Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride

Chemosphere xxx (2013) xxx–xxx

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Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride Haiquan Zhao a,b,1,⇑, Jie Hong a,1, Xiaohong Yu a,b,c,d,1, Xiaoyang Zhao a,1, Lei Sheng a, Yuguan Ze a, Xuezi Sang a, Suxin Gui a, Qingqing Sun a, Ling Wang a, Fashui Hong a,c,d,⇑ a

Medical College of Soochow University, Suzhou 215123, China College of Life Sciences, Anhui Agriculture University, Hefei 230036, China Jiangsu Province Key Laboratory of Stem Cell Research, Soochow University, 708 Renmin Road, Suzhou 215007, China d Cultivation Base of State Key Laboratory of Stem Cell and Biomaterials built together by Ministry of Science and Technology and Jiangsu Province, Suzhou 215007, China b c

h i g h l i g h t s  Exposure to lanthanides impaired renal function in mice.  Exposure to lanthanides induced cell necrosis in kidney.  Exposure to lanthanides promoted ROS accumulation in kidney.  Exposure to lanthanides caused the reduction of antioxidant capacity in kidney.  The order of kidney damages was Ce exposure > Nd exposure > La exposure.

a r t i c l e

i n f o

Article history: Received 31 March 2012 Received in revised form 17 May 2013 Accepted 21 May 2013 Available online xxxx Keywords: Lanthanides Kidney Biochemical function Histopathological changes Oxidative stress

a b s t r a c t Environmental pollution from lanthanides (Ln) has been recognized as a major problem due to a grab exploitation of Ln mine in China. Exposure to Ln has been demonstrated to cause the nephrotoxicity, very little is known about the mechanism of oxidative damage to kidney in animals. In order to understand Ln-induced nephrotoxicity, various biochemical and chemical parameters were assayed in mouse kidney. Intragastric exposures of LaCl3, CeCl3, and NdCl3 at doses of 2, 5, and 10 mg kg1 BW for 90 consecutive days caused nephritis or epithelial cell necrosis and oxidative stress to kidney. An increase in coefficients of the kidney, La, Ce, and Nd accumulation and histopathological changes in the kidney could be observed, followed by increased reactive oxygen species production and peroxidation levels of lipid, protein and DNA, and decreased activities of superoxide dismutase, catalase, glutathione-S-transferase and glutathione reductase as well as antioxidants such as glutathione, ascorbic acid and thiol contents. Furthermore, La, Ce, and Nd significantly suppressed expression of genes and proteins of these antioxidative enzymes in mouse kidney. In addition, kidney functions were disrupted, including an increase of the creatinine, and reductions of uric acid, urea nitrogen, calcium and phosphonium. These findings suggest that nephritis generation or epithelial cell necrosis in mice following exposure to Ln is closely associated with oxidative stress. Of these damages, the most severe was in the Ce3+-exposed kidneys, next in the Nd3+-exposed kidneys, and the least in the La3+-exposed kidneys, which may be attributed to the 4f electron of Ln. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction China has the largest deposits of lanthanides (Ln) and supplies >90% of these elements to the rest of the world. As we know, Ln belongs to the IIIB family in the periodic table of elements. Because of their diverse physical, chemical and biological ⇑ Corresponding authors. Address: Medical College of Soochow University, Suzhou 215123, China. Tel.: +86 0512 61117563; fax: +86 0512 65880103 (F. Hong). E-mail address: [email protected] (F. Hong). 1 These authors contributed equally to this work.

effects, Ln has been used industrially in various areas, such as metallurgy, color TV, lasers, photographic cameras, semiconductors, binoculars and movie films; and also in medicine as anticancer, anti-inflammatory and antiviral agents (Kostova, 2005; Wason and Zhao, 2013). Ln-enriched fertilizers (mainly consisted of lanthanum, cerium, and neodymium nitrates) are known to be able to increase the yields of crops in China (Ni, 2002; Hu et al., 2004). The rapid development and widespread application of novel Ln technologies in the industrialized countries requires additional information on the potential health effects derived from possible exposure to Ln compounds.

0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.05.034

Please cite this article in press as: Zhao, H., et al. Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.034

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Recently, environmental pollution from Ln has been recognized as a major problem due to a grab exploitation of Ln mine in China (Wu et al., 2003; Tong et al., 2004; Zhao et al., 2008). Ln-related pneumoconiosis in Chinese mineworkers has been reported (Chen et al., 2005). However, the Ln-related health impacts are still not fully understood and there still lacks the systemic and basic information (He and Rambeck, 2000; Yu et al., 2007; He et al., 2010). Numerous studies have definitely showed that Ln exposure are able to cause injuries in various animal organ types, including lung (Li et al. 2010), liver (Cheng et al., 2011, 2012a; Fei et al., 2011a,b; Ze et al., 2011; Li et al., 2013), spleen (Liu et al., 2010; Cheng et al., 2011) and brain (He et al., 2008; Zhao et al., 2011a,b; Cheng et al., 2012b, 2013; Wang et al., 2013). The toxicity of Ln to kidneys has also been reported. Hayashi et al. (2006) and Tanida et al. (2009) had found that scandium and yttrium were accumulated in the kidney, leading to damage of renal functions in rats. We speculated that the Ln-induced renal damage might be associated with oxidative stress of kidney. Kidney is also equipped with an advanced defence system of enzymatic and non-enzymatic antioxidants, which are known as scavengers of reaction oxygen species (ROS). Renal injury following exposure to Ln may occur when the balance between oxidant production and the antioxidant system is altered. In this study we investigated the effect of LaCl3, CeCl3, and NdCl3 on mouse kidney after intragastric administrations for 90 consecutive days; the oxidative injury in the kidney was assessed by histopathological tests, and measuring Ln accumulation and biochemical parameters of renal functions, the production of O 2 and H2O2, and the level of lipid, protein and DNA peroxidation. The intracellular levels of GSH, AsA and thiol, and the activities and expressions of various antioxidant enzymes that are known to regulate cellular oxidative tone are also examined in the mouse kidney.

2. Materials and method 2.1. Reagent LaCl3, CeCl3, and NdCl3 were purchased from Shanghai Chem. Co. (China) and were analytical-grade.

2.2. Animal and treatment Four-week-old male ICR mice were purchased from Soochow University Experimental Animal Center. (Suzhou, China). All mice were housed in stainless steel cages in a ventilated animal room. Room temperature of the housing facility was maintained at 24 ± 2 °C with a relative humidity of 60 ± 10% and a 12-h light/dark cycle. Distilled water and sterilized food were available for mice ad libitum. Prior to dosing, the mice were acclimated to this environment for 5 d. All procedures used in animal experiments conformed to the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH, 1996). 150 mice were randomly divided into 10 groups: one normal control group, La-treated, Ce-treated, Nd-treated groups of three gavage doses (2, 5 and 10 mg kg1 body weight [BW], n = 30 in each group). Ln chloride solutions were administered by gavage to mice daily for 90 d. And controls were administered with equivalent amount of distilled water. Any symptoms or mortality were observed and recorded carefully everyday during various experimental stages. After 90 d, all mice were weighed firstly, and then sacrificed after being anesthetized using ether. Blood samples were collected from the eye vein by removing the eyeball quickly. Serum was collected by centrifuging blood at 2500 rpm for 10 min. Kidneys were collected and weighed.

2.3. Coefficients of kidney After weighing the body and tissues, the coefficients of kidney to body weight were calculated as the ratio of tissues (wet weight, mg) to body weight (g). 2.4. Biochemical analysis of renal functions Renal functions were determined by uric acid (UA), blood urea nitrogen (BUN), creatinine (Cr), calcium (Ca) and phosphonium (P). All biochemical assays were performed using a clinical automatic chemistry analyzer (Type 7170A, Hitachi, Japan). 2.5. Histopathological examination For the pathologic studies, all histopathologic examinations were performed using standard laboratory procedures. The kidneys were embedded in paraffin blocks, then sliced (5 lm thick) and placed onto glass slides. After hematoxylin-eosin (HE) staining, the stained sections were evaluated by a histopathologist unaware of the treatments, using an optical microscope (Nikon U-III Multipoint Sensor System, Japan). 2.6. Ln content analysis of kidney The kidneys were thawed and approximately 0.3 g of the kidney was weighed, digested and analyzed for lanthanum, cerium and nedymium contents. Inductively coupled plasma-mass spectrometry (ICP-MS, Thermo Elemental X7, Thermo Electron Co., USA) was used to analyze the lanthanum, cerium and nedymium concentrations in the samples. An Indium concentration of 20 ng mL1 was utilized as an internal standard, and the detection limit of lanthanum, cerium and nedymium was 0.089 ng mL1. The data were expressed as nanograms per gram fresh tissue. 2.7. Oxidative stress assay of kidney ROS (O 2 and H2O2) production and levels of malondialdehyde (MDA), protein carbonyl (PC), and 8-hydroxy deoxyguanosine (8OHdG) in the kidney tissues were assayed using commercial enzyme-linked immunosorbent assay kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) according to the manufacturer’s instructions. 2.8. Assay of antioxidant enzymes and antioxidants of kidney The kidneys were homogenized in 1 mL of ice-cold 50 mM sodium phosphate (pH 7.0) that contained 1% polyvinyl polypyrrolidone (PVPP). The homogenate was centrifuged at 30,000g for 30 min and the supernatant was used for assays of the actives of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase (GST), and glutathione reductase (GR). The activity of SOD was assayed by monitoring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). Each 3-mL reaction mixture contained 50 mM sodium phosphate (pH 7.8), 13 lM methionine, 75 lM NBT, 2 lM riboflavin, 100 lM EDTA, and 200 lL of the enzyme extract. Monitoring the increase in absorbance at 560 nm followed the production of blue formazan (Beauchamp and Fridovich, 1971). The CAT activity was measured by the decrease in the H2O2 concentration for 15 s, reading the absorbance at 240 nm on a UV3010 absorption spectrophotometer according to Claiborne (1985). The reaction volume was 1 mL and contained 500 lL of sample homogenate and 500 lL of sodium phosphate buffer 50 mM, pH 7.0 and 15 mM H2O2. The control was assayed without

Please cite this article in press as: Zhao, H., et al. Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.034

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H2O2. One unit of enzyme activity was defined as a decrease in absorbance of 0.001/min at 240 nm. GST activity was measured following the method of Habig and Jakoby (1981). The reaction buffer solution contained 100 mM Na-phosphate buffer (pH 6.5), 1 mM 1-chloro-2, 4-dinitrobenzene (CDNB) and 1 mM reduced glutathione (GSH). The reaction was started by the addition of sample solution to the reaction buffer solution. The activity was calculated from the increase in absorbance at 340 nm for 1 min due to CDNB–GSH conjugation when the extinction coefficient was 9.6 mM1 cm1. GR activity was determined by a modified coupled assay procedure of Paglia and Valentine (1967). This method measures the rate of glutathione oxidation by hydrogen peroxide as catalyzed by GR present in the sample by the addition of endogenous GR and NADPH that converts oxidized glutathione (GSSG) to the reduced form. The rate of GSSG formation was measured by following the decrease in absorbance of the reaction mixture at 340 nm as NADPH was converted to NADP. Selenium dependent GR was measured using hydrogen peroxide as substrate. The activity of GR was calculated as the amount of NADPH oxidized per minute using the molar absorption coefficient of 6.22  106 for NADPH. Enzyme activity was expressed as nanomole of NADPH oxidized/mg protein/min. In order to determine GSH and GSSG levels, the kidneys were homogenized as described above. GSH and GSSG contents were estimated using the method of Hissin and Hilf (1976). The reaction mixture contained 100 mL of supernatants, 100 mL o-phthaldehyde (1 mg mL1), and 1.8 mL phosphate buffer (0.1 M sodium phosphate, 0.005 M EDTA, pH8). Fluorometry was performed using a F-4500 fluorometer (F-4500, Hitachi Co., Japan) with excitation at 350 nm and emission at 420 nm. Ascorbate acid (AsA) and dehydroascorbic acid (DHA) determination was determined as described by Jacques-Silva et al. (2001). Proteins were precipitated in 10 volumes of a cold 4% trichloroacetic acid (TCA) solution. An aliquot of homogenized sample (300 mL), in a final volume of 1 mL of the solution, was incubated at 38 °C for 3 h, then 1 mL H2SO4 65%(v/v) was added to the medium. The reaction product was determined using color reagent containing 4.5 mg mL1 dinitrophenyl hydrazine and CuSO4 (0.075 mg mL1). Thiol and disulfide contents in the kidneys were determined by the procedure described by Shiau and Yeh (2004). Maize roots were ground and homogenized in 100 mM Tris–HCl buffer (pH 8.6) containing 1 mM EDTA. After centrifuging at 9,500g for 10 min, the supernatant was used for the determination of thiol and disulfide contents. For the determination of thiol content, 100 mL of supernatant was incubated in 880 lL of 8 M urea Tris– HCl buffer (pH 8.2) with 20 lL of 10 mM 5,5´-dithiobis(2-nitrobenzoic acid) (DTNB) for 30 min at room temperature. Similarly, sample and reagent blanks were prepared for each determination. The absorbance was recorded at 412 nm and thiol content was calculated using the extinction coefficient of 13.6 mM1 cm1. The disulfide bonds were reduced to thiol groups by adding 80 lL of

0.6 M NaBH4 in 8.0 M urea into 100 lL of supernatant. 2 mL no-ctyl alcohol was added to the mixture to avoid foaming. The mixture was then incubated for 2 h in a water bath at 25 °C. The residual NaBH4 was reacted with 20 lL of 2.0 N HCl and then 778 lL of 8 M urea Tris–HCl buffer (pH 8.2) was added to it. The mixture was incubated with 20 lL of 10 mM DTNB for 30 min at room temperature. Sample and reagent blanks were prepared for each determination. The absorbance was recorded at 412 nm in order to evaluate the total thiol content, which is the summation of amounts of thiol and (disulfide)/2. The disulfide content was calculated as the half of the difference between the total thiol content and thiol content. The content of the proteins was determined following the Lowry et al. (1951) method. Each parameter was determined in five animals. 2.9. Assay of antioxidant enzyme expression The levels of mRNA expression of SOD, CAT, GST, and GR in the kidneys (N = 5) were determined using real-time quantitative RT polymerase chain reaction (RT-PCR) (Liu and Saint 2002). Synthesized complimentary DNA was generated by qRT-PCR with primers designed with Primer Express Software (Applied Biosystems, Foster City, CA, USA) according to the software guidelines, and PCR primer sequences are listed Table 1. To determine SOD, CAT, GST, and GR levels in mouse kidney tissues, ELISA was performed using commercial kits that were selective for each respective protein (R&D Systems, USA), following the manufacturer’s instructions. The absorbance was measured on a microplate reader at 450 nm (Varioskan Flash, Thermo Electron, Finland), and the concentrations of SOD, CAT, GST, and GR were calculated from a standard curve for each sample. 2.10. Statistical analysis All results are expressed as means ± standard error of the mean (SEM). The significant differences were examined by unpaired Student’s t-test using SPSS 19 software (USA). A p-value <0.05 was considered statistically significant. 3. Results 3.1. Coefficient of kidney to body weight and Ln content After the 90-d exposure, the mice were sacrificed after weighing and kidneys were collected. Fig. 1 shows net increase of body weight and the coefficients of the kidney to body weight. Significant reductions were found in the net increase of body weight of La3+-treated, Ce3+-treated, and Nd3+-treated groups (p > 0.05), and significant increases were observed in the coefficients of kidney following exposure to La3+, Ce3+, and Nd3+ compared with the

Table 1 Real time PCR primer pairs. PCR primers used in the gene expression analysis. Gene name

Description

Primer sequence

Refer-actin

mactin-F mactin-R mSOD-F mSOD-R mCAT-F mCAT-R mGST-F mGST-R mGR-F mGR-R

5-GAGACCTTCAACACCCCAGC-3 5-ATGTCACGCACGATTTCCC-3 50 -CTGGACAAACCTGAGCCCTAA-30 50 -TCCCCAGCAGCGGAATAA-30 50 -AGCGACCAGATGAAGCAGTG-30 50 -GGGTGACCTCAAAGTATCCAA-30 50 -CCGCTCTTTGGGGCTTTAT-30 50 -GGTTCTGGGACAGCAGGGT-30 50 -TCAGCCCTGGGTTCTAAGAC-30 50 -TGTGACCAGCTCTTCTGAAG-30

SOD CAT GST GR

Primer size (bp) 263 242 241 191 231

Please cite this article in press as: Zhao, H., et al. Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.034

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increased Ln doses, the Cr level of renal function parameter was increased, but the UA, BUN, Ca and P were decreased gradually (p < 0.05), respectively. Among the Ln-treated groups, damages of renal functions were the most severe in the Ce-treated groups, next in the Nd-treated groups, last in the La-treated groups. 3.3. Histopathological evaluation

Fig. 1. Effect of Ln on body weight and coefficients of kidney of mouse by intragastric administrations with Ln solutions for 90 consecutive days. (1) Control (unexposed mice), (2) La: 2 mg kg1 BW, (3) La: 5 mg kg1 BW, (4) La: 10 mg kg1 BW, (5) Ce: 2 mg kg1 BW, (6) Ce: 5 mg kg1 BW, (7) Ce: 10 mg kg1 BW, (8) Nd: 2 mg kg1 BW, (9) Nd: 5 mg kg1 BW, (10) Nd: 10 mg kg1 BW. p < 0.05, p < 0.01,  p < 0.001. Values represent means ± SEM (N = 5).

control (p < 0.05). The deceases of body weight or increases of coefficients of kidney were greatest in the Ce3+-treated groups, medium in the Nd3+-treated groups, smallest in the La3+-treated groups. The elevated coefficients of kidney indicate that the injury might be induced in mice after Ln treatments, which is confirmed by the further morphological examination of kidney. With increased Ln doses, the Ln contents in the kidney were significantly increased, Ce3+ accumulation was greatest, Nd3+ was medium, and La3+ was smallest (p < 0.05, Fig. 2). In the control group, however, the Ln contents were not detectable. This observation suggests that Ln accumulation in the kidney was closely related to the coefficients of the kidney and the renal injury of mice.

Fig. 3 presents the histopathological changes of kidneys in mice following exposure to Ln. In the 2, 5 and 10 mg kg1 BW LaCl3 treated groups, the nephric tissues are significantly shown to congestion of vein, mesenchyme blood vessel, and inflammatory cell infiltration (Fig. 3), respectively. In the 2, 5 and 10 mg kg1 BW CeCl3 treated groups, a large area of spotty necrosis of renal tubular epithelial cells were significantly observed (Fig. 3), respectively. In the 2, 5 and 10 mg kg1 BW NdCl3 treated groups, the nephric tissues are significantly presented to a large area of ambiguity of tissue structure, inflammatory cell infiltration and spotty necrosis of renal tubular epithelial cell (Fig. 3), respectively. The findings indicate that the kidney injury was related to a dose-dependent manner of Ln exposure. Of these injuries, the most severe damages were in the Ce-treated kidneys, medium were in the Nd-treated kidneys, the least were in the La-treated kidneys. 3.4. Oxidative stress The effects of Ln on the production of O 2 and H2O2 in the kidney tissues are shown in Table 2. With increased Ln doses, the rate of ROS generation in the Ln-exposed groups was significantly elevated (p < 0.05 or 0.01), suggesting Ce > Nd > La > control (p < 0.05). To further demonstrate the effects of Ln on ROS production in mouse kidney tissue, the levels of MDA, PC, and 8-OHdG were examined. As shown in Table 2, levels of MDA, PC, and 8-OHdG in kidney tissues from the Ln-exposed groups were markedly elevated (p < 0.05 or 0.01), showing Ce > Nd > La > control (p < 0.05 or 0.01). 3.5. Antioxidant capacity

3.2. Biochemical parameters in serum The changes of biochemical parameters in the blood serum of mice kidney caused by Ln exposure are presented in Table 1. With

The activities of antioxidative enzymes in mouse kidney following exposure to Ln are listed in Table 3. In the Ln-treated groups, the activities of SOD, CAT, GST and GR were lower than the control (p < 0.05 or 0.01), which was lowest in the Ce-treated groups, next in the Nd-treated groups, highest in the La-treated groups. The results mentioned above indicated that Ln inhibited the activities of antioxidative enzymes in mouse kidney. To further explore the effects of Ln exposure on antioxidant capacity, the redox states of GSH–GSSG, AsA–DHA, and thiol–disulfide in the kidneys were examined and shown in Fig. 4. It shows significant decreases of GSH, AsA and thiol in the Ln-treated kidneys for 90 consecutive days (p < 0.05 or 0.01), which was lowest in the Ce-treated groups, medium in the Nd-treated groups, greatest in the La-treated groups, suggesting that Ln decreased the capability of ROS removal in the kidney. 3.6. Expressions of SOD, CAT, GST and GR

Fig. 2. The contents of Ln in kidney of mice after intragastric administrations with Ln solutions for 90 consecutive days. Additional details as in Fig. 1.

The oxidative damages occurred in the kidney according to the mentioned above results. To confirm expression levels of the oxidative stress genes and their proteins including SOD, CAT, GST and GR in the Ln-induced kidney injury, real-time quantitative RT-PCR and ELISA were used to demonstrate the changes of the oxidative stress genes and their proteins levels in the Ln-treated mice (Tables 5 and 6). It can be seen that Ln significantly decreased levels of SOD, CAT, GST and GR expression in the Ln-exposed mouse kidney (p < 0.05), which was lowest in the Ce-exposed

Please cite this article in press as: Zhao, H., et al. Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.034

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Fig. 3. Histopathology of the kidney tissue in kidney of mice after intragastric administrations with Ln solutions for 90 consecutive days. Control group (unexposed mice) presents integrated glomerulars and normal kidney tubulars; 2 mg kg1 BW LaCl3 group vein congestion (black arrows); 5 mg kg1 BW LaCl3 group indicates vein congestion (black arrows), congestion of mesenchyme blood vessel (blue arrows); 10 mg kg1 BW LaCl3 group indicates inflammatory cell infiltration (green cycles); 2 mg kg1 BW CeCl3 group presents spotty necrosis of renal tubular epithelial cell (yellow cycles); 5 mg kg1 BW CeCl3 group indicates spotty necrosis of renal tubular epithelial cell (yellow cycle); 10 mg kg1 BW CeCl3 group indicates severe necrosis of renal tubular epithelial cell (yellow cycles); 2 mg kg1 BW NdCl3 group presents a large area of ambiguity of tissue structure; 5 mg kg1 BW NdCl3 group indicates inflammatory cell infiltration (green cycle), vein congestion (black arrows) and spotty necrosis of renal tubular epithelial cell (green cycle); 10 mg kg1 BW NdCl3 group indicates spotty necrosis of renal tubular epithelial cell (green cycles). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

groups, medium in the Nd-exposed groups, greatest in the La-exposed groups. The alterations of these genes and their proteins expression are in agreement with the trends on the histological changes of the kidney and alterations of enzyme activities in the Ln-exposed mice.

4. Discussion In this study, effects of Ln on the mouse kidney were studied. After intragastric administrations with 2, 5, and 10 mg kg1 BW of LaCl3, CeCl3 and NdCl3 for 90 consecutive days, significant

Please cite this article in press as: Zhao, H., et al. Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.034

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Table 2 The changes of biochemical parameters in the blood serum of mice after intragastric administrations with Ln for 90 consecutive days. Ln (mg kg1 BW)

UA (lmol L1)

Cr (lmol L1)

BUN (mmol L1)

Ca (mmol L1)

P (mmol L1)

0 2 5 10

222.56 ± 11.13 160.21 ± 8.01* 110.88 ± 5.54** 96.76 ± 4.84***

8.81 ± 0.44 9.75 ± 0.49* 11.68 ± 0.58** 13.19 ± 0.66***

9.28 ± 0.46 8.11 ± 0.41* 7.05 ± 0.35** 6.32 ± 0.32***

2.79 ± 0.14 2.58 ± 0.13* 2.41 ± 0.12** 2.12 ± 0.11***

3.68 ± 0.18 3.52 ± 0.18* 3.40 ± 0.17** 3.18 ± 0.16**

Ce

2 5 10

103.55 ± 5.18** 81.23 ± 4.06*** 60.79 ± 3.04***

12.81 ± 0.64** 15.22 ± 0.76*** 18.68 ± 0.93***

7.16 ± 0.36** 6.09 ± 0.30*** 5.01 ± 0.25***

2.10 ± 0.11** 1.89 ± 0.09*** 1.68 ± 0.08***

3.17 ± 0.16** 2.75 ± 0.14*** 2.25 ± 0.11***

Nd

2 5 10

130.19 ± 6.51** 98.76 ± 4.94*** 80.88 ± 4.04***

10.75 ± 0.54** 13.09 ± 0.65*** 15.11 ± 0.76***

7.89 ± 0.39** 6.85 ± 0.34*** 5.93 ± 0.30***

2.31 ± 0.12** 2.13 ± 0.11*** 1.96 ± 0.10***

3.31 ± 0.17** 3.08 ± 0.15*** 2.67 ± 0.13***

La

Values represent means ± SEM (N = 5). * p < 0.05. ** p < 0.01. *** p < 0.001.

Table 3 The generating rate of ROS and lipid, protein and DNA peroxidation in mouse kidney by intragastric administrations with Ln solutions for 90 consecutive days. Ln (mg kg1 BW) La

Ce

Nd

0 2 5 10 2 5 10 2 5 10

H2O2 (nmol mg1 prot min)

1 O prot min) 2 (nmol mg

MDA (lmol g1 tissue)

PC (lmol mg1 prot)

8-OHdG (mg g1 tissue)

14.52 ± 0.73 16.00 ± 0.80* 17.82 ± 0.89** 21.94 ± 1.10*** 19.88 ± 0.99** 23.62 ± 1.23*** 28.92 ± 1.45*** 17.64 ± 0.88** 19.48 ± 0.97** 22.94 ± 1.15***

117.59 ± 5.88 131.46 ± 6.57* 140.55 ± 7.03** 154.06 ± 7.69*** 151.43 ± 7.47** 170.15 ± 8.87*** 196.68 ± 9.83*** 141.79 ± 7.09** 153.64 ± 7.68** 177.51 ± 9.88***

2.21 ± 0.11 2.43 ± 0.12* 2.95 ± 0.15** 3.31 ± 0.17** 2.79 ± 0.14** 3.57 ± 0.18** 4.71 ± 0.24*** 2.63 ± 0.13** 3.19 ± 0.16** 4.12 ± 0.21***

0.48 ± 0.024 0.95 ± 0.048** 1.47 ± 0.07*** 2.27 ± 0.11*** 2.08 ± 0.1*** 3.16 ± 0.16*** 4.55 ± 0.23*** 1.51 ± 0.08*** 2.41 ± 0.12*** 3.22 ± 0.16***

0.31 ± 0.016 1.76 ± 0.088** 3.69 ± 0.19*** 4.97 ± 0.25*** 4.95 ± 0.25*** 6.86 ± 0.34*** 9.69 ± 0.48*** 3.72 ± 0.19*** 5.93 ± 0.3*** 7.08 ± 0.35***

Values represent means ± SEM (N = 5). * p < 0.05. ** p < 0.01. *** p < 0.001.

Fig. 4. Effect of Ln on the ratios of AsA versus DHA, GSH versus GSSG and thiol versus disulfide of the mouse kidney by intragastric administrations with Ln solutions for 90 consecutive days. Additional details as in Fig. 1.

reductions of body weight, and increases of the kidney indices (Fig. 1) and Ln accumulation in mouse kidneys (Fig. 2) were observed, coupled with an increase of Cr level, decreases of BUN, UA, Ca and P levels in serum (Table 2), and induced inflammatory response and necrosis of kidneys (Fig. 3). Of Ln exposures, the Ce3+treated groups exhibited the most severe damages to kidney of

mice, next in the Nd3+-treated groups, and the least in the La3+treated groups. All lanthanides form stable triple-charged state when they lose outer electrons and the electron configuration of Ln3+ ions extends from f0 (La3+) to f14 (Lu3+) regularly. Thus La3+ has no f electron, Ce3+ has one and Nd3+ has three f electrons, respectively. Moreover, according to the Hund’s rule, the empty (f0), half-filled (f7) and the completely filled shell (f14) are in stable state. So Ce3+ (f1) can easily lose an electron to be oxidized to Ce4+ (f0) (Tsuchiya et al. 1999; Ni 2002; Arnd and Horst 2006). Ln as the 4f group elements varied only in the number of 4f electrons, their chemical properties are similar. Numerous reports proposed that the difference in the number of 4f electrons leads to quite different biological properties of Ln (Kostova et al., 2008; Li et al., 2010; Liu et al., 2010; Fei et al., 2011a,b; Cheng et al., 2011, 2012a; Zhao et al., 2011a,b). The differences from nephric injuries of mice caused by Ln were probably determined by the 4f electron of Ln. It is well known that serum BUN and UA were excreted out through the renal glomerulus by blood transportation and Cr is a breakdown product of creatinine phosphate and is usually produced by the body and excreted by the kidneys at a fairly constant rate (Bond, 1996). The changes in serum BUN, UA and Cr concentrations can be used as indicators of glomerular filtration rate, with a reduction in BUN or UA, and a rise in Cr levels indicating damage to nephrons. In this study, inflammatory response and necrosis in nephric tissue were observed due to the accumulated Ln, which in turn led to the high Cr level and the low BUN, UA, Ca and P concentrations in the serum (Table 2) and the serious pathological changes of kidneys (Fig. 3), and the mouse nephritis and cell necrosis was triggered by Ln ROS accumulation that resulted in the disruption of the nephric tissue.

Please cite this article in press as: Zhao, H., et al. Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.034

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H. Zhao et al. / Chemosphere xxx (2013) xxx–xxx Table 4 The activities of antioxidative enzyme of kidney of mice by intragastric administrations with Ln solutions for 90 consecutive days. Ln (mg kg1 BW) La

Ce

Nd

0 2 5 10 2 5 10 2 5 10

SOD (U mg1 prot min)

CAT (U mg1 prot min)

GST (U mg1 prot min)

GR (U mg1 prot min)

63.86 ± 3.19 53.21 ± 2.66* 46.50 ± 2.38** 41.80 ± 2.10** 44.99 ± 2.25** 32.96 ± 1.65*** 21.71 ± 1.09*** 50.60 ± 2.53* 41.27 ± 2.09** 36.26 ± 1.81***

434.72 ± 21.74 420.13 ± 21.02* 406.55 ± 20.33** 327.26 ± 16.36** 341.46 ± 17.07** 317.42 ± 20.87*** 246.86 ± 12.34*** 407.48 ± 20.34** 384.04 ± 19.20** 302.31 ± 15.12***

51.26 ± 2.56 45.03 ± 2.25 37.66 ± 1.88* 30.07 ± 1.50** 32.72 ± 1.64** 23.68 ± 1.18*** 17.68 ± 0.88*** 38.79 ± 1.94* 29.43 ± 1.52** 24.19 ± 1.21***

622.74 ± 31.14 598.26 ± 29.91* 528.96 ± 26.45** 464.28 ± 23.21** 424.38 ± 21.22** 341.34 ± 17.07*** 280.02 ± 14.00*** 483.84 ± 24.19** 451.26 ± 22.56** 398.40 ± 19.92***

Values represent means ± SEM (N = 5). * p < 0.05. ** p < 0.01. *** p < 0.001.

Table 5 mRNAs expressions of antioxidative enzymes in mouse kidney after intragastric administration of Ln for 90 consecutive days. Ln (mg kg1 BW) La

Ce

Nd

0 2 5 10 2 5 10 2 5 10

SOD (gene/actin)

CAT (gene/actin)

GST (gene/actin)

GR (gene/actin)

0.55 ± 0.028 0.45 ± 0.022* 0.39 ± 0.02** 0.27 ± 0.014*** 0.22 ± 0.01*** 0.16 ± 0.008*** 0.10 ± 0.005*** 0.29 ± 0.015* 0.24 ± 0.012** 0.16 ± 0.008***

0.62 ± 0.031 0.52 ± 0.026* 0.42 ± 0.021** 0.33 ± 0.017*** 0.26 ± 0.013*** 0.21 ± 0.011*** 0.15 ± 0.008*** 0.37 ± 0.019** 0.30 ± 0.015*** 0.23 ± 0.012***

0.39 ± 0.02 0.30 ± 0.015* 0.25 ± 0.012** 0.21 ± 0.011*** 0.19 ± 0.01*** 0.13 ± 0.007*** 0.07 ± 0.004*** 0.25 ± 0.013** 0.18 ± 0.009*** 0.13 ± 0.007***

0.27 ± 0.014 0.21 ± 0.011* 0.16 ± 0.008** 0.12 ± 0.006*** 0.12 ± 0.006*** 0.08 ± 0.004*** 0.06 ± 0.003*** 0.16 ± 0.008** 0.13 ± 0.007*** 0.09 ± 0.005***

Values represent means ± SEM (N = 5). * p < 0.05. ** p < 0.01. *** p < 0.001.

Kidney injury following exposure to Ln is probably because an imbalance between ROS production and their removal damages macromolecules and membranes, thus leading to increases of kidney indices, mouse nephritis, and cell necrosis. It had been demonstrated that Ln stimulates ROS production in mouse lung (Li et al. 2010), liver (Fei et al. 2011a; Zhao et al. 2012), spleen (Liu et al. 2010; Cheng et al. 2011) and brain (Zhao et al., 2011a; Cheng et al., 2012b, 2013). In contrast, numerous studies have also shown positive biological effects of Ln compounds and nano-particular CeO2, primarily through the protection of cells against damage by free radicals and ROS (Wang et al., 1997; Wang et al., 2000; Liu et al., 2006; Schubert et al., 2006; Ju-Nam and Lead, 2008; Ling and Hong, 2010). Lanthanum-containing KP772 widely inhibited H2O2-mediated ROS formation confirming the radical scavenging properties for KP772 in human cell lines (Heffeter et al., 2009). CeO2 nanoparticles can protect against cigarette smoke extract-induced oxidative stress and inflammation in cultured rat H9c2 cardiomyocytes (Niu et al., 2011), and may be a useful antioxidant treatment for oxidative stress (Hirst et al., 2011; Wason and Zhao, 2013). The present data show that the ROS production in mouse kidney following exposure to LaCl3, CeCl3, and NdCl3 was significantly elevated (Table 3), suggesting that the kidney suffered from oxidative stress. The reasons for the generation of ROS are not understood. However, these ROS might be either from Ln-damaged mitochondria or other Ln-triggered signal pathways (Liu et al., 2001, 2003). Few studies indicated that Ln can increase the intracellular Ca2+ level by increasing the Ca influx, which indirectly induced ROS production (Wang et al., 1999;  Wu et al., 2013). Interaction between H2O2 and O 2 can create OH and 1O2, which are far more destructive and can peroxidate the unsaturated lipid of the cell membrane (Fridovich, 1978). As one kind of peroxide, MDA can intensively react with various cellular

components; therefore, enzymes and membranes are seriously damaged and membranous electric resistance and fluidity fall, and this eventually cause the destruction of the membrane structure and physiological integrality (John and Scandalios, 1993). In this study, the lipid peroxidation of the kidney membrane along with increased LaCl3, CeCl3, and NdCl3 doses was demonstrated by a significant increase in MDA content (Table 3). Protein peroxidation is often generated in organisms, which results in protein structure damage and dysfunction. The carbonyl level of proteins is widely used as an indicator of oxidative protein damage (Stadtman and Levine, 2003). Elevated carbonyl level has been demonstrated in organisms under various oxidative stresses (Kasai and Nishimura, 1986; Chevion et al., 2000). Our results show an increased carbonyl level in the LaCl3, CeCl3, and NdCl3-exposed mouse kidney (Table 3). Furthermore, ROS over production in cells cannot only result in oxidative damages in lipids and proteins, but also conduct to oxidative damage of DNA. Various reagent chemicals are effective in the hydroxylation of the deoxyguanosine residue in DNA. H2O2 generation is demonstrated to induce 8-OHdG or 2, 6-diamino-4-hydroxy5-formamidopyrimidine formation (Grollman and Moriya 1993; Evans et al. 2004), while 8-OHdG is by far the most studied oxidative DNA lesion and has caused much attention because of its mutagenic potential (Pilger and Rüdiger 2006). The oxidized guanine residue 8oxoguanine can pair both in Watson–Crick mode with cytosine and in Hoogsteen mode with adenine. The latter causes G:C ? T:A transversion in cells. As suggested in cells of patients with Cockayne syndrome, the deficiency in nucleotide excision repair causes a low level of 8-OHdG repair and a high frequency of G:C ? T:A transversion at the site of the lesion. Besides, the transversions are frequent in human cancers and are especially prevalent in the mutational spectrum of the tumor suppressor gene p53. It suggests the significance

Please cite this article in press as: Zhao, H., et al. Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.034

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Table 6 Proteins expressions of antioxidative enzymes in mouse kidney after intragastric administration of Ln for 90 consecutive days. Ln (mg kg1 BW) La

Ce

Nd

0 2 5 10 2 5 10 2 5 10

SOD (ng g1 tissue)

CAT (ng g1 tissue)

GST (ng g1 tissue)

GR (ng g1 tissue)

122.65 ± 6.13 97.9 ± 3.9* 76 ± 3.8** 53 ± 2.65*** 49.22 ± 1.61*** 36.53 ± 1.83*** 22.69 ± 1.13*** 72.78 ± 2.64** 55.23 ± 2.76*** 39.81 ± 1.99***

138.26 ± 6.91 110.9 ± 4.84* 85 ± 4.25** 62 ± 3.1*** 56.54 ± 2.33*** 39.39 ± 1.97*** 26.38 ± 1.32*** 87.34 ± 3.37** 62.38 ± 3.12*** 42.83 ± 2.14***

86.97 ± 4.35 67.0 ± 2.85* 52 ± 2.6** 41 ± 2.05*** 44.01 ± 1.7*** 32.16 ± 1.61*** 17.89 ± 0.89*** 55.5 ± 2.28** 41.27 ± 2.06*** 27.93 ± 1.4***

60.21 ± 3.01 50.9 ± 2.0* 38 ± 1.9*** 28 ± 1.4*** 31.48 ± 1.57c*** 22.17 ± 1.11*** 15.28 ± 0.76*** 41.12 ± 1.46** 31.76 ± 1.59*** 21.96 ± 1.1***

Values represent means ± SEM (N = 5). * p < 0.05. ** p < 0.01. *** p < 0.001.

of 8-OHdG as an endogenous mutagen and its possible role in the process of carcinogenesis (Fortunato et al., 2006). Accordingly, 8-OHdG level in the LaCl3, CeCl3, and NdCl3-exposed mouse kidney was greatly elevated compared to control (Table 3). Therefore, long-term exposure to Ln was demonstrated to cause severe oxidative damages in mouse kidney. Organisms possess their own active antioxidant defense systems (antioxidative enzymes such as SOD, CAT, GST, and GR, as well as non-enzymatic antioxidants such as GSH, AsA, and thiol) through which production and removal of ROS is in balance. SOD can convert O 2 into H2O2 and O2; moreover, CAT can reduce H2O2 into H2O and O2 (Beauchamp and Fridovich 1971; Buege and Aust 1978). GR plays a key role in oxidative stress by converting the GSSG into GSH. The elevated levels of GR activity could increase the GSH/GSSG ratio, which is required for AsA regeneration (Crawford et al., 2000). GST catalyzes the detoxification of lipid peroxides and xenobiotics by conjugating them with GSH (Noctor et al., 2002). Therefore, SOD, CAT, GST, and GR can keep a low level of ROS and prevent ROS toxicity and protect cells. AsA directly interact with and detoxify oxygen free radicals and thus contribute significantly to non-enzymatic ROS scavenging. GSH is a key component of the antioxidant network that scavenges ROS either directly or indirectly by participating in the AsA–GSH cycle (Noctor and Foyer, 1998; Smirnoff, 2005). The central role of GSH in the antioxidant defense system is due to its ability to regenerate AsA through reduction of dehydroascorbate via the AsA–GSH cycle (Noctor and Foyer, 1998). GSH also plays a vital role in the antioxidant defense system by acting as a substrate or cofactor for some enzymes. Furthermore, GSH plays a protective role in Ln-induced toxicity by the maintenance of the redox state (Liu et al., 2010; Fei et al., 2011a). While thiol is considered to be a highly reactive constituent of protein molecules, and it acts as an antioxidant and participates in the detoxification of xenobiotics and toxic substances. Increased thiol contents under adversity (Bartoli et al., 1999; Agrawal and Rathore, 2007) indicate that oxidative stress might induce the oxidation of thiol group to disulfide group, resulting in alteration of the cellular thiol–disulfide redox state. Thiol– disulfide interconversion plays a major role in the regulation of different physiological processes, and depends strongly on the redox state of the thiol pool (Smirnoff, 2005). Kawagoe et al. (2005) suggested that orally administered cerium increased metallothionein and GSH content as an antioxidant in mouse liver. 1.5 mg kg1 BW CeCl3 treatment was also demonstrated to increase antioxidant capacity via increasing activities of SOD, CAT, APx, and GSHPx, and increasing contents of GSH and ASA in the liver of silver crucian carp under Pb2+-induced toxicity (Ling and Hong, 2010), which is attributed to Hormesis effects of Ln. In our study, however, the

reductions of SOD, CAT, GST and GR activities (Table 4) and their genes and proteins expression (Tables 5 and 6), GSH, AsA, and thiol contents (Fig. 4) in mouse kidney caused by Ln exposure were observed, these reductions from CeCl3-exposed groups were higher than those of exposure to NdCl3, and those of exposure to NdCl3 was higher than those of exposure to LaCl3 (Tables 5 and 6, Fig. 4), which was associated with increases in ROS, MDA, PC and 8-OHdG (Table 3). It suggests that the decreases of antioxidant capacity in kidney may be attributable to 4f electron shell and alterable valence characteristics of Ln. Ca is not only the important component of cell structure, but also participates in many physiological and biochemical process to regulate the cells. Furthermore, Ca2+ can activate SOD, CAT, GST, and GR, decrease oxidative stress in vivo (Buchanan et al., 2002; Michaela et al., 2002; Chaney et al., 2008; Wu et al., 2008). As we know, Ln3+ properties are similar to Ca2+, and after entering the body Ln3+ can more easily occupy or substitute for the position of Ca2+, thus resulting in the oxidative stress and biochemical dysfunction in mouse organ (Guo et al., 1990; Tsuchiya et al., 1999). The previous reports demonstrated that the Ca levels in rat hippocampus and mouse brain significantly decreased by exposure to La or Ce, and the imbalance of the Ca level and Ca2+ binding protein can impair the ion homeostasis and lead to a series of physiological disorders in the brain (Feng et al., 2006; Zhao et al., 2011b). In the study, our data suggest that Ln exposure decreased Ca and P levels in serum (Table 2), which associated with Ln accumulation in the kidney (Fig. 2) and inhibition of mouse growth (Fig. 1). Of three rare earths, the Ce3+ chemical properties are most similar to Ca2+, i.e. Ce3+ radius is at 101– 120 pm when its coordination number is at 6–9, and Ca2+ radius is at 100–118 pm when its coordination number is at 6–9; and Ce3+ can more easily occupy or substitute for the position of Ca2+ than La3+ (radius is at 103–122 pm) and Nd3+(radius is at 98– 116 pm); which may be why Ce that has 4f electron shell and alterable valence characteristics has the most severe oxidative stress in mouse kidney. In addition, Ce also participates in a series of redox reaction through valence alteration between +3 and +4 in vivo, which results in a series of changes of biological functions (Liu et al., 2006). As 4f electron shell orbit of Nd is located at the inner shell compared with natural valence electronic orbit, their bonding ability is weak, so Nd do not participate in bonding reaction under natural conditions. However, 4f electron still of Nd shows some low bonding ability, which may play a role of ‘‘residual chemical bond or residual valence’’, and this ability is just what catalyst requires (Guo et al., 1990; Tsuchiya et al., 1999). Furthermore, Nd3+ radius is more similar to Ca2+ than La3+. Therefore, Nd3+ exhibited stronger oxidative stress to mouse kidney than La3+ without 4f electron.

Please cite this article in press as: Zhao, H., et al. Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.034

H. Zhao et al. / Chemosphere xxx (2013) xxx–xxx

5. Conclusion The results of this study added to a growing body of knowledge of Ln-induced kidney toxicity and antioxidative responses in mouse kidney. We suggested that Ln exposure could cause obvious ROS accumulation, lipid, protein and DNA peroxidation, which was attributed to the decrease of antioxidative defence, resulting in serious injuries of kidney. Of the three rare earths, the Ce3+-treated groups exhibited the most severe oxidative stress, next in the Nd3+treated groups, and the least in the La3+-treated groups. The discrepancy of oxidative stress in mouse kidney following exposure to Ln was probably determined by the 4f electron of Ln. Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant No. 81273036, 30901218), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institution, National New Ideas Foundation of Student of China (Grant No. 111028534), and the National Bringing New Ideas Foundation of Student of China (Grant No. 57315427, 57315927). References Agrawal, S.B., Rathore, D., 2007. Changes in oxidative stress defense system in wheat (Triticum aestivum L.) and mung bean (Vigna radiata L.) cultivars grown with and without mineral nutrients and irradiated by supple-mental ultraviolet-B. Environ. Exp. Bot. 59, 21–33. Arnd, V., Horst, K., 2006. Excited state properties of lanthanide complexes: beyond ff states. Inorg. Chim. Acta 359, 4130–4138. Bartoli, C.G., Simontacchi, M., Tambussi, E., Beltrano, J., Montaldi, E., Puntarulo, S., 1999. Drought and watering-dependent oxidative stress: effect on antioxidant content in Triticum aestivum L. leaves. J. Exp. Bot. 50, 375–383. Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and assay applicable to acrylamide gels. Anal. Biochem. 44, 276–286. Buchanan, B.B., Gruissem, W., Johones, R.L., 2002. Biochemistry and Molecular Biology of Plants. Science Press, American Society of Plant Physiology, Beijing. pp. 962–9781. Bond, C.E., 1996. Biology of Fishes, third ed. Saunders College Publishing, Orlando, FL. Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. Methods Enzymol. 52, 302–310. Chaney, L.R., Chen, K.Y., Li, Y.M., Scott, J.A., Baker, J.M.A., 2008. Effects of calcium on nickel tolerance and accumulation in Alyssum species and cabbage grown in nutrient solution. Plant Soil 311, 131–140. Chen, X.A., Cheng, Y.E., Rong, Z., 2005. Recent results from a study of thorium lung burdens and health effects among miners in China. J. Radiol. Prot. 25, 451–460. Cheng, J., Li, N., Cheng, Z., Hu, R.P., Sun, Q.Q., Gui, S.X., Sang, X.Z., Wang, L., Hong, F.S., 2012a. Organ histopathological changes and its function damage in mice following long-term exposure to lanthanides chloride. Biol. Trace Elem. Res. 145, 361–368. Cheng, Z., Li, N., Cheng, J., Hu, R., Gao, G.D., Cui, Y.L., Gong, X.L., Wang, L., Hong, F.S., 2012b. Signal pathway of hippocampal apoptosis and cognitive impairment of mice caused by cerium chloride. Environ. Toxicol. 27, 707–718. Cheng, J., Li, N., Cheng, Z., Hu, R.P., Cai, J.W., Si, W.H., Hong, F.S., 2011. Splenocyte apopotic pathway in mice following exposure to cerium chloride. Chemosphere 8, 612–617. Cheng, Z., Zhao, H.Q., Ze, Y.G., Su, J.J., Li, B., Sheng, L., Zhu, L.Y., Guan, N., Gui, S.X., Sang, X.Z., Zhao, X.Y., Sun, Q.Q., Wang, L., Cheng, J., Hu, R.P., Hong, F.S., 2013. Gene-expression changes in cerium chloride-induced injury of mouse hippocampus. PlosOne 4 (8), e60092. Cheng, J., Fei, M., Sang, X.Z., Cheng, Z., Gui, S.X., Zhao, X.Y., Sheng, L., Sun, Q.Q., Hu, R.P., Wang, L., Hong, F.S., in press. Gene expression profile in chronic mouse liver injury caused by long-term exposure to CeCl3, 2013. Environ. Toxicol. http://dx.doi.org/10.1002/tox.21826. Chevion, M., Berenshtein, E., Stadtman, E.R., 2000. Human studies related to protein oxidation: protein carbonyl content as a marker of damage. Free Radical Res. 33, S99–S108. Claiborne, A., 1985. Catalase Activity. In: Greenwaid, A. (Ed.), Handbook of Methods for Oxygen Free Radical Research. CRC R, Boca Raton, FL. Crawford, N.M., Kahn, M.L., Leustek, T., Long, S.R., 2000. Nitrogen and Sulfur. In: Buchanan, B.B., Gruissem, W., Jones, R.L. (Eds.), Biochemistry and Molecular Biology of Plants. Rockville: ASPP, pp. 786–849. Evans, M.D., Dizdaroglu, M., Cooke, M.S., 2004. Oxidative DNA damage and disease: induction, repair and significance. Mutat. Res. 567, 1–6.

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Please cite this article in press as: Zhao, H., et al. Oxidative stress in the kidney injury of mice following exposure to lanthanides trichloride. Chemosphere (2013), http://dx.doi.org/10.1016/j.chemosphere.2013.05.034