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 (t_1/2 = 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 (t_1/2 = 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 (t_1/2 = 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 (t_1/2 = 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 (Fe₄^III[Fe^II(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 (T_1/2 = 64.8 d) was used instead of ⁹⁰Sr because ⁸⁵Sr emits gamma rays that can be measured easily.

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.