Correspondence: Masahiko Satoh (E-mail: masahiko@dpc.agu.ac.jp)

Glutathione has a more important role than metallothionein-I/II against inorganic mercury-induced acute

renal toxicityMaki Tokumoto1, Jin-Yong Lee1, Akinori Shimada2, Chiharu Tohyama3,4,5

and Masahiko Satoh1,4

1Laboratory of Pharmaceutical Health Sciences, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya, Aichi 464-8650, Japan

2Laboratory of Pathology, Department of Medical Technology, School of Life and Environmental Science, Azabu University, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa 252-5201, Japan

3Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan4National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan

5Program of Environmental Toxicology, School of Public Health, China Medical University, No 77 Puhe Road, Shenyang North New Area, Shenyang, Liaoning 110122, China

(Received December 15, 2017; Accepted January 27, 2018)

ABSTRACT — Inorganic mercury is a harmful heavy metal that causes severe kidney damage. Glutath-ione (GSH), a tripeptide comprising L-glutamic acid, glycine and L-cysteine, and metallothionein (MT), a cysteine-rich and metal-binding protein, are biologically important protective factors for renal toxic-ity by inorganic mercury. However, the relationship between GSH and MT for the prevention of renal toxicity by inorganic mercury is unknown. We examined the sensitivity of the mice depleted in GSH by treatment with L-Buthionine-SR-sulfoximine (L-BSO), and MT-I/II null mice genetically deleted for MT-I and MT-II, to inorganic mercury (HgCl2). Kidney damage was not induced in the wild-type mice treat-ed with HgCl2 (30 μmol/kg). In the MT-I/II null mice, renal toxicity was induced by HgCl2 at a dose of 30 μmol/kg but not 1.0 μmol/kg. All GSH-depleted mice of both strains were dead following the injection of HgCl2 (30 μmol/kg). GSH-depleted wild-type mice treated with HgCl2 (1.0 μmol/kg) developed kidney damage similar to MT-I/II null mice treated with HgCl2 (30 μmol/kg). Moreover, renal toxicity induced by HgCl2 (1.0 μmol/kg) was more severe in GSH-depleted MT-I/II null mice compared with GSH-deplet-ed wild-type mice. The present study found that GSH and MT-I/II play cooperatively an important role in the detoxification of severe kidney damage caused by inorganic mercury. In addition, GSH may act as a primary protective factor against inorganic mercury-induced acute renal toxicity, because GSH-depleted mice were more sensitive to inorganic mercury than MT-I/II null mice. Key words: Inorganic mercury, Metallothionein, Glutathione, Nephrotoxicity

INTRODUCTION

Mercury exists in the environment (soil, air, and water) and induces harmful effects in organisms (WHO, 1991). Mercury exists in three forms: metal mercury, organic mercury, and inorganic mercury. Organic mercury, espe-cially methylmercury, causes Minamata disease, a cen-tral nervous system disorder (Harada, 1995). On the oth-er hand, acute and chronic exposure to inorganic mercury causes severe nephrotoxicity (Zalups, 1997).

Glutathione (GSH), a tripeptide comprising L-glutam-ic acid, glycine and L-cysteine, might be a cellular fac-tor that protects against inorganic mercury (Naganuma et al., 1990; Bohets et al., 1995). L-Glutamic acid binds to L-cysteine by γ-glutamylcysteine synthetase, and then glycine is associated by glutathione transferase (Wu et al., 2004). L-Buthionine-SR-sulfoximine (L-BSO) depleted endogenous GSH by the inhibition of γ-glutamylcysteine synthetase (Griffith and Meister, 1979). The mice deplet-ed in GSH by treatment with L-BSO were highly sensi-

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tive to inorganic mercury (Naganuma et al., 1990; Bohets et al., 1995).

Metallothionein (MT) is cysteine-rich and low molec-ular weight protein with a high affinity for metals such as mercury, cadmium, and zinc (Wiśniewska et al., 1970; Nordberg and Nordberg, 1975; Cherian, 1977). MT has four isoforms: MT-I, MT-II, MT-III and MT-IV (Vašák and Meloni, 2011). The MT-I and MT-II (MT-I/II) forms are expressed in most tissues and have a defensive func-tion against toxicity induced by heavy metals such as inorganic mercury and cadmium (Chan et al., 1992). Heavy metals including inorganic mercury can induce MT-I/II expression (Chan et al., 1992; Satoh et al., 1997). A previous study demonstrated that sensitivity to renal toxicity by inorganic mercury was increased in the MT-I/II null mice, which are transgenic mice deficient in MT-I and MT-II (Satoh et al., 1997).

Although GSH and MT-I/II, each, plays a protective role in renal toxicity caused by inorganic mercury, the relationship of biological protective function between endogenous GSH and MT-I/II has not been studied. In the present study, the sensitivity of GSH-depleted mice and MT-I/II null mice to inorganic mercury was examined to compare the protective roles of GSH and MT-I/II against acute renal toxicity caused by inorganic mercury.

MATERIALS AND METHODS

Animals and chemicalsMT-I/II null mice with null mutations of the MT-I and

MT-II genes, and wild-type mice were kindly provid-ed by Dr. K.H.A. Choo (Murdoch Institute for Research into Birth Defects, Royal Children’s Hospital, Parkville, Australia) (Michalska and Choo, 1993) and were of a mixed genetic background of 129 Ola and C57BL/6 strains. F1 hybrid mice were mated with C57BL/6J mice (CLEA Japan, Tokyo, Japan) and their offspring were backcrossed to C57BL/6J for six generations. Both MT-I/II null mice and wild-type mice were generated by mat-ing with heterozygous (MT+/-) mice. The mice were rou-tinely bred in the vivarium of the National Institute for Environmental Studies, reproducing normally and dis-playing no overt abnormalities related to physical state or behavior. Both strains of mice were housed in cages in ventilated animal rooms with a controlled temperature of 23 ± 1°C, a relative humidity of 55 ± 10%, and a 12 hr light/dark cycle. They were maintained on standard lab-oratory chow and tap water ad libitum, and received humane care throughout the experiment according to the guidelines of the National Institute for Environmental Studies.

L-BSO was purchased from Sigma-Aldrich (St. Louis, MO, USA). Paraffin, hematoxylin, and eosin were procured from Sakura Finetek Japan (Tokyo, Japan). HgCl2 and other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan).

TreatmentsEight-week-old male MT-I/II null mice and wild-type

mice (20-24 g) were randomized into control and experi-mental groups (4 mice in each group), respectively. Mice were given a single s.c. injection of L-BSO (2.5 mmol/kg) or saline. Four hours later, the mice were s.c. injected with HgCl2 (1.0 or 30 μmol/kg) or saline and sacrificed under diethyl ether anesthesia at 24 hr after HgCl2 treat-ment. To assess the renal toxicity of HgCl2, blood and kidney were removed from each mouse.

MT and GSH concentrationsMT-I/II and GSH concentrations in the kidney were

measured by radioimmunoassay (Tohyama and Shaikh, 1981) as modified by Nishimura et al. (1990), and Bioxytech GSH-400 Assay kit (Percipio Biosciences, Burlingame, CA, USA) using DTNB-recycling methods, respectively. MT-I/II and GSH concentrations were deter-mined at the point of HgCl2 treatment.

Renal toxicity evaluationAs an indicator of renal toxicity, blood urea nitrogen

(BUN) and creatinine values in the serum were deter-mined using an automatic dry-chemistry analyzer system (Spotchem SP-4410; Arkray, Kyoto, Japan).

Histochemical stainingThe kidney of mice treated with HgCl2 at a dose of

1.0 μmol/kg was fixed in 10% buffered formalin (pH 7.4) and embedded in paraffin. Deparaffinized kidney sections, sectioned at 5 μm thickness, were stained with hematoxy-lin/eosin for histopathological analysis.

Mercury concentrationsMercury concentrations in the kidney were meas-

ured by the reduction-aeration method using a cold vapor atomic absorption spectrophotometer (RA-2A Mercury Analyzer; Nippon Instruments, Tokyo, Japan) after diges-tion of kidney specimens with mixture of concentrated acids [HNO3:HClO4 = 1:3 (v/v)].

Statistical analysisData are expressed as the mean ± S.D. for 4 mice. Sta-

tistical analysis was performed using the Student’s t-test and one-way analysis of variance followed by Fisher’s

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least signifi cant difference tests for post hoc comparison. Differences between groups were considered signifi cant at P < 0.05.

RESULTS

MT and GSH concentrationsTable 1 shows MT-I/II and GSH concentrations in the

kidney of MT-I/II null mice and wild-type mice treat-ed with L-BSO. The basal MT-I/II level in the kidney of wild-type mice was 4.27 ± 1.20 μg/g tissue and was not affected by L-BSO treatment. The amount of renal MT-I/II was below the detection limit (under 0.2 μg/g tissue) in the saline-treated MT-I/II null mice and could not be induced by L-BSO treatment. The basal GSH level in kidney was not signifi cantly different between the MT-I/II null mice and wild-type mice. At 4 hr after L-BSO treatment (at the point of HgCl2 treatment), the renal GSH levels in MT-I/II null mice and wild-type mice were decreased to approximately 20% of the control levels.

Renal toxicity evaluationBUN (Fig. 1A) and creatinine (Fig. 1B) values in the

serum were used as an indicator of renal toxicity. BUN and creatinine values in the serum of wild-type mice treated with HgCl2 at a dose of 30 μmol/kg were simi-lar to those of the saline-treated wild-type mice. Howev-er, both indicator values in the serum of MT-I/II null mice were signifi cantly increased by the injection of HgCl2 at a dose of 30 μmol/kg but not at 1.0 μmol/kg. Furthermore, all L-BSO-treated mice of both strains were dead follow-ing the injection of 30 μmol/kg HgCl2 (data not shown). BUN and creatinine values in the serum of L-BSO-treat-ed mice of both strains were signifi cantly increased by the injection of 1.0 μmol/kg HgCl2, whereas the increase of both indicator values was more extensive in the MT-I/II null mice than the wild-type mice. To increase of values of BUN and creatinine in the serum, MT-I/II null mice required 30 μmol/kg HgCl2 and L-BSO-treated wild-type mice required 1.0 μmol/kg HgCl2.

Histopathological changesNo pathological changes were detected in the renal

cortex of MT-I/II null mice and wild-type mice treat-ed with 1.0 μmol/kg HgCl2 (Figs. 2B and 2E). HgCl2 (1.0 μmol/kg) caused tubular damage such as degenera-tion and necrosis in the tubules and urinary casts in the tubular lumen of L-BSO-treated mice of both strains.

Table 1. MT-I/II and GSH concentrations in the kidneys of mice treated with L-BSO.MT-I/II (μg/g tissue) GSH (μmol/g tissue)

Wild-type MT-I/II null Wild-type MT-I/II nullControl 4.27 ± 1.20 < 0.2 3.93 ± 0.18 4.16 ± 0.28L-BSO 4.65 ± 0.97 < 0.2 0.71 ± 0.03 * 0.82 ± 0.09 *Values are the mean ± S.D. for 4 mice.*Signifi cantly different from the corresponding control (P < 0.05).

Fig. 1 . Effect of pretreatment with L-BSO on renal toxicity caused by mercury(II) chloride in wild-type mice and MT-I/II null mice. HgCl 2 was s.c. injected to mice (left panel; 1.0 μmol/kg, right panel; 30 μmol/kg) 4 hr af-ter L-BSO treatment. Twenty-four hours later, serum (A) BUN and (B) creatinine values were determined. Values are the mean ± S.D. for 4 mice. *Signifi cantly different from the corresponding control ( P < 0.05). # P < 0.05. BSO: L-BSO, Hg: HgCl 2 .

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However, tubular damage of L-BSO-treated MT-I/II null mice by injection of 1.0 μmol/kg HgCl2 was more severe than that in the L-BSO-treated wild-type mice (Figs. 2C and 2F).

Mercury concentrationsMercury concentrations are shown in Fig. 3. In the

HgCl2-treated group, mercury accumulated in the kid-ney of mice compared with the corresponding controls. This accumulation was significantly lower in the MT-I/II null mice compared with wild-type mice treated with HgCl2 alone. Mercury accumulation in the wild-type mice treated with HgCl2 was decreased by pretreatment with L-BSO, but this effect was not observed in MT-I/II null mice.

DISCUSSION

GSH and MT-I/II have been shown to protectively act against renal toxicity by inorganic mercury (Naganuma et al., 1990; Bohets et al., 1995; Chan et al., 1992). The current study also indicates that susceptibility to acute renal toxicity by inorganic mercury is increased by the depletion of GSH or a deficiency in MT-I/II (Figs. 1 and 2). Because mercury binds to thiols in cysteine, GSH and MT-I/II may prevent inorganic mercury-induced renal toxicity by trapping mercury. Moreover, sensitivi-ty to acute renal toxicity by inorganic mercury was great-ly enhanced by a deficiency of both GSH and MT-I/II (Figs. 1 and 2). The kidney toxicity by inorganic mercu-ry in either depletion of GSH or MT-I/II is less than that

Fig. 2. Effect of pretreatment with L-BSO on histopathological changes caused by mercury(II) chloride in wild-type mice and MT-I/II null mice. HgCl2 (1.0 μmol/kg) was s.c. injected to wild-type mice and MT-I/II null mice 4 hr after L-BSO treat-ment. Kidney was collected 24 hr later and tissue sections were stained with hematoxylin-eosin. (A) Control, (B) HgCl2 and (C) L-BSO + HgCl2 groups of wild-type mice. (D) Control, (E) HgCl2 and (F) L-BSO + HgCl2 groups of MT-I/II null mice. Magnification, × 400.

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in the depletion of both GSH and MT-I/II. These results indicate that they may compensate for one another when either GSH or MT-I/II is depleted. In addition, GSH and MT-I/II also played a major role in the accumulation of mercury in the kidney (Fig. 3). These results suggest that GSH and MT-I/II, which are biological factors that include thiol, play cooperatively an important role in the detoxification of severe kidney damage caused by inor-ganic mercury.

Endogenous level of GSH is 100-1,000 times high-er than that of MT-I/II in the kidney. When comparing GSH and MT, GSH may act as a primary protective fac-tor against inorganic mercury-induced acute nephrotoxic-ity compared with MT-I/II, because GSH-depleted mice were more sensitive to inorganic mercury than MT-I/II null mice (Figs. 1 and 2), and endogenous GSH level in the kidney was higher than MT-I/II level (Table 1). How-ever, MT synthesis is easily induced by various metals, cytokines, glucocorticoids, some stressors and many oth-er factors (Kägi and Schäffer, 1988; Sato and Bremner, 1993) although the endogenous MT content in the kidney is lower than the amount of GSH. Thus, MT-I/II may act as a secondary detoxifying factor against acute renal tox-icity caused by inorganic mercury in cases where the kid-ney is deficient in GSH. Few studies using MT-I/II null mice have compared the protective effects of GSH and MT-I/II against various kinds of toxicants and harmful factors. The present study demonstrates that GSH is the major preventive factor for inorganic mercury-induced

acute renal toxicity compared with MT-I/II. However, our previous study revealed that GSH and MT-I/II had a sim-ilar protective potency against cisplatinum-induced acute renal toxicity (Satoh et al., 2000). Therefore, the protec-tive effects of GSH and MT-I/II may differ for each type of chemical substance.

GSH and MT-I/II levels in humans differ under vari-ous conditions and life-styles. For example, GSH lev-els in the kidney have a circadian rhythm (Davies et al., 1983). Furthermore, some people have SNPs of MT-I/II genes (Kita et al., 2006; Hattori et al., 2016), or gen-erate low levels of MT-I/II (Yoshida et al., 1998). Thus, variations in sensitivity to renal toxicity caused by inor-ganic mercury may be explained by alterations of endog-enous renal GSH and/or MT-I/II levels (Li et al., 2015; Bose-O'Reilly et al., 2017).

In conclusion, acute renal toxicity induced by inor-ganic mercury is enhanced by the depletion of GSH or a deficiency of MT-I/II, and is greatly accelerated by the absence of both GSH and MT-I/II. Interestingly, GSH-depleted mice were more sensitive to inorganic mercury compared with MT-I/II null mice. These results suggest that GSH and MT-I/II cooperatively play as preventive factors against acute renal toxicity by inorganic mercury, and that GSH has a primary role compared with MT-I/II.

Conflict of interest---- The authors declare that there is no conflict of interest.

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