Evaluation of Chlorella as a Decorporation Agent to Enhance the Elimination of Radioactive Strontium from Body.

BACKGROUND: Release of radionuclides, such as 137Cs and 90Sr, into the atmosphere and the ocean presents an important problem because internal exposure to 137Cs and 90Sr could be very harmful to humans. Chlorella has been reported to be effective in enhancing the excretion of heavy metals; thus, we hypothesized that Chlorella could also enhance the elimination of 137Cs or 90Sr from the body. We evaluated the potential of Chlorella as a decorporation agent in vitro and in vivo, using 85Sr instead of 90Sr.
METHODS: In vitro experiments of adsorption of 137Cs and 85Sr to Chlorella were performed under wide pH conditions. The maximum sorption capacity of Chlorella to strontium was estimated using the Langmuir model. A 85Sr solution was orally administrated to mice pretreated with Chlorella. At 48 h after 85Sr administration, the biodistribution of radioactivity was determined.
RESULTS: In the in vitro experiments, although 85Sr barely adsorbed to Chlorella at low pH, the 85Sr adsorption ratio to Chlorella increased with increasing pH. The maximum sorption capacity of Chlorella to strontium was 9.06 mg / g. 137Cs barely adsorbed to Chlorella under any pH conditions. In the biodistribution experiments, bone accumulation of radioactivity after 85Sr administration was significantly decreased in the Chlorella pretreatment group compared with the non-treatment control group.
CONCLUSIONS: In conclusion, these results indicated that Chlorella could inhibit the absorption of 90Sr into the blood and enhance the elimination of 90Sr from the body through adsorption in intestine. Further studies are required to elucidate the mechanism and the components of Chlorella needed for adsorption to strontium and could promote the development of more effective decorporation agents.

Introduction

Radionuclides released into the atmosphere and the ocean by nuclear power plant accidents, such as the Chernobyl and Fukushima disasters, are important problems for the ecosystems and humans [1-5]. Among the released radionuclides, cesium-137 (137Cs) and strontium-90 (90Sr) are considered to be harmful to humans because both have a long half-life and can contaminate aquatic ecosystems and food products due to their high solubility in water [3].

137Cs (t1/2 = 30.1 y) is one of the most important nuclear fission elements and decays to barium-137m (137mBa) with the emission of β-particles (0.51 MeV). 137mBa in turn decays to the stable isotope 137Ba with the emission of gamma rays (0.66 MeV) by isomeric transition. Because the physical half-life of 137mBa (t1/2 = 2.55 m) as a daughter radionuclide is sufficiently short compared with that of 137Cs, a radioactive equilibrium is established. Cesium is an alkaline metal and a member of the IA family of the periodic table. As its chemical properties are similar to those of potassium, cesium is immediately absorbed after intake and is fairly uniformly distributed throughout body [6]. In particular, cesium shows high accumulation in muscle and is retained in the entire body, with a biological half-life of approximately 100 days in healthy adult male humans [7].

90Sr (t1/2 = 29.1 y) is another important nuclear fission radionuclide. 90Sr decays to yttrium-90 (90Y) with the emission of β-particles (0.54 MeV). 90Y in turn decays to the stable isotope zirconium-90 (90Zr) with the emission of high energy β-particles (2.27 MeV). Because the physical half-life of 90Y (t1/2 = 2.67 d) as a daughter radionuclide is sufficiently short compared with that of the parent radionuclide 90Sr, a radioactive equilibrium is established. Strontium (Sr) is an alkaline earth metal and a member of the IIA family of the periodic table. Therefore, strontium acts as a calcium mimic and shows high accumulation in bone through incorporation into mineralizing collagen during new bone formation [8,9]. The biological half-life of 90Sr is very long [10]. Therefore, internal exposure to 90Sr is associated with the development of leukemia and osteosarcoma [11,12].

Because of the properties mentioned above, internal exposure to 137Cs and 90Sr radionuclides can be very harmful to humans. Compounds that inhibit the absorption of these radionuclides from the gastrointestinal tract into the blood and enhance their elimination after intake are useful in decreasing the absorbed radiation dose when people are exposed to these radionuclides. Indeed, insoluble ferric(III) hexacyanoferrate (Fe4III[FeII(CN)6]3)·xH2O (Radiogardase®, Prussian blue insoluble, PB), a compound that promotes the elimination of 137Cs from the body, is useful for the treatment of radiocesium poisoning and has been approved for use by regulatory bodies, such as U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). In the case of 90Sr, some compounds such as alginate promote excretion as reported from basic research [13,14].

Chlorella is a genus of single-cell green algae that grows in fresh water. It is known as a health food composed of approximately 1%–4% chlorophyll, 55%–67% protein, 9%–18% dietary fiber, and large amounts of minerals and vitamins [15]. In a previous study, Chlorella was shown to enhance the excretion of cadmium (Cd) by inhibiting intestinal Cd absorption in rats [16]. In another report, lead (Pb) concentration in blood was markedly reduced when Pb and Chlorella vulgaris extract were simultaneously administrated compared with when only Pb was administrated [17]. Furthermore, another study reported enhancement of mercury (Hg) elimination by Chlorella [18]. Because Chlorella has been shown to be effective in enhancing the excretion of heavy metals, we hypothesized that Chlorella could also inhibit the absorption and enhance the elimination of radioactive cesium or strontium from the body after their oral intake. We evaluated the potential of Chlorella as a decorporation agent for radioactive cesium and strontium in vitro and in vivo. In this study, 85Sr (T1/2 = 64.8 d) was used instead of 90Sr because 85Sr emits gamma rays that can be measured easily.

Materials and Methods

Materials

85SrCl2 (> 111 GBq/g) was purchased from PerkinElmer (Waltham, MA, USA). 137CsCl was purchased from Eckert & Ziegler Isotope Products Inc. (Valencia, CA, USA). Chlorella powder was supplied by Daesang Corp. (Seoul, Korea). Other reagents were of reagent grade and used as received.

In vitro experiments of adsorption of 137Cs and 85Sr to Chlorella

Chlorella (10, 30, or 100 mg) was suspended and 137Cs or 85Sr was added in 1 mL of the first test solution (artificial gastric juice, pH 1.2) or the second test solution (artificial intestinal juice, pH 6.8) defined in the Japanese Pharmacopoeia. After shaking the suspension at 1,000 rpm and 37°C for 1 h using a shaking incubator (SI-300C; AS ONE Corp., Osaka, Japan), the samples were centrifuged at 10,000g and room temperature for 10 min. The radioactivity of the supernatant was measured using an auto-well gamma counter (ARC-380; Hitachi Aloka Medical, Ltd., Tokyo, Japan), and the counts were corrected for background radiation. Control experiments were performed using the same procedure but without Chlorella. The binding ratios were determined as follows:

Binding ratio to Chlorella (%) = [1 − (radioactivity of the supernatant of each sample) / (radioactivity of the supernatant of the respective control)] × 100

pH dependence of in vitro adsorption of 137Cs and 85Sr to Chlorella

Chlorella (30 mg) was suspended and 137Cs or 85Sr was added in 1 mL of 0.01 M HEPES buffer solution (pH 2–13), and shaking, centrifugation, and radioactivity measurement were conducted as described above. The pH of each suspension after shaking was measured.

Reversibility of the adsorption potential between Chlorella and 85Sr with pH variation was evaluated. Chlorella (30 mg) was suspended in 1 mL of the first test solution (pH 1.2) defined in the Japanese Pharmacopoeia and then shaken at 1,000 rpm and 37°C for 1 h. After centrifugation at 10,000g and room temperature for 10 min, 800 μL of the supernatant was removed. Following this, 23, 25, or 27 μL of 1M NaOH solution and 777, 775, or 773 μL of 0.01M HEPES buffer solution (pH 8) were added to the Chlorella suspension. Thereafter, 85Sr was added to the Chlorella suspension, and the suspension was shaken at 1,000 rpm and 37°C for 1 h. After centrifugation at 10,000g and room temperature for 10 min, the radioactivity and pH of the supernatant were measured as described above.

Langmuir model

Chlorella suspension samples (10 mg/mL) containing 1, 10, 50, 100, 200, 500, and 1000 ppm of Sr and 832.5 kBq/mL of 85Sr as a final concentration in 0.02 M HEPES buffer (pH 7.4) were prepared by mixing radioactive strontium and non-radioactive strontium. After shaking at 1,000 rpm and 37°C for 1 h, the binding ratio of each sample to Chlorella was determined using the method described above.

Effects of cations (Na+, K+, or Ca2+) on in vitro adsorption of 85Sr to Chlorella

Chlorella (10 mg) was suspended and 85Sr was added in 1 mL of 0.02 M HEPES buffer solution containing 500, 1000, or 10000 ppm of Na+, K+, or Ca2+, which were prepared by dissolution of NaCl, KCl, or CaCl2. After shaking at 1,000 rpm and 37°C for 1 h, the binding ratio of each sample to Chlorella was determined using the method described above.

In vivo Chlorella experiments for promoting excretion of 85Sr from the body

Experiments with animals were conducted in strict accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University. The animal experimental protocols used were approved by the Committee on Animal Experimentation of Kanazawa University (Permit Number: AP-143039). The animals were housed with free access to food and water at 23°C with a 12-hour alternating light/dark schedule. Chlorella suspended in a 5% aqueous glucose solution (75 mg / 500 μL) was orally administrated to 6-week-old male ddY mice (n = 19, 27–30 g, Japan SLC, Inc., Hamamatsu, Japan) 5 times every 90 min. The mice were fasted for 36 h after the first administration of Chlorella. Thirty minutes after the final administration of the Chlorella suspension, a 85Sr solution (24–40 kBq / 100 μL) was orally administrated to the mice. In the control group (n = 28), 500 μL of a 5% aqueous glucose solution was administrated instead of the Chlorella suspension. In the alginate group (n = 14), sodium alginate in a 5% aqueous glucose solution (15 mg / 500 μL) was administrated instead of the Chlorella suspension. Mice were sacrificed 48 h after 85Sr administration. Tissues of interest were removed and weighed, and radioactivity counts were measured. Complete left femurs were isolated as representative bone samples. Whole-body in Table 1 means the sum of whole-body radioactivity except the gastrointestinal tract.

Table 1

Biodistribution of radioactivity at 48 h after oral administration of 85Sr in mice with pretreatment of Chlorella or alginate.

Tissue	Control	Chlorella	Alginate
Blood	0.02 (0.01)	0.02 (0.02)	0.02 (0.01)
Liver	0.01 (0.00)	0.01 (0.00)	0.01 (0.00)
Kidney	0.03 (0.01)	0.02 (0.01)	0.02 (0.01)
Small-intestine*	0.09 (0.04)	0.08 (0.03)	0.24 (0.23)†
Large-intestine*	0.16 (0.07)	0.14 (0.07)	0.50 (0.47)†
Spleen	0.05 (0.06)	0.02 (0.04)	0.01 (0.01)†
Pancreas	0.02 (0.02)	0.02 (0.02)	0.02 (0.01)
Lung	0.03 (0.01)	0.03 (0.03)	0.02 (0.01)
Heart	0.02 (0.02)	0.02 (0.03)	0.01 (0.01)
Stomach*	0.04 (0.02)	0.03 (0.02)	0.73 (0.91)†
Bone	29.28 (5.15)	25.96 (4.32)†	27.79 (4.08)
Muscle	0.03 (0.04)	0.02 (0.03)	0.01 (0.01)
Whole-body*	26.83 (4.60)	23.14 (3.22)†	28.58 (4.88)

Expressed as % injected dose per gram. Each value represents the mean (SD) for twenty-eight (control), nineteen (Chlorella), or fourteen (alginate) animals.

*Expressed as % injected dose.

†p < 0.05 vs. Control

Statistical evaluation

Data are expressed as means ± standard deviations where appropriate. Dunnett’s multiple comparison test compared with the control group was used for in vivo experiments. Results were considered statistically significant at p < 0.05.

Results

The adsorption ratios of 137Cs and 85Sr to Chlorella are shown in Fig 1. 85Sr adsorbed to Chlorella in a quantity-dependent manner under a neutral pH condition. However, it barely adsorbed to Chlorella under an acidic pH condition. 137Cs barely adsorbed to Chlorella under both neutral and acidic pH conditions.

Fig 1

Adsorption of radionuclides to Chlorella.

Binding ratios of 85Sr at pH 1.2 (closed circles), 85Sr at pH 6.8 (open circles), 137Cs at pH 1.2 (closed diamonds), or 137Cs at pH 6.8 (open diamonds) to Chlorella depended on the Chlorella concentration. Data are expressed as the mean ± SD of three samples.

pH dependence of in vitro adsorption of 85Sr to Chlorella

The open circles in Fig 2 show the pH dependence of 85Sr adsorption to Chlorella. Although 85Sr barely adsorbed to Chlorella at low pH, its adsorption ratio to Chlorella increased with increasing pH. The closed circles in Fig 2 show 85Sr adsorption to Chlorella at pH 6–7 after exposure to acidic conditions. It was confirmed that 137Cs was barely adsorbed to Chlorella under any pH conditions (open diamonds).

Fig 2

pH dependence of Chlorella adsorption.

Binding ratios of 85Sr (open circles) or 137Cs (open diamonds) to Chlorella depended on the pH conditions. Binding ratios of 85Sr (closed circles) after exposure to acidic conditions. Data are expressed as the mean ± SD of three samples.

Adsorption capacity of Chlorella with Sr was evaluated by the Langmuir model, defined as follows: where q (mg / g) represents Sr binding per Chlorella, Ceq (mg / L) is the equilibrium concentration of strontium, qmax (mg / g) is the maximum sorption capacity, and a (L / mg) is the sorption constant.

Fig 3 shows that the binding of strontium to Chlorella increased at low metal concentrations and began to level off at high concentrations, indicating saturation of the adsorption sites. These data showed an excellent fit with the Langmuir model, indicating the correct assumption of a uniform surface with finite identical sites and monolayer adsorption of the adsorbate. The maximum sorption capacity was estimated to be 9.06 mg / g.

Fig 3

Langmuir model.

Adsorption capacity of Chlorella to strontium. Data are expressed as the mean ± SD of three samples.

Fig 4 shows that the binding of strontium to Chlorella was decreased by the presence of cations. Although Na+ and K+ hardly decreased the binding of strontium to Chlorella at low concentration of the cations, but the addition of Ca2+ remarkably decreased the binding of strontium to Chlorella even at low concentration of Ca2+.

Fig 4

Adsorption of 85Sr to Chlorella in the presence of cations.

Binding ratios of 85Sr to Chlorella in the presence of Na+ (closed circles), K+ (open circles), or Ca2+ (closed diamonds). Data are expressed as the mean ± SD of three samples.

In vivo experiments for promoting excretion of 85Sr from the body

Table 1 lists the biodistribution of radioactivity at 48 h after oral administration of 85Sr with pretreatment of Chlorella or sodium alginate, or without pretreatment as a control group. Pretreatment with Chlorella significantly decreased the accumulation of radioactivity in the bone and the whole-body (excluding the gastrointestinal tract) compared to the control group. Meanwhile, the difference between radioactivity in bone and whole body was not statistically significant between control group and sodium alginate treated group. Little radioactivity existed in almost all tissues except the bone.

Discussion

In in vitro adsorption experiments, Chlorella did not adsorb 137Cs at any pH range or concentration. Therefore, in vivo experiments were not performed using 137Cs. On the other hand, we had anticipated that Chlorella could adsorb radioactive strontium because a column packed with Chlorella vulgaris had previously been proposed as a means to remove strontium cations from contaminated aqueous solutions [19]. 85Sr adsorbed to Chlorella under neutral and weak basic pH conditions, whereas it barely adsorbed under acidic pH conditions. The low pH of the gastric juice must prevent the adsorption of 85Sr to Chlorella. Therefore, any inhibition of absorption or enhanced excretion of radiostrontium observed in vivo must have been the result of adsorption of strontium to Chlorella in the intestine. Thus, we investigated whether the Chlorella sample that was exposed to an acidic condition and then adjusted to neutral pH could adsorb 85Sr or not. The results indicated that after exposure to an acidic solution, Chlorella still adsorbed 85Sr to the same degree at neutral pH compared with the samples not exposed to the acidic solution (Fig 2). These results indicated that Chlorella could bind radiostrontium in the intestine after passing through the acidic conditions in the stomach.

From the results of in vitro experiments, it was assumed that abundant Chlorella in the intestine could adsorb radioactive strontium and inhibit absorption, resulting in enhanced excretion of radioactive strontium. Thus, in vivo experiments to enhance the elimination of radiostrontium were designed using mice given multiple oral administrations of Chlorella. The results of the in vivo experiments showed that the accumulation of radioactivity in the bones and the whole-body except the gastrointestinal tract in the Chlorella treatment group was significantly lower than that in the control group. As described in the Introduction section, it has been known that orally administrated strontium accumulates at high levels in the bone after absorption into the blood. The bone accumulation of radioactivity after intake of radioactive Sr must be correlated to AUC of radioactivity level in blood (time-activity curve) because Sr must not be metabolized and Sr is not released from bone after adsorption [20,21]. Therefore, the bone accumulation could be index of the radioactivity level of Sr in blood. The difference in the biodistribution after 85Sr administration between the Chlorella treatment group and the control group indicates that Chlorella inhibited 85Sr absorption into blood and enhanced its excretion as a result of adsorption between Chlorella and 85Sr in the intestine.

It has been reported that alginate reduces absorption and enhances excretion of strontium [22-24], but the alginate did not inhibit absorption of 85Sr in in vivo experiments of this study because the bone accumulation of radioactivity in the alginate treated group was not significantly reduced compared to that in the control group. Although the reason for this is not known, in a previous report using rats, whole-body retention of 85Sr was not significantly changed by treatment of single oral administration of alginate (2%, 1 mL) just before oral administration of 85Sr. Meanwhile, when 85Sr was orally administrated after feeding on 10% alginate containing diet for 10 days, the whole-body retention of 85Sr in alginate-treated rats was decreased sharply compared with that in control rats [23]. Thus, preadministration for a longer period of time or preadministration of a larger amount alginate might be necessary to enhance excretion of 85Sr in this study.

As mentioned above, 85Sr absorption was significantly inhibited in the Chlorella treatment group compared with the control group, but the inhibitory effects of Chlorella were smaller than expected. This finding seems to be derived from some of following causes. First, it has been reported that Chlorella promotes dioxin excretion [15]. As some of the dioxin that accumulates in the body is secreted with bile into the intestine [25], Chlorella could inhibit not only dioxin absorption but also dioxin reabsorption in the intestine, thereby promoting excretion of dioxin from the body into the feces. On the other hand, strontium barely enters the enterohepatic circulation. It is known that after absorption into blood, strontium mainly accumulates in the bones or is mainly excreted into the urine via kidney. Therefore, the strategy of using Chlorella as a decorporation agent for radioactive strontium can be applied only before absorption; thus, Chlorella has a lower chance of encountering strontium compared with other target compounds that enter the enterohepatic circulation. Second, given that Chlorella has been used as a health food or functional food in USA, Japan, and other countries, its safety after administration must be promising. However, the maximum sorption capacity of Chlorella to strontium is not very high (9.06 mg/g). In contrast, it has been reported that the maximum sorption capacity of Prussian blue to cesium is 16.5 mg/g in synthetic intestinal fluid pH 8.6 [26], 238 mg/g at pH 7.5 [27], and 715 mg/g at pH 7.5 [28]. Finally, the binding selectivity of Chlorella to strontium is not clear. In in vitro experiments, only strontium and some endogenous metals in Chlorella exist in the experimental system. However, because many types of metals exist under in vivo circumstances, the effects of some cations (Na+, K+, or Ca2+) on in vitro adsorption of 85Sr to Chlorella were evaluated (Fig 4). As the presence of the cations, especially Ca2+, decreased adsorption of 85Sr to Chlorella probably because of the chemical similarity between Sr and Ca, some metals, such as calcium, must compete with strontium for the Chlorella adsorption sites.

In summary, Chlorella did not adsorb cesium at any pH range but adsorbed strontium under neutral and weak basic pH conditions in in vitro experiments. Bone accumulation of 85Sr in the Chlorella pretreatment group was significantly lower than that in the non-treatment control group. Therefore, these results indicated that Chlorella could inhibit strontium absorption and enhance strontium elimination from the body through adsorption between Chlorella and strontium in the intestine. Further studies are required to elucidate the mechanism and the components of Chlorella needed for adsorption to strontium and could promote the development of more effective decorporation agents.