The Protective Role of Zinc against Acute Toxicity of Depleted Uranium in Rats

Authors

  • Yuhui Hao,

    1. State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing, China
    Search for more papers by this author
  • Jiong Ren,

    1. State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing, China
    Search for more papers by this author
  • Jing Liu,

    1. State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing, China
    Search for more papers by this author
  • Shenglin Luo,

    1. State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing, China
    Search for more papers by this author
  • Ting Ma,

    1. State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing, China
    Search for more papers by this author
  • Rong Li,

    Corresponding author
    • State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing, China
    Search for more papers by this author
  • Yongping Su

    Corresponding author
    • State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, Chongqing Engineering Research Center for Nanomedicine, College of Preventive Medicine, Third Military Medical University, Chongqing, China
    Search for more papers by this author

Author for correspondence: Rong Li and Yongping Su, State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, College of Preventive Medicine, Third Military Medical University, Chongqing, China (fax +86 23 68752009, e-mail yuhuihao@tmmu.edu.cn).

Abstract

Depleted uranium (DU) has been widely used in both civilian and military activities and contributes to health problems. This study was undertaken to evaluate the protective role of zinc against acute toxicity of DU. Sprague Dawley rats were injected with DU (10 mg/kg, i.p.) to create a toxicity model (DU group). Before and after the injection of DU, zinc sulphate (10 mg/kg, i.p.) was administered once a day for 2 days. The survival rates at 30 days post DU administration and the effects of zinc at 4 days post DU administration were evaluated. Our data indicate that zinc has obvious protective effects, especially pre-treatment with zinc. Rats pre-treated with zinc had significantly higher survival rates than rats in the DU group, with 60.03% more surviving. In addition, at 4 days post DU administration, the former had lower kidney uranium content, insignificant renal tubular epithelial cell necrosis and less transparent tubes. Meanwhile, blood urea nitrogen, creatinine and urine N-acethyl-β-d-glucosaminidase concentrations were significantly decreased; the gene expression levels of metallothionein (MT) in kidney tissues were significantly increased; and catalase levels were increased and malondialdehyde levels were decreased. In conclusion, pre-treatment with zinc significantly alleviated acute toxicity of DU, and the mechanism appeared to be related to the induction of MT synthesis and enhancement of the antioxidant function.

Depleted uranium (DU) is the by-product of uranium (235U) enrichment from natural uranium, as having 235U content lower than 0.7112%, which emits α and β particles with high linear energy transfer. Therefore, DU has the dual effects of radioactive toxicity and heavy metal toxicity, with heavy metal toxicity pre-dominating [1]. Owing to its efficient penetration and affordability, DU has recently been widely used in counterweights, radiation protection and military activities (such as armour material and ammunition components) [2]. However, unregulated release of DU into the environment could become a threat to human health [3, 4].

The chemical toxicity of acute, high DU doses especially targets the kidneys, causing severe renal tubular necrosis [5]. Kidney damage is caused when a body's uranium intake is higher than 2 mg/kg [6] or when kidney uranium deposition reaches 3 μg/g [7]. Although low-dose chronic exposure to DU may not lead to clear clinical symptoms in the kidneys, it can cause harmful effects elsewhere, including abnormalities in neural activity, immunotoxicity and liver toxicity [8, 9]. In addition to the dose of exposure, the biological effects of DU are affected by the exposure duration, the exposure pathway and many other factors [10].

Until now, the prevention and treatment of DU intoxication have primarily relied on shielding the subject from exposure and providing supportive treatment for symptom relief. Yapar et al. [11] showed that ginkgo leaf extract significantly improved liver and kidney functions in mice after uranium ingestion. Pourahmad et al. [12] confirmed the antioxidant and radical scavenging activity of β-(1 to > 3)-d-glucan could successfully protect the isolated rat hepatocytes against cell lysis and all oxidative stress cytotoxicity end-points caused by DU, and the in vivo effect needs to be verified in future. In addition, many chelating agents have been examined in animal studies over the years to determine their efficacies in removing uranium [13-17]. The compound catechol-3,6-bis(methyleiminodiacetic acid) (CBMIDA) has shown promise by reducing uranium burden without causing renal damage and may be administered orally, but it also has some deficiency more or less, such as low gastrointestinal absorption and the effect related to pH [15]. Indeed, CBMIDA had no uranium removal effect when the uranium was dissolved in a solution with pH 7.

Briner [18] has proposed that lead and DU share many physical and biochemical properties, indicating the usefulness of lead as a model in DU studies. Numerous papers [19-21] have reported that zinc supplements reduced lead absorption and lead deposition in tissues, with metallothionein (MT) serving a major role in the lead detoxification. Zinc has also been shown to have cadmium detoxification effects that are linked to the promotion of MT synthesis in vivo [22]. MT is a low-molecular weight, thiol-containing protein that is found throughout the body and that participates in homoeostasis and heavy metal detoxification [23-25]. In vertebrates, four isoforms of MT have been reported: MT-1, MT-2, MT-3 and MT-4. MT-1 and MT-2 are ubiquitously expressed. MT-3 is only expressed in animal nerve tissue, and MT-4 is primarily expressed in keratinised epithelial cells [24-26]. So, MT induced by zinc may have DU detoxification effects. Jiang et al. [27] used the nematode, Caenorhabditis elegans, to evaluate the toxicity of DU and its effects in knockout strains of MT, and indicated that MT was protective against DU exposure.

In this study, we report the protective role of zinc against acute toxicity of DU and propose a mechanism for its action. We tested whether the effects occur via MT synthesis. We observed changes in the 30-day survival rate after acute DU intoxication in the presence of zinc. At 4 days post DU exposure, we examined the uranium content in tissues, serum and urine biochemical markers relevant to liver and kidney functions, the proliferation of splenic lymphocytes, the histopathology, the expressions of cytokine, MT-1 and MT-2 at the transcriptional level in kidney tissue, and the changes in catalase (CAT) and malondialdehyde (MDA) content. The present report provides a new approach for the prevention and treatment of DU acute intoxication.

Materials and Methods

Animals

Male Sprague Dawley (SD) rats at 6–8 weeks of age were obtained from the Institute of Daping Zoology [The Third Military Medical University, SCXK (Chongqing) 2002003, China] and bred under controlled conditions with a 12-hr light/dark cycle, a temperature of 23°C and a relative humidity of 55%. Food and water were provided ad libitum. After a quarantine period of 5 days, the rats weighing 180–200 g were selected for experiment. Food intake, water intake and health status were recorded daily. The protocol was carried out according to the National Institute of Health Guide for Care and Use of Laboratory Animals and was approved by Chinese legislation regarding the ethical use of animals.

Treatments

The rats were randomly divided into five groups: (i) DU group: The rats were exposed to DU [The source and constituent of DU was as the previous study [28], with U238 = 99.75%, U235 = 0.20%, trace U234] in the form of uranyl nitrate (pH = 6.8–7.0) at a single dose of 10 mg/kg body-weight. The LD50 of uranium for human beings has been calculated to be about 14 mg/kg, depending on the chemical form [29]. Rats exposed to uranyl acetate (1 mg/kg injected) demonstrated tubular necrosis and changes in blood chemistry reflecting renal compromise [5]. To clarify the acute toxicity of DU and the protective effects of zinc, the dose of 10 mg/kg was used to create a toxicity model. (ii) Control group: Compared with the DU group, the rats were exposed to equal volume of saline in place of DU. (iii) Zn + DU group: The rats were treated with the solution of zinc sulphate (Sigma, Santa Clara, CA, USA) at a dose of 10 mg/kg once a day for 2 days. Then, the rats were exposed to DU on the third day. (iv) DU + Zn group: The rats were treated with the solution of zinc sulphate (10 mg/kg) at 30 min. post DU exposure. This administration time was the same with Fukuda et al. [30]. Then, the rats were treated with the solution of zinc sulphate (10 mg/kg) again on the second day. (v) Zn group: The administration manner was the same as the Zn + DU group but exposure to equal volume of saline in place of DU on the third day. All of the drugs were administered by intraperitoneal injection (i.p.).

Each group consisted of 40 animals, and the tests were as follows: (i) Thirty rats per group were used to evaluate the survival rates of 30 days after exposure to DU and (ii) the other 10 rats per group were killed at 4 days after DU injection. Blood was collected, and sensitive organs were removed for further determinations as described later.

Survival rate

After exposure to DU, we were checking the death of rats on time twice a day, and making a record for a month. The survival rates of the 30 rats per group were analysed.

Uranium analysis

The other 10 rats per group were additionally kept individually in a metabolic glass cage and urine was collected for 4 days after DU injection. At 4 days after DU injection, the rats were killed by rapid decapitation. Uranium content was measured in the kidney, liver and spleen by inductively coupled plasma mass spectrometry. The methods were followed as in the previous study [31].

Serum and urinary biochemical parameters

The blood from femoral artery was obtained under the xylazine hydrochloride (anaesthesia). We used an automated Konelab 20 to measure plasma creatinine (Cr), blood urea nitrogen (BUN), alanine amino-transferase (ALT), aspartate amino-transferase (AST) in the serum and N-acethyl-β-d-glucosaminidase (NAG) in the urine (Biological chemistry reagents; Jiancheng Institute of Bioengineering, Nanjing, China).

Lymphocyte proliferation test

Spleens were removed and homogenised by pressing the organs gently through a metal net. Splenocyte concentration was adjusted to 2 × 106 cell/ml in RPMI-1640 supplemented by 10% foetal calf serum, 25 mM HEPES and 2 mM l-glutamine. One hundred microlitres of diluted cell suspensions was dispensed into 96-well culture plates. Lipopolysaccharide (LPS) or concanavalin A (ConA) was then added at 5 μg/ml final concentration to each well, and the volume was adjusted to 0.2 ml. After incubating for 72 hr at 37°C and 5% CO2 in humid incubator, cell proliferation was determined by MTT [3-(4, 5-diamethyl-2-thiazolyl) 2, 5-diphenyl-2H-tetrazolium] based assay [32]. Briefly, 10% of MTT (5 mg/ml) was added to each well and incubated at 37°C in CO2 humid incubator for 4 hr. The blue formazan precipitate was then dissolved in acidic isopropanol and its optical density was measured at 570 nm by using Stat-FaxTM Elisa Reader (Awareness Technology Inc., Palm City, USA). Each sample was examined in triplicate. LPS was used to measure B lymphocyte proliferation, and ConA was used to measure T lymphocyte proliferation. Stimulation Index (S.I.) was calculated for each sample as: S.I. = At/Ac, where At is the absorbance of test sample stimulated by ConA or LPS, and Ac is the absorbance of the test sample without stimulation by ConA or LPS.

Histopathology and light microscopy

Spleen, liver and kidney were dissected and fixed with 4% formaldehyde, dehydrated and embedded in paraffin for sectioning at 5 μm. Haematoxylin and eosin staining (Beyotime, Haimen, Jiangsu, China) was used for light microscopic examination.

Cytokine assays

TNF-α and IL-6 secretions in serum were measured using specific enzyme-linked immunosorbent assays (ELISA) according to the respective manufacturer instructions. The rat TNF-α and IL-6 ELISA kit were all from R&D (R&D Systems Inc., Minneapolis, MN, USA). Sensitivity announced by the manufacturers was < 15 and 8 pg/ml for TNF-α and IL-6, respectively.

RT-PCR

To clarify whether zinc could induce MT synthesis, we also established another group (Zn group, n = 10) whose administration manner was the same as the Zn + DU group, but the animals were killed on the third day without DU injection. RT-PCR was used to analyse the mRNA level of the MT-1 and MT-2 using the methods as previously described [33]. Total RNA from the kidney was isolated using RNeasy total RNA isolation Kit (Takara, Kyoto, Japan). Synthesis of single-stranded complementary DNA (cDNA) was performed using an oligo(dT)20 primer with the SuperScript III First Strand cDNA Synthesis kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The specific primer sequences are shown in table 1. The PCR products were run on a 2% agarose gel supplemented with ethidium bromide. Densitometry analysis was performed using Gene Genius Bio-Imaging system and GeneSnap software (Gene Tools, Syngene, MD, USA). Relative quantification of mRNA levels was determined by standardising gene expression against the non-variable control gene, β-actin. All determinations were replicated at least three times.

Table 1. Primer sequences
GenePrimer sequences
MT-1 forward5′-ACT GCC TTC TTG TCG CTT A-3′
MT-1 reverse5′-TGG AGG TGT ACG GCA AGA CT-3′
MT-2 forward5′-CCA ACT GCC GCC TCC ATT CG-3′
MT-2 reverse5′-GAA AAA AGT GTG GAG AAC CG-3′
β-actin forward5′-CCG TGA AAA GAT GAC CCA GAT-3′
β-actin reverse5′-CAT TGC CGA TAG TGA TGA CCT-3′

CAT and MDA analysis

The kidney tissues of each animal were homogenised in ice-cold saline. Then, the homogenates were centrifuged at 5000 × g for 15 min. at 4°C. The supernatants were collected for analysis. Tissue CAT and MDA contents were colorimetrically measured by the methods of Verhaeghe et al. [34] and Yoshoika et al. [35], respectively, using an ultraviolet/visible spectrophotometer (UVmini-1240; Shimadzu, Kyoto, Japan). CAT and MDA activities were determined using commercial kits (Jiancheng Institute of Bioengineering, Nanjing, China).

Statistical analysis

All data were analysed with SPSS 12.0 (SPSS Inc., Chicago, IL, USA). Statistical analysis was performed by one-way anova. Tukey's HSD was used to perform comparisons between the groups. All data were expressed in the text as means or means ± standard deviation (S.D.) of several separate experiments. Results were considered to be statistically significant at < 0.05 (two-sided).

Results

Survival rate

After DU administration, rats in the DU group exhibited a decrease in drinking and feeding, and their urine output was decreased or non-existent. Over time, the fur of these rats became ruffled, and they exhibited less physical activity. Recovery started around day 20. At 30 days post DU administration, the survival rates of each group (n = 30) displayed significant differences, with the DU group being the lowest (fig. 1). The survival rate of the Zn group was 100%, suggesting that 10 mg/kg of zinc had no obvious deleterious effects by day 30. The survival rate of the Zn + DU group was significantly higher than that of the DU group, with 60.03% more surviving, indicating that zinc pre-administration reduced the damage caused by DU. The survival rate of the DU + Zn group was between those of the DU and the Zn + DU groups, suggesting that the protective effects of zinc treatment after acute DU intoxication is significantly less effective than that obtained by preventative pre-administration of zinc. In addition, the peak time of death in the DU group was 7 days post DU administration, whereas the peak time of death in the Zn + DU group was 4 days after that of the DU group, indicating that zinc pre-administration may also delay death.

Figure 1.

Zinc pre-treatment improved the survival rates after exposure to Depleted uranium (DU). This figure illustrates the survival rates of each group (n = 30) during 30 days post DU administration (10 mg/kg, i.p.). Zn + DU group: Before the injection of DU, zinc sulphate (10 mg/kg, i.p.) was administered once a day for 2 days; DU + Zn group: After the injection of DU, zinc sulphate (10 mg/kg, i.p.) was administered once a day for 2 days.

Measurement of uranium content

After entering the animal body, uranium was distributed unevenly amongst different tissues, with the content in kidney tissues being the highest (fig. 2A). At 4 days post DU exposure, the uranium content in kidney tissues of the Zn + DU and DU + Zn groups was significantly lower (approximately 62.14% and 48.44%) than that of the DU group, with the uranium content of the Zn + DU group being the lowest. There was a significant difference in the liver and spleen uranium contents of the DU and Zn + DU groups, but there was no significant difference between the DU and DU + Zn groups. The tissue uranium content in the Zn group was not significantly different from the control group. It is well known that uranium can be excreted from the body by the kidneys through urine. The urinary uranium excretion rate of the Zn + DU group was significantly higher than that of the DU group [(12.66 ± 1.39%) versus (5.44 ± 0.83%), n = 10, < 0.05], suggesting that zinc pre-treatment enhanced DU excretion through urine (fig. 2B).

Figure 2.

Zinc pre-treatment decreased uranium accumulation in the organs and increased the excretion rates of uranium. This figure illustrates urinary uranium excretion rates for 4 days post Depleted uranium (DU) administration (B) and the uranium contents in the kidney, liver and spleen of each group at 4 days post DU exposure (A), as measured by inductively coupled plasma mass spectrometry. Data are expressed as means ± S.D. (n = 10), and error bars represent one S.D. Compared with the control group, # < 0.05; Compared with the DU group, @< 0.05.

Serum, urine biochemical parameters and lymphocyte proliferation tests

At 4 days post DU exposure, biochemical parameters of liver (table 2), such as serum ALT and AST showed no significant differences amongst the different groups. However, the DU group had significantly higher renal biochemical parameters (serum Cr, BUN and urine NAG) than the control group. Although the values of the Zn + DU group were still significantly higher than those of the control group, the Zn + DU group had significantly lower values than the DU group. The Zn group displayed no significant differences from the control group in all the previously mentioned parameters.

Table 2. The examination of organ function after 4 days of exposure
GroupCrBUNNAGALTASTT S.I.B S.I.
  1. Values are expressed as the mean ± S.D. (n = 10). Statistical analysis was performed by one-way anova, and Tukey's HSD was used to perform comparisons between the groups.

  2. a

    < 0.05 significantly different from the values of the control group.

  3. b

    < 0.05 significantly different from the values of the Depleted uranium (DU) group.

  4. Cr, creatinine; BUN, blood urea nitrogen; NAG, N-acethyl-β-D- glucosaminidase; ALT, alanine amino-transferase; AST, aspartate amino-transferase; T S.I., Stimulation Index of T cell; B S.I., Stimulation Index of B cell.

Control12.71 ± 5.16b 6.87 ± 1.73b 1.19 ± 0.34b 47.20 ± 9.19136.90 ± 23.271.93 ± 0.121.83 ± 0.11b
DU361.40 ± 60.79a 83.80 ± 35.13a 6.85 ± 1.78a 51.70 ± 12.45169.70 ± 46.871.59 ± 0.391.23 ± 0.14a
Zn + DU251.74 ± 40.41a , b 49.39 ± 10.73a , b 3.47 ± 1.12a , b 51.44 ± 3.13171.56 ± 39.651.89 ± 0.061.65 ± 0.12b
DU + Zn345.79 ± 71.05a 44.46 ± 13.55a , b 4.00 ± 1.62a 50.40 ± 8.41174.89 ± 51.211.83 ± 0.461.67 ± 0.15b
Zn11.12 ± 3.51b 9.73 ± 2.27b 1.10 ± 0.45b 54.50 ± 9.23148.67 ± 20.461.96 ± 0.181.85 ± 0.29b

Changes in splenic lymphocyte proliferation ability were tested using an MTT assay (table 2). Interestingly, the T lymphocyte S.I. showed no significant differences between the DU group and the other four groups, but the B lymphocyte S.I. of the DU group was significantly lower than the other four groups. There were no significant differences amongst the other four groups.

Histopathology and light microscopy

When investigated by light microscopy, each group showed different degrees of kidney damage. The DU group had the most severe damage, exhibiting renal tubular epithelial cell hyalinosis, cell vacuolisation, cell shedding and necrosis. A large amount of urinary casts were also observed in the DU group (fig. 3A). In contrast, the Zn and control groups both showed no observable kidney damage. The Zn + DU group showed less severe damage than the DU group, with no obvious renal tubular degeneration or necrosis and almost no urinary casts (fig. 3B). In the DU + Zn group, both urinary casts and renal tubular epithelial cell degeneration were observed.

Figure 3.

Zinc pre-treatment significantly alleviated the pathological damage at 4 days post Depleted uranium (DU) exposure. This figure illustrates the pathological section in each group, stained by H&E. (A, B) Kidney (×400); (C, D) spleen (×400); (E, F) liver (×200). A, C and E were from the DU group, and B, D and F were from the Zn + DU group.

Each group's degree of spleen damage corresponded with that observed for the kidneys. In the DU group, no obvious germinal centres were observed in the splenic corpuscles, whereas the periarterial lymphatic sheaths and red pulp cords were reduced, and sinusoidal dilatation and congestion (fig. 3C) were observed. The Zn + DU group displayed a reduction in splenic white pulp and a decrease in the number and volume of splenic corpuscles, but germinal centres were still visible (fig. 3D). All groups displayed only minor liver damage, with little difference between groups. There was minor liver cell damage with only minor cloudy swelling in both the DU group (fig. 3E) and the Zn + DU group (fig. 3F).

Cytokine detection and MT gene expression

Both IL-6 and TNF-α were able to induce MT synthesis in vivo and in vitro [36, 37], and the levels of cytokines could indirectly reflect the content of MT. The changes in serum cytokines were measured by ELISA to assess the influence of DU on cytokine secretion (fig. 4). Both the IL-6 and TNF-α levels of the DU group were significantly lower (approximately 62.14% and 48.44%) than those of the control, but there were no significant differences between the control and Zn + DU groups (45.82 ± 8.23 ng/l versus 31.46 ± 8.90 ng/l, n = 10, > 0.05; 90.33 ± 7.59 ng/l versus 72.97 ± 23.98 ng/l, n = 10, > 0.05).

Figure 4.

Zinc pre-treatment prevented the down-regulation of cytokine in the serum at 4 days post Depleted uranium (DU) exposure. This figure illustrates the levels of IL-6 and TNF-α in serum of each group, as measured by ELISA. Data are expressed as means ± S.D. (n = 10), and error bars represent one S.D. Compared with the control group, # < 0.05; Compared with the DU group, @< 0.05.

Using β-actin as an internal control, RT-PCR was used to detect the transcription levels of the MT gene in the kidneys of all groups (fig. 5). Compared with the control group, the expression levels of MT-1 and MT-2 in the DU group significantly reduced, especially the MT-1 decrease (about 31.77%), whilst those in the Zn group markedly increased (about 100%), and those in the Zn + DU group did not significantly decrease. In addition, the expression levels of MT-1 and MT-2 of the Zn group were also not significantly different from the control group.

Figure 5.

Zinc pre-treatment improved MT gene expressions. This figure illustrates gene expressions of MT-1 (A) and MT-2 (B) in the kidney of each group, as measured by RT-PCR. Data are expressed as means ± S.D. (n = 10), and error bars represent one S.D. Compared with the control group, #< 0.05; Compared with the Depleted uranium (DU) group, @< 0.05.

CAT and MDA measurements

The cytotoxicity of DU is related to the generation of reactive oxygen species (ROS) and lipid peroxidation [38], and it could cause a significant increase in MDA levels [11]. In this study, the CAT and MDA contents in kidney tissues from all groups were measured at 4 days post DU exposure (fig. 6). The CAT contents in the DU and DU + Zn groups were significantly lower (approximately 52.84% and 23.54%) than that of the control group, whereas there were no significant differences between the control and Zn + DU groups (23.96 ± 1.19 U/mg protein versus 21.66 ± 6.18 U/mg protein, n = 10, > 0.05). On the other hand, MDA content in the DU group was clearly higher (about three times) than that of the control groups. Although the MDA content of the Zn + DU group remained significantly higher (approximately 127%) than the control group, it was greatly reduced compared with the DU group (6.47 ± 2.09 mmol/mg protein versus 11.23 ± 3.12 mmol/mg protein, n = 10, < 0.05). The MDA contents of the Zn and control groups had no significant differences (2.82 ± 0.31 mmol/mg protein versus 2.84 ± 0.16 mmol/mg protein, n = 10, > 0.05).

Figure 6.

This Zinc pre-treatment enhanced the antioxidant abilities at 4 days post DU exposure. This figure illustrates the contents of CAT and MDA in the kidney of each group, as measured by colourimetry. CAT and MDA reflect the organism's antioxidant ability and lipid peroxidation, respectively. Data are expressed as means ± S.D. (n = 10), and error bars represent one S.D. Compared with the control group, #< 0.05; Compared with the DU group, @< 0.05.

Discussion

Depleted uranium has been widely used in both civilian and military activities and has been claimed to contribute to health problems [3, 4, 39]. Numerous reports have confirmed that the major target organs are the kidneys, bones, liver, lungs and central nervous system exposure to DU, and renal insufficiency is the primary cause of death [5, 40, 41]. So, the prevention and treatment of DU intoxication are worth attention. To our knowledge, the present study is the first to investigate the effectiveness of zinc in preventing acute DU toxicity in SD rats. The results show that zinc effectively alleviated bodily damage caused by DU and greatly improved the survival rate after acute DU intoxication. The mechanism appears to be related to the induction of MT synthesis and markedly enhanced antioxidant ability.

The survival rates at 30 days post DU administration well indicated that zinc played an important role in protecting the body from DU toxicity. When given before DU administration, zinc increased the survival rate by 60.03% and postponed the peak time of death for about 4 days (fig. 1). Zinc had some protective effects when administered after DU exposure (compared with the DU group), but its efficiency was significantly decreased. This phenomenon is similar to those reported in studies [21-23] of zinc detoxification of other heavy metals (lead, cadmium, mercury, etc.), in which the pre-administration of zinc or simultaneous intake of zinc and heavy metal effectively prevented heavy metal toxicity.

Inductively coupled plasma mass spectrometry results show that zinc pre-treatment enhanced the excretion of uranium from the body and decreased uranium accumulation in organs (fig. 2). Uranium is excreted into the urine via the kidneys, leading to the accumulation of highly concentrated uranium in those organs [5, 42]. The results also confirmed that kidney tissue had the highest uranium content than other tissues. The kidney uranium content of the Zn + DU group was 62.14% lower than that of the DU group, whereas the urinary uranium excretion rate of Zn + DU group was two times higher than that of the DU group. This suggests that one important protective mechanism of zinc is to increase uranium excretion in the urine.

Not only did zinc pre-administration increase uranium excretion in the urine, it also decreased the organ function (biochemical parameters) damage caused by uranium (table 2). Consistent with the renal biochemical parameters (NAG, BUN and Cr), the pathological results (fig. 3A,B) indicated that zinc could effectively protect kidney from DU toxicity. Alscher et al. [43] demonstrated that, by inducing MT synthesis, zinc administration antagonised kidney damage caused by oxidative stresses and therefore protected renal functions. However, an in vitro study by Wan et al. [44] confirmed that short-term but large doses of DU disrupted the interaction between macrophages and T cells and their immune functions. Results of the MTT experiment (table 2) shows that the splenic B lymphocyte stimulation index of the DU group significantly decreased, implying that B lymphocytes are more susceptible to DU damage, a conclusion requiring further investigation. In combination with the pathological results obtained from spleen tissue (fig. 3C,D), the damage in the Zn + DU group was obviously less severe than that of the DU group. These results indicate that zinc can also antagonise the immune dysfunction caused by DU toxicity.

Many studies [45-47] have shown that zinc induces MT synthesis and subsequently plays a role in antioxidation. MT binds to heavy metal ions and expels them from the cytoplasm, thereby protecting cells from oxidative damage [25, 48, 49]. Zinc-induced MT has reportedly been used to treat copper-storage disease [50, 51] and renal insufficiency [43]. To clarify whether the protective role of zinc is related to MT synthesis, we examined the concentration of cytokines relevant to MT synthesis and MT expression on the transcriptional level in kidney tissues.

Both IL-6 and TNF-α were related to induce MT synthesis, and IL-6 was the major cytokine regulating MT expression in the central nervous system [52]. In the present study, both IL-6 and TNF-α levels in serum significantly decreased at 4 days post DU exposure, suggesting that MT synthesis may also decrease, and zinc pre-administration markedly prevents this decrease (fig. 4). This was likely due to the pre-administration of zinc that enhanced MT synthesis and antagonised DU toxicity. Interestingly, the IL-6 and TNF-α levels in the Zn group were not significantly higher than those of the control group, suggesting that Zn-induced MT synthesis may be a timed event, and five days later, the MT levels return to normal.

Previous studies [53, 54] have shown that DU administration caused macrophages to abnormally express IL-6 and TNF-α, although this result is still controversial. Consistent with our results, Dublineau et al. [55] showed a decrease in TNF-α expression after a chronic low-level of DU exposure. However, other studies [54, 56] reported that the mRNA and protein levels of TNF-α increased after DU exposure. The same inconsistency exists in cadmium toxicity studies [57-59]. We believe that the main causes of the inconsistency are related to the DU dose, exposure duration, exposure pathway and the time interval after DU exposure. If the previously mentioned factors cause the body to be in the compensatory stage, serum cytokine levels will increase, but if the body is in the decompensatory stage, serum cytokine levels will decrease. This hypothesis requires further investigation.

It has been reported that MT-1 and MT-2 are mainly distributed in liver and kidney tissues and are closely related to heavy metal (such as cadmium and copper) toxicity [60, 61]. Therefore, we focused on examining MT-1 and MT-2 gene expressions in the kidney. The results showed that the kidney expression levels of MT-1 and MT-2 were significantly decreased in the DU group at 4 days post DU exposure, but showed no significant decrease in the Zn + DU group, compared with the control group (fig. 5). These results are consistent with the results obtained for cytokines. In addition, the expression levels of MT-1 and MT-2 of the Zn group were significantly higher than those of the control group. Thus, it appears that short-term exposure to a large dose of DU results in insufficient MT synthesis, and that zinc is a stimulant that induces MT synthesis.

Pourahmad et al. [38] found that uranium (VI) was the major toxicity carrier inside organisms, and the mechanism of its toxicity was to reduce the body's metabolism by generating ROS. Generally, uranium (VI) and uranium (IV) are stable forms of uranium, but soluble uranium (VI) may generate more ROS than insoluble uranium (IV) in the body. Measurements of CAT and MDA should reflect the organism's antioxidant ability and lipid peroxidation, providing a means to assess zinc's protective effects on DU intoxication. The results clearly showed that the antioxidant abilities of rats in the Zn + DU group were significantly higher than those of the DU group, whereas the lipid peroxide levels were significantly lower than those of the DU group (fig. 6). Consistent with other reports [43, 62, 63], our results show that zinc might induce MT production to antagonise ROS and alleviate oxidative damage, so as to reduce organ functional and histopathology damage and greatly improve the survival rate post DU administration. This may be another important protective mechanism of zinc. The limitation of this study is that only one dose of DU and zinc was used, so it could not fully reflect the toxicology of DU, and the dose-effect relationship about the protective role of zinc.

In summary, our experimental results clearly show that Zn improved rat survival rates after a large dose of acute DU intoxication. Zinc pre-administration increased uranium excretion and decreased organ uranium accumulation, thereby significantly reducing organ functional damage caused by uranium. In addition, the dose of Zn used (10 mg/kg) did not cause adverse effects to the organism. The protective effects were related to the induction of MT synthesis and the marked enhancement of the organism's antioxidant ability. It is noteworthy that the protective effects of the Zn + DU group surpassed those of the DU + Zn group in all examinations throughout the experiment. Presumably, in the DU + Zn group, DU administration had already damaged the organism and led to organ dysfunction, which affected the MT production and secretion functions of the organs when zinc was used to induce MT synthesis. Therefore, it is ineffective to administer Zn after DU exposure.

Zinc is an essential trace element in the human body and an important component of many enzymes. It can antagonise the absorption of many other metals in the human body [20, 64]. Therefore, it remains to be clarified whether the protective role of Zn against DU intoxication depends solely on the induction of MT synthesis or whether it has additional functions. Meanwhile, to further understand the roles of different MT subtypes in protection, it is necessary to study the mechanisms of DU damage and protective role in MT transgenic animals in the future.

Acknowledgements

We thank the National Natural Science Fund of China (no. 30970678) and State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, China (no. SKLZZ200809) for the financial support of this study.

Conflict of Interest

The authors report no conflicts of interest.

Ancillary