Effect of 1,2-dibromoethane (DBE) on meiotic chromosomes of tradescantia

251

Mutatton Research, 58 ( 1 9 7 8 ) 2 5 1 - - 2 5 8 © E l s e w e r / N o r t h - H o l l a n d B m m e d m a l Press

EFFECT OF 1,2-DIBROMOETHANE (DBE) ON MEIOTIC CHROMOSOMES OF TRADESCANTIA *

T E - H S I U M A * * , A R N O L D H S P A R R O W * * * , L L O Y D A. S C H A I R E R a n d A N N E F. N A U M A N

Department o f B~ology, Brookhaven Natmnal Laboratory, Associated Unwers~tzes, Inc , Upton, N Y 11973 ( U S A ) (Received 3 August 1977) (Accepted 5 June 1978)

Summary The production of mmronuclei (MCN) in tetrads of mmrosporogenesls of Tradescantla clone 4430 by a 6-h treatment with 1,2-dlbromoethane (DBE) gas showed a pomti~e correlation with the concentration of the gas administered. A range of 0.001--0.002 MCN/tetrad/ppm-h was established by dose--effect experiments. The dose--response curve of the present study resembles that of a previous study on pink mutation m stamen hairs of the same clone of plants. The mutagenic cffmiency of DBE for induction of MCN in tetrads is about 36 tlm~s t h a t of DBE for reduction of pink m u t a t m n in stamen hairs ff comparison is made on the basis of MCN/tetrad and pmk m u t a t m n / s t a m e n h a m

Introduction Halogenated hydrocarbons comprise a large group of environmental pollutants whmh are mutagenm and/or carcinogenic, and some are teratogenm. Ethylene dlbromide or 1,2-dlbromoethane (DBE) is one of the members of this group whmh demands special a t t e n h o n because of its wide distribution through gasohne additives, farm fumigants, and household pestmldes [4]. Recent studies in Neurospora [2], stamen hmrs of Tradescantia [5,6,12] and Drosophila [18] proved that DBE at very low concentrations induced mutations. Brem et al. [1] * Research supported m part by the U.S Energy Research and Development Admlmstratlon and m part by Nahonal Institute of Environmental Health Semnces. ** Visiting Semnhst, Department of Biological Scmnces, Western Ilhnols Umverslty, Macomb, IL 61455 (U S.A.) *** Deceased on June 25, 1976

Abbrewatlons DBE, 1,2-dlbromoethane, MCN, mmronuclel

252 reported that DBE inhibited growth of DNA-polymerase-deflcmnt (pol A~)

Escherichia coli. In rats and mice, stomach cancers were induced by chronic exposure to low doses of DBE [7]. When barley kernels were soaked m DBE solution, mutations were scored in mature plants [3], and chromosome damage was observed m root tips of germinating kernels [3]. Based upon the ethylatmg reaction kinetic studies of DBE, Ehrenberg [3] proposed that mutageniclty of DBE could be related to rater-strand cross-links and single-strand breakage of DNA. More cnticai studms are needed to ascertain the relatmnship between mutagenicity and DNA damage. Extensive studies on DBE-induced mutations in stamen hmrs of Tradescantm in this laboratory in recent years [ 5,6,12], indicated that the dose--response curves was similar to that of X-rays. The mutagenic efficiency of 1 ppm of DBE equals roughly that of 10 rad of X-rays. Tradescantia clone 4430 is more sensitive to DBE m m u t a t i o n p r o d u c t m n than other clones tested. The present study is designed to determine DBE-mduced chromosome damage in meiocytes of this clone in order to cross check the mutation studms in the same system. The first portmn of this investigation was devoted to the establishment of a new scheme for detection of damage to meiotic chromosomes through micronuclei (MCN) production in early tetrad stage of microsporogenesls, and a simple cause-and-effect relatmnship between DBE and MCN production. The second portion was devoted to the establishm e n t of a dose--response curve. Materials and methods Tradescantia clone 4430 (T. hirsutiflora × T. subcaulis) was utilized throughout this investigation. Cuttings (about 20 cm) from plants grown in environmental growth chambers [17] were selected for uniformly matured influorescences which bear pollen in the first bud and meiotic pollen m o t h e r cells in the 6th or 7th bud. The cuttings, in groups of 20--50, were maintained m Hoagland's solution in an environmental growth chamber for 2--3 h before treatment. Treatments were made in a series of chambers which contained different concentrations of 1,2-dibromoethane (DBE) gas. The rate of flow of the mixture of mr and gas through the chamber was 2 1/min, and the concentrations were m o m t o r e d with a Beckman G6-5 gas chromatographic system with a flame lomzation detector at hourly intervals. The temperature and relative humidity of each chamber were also recorded (around 26°C and 76% RH) at similar intervals. The control groups were placed in similar chambers containing only air with the same flow rate. The gas concentration generally reached a plateau within 20--30 min, and the actual average concentration in each chamber was slightly lower than the intended concentration as shown in Table 1. After exposure, treated and control cuttings were transferred into fresh Hoagland's solution and returned to the environmental growth chamber to allow the treated meiocytes to go through meiosis. Whole influorescences of each cutting were fixed in Newcomer's fluid 24 h later. The fixed influorescences were transferred into 70% ethanol after 24--48 h of fixation. The 70% ethanol preserving solution was changed twice in the next 48 h to wash away the residual fixative. Fixed influorescences which were composed of 11--12 discernible buds were

253 TABLE 1 C O M P A R I S O N O F M U T A G E N I C E F F I C I E N C I E S O F 6-h D B E E X P O S U R E S O N I N D U C T I O N O F P I N K MUTATIONS IN STAMEN HAIRS, AND ON INDUCTION OF MICRONUCLEI IN MEIOCYTES OF TRADESCANTIA CLONE 4430 DBE conch (ppm)

Mutahons per 100 stamen hmrs (minus control) a

Mutagemc effmlency (mut /halr/ppm-h)

DBE conch (ppm)

MCN per 100 tetrads (minus control)

Mutagemc effmlency (MCN/tetrad/ppm-h)

36 108 19 3 36 1 60 2 783 148 2

004 010 0 53 1 08 2 53 246 4 57

1 1 4 4 7 5 5

46 + 037 94-+ 098 1 8 6 +- 1 51 37 3-+ 3 60 58 6+- 4 37 77 5 + 5 5 0

6 6 9 1 16 9 253 410 639

239 ×10 1 61×10 1 51 X 1 0 1 13 X 1 0 1 17 X 1 0 1 37 × 1 0

Mean

85X10 54X10 58X10 99 X 1 0 00 X10 24X10 14 × 10

-5 -s -5 -5 -5 -5 -5

4 33 X 10 -5

-3 -3 -3 -3 -3 -3

1 53 X 10 -3

a D a t a f r o m N a u m a n e t al [ 6 ]

dissected under a magnifying lens to obtain early tetrads. Generally, the appropriate stage of tetrads can be found in one out of 3--4 influorescences. The early tetrads, usually accompanied by dyads, were stained in aceto-carmme, heated and pressed under the coverglass to allow the cells to stick to the microslides. The dry-ice quick-freezing m e t h o d was utilized to remove the coverglass in order to destaln and dehydrate the cells to make permanent preparations. Only intact tetrads were selected for scoring MCN and the MCN or fragments in anaphase I or anaphase iI were n o t scored. The number of MCN, ranging from 0 to 6, from each tetrad were recorded in separate columns of a sconng sheet. The total number of MCN from about 500 tetrads per slide was dJvlded by the actual number of tetrads scored to derive the percentage values, or number of MCN per 100 tetrads. After a number of preliminary tests, two series of experiments were carried out in this investigation. Preliminary tests were designed to determine the sensitive stage for treatment and proper fixation time for tetrads contmning induced MCN. According to radiation studies of Sax [8,10] and Taylor [16] meiocytes of microsporogenesis of Tradescantia have a sensitive peak centered around early prophase. Series of fixations, at 2-h intervals within the duration of 30 h of meiosis, were made in order to determine the specific portion of the tetrad stage which yields relatively high frequency of MCN. The first series of 3 repeated experiments with smaller number of samples (3--10) were conducted consecutively to determine the cause-and-effect relationship between DBE treatment and MCN production. Samples were treated at about 20, 40, 80 and 160 ppm of DBE for 6 h. Early and late tetrads were observed for frequencies of MCN. A more extensive study, with larger number of samples (15--27) per treatment group, was carried out to establish the dose--response curve. Samples in this study were treated at about 5, 10, 20, 40, 60 and 80 ppm of DBE for 6 h. Results and discussion A total of approximately 1.1 × 10 s tetrads were scored in this investigation. The general morphology of MCN observed under 320× magnification varied

254 greatly according to the DBE dosage, the stage of tetrads and the initial size of the deletion from the chromosome or group of chromosomes involved (Fig. 1). Generally, larger sized MCN were produced after exposure to higher concentrations of DBE. Occasionally the size of a MCN was about the same as that of the

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255

m o t h e r nucleus. In other instances, the size of MCN was quite small. The MCN of early stage tetrads from low-dose groups appeared to be composed of loosely mterwoven chromosomes, sometimes superimposed over or under the mother nucleus of similar appearance. The MCN of high-dose groups were usually condensed and coalesced m t o a sohd mass, and were clearly distinguishable from the condensed m o t h e r nucleus {Fig. 1). The p h e n o m e n o n was later determined to be a sign of death of the cell. The relatively large MCNs could have involved a group of acentrlc fragments whmh had adhered together or could have been comprised of 2--3 entire centric chromosomes out of the total complement of 6 chromosomes by the process of polyspindle formation. In roughly 2--5% of the tetrads observed, there were MCN twins. These were MCN of exactly the same size and morphology, §ymmetrlcally situated between two sister nuclei of a tetrad. They were either chromosome breaks of prophase I which were separated during the second division, or chromatid breaks of prophase I or II. In some rare cases, MCN quadruplets were observed. All of the MCN appearing as twins or quadruplets were counted individually without concern for the initial number of breakages involved. Preliminary tests using serial fixation technique found t h a t fixation of early tetrads 24 h after treatment give relatively higi~ frequency of MCN. Based upon the durations of meiotic stages of Tradescantia reported by Sax [8,10] and of Trillium reported by Sax [9] and Sparrow [11], the meiotic stage effectively damaged by DBE was around pachytene, presumably d u n n g the process of crossing-over. The results of three repeated experiments m the first series of this study are shown in Fig. 2. The data of experiment A, derived from a total of 23 samples m 5 experimental groups, roughly demonstrated the cause-and-effect relationship m the first 3 concentrations (20, 40 and 80 ppm) of DBE, while the 160 ppm concentratmn exhibited a decline of MCN frequency. This was probably

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256

due to me]otic delay whmh caused the late arrival of MCN-bearing tetrads. Most of the tetrads observed in this group were the melotm products of later prophase meiocytes which were relatwely msens~twe to treatment. Furthermore, tetrads which were scored from the 160 ppm group were late-stage tetrads. It was assumed that some of the early-stage tetrads which bear MCN had not yet arrived, or never would be able to proceed to the tetrad stage because of lethallty under high dose treatment. It was also noted that the cuttings treated at 160 ppm dmd and dried completely at the end of 48--72 h after treatment. The high DBE dosage--MCN frequency dechne p h e n o m e n o n and the explanation proposed above could also be utilized to explmn the decline of m u t a t i o n rates m stamen hmrs after high dosages of DBE [6]. Based upon the observations made and the information obtained from experiment A, adjustments were made to compensate for this over-dose effect. This was done by sconng MCN only from early tetrads. Presumably, the early-treated stage may last a few hours and could accommodate the delayed and nondelayed meiotm nuclei and show roughly the appropriate frequencies of MCN m low as well as high-dose treatment groups. In order to obtain early tetrads, shdes were prepared only from samples exhibiting a mLxture of dyads and tetrads. A higher degree of mixture was apparent m high-dose treated samples than m low-dose treated and control samples. This may have been because the treatment disturbed the normally high degree of synchrony of meiotic events m Tradescantla. The results of expenments B and C (Fig. 2) were obtamed from 20 and 37 samples respectively. They both demonstrated a positive correlation between dose and MCN frequency. Great varmnce as denoted by the standard errors of all three expermlents was contributed by (a) the nature of the gaseous mutagen effect [5,6] which may be attributed to the rate of penetratmn and the

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257 threshold tolerance, (b) physiologmal state of plants [13--15], (c) stage difference of tetrads scored whmh created a hit or miss situation m reference to the sensitive stage, and (d) meiotm delay which further changes the h m e of arrival of MCN-beanng tetrads. The results of the dose--response experiment were obtamed from a larger number of samples (15--27 samples per group with a total of 146 samples) treated at about 5, 10, 20, 40, 60 and 80 ppm concentrations. Frequencms of MCN, expressed as MCN per 100 tetrads, were plotted agmnst concentrahons of DBE, expressed m ppm (Fig. 3). The dose--response curve shows a positive correlation and hnearity with the coefficient of correlation equal to 0.99, and the slope equal to 0.754. The standard errors are smaller than those of the preh m m a r y experiments. It is clear that the magmtude of variance was m proportion to the dosages of DBE applied. The magnitude of damage expressed m terms of damage done in a scoring unit by 1 ppm-h of DBE fell in the range of 0.0014--0.0024 MCN/tetrad/ppm-h of DBE. A comparison was made between the mutagenic efficiencies of DBE for MCN induction m this study and for pink m u t a t i o n reduction from a previous study [6] which was carried out under similar experimental conditions. Mutagenm efficiencies at different dosages, expressed in terms of damage done m a scormg u m t by 1 ppm-h of DBE, for both studies, are listed in Table 1. The effmmncy in m u t a t i o n induction is proportmnally increased as the dosages are increased, while the efficmncy m MCN m d u c t m n is in reverse relationship. The average mutagenic efficiency in MCN induction is about 36 times as great as in m u t a t m n reduction. This is mmnly due to the fact that there are numerous sites m each of the 12 chromosomes of the m e m c y t e for chromosome or chromatid aberrations resultmg m MCN, but only 1 site in 12 chromosomes of each of the cells of a stamen hmr at the time of treatment that will permit expressmn of a pink m u t a t m n . In conclusmn, it is important to note that the present new testing scheme for gaseous pollutants effects on eukaryotm chromosomes can ymld easily visible (under 320× magnificatmn) quantitative and qualitative ewdences of damage from a large population of cells m a relatively short time. The ease of scoring MCN rather than chromatid or chromosome aberratmns has made this a more efficient scheme. Memtic pollen m o t h e r cells, unhke the microspores, are capable of absorbing chemical solutions through the stem and pedicel of the inflorescence, thus facilitating the testing of pollutants or drugs m hquld as well as gaseous form. Large numbers of meiocytes of a given stage can be obtained for use in bmchemical or ultrastructural studies of altered DNA or other constituents of the cell.

Acknowledgements The authors wish to express their appreciation to Mr. Paul J. Klotz for his technical assistance in DBE dosimetry, Mr. Rmhard C. Sautkuhs for his assistance in treating and caring for the plants, Ms. Vlrgima Pond for her assistance in slide preparation and scormg, and Drs. Jack van't Hof and Charles H. Nauman for their comments and suggestions in the course of this investigation and in preparation of the manuscript.

258

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