- Email: [email protected]
Radioprotective effects of ascorbic acid in barley seeds
Radiation
Botany,
1975, Vol.
15, pp. 39 to 48. Pergamon
RADIOPROTECTIVE
Press. Printed
in Great
Britain.
EFFECTS OF ASCORBIC BARLEY SEEDS*
ACID
IN
37830,
U.S.A.
B. V. CONGER UT-AEC
Comparative
Animal
Research
Laboratory,
(Received
29
3t4
Oak
Ridge,
Tennessee
1974)
B. V. Radioprotective effects of ascorbic acid in barley seeds. RADIATION BOTANY 15, 39-48, 1975.-Experiments were conducted to test the radioprotective effects of a naturally occurring reducing agent, ascorbic acid, on seeds (caryopses) of barley, Horakum vulgare L. emend Lam. Seeds were soaked either before or after gamma or fission neutron irradiation in distilled water or ascorbic acid solutions ranging in concentration from 0.01 to 1.00 M. Results are reported as percentage germination, seedling height, seedling growth reduction, and (in one experiment) per cent of cells with chromosome aberrations. As evidenced by both reduced germination and seedling growth, ascorbic acid was toxic when seeds were soaked for 1 hr at ambient temperature prior to irradiation and then planted immediately. When seeds were soaked in ascorbic acid before irradiation and soaked after irradiation in air-bubbled water at 0°C for 18 hr, the toxicity disappeared, and a protective effect (which increased with increasing ascorbic acid concentration) was observed for gamma and, to a lesser extent, for neutron irradiation. Additional studies suggested that the protective effect was related to reduced hydration of the embryos of seeds soaked in ascorbic ‘acid. Also, no radioprotective effect was observed when seeds were presoaked for 2 or 16 hr in 0.01 M ascorbic acid solutions buffered at pH 3 or pH 7. A protective effect was observed for seeds of 1.5 per cent water content soaked after irradiation in an oxygen-bubbled ascorbic acid solution of 0.5 M but was not observed for seeds soaked in nitrogen-bubbled ascorbic acid. The protective effect against oxygen-dependent damage may be a result of interaction of ascorbic acid with radiation-induced free radicals. CONGER
THE acid
INTRODUCTION RADIOPROTECTIVE properties (a naturally
occurring
reducing
marked protection against radiation damage when present in cell suspensions containing radiosensitizing tetracycline antibiotics.(14) Ascorbate, however, protects against radiation damage in plant seeds as measured both by chromosome aberrations(ls) and seedling growth reduction.(3*s) The protective effect existed only when ascorbate was applied as a preirradiation
of ascorbic agent) have
been studied in several biological systems, and varying results have been reported. Sodium ascorbate did not protect against radiationinduced chromosome aberrations in Viciafaba. w Also, in spite of a few early favorable reports, most experiments dealing with the radioprotective effects of ascorbic acid in mammalian systems have yielded negative results. t2) Although ascorbic acid has no significant radiomodifying properties in Escherichia coli, it does provide *Research the University
sponsored by the U.S. of Tennessee.
Atomic
Energy
treatment.tsls)
Postirradiation
treatments
were
ineffective. There was no interaction between ascorbic acid and sodium azide, an agent known to enhance radiation damage in seeds,(r7) when applied to barley seeds either before or after
Commission 39
under
Contract
No.
AT-40-l-GEN-242
with
40
B.V.CONGER
gamma irradiationto) Ascorbic acid also had to be present during irradiation to protect against the radiosensitizing effect of oxytetracycline in E . coli . (14) MATERLUS AND METHODS Caryopses (hereafter referred to as seeds) of barley Hordeum vulgar-e L. emend Lam. cv. Himalaya were adjusted to a water content of N 13 per cent by storage in a desiccator over 60 per cent glycerol and 40 per cent water as previously described.02) In Experiment VII, the seeds were dried to 1.5 per cent water content by storage in a vacuum desiccator over CaSO, on the particular for -2 years. Depending experiment, seeds were soaked either prior to or after irradiation in distilled water or different concentrations of ascorbic acid ranging from O-01 M to I*00 M. Except for Experiment V, the pH of the solutions was not adjusted and was measured to be from 2-7 for O-06 M to 2-2 for 1.00 M. (Procedure details for individual experiments are described in the Results section.) The seeds were irradiated with e°Co y-radiation at irradiation facility the variable-dose-rate (VDRIF) of the UT-AEC Comparative Animal Research Laboratory(s) or with fission neutrons (Nr) (Experiment III) at the Health Physics Research Reactor (HPRR) of Oak Ridge National Laboratory. (1) Exposures for the y-radiation were determined by LiF thermoluminescent and radiochromic liquid dosimeters (courtesy ofDr. LEO WADE, JR., UT-AEC-CARL) . Dosimetry for the neutron irradiations was done by personnel at the HPRR, according to procedures of JOHNSON and POSTON. The doses absorbed by the seeds (in this case, O-70 of the tissue equivalent in air dose) were calculated from the energy spectrum of the reactor and the chemical composition of the seeds. Except for Experiments I and V, the seeds were soaked after irradiation in air-bubbled water at 0°C for N 18 hr. The seeds were planted in blotter sandwiches, and the seedlings were cultured 5 days before measurement as previously described.(13) The results are presented as percentage germination, seedling height (cm), and injury (percentage reduction in growth of seedlings grown from irradiated seeds compared
to that of seedlings grown from nonirradiated seeds). In Experiment II, the frequency of shoot-tip cells possessing bridges and/or fragments at the anaphase stage of the first mitotic division is also listed. Each treatment consisted of three replications of 50 seeds each for the seedling injury studies, and 300 cells per treatment were scored in Experiment II for chromosome aberrations. RESULTS Exberiment I Seeds were soaked prior to irradiation for 1 hr at 22°C in distilled water or ascorbic acid solutions ranging from 0.06 to 1 .O” M. After soaking, the seeds were rinsed and blotted dry. Half of the treatments received 20 kR of 60Co y-radiation at -2600 R/min while the other half was not irradiated. The seeds were planted after irradiation. The results immediately (Table 1) show that ascorbic acid exerted a toxic effect by reducing both seedling growth and percentage germination. The effect was concentration dependent. Soaking in I.00 M ascorbic acid reduced seedling growth to approximately the same degree as seeds receiving 20 kR of y-radiation, but only 33 per cent of the nonirradiated seeds and 43 per cent of the irradiated seeds germinated. When seedling growth of irradiated treatments was compared, little effect of ascorbic acid was found (except for the reduction at the highest concentration). However, a comparison of the irradiated treatments with the corresponding nonirradiated treatments at the same concentration showed a protective effect as measured by seedling injury. Experiment II This experiment was conducted similarly to Experiment I, except that (1) the seeds were soaked at 24°C (2) two exposures of y-radiation (20 and 25 kR) were used, and (3) following irradiation, the seeds were soaked in air-bubbled water at 0°C for N 18 hr before planting. Contrary to the results of the first experiment, ascorbic acid had little effect on seedling growth and germination of nonirradiated seeds (Table 2). Germination was also high in the irradiated seeds soaked in ascorbic acid, regardless of concentration. A protective effect, which
RADIOPROTECTIVE
EFFECTS
OF ASCORBIC
ACID
IN BARLEY
SEEDS
Table 1. Effect of ascorbic acid at d$erent concentrations on y-radiation-induced seedling injury and germination. presoakedfor 1 hrat 22°C in various concentrations of ascorbic acid, exfiosed to 20 kR of e°Co y-radiation andplanted immediately after irradiation
0 kR Ascorbic acid cone (M)
Seedling height (cm + S.E.)
41
Seeds were in blotters
20 kR
Injurya %
Seedling height (cm ? S.E.)
Germination %
In juryb %
Germination %
0
13-l
+ o-2
o-0
100
8.5
f O-2
35-1
93
O-06
12.5
+ O-2
4.6
97
7.8
+ O-2
37.6
89
o-12
12.0
c 0.2
B-4
95
8.8
f o-2
26.7
89
O-25
11.5
+ o-3
12.2
87
8.1
t 0.2
29.6
71
o-50
9.5
+ o-3
27 * 5
67
B-6 + o-3
9-5
80
l-00
8-3
c O-4
36.6
33
7-l
14-5
43
*Compared Wornpared
2 o-4
to nonirradiated, nonascorbic acid control, i.e. to 13.1 cm. to seedlings from nonirradiated, ascorbic acid treated seeds.
Table 2. Effect of axorbit acid at dxerent concentrations on y-radiation-induced seedling injtq, chromosome aberrations (percentages of cells with bridges andlorfragments at thefirst mitotic anaphase) and germination. See& were presoakedfor 1 hr at 24”Cin various concentrations of ascorbic acid, exposed to 20 or 25 kR of @oCo y-radiation and then soaked after irradiation in aerated waterfor 18 hr at 0°C 0 kR acid ccmc
height CM)
(cm
Injurya
I S.E.)
%
25
20 kR
Seedling
Ascorbic
Germination %
Seedling height (cm t S.E.)
Injuryb 1:
20
kR
Cells Germination %
Seedling height (cm t S.E.)
Injuryb z
Germination Y.
aberrations !:
0
15-l
Ia.:
o-o
97
6,7
i 0.2
55.6
89
4.6
! O*I
69.5
95
48.7
O-06
15.0
t 0.2
0.7
98
7.5
t 0.2
50.0
97
4.6
2 O*l
69.3
99
39.0
0.12
15.1
f 0.2
0.0
96
7-9
+ 0.2
47.7
9R
5.1
i 0.1
66.2
98
51.7
O-25
15.0-i
0.2
0.7
98
8.8
1 0.2
41.3
95
6.3
? 0.1
58.0
99
so*0
0.50
14.7
i 0.2
2.7
95
9.8
t 0.2
33-3
98
6.7
1 0.2
54.1.
91
39.0
1.00
14.8
t O-2
2.0
95
9.8
i 0.2
33.8
94
7.7
f 0.2
4i.6
94
38.0
“Compared bCompared
to nonirradiated, nonascorbic acid control, i.e. to 15-I cm. to seedlings from nonirradiated, ascorbic acid treated seeds.
kR
with
chromosome
B. V. CONGER
42
was concentration dependent, resulted from the I-hr soak in ascorbic acid. In the 20-kR treatments, the mean height of seedlings from seeds soaked only in distilled water was 6.7 cm and the injury level was 55.6 per cent. For seeds soaked in I*00 M ascorbic acid, these values were 9.8 cm and 33.8 per cent, respectively. A more than 20 per cent decrease in injury was also observed for seeds exposed to 25 kR. The protection, however, was less clear for induction of chromosome aberrations in shoot-tips as measured by the percentage of cells with bridges and/or fragments at the first mitotic anaphase. Approximately 40 to 50 per cent of the cells had aberrations, regardless of ascorbic acid concentration. Exberiment
III
This experiment was designed to test the protective effects of ascorbic acid on neutron radiation-induced damage. Seeds were soaked in distilled water or ascorbic acid solutions of 0.12, O-50, or 1.00 M for 1 hr at 24°C. The seeds were blotted dry and irradiated with 276, 412, or 552 rads of unmoderated fission neutrons. After irradiation, the seeds were soaked in aerated water at 0°C for N 18 hr and planted in blotter sandwiches. Again, ascorbic acid had little influence on seedling growth of nonirradiated seeds or the germination of either nonirradiated or irradiated seeds. Thus, injury values only for the three neutron doses are given (Table 3). Increasing
the concentration of ascorbic acid decreased injury at all neutron doses, but the total effect was not as great as for y-radiation. The decrease in injury for seeds soaked in 1 .OO M ascorbic acid vs those soaked in distilled water was approximately 8 to 10 per cent. Experiment
IV
This experiment was conducted to determine if the radioprotective effect was at least partly caused by reduced hydration of the seeds presoaked in ascorbic acid. A comparable batch of seeds was presoaked in a range of molar concentrations of CaCl, identical to those used for ascorbic acid. The water content of both whole seeds and embryos was measured after 1 hr of soaking at 22°C in water and in each of the three molar concentrations of the two solutions. Seed water content was determined by grinding whole seeds (20-mesh screen) and drying the ground sample in an air oven at 130°C for 1 hr. Embryo water contents were determined by excising the embryos, weighing, and drying. The water contents reported are percentages of the wet weights. These data, along with seedling height and injury data for nonirradiated seeds and seeds exposed to 20 kR, are presented in Table 4. Soaking in either ascorbic acid or CaCl, reduced the water uptake of whole seeds (and especially embryos) as compared with seeds soaked in water. The effect was concentration
Table 3. Efect of ascorbic acid at d@rent concen&ations on&ion-net&on-induced seedling ascorbic acid solutions for 1 hr al 24”C, irradiated with&ion neutrons, and then soaked afkr aerated water
Ascorbic acid cone (M)
0
Per 276
rads
lO*O
cent injury 412 rads
injury. See& were presoaked in irradiation for 18 hr al 0°C in
552
rads
22*7
62* 1 59-2
0.12
4-7
16.9
o-50
3-7
li-
1.00
3.4
12.8
a
57-5 54.6
RADIOPROTECTIVE Table
4. Embryo
and whole seed water I hr at 22°C.
CaCI, or ascorbic acidfor
Trt
(M)
Water
EFFECTS
Seed water content %
contents Seedling
OF ASCORBIC
ACID
IN BARLEY
SEEDS
43
of Himalaya
barley seeds soaked in water or d$erent molar concentrations of height and injury valuesfor nonirradiated seeds and seeds receiving 20 kR are also given
Embryo water content %
0 kR Seedling height Injury (cm + S.E.) %
20 kR Seedling height Injury” (cm + S.E.) %
19.9
47-l
14-l
* 0.1
o-0
4.4
F o-1
68.8
0.06
18-l
45-o
14.3
!I O-1
O-0
5.4
2 o-1
62.2
0.25
17.7
39.8
13.5
2 0.1
o-0
6-l
f O-1
54.8
l-00
15.9
31.9
13-3
+ 0.1
o-0
7.2
f O-1
45.9
0.06
19.5
45-3
13.3
I! 0.1
o-0
5.1
+ o-1
61.6
0.25
17.6
41-7
13-S
? o-1
0.0
6.5
+ 0.1
52.9
1.00
18.6
33-l
13.4
+ 0.1
o-0
7.1
+ o-2
47.0
CaC12
Ascorbic acid
acompared
to seedlings from nonirradiated,
CaCl, or ascorbic acid treated seeds.
dependent, as was the reduction in seedling injury, following 20 kR of Wo y-radiation. The reduction was about the same for seeds soaked in the two kinds of solutions. Heights of seedlings
grown from seedsexposed to 20 kR were less than reported for the other experiments because of a 2-hr interval between the end of the soaking period and irradiation. ExperimentV The previous experiments involved relatively high concentrations of ascorbic acid and soaking periods of 1 hr in nonbuffered solutions prior to irradiation. In this experiment, soaking periods of 2 or 16 hr at 25°C were used; the ascorbic acid concentration was reduced to 0.01 M; and the solutions were buffered with O-1Mphosphate at pH 3 or pH 7 and bubbled with nitrogen gas.
After soaking, the seedswere briefly rinsed in running tap water. The radiation exposures were 20 and 3 kR for seedssoaked 2 and 16 hr, respectively, and the seeds were planted in blotters immediately after irradiation. Seedling height and germination were reduced -30 per cent for nonirradiated seedssoakedfor 16 hr at pH 3. The reduction was the samefor both the 0 and 0.01 M ascorbic acid solutions. There was no reduction in seedling height or germination of nonirradiated seedsof the other treatment combinations (data not shown). Seedling injury values for the irradiated treatments (Table 5) showed no protective effect of ascorbic acid for either soaking period at either pH. In fact, there appeared to be a detrimental effect of ascorbic acid for the 164~ presoak period, especially at pH 7 where the seedling
B. V. CONGER
44 Table 5. Seedling injury (%) buffs solutions at pH 3 or pH
of Himalaya barley seea? soakedfor 2 or 16 hr at 25°C in nitrogen-bubbled 7 wilh 0 or WI1 M ascorbic acid before exposure to 20 kR or 3 kR of Wo 2 hr, 20 kR Ascorbic acid cone, M 0 O*Ol
0.1 M phosphale gamma radiation
16 hr, Ascorbic 0
3 kR
acid cone, M o-01
PB 3
48.5
48-O
38-3
42-l
PB 7
48-3
49*_8
45-5
56-7
injury was 45.5 per cent for seedsnot soaked in if a postirradiation treatment with ascorbic acid ascorbic acid and 56.7 per cent for seedssoaked would reduce oxygen-dependent damage in very in O-01 M ascorbic acid. dry seeds.Thus, the experiment was conducted with seedsof 1.5 per cent water content. The seedswere exposed (in UUGUU) to three levels of ExperimentVI s°Co y-radiation and then soaked in nitrogenSince one of the hypothesesfor the mechanism or oxygen-bubbled water or 0.5 ascorbic acid of action of ascorbic acid is that of a free radical for N 18 hr after irradiation. A much lower scavenger, this experiment was designed to test exposure serieswas usedfor seedsto be soakedin the influence of soaking in oxygen- vs nitrogenoxygen-bubbled water or ascorbic acid because bubbled distilled water or ascorbic acid solutions of the high degree of oxygen enhancement of of O-12, O-50, or 1.00 M at 24°C for 1 hr prior to damage in very dry seeds.(s) irradiation. The seedswere rinsed, blotted dry, Results of this experiment (Table 7) show and exposed to either 15 kR of Wo y-radiation germination was N 13 and 16 per cent lessfor or not irradiated. The seedswhich had been seeds soaked in nitrogen- or oxygen-bubbled presoaked in oxygen-bubbled solutions were O-5 M ascorbic acid, respectively, than for seeds then soaked after irradiation in oxygen-bubbled soaked in nitrogen- or oxygen-bubbled water. water at 0°C for 18 hr; those presoaked in The germination of nonirradiated seedswas not nitrogen-bubbled solutions were soaked likewise as good as in the other experiments, but it was in nitrogen-bubbled water after irradiation. still more than 90 per cent in all but one instance, The resultsshowed little difference in seedling where it was 85 per cent. The reduced germinaheights (Table 6) among the nonirradiated tion may be partly due to the very low water treatments, regardless of whether they were content of the seeds. soaked in oxygen- or nitrogen-bubbled solutions There was no difference in seedling growth of of ascorbic acid. Germination also was not seedssoaked in nitrogen-bubbled O-5 M ascorbic affected (data not shown). There was a protective acid as compared with those soaked in nitrogeneffect of about the samemagnitude with increasbubbled water. The seedling heights and injury ing ascorbic acid concentration for seedssoaked levels were approximately the same for each of in either oxygen- or nitrogen-bubbled ascorbic the three radiation exposures and the controls. acid solutions prior to irradiation. The damage The seedling heights were greater and the injury levels, however, were greater for seedssoaked levels lower if seeds were soaked in oxygenin oxygen-bubbled ascorbic acid at each bubbled O-5 M ascorbic acid than if they were concentration. soaked in oxygen-bubbled water. The injury levels were about 10 per cent lessfor each of the three radiation levels, and there wasno difference Ex@riment VII This experiment was conducted to determine in the controls.
RADIOPROTECTIVE
EFFECTS
OF ASCORBIC
ACID
IN BARLEY
SEEDS
45
Table 6. Effect of soaking in oxygen- or nitrogen-bubbled water or ascorbic acid solutions of di$erent concentrations firior to irradiation on radiation-induced seedling injury. See&presoaked in oxygen-bubbled solutions were postsoaked in oxygen-bubbled water at 0°C for 18 hr and those presoaked in nitrogen-bubbled solutions were postsoaked in nitrogen-bubbled water at 0°C fir 18 hr
Ascorbic acid cone (M)
OkR Seedling height (cm -I S.E.)
15 kR Seedling height (cm f S.E.)
Injury %.
02-bubbled
water
or
ascorbic
acid
Injurya %
0
13-4
f
o-2
o*o
7-2
+ O-2
46.3
0*12
13-3
+ o-1
o*o
8-O
LIZ 0.2
39.9
O-50
13.3
2 o-1
O-0
9.1
* 0.2
31.6
1.00
13.5
+ o-2
o*o
9-8
+ 0.2
27.4
N2-bubbled
water
or
ascorbic
acid
0
13-2
f
O-1
o*o
7-9
+ o-2
40*2
o-12
13*5
+ O*l
o*o
8.8
+ 0*2
34-8
0*50
13-2
+ O-2
0.0
9-6
t
O-2
27-3
l*OO
13-l
+ 0*2
* o-2
15.3
*Compared
to seedlings from nonirradiated,
-
o*o
11-l
ascorbic acid treated seeds.
DISCUSSION compounds, have been used in numerous Pulse radiolysis and associatedcellular studies radiation-protection experiments. The pro-
have indicated that the radiosensitizing and oxidative properties of many solutes are closely related. Thus, a priori, a relationship between radioprotective and reducing properties might also be expected. Model experiments have shown that ascorbic acid is an efficient protector for hydroxyl radicals and can repair radiationinduced organic radicals.(16) The fact that ascorbic acid does not protect efficiently in all systems does not invalidate the general correlation between radioprotection and the reducing phenomenon. Reducing agents, particularly sulphydryl
tective effects of reducing compounds other than sulphydryl have been studied; however, the effects of ascorbic acid have been variable. The study of this compound is certainly warranted, however, if for no other reason than its use asan alternative protective agent to sulphydryl compounds.(16) Furthermore, ascorbic acid is a naturally occurring agent, and its possible beneficial uses for other purposes have been widely publicized. The barley seed provides the opportunity of study with a dormant biological system in which, depending on the water content,
B. V. CONGER
46 Table barley
I. Effect of 0.5 M ascorbic acid on germination and seedling growth see&. The seea2 were exposed ta &j?erent levels of e°Co y-radiation bubbled water or 0.5 M ascorbic acidfor
Exposure kR
Germination %
Water Seedling height (cm + S.E.) Posttreatment,
cent wakr
conknt)
in vacua and then soaked in oqgen-
18hr
water
Himalva or nitrogen-
at 0°C
O-5 M ascorbic acid Seedling height Germination (cm k S.E.) %
Injurya %
Og-bubbled
of very dy (1.5 per
or ascorbic
In jurya 5
acid
0
85
12.3
f 0.2
0.0
79
12.4
+ 0.2
4
91
10.1
f 0.3
17-9
73
11.6
f 0.2
6
92
8-9
!I 0.2
27.6
76
10.2
-i 0.2
17-l
8
93
7.3
f 0.2
40.7
74
8*4 f 0.2
31.7
Posttreatment,
0
Np-bubbled + O-2
5.7
or ascorbic acid
0.0
79
11.6
f O-4
1.7
5-9
85
11.3
+ 0.3
4.2
77
8.2 2 O-3
30.5
81
6.2 f 0.2
47.5
96
11.8
30
91
11.1 + 0.2
45
93
8.0
t 0.3
32.2
60
91
6.1 + 0.2
48.3
*Compared to nonirradiated, nonascorbic nitrogentreatments, respectively.
water
-0.8
acid treated controls, i.e. to 12.3 and 11.8 cm for oxygen and
radiation-induced free radicals can be trapped and retained almost indefinitely.(s) Upon soaking, normal metabolic processesare initiated. The flexibility of this system should, therefore, provide some basic information relative to the effects of ascorbic acid on radiation-induced oxygen-dependent and oxygen-independent damage. Results of the first experiment showed little difference in seedling heights from seedsexposed to 20 kR of y-radiation, regardless of ascorbic acid concentration. However, ascorbic acid alone was quite toxic as evidenced by a reduction in seedling growth with increased ascorbic acid concentration in the nonirradiated seedsand decreased germination in both irradiated and nonirradiated seeds. Thus, a protective effect was apparent when the irradiated treatments were compared to the nonirradiated treatments
at the same ascorbic acid concentration. Considerable toxicity was also observed in Serratia tnarcescensunder certain conditions and was attributed to autoxidation catalyzed by metal ions.(15) Toxicity in the present experiment appeared to be causedby the residual ascorbic acid in or on the seed(probably the embryo) since a thorough rinsing of the seeds, by soaking overnight in distilled water at O”C, reduced the toxicity to almost zero. In this case, a sizable protective effect, as measured by seedling growth, was observed with increasing ascorbic acid concentration for each of the two exposures. The results for chromosome aberrations, however, were variable, and no conclusive statements can be made regarding radiation protection. The lack of a definitive protective effect is consistent with previous findings. Ascorbate also did not
RADIOPROTECTIVE
EFFECTS
OF ASCORBIC
reduce the frequency of induced mutations in barley.c3) The finding, however, of a protective effect against seedling injury is consistent with previous reports. c3sG) Since the effects of neutron irradiation on seeds are modified to a lesser extent by factors such as oxygen, seed water content, etc., than those of y-radiation, a smaller protective effect of ascorbic acid was expected. There was, however, a concentration-dependent protective effect at all three neutron doses. The protective effect, however, for both neutron and y-radiation may be primarily or even entirely due to reduced hydration of seeds soaked in ascorbic acid. Soaking seeds prior to . . . y-irradiation in an identical concentration series of a salt solution (CaCl,) gave almost identical results. Barley seeds increase in radiosensitivity as the water content increases above 14 to 16 per cent. c4p7, The increase in radiosensitivity with an increase in water content is less for neutron than for y-radiation,(7) which is also consistent with the present results. GILLET found that soaking barley seeds in increasing concentrations of CaCl, prior to irradiation gave decreasing seedling injury. The water content of the seeds also decreased. He concluded, however, that the main factor is related to the lack of leaching of natural chemical protectors in the seed when seeds are soaked in salt solutions. In the present study, the reduced hydration of seeds (and especially embryos of seeds) soaked in either ascorbic acid or CaCl, appears to be directly related to the decrease in radiosensitivity. This would indicate that the radioprotective effect is caused mainly by reduced water uptake of embryos soaked in a solution of increased osmotic potential. Thus, radioprotection may not be due to the reducing properties of ascorbic acid or lack of leaching of radioprotectants, although the possibility of either of these mechanisms playing a minor role cannot be completely ruled out. When the ascorbic acid concentration was reduced to O-01 M to minimize the reduced hydration effect and used in phosphate buffer solutions at pH 3 or pH 7, there also was no observable protective effect when seeds were soaked for either 2 or 16 hr prior to irradiation. These results are in contrast to those reported by D
ACID
IN BARLEY
SEEDS
47
SELIMBEKOVA,(~~) who observed radioprotective effects in Allium with ascorbic acid concentrations as low as 1 X 1O-5 pg/ml. Studies with rat organs indicated that ascorbic acid serves as a free radical scavenger.(lO) Also, iis action may reduce oxygen tension in the cells. The fact that ascorbic acid is effective only when present during irradiation in bacteria(i4) is consistent with the free radical hypothesis. Because of the two above hypotheses, two additional experiments were performed, Soaking seeds in oxygen-bubbled water or ascorbic acid prior to irradiation enhanced damage ~5 to 10 per cent at all concentrations as compared with soaking in nitrogen-bubbled water or ascorbic acid. A protective effect was observed in both cases, however, by increasing ascorbic acid concentration. The increased radiosensitivity of seeds soaked in oxygen-bubbled water is probably due to oxygen enhancement resulting from the interaction of free radicals with oxygen, while the protective effect of ascorbic acid is, again, probably related to reduced embryo hydration. Ascorbic acid did protect against postirradiation oxygen-dependent damage when very dry seeds (I.5 per cent water content) were soaked in 0.5 M ascorbic acid instead of distilled water. Since postirradiation oxygen enhancement is very great in barley seeds of low water content, presumably because of a high concentration of free radicals which react with the oxygen,@) these results suggest that ascorbic acid may act as a radical scavenger or in some other way interfere with the interaction of free radicals with oxygen. The lack of a difference in seedling growth of seeds soaked after irradiation in nitrogen-bubbled water vs 0.5 M ascorbic acid indicates that there was no protection against oxygen-independent damage. These results are consistent with previous observations in seeds of higher water content ( N 13 per cent) when postirradiation oxygen enhancement is minimized or eliminated. In that case ascorbic acid was not effective when applied after irradiation.@~s) In conclusion, the radioprotective effects of ascorbic acid observed when seeds are soaked prior to irradiation may be primarily or even entirely due to reduced hydration of the embryos. The postirradiation protective effect against
48
B. V. CONGER
oxygen-dependent damage in very dry seeds may be scavenging of free radicals or some other mechanism. Residual ascorbic acid either on or in the embryo is quite toxic. Acknowledgement-The technical assistance of Mr. J. V. -IA in conducting the experiments is gratefully acknowledged. REFERENCES 1. AUYCIERJ. A. (1966) The Health Physics Research Reactor. Health Physics 11,89-93. 2. BACQ Z. M. (1965) Chemical protection against ionizing radiation, Charles Thomas, Springfield, Ill., 328 p. 3. BISWA.SS. and MATSUO T. (1966) Protective and recovery effects of chemicals on plant growth, chromosome aberration and mutation in irradiated seed of crop plants. Radiation Botany 6, 575-587. 4. CALDECOTT R. S. (1955) Effects of hydration on X-ray sensitivity in Hordeurn. Radiation Res. 3, 316-330. 5. CHBKA J. S., ROBINSON E. M., WADE Lao JR. and GRAMLY W. A. (1971) The UT-AEC Agricultural Research Laboratory variable gamma dose rate facility. Health Physics 20,339-343. 6. CONGER B. V. (1973) The effects of ascorbic acid and sodium azide on seedling growth of irradiated and nonirradiated seeds of barley. Radiation Botany 13,375-379. 7. CONGER B. V. and CARASIA J. V. (1972) Modification of the effectiveness of fission neutrons versus Ylo gamma radiation in barley seeds by oxygen and seed water content. Radiation Botany 12,41 I-420. 8. CONGER B. V., NILAN R. A., KONZAK C. F. and MmT'ER S. (1966) The influence of seed water content on the oxygen effect in irradiated barley see&. Radiation Botuny 6, 129-144.
9. GILLET C. (1965) Modification of radiationinduced injury in barley by pretreatment with solutions ofdifferent osmotic potential. Nature207, 99-100. 10. GL.AWVD J. and FABER M. (1966) Antioxidants and lipoperoxides in the organs of irradiated rats. II. The influence of ionizing radiation on the amounts of the individual antioxidants in rat organs. Int. J. Radiation Biol. 11,445448. 11. JOHNSON D. R. and POSTONJ. W. (1967) Radiation dosimetry studies at the Health Physics Research Reactor. ORNL-4113, Oak Ridge National Laboratory, Oak Ridge, Tenn. U.S.A. 12. KONZAK C. F., BOTTINO P. J., NILAN R. A. and CONGER B. V. (1968) Irradiation of seeds: A review of procedures employed at Washington State University. pp. 83-96. In, .Neutron irradiation of See& II. Intern. At. Energy Agency Tech. Rept. Ser. 92, IAEA, Vienna. 13. MYHILL R. R. and KONZAIC C. F. (1967) A new technique for culturing and measuring barley seedlings. Crop Sci. 7,275-277. 14. PITTILLO R. F. and LUCAS M. B. (1967) Ascorbic acid protection of Escherichia coli against radiation lethality in the presence of tetracyclines and other radiosensitizers. Radiation Res. 31, 36-46. 15. RETDPATH J. L. and WILLSON R. L. (1973) Reducing compounds in radioprotection and radiosensitization : Model experiments using ascorbic acid. Int. 3. Radiation Biol. 23,51-65. 16. SELIMBEKOVA D. D. (1969) Radioprotective properties of ascorbic acid. Dokl. Akad. .TVauk Azerb. SSR. 25,78-80. 17. SIDERI~ E. G., NILAN R. A. and KONZAK C. F. (1969) Relationship of radiation-induced damage in barley seeds to the inhibition of certain oxidoreducatases by sodium azide. pp. 313-322. In, Induced mutations in plants. IAEA, Vienna. 18. WOLFF S. (1953) Some aspects of the chemical protection against radiation damage to Vicia faba chromosomes. Genetics 39,356-364.
Botany,
1975, Vol.
15, pp. 39 to 48. Pergamon
RADIOPROTECTIVE
Press. Printed
in Great
Britain.
EFFECTS OF ASCORBIC BARLEY SEEDS*
ACID
IN
37830,
U.S.A.
B. V. CONGER UT-AEC
Comparative
Animal
Research
Laboratory,
(Received
29
3t4
Oak
Ridge,
Tennessee
1974)
B. V. Radioprotective effects of ascorbic acid in barley seeds. RADIATION BOTANY 15, 39-48, 1975.-Experiments were conducted to test the radioprotective effects of a naturally occurring reducing agent, ascorbic acid, on seeds (caryopses) of barley, Horakum vulgare L. emend Lam. Seeds were soaked either before or after gamma or fission neutron irradiation in distilled water or ascorbic acid solutions ranging in concentration from 0.01 to 1.00 M. Results are reported as percentage germination, seedling height, seedling growth reduction, and (in one experiment) per cent of cells with chromosome aberrations. As evidenced by both reduced germination and seedling growth, ascorbic acid was toxic when seeds were soaked for 1 hr at ambient temperature prior to irradiation and then planted immediately. When seeds were soaked in ascorbic acid before irradiation and soaked after irradiation in air-bubbled water at 0°C for 18 hr, the toxicity disappeared, and a protective effect (which increased with increasing ascorbic acid concentration) was observed for gamma and, to a lesser extent, for neutron irradiation. Additional studies suggested that the protective effect was related to reduced hydration of the embryos of seeds soaked in ascorbic ‘acid. Also, no radioprotective effect was observed when seeds were presoaked for 2 or 16 hr in 0.01 M ascorbic acid solutions buffered at pH 3 or pH 7. A protective effect was observed for seeds of 1.5 per cent water content soaked after irradiation in an oxygen-bubbled ascorbic acid solution of 0.5 M but was not observed for seeds soaked in nitrogen-bubbled ascorbic acid. The protective effect against oxygen-dependent damage may be a result of interaction of ascorbic acid with radiation-induced free radicals. CONGER
THE acid
INTRODUCTION RADIOPROTECTIVE properties (a naturally
occurring
reducing
marked protection against radiation damage when present in cell suspensions containing radiosensitizing tetracycline antibiotics.(14) Ascorbate, however, protects against radiation damage in plant seeds as measured both by chromosome aberrations(ls) and seedling growth reduction.(3*s) The protective effect existed only when ascorbate was applied as a preirradiation
of ascorbic agent) have
been studied in several biological systems, and varying results have been reported. Sodium ascorbate did not protect against radiationinduced chromosome aberrations in Viciafaba. w Also, in spite of a few early favorable reports, most experiments dealing with the radioprotective effects of ascorbic acid in mammalian systems have yielded negative results. t2) Although ascorbic acid has no significant radiomodifying properties in Escherichia coli, it does provide *Research the University
sponsored by the U.S. of Tennessee.
Atomic
Energy
treatment.tsls)
Postirradiation
treatments
were
ineffective. There was no interaction between ascorbic acid and sodium azide, an agent known to enhance radiation damage in seeds,(r7) when applied to barley seeds either before or after
Commission 39
under
Contract
No.
AT-40-l-GEN-242
with
40
B.V.CONGER
gamma irradiationto) Ascorbic acid also had to be present during irradiation to protect against the radiosensitizing effect of oxytetracycline in E . coli . (14) MATERLUS AND METHODS Caryopses (hereafter referred to as seeds) of barley Hordeum vulgar-e L. emend Lam. cv. Himalaya were adjusted to a water content of N 13 per cent by storage in a desiccator over 60 per cent glycerol and 40 per cent water as previously described.02) In Experiment VII, the seeds were dried to 1.5 per cent water content by storage in a vacuum desiccator over CaSO, on the particular for -2 years. Depending experiment, seeds were soaked either prior to or after irradiation in distilled water or different concentrations of ascorbic acid ranging from O-01 M to I*00 M. Except for Experiment V, the pH of the solutions was not adjusted and was measured to be from 2-7 for O-06 M to 2-2 for 1.00 M. (Procedure details for individual experiments are described in the Results section.) The seeds were irradiated with e°Co y-radiation at irradiation facility the variable-dose-rate (VDRIF) of the UT-AEC Comparative Animal Research Laboratory(s) or with fission neutrons (Nr) (Experiment III) at the Health Physics Research Reactor (HPRR) of Oak Ridge National Laboratory. (1) Exposures for the y-radiation were determined by LiF thermoluminescent and radiochromic liquid dosimeters (courtesy ofDr. LEO WADE, JR., UT-AEC-CARL) . Dosimetry for the neutron irradiations was done by personnel at the HPRR, according to procedures of JOHNSON and POSTON. The doses absorbed by the seeds (in this case, O-70 of the tissue equivalent in air dose) were calculated from the energy spectrum of the reactor and the chemical composition of the seeds. Except for Experiments I and V, the seeds were soaked after irradiation in air-bubbled water at 0°C for N 18 hr. The seeds were planted in blotter sandwiches, and the seedlings were cultured 5 days before measurement as previously described.(13) The results are presented as percentage germination, seedling height (cm), and injury (percentage reduction in growth of seedlings grown from irradiated seeds compared
to that of seedlings grown from nonirradiated seeds). In Experiment II, the frequency of shoot-tip cells possessing bridges and/or fragments at the anaphase stage of the first mitotic division is also listed. Each treatment consisted of three replications of 50 seeds each for the seedling injury studies, and 300 cells per treatment were scored in Experiment II for chromosome aberrations. RESULTS Exberiment I Seeds were soaked prior to irradiation for 1 hr at 22°C in distilled water or ascorbic acid solutions ranging from 0.06 to 1 .O” M. After soaking, the seeds were rinsed and blotted dry. Half of the treatments received 20 kR of 60Co y-radiation at -2600 R/min while the other half was not irradiated. The seeds were planted after irradiation. The results immediately (Table 1) show that ascorbic acid exerted a toxic effect by reducing both seedling growth and percentage germination. The effect was concentration dependent. Soaking in I.00 M ascorbic acid reduced seedling growth to approximately the same degree as seeds receiving 20 kR of y-radiation, but only 33 per cent of the nonirradiated seeds and 43 per cent of the irradiated seeds germinated. When seedling growth of irradiated treatments was compared, little effect of ascorbic acid was found (except for the reduction at the highest concentration). However, a comparison of the irradiated treatments with the corresponding nonirradiated treatments at the same concentration showed a protective effect as measured by seedling injury. Experiment II This experiment was conducted similarly to Experiment I, except that (1) the seeds were soaked at 24°C (2) two exposures of y-radiation (20 and 25 kR) were used, and (3) following irradiation, the seeds were soaked in air-bubbled water at 0°C for N 18 hr before planting. Contrary to the results of the first experiment, ascorbic acid had little effect on seedling growth and germination of nonirradiated seeds (Table 2). Germination was also high in the irradiated seeds soaked in ascorbic acid, regardless of concentration. A protective effect, which
RADIOPROTECTIVE
EFFECTS
OF ASCORBIC
ACID
IN BARLEY
SEEDS
Table 1. Effect of ascorbic acid at d$erent concentrations on y-radiation-induced seedling injury and germination. presoakedfor 1 hrat 22°C in various concentrations of ascorbic acid, exfiosed to 20 kR of e°Co y-radiation andplanted immediately after irradiation
0 kR Ascorbic acid cone (M)
Seedling height (cm + S.E.)
41
Seeds were in blotters
20 kR
Injurya %
Seedling height (cm ? S.E.)
Germination %
In juryb %
Germination %
0
13-l
+ o-2
o-0
100
8.5
f O-2
35-1
93
O-06
12.5
+ O-2
4.6
97
7.8
+ O-2
37.6
89
o-12
12.0
c 0.2
B-4
95
8.8
f o-2
26.7
89
O-25
11.5
+ o-3
12.2
87
8.1
t 0.2
29.6
71
o-50
9.5
+ o-3
27 * 5
67
B-6 + o-3
9-5
80
l-00
8-3
c O-4
36.6
33
7-l
14-5
43
*Compared Wornpared
2 o-4
to nonirradiated, nonascorbic acid control, i.e. to 13.1 cm. to seedlings from nonirradiated, ascorbic acid treated seeds.
Table 2. Effect of axorbit acid at dxerent concentrations on y-radiation-induced seedling injtq, chromosome aberrations (percentages of cells with bridges andlorfragments at thefirst mitotic anaphase) and germination. See& were presoakedfor 1 hr at 24”Cin various concentrations of ascorbic acid, exposed to 20 or 25 kR of @oCo y-radiation and then soaked after irradiation in aerated waterfor 18 hr at 0°C 0 kR acid ccmc
height CM)
(cm
Injurya
I S.E.)
%
25
20 kR
Seedling
Ascorbic
Germination %
Seedling height (cm t S.E.)
Injuryb 1:
20
kR
Cells Germination %
Seedling height (cm t S.E.)
Injuryb z
Germination Y.
aberrations !:
0
15-l
Ia.:
o-o
97
6,7
i 0.2
55.6
89
4.6
! O*I
69.5
95
48.7
O-06
15.0
t 0.2
0.7
98
7.5
t 0.2
50.0
97
4.6
2 O*l
69.3
99
39.0
0.12
15.1
f 0.2
0.0
96
7-9
+ 0.2
47.7
9R
5.1
i 0.1
66.2
98
51.7
O-25
15.0-i
0.2
0.7
98
8.8
1 0.2
41.3
95
6.3
? 0.1
58.0
99
so*0
0.50
14.7
i 0.2
2.7
95
9.8
t 0.2
33-3
98
6.7
1 0.2
54.1.
91
39.0
1.00
14.8
t O-2
2.0
95
9.8
i 0.2
33.8
94
7.7
f 0.2
4i.6
94
38.0
“Compared bCompared
to nonirradiated, nonascorbic acid control, i.e. to 15-I cm. to seedlings from nonirradiated, ascorbic acid treated seeds.
kR
with
chromosome
B. V. CONGER
42
was concentration dependent, resulted from the I-hr soak in ascorbic acid. In the 20-kR treatments, the mean height of seedlings from seeds soaked only in distilled water was 6.7 cm and the injury level was 55.6 per cent. For seeds soaked in I*00 M ascorbic acid, these values were 9.8 cm and 33.8 per cent, respectively. A more than 20 per cent decrease in injury was also observed for seeds exposed to 25 kR. The protection, however, was less clear for induction of chromosome aberrations in shoot-tips as measured by the percentage of cells with bridges and/or fragments at the first mitotic anaphase. Approximately 40 to 50 per cent of the cells had aberrations, regardless of ascorbic acid concentration. Exberiment
III
This experiment was designed to test the protective effects of ascorbic acid on neutron radiation-induced damage. Seeds were soaked in distilled water or ascorbic acid solutions of 0.12, O-50, or 1.00 M for 1 hr at 24°C. The seeds were blotted dry and irradiated with 276, 412, or 552 rads of unmoderated fission neutrons. After irradiation, the seeds were soaked in aerated water at 0°C for N 18 hr and planted in blotter sandwiches. Again, ascorbic acid had little influence on seedling growth of nonirradiated seeds or the germination of either nonirradiated or irradiated seeds. Thus, injury values only for the three neutron doses are given (Table 3). Increasing
the concentration of ascorbic acid decreased injury at all neutron doses, but the total effect was not as great as for y-radiation. The decrease in injury for seeds soaked in 1 .OO M ascorbic acid vs those soaked in distilled water was approximately 8 to 10 per cent. Experiment
IV
This experiment was conducted to determine if the radioprotective effect was at least partly caused by reduced hydration of the seeds presoaked in ascorbic acid. A comparable batch of seeds was presoaked in a range of molar concentrations of CaCl, identical to those used for ascorbic acid. The water content of both whole seeds and embryos was measured after 1 hr of soaking at 22°C in water and in each of the three molar concentrations of the two solutions. Seed water content was determined by grinding whole seeds (20-mesh screen) and drying the ground sample in an air oven at 130°C for 1 hr. Embryo water contents were determined by excising the embryos, weighing, and drying. The water contents reported are percentages of the wet weights. These data, along with seedling height and injury data for nonirradiated seeds and seeds exposed to 20 kR, are presented in Table 4. Soaking in either ascorbic acid or CaCl, reduced the water uptake of whole seeds (and especially embryos) as compared with seeds soaked in water. The effect was concentration
Table 3. Efect of ascorbic acid at d@rent concen&ations on&ion-net&on-induced seedling ascorbic acid solutions for 1 hr al 24”C, irradiated with&ion neutrons, and then soaked afkr aerated water
Ascorbic acid cone (M)
0
Per 276
rads
lO*O
cent injury 412 rads
injury. See& were presoaked in irradiation for 18 hr al 0°C in
552
rads
22*7
62* 1 59-2
0.12
4-7
16.9
o-50
3-7
li-
1.00
3.4
12.8
a
57-5 54.6
RADIOPROTECTIVE Table
4. Embryo
and whole seed water I hr at 22°C.
CaCI, or ascorbic acidfor
Trt
(M)
Water
EFFECTS
Seed water content %
contents Seedling
OF ASCORBIC
ACID
IN BARLEY
SEEDS
43
of Himalaya
barley seeds soaked in water or d$erent molar concentrations of height and injury valuesfor nonirradiated seeds and seeds receiving 20 kR are also given
Embryo water content %
0 kR Seedling height Injury (cm + S.E.) %
20 kR Seedling height Injury” (cm + S.E.) %
19.9
47-l
14-l
* 0.1
o-0
4.4
F o-1
68.8
0.06
18-l
45-o
14.3
!I O-1
O-0
5.4
2 o-1
62.2
0.25
17.7
39.8
13.5
2 0.1
o-0
6-l
f O-1
54.8
l-00
15.9
31.9
13-3
+ 0.1
o-0
7.2
f O-1
45.9
0.06
19.5
45-3
13.3
I! 0.1
o-0
5.1
+ o-1
61.6
0.25
17.6
41-7
13-S
? o-1
0.0
6.5
+ 0.1
52.9
1.00
18.6
33-l
13.4
+ 0.1
o-0
7.1
+ o-2
47.0
CaC12
Ascorbic acid
acompared
to seedlings from nonirradiated,
CaCl, or ascorbic acid treated seeds.
dependent, as was the reduction in seedling injury, following 20 kR of Wo y-radiation. The reduction was about the same for seeds soaked in the two kinds of solutions. Heights of seedlings
grown from seedsexposed to 20 kR were less than reported for the other experiments because of a 2-hr interval between the end of the soaking period and irradiation. ExperimentV The previous experiments involved relatively high concentrations of ascorbic acid and soaking periods of 1 hr in nonbuffered solutions prior to irradiation. In this experiment, soaking periods of 2 or 16 hr at 25°C were used; the ascorbic acid concentration was reduced to 0.01 M; and the solutions were buffered with O-1Mphosphate at pH 3 or pH 7 and bubbled with nitrogen gas.
After soaking, the seedswere briefly rinsed in running tap water. The radiation exposures were 20 and 3 kR for seedssoaked 2 and 16 hr, respectively, and the seeds were planted in blotters immediately after irradiation. Seedling height and germination were reduced -30 per cent for nonirradiated seedssoakedfor 16 hr at pH 3. The reduction was the samefor both the 0 and 0.01 M ascorbic acid solutions. There was no reduction in seedling height or germination of nonirradiated seedsof the other treatment combinations (data not shown). Seedling injury values for the irradiated treatments (Table 5) showed no protective effect of ascorbic acid for either soaking period at either pH. In fact, there appeared to be a detrimental effect of ascorbic acid for the 164~ presoak period, especially at pH 7 where the seedling
B. V. CONGER
44 Table 5. Seedling injury (%) buffs solutions at pH 3 or pH
of Himalaya barley seea? soakedfor 2 or 16 hr at 25°C in nitrogen-bubbled 7 wilh 0 or WI1 M ascorbic acid before exposure to 20 kR or 3 kR of Wo 2 hr, 20 kR Ascorbic acid cone, M 0 O*Ol
0.1 M phosphale gamma radiation
16 hr, Ascorbic 0
3 kR
acid cone, M o-01
PB 3
48.5
48-O
38-3
42-l
PB 7
48-3
49*_8
45-5
56-7
injury was 45.5 per cent for seedsnot soaked in if a postirradiation treatment with ascorbic acid ascorbic acid and 56.7 per cent for seedssoaked would reduce oxygen-dependent damage in very in O-01 M ascorbic acid. dry seeds.Thus, the experiment was conducted with seedsof 1.5 per cent water content. The seedswere exposed (in UUGUU) to three levels of ExperimentVI s°Co y-radiation and then soaked in nitrogenSince one of the hypothesesfor the mechanism or oxygen-bubbled water or 0.5 ascorbic acid of action of ascorbic acid is that of a free radical for N 18 hr after irradiation. A much lower scavenger, this experiment was designed to test exposure serieswas usedfor seedsto be soakedin the influence of soaking in oxygen- vs nitrogenoxygen-bubbled water or ascorbic acid because bubbled distilled water or ascorbic acid solutions of the high degree of oxygen enhancement of of O-12, O-50, or 1.00 M at 24°C for 1 hr prior to damage in very dry seeds.(s) irradiation. The seedswere rinsed, blotted dry, Results of this experiment (Table 7) show and exposed to either 15 kR of Wo y-radiation germination was N 13 and 16 per cent lessfor or not irradiated. The seedswhich had been seeds soaked in nitrogen- or oxygen-bubbled presoaked in oxygen-bubbled solutions were O-5 M ascorbic acid, respectively, than for seeds then soaked after irradiation in oxygen-bubbled soaked in nitrogen- or oxygen-bubbled water. water at 0°C for 18 hr; those presoaked in The germination of nonirradiated seedswas not nitrogen-bubbled solutions were soaked likewise as good as in the other experiments, but it was in nitrogen-bubbled water after irradiation. still more than 90 per cent in all but one instance, The resultsshowed little difference in seedling where it was 85 per cent. The reduced germinaheights (Table 6) among the nonirradiated tion may be partly due to the very low water treatments, regardless of whether they were content of the seeds. soaked in oxygen- or nitrogen-bubbled solutions There was no difference in seedling growth of of ascorbic acid. Germination also was not seedssoaked in nitrogen-bubbled O-5 M ascorbic affected (data not shown). There was a protective acid as compared with those soaked in nitrogeneffect of about the samemagnitude with increasbubbled water. The seedling heights and injury ing ascorbic acid concentration for seedssoaked levels were approximately the same for each of in either oxygen- or nitrogen-bubbled ascorbic the three radiation exposures and the controls. acid solutions prior to irradiation. The damage The seedling heights were greater and the injury levels, however, were greater for seedssoaked levels lower if seeds were soaked in oxygenin oxygen-bubbled ascorbic acid at each bubbled O-5 M ascorbic acid than if they were concentration. soaked in oxygen-bubbled water. The injury levels were about 10 per cent lessfor each of the three radiation levels, and there wasno difference Ex@riment VII This experiment was conducted to determine in the controls.
RADIOPROTECTIVE
EFFECTS
OF ASCORBIC
ACID
IN BARLEY
SEEDS
45
Table 6. Effect of soaking in oxygen- or nitrogen-bubbled water or ascorbic acid solutions of di$erent concentrations firior to irradiation on radiation-induced seedling injury. See&presoaked in oxygen-bubbled solutions were postsoaked in oxygen-bubbled water at 0°C for 18 hr and those presoaked in nitrogen-bubbled solutions were postsoaked in nitrogen-bubbled water at 0°C fir 18 hr
Ascorbic acid cone (M)
OkR Seedling height (cm -I S.E.)
15 kR Seedling height (cm f S.E.)
Injury %.
02-bubbled
water
or
ascorbic
acid
Injurya %
0
13-4
f
o-2
o*o
7-2
+ O-2
46.3
0*12
13-3
+ o-1
o*o
8-O
LIZ 0.2
39.9
O-50
13.3
2 o-1
O-0
9.1
* 0.2
31.6
1.00
13.5
+ o-2
o*o
9-8
+ 0.2
27.4
N2-bubbled
water
or
ascorbic
acid
0
13-2
f
O-1
o*o
7-9
+ o-2
40*2
o-12
13*5
+ O*l
o*o
8.8
+ 0*2
34-8
0*50
13-2
+ O-2
0.0
9-6
t
O-2
27-3
l*OO
13-l
+ 0*2
* o-2
15.3
*Compared
to seedlings from nonirradiated,
-
o*o
11-l
ascorbic acid treated seeds.
DISCUSSION compounds, have been used in numerous Pulse radiolysis and associatedcellular studies radiation-protection experiments. The pro-
have indicated that the radiosensitizing and oxidative properties of many solutes are closely related. Thus, a priori, a relationship between radioprotective and reducing properties might also be expected. Model experiments have shown that ascorbic acid is an efficient protector for hydroxyl radicals and can repair radiationinduced organic radicals.(16) The fact that ascorbic acid does not protect efficiently in all systems does not invalidate the general correlation between radioprotection and the reducing phenomenon. Reducing agents, particularly sulphydryl
tective effects of reducing compounds other than sulphydryl have been studied; however, the effects of ascorbic acid have been variable. The study of this compound is certainly warranted, however, if for no other reason than its use asan alternative protective agent to sulphydryl compounds.(16) Furthermore, ascorbic acid is a naturally occurring agent, and its possible beneficial uses for other purposes have been widely publicized. The barley seed provides the opportunity of study with a dormant biological system in which, depending on the water content,
B. V. CONGER
46 Table barley
I. Effect of 0.5 M ascorbic acid on germination and seedling growth see&. The seea2 were exposed ta &j?erent levels of e°Co y-radiation bubbled water or 0.5 M ascorbic acidfor
Exposure kR
Germination %
Water Seedling height (cm + S.E.) Posttreatment,
cent wakr
conknt)
in vacua and then soaked in oqgen-
18hr
water
Himalva or nitrogen-
at 0°C
O-5 M ascorbic acid Seedling height Germination (cm k S.E.) %
Injurya %
Og-bubbled
of very dy (1.5 per
or ascorbic
In jurya 5
acid
0
85
12.3
f 0.2
0.0
79
12.4
+ 0.2
4
91
10.1
f 0.3
17-9
73
11.6
f 0.2
6
92
8-9
!I 0.2
27.6
76
10.2
-i 0.2
17-l
8
93
7.3
f 0.2
40.7
74
8*4 f 0.2
31.7
Posttreatment,
0
Np-bubbled + O-2
5.7
or ascorbic acid
0.0
79
11.6
f O-4
1.7
5-9
85
11.3
+ 0.3
4.2
77
8.2 2 O-3
30.5
81
6.2 f 0.2
47.5
96
11.8
30
91
11.1 + 0.2
45
93
8.0
t 0.3
32.2
60
91
6.1 + 0.2
48.3
*Compared to nonirradiated, nonascorbic nitrogentreatments, respectively.
water
-0.8
acid treated controls, i.e. to 12.3 and 11.8 cm for oxygen and
radiation-induced free radicals can be trapped and retained almost indefinitely.(s) Upon soaking, normal metabolic processesare initiated. The flexibility of this system should, therefore, provide some basic information relative to the effects of ascorbic acid on radiation-induced oxygen-dependent and oxygen-independent damage. Results of the first experiment showed little difference in seedling heights from seedsexposed to 20 kR of y-radiation, regardless of ascorbic acid concentration. However, ascorbic acid alone was quite toxic as evidenced by a reduction in seedling growth with increased ascorbic acid concentration in the nonirradiated seedsand decreased germination in both irradiated and nonirradiated seeds. Thus, a protective effect was apparent when the irradiated treatments were compared to the nonirradiated treatments
at the same ascorbic acid concentration. Considerable toxicity was also observed in Serratia tnarcescensunder certain conditions and was attributed to autoxidation catalyzed by metal ions.(15) Toxicity in the present experiment appeared to be causedby the residual ascorbic acid in or on the seed(probably the embryo) since a thorough rinsing of the seeds, by soaking overnight in distilled water at O”C, reduced the toxicity to almost zero. In this case, a sizable protective effect, as measured by seedling growth, was observed with increasing ascorbic acid concentration for each of the two exposures. The results for chromosome aberrations, however, were variable, and no conclusive statements can be made regarding radiation protection. The lack of a definitive protective effect is consistent with previous findings. Ascorbate also did not
RADIOPROTECTIVE
EFFECTS
OF ASCORBIC
reduce the frequency of induced mutations in barley.c3) The finding, however, of a protective effect against seedling injury is consistent with previous reports. c3sG) Since the effects of neutron irradiation on seeds are modified to a lesser extent by factors such as oxygen, seed water content, etc., than those of y-radiation, a smaller protective effect of ascorbic acid was expected. There was, however, a concentration-dependent protective effect at all three neutron doses. The protective effect, however, for both neutron and y-radiation may be primarily or even entirely due to reduced hydration of seeds soaked in ascorbic acid. Soaking seeds prior to . . . y-irradiation in an identical concentration series of a salt solution (CaCl,) gave almost identical results. Barley seeds increase in radiosensitivity as the water content increases above 14 to 16 per cent. c4p7, The increase in radiosensitivity with an increase in water content is less for neutron than for y-radiation,(7) which is also consistent with the present results. GILLET found that soaking barley seeds in increasing concentrations of CaCl, prior to irradiation gave decreasing seedling injury. The water content of the seeds also decreased. He concluded, however, that the main factor is related to the lack of leaching of natural chemical protectors in the seed when seeds are soaked in salt solutions. In the present study, the reduced hydration of seeds (and especially embryos of seeds) soaked in either ascorbic acid or CaCl, appears to be directly related to the decrease in radiosensitivity. This would indicate that the radioprotective effect is caused mainly by reduced water uptake of embryos soaked in a solution of increased osmotic potential. Thus, radioprotection may not be due to the reducing properties of ascorbic acid or lack of leaching of radioprotectants, although the possibility of either of these mechanisms playing a minor role cannot be completely ruled out. When the ascorbic acid concentration was reduced to O-01 M to minimize the reduced hydration effect and used in phosphate buffer solutions at pH 3 or pH 7, there also was no observable protective effect when seeds were soaked for either 2 or 16 hr prior to irradiation. These results are in contrast to those reported by D
ACID
IN BARLEY
SEEDS
47
SELIMBEKOVA,(~~) who observed radioprotective effects in Allium with ascorbic acid concentrations as low as 1 X 1O-5 pg/ml. Studies with rat organs indicated that ascorbic acid serves as a free radical scavenger.(lO) Also, iis action may reduce oxygen tension in the cells. The fact that ascorbic acid is effective only when present during irradiation in bacteria(i4) is consistent with the free radical hypothesis. Because of the two above hypotheses, two additional experiments were performed, Soaking seeds in oxygen-bubbled water or ascorbic acid prior to irradiation enhanced damage ~5 to 10 per cent at all concentrations as compared with soaking in nitrogen-bubbled water or ascorbic acid. A protective effect was observed in both cases, however, by increasing ascorbic acid concentration. The increased radiosensitivity of seeds soaked in oxygen-bubbled water is probably due to oxygen enhancement resulting from the interaction of free radicals with oxygen, while the protective effect of ascorbic acid is, again, probably related to reduced embryo hydration. Ascorbic acid did protect against postirradiation oxygen-dependent damage when very dry seeds (I.5 per cent water content) were soaked in 0.5 M ascorbic acid instead of distilled water. Since postirradiation oxygen enhancement is very great in barley seeds of low water content, presumably because of a high concentration of free radicals which react with the oxygen,@) these results suggest that ascorbic acid may act as a radical scavenger or in some other way interfere with the interaction of free radicals with oxygen. The lack of a difference in seedling growth of seeds soaked after irradiation in nitrogen-bubbled water vs 0.5 M ascorbic acid indicates that there was no protection against oxygen-independent damage. These results are consistent with previous observations in seeds of higher water content ( N 13 per cent) when postirradiation oxygen enhancement is minimized or eliminated. In that case ascorbic acid was not effective when applied after irradiation.@~s) In conclusion, the radioprotective effects of ascorbic acid observed when seeds are soaked prior to irradiation may be primarily or even entirely due to reduced hydration of the embryos. The postirradiation protective effect against
48
B. V. CONGER
oxygen-dependent damage in very dry seeds may be scavenging of free radicals or some other mechanism. Residual ascorbic acid either on or in the embryo is quite toxic. Acknowledgement-The technical assistance of Mr. J. V. -IA in conducting the experiments is gratefully acknowledged. REFERENCES 1. AUYCIERJ. A. (1966) The Health Physics Research Reactor. Health Physics 11,89-93. 2. BACQ Z. M. (1965) Chemical protection against ionizing radiation, Charles Thomas, Springfield, Ill., 328 p. 3. BISWA.SS. and MATSUO T. (1966) Protective and recovery effects of chemicals on plant growth, chromosome aberration and mutation in irradiated seed of crop plants. Radiation Botany 6, 575-587. 4. CALDECOTT R. S. (1955) Effects of hydration on X-ray sensitivity in Hordeurn. Radiation Res. 3, 316-330. 5. CHBKA J. S., ROBINSON E. M., WADE Lao JR. and GRAMLY W. A. (1971) The UT-AEC Agricultural Research Laboratory variable gamma dose rate facility. Health Physics 20,339-343. 6. CONGER B. V. (1973) The effects of ascorbic acid and sodium azide on seedling growth of irradiated and nonirradiated seeds of barley. Radiation Botany 13,375-379. 7. CONGER B. V. and CARASIA J. V. (1972) Modification of the effectiveness of fission neutrons versus Ylo gamma radiation in barley seeds by oxygen and seed water content. Radiation Botany 12,41 I-420. 8. CONGER B. V., NILAN R. A., KONZAK C. F. and MmT'ER S. (1966) The influence of seed water content on the oxygen effect in irradiated barley see&. Radiation Botuny 6, 129-144.
9. GILLET C. (1965) Modification of radiationinduced injury in barley by pretreatment with solutions ofdifferent osmotic potential. Nature207, 99-100. 10. GL.AWVD J. and FABER M. (1966) Antioxidants and lipoperoxides in the organs of irradiated rats. II. The influence of ionizing radiation on the amounts of the individual antioxidants in rat organs. Int. J. Radiation Biol. 11,445448. 11. JOHNSON D. R. and POSTONJ. W. (1967) Radiation dosimetry studies at the Health Physics Research Reactor. ORNL-4113, Oak Ridge National Laboratory, Oak Ridge, Tenn. U.S.A. 12. KONZAK C. F., BOTTINO P. J., NILAN R. A. and CONGER B. V. (1968) Irradiation of seeds: A review of procedures employed at Washington State University. pp. 83-96. In, .Neutron irradiation of See& II. Intern. At. Energy Agency Tech. Rept. Ser. 92, IAEA, Vienna. 13. MYHILL R. R. and KONZAIC C. F. (1967) A new technique for culturing and measuring barley seedlings. Crop Sci. 7,275-277. 14. PITTILLO R. F. and LUCAS M. B. (1967) Ascorbic acid protection of Escherichia coli against radiation lethality in the presence of tetracyclines and other radiosensitizers. Radiation Res. 31, 36-46. 15. RETDPATH J. L. and WILLSON R. L. (1973) Reducing compounds in radioprotection and radiosensitization : Model experiments using ascorbic acid. Int. 3. Radiation Biol. 23,51-65. 16. SELIMBEKOVA D. D. (1969) Radioprotective properties of ascorbic acid. Dokl. Akad. .TVauk Azerb. SSR. 25,78-80. 17. SIDERI~ E. G., NILAN R. A. and KONZAK C. F. (1969) Relationship of radiation-induced damage in barley seeds to the inhibition of certain oxidoreducatases by sodium azide. pp. 313-322. In, Induced mutations in plants. IAEA, Vienna. 18. WOLFF S. (1953) Some aspects of the chemical protection against radiation damage to Vicia faba chromosomes. Genetics 39,356-364.