Cadmium-Induced Microsatellite Instability in the Kidneys and Leukocytes of C57BL/6J Mice Xiaoyan Du,1* Tianfeng Lan,2* Bao Yuan,1 Jian Chen,1 Jinping Hu,1 Wenzhi Ren,1 Zhenwen Chen2 1

Department of Laboratory Animal Science, School of Basic Medical Science, Capital Medical University, Beijing 100069, China 2

Laboratory Animal Center, Jilin University, Changchun, Jilin, 130062, China

Received 5 September 2013; revised 15 December 2013; accepted 21 December 2013 ABSTRACT: Cadmium is a cytotoxic, carcinogenic, and mutagenic industrial product or byproduct. The correlation between metal exposure and microsatellite instability (MSI) has been reported by several groups. In the present study, 50 C57BL/6J mice at 6 weeks of age were divided into five groups and intraperitoneally injected with 0, 0.25, 0.5, 1, or 2 mg/kg cadmium chloride quaque die alterna for 4 weeks. Then, the liver, kidney, testis, leukocytes, bone marrow, and small intestine were collected from the treated mice and weighed. Portions of these tissues were fixed for further histological analysis, and the remaining tissues were subjected to genomic DNA extraction for the analysis of a panel of 42 microsatellite markers. The liver and testis weight coefficients were significantly changed in the 1 and 2 mg/kg cadmium chloride-treated groups compared with the control group. Simultaneously, severe histopathologic changes in the liver and kidneys, along with a complete disorganization of testicular structure and obvious severe necrosis in the testes were observed in the cadmium-treated group. The cadmium accumulated in the liver and kidneys of the mice in all cadmium-treated groups; the tissue cadmium concentrations were significantly higher than those in the control group. After STR scanning, MSI was found at three loci (D15Mit5, D10Mit266, and DxMit172) in the kidneys and leukocytes of mice in the lower dose groups (0.25 and 0.5 mg/kg). In summary, we have successfully established a sub-chronic cadmium exposure model and confirmed that cadmium exposure can induce MSI in mice. We also identified two loci C 2014 Wiley Periodicals, Inc. that could be regarded as “hotspots” of microsatellite mutation in mice. V Environ Toxicol 00: 000–000, 2014.

Keywords: cadmium chloride; microsatellite instability; C57BL/6J mice

INTRODUCTION Additional Supporting Information may be found in the online version of this article. Correspondence to: Z. Chen; e-mail: [email protected] or [email protected] *These authors contributed equally to this work. Contract grant sponsor: National Science Foundation of China. Contract grant numbers: 30970420, 31172168, and 31071988. Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/tox.21946

Cadmium is a heavy metal and an environmental contaminant. It is used in many industrial processes, such as electroplating (as a deoxidizer in nickel plating) and engraving processes, and in many products, including easily fusible alloys, soft solder, electrodes for vapor lamps, photoelectric cells, and nickel–cadmium storage batteries (Pilgrim et al., 2005). Food and cigarette smoke are two major sources of human exposure to Cd (Schwartz and Reis, 2000; Mannino

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et al., 2004). Cadmium is eliminated from most organs very slowly, with a long half-life (15–20 years) (Jin et al., 1998). No effective treatment or cure for chronic cadmium intoxication has been reported to date (Li et al., 2010). In 1993, the International Agency for the Research on Cancer classified cadmium as a confirmed human carcinogen (IARC, 1993). It has been reported that there might be a causal relationship between Cd exposure and the occurrence of lung cancer in humans, and exposure to Cd has been shown to cause tumors in multiple organs and tissues in experimental animals (Waalkes et al., 1999, 2000; Qu et al., 2012). Nevertheless, the mechanism underlying Cd carcinogenesis has not yet been elucidated. Possible mechanisms involved in Cd carcinogenesis can be broadly categorized as aberrant gene expression, DNA damage repair inhibition, apoptosis inhibition, and oxidative stress induction (Joseph, 2009). Cadmium inhibits several types of DNA-repair mechanism, i.e. base excision (Fatur et al., 2003), nucleotide excision (Hartwig and Schwerdtle, 2002), and the limination of the pre-mutagenic DNA precursor 7,8-dihydro8-oxoguanine (Bialkowski and Kasprzak, 1998). Furthermore, chronic exposure of yeast to very low cadmium concentrations results in hypermutability; and in human cell extracts, cadmium has been shown to inhibit DNA-mismatch repair (Jin et al., 2003). Microsatellites, or short tandem repeats (STRs), are short sequences of DNA, normally of one to six base pairs in length. Microsatellites are abundant in both noncoding and coding regions of mammalian genomes (Weber, 1990; Stallings et al., 1991). They are useful DNA markers for deciphering genetic variability due to their abundance, random occurrence and high degree of polymorphism (McDonald and Potts, 1997). Due to microsatellites’ repetitive nature, they are particularly prone to slippage during DNA replication, which causes size variations called microsatellite instability (MSI) (Thibodeau et al., 1993; Lengauer et al., 1997). Microsatellites lose or gain repeats when MMR (mismatch repair) proteins, such as MLH1, MSH2, MSH3, MSH6, PMS1, and PMS2, are absent or display reduced activity in specific organs or tissues (Aquilina et al., 1994; Palombo et al., 1995; Strand et al., 1995; Acharya et al., 1996; Culligan et al., 2000; Staebler et al., 2000; Acharya et al., 2003; Kunkel and Erie, 2005; Jascur and Boland, 2006; Ling et al., 2010). These phenomena are often caused by germline and/ or somatic mutations. Cadmium has been shown to impair DNA mismatch repair. Jin et al. (2003) took advantage of a yeast model system to demonstrate that low concentrations of cadmium ions inhibit MMR in yeast cells and in vitro cellfree extracts. This inhibition occurs through the Msh2–Msh3 and Msh2–Msh6 protein complexes. Clark and Kunkel (2004) suggested that Cd suppressed both eukaryotic MSH2MSH6- and MSH2-MSH3-dependent human MMR activity in vitro. They also demonstrated that Cd attenuated the DNA binding affinity of MSH2-MSH6 and impaired its ability to discriminate between mismatched and matched duplexes.

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Few reports have investigated whether cadmium induces MSI in humans or experimental animals. Recently, ElGhor et al. (2010) investigated MSI at three microsatellite loci (D6mit3, D9mit2, and D15Mgh1) in leukocytes isolated from cadmium-exposed rats. Their findings demonstrated that the analysis of microsatellites in fragile sites provides an efficient assay system for the detection of rare mutations in rat. Oliveira et al. (2012) used a panel of six microsatellite loci to detect the genotoxic effects of cadmium in murine testes. They detected MSI in two of the five tested microsatellite markers in cadmium-exposed mouse testes. In the present study, we tested the mutagenic effects of cadmium on a panel of 42 loci in murine liver, kidney, testis, leukocytes, bone marrow, and small intestine. Zuo et al. (2012) have used these 42 microsatellite loci to determine the relationship between MSI and various gene knockout phenotypes. The microsatellite loci in this panel exhibited changes from monomorphism to polymorphism in various knockout mice. Du et al. have also used this panel to detect genetic alterations (GAs) in mice and found MSI in transgenic mice and ENU-treated mice (Du et al., 2013).

MATERIALS AND METHODS Ethics Statement All the experiments and animal procedures were conducted in accordance with the Institutional Guidelines for Ethics in Animal Research, Changchun, China. The experiment protocol and project were approved by the ethics committee of Jilin University and the license number for this research is 2011035.

Animals and Treatment Fifty male C57BL/6J (B6) mice at 6 weeks of age, weighing 16 to 18 g each, were purchased from Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). The animals were housed in an IVC system at a constant temperature (23 6 2 C) and relative humidity (55% 6 5%) on a 12 h light/12 h dark cycle. Sterile water and food were provided ad libitum. The mice were allowed to acclimate to the environment for 1 week prior to the start of the experiments. Animals were randomly divided into four experimental groups of 10 mice each. Before treatment, the tails of all animals were sheared to approximately 1.5 cm long as a background control. The mice were weighed and then administered 0.25, 0.5, 1, or 2 mg CdCl2/kg bw via intraperitoneal injection every 2 days. Ten mice from the control group were injected with the solvent (distilled water). After 28 days of exposure, the mice were anaesthetized; then, blood samples were collected by retro-orbital puncture, the

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mice were euthanized, and tissues were collected for subsequent analyses.

Weight and Cd Concentration Determination After the mice were euthanized, their livers, kidneys, and testes were removed and weighed. The weight coefficients of the liver, kidneys, and testes were determined. The Cd concentrations in the liver, kidneys and testes were determined by atomic absorption spectrometry.

DNA Extraction Genomic DNA was extracted from the frozen liver, kidney, testis, leukocyte, bone marrow, and small intestine samples and their corresponding controls (pre-treated tails) using a standard phenol-chloroform extraction and ethanol precipitation method (Du et al., 2010). For the blood sample, WBCs were obtained, and total genomic DNA was isolated from the WBCs as described by El-Ghor (2010). The concentration and purity of each DNA sample were determined using a micro-volume spectrophotometer (Thermo Scientific NanoDrop 2000) and further evaluated by agarose gel electrophoresis. The DNA was diluted to a concentration of 100 ng/mL and stored at 220 C for later use as a PCR template.

Microsatellite Analysis Forty-two microsatellite loci that had previously been identified as becoming polymorphic in genetically modified mice and in chemically treated mice (Zuo et al., 2012; Du et al., 2013) were used to evaluate microsatellite instability in this study. The loci were evaluated in each mouse using PCR and electrophoresis. PCR reaction samples were prepared in a total volume of 20 lL as follows: 2 lL 103 buffer, 0.5 lmol/L each primer, 125 lmol/L dNTPs, 1.0 U Taq DNA polymerase, 1.5-2.5 mmol/L MgCl2, and 100 ng template DNA. The PCR amplification was performed in a gradient thermal cycler (BIO-RAD Inc. ALS1296) using the following protocol: pre-denaturation at 94 C for 5 min; 35 cycles of denaturation at 94 C for 30 s, annealing at the appropriate gradient temperature for each microsatellite for 30 s, and extension at 72 C for 30 s; and a final extension at 72 C for 5 min. The PCR products were analyzed on 2% agarose gels stained with ethidium bromide and visualized using a UV transilluminator (Vil Ber Lou RMAT Inc.). If the PCR products from all the animals of a specific strain were amplified successfully, then the samples were stored at 4 C for further STR scanning. The primer sequences and optimized annealing temperatures for each locus are shown in Supporting Information Table S1. The forward primer for each locus was tagged at the 50 end with a FAM, HEX, or TAMRA fluorescent marker. Three types of PCR products that were synthesized from fluorescently labeled primers were mixed at a ratio of 1: 2: 3 (FAM: HEX: TAMRA). A 1 lL aliquot of each mixture was

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gently mixed with 25 lL formamide and then visualized using capillary electrophoresis (CE) on an ABI-3730XL DNA Analyzer system (PE Biosystems). The peak height of the waves for each product was determined using GeneMarker software. For sequencing, the PCR products (total volume of 50 mL) were purified using an ABI BigDye Terminator v3.1 Cycle Sequencing Kit, cloned into the PMD18T vector (TAKARA), and then sequenced using an ABI 3730XL DNA Sequencer.

Histological Analysis Slices of the left liver lobe and the left kidney and testes were fixed in 10% buffered formalin, dehydrated and embedded in paraffin wax. Horizontal sectioning was performed using a microtome (MICROW) to prepare 4 to 6 lm slices. Subsequently, the tissue sections were stained with hematoxylin-eosin and examined histologically under a microscope (AE30 MOTIC). The individual(s) performing histological outcomes were blinded to the treatment group.

Statistical Analysis Weight results are expressed as the mean 6 SD. Statistical comparisons were performed using SPSS13.0 (SPSS Inc.). The weights of control and CdCl2-treated groups were compared using one-way analysis of variance. The level of statistical significance was set at p  0.05. The amounts of Cd in the liver, kidney, and testes in the control and CdCl2-treated groups were also compared using one-way analysis of variance. The level of statistical significance was set at p  0.01.

RESULTS Microsatellite Analysis We analyzed 42 microsatellite loci in the liver, kidney, testis, leukocyte, bone marrow, and small intestine of exposed mice. PCR products from each locus in each tissue of every animal were analyzed by STR scanning. Uniform microsatellite patterns were found for all markers in the pretreated mice tails and showed that the genetic backgrounds of all animals in the present study were identical for the 42 microsatellite loci. However, compared with the background control and blank control groups, the CdCl2-treated groups exhibited MSI at 3 loci (7.14%, 3 of 42 loci) in two mice in the two lower dose groups (0.25 mg/kg and 0.5 mg/kg). Two MSI loci (4.76%, 2 of 42) were found in one mouse in group 1 (10%, 1/10). Only one MSI locus (2.38%, 1 of 42) was found in one mouse in group 2 (10%, 1 of 10). The other two groups of higher doses were MSI negative. As shown in Figure 1, both D15Mit5 and D10Mit266 exhibited MSI in mouse number 10 in group 1 (0.25 mg/kg) in the kidney and leukocytes, respectively. For the blank control, the pre- vs. post-treatment allele sizes of D15Mit5 and D10Mit266 were

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Fig. 1. STR scanning results of the D15Mit5 (kidney), D10Mit266 (leukocytes), and DxMit172 (leukocytes) loci showed microsatellite instability in post-treated mice (number 10 and 20) compared with pretreated mice and blank control mice. The allele sizes of D15Mit5 in pre- and post-treated mouse number 10 and control mice were 100 bp (Aa), 102 bp (Ab), and 100 bp (Ac) respectively, with 2 bp insertion in Cd-treated mouse. The allele sizes of D10Mit266 in pre- and post-treated mouse number 10 and in control mice were 92 bp (Ba), 90 bp (Bb), and 92 bp (Bc) respectively, with 2 bp deletion in Cd treated mouse. The allele sizes of DxMit172 in pre- and post-treated mouse number 20 and control mice were 148 bp (Ca), 146 bp (Cb), and 148 bp (Cc) respectively, with 2 bp insertion in Cd treated mouse. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

100 bp vs. 102 bp and 93 bp vs. 91 bp, respectively. DxMit172 showed MSI in mouse number 20 in group 2 (0.5 mg/kg) in leukocytes. For the blank control, the pre- vs. post-treatment allele sizes at this locus were 148 bp vs. 146 bp. We analyzed MSI by cloning these sequences as well. A photograph of typical sequence results is presented in Figure 2; this image shows that the repeat motif in the sequence of the D15Mit5 locus is 2 bp longer in the treated mouse than in the background control.

et al., 2013). Thereinto, loci D10Mit266 and DXMit172 exhibited MSI in transgenic mouse strains (Du et al., 2013), gene knockout mouse strains (Zuo et al., 2012), and our CdCl2-induced strain. Similarly, the D15Mit5 locus exhibited MSI in transgenic mouse strains, an ENU-induced mutation strain (Du et al., 2013), and our CdCl2-induced strain (see Table I). Thus, these three loci could be regarded as “hot spots” of microsatellite mutation in mice.

Cadmium Concentration Determination MSI Loci were Detected Simultaneously in Cd-Induced Mice and GA Mice We found that among the microsatellites investigated in this study, three loci which exhibited MSI in Cd-treated mouse are shared MSI in our previous study (Zuo et al., 2012; Du

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The amounts of Cd in the liver, kidney, and testis were measured by atomic absorption spectrometry; the results are shown in Figure 3. The cadmium concentrations in the liver and kidneys increased with the increase in the cadmium dose. The cadmium accumulation levels in the liver and

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Fig. 2. The DNA sequences of the D15Mit5 locus (positive for MSI). Sequencing showed that the repeat motif is 2 bp shorter in the background control (top) than in the treated mouse (bottom). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

kidneys were significantly higher in all treated groups than in the control group (p 5 0.000, p < 0.01). The testis cadmium concentrations were significantly higher in group 3 and group 4 than in the control group (p 5 0.000, p < 0.01). No significant difference in testis cadmium was observed among group 1, group 2, and the control group. The cadmium concentration was higher in the liver than in the kidneys or testes.

Histopathology We also examined the effects of CdCl2 exposure on the liver, kidneys, and testes using histological methods (Fig. 4). The livers of the control group mice showed a normal structure (Fig. 4a-A), but the administration of Cd altered this structure. Congestion and swelling of the veins were noted in the hepatic lobules of the higher dose group (2 mg/kg). Severe granular degeneration was also noted in the cells

adjacent to the central vein zone. Meanwhile, the necrotic changes in the liver cells became more serious with increasing Cd concentration. Dilatation and congestion were observed in the renal tubular interstitial capillaries of the kidneys in treated mice. Epithelial cells were swollen and were filled with pink granules, indicating degeneration. Renal tubular epithelial cell necrosis and nuclear condensation were observed in the higher dose group (2 mg/kg) [Fig. 4(b)]. The testis tissue was deeply affected by CdCl2 exposure, particularly at the doses of 1 and 2 mg/kg bw. The highest dose of CdCl2 (2 mg/kg bw) induced severe alterations in the testes; pyknotic nuclei and dense and deeply stained cytoplasm were observed in the spermatogonia, and multinucleated giant cells were visible [Fig. 4(c)]. Seminiferous tubule atrophy, seminiferous epithelium thinning, and the dissociation of spermatogenic cells were also observed in the high-dose group.

TABLE I. MSI detected simultaneously in genetically altered mice strains and Cd-induced mice

STR Loci With MSIs D10Mit266 D11mit258 D13mit3 D14mit102 DXmit172 D15mit5

Transgenic Mouse Strains

Gene Knockout Mice Strains

ENU-Induced Mutation Mice Strains

Cd-Induced Mutation Mice Strains

1 1 1 1 1 1

1 1 1 1 1 2

2 2 2 1 2 1

1 2 2 2 1 1

Loci D10Mit266 and DXMit172 exhibited MSI in transgenic mouse strains, gene knockout mice strains, and CdCl2-induced strain Similarly, the D15Mit5 locus exhibited MSI in transgenic mouse strains, an ENU-induced mutation strain, and our CdCl2-induced strain. STR loci: the number of microsatellite loci with MSI; “1”: MSI positive; “2”: MSI negative.

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Fig. 3. Cadmium concentrations in the kidneys, testis, and liver. The cadmium concentrations in the liver and kidneys increased with the increase in the cadmium dose. The cadmium accumulation levels in the liver and kidneys were significantly higher in all treated groups than in the control group. The testis cadmium concentrations were significantly higher in group 3 and group 4 than in the control group.

Body Weight and Weight Coefficients of the Liver, Kidneys, and Testes After 28 days of Cd exposure, all animals were weighed and euthanized. The liver, kidneys, and testes of all mice were collected and weighed. Compared with the control group, at the end of experiment, the body weights of the mice in the control group (29.26 6 0.47) were significantly higher than those in group 2 (24.24 6 4.33), group 3 (22.27 6 4.22), and group 4 (17.92 6 3.35) (p 5 0.003, p < 0.01). Only group 1 has no significant difference with control group (25.46 6 2.54), indicating that lowest dose has less impact on body weights of the mice than higher doses. The weight coefficient of the liver and testis (WCL and WCT) were only significantly increased in mice that had been exposed to 2 mg CdCl2/kg bw (0.0782 6 0.0020 and 0.0078 6 0.0011, respectively) compared to control mice (0.0584 6 0.0011 and 0.0056 6 0.0001, respectively) (p 5 0.000, p < 0.05). No significant difference exhibited in other three groups and control group. However, the kidney weight coefficients were unaffected in all treatment groups (Table II).

DISCUSSION Cadmium pollution incidents have raised public concerns in recent years. Human sources of cadmium exposure include environmental and occupational exposures. Occupational exposures are of particular concern for workers in battery

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factories, pigment plants, and zinc smelters, as well as for welders. Environmental exposures occur mainly through smoking and through the consumption of food grown in cadmium-contaminated areas (World Health Organization, 1992). A correlation between metal exposure and MSI has been reported by some studies. Zienolddiny et al. (2000) found that nickel can promote microsatellite mutations in human cell lines. Slebos et al. (2006) showed that exposure to cadmium significantly increased MSI in the human fibrosarcoma cell line HT1080-C1. There are three MSI loci that were altered in different organs in the mice in the lowerdose groups (0.25 and 0.5 mg/kg). No MSI was observed in the higher-dose groups (1 and 2 mg/kg bw). We analyzed all of the loci in the liver, kidney, testis, leukocyte, bone marrow, and small intestine of exposed mice and observed MSI in the kidneys and leukocytes only. Only 1 of 10 mice tested (10%) presented MSI in the 0.25 and 0.5 mg/kg groups. In general, the frequencies of MSI are 7.14% (3 of 42) for all detected loci and 5% (2 of 40) for all treated mice, representing a lower frequency than that in the previous report (Oliveira et al., 2012). To exclude the spontaneous origin of these MSI, every locus determined to be MSI was compared with the PCR results of the background control DNA and blank control DNA. To exclude the influence of genetic variance from sampling, we examined 10 C57BL/6J(B6) mice, which belonged to three consecutive generations, for their microsatellite stability at 42 loci that were to be applied in this study. The results showed that there were no variations

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Fig. 4. Histopathological section of liver (a), kidney (b), and testis (c) (A, control group; B, group 1; C, group 2; D, group 3; and E, group 4; 3400, F200). Severe histopathologic changes in the liver and kidneys, along with a complete disorganization of testicular structure and obvious severe necrosis in the testes, were observed in the cadmium-treated group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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TABLE II. Effects of cadmium chloride on the weight coefficient of liver, kidneys, and testis in mice at the end of 28 days of exposure to CdCl2 Groups Control Group 1 (0.25 mg/kg bw) Group 2 (0.5 mg/kg bw) Group 3 (1.0 mg/kg bw) Group 4 (2.0 mg/kg bw)

BW (g)

WCL

WCK

WCT

29.26 6 0.47 25.46 6 2.54 24.24 6 4.33a 22.27 6 4.22a 17.92 6 3.35a

0.0584 6 0.0011 0.0598 6 0.0008 0.0660 6 0.0019 0.0521 6 0.0161 0.0782 6 0.0020b

0.0162 6 0.0001 0.0148 6 0.0001 0.0162 6 0.0013 0.0202 6 0.0070 0.0190 6 0.0026

0.0056 6 0.0001 0.0054 6 0.0002 0.0060 6 0.0012 0.0044 6 0.0002 0.0078 6 0.0011c

Compared with control group, body weights of mice were significantly lower in doses of 0.5, 1.0, and 2.0 mg/kg bw group. The liver and testis weight coefficients were significant increased in mice of group 4. a Significant difference between the control and group 2, group 3, or group 4 (CdCl2-treated mice) (p 5 0.003, p < 0.01). b Significant difference between the control group and group 4 (p 5 0.043, p < 0.05). c Significant difference from the control group (p 5 0.041, p < 0.05). BW, body weight; WCL, the weight coefficient of the liver; WCK, the weight coefficient of the kidney; WCT, the weight coefficient of the testis.

among all three generations of mice at every microsatellite locus and that the genotypes of these loci were homologous at 42 loci (Supporting Information Fig. 1). The possible explanation for the lower MSI frequencies is because higher doses were used in our study. The lowest dose used here of 0.25 mg/kg represents a total exposure of 3.5 mg/kg, which is higher than the highest dose used by Oliveira et al. (2012). It is difficult to reconcile such high/cytotoxic doses with a mutagenic effect. Meloni et al. (1998) provided evidence for the mutagenic effects of low, environmentally relevant levels of cadmium in intact human cells. Oliveira et al. (2012) reported MSI in the testes of mice treated with a low single dose (1 mg/kg bw) of CdCl2. Our results in the present study also illustrated that a low dose of cadmium can induce MSI. Another explanation is that the panel of microsatellites that we used might be more sensitive to genetic modification and chemical induction (Zuo et al., 2012; Du et al., 2013). Oliveira et al. used a panel of microsatellites that was highly sensitive in detecting MSI in murine tumors. Among the microsatellites that we investigated in this study, the D15Mit5 locus exhibited MSI in transgenic mouse strains, an ENU-induced mutation strain (Du et al., 2013), and our CdCl2-induced strain. Similarly, DXMit172 also exhibited MSI in transgenic mouse strains, gene knockout mouse strains, and our CdCl2-induced strain (Zuo et al., 2012). These two loci could be regarded as “hot spots” of microsatellite mutation in mice. To date, there have been few studies in cadmiumexposed mouse models (Albarellos et al., 2008). In the present study, mice were injected with different doses of cadmium chloride, and we successfully established a cadmium exposure model of correlative damage. The results of this study will serve as a good reference for investigations of human exposure. In the present work, we used C57BL/6J mice. This inbred mouse line has a homogeneous genetic background, which eliminates the issue of genetic heterogeneity among individuals. The distributions of cadmium in mice depend on the form of the administered cadmium compound. Cherian (1983) compared CdCl2- and CdMt-treated

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mice and found that the major accumulation organ was the liver in the CdCl2 group, while the kidney was the target organ in the CdMt group. We also measured the cadmium concentrations in the kidneys, testes, and liver. Our results showed that the liver has a strong accumulation capacity for cadmium. These results also proved that CdCl2 accumulated preferentially in the liver; less CdCl2 accumulated in other organs, such as the testes and kidneys. The organ weight coefficients and body weight gain reflect, to a certain extent, the impact of the metal on the body/organs (Hoffman and Boast, 1995). As indicated by the weight growth table, weight gain was significantly inhibited in the treated mice compared to the control group as a result of the administration of cadmium chloride (p 5 0.003, p < 0.01), and the inhibition was both dose- and time-dependent. In this study, both the liver and testis weight coefficients in group 4 (2 mg/kg) were higher than that in the control group, which showed that higher doses of cadmium treatment had a greater effect on the weight coefficients of the liver and testis. As the major organ involved in cadmium chloride accumulation, the liver exhibited swelling and congestion as well as an increased weight coefficient. Cadmium-induced degeneration and necrosis of the hepatocytes could be observed histologically. Previous studies (Hatch et al., 2007; Martin et al., 2007) have shown that the testes were highly sensitive to cadmium. Gabbiani et al. (1974) found that at higher exposure concentrations (10 mg/kg), the testicular endothelial tissue was the primary target tissue of cadmium chloride, while the peritubular cells and epithelial cells in seminiferous tubules were more sensitive to lower concentrations. In this experiment, although the exposure concentration in each Cd-treated group did not exceed 2 mg/kg, the damage observed in the tissue (liver, kidney, and testis) included the dissociation of spermatogenic cells, an increase in nuclear density staining, the thinning of the seminiferous epithelium, and pathological changes. These changes proved that the testis is a target organ of Cd, even though lower concentrations of cadmium affected mainly the peritubular cells. However, Oliveira et al. (2012) reported that a dose of 1 mg CdCl2/kg bw was

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associated with no histopathologic changes in the testes. These results are very different from ours. The discrepancy may be due to differences in the mouse strains used in our studies or to fact that the accumulated doses we used were higher than what Oliveira et al. used. Intronic microsatellite loci can regulate gene transcription and can affect gene expression. Meloni et al. (1998) reported that a (TCAT)n sequence located in the intron acts as a transcription regulatory element in vitro. Observations from Akai et al. (1999) also indicated that transcriptional activity of the COL1A2 (human type I collagen alpha 2) gene was enhanced by the simultaneous presence of a (CA)n repeat in the 50 flanking region of the gene and a (GT)n tract in the first intron. In this study, we found one locus with MSI (D15mit5) in an intron of the Gm10373 gene (a predicted gene). This microsatellite may have some relationship with Gm10373 gene regulation; further study is needed to reveal whether and how the cadmium-induced mutation at this locus affects gene expression. Although we detected six kinds of organ of animals, only kidney and leukocytes from two lower doses groups exhibited MSI, the rest were MSI negative. However, regarding Cd concentration, the liver is the major accumulation organ. Less CdCl2 accumulated in other organs, such as the testes and kidneys. These results indicated that MSI is prone to occur in lower dose of Cd. And the impact of Cd concentration in organ may be less than biological effect because kidney is one of important Cd target organs (Thijssen et al, 2007). Histological results demonstrated that most severe injury in liver, kidney and testis existed in higher dose groups (1 mg/kg and 2 mg/ kg). Moreover, body weight is significant lower in higher dose groups (2 mg/kg, 1 mg/kg, and 0.5 mg/kg) than that in the lowest dose group and control group, which means the body weight variation result is identical to histological results.

CONCLUSIONS C57BL/6J mice intraperitoneally injected with 2 mg/kg cadmium chloride quaque die alterna for 4 weeks can be used as an animal model of subchronic cadmium poisoning. Our study also provided an experimental model for investigating the mechanisms of cadmium-induced hepatic, renal, and testicular damage. Cadmium could induce MSI in inbred mice at low doses. The results of the present study confirmed that two microsatellite loci (D15Mit5 and DXMit172) could be regarded as “hot spots” of microsatellite mutation in mice.

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