Further multiple fixation, chromosome aberration, dose fractionation studies on Vicia faba: The effect of cycloheximide

Mutation Research, 18 (1973) 163-17 ° © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - P r i n t e d in The N e t h e r l a n d s

163

F U R T H E R M U L T I P L E F I X A T I O N , CHROMOSOME A B E R R A T I O N , DOSE F R A C T I O N A T I O N S T U D I E S ON VICIA F A B A : T H E E F F E C T OF CYCLOHEXIMIDE*

M O R T O N W. M I L L E R AND C H R I S T O P H E R W E I S S

Department of Radiation Biology and Biophysics, School of Medicine and Dentistry, The University of Rochester, Rochester, N, Y. z4 642 (U.S,A.) (Received October 23rd, 1972)

SUMMARY

Soaked seeds of Vicia faba were irradiated with 800 or 400 R, or two 4oo-R exposures, both with VAC (vacuum) but one with a concomitant treatment of CHM (cycloheximide) (i #g/ml). The chromosome aberration yields from each regimen varied with fixation time such that a unique and characteristic aberration yield for each regimen relative to the others was not obtained. Were single fixations employed one could obtain yields which would indicate no, some, or m a x i m u m repair. A single fixation would lead to an incorrect estimation of chromosome damage repair.

INTRODUCTION

Chromosome repair studies have involved dose fractionation regimens on a wide variety of systemsl~,l*,17,18,~l,22,~3,3~,3~,48. The general theory for studying repair of chromosome damage involves a comparison of the yields from full-dose, split-dose, and half-dose regimens. Briefly stated, if the breaks from the first dose have rejoined (repaired) before the second dose is administered the yield of two-hit aberrations will be the sum of those produced by each of the two-dose fractions--i.e., the yield will be low. If the breaks remain open the yield should be the square of tile sum of tile square roots of the yields produced by each fraction--i.e., the yield will be high47, 4s, provided the kinetics of aberration production is dose squared. Thus, the yield of rings and dicentrics (two-hit aberrations) has been used to indicate the presence or absence of repair. Throughout much of the chromosome repair literature single fixations have been used. The reports of SAXa3 and others n,29 have indicated a constant radiosensitivity in G~ for Tradescantia microspores. Thus, this synchronous G~ population with its uniform radiosensitivity can be studied with single fixation regimens since the chromosome aberration yield is characteristic for the system. The germinating root * This p a p e r is based on w o r k performed u n d e r c o n t r a c t w i t h the U.S. Atomic E n e r g y Commission at The University of Rochester Atomic E n e r g y Project and has been assigned R e p o r t No. UR. 3490-205 . A b b r e v i a t i o n s : CHM, cycloheximide; LAM, labeled a m i n o acid m i x t u r e ; V, VAC, v a c u u m .

lO4

M . W . MILLER, C. WEISS

tip meristem in seeds is a system which has been assumed to contain G1 cells of uniform sensitivity. This assumption has been proved incorrect for Vicia faba12,21, 2~ and Hordeum vulgare 3~ root tip cells. In two previous papers21, 22 it was shown that (i) the frequencies of two-hit chromosome aberrations, induced by irradiating primary roots of germinating seeds prior to their first mitosis, varied with fixation time and (2) the respective 6-h peak 21 and 2-h "trough ''~ yields showed fixation time dependency. In this paper data on the effect of a known metabolic inhibitor, cycloheximide, on chromosome aberration yields at various fixation times is presented and the bearing of these results upon the concept of chromosome break repair is discussed. MATERIALS AND METHODS

Seeds of Vicia faba obtained from the Kellogg Seed Company (Ventura, Calif. 93oo1) were used for this experiment. In all cases, the seeds were soaked for 16 h in running tap water (19 ± I°), peeled and grown for various lengths of time in sandwiches (consisting of a 17 cm × 12 cm aluminum sheet, cotton, paper toweling, the seeds, paper toweling, cotton, and aluminum) at 19 -L I ° (ref. 45). The seeds were maintained upiight near the top of the sandwiches and kept moist by having the bottom edge of the sandwich in about I cm of water. Irradiations were from a G.E. Maxitron operated at 3oo kVp at 20 mA; the h.v.1, was 1.5 mm Cu and the intensity was 14o R/min. Four radiation regimens were utilized: (z) a single 4oo R (half dose) exposure; (2) a single 8oo-R (full dose) exposure ; (3) a split dose of 4oo R in vacuum plus a second 4oo R; and (4) a split dose of 4oo R in vacuum, then CHM plus a second 4oo R; the vacuum was present only for the first dose. The concentration of CHM utilized was I Fg/ml. A scheme of the overall sequence of events for the chromosomal analyses is given in Fig. I. All seeds were peeled just before the first exposure. Prior to fixation in 3:1 alcohol: acetic acid the roots were excised and soaked for 2 h in an aerated 0.4% colchicine solution. The root tips were Feulgen stained, squashed and prepared for microscopic examination. The frequency of rings and dicentrics per cell as an unweighted mean was determined for each exposure regimen and each fixation time, using four roots per treatment and scoring 5o cells per root. BEGIN GERMINATION

IIRRADIATIONSI I WlTHIN THIS I

.... I r~..b_o - .......

PERIOD

j .........

400 R + (HALF DOSE)

0

800 R + (FULL DOSE)

0

400 R* VAC +

I j

FIXATION TIMES

L

400 R

400 R* VAC* CHM*400 R 0

J

18"

53 55 57 61 63 65

HOURS • SEEDS PEELEDAT

16HRS.{BEFOREIRRADIATIONI,GROWN IN "SANDWICHES" UNTIL APPROPRIATEFIXATION INTERVAL.

Fig. I. Germination, radiation, cultivation, fixation regimens for seeds of Vicia faba.

EFFECT OF CYCLOHEXIMIDE ON

I65

V. faba

The effect of CHM on cellular incorporation of an all-labeled L-amino acid (G) mixture (New England Nuclear: i/~Ci/ml) was determined. The roots and a small part of the attached cotyledons from 16 h germinated seeds were employed. The treatments were as follows : o R plus LAM ; 4o0 R plus LAM and 400 R plus CHM simultaneously with LAM. All treatments were begun on I6-h germination and all were subjected to an evacuation before irradiation and/or application of the CHM:LAM; the duration of CHM-LAM treatments was 2 h. At 18 h germination the cells were fixed overnight in 3 : I alcohol : acetic acid, then Feulgen stained, squashed and dipped in Kodak's NTB-3 liquid emulsion. After drying the slides were stored in the dark at 4 ° for three weeks: grain counts were made over about 15o meristematic cells from each of three root tips for each treatment. RESULTS

The chromosome aberration yield results of the four radiation regimens are shown in Table I and Fig. 2. Among the 8oo-R samples the 53-h yield is statistically TABLE I THE PERCENT GERMINATION

YIELD OF RINGS AND D;CENTRICS TIMES e

Radiation

53

55

PER CELL (MEAN

-~ S . E . )

57

59

6~

FOR FOUR RADIATION

REGIMENS

63

65

AT VARIOUS

Average

regimen (h) 400 R (half dose) 12.5 i 4-6 800 R (full dose) 39.0 ± 2.9 a 400 R + V + 400 R __b a 400 R q - V + CHM+4ooR61.5±5.6

13,o ±

3.1

12.5 i 1.7

14.5 ~ 3.0

29.5 ~

3-9

36.5 ± 4 . 8

31.O i

45,5 ±

5.9

59.o±9-9

44.5±7.1

57.3±

3.7

4 4 . 0 ± 3 .8

62.5£13.4

5o.216.7

91. 7 ± 15.2

81.5±1.7

5 3 . 5 4 - 4 .0

65.54-

5.4

56.5=ci.5

93.5~

73.7i6.I

26.8±

41.5 d- 5.4

52.0 i 3.9

69. 5 ± 16.9

69.5 2:- 4.2

77.5 ~- 13.2

4.9

5 .0

8.5

23.4 ± 3-7

56.9 ~ 7.7

a D o s e fractionation. b No m e t a p h a s e s a m o n g four r o o t tips. e 200 m e t a p h a s e s / i n t e r v a l (5 ° m e t a p h a s e s / r o o t x four roots).

,380 J

•...<, ..,.. ,.,-I

060 J

04O

.......r

-t- ........... ilIil

...........

y....-'" ± .............

/";

.............

~5

..............

~-,7

.....

59

/:,~

63

65

AVERAGE

GERMINATION HOJJRS

Fig. 2. The yield of ring and dicentric c h r o m o s o m e aberrations in Viciafaba r o o t tip cells exposed to 8oo R or 4oo R and t h e n scored for aberrations at 2-h i n t e r v a l s f r o m 53 to 65 h g e r m i n a t i o n . The e x p e r i m e n t w a s carried o u t using seeds s o a k e d for 16 h before irradiation. O,8oo R (full dose) ; A, 4oo R (half dose).

166

~t. w. MILLER, C. WEISS

significantly (at least 5 % level) different from tlle 57-h, 6i-h, and 65-h yields; the 55-h yield is statistically significantly different from the 6i-h yield. Among the 4oo-R samples the 53- and 57-h yields are significantly different from the 6i-, 63- and 65-h yields. Thus, within either of the two single exposure regimens (400 R or 800 R) there are significant differences in yields among the fixation intervals. A somewhat similar pattern occurs from single radiation regimens administered at 18 h germination 21. The data collectively reemphasize the lack of an apparent uniform sensitivity of the root tip cells during the first mitotic peak. The chromosome aberration yields from the full-dose (800 R) and split-dose (V+CHM) regimens are not significantly different from each other at the 59-, 61-, and 65-h fixations and are significantly different (at least 5 % level) at the 53-, 55-, 57-, and 63-h fixations. A somewhat similar pattern applies to the split-dose (+V) and splitdose (V+CHM) regimens (Table I) : not significantly different yields for tile 59-, 6I-, and 65-h fixations and significantly different yields for the 55-, 57-, and 63-h fixations. The full-dose (800 R) and split-dose (v-V) yields with the exception of that of the 59-h fixation are all significantly different from each other. On average, tile full-dose (800 R) yield (50.2 ~_ 6.7) is about twice that of tile half-dose (400 R) yield (23.4 ± 3.7) and equal to that of the split-dose (V+CHM) yield (56. 9 ~ 7.7). A puzzling and complicating fact is that the yield of the split-dose (+V) is very significantly greater than either the full-dose (800 R) or split-dose ( V + CHM) yields. The autoradiographic data (Table II) indicate a sharp reduction in grain counts TABLE

11

OF CYCLOHEXIMIDE [CHM] (i i~g/ml) on ACID MIXTURE[LAM] (3.2 /~Ci/ml)a THE

EFFECT

CELLULAR

UPTAKE

OF

A LABELED

Treatment

Number of cells

Number of grains

Mean (grains/cell)

o R+LAM 400 R+LAM 400 R+LAM+CHM

460 5o4 653

5325 6049 941

11.6 12.o 1.4

AMINO

a The average specific activity is derived by weighting the specific activity of each amino acid by its molarity and then calculating an average for the 15 labeled amino acids in LAM. --~ l o o ~

.

.

.

.

080

020

t 53

' 5~

57

59

6

03

65

AVFRAGE

OER/fdNAlfOxJ HOURS

Fig. 3. A multiple fixation study of the chromosome aberration yield of the full-dose (800 R) and split-dose (+V+CHM) regimens. O, 800 R (full dose); O, 400 R+V+CHM+4oo R.

EFFECT OF CYCLOHEXIMIDE

ON

V. faba

16 7

per cell with the application of CHM; for the control (o R + L A M ) 11.6, for the 40o R + L A M sample 12.o, and for the 400 R + L A M + C H M 1. 4. DISCUSSION

A cell system with a uniform sensitivity allows one to obtain a characteristic aberration yield from a single sample of first division metaphases at any post-treatment fixation time. Unfortunately, the root meristem of Vicia faba is heterogeneous with regard to radiosensitivity and time such that a single fixation cannot give a specific aberration yield characteristic of a given dose21, 2~, From a single exposure one can obtain widely oscillating aberration yields, obtaining low and high aberration yields with fixation time dependencies. Irradiation of a biological system perturbs it, and the degree of perturbation is dose dependent to some extent ; for example, the depression of mitotic index with Xray exposureS,9,13,2~,~9, 49. When split-dose regimens are employed the perturbation is different from that produced when just one full dose is utilized: the chromosome aberration yields from the full-dose and split-dose regimens are hardly uniformly similar when time-yield curves are compared~l,2L even though both treatments involve the same total exposure. Furthermore, when metabolic inhibitors and other types of treatments (e.g., vacuum) are employed additional perturbing factors are introduced as evidenced by fixation time-chromosome aberration yield comparisons among the full-dose and split-dose (V and V + C H M ) regimens (Table I). Thus, in telms of data analyses, single fixations are hardly likely to yield meaningful results. The problem of analysis becomes even more acute when one realizes that cells in different parts of their cycle m a y have different radiosensitivities as well as durations~,31; such factors render considerable complication to the shape and positions of peaks in yield-time aberration curves. The root tip is a highly organized and complex organ. When, for example, a single specific fixation time is employed in a split-dose radiation regimen, the chromosome yield pattern is highly reproducible 21. When the same radiation regimens are utilized and a multiple fixation scheme employed the aberration yields show no consistent relation to a different radiation regimen. The yields from a full- and half-dose regimen do not have a consistent iatio at different times; similar variations occur with yields from split-dose vs. full-dose regimens. We are emphasizing the differences in aberration yields between full- and splitdose regimens in terms of their bearing upon the concept of chromosome break repair. An alternative method would have been to compare split-dose yields with those obtained from appropriate half-dose regimens (e.g., 4oo R + V A C ; 4oo R + C H M ; 4oo R + V A C + C H M ; V A C + C H M + 4 0 0 R; VAC+400 R, and C H M + V A C + 4 0 0 R). Because this latter procedure would have been extremely cumbersome in terms of multiple fixation regimens it was not undertaken ; we also feel it would not have resulted in insight above that gained from comparisons to a full-dose regimen. We do not feel that second mitotic cells are significantly affecting our yield-time curves; second mitotic cells, if present, are not present in the early fixation interval and are less than 10% of the cells at the 65-h fixations 22. The experiments reported here further confirm the heterogeneity of the Vicia faba root tip system and clearly point to the hazard of evaluating biological processes

~68

M . W . MILLER, C. WEISS

(e.g., chromosome repair) from single fixation experiments in which there is not a uniform radiosensitivity. For example, WOLEF~4 has reported on the basis of single fixation experiments that protein synthesis is essential to repair of chromosome damage in Viciafaba. His conclusion was based upon the observation that the yield of a single full dose was similar to that of split dose for that one fixation time. WOLFF'S interpretation m a y well be correct in that on average our data indicate similar yields for full-dose and split-dose (V+CHM) regimens. However, our multiple fixation chromosome aberration yield data indicate on average our single doses are additive, which m a y vitiate any arguments about repair, breaks open or not. Were we to draw our conclusion from one of several fixation times we could argue for no (55-h) or positive (59-tl, Table I) repair. Clearly, with Viciafaba primary roots, because the yields from individual radiation regimens oscillate over the first post-radiation mitosis, a valid comparison at specific fixation times is not possible. For example, a comparison between the yields at various fixation times indicates significant differences between full-dose (Boo R) and s p l i t - d o s e + V + C H M regimens. If protein synthesis is necessary for repair one would anticipate that the yields would be identical and uniformly high with all fixation intervals, provided the different regimens perturbed similarly. If the split-dose regimen greatly alters the cells' progression and radiosensitivity sufficiently from the full-dose regimen then conclusions drawn from single fixation experiments (are the yields high, low, or identical ?) must be viewed with considerable caution. It is quite clear that the CHM is affecting some cellular processes in the treated root tips. The grain counts from all CHlVI-treated cells were significantly less than their appropriate control for uptake of the LAM (Table II). CHM has been found to inhibit in vivo (refs. I5,I9,27,4o ) and in vitro38, 4a protein synthesis; the inhibition occurs at either the transfer of amino-sRNA to the ribosomes or the formation of the peptide bond3,6,1°, 26. In some instances the effect is reported to be stage specific%2~,28,3~,37,41, irreversiblel, L16,23, in others reversible2,8,2°,24,~,4°,42, 46. Our data suggest a reversible effect in that the post CHM-treated cells proceeded to mitosis. It seems reasonable to assume that at least some perturbation--perhaps for the duration of the CHM treatment must be considered when attempting to make valid comparisons among treated and control regimens. Under any circumstances, the root tip of Vicia faba while possessing large and few chromosomes which renders light microscopy scoring "easy", is not a simple homogeneous system and therefore information about cellular processes based on single fixation regimens must be considered with caution. CONCLUSIONS

(~) Irradiation of 16 h germinated seeds with full-, split-, and half-dose regimens induced yields of chromosome aberrations which varied with fixation time. (2) When the seeds were treated with cycloheximide for the 2 h between the dose fractions the yields also varied. (3) A true estimate of repair or lack of repair was not possible since relatively low and high yields of aberrations were obtained from the split-dose regimens relative to the full dose.

EFFECT OF CYCLOHEXIMIDE ON V.

faba

169

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31 32 33 34 35 36 37 38 39 4° 41 42 43 44 45 46 47 48 49

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