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.


Methods
In vitro experiments of adsorption of 137 Cs and 85 Sr to Chlorella were performed under wide pH conditions. The maximum sorption capacity of Chlorella to strontium was estimated using the Langmuir model. A 85 Sr solution was orally administrated to mice pretreated with Chlorella. At 48 h after 85 Sr administration, the biodistribution of radioactivity was determined.

Results
In the in vitro experiments, although 85 Sr barely adsorbed to Chlorella at low pH, the 85 Sr adsorption ratio to Chlorella increased with increasing pH. The maximum sorption capacity of Chlorella to strontium was 9.06 mg / g. 137 Cs barely adsorbed to Chlorella under any pH conditions. In the biodistribution experiments, bone accumulation of radioactivity after 85 Sr 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 90 Sr into the blood and enhance the elimination of 90 Sr from the body through adsorption in intestine.

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][2][3][4][5]. Among the released radionuclides, cesium-137 ( 137 Cs) and strontium-90 ( 90 Sr) 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]. 137 Cs (t 1/2 = 30.1 y) is one of the most important nuclear fission elements and decays to barium-137m ( 137m Ba) with the emission of β-particles (0.51 MeV). 137m Ba in turn decays to the stable isotope 137 Ba with the emission of gamma rays (0.66 MeV) by isomeric transition. Because the physical half-life of 137m Ba (t 1/2 = 2.55 m) as a daughter radionuclide is sufficiently short compared with that of 137 Cs, 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]. 90 Sr (t 1/2 = 29.1 y) is another important nuclear fission radionuclide. 90 Sr decays to yttrium-90 ( 90 Y) with the emission of β-particles (0.54 MeV). 90 Y in turn decays to the stable isotope zirconium-90 ( 90 Zr) with the emission of high energy β-particles (2.27 MeV). Because the physical half-life of 90 Y (t 1/2 = 2.67 d) as a daughter radionuclide is sufficiently short compared with that of the parent radionuclide 90 Sr, 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 90 Sr is very long [10]. Therefore, internal exposure to 90 Sr is associated with the development of leukemia and osteosarcoma [11,12]. Because of the properties mentioned above, internal exposure to 137 Cs and 90 Sr 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 (Fe 4 III [Fe II (CN) 6 ] 3 )ÁxH 2 O (Radiogardase 1 , Prussian blue insoluble, PB), a compound that promotes the elimination of 137 Cs 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 90 Sr, 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, 85 Sr (T 1/2 = 64.8 d) was used instead of 90 Sr because 85 Sr emits gamma rays that can be measured easily. In vitro experiments of adsorption of 137 Cs and 85 Sr to Chlorella Chlorella (10, 30, or 100 mg) was suspended and 137 Cs or 85 Sr 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:

Materials and Methods
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 137 Cs and 85 Sr to Chlorella Chlorella (30 mg) was suspended and 137 Cs or 85 Sr 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 85 Sr 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, 85 Sr 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 85 Sr 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 Ca 2+ ) on in vitro adsorption of 85 Sr to Chlorella Chlorella (10 mg) was suspended and 85 Sr was added in 1 mL of 0.02 M HEPES buffer solution containing 500, 1000, or 10000 ppm of Na + , K + , or Ca 2+ , which were prepared by dissolution of NaCl, KCl, or CaCl 2 . 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 85 Sr 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 85 Sr 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 85 Sr 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.

In vitro experiments of adsorption of 137 Cs and 85 Sr to Chlorella
The adsorption ratios of 137 Cs and 85 Sr to Chlorella are shown in Fig 1.

Langmuir model
Adsorption capacity of Chlorella with Sr was evaluated by the Langmuir model, defined as follows: where q (mg / g) represents Sr binding per Chlorella, C eq (mg / L) is the equilibrium concentration of strontium, q max (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. Although Na + and K + hardly decreased the binding of strontium to Chlorella at low concentration of the cations, but the addition of Ca 2+ remarkably decreased the binding of strontium to Chlorella even at low concentration of Ca 2+ .

Effects of cations (Na + , K + , or Ca 2+ ) on in vitro adsorption of 85 Sr to Chlorella
In vivo experiments for promoting excretion of 85 Sr from the body Table 1 lists the biodistribution of radioactivity at 48 h after oral administration of 85 Sr 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

Discussion
In in vitro adsorption experiments, Chlorella did not adsorb 137 Cs at any pH range or concentration. Therefore, in vivo experiments were not performed using 137 Cs. 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]. 85 Sr 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 85 Sr 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 85 Sr or not. The results indicated that after exposure to an acidic solution, Chlorella still adsorbed 85 Sr 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 85 Sr administration between the Chlorella treatment group and the control group indicates that Chlorella inhibited 85 Sr absorption into blood and enhanced its excretion as a result of adsorption between Chlorella and 85 Sr in the intestine. It has been reported that alginate reduces absorption and enhances excretion of strontium [22][23][24], but the alginate did not inhibit absorption of 85 Sr 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 85 Sr was not significantly changed by treatment of single oral administration of alginate (2%, 1 mL) just before oral administration of 85 Sr. Meanwhile, when 85 Sr was orally administrated after feeding on 10% alginate containing diet for 10 days, the whole-body retention of 85 Sr 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 85 Sr in this study.
As mentioned above, 85 Sr 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 Ca 2+ ) on in vitro adsorption of 85 Sr to Chlorella were evaluated (Fig 4). As the presence of the cations, especially Ca 2+ , decreased adsorption of 85 Sr 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 85 Sr 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.

Author Contributions
Conceived and designed the experiments: KO TF AO. Performed the experiments: TF. Analyzed the data: KO TF. Contributed reagents/materials/analysis tools: JH YK KS. Wrote the paper: KO.