Uptake of tritiated 1,2-dibromoethane by Tradescantia floral tissues: Relation to induced mutation frequency in stamen hair cells

Emi~,,m.,pmd and E~p<'tim,,ntal Botany, \'ol. 19, pp, 2111 t,, 215. I

0098-8472/79/0S01-0201 $02.00/0

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UPTAKE OF T R I T I A T E D 1,2-DIBROMOETHANE BY TRADESCANTL4 FLORAL TISSUES: RELATION TO INDUCED MUTATION FREQUENCY IN STAMEN HAIR CELLS C. H. NAUMAN*, P. j . KLOTZ~ and L. A. SCHAIRER*++ Departments of Biology* and Energy and Environment~, Brookhaven National Laboratory, Upton, New York 11973, U.S.A.

(Received 13 September 1978; in revisedform I I December 1978; accepted 13 December 1978)

NAUMAN C. H., KLOTZ P. J. and SCHAIRER L. A. Uptake of tritiated 1,2-dibromoethane by Tradescantia jloral tissues: relation to induced mutation frequency in stamen hair cells. ENVIRONMENTAL AND EXPERIMENTAL BOTANY 19, 201--215, 1979.--Inflorescences of two clones of Tradescantia (02 and 4430) have been exposed to the gaseous form of tritium-labeled 1,2-dibromoethane (DBE). A comparison of chemical exposure concentration and tissue dose for various exposure periods indicates that DBE readily and rapidly penetrates through the outer sepal and petal tissues to the critical stamen hair cells--the targets for mutation induction. Bud and open tlower tissues of both clones contain generally similar amounts of [3H]-DBE after similar exposures; thus, a dill~rential penetration or uptake of the mutagen into the tissues of these clones cannot account for the 7-9-fold difference between clones in pink mutation frequency elicited by DBE exposure. Autoradiographs of stamen hair cells show clearly that the DBE is not localized, but distributed randomly throughout the cytoplasm and nucleus. Compal'ison of [3H]-DBE-induced pink mutation-response curves to those derived previously with unlabeled DBE reveals that the rad dose from tritium could not account entirely for the elevated mutation response following exposure to the [3H]-DBE. Plots of the total exposure to [3H]DBE vs both tissue molar concentration of [3H]-DBE and pink mutation incidence following exposure to DBE makes possible the construction of true target-tissue dose-response curves. INTRODUCTION

DURING THE past several years Tradescantia clones have been utilized in a variety of gaseous chemical mutagen e x p e r i m e n t s J 2~' 31' 32' 44' 45~ Those chemicals that have proven to be significantly mutagenic include the industrial compounds ethyl methanesulfonate, 1,2-dibromoethane, trichloroethylene, trimethyl phosphate, h e x a m e t h y l p h o s p h o r a m i d e , vinyl chloride, vinyl bromide, sodium azide, benzene, 2-bromoethanol and Freon-22, and the air pollutants sulfur dioxide, nitrogen dioxide, nitrous oxide and ozone. T o date, the compounds 1,1dibromoethane, vinylidine chloride, Freon-12, +To whom reprint requests should be addressed.

caffeine, atrazine, d i m e t h y l a m i n e hydrochloride and V a p o n a have not proved to be significantly mutagenic in TradescantiaJ 37) T h e clwmical most frequently utilized in our studies has been 1,2-dibromoethane (ethylene dibroxnide), hereafter referred to as DBE, and will be the subject of this paper. DBE has been an ideal m u t a g e n fbr use in the gaseous phase: it is sufficiently volatile, does not condense out on the exposure c h a m b e r walls nor on the p l a n t material as does ethyl methanesulfonate, and is a fairly potent m u t a g e n that elicits good exposure-response d a t a in experiments with Tr adescantia. DBE is a c o m p o u n d that is used heavily in and by industry, and thus is one to which m a n y 201

202

C. H. NAUMAN, P. J. KLOTZ and L. A. SCHAIRER

persons are exposed during manufacture and during usage in everyday life. Its primary uses include that as a broad-spectrum pesticide-both as a soil fumigant (nematocide) and as a fumigant for stored grains, fruits and nuts--as an intermediate in the production of" dyes and pharmaceuticals, and as a lead scavenging antiknock agent in gasoline. Ix7'29) For this latter function, DBE is often mixed with 1,2dichloroethaneJ 1~) DBE is an alkylating agent and has been found to be mutagenic in E. coli,{4,38)

Salmonella,~4) Aspergillus,(3s) A.eurospora,{S. 26) Hordeum,~XO) Tradescantia(31, 32, ,,,,, 45) and Drosophila. (49) In addition, DBE has been shown to cause cancers in rats interest that when both the same carbon, as in compound is without

and mice. {35' so) It is of bromine atoms are on 1,1-dibromoethane, the mutagenic activity in

Tradescantia.~3 v) In all of the experiments that we have performed with gaseous DBE, dosimetry has been performed with gas chromatography: exposures were read as ppm of DBE in air to which the plant material is exposed, while the mutagen is continuously passed through the exposure chambers. However, we were unsure of the true tissue dose that was received by the target tissues, i.e. the stamen hair cells in which pink mutations were induced and ultimately registered. The purpose of this study was to investigate the kinetics of DBE penetration through the inflorescence tissues, and to determine the relative tissue dose in inflorescences of Tradescantia clones 02 and 4430 tbllowing exposure to tritium-labeled DBE. It was hoped to determine whether differential rates of uptake and/or tissue dose would account for the previously determined 7-9-fold difference in mutation frequency exhibited by these two clones following exposure to DBE33a'44) Preliminary results of this study have appeared previously. ¢3°) MATERIALS AND METHODS

Cuttings of two diploid clones of Tradescantia, designated 02 and 4430, were used in these experiments. Both clones are equally sensitive to X-ray-induced pink mutations in stamen hair

cells, but clone 4430 is 7-9 times more sensitive to at least two chemical mutagens. ¢31'44) Both clones are diploid ( 2 n = 1 2 ) and heterozygous for flower color. The origins of these clones and the details of their propagation, maintenance and handling have been described elsewhere. (31, 44. 47) Tritiated DBE was synthesized by and purchased from Biochemical and Nuclear Corporation, Burbank, California (specific activity determined to be 1.1 Ci/ml on receipt). Gas chromatograph analyses of the tritiated DBE indicated the chemical purity to be high and with minimal decomposition. Unlabeled DBE was purchased fi'om Fisher Scientific Company, Fair Lawn, New Jersey, and was further purified by distillation. Exposure of cuttings to both forms of DBE was performed by a continuous flow-through of constant levels of the gas phase of the chemical, as described previouslyJ 31'44) The concentration of" mutagen (ppm, by volume in air) was monitored at both the input and exhaust ports of the exposure chamber during all exposure periods by a Beckman GC-5 gas chromatograph equipped with a flame ionization detector, Controls were exposed to a flow of filtered air and under the same conditions as experimental groups. Cuttings of both clones were maintained in controlled-environment growth chambers before and after exposure to DBE: 2 0 + 1 ~ C during an 18-hr day with a light intensity of about 1.8 x 104 lux, and 1 8 + 1 ° C at night. Following exposure, floral tissues including sepals, petals, anthers, stamens minus anthers (hereafter referred to as stamens for simplicity) and ovaries, were dissected fi'om buds corresponding to those most sensitive to mutation induction by the mutagen. Initial dissection o[ buds was performed 30 rain post-exposure and on subsequent days up to day 14. Tissues could not be dissected earlier than 30 min postexposure due to tile time required to remove cuttings from the exposure chambers, wash them, and return them to an area for dissection. Tissues were also dissected from flowers that opened on various days during the period 1 24 days subsequent to exposure. All dissected tissues were placed in 20-ml glass scintillation vials, in quantities predeter-

FIG. 1. Inflorescence of clone 02, including developing buds where mutations are induced, and a mature flower, in the stamen hairs of which the mutations are expressed.

203

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204

UPTAKE OF 3H-1,2-DIBROMOETHANE mined to give good counting statistics, and digested with a mixture of 70°o perchloric acid (HC104) and 30~o hydrogen peroxide ( H 2 0 2 ) at 65°(: tbr 1 hr. Initially 0.5 ml each of the digesting agents was utilized, followed by cooling to room temperature and addition of 3 ml water and 11 ml Aquasol (New England Nuclear). Subsequently it was found that 0.1 ml each of H C 1 0 4 and H 2 0 2 was sufficient to digest the plant materials; later experiments utilized this digest mixture, followed by addition of 1 ml of water and 14 ml Aquasol. Both regimens provided a medium in which the plant tissue was completely dissolved and in which phase separation did not occur. Quench curves were determined tbr each digestscintillation mixture, the latter mixture being significantly less quenching. Heating of the digesting mixture was necessary, since results of preliminary trials showed that digestion of Tradescantia tissues was not completed in up to two weeks at room temperature. The digesting reagents and technique were suggested by the studies of MArtIN and LOFBERG.(25) Complete digestion was necessary to avoid errors due to self absorption of the weak tritium beta rays. The inclusion of heating in the technique necessitated a test for loss of radioactivity through oxidation to volatile products during heating at 65°C. Tests including both mixtures of HC10,~ and H 2 0 2 showed that there was no significant loss of radioactivity due to the heating. Internal standardizations were performed at intervals during all experiments, with samples selected at random, to ensure that results would be quantitatively accurate. An average of 1.03~o difference was found between results based on the appropriate quench curve and those based on the internal standardization (range 0-2.6~!0 ). Samples were counted at least twice in a Beckman CPM-100 liquid scintillation counter for times sufficient to yield counting data with less than 1'!o error. All data were corrected for D P M according to the appropriate quench curve and converted to moles DBE per mg plant tissue via a computer program written by Mr. K. H. Thompson for the C D C 6600 and 7600 computers at BNL.

205 RESULTS

Figure 1 is a photograph of a typical inflorescence of" clone 02, similar to those exposed to gaseous DBE. It is a little less than one cm wide and is composed of a series of about 20 visible buds which increase in size and developmental state as they approach the top of the inflorescence. New buds are formed more or less continually at the base of the inflorescence as the upper buds bloom. Mutations are induced in the young developing buds, either with ionizing radiation or with chemical mutagens. Induced and spontaneously occurring mutations become visible several days later in the petals and stamen hairs of open flowers, such as the one shown in Fig. 1. The inflorescence of clone 4430 is similar in general structure. Radiation will, of course, readily penetrate the bud tissues and dosimetry to determine the tad dose to the tissue is relatively easily performed. However, with gaseous chemicals, penetration to the target tissues, the stamen hairs, inside the buds, is questionable because the buds are somewhat tightly packed and several layers of sepal and petal tissue form an outer covering. This may be seen more clearly in Fig. 2, which is a histological preparation of a longitudinal section through an inflorescence of clone 02 (a clone 4430 inflorescence would appear generally similar). Each of the larger enclosed compartments is a bud; all components are not visible in each bud, but in two of them the complete developing stamen can be seen (lower buds, right side). Stamen hairs are very small at this stage and not readily visible in Fig. 2, but stamen hairs for stamens of about this stage and slightly older are the target tissues where mutations are induced. Several layers of sepal and petal tissue, approximately 0.25 mm thick, cover the internal bud tissues. The first set of results following bud dissection at 30min post-exposure is shown for clone 02 in Fig. 3a and for clone 4430 in Fig. 3b. Exposure to tritiated DBE is expressed as the product of concentration and time; tissue content is in units of moles per mg tissue weight. All data for clone 02 are from a single experiment in which exposures r a n g e d from 9.6ppm-

206

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FIG. 3. Bud tissue contents of [3H]-DBE as a function of exposure. Tissues were sampled at 30 rain post-exposure. (a) Clone 02 with slopes averaging 0.90 (b) Clone 4430 with slopes averaging 1.13. hr (4.8 ppm for 2 hr) to 288 ppm-hr (57.6 ppm for 5 hr). Clone 02 stamen tissues contained the greatest concentration of [3H]-DBE, and sepals the least, with the other tissues being inter° mediate. Tissues were also dissected from buds of clone 02 on days 1, 3, 6 and 9 post-exposure (data not shown), and this same ranking of tissue contents and slopes averaging 0.90 were maintained. Data for clone 4430 (Fig. 3b) are from three separate experiments in which different exposure concentrations and exposure durations were employed. In this case, exposures ranged from 4.3 p p m - h r (17 ppm for 15 min) to 337.8 ppm-hr (56.3 ppm for 6 hr). Stamens once again show the highest DBE content and petals the least; however, the regression lines are much more closely grouped and have steeper slopes, not significantly different from one another {p > 0.05), and averaging 1.13. Parameters for all exposures of both clones are given in Table 1.

Tissues were also dissected from buds of clone 4430 on days 1, 3, 6, 8, 10, 12 and 14 postexposure (data not shown): the same close grouping of regression lines for the different tissues was maintained, but with slopes slightly decreased. Since the same close grouping was maintained for the five tissues of clone 4430 on days 1-14, data for all tissues were lumped on each of these days (1, 3, 6, 8, 12, 14) and data plotted in Fig. 4. As may be seen from this figure, slopes decreased to 0.99 on day 1, and averaged 1.00 for the 7 days sampled. There is a continual decline in DBE in the tissues with time, as would be expected due to dilution of the label in the increasing mass due to tissue growth. This decline in DBE content is graphed in Fig. 5 for the five lowest exposures. In all cases there is an increase between day 0 (30 min post-exposure) and day 1, followed by a continuous decline. The tissue content on day 14 averages about 24'!.0 of that on

UPTAKE OF 3H-1,2-DIBROMOETHANE

207

Table 1. [3H]-DBE exposure parameters and resulting pink mutationfrequencies Exposure concentration (ppm)

Clone 02

Exposure duration (hr)

Total exposure (ppm-hr)

Pink mutations/100 hairs (minus control)_+ SE*

2.0 2.0 5.0 5.0 0.25 0.50 1.0 2.0 6.0 4.0 6.0 6.0 6.0 6.0

9.6 28.4 97.5 288.0 4.3 11.2 25.2 56.2 78.0 112.8 162.6 279.6 337.8 532.8

0.090 +0.025 0.285 3- 0.031 1.202 3- 0.071 3.313 + 0.165 0.075 _+0.055 0.1243-0.050 0.428 -t-0.058 0.995 _+0.085 1.591 -+0.130 2.159_+0.125 3.930-+0.210 6.436 + 0.259 8.766 -+0.493 16.538 3- 2.297

4.8 14.2 19.5 57.6 17.0 22.3 25.2 28.1 13.0 28.2 27.1 46.6 56.3 88.2~"

4430

*Mutation frequencies are the mean of peak response days 12-16 after exposure for clone 02, and da'~s 10 15 lbr clone 4430. tNo tissue samples were taken for scintillation counting at this exposure--only pink mutations were scored. 10-9

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Fro. 4. [~H]-DBE contained in the combined bud tissues of clone 4430, for days 1-14 after exposure. Slopes average 1.0. d a y 1 post-exposure. Limited d a t a tbr clone 02 (not shown) indicate that the increase in tissue content of [3H]-DBE from d a y 0 to d a y 1 is not as m a r k e d as in clone 4430, and that tissue

content on d a y 9 is reduced to about 55",, of that on d a y 1 post-exposure. Tissues were also dissected from open tlowers subsequent to exposure. D a t a for clones 02 and 4430 sepals, petals, anthers, stamens and ovaries on d a y 1 are shown in Figs. 6a and 6b. In both clones tissue content is similar, especially at the higher exposures; [3H]-DBE contents vary widely a m o n g tissues, anthers containing the greatest amount, followed by ovaries, stamens, petals and sepals. This p a t t e r n holds for both clones. As in the case of bud tissues, slopes of the curves for clone 02 are less, averaging 0.83, than those for clone 4430 where they average 0.96. T h e same tissues were also dissected from open flowers in clone 02 on days 3, 6 and 9 post-exposure, and from open flowers of clone 4430 on various days from 2 to 24 (data not shown). O n these subsequent days the same positioning and spacing of the regression lines was observed and with slopes that consistently averaged 0.84 for clone 02 and 0.99 for clone 4430. A u t o r a d i o g r a p h s were p r e p a r e d with stamen tissues of both clones to determine whether the [3H]-DBE was bound specifically to the cell

208

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Fro. 5. Combined clone 4430 bud tissue contents of [3H]-DBE for the five lower exposures from Fig. 4, as a function of time after exposure. Tissue content declines fiom day I to 14 as would be expected due to dilution of chemical in the growing tissue mass. nucleus or elsewhere. All preparations showed clearly that the label was distributed completely at r a n d o m over cytoplasm and nucleus; results were qualitatively the same when tissues were sampled i m m e d i a t e l y after exposure or at days 1, 3, 6, 8 or 12 after exposure. Pink mutation frequencies were determined from stamen hairs of open flowers after all experiments with [3H]-DBE. T h e [3H]-DBE exposures and resulting pink mutation frequencies are listed in T a b l e 1. These d a t a are plotted as mutation-response curves and are c o m p a r e d to those previously determined with unlabeled DBE Or) in Figs. 7a and b. T h e [3H]DBE mutation-response curves tbr clone 02 and for clone 4430 are elevated over those determined after exposure to non-labeled DBE, possibly for one or more of several reasons which will be discussed. FoE" any given exposure

the mutation frequency in clone 02 exposed to [3H]-DBE is increased by a factor of about 22 over that in those inflorescences exposed to unlabeled DBE; similarly, the mutation frequency in clone 4430 is increased by a factor of from 4.6 (.at high exposures) to 9.3 (at low exposures). W h e n related to previously determined X - r a y dose-response curves, (31' 33, 44) the m a g n i t u d e of the enhanced mutation response to [3H]-DBE in clone 02 corresponds to that which would be induced by from about 14 rad to about 35 rad in the range of exposures where the curves overlap vertically (97.5-288 p p m - h r ) ; corresponding values for clone 4430 vary ti"om about 6.5, to 51, and to 74 rad (at 25.2, 337.8 and 532.8 p p m - h r , respectively). For comparison to these values we have calculated the rad dose from tritium decay that would be expected in stamen tissues. Assuming that tritium is uniformly distributed in stamen tissue, as is indicated by our a u t o r a d i o g r a p h i c studies, we would expect a dose of 12.18 lnrad/hr//~Ci/g stamen tissue (our calculations and those in ref. 12). Using this value and making the most liberal assumptions (i.e. (1) that a 12-day exposure period contributed to the mutation response, (2) that the m a x i m a l tissue #Ci content from d a y 1 was present throughout the exposure period, and (3) that the mass of stamen tissue was m a x i m a l and equal to that on day 1 of flowering), we calculate that the tritium would have contributed a dose of 0.66 rad at the 97.5 p p m - h r exposure and 1.20 t a d at the 288 p p m - h r exposure of clone 02. C o m p a r a b l e values for clone 4430 would be 0.11 rad at the 25.2 p p m - h r exposure to 1.33 t a d at the 337.8 p p m - h r exposure. T h e curves in Fig. 7 can also be used to determine an equal effect ratio (EER), defined as the ratio of exposures producing the same level of effect, for the two mutagens. For clone 02 the E E R is essentially constant at about 17 for all levels of eft>ct; for clone 4430 E E R values range from about 6.8 to 3.4 as the curves converge and as the mutation level increases from 0.05 to 5.0 pink mutations/100 hairs. T h e main purpose of this investigation has been to determine the tissue dose of a gaseous chemical mutagen. T o this end, pink mutation

UPTAKE OF 3H-1,2-DIBROMOETHANE

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frequency is plotted as a function of the tissue dose o f DBE for both clones in Figs. 8a and b. This was achieved by plotting the n u m b e r of pink mutations induced by each unlabeled-DBE exposure (Figs. 7a for clone 02, 7b for clone 4430) as a function of the n u m b e r of moles DBE in stamens after the same exposure to labeled DBE {Figs. 3a tbr clone 02, 3b tor clone 4430). Since mutations are induced in stamen tissue, the curves are plotted as a function of the DBE content of stamen tissue only. T h e r e are two curves for each clone in Fig. 8 because two values for the molar content of stamen tissue were considered: that at 30 rain postexposure and that at 24 hr post-exposure.

DISCUSSION

T h e present study has shown that it is possible to relate a c c u m u l a t e d tissue content of a chemical mutagen to the exposure concentration of that mutagen, over a relatively wide range of exposure concentrations and times, and to derive true d o s e - m u t a t i o n - r e s p o n s e relationships for target tissues of a complex organism. Experimental d a t a have been expressed as a function of the "total exposure" of DBE to which inflorescences were subjected; total exposure is designated herein as the product of concentration ( p p m ) and time (hr), and is given in units of p p m - h r . This convention per-

210

C . H . NAUMAN, P. J. KLO'FZ and L. A. SCHAIRER i

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mits the plotting of d a t a from experiments in which different exposure concentrations and durations were utilized in one overall graphic relationship. T h e use of this product to represent exposure or dose has been utilized relatively infrequently in recent chemical mutation studies; (1'6'2°'21'461 to the best of our knowledge we are among the very few authors who have used this product to express exposure for chemicals in the gaseous form. 19' 32, 3,*, 3"7} By way of analogy, the units of radiation exposure and dose, the Roentgen and the rad, are themselves the produc[s of similar parameters: intensity and time. 1221 T h e presem experiments show clearly that the tissues of the two clones accumulate similar amounts of DBE alter similar total exposures. This is especially true at higher total exposures and for tissues.li'om open flowers, where tissue weights were quite accurately determinable. Thus, we cannot attribute the 7-9-fold increased DBE sensitivity of clone 4430 over clone

02 (31'44) to an increased uptake of the mutagen.

In fact, the more sensitive clone 4430 tends to have accumulated slightly less mutagen at most exposures. W h e n c o m p a r i n g tissue content of DBE in buds and open flowers (Figs. 3 and 6),. it is evident that there has been an alteration in the ranking of the various tissues. For example, stamen tissue from buds contains the greatest relative amount of [3H]-DBE, but among tissues ti'om open fowers the anthers contain by fhr the largest concentration of mutagen. T h e reason for this is unknown, but it is possible that there are changes in affinities of the various tissues for nonpolar substances like DBE as they synthesize more or new substances during development. It is known, for example, that the anther wall thickens considerably during maturation. (z3) T h e material synthesized for the thickened wall m a y have a high affinity for DBE. For clone 4430 there is a consistently higher slope tbr all regressions of tissue content on total

UPTAKE OF 3H-1,2-DIBROMOETHANE

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exposure than for clone 02. This is consistent with and probably a cause for the higher slope of the clone 4430 exposure-mutation-response curve (Fig. 7). Nevertheless, tissue content for both clones at 30 min post-exposure (Fig. 3) is nearly directly proportional to total exposure (slopes averaging 0.9 for clone 02 and 1.1 for clone 4430). Considering that there are some very low exposure concentrations and exposure times (Table 1) contributing to these regressions, penetration of the chemical must be very rapid. A rapid penetration might be expected f o r a low molecular weight and relatively nonpolar molecule (dielectric constant

=4.78). (2v) Further, the constant decline in [3H]-DBE content with dilution in the increased tissue mass resulting from growth (e.g. Fig. 4 tbr clone 4430) and the maintenance of regression slope and tissue rankings for samples taken on many days after exposure indicate that the [3H]-DBE is tightly bound in the tissues. The consistent increase in bud tissue [3H]DBE content on day 1, relative to that at 30 min post-exposure (Fig. 5), is perplexing but could indicate a preferential translocation of the [3H]-DBE into bud tissues via the transport elements of the stem during the first 24 hr alter exposure.

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C. H. NAUMAN, P.J. KLOTZ and L. A. SCHAIRER

Beta radiation, both from an external 9°Sr/9°Y source (3) and from incorporated tritium, O6) have been shown previously to be capable of inducing pink mutations in Tradescantia. Obviously, however, the increased effectiveness of the tritium-labeled DBE over unlabeled DBE in the present study is tar greater than that which can be accounted tbr by the rad dose ti'om the beta decay of tritium alone. O t h e r factors that are possibly contributing to the nmtagenicity of [aH]-DBE include transmutation of the tritium nucleus, a n d / o r a synergism between the beta radiation and the chemical mutagen. T r a n s m u t a t i o n of the tritium nucleus to helium as a consequence of the radioactive decay can lead to such local effects as recoil of the decaying nucleus, electronic excitation energy of the newly arising atom, and build-up of charged states. O2' 14) These instabilities are known to contribute to the biological effects of tritiu'm decay, but the extent to which transmutation contributes to the mutation response in Tradescantia is quite uncertain. The B E I R COMMITTEE(2) concluded that beta decay from incorporated [3H]-thymidine is the major cause of d a m a g e in biological systems, while the transmutation effect is of minor importance. However, it is known that tritium transmutation causes D N A strand breaks and can play a key role in producing genetic effects/TM W h e n , 3H decays in small molecules, such as DBE, the c a r b o n - t r i t i u m bond becomes the thermod y n a m i c a l l y unstable carbon SHe + bond. This bond breaks in less than 10 - 4 sec leaving a carbocation, C ÷, which is a very reactive electrophile that will undergo manifold fast reactions. O4) Thus, transmutation of tritium in DBE could be a significant factor; alternatively, or in addition, there m a y be some synergism (greater than additive effect) between the beta radiation and the chemical mutagen. Exposures to radiation plus chemical are known to result in synergistic responses for mutation induction,(19, 41, ,*2) as well as other endpoints such as tumor induction, (4a) cell survival (5) and chromosome aberrations. Iv) T h e explanation often offered for this p h e n o m e n o n is that one or both o f the agents interferes with the recovery fi'om or repair process for d a m a g e induced by the other agent.(t 8, 48)

W h a t e v e r the true explanation, the enhanced mutation response to [aH]-DBE over that which would be expected due to the rad dose from incorporated t r i t i u m - - w h i c h we have expressed variously as a factor increase, as the external Xray dose required to produce the enhanced response, and as the EER---is an example of how a biological organism, acting as a biological dosimeter, responds t~tr in excess of that which would have been predicted by an estimate based oil measured dose. A somewhat less dramatic parallel to this is seen in the work of ICHIKAWA,(161 where it was shown that more pink mutations were induced by scattered radiation than with direct g a i n m a rays plus scattered radiation per unit of exposure. This effect was presumably due to the difference in energy (and higher RBE for this genetic effect) of the scattered r a d i a t i o n - - w h i c h was nevertheless recorded by thermo-luminescent dosimeters as the same physical dose. W i t h regard to the dose-response curves shown in Fig. 8, we might assume the regression tor d a t a collected from bud tissues at the earliest time possible after exposure (30 min) to be the most i m p o r t a n t in regard to representing the true dose of DBE. W e do not, however, know the length of time that DBE functions as a mutagen in Tradescantia tissues. Best estimates of the biological half life of DBE range from less than 2 hr in rats and less than 12 hr in chicks 128) to less.than 48 hr in guinea pigsJ TM A related complicating factor is the indication that DBE m a y be metabolically activated by Tradescantia tissues, lain Thus, its presence in tissues at 24 hr (and possibly later times) m a y also contribute to mutation frequency. Since DBE is an alkylating agent, its presence in stamen hair cells as a covalently bound a d d u c t is not limited to DNA. O t h e r cellular constituents, including protein and R N A , could also be expected to be alkylated. A u t o r a d i o g r a p h s p r e p a r e d from experimental material used in the present experiments would indicate this to be the case in Tradescantia stamen hair cells. Thus, even though we now have true d o s e - m u t a t i o n respgnse curves (Fig. 8), we do not know the proportion of the DBE binding to constituents other than DNA, nor to what extent this n o n - D N A binding may eontri-

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(1973) Relative biological effectiveness of fl, ), and X irradiation for seedling growth and survival in barley and somatic mutations in Tradescantia. Radiat. Res. 55, 602. 4. BREM H., STEIN A. B. and ROSENKRANZ H. S. (1974) The mutagenicity and DNA-modifying effect of haloalkanes. Cancer Res. 34, 2576--2579. 5. CHADWICKK. H., LEENH(~UTSH. P., SZUMIEL I. and NIAs A. H. W. (1976) An analysis of the interaction of a platinum complex and radiation with CHO cells using the molecular theory of cell survival. Int. J. Radiat. Biol. 311, 511-524. 6. CHANET R., IZARD C. and MOUSTACCHI E. (1976) Genetic effects of formaldehyde in yeast. II. Influence of ploidy and of mutations affecting radiosensitivity on its lethal effect. Murat. Res. 35, 29-38. 7. COHN N. S. (1964)The ett~ct of hydroxylamine on the rejoining of X-ray-induced chromatid breaks in Viciafaba. Mutat. Res. 1, 409-413. 8. DE SERRES F. J. and MALLING H. V. 11970) Genetic analysis of ad-3 mutants of Neurospora crassa induced by ethylene dibromide---a comAcknowledgements We very gratefully acknowledge monly used pesticide. EMS Newsletter 3, 36-37. the technical assistance of Ms. S. W. BEATTY, Mr. E. 9. EHRENBERG L., HIESCHE K. D., OSTERMANE. KLUG, Ms. A. F. NAUMAN,Ms. V. POND, Mr. R. GOLKAR S. and WENNBERG I. (1974) Evaluation C. SAUTKULIS and Ms. L. N. SCOTTI. Ms. C. A. of genetic risks of alkylating agents: tissue doses BJERKNES and Dr. L. EVANS provided initial assisin the mouse from air contaminated with tance with liquid scintillation counting, and Mr. J. ethylene oxide. Mutat. Res. 24, 83-103. R. STEIMERS and Dr. R. E. EHRENKAUFERprovided assistance and advice in determining the specific 10. EHRENBERG L., OSTERMAN-GoI.KARS., SINGH D. and LUNDQVIST U . (1974) On the reaction kiactivity of the tritiated DBE. Dr. L. F. PHILLIPS netics and mutagenic activity of methylating and confirmed calculation of the radiation dose from fl-halogenoethylating gasoline additives. Radiat. tritium, and Mr. K. H. 'FHOMPSON carried out an Bot. 15, 185 194. extensive computer analysis of the data. Dr. J. W, BAUM and Ms. A. F. NAUMANmade helpful sugges- 11. FISHBEINL. (1976) Industrial mutagens and potential mutagens. I. Halogenated aliphatic detions concerning the manuscript. This study was rivatives. Murat. Res. 32, 267-308. initiated as a part of the research program of the late Dr. A. H. SPARROW, and was supported in part by 12. GLUBRECHT H. (1965) Mode of action of incorporated nuclides (comparison of external and the National Institute of Environmental Health internal irradiation). Pages 91-99 in The use oJ Sciences and in part hy the U.S. Department of induced mutations in plant breeding, Radiat. Bot. Energy. (Suppl.) 5, Pergamon Press, Oxford. 13. GRAY L. H. and DOUGLAS G. M. (1964) The rate of consumption of oxygen by anthers of REFERENCES Tradescantia paludosa. Radiat. Bot. 4, 233-245. 1. AUERBACHC. and RAMSAYD. (1968) Analysis of 14. HALPERN A. and ST6CKLIN G. (1977) Chemical a case of mutagen specificity in Neurospora crassa. and biological consequences of fl-decay, Part 1. I. Dose response curves. Molec. Gen. Genet. 103, Radiat. Environ. Biophys. 14, 167-183. 72-104. 15. HALPERN A. and ST6CKLIN G. (1977) Chemical 2. BEIR COMMITTEE (1972) The effects on popuand biological consequences of fl-decay, Part 2. lations of exposure to low levels of ionizing Radiat. Environ. Biophys. 14, 257--274. radiation. National Academy of Sciences-- 16. ICHIKAWA S. (1973) Comparison of genetic efNational Research Council, Washington, D.C., fects of scattering radiation with direct gamma 217 p. rays in the stamen hairs of Tradescantia ohiensis 3. B/)TTINO P. J., BORES R. J. and SPARRO'¢¢A. H. KU 7 clone, japan, j . Genet. 48~ 35~1-0.

bute to the mutation response. Even in studies where alkylation of D N A by a m u t a g e n has been measured, (39' 4o} it has been felt that relatively few of the alkylations are of significance in m u t a t i o n induction; apparently relatively rare alkylations or combinations of alkylations are those of genetic significance.(23' 39, 40) This investigation has shown that DBE penetrates plant tissues rapidly and is b o u n d tightly by tloral tissues. Cellular b i n d i n g of DBE is nonspecific, nuclear and cytoplasmic cellular constituents of stamen hair cells both being labeled. Further, a differential target tissue dose is not responsible for the differential sensitivity to DBE exhibited by these two clones; however, experiments in progress may elucidate the nature of this ditterential sensitivity, both with regard to DBE as well as other inutagens already tested.

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