Acute and chronic immobilization stress can affect elements homeostasis and change their distribution in the different parts of the body, especially in the brain [1, 2]. Immobilization stress was decreased endogenous Zn2+ and Fe2+/3+ concentrations in different parts of the brain such as hippocampus 24 hours after the stress induction . On the other hand, trace elements such as Mg2+, Zn2+, Fe2+/3+ and Ca2 are essential for living cells and body systems functions[4-6] and can change balance of each other in the body[7,8]. Magnesium and zinc have key roles in the central nervous system and hippocampus functions [9, 10].
Both of them work on some similar receptors, like N-methyl-D-aspartate (NMDA), that block or reduce its activity . It has been shown that in rats under Zn2+ restriction Mg2+, Fe2+/3+ and Ca2 levels in the serum have increased significantly and decreased Zn2+ concentration in the serum and hippocampus, while could not affect Mg2+, Fe2+/3+ and Ca2+ levels in the rat’s hippocampus.Magnesium oxide and zinc oxide nanoparticles (MgO NPs and ZnO NPs), as novel sources of Mg2+ and Zn2+, are widely used in medicine and pharmacology, by the development of nanotechnology [13, 14]. Benefit and toxic effects of these components have been investigated in the various living cells and systems, because of the unique properties of them [13, 15-18].
Some studies have shown that nanoparticles can change ions level in the central nervous system and neural cells [16, 19, 20]. Amaraet al. (2013) have shown that in the rat brainZnO NPs change elements levels includingFe2+/3+, Zn2+, and Ca2+ . Also, ZnO NPs increase intracellular Ca2+ level and affect neural cell by increasing Zn2+ ions [16, 20]. Ion releasing is one of the most important cytotoxic factors of metal oxide nanoparticles and their primary particle size or surface area did not aﬀect cellular functions directly .
Ben-Slama and et al. (2015) have indicated that oral exposure to ZnO NPs decreased the brain Ca2+ concentration . Our previous study has shown that ZnO NPs can increase Zn2+ level in the serum and decrease anxiety-like behaviors in animal models .
In this work we investigated and compared the MgO NPs and ZnO NPs effectson Mg2+, Zn2+, Fe2+/3+, and Ca2+ level changes in the serum/ hippocampus of adult male rats attwo different acute times following acute stress induction.
MATERIAL AND METHODS
Animals grouping and treatments
In this experimental work, male Wistar rats (220 ± 10 g) were purchased from animal house of faculty of veterinary in the Shahid Chamran University of Ahvaz.Experiments were carried out under ethical code of EE/22.214.171.124369/scu.ac.ir. MgO and ZnO nanoparticles (USnano., CO, USA) (Fig. 1), suspensions were prepared before experiments and injected intraperitoneally in a single dose of 5 mg/kg and a volume of 1 mL/kg [22, 23]. Both nanoparticles didn’t formed large aggregates that blocked the syringe during injections. Rats were divided into 16 groups, which included two sub-groups, 1) non- restraint rats: control,MgO NPsand ZnO NPs 5 mg/kgand2) restraint rats: restrained for90 , 180 and 360 min+ saline (ST 90 min, ST 180 min and ST 360 min) andST 90 min+ MgO orZnO NPs 5 mg/kg. In all restraint groups, components were injected immediately after restraint stress induction, then in on main group(including, 8 groups) 30 min and in the other (including, 8 groups) 120 min after components injections or restraint stress induction animals were killed for a measure of ions concentrations in the serum and hippocampus.The number of rats in each group was six.
Acute stress induction
Rats were restrained for 90, 180 and 360 min in the plexiglass tubes, then immediately received saline (1ml/kg) or MgO NPs and ZnO NPs 5 mg/kg.
Serum/ hippocampus sampling and assessment of elements contents
In the first 8 groups after 30 min and in the second 8 groups after 120 min, all rats scarified then serums and hippocampus homogenates of them obtained.Elements contents measured by a ﬂame atomic absorption spectrophotometer apparatus in all samples, and results expressed as a μg/ mL of the serum and mg/g of wet hippocampus tissue.
One way ANOVA with Tukey post-hoc was used for comparing among groups and Student’s t-test was used for comparing the means of unpaired data by using SPSS 16 software. Pearson correlation coefficient was calculated between elements contents of the serum/ hippocampus in 2 acute different times. Differences with a p value of <0.05 was considered statistically significant. Results are presented as the mean ± standard error of the mean and graphs are plotted with the Excel software.
RESULTS AND DISCUSSION
Nanoparticles size detection by XRD patterns
Fig. 1 is XRD patterns of the MgO NPs (A) and ZnO NPs (B) and indicates that the sizes of both nanoparticles were lower than 100 nm before injections.
Assessment of Mg2+concentration
As seen in Fig. 2A, MgO NPs (30 min (P=0.002) and 120 min (P<0.0001)) and ZnO NPs (30 min (P<0.001) and 120 min (P=0.003)) significantly increased Mg2+ level in the serum 30 and 120 min after injections. Also, in MgO NPs group Mg2+level 120 min after injection was significantly higher than 30 min (P=0.0018).Level of Mg2+ in serum was increased 30 min after ST 90 (P=0.0064) and ST 180 min induction (P=0.038) and was decreased and reached to the control group at 120 min. ST 360 min did not affect the level of Mg2+ significantly after 30 and 120 min compared to the control groups but was seen significant difference between 30 and 120 min (P=0.0092).
In the restraint groups, MgO NPs and ZnO NPs did not change Mg2+ level in comparison with ST 90 min group after 30 min and 120 min, but following injection of both nanoparticles Mg2+level after 120 min was decreased in comparison with after 30 min (P=0.042). Data analysis showed that ST 90 min has a reductive effect on the MgO NPs (P<0.0001) and ZnO NPs (P=0.0167) effects on serum magnesium concentration 120 min after injection.
Fig. 3B showed that MgO NPs (P=0.0004) and ST 90 min (P=0.041) increased hippocampus magnesium level after 120 min. While MgO NPs acted significantly in opposite directions in the presence of ST 90 min at two different times of 30 and 120 min (P=0.0030). MgO NPs in the presence of stress after 30 min reduced magnesium compared with the administration of MgO NPs alone but increased it after 120 min. This effect was not observed for ZnO NPs.
These results show that time duration after MgO NPs injection is an important factor in the release of Mg2+ from it in the serum and hippocampus. Also, stress has a dual role in the release of Mg2+, in a short time can increase Mg2+ in the serum and while with passing the time increase it in the hippocampus.
Assessment of Zn2+ concentration
MgO NPs significantly decreased Zn2+ level after 120 min (P=0.0002), while ZnO NPs increased Zn2+ level in the serum after 30 (P=0.013) and 120 min (P=0.040). Stress was decreased Zn2+level in a time-dependent manner 120 min after induction (ST 90 min (P<0.05), ST180 min (P<0.01) and ST 360 min (P<0.001)). In the restraint groups, MgO NPs (P=0.0075) and ZnO NPs (P=0.030) have increased Zn2+ level after 30 min, while decreased it after 120 min in comparison with 30 min after injections (MgO NPs (P<0.001) and ZnO NPs (P<0.05)). Stress 90 min decreased the serum zinc level in the ZnO NPs group (P=0.013) after 120 min that show negative effect of stress on ZnO NPs efficacy especially (Fig. 3A).
ZnO NPs was decreased Zn2+ level in the hippocampus after 30 min (P=0.015). Stress increased partially Zn2+ level 30 and 120 min after induction in all groups and it was significant 30 min after induction of ST 360 min (P=0.0058). In restraint groups, MgO NPs and ZnO NPs have increased Zn2+ level after 120 min and it was significant in the ZnO NPs group (P=0.044) (Fig. 3B).
These results show that except in one group (ZnO NPs) in all the other groups decrease of Zn2+ in serum was parallel to increase of it in the hippocampus, 120 min after treatments.
Assessment of Fe2+/3+ concentration
MgO NPs increased serum Fe2+/3+level in the stressed (P<0.0001)and non-stressed(P=0.0005) animals after 30 min, while ZnO NPs decreased Fe2+/3+ level partially. Ironlevel decreased after 120 min in ST 360 min group (P=0.011). In the restraint animals MgO NPs significantly decreased Fe2+/3+ level after 120 min in comparison with 30 min (P<0.0001) (Fig. 4A).
MgO NPs (P<0.0076) and ZnO NPs (P=0.0018) significantly increased Fe2+/3+ level in the hippocampus of non-restraint animals after 30 min.Also stress increased Fe2+/3+ level in all duration after 30 min (ST 90 and 180 min (P<0.001) and ST 360 min (P<0.05)) and decreased it after 120 min. In the restraint animals MgO NPs decreased Fe2+/3+ level after 120 min in compared with 30 min (P=0.0012). On the other hand, ZnO NPs significantly decreased Fe2+/3+ level in the non-restraint (P=0.0031) after 120 min (Fig. 4B).
According to these results, it seems that acute injection of both nanoparticles could change the Fe2+/3+ balance in two of measurement sites and stress influences their effects.
Assessment of Ca2+ concentration
In the serum, MgO NPs increased Ca2+ concentration after 30 min (P=0.0106), while ZnO NPs increased it after 120 min (P=0.012). The ST 90 min significantly decreased Ca2+ in 120 and 30 min after induction (P<0.01), while ST 360 min increased it and was significant after 30 min (P<0.05). In the restraint animals, MgO NPs was increased Ca2+ level after 30 min and decreased it after 120 min (P<0.0001), while in these animals ZnO NPs was increased Ca2+ level in both times after injection (30 min (P<0.0001) and 120 min (P=0.0021)). Also, in MgO NPs (P=0.0002) and ZnO NPs recipients groups stress had negative effect on calcium level and decreased it in comparison with nanoparticles injections alone after 120 min (P=0.0106).
MgO NPs significantly was increased Ca2+ level in the hippocampus, after 120 min (P=0.008), while ZnO NPs increased it after 30 min (P=0.0054) (Fig. 5 A and B). Stress in all duration increased Ca2+ level after 30 min and it was significant in the ST 180 min group (P=0.037). In the restraint animals, MgO NPs decreased Ca2+ level after 30 min (P=0.0105), while ZnO NPs decreased it after 120 min (P=0.0042) (Fig. 5B).
Based on these findings, MgO NPs and ZnO NPs effects on Ca2+ level balance, in the restraint and non-restraint rats, were completely adverse in two different acute times.
Assessment of Pearson correlation between ions concentration in two acute times (30 and 120 min)
Data on Table 1 show that there were significant positive correlations between both of Fe2+/3+ (R=0.0607, P=0.008) and Ca2+ (R=0.626, P=0.005) level changes in the serum, 30 and 120 min after stress induction. There was a negative correlation between Fe2+/3+ contentof the hippocampus in two different times after stress induction (R=-0.471, P=0.049).Also, in ZnO NPs group there was a positive correlation between Ca2+ level changes in the hippocampus, at two different acute times (R=0.844, P<0.035).
Results have indicated that efficacy of MgO and ZnO NPs on ions level changes in the serum and hippocampus could be different with passing the time. Since metal oxide nanoparticles are dissolved easily in acidic environments, probably ZnO NPs can dissolve in the lysosomes and release Zn2+ ions [18, 24, 25].This is a possible way to increase of Mg2+ and Zn2+ in the serum during the times after nanoparticles injections.Oxide salts are less reactive and release of ions from them is slow . Previously we have indicated that Zn2+ concentration increased in the serum of male rats following injection of ZnO NPs and conventional ZnO and retention of Zn2+ ions in the serum of ZnO NPs group was higher than conventional ones after 24 hours; so that probably in the rat body ZnO NPs clearance was less than conventional forms . This retention of nanoparticles in the body can affect their efficacy with passing the time.
Injections of nanoparticles could change the balance of elements too. Magnesium is a natural Ca2+ antagonist and can regulate Ca2+ channels with an important role in the active transport of Ca2+ ions through the cell membranes [27, 28]. On the other hands, there is a divalent metal transporter 1 that transports divalent metals including Mg2+, Zn2+ and Ca2+ by a proton-coupled mechanism . Some Zn2+ transporter proteins can facilitate non-transferrin bound Fe2+/3+ -mediated delivery in cultured cells and similar trans membrane pores conduct Fe2+/3+ and Ca2+ through the membranes . Consumption of a Ca2+ supplement decreased the total Fe2+/3+absorption, primarily by reducing the initial uptake of heme Fe2+/3+. Transferrin receptor (TfR)-mediated Fe2+/3+ transport by the blood-brain-barrier andFe2+/3+ concentration ishigh in the hippocampus of the normal brain and TfR in the cerebral endothelial of the hippocampus is about 3–7 folds higher than in the cortex .
All of these studies indicated that MgO NPs and ZnO NPs could affect the balance of other elements in the serum and hippocampus, it’s while maybe nanoparticles directly change the balance of elements, that this needs too more investigation.At the following results have indicated that restraint stress has different effects on elements changes in the serum and hippocampus depend on the acute time passing after stress induction.
Karakoc and et al. (2003) have shown that acute immobilization stress causes endogenous Zn2+ release from the brain and may enhance production of the brain iron transport proteins . In all over the world iron deficiency anemia is a popular nutritional deficiency anemia and it has been reported that Zn2+ supplementation prevents stress effects and a stress-induced decrease in Fe2+/3+ level [32, 33]. However, usage of a modest Zn2+ supplement induce a cellular Fe2+/3+ deﬁciency and probably further reduce of the Fe2+/3+ statue .
Also, it has been shown that in the rat hippocampus stress increases Ca2+ current amplitude . In this study MgO NPs and ZnO NPs could improve Fe2+/3+ and Ca2+ concentration changes in the serum and hippocampus following stress induction and their effects depend on the acute time passing after injection.
It seems that rather than the elements level changes by nanoparticles, the efficacy of MgO and ZnO NPs on ions level imbalance induced by restraint stress depend on the acute time passing after stress induction. But more investigation needs to find exact effects of nanoparticles on body ions balance in healthy and stressful situations as well as in different acute and chronic times.
This study is supported financially by the Research Council of the Shahid Chamran University of Ahvaz (Grant: 96/3/02/16670).
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interest.
1. Karakoc Y, Yurdakos E, Gulyasar T, Mengi M, Barutcu UB. Experimental stress-induced changes in trace element levels of various tissues in rats. The Journal of Trace Elements in Experimental Medicine. 2003;16(1):55-60.
3. Kamal Z, Najimi M, Chigr M, El Ouahli M, Er-Raoui G, Chigr F. Trace elements distribution in the brain of stressed rats. American Journal of Neuroscience. 2012; 3(2): 79-86.
5. Li W, Yu J, Liu Y, Huang X, Abumaria N, Zhu Y, et al. Elevation of brain magnesium prevents synaptic loss and reverses cognitive deficits in Alzheimer’s disease mouse model. Molecular Brain. 2014;7(1).
7. Baltaci AK, Mogulkoc R, Belviranli M. Serum levels of calcium, selenium, magnesium, phosphorus, chromium, copper and iron--their relation to zinc in rats with induced hypothyroidism. Acta Clinica Croatica. 2013; 52(2):151-156.
8. Bicer M, Akil M, Sivrikaya A, Kara E, Baltaci AK, Mogulkoc R. Effect of zinc supplementation on the distribution of various elements in the serum of diabetic rats subjected to an acute swimming exercise. Journal of Physiology and Biochemistry. 2011;67(4):511-7.
9. Yang Y, Jing X-P, Zhang S-P, Gu R-X, Tang F-X, Wang X-L, et al. High Dose Zinc Supplementation Induces Hippocampal Zinc Deficiency and Memory Impairment with Inhibition of BDNF Signaling. PLoS ONE. 2013;8(1):e55384.
10. Yorulmaz H, Şeker FB, Demir G, Yalçın İE, Öztaş B. The Effects of Zinc Treatment on the Blood–Brain Barrier Permeability and Brain Element Levels During Convulsions. Biological Trace Element Research. 2012;151(2):256-62.
11. Sowa-Kućma M, Szewczyk B, Sadlik K, Piekoszewski W, Trela F, Opoka W, et al. Zinc, magnesium and NMDA receptor alterations in the hippocampus of suicide victims. Journal of Affective Disorders. 2013;151(3):924-31.
12. Doboszewska U, Szewczyk B, Sowa-Kućma M, Noworyta-Sokołowska K, Misztak P, Gołębiowska J, et al. Alterations of Bio-elements, Oxidative, and Inflammatory Status in the Zinc Deficiency Model in Rats. Neurotoxicity Research. 2015;29(1):143-54.
13. Horie M, Fujita K, Kato H, Endoh S, Nishio K, Komaba LK, et al. Association of the physical and chemical properties and the cytotoxicity of metal oxide nanoparticles: metal ion release, adsorption ability and specific surface area. Metallomics. 2012;4(4):350.
14. Teymuri Zamaneh H,Kesmati M, Malekshahi Nia H, Najafzadeh Varzi H, Torabi M. Investigating the effects of chronic magnesium oxide nanoparticles on aerobic exercise-induced antinociception in adult male rats. International Journal of Green Pharmacy. 2017; 11(4) (Suppl) S892.
15. Ghobadian M, Nabiuni M, Parivar K, Fathi M, Pazooki J. Toxic effects of magnesium oxide nanoparticles on early developmental and larval stages of zebrafish (Danio rerio). Ecotoxicology and Environmental Safety. 2015;122:260-7.
17. Moeini-Nodeh S, Rahimifard M, Baeeri M, Abdollahi M. Functional Improvement in Rats’ Pancreatic Islets Using Magnesium Oxide Nanoparticles Through Antiapoptotic and Antioxidant Pathways. Biological Trace Element Research. 2016;175(1):146-55.
19. Amara S, Slama IB, Omri K, Ghoul JEL, Mir LEL, Rhouma KB, et al. Effects of nanoparticle zinc oxide on emotional behavior and trace elements homeostasis in rat brain. Toxicology and Industrial Health. 2013;31(12):1202-9.
21. Ben-Slama I, Mrad I, Rihane N, EL Mir L, Sakly M. Amara S. Sub-acute oral toxicity of Zinc Oxide nanoparticles in male rats. Journal of Nanomedicine and Nanotechnology. 2015; 6(3): 1-6.
23. Kesmati M, Zadehdarvish F, Jelodar Z, Torabi M. Vitamin C potentiate sedative effect of magnesium oxide nanoparticles on anxiety and nociception in the postpartum depression model. Nanomedicine Journal. 2017; 4(1): 17-24.
24. Bannunah AM, Vllasaliu D, Lord J, Stolnik S. Mechanisms of Nanoparticle Internalization and Transport Across an Intestinal Epithelial Cell Model: Effect of Size and Surface Charge. Molecular Pharmaceutics. 2014;11(12):4363-73.
25. Cho W-S, Duffin R, Howie SEM, Scotton CJ, Wallace WAH, MacNee W, et al. Progressive severe lung injury by zinc oxide nanoparticles; the role of Zn2+ dissolution inside lysosomes. Particle and Fibre Toxicology. 2011;8(1):27.
26. Veldkamp T, van Diepen JTM, Bikker P. The bioavailability of four Zn2+ Oxide Sources andZn2+ sulfate in broiler chickens; Lelystad, Wageningen UR (University & Research center) LivestockResearch, Confidential Livestock Research Report. 2014; 806 pages: 27
29. Nadadur SS, Srirama K, Mudipalli A. Fe2+/3+ transport & homeostasis mechanisms: Their role in health & disease. Indian Journal of Medical Research.2008; 128: 533-544.
30. Roughead ZK, Zito CA, Hunt JR. Inhibitory effects of dietary calcium on the initial uptake and subsequent retention of heme and nonheme iron in humans: comparisons using an intestinal lavage method. The American Journal of Clinical Nutrition. 2005;82(3):589-97.
32. Li Y, Zheng Y, Qian J, Chen X, Shen Z, Tao L, et al. Preventive Effects of Zinc Against Psychological Stress-Induced Iron Dyshomeostasis, Erythropoiesis Inhibition, and Oxidative Stress Status in Rats. Biological Trace Element Research. 2012;147(1-3):285-91.
35. Joels M, Velzing E, Nair S, Verkuyl JM, Karst H. Acute stress increases calcium current amplitude in rat hippocampus: temporal changes in physiology and gene expression. European Journal of Neuroscience. 2003;18(5):1315-24.