Effects of leaf aging on the distribution and metabolism of technetium-99 in Spinacea oleracea L.

Environmental andExperimental Botany, Vol. 25, No. 4, pp. 361 368, 1985. Printed in Great Britain

009g-8472/85 $3.00 + 0.00 © 1985 Pergamon Press Ltd.

EFFECTS OF LEAF AGING ON THE DISTRIBUTION AND M E T A B O L I S M OF T E C H N E T I U M - 9 9 IN SPINACEA OLERACEA L. j. F. LEMBRECHTS and G. M. DESMET R.i.V.M,, Lab. of Radiation Research, c/o Association EURATOM-ITAL Keyenbergseweg 6, 6704 PJ Wageningen, The Netherlands

(Received l 3 November 1984; acceptedin revisedform 26 February 1985) LEMBRECZHTSJ.F. and DESMETG. M. Effects of leaf aging on the distribution and metabolism of technetium99 in Spinacea oleracea L. ENVIRONMENTAI~ANn EXPERIMENTALBOTANY 2.5, 361--368, 1985.Results of gelfiltration chromatographic separation of the major groups of technetium (Tc) compounds in spinach leaves (Spinaceaoleracea L. cv. Verbeterd Breedblad) are described. When grown on a nutrient solution contaminated with NH4TcO4 (10 8 to 10 ~ M), free ToO 4 was present in the leaves. Its level remained fairly constant as the leaves grew older and was proportional to the TcO,, content o["the imtrient solution. The relative rates of uptake and incorporation were highest in the young leaves. Technetium was essentially ( > 75°S) incorporated in low molecular weight compounds ( < 6000 g/tool). Most of the Tc-labeted complexes are postulated to be formed simultaneously and independently. They were present in different proportions in the distinct leaves; the smallest Tc bio-organic complexes predominated in the cotyledons and first leaves, whereas increasing quantities of larger compounds were produced in subsequent emerging leaves.

INTRODUCTION Rls~-asscssment and m o d e l i n g in r a d i a t i o n protection studies concerning radionuclides has generally been confined to the q u a n t i l i c a t i o n of their transfi'r t h r o u g h food chains u n d e r v a r y i n g e n v i r o n m e n t a l conditions. Construction of a workable and more general model, however, requires a qualitative description of possible r a d i o n u c l i d e transtbrmations in order to account tbr observed distribution patterns. This is especially true tbr elements such as technetium, with a complex physicochemical behavior. O*As'I"7I l)iw:rgcnt rcsuhs on the mobility and availability of Tc in soils, Iv'o'ls) and experiments o n 99m~I'('labeled radiopharmaceuticals (z'8,x2~ clearly dem o n s u a t c the nccd for such models. This inw'stigation is part of a research project on the biochemical b e h a v i o r of 99Tc in plants. It 361

describes the m u t u a l relationship between d~e uptake of Tc, supplied as T c O , , and its incorp o r a t i o n in the m a j o r groups of bio-organic Tc complexes. This relation is investigated for difl~rent leaves of Spbmcea oleracea L. cv. V e r b e t e r d Breedhlad as a function of administration time and age of the plants. T h e experiments are based on a gel tihration chromatographic technique described by LEMBRECHTS el a[., ~1~) which discriminates between fiee T c O 4 and bound Tc, and a m o n g tile most preI)onderant Tc bio-organic molecules.

MATERIALS AND METHODS G r o u p s of 10 seedlings, g e r m i n a t e d in moist t)erlite, were grown in 16 I of aerated nutrient solution(l 6) at pH 6.3. T h c y werc kept in a climate

362

J . F . I,EMBRECHTS and G. M. DESMET

room with a day/night regime of l 1/13 hr and a light intensity of 40 W m -2. Relative humidity was approximately 60°.{, during the day and 8()~Ii, during the night. Loss of weight of the solution was not measured. Four ditt~rent experiments were performed with respect to the duration, timing and level of ' I ' c O 4 application (Fig. 1). " A g e " reti~rs to the n u m h e r of days after transt~'r to the nutrient solution. To minimize transport o f T c O 4 towards tile leaves in the last part of experiment C, the roots of the plants and the perlbrated plastic tubes in which the plants were grown to kee f) the rootsystems separated, were rinsed tbr 5 rain with running tapwater prior to transfier to a Tc-ti'ee nutrient solution. I)ifli~rcnt sets of opposite leaves of each plant were harvested separately without petiole. Each lamina was weighed, and comparable pairs of leaves tiom different plants were pooled. Rools, and some petioles of the second and third leaw's were harvested once ill experiment A (at day 29). All samples were ground by means of a Virtis23 homogenizer in an extraction medium with sucrose (0.5 M), TES (0.05 M), E D T A (0.5 mM) and 2-mercaptoethanol (1 mM), at p H 7.5. The ground material was strained through one layer of 60/~m nylon net. The homogenate passing the net was ccntritilged tor 2(/ rain at 12,000 .q. The

exp A I

supernatant was ti'ozen in liquid N 2 and stored at -- 20°C, until used for chromatographic separation. Three ml of supernatant were placed on a gel filtration column (Pharmacia K 16/70) filled with 125 ml polyacrylamide gel (BioGel P6, 100 200 mesh, fractionation range 6000 1000 g/mol), and separated with the extraction butt;er as an eluant (16 ml/hr). The samples of experiment 1) (350 yl) were separated with the same eluant on a Bio-Rad Econo-column (i.d. = 1.0 cm) filled with 4.7 ml potyacrylamide gel (BioGel P2, 200 400 mesh, fractionation range 1800 100), resuhing in a separation of fiee TcO47 and bound Tc. Extraction and separation were pcrtormed at 0 4°C. The '~Tc COlltent of the nutrient solutions, tile ground material, the supernant and rcsuspended pellet and of the ti'actionated effluent were determined by liquid scintillation counting (Packard Tri-Carb 4530). The standard addition technique was used tbr computation of counting ctIiciency. RESULTS

The coml)ined effects of transpiration by the leaves and free surface evaporation enhanced t)y aeration were in equilibriuln with or slightly

t

~

•

= m dorknes~

\ germination /

exp B

~

= pn fhe hght

~

= w~thouf

ToO.

gro\wfh on exp

D

~

tdifferentT(O~(on~:enfrahons

nutrient sol.ulion

~0

10

20

harvest

30

time ( d a y s ) ~

Fl~;. 1. (luhivadon and samt)lin ~ schedule ust'd to determine the uptake and incorporation of 9')q'c()~ by spimwh plants as a timclion of lime.

EFFECTS O1" LEA}' AGING IN S. exceeded the uptake of Tc() 4 tbr all contamination levels, and for cultures up to 3 weeks of age. Then accumulation of T c O 4 began to exceed evapotranspiration (Fig. 2). Pertechnetate present in the uncontaminated solution of experiment C partly originated from the tubes in which each plant was growing. Although rinsed when the plants were transferred, they remained clearly contaminated and part of the adsorbed Tc was released to the fresh medium. Table 1 represents for every harvest the n u m b e r and mean weights of the leaves fi'om each homogenate, and the weight data of the petiole and root-system samples. After 2 weeks of growth, the cotyledons are full-grown, whereas the lirst and second leaves reach maturity towards the end of the" experiment. W h e n the plants are 4 weeks old, the cotyledons start fading, while the third and subsequent emcrging leaves are still growing exponentially. Table 2 shows the quantity of '['c in the supernatant per gram homogenized material tbr lhe different leaves and harvests, and represents the contritmtion o l T c O , to the total Tc content and.the results tbr roots and petioles. This conccntration o f Tc should not be considered the

OLERACEA

i,.

total quantity present in the leaves since part of the radioactive material is retained by the nylon net and about 501, is pelleted during centrifligation. In experiment A the Tc content per g ft'esh weight increased continuously tbr all leaves o v e r the 4 weeks of growth, hut the resuhs for cotyledons and primary leaves suggest the T c O 4 concentration stabilizes after an initial increase. When 4-week-old plants were contaminated with TcOa7 (experiment B), younger leaves clearlx accumulated and metabolized more '['cO 4 than the older (rues. When after 18 {lays ofgr()wth on a contaminated solution, the 'l'cO,~ supply was diminished (experiment C), the total Tc content per g ft'esh weight remained constant in the fullgrown cotyledons and decreased in the first and second leaves, as a result of the dominance of growth-dilution over uptake• The second leaves gained about 40% in weight between day 19 and 25, and the third more than 300°<~), the mean Tc content per leaf increased with 20% respectively 100% . The TcO~- content diminished, however, in all leaves. TcO2 was the preponderant compound in roots and petioles, both present in comparable quantities. T h e relative share of the four major Tc-lat)eled

CICo 1,20

1,10 , ~LJ,-

J J

i

+

1,00

090

080

005 4

20

l"l¢;. '2. Evolution of thc 9~'l'c

363

('011[C[1[

(~-~15"t,

25

30 time(dQys)

colllid, limits) of

the nulrienl solution, expressed as a thnclion of the initial concentration Co ( Bq,'ml ).

364

,I- F. L E M B R E C H T S

a n d G, M. D E S M E T

-H II

0

~ 0 ~

+1 II +1 II +1 II

+1 II +1 II +1 II

0

+1 II -FI II -FI II +l

II -H II -H II -FI [I -FI II +t

II

d

-H II -FI II -FI II -FI II -FI II -FI II -H II -FI II +1 II HH II 0

+1 II +1 II +1 II +1 II +1 II +1 II +1 II +1 II +1 11 +1 II

+L

EFFECTS OF LEAF AGING IN S. OLERACEA 1,.

365

Table 2. Extracted Tc quantity and absolute (italics) and relative quantity of free TcO2 in the supernatant (Bq/g homogenized material)for the different organs as a function of time

Experiment A

A

Time (days) 8

15

Cotyledon

1° Leaf

1871

902

705

478

38 °; /O 3085

53% 2382

959

779

339

1101

36% A

A

B

C

22

29

30

18

19

25

1)2

15

15

Petiole

Root

3371, 700

277

279

178

163

35% 1514

827

368

697

811

492

232

120

18% 410t

29% 3509

677

681

479

348

215

1700 145

19%~ 298

t9°4, 609

27°/,, 834

31";, 555

113

197

382

448

315

78% 2641

66% 2060

37",,, 2488 27~i, 2568 324

DI

4 ° Leaf

2758

672

C

3° Leaf

3915

985

C

33%

2° Leaf

13°J 21.7

863

42'~{, 1934 56"1

29~}i, 1671 197

12°i~ 21.0

8.4

6.9

39o.i, 246

33" °0. 207

96

77

39°i,

370,~,

c o m p o u n d s or groups of compounds, which were distinguished in a BioGel P6-radiochromatogram as described by LEMBRECHTS et al., °1~

is represented in Figs 3-5. They demonstrate the relative decrease of the TOO,7 fraction as a function of time, due to the continuous metabolism of previously a c c u m u l a t e d TcO,~- (experiments A and C). T h e ett~ct of leafage on the incorporation of T c , as it was deduced from T a b l e 2, is also a p p a r e n t (experiment B). Younger leaves clearly produce more Te-labeled metabolites. C o m p a r a b l e ratios between fi-ee To() 4 and b o u n d Tc were found in plants grown on dittbrent concentrations o f T c O 4 for 15

32°:o 2559

63% 988

28<';o 1288

54%

64o.;,

58o,

57%

511

52°~, 687 226"

33°;, 303 36 1')'~/

8.4 3.6

42 <'' ~ 84.5 43.9

52'Ii,

days (Table 2). T h e ratios of the various Tc mctabolites are also not the same tbr different leaves. T h e relative portion of low molecular weight Tc bio-organic complexes ( < 1 0 0 0 g/tool) is highest in the cotyledon. A decrease in lhe relative a m o u n t of these compounds in the subsequent leaves is c o u n t e r b a l a n c e d by a general increase in high molecular weight T c complexes.

DISCUSSION It was shown that the last radioactive comp o u n d of the BioGel P6 (M) radiochromatograms is Tc02 3' ~J It is the p r e d o m i n a n t Tc c o m p o u n d

366

.I.F.

I,EMBRFCHTS

and (;. M. D E S M E T

100

]

Tc 04

]

M W < 1000

O/o MW ]

> <

I O OO 6 000

MW>6000

8

15 22 29

8

15 2 2

29

15 22 29

22 29

2

3

1

22 29doy 4

reof

l:m. 3. l';xpcrimcnt .\: rclalivc distribution o14 '~9Tc-lahclcd ,groups o1") compounds in dill~'rcnl spinach leaves as it thncfion of time.

in the homo£cnatc ot'the root-system and pctioh's. This resuh is in agreement wilh the observation of CATAI.1)O
100

[~

TC04

%

~]

MW< 1000 > 100Q MW < 6000

hAW> 6000

50-

C

1

2 30

3

4 leof doy

l"I~;, t~. Exp,..rim.cnt B: :q'lati\c di'-;Irilmlion ( / 4 "°'l'clalmlcd (£iOUl)S of'., COml)OUnds in h'avcs of spinach

ph:mts.

is no artifiwt, which is indirectly conlirmed by the tbllowin< results: (1', tilt" relative a m o u n t ()f To() 4 iIl Ill(: total Tc ('Olltell[ Of Ihc l ( ' a v c s c o n t i n u o u s l ) decreases as a [illlCliOll Ot" thnc in CXlx'rimenls A and (:, which would bc duc to tt~(' coHlinuous trailslbrmation of acctnnulaled T O O . ; (2) the rate of metabolic use, within tlw concctm'a/ion range used, seems to bc directly proportional to the rate of Ul)takc. and thus related to the external concentration (Table 2A and 1)3; (3) the sharp titll in the concentration of free T ( ( ) 4 in the h':tves, when its SUl)pl5 is diminished (Tal)lc 2C', can t)e inlcrprcled as an ,tdat)t,Hion tmvard a n e w equilibrium l)ctwcen rate of uptake and rate of transl'o,nlation. The high aflinit 3 of phtnts tbr lcchnctiurn 13.,*~ and its ])ronounc('d toxiciI,, x1'6'7'1°) sug,gest an intensive and actixe interlbr('nce of T o O 4 with o n e or m o r e b i o c h e m i c a l processes, possibly resulting in the lbrmation o l " l ' c I)io-organic co m p h 'xe s. T h e t)rescncc o f various T(-lal)ch'd ruolc(ulcs hi phmts has been reported. (5'13) T h e results of cxtx'vimcnt B demonstrate I h a l 1]1(' I'~-tl.co t l o r m a lion of these products is a t'tnlclion of the a / e of the l e a l and thus of age-dependent physiological processes. S i m e toxMty is an agc-dependem t)henomenon, ~1'°~ il mi.ghl I)e directl,, associated with the process of incorporation.

E F F E C T S OF I,EAI" AGING IN S. OLI:,RA()EA 1+. 100

--

olo

[]

+co~

[]

MW < 1000

]

> 1000 MW<600O

367

MW > 6 0 0 0

5(3-

/,

"/,,

O- 18

!

+ i

19 25 C

18 19 25 1

1B 19 25 doy 2 leQf

Fro. 5. Experiment C: relative distribution ot"4 99To-labeled (groups of'l compounds in ditt~_'rcntspinach leaves as a thnction of time.

The ilnportancc of low molecular weight compounds ( < 6 0 0 0 g/moll for the fixation of technetium is apparent, since their share in tile total a m o u n t of T0-1abeled complexes was always more than 75,1,. Even a diminished supply o f T c O 2 tbr one week (Fig. 5) did not alter this proportion. ,.'ks can be deduced from experiments A (Fig. 3) and (1 (F'ig. 5), the proportion between the various '['c-lal/eled complexes is not the same in difli~rent h'avcs. It is, however, impossible to deIine whether this is due to f u n d a i n e n t a l structural and physiological dill~rences between tile leaves, to lhe c h a n g i n g competition between leaves as the total sink tbr Tc increases, or to the fact that all leaves do not pass the same stages of development during the experiment. If some of the Tc-labeled products are successi\'c internwdiates of the same reaction pathways, their deph'tion has to be expected alier one week of reduced T c O 4 supply (experiment C). Because consiclerabh" quantities of all To-labeled products are, however, still present at the last harvest (Fi R. 5), most of them will probably be synthesized i n d e p e n d e n t l y of one another. Since the final object of the investigation is the t)urilication and characterization of individual To-labeled complexes, the results of the experitnents discussed in this paper call be considered as

guidelines ti)r the selection of proper cultivation schedules and plant material. T h e gel fihration chronaatograt~hic technique can further I)e used for a tirst characterization of this material. The tirst author wishes to drank flw Commission of the European (k)mmunitics tbr a Research ti:llowship. \.Vc gratcfidly acknowl('dgc Proll l)r. So. O. Vandcrborglu tbr valuable criticism of the manuscript. Publication No. 2169 of tlw COll/ract No. BIO 325 NL of tile: Radiation Protection Piogrammc. DG XII of the Commission of the European Communities.

Acknowledgements

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