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Use of INAA to study Se, Sb, Zn and Co levels of yeast cells
~
Pergamon
Appl. Radiat. Isot. Vol. 46, No. 12, pp. 1295--1298,1995 Copyright© 1995ElsevierScienceLtd 0969-8043(95)00232-4 Printed in Great Britain.All rights reserved 0969-8043/95 $9.50+0.00
Use of INAA to Study Se, Sb, Zn and Co Levels of Yeast Cells M. C Z A U D E R N A ,
S. S I E R A K O W S K A
a n d B. S I T O W S K A
The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-1 I0 Jabtonna near Warsaw, Poland (Received 30 May 1995)
Yeast cells, Saccharomyces cerevisiae, were exposed to Sb(V) (10 -5 M) and SeO2(10 -4 M) or seleno-cystine (CySe)2 (5 x 10-5 M). Se, Sb, Zn and Co levels of the yeast were measured by instrumental neutron activation analysis. The results obtained show that in the absence of Se, Sb is taken up by the cells and the highest concentration of Sb in the yeast was observed during the initial 2.5 h of incubation. Both Se-compounds resulted, in general, in a minute decrease of uptake yield of Sb by the cells. This effect can be particularly observed in the presence of SeO2. The presence of Sb in the yeast medium slightly increased the Se level only after long incubation times. Se uptake by the yeast was higher (regardless of Sb dosage) when the yeast was incubated in the medium containing (CySe)2 (in comparison with SeO2). The presence of Se-compounds and/or Sb caused decrease in the levels of Zn found in the cells. While SeO2 presence resulted in minor changes of the Co level of the yeast, the combined presence of Sb and Se-compounds produced the significant enhancement of Co abundance. The similar effect was noted in the yeast incubated in a medium containing only (CySe)z or Sb.
Introduction There is increasing recognition that selenium (Se) is an important metalloid with industrial, environmental, biological and toxicological significance. Se is an essential element in many species, including humans (Aggett, 1985; Combs and Combs, 1986; Fox, 1992; Stadtman, 1990; Zingaro and Cooper 1974). The chemistry of Se suggests that, in biological systems, it is most likely present as the selenol (R-Sell) or, as the Se analogous to sulfur in the amino-acids (Zingaro and Cooper, 1974; Reddy and Massaro, 1983). Selenols are stronger acids than thiols and, at physiological pH, exist mainly in anionic form (R-Se-) whereas the sulfhydryl group exists mainly in the protonated form. The anionic form of the selenohydryl group is not only a very good nucleophile but also a very good leaving group (Reddy and Massaro, 1983). In addition, the anionic form of this group binds heavy metals strongly (Baldew et al., 1991; Imura, 1989; Nuttal and Allen, 1984; Kabata-Pendias and Pendias, 1993). This is the principle behind the usage of Se compounds in heavy metal detoxification (e.g. antimony, arsenic, cadmium, mercury, copper, silver or lead) (Baldew et al., 1991; Chutke et al., 1993; Imura, 1989; KabataPendias and Pendias, 1993; Nuttal and Allen, 1984; Witkowska et aL, 1991; Reddy and Massaro, 1983; Zingaro and Cooper, 1974). Likewise, compounds of these metals have been reported to counteract the
toxic effects of Se (Kabata-Pendias and Pendias, 1993; Zingaro and Cooper, 1974). Se and antimony (Sb) are potentially toxic and carcinogenic in nature (Berman, 1989; Elinder, 1984; Riihling et al., 1987). In biogeochemical cycling of Se and Sb microorganism biomass have been very effective (Czauderna et al., 1993, 1994; Gol~tb et al., 1990; Nakajima and Sakaguchi, 1986). Thus, in order to safeguard the public health it is essential to study the interaction between Sb (as Sb205) and SeO2 or seleno-cystine [(CySe)2 ]. Furthermore, considering the above-mentioned facts we found reasonable to use Saccharomyces cerevisiae as an experimental system to detect the shift in relative contents of Se, Sb, Zn and Co as induced by dosed compounds. Instrumental neutron activation analysis (INAA) was applied as the analytical method.
Experimental Reagents
The uptake of Sb was carried out from solutions prepared from Sb2Os--made by E. Merck. Selenocystine (CySe)2, was purchased from Sigma and SeO2 from Polish POCh works. The remaining chemicals, Co(NO3)2'6H20, FeSO4.7H20 and Zn(NO3)2.6H20 were obtained from Fluka AG, Buchs SG. Water was carefully purified by four distillations.
1295
M. Czauderna et al.
1296
Yeast strain and growth conditions
Table 1. Limitsof qualitative (Lo) and quantitative (Lo) determinationfor Sb, Se, Zn and Co
The strain SI of Saccharomyces cerevisiae used in all studies had the wild genotype. The suspension of the yeast was incubated for 24 h in YEPGlu liquid medium (1% yeast extract, 1% Bacto peptone and 2% glucose) at 28°C. The final culture density was about (1-2) x 10 7 cells/mL of the medium. Non-toxic concentrations of the selenium compounds in the yeast culture were selected experimentally, so as to obtain the least survival drop after 48 h of incubation. The suitable dilutions of test cultures and the control culture were plated on YEPGIu + agar medium (to test survivals) and quantities of the yeast were computed. After 24 h of incubation, the culture was divided into 12 equal aliquots (320mL). To 10 of these aliquots were added 5 mL of aqueous solution of appropriate doses of the test compounds. The incubation temperature of the yeast was 4°C (without shaking) to create conditions to prevent growth. The ceils were harvested (64 mL of the yeast suspension from each group) after 2.5 (a), 8 (b), 27 (c), 50 (d) and 75 (e)h of the incubation in the medium containing the appropriate compounds:
Element
Group I - - n o additions (the control group); Group II--SeO 2 (10 -4 M) and Sb (10 -5 M); Group IEt--SeO2 (10 -4 M); Group III---(CySe)2 (5 x 10 -SM) and (10 -5 M); Group III~--(CySe)2 (5 x 10 -5 M); Group IV--Sb (10 -5 M).
Sb
The cells suspension were centrifuged at 2000~000g for 20 rain. The resulting cells were washed once with 10mL of distilled water and, then, freeze-dried (for 20-30 h at 4 Pa). Experiments were repeated two times and data (Tables 2--4) are presented as the mean.
Sb Se Zn Co
Lo (/ag)
LQ (pg)
0.37 0.I 1 0.41 0.006
0.74 0.22 0.81 0.012
samples were irradiated for 101 h in the nuclear reactor "EWA" (Swierk, Poland) at a well thermalized neutron flux (tp) of 2.3 x 1 0 n n . c m - 2 - s - l . The contribution of the epithermal neutron flux was less than 1% of the total flux. After 5-6 wk of cooling each irradiated biological sample were transferred into a new container and then weighed. All samples were placed on the top face of the Ge(Li) detector having 3 mm glass entrance window and the resolution of 6.2 keV at 1332.5 keV. Se, Sb, Zn and Co in the irradiated samples were determined after 6 wk of cooling by the following ~,-rays: 7SSe (264.5 keV), 124Sb (602.7keV), 65Zn (lll5.5keV) and 6°Co (1332.5keV) (IAEA, 1987). The qualitative and quantitative detection limits (Rogers, 1970) for determined elements in the absence of interfering activities are summarized in Table 1. The biological reference material (SRM 1577b bovine liver) was used for the evaluation of the accuracy of applied analytical procedure. Each sample was counted for 2000--4000 s live time on the Ge(Li) detector (active volume 36 em 3) connected to a 4096 channel pulse-height analyzer (1024-NTA, 4 K, Hungary) based on the CAMAC interface and the IBM PC computer. The total deadtime correction was always less than 3%. The concentrations of Se, Sb, Zn and Co were determined by comparison with standards having similar amounts of the elements as the samples. The precision of the measurements was calculated as the statistical counting error of the net peak area (Amid, 1981).
Irradiation and activity measurements The dry cells of mass (63-218 rag) and standards (0.011-98 #g) were wrapped in an aluminium foil. (The foil had been heated for 100 h at 140°C.) The
ResuRs
and Discussion
The yeast cells of Saccharomyces cerevisiae were exposed to Sb and SeO2 or (CySe): but no lesions or
Table2. Uptake yieldof Sb by the yeastcells(/~g/g)and the "percentagechanges''a (in parentheses)as a functionof the chemical form of the So-compoundin the culturemedium,and of the exposure time Initial concentration Incubationtime (h) of Sb, Se
Control group
(tool/L) in medium No addition
2,5 IA] .
II
Sb SeO2
10-5 10-4
78.7 _+4.8 (-5.5%)
68.8 + 2.8 ( - 13,6%)
62.3 + 2.6 (-17.2%)
61.2 + 2,3 (-12.3%)
73.9 + 2.3 (-6.02%)
III
Sb (CySe)2
10-5 5 x 10-5
75.4 ± 5.3 (-9.5%)
69.7 + 2.4 (-14.2%)
66.4 ± 6.9 (-11.7%)
63.1 + 2,4 (-9.6%)
82.2 4-0.8 (4.3%)
79.6 ± 2.9
75.2 ± 1.7
69.8 +_2.6
78.8 + 2.4
No. Group I
Sb I0-~ 83.3 + 2.4 no ~ 'Percentage changes relative to absence of Se (i.e. group lV ). IV
8 [131 .
.
27 [C] .
50 [D]
75 [El
.
Use of INAA
1297
Table 3. Uptake yield of Sea by the yeast ceils (/,tg/g) and the "'percentage changes''b (in parentheses) as a function of Sb and the chemical form of the Se-compound in the culture medium, and of the exposure time Initial concentration of Sb, Se (mol/L) in medium
No. Group I
Incubation time (h) 2.5 [A]
8 IBI .
27 IC] .
.
50 ID]
75 [El
Control group c
No addition
.
11
Sb SeO 2
10 -5 10 4
28.7±4.8 (-4.3%)
27.7±3.2 (14.5%)
38.3±4.2 (0%)
37.4±0.4 (32.2%)
40.3_+4.8 (38.6%)
Iht
SeO 2
10 4
30.0±0.6
24.2±0.8
38.3±5.1
28.3±0.9
29.1 ± 1.3
III
Sb (CySe) 2
10 ~ 5 x 10 ~
34.4±2.5 (-2.9%)
36.8±2.1 (-11.9%)
44.9±0.8 (-3.7%)
38.7_+0.5 (1.6%)
4 2 . 4 ± 1.7 (15.6%)
III~
(CySe)2
5 × 10 5
35.4 ± 2.5
38.1 ± 1.7
35.8 ± 2.0
IV c
Sb no Se
10 -5
.
41.8 ± 1.4 .
.
46.6 _++1.2
.
.
.
aPercentage changes relative to no added Sb (i.e. groups ct). bThe Se concentration (#g/g) in SRM 1577b bovine liver--found: 0.75 ± 0.07 (certified: 0.73 ± 0.06). ¢Se content in the cells below the quantitative detection limit of Se.
symptoms of Sb or Se intoxication were found. The data in Table 2 show that in the absence of selenium, Sb is taken up by the cells and the highest concentration of Sb in the yeast was observed during the initial 2.5 h of the incubation. From the data shown in Table 2 it follows that the both Se-compounds, in general, in the minute decrease of the Sb level of the cells (only for the 75th h of the incubation the presence of (CySe)2 produced slight enhancement of the concentration of Sb in the cells). This phenomenon can be particularly observed in the presence of SeO 2: the Sb level of the yeast was regularly lower in comparison with the single Sb-supply. The present results strongly suggest that the Sb dose (i.e. 10 -5 M) in the medium was too small to produce a marked effect on the Se level of the cells (Table 3). Indeed, the presence of Sb in the medium increased the Se level only after long incubation times. The presence of SeO2 and Sb in the medium
produced observable enhancement of the Se level of the cells in the 50th and 75th h of the incubation. This phenomenon was altered by the presence of (CySe)2: the concentration of Se in the yeast was slightly higher in the 75th h of the incubation (the mutual interaction between Se and Sb). Table 3 also illustrates the Se intake from the culture medium containing only SeO2 or (CySe)2. It was found that the yield of the Se uptake by the cells (regardless of Sb dosage) was less when the yeast was incubated in the medium containing SeO2. The present work proved that (CySe)2 resulted in a higher uptake yield of Se by the yeast in comparison with SeO2. Zinc and cobalt
The results in Table 4 clearly proved that the presence of Se-compounds and/or Sb induced changes in the Zn and Co levels of the yeast. The
Table 4. Zn and Co level of the yeast ceils per unit dry mass (/~g/g)' Incubation time (h) No. Group
2.5 [A]
8 [B]
27 [C]
50 [D]
75 [El
Control I
Zn Co
310 ± 8 0.92 _+ 0.05
410 ± 9 0.98 ± 0.04
314 ± 2 0.98 ± 0.04
281 ± 8 0.79 ± 0.04
305 ± 8 0.99 ± 0.05
II SeO2 + Sb b
Zn Co
2 9 1 ± II 1.47 ± 0.12
284±5 1.60 ± 0.08
290__.6 1.30 ± 0.10
300±6 1.30 ± 0.05
285_+11 1.31 ± 0 . 0 7
Iht SeO 2
Zn Co
325 ± 7 0.86 ± 0.05
317 ± 3 1.29 __.0.04
283 _+ 13 1.01 ± 0.09
291 ± 17 1.05 ± 0.08
264 ± 12 0.94 ± 0.08
IIl (CySe)2 + Sb b
Zn Co
289 ± 6 1.36 ± 0.05
281 ± 8 1.38 ± 0.05
246 __. 7 1.33 ± 0.07
254 ± I 1 1.36 ± 0.06
277 ± 8 1.39 ± 0.05
Ill~t (CySe)2
Zn Co
286 ± 8 1.27 ± 0.04
265 ± 8 1.38 ± 0.13
274 ± 9 1.54 ± 0.10
249 ± 8 1.44 ± 0.06
255 ± 9 1.41 ± 0.07
IV Sb b
Zn Co
2 5 6 ± 12 1.77 ± 0.06
2 6 0 ± 13 1.75 + 0.06
2 4 3 + 11 1.65 ± 0.05
2 6 0 ± 12 1.80 + 0.08
264_+ 15 1.80 ± 0.06
'The concentration of Sb (as Sb205) in the yeast medium: 10 -s M. bThe Zn and Co concentrations (/~g/g) in SRM 1577b bovine liver--found: 114 ± 2 and 0.24 ± 0.01, respectively (certified--Zn: 127 ± 16 and Co: ~0.25).
M. Czauderna et al.
1298
single SeO2 supply had only a minor antagonistic effect by causing a slight (however irregular) decrease in the Zn level of the yeast. The addition of (CySe)2 alone in the medium markedly diminished the Zn level, so it seems reasonable to suggest that the organic form of Se interacts efficiently with Zn. The Sb dosage alone produced the regular and significant diminishment of the content of Zn in the ceils. This phenomenon can also be generally observed in the combined presence of the Sb and SeO2 or (CySe) 2. No significant changes of Co were found in the cells, when the yeast was incubated in the medium containing only SeO2. For the yeast exposed to both SeO2 and Sb, the amount of Co in the cells markedly increased. The addition of (CySe)2 alone or in combination with Sb resulted in a significant enhancement of Co abundance in the yeast. The presence of Sb alone in the medium was accompanied by the highest increase in the Co level of the cells.
Conclusion INAA is suitable for the instrumental determination of trace amounts of Se, Sb, Co and Zn in the yeast. Moreover, it has been employed for the simultaneous determination of changes of Se, Sb, Zn and Co levels of the cells when the yeast was incubated in the medium containing tested compounds. Thus, INAA is particularly useful for studying interactions between trace elements in microorganisms. INAA combines a true multi-element capability, high accuracy, good sensitivity and moderate price with applicability to yeast samples (De Bruin, 1992). Acknowledgement--This work was supported by a grant
from KBN No. 4 4282 91 02.
References Aggett P. J. (1985) Physiology and metabolism of essential trace element: an outline. Clin. Endocrinol. Metab. 14, 513. Amiel S. (1981) Nondestructive Activation Analysis. Elsevier, Amsterdam. Baldew G. S., Mol J. G. J., De Kanter F. J. J., Van Baar B., De Goeij J. J. M. and Vermeulen N. P. E. (1991) The
mechanism of interaction between cisplatin and selenite. Biochem. Pharmacol. 41, 1429. Berman E. (1980) Toxic Metals and Their Analysis. Heyden & Sons, London. De Bruin M. (1992) Present and future position of neutron activation analysis. J. Radioanal. Nucl. Chem. 131, 31. Chutke N. L., Ambulkar M. N. and Garg, A. N. (1993) A simultaneous radiochemical solvent extraction method for Se(IV) and Sb(III) using 75Se and ~24Sbtraces. Chem. Anal. (Warsaw) 38, 733. Combs G. F. and Combs St G. (1986) The Role of Selenium in Nutrition. Academic Press, Inc., Orlando. Elinder C. G. (1984) Changing Metal Cycles and Human Health (Edited by Nriagu J. O.) Dahlem Koferenzen 1984. Springer-Verlag, Berlin. Fox J. M. (1992) Selenium: nutritional implications and prospects for therapeutic medicine. Meth. Find. Exp. Clin. Pharmacol. 14, 275. Golob Z., Orlowska M., Glubiak M. and Olejnik K. (1990) Uranium and lead accumulation in cells of Streptomyces sp. Acta Microbiol. Polon. 39, 177. IAEA (1987) Handbook on Nuclear Activation Data. Technical Reports, Series No. 273. International Atomic Energy Agency, Vienna. Imura N. (1989) New Concepts and Developments in Toxicology (Edited by Chambers P. L., Gehring P. and Sakai F.). Elsevier, Biomedical Division. Kabata-Pendias A. and Pendias H. (1993) Biogeochemia Pierwiastk(~w ,~ladowych, Wydawnictwo Naukowe PWN, Warszawa. Nakajima A. and Sakaguchi T. (1986) Selective accumulation of heavy metals by microorganisms. Appl. Microbiol. Biotechnol. 24, 59. Nuttal K. L. and Allen T. S. (1984) Redox reactions of hydrogen selenite ion. Inorg. Chim. Acta 92, 187. Reddy C. C, and Massaro E. J. (1983) Biochemistry of selenium:a brief overview. Fundam. Appl. Toxicol. 3, 431. Rogers V. S. (1970) Detection limits for gamma-ray spectral analysis. Anal. Chem. 42, 455. Rfihling A., Rasmussen L. and Pilegaard K. (1987) Survey of atmospheric heavy metal deposition in Nordic countries in 1985. Report for the Nordic Council of Minstrs. Kobenhavn. Stadtman T. C. (1990) Selenium biochemistry. Annu. Rev. Biochem. 59, 11I. Tsezos M. (1985) The selective extraction of metals from solution by microorganisms. A brief overview. Can. MetalL Q. 24, 141. Witkowska J., Czerwifiska D., Kiepurski A. and Roszkowski W. (1991) Harmful elements versus iron, zinc and copper--interactions in animal and human organism. Part I. Mercury, tin, nickel, selenium, fluorine, aluminium. Roczn. P Z H XLII, 15. Zingaro R. A. and Cooper W. C. (1974) Selenium. Van Nostrand Reinhold, New York.
Pergamon
Appl. Radiat. Isot. Vol. 46, No. 12, pp. 1295--1298,1995 Copyright© 1995ElsevierScienceLtd 0969-8043(95)00232-4 Printed in Great Britain.All rights reserved 0969-8043/95 $9.50+0.00
Use of INAA to Study Se, Sb, Zn and Co Levels of Yeast Cells M. C Z A U D E R N A ,
S. S I E R A K O W S K A
a n d B. S I T O W S K A
The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences, 05-1 I0 Jabtonna near Warsaw, Poland (Received 30 May 1995)
Yeast cells, Saccharomyces cerevisiae, were exposed to Sb(V) (10 -5 M) and SeO2(10 -4 M) or seleno-cystine (CySe)2 (5 x 10-5 M). Se, Sb, Zn and Co levels of the yeast were measured by instrumental neutron activation analysis. The results obtained show that in the absence of Se, Sb is taken up by the cells and the highest concentration of Sb in the yeast was observed during the initial 2.5 h of incubation. Both Se-compounds resulted, in general, in a minute decrease of uptake yield of Sb by the cells. This effect can be particularly observed in the presence of SeO2. The presence of Sb in the yeast medium slightly increased the Se level only after long incubation times. Se uptake by the yeast was higher (regardless of Sb dosage) when the yeast was incubated in the medium containing (CySe)2 (in comparison with SeO2). The presence of Se-compounds and/or Sb caused decrease in the levels of Zn found in the cells. While SeO2 presence resulted in minor changes of the Co level of the yeast, the combined presence of Sb and Se-compounds produced the significant enhancement of Co abundance. The similar effect was noted in the yeast incubated in a medium containing only (CySe)z or Sb.
Introduction There is increasing recognition that selenium (Se) is an important metalloid with industrial, environmental, biological and toxicological significance. Se is an essential element in many species, including humans (Aggett, 1985; Combs and Combs, 1986; Fox, 1992; Stadtman, 1990; Zingaro and Cooper 1974). The chemistry of Se suggests that, in biological systems, it is most likely present as the selenol (R-Sell) or, as the Se analogous to sulfur in the amino-acids (Zingaro and Cooper, 1974; Reddy and Massaro, 1983). Selenols are stronger acids than thiols and, at physiological pH, exist mainly in anionic form (R-Se-) whereas the sulfhydryl group exists mainly in the protonated form. The anionic form of the selenohydryl group is not only a very good nucleophile but also a very good leaving group (Reddy and Massaro, 1983). In addition, the anionic form of this group binds heavy metals strongly (Baldew et al., 1991; Imura, 1989; Nuttal and Allen, 1984; Kabata-Pendias and Pendias, 1993). This is the principle behind the usage of Se compounds in heavy metal detoxification (e.g. antimony, arsenic, cadmium, mercury, copper, silver or lead) (Baldew et al., 1991; Chutke et al., 1993; Imura, 1989; KabataPendias and Pendias, 1993; Nuttal and Allen, 1984; Witkowska et aL, 1991; Reddy and Massaro, 1983; Zingaro and Cooper, 1974). Likewise, compounds of these metals have been reported to counteract the
toxic effects of Se (Kabata-Pendias and Pendias, 1993; Zingaro and Cooper, 1974). Se and antimony (Sb) are potentially toxic and carcinogenic in nature (Berman, 1989; Elinder, 1984; Riihling et al., 1987). In biogeochemical cycling of Se and Sb microorganism biomass have been very effective (Czauderna et al., 1993, 1994; Gol~tb et al., 1990; Nakajima and Sakaguchi, 1986). Thus, in order to safeguard the public health it is essential to study the interaction between Sb (as Sb205) and SeO2 or seleno-cystine [(CySe)2 ]. Furthermore, considering the above-mentioned facts we found reasonable to use Saccharomyces cerevisiae as an experimental system to detect the shift in relative contents of Se, Sb, Zn and Co as induced by dosed compounds. Instrumental neutron activation analysis (INAA) was applied as the analytical method.
Experimental Reagents
The uptake of Sb was carried out from solutions prepared from Sb2Os--made by E. Merck. Selenocystine (CySe)2, was purchased from Sigma and SeO2 from Polish POCh works. The remaining chemicals, Co(NO3)2'6H20, FeSO4.7H20 and Zn(NO3)2.6H20 were obtained from Fluka AG, Buchs SG. Water was carefully purified by four distillations.
1295
M. Czauderna et al.
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Yeast strain and growth conditions
Table 1. Limitsof qualitative (Lo) and quantitative (Lo) determinationfor Sb, Se, Zn and Co
The strain SI of Saccharomyces cerevisiae used in all studies had the wild genotype. The suspension of the yeast was incubated for 24 h in YEPGlu liquid medium (1% yeast extract, 1% Bacto peptone and 2% glucose) at 28°C. The final culture density was about (1-2) x 10 7 cells/mL of the medium. Non-toxic concentrations of the selenium compounds in the yeast culture were selected experimentally, so as to obtain the least survival drop after 48 h of incubation. The suitable dilutions of test cultures and the control culture were plated on YEPGIu + agar medium (to test survivals) and quantities of the yeast were computed. After 24 h of incubation, the culture was divided into 12 equal aliquots (320mL). To 10 of these aliquots were added 5 mL of aqueous solution of appropriate doses of the test compounds. The incubation temperature of the yeast was 4°C (without shaking) to create conditions to prevent growth. The ceils were harvested (64 mL of the yeast suspension from each group) after 2.5 (a), 8 (b), 27 (c), 50 (d) and 75 (e)h of the incubation in the medium containing the appropriate compounds:
Element
Group I - - n o additions (the control group); Group II--SeO 2 (10 -4 M) and Sb (10 -5 M); Group IEt--SeO2 (10 -4 M); Group III---(CySe)2 (5 x 10 -SM) and (10 -5 M); Group III~--(CySe)2 (5 x 10 -5 M); Group IV--Sb (10 -5 M).
Sb
The cells suspension were centrifuged at 2000~000g for 20 rain. The resulting cells were washed once with 10mL of distilled water and, then, freeze-dried (for 20-30 h at 4 Pa). Experiments were repeated two times and data (Tables 2--4) are presented as the mean.
Sb Se Zn Co
Lo (/ag)
LQ (pg)
0.37 0.I 1 0.41 0.006
0.74 0.22 0.81 0.012
samples were irradiated for 101 h in the nuclear reactor "EWA" (Swierk, Poland) at a well thermalized neutron flux (tp) of 2.3 x 1 0 n n . c m - 2 - s - l . The contribution of the epithermal neutron flux was less than 1% of the total flux. After 5-6 wk of cooling each irradiated biological sample were transferred into a new container and then weighed. All samples were placed on the top face of the Ge(Li) detector having 3 mm glass entrance window and the resolution of 6.2 keV at 1332.5 keV. Se, Sb, Zn and Co in the irradiated samples were determined after 6 wk of cooling by the following ~,-rays: 7SSe (264.5 keV), 124Sb (602.7keV), 65Zn (lll5.5keV) and 6°Co (1332.5keV) (IAEA, 1987). The qualitative and quantitative detection limits (Rogers, 1970) for determined elements in the absence of interfering activities are summarized in Table 1. The biological reference material (SRM 1577b bovine liver) was used for the evaluation of the accuracy of applied analytical procedure. Each sample was counted for 2000--4000 s live time on the Ge(Li) detector (active volume 36 em 3) connected to a 4096 channel pulse-height analyzer (1024-NTA, 4 K, Hungary) based on the CAMAC interface and the IBM PC computer. The total deadtime correction was always less than 3%. The concentrations of Se, Sb, Zn and Co were determined by comparison with standards having similar amounts of the elements as the samples. The precision of the measurements was calculated as the statistical counting error of the net peak area (Amid, 1981).
Irradiation and activity measurements The dry cells of mass (63-218 rag) and standards (0.011-98 #g) were wrapped in an aluminium foil. (The foil had been heated for 100 h at 140°C.) The
ResuRs
and Discussion
The yeast cells of Saccharomyces cerevisiae were exposed to Sb and SeO2 or (CySe): but no lesions or
Table2. Uptake yieldof Sb by the yeastcells(/~g/g)and the "percentagechanges''a (in parentheses)as a functionof the chemical form of the So-compoundin the culturemedium,and of the exposure time Initial concentration Incubationtime (h) of Sb, Se
Control group
(tool/L) in medium No addition
2,5 IA] .
II
Sb SeO2
10-5 10-4
78.7 _+4.8 (-5.5%)
68.8 + 2.8 ( - 13,6%)
62.3 + 2.6 (-17.2%)
61.2 + 2,3 (-12.3%)
73.9 + 2.3 (-6.02%)
III
Sb (CySe)2
10-5 5 x 10-5
75.4 ± 5.3 (-9.5%)
69.7 + 2.4 (-14.2%)
66.4 ± 6.9 (-11.7%)
63.1 + 2,4 (-9.6%)
82.2 4-0.8 (4.3%)
79.6 ± 2.9
75.2 ± 1.7
69.8 +_2.6
78.8 + 2.4
No. Group I
Sb I0-~ 83.3 + 2.4 no ~ 'Percentage changes relative to absence of Se (i.e. group lV ). IV
8 [131 .
.
27 [C] .
50 [D]
75 [El
.
Use of INAA
1297
Table 3. Uptake yield of Sea by the yeast ceils (/,tg/g) and the "'percentage changes''b (in parentheses) as a function of Sb and the chemical form of the Se-compound in the culture medium, and of the exposure time Initial concentration of Sb, Se (mol/L) in medium
No. Group I
Incubation time (h) 2.5 [A]
8 IBI .
27 IC] .
.
50 ID]
75 [El
Control group c
No addition
.
11
Sb SeO 2
10 -5 10 4
28.7±4.8 (-4.3%)
27.7±3.2 (14.5%)
38.3±4.2 (0%)
37.4±0.4 (32.2%)
40.3_+4.8 (38.6%)
Iht
SeO 2
10 4
30.0±0.6
24.2±0.8
38.3±5.1
28.3±0.9
29.1 ± 1.3
III
Sb (CySe) 2
10 ~ 5 x 10 ~
34.4±2.5 (-2.9%)
36.8±2.1 (-11.9%)
44.9±0.8 (-3.7%)
38.7_+0.5 (1.6%)
4 2 . 4 ± 1.7 (15.6%)
III~
(CySe)2
5 × 10 5
35.4 ± 2.5
38.1 ± 1.7
35.8 ± 2.0
IV c
Sb no Se
10 -5
.
41.8 ± 1.4 .
.
46.6 _++1.2
.
.
.
aPercentage changes relative to no added Sb (i.e. groups ct). bThe Se concentration (#g/g) in SRM 1577b bovine liver--found: 0.75 ± 0.07 (certified: 0.73 ± 0.06). ¢Se content in the cells below the quantitative detection limit of Se.
symptoms of Sb or Se intoxication were found. The data in Table 2 show that in the absence of selenium, Sb is taken up by the cells and the highest concentration of Sb in the yeast was observed during the initial 2.5 h of the incubation. From the data shown in Table 2 it follows that the both Se-compounds, in general, in the minute decrease of the Sb level of the cells (only for the 75th h of the incubation the presence of (CySe)2 produced slight enhancement of the concentration of Sb in the cells). This phenomenon can be particularly observed in the presence of SeO 2: the Sb level of the yeast was regularly lower in comparison with the single Sb-supply. The present results strongly suggest that the Sb dose (i.e. 10 -5 M) in the medium was too small to produce a marked effect on the Se level of the cells (Table 3). Indeed, the presence of Sb in the medium increased the Se level only after long incubation times. The presence of SeO2 and Sb in the medium
produced observable enhancement of the Se level of the cells in the 50th and 75th h of the incubation. This phenomenon was altered by the presence of (CySe)2: the concentration of Se in the yeast was slightly higher in the 75th h of the incubation (the mutual interaction between Se and Sb). Table 3 also illustrates the Se intake from the culture medium containing only SeO2 or (CySe)2. It was found that the yield of the Se uptake by the cells (regardless of Sb dosage) was less when the yeast was incubated in the medium containing SeO2. The present work proved that (CySe)2 resulted in a higher uptake yield of Se by the yeast in comparison with SeO2. Zinc and cobalt
The results in Table 4 clearly proved that the presence of Se-compounds and/or Sb induced changes in the Zn and Co levels of the yeast. The
Table 4. Zn and Co level of the yeast ceils per unit dry mass (/~g/g)' Incubation time (h) No. Group
2.5 [A]
8 [B]
27 [C]
50 [D]
75 [El
Control I
Zn Co
310 ± 8 0.92 _+ 0.05
410 ± 9 0.98 ± 0.04
314 ± 2 0.98 ± 0.04
281 ± 8 0.79 ± 0.04
305 ± 8 0.99 ± 0.05
II SeO2 + Sb b
Zn Co
2 9 1 ± II 1.47 ± 0.12
284±5 1.60 ± 0.08
290__.6 1.30 ± 0.10
300±6 1.30 ± 0.05
285_+11 1.31 ± 0 . 0 7
Iht SeO 2
Zn Co
325 ± 7 0.86 ± 0.05
317 ± 3 1.29 __.0.04
283 _+ 13 1.01 ± 0.09
291 ± 17 1.05 ± 0.08
264 ± 12 0.94 ± 0.08
IIl (CySe)2 + Sb b
Zn Co
289 ± 6 1.36 ± 0.05
281 ± 8 1.38 ± 0.05
246 __. 7 1.33 ± 0.07
254 ± I 1 1.36 ± 0.06
277 ± 8 1.39 ± 0.05
Ill~t (CySe)2
Zn Co
286 ± 8 1.27 ± 0.04
265 ± 8 1.38 ± 0.13
274 ± 9 1.54 ± 0.10
249 ± 8 1.44 ± 0.06
255 ± 9 1.41 ± 0.07
IV Sb b
Zn Co
2 5 6 ± 12 1.77 ± 0.06
2 6 0 ± 13 1.75 + 0.06
2 4 3 + 11 1.65 ± 0.05
2 6 0 ± 12 1.80 + 0.08
264_+ 15 1.80 ± 0.06
'The concentration of Sb (as Sb205) in the yeast medium: 10 -s M. bThe Zn and Co concentrations (/~g/g) in SRM 1577b bovine liver--found: 114 ± 2 and 0.24 ± 0.01, respectively (certified--Zn: 127 ± 16 and Co: ~0.25).
M. Czauderna et al.
1298
single SeO2 supply had only a minor antagonistic effect by causing a slight (however irregular) decrease in the Zn level of the yeast. The addition of (CySe)2 alone in the medium markedly diminished the Zn level, so it seems reasonable to suggest that the organic form of Se interacts efficiently with Zn. The Sb dosage alone produced the regular and significant diminishment of the content of Zn in the ceils. This phenomenon can also be generally observed in the combined presence of the Sb and SeO2 or (CySe) 2. No significant changes of Co were found in the cells, when the yeast was incubated in the medium containing only SeO2. For the yeast exposed to both SeO2 and Sb, the amount of Co in the cells markedly increased. The addition of (CySe)2 alone or in combination with Sb resulted in a significant enhancement of Co abundance in the yeast. The presence of Sb alone in the medium was accompanied by the highest increase in the Co level of the cells.
Conclusion INAA is suitable for the instrumental determination of trace amounts of Se, Sb, Co and Zn in the yeast. Moreover, it has been employed for the simultaneous determination of changes of Se, Sb, Zn and Co levels of the cells when the yeast was incubated in the medium containing tested compounds. Thus, INAA is particularly useful for studying interactions between trace elements in microorganisms. INAA combines a true multi-element capability, high accuracy, good sensitivity and moderate price with applicability to yeast samples (De Bruin, 1992). Acknowledgement--This work was supported by a grant
from KBN No. 4 4282 91 02.
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