Effects of ZnO Nanoparticles on Dimethoate-Induced Toxicity in Mice

Sep 3, 2015 - The extensive applications of ZnO nanoparticles (nano ZnO) and dimethoate have increased the risk of people's coexposure to nano ZnO and...
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Effects of ZnO Nanoparticles on Dimethoate-Induced Toxicity in Mice Xincheng Yan, Rui Rong, Shanshan Zhu, Mingchun Guo, Shang Gao, Shasha Wang, and Xiaolong Xu* Department of Chemistry, University of Science and Technology of China, Hefei, 230026, P. R. China S Supporting Information *

ABSTRACT: The extensive applications of ZnO nanoparticles (nano ZnO) and dimethoate have increased the risk of people’s coexposure to nano ZnO and dimethoate. Therefore, we evaluated in this study the effects of nano or bulk ZnO on dimethoateinduced toxicity in mice. The serum biochemical parameters, biodistributions, oxidative stress responses, and histopathological changes in mice were measured after intragastric administration of nano or bulk ZnO and/or dimethoate for 14 days. Oral administration of nano or bulk ZnO at a dose of 50 mg/kg did not cause obvious injury in mice. In contrast, oral administration of dimethoate at a dose of 15 mg/kg induced observable oxidative damage in mice. Co-administration of nano or bulk ZnO with dimethoate significantly increased Zn accumulation by 30.7 ± 1.7% or 29.7 ± 2.4% and dimethoate accumulation by 42.8 ± 2.1% or 46.6 ± 2.9% in the liver, respectively. The increased accumulations of dimethoate and Zn in the liver reduced its cholinesterase activity from 5.64 ± 0.45 U/mg protein to 4.67 ± 0.42 U/mg protein or 4.76 ± 0.45 U/mg protein for nano or bulk ZnO, respectively. Furthermore, the accumulations of dimethoate and Zn in liver also increased hepatic oxidative stress, resulting in severe liver damage. Both nano and bulk ZnO dissolved quickly in acidic gastric fluid, regardless of particle size; therefore, they had nearly identical enhanced effects on dimethoate-induced toxicity in mice. KEYWORDS: ZnO nanoparticles, dimethoate, oxidative stress, combined toxicity

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INTRODUCTION ZnO nanoparticles (nano ZnO) are widely utilized in commercial products, including toothpaste, pigments, medicine, drug carriers, and bioimaging probes.1−3 In addition, nano ZnO is commonly used in the food industry as dietary supplements, food additives, and food packaging components owing to its antimicrobial property.4,5 Currently, several cosmetics and food additives containing nano ZnO are actually on the market.6 Nano ZnO, as a fungicide, has a potential application in agriculture.7 As a consequence, human beings have a higher chance of exposure to nano ZnO in food-related products than other nanomaterials.8 Furthermore, the wide applications of nano ZnO increase the potential risk for its release to the environment.9 Gottschalk et al. reported that the modeled nano ZnO concentrations are 430 ng/L in treated wastewater and 10 ng/L in natural surface water in Europe.10 Previous studies have shown that nano ZnO has relatively high toxicity to biological systems.11−15 On the other hand, dimethoate (DM), a popular organophosphorus insecticide, is widely used in developing countries for housefly control and against a great variety of insects in agriculture owing to its high efficiency.16−18 The residue and analog of DM have been found in soil, crops, water, and foods, including cow’s milk.19−25 The main toxicity of DM involves the inhibition of cholinesterase (ChE), thereafter causing dysfunction at the neuromuscular junction and blocking of nerve conduction.26 The extensive applications of both nano ZnO and DM have increased the risk of humans’ coexposure to nano ZnO and DM. For example, in some developing countries, most of the houses in rural, less developed regions do not have piped water. Some farmers drink the natural water and cow’s milk and eat fruits and vegetables, which may be contaminated with DM residue and nano ZnO. In addition, some farmers may eat the © 2015 American Chemical Society

foods that contain nano ZnO additive. Therefore, it is critical to assess the effects of nano ZnO on DM-induced toxicity in animals and humans. Although many published reports have focused on the toxicity of either nano ZnO or DM alone, the combined effect of nano ZnO and DM on animals is still unknown. Nano ZnO can be ingested directly when used in food, food packaging, drug delivery, cosmetics, and denture adhesives.27,28 A majority of the population in some developing countries is exposed to low doses of DM via food and contaminated drinking water.29 This study was designed to assess the effects of nano or bulk ZnO on DM-induced toxicity in mice by repeated intragastric administration. The results show that nano ZnO at a dose of 50 mg/kg BW has low toxicity to mice. However, in case of coexposure with DM, our findings demonstrate that nano ZnO significantly enhances DMinduced damage in mice liver. These results provide useful information concerning the adverse effects of nano ZnO in the case of coexposure with DM.

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MATERIALS AND METHODS

Chemicals. Bulk and nano ZnO used in our experiments are commercially available from Sigma and were characterized with TEM (JEM-2010, JEOL Ltd.) [Figure S1, Supporting Information (SI)]. The spherical particles of nano ZnO had a mean size of 30.1 ± 1.1 nm with unimodal distribution. The particles of bulk ZnO had an approximately rod morphology with different sizes ranging from 100 to 600 nm. DM (99.3%, batch No: 40606) was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). Nano or bulk ZnO Received: Revised: Accepted: Published: 8292

April 20, 2015 September 1, 2015 September 3, 2015 September 3, 2015 DOI: 10.1021/acs.jafc.5b01979 J. Agric. Food Chem. 2015, 63, 8292−8298

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Journal of Agricultural and Food Chemistry

Statistical Analysis. Data were analyzed by one-way analysis of variance (ANOVA) test by using SPSS 18.0 software (SPSS Inc.). The statistical significance was set at p < 0.05 for all tests.

and DM were suspended in aqueous 0.15 M NaCl before administration in mice. Animals and Treatment. Male mice (21 ± 1 g, 7 weeks old) were obtained from Kunming Institute of Zoology. The mice were treated as described previously.30 All animal experiments were done in compliance with Animal Ethical Committee of University of Science and Technology of China. Sixty male mice were divided randomly into six groups: one control group (0.15 M NaCl) and five experimental groups (50 mg/kg BW nano ZnO, 50 mg/kg BW bulk ZnO, 15 mg/kg BW DM, 50 mg/kg BW nano ZnO + 15 mg/kg BW DM, and 50 mg/ kg BW bulk ZnO + 15 mg/kg BW DM). The animals were intragastrically administered with chemicals at a dose of 0.1 mL/10 g BW, respectively, for 14 consecutive days. After 2 weeks, the samples of blood were collected from the tail vein after the mice were anaesthetized by ether. The brain, heart, lung, liver, kidney, and spleen were excised, washed with 0.15 M NaCl, and weighed for determination of the coefficients of organs. Serum Biochemical Parameters Assay. Liver function was determined by the serum levels of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), alkaline phosphatase (ALP), total protein (TP), albumin protein (ALB), and albumin/globulin (A/G). Nephrotoxicity was evaluated with creatinine (CRE) and blood urea nitrogen (BUN). All of the biochemical parameters were determined by an automatic biochemical analyzer (Roche Modular DPP System) using commercial kits (Roche Diagnostics, Mannheim, Germany). Histopathological Examination. Tissues of the kidney, liver, heart, lung, brain, and spleen were recovered from the necropsy. Their histological examinations were performed using an optical microscope as described previously.30 Measurement of Oxidative Stress Makers. The kidney and liver tissues were assayed for the malondialdehyde (MDA) level and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxide (GPx) using the commercial kits (Nanjing Jiancheng Bioeng Inst.) as described previously.30 Measurement of ChE Activity. ChE activity was determined in liver tissue by the hydroxylamine−ferric chloride method using acetylcholine as a substrate.31 The rate of acetylcholine hydrolysis was determined through the measurement of the remaining acetylcholine after hydrolysis catalyzed by ChE. The remaining acetylcholine reacted with NH2OH to form acethydroximic acid, which reacted with Fe3+ to produce the purple-brown color of the ferric−acethydroximic acid compound. This compound could be spectrophotometrically determined at 520 nm. One unit of ChE activity is the amount of protein that decomposes 60 μmol of acetylcholine per hour at 37 °C. Analysis of Biodistributions of Zn and DM. The liver, kidney, heart, lung, brain, or spleen tissue (∼0.2 g) was digested with ultrapure HNO3 solution for 12 h. The solutions were then mixed with 0.5 mL of H2O2 and heated at 130 °C to remove the remaining HNO3. The remaining solutions were diluted to 5 mL with 2% (v/v) HNO3. The Zn contents in the solutions were measured by inductively coupled plasma-atomic emission spectrometry (ICP-AES) (Optima 7300 DV, PerkinElmer Corp.). The DM contents in the organs were measured by gas chromatography−mass spectrometry (GC−MS).32 The liver, kidney, heart, lung, brain, or spleen tissue (0.1−0.2 g) was homogenized and centrifuged at 4500 rpm for 25 min at 4 °C. The supernatant was added to a CNWBOND C18 SPE column. The column was washed with 10 mL of 5% CH3OH and then eluted with 10 mL of CH3OH. The eluate was evaporated to dryness by a nitrogen stream at 25 °C. The dry extract was dissolved in 50 μL of CH3OH, and 1 μL of the solution was injected into a Thermo Trace GC-ISQ MS system (Thermo Fisher Scientific). Assays for Stability of Nanoparticles. To evaluate the stability of nanoparticles in the stomach of mice, 5−40 mg of nano ZnO or bulk ZnO was incubated with 5 mL of artificial gastric fluid (AGF) (0.32% pepsin, 0.2% NaCl, pH 1.5) with gentle shaking for up to 4 h at 37 °C. Supernatants were collected after centrifugation at 12 000g for 20 min. The Zn concentration in supernatants was determined by ICP-AES.

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RESULTS AND DISCUSSION Coefficients of Organs. No abnormal daily behaviors were seen in the control, nano ZnO, and bulk ZnO groups during the entire experimental period. A few signs of acute organophosphate poisoning, including anorexia and reduced activity, were observed in the DM group. The mice coadministrated with DM and nano or bulk ZnO showed varying degrees of signs such as loss of appetite, mild tremor, and piloerection. As shown in Figure 1, the body weights of the

Figure 1. Changes of the body weight in mice (n = 10) during the administration period.

mice in the nano and bulk ZnO groups increased a little more slowly than those in the control group, whereas the body weight of mice administrated with DM increased much more slowly, as compared with the control. Co-administration of DM with nano or bulk ZnO had a significantly enhanced adverse effect on the weight gain. As shown in Table 1, compared with the control, the coefficients of the liver of the nano and bulk ZnO groups did not change significantly, but a significantly higher coefficient of the liver was observed in the DM group (p < 0.05). In the nano ZnO + DM group, a significant increase was seen in the coefficient (p < 0.05) of the liver compared with either the nano ZnO or the DM group, revealing that nano ZnO and DM enhanced each other in affecting the coefficient of the liver. Similarly, co-administration of DM with bulk ZnO also had an enhanced influence on the coefficient of the liver. Compared with the control, the coefficients of the brain, kidney, heart, lung, and spleen of all exposure groups did not change significantly. Combined Effects on Biodistributions of Zn and DM. Figure 2A shows the biodistribution of Zn in the mice after administration with nano or bulk ZnO and/or DM. A major percentage of Zn accumulated in the liver, lung, and spleen of mice after oral administration with nano or bulk ZnO. The biodistribution of Zn in the DM group was similar to that in the control group. Co-administration with nano ZnO and DM significantly increased the biodistribution densities of Zn in the liver and lung (p < 0.05) compared to the nano ZnO group. Similarly, co-administration with bulk ZnO and DM also significantly increased the biodistribution densities of Zn in the liver and lung compared to the bulk ZnO group. DM is lipophilic in nature and prone to interacting with the cell membrane, which results in a disturbance in the phospholipids bilayer structure,33 allowing Zn2+ to easily pass through the membrane and distribute into the tissues. As a result, DM 8293

DOI: 10.1021/acs.jafc.5b01979 J. Agric. Food Chem. 2015, 63, 8292−8298

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Journal of Agricultural and Food Chemistry Table 1. Coefficients of the Major Organs in Micea index liver/BW (mg/g) kidney/BW (mg/g) heart/BW (mg/g) lung/BW (mg/g) spleen/BW (mg/g) brain/BW (mg/g)

control 54.8 14.2 4.53 7.96 4.42 18.3

± ± ± ± ± ±

1.9 0.5 0.35 0.24 0.68 1.6

nano ZnO 55.8 14.5 4.45 8.56 4.38 18.6

± ± ± ± ± ±

1.8 0.6 0.28 0.56 0.35 1.1

bulk ZnO 55.3 14.2 4.49 8.90 4.54 17.9

± ± ± ± ± ±

1.5 0.3 0.32 0.41 0.38 0.7

DM 60.1 14.5 4.46 8.64 4.38 18.3

± ± ± ± ± ±

1.7b 0.5 0.29 0.36 0.46 0.6

nano ZnO + DM 64.3 14.8 4.52 9.02 4.50 18.7

± ± ± ± ± ±

3.3b,c,d 0.5 0.27 0.56 0.39 0.5

bulk ZnO + DM 63.9 14.6 4.55 9.21 4.49 18.1

± ± ± ± ± ±

2.2b,e,d 0.4 0.24 0.51 0.52 0.8

a The mice were intragastrically administered with nano or bulk ZnO and/or DM for 14 days. Data shown are means ± SD (n = 10). bp < 0.05 vs the control. cp < 0.05 vs the nano ZnO group. dp < 0.05 vs the DM group. ep < 0.05 vs the bulk ZnO.

Figure 3. Pathological images of the mice liver tissue following daily intragastrical administrations of nano or bulk ZnO and/or DM for 14 days. No abnormalities were seen in the control, bulk ZnO, and nano ZnO groups. In the DM group, the infiltration of inflammatory cells (arrow) was seen around the central vein. In the nano ZnO + DM and bulk ZnO + DM groups, multiple areas of spotty necrosis (arrows) were induced around the central vein.

Figure 2. Biodistributions of DM and Zn in the mice tissues determined by GC−MS and ICP-AES. The male mice were intragastrically administered with nano or bulk ZnO and/or DM for 14 days. (A) The Zn contents in the different tissues of the mice. (B) The DM contents in different tissues of mice. Statistical significance: a, p < 0.05 vs the control; b, p < 0.05 vs the nano ZnO (A) or DM (B) group. Values represent means ± SD, n = 10.

accumulation of DM and Zn in the liver. Similarly, multiple areas of spotty necrosis were also found in the liver in the bulk ZnO + DM group, indicating that co-administration with bulk ZnO and DM also increased DM-induced pathological damages in the liver. Compared with the control, no apparent abnormal changes were found in the cellular structures of heart, spleen, kidney, brain, and lung tissues in all administrated mice (Figure S2, SI). Effects of Co-Administration on Liver and Kidney Functions. The combined toxicity of nano ZnO and DM in mice was analyzed by a blood biochemical assay. As shown in Table 2, administration with nano or bulk ZnO did not significantly effect the serum biochemical parameters of mice, except that their LDH that significantly increased (p < 0.05) compared with the control value, revealing that nano or bulk ZnO did not influence the kidney function but slightly influenced the liver function. On the contrary, administration with DM significantly increased the AST, ALP, LDH, and TBIL levels compared with the control (p < 0.05), indicating DMinduced liver dysfunction. Co-administration with nano ZnO and DM significantly increased the AST, ALT, ALP, LDH, and TBIL levels (p < 0.05), compared with either the nano ZnO or the DM group, suggesting that co-administration with nano ZnO and DM had an enhanced influence on the liver dysfunction, which is attributed to the co-administrationinduced synergistic accumulation of DM and Zn in the liver.

significantly increases the biodistribution density of Zn in mice liver. As shown in Figure 2B, the majority of DM was accumulated in the liver, kidney, and spleen after administration with DM. Interestingly, co-administration with nano ZnO and DM significantly increased DM accumulation in the liver and kidney compared to the DM group. Similarly, coexposure to bulk ZnO and DM also significantly increased DM accumulation in the liver and kidney compared to the DM group. These results together indicate that co-administration of DM and nano or bulk ZnO results in the enhanced accumulation of Zn and DM in the liver. Effects of Co-Administration on Pathological Changes in Tissues. As shown in Figure 3, in the nano and bulk ZnO groups, no abnormalities were observed in the liver. In the DM group, the infiltration of inflammatory cells around the central vein was observed in the liver. In the nano ZnO + DM group, multiple areas of spotty necrosis were found in the liver. These results indicate that co-administration with nano ZnO and DM induces more pathological damage in the liver than administration of either nano ZnO or DM alone, which is attributed to the co-administration-induced increases in the 8294

DOI: 10.1021/acs.jafc.5b01979 J. Agric. Food Chem. 2015, 63, 8292−8298

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Journal of Agricultural and Food Chemistry Table 2. Changes in Biochemical Parameters in Mice Seruma index AST (U/L) ALT (U/L) ALP (U/L) LDH (U/L) TBIL (μmol/L) ALB (g/L) TP (g/L) CRE (μmol/L) BUN (mmol/L)

control 93 31.2 136 613 0.98 36.4 52.4 51.2 8.63

± ± ± ± ± ± ± ± ±

10 2.8 15 23 0.13 2.5 3.5 3.2 0.58

nano ZnO 112 33.2 139 809 1.08 37.8 51.6 50.8 8.26

± ± ± ± ± ± ± ± ±

9 4.8 11 27b 0.11 1.9 2.9 3.0 0.36

bulk ZnO 109 31.9 133 797 1.03 36.9 52.8 50.2 8.94

± ± ± ± ± ± ± ± ±

DM

9 5.6 17 33b 0.09 2.6 2.8 2.2 0.44

135 34.9 174 838 1.39 40.4 54.5 49.5 8.57

± ± ± ± ± ± ± ± ±

14b 7.1 23b 36b 0.12b 2.3 3.9 4.7 0.39

nano ZnO + DM 158 62.0 218 987 1.61 39.5 52.6 49.1 8.73

± ± ± ± ± ± ± ± ±

16b,c,e 11.9b,c,e 28b,c,e 52b,c,e 0.08b,c,e 3.5 4.4 2.8 0.66

bulk ZnO + DM 153 60.3 226 973 1.58 38.8 53.8 50.7 8.51

± ± ± ± ± ± ± ± ±

12b,d,e 10.2b,d,e 12b,d,e 37b,d,e 0.16b,d,e 3.4 3.1 4.0 0.45

The mice were intragastrically administered with nano or bulk ZnO and/or DM for 14 days. Data shown are means ± SD (n = 10). bp < 0.05 vs the control. cp < 0.05 vs the nano ZnO group. dp < 0.05 vs the bulk ZnO group. ep < 0.05 vs the DM group. a

of antioxidative enzyme is related to increased oxidative stress.35 Figure 5 shows that administration with nano or bulk ZnO did not induce apparent oxidative stress in mice liver. On the contrary, administration with DM caused significant decreases in GPx and CAT activities in mice liver, indicating that DM induced significant oxidative stress in the liver. The oxidative stress is owing to a high accumulation of DM in the liver after administration with DM. It is worthwhile to note that administration with DM caused an increase of SOD activity in mice liver. The increased SOD activity is accounted for by a DM-activated compensatory mechanism.36 Co-administration with nano ZnO and DM led to significant decreases (p < 0.05) in GPx, CAT, and SOD activities in mice liver compared to that in either nano ZnO or DM group, revealing that coadministration with nano ZnO and DM causes more severe oxidative stress in mice liver than administration with either nano ZnO or DM alone, which is attributed to the coadministration-induced enhanced accumulation of DM and Zn in the liver. Similarly, co-administration with bulk ZnO and DM also significantly enhanced the DM-caused oxidative stress in mice liver. MDA is the final product of lipid peroxidation, and the MDA level indicates the level of oxidative damage in organs. Figure 5D shows that administration with nano or bulk ZnO did not significantly effect on the level of MDA in mice liver, compared to that in the control. By contrast, administration with DM induced a significant increase of the MDA level in mice liver (p < 0.05) than that in control group, indicating that the lipid peroxidation of the liver was induced by DM. The oxidative damage in mice liver resulted in its pathological change (Figure 3) and dysfunction (Table 2). Co-treatment with nano ZnO and DM resulted in a significantly higher concentration of MDA (p < 0.05) in mice liver compared to that in either the nano ZnO or the DM group, indicating an enhanced influence of nano ZnO and DM on lipid peroxidation in mice liver, which may be explained by the fact that DM and nano ZnO significantly enhanced the accumulation of each other in the liver. Similarly, co-administration with bulk ZnO and DM also significantly enhanced the DM-induced lipid peroxidation in the liver. Stability of Nano or Bulk ZnO in Artificial Gastric Fluid. When nanoparticles are administrated orally to animals, the dissolution behaviors in gastric fluid should be checked. However, there are several conflicting results on the dissolution of nano ZnO in AGF. Seok et al. reported that around 98% of nano ZnO (40 nm) had dissolved in AGF (pH 1.7).4 Cho et al. showed that all nano ZnO (89.3 nm) dissolved in AGF (pH 1.5) within 5 min.8 However, Wang et al. found that only 3.3%

Similarly, co-administration with bulk ZnO and DM also had an enhanced influence on the liver dysfunction. The significant increase in the LDH level was caused by hepatocellular necrosis after co-administration of nano or bulk ZnO with DM. The kidney function markers CRE and BUN did not change obviously in all administrated mice, suggesting that coadministration with nano or bulk ZnO and/or DM did not significantly influence the renal function. Effects of Co-Administration on ChE Activity. The combined toxicity of nano ZnO and DM in mice was investigated by ChE assay. As reported previously, DM is a ChE inhibitor.26,34 Figure 4 shows the ChE activities in the liver

Figure 4. ChE activity of mice liver following daily intragastrical administrations of nano or bulk ZnO and/or DM for 14 days. Statistical significance: a, p < 0.05 vs the control; b, p < 0.05 vs the nano ZnO group; c, p < 0.05 vs the bulk ZnO; d, p < 0.05 vs the DM group. Values represent means ± SD, n = 10.

of the mice after administration with nano or bulk ZnO and/or DM. In the nano and bulk ZnO groups, the ChE activity did not significantly change compared with the control value. Administration with DM resulted in marked inhibition in the ChE activity in the liver. In the nano ZnO + DM group, ChE activity in the liver was significantly inhibited compared to either the nano ZnO or the DM group, revealing that nano ZnO significantly enhances the inhibition of the ChE activity by DM, which is attributed to an increase in DM and/or nano ZnO accumulation in the liver after co-administration with nano ZnO and DM. Similarly, co-administration with bulk ZnO and DM also significantly enhanced the inhibition of the ChE activity by DM. Effects of Co-Administration on Oxidative Stress in Liver. The combined effect of co-administration of nano ZnO with DM on the oxidative damage in mice liver was investigated by analysis of the endogenous antioxidative enzymes, GPx, SOD, and CAT. Previous studies revealed that a decreased level 8295

DOI: 10.1021/acs.jafc.5b01979 J. Agric. Food Chem. 2015, 63, 8292−8298

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Journal of Agricultural and Food Chemistry

Figure 5. Activities of GPx, CAT, and SOD and the MDA level in the mice liver after daily intragastrical administrations with nano or bulk ZnO and/or DM for 14 days. Statistical significance: a, p < 0.05 vs the control; b, p < 0.05 vs the nano ZnO group; c, p < 0.05 vs the DM group; d, p < 0.05 vs the bulk ZnO. Data were shown as means ± SD (n = 10).

of nano ZnO (20 nm) had dissolved in AGF (pH 1.2).37 Paek et al. demonstrated that about 13−14% of zinc ions in nano ZnO (20 and 70 nm) was released into the AGF (pH 1.5), and the release level was not influenced by size.38 To analyze the stability of nano or bulk ZnO in gastric fluid, different amounts of bulk ZnO or nano ZnO were incubated with acidic AGF. When the concentration of bulk or nano ZnO was less than or equal to 5 mg/mL, the milk-white nano or bulk ZnO solution became clear within a few minutes after incubation with the AGF, indicating that all of nano or bulk ZnO mass had dissolved in the AGF. The ICP-AES data show that 98.4% of Zn2+ ions in nano ZnO and 97.8% of Zn2+ ions in bulk ZnO were released into supernatant after incubation of nano or bulk ZnO (5 mg/mL) with the AGF for 4 h (Figure 6B), further confirming that almost all of the nano or bulk ZnO dissolved in the AGF. However, when the concentration of nano or bulk ZnO exceeded 5 mg/mL, the milk-white nano or bulk ZnO solutions were still unclear after incubation with the AGF for 4 h, suggesting that only part of nano or bulk ZnO dissolved in the AGF. As shown in Figure 6A, the concentration of Zn2+ in AGF was almost constant with the increases of the concentrations of nano or bulk ZnO from 5 to 8 mg/mL, indicating that the dissolved Zn2+ concentration tended to equilibrium. These results reveal that the dissolution of bulk ZnO in AGF was similar to that of nano ZnO. Nano or bulk ZnO at low concentration (≤5 mg/mL) almost completely dissolved in the AGF. However, nano or bulk ZnO at high concentration (>5 mg/mL) partly dissolved in the AGF. The previous conflicting results on the dissolution of nano ZnO in AGF should be due to the concentrations of nano ZnO used in each experiment being different.4,8,37,38 In the present study, the concentration of administered nano or bulk ZnO was 0.5 mg/mL, which was much less than 5 mg/ mL. After administration, the solution of nano or bulk ZnO was diluted with the gastric fluid and its concentration should further decrease. It appears that both bulk and nano ZnO administered by the oral route must dissolve completely in the

Figure 6. Zn2+ ions released from nano or bulk ZnO in AGF. The content of Zn2+ in AGF (A) and the percentage of dissolution of Zn2+ ion (B) were measured using ICP-AES after incubation of nano or bulk ZnO with the artificial gastric fluid (0.32% pepsin, 0.2% NaCl, pH 1.5) for 4 h. Data shown are means ± SD (n = 3).

stomach of mice. The dissolved Zn2+ ions will be absorbed through the small intestine and then enter into the systemic circulation.39 The fast dissolution of both bulk and nano ZnO in acidic gastric fluid results in that bulk ZnO and nano ZnO have the nearly identical, slight effects on the liver function in the mice after oral administration, regardless of particle size. In addition, the results of the function and histopathology of mice liver show that both nano and bulk ZnO have nearly identical enhancement effects on the DM toxicity in the liver after oral 8296

DOI: 10.1021/acs.jafc.5b01979 J. Agric. Food Chem. 2015, 63, 8292−8298

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Journal of Agricultural and Food Chemistry Author Contributions

co-administration with DM. The data from the oxidative stress measurement and ChE activity assay also confirm that both nano and bulk ZnO have the nearly identical enhanced effects on DM-induced toxicity in mice liver. Nano ZnO represents an important class of toxicant, because nano ZnO is unstable in aqueous solutions and releases Zn2+ and the high level of Zn2+ can induce oxidative stress inside cells.40,41 It is well-known that free Zn2+ is highly reactive and toxic.4 Our results demonstrated that oral administration of nano ZnO (30.1 nm) at a dose of 50 mg/kg BW for 14 days did not cause observable injury in mice, suggesting that most Zn2+ ions released from nano ZnO may form compounds with biomolecules and the amount of free Zn2+ is insufficient to cause injury in mice. However, Sharma et al. showed that oral administration of nano ZnO (30 nm) at a dose of 300 mg/kg BW for 14 consecutive days caused significant injury in mice liver.27 Similarly, Esmaeillou et al. reported that exposure of nano ZnO (20−30 nm) through oral gavage at a single dose (333.33 mg/kg BW) had severe toxicological effects on the liver, kidney, and lung of mice.42 These findings together with present results indicate that the low amount of nano ZnO is biocompatible; however, nano ZnO at high doses displays hazardous effects. Therefore, the content of nano ZnO in food and food supplements is the critical matter for its safe application. Nano ZnO at low dose is safe for application in food and food supplements. However, in the case of coexposure with DM, our findings demonstrate that nano ZnO significantly enhances DM-induced damage in mice liver, which is attributed to the enhanced accumulation of Zn and DM in the liver. People may be exposed to nano ZnO and/or DM at different doses in different situations. The dose of nano ZnO or DM used in our experiments do not necessarily reflect the actual concentration of nano ZnO or DM found in real situations; however, the data can be used to assess the health risks from coexposure to nano ZnO and DM. In the present study, we have made the first attempt to evaluate the effect of nano ZnO on DM-induced toxicity in mice. Further evaluation of the combined toxicity of nano ZnO and DM in animals at various doses is needed. In summary, although nano ZnO at a dose of 50 mg/kg BW is of low toxicity to male mice, co-administration with nano ZnO and DM significantly enhances DM-induced injury in mice liver, which is due to the enhanced accumulation of Zn and DM in mice liver. The increased accumulation of Zn and DM causes marked oxidative stress in mice liver, which results in severe liver damage. These findings suggest that close attention should be paid to the combined toxicity of nanoparticles and pesticides with the increasing use of nanomaterials in the world.

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X.Y. and R.R. contributed equally to this work. Funding

This study was supported by grants from the National Natural Science Foundation of China (Grant Nos. 20571069, 20871111, 21171157). Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED BW, body weight; DM, dimethoate; nano ZnO, ZnO nanoparticle; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; AST, aspartate aminotransferase; TP, total protein; ALP, alkaline phosphatase; TBIL, total bilirubin; ALB, albumin protein; BUN, blood urea nitrogen; A/G, albumin/ globulin; CRE, creatinine; MDA, malondialdehyde; ChE, cholinesterase; SOD, superoxide dismutase; GPx, glutathione peroxidase; CAT, catalase; BSA, bovine serum albumin; TEM, transmission electron microscopy; ICP-AES, inductive coupled plasma atomic emission spectrometer; ROS, reactive oxygen species; GC−MS, gas chromatography−mass spectrometry; AGF, artificial gastric fluid

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b01979. TEM images of nano or bulk ZnO (Figure S1) and histopathological examination (Figure S2) (PDF)

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DOI: 10.1021/acs.jafc.5b01979 J. Agric. Food Chem. 2015, 63, 8292−8298

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DOI: 10.1021/acs.jafc.5b01979 J. Agric. Food Chem. 2015, 63, 8292−8298