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Effects of zinc oxide nanoparticles and/or zinc chloride on biochemical parameters and mineral levels in rat liver and kidney S Amara, I Ben Slama, I Mrad, N Rihane, W Khemissi, L El Mir, K Ben Rhouma, H Abdelmelek and M Sakly Hum Exp Toxicol published online 5 February 2014 DOI: 10.1177/0960327113510327 The online version of this article can be found at: http://het.sagepub.com/content/early/2014/02/04/0960327113510327
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Article
Effects of zinc oxide nanoparticles and/or zinc chloride on biochemical parameters and mineral levels in rat liver and kidney
Human and Experimental Toxicology 1–8 ª The Author(s) 2014 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327113510327 het.sagepub.com
S Amara1, I Ben Slama1, I Mrad1, N Rihane1, W Khemissi1, L El Mir2,3, K Ben Rhouma1, H Abdelmelek1 and M Sakly1
Abstract The aim of this study was to assess the potential subacute toxicity of zinc oxide (ZnO) nanoparticles (NPs) in Wistar rats in comparison with reference toxicant, zinc chloride (ZnCl2), of a non-nanoparticulate form. We therefore studied the relationships between zinc (Zn) accumulation, liver and kidney trace element levels, and plasmatic biochemical parameters. Rats in all groups were treated by intraperitoneal injection of ZnO NPs and/ or ZnCl2 solution (25 mg/kg) every other day for 10 days. The contents of trace element in the liver and kidney were slightly modulated after ZnO NPs and/or ZnCl2 solution exposure. The same treatment increased the aspartate aminotransferase activity and uric acid concentration. However, ZnO NPs or ZnCl2 solution decreased the creatinine levels, whereas the combined intake of ZnO NPs and ZnCl2 decreased the glucose concentration. Interestingly, the analysis of the lyophilized powder of liver using the x-ray diffractometer showed the degradation of ZnO NPs in ZnO-treated group, instead there is a lack of NPs ZnO biosynthesis from the ZnCl2 solution injected in rats. These investigations suggest that combined injection of ZnO NPs and ZnCl2 solution has a possible toxic effect in rats. This effect could be related to Zn2þ ion release and accumulation of this element in organs. Our findings provide crucial information that ZnO appeared to be absorbed in the organs in an ionic form rather than in a particulate form. Keywords ZnO nanoparticles, ZnCl2 solution, liver, kidney, trace element, rats
Introduction There are various definitions on nanomaterials in the literature, and it should be mentioned that the International Organization for Standardization (ISO) has recently developed a technical specification on the terminology and definitions of nanoobjects.1 The specification of the term nanomaterial describes the nanoobject as a material with dimensions being the size range from approximately 1 to 100 nm.1 There is growing community and scientific concern that the desirable technological characteristics of nanoparticles (NPs) may be offset by increased health and environmental risks, with the potential for exposure resulting in reactions at a cellular level that have not been observed with macroscopic materials.2 The small size of NPs infers both greater mobility as well as potentially enhanced uptake across biological
membranes.3 Zinc oxide (ZnO) NPs have a wide variety of applications in industry, including agriculture, medicine, and cosmetics. With increased use of ZnO NPs, exposure to these NPs has been rising steadily, resulting in more attention being paid to their potential toxicity, including cytotoxic, genotoxic, and
1
Laboratoire de Physiologie Inte´gre´e, Faculte´ des Sciences de Bizerte, Jarzouna, Tunisia 2 Al-Imam Mohammad Ibn Saud Islamic University (IMSIU), College of Sciences, Department of Physics, Riyadh, Saudi Arabia 3 Laboratory of Physics of Materials and Nanomaterials Applied at Environment, College of Sciences in Gabes, Zirig, Tunisia Corresponding author: S Amara, Laboratoire de Physiologie Inte´gre´e, Faculte´ des Sciences de Bizerte, Jarzouna 7021, Tunisia. Email: [email protected]
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proinflammatory effects.4,5 Several studies have reported that ZnO NPs at a high dose of 1–5 g/kg can cause acute toxicity and apoptosis in murine liver cells in vivo.6 Study of the toxicological effects of ZnO NPs in biological systems has lagged behind the speed of their mass production and applications in various fields. Furthermore, ZnO is generally considered to be a material with low toxicity, because zinc (Zn) is an essential trace element in the human body and is commonly present in foods or added as a nutritional supplement, so Zn attracts little attention during assessment of toxicity of NPs.7,8 ZnO is slightly soluble and can release Zn2þ ions in solution. Some researchers considered that dissolved Zn ions in the toxicity of ZnO NPs played an important role.9 Brunner et al.10 inferred that toxic effects of ZnO NPs on cells may be attributed to the dissolution of Zn2þ ions. Deng et al.11 found that ZnO NPs and zinc chloride (ZnCl2) had the similar toxic effect on mouse. However, they did not determine the content of dissolved Zn2þ ions. Understanding how nanomaterials are distributed in the body after exposure is important for assessing whether they are safe.12 At present, there are few reports about the toxicity of ZnO NPs and/or ZnCl2 solution. The present study was undertaken to investigate the subacute toxicity of ZnO NPs and/or ZnCl2 solution. For this, rats were exposed to ZnO NPs and/or ZnCl2 solution via intraperitoneal injection and the distribution of Zn in liver and kidney was investigated. The effects on tissue weight, plasmatic biochemical parameters, and trace element homeostasis in liver and kidney were also examined. Additionally, to understand the fates of ZnO NPs and/or ZnCl2 solution in rat tissues following intraperitoneal injection, we analyzed the lyophilized powder of liver using the x-ray diffractometer.
Materials and methods Animals and treatment Male Wistar rats (SIPHAT, Bin Arous, Tunisia), weighing 150–155 g at the beginning of the experiment, were housed in groups of 5/7 in cages at 25 C under a 12-h/12-h light/dark cycle (lights on at 07:00 h) with free access to food and water. ZnO is an inorganic compound appearing as a white powder. We prepared ZnO suspension using physiological saline solution (0.9% sodium chloride). The powdered ZnO NPs (20–30 nm) were dispersed in the fresh sterilized physiological saline solution, and the suspension was ultrasonicated for 20 min to
disperse completely as well as possible (ultrasonic liquid processor: sonicator 4000, Qsonica, USA). ZnO suspension was vortexed for 1 min before injections. Animals were then randomly divided into four groups (n ¼ 7 in each group). Group 1 (control) received physiological saline solution (0.9% sodium chloride), group 2 (Zn treated) received injections of ZnCl2 solution (25 mg/kg body weight) dissolved in 0.9% physiologic saline solution, group 3 (ZnO-treated) received injections of ZnO NPs (25 mg/kg body weight), and group 4 (ZnO þ ZnCl2 treated) received injections of ZnO NPs (25 mg/kg body weight) and injections of ZnCl2 solution (25 mg/kg body weight). Twenty-four hours after the final injection, treated and control animals were killed in compliance with the code of practice for the Care and Use of Animals for Scientific Purposes. The experimental protocols were approved by the Faculty Ethics Committee. Rats in all groups were treated by intraperitoneal injection every other day for 10 days.
NPs preparation and characterization techniques ZnO NPs were provided by the Laboratory of Physics of Materials and Nanomaterials Applied to Environment, College of Sciences in Gabes, Zirig, Tunisia.13,14 The preparation of nanocrystalline ZnO aerogels were prepared by dissolving 2 g of zinc acetate dehydrate in 14 ml of methanol under magnetic stirring for 2 h. The water for hydrolysis was slowly released by esterification of acetate with methanol. NP aerogel as obtained by supercritical drying in ethyl alcohol (EtOH).13,14 The crystalline data were obtained by x-ray diffractometry (XRD; D8 Advance, Bruker Biosciences Coprporation, Billerica, Massachusetts, USA; 40 kV, 30 mA). To determine the lattice parameters of the different phases, the diffraction x-rays were collected by scanning between 2 ¼ 5 and 70 in 0.02 steps. To examine the size and morphology of oxide nanocrystals, transmission electron microscopy (TEM; JEM-200 CX, Jeol, Tokyo, Japan) was used (Figure 1). The sample preparation for TEM observation was as follows: the powder was first put in EtOH, and then the ultrasonic dispersed solution was dropped on a copper (Cu) net. For photoluminescence (PL) measurements, the 337.1 nm laser line of a Laser Photonics LN 100 nitrogen laser (Stanford Research Systems, Sunnyvale, CA) was used as an excitation source. The emitted light from the sample, collected by an optical fiber on the same side as the excitation, was analyzed with a Jobin-Yvon (French
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Figure 1. TEM images of synthesized ZnO NPs. TEM: Transmission electron microscopy; ZnO: zinc oxide; NPs: nanoparticles.
company of optical instrumentation and Spectroscopy) Spectrometer HR460 and a multichannel charge-coupled device detector (2000 pixels). The PL excitation measurements were performed on Jobin-Yvon Fluorolog 3-2 spectrometer and 450 xenon lamp as the excitation source. The emission spectra were corrected for the spectral response of the excitation source. The low-temperature experiments were carried out in a Janis (France) VPF-600 Dewar with variable temperature controlled between 78 and 350 K13,14 (Figure 2).
Determination of trace element content The concentrations of trace element (Zn, iron (Fe), calcium (Ca), sodium (Na), potassium (K), magnesium (Mg), manganese (Mn), and Cu) in rat liver and kidney were determined with an atomic absorption spectrometer (Avanta, GBC, Australia). The standard solution of Zn used in this assay resulted by the dissolution of ZnCl2 in deionized water. Liver and kidney tissues were lyophilized, weighed, and digested in 2 ml of concentrated nitric acid in pressurized Teflon containers at 160 C for 3 h. After cooling at room temperature, samples were diluted with 10 ml of deionized water.15 Analyses of trace elements were performed using acetylene gas as fuel and air as an oxidizer. Operational conditions were adjusted to yield optimal determination. The calibration curves were prepared separately for all the trace elements by running suitable concentrations of the standard solutions. Digested samples were aspirated into the
Figure 2. EDX spectra of ZnO-NPs representing the elemental composition of nanoparticles samples. EDX: Energy dispersive x-ray; ZnO: zinc oxide; NPs: nanoparticles.
fuel-rich air–acetylene flame, and the concentrations of the trace element were determined from the calibration curves. Average values of three replicates were taken for each determination. Suitable blanks were also prepared and analyzed in the same manner. The detection limits for Fe, Zn, Ca, Na, K, Mg, Mn, and Cu were 0.05, 0.008, 0.025, 0.04, 0.05, 0.05, 0.04, and 0.05 ppm, respectively. Trace elements concentration was expressed in microgram per gram of the dry mass of tissues.
Biochemistry panel analysis All animals were killed at the same time. Blood samples were taken from all rats in heparinized tubes and then centrifuged. In the present study, we chose plasmatic biochemical parameters related to liver and kidney function. We determined the glucose content, uric acid, creatinine, and levels of enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT). These enzymes were examined by routine colorimetric methods using commercial kits. Organs of the control and treated groups were harvested immediately. Organs were rinsed with icecold deionized water and dried with filter paper. After weighing the body and tissues, the coefficients of liver and kidneys to body weight were calculated as the ratio of tissues (wet weight, in milligram) to body weight (in gram).
X-Ray characterization of the liver powder Liver powder was produced by mechanical method. Rat liver was lyophilized, and the analyzed material is finely ground, homogenized, and average bulk
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Table 1. Effect of ZnO nanoparticles and/or ZnCl2 solution treatment on the coefficient of liver and kidney.a Liver C ZnCl2 ZnO ZnO þ ZnCl2
43.3 + 41.75 + 42.27 + 40.86 +
Kidney 0.69 1.12 0.77 0.74
8.82 9.42 8.99 8.46
+ 0.13 + 0.2 + 0.22 + 0.13
injection decreased significantly the glucose concentration (p < 0.05).
Tissue distribution of trace element
composition is determined. X-Ray powder diffraction is a rapid analytical technique primarily used for phase identification of a crystalline material and can provide information on unit cell dimensions. The crystalline characterization of the liver powder was obtained using XRD (Bruker D8 Advance; 40 kV, 30 mA). To determine the lattice parameters of the different phases, the diffraction x-rays were collected by scanning between 2 ¼ 5 and 70 in 0.02 steps.13,14
The tissue distribution of ZnO NPs and ZnCl2 in solution was determined in the liver and kidney by measuring the total Zn level with flame emission atomic absorption. When the concentrations of total Zn levels were compared between the experimental groups treated by ZnO NPs or ZnCl2 solution and the control group, no statistically significant increases were observed in the liver and kidney (Tables 3 and 4). However, the combined effect of ZnO NPs and ZnCl2 solution showed a significant increase in total Zn accumulation in rat liver (Table 3). A slight decrease in trace element content was detected in the liver of rats injected by ZnO NPs and/or ZnCl2 solution, but the difference did not reach statistical significance. However, the combined effect of ZnO NPs and ZnCl2 solution showed a significant increase in Cu, Mn, and Na contents in rat kidney (Table 4).
Data presentation and statistical analysis
XRD of the liver powder
Results were expressed as the mean + SEM, and data were analyzed by means of one-way analysis of variance with the post hoc test to determine significant relevance to the unexposed control using Statistica 5.0 software (StatSoft). In all cases, p < 0.05 was considered significant.
We investigated the biodistribution and the fate of ZnO NPs and ZnCl2 solution in rat liver. The spectra of liver powder in ZnO NPs and ZnCl2 solutiontreated rats were similar and obviously differed from the reference ZnO NPs spectrum. This suggests that most of the ZnO NPs injected in rats were dissolved in liver. Moreover, our data indicate the lack of biosynthesis of ZnO NPs types from ZnCl2 in rat liver (Figure 3(a) to (f)).
ZnO: zinc oxide; ZnCl2: zinc chloride; C: control; SEM: standard error mean. a Data represent the means + SEM of seven animals per group.
Results Animal observation and coefficients of organs No toxic signs or mortality was observed related to ZnO NPs and/or ZnCl2 solution injection in rats. Also, no obvious significant differences were observed in the coefficients of liver and kidney between the exposed rats and the control group (Table 1).
Biochemical measurements Table 2 showed the changes inf plasmatic biochemical parameters induced by ZnO NPs and/or ZnCl2 solution injection in adult Wistar rats. Experimental groups treated by intraperitoneal injection of ZnO NPs and/or ZnCl2 solution showed a significant increase in AST and uric acid but decreased creatinine levels compared with the control group (p < 0.05). However, the ALT activity remained unchanged (p > 0.05). Interestingly, ZnO NPs and ZnCl2 solution
Discussion The present in vivo study in Wistar rats was aimed to investigate how different sources of Zn such as ZnO NPs and/or ZnCl2 in solution influence first the biochemical parameters related to liver and kidney function and second the distribution of trace element in rat tissues. Moreover, this article describes the fates of ZnO NPs and/or ZnCl2 solution in rat liver determined using XRD. Several NPs exposure routes were chosen for experimental animal studies. These include oral, inhalation, intratracheal, subcutaneous injection, intravenous, and intraperitoneal administration. Recently, Li et al.16 showed that intraperitoneally injected ZnO NPs (2.5 g/kg) were absorbed into circulation (within 30 min postdosing), then biodistributed to the liver, spleen, and kidney. The injected
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Table 2. Effect of ZnO nanoparticles and/or ZnCl2 solution treatment on rat plasmatic biochemical parameters.a AST (IU/l) C ZnCl2 ZnO ZnO þ ZnCl2
25.8 + 68.1 + 85.74 + 75.9 +
ALT (IU/l)
2.262 7.86b 6b 5.9b
23.33 + 29.73 + 36.45 + 42.91 +
Creatinine (mmol/l) 62.36 + 44.54 + 38.77 + 47.58 +
3.37 2.82 12.11 5.08
Uric acid (mg/l)
3.4 5.18b 3.57b 5.66
38.37 76.88 75.80 97.82
+ 7.72 + 10.61b + 8.39b + 10.6b
Glucose (g/l) 1.542 1.42 1.35 1.238
+ 0.079 + 0.078 + 0.079 + 0.043b
ZnO: zinc oxide; ZnCl2: zinc chloride; C: control; AST: aspartate aminotransferase; ALT: alanine aminotransferase; SEM: standard error mean. a Data represent the means + SEM of seven animals per group. b p < 0.05 compared with the control.
Table 3. Liver distribution of trace element in rats after subacute exposure to ZnO nanoparticles and/or ZnCl2 solution.a C Zn Fe Na K Ca Mg Mn Cu
416.61 + 111.123 + 138.88 + 779.62 + 49.38 + 160.61 + 3.7 + 2.95 +
ZnCl2 18.52 19.922 4.79 28.32 5.4 22.13 0.86 0.93
441.03 + 65.33 + 129.64 + 709.87 + 37.73 + 126.47 + 2.46 + 2.48 +
ZnO
20.07 15.53 23.31 115.07 5.52 14.49 0.58 0.18
442.88 79.13 125.94 666.71 39.33 64.45 2.98 1.36
+ 39.69 + 20.83 + 16.17 + 56.14 + 8.26 + 7.47b + 0.27 + 0.48
ZnO þ ZnCl2 587.28 + 97.23 + 128.19 + 728.74 + 52.78 + 124.02 + 2.72 + 1.09 +
37.06b,c,d 11.77 10.12 71.09 13.11 19.55 0.32 0.03
Zn: zinc; Fe: iron; Na: sodium; K: potassium; Ca: calcium; Mg: magnesium; Mn: manganese; Cu: copper; ZnO: zinc oxide; ZnCl2: zinc chloride; C: control; SEM: standard error mean. a Data represent the means + SEM of three animals per group. b Statistical differences p < 0.05 compared with the control. c Statistical differences p < 0.05 compared with ZnCl2 group. d Statistical differences p < 0.05 compared with ZnO nanoparticles.
Table 4. Kidney distribution of trace element in rats after subacute exposure to ZnO nanoparticles and/or ZnCl2 solution.a C Zn Fe Na K Ca Mg Mn Cu
235.09 105.14 312.44 1200.08 148.12 112.53 3.55 1.06
ZnCl2
+ 18.68 + 18.03 + 3.74 + 44.32 + 37.06 + 11.54 + 0.12 + 0.07
465.75 + 57.75 + 274.23 + 826.09 + 115.61 + 66.54 + 2.85 + 0.49 +
289.78 2.19 6.6 29.08 11.56 0.65 0.55 0.04
ZnO 293.47 + 45 62.59 + 21.4 346.23 + 47.62 819.89 + 97.72 173.68 + 28.77 61.12 + 26.95 3.99 + 0.31 1.94 + 0.34
ZnO þ ZnCl2 420 80.99 403.74 1234.42 145.95 81.39 4.81 4.23
+ 79.22 + 3.59 + 15.32b + 120.44 + 26.69 + 17.86 + 0.34b + 1.08b,c,d
Zn: zinc; Fe: iron; Na: sodium; K: potassium; Ca: calcium; Mg: magnesium; Mn: manganese; Cu: copper; ZnO: zinc oxide; ZnCl2: zinc chloride; C: control; SEM: standard error mean. a Data represent the means + SEM of three animals per group. b p < 0.05: statistical differences compared with ZnCl2 group. c p < 0.05: statistical differences compared with the control. d p < 0.05: statistical differences compared to ZnO nanoparticles.
ZnO NPs remained in serum for 72 h and could more effectively spread to the heart, lung, and testes. In our study, the ZnO NPs and ZnCl2 solution (25 mg/kg) were administered to rats through the intraperitoneal
route. Treated rats showed no obvious significant differences in the coefficients of liver and kidney. However, the liver and kidney could be the possible target organs for accumulation and toxicity of ZnO
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Figure 3. Diffraction patterns for different samples. (a) X-ray spectra for ZnO NPs þ sample holder, (b) x-ray spectra for sample holder, (c) x-ray liver powder diffractogram (control group), (d) ZnCl2 group, (e) ZnO NPs group, and (f) ZnO NPs group þ ZnCl2 group. Results are commonly presented as peak positions at 2 and x-ray counts (intensity). ZnO: zinc oxide; ZnCl2: zinc chloride; NPs: nanoparticles
NPs and/or ZnCl2 solution. This was supported by the modulation of plasmatic AST activity and the creatinine and uric acid levels in rats. These results support the findings of Wang et al.17 who observed liver damage in mice after an oral exposure of ZnO NPs although at a higher dose of 5 g/kg. Interestingly, subacute exposure to ZnO NPs and ZnCl2 solution decreased plasmatic glucose concentration in rats. Yaghmaei et al.18 reported that Zn has a role in the synthesis, storage, and secretion of insulin and has been suggested to be beneficial when used in the
diabetic state. The NPs when ingested into the body can be distributed to different regions because of their small size. It is important to find out information about their biodistribution. Lee et al.12 reported that ZnO NPs are absorbed into the blood, distributed into various organs, and cleared from the body via the urine. Furthermore, according to radioactive ZnO experiments, NPs primarily showed retention in the lung, followed by retention in the liver and kidney after intravenous administration.19 In case of our investigation, the tissue distribution of ZnO NPs and
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ZnCl2 solution were determined in the liver and kidney by measuring the total zinc levels with flame emission atomic absorption. No remarkable difference in distribution of Zn in the liver was seen in rats treated with ZnO NPs or ZnCl2 solution. However, significantly elevated Zn levels were observed in the liver of rats after ZnO NPs and ZnCl2 solution exposure. The ionic form (Zn2þ) would be taken up by the liver via the first-pass effects and then redistributed from the liver to the other organs.20 However, very little is known about the size, dose, and exposure dependence of in vivo NP processing biodistribution to every body organ and tissue. Furthermore, there are currently no data available regarding the systemic circulation and accumulation of ZnO NPs in rat liver following repeated intraperitoneal administration. Several studies show that interstitially injected NPs pass preferentially through the lymphatic system and not the circulatory system, probably due to permeability differences.21,22 The accumulation of Zn in the liver could be related probably to the induction of tissues metallothioneins in ZnO NPs and ZnCl2 solution-treated rats, since these proteins have been suspected to control metal homeostasis and to maintain cell survival in response to various stimuli.23 Morover, a slightly higher amount of Zn was detected in the kidney of rats treated with ZnO NPs and/or ZnCl2 solution, but the difference did not reach statistical significance. Lee et al.12 showed that when ZnO NPs were administered to rats, some of them were absorbed into the blood and then mainly distributed in the liver and finally cleared from the body in the kidney via the urine. The accumulation of Zn in rat liver or kidney for different origins such as ZnCl2 in solution or particulate ZnO is similar. Interestingly, this finding showed that animals do not assimilate more Zn when it is added as dissolved ZnCl2 in comparison with ZnO NPs. In order to evaluate the tissue accumulation and distribution of intraperitoneally injected ZnO NPs and/or ZnCl2 solution, we carried out the XRD analysis on the lyophilized liver powder dissected from rats. The x-ray spectrum in ZnO NPs and/or ZnCl2 groups looked similar to the control groups, and no ZnO NPs types were found in rat liver. This result confirms that ZnO NPs were dissolved in liver tissues giving Zn2þ ions in solution. Accordingly, previous investigation has reported that ZnO NPs could not be observed in the TEM images of the liver and kidney, whereas accumulation of ZnO NPs was evident on inductively coupled plasma atomic emission spectroscopy analysis, implying an ionized
fate in the tissues.7 On the other hand, the XRD analysis clearly showed the lack of biosynthesis of ZnO NPs from ZnCl2in rat liver. The possible biosynthesis of ZnO NPs in ZnCl2-treated rats could be assigned as Zn–ligand interactions between Zn2þ ions and proteins, such as metallothionein, with sulfur-rich ligands, which is consistent with previous evidence for Zn2þ ions bonding to sulfur in the liver as determined by x-ray absorption spectra analysis.1,24 Furthermore, the synthetic method of synthesizing ZnO NPs from Zn hyperaccumulator tissues could provide in the future a new finding. In order to test the hypothesis that ZnO NPs and/or ZnCl2 solution altered the homeostasis of trace element in rat tissues, the contents of Ca, Mg, Mn, Na, K, Cu, and Fe were studied in treated rat liver and kidney. Increasing evidence suggested that Zn accumulation in tissues may involve disruptions of trace elements homeostasis. Our data suggest that dissolution of Zn2þ ions in rat liver decreased slightly the trace element content in this organ. By contrast, the combined effect of ZnO NPs and ZnCl2 solution showed a significant increase of Cu, Mn, and Na content in the kidney. This effect is probably favored by the urinary excretion, the excretion profiles for trace element via urine under ZnO NPs and/or ZnCl2 solution is currently underway. Our results demonstrate that subacute intraperitoneal injection of ZnO NPs (20–30 nm) and/or ZnCl2 solution (25 mg/kg) in rats leads to the modulation of plasmatic biochemical parameters resulting probably to cellular injury. Interestingly, animals do not assimilate more Zn when it is added as dissolved ZnCl2 in comparison with particulate ZnO, and the accumulation patterns for different origins of Zn are similar. Dissolved Zn2þ ions in ZnO NPs and/or ZnCl2 groups did not show remarkable perturbation of trace element homeostasis in rat liver and kidney. On the basis of total Zn concentrations in rat liver and kidney, the subacute toxicity of ZnO NPs was statistically similar to that of ZnCl2 in solution. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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Effects of zinc oxide nanoparticles and/or zinc chloride on biochemical parameters and mineral levels in rat liver and kidney S Amara, I Ben Slama, I Mrad, N Rihane, W Khemissi, L El Mir, K Ben Rhouma, H Abdelmelek and M Sakly Hum Exp Toxicol published online 5 February 2014 DOI: 10.1177/0960327113510327 The online version of this article can be found at: http://het.sagepub.com/content/early/2014/02/04/0960327113510327
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Article
Effects of zinc oxide nanoparticles and/or zinc chloride on biochemical parameters and mineral levels in rat liver and kidney
Human and Experimental Toxicology 1–8 ª The Author(s) 2014 Reprints and permission: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0960327113510327 het.sagepub.com
S Amara1, I Ben Slama1, I Mrad1, N Rihane1, W Khemissi1, L El Mir2,3, K Ben Rhouma1, H Abdelmelek1 and M Sakly1
Abstract The aim of this study was to assess the potential subacute toxicity of zinc oxide (ZnO) nanoparticles (NPs) in Wistar rats in comparison with reference toxicant, zinc chloride (ZnCl2), of a non-nanoparticulate form. We therefore studied the relationships between zinc (Zn) accumulation, liver and kidney trace element levels, and plasmatic biochemical parameters. Rats in all groups were treated by intraperitoneal injection of ZnO NPs and/ or ZnCl2 solution (25 mg/kg) every other day for 10 days. The contents of trace element in the liver and kidney were slightly modulated after ZnO NPs and/or ZnCl2 solution exposure. The same treatment increased the aspartate aminotransferase activity and uric acid concentration. However, ZnO NPs or ZnCl2 solution decreased the creatinine levels, whereas the combined intake of ZnO NPs and ZnCl2 decreased the glucose concentration. Interestingly, the analysis of the lyophilized powder of liver using the x-ray diffractometer showed the degradation of ZnO NPs in ZnO-treated group, instead there is a lack of NPs ZnO biosynthesis from the ZnCl2 solution injected in rats. These investigations suggest that combined injection of ZnO NPs and ZnCl2 solution has a possible toxic effect in rats. This effect could be related to Zn2þ ion release and accumulation of this element in organs. Our findings provide crucial information that ZnO appeared to be absorbed in the organs in an ionic form rather than in a particulate form. Keywords ZnO nanoparticles, ZnCl2 solution, liver, kidney, trace element, rats
Introduction There are various definitions on nanomaterials in the literature, and it should be mentioned that the International Organization for Standardization (ISO) has recently developed a technical specification on the terminology and definitions of nanoobjects.1 The specification of the term nanomaterial describes the nanoobject as a material with dimensions being the size range from approximately 1 to 100 nm.1 There is growing community and scientific concern that the desirable technological characteristics of nanoparticles (NPs) may be offset by increased health and environmental risks, with the potential for exposure resulting in reactions at a cellular level that have not been observed with macroscopic materials.2 The small size of NPs infers both greater mobility as well as potentially enhanced uptake across biological
membranes.3 Zinc oxide (ZnO) NPs have a wide variety of applications in industry, including agriculture, medicine, and cosmetics. With increased use of ZnO NPs, exposure to these NPs has been rising steadily, resulting in more attention being paid to their potential toxicity, including cytotoxic, genotoxic, and
1
Laboratoire de Physiologie Inte´gre´e, Faculte´ des Sciences de Bizerte, Jarzouna, Tunisia 2 Al-Imam Mohammad Ibn Saud Islamic University (IMSIU), College of Sciences, Department of Physics, Riyadh, Saudi Arabia 3 Laboratory of Physics of Materials and Nanomaterials Applied at Environment, College of Sciences in Gabes, Zirig, Tunisia Corresponding author: S Amara, Laboratoire de Physiologie Inte´gre´e, Faculte´ des Sciences de Bizerte, Jarzouna 7021, Tunisia. Email: [email protected]
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proinflammatory effects.4,5 Several studies have reported that ZnO NPs at a high dose of 1–5 g/kg can cause acute toxicity and apoptosis in murine liver cells in vivo.6 Study of the toxicological effects of ZnO NPs in biological systems has lagged behind the speed of their mass production and applications in various fields. Furthermore, ZnO is generally considered to be a material with low toxicity, because zinc (Zn) is an essential trace element in the human body and is commonly present in foods or added as a nutritional supplement, so Zn attracts little attention during assessment of toxicity of NPs.7,8 ZnO is slightly soluble and can release Zn2þ ions in solution. Some researchers considered that dissolved Zn ions in the toxicity of ZnO NPs played an important role.9 Brunner et al.10 inferred that toxic effects of ZnO NPs on cells may be attributed to the dissolution of Zn2þ ions. Deng et al.11 found that ZnO NPs and zinc chloride (ZnCl2) had the similar toxic effect on mouse. However, they did not determine the content of dissolved Zn2þ ions. Understanding how nanomaterials are distributed in the body after exposure is important for assessing whether they are safe.12 At present, there are few reports about the toxicity of ZnO NPs and/or ZnCl2 solution. The present study was undertaken to investigate the subacute toxicity of ZnO NPs and/or ZnCl2 solution. For this, rats were exposed to ZnO NPs and/or ZnCl2 solution via intraperitoneal injection and the distribution of Zn in liver and kidney was investigated. The effects on tissue weight, plasmatic biochemical parameters, and trace element homeostasis in liver and kidney were also examined. Additionally, to understand the fates of ZnO NPs and/or ZnCl2 solution in rat tissues following intraperitoneal injection, we analyzed the lyophilized powder of liver using the x-ray diffractometer.
Materials and methods Animals and treatment Male Wistar rats (SIPHAT, Bin Arous, Tunisia), weighing 150–155 g at the beginning of the experiment, were housed in groups of 5/7 in cages at 25 C under a 12-h/12-h light/dark cycle (lights on at 07:00 h) with free access to food and water. ZnO is an inorganic compound appearing as a white powder. We prepared ZnO suspension using physiological saline solution (0.9% sodium chloride). The powdered ZnO NPs (20–30 nm) were dispersed in the fresh sterilized physiological saline solution, and the suspension was ultrasonicated for 20 min to
disperse completely as well as possible (ultrasonic liquid processor: sonicator 4000, Qsonica, USA). ZnO suspension was vortexed for 1 min before injections. Animals were then randomly divided into four groups (n ¼ 7 in each group). Group 1 (control) received physiological saline solution (0.9% sodium chloride), group 2 (Zn treated) received injections of ZnCl2 solution (25 mg/kg body weight) dissolved in 0.9% physiologic saline solution, group 3 (ZnO-treated) received injections of ZnO NPs (25 mg/kg body weight), and group 4 (ZnO þ ZnCl2 treated) received injections of ZnO NPs (25 mg/kg body weight) and injections of ZnCl2 solution (25 mg/kg body weight). Twenty-four hours after the final injection, treated and control animals were killed in compliance with the code of practice for the Care and Use of Animals for Scientific Purposes. The experimental protocols were approved by the Faculty Ethics Committee. Rats in all groups were treated by intraperitoneal injection every other day for 10 days.
NPs preparation and characterization techniques ZnO NPs were provided by the Laboratory of Physics of Materials and Nanomaterials Applied to Environment, College of Sciences in Gabes, Zirig, Tunisia.13,14 The preparation of nanocrystalline ZnO aerogels were prepared by dissolving 2 g of zinc acetate dehydrate in 14 ml of methanol under magnetic stirring for 2 h. The water for hydrolysis was slowly released by esterification of acetate with methanol. NP aerogel as obtained by supercritical drying in ethyl alcohol (EtOH).13,14 The crystalline data were obtained by x-ray diffractometry (XRD; D8 Advance, Bruker Biosciences Coprporation, Billerica, Massachusetts, USA; 40 kV, 30 mA). To determine the lattice parameters of the different phases, the diffraction x-rays were collected by scanning between 2 ¼ 5 and 70 in 0.02 steps. To examine the size and morphology of oxide nanocrystals, transmission electron microscopy (TEM; JEM-200 CX, Jeol, Tokyo, Japan) was used (Figure 1). The sample preparation for TEM observation was as follows: the powder was first put in EtOH, and then the ultrasonic dispersed solution was dropped on a copper (Cu) net. For photoluminescence (PL) measurements, the 337.1 nm laser line of a Laser Photonics LN 100 nitrogen laser (Stanford Research Systems, Sunnyvale, CA) was used as an excitation source. The emitted light from the sample, collected by an optical fiber on the same side as the excitation, was analyzed with a Jobin-Yvon (French
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Figure 1. TEM images of synthesized ZnO NPs. TEM: Transmission electron microscopy; ZnO: zinc oxide; NPs: nanoparticles.
company of optical instrumentation and Spectroscopy) Spectrometer HR460 and a multichannel charge-coupled device detector (2000 pixels). The PL excitation measurements were performed on Jobin-Yvon Fluorolog 3-2 spectrometer and 450 xenon lamp as the excitation source. The emission spectra were corrected for the spectral response of the excitation source. The low-temperature experiments were carried out in a Janis (France) VPF-600 Dewar with variable temperature controlled between 78 and 350 K13,14 (Figure 2).
Determination of trace element content The concentrations of trace element (Zn, iron (Fe), calcium (Ca), sodium (Na), potassium (K), magnesium (Mg), manganese (Mn), and Cu) in rat liver and kidney were determined with an atomic absorption spectrometer (Avanta, GBC, Australia). The standard solution of Zn used in this assay resulted by the dissolution of ZnCl2 in deionized water. Liver and kidney tissues were lyophilized, weighed, and digested in 2 ml of concentrated nitric acid in pressurized Teflon containers at 160 C for 3 h. After cooling at room temperature, samples were diluted with 10 ml of deionized water.15 Analyses of trace elements were performed using acetylene gas as fuel and air as an oxidizer. Operational conditions were adjusted to yield optimal determination. The calibration curves were prepared separately for all the trace elements by running suitable concentrations of the standard solutions. Digested samples were aspirated into the
Figure 2. EDX spectra of ZnO-NPs representing the elemental composition of nanoparticles samples. EDX: Energy dispersive x-ray; ZnO: zinc oxide; NPs: nanoparticles.
fuel-rich air–acetylene flame, and the concentrations of the trace element were determined from the calibration curves. Average values of three replicates were taken for each determination. Suitable blanks were also prepared and analyzed in the same manner. The detection limits for Fe, Zn, Ca, Na, K, Mg, Mn, and Cu were 0.05, 0.008, 0.025, 0.04, 0.05, 0.05, 0.04, and 0.05 ppm, respectively. Trace elements concentration was expressed in microgram per gram of the dry mass of tissues.
Biochemistry panel analysis All animals were killed at the same time. Blood samples were taken from all rats in heparinized tubes and then centrifuged. In the present study, we chose plasmatic biochemical parameters related to liver and kidney function. We determined the glucose content, uric acid, creatinine, and levels of enzymes such as aspartate aminotransferase (AST) and alanine aminotransferase (ALT). These enzymes were examined by routine colorimetric methods using commercial kits. Organs of the control and treated groups were harvested immediately. Organs were rinsed with icecold deionized water and dried with filter paper. After weighing the body and tissues, the coefficients of liver and kidneys to body weight were calculated as the ratio of tissues (wet weight, in milligram) to body weight (in gram).
X-Ray characterization of the liver powder Liver powder was produced by mechanical method. Rat liver was lyophilized, and the analyzed material is finely ground, homogenized, and average bulk
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Table 1. Effect of ZnO nanoparticles and/or ZnCl2 solution treatment on the coefficient of liver and kidney.a Liver C ZnCl2 ZnO ZnO þ ZnCl2
43.3 + 41.75 + 42.27 + 40.86 +
Kidney 0.69 1.12 0.77 0.74
8.82 9.42 8.99 8.46
+ 0.13 + 0.2 + 0.22 + 0.13
injection decreased significantly the glucose concentration (p < 0.05).
Tissue distribution of trace element
composition is determined. X-Ray powder diffraction is a rapid analytical technique primarily used for phase identification of a crystalline material and can provide information on unit cell dimensions. The crystalline characterization of the liver powder was obtained using XRD (Bruker D8 Advance; 40 kV, 30 mA). To determine the lattice parameters of the different phases, the diffraction x-rays were collected by scanning between 2 ¼ 5 and 70 in 0.02 steps.13,14
The tissue distribution of ZnO NPs and ZnCl2 in solution was determined in the liver and kidney by measuring the total Zn level with flame emission atomic absorption. When the concentrations of total Zn levels were compared between the experimental groups treated by ZnO NPs or ZnCl2 solution and the control group, no statistically significant increases were observed in the liver and kidney (Tables 3 and 4). However, the combined effect of ZnO NPs and ZnCl2 solution showed a significant increase in total Zn accumulation in rat liver (Table 3). A slight decrease in trace element content was detected in the liver of rats injected by ZnO NPs and/or ZnCl2 solution, but the difference did not reach statistical significance. However, the combined effect of ZnO NPs and ZnCl2 solution showed a significant increase in Cu, Mn, and Na contents in rat kidney (Table 4).
Data presentation and statistical analysis
XRD of the liver powder
Results were expressed as the mean + SEM, and data were analyzed by means of one-way analysis of variance with the post hoc test to determine significant relevance to the unexposed control using Statistica 5.0 software (StatSoft). In all cases, p < 0.05 was considered significant.
We investigated the biodistribution and the fate of ZnO NPs and ZnCl2 solution in rat liver. The spectra of liver powder in ZnO NPs and ZnCl2 solutiontreated rats were similar and obviously differed from the reference ZnO NPs spectrum. This suggests that most of the ZnO NPs injected in rats were dissolved in liver. Moreover, our data indicate the lack of biosynthesis of ZnO NPs types from ZnCl2 in rat liver (Figure 3(a) to (f)).
ZnO: zinc oxide; ZnCl2: zinc chloride; C: control; SEM: standard error mean. a Data represent the means + SEM of seven animals per group.
Results Animal observation and coefficients of organs No toxic signs or mortality was observed related to ZnO NPs and/or ZnCl2 solution injection in rats. Also, no obvious significant differences were observed in the coefficients of liver and kidney between the exposed rats and the control group (Table 1).
Biochemical measurements Table 2 showed the changes inf plasmatic biochemical parameters induced by ZnO NPs and/or ZnCl2 solution injection in adult Wistar rats. Experimental groups treated by intraperitoneal injection of ZnO NPs and/or ZnCl2 solution showed a significant increase in AST and uric acid but decreased creatinine levels compared with the control group (p < 0.05). However, the ALT activity remained unchanged (p > 0.05). Interestingly, ZnO NPs and ZnCl2 solution
Discussion The present in vivo study in Wistar rats was aimed to investigate how different sources of Zn such as ZnO NPs and/or ZnCl2 in solution influence first the biochemical parameters related to liver and kidney function and second the distribution of trace element in rat tissues. Moreover, this article describes the fates of ZnO NPs and/or ZnCl2 solution in rat liver determined using XRD. Several NPs exposure routes were chosen for experimental animal studies. These include oral, inhalation, intratracheal, subcutaneous injection, intravenous, and intraperitoneal administration. Recently, Li et al.16 showed that intraperitoneally injected ZnO NPs (2.5 g/kg) were absorbed into circulation (within 30 min postdosing), then biodistributed to the liver, spleen, and kidney. The injected
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Table 2. Effect of ZnO nanoparticles and/or ZnCl2 solution treatment on rat plasmatic biochemical parameters.a AST (IU/l) C ZnCl2 ZnO ZnO þ ZnCl2
25.8 + 68.1 + 85.74 + 75.9 +
ALT (IU/l)
2.262 7.86b 6b 5.9b
23.33 + 29.73 + 36.45 + 42.91 +
Creatinine (mmol/l) 62.36 + 44.54 + 38.77 + 47.58 +
3.37 2.82 12.11 5.08
Uric acid (mg/l)
3.4 5.18b 3.57b 5.66
38.37 76.88 75.80 97.82
+ 7.72 + 10.61b + 8.39b + 10.6b
Glucose (g/l) 1.542 1.42 1.35 1.238
+ 0.079 + 0.078 + 0.079 + 0.043b
ZnO: zinc oxide; ZnCl2: zinc chloride; C: control; AST: aspartate aminotransferase; ALT: alanine aminotransferase; SEM: standard error mean. a Data represent the means + SEM of seven animals per group. b p < 0.05 compared with the control.
Table 3. Liver distribution of trace element in rats after subacute exposure to ZnO nanoparticles and/or ZnCl2 solution.a C Zn Fe Na K Ca Mg Mn Cu
416.61 + 111.123 + 138.88 + 779.62 + 49.38 + 160.61 + 3.7 + 2.95 +
ZnCl2 18.52 19.922 4.79 28.32 5.4 22.13 0.86 0.93
441.03 + 65.33 + 129.64 + 709.87 + 37.73 + 126.47 + 2.46 + 2.48 +
ZnO
20.07 15.53 23.31 115.07 5.52 14.49 0.58 0.18
442.88 79.13 125.94 666.71 39.33 64.45 2.98 1.36
+ 39.69 + 20.83 + 16.17 + 56.14 + 8.26 + 7.47b + 0.27 + 0.48
ZnO þ ZnCl2 587.28 + 97.23 + 128.19 + 728.74 + 52.78 + 124.02 + 2.72 + 1.09 +
37.06b,c,d 11.77 10.12 71.09 13.11 19.55 0.32 0.03
Zn: zinc; Fe: iron; Na: sodium; K: potassium; Ca: calcium; Mg: magnesium; Mn: manganese; Cu: copper; ZnO: zinc oxide; ZnCl2: zinc chloride; C: control; SEM: standard error mean. a Data represent the means + SEM of three animals per group. b Statistical differences p < 0.05 compared with the control. c Statistical differences p < 0.05 compared with ZnCl2 group. d Statistical differences p < 0.05 compared with ZnO nanoparticles.
Table 4. Kidney distribution of trace element in rats after subacute exposure to ZnO nanoparticles and/or ZnCl2 solution.a C Zn Fe Na K Ca Mg Mn Cu
235.09 105.14 312.44 1200.08 148.12 112.53 3.55 1.06
ZnCl2
+ 18.68 + 18.03 + 3.74 + 44.32 + 37.06 + 11.54 + 0.12 + 0.07
465.75 + 57.75 + 274.23 + 826.09 + 115.61 + 66.54 + 2.85 + 0.49 +
289.78 2.19 6.6 29.08 11.56 0.65 0.55 0.04
ZnO 293.47 + 45 62.59 + 21.4 346.23 + 47.62 819.89 + 97.72 173.68 + 28.77 61.12 + 26.95 3.99 + 0.31 1.94 + 0.34
ZnO þ ZnCl2 420 80.99 403.74 1234.42 145.95 81.39 4.81 4.23
+ 79.22 + 3.59 + 15.32b + 120.44 + 26.69 + 17.86 + 0.34b + 1.08b,c,d
Zn: zinc; Fe: iron; Na: sodium; K: potassium; Ca: calcium; Mg: magnesium; Mn: manganese; Cu: copper; ZnO: zinc oxide; ZnCl2: zinc chloride; C: control; SEM: standard error mean. a Data represent the means + SEM of three animals per group. b p < 0.05: statistical differences compared with ZnCl2 group. c p < 0.05: statistical differences compared with the control. d p < 0.05: statistical differences compared to ZnO nanoparticles.
ZnO NPs remained in serum for 72 h and could more effectively spread to the heart, lung, and testes. In our study, the ZnO NPs and ZnCl2 solution (25 mg/kg) were administered to rats through the intraperitoneal
route. Treated rats showed no obvious significant differences in the coefficients of liver and kidney. However, the liver and kidney could be the possible target organs for accumulation and toxicity of ZnO
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Figure 3. Diffraction patterns for different samples. (a) X-ray spectra for ZnO NPs þ sample holder, (b) x-ray spectra for sample holder, (c) x-ray liver powder diffractogram (control group), (d) ZnCl2 group, (e) ZnO NPs group, and (f) ZnO NPs group þ ZnCl2 group. Results are commonly presented as peak positions at 2 and x-ray counts (intensity). ZnO: zinc oxide; ZnCl2: zinc chloride; NPs: nanoparticles
NPs and/or ZnCl2 solution. This was supported by the modulation of plasmatic AST activity and the creatinine and uric acid levels in rats. These results support the findings of Wang et al.17 who observed liver damage in mice after an oral exposure of ZnO NPs although at a higher dose of 5 g/kg. Interestingly, subacute exposure to ZnO NPs and ZnCl2 solution decreased plasmatic glucose concentration in rats. Yaghmaei et al.18 reported that Zn has a role in the synthesis, storage, and secretion of insulin and has been suggested to be beneficial when used in the
diabetic state. The NPs when ingested into the body can be distributed to different regions because of their small size. It is important to find out information about their biodistribution. Lee et al.12 reported that ZnO NPs are absorbed into the blood, distributed into various organs, and cleared from the body via the urine. Furthermore, according to radioactive ZnO experiments, NPs primarily showed retention in the lung, followed by retention in the liver and kidney after intravenous administration.19 In case of our investigation, the tissue distribution of ZnO NPs and
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ZnCl2 solution were determined in the liver and kidney by measuring the total zinc levels with flame emission atomic absorption. No remarkable difference in distribution of Zn in the liver was seen in rats treated with ZnO NPs or ZnCl2 solution. However, significantly elevated Zn levels were observed in the liver of rats after ZnO NPs and ZnCl2 solution exposure. The ionic form (Zn2þ) would be taken up by the liver via the first-pass effects and then redistributed from the liver to the other organs.20 However, very little is known about the size, dose, and exposure dependence of in vivo NP processing biodistribution to every body organ and tissue. Furthermore, there are currently no data available regarding the systemic circulation and accumulation of ZnO NPs in rat liver following repeated intraperitoneal administration. Several studies show that interstitially injected NPs pass preferentially through the lymphatic system and not the circulatory system, probably due to permeability differences.21,22 The accumulation of Zn in the liver could be related probably to the induction of tissues metallothioneins in ZnO NPs and ZnCl2 solution-treated rats, since these proteins have been suspected to control metal homeostasis and to maintain cell survival in response to various stimuli.23 Morover, a slightly higher amount of Zn was detected in the kidney of rats treated with ZnO NPs and/or ZnCl2 solution, but the difference did not reach statistical significance. Lee et al.12 showed that when ZnO NPs were administered to rats, some of them were absorbed into the blood and then mainly distributed in the liver and finally cleared from the body in the kidney via the urine. The accumulation of Zn in rat liver or kidney for different origins such as ZnCl2 in solution or particulate ZnO is similar. Interestingly, this finding showed that animals do not assimilate more Zn when it is added as dissolved ZnCl2 in comparison with ZnO NPs. In order to evaluate the tissue accumulation and distribution of intraperitoneally injected ZnO NPs and/or ZnCl2 solution, we carried out the XRD analysis on the lyophilized liver powder dissected from rats. The x-ray spectrum in ZnO NPs and/or ZnCl2 groups looked similar to the control groups, and no ZnO NPs types were found in rat liver. This result confirms that ZnO NPs were dissolved in liver tissues giving Zn2þ ions in solution. Accordingly, previous investigation has reported that ZnO NPs could not be observed in the TEM images of the liver and kidney, whereas accumulation of ZnO NPs was evident on inductively coupled plasma atomic emission spectroscopy analysis, implying an ionized
fate in the tissues.7 On the other hand, the XRD analysis clearly showed the lack of biosynthesis of ZnO NPs from ZnCl2in rat liver. The possible biosynthesis of ZnO NPs in ZnCl2-treated rats could be assigned as Zn–ligand interactions between Zn2þ ions and proteins, such as metallothionein, with sulfur-rich ligands, which is consistent with previous evidence for Zn2þ ions bonding to sulfur in the liver as determined by x-ray absorption spectra analysis.1,24 Furthermore, the synthetic method of synthesizing ZnO NPs from Zn hyperaccumulator tissues could provide in the future a new finding. In order to test the hypothesis that ZnO NPs and/or ZnCl2 solution altered the homeostasis of trace element in rat tissues, the contents of Ca, Mg, Mn, Na, K, Cu, and Fe were studied in treated rat liver and kidney. Increasing evidence suggested that Zn accumulation in tissues may involve disruptions of trace elements homeostasis. Our data suggest that dissolution of Zn2þ ions in rat liver decreased slightly the trace element content in this organ. By contrast, the combined effect of ZnO NPs and ZnCl2 solution showed a significant increase of Cu, Mn, and Na content in the kidney. This effect is probably favored by the urinary excretion, the excretion profiles for trace element via urine under ZnO NPs and/or ZnCl2 solution is currently underway. Our results demonstrate that subacute intraperitoneal injection of ZnO NPs (20–30 nm) and/or ZnCl2 solution (25 mg/kg) in rats leads to the modulation of plasmatic biochemical parameters resulting probably to cellular injury. Interestingly, animals do not assimilate more Zn when it is added as dissolved ZnCl2 in comparison with particulate ZnO, and the accumulation patterns for different origins of Zn are similar. Dissolved Zn2þ ions in ZnO NPs and/or ZnCl2 groups did not show remarkable perturbation of trace element homeostasis in rat liver and kidney. On the basis of total Zn concentrations in rat liver and kidney, the subacute toxicity of ZnO NPs was statistically similar to that of ZnCl2 in solution. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
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