253

Mutation Research, 71 (1980) 253--261 © Elsevier/North-Holland Biomedical Press

THE EFFECTS OF SODIUM AZIDE ON MAMMALIAN CELLS CULTIVATED IN VITRO

D. SLAME~IOV.~ and A. GABELOV~

Cancer Research Institute, Slovak Academy of Sciences, Department of Mutagenesis, Bratislava (Czechoslovakia)

(Received 28 January 1979) (Revision received 16 January 1980) (Accepted 19 February 1980)

Summary

Sodium azide acted cytostatically to cytotoxical ly on 2 lines of mammalian cells. After application of the substance in an acid environment the highest cytostat ic effect was noted. The results of the DNA-synthesis inhibition test suggest that sodium azide does not damage the DNA of the observed fibroblasts with any of the tested modes of application. In Chinese hamster cells, neither 20-h t reatment in medium nor 60omin t reatment in an acid environment gave rise to significantly increased occurrence of 6-TG-resistant mutations.

The results of the DNA-synthesis inhibition test, as well as the mutagenicity testing, do no t suggest the possibility that t reatment with sodium azide might induce DNA damage in the observed human and Chinese hamster cells. The cytostat ic effect of sodium azide on the fibroblasts studied is probably not accompanied by a genotoxic effect.

The effects of sodium azide (NAN3) on mammalian cells have so far no t been studied in great detail, even though this substance is often used in industry, agriculture and medicine. Tests on experimental animals have shown that sodium azide has no carcinogenic effect [18]; however, in several systems, mutagenic effects o f this drug have been demonstrated [5,6,8--11,14,15], which cannot be predicted. In bacterial DNA in vivo, sodium azide induces damage that is recognized and repaired by the excision-repair system [8]. How- ever, the character of such damage to DNA remains unknown. In investigations on the reactions of sodium azide with DNA in vitro, different results have been obtained in DNA of different origins. Bacterial DNA [7] and DNA isolated from calf thymus [19] appeared unreactive to sodium azide irrespective of the environmental pH. DNA isolated from non-germinating seeds of barley [19] and DNA isolated from salmon sperm [16] reacted with sodium azide in an

254

acid environment: in the former the results of the reaction were mainly single- strand DNA breaks, in the latter, DNA depurination. It has lately been sug- gested that extracts from sodium azide-treated embryos of Himalaya barley seeds exhibit mutagenic activity in excision-deficient Salmonella typhimurium [12] and Saccharomyces cerevisiae [20]. These experiments indicate the presence of a mutagenic metaboli te formed in vivo in barley embryos exposed to sodium azide. On the other hand, efforts to activate sodium azide in the presence of the liver microsomal fraction were unsuccessful; in the Salmonella typhimurium/mammalian microsome test of Ames, sodium azide reduced the mutagenic activity [3].

In the present paper we report our investigation of the effects of sodium azide on human EUE fibroblastoid cells and on V79 hamster cells. We con- cerned ourselves with studying the growth activity of influenced cells, with changes in the intensity of daughter DNA synthesis and with induction of 6-thioguanine-resistant mutations.

Material and methods

Cell lines. We used heteroploid human EUE fibroblasts (obtained from Dr. A. Abbondandolo , Laboratory of Mutagenesis, Pisa, Italy) and V79 hamster cells (obtained from Prof. Geissler, Department of Somatic Cell Genetics, Cen- tral Institute of Molecular Biology, Berlin, GDR). Human cells were cultivated in Parker's medium with the addition of 20% calf serum, hamster cells in MEM Eagle with the addition of 10% calf serum and 2.5% foetal calf serum. To both kinds of medium were added penicillin (200 U/ml) and streptomycin (100 #g/ml). Cells were cultivated in a humidified atmosphere of 5% CO2 and air at 37°C in glass petri dishes. The monolayer of cells was removed from the glass with 0.25% trypsin. Cell stocks were kept over liquid nitrogen. Both lines were checked in an electron microscope. No contamination with PPLO was noted.

Influencing of cells. Cells exponentially growing on petri dishes were influ- enced with sodium azide: namely a, continuously (NAN3 added to the culture medium), b, for 20 or 24 h (NAN3 added to the culture medium), c, for 30 or 60 min (NAN3 added to Dulbecco PBS buffer, pH 4.2--7.2). In all experiments, cells were removed with 0.25% trypsin and counted in a haemocytometer .

DNA synthesis inhibition test. We used the method described by Painter [13], modified in regard to the conditions of cultivation of EUE and V79 cells'. In brief, exponentially growing cells were cultivated for 24 h in the presence of radioactive thymidine (14C-Tdr at 0.02 #Ci/ml). At the same time the parental DNA was labelled radioactively. Pre-labelled cells were influenced with various NaN3 concentrations in PBS buffer (30--60 min at pH 5.3) or in growth medi- um (20 h). As a positive DNA<lamaging factor we used UV radiation (Phillips TUV 15-W lamp, emitting 90% radiation at 253.7 nm). Cells were irradiated in the monolayer; before irradiation the medium had been sucked off and cells were rinsed in PBS buffer. After the influencing, cells were washed and further cultivated in fresh growth medium. At 0, 30, 60, 9 0 , 1 2 0 and 180 min we took one sample from each group and followed daughter DNA synthesis according to 10-min pulse labelling with 3H-Tdr (10/~Ci/ml). After termination of pulse- labelling, the cells were processed as follows. Labelling was terminated by wash-

255

ing the cells with 3 ml SSC buffer (0.15 M sodium chloride + 0.015 M sodium citrate) and adding 3 ml 5% trichloroacetic acid (TCA) to the monolayer of cells from which the radioactive medium had already been sucked off. Both solutions were pre-cooled to 0°C. After 24-h storage in 5% TCA in the refrig- erator, cells were removed from the glass with a silicone scraper and resus- pended in TCA. Aliqu0ts of 2 ml were filtered through a SYNPOR membrane filter (0.44 pm), washed in 5% TCA, distilled water, 70% ethanol and 96% ethanol resp., and dried. Activities of samples were determined in a Packard liquid-scintillation counter.

Detection of 6-thioguanine-resistant mutations. V79 cells ( 3 . l 0 s) were plated in petri dishes and cultivated for 24 h. The medium was sucked off and replaced either by a PBS buffer (pH 4.2 or 5.3) containing 0, 10, 25 or 50 gg NaN3/ml in which cells were cultivated for 20--60 min; or by a growth medi- um containing 0, 30, 100 or 300 #g NaN3/ml in which cells were cultivated for 20 h. As a positive mutagenic factor for demonstrating the ability to measure muta t ion frequency in V79 cells, we used UV irradiation. The conditions of UV irradiation were similar to those for the DNA-synthesis inhibition test. For the detect ion of 6-TG-resistant mutat ions we used the respreading mutat ion assay proposed by Chasin [2] and modified by Abbondandolo [1]. In short, influ- enced cells as well as control cells were plated on 5--10 dishes, q) 10 cm (5 • l 0 s cells per dish). After attaching for 2 h, 6-TG was added to these cells to a 5 pg/ml final concentration. Next, 2 • 102--1 • 103 cells were plated to deter- mine plating efficiency. The remaining influenced and control cells were replated in several petri dishes, ~b 10 cm, 5 • 10 s cells per dish. These cells were processed after 24 or 48 h. The same procedure was repeated at 48-h intervals. We observed the populat ion of treated and control cells for up to 8--12 days. The respreading mutat ion assay, in which cells are maintained by regular pas- saging at a certain cell density per surface unit, ensured that cells did not become overcrowded and start metabolic co-operation.

Results and discussion

Sodium azide has a comparatively stable structure which makes possible a s tudy of its long-term influence on cells. After prolonged application in culture medium, this substance acted on human EUE fibroblasts cytostatically (Fig. 1). After 72 h, cells died at all concentrations used. The cytostat ic effects of lesser concentrations of sodium azide (<100 /~g/ml) are, after 24 h, still reversible (Fig. 2).

When we studied the short-term influence of NaN3 on EUE fibroblasts, it was applied in Dulbecco PBS buffer, with the pH adjusted to 5.3--7. When the drug was applied at a neutral pH, no cytostat ic effect was noted during the post-NaN3 t reatment (Fig. 3). After application of NaN3 at reduced pH, how- ever, we observed a pronounced cytostat ic to cyto toxic effect (Fig. 4). The dependence of the cyto toxic i ty of NaN3 on environmental acidity is shown in Fig. 5, which illustrates the effects of a 60-min t reatment on growth activity of EUE cells influenced by a concentrat ion of 50 pg/ml over a greater range of pH values. During post-NaN3 t reatment in fresh medium there were manifested vari- ous damaging effects of one and the same NaN3 concentrat ion depending on

2 5 6

lx106

m

~ 1,105 - I -

1N104~

i

0

lx106 t

1 x105 .j ~ -

-4

lx104

1=104 24 /~8 72 ;6 ll.lt, 24

HOURS OF CULTIVATION WITH NoN 3 POST-NoN 3 TREATMENT(HOURS}

Fig. 1. The effect of cont inuous act ion of various NaN 3 concentrat ions on growth activity of EUE cells. Control, o; 25/~g NaN3/m], e; 50/~g NaN3/ml, v; 100/Jg NaN3/ml, e; 250/~g NaN3/ml, 4.

Fig. 2. The effect of 24-h act ion of various NaN 3 concentrat ions on the growth activity of EUE cells. Control, o; 25/~g NaN3/ml, o; 50 ~g NaN3/ml, o; 100 ~g NaN3/ml, s; 250 #g NaN3/ml, 4.

lxi06 • I

i

I

_

-

-

~ -

~ - g

1 ~ 1 0 5 -

. - 4

lx106 1 i

i

I

mlxl0 -- Z

, ~ -

24 48 7u2 g6 POST-NoN 3 TREATMENT(HOURS) POST-NaN 3 TREATMENT (HOURS)

Fig. 3. Growth act ivi ty of EUE cells after 60-min incubat ion with various concentrat ions of NaN 3 at pH 7. Control, o; 25/~g NaN3/ml, o; 50/~g NaN3/ml, D; 100/~g NaN3/ml, e.

Fig. 4. Growth activity of EUE cells after 60-rain incubat ion with various concentra t ions of NaN 3 at pH 5.3. Control, o; 25 , g NaN3/ml, o; 50/~g NaN3/ml, v; 100/~g NaN3/ml, e.

257

1~106 1

~1~10 5 --

1=104

1ooi 90-

B0-

70-

60-

5O l

30-

20-

10-

4'8 7'2 9'6 o' 3'o' 66' 9'0' i~o' I;O' i~o' POST-NaN 3 TREATMENT(HOURS) TIME OF PULSE LABELING DURING POSTTREATMENT

Fig. 5. G r o w t h act iv i ty o f E U E cel ls a f ter 60-rain i n c u b a t i o n w i t h 50/zg NaN3/ml , pH 5.3--7.2. Contro l , pH 7.2, o; control , pH 5.3, o; 50 /~g NaN3/ml, pH 7.2, ~; 50 ktg NaN3/ml, pH 6.8, A; 50/~g NaN3/ml, pH 6.3, A; 50/~g NaN3/ml, pH 5.8, ~; 50 ktg NaN3/ml, pH 5.3, m.

Fig. 6. Resul t s o f the D N A - s y n t h e s i s inh ib i t ion t e s t a f ter shor t - t erm t r e a t m e n t o f E U E cel l s w i t h N a N 3 in an acid e n v i r o n m e n t or a f ter U V irradiat ion. Pre-label led cel ls w e r e treated for 3 0 rain w i t h var ious NaN 3 c o n c e n t r a t i o n s in PBS b u f f e r (pH 5.3) or were UV irradiated. Treated and c o n t r o l cel ls w e r e further culti- vated in fresh g r o w t h m e d i u m , and at regular intervals t h e y w e r e pulse- label led with 3H-Tdr (10 ~Ci/ml, 10 rain). The 3H/14C ratio was ca lcu la ted and the result ing values w e r e ex pres sed in percentages . The 3H/14C ratio at single t i m e intervals in contro l s was t a k e n as 100%. 10 #g NaN3/ml, o; 25 #g NaN3/ml, A; 50 ~g NaN3/ml, m; 100 pg NaN3/ml, v ; 50 erg/mm 2, ~; 100 erg/mm 2, o; 200 erg/mm 2, m. Mean values from 3 Expts .

the acidity of the PBS buffer in which the drug had been dissolved. The dependence of the cytotoxic effect of NaN3 on environmental acidity

can be explained either by enhanced permeability of cells or by their higher sensitivity to the effects of sodium azide. In an acid environment, NaN3 acts as a powerful inhibitor of oxidoreductases [17] . Moreover, in mammalian cells, RNA and protein syntheses are inhibited at pH values lower than 6. In normal circumstances this effect is reversible, and the viability of cells is not influ- enced. However, cells are likely to react by losing their viability if the influence of lower pH is accompanied by the influence of sodium azide, which is an inhibitor of respiratory processes.

In view of the marked effects of NaN3 in an acid environment, EUE and V79 cells influenced with NaN3 at pH 5.3 were assayed by the DNA-synthesis inhi- bition test. From the results it is possible to judge whether this substance will induce damage to the DNA of tested cells. If it acts as a metabolic inhibitor without injuring DNA shortly after termination of the inhibitory influence, parental DNA becomes capable of serving as a template for renewal of DNA

TA

BL

E 1

OC

CU

RR

EN

CE

O

F 6

-TG

r M

UT

AT

ION

S

IN C

UL

TU

RE

D

V7

9 A

FT

ER

U

V I

RR

AD

IAT

ION

A

ND

D

IFF

ER

EN

T

Na

N 3

TR

EA

TM

EN

T

t~

~n

Go

Ex

pre

ssio

n

UV

irr

ad

iati

on

(e

rg]m

m 2

) N

aN

3

20

-h t

rea

tme

nt

in M

EM

N

aN

3 6

0-m

in t

rea

tme

nt

in

tim

e

(/~

g/m

l)

PB

S (

/~g

/ml)

pH

5

.3

(da

ys)

0

25

5

0

75

0 3

0

I00

3

00

0

I0

25

5

0

Na

N 3

2

5 D

g/r

nl

in P

BS

pH

4.2

5 (

min

)

+C

2

0

40

6

0

0 a

11

4

58

2

7

16

8

4

80

b 0

.2

0.3

c

1 1

.5

1 a

84

7

0

b e

2 a

87

5

5

31

4

1

94

8

2

b 0

.1

c 1

3 a

47

6

0

b 0

.3

0.5

c

1 1

.6

4 a

90

7

7

60

7

1

b 0

.2

1.5

2

.1

2.6

c 1

7 9

.6

12

5 a

97

9

1

b 0

.4

c 1

8 a

93

6

2

85

7

9

99

6

9

b 0

.3

5.9

9

.2

10

.6

0.6

•

I 1

6.6

2

5.5

2

9.8

1

10

a

98

4

3

45

9

2

b 0

.2

8 2

5.8

1

6

c 1

40

.3

12

8

80

.1

12

a b b

82

5

8

0.1

0

.3

0.5

1

.5

77

7

4

89

0

.3

1.5

46

0.I

0

.3

67

0.3

1.5

71

0.1

0

.3

80

8

4

0.1

0

.16

0.2

0

.4

84

9

9

0.0

8

0.2

2

0.1

3

0.3

6

94

6

8

46

3

8

78

7

8

66

5

7

0.2

0

.3

0.9

0

.5

1 1

.5

4.5

2

.5

69

8

8

86

7

5

0.5

0

.8

1.3

0

.9

1 1

.6

2.6

1

.8

93

6

8

57

6

2

0.2

0

.8

1 0

.9

1 4

5 4

.5

70

5

8

65

5

6

0.7

2

.8

2.5

1

1 4

3.5

1

.42

78

6

7

53

4

6

0.3

1

.1

1.6

1

.7

1 3

.7

5.3

5

.6

76

8

8

56

3

5

93

8

6

84

7

3

0.4

0

.5

0.6

1

.2

1 1

.2

1.5

3

97

8

5

10

0

87

0

.8

0.2

2

0.8

1 0

.2

2.5

1

86

9

2

74

8

7

0.9

0

.2

2 0

.4

1 0

.2

2.2

0

.4

95

9

4

97

9

8

1 0

.3

1 0

.7

1 0

.3

1 0

.7

a, p

lati

ng

e

ffic

ien

cy

(%

); b

, n

um

be

r o

f 6

-TG

r c

olo

nie

s p

er

1 -

10

5

surv

ivo

rs;

c, r

ati

o o

f in

du

ce

d

6-T

G r

mu

tati

on

s to

sp

on

tan

eo

us

6-T

G r

mu

tati

on

s; +

C,

co

ntr

ol

cell

s k

ep

t fo

r 6

0 m

in i

n P

BS

, p

H 4

.26

.

259

synthesis [13]. From the results of this test (Figs. 6 and 7) it follows that initial inhibition of DNA synthesis (induced by treatment with NAN3) had been gradually removed and during post-NaN3 treatment cells had re-acquired their normal capacity to synthesize DNA. These results are characteristic of sub- stances that do no t damage DNA. UV-irradiated EUE and V79 cells (Figs. 6 and 7) manifested the typical positive reactions characteristic of DNA-dam- aging factors.

V79 cells cultivated for 20 h in the presence of NaN3 were also assayed by the DNA synthesis inhibition test (Fig. 8). The results of this test corresponded with the results obtained on human EUE and hamster V79 cells treated in an acid environment.

When studying the mutagenic capacity of NAN3, we fol lowed the occurrence of 6-TG-resistant colonies in a V79 cell populat ion cultivated either for 20--60 min in an acid environment or for 20 h in a neutral environment (MEM, Eagle) with various NaN3 concentrations. The occurrence of 6-TG-resistant forms had also been evaluated in the progeny of influenced and control cells. The results show that NaN3 treatment will not induce significantly increased occurrence of 6-TG-resistant colonies (Table 1).

The non-conforming data gained in the field of biological research of sodium

100-

c~

g

8 - --o 10

, i i i i i

(]" 30' 60" 90' 120' 150' 1~0' TIME OF PULSE LABELING DURING POSTTREATMENT

s°--I~ /

1 0 1 - - i I I ! I

0' 30" 50' 90' 120' 150' I

180' TIME OF PULSE LABELLING DURING POST-NoN 3 TREATMENT

Fig. 7. Resu l t s o f t h e D N h - s y n t h e s i s i n h i b i t i o n t e s t a f t e r s h o r t - t e r m t r e a t m e n t o f V79 cells w i t h N a N 3 in an acid e n v i r o n m e n t or a f t e r UV i r r ad i a t i on . Pre- label led cells w e r e t r e a t e d fo r 60 r a i n w i t h 25, 50 or 1 0 0 ~g N a N 3 / m l a t p H 5.3; o r were U V i r r ad i a t ed . A f t e r t e r m i n a t i o n o f t r e a t m e n t , cells w e r e p r o c e s s e d in t h e m a n n e r d e s c r i b e d f o r Fig. 6. 25 /~g N a N 3 / m l , s ; 50 ~g N a N 3 / m l , $; 100 /~g N a N 3 / m l , A; 100 e r g / m m 2, o. M e a n va lues f r o m 3 Exp t s .

F ig . 8. Resu l t s o f t h e D N A - s y n t h e s i s i n h i b i t i o n t e s t a f t e r l o n g - t e r m t r e a t m e n t o f V 7 9 cellg w i t h va r i ous c o n c e n t r a t i o n s o f N a N 3. Pre- label led cells were c u l t i v a t e d fo r 20 h w i t h 1 0 0 and 3 0 0 ~g N a N 3 / m l resp . A f t e r t e r m i n a t i o n o f t r e a t m e n t cells w e r e p r o c e s s e d in the m a n n e r d e s c r i b e d in Fig . 6. 100 ~g N a N 3 / m l , 0; 3 0 0 ~g N a N 3 / m l , D. M e a n va lues f r o m 2 Exp t s .

2 6 0

azide place it in the group of substances with an obscure mechanism of action. The results described in the present study suggest that the cytostatic and cyto- toxic effects of sodium azide noted in 2 cell lines have no genotoxic effects. However, the possibility cannot be excluded that, in DNA-excision-proficient EUE and V79 cells, azide induces such a type of injury (ionizing-like type) which becomes, by the excision-repair mechanism, effectively and accurately repaired in a short time after termination of treatment. These changes could not be detected either with the aid of the DNA synthesis inhibition test or by induction of 6-TG-resistant mutations.

We believe that a deeper comprehension of the mutagenic capacity of NaN3 on mammalian cells will promote studies of the mutagenic and DNA-inhibitory effects of the NaN3 metabolite which is believed to form in treated barley seeds. The assumed NaN3 metab01ite, mutagenic in excision-deficient Salmo- nella typhimurium and Saccharomyces cerevisiae cells, has perhaps some geno- toxic effect on excision<leficient mammalian cells cultivated in vitro.

Acknowledgement

We are greatly indebted to Dr. Ma~o~ka for electron microscopy of EUE and V79 cell lines.

References

1 A b b o n d a n d o l o , A., S. Bonat t i , C. Colella, G. Corti , F. Matteucei , A. Mazacarro and G. Ralnaldi , A compara t ive s tudy of d i f fe ren t exper imenta l p ro toco l s for mutagenes is assays wi th the Soazaguaulne resistance sys tem in cu l tured Chinese h ams t e r cells, Muta t ion Res., 37 (1976) 293 - -306 .

2 Chasin, L.A.° The effect of p lo ldy on chemical mutagenes ls in cu l tured Chinese hams te r cells, J. Cell. Physiol . , 82 (1973) 299 - -308 .

3 De Flora , S., MetaboLic deac t iva t ion of mutagens in the Salmonel la-microsome test , Nature (London) , 271 (1978) 4 5 5 - - 4 5 6 .

4 Freese, E.B., Transi t ions and transvef-sions induced b y depur ina t ing agents, Genetics , 47 (1961) 5 4 0 - - 545.

5 Hadwiger , L.A. , C. Sander, J. Eddyvean and J. Ra ls ton , Sodium azide- induced m u t a n t s of peas t ha t accumula te plsat in, P h y t o p a t h o l o g y , 66 (1976) 629---630.

6 Kleinhofs , A., C. Sander , R.A. Niian and C.F. Konzak° Azide mutagen ie i ty mechan i sm and na tu re o f m u t a n t s p roduced , in: Po lyp lo tdy and Induced Muta t ions and Plant Breeding, STI /PUB/S59, In tern . Atomic Energy Agency , Vienna, 1974 , pp. 195 - -199 .

7 Kleinhofs , A., M. Kle inschmidt , D. Sciaky and S. yon Broembsen, Azide mutagenesls , In vi t ro studies, Muta t ion Res., 29 (1975) 4 9 7 - - 5 0 0 .

8 Kleinhofs , A., and J .A. Smith , Effec t o f excis ion repair on azide- induced mutagenesls , Muta t ion Res., 41 (1976) 2 3 3 - - 2 4 0 .

9 Konzak , C.F., M. Niknejad, I. Wickhara and E. Dona ldson , Mutagenic in t e rac t ion of sod ium azide on m u t a t i o n s induced in bar ley seeds wi th d ie thy l su lphate or N-methyl -N-u l t rosourea , Muta t ion Res., 30 (1975) 55--62 .

10 Nilan, R.A. , E.G. Siderls, A. Kleinhofs , C. Sander and C.F. Konzak , Azide - - a p o t e n t mu tagen , Muta- t ion Res.° 17 (1973) 1 4 2 - - 1 4 4 .

11 Nilan, R.A. , A. Kleinhofs and C. Sander , Azide mutagenes ls in bar ley , in: H. Gaul (Ed.), Barley Genet- ics, III, Thiemig, Munich, 1976 , pp . 113 - -122 .

12 Owals, W.M., M.A. Zarowitz , R.A. Gunovich , A.L. H o d g d o n , A. K/einhofs and R.A. Hi/an, A muta - genie in vivo metabol i t e o f sod ium azide, Muta t ion Res., 53 (1978) 355 - -358 .

13 Painter , R.B., Rap id test to de tec t agents tha t damage h u m a n DNA, Nature (London) , 265 (1977) 6 5 0 - - 6 5 1 .

14 Sander , C., and F.J . Muehlbauer , Mutagenic effects of sod ium azide and g a m m a i r rad ia t ion in Pisum, Environ. Exp. Bo tany , 17 (1977) 43 - -47 .

15 Siderls, E.G., R.A. Nilan and T.P. Bogyo , Different ial ef fect of sod ium azide on the f r equency of rad ia t ion- induced c h r o m o s o m e aber ra t ions vs. the f r equency of rad ia t lon- induced ch lo rophy l l mu ta - t i ons in H o r d e , , m v,,Igare, Radia t . Bo tany , 13 (1973) 315 - -322 .

261

16 Sideris, E.G., and M. Argyrakis, Chemical al terat ions induced in DNA and DNA components by the mutagenic agent azide, Biochim. Blophys. Aeta, 366 (1974) 367--373.

17 Sideris, E.G., R.A. Nilan and C.F. Konzak, Relat ionship of radiat ion-induced damage in barley seeds to the inhibi t ion of certain oxidoreductases by sodium azlde, in: Induced Mutat ions in Plants, IAEA, Vienna, 1969, pp. 313--322.

18 Ulland, B., E.K. Weisbu~ger and J.H. Weisburger, Chronic toxic i ty and carcinogenicity of industr ial chemicals and pesticides, Toxicol. Appl. Pharmacol., 25 (1973) 446 (Abstr.).

19 Veleminsk~, J., T. Giehner and V. Pokorn~,, Induct ion of single-strand breaks in barley by sodium azide applied at pH 3, Mutat ion Res., 42 (1977) 65--70.

20 Veleminsk~, J., L. Silh~mkov~ and T. Gichner, Mutagenesis of Saccharomyces cerevisiae by sodium azide activated in barley, Mutat ion Res., 61 (1979) 197--205.