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Tradescantia assay system for gaseous mutagens
Mutanon Research, 99 (1982) 303-315 Elsevier Biomedical Press
303
Tradescantia assay system for gaseous mutagens A report of the U.S. Environmental Protection Agency Gene-Tox Program * J. Van't Hof and L.A. Schairer Department of Biology, Brookhaven National Laboratory **, Upton, N Y 11973 (U.S.A.)
(Received 7 April 1982) (Accepted 8 April 1982)
Summary The hybrid diploid clone 4430 of the p l a n t T r a d e s c a n t i a is heterozygous for a flower color locus. Its blue p h e n o t y p e is the p r o d u c t of the d o m i n a n t blue allele, a n d its p i n k p h e n o t y p e is controlled b y a recessive p i n k allele u n m a s k e d by m u t a t i o n or deletion of the blue allele. Somatic cell m u t a n t s in the filamentous stamen hair are scored to measure mutagenic effect. The p l a n t is used for tests c o n d u c t e d in the field a n d laboratory alike, and it is u n i q u e l y suited for the detection of gaseous mutagens.
Research in the past several decades with the p l a n t T r a d e s c a n t i a has yielded i n f o r m a t i o n a b o u t c h r o m o s o m e structure a n d c o n t r i b u t e d to cytogenetics a n d radiation biology (Swanson, 1957; U n d e r b r i n k et al., 1973). A few years ago the p l a n t By acceptance of this article the publisher or recipient acknowledges the U.S. Government's right to retain a nonexclusive,royalty-free hcense in and to any copyright covering the article. * Work Group Report prepared for the Gene-Tox Program (Office of Toxic Substances, Office of Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washingtofi, DC). The authors are members of the Gene-Tox Work Group on Higher Plant Genetic and Cytogenetic Assays. ** Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Department of Energy. Although the review described in this article has been funded wholly or in part by the United States Environmental Protection Agency through Interagency Agreement DOE 40-1123-80, EPA No. 80-DX0953, to the Oak Ridge National Laboratory, it has not been subjected to the Agency's required peer and policy review and, therefore, does not necessarily reflect the views of the Agency and no official endorsement should be inferred. The protocols stated, suggested use of the assay in a screening program, and research recommended should not be taken to represent Agency policy on these matters. 0165-1110/82/0000-0000/$02.75 © Elsevier BiomedicalPress
304 acquired still another useful purpose as a detector of mutagens in the environment (Schairer et al., 1978). The Tradescantia assay involves a phenotypic change in flower color from blue to pink expressed and detected either as sectors in the petals or cells in the stamen hairs. A distinctive feature of this assay system is its ability to detect mutagenic pollutants in the atmosphere. Because of their high sensitivity, the plants need only to be exposed to contaminated ambient air to produce an effect: collection. concentration, or modification of the airborne molecules is unnecessary. The utility of the system has been tested by rigorous environmental and laboratory experimentation. Field trials, which were conducted throughout the continental United States, included a control site with putative clean air, industrial urban areas, and rural locations. The results showed that the air in the atmosphere about the south rim of the Grand Canyon was free of mutagenic substances, while that of many industrial sites contained gaseous mutagens. Thus, besides validation of the Tradescantia assay system, the field trials provided a good estimation of the background mutation frequency for clone 4430. Tradescantia clone 4430 is the only clone with a known genotype, and therefore it is the most useful clone for chemical mutagenesis work. Reference is made in the text to published papers that contain details applicable to clone 4430 as well as to other Tradescantia species and clones. Underbrink et al. (1973), Sparrow et al. (1974), and Schairer et al. (1978), respectively, describe the biology and general procedures of calibration, sample treatment, and measurement of mutagenic effects of gaseous agents in the laboratory, and the equipment needed for field tests of atmospheric pollutants. Other publications, located in the Environmental Mutagen Information Center (EMIC) files dealing with Tradescantia, such as clone 02, were excluded from consideration because the plants used in the work were of unknown, questionable, or untested genotypes, because the mutations measured were produced by sources other than chemicals, or because the experiments were performed under unspecified conditions in the field.
Test description Genetic basis of effect detected Clone 4430 is an interspecific, diploid hybrid produced at Brookhaven National Laboratory by a cross between Tradescantia hirsutiflora, which is homozygous blue, and T. subacaulis, which is homozygous pink, at the flower color locus. The flower of clone 4430 is blue (dominant), and it is heterozygous for this locus. Reciprocal backcrosses of clone 4430 to its T. subacaulis parent confirmed its heterozygosity. These crosses, as well as those between heterozygous siblings of 4430, demonstrated that both the blue and pink genes segregate as Mendelian units (Emmerling-Thompson and Nawrocky, 1980). In Tradescantia at least 5 genes govern pigment production and color coding (Emmerling-Thompson and Nawrocky, 1979). Only pink sectors, however, are considered in the mutagenicity test. In the heterozygotes, a mutation (or loss) of the blue gene results in a phenotypic change from blue to pink
305 in the affected cells of flower petals and stamen hairs. To guard against false positives, inclusion in the assay of a diploid homozygous blue clone is useful. T. hirsutiflora (2461) and T. subacaulis (4468) are appropriate clones. The expected frequency of pink cells of the homozygous clone is the square of the mutation frequency in clone 4430, all other factors being equal. If any pink cells are observed in the treated homozygous clone, the investigator would be alerted to the possibility that nongenetic factors were influencing the results. Pink cells in the homozygous clone are very rare; an expected frequency would be less than 1 per 100000 hairs.
The clone and procedures for testing in the laboratory Mature plants vary from about 20 to 45 cm in height. They produce many flowering shoots or branches, each with an inflorescence composed of a series of flower buds of a range of ages such that, once blooming starts, about 1 flower opens daily. For experimental use, large plant populations are grown in controlled environment chambers. An 18-h day is maintained with 1650-4-50 footcandles of illumination from Sylvania F R 9 6 T 1 2 / C W / V H O / 1 3 5 ° bulbs supplemented by about 20% incandescent light under day/night temperatures of 20 ± 0 . 5 ° C and 18---0.5°C. Fresh cuttings, each bearing a young inflorescence (Fig. 1), are treated with gaseous or liquid mutagens for periods of a few hours to several days. If prolonged observations are required, potted plants can be used also. After exposure, the cuttings are placed in containers of aerated Hoagland's nutrient solution and kept in a growth chamber, under the standard conditions described above, until observations are completed. The inflorescence is determinate and composed of 18-20 flower buds arranged as a gradient of developmental age, with the oldest topmost on the inflorescence. Each stamen hair is a filament derived from a single epidermal cell increasing in cell number by mitosis of primarily the apical and subapical cells. At maturity and under optimal growth conditions, untreated stamens have between 40 and 75 hairs, each with an average of 24 cells. A mutant cell, produced early in development, is capable of having several progeny, seen in the hair as a string of pink cells. Because the multiple mutant cells in a line are due to a single mutational event, we score such a line as a single mutation. The flower is trimerrous so that counting hairs on one of each of the 3 antipetalous and antisepalous stamens provides an index of hair number for an average flower. When exposed to chemical or physical mutagens, some meristematic cells are mutated. Since the mutant cell may divide thereafter, it is possible for its progeny to form a sector in a mature hair of 2 or more cells in length. On the other hand, if the lesion produced is lethal, cell division would cease and a stunted hair would result. By counting the number of cells in the shortened hair, a survival curve can be constructed that is useful for analyzing a dose-response effect. Tradescantia cuttings can be exposed to liquid mutagens by simple immersion of the inflorescences or to gaseous mutagens by fumigation in open air or in gas-tight chambers. In such chambers, it is possible to give simultaneous or sequential
306
Fig. 1. Young inflorescences of Tradescantia clone 4430. are exposed to chemical mutagens in either the liquid or vapor phase. Each inflorescence contains flower buds ranging m age from premeiotic (lower right) to fully pigmented (upper right).
treatments with gaseous chemical and physical mutagens. Exposure to gaseous chemical mutagens is a relatively new phase of mutation research requiring development of exposure and dosimetric techniques. The chamber used for gaseous exposure of Tradescantia cuttings is shown in Fig. 2. When using a chemical mutagen such as ethyl methanesulfonate (EMS), more
307
Fig. 2. The exposure chamber used for treating Tradescantia cuttings with gaseous chemical mutagens. The chemical mutagen is introduced into this chamber by bubbling filtered air through a reservoir of the aqueous solution of the mutagen in the glass impinger tube located at the left of the exposure chamber. The mutagen is exhausted through a copper oxide furnace, which oxidizes the chemical mutagen, and the gaseous effluent is released to the atmosphere through a 90-m stack. Cuttings are exposed in a beaker of water containing as m a n y as 30-50 cuttings per treatment.
complete tissue permeation is achieved by evacuating the air in the exposure chamber for approximately 10 min and then slowly returning it to 1 atmosphere of pressure by bubbling filtered air through an aqueous reservoir of the chemical mutagen. The liquid chemical mutagen is in the glass impinger tubes located at the left of the exposure chamber in Fig. 2. The maximum chemical concentration is determined by the vapor pressure of the compound and is controlled by varying the amount of air and the flow rate through the liquid mutagen and by dilution of the mutagen-saturated atmosphere with filtered 'clean air'. A steady flow of gaseous mutagen is maintained through the chamber, with the concentration measured at both inlet and exhaust ports by standard gas chromatographic techniques. The gas is exhausted through a copper oxide furnace, shown at the extreme right in Fig. 2, which oxidizes the mutagen, rendering it harmless. The oxidized effluent is then exhausted to the outdoor atmosphere through a 90-m stack. Thus, the plants may be
308 exposed to a constant level of gaseous chemical mutagen for a period of a few hours or up to several days or even weeks. This fumigation technique simulates the infinite pollution source of chronic exposure to the ambient atmosphere and minimizes the problems of chemical sedimentation, absorption, adsorption, biological half-life, etc.
Techniques for field work The requirements for monitoring air pollution for mutagenicity include: (1) a roadworthy vehicle to house the test organism during exposures; (2) exposure of the test organism under suitable culture conditions: (3) a constant flow of untempered ambient air; and (4) a protracted exposure to simulate natural exposures to plants and animals. The vehicle used for the mobile monitoring project was a 24-ft Clark mini-van trailer that was insulated and air-conditioned to permit year-round operation of the laboratory. In order to maintain a semiclean environment in the field, the trailer air was recirculated through activated charcoal and HEPA particulate filters. 3 Model M-13 growth chambers (Environmental Growth Chambers, Chagrin Falls, O H ) were installed. One of the chambers serves as a clean air control, the second is used for ambient air exposures, and the third is a back-up unit for either control or ambient air exposures. The chambers, located against the rear doors (ambient air), the side wall (back-up unit), and the forward deck (control), are designed to maintain any desired standard laboratory condition or to simulate fluctuations in the temperature and relative humidity of the ambient air outside. Ambient air is drawn into the fumigation chamber through a 4-in. glass duct at continuous flow rates up to about 18 ft3/min, a maximum of one air change every 2min. Each chamber is equipped with an air filter train composed of activated charcoal and H E P A particulate filters with the option of adding a canister of a chemical catalytic filter. The filter train is used to scrub the air continously in the chamber serving as the concurrent control. The total external electrical power requirement for the trailer air-conditioning and chamber operation is a 100-A, 220-V service. Field exposures were accomplished in the following manner: fresh cuttings of Tradescantia clone 4430 were made from stock plants grown in controlled environment chambers; they were hand-carried to the test site by car or airplane; cuttings were placed in the chambers in glass containers filled with Hoagland's nutrient solution, and exposures were made for a 10-day period. At the end of the exposure the cuttings were taken back to the laboratory for analysis of the flowers as they bloomed each day. Exposures of a few hours to several weeks could be made, but 10 days was chosen for the Tradescantia plants because it was long enough to maximize the sensitivity of the system and to simulate protracted exposures, yet short enough to permit flower analysis to be done after returning the cuttings to Brookhaven National Laboratory. The peak period, following a 10-day exposure, is 11-17 days after the start of the exposure. The mutant frequency for the 7-day scoring period is based on an average stamen hair population between 300000 and 400000. 300 cuttings in each ambient air chamber will yield enough data to resolve as little as a 10% increase in mutations to pink cells over the background frequency.
309
Data collection, presentation, and interpretation The post-treatment scoring period is important because the assay is based on a determinate developmental system. A daily plot of the frequency of induced pink events found during the several weeks following an acute exposure to a mutagen has 3 stages: (1) an initial lag period of low, approximately spontaneous frequency, (2) several days of increasing frequency reaching a peak or plateau, and (3) a subsequent decline. The reason for the 3 stages is now understood (Kudirka and Van't Hof, 1980). To reiterate, the buds arranged into inflorescences are characterized by a size gradient that reflects developmental age (Fig. 1). In the younger buds the larger portion of the cells are in GI, and the others are distributed in S, G 2, and M. There is an inverse relation between cell number and the proportion of cells in mitosis with increasing bud age. Likewise, the number of ceils in S first decreases with age, but after mitosis it rises again just prior to pigmentation. This last wave of DNA synthesis signals the mass movement of the cells from G 1 to G 2, where they stop. Hence, during bud development there are 2 periods of DNA synthesis: an early one in younger buds associated with mitosis and a later one that occurs as cell division ceases 5-12 days before blooming. Both periods of DNA synthesis occur prior to pigmentation, which begins about 3 days before blooming. Given these cellular changes and an acute treatment with a chemical mutagen that is most effective during DNA synthesis, the induced mutant frequency curve should have 3 stages: (1) in the older buds a low frequency that reflects the fact that they were pigmented before treatment; (2) a peak frequency composed of cells that were progressing from GI --, S --, G 2 in the buds of an intermediate age; and (3) a decline to a plateau that represents previously proliferating cells that were in the S period when the buds were very young. Thus, the shape of the mutant frequency curves after a chemical treatment mirrors the DNA synthetic activity of the target cells. The upper limit for dosage selection for the assessment of the mutagenicity of a new chemical is based on stamen hair-cell mortality. Mature hairs with less than 75% of the control cell number are considered stunted because of cell death. If an effect is not observed with dosages that inhibit growth in 50% of the hairs, a chemical is considered non-mutagenic. The lower limit for dose-response determinations is governed by either dosimetric techniques or by the number of people needed to analyze large populations of flowers. Low-dose considerations are very important, however, since dose-response curves for 1,2-dibromoethane show slope changes at low dosages (see Fig. 3). It is evident that extrapolation from the high-dose portion of the curve to the low-dose region would result in an underestimate of the mutagenicity of a chemical. Suitable doses nearly always are selected empirically, but Sparrow et al. (1974) settled on a standard treatment time of 6 h at concentrations between 1 and 150 ppm of mutagen for potent compounds such as EMS and 1,2-dibromoethane. The choice was based on the observation that a 'straight-line' relationship existed between the number of mutants produced and the duration of the treatment. The linear relation is valid for exposures from 4 h to approximately 20 days.
310 20.0
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Fig. 3. Typical dose-response curve for Tradescantia clone 4430 following a 6-h exposure to gaseous 1,2-dlbromoethane. Extrapolation from high- to low-dose portions of the curve would underestimate the response. Both E M S and 1,2-dibromoethane are c o m p o u n d s suitable to use as positive controls, should an investigator need to demonstrate an effect. D i m e t h y l sulfoxide ( D M S O ) , on the other hand, at concentrations up to 1000 ppm. is a good c o m p o u n d to use as a negative control. Also, because D M S O is not mutagenic, it is the solvent of choice whenever one is needed for s o m e chemicals (those not water-soluble). Raw data are collected over a 5 - 7 - d a y period. The flowers are collected daily. and the stamens are removed, m o u n t e d for microscopic examination, and scored for pink cells a m o n g the blue ones. The number of mutant cells, the number of hairs, and the total cell counts are recorded directly on c o m p u t e r input sheets for analyses. The data gathered are expressed as the number of pink sectors per I00 or 1000 hairs or pink cells per total cells scored. The m e a n number of mutations per hair is estimated by s u m m i n g the number of pink sectors from individual flowers and dividing by the number of flowers multiplied by an estimate of the number of hairs per flower. The number of hairs per flower is obtained by counting the hairs of several stamens and is taken to be a constant for that particular day and treatment. A hair is counted as a single mutant event if it has one or m o r e pink cells. Each flower has a score equal to the total n u m b e r of hairs with mutations observed in that flower. Thus if H = estimated number of hairs per flower, and E, = the number of mutations in the l th flower, then X, = E,/H is the mutant frequency of the i th flower per hair, where i = 1,2 . . . . N. The m e a n number of events per hair is given as .~ = EX,/N, and the standard error of x is calculated as
s.~=[Y~( X,--.2)Z/( N - 1)(N)] '/2
311 When the mean number of mutations per hair of the control, c, is subtracted from a treatment, t, the standard error of the difference of the mean is calculated as 1/2
Evidence based on nearly 100 experiments with Tradescantia exposed to 0-500 rads shows that ~ is distributed as a Poisson variable. This distribution is used in determining theoretical sample sizes for a predetermined accuracy. In testing hypotheses, however, the normal approximation of the Poisson distribution was used. To present data as a dose-response curve after acute and chronic exposures, a mean peak value is used on a log-log plot of mutant frequency versus chemical concentration in parts per million. In every experiment both treated and untreated plants are scored, so the tabulation of mutant frequencies includes spontaneous and induced mutations. The net mutant frequency of the treated cells is then obtained by subtraction of the background (untreated) frequency. For clone 4430 the background mutation frequency is 3.31 - 0 . 1 4 per 1000 hairs. For further information on data collection and its presentation, the reader may consult Underbrink et al. (1973). Here detailed logistics of scoring are provided along with graphical data for predicting the population size required to obtain various mutant frequencies with a standard error that is a predetermined percentage of the mean.
Test performance Given in Table 1 are the chemicals tested with the Tradescantia clone 4430 assay. The list includes inorganic and organic compounds, roughly two-thirds of which were tested in the vapor phase. The table is for the most part self-explanatory, and it shows that carcinogenic substances are mutagenic at statistical levels of significance of 2% or less. The converse, however, is not true; that is, some substances such as sulfur dioxide, nitrous oxide, vinyl bromide, sodium azide, and hydrazoic acid, though mutagenic at the 1% level of significance, are not presently considered carcinogens. There is no simple formula for confirming that a compound is mutagenic. Using the statistical evaluation described above, the average standard error, based on observations of 300 flowers, is less than 5% of the mean mutation frequency. Using the rule of thumb that non-overlapping 95% confidence limits indicate statistical significance at the 5% level, it may be inferred that this stamen-hair assay can discriminate between a 10% increase in mutation frequency and the background level. Allowing for unforeseen biological variability, a 20% increase over background mutation frequency would establish the mutagenicity of a chemical at the 1% level of significance. Mutation increases between 5% and 20% would be sufficient reason to initiate further testing; less than 5% would be considered insignificant in this assay.
TABLE
1
TRADESCANTIA
Vapor Vapor Vapor vapor Vapor Vapor Vapor
187.88
1.2-Dtbromoethane
Trrmethylphosphate Trichloroethylene Vinyl chloride Vinylidene chloride
Vmyl bromide 2-Bromoethanol Dtchlorodifluoromethane (Freon- 12) Chlorochfluoromethane (Freon-22) Hexamethylphosphoramide
6
7 8 9 10
11 12 13
15
14
I
Vapor Vapor
Vapor
120.92
84.48 179.20
Vapor Vapor
106.96 124.98
140.08 131.40 62.5 96.95
Vapor Vapor Vapor Vapor Vapor
48 64.07 46.01 44.02 108.16
Ozone Sulfur dtoxtde Nitrogen droxtde Nitrous oxide Ethyl methanesulfonate
2 3 4 5
No.
Phase
signtficance.
-
6 6
6
24 6
6 144 6 6 6 6 24
6 6 6 6 6
(h)
ppm ppm ppm ppm
I94 ppm Saturated
50 ppm 24 ppm
22 wm
1 ppm 0 I4 ppm 13 ppm 0.5 ppm 75 ppm
5 ppm
5 40 50 250
392 ppm
1288 ppm
EFFECT.
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r
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StatIstIcal sigmftcance
MUTAGENIC
Maxtmum h concentratton for effect
OF TREATMENT.
Mtnunum * concentration
CONDITIONS
Exposure
WAS TREATED.
Molecular wetght
effect is gtven tn terms of stattsttcal
WITH WHICH
Chemical
Mutagentc
CHEMlCALS ICITY
+(I)
+(I) +(I) +(I)
+(I) +
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Carcinogentc
75-45-6 680-3 l-9
75-7 l-8
593-60-2 540-5 l-2
106-93-4 106-93-4 512-56-I 79-o I-6 75-01-4 75-35-4 75-35-4
102X- 15-6 7 446-09-5 IO 102-44-o 10024-97-2 62-50-O
CAS ’ regtstry number
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Conclusions
Strengths and weaknesses of the assay Strengths a. High sensitivity; can detect some mutagens at concentrations down to 0.1 ppm. b. Proven performance in the laboratory for both liquid and gaseous exposures and in the field with unique advantages for vapor phase studies. c. A short-term assay system. d. Freedom from constraints required by sterile culture conditions. e. Useful additional end points. (i) Chromosome aberrations and somatic mutations can be scored in the same cells. (ii) Chromosome aberrations can be scored in meiotic and somatic cells. (iii) Gametic and somatic mutation frequencies can be measured.
Limztations a. Flower analysis is tedious and, at present, cannot be automated. b. Only a limited number of alleles are available. c. Genetic nature of mutations is poorly defined. d. Structure of pigment molecules is unknown. e. Chromosomal structure, banding patterns, linkage groups, etc., are virtually unexplored. Role of the assay in a mutagenicity testing program Tradescantia clone 4430 holds a unique position among assay systems. It is the only system that can detect the presence of gaseous mutagens in the atmosphere and the only one with which an environmental experiment was successfully conducted (Schairer et al., 1978). For laboratory tests it is equally well suited to measure mutagenicity of compounds in either gaseous or liquid forms (Sparrow et al., 1974; Schairer et al., 1978). It is a relatively inexpensive assay with the capability of indicating where additional, more expensive assays need to be performed. Recommendattons for research, development, and vahdation of the system The genetics should be pursued with the idea of introducing new specific markers that would be useful to measure gametic mutation frequencies. Such genes would provide the tools for the comparison of somatic and gametic genetic effects. The comparison will tell us whether or not somatic mutation frequencies are reliable indices for gametic transmission of the same lesion. The obvious comparison would be the somatic and gametic mutation frequencies of the blue locus of clone 4430 after treatment with a chemical mutagen. The second category of research should focus on the pigments responsible for the blue and pink phenotypes. The molecules involved need to be identified along with the modifications associated with particular phenotypes and genotypes. This work would provide the superstructure for an automated scoring device based on pigment
315 identification. Such a device w o u l d greatly reduce m u t a t i o n frequencies. Finally, the third c a t e g o r y of research should cytogenetics of T r a d e s c a n t i a chromosomes. R e l i a b l e tion with the gametic a n d somatic m u t a t i o n w o r k further characterize the actions of mutagens.
the l a b o r n e e d e d to m e a s u r e encompass modern molecular c h r o m o s o m e m a p s in conjuncw o u l d be a p o w e r f u l tool to
References Emmerling-Thompson, M., and M.M. Nawrocky (1979) J. Hered.. 70, 115-122. Emmerhng-Thompson, M., and M.M. Nawrocky (1980) J. Hered., 71,261-265. Kudirka, D.T., and J. Van't Hof (1980) Exp. Cell Res., 130, 443-450. Schairer. L.A., J. Van't Hof, C.G. Hayes, R.M. Burton and F.J. de Serres (1978) Environ. Health Perspect., 27, 51-60. Sparrow, A.H., L.A. Schairer and R. Viilalobos-Pietrini (1974) Mutation Res., 26, 265-276. Swanson, C.P. (1957) Cytology and Cytogenetics, Prentice-Hall, Englewood Cliffs, NJ. Underbrink, A.G., L.A. Schairer and A.H. Sparrow (1973) in: A. Hollaender (Ed.), Chemical Mutagens; Principles and Methods for their Detection, Vol. 3, Plenum, New York. pp. 171-207.
303
Tradescantia assay system for gaseous mutagens A report of the U.S. Environmental Protection Agency Gene-Tox Program * J. Van't Hof and L.A. Schairer Department of Biology, Brookhaven National Laboratory **, Upton, N Y 11973 (U.S.A.)
(Received 7 April 1982) (Accepted 8 April 1982)
Summary The hybrid diploid clone 4430 of the p l a n t T r a d e s c a n t i a is heterozygous for a flower color locus. Its blue p h e n o t y p e is the p r o d u c t of the d o m i n a n t blue allele, a n d its p i n k p h e n o t y p e is controlled b y a recessive p i n k allele u n m a s k e d by m u t a t i o n or deletion of the blue allele. Somatic cell m u t a n t s in the filamentous stamen hair are scored to measure mutagenic effect. The p l a n t is used for tests c o n d u c t e d in the field a n d laboratory alike, and it is u n i q u e l y suited for the detection of gaseous mutagens.
Research in the past several decades with the p l a n t T r a d e s c a n t i a has yielded i n f o r m a t i o n a b o u t c h r o m o s o m e structure a n d c o n t r i b u t e d to cytogenetics a n d radiation biology (Swanson, 1957; U n d e r b r i n k et al., 1973). A few years ago the p l a n t By acceptance of this article the publisher or recipient acknowledges the U.S. Government's right to retain a nonexclusive,royalty-free hcense in and to any copyright covering the article. * Work Group Report prepared for the Gene-Tox Program (Office of Toxic Substances, Office of Pesticides and Toxic Substances, U.S. Environmental Protection Agency, Washingtofi, DC). The authors are members of the Gene-Tox Work Group on Higher Plant Genetic and Cytogenetic Assays. ** Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Department of Energy. Although the review described in this article has been funded wholly or in part by the United States Environmental Protection Agency through Interagency Agreement DOE 40-1123-80, EPA No. 80-DX0953, to the Oak Ridge National Laboratory, it has not been subjected to the Agency's required peer and policy review and, therefore, does not necessarily reflect the views of the Agency and no official endorsement should be inferred. The protocols stated, suggested use of the assay in a screening program, and research recommended should not be taken to represent Agency policy on these matters. 0165-1110/82/0000-0000/$02.75 © Elsevier BiomedicalPress
304 acquired still another useful purpose as a detector of mutagens in the environment (Schairer et al., 1978). The Tradescantia assay involves a phenotypic change in flower color from blue to pink expressed and detected either as sectors in the petals or cells in the stamen hairs. A distinctive feature of this assay system is its ability to detect mutagenic pollutants in the atmosphere. Because of their high sensitivity, the plants need only to be exposed to contaminated ambient air to produce an effect: collection. concentration, or modification of the airborne molecules is unnecessary. The utility of the system has been tested by rigorous environmental and laboratory experimentation. Field trials, which were conducted throughout the continental United States, included a control site with putative clean air, industrial urban areas, and rural locations. The results showed that the air in the atmosphere about the south rim of the Grand Canyon was free of mutagenic substances, while that of many industrial sites contained gaseous mutagens. Thus, besides validation of the Tradescantia assay system, the field trials provided a good estimation of the background mutation frequency for clone 4430. Tradescantia clone 4430 is the only clone with a known genotype, and therefore it is the most useful clone for chemical mutagenesis work. Reference is made in the text to published papers that contain details applicable to clone 4430 as well as to other Tradescantia species and clones. Underbrink et al. (1973), Sparrow et al. (1974), and Schairer et al. (1978), respectively, describe the biology and general procedures of calibration, sample treatment, and measurement of mutagenic effects of gaseous agents in the laboratory, and the equipment needed for field tests of atmospheric pollutants. Other publications, located in the Environmental Mutagen Information Center (EMIC) files dealing with Tradescantia, such as clone 02, were excluded from consideration because the plants used in the work were of unknown, questionable, or untested genotypes, because the mutations measured were produced by sources other than chemicals, or because the experiments were performed under unspecified conditions in the field.
Test description Genetic basis of effect detected Clone 4430 is an interspecific, diploid hybrid produced at Brookhaven National Laboratory by a cross between Tradescantia hirsutiflora, which is homozygous blue, and T. subacaulis, which is homozygous pink, at the flower color locus. The flower of clone 4430 is blue (dominant), and it is heterozygous for this locus. Reciprocal backcrosses of clone 4430 to its T. subacaulis parent confirmed its heterozygosity. These crosses, as well as those between heterozygous siblings of 4430, demonstrated that both the blue and pink genes segregate as Mendelian units (Emmerling-Thompson and Nawrocky, 1980). In Tradescantia at least 5 genes govern pigment production and color coding (Emmerling-Thompson and Nawrocky, 1979). Only pink sectors, however, are considered in the mutagenicity test. In the heterozygotes, a mutation (or loss) of the blue gene results in a phenotypic change from blue to pink
305 in the affected cells of flower petals and stamen hairs. To guard against false positives, inclusion in the assay of a diploid homozygous blue clone is useful. T. hirsutiflora (2461) and T. subacaulis (4468) are appropriate clones. The expected frequency of pink cells of the homozygous clone is the square of the mutation frequency in clone 4430, all other factors being equal. If any pink cells are observed in the treated homozygous clone, the investigator would be alerted to the possibility that nongenetic factors were influencing the results. Pink cells in the homozygous clone are very rare; an expected frequency would be less than 1 per 100000 hairs.
The clone and procedures for testing in the laboratory Mature plants vary from about 20 to 45 cm in height. They produce many flowering shoots or branches, each with an inflorescence composed of a series of flower buds of a range of ages such that, once blooming starts, about 1 flower opens daily. For experimental use, large plant populations are grown in controlled environment chambers. An 18-h day is maintained with 1650-4-50 footcandles of illumination from Sylvania F R 9 6 T 1 2 / C W / V H O / 1 3 5 ° bulbs supplemented by about 20% incandescent light under day/night temperatures of 20 ± 0 . 5 ° C and 18---0.5°C. Fresh cuttings, each bearing a young inflorescence (Fig. 1), are treated with gaseous or liquid mutagens for periods of a few hours to several days. If prolonged observations are required, potted plants can be used also. After exposure, the cuttings are placed in containers of aerated Hoagland's nutrient solution and kept in a growth chamber, under the standard conditions described above, until observations are completed. The inflorescence is determinate and composed of 18-20 flower buds arranged as a gradient of developmental age, with the oldest topmost on the inflorescence. Each stamen hair is a filament derived from a single epidermal cell increasing in cell number by mitosis of primarily the apical and subapical cells. At maturity and under optimal growth conditions, untreated stamens have between 40 and 75 hairs, each with an average of 24 cells. A mutant cell, produced early in development, is capable of having several progeny, seen in the hair as a string of pink cells. Because the multiple mutant cells in a line are due to a single mutational event, we score such a line as a single mutation. The flower is trimerrous so that counting hairs on one of each of the 3 antipetalous and antisepalous stamens provides an index of hair number for an average flower. When exposed to chemical or physical mutagens, some meristematic cells are mutated. Since the mutant cell may divide thereafter, it is possible for its progeny to form a sector in a mature hair of 2 or more cells in length. On the other hand, if the lesion produced is lethal, cell division would cease and a stunted hair would result. By counting the number of cells in the shortened hair, a survival curve can be constructed that is useful for analyzing a dose-response effect. Tradescantia cuttings can be exposed to liquid mutagens by simple immersion of the inflorescences or to gaseous mutagens by fumigation in open air or in gas-tight chambers. In such chambers, it is possible to give simultaneous or sequential
306
Fig. 1. Young inflorescences of Tradescantia clone 4430. are exposed to chemical mutagens in either the liquid or vapor phase. Each inflorescence contains flower buds ranging m age from premeiotic (lower right) to fully pigmented (upper right).
treatments with gaseous chemical and physical mutagens. Exposure to gaseous chemical mutagens is a relatively new phase of mutation research requiring development of exposure and dosimetric techniques. The chamber used for gaseous exposure of Tradescantia cuttings is shown in Fig. 2. When using a chemical mutagen such as ethyl methanesulfonate (EMS), more
307
Fig. 2. The exposure chamber used for treating Tradescantia cuttings with gaseous chemical mutagens. The chemical mutagen is introduced into this chamber by bubbling filtered air through a reservoir of the aqueous solution of the mutagen in the glass impinger tube located at the left of the exposure chamber. The mutagen is exhausted through a copper oxide furnace, which oxidizes the chemical mutagen, and the gaseous effluent is released to the atmosphere through a 90-m stack. Cuttings are exposed in a beaker of water containing as m a n y as 30-50 cuttings per treatment.
complete tissue permeation is achieved by evacuating the air in the exposure chamber for approximately 10 min and then slowly returning it to 1 atmosphere of pressure by bubbling filtered air through an aqueous reservoir of the chemical mutagen. The liquid chemical mutagen is in the glass impinger tubes located at the left of the exposure chamber in Fig. 2. The maximum chemical concentration is determined by the vapor pressure of the compound and is controlled by varying the amount of air and the flow rate through the liquid mutagen and by dilution of the mutagen-saturated atmosphere with filtered 'clean air'. A steady flow of gaseous mutagen is maintained through the chamber, with the concentration measured at both inlet and exhaust ports by standard gas chromatographic techniques. The gas is exhausted through a copper oxide furnace, shown at the extreme right in Fig. 2, which oxidizes the mutagen, rendering it harmless. The oxidized effluent is then exhausted to the outdoor atmosphere through a 90-m stack. Thus, the plants may be
308 exposed to a constant level of gaseous chemical mutagen for a period of a few hours or up to several days or even weeks. This fumigation technique simulates the infinite pollution source of chronic exposure to the ambient atmosphere and minimizes the problems of chemical sedimentation, absorption, adsorption, biological half-life, etc.
Techniques for field work The requirements for monitoring air pollution for mutagenicity include: (1) a roadworthy vehicle to house the test organism during exposures; (2) exposure of the test organism under suitable culture conditions: (3) a constant flow of untempered ambient air; and (4) a protracted exposure to simulate natural exposures to plants and animals. The vehicle used for the mobile monitoring project was a 24-ft Clark mini-van trailer that was insulated and air-conditioned to permit year-round operation of the laboratory. In order to maintain a semiclean environment in the field, the trailer air was recirculated through activated charcoal and HEPA particulate filters. 3 Model M-13 growth chambers (Environmental Growth Chambers, Chagrin Falls, O H ) were installed. One of the chambers serves as a clean air control, the second is used for ambient air exposures, and the third is a back-up unit for either control or ambient air exposures. The chambers, located against the rear doors (ambient air), the side wall (back-up unit), and the forward deck (control), are designed to maintain any desired standard laboratory condition or to simulate fluctuations in the temperature and relative humidity of the ambient air outside. Ambient air is drawn into the fumigation chamber through a 4-in. glass duct at continuous flow rates up to about 18 ft3/min, a maximum of one air change every 2min. Each chamber is equipped with an air filter train composed of activated charcoal and H E P A particulate filters with the option of adding a canister of a chemical catalytic filter. The filter train is used to scrub the air continously in the chamber serving as the concurrent control. The total external electrical power requirement for the trailer air-conditioning and chamber operation is a 100-A, 220-V service. Field exposures were accomplished in the following manner: fresh cuttings of Tradescantia clone 4430 were made from stock plants grown in controlled environment chambers; they were hand-carried to the test site by car or airplane; cuttings were placed in the chambers in glass containers filled with Hoagland's nutrient solution, and exposures were made for a 10-day period. At the end of the exposure the cuttings were taken back to the laboratory for analysis of the flowers as they bloomed each day. Exposures of a few hours to several weeks could be made, but 10 days was chosen for the Tradescantia plants because it was long enough to maximize the sensitivity of the system and to simulate protracted exposures, yet short enough to permit flower analysis to be done after returning the cuttings to Brookhaven National Laboratory. The peak period, following a 10-day exposure, is 11-17 days after the start of the exposure. The mutant frequency for the 7-day scoring period is based on an average stamen hair population between 300000 and 400000. 300 cuttings in each ambient air chamber will yield enough data to resolve as little as a 10% increase in mutations to pink cells over the background frequency.
309
Data collection, presentation, and interpretation The post-treatment scoring period is important because the assay is based on a determinate developmental system. A daily plot of the frequency of induced pink events found during the several weeks following an acute exposure to a mutagen has 3 stages: (1) an initial lag period of low, approximately spontaneous frequency, (2) several days of increasing frequency reaching a peak or plateau, and (3) a subsequent decline. The reason for the 3 stages is now understood (Kudirka and Van't Hof, 1980). To reiterate, the buds arranged into inflorescences are characterized by a size gradient that reflects developmental age (Fig. 1). In the younger buds the larger portion of the cells are in GI, and the others are distributed in S, G 2, and M. There is an inverse relation between cell number and the proportion of cells in mitosis with increasing bud age. Likewise, the number of ceils in S first decreases with age, but after mitosis it rises again just prior to pigmentation. This last wave of DNA synthesis signals the mass movement of the cells from G 1 to G 2, where they stop. Hence, during bud development there are 2 periods of DNA synthesis: an early one in younger buds associated with mitosis and a later one that occurs as cell division ceases 5-12 days before blooming. Both periods of DNA synthesis occur prior to pigmentation, which begins about 3 days before blooming. Given these cellular changes and an acute treatment with a chemical mutagen that is most effective during DNA synthesis, the induced mutant frequency curve should have 3 stages: (1) in the older buds a low frequency that reflects the fact that they were pigmented before treatment; (2) a peak frequency composed of cells that were progressing from GI --, S --, G 2 in the buds of an intermediate age; and (3) a decline to a plateau that represents previously proliferating cells that were in the S period when the buds were very young. Thus, the shape of the mutant frequency curves after a chemical treatment mirrors the DNA synthetic activity of the target cells. The upper limit for dosage selection for the assessment of the mutagenicity of a new chemical is based on stamen hair-cell mortality. Mature hairs with less than 75% of the control cell number are considered stunted because of cell death. If an effect is not observed with dosages that inhibit growth in 50% of the hairs, a chemical is considered non-mutagenic. The lower limit for dose-response determinations is governed by either dosimetric techniques or by the number of people needed to analyze large populations of flowers. Low-dose considerations are very important, however, since dose-response curves for 1,2-dibromoethane show slope changes at low dosages (see Fig. 3). It is evident that extrapolation from the high-dose portion of the curve to the low-dose region would result in an underestimate of the mutagenicity of a chemical. Suitable doses nearly always are selected empirically, but Sparrow et al. (1974) settled on a standard treatment time of 6 h at concentrations between 1 and 150 ppm of mutagen for potent compounds such as EMS and 1,2-dibromoethane. The choice was based on the observation that a 'straight-line' relationship existed between the number of mutants produced and the duration of the treatment. The linear relation is valid for exposures from 4 h to approximately 20 days.
310 20.0
i
I
i
i
I
I
I
I
I
2
5
I0
20
50
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8
m
5.0
z m
2.0
I 0 o
LO
~, 0.5 o
,~
0.2
I00
DBE CONCENTRATION (pprn)
Fig. 3. Typical dose-response curve for Tradescantia clone 4430 following a 6-h exposure to gaseous 1,2-dlbromoethane. Extrapolation from high- to low-dose portions of the curve would underestimate the response. Both E M S and 1,2-dibromoethane are c o m p o u n d s suitable to use as positive controls, should an investigator need to demonstrate an effect. D i m e t h y l sulfoxide ( D M S O ) , on the other hand, at concentrations up to 1000 ppm. is a good c o m p o u n d to use as a negative control. Also, because D M S O is not mutagenic, it is the solvent of choice whenever one is needed for s o m e chemicals (those not water-soluble). Raw data are collected over a 5 - 7 - d a y period. The flowers are collected daily. and the stamens are removed, m o u n t e d for microscopic examination, and scored for pink cells a m o n g the blue ones. The number of mutant cells, the number of hairs, and the total cell counts are recorded directly on c o m p u t e r input sheets for analyses. The data gathered are expressed as the number of pink sectors per I00 or 1000 hairs or pink cells per total cells scored. The m e a n number of mutations per hair is estimated by s u m m i n g the number of pink sectors from individual flowers and dividing by the number of flowers multiplied by an estimate of the number of hairs per flower. The number of hairs per flower is obtained by counting the hairs of several stamens and is taken to be a constant for that particular day and treatment. A hair is counted as a single mutant event if it has one or m o r e pink cells. Each flower has a score equal to the total n u m b e r of hairs with mutations observed in that flower. Thus if H = estimated number of hairs per flower, and E, = the number of mutations in the l th flower, then X, = E,/H is the mutant frequency of the i th flower per hair, where i = 1,2 . . . . N. The m e a n number of events per hair is given as .~ = EX,/N, and the standard error of x is calculated as
s.~=[Y~( X,--.2)Z/( N - 1)(N)] '/2
311 When the mean number of mutations per hair of the control, c, is subtracted from a treatment, t, the standard error of the difference of the mean is calculated as 1/2
Evidence based on nearly 100 experiments with Tradescantia exposed to 0-500 rads shows that ~ is distributed as a Poisson variable. This distribution is used in determining theoretical sample sizes for a predetermined accuracy. In testing hypotheses, however, the normal approximation of the Poisson distribution was used. To present data as a dose-response curve after acute and chronic exposures, a mean peak value is used on a log-log plot of mutant frequency versus chemical concentration in parts per million. In every experiment both treated and untreated plants are scored, so the tabulation of mutant frequencies includes spontaneous and induced mutations. The net mutant frequency of the treated cells is then obtained by subtraction of the background (untreated) frequency. For clone 4430 the background mutation frequency is 3.31 - 0 . 1 4 per 1000 hairs. For further information on data collection and its presentation, the reader may consult Underbrink et al. (1973). Here detailed logistics of scoring are provided along with graphical data for predicting the population size required to obtain various mutant frequencies with a standard error that is a predetermined percentage of the mean.
Test performance Given in Table 1 are the chemicals tested with the Tradescantia clone 4430 assay. The list includes inorganic and organic compounds, roughly two-thirds of which were tested in the vapor phase. The table is for the most part self-explanatory, and it shows that carcinogenic substances are mutagenic at statistical levels of significance of 2% or less. The converse, however, is not true; that is, some substances such as sulfur dioxide, nitrous oxide, vinyl bromide, sodium azide, and hydrazoic acid, though mutagenic at the 1% level of significance, are not presently considered carcinogens. There is no simple formula for confirming that a compound is mutagenic. Using the statistical evaluation described above, the average standard error, based on observations of 300 flowers, is less than 5% of the mean mutation frequency. Using the rule of thumb that non-overlapping 95% confidence limits indicate statistical significance at the 5% level, it may be inferred that this stamen-hair assay can discriminate between a 10% increase in mutation frequency and the background level. Allowing for unforeseen biological variability, a 20% increase over background mutation frequency would establish the mutagenicity of a chemical at the 1% level of significance. Mutation increases between 5% and 20% would be sufficient reason to initiate further testing; less than 5% would be considered insignificant in this assay.
TABLE
1
TRADESCANTIA
Vapor Vapor Vapor vapor Vapor Vapor Vapor
187.88
1.2-Dtbromoethane
Trrmethylphosphate Trichloroethylene Vinyl chloride Vinylidene chloride
Vmyl bromide 2-Bromoethanol Dtchlorodifluoromethane (Freon- 12) Chlorochfluoromethane (Freon-22) Hexamethylphosphoramide
6
7 8 9 10
11 12 13
15
14
I
Vapor Vapor
Vapor
120.92
84.48 179.20
Vapor Vapor
106.96 124.98
140.08 131.40 62.5 96.95
Vapor Vapor Vapor Vapor Vapor
48 64.07 46.01 44.02 108.16
Ozone Sulfur dtoxtde Nitrogen droxtde Nitrous oxide Ethyl methanesulfonate
2 3 4 5
No.
Phase
signtficance.
-
6 6
6
24 6
6 144 6 6 6 6 24
6 6 6 6 6
(h)
ppm ppm ppm ppm
I94 ppm Saturated
50 ppm 24 ppm
22 wm
1 ppm 0 I4 ppm 13 ppm 0.5 ppm 75 ppm
5 ppm
5 40 50 250
392 ppm
1288 ppm
EFFECT.
2 I
r
2
5
c
2
(8)
StatIstIcal sigmftcance
MUTAGENIC
Maxtmum h concentratton for effect
OF TREATMENT.
Mtnunum * concentration
CONDITIONS
Exposure
WAS TREATED.
Molecular wetght
effect is gtven tn terms of stattsttcal
WITH WHICH
Chemical
Mutagentc
CHEMlCALS ICITY
+(I)
+(I) +(I) +(I)
+(I) +
+(I)
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Carcinogentc
75-45-6 680-3 l-9
75-7 l-8
593-60-2 540-5 l-2
106-93-4 106-93-4 512-56-I 79-o I-6 75-01-4 75-35-4 75-35-4
102X- 15-6 7 446-09-5 IO 102-44-o 10024-97-2 62-50-O
CAS ’ regtstry number
AND CARCINOGEN-
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314
Conclusions
Strengths and weaknesses of the assay Strengths a. High sensitivity; can detect some mutagens at concentrations down to 0.1 ppm. b. Proven performance in the laboratory for both liquid and gaseous exposures and in the field with unique advantages for vapor phase studies. c. A short-term assay system. d. Freedom from constraints required by sterile culture conditions. e. Useful additional end points. (i) Chromosome aberrations and somatic mutations can be scored in the same cells. (ii) Chromosome aberrations can be scored in meiotic and somatic cells. (iii) Gametic and somatic mutation frequencies can be measured.
Limztations a. Flower analysis is tedious and, at present, cannot be automated. b. Only a limited number of alleles are available. c. Genetic nature of mutations is poorly defined. d. Structure of pigment molecules is unknown. e. Chromosomal structure, banding patterns, linkage groups, etc., are virtually unexplored. Role of the assay in a mutagenicity testing program Tradescantia clone 4430 holds a unique position among assay systems. It is the only system that can detect the presence of gaseous mutagens in the atmosphere and the only one with which an environmental experiment was successfully conducted (Schairer et al., 1978). For laboratory tests it is equally well suited to measure mutagenicity of compounds in either gaseous or liquid forms (Sparrow et al., 1974; Schairer et al., 1978). It is a relatively inexpensive assay with the capability of indicating where additional, more expensive assays need to be performed. Recommendattons for research, development, and vahdation of the system The genetics should be pursued with the idea of introducing new specific markers that would be useful to measure gametic mutation frequencies. Such genes would provide the tools for the comparison of somatic and gametic genetic effects. The comparison will tell us whether or not somatic mutation frequencies are reliable indices for gametic transmission of the same lesion. The obvious comparison would be the somatic and gametic mutation frequencies of the blue locus of clone 4430 after treatment with a chemical mutagen. The second category of research should focus on the pigments responsible for the blue and pink phenotypes. The molecules involved need to be identified along with the modifications associated with particular phenotypes and genotypes. This work would provide the superstructure for an automated scoring device based on pigment
315 identification. Such a device w o u l d greatly reduce m u t a t i o n frequencies. Finally, the third c a t e g o r y of research should cytogenetics of T r a d e s c a n t i a chromosomes. R e l i a b l e tion with the gametic a n d somatic m u t a t i o n w o r k further characterize the actions of mutagens.
the l a b o r n e e d e d to m e a s u r e encompass modern molecular c h r o m o s o m e m a p s in conjuncw o u l d be a p o w e r f u l tool to
References Emmerling-Thompson, M., and M.M. Nawrocky (1979) J. Hered.. 70, 115-122. Emmerhng-Thompson, M., and M.M. Nawrocky (1980) J. Hered., 71,261-265. Kudirka, D.T., and J. Van't Hof (1980) Exp. Cell Res., 130, 443-450. Schairer. L.A., J. Van't Hof, C.G. Hayes, R.M. Burton and F.J. de Serres (1978) Environ. Health Perspect., 27, 51-60. Sparrow, A.H., L.A. Schairer and R. Viilalobos-Pietrini (1974) Mutation Res., 26, 265-276. Swanson, C.P. (1957) Cytology and Cytogenetics, Prentice-Hall, Englewood Cliffs, NJ. Underbrink, A.G., L.A. Schairer and A.H. Sparrow (1973) in: A. Hollaender (Ed.), Chemical Mutagens; Principles and Methods for their Detection, Vol. 3, Plenum, New York. pp. 171-207.