Journal of Nuclear and Radiochemical Sciences, Vol. 6, No. 1, pp. 111-113, 2005

Accepted
Uptake of Radiocesium by Hypha of Basidiomycetes– Radiotracer Experiments –Tadaaki Ban-nai,*, a Satoshi Yoshida,a Yasuyuki Muramatsu,b and Akira Suzukic
aNational Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi, Chiba 263-8555, Japan
Gakushuin University, Mejiro 1-5-1, Toshima-ku, Tokyo 171-8588, Japan
cChiba University, 1-33 Yayoi-cho, Inage-ku, Chiba-shi, Chiba 263-8522, Japanb

Received: November 15, 2004; In Final Form: April 21, 2005
Uptake of Cs and its effects on mushroom growth were studied using a simulated medium in which K+ and NH4+
concentrations were lowered as much as possible. The hyphae of H. vinosophyllum did not grow in the simulated
medium with 2 mmol CsCl. The growth increased with the addition of K, indicating that K lowers the toxicity ofCs to mushrooms in the simulated medium. The inhibition of mushroom growth by Cs was greater than that byRb. The uptake of Cs was reduced by the addition of the monovalent cations of K+, Rb+, and NH4+.

1. Introduction
Many papers1−4 have reported high concentrations of radiocesium in mushrooms collected in Ukrainian and European
forests after the Chernobyl accident. The concentrations of137
Cs in forest mushrooms were markedly higher than those inautotrophic plants.5 Mushrooms sometimes affect the behaviorof Cs in the environment.6 Lindner et al.7 surveyed roe deercontamination with radiocesium isotopes in the rural prealpine
area of Oberschwaben in southwestern Germany beginning in
the autumn of 1986. They identified characteristic regionaland seasonal patterns of contamination that resulted from its
transfer from the soil to the grazing plants of these animals.
They suggested that the periodic maximum observed in roe
deer in the autumn was correlated with the mushroom season
in the grazing forests.
Since edible mushrooms for humans also have higher 137Csconcentrations than other foodstuffs, they are important for anestimation of internal radiation doses from 137Cs through foods.
Skuterud et al.8 reported dietary surveys and whole body monitoring at two sites in the Bryansk Region of the RussianFederation in order to estimate the transfer of Chernobyl radiocesium to humans. They found mushroom consumption was
the predominant factor in a 60 – 70% mean increase in radiocesium activity concentrations in humans in the autumn. The
contribution from a high concentration of 137Cs in mushrooms
was found worldwide because of atmospheric nuclear tests. In
our most recent study,9 the contribution of mushrooms to thetotal intake of 137Cs in Japanese was estimated to be 28% (onaverage) of the total intake of 137Cs, while that of 40K was estimated to be only 1.9%. Gaso et al.10 calculated the intake of137
Cs through 30 local mushroom species collected from 1993
to 1999 in a semi-natural temperate forest in Mexico and foundthat the mushrooms contributed 37% of 137Cs and 4% of 40K to
the total dietary intake of these contaminants. We suggest that
it is unreliable to neglect 137Cs in mushrooms when estimatinginternal doses of 137Cs. Therefore, the accumulation behaviorof radiocesium in mushrooms should be elucidated for understanding radiation protection and radioecology.
Accumulation of radiocesium in fruit bodies has been
observed by laboratory experiments. We carried out cultivation experiments for four basidiomycetes (Hebeloma vinoso*Corresponding author. E-mail: E-mail: t_bannai@nirs.go.jp.
FAX: +81-43-206-3267.

phyllum, Flammulina velutipes, Coprinus phlyctidosporus, andAgrocybe cylindracea) in a solid growth medium including
137
Cs as a radiotracer.11, 12 Our results showed that fruit bodiesof the four basidiomycetes accumulated Cs from the medium,
although there were large differences in Cs concentration
among mushroom species.11 The effects of Cs in the medium
on the accumulations by the fruit body of H. vinosophyllum
were investigated, and it was found that the concentration
ratios of 137Cs were not highly influenced by coexisting Cs in
the medium.12
On the other hand, some references have reported that Cs is
toxic to organisms. For example, Nishita et al. 13 cultivatedbean plants in nutrient solution at various Cs concentrations
and a constant K concentration of 20 ppm. Visibly reduced
plant growth occurred when the substrate Cs concentration was5 ppm. At a Cs concentration of 20 ppm, the fruits weredwarfed and deformed. No fruit was produced at a Cs concentration of 30 ppm. Matsudaira et al.14 cultivated the cellularslime mold, Dictyostelium discodeum A-3, in the usual
medium with 1 mM CsCl added. They found that the cell
doubling time in this medium was 1.1 times as much as thatwhen cultivated in the medium without Cs. The Merck index15
reports the LD50 (medium lethal dose, the quantity of a chemical that is estimated to be fatal to 50% of the organismstested) of CsCl to be 1.5 g/kg, or about 8.9 mmol/kg, intraperitoneally in rats, but values for RbCl and KCl are not given
there.
Though it is known that mushrooms concentrate Cs, there
are few reports on the role and the toxicity of Cs for mushrooms. It is difficult to evaluate the effects on growth andconcentration of Cs by varying the concentrations of monovalent ions in the medium because the usual media for cultivationof fungi have high concentrations of K+ and NH4+. Therefore,
we studied uptake of Cs and its effects on mushroom growthusing a synthetic medium in which K+ and NH4+ concentrations were reduced as much as possible.
2. Experimental
Hebeloma vinosophyllum NBRC31231 (IFO31231) wasused for the cultivation experiment. The medium compositionis shown in Table 1. We used a medium proposed by Suzuki16
with modifications designed to reduce K+ and NH4+ concentrations. A portion of the KH2PO4 was replaced with Na2HPO4 inorder to lower the K content in the medium. Glycine© 2005 The Japan Society of Nuclear and Radiochemical Sciences
Published on Web 07/31/2005

Ban-nai112 J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005
TABLE 1: Components of Media
Blank

MaltoseGlycineKH2PO4
Na2HPO412H2O
MgSO47H2O
FeSO4
CaCl2
ZnSO47H2O
CuSO45H2O
MnSO45H2O(NH4)6Mo7O24H2O
ThiaminNicotinamide

40 g
1g
0.14 g
2.7 g
0.3 g
0.01 g
1 mg
0.3 mg
0.1 mg
0.1 mg0.02 mg
0.5 mg
0.1 mgNH 4Cl (0.2 mmol)
NH 4Cl (2 mmol)
KCl (0.2 mmol)
KCl (2 mmol)
RbCl (0.2 mmol)
RbCl (2 mmol)
CsCl (0.2 mmol)

CsCl + KCl (1 mol)
CsCl +NH4Cl (1 mol)00.10.15

Figure 1. Growth of hyphae of Hebeloma vinosophyllum in various
media. Arithmetic mean (●) and range of standard deviation (solidlines). Original data are shown by ◆, if only two data are available.

(H2NCH2COOH) was added in exchange for NH4Cl in order toreduce NH4+ in the medium. We assumed that cells uptake
glycine through different channels from the NH 4+ channels
since glycine is an organic substance. Cells can make NH 4+
from glycine in the cell by using glycine synthase (see eq 1).17
Therefore, the amount of glycine was decided by equating thenitrogen content to the NH4Cl content and assuming that allglycine in the medium was converted into NH4+. The amountof Ca was reduced in order to prevent the precipitation of Ca
phosphate in the medium.

CO2 + NH4+ + 5,10- methylene H4 folic acid + NADH

0.05

Weight (g- wet)Distilled Water 1 L

H3+NCH2COO− + H4 folic acid + NAD+ →No GrowthCsCl (2 mmol)(1)Concentrations of K+, Rb+, Cs+, and NH4+ in several medium
solutions were controlled by adding additional amounts ofKCl, RbCl, CsCl, and NH4Cl (see Table 2). We refer to thebasic medium without the additional chlorides as “blankmedium.” The amount of K in the blank medium was successfully reduced to 1/25 of that in the medium that we had usedpreviously.8 Each medium solution was sterilized by filtration
through a membrane filter. We poured 25 mL of each medium
solution into bottles (50 mL) and added the radiotracer, 137Cs(661.7 keV, half life: 30.07 y), as 137CsCl. Since the 137Cs used
was carrier-free, the amount of elemental Cs added as 137Cs
was negligible. Two or three bottles were prepared for eachmedium solution. The fungus was pre-cultivated on themedium in a petri dish. After pre-cultivation, the fungus with
its accompanying medium was taken out using a cork borer
and placed on the medium in the bottles. After incubating thebottle contents at 25 ˚C for three weeks, the hyphae wereremoved using tweezers. The hyphae were washed with radiotracer-free medium by placing them on a membrane filter in asuction funnel.
TABLE 2: Extra Salt in Medium (medium name in paper)1) nothing(Blank medium)(0.2 mmol NH4Cl medium)2) 0.2 mmol NH4Cl(2.0 mmol NH4Cl medium)3) 2.0 mmol NH4Cl
4) 0.2 mmol KCl(0.2 mmol KCl medium)5) 2.0 mmol KCl(2.0 mmol KCl medium)6) 0.2 mmol RbCl
(0.2 mmol RbCl medium)7) 2.0 mmol RbCl
(2.0 mmol RbCl medium)8) 0.2 mmol CsCl
(0.2 mmol CsCl medium)9) 2.0 mmol CsCl
(2.0 mmol CsCl medium)10) 1.0 mmol CsCl + 1.0 mmol KCl(1 mmol CsCl+KCl medium)11) 1.0 mmol CsCl + 1.0 mmol NH4Cl (1 mmol CsCl+NH4Cl medium)

The hyphae were placed in polyethylene vials and weighed.
The concentrations of 137 Cs in hyphae were determined bymeans of a NaI scintillation counter (Aloka ARS-380). Decay
corrections were made at the start of the measurement. The
concentration ratio was calculated as “activity of 137 Cs inhyphae of mushroom (Bq/g, wet wt.)” divided by “activity of137
Cs in medium (Bq/g, wet wt.)”.
3. Results and Discussion
Effect on growth of hyphae. The hyphal growth in various
medium solutions is shown in Figure 1. The growth of thehyphae is usually described as an extension or weight increaseof the hyphae; we used the wet weight increase in this report.
The growth of H. vinosophyllum in 0.2 mmol NH4Cl mediumand 2 mmol KCl medium was larger than that in the blankmedium. Addition of NH4+ and K+ promoted the growth ofhyphae.
No hyphal growth in 2 mmol CsCl medium was observedby visual inspection. The hyphal growth in 0.2 mmol CsCl
medium was less than that in the blank medium. The growth
was inhibited by increasing the Cs concentration of the medium.
Hyphae grew in the media that contained more than 2 mol Cs
if there were sufficient amounts of NH 4 + and K + present. 18Hence, not only the toxicity of Cs but also the low NH4+ and K+
levels contributed to the inhibition of hyphal growth.
The hyphal growth in the 1 mmol CsCl + KCl medium waslarger than that in the 0.2 mmol CsCl medium. In terms of low
K content, the hyphal growth in 1 mmol CsCl medium shouldbe less than that in 0.2 mmol CsCl medium. Our results indicate that the toxicity of Cs+ decreased with the addition of K+.
We thought that the obstruction by Cs of enzyme activities was
one of the causes for the growth inhibition of mushrooms byCs. Some enzymes, such as rabbit liver fructose-1,6-bisphosphatase,19 have been found to lose activity in the presence ofCs rather than K. The influence of Cs increased as the ratio ofCs to K increased.
The hyphal growth of H. vinosophyllum in 0.2 mmol CsCl
medium was less than that in 0.2 mmol RbCl medium. The
hyphae of H. vinosophyllum in 2 mmol RbCl medium grewslightly while the hyphae in 2 mmol CsCl medium did not
grow at all. The hyphal growth was inhibited more strongly by
Cs than by Rb.
The effect of the cultivation medium on the concentration ratio of 137Cs. The concentration ratio is one parameterthat shows the uptake of elements from a medium to anorganism. The results for the concentration ratio of 137Cs inhyphae cultivated in various medium solutions are shown inFigure 2.

J. Nucl. Radiochem. Sci., Vol. 6, No. 1, 2005 113Uptake of Radiocesium by Hypha of Basidiomycetes

cultivated in 0.2 mmol RbCl medium whereas the hyphal
growth decreased in the former.
The Cs concentration ratio in the hyphae in this study was
lower than the concentration ratio in mushroom fruit bodies inour previous study11 even taking into account the difference ofmedia. However, Cs might be a physiologically non-essential
element for H. vinosophyllum. It has been reported that fruit
bodies have an ability to change Cs from an ionic form tocomplexes with pigment; Aumann et al.22 reported complexation of 137Cs by the cap pigments (norbadione A) of the bay
boletus (Xerocomus badius).

Blank
NH 4Cl (0.2 mmol)
NH 4Cl (2 mmol)
KCl (0.2 mmol)
KCl (2 mmol)
RbCl (0.2 mmol)
RbCl (2 mmol)
CsCl (0.2 mmol)
CsCl (2 mmol)

No Growth

References

CsCl + KCl (1 mol)
CsCl +NH 4Cl (1 mol)02468Concentration ratio of 137 Cs

Figure 2. Concentration ratio of 137Cs in hyphae of Hebeloma vinosophyllum cultivated in various media. Arithmetic mean (●) and rangeof standard deviation (solid lines). Original data are shown by ◆, if
only two data are available.

The concentration ratio of 137Cs in hyphae cultivated in theblank medium was about 5.3 ± 1.2. The concentration ratio(5.8 ± 1.8) of 137Cs in hyphae cultivated in the 0.2 mmol CsClmedium was just slightly different from that in the blankmedium. This indicates that the concentration ratios of 137Cs in
the hyphae were not affected by that of Cs+ in the medium.
The concentration ratios of 137Cs in hyphae cultivated in themedium solutions containing KCl, RbCl, or NH4Cl were lower
than the ratio of 137 Cs in hyphae cultivated in the blankmedium. This indicates that the concentration ratio of 137Cs
was inhibited by monovalent cations other than Cs +. In thecase of the addition of 2 mmol cations to the medium, theconcentration ratio of 137Cs was in the order NH4+ > K+ > Rb+ >Cs+. Ammonia ions inhibited the uptake of 137Cs, though NH4+
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medium were larger than that in the 0.2 mmol CsCl medium(Figure 1). This was caused by decreasing the absorption ofCs through the addition of NH4+ or K+ in the medium. As aresult, the toxicity of Cs in the medium decreased.
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mechanism of radiocesium. The concentration ratio of 137Cs inhyphae decreased with increasing K or Rb concentrations.
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suggesting a decrease in the accumulation of K, P, and Fe by
Saccharomyces cerevisiae during Cs uptake. These results
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Cs concentration in the media. This might be caused by thedifference of species and by the K concentration in themedium. The concentration ratio of 137Cs in hyphae cultivatedin 2 mmol KCl medium was higher than that cultivated in 0.2mmol KCl medium. This difference in the concentration ratio
was caused by better hyphal growth.
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