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

Abstract

Inorganic mercury is a harmful heavy metal that causes severe kidney damage. Glutathione (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 toxicity 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 treated 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-depleted 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.

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, especially methylmercury, causes Minamata disease, a central nervous system disorder (Harada, 1995). On the other hand, acute and chronic exposure to inorganic mercury causes severe nephrotoxicity (Zalups, 1997).

Glutathione (GSH), a tripeptide comprising L-glutamic acid, glycine and L-cysteine, might be a cellular factor 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 depleted in GSH by treatment with L-BSO were highly sensitive to inorganic mercury (Naganuma et al., 1990; Bohets et al., 1995).

Metallothionein (MT) is cysteine-rich and low molecular 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 function 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 chemicals

MT-I/II null mice with null mutations of the MT-I and MT-II genes, and wild-type mice were kindly provided 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 mating with heterozygous (MT+/-) mice. The mice were routinely bred in the vivarium of the National Institute for Environmental Studies, reproducing normally and displaying 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 laboratory 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).

Treatments

Eight-week-old male MT-I/II null mice and wild-type mice (20-24 g) were randomized into control and experimental 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 treatment. To assess the renal toxicity of HgCl₂, blood and kidney were removed from each mouse.

MT and GSH concentrations

MT-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 determined at the point of HgCl₂ treatment.

Renal toxicity evaluation

As an indicator of renal toxicity, blood urea nitrogen (BUN) and creatinine values in the serum were determined using an automatic dry-chemistry analyzer system (Spotchem SP-4410; Arkray, Kyoto, Japan).

Histochemical staining

The 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 hematoxylin/eosin for histopathological analysis.

Mercury concentrations

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

Statistical analysis

Data are expressed as the mean ± S.D. for 4 mice. Statistical analysis was performed using the Student’s t-test and one-way analysis of variance followed by Fisher’s least significant difference tests for post hoc comparison. Differences between groups were considered significant at P < 0.05.

RESULTS

MT and GSH concentrations

Table 1 shows MT-I/II and GSH concentrations in the kidney of MT-I/II null mice and wild-type mice treated 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 significantly 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.

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 null
Control	4.27	±	1.20	< 0.2		3.93	±	0.18	4.16	±	0.28
L-BSO	4.65	±	0.97	< 0.2		0.71	±	0.03 *	0.82	±	0.09 *

Values are the mean ± S.D. for 4 mice.

*Significantly different from the corresponding control (P < 0.05).

Renal toxicity evaluation

BUN (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 similar to those of the saline-treated wild-type mice. However, both indicator values in the serum of MT-I/II null mice were significantly 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 following the injection of 30 µmol/kg HgCl₂ (data not shown). BUN and creatinine values in the serum of L-BSO-treated mice of both strains were significantly 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.

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. HgCl₂ was s.c. injected to mice (left panel; 1.0 µmol/kg, right panel; 30 µmol/kg) 4 hr after 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. *Significantly different from the corresponding control (P < 0.05). ^#P < 0.05. BSO: L-BSO, Hg: HgCl₂.

Histopathological changes

No pathological changes were detected in the renal cortex of MT-I/II null mice and wild-type mice treated with 1.0 µmol/kg HgCl₂ (Figs. 2B and 2E). HgCl₂ (1.0 µmol/kg) caused tubular damage such as degeneration and necrosis in the tubules and urinary casts in the tubular lumen of L-BSO-treated mice of both strains. 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).

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 treatment. 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.

Mercury concentrations

Mercury concentrations are shown in Fig. 3. In the HgCl₂-treated group, mercury accumulated in the kidney 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.

Fig. 3

Mercury concentrations in the kidney of wild-type mice and MT-I/II null mice treated with mercury(II) chloride and/or L-BSO. HgCl₂ was s.c. injected to mice (1.0 µmol/kg) 4 hr after L-BSO treatment. Kidney was collected 24 hr later and digested with a concentrated acid mixture [HNO₃:HClO₄ = 1:3 (v/v)]. Values are the mean ± S.D. for 4 samples. *Significantly different from corresponding control (P < 0.05). ^#P < 0.05. BSO: L-BSO, Hg: HgCl₂.

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, sensitivity to acute renal toxicity by inorganic mercury was greatly enhanced by a deficiency of both GSH and MT-I/II (Figs. 1 and 2). The kidney toxicity by inorganic mercury in either depletion of GSH or MT-I/II is less than that 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 inorganic mercury.

Endogenous level of GSH is 100-1,000 times higher than that of MT-I/II in the kidney. When comparing GSH and MT, GSH may act as a primary protective factor against inorganic mercury-induced acute nephrotoxicity 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). However, MT synthesis is easily induced by various metals, cytokines, glucocorticoids, some stressors and many other 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 toxicity caused by inorganic mercury in cases where the kidney 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 similar protective potency against cisplatinum-induced acute renal toxicity (Satoh et al., 2000). Therefore, the protective 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 various conditions and life-styles. For example, GSH levels 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 generate low levels of MT-I/II (Yoshida et al., 1998). Thus, variations in sensitivity to renal toxicity caused by inorganic mercury may be explained by alterations of endogenous 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 inorganic 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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