United States Environmental Protection Agency Health Effects Research Laboratory Research Triangle Park, NC 2771 1 Research and Development EPA-600/S1-83-005 Sept. 1983 &EPA Project Summary Detection of Carcinogenicity Based on Mutagenicity in Arabidopsis George Redei and Gregona N. Acedo Thirty-seven synthetic chemicals plus two mycotoxins were tested for mu- tagenicity in an Arabidopsis embryo system. The results of this test, pro- karyotic repair tests, bacterial mutation assays, eukaryotic cell systems and in vivo tests were compared to the car- cinogenicity classifications of the chemicals. Thirty-two of the 37 chemicals tested were correctly identified as either mu- tagenic or nonmutagenic in the Arabi- dopsis assay. To com pa re these results with those of the other assays, we defined three criteria. "Sensitivity" in- dicated the percentage of tested car- cinogens that were mutagenic in a system. Of 20 carcinogens tested in the Arabidopsis assay, 1 9 were mu- tagenic (sensitivity 95%). For the other assays, sensitivities ranged from 16% to 88%. "Specificity" indicated the percentage of noncarcinogens that were nonmutagenic in a system. Three of twelve noncarcinogens were non- mutagenic to Arabidopsis (specificity 25%). For the other assays, specificities ranged from 20% to 100%. The non- carcinogens pyrene, 4-acetylamino- fluorene, 1-naphthylamine, isopropyl- N-(chlorophenyl) carbamate, azoxyben- zene, and ascorbic acid were mutagenic in the Arabidopsis assay and other assays. "Accuracy" indicated the per- centage of correctly identified chemi- cals based on the predicted classifica- tion. For the Arabidopsis assay, 22 of 32 chemicals were correctly identified (accuracy 69%). Overall accuracies for the other assays ranged from 57% to 62%. The Arabidopsis assay was the most sensitive and accurate short-term test in our comparison. Therefore, Arabidopsis can be used to supplement a battery of short-term tests for identifying car- cinogens. This Project Summary was developed by EPA's Health Effects Research Lab- oratory, Research Triangle Park, NC. to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report ordering information at back). Introduction In an international program sponsored by the British Medical Research Council, the U S. National Institute of Environmental Health Sciences, the U.S Environmental Protection Agency (EPA), and the National Cancer Institute of Japan, workers from 63 laboratories evaluated the mutagenic effects of 42 chemicals using different short-term biological assays, such as pro- karyotic repair tests, bacterial mutation assays, in vitro eukaryotic cell assays, and in vivo tests with Drosophila and mice (the results of these studies were published in Evaluation of Short- Term Tests for Car- cinogens, F.D. deSerresandJ Ashby, eds., 1981, Elsevier/North Holland, New York). From the results of previous animal assays, the chemicals were classified as proven carcinogens, harmless analogues of car- cinogens, and noncarcinogens This pro- gram was intended to help researchers select the most effective series of short- term tests. Because no single system consistently identified all the carcinogens and noncarcinogens, we retested 37 chemicals using an Arabidopsis assay and compared the results to those of the other assays. ------- Arabidopsis thaliana (L) Heynh. seeds were soaked in water for 24 hr and then treated for 1 5 hr with chemicals dissolved in either medium with nutrients and minerals or dimethylsulfoxide (DMSO). As a control, some seeds were concurrently exposed only to the solvents. Approximately 250 seeds per treatment were planted on Promix medium, moistened, placed in covered glass vessels, and incubated at 24° C under a light intensity of 800 foot-candles. After germination, 1 00 to 200 plants per culture vessel were examined for mutations (fruits with pale or white embryos) The mutation frequency for exposed seeds minus control frequency was recorded as "mutation %." To compare the assays, we defined three criteria "Sensitivity" indicated the percentage of tested carcinogens that were mutagenic to a system. "Specificity" indi- cated the percentage of noncarcmogens that were nonmutagenic to a system. "Ac- curacy" indicated the percentage of correctly identified chemicals based on the predicted classification The Arabidopsis assay has many advan- tages: (i) it is a eukaryotic test, (n) forward mutations are scored at more than 10,000 loci, which ensures a representative sample of the genome, (in) the results of the embryo assays can be supplemented by progeny tests, (iv) the assay does not rely on any supplement for activation; the plant seems to convert most promutagens into ultimate mutagens, (v) the test is less expensive than most higher eukaryotic assays, and (vi) according to literature survey, 88% of the tested carcinogens are mutagenic to Arabidopsis. Results Thirty-two of the 37 chemicals tested with the Arabidopsis assay could be cor- rectly identified as mutagens or nonmuta- gens. Of these 32 chemicals, 28 were mutagenic and four were nonmutagenic. Nineteen of 20 carcinogens were mu- tagenic in the Arabidopsis assay (sensitivity 95%) The sensitivity ranges for the other tests were prokaryotic repair tests, 1 6% to 73% , bacterial mutation assays, 27% to 76%, eukaryotic cell systems, 8% to 88%, and in vivo tests, 1 7% to 52%. Table 1 compares the sensitivities of all assays combined, bacterial mutation as- says, and the Arabidopsis assay. Of the 2 5 carcinogens, 14 (Group I) were correctly identified as mutagens by all assays com- bined (sensitivity 60% or better) and bac- terial mutation assays (sensitivity 70% or better). The Arabidopsis assay identified 10 of the 14 Group I compounds as mutagens (sensitivity 71 %) The identifica- Table 1 A comparison of the sensitivities of all assays combined, the bacterial mutation tests, and the Arabidopsis assay for 25 carcinogens. Sensitivity is defined as the percentage of carcinogens that are mutagenic in a system. The symbols •, -, 7, and NT indicate mutagenicity, nonmutagenicity, inconclusive results, and not tested, respectively Carcinogens Group I fi-Propiolactone 4-Nitroqumoline-N-oxide 2-Acetylammofluorene Benzo(a)pyrene Epichlorohydnn Methylazoxymethanolacetate Methylene bis(2- chloroaniline) 2-Naphtylamine Cyclophosphamide Hydrazine sulphate Dimethylanthracene Benzidme Dimethylcarbamoyl chloride Nitrosomorpholme Group II Auramme Dimethylammobenzene O- Toluidme Hexamethylphosphoramide Safrole Urethane Ethylenethiourea Ethionme Diethylstilbestrol Chloroform Ammotnazole All assays 93 89 86 83 82 81 77 75 74 71 70 68 67 60 49 48 44 38 38 38 32 28 24 20 20 Sensitivity Bacterial mutation 100 700 700 700 95 73 84 95 74 80 88 85 76 70 48 45 33 7; 20 26 20 14 14 11 70 Arabidopsis • NT • • • • • ? • • ? • 7 • • • • 7 • • • • • • - tion of Group II carcinogens was more difficult. The bacterial mutation and com- bined assay sensitivities were below 49% for these carcinogens. However, Arabidopsis identified 9 of 1 1 Group II carcinogens as mutagens (sensitivity 82%). Only three of the 1 2 presumably non- carcinogenic chemicals were nonmutagenic in the Arabidopsis assay (specificity 25%) Specificity ranges for the other assays were: prokaryotic repair tests, 35% to 75%; bacterial mutation assays, 59% to 82%, eukaryotic systems, 23% to 85%; and in vivo tests, 20% to 1 00%. Although classified as noncarcmogens, pyrene, 1- naphthylamme, 4-acetylammofluorene, isopropyl-N-(chlorophenyl) carbamate, azoxybenzene, and ascorbic acid were mutagenic in the Arabidopsis assay and other assays. Table 2 compares the pooled accuracies of four assay systems with the accuracies of \he Arabidopsis assay for 42 chemicals. The Arabidopsis assay correctly identified 69% of the chemicals and was the most accurate assay. Overall accuracies for the other systems were: prokaryotic repair tests, 58%, bacterial mutation assays, 62%; eukaryotic tests, 60%; and in vivo tests, 57%. Conclusion A higher percentage of carcinogens and presumed noncarcmogens were mutagenic to Arabidopsis than to the prokaryotic repair systems, bacterial mutation assays, eukaryotic tests, and in vivo tests. This high sensitivity can be attributed to the large number of target loci in the Arabidop- sis genome. Also, the Arabidopsis system correctly identified carcinogens and non- carcinogens more consistently than the other assays. Therefore, the Arabidopsis assay could be a valuabe addition to a battery of short-term tests for identifying carcinogens 2 ------- Table 2 A comparison of the pooled accuracies of four assay systems and the accuracy of the Arabidopsis assay for 42 chemicals Accuracy is defined as the percentage of correctly identified chemicals based on the predicted classification Jhesymbols •, -, NT, and ? indicate carcinogen/city, noncarcmogenicity, not tested, and inconclusive results, respectively. Accuracy (%) Chemicals Nitroquinolme-N-oxide Methyl nit roquinol me N-oxide Benzidme Tetramethylbenzidme Dimethyl ammobenzene D/methylammobenzene sulphonic acid-Na I'rPropiolactone j-Butyrolactone Benzofajpyrene Pyrene Ethionine Methionine Chloroform Tnchloroethane 2-Acetylammofluorene 4-Acetylammofluorene 2-Naphthylamme 1-Naphthylamme Nitrosomorpholine Diphenylnitrosomme Urethane lsopropyl-N-(3-chlorophenyl) carbamate Methylazoxymethanol- acetate Azoxybenzene Dinitrosopentamethylene tetramme Hydrazine sulphate Hexamethylphosphoramide Safrole Diethy/stilbestrol Cyclophosphamide Epichlorohydrme Auramme Methylene bis(2-chloro- aniline) To/uidine Ethylenethiourea Aminotnazole Dimethylcarbamoyl chloride Dimethylformamide D/methy /anthracene Anthracene Sucrose Ascorbate Repair tests • 100 0 • 57 71 • 0 77 • 700 67 • 71 - 75 • 29 - 67 • 17 - 83 • 71 - 33 • 71 - 50 • 43 - 57 • 38 - 50 • 86 17 17 83 17 86 29 67 80 57 100 43 67 77 • 77 - 700 • 57 - 63 - 700 67 Bacterial mutation 100 0 85 95 45 45 100 90 100 57 14 90 11 94 100 0 95 30 70 70 26 100 73 37 90 80 11 20 14 74 95 48 84 33 20 10 76 82 88 89 100 84 Eukaiyot/c systems 91 5 68 88 64 65 92 71 77 53 35 76 31 67 73 50 75 36 56 65 41 38 83 46 56 21 48 39 26 73 20 50 54 64 38 46 64 76 70 92 75 58 In vivo tests 20 60 40 88 40 100 33 100 83 100 40 100 0 100 100 100 29 78 50 80 100 100 100 67 75 0 88 20 33 100 20 0 66 0 0 0 40 100 0 100 100 100 Arabidopsis assays NT NT 100 7 100 NT 100 7 100 0 100 100 100 0 100 0 7 0 100 0 100 0 100 0 7 100 7 100 100 100 700 700 700 700 700 0 7 0 7 100 100 0 Total 89 8 68 88 48 62 93 81 83 62 28 81 20 81 86 27 75 41 60 66 38 67 81 38 67 71 38 38 34 74 82 49 77 44 32 20 67 82 70 85 92 72 Accuracy of the systems 583 62 1 595 573 688 608 ------- George Redei and Gregoria N. Acedo are with the University of Missouri, Columbia, MO 65211. Shahbeg S. Sandhu is the EPA Project Officer (see below). The complete report, entitled "Detection of Carcinogenicity Based on Mutagenicity in Arabidopsis," (Order No. PB 83-225 078, Cost: $10.00, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officer can be contacted at: Health Effects Research Laboratory U S Environmental Protection Agency Research Triangle Park, NC 27711 -,-US GOVERNMENT PRINTING OFFICE 1983-659-017/7186 United Stales Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Postage and Fees Paid Environmental Protection Agency EPA 335 Official Business Penalty for Private Use S300 U 5 tNVIK KKUTtCriUN U* 5 H6KAKY o D£AkbOK,v SIRtEF CHICAGO IL 606U4 ------- |