 6. McCann, J., Simmon, V., Streitwieser, D., and Ames, B. N. Mutagenicity CI CI CIpropene of Chboroacetabdehyde, a Possible Metabolic Product of 1,2-Dichboro Chlorohydrin),Chart ethane (Ethylene Dichbonide), Chboroethanol (Ethylene 1.Vinyl Chloride, Cyclophosphamide. Proc. NatI. Acad. Sci. U. S.. 72: 1975.harmful. 3190-3193, McCann, J., Spingam, N. E., Koboni, J., and Ames, B. N. Detection of These considerations intensify when applied to the7. Carcinogens as Mutagens: Bacterial Tester Strains with R-Factor Plas safety provisions ofthe factories producing this compound. Proc. NatI. Aced. Sci. U. S., 72: 979-983, 1975. 8. Mc Kenry, M. V., and Thomason, I. J. 1,3 Dichloropropene and 1,2 The relationship between mutagenicity and carcinogenic Dibnomcethane: I. Movement and Fate as Affected by Various Conditions ity1974.recent of chemical compounds has been well documented inmids. in Several Soils. Hilgardla, 42: 392-420, years. Ames et al. (3) have shown that a great number U., Johannon, A., Ramel, C., and Wachtmeister, C. A. The Mutagenicity of Vinyl Chloride after Metabolic Activation. Ambio, 3: 194â€” of1974.bacterial webb-known chemical carcinogens are mutagens on theRannug,197, Whatmutagenictester strains. The results of our studies on the10. Thomason, L. J., Castro, C. E., Raines, A. C., and Mankau, R. to Soil Fumigants after Nematode Control? Calif. Agr. 25: 10- effect of 1,3-dichboropropene and of some me 13, 1971. bated compounds can be compared to the mutagenic andHappensi i . Van Dijk, H. Degradation of 1,3 Dichboropropenes in the Soil. Agro carcinogenic effect of vinyl chloride (see Chart 1 and Refs. Ecosistem, 1: 193-204, 1974. 12. Viola, P. L., Bigotti, A., and Caputo, A. Oncogenic Response of Rat 4, 6, 9, and 12). The structural similarity between these Skin Lungs and Bones to Vinyl Chloride. Cancer Res., 31: 516-522, compounds suggests a similar mechanism of action. Stud 1971.

JUNE 1977 1917

Francesco De Lorenzo, Sandro Degl'Innocenti, Aldo Ruocco, et al.

Cancer Res 1977;37:1915-1917.

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