Oleanolic Acid3-( 1 ′ 2 ′ Orthoacetate-Glucoside )-28-Glucoside Alleviates Methylmercury Toxicity in Vitro and in Vivo

Methylmercury (MeHg) is a widely distributed compound in nature and one of the most toxic environmental pollutants that causes neurotoxic effects.1,2) MeHg is absorbed in the intestines, distributed to all tissues in the body, and accumulated in various organs such as brain, liver, kidney, and developing fetus.3,4) It is important to understand the effects of low-dose MeHg to which humans are exposed to through food intake as well as those of high-dose exposure. Recently, we have suggested that the immunotoxic effect on T helper 2 (Th2) responses to low-dose MeHg is negligible in ovalbuminor mite-induced Th2 allergy mouse models.5) Moreover, we have shown that a low concentration of MeHg activated autophagy, which functions as protective response for cell survival, in various cell types.6,7) To date, very little is understood about the effects of low-dose MeHg exposure on animals and humans, especially through daily food intake. Furthermore, anti-MeHg medicines are in demand. Saponins are natural glycosides of steroid or triterpene, which exhibit many different biological and pharmacological actions such as immunomodulatory, antitumor, and antiinflammatory.8–11) Saponins have a diverse range of characteristics, which include sweetness, bitterness,12–14) foaming, and hemolytic properties.15,16) Wide application of saponins in beverages and confectionery as well as in pharmaceutical products is not uncommon.8–11) It is believed that saponins form the main constituents of many phototherapies and folk medicines and they are considered to be responsible for numerous pharmacological properties.16) Onjisaponins, which are derived from the roots of Polygala tenuifolia Willd., are saponin derivatives with OA as an aglycon and have various pharmacological activities such as induction of nerve growth factor (NGF) synthesis and enhancement of autophagy.17,18) Since NGF and autophagy contribute to cell protection against MeHg toxicity,6,19) these activities of onjisaponins suggest the potential of OA saponin derivatives to have anti-MeHg poisoning properties. However, it is difficult to isolate enough target saponin compound from plant material for clinical and animal experiments due to the structural similarity of onjisaponins. Therefore, in the previous study, we synthesized OA saponin derivatives and compared their antiMeHg activity; as a result, we demonstrated that OA 3-glucoside (OA3Glu) in which glucose is bound to the C3 position of OA exhibits anti-MeHg activity.20) In the process of OA saponin derivative synthesis, we obtained OA-3-(1′2′orthoacetateglucoside)-28-glucoside [OA-3-(1′2′orthoacetate-Glu)-28Glu], an orthoester saponin that differs from OA3Glu in the glucose binding form to the C3 position of OA, as a by-product. In this study, to further understand how anti-MeHg activReport


INTRODUCTION
Methylmercury (MeHg) is a widely distributed compound in nature and one of the most toxic environmental pollutants that causes neurotoxic effects. 1,2) MeHg is absorbed in the intestines, distributed to all tissues in the body, and accumulated in various organs such as brain, liver, kidney, and developing fetus. 3,4) It is important to understand the effects of low-dose MeHg to which humans are exposed to through food intake as well as those of high-dose exposure. Recently, we have suggested that the immunotoxic effect on T helper 2 (Th2) responses to low-dose MeHg is negligible in ovalbumin-or mite-induced Th2 allergy mouse models. 5) Moreover, we have shown that a low concentration of MeHg activated autophagy, which functions as protective response for cell survival, in various cell types. 6,7) To date, very little is understood about the effects of low-dose MeHg exposure on animals and humans, especially through daily food intake. Furthermore, anti-MeHg medicines are in demand.
Saponins are natural glycosides of steroid or triterpene, which exhibit many different biological and pharmacological actions such as immunomodulatory, antitumor, and antiinflammatory. [8][9][10][11] Saponins have a diverse range of characteristics, which include sweetness, bitterness, [12][13][14] foaming, and hemolytic properties. 15,16) Wide application of saponins in bev-erages and confectionery as well as in pharmaceutical products is not uncommon. [8][9][10][11] It is believed that saponins form the main constituents of many phototherapies and folk medicines and they are considered to be responsible for numerous pharmacological properties. 16) Onjisaponins, which are derived from the roots of Polygala tenuifolia Willd., are saponin derivatives with OA as an aglycon and have various pharmacological activities such as induction of nerve growth factor (NGF) synthesis and enhancement of autophagy. 17,18) Since NGF and autophagy contribute to cell protection against MeHg toxicity, 6,19) these activities of onjisaponins suggest the potential of OA saponin derivatives to have anti-MeHg poisoning properties. However, it is difficult to isolate enough target saponin compound from plant material for clinical and animal experiments due to the structural similarity of onjisaponins. Therefore, in the previous study, we synthesized OA saponin derivatives and compared their anti-MeHg activity; as a result, we demonstrated that OA 3-glucoside (OA3Glu) in which glucose is bound to the C3 position of OA exhibits anti-MeHg activity. 20) In the process of OA saponin derivative synthesis, we obtained OA-3-(1′2′orthoacetateglucoside)-28-glucoside [OA-3-(1′2′orthoacetate-Glu)-28-Glu], an orthoester saponin that differs from OA3Glu in the glucose binding form to the C3 position of OA, as a by-product. In this study, to further understand how anti-MeHg activ-ity of OA derivatives differing in the binding form of glucose at the C3 position of OA varies, the anti-MeHg activity of OA-3-(1′2′orthoacetate-Glu)-28-Glu was examined in vitro and in vivo.

Cell Culture
The human colon carcinoma (Caco-2) cell line was obtained from the European Collection of Cell Cultures (ECACC number 86010202; UK). The cells were maintained in minimum essential medium supplemented with 20% (v/v) fetal bovine serum, 0.1 mM non-essential amino acids, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. The cells were incubated at 37 °C in an atmosphere with 95% relative humidity and 5% CO 2 .

Measurement of Hg Content in Animal Organs
The kidneys and liver were excised, weighed, and homogenized in 3 mL of 10 mM potassium phosphate buffer (pH 7.4). The homogenates were digested with a concentrated acid mixture (nitric acid:perchloric acid = 4:1) for 2 h at 160 °C, and the total Hg content was measured using an atomic absorption spectrometry analyzer HG-310 (Hiranuma, Ibaraki, Japan). All results are expressed as ng mg -1 (ppm) dry weight.
Quantification of Brain Cytokine Levels by Enzyme Linked Immunosorbent Assay (ELISA) Brains were excised, weighed, flash frozen in liquid nitrogen, and stored at −80°C until required for further processing. Frozen brain tissues were homogenized in 3 ml of 10 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM ethylenediaminetetraacetic acid (Sigma Aldrich, St. Louis, MO), 0.1 mM phenylmethanesulfonyl fluoride (Nacalai Tesque), 1 µM pepstatin A (Peptide Institute, Osaka, Japan), and 2 µM leupeptin (Peptide Institute). The homogenates were then centrifuged at 105,000 × g for 1 h. The supernatants were stored at −80°C until analysis by ELISA. This analysis was done to measure interleukin (IL)-1β (R&D Systems, Minneapolis, MN, USA) and IL-6 (Biolegend, San Diego, CA, USA) in brain homogenates, according to the manufacturer's instructions. The absorbance was read in a microplate reader at 450 nm. The control reading at 550 nm was then subtracted. The ELISA results were converted to pg/mL, using values obtained from standard curves generated by assay of varying concentrations of recombinant IL-1β and IL-6.
Statistical Analyses The data are presented as mean ± standard error of the mean (SE). Differences between groups were evaluated by one-way analysis of variance (ANOVA). If the differences were significant, Tukey-Kramer post hoc test was used to compare each treatment group with the vehicle control. The results with a p-value of <0.05 were considered statistically significant.
To e v a l u a t e t h e t h e r a p e u t i c e f f e c t s o f O A -3 -(1′2′orthoacetate-Glu)-28-Glu in vivo, we treated the mice in our test groups with OA-3-(1′2′orthoacetate-Glu)-28-Glu, and then exposed them to low dose (0.02 mg·kg -1 ·d -1 : a dose that is at human exposure levels in Hg-contaminated areas), intermediate dose (1 mg·kg -1 ·d -1 : a dose that did not exhibit apparent toxicity in NC/Nga mice in a previous study), 5) or high dose (5 mg·kg -1 ·d -1 : a dose at which toxicity appears in this line of mice) of MeHg. After the experimental period, the total Hg content was measured in the liver and kidneys. At all MeHg exposure concentrations, the total Hg accumulation in the OA-3-(1′2′orthoacetate-Glu)-28-Glu-cotreated group was lower than that in the MeHg group in both organs. Especially, under the low-dose MeHg exposure condition, Hg accumulation in the OA-3-(1′2′orthoacetate-Glu)-28-Glu-cotreated group was significantly lower than that in the OA-3-(1′2′orthoacetate-Glu)-28-Glu-untreated group in both organs. In the OA-3-(1′2′orthoacetate-Glu)-28-Glu-cotreated group, the total Hg content in the liver and kidney was 2.6% and 11.8% compared with that in the OA-3-(1′2′orthoacetate-Glu)-28-Glu-untreated group, respectively (Table 1). These results indicate a possibility that OA-3-(1′2′orthoacetate-Glu)-28-Glu alleviates low-dose MeHg toxicity by suppressing Hg accumulation. We previously reported that OA3Glu suppressed Hg accumulation in organs under intermediate-dose MeHg exposure condition, while OA3Glu hardly affected Hg levels in organs under low-dose MeHg exposure condition. 20) The results suggested that OA-3-(1′2′orthoacetate-Glu)-28-Glu is more effective than OA3Glu against the toxicity of low concentration MeHg exposure to which humans are exposed through daily food intake.
In this study, OA-3-(1′2′orthoacetate-Glu)-28-Glu reduced the cellular accumulation of MeHg (Fig. 1B). As it has been reported that transporters such as multidrug resistance-associated proteins and the L-type amino acid transporters are involved in the intake or excretion of cellular MeHg, [24][25][26][27][28] it is possible that OA-3-(1′2′orthoacetate-Glu)-28-Glu affects  MeHg uptake mediated by these transporters. Because some onjisaponins also reportedly induced NGF production and autophagy in various cell types, 17,18) it is important to examine the effects of OA-3-(1′2′orthoacetate-Glu)-28-Glu on these growth factors to clarify the mechanism of their anti-MeHg accumulation action. In addition, OA-3-(1′2′orthoacetate-Glu)-28-Glu has two glucose units in its structure, which could possibly allow its transfer into cells via glucose transporters and exert anti-MeHg effects. This possibility is supported by our previous results that α-hederin, which has arabinose and rhamnose instead of glucose, showed no anti-MeHg activity. 20) Further investigation is necessary to elucidate the underlying mechanisms of OA-3-(1′2′orthoacetate-Glu)-28-Glu's anti-MeHg activity. In our previous study, OA 3-glucoside, with glucose at the C3 position of OA exhibited anti-MeHg activity, while OA 3,28-diglucoside, with glucose at the C3 and C28 positions of OA, had no anti-MeHg activity. 20) These results indicate that glucose at the C3 position of OA is important for anti-MeHg activity of OA saponin compounds, while its action may be hindered by glucose at the C28 position of OA. On the contrary, in this study, OA-3-(1′2′orthoacetate-Glu)-28-Glu, with glucose at the C3 and C28 positions of OA, similar to OA 3,28-diglucoside, exhibited anti-MeHg activity. These results indicate that a unique binding form of the orthoester of OA-3-(1′2′orthoacetate-Glu)-28-Glu may prevent the inhibition of anti-MeHg activity of glucose of C28 of OA. It is necessary to study differences in the steric structure of the compound and the direct interaction between the compound and MeHg.
It has been reported that prenatal exposure to MeHg adversely affects neurobehavioral function in humans. 29) In addition, low-level MeHg exposure (an amount corresponding to the provisional tolerable weekly intake (PTWI) from fish consumption in Japan) during adulthood has been reported to induce temporary sympathodominant state. 17) Our results indicate that OA-3-(1′2′orthoacetate-Glu)-28-Glu treatment can effectively suppress MeHg toxicity under conditions of repeated exposure to lower concentrations of MeHg, which corresponds to situations faced in daily life, through regular ingestion of aquatic and marine animals with considerable MeHg accumulation.