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Tradescantia stamen hair mutation bioassay
Fundamental and Molecular Mechanisms of Mutagenesis
Mutation Research 310 (1994) 211-220
ELSEVIER
Tradescantia stamen hair mutation bioassay T.-H. Ma a,. G.L. Cabrera
b A. Cebulska-Wasilewska
A.L. Vandenberg
c, R . C h e n
e, M . F . S a l a m o n e
d F. Loarca
b
e
a Department of Biological Sciences, 525 Meadow Drive, Western Illinois University, Macomb, IL 61455, USA b Centro de Estudios Academicos sobre Contaminacion Ambiental, Universidad Autonoma de Queretaro, Queretaro, QRQ, Mexico c Radiobiology Department, Institute of Nuclear Physics, ul. Radzikowskiego 152, 31-342 Cracow, Poland d Guangxi Institute of Botany, Yanshan, Guilin 541006, People's Republic of China c Ministry of Environment and Energy, Biohazard Unit, 125 Resources Road, Etobicoke, Ont. M9P 3V6, Canada
Accepted 16 November 1993
Abstract
The Tradescantia stamen hair mutation (Trad-SH) assay (clone 4430) was evaluated for its efficiency and reliability as a screen for mutagens in an IPCS collaborative study on plant systems. Four coded chemicals, i.e. azidoglycerol (AG, 3-azido-l,2-propanediol), N-methyl-N-nitrosourea (MNU), sodium azide (NaN 3) and maleic hydrazide (MH) were distributed by the Radian Corporation to the five laboratories in five different countries for testing mutagenicity. Pink mutations were scored between the 7th and 14th day according to a standard protocol. Test results from the five individual laboratories were analyzed and compared after decoding. One out of the two laboratories that conducted tests on AG demonstrated that AG is a mutagen with genetically effective doses ranging from 50 to 100/~g/ml. MH yielded positive responses in all laboratories but no linear dose-response pattern was observed. The effective dose range for MH was between 1 and 45/zg/ml. The mutagenicity of MNU was reported by five laboratories in the dose range between 10 and 80/zg/ml. NAN3, which exhibited a relatively high degree of toxicity, elicited a positive mutagenic response in three of the five laboratories in which it was tested. As with MNU the effective dose for NaN 3 ranged between 3 and 80/xg/ml. The results from the current study substantiate the Trad-SH assay as a reliable system for screening chemicals for their potential mutagenic effects. Although the study was carried out exclusively under laboratory conditions, a survey of the current literature would indicate that the Trad-SH assay could be an effective in situ monitor of gaseous, liquid, and radioactive pollutants as well. Keywords: Tradescantia clone 02; Tradescantia clone 4430; Maleic hydrazide; Azidoglycerol; N-Methyl-N-nitrosourea;
Sodium azide I. Introduction
T h e Tradescantia stamen hair m u t a t i o n (TradSH) test was developed by Dr. A r n o l d H. Spar-
* Corresponding author.
row and colleagues during the late 1950s and t h r o u g h o u t the 1960s at the Brookhaven National Laboratory, L o n g Island, NY. Early studies with Tradescantia c o n c e n t r a t e d on the genetic effects induced by nuclear radiation (Sparrow et al., 1972). Subsequently, the assay was a d a p t e d for the detection of mutagenic airborne agents and
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volatile organic c o m p o u n d s (Sparrow and Schairer, 1971) and still later for the evaluation of chemical agents in liquid form (Schairer et al., 1982). Since the discovery of its high sensitivity to gaseous mutagens, the Trad-SH assay has been a major part of the in situ air monitoring program at several industrial areas in the USA (Schairer and Sautkulis, 1982; Schairer et al., 1982). Furthermore, the Trad-SH assay has been used to test a wide range of chemicals such as pesticides (Ahmed and Grant, 1972), sewage sludge (Hopke et al., 1982), soil samples in the vicinity of a lead smelter (Lower et al., 1983) and engine exhaust fumes (Ma et al., 1983). Recently, the Trad-SH assay has been used in the in situ monitoring of waterways and aquatic effluents (Grant et al., 1992). The Trad-SH is a simple, cost effective shortterm bioassay that yields data within 11-14 days and is easily adapted for either in house or in situ monitoring. The principal cells in this assay are the mitotic stamen hair cells developing in the young flower buds. The assay is based on the fact that these stamen hair ceils in Tradescantia clone 4430 (and other clones) are heterozygous for flower color with the visible marker being the phenotypic change in pigmentation from blue (dominant) to pink (recessive). Thus, most mutations at the blue allele will produce a recessive pink cell among the blue cells (a single mutant event). A pink mutation occurring at this time may continue to divide and give rise to a contiguous series of pink cells. Such a series of pink cells separated by blue cells is also considered only a single mutant event. A stamen hair that has two pink cells separated by one or more blue cells is considered to have undergone two mutant events (Underbrink et al., 1973; Schairer et al., 1978). In general, the mutation rate is generally calculated on the bases of mutation events per 1000 hairs. This study was established under the sponsorship of the International Programme on Chemical Safety (IPCS). The purpose was to evaluate the effectiveness and reliable performance of some commonly used plant bioassays of which the Trad-SH was selected as one. The five laboratories selected to participate in this study were the Biohazard Unit of the Ministry of Environment,
Toronto, Canada (CAN); the Laboratory of Nuclear Physics, National Academy of Science, Poland (POL); the Guangxi Institute of Botany, the People's Republic of China (CHN); the Centro de Estudios Academicos sobre Contaminacion Ambiental, Universidad Autonoma de Queretaro, Mexico (UAQ); and the Laboratory of Environmental Mutagenesis, Western Illinois University, USA (US-WIU). The effectiveness and reliability of the Trad-SH assay were judged by evaluating the uniformity in the results of the five different laboratories. The sensitivity of the test system was judged by evaluating Tradescantia's effectiveness in detecting mutagens as positive on the assay, and by evaluating both the minimum effective dose (MED) for a positive response, and the response to toxic effects.
2. Materials and methods
Tradescantia clone 4430 (heterozygous for the b l u e / p i n k alleles in their stamen hair cells) was chosen for this collaborative study for its high sterility and sensitivity to chemical mutagens. Since the clone is vegetatively propagated it retains its genetic homogeneity. A detailed account of the principles and experimental procedures has been published previously (Underbrink et al., 1973; Schairer et al., 1982). For practical purposes, an outline of this assay is given in Appendix 1 of this paper.
Selection of test chemicals The chemicals used in the study were azidoglycerol (AG, 3-azido-l,2-propanediol; CAS No. 73018-98-1), N-methyl-N-nitrosourea (MNU, CAS No. 684-93-5), sodium azide (NaN 3, CAS. No. 26628-22-8) and maleic hydrazide (MH, CAS No. 123-33-1). Plant cuttings were treated with each chemical dissolved in distilled water using three increasing dosages. Each of the five participating laboratories, except the laboratory of the People's Republic of China, conducted at least three trials with each chemical. The CHN laboratory conducted only two rounds of tests for each of the chemicals but used a large number of plant
T.-H. Ma et al. / Mutation Research 310 (1994) 211-220
samples to attain high reliability. Treatments were generally applied for 24-30 h followed by a recovery period of 7-14 days before scoring for pink mutation events.
Statistical analysis of data Data were analyzed with a computer software program called 'CoStat' which contains the A N O V A F-test for variance and the StudentNewman-Keuls t-test. The significance of differences among one control with three test groups was determined by Dunnett's t statistic at the 0.05 probability level.
3. Results and discussion
The results from all five laboratories in this study are summarized in Tables 1-4. In most Table 1 Mean pink mutation rates of maleic hydrazide (MH) tested with the Trad-SH assay among five laboratories (mutations/ 1000 hairs) Laboratory code a
Dose (/zg/ml)
SH frequency (mean + SD)
CAN
0 10 20 40
8.4 + 24.5 + 28.0 + 30.4+
3.4 6.3 * 6.0 * 4.5 *
POL
0 15 50 80
1.4+ 13.6+ 14.7+ 23.7±
0.6 4.3 * 4.4 * 6.8 *
CHN
0 10 40
0.6+ 0.1 3.2+ 0.3 * 4.6± 0.2 *
UAQ
0 10 15 30
2.3 + 24.7+ 21.7 + 31.3 +
US-WlU
0 4 10 45
2.9 + 8.3 ± 14.2± 16.5 ±
2.2 11.7 11.7 11.4 * 0.3 4.4 9.3 0.78 *
a Laboratory codes: CAN, Canada; CHN, People's Republic of China; POL, Poland; UAQ, Universidad Autonoma de Queretaro; US-WlU, Western Illinois Unversity. * Positive response according to Dunnett's t-test ( P < 0.05).
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cases (except for the CHN laboratory where each chemical was tested only twice) the values presented represent the average result of three trials. For analysis, the data from the different repetitions were combined. Thus, it is possible that, even if two out of three individual trials generated a significant positive result, when combined, the overall response for a given dose may be negative in the Dunnett t-test. Based upon this fact, it is significant to note the high degree of congruity in the results for the five laboratories. All five laboratories detected MH as positive in the assay system, while four out of five (80%) observed MNU as a positive mutagen of pink events. One out of two laboratories detected A G as a mutagen and three out of five (60%) detected NaN 3 as a mutagen. That totals to 13 out of 17 results or 76.5% being positive. Furthermore, of the 51 doses tested by all the laboratories, including the lowest non-zero doses, 45% were positive. The low MED values for NaN 3 (3 /zg/ml), M H and MNU ( 1 0 / z g / m l ) indicate that the Trad-SH assay is very sensitive to these compounds. As well, there was a high level of agreement in the results among the participating laboratories with regard to the qualitative analysis of a positive response, although quantitatively, there was not full agreement.
Maleic hydrazide As indicated, M H induced a significant elevation in the frequency of pink mutations in all five laboratories (Table 1). In fact, 12 of the 14 treatment doses, or 86%, were classified as positive responses, although dose-effect related responses were not produced in all laboratories. The effective range for MH among the five laboratories was 1 0 - 8 0 / z g / m l . These results with MH are in agreement with earlier studies where M H has been found to be a strong mutagen in tobacco plants (Nicotiana tabacum, Brisza et al., 1984), tomato seeds (Lycopersicon esculentum, Grant and Harney, 1960), barley (Hordeum uulgare, Kak and Kaul, 1975; Malepszy et al., 1973), Tradescantia (Gichner et al., 1982), Zea mays (Plewa and Wagner, 1981), Drosophila melanogaster (Nasrat, 1965) and on the TK locus of mouse lymphoma cells (Paschin, 1981). As well, M H was
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Table 2 Mean pink mutation rates of methyl nitrosourea (MNU) tested with the Trad-SH assay among five laboratories (mutations/ 1000 hairs) Laboratory dose a
Dose (~g/ml)
SH frequency (mean±SD)
CAN
0 8.4 16.7 33.4
5.9±1.8 11.3±5.1 13.6±5.6" 11.9±3.1
POL
0 4 12.5 50
2.4±0.9 3.4±3.6 2.8±2.8 8.7±5.7
CHN
0 40 60 80
0.6±0.2 1.2±0.5 1.1±0.7 20.9±2.9*
UAQ
0 25 45 55
2.5+1.6 6.2±3.0 9.0±3.5 12.5±8.0"
US-WIU
0 5 10 50
1.5±0.5 2.9±3.0 4.2±2.1" 5.2±1.5"
Laboratory codes: CAN, Canada; CHN, People's Republic of China; POL, Poland; UAQ, Universidad Autonoma de Queretaro; US-WlU, Western Illinois Unversity. * Positive response according to Dunnett's t-test (P < 0.05).
shown to be a strong clastogen in the current collaborative study in both the Tradescantia stamen hair and Vicia faba root assays.
were no dose-effect related responses observed in any of the laboratories. The mutagenic effects of M N U observed here agree with the results of the Arabidopsis mutation test in this collaborative study, as well as the finding in previous mutation studies with Arabidopsis thaliana (Gichner et al., 1982), barley (Gichner et al., 1982), Drosophila melanogaster (Vogel and Natarajan, 1979), Saccharomyces cerevisiae (Schwaier et al., 1968), Salmonella typhimurium (Gichner et al., 1982) and the Trad-SH assay (Gichner et al., 1982).
Sodium azide N a N 3 exhibited positive responses for the stamen hair assay in three of the five laboratories in which it was tested (Table 3). In each of the three laboratories, however, the positive response was
Table 3 Mean pink mutation rates of sodium azide (NaN 3 tested with the Trad-SH assay among five laboratories (mutations/1000 hairs) Laboratory code a
Dose (~g/ml)
SHfrequen~ (mean±SD)
CAN
0 10 20 80
3.5±0.8 7.2±3.3 8.9±3.6 14.0±2.3"
POL
0 8 10 25
2.4+0.9 6.1±2.8 5.9±3.1 5.2±2.4
CHN
0 4 6 8
UAQ
0 5 10 18
1.8±0.8 2.2±0.7 3.9±2.7 2.0±1.0
US-WIU
0 3 6 20
1.8±0.5 3.2±0.6" 1.7±0.8 2.4±1.0
N-Methyl-N-nitrosourea M N U showed the next highest degree of congruity among the laboratories in that four out of the five laboratories observed M N U to induce a positive response (Table 2). In the one laboratory in which it was not detected positive, the exposure time was considerably less than in the other four laboratories. Unlike MH, however, mutagenicity of M N U was not as easily detected. Only 33% (five out of 15) treatment doses were signified positive by the Dunnett t-test. The minimum effective dose for M N U was 10 /.~g/ml and its positive dose range went up to 80 t z g / m l . There
1.6±0.4 2.4±0.1" 2.0±0.7 2.5±0.5
a Laboratory codes: CAN, Canada; CHN, People's Republic of China; POL, Poland; UAQ, Universidad Autonoma de Queretaro; US-WIU, Western Illinois Unversity. * Positive response according to Dunnett's t-test (P < 0.05).
T.-H. Ma et al. / Mutation Research 310 (1994) 211-220 Table 4 Mean pink mutation rates of 3-azidoglycerol (AG) tested with the Trad-SH assay among two laboratories (mutations/1000 hairs) Laboratory code a
Dose (/xg/ml)
SH frequency (mean + SD)
CAN
0 5 50 100
5.4 _+3.2 6.6+2.3 5.7 + 1.7 7.5 _+4.3
US-WlU
0 10 50 100
1.7 5:0.3 2.9_+0.9 4.2+2.0 * 5.1 -+ 1.4 *
a Laboratory codes: CAN, Canada; US, US-WIU, Western Illinois Unversity. * Positive response according to D u n n e t t ' s t-test ( P < 0.05).
restricted to a single dose which differed in each laboratory and which ranged between 3 and 80 /xg/ml. These data support the general published results for NaN 3. NaN 3 has been a well-known mutagen for many years and has induced mutations in a number of plants including barley (Nilan et al., 1976), black gram (Phaseolus mungo L., Mahna et al., 1989), pea (Pisum sativum, Kleinhofs et al., 1978), petunia (Petunia hybrida, Khalatkar and Kashikar, 1980) and oats (Arena sativa, Rines, 1985). NaN 3 also appears to be able to induce nondisjunction (Vig, 1973) but has been reported to be a nonclastogen (Sander et al., 1978; Ma, 1979). However, in some cases it has been shown to be a very weak inducer of chromosome aberrations but this was only at high doses and at pH 3 (Veleminslo) et al., 1977). Since NaN 3 is considered a mutagen without potent clastogenic properties, it is of interest to note that it was as easily, if not more easily, detected in this collaborative study with the Trad.MCN assay as with the Trad-SH assay.
Azidoglycerol Special attention has been given to the genotoxicity of A G in recent years owing to the fact that it may have similar pharmacokinetics as A Z T (3'-azido-3'-deoxythymidine), a popular drug for AIDS (Phillips et al., 1991). In this study A G was
215
detected as a mutagen. In only one of the two laboratories in which it was tested it produced positive responses (Table 4). In these laboratories, two of the six concentrations were considered significantly different from the control, while most of the concentrations around 5 0 - 1 0 0 / z g / m l showed a trend towards high mutation rates.
4. Conclusions and recommendations
A comparative analysis of the results of the Trad-SH tests from the five laboratories indicates that all four chemicals were mutagens. In addition, there was a relatively high congruity among the five laboratories for all four chemicals. M H was the most potent and easily detected of the four compounds, while NaN 3 had the lowest detection rate as only three out of 15 doses were significantly different from the control. Based upon the results with the Trad.MCN assay in this study, the genotoxic nature of NaN 3 may not be as clear as indicated in the literature and further studies should be initiated to verify the picture regarding the genotoxic action of NaN 3 in general and specifically with Tradescantia. This study substantiates A G as a mutagen, as shown in the Trad-SH assay, although it is not a strong clastogen as shown by the results of the Trad.MCN assay in this collaborative study. The appraisals of the results from earlier data bases and this study further document the TradSH assay as a sensitive, reliable system for monitoring external radiation, screening radioactive pollutants as well as gaseous and liquid mutagens in the laboratory. The Trad-SH assay has a broad data base from radiation biology and is a wellvalidated monitor of nuclear radiation both in the laboratory and in the field (Sparrow and Schairer, 1971; Sparrow et al., 1972; Schairer et al., 1982; Ma, 1979, 1992). In addition, the literature indicates this assay is well suited for in situ monitoring of the mutagenicity of air and water pollutants (Grant et al., 1989, 1992; Ma, 1992) as well as for external radiation sources (Sparrow et al., 1972; Ichikawa, 1981; Shevchenko, 1989).
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Acknowledgements The authors express their deepest appreciation to the International Progamme on Chemical Safety of the World Heath Organization, the United Nations Environmental Programme and the US Environmental Protection Agency for their financial support for conferences and chemicals, and to Dr. William F. Grant of McGill University for his assistance in the preparation of the manuscript.
References Ahmed, M. and W.F. Grant (1972) Cytological effects of the pesticides phosdrin and bladex on Tradescantia and Vicia faba root tips, Can. J. Genet. Cytol., 14, 157-165. Brisza, J., T. Gichner and J. Velemlnsk~ (1984) Somatic mutation in tobacco plants after chronic exposure to maleic hydrazide and its diethanolamine and potassium salts, Mutation Res., 139, 25-28. Gichner, T., J. Velemlnsk~ and V. Pokorny (1982) Somatic mutations induced by maleic hydrazide and its potassium and diethanolamine salts in the Tradescantia mutation assay, Mutation Res., 103, 289-293. Grant, W.F. and P.M. Harney (1960) Cytogenetic effects of maleic hydrazide treatment of tomato seed, Can. J. Genet. Cytol., 2, 162-174. Grant, W.F., H.G. Lee, D.M. Logan and M.F. Salamone (1989) The use of Tradescantia and Vicia faba bioassays for the in situ detection of mutagens in an aquatic environment, Environ. Mol. Mutagen., 14 (Suppl. 15), 75 (Abstract). Grant, W.F., H.G. Lee, D.M. Logan and M.F. Salamone (1992) The use of Tradescantia and Vicia faba bioassays for the in situ detection of mutagens in an aquatic environment, Mutation Res., 270, 53-64. Hopke, P.K., M.J. Plewa, J.B. Johnston, D. Weaver, S.G. Wood, R.A. Larson and T. Hinesly (1982) Multitechnique screening of Chicago municipal sewage sludge for mutagenic activity, Environ. Sci. Technol., 16, 140-147. Ichikawa, S. (1981) In situ monitoring with Tradescantia around nuclear power plants, Environ. Health Perspect., 37, 145-164. Kak, S.N. and B.L. Kaul (1975) Mutagenic activity of hydrazine and its combinations with maleic hydrazide and X-rays in barley, Cytobios, 12, 123-128. Khalatkar, A.S. and S.G. Kashikar (1980) Sodium azide mutagenicity in Petunia hybrida, Mutation Res., 79, 81-85. Kleinhofs, A., R.L. Warner, F.J. Muehlbauer and R.A. Nilan (1978) Induction and selection of specific gene mutations in Hordeum and Pisum, Mutation Res., 51, 29-35.
Lower, W.R., W.A. Thompson, V.K. Drobney and A.F. Yanders (1983) Mutagenicity in the vicinity of a lead smelter, Teratogen. Carcinogen. Mutagen., 3, 231-253. Ma, T.-H. (1979) Micronuclei induced by X-rays and chemical mutagens in meiotic pollen mother cells of Tradescantia. A promising mutagen test system, Mutation Res., 64, 307-313. Ma, T.-H. (1990) Tradescantia-micronucleus test in clastogens and in situ monitoring, in: M.L. Mendelson and R.J. Albertini (Eds.), Mutation and the Environment, Part E, Environmental Genotoxicity, Risk and Modulation, Wiley-Liss, New York, pp. 83-90. Ma, T.-H. (1992) Monitoring of genetic toxicity of the gaseous agents and leachates from a landfill and the ambient air quality around an incinerator, Report submitted to the Office of Solid Waste Research, Institute for Environmental Studies, University of Illinois, Urbana, IL, 17 pp. (unpublished). Ma, T.-H., W.R. Lower, F.D. Harris, J. Poku, V.A. Anderson, M.M. Harris and J.L. Bare (1983) Evaluation by the Tradescantia-micronucleus test of the mutagenicity of internal combustion engine exhaust fumes from diesel and diesel-soybean oil mixed fuels, in: M.D. Waters, S.S. Sandhu, J. Lewtas, L. Claxton, N. Chernoff and S. Nesnow (Eds.) Short-Term Bioassays in the Analysis of Complex Environmental Mixtures III, Plenum, New York, pp. 8999. Ma, T.-H., G.L. Cabrera, R. Chen, B.S. Gill, M.F. Salamone and A.L. Vandenberg (1994) Tradescantia-micronucleus bioassay, Mutation Res., this issue. Mahna, S.K., R. Garg and M. Parvateesam (1989) Mutagenic effects of sodium azide on black gram (Phaseolus mungo L.), Curr. Sci., 58, 582-584. Malepszy, S., J. Eberhardt and M. Maluszynski (1973) Mutagenic effects of NMH (N-nitroso-N-methylurea), NEH (N-nitroso-N-ethylurea) and MH (maleic hydrazide) in barley, Genet Polon., 14, 47-59. Nasrat, G.E. (1965) Maleic hydrazide, a chemical mutagenic in Drosophila melanogaster, Nature, 207, 439. Nilan, R.A., A. Kleinhofs and C. Sander (1976) Azide mutagenesis in barley, in: H. Gaul (Ed.), Barley Genetics III, Proc. Third Int. Barley Genet. Symp., Theimig, Munich, pp. 113-122. Paschin, Y.V. (1981) Mutagenicity of maleic hydrazide for the TK locus of mouse lymphoma cells, Mutation Res., 91, 359-362. Phillips, M.D., B. Nascimbeni, R.R. Tice and M.D. Shelby (1991) Induction of micronuclei in mouse bone marrow cells: An evaluation of nucleoside analogues used in the treatment of AIDS, Environ. Mol. Mutagen., 18, 168-183. Plewa, M.J. and E.D. Wagner (1981) Germinal cell mutagenesis in specially designed maize genotypes, Environ. Health Perspect., 37, 61-73. Rines, H.W. (1985) Sodium azide mutagenesis in diploid and hexaploid oats and comparison with ethyl methanesulfonate treatments, Environ. Exp. Bot., 25, 7-16.
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Sander, C., R.A. Nilan, A. Kleinhofs and B.K. Vig (1978) Mutagenic and chromosome breaking effects of azide in barley and human leukocytes, Mutation Res., 50, 67-75. Schairer, L.A. and R.C. Sautkulis (1982) Detection of ambient levels of mutagenic atmospheric pollutants with the higher plant Tradescantia, in: E.J. Klekowski Jr. (Ed.) Environmental Mutagenesis, Carcinogenesis, and Plant Biology, Vol. 2, Praeger, New York, pp. 153-194. Schairer, L.A., R.C. Sautkulis and N.R. Tempel (1982) Monitoring ambient air for mutagenicity using the higher plant Tradescantia, in: R.R. Tice, D.L. Costa and K.M. Schaich (Eds.), Genotoxic Effects of Airborne Agents, Plenum, New York, pp. 123-140. Schwaier, R., N. Nashed and F.K. Zimmermann (1968) Mutagen specificity in induction of karyotic versus cytoplasmic respiratory deficient mutants in yeast by nitrous acid and alkylating nitrosamines, Mol. Gen. Genet., 102, 290-300. Shevchenko, V.A. (1989) Some aspects of the genetic consequences of the Chernobyl disaster, in: Transactions of the 10th Int. Conf. on Structural Mechanics in Reactor Technology, Vol. D, pp. 245-249. Sparrow, A.H. and L.A. Schairer (1971) Mutation response in Tradescantia after an accidental exposure to a chemical mutagen, EMS Newslett., 5, 16-19. Sparrow, A.H., A.G. Underbrink and H.H. Rossi (1972) Mutation induced in Tradescantia by small doses of X-rays and neutrons: Analysis of dose-response curves, Science, 176, 916-918. Underbrink, A.G., LA. Schairer and A.H. Sparrow (1973) Tradescantia stamen hairs: A radiobiological test system applicable to chemical mutagenesis, in: A. Hollaender (Ed.), Chemical Mutagens: Principles and Methods for Their Detection, Vol. 3, Plenum, New York, pp. 171-207. Velemlnsk~, J., T. Gichner and V. Pokorny (1977) Induction of DNA single-strand breaks in barley by sodium azide applied at pH 3, Mutation Res., 42, 65-70. Vig, B.K. (1973) Somatic crossing over in Glycine max (L.) Merrill: Mutagenicity of sodium azide and lack of synergistic effect with caffeine and mitomycin C, Genetics, 75, 265-277. Vogel, E. and A.T. Natarajan (1979) The relation between reaction kinetics and mutagenic action of mono-functional alkylating agents in higher eukaryotic systems. I. Recessive lethal mutations and translocations in Drosophila, Mutation Res., 62, 51-100.
Appendix 1: Protocol for the Tradescantia stamen hair mutation (Trad-SH) assay
1. Raising Tradescantia plants Potting soil and other adequate growing media An adequate soil mixture for this plant is composed of 2 parts sand, 1 part peat moss, and 4
217
parts soil. Plants can be grown hydroponically with gravel, Vermiculite or Perlite as supporting medium or in nutrient solution (Hoagland's solution at a 3 × dilution; Hoagland and Arnon, 1950) alone. Liquid fertilizer should be applied to the soil mixture in the pots twice per month, or more often if Vermiculite or Perlite is used. Plants should be lightly watered daily. Heavily chlorinated water may increase the background mutation rate.
The plant Tradescantia clones 4430 and 0 2 are interspecific hybrids which are both suitable for this mutation test. They are heterozygous for b l u e / p i n k alleles in their floral parts. Clone 4430 is more sensitive for the detection of chemical mutagens than clone 0 2 (Nauman et al., 1979). Clone 4430 is a sterile hybrid between T. hirsutiflora (blue floral parts) and T. subcaulis (pink floral parts) with a dominant blue phenotype in all flower parts including the stamen hair cells. Mutagen induced somatic mutation in the ceils is from blue to pink and readily recognizable in the diploid cells of the stamen hairs under a dissecting microscope.
Adequate population size About 150-200 pots (15 cm diameter) of wellgrown plants are needed to supply 100-150 plant cuttings bearing young inflorescences once a week to conduct a test of three or four different treated and control groups (generally, about 20 cuttings are needed for a sample population).
Optimal growing conditions Plants can be grown in a growth chamber or in a greenhouse for year round supply of experimental materials. If grown in a growth chamber, the intensity of the fluorescent light should be about 378 E / m Z / s (1800 ft candies) and the incandescent light should be around 38 E / m Z / s (180 ft candles) measured at the tip of the plants. To induce flowering a 1 6 / 8 h (light/dark) photoperiod is necessary. If grown in the greenhouse, supplemental light is needed during the short day season. The daytime temperature should be around 21-25°C and nighttime temperature
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T.-H. Ma et al./ Mutation Research310 (1994) 211-220
around 16°C. The relative humidity should be around 60-80%.
Propagation procedure Since clone 4430 is sterile, any crowded pots can be subdivided into two to four clones. During this vegetative process, some of the old plants should be removed or the tops cut off to allow the younger tillers to grow. The subdivided clones should not be less than five per pot. 2. Treatment procedure Selection of plant cuttings Only young inflorescences (composed of at least nine exposed buds) are selected for treatment. Generally, 25 cuttings per experimental group are needed to provide sufficient material for mutation data. Maintenance of the plant cuttings during the recovery period Plant cuttings with a stem length of 15-20 cm can be maintained in tall plastic cups filled with Hoagland's solution (3 x dilution). If the chemical under test is reactive or absorbed by the plastics, beakers or glass jars are recommended. Treatment with gaseous agents Plant cuttings may be treated in a dynamic gas-flow chamber. The gaseous agents may be pumped through the chamber at a constant rate and the concentration of the gas determined by analyzing the gas samples using gas chromatography and a flame ion detection system. This operation is very expensive. A simplified method to estimate the gas concentration can be used to make it more cost effective and repeatable for treatment purposes. This may be done by either supplying the known gaseous agent with the known quantity into a dynamic flow chamber or supplying a known quantity of volatile liquid from a source bottle. The molarity of the liquid used can be converted into a gaseous volume through Avogadro's gas law. Similar gaseous agents could be generated through a known chemical reaction, and the gaseous volume can be derived from
Avogadro's gas law. The treatment chamber volume and flow rate of the gas per unit time are used to derive the estimated concentration of the gas and dosages (Ma et al., 1984). The duration of gaseous treatment usually ranges from 5 - 6 h to several days.
In situ monitoring For monitoring polluted, gaseous or liquid agents in an indoor or outdoor setting, a set of plant cuttings could be transported to and from a site with a clean air chamber equipped with a charcoal filtered window and a glass roof-top window so that the plants can generate oxygen in the enclosed chamber. Plant cuttings may also be transported in sealed plastic bags. Earlier practices in outdoor air monitoring carried out by transporting plant cuttings in an air-conditioned independently powered trailer unit are not only very costly but also the diesel engine powered trailer provides its own pollutants which are known to be potent polluting mixtures in urban streets and truck stops (Ma et al., 1982, 1983). The duration of exposure may be from a few hours to several days. Treatment with liquid agents A set of 20 plant cuttings could be maintained in an aqueous solution of the chemical if the agent is water soluble. Water insoluble agents may be dissolved in dimethyl sulfoxide (DMSO) or ethanol ( E t O H ) and then diluted with Hoagland's solution to treat plant cuttings. The duration of treatment usually ranges from a few hours to several days. 3. Recovery time Although the scoring of pink mutations from the flowers of plant cuttings could be done after the initial lag stage (around 7 days), the peak mutation period occurs between 8 and 14 days after acute treatment. Therefore, for high efficiency of this test system, the major scoring should be done during this peak period. The recovery time should be adjusted accordingly if a chronic treatment of several days is applied.
T.-H. Ma et a l . / Mutation Research 310 (1994) 211-220
4. Scoring for induced mutations Time schedule Since the flowers and stamen hairs cannot be fixed for observations at a later time, each treated and control group should be carefully labeled and a specific scoring date should be assigned. Usually several scorers are needed in order to accomplish the task within a limited time. Roughly 10-15 flowers could be expected from 25 plant cuttings in each group per day. They should be scored as soon as possible after removal of the flowers from the stem.
Slide preparation Fully opened flowers are removed from the stem and three out of the six stamens are dissected out for counting of the number of hairs per stamen and the estimation of hair number per flower. An average hair number per flower can be derived from counting 7-10 flowers collected at random. This average number of hairs per flower could be used in the tests for a period of weeks or months until this average number is noticeably changed due to seasonal growing conditions or due to high toxicity of the treatment. The excised flower should be cooled in a refrigerator for 30-60 min to make the stamen hair more turgid for easy combing. The anthers are first removed and then the stamens and pistil are removed with a pair of fine forceps at their base. Each dislodged filament is placed in a drop of glycerin on a glass plate large enough to accommodate the six stamens of one flower. The filaments should be placed with their adaxial side down and viewed under a dissecting microscope at 25 x magnification. A pair of fine needles are needed to comb the hair so that the stamen hairs are untangled and aligned orderly to facilitate the scoring of pink mutations.
Scoring procedure The stamen hairs should be viewed against a white background to reveal the true color of the cells. Each flower contains six stamens, each stamen bears 40-75 hairs, and each hair is composed of 24 cells, on average. Cells of each sta-
219
men hair originate from a single epidermal cell of the filament. All the cells in each hair are derived by mitosis from primarily the apical or subapical cells. For this reason, pink mutations occur more frequently in the early stage of stamen hair development. Mutant cells which occur early in stamen hair development may divide repeatedly and give rise to a string of pink cells. This results from the fact that each string of pink cells originates from a single mutant cell. This cluster of mutants is considered to be a single mutation event when scoring. In order to increase the efficiency of scoring, the pink mutation rate is calculated on the basis of the number of pink mutant events divided by the average number of hairs per flower and expressed in terms of pink mutant events per 1000 hairs.
5. Data collection and statistical analysis Data collection Generally, 10-12 flowers are needed for mutation data collection for each day. This number of flowers could be provided from 20 cuttings in each experimental group. Each flower contains 300 stamen hairs, on average, and each flower is considered to be a sample population. The data gathered are expressed in terms of pink mutant events per 1000 hairs.
Statistical analysis The mutation rate for each experimental group is derived from the total number of mutant events scored from 10-12 flowers divided by the total number of hairs scored in one of the peak mutation days. The mean and standard deviation of the mutation rate of an experimental group is derived from the mutation rates of the three peak mutation days. For greater confidence, experiments should be repeated three times and the mean and standard deviation of a specific experimental group should be derived from nine individual mutation rates. The difference between treated groups and one control group at the 0.05 level of significance is determined generally by F analysis of variance and Dunnett's t-statistic.
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References Hoagland, D.R. and D.I. Arnon (1950) The water-culture method for growing plants without soil, Univ. Calif. Agric. Exp. Stn., Berkeley, CA, Circular No. 347, pp. 1-39. Ma, T.-H., V.A. Anderson and I. Ahmed (1982) Environmental clastogens detected by meiotic pollen mother cells of Tradescantia, in: R.R. Tice, D.L. Costa and K.M. Schaich (Eds.), Genotoxic Effects of Airborne Agents, Plenum, New York, pp. 141-157. Ma, T.-H., W.R. Lower, F.D. Harris, J. Poku, V.A. Anderson, M.M. Harris and J.L. Bare (1983) Evaluation by the Tradescantia-micronucleus test of the mutagenicity of internal combustion engine exhaust fumes from diesel and
diesel-soybean oil mixed fuels, in: M.D. Waters, S.S. Sandhu, J. Lewtas, L. Claxton, N. Chernoff and S. Nesnow (Eds.), Short-Term Bioassays in the Analysis of Complex Environmental Mixtures III, Plenum, New York, pp. 8999. Ma, T.-H., M.M. Harris, V.A. Anderson, I. Ahmed, K. Mohammad, J.L. Bare and G. Lin (1984) Tradescantia-micronucleus (Trad-MCN) test on 140 health-related agents, Mutation Res., 138, 157-167. Nauman, C.H., P.J. Klotz and L.A. Schairer (1979) Uptake of tritiated 1,2-dibromoethane by Tradescantia floral tissues: Relation to induced mutation frequency in stamen hair cells, Environ. Exp. Bot., 19, 201-215.
Mutation Research 310 (1994) 211-220
ELSEVIER
Tradescantia stamen hair mutation bioassay T.-H. Ma a,. G.L. Cabrera
b A. Cebulska-Wasilewska
A.L. Vandenberg
c, R . C h e n
e, M . F . S a l a m o n e
d F. Loarca
b
e
a Department of Biological Sciences, 525 Meadow Drive, Western Illinois University, Macomb, IL 61455, USA b Centro de Estudios Academicos sobre Contaminacion Ambiental, Universidad Autonoma de Queretaro, Queretaro, QRQ, Mexico c Radiobiology Department, Institute of Nuclear Physics, ul. Radzikowskiego 152, 31-342 Cracow, Poland d Guangxi Institute of Botany, Yanshan, Guilin 541006, People's Republic of China c Ministry of Environment and Energy, Biohazard Unit, 125 Resources Road, Etobicoke, Ont. M9P 3V6, Canada
Accepted 16 November 1993
Abstract
The Tradescantia stamen hair mutation (Trad-SH) assay (clone 4430) was evaluated for its efficiency and reliability as a screen for mutagens in an IPCS collaborative study on plant systems. Four coded chemicals, i.e. azidoglycerol (AG, 3-azido-l,2-propanediol), N-methyl-N-nitrosourea (MNU), sodium azide (NaN 3) and maleic hydrazide (MH) were distributed by the Radian Corporation to the five laboratories in five different countries for testing mutagenicity. Pink mutations were scored between the 7th and 14th day according to a standard protocol. Test results from the five individual laboratories were analyzed and compared after decoding. One out of the two laboratories that conducted tests on AG demonstrated that AG is a mutagen with genetically effective doses ranging from 50 to 100/~g/ml. MH yielded positive responses in all laboratories but no linear dose-response pattern was observed. The effective dose range for MH was between 1 and 45/zg/ml. The mutagenicity of MNU was reported by five laboratories in the dose range between 10 and 80/zg/ml. NAN3, which exhibited a relatively high degree of toxicity, elicited a positive mutagenic response in three of the five laboratories in which it was tested. As with MNU the effective dose for NaN 3 ranged between 3 and 80/xg/ml. The results from the current study substantiate the Trad-SH assay as a reliable system for screening chemicals for their potential mutagenic effects. Although the study was carried out exclusively under laboratory conditions, a survey of the current literature would indicate that the Trad-SH assay could be an effective in situ monitor of gaseous, liquid, and radioactive pollutants as well. Keywords: Tradescantia clone 02; Tradescantia clone 4430; Maleic hydrazide; Azidoglycerol; N-Methyl-N-nitrosourea;
Sodium azide I. Introduction
T h e Tradescantia stamen hair m u t a t i o n (TradSH) test was developed by Dr. A r n o l d H. Spar-
* Corresponding author.
row and colleagues during the late 1950s and t h r o u g h o u t the 1960s at the Brookhaven National Laboratory, L o n g Island, NY. Early studies with Tradescantia c o n c e n t r a t e d on the genetic effects induced by nuclear radiation (Sparrow et al., 1972). Subsequently, the assay was a d a p t e d for the detection of mutagenic airborne agents and
0027-5107/94/$07.00 © 1994 Elsevier Science B.V, All rights reserved SSDI 0027-5107( 94)00104 - D
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T.-H. Ma et aL /Mutation Research 310 (1994) 211-220
volatile organic c o m p o u n d s (Sparrow and Schairer, 1971) and still later for the evaluation of chemical agents in liquid form (Schairer et al., 1982). Since the discovery of its high sensitivity to gaseous mutagens, the Trad-SH assay has been a major part of the in situ air monitoring program at several industrial areas in the USA (Schairer and Sautkulis, 1982; Schairer et al., 1982). Furthermore, the Trad-SH assay has been used to test a wide range of chemicals such as pesticides (Ahmed and Grant, 1972), sewage sludge (Hopke et al., 1982), soil samples in the vicinity of a lead smelter (Lower et al., 1983) and engine exhaust fumes (Ma et al., 1983). Recently, the Trad-SH assay has been used in the in situ monitoring of waterways and aquatic effluents (Grant et al., 1992). The Trad-SH is a simple, cost effective shortterm bioassay that yields data within 11-14 days and is easily adapted for either in house or in situ monitoring. The principal cells in this assay are the mitotic stamen hair cells developing in the young flower buds. The assay is based on the fact that these stamen hair ceils in Tradescantia clone 4430 (and other clones) are heterozygous for flower color with the visible marker being the phenotypic change in pigmentation from blue (dominant) to pink (recessive). Thus, most mutations at the blue allele will produce a recessive pink cell among the blue cells (a single mutant event). A pink mutation occurring at this time may continue to divide and give rise to a contiguous series of pink cells. Such a series of pink cells separated by blue cells is also considered only a single mutant event. A stamen hair that has two pink cells separated by one or more blue cells is considered to have undergone two mutant events (Underbrink et al., 1973; Schairer et al., 1978). In general, the mutation rate is generally calculated on the bases of mutation events per 1000 hairs. This study was established under the sponsorship of the International Programme on Chemical Safety (IPCS). The purpose was to evaluate the effectiveness and reliable performance of some commonly used plant bioassays of which the Trad-SH was selected as one. The five laboratories selected to participate in this study were the Biohazard Unit of the Ministry of Environment,
Toronto, Canada (CAN); the Laboratory of Nuclear Physics, National Academy of Science, Poland (POL); the Guangxi Institute of Botany, the People's Republic of China (CHN); the Centro de Estudios Academicos sobre Contaminacion Ambiental, Universidad Autonoma de Queretaro, Mexico (UAQ); and the Laboratory of Environmental Mutagenesis, Western Illinois University, USA (US-WIU). The effectiveness and reliability of the Trad-SH assay were judged by evaluating the uniformity in the results of the five different laboratories. The sensitivity of the test system was judged by evaluating Tradescantia's effectiveness in detecting mutagens as positive on the assay, and by evaluating both the minimum effective dose (MED) for a positive response, and the response to toxic effects.
2. Materials and methods
Tradescantia clone 4430 (heterozygous for the b l u e / p i n k alleles in their stamen hair cells) was chosen for this collaborative study for its high sterility and sensitivity to chemical mutagens. Since the clone is vegetatively propagated it retains its genetic homogeneity. A detailed account of the principles and experimental procedures has been published previously (Underbrink et al., 1973; Schairer et al., 1982). For practical purposes, an outline of this assay is given in Appendix 1 of this paper.
Selection of test chemicals The chemicals used in the study were azidoglycerol (AG, 3-azido-l,2-propanediol; CAS No. 73018-98-1), N-methyl-N-nitrosourea (MNU, CAS No. 684-93-5), sodium azide (NaN 3, CAS. No. 26628-22-8) and maleic hydrazide (MH, CAS No. 123-33-1). Plant cuttings were treated with each chemical dissolved in distilled water using three increasing dosages. Each of the five participating laboratories, except the laboratory of the People's Republic of China, conducted at least three trials with each chemical. The CHN laboratory conducted only two rounds of tests for each of the chemicals but used a large number of plant
T.-H. Ma et al. / Mutation Research 310 (1994) 211-220
samples to attain high reliability. Treatments were generally applied for 24-30 h followed by a recovery period of 7-14 days before scoring for pink mutation events.
Statistical analysis of data Data were analyzed with a computer software program called 'CoStat' which contains the A N O V A F-test for variance and the StudentNewman-Keuls t-test. The significance of differences among one control with three test groups was determined by Dunnett's t statistic at the 0.05 probability level.
3. Results and discussion
The results from all five laboratories in this study are summarized in Tables 1-4. In most Table 1 Mean pink mutation rates of maleic hydrazide (MH) tested with the Trad-SH assay among five laboratories (mutations/ 1000 hairs) Laboratory code a
Dose (/zg/ml)
SH frequency (mean + SD)
CAN
0 10 20 40
8.4 + 24.5 + 28.0 + 30.4+
3.4 6.3 * 6.0 * 4.5 *
POL
0 15 50 80
1.4+ 13.6+ 14.7+ 23.7±
0.6 4.3 * 4.4 * 6.8 *
CHN
0 10 40
0.6+ 0.1 3.2+ 0.3 * 4.6± 0.2 *
UAQ
0 10 15 30
2.3 + 24.7+ 21.7 + 31.3 +
US-WlU
0 4 10 45
2.9 + 8.3 ± 14.2± 16.5 ±
2.2 11.7 11.7 11.4 * 0.3 4.4 9.3 0.78 *
a Laboratory codes: CAN, Canada; CHN, People's Republic of China; POL, Poland; UAQ, Universidad Autonoma de Queretaro; US-WlU, Western Illinois Unversity. * Positive response according to Dunnett's t-test ( P < 0.05).
213
cases (except for the CHN laboratory where each chemical was tested only twice) the values presented represent the average result of three trials. For analysis, the data from the different repetitions were combined. Thus, it is possible that, even if two out of three individual trials generated a significant positive result, when combined, the overall response for a given dose may be negative in the Dunnett t-test. Based upon this fact, it is significant to note the high degree of congruity in the results for the five laboratories. All five laboratories detected MH as positive in the assay system, while four out of five (80%) observed MNU as a positive mutagen of pink events. One out of two laboratories detected A G as a mutagen and three out of five (60%) detected NaN 3 as a mutagen. That totals to 13 out of 17 results or 76.5% being positive. Furthermore, of the 51 doses tested by all the laboratories, including the lowest non-zero doses, 45% were positive. The low MED values for NaN 3 (3 /zg/ml), M H and MNU ( 1 0 / z g / m l ) indicate that the Trad-SH assay is very sensitive to these compounds. As well, there was a high level of agreement in the results among the participating laboratories with regard to the qualitative analysis of a positive response, although quantitatively, there was not full agreement.
Maleic hydrazide As indicated, M H induced a significant elevation in the frequency of pink mutations in all five laboratories (Table 1). In fact, 12 of the 14 treatment doses, or 86%, were classified as positive responses, although dose-effect related responses were not produced in all laboratories. The effective range for MH among the five laboratories was 1 0 - 8 0 / z g / m l . These results with MH are in agreement with earlier studies where M H has been found to be a strong mutagen in tobacco plants (Nicotiana tabacum, Brisza et al., 1984), tomato seeds (Lycopersicon esculentum, Grant and Harney, 1960), barley (Hordeum uulgare, Kak and Kaul, 1975; Malepszy et al., 1973), Tradescantia (Gichner et al., 1982), Zea mays (Plewa and Wagner, 1981), Drosophila melanogaster (Nasrat, 1965) and on the TK locus of mouse lymphoma cells (Paschin, 1981). As well, M H was
T.-H. Ma et al. / Mutation Research 310 (1994) 211-220
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Table 2 Mean pink mutation rates of methyl nitrosourea (MNU) tested with the Trad-SH assay among five laboratories (mutations/ 1000 hairs) Laboratory dose a
Dose (~g/ml)
SH frequency (mean±SD)
CAN
0 8.4 16.7 33.4
5.9±1.8 11.3±5.1 13.6±5.6" 11.9±3.1
POL
0 4 12.5 50
2.4±0.9 3.4±3.6 2.8±2.8 8.7±5.7
CHN
0 40 60 80
0.6±0.2 1.2±0.5 1.1±0.7 20.9±2.9*
UAQ
0 25 45 55
2.5+1.6 6.2±3.0 9.0±3.5 12.5±8.0"
US-WIU
0 5 10 50
1.5±0.5 2.9±3.0 4.2±2.1" 5.2±1.5"
Laboratory codes: CAN, Canada; CHN, People's Republic of China; POL, Poland; UAQ, Universidad Autonoma de Queretaro; US-WlU, Western Illinois Unversity. * Positive response according to Dunnett's t-test (P < 0.05).
shown to be a strong clastogen in the current collaborative study in both the Tradescantia stamen hair and Vicia faba root assays.
were no dose-effect related responses observed in any of the laboratories. The mutagenic effects of M N U observed here agree with the results of the Arabidopsis mutation test in this collaborative study, as well as the finding in previous mutation studies with Arabidopsis thaliana (Gichner et al., 1982), barley (Gichner et al., 1982), Drosophila melanogaster (Vogel and Natarajan, 1979), Saccharomyces cerevisiae (Schwaier et al., 1968), Salmonella typhimurium (Gichner et al., 1982) and the Trad-SH assay (Gichner et al., 1982).
Sodium azide N a N 3 exhibited positive responses for the stamen hair assay in three of the five laboratories in which it was tested (Table 3). In each of the three laboratories, however, the positive response was
Table 3 Mean pink mutation rates of sodium azide (NaN 3 tested with the Trad-SH assay among five laboratories (mutations/1000 hairs) Laboratory code a
Dose (~g/ml)
SHfrequen~ (mean±SD)
CAN
0 10 20 80
3.5±0.8 7.2±3.3 8.9±3.6 14.0±2.3"
POL
0 8 10 25
2.4+0.9 6.1±2.8 5.9±3.1 5.2±2.4
CHN
0 4 6 8
UAQ
0 5 10 18
1.8±0.8 2.2±0.7 3.9±2.7 2.0±1.0
US-WIU
0 3 6 20
1.8±0.5 3.2±0.6" 1.7±0.8 2.4±1.0
N-Methyl-N-nitrosourea M N U showed the next highest degree of congruity among the laboratories in that four out of the five laboratories observed M N U to induce a positive response (Table 2). In the one laboratory in which it was not detected positive, the exposure time was considerably less than in the other four laboratories. Unlike MH, however, mutagenicity of M N U was not as easily detected. Only 33% (five out of 15) treatment doses were signified positive by the Dunnett t-test. The minimum effective dose for M N U was 10 /.~g/ml and its positive dose range went up to 80 t z g / m l . There
1.6±0.4 2.4±0.1" 2.0±0.7 2.5±0.5
a Laboratory codes: CAN, Canada; CHN, People's Republic of China; POL, Poland; UAQ, Universidad Autonoma de Queretaro; US-WIU, Western Illinois Unversity. * Positive response according to Dunnett's t-test (P < 0.05).
T.-H. Ma et al. / Mutation Research 310 (1994) 211-220 Table 4 Mean pink mutation rates of 3-azidoglycerol (AG) tested with the Trad-SH assay among two laboratories (mutations/1000 hairs) Laboratory code a
Dose (/xg/ml)
SH frequency (mean + SD)
CAN
0 5 50 100
5.4 _+3.2 6.6+2.3 5.7 + 1.7 7.5 _+4.3
US-WlU
0 10 50 100
1.7 5:0.3 2.9_+0.9 4.2+2.0 * 5.1 -+ 1.4 *
a Laboratory codes: CAN, Canada; US, US-WIU, Western Illinois Unversity. * Positive response according to D u n n e t t ' s t-test ( P < 0.05).
restricted to a single dose which differed in each laboratory and which ranged between 3 and 80 /xg/ml. These data support the general published results for NaN 3. NaN 3 has been a well-known mutagen for many years and has induced mutations in a number of plants including barley (Nilan et al., 1976), black gram (Phaseolus mungo L., Mahna et al., 1989), pea (Pisum sativum, Kleinhofs et al., 1978), petunia (Petunia hybrida, Khalatkar and Kashikar, 1980) and oats (Arena sativa, Rines, 1985). NaN 3 also appears to be able to induce nondisjunction (Vig, 1973) but has been reported to be a nonclastogen (Sander et al., 1978; Ma, 1979). However, in some cases it has been shown to be a very weak inducer of chromosome aberrations but this was only at high doses and at pH 3 (Veleminslo) et al., 1977). Since NaN 3 is considered a mutagen without potent clastogenic properties, it is of interest to note that it was as easily, if not more easily, detected in this collaborative study with the Trad.MCN assay as with the Trad-SH assay.
Azidoglycerol Special attention has been given to the genotoxicity of A G in recent years owing to the fact that it may have similar pharmacokinetics as A Z T (3'-azido-3'-deoxythymidine), a popular drug for AIDS (Phillips et al., 1991). In this study A G was
215
detected as a mutagen. In only one of the two laboratories in which it was tested it produced positive responses (Table 4). In these laboratories, two of the six concentrations were considered significantly different from the control, while most of the concentrations around 5 0 - 1 0 0 / z g / m l showed a trend towards high mutation rates.
4. Conclusions and recommendations
A comparative analysis of the results of the Trad-SH tests from the five laboratories indicates that all four chemicals were mutagens. In addition, there was a relatively high congruity among the five laboratories for all four chemicals. M H was the most potent and easily detected of the four compounds, while NaN 3 had the lowest detection rate as only three out of 15 doses were significantly different from the control. Based upon the results with the Trad.MCN assay in this study, the genotoxic nature of NaN 3 may not be as clear as indicated in the literature and further studies should be initiated to verify the picture regarding the genotoxic action of NaN 3 in general and specifically with Tradescantia. This study substantiates A G as a mutagen, as shown in the Trad-SH assay, although it is not a strong clastogen as shown by the results of the Trad.MCN assay in this collaborative study. The appraisals of the results from earlier data bases and this study further document the TradSH assay as a sensitive, reliable system for monitoring external radiation, screening radioactive pollutants as well as gaseous and liquid mutagens in the laboratory. The Trad-SH assay has a broad data base from radiation biology and is a wellvalidated monitor of nuclear radiation both in the laboratory and in the field (Sparrow and Schairer, 1971; Sparrow et al., 1972; Schairer et al., 1982; Ma, 1979, 1992). In addition, the literature indicates this assay is well suited for in situ monitoring of the mutagenicity of air and water pollutants (Grant et al., 1989, 1992; Ma, 1992) as well as for external radiation sources (Sparrow et al., 1972; Ichikawa, 1981; Shevchenko, 1989).
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Acknowledgements The authors express their deepest appreciation to the International Progamme on Chemical Safety of the World Heath Organization, the United Nations Environmental Programme and the US Environmental Protection Agency for their financial support for conferences and chemicals, and to Dr. William F. Grant of McGill University for his assistance in the preparation of the manuscript.
References Ahmed, M. and W.F. Grant (1972) Cytological effects of the pesticides phosdrin and bladex on Tradescantia and Vicia faba root tips, Can. J. Genet. Cytol., 14, 157-165. Brisza, J., T. Gichner and J. Velemlnsk~ (1984) Somatic mutation in tobacco plants after chronic exposure to maleic hydrazide and its diethanolamine and potassium salts, Mutation Res., 139, 25-28. Gichner, T., J. Velemlnsk~ and V. Pokorny (1982) Somatic mutations induced by maleic hydrazide and its potassium and diethanolamine salts in the Tradescantia mutation assay, Mutation Res., 103, 289-293. Grant, W.F. and P.M. Harney (1960) Cytogenetic effects of maleic hydrazide treatment of tomato seed, Can. J. Genet. Cytol., 2, 162-174. Grant, W.F., H.G. Lee, D.M. Logan and M.F. Salamone (1989) The use of Tradescantia and Vicia faba bioassays for the in situ detection of mutagens in an aquatic environment, Environ. Mol. Mutagen., 14 (Suppl. 15), 75 (Abstract). Grant, W.F., H.G. Lee, D.M. Logan and M.F. Salamone (1992) The use of Tradescantia and Vicia faba bioassays for the in situ detection of mutagens in an aquatic environment, Mutation Res., 270, 53-64. Hopke, P.K., M.J. Plewa, J.B. Johnston, D. Weaver, S.G. Wood, R.A. Larson and T. Hinesly (1982) Multitechnique screening of Chicago municipal sewage sludge for mutagenic activity, Environ. Sci. Technol., 16, 140-147. Ichikawa, S. (1981) In situ monitoring with Tradescantia around nuclear power plants, Environ. Health Perspect., 37, 145-164. Kak, S.N. and B.L. Kaul (1975) Mutagenic activity of hydrazine and its combinations with maleic hydrazide and X-rays in barley, Cytobios, 12, 123-128. Khalatkar, A.S. and S.G. Kashikar (1980) Sodium azide mutagenicity in Petunia hybrida, Mutation Res., 79, 81-85. Kleinhofs, A., R.L. Warner, F.J. Muehlbauer and R.A. Nilan (1978) Induction and selection of specific gene mutations in Hordeum and Pisum, Mutation Res., 51, 29-35.
Lower, W.R., W.A. Thompson, V.K. Drobney and A.F. Yanders (1983) Mutagenicity in the vicinity of a lead smelter, Teratogen. Carcinogen. Mutagen., 3, 231-253. Ma, T.-H. (1979) Micronuclei induced by X-rays and chemical mutagens in meiotic pollen mother cells of Tradescantia. A promising mutagen test system, Mutation Res., 64, 307-313. Ma, T.-H. (1990) Tradescantia-micronucleus test in clastogens and in situ monitoring, in: M.L. Mendelson and R.J. Albertini (Eds.), Mutation and the Environment, Part E, Environmental Genotoxicity, Risk and Modulation, Wiley-Liss, New York, pp. 83-90. Ma, T.-H. (1992) Monitoring of genetic toxicity of the gaseous agents and leachates from a landfill and the ambient air quality around an incinerator, Report submitted to the Office of Solid Waste Research, Institute for Environmental Studies, University of Illinois, Urbana, IL, 17 pp. (unpublished). Ma, T.-H., W.R. Lower, F.D. Harris, J. Poku, V.A. Anderson, M.M. Harris and J.L. Bare (1983) Evaluation by the Tradescantia-micronucleus test of the mutagenicity of internal combustion engine exhaust fumes from diesel and diesel-soybean oil mixed fuels, in: M.D. Waters, S.S. Sandhu, J. Lewtas, L. Claxton, N. Chernoff and S. Nesnow (Eds.) Short-Term Bioassays in the Analysis of Complex Environmental Mixtures III, Plenum, New York, pp. 8999. Ma, T.-H., G.L. Cabrera, R. Chen, B.S. Gill, M.F. Salamone and A.L. Vandenberg (1994) Tradescantia-micronucleus bioassay, Mutation Res., this issue. Mahna, S.K., R. Garg and M. Parvateesam (1989) Mutagenic effects of sodium azide on black gram (Phaseolus mungo L.), Curr. Sci., 58, 582-584. Malepszy, S., J. Eberhardt and M. Maluszynski (1973) Mutagenic effects of NMH (N-nitroso-N-methylurea), NEH (N-nitroso-N-ethylurea) and MH (maleic hydrazide) in barley, Genet Polon., 14, 47-59. Nasrat, G.E. (1965) Maleic hydrazide, a chemical mutagenic in Drosophila melanogaster, Nature, 207, 439. Nilan, R.A., A. Kleinhofs and C. Sander (1976) Azide mutagenesis in barley, in: H. Gaul (Ed.), Barley Genetics III, Proc. Third Int. Barley Genet. Symp., Theimig, Munich, pp. 113-122. Paschin, Y.V. (1981) Mutagenicity of maleic hydrazide for the TK locus of mouse lymphoma cells, Mutation Res., 91, 359-362. Phillips, M.D., B. Nascimbeni, R.R. Tice and M.D. Shelby (1991) Induction of micronuclei in mouse bone marrow cells: An evaluation of nucleoside analogues used in the treatment of AIDS, Environ. Mol. Mutagen., 18, 168-183. Plewa, M.J. and E.D. Wagner (1981) Germinal cell mutagenesis in specially designed maize genotypes, Environ. Health Perspect., 37, 61-73. Rines, H.W. (1985) Sodium azide mutagenesis in diploid and hexaploid oats and comparison with ethyl methanesulfonate treatments, Environ. Exp. Bot., 25, 7-16.
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Sander, C., R.A. Nilan, A. Kleinhofs and B.K. Vig (1978) Mutagenic and chromosome breaking effects of azide in barley and human leukocytes, Mutation Res., 50, 67-75. Schairer, L.A. and R.C. Sautkulis (1982) Detection of ambient levels of mutagenic atmospheric pollutants with the higher plant Tradescantia, in: E.J. Klekowski Jr. (Ed.) Environmental Mutagenesis, Carcinogenesis, and Plant Biology, Vol. 2, Praeger, New York, pp. 153-194. Schairer, L.A., R.C. Sautkulis and N.R. Tempel (1982) Monitoring ambient air for mutagenicity using the higher plant Tradescantia, in: R.R. Tice, D.L. Costa and K.M. Schaich (Eds.), Genotoxic Effects of Airborne Agents, Plenum, New York, pp. 123-140. Schwaier, R., N. Nashed and F.K. Zimmermann (1968) Mutagen specificity in induction of karyotic versus cytoplasmic respiratory deficient mutants in yeast by nitrous acid and alkylating nitrosamines, Mol. Gen. Genet., 102, 290-300. Shevchenko, V.A. (1989) Some aspects of the genetic consequences of the Chernobyl disaster, in: Transactions of the 10th Int. Conf. on Structural Mechanics in Reactor Technology, Vol. D, pp. 245-249. Sparrow, A.H. and L.A. Schairer (1971) Mutation response in Tradescantia after an accidental exposure to a chemical mutagen, EMS Newslett., 5, 16-19. Sparrow, A.H., A.G. Underbrink and H.H. Rossi (1972) Mutation induced in Tradescantia by small doses of X-rays and neutrons: Analysis of dose-response curves, Science, 176, 916-918. Underbrink, A.G., LA. Schairer and A.H. Sparrow (1973) Tradescantia stamen hairs: A radiobiological test system applicable to chemical mutagenesis, in: A. Hollaender (Ed.), Chemical Mutagens: Principles and Methods for Their Detection, Vol. 3, Plenum, New York, pp. 171-207. Velemlnsk~, J., T. Gichner and V. Pokorny (1977) Induction of DNA single-strand breaks in barley by sodium azide applied at pH 3, Mutation Res., 42, 65-70. Vig, B.K. (1973) Somatic crossing over in Glycine max (L.) Merrill: Mutagenicity of sodium azide and lack of synergistic effect with caffeine and mitomycin C, Genetics, 75, 265-277. Vogel, E. and A.T. Natarajan (1979) The relation between reaction kinetics and mutagenic action of mono-functional alkylating agents in higher eukaryotic systems. I. Recessive lethal mutations and translocations in Drosophila, Mutation Res., 62, 51-100.
Appendix 1: Protocol for the Tradescantia stamen hair mutation (Trad-SH) assay
1. Raising Tradescantia plants Potting soil and other adequate growing media An adequate soil mixture for this plant is composed of 2 parts sand, 1 part peat moss, and 4
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parts soil. Plants can be grown hydroponically with gravel, Vermiculite or Perlite as supporting medium or in nutrient solution (Hoagland's solution at a 3 × dilution; Hoagland and Arnon, 1950) alone. Liquid fertilizer should be applied to the soil mixture in the pots twice per month, or more often if Vermiculite or Perlite is used. Plants should be lightly watered daily. Heavily chlorinated water may increase the background mutation rate.
The plant Tradescantia clones 4430 and 0 2 are interspecific hybrids which are both suitable for this mutation test. They are heterozygous for b l u e / p i n k alleles in their floral parts. Clone 4430 is more sensitive for the detection of chemical mutagens than clone 0 2 (Nauman et al., 1979). Clone 4430 is a sterile hybrid between T. hirsutiflora (blue floral parts) and T. subcaulis (pink floral parts) with a dominant blue phenotype in all flower parts including the stamen hair cells. Mutagen induced somatic mutation in the ceils is from blue to pink and readily recognizable in the diploid cells of the stamen hairs under a dissecting microscope.
Adequate population size About 150-200 pots (15 cm diameter) of wellgrown plants are needed to supply 100-150 plant cuttings bearing young inflorescences once a week to conduct a test of three or four different treated and control groups (generally, about 20 cuttings are needed for a sample population).
Optimal growing conditions Plants can be grown in a growth chamber or in a greenhouse for year round supply of experimental materials. If grown in a growth chamber, the intensity of the fluorescent light should be about 378 E / m Z / s (1800 ft candies) and the incandescent light should be around 38 E / m Z / s (180 ft candles) measured at the tip of the plants. To induce flowering a 1 6 / 8 h (light/dark) photoperiod is necessary. If grown in the greenhouse, supplemental light is needed during the short day season. The daytime temperature should be around 21-25°C and nighttime temperature
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around 16°C. The relative humidity should be around 60-80%.
Propagation procedure Since clone 4430 is sterile, any crowded pots can be subdivided into two to four clones. During this vegetative process, some of the old plants should be removed or the tops cut off to allow the younger tillers to grow. The subdivided clones should not be less than five per pot. 2. Treatment procedure Selection of plant cuttings Only young inflorescences (composed of at least nine exposed buds) are selected for treatment. Generally, 25 cuttings per experimental group are needed to provide sufficient material for mutation data. Maintenance of the plant cuttings during the recovery period Plant cuttings with a stem length of 15-20 cm can be maintained in tall plastic cups filled with Hoagland's solution (3 x dilution). If the chemical under test is reactive or absorbed by the plastics, beakers or glass jars are recommended. Treatment with gaseous agents Plant cuttings may be treated in a dynamic gas-flow chamber. The gaseous agents may be pumped through the chamber at a constant rate and the concentration of the gas determined by analyzing the gas samples using gas chromatography and a flame ion detection system. This operation is very expensive. A simplified method to estimate the gas concentration can be used to make it more cost effective and repeatable for treatment purposes. This may be done by either supplying the known gaseous agent with the known quantity into a dynamic flow chamber or supplying a known quantity of volatile liquid from a source bottle. The molarity of the liquid used can be converted into a gaseous volume through Avogadro's gas law. Similar gaseous agents could be generated through a known chemical reaction, and the gaseous volume can be derived from
Avogadro's gas law. The treatment chamber volume and flow rate of the gas per unit time are used to derive the estimated concentration of the gas and dosages (Ma et al., 1984). The duration of gaseous treatment usually ranges from 5 - 6 h to several days.
In situ monitoring For monitoring polluted, gaseous or liquid agents in an indoor or outdoor setting, a set of plant cuttings could be transported to and from a site with a clean air chamber equipped with a charcoal filtered window and a glass roof-top window so that the plants can generate oxygen in the enclosed chamber. Plant cuttings may also be transported in sealed plastic bags. Earlier practices in outdoor air monitoring carried out by transporting plant cuttings in an air-conditioned independently powered trailer unit are not only very costly but also the diesel engine powered trailer provides its own pollutants which are known to be potent polluting mixtures in urban streets and truck stops (Ma et al., 1982, 1983). The duration of exposure may be from a few hours to several days. Treatment with liquid agents A set of 20 plant cuttings could be maintained in an aqueous solution of the chemical if the agent is water soluble. Water insoluble agents may be dissolved in dimethyl sulfoxide (DMSO) or ethanol ( E t O H ) and then diluted with Hoagland's solution to treat plant cuttings. The duration of treatment usually ranges from a few hours to several days. 3. Recovery time Although the scoring of pink mutations from the flowers of plant cuttings could be done after the initial lag stage (around 7 days), the peak mutation period occurs between 8 and 14 days after acute treatment. Therefore, for high efficiency of this test system, the major scoring should be done during this peak period. The recovery time should be adjusted accordingly if a chronic treatment of several days is applied.
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4. Scoring for induced mutations Time schedule Since the flowers and stamen hairs cannot be fixed for observations at a later time, each treated and control group should be carefully labeled and a specific scoring date should be assigned. Usually several scorers are needed in order to accomplish the task within a limited time. Roughly 10-15 flowers could be expected from 25 plant cuttings in each group per day. They should be scored as soon as possible after removal of the flowers from the stem.
Slide preparation Fully opened flowers are removed from the stem and three out of the six stamens are dissected out for counting of the number of hairs per stamen and the estimation of hair number per flower. An average hair number per flower can be derived from counting 7-10 flowers collected at random. This average number of hairs per flower could be used in the tests for a period of weeks or months until this average number is noticeably changed due to seasonal growing conditions or due to high toxicity of the treatment. The excised flower should be cooled in a refrigerator for 30-60 min to make the stamen hair more turgid for easy combing. The anthers are first removed and then the stamens and pistil are removed with a pair of fine forceps at their base. Each dislodged filament is placed in a drop of glycerin on a glass plate large enough to accommodate the six stamens of one flower. The filaments should be placed with their adaxial side down and viewed under a dissecting microscope at 25 x magnification. A pair of fine needles are needed to comb the hair so that the stamen hairs are untangled and aligned orderly to facilitate the scoring of pink mutations.
Scoring procedure The stamen hairs should be viewed against a white background to reveal the true color of the cells. Each flower contains six stamens, each stamen bears 40-75 hairs, and each hair is composed of 24 cells, on average. Cells of each sta-
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men hair originate from a single epidermal cell of the filament. All the cells in each hair are derived by mitosis from primarily the apical or subapical cells. For this reason, pink mutations occur more frequently in the early stage of stamen hair development. Mutant cells which occur early in stamen hair development may divide repeatedly and give rise to a string of pink cells. This results from the fact that each string of pink cells originates from a single mutant cell. This cluster of mutants is considered to be a single mutation event when scoring. In order to increase the efficiency of scoring, the pink mutation rate is calculated on the basis of the number of pink mutant events divided by the average number of hairs per flower and expressed in terms of pink mutant events per 1000 hairs.
5. Data collection and statistical analysis Data collection Generally, 10-12 flowers are needed for mutation data collection for each day. This number of flowers could be provided from 20 cuttings in each experimental group. Each flower contains 300 stamen hairs, on average, and each flower is considered to be a sample population. The data gathered are expressed in terms of pink mutant events per 1000 hairs.
Statistical analysis The mutation rate for each experimental group is derived from the total number of mutant events scored from 10-12 flowers divided by the total number of hairs scored in one of the peak mutation days. The mean and standard deviation of the mutation rate of an experimental group is derived from the mutation rates of the three peak mutation days. For greater confidence, experiments should be repeated three times and the mean and standard deviation of a specific experimental group should be derived from nine individual mutation rates. The difference between treated groups and one control group at the 0.05 level of significance is determined generally by F analysis of variance and Dunnett's t-statistic.
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References Hoagland, D.R. and D.I. Arnon (1950) The water-culture method for growing plants without soil, Univ. Calif. Agric. Exp. Stn., Berkeley, CA, Circular No. 347, pp. 1-39. Ma, T.-H., V.A. Anderson and I. Ahmed (1982) Environmental clastogens detected by meiotic pollen mother cells of Tradescantia, in: R.R. Tice, D.L. Costa and K.M. Schaich (Eds.), Genotoxic Effects of Airborne Agents, Plenum, New York, pp. 141-157. Ma, T.-H., W.R. Lower, F.D. Harris, J. Poku, V.A. Anderson, M.M. Harris and J.L. Bare (1983) Evaluation by the Tradescantia-micronucleus test of the mutagenicity of internal combustion engine exhaust fumes from diesel and
diesel-soybean oil mixed fuels, in: M.D. Waters, S.S. Sandhu, J. Lewtas, L. Claxton, N. Chernoff and S. Nesnow (Eds.), Short-Term Bioassays in the Analysis of Complex Environmental Mixtures III, Plenum, New York, pp. 8999. Ma, T.-H., M.M. Harris, V.A. Anderson, I. Ahmed, K. Mohammad, J.L. Bare and G. Lin (1984) Tradescantia-micronucleus (Trad-MCN) test on 140 health-related agents, Mutation Res., 138, 157-167. Nauman, C.H., P.J. Klotz and L.A. Schairer (1979) Uptake of tritiated 1,2-dibromoethane by Tradescantia floral tissues: Relation to induced mutation frequency in stamen hair cells, Environ. Exp. Bot., 19, 201-215.