Patho-biochemical Markers of Renal Toxicity in Homing Pigeons (Columba Livia f. Urbana) Induced by Nano-ZnO Particles
محورهای موضوعی :
Mehran Arabi
1
(Department of Animal Sciences, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran)
Hamid-Reza Naseri
2
(Department of Animal Sciences, Faculty of Basic Sciences, Shahrekord University, Shahrekord, Iran)
کلید واژه: Birds, Nanotoxicity, Renal toxicity, Oxidative stress,
چکیده مقاله :
Nanotechnology, the main technology in the twenty-first century, is the perception and control of matter at the dimensions 1-100 nanometers which revolutionized many aspects of modern life. Birds have not been used as commonly animal models in experimental toxicology but have proven useful in monitoring of environmental quality. This study aimed to evaluate zinc oxide nanoparticle (nano-ZnO) toxicity on kidneys in the homing pigeons (Columba livia f. urbana). The experimental groups orally received doses of 0, 30, 50, and 75 mg kg-1 b.w. of nano-ZnO (1 ml per bird, everyday) for time periods of 7 and 14 days. The lipid peroxidation (MDA/LPO) content, catalase (CAT) activity, and carbonyl protein (CP) content were increased and the total antioxidant capacity (TAC) level was lowered in kidney samples. The plasma levels of uric acid, urea, and creatinine were also slightly elevated (p>0.05). Histopathological examinations showed glomerular nanoparticle aggregation and tubular necrosis lines in the kidney parenchyma. In brief, nano-ZnO affected kidney function and structure in homing pigeons through the induction of oxidative stress.
Nanotechnology, the main technology in the twenty-first century, is the perception and control of matter at the dimensions 1-100 nanometers which revolutionized many aspects of modern life. Birds have not been used as commonly animal models in experimental toxicology but have proven useful in monitoring of environmental quality. This study aimed to evaluate zinc oxide nanoparticle (nano-ZnO) toxicity on kidneys in the homing pigeons (Columba livia f. urbana). The experimental groups orally received doses of 0, 30, 50, and 75 mg kg-1 b.w. of nano-ZnO (1 ml per bird, everyday) for time periods of 7 and 14 days. The lipid peroxidation (MDA/LPO) content, catalase (CAT) activity, and carbonyl protein (CP) content were increased and the total antioxidant capacity (TAC) level was lowered in kidney samples. The plasma levels of uric acid, urea, and creatinine were also slightly elevated (p>0.05). Histopathological examinations showed glomerular nanoparticle aggregation and tubular necrosis lines in the kidney parenchyma. In brief, nano-ZnO affected kidney function and structure in homing pigeons through the induction of oxidative stress.
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Nanoparticles vs Pigeons
ABSTRACT: Nanotechnology, the main technology in the twenty-first century, is the perception and control of matter at the dimensions 1-100 nanometers which revolutionized many aspects of modern life. Birds have been less favored as laboratory models but have proven valuable as natural models with immediate relevancy to risk assessment. This study was aimed to evaluate zinc oxide nanoparticles (nano-ZnO) toxicity on renal function and structure in homing pigeon (Columba livia f. urbana). The experimental groups were orally received 0, 30, 50, and 75 mg kg-1 b.w. of nano-ZnO through oral gavage (1 ml/bird, daily) for 7 and 14 consecutive days. The lipid peroxidation (MDA/LPO) content, catalase (CAT) activity, and carbonyl protein (CP) content were increased and total antioxidant capacity (TAC) level lowered in kidney samples. The levels of plasma urea, uric acid, and creatinine were also slightly elevated (p>0.05). Histopathological examinations showed glomerular nanoparticle aggregation and tubular necrosis lines in kidney parenchyma. In brief, nano-ZnO affected kidney function and structure in homing pigeons through the induction of oxidative stress.
KEYWORDS Birds; Nanotoxicity; Renal toxicity; Oxidative stress
INTRODUCTION
Nanotechnology, the main technology in the twenty-first century, is the perception and control of matter at the dimensions 1-100 nanometers which revolutionized many aspects of modern life. Nanotechnologies are based on the manipulation and integration of atoms and molecules to form different structures and systems at the nanoscale. Nanoparticles (NPs) are present in the environment from both natural and anthropogenic sources. Many studies have implicated that NPs due to high surface area to volume ratio are more toxic to organisms. The NPs may leak into the environment in their life cycles including production, consumption, and disposal processes [1,2]. Induction of oxidative stress (OS) as a possible mechanism for damaging cells by NPs is well evidenced. The high chemical reactivity of NPs results in increased production of reactive oxygen species (ROS), including free radicals. ROS and free radical production is one of the primary mechanisms of NP toxicity; it may result in OS, and consequent damage to proteins, lipids, and DNA [2]. The appropriate physiological level of ROS is managed by antioxidant molecules such as glutathione (GSH), vitamin E, catalase (CAT), superoxide dismutase (SOD), etc. Following occurrence of OS, lipid peroxidation (LPO) process and malondialdehyde (MDA) are the initial steps of cellular membrane damage. Any accumulation of MDA in cells leads to disruption in cell membrane [3,4].
The metallic NPs such as nano-Zinc oxide particles (nano-ZnO) are attractive because of their wide range of uses. Nano-ZnO are used generally in the veterinary applications due to their wound healing, antibacterial, and angiogenic properties. The nano-ZnO dissolves in the extracellular region, which in turn increases the intra cellular [Zn2+] level. Cytotoxicity, increased ROS and OS, decreased mitochondrial membrane potential, and apoptosis, are accounted as the main symptoms following exposure to nano-ZnO in cells. Nano-ZnO have the ability to induce the production of ROS in addition to that nano-ZnO harm DNA and induce apoptosis, as nano-ZnO are characterized by excitation of toxicity with easily penetration and accumulation in the organism. Nano-ZnO penetrate the cell membrane via ion channels in the cell membrane and mitochondrial membrane, specific receptors, and endocytosis. The more nano-ZnO internalize in the cell, the more distribute in cell organelles [5].
Many species of birds are also known to have measurable responses to persistent contaminants, ranging from residue accumulation to population decline. Apart from the species, other factors such as sex, age, feeding habits, migratory habits, etc. influence the exposure of contaminants into the organisms, and consequently the accumulation in tissues, fluids, and bird products [6]. The heavy metal toxicosis is commonly seen in birds, with zinc toxicity being the most frequently diagnosed in pet birds [7,8]. The pigeon for many centuries is one of the avian species more used for human nutrition. The homing pigeon (Columba livia f. urbana) is one of the recommended model organisms that are used to assess environmental pollution [9]. Birds exposed to NPs show adverse alterations in the function of certain tissues [10], but the ecotoxicological data on nano-ZnO are just emerging and scanty. The avian renal system excretes nitrogenous waste products, and is central to body water and solute homeostasis. In general, avian kidneys comprise 1 to 2.6 % of body weight compared to an average of 0.5% of body weight in mammals [11].
There are little data concerning the toxicological effects of nano-ZnO on renal function and structure in homing pigeons (Columba livia f. urbana). Thus, the present study was aimed to evaluate these effects in homing pigeons under the standard laboratory conditions.
MATERIALS AND METHODS
Chemicals and Experimental design
The nano-ZnO with a diameter size of < 20 nm, spherical in shape, and 99% purity was purchased from Nano-shop Company, Tehran, Iran. Other chemicals in reagent grade were purchased from Sigma-Aldrich Chemical CO. (St. Louis, MO, USA). The nano-ZnO suspension in deionized water was sonicated for 20 min and vortexed for 1 min before every administration [12].
Homing pigeons (Columba livia f. urbana), with the mean weight about 300-350 gr were acclimatized to the laboratory conditions with a photoperiod of 12:12 hr at 20±2 ˚C for 14 days. Food and water were provided ad libitum. Then, pigeons were randomly divided into three experimental groups (in triplicate) and received 30, 50, and 75 mg kg-1 b.w. of nano-ZnO solutions for 7 and 14 consecutive days (1 mL/bird) through oral gavage (Figure 1).
Figure 1. Nano-ZnO administration through oral gavage.
Preparation of kidney samples
The pigeons were euthanized by cervical dislocation. The kidney samples were taken out and homogenized in chilled 50 mM potassium phosphate buffer (1:8, w/v; pH 7.0). The resulting homogenate was divided into two parts, i) for measuring MDA, TAC, CP, and protein content, and ii) centrifuged at 10,000 rpm for 10 min at 4˚C to obtain the post-mitochondrial supernatant for CAT assay [4].
Content of lipid peroxidation (LPO/MDA)
In this assay, MDA as a reliable marker of LPO reacted with thiobarbituric acid (TBA) forming TBA-reactive substances (TBARS). The chilled 30% (w/v) trichlroacetic acid (TCA) were added to supernatants and centrifuged at 5000 ×g for 10 min. Then, resulting aliquot reacted with TBA and MDA (TBARS) absorbance recorded at 532 nm by use of UV-1700 spectrophotometer (Shimadzu, Japan). The MDA level was expressed as n moles MDA per mg protein. min. A range of MDA was also freshly prepared from tetramethoxypropane as standard. A molar extinction coefficient of 1.56×105 M-1cm-1 was used for calculations [13].
Catalase (CAT) activity
A spectrophotometric method was applied to measure the breakdown rate of hydrogen peroxide (H2O2) was in assessing the changes in CAT (EC 1.11.1.6) activity at 240 nm. To start the reaction, H2O2 in 50 mM potassium phosphate buffer (pH 7.0) was added to 10 % supernatants. Any decrease in the optical density of assay mixture (H2O2 degradation) was recorded at 240 nm (every 20 s) for 3 min. CAT activity is defined in unit equals the amount of CAT that is needed to decompose H2O2 (1 mM/min). The calculated activity of CAT was expressed as IU H2O2 decomposed per mg protein. min, using absorption coefficient for H2O2 of 0.04 m mol−1 cm−1 [14].
Content of carbonylated proteins (CP)
Supernatant was added to 10 mM 2, 4-dinitrophenylhydrazine (DNPH) in 2 M HCl. After 1 h, 10% trichloroacetic acid (TCA) was added and centrifuged at 3,000 ×g for 20 min. The pellet was solubilized in guanidine and potassium phosphate, the resulting solution was incubated at 37 °C for 15 min. Carbonyl concentrations were determined at 370 nm and expressed as n mol CP per mg protein [15].
Value of total antioxidant capacity (TAC)
Ferric reducing ability of plasma (FRAP) test was used. During the assay, yellow ferric tripyridyltriazine complex (Fe (III)-TPTZ) is reduced to blue ferrous complex (Fe (II)-TPTZ). FRAP reagent was mixed with 0.1 mL of diluted kidney sample (1:10) and incubated at 37 °C for 10 min. The resulting blue color is linearly related to the total reducing capacity of antioxidants in kidney samples, measured at 593 nm for 4 min and expressed as m moles Fe2+ produced per mL [16].
Blood plasma biochemistry
Blood sample was collected via puncturing from brachial vein and centrifuged at 10000 ×g for 10 min at 4˚C, containing 0.02 ml heparin/ml blood. The resulting plasma was stored at −80 ˚C. The changes in the levels of urea, uric acid, and creatinine were measured by a Roche Hitachi Model 917 Multichannel Analyzer using commercial kits.
Histopathological examinations
The kidney tissues have been taken and placed into formalin 10%. Serial sections of 5 microns were prepared and stained with hematoxylin and eosin (H&E) technique. The structural changes of the sections were assessed by optical microscopy.
Statistical analyses
The standard ANOVA techniques was carried out with Tukey’s HSD post hoc test (P<0.05: significant probability level), using SPSS software. Data were presented as Mean±Standard Deviation (SD) (n=10/group). The Pearson’s correlation coefficient analysis was performed to determine the strength of the correlation among parameters.
RESULTS
Content of LPO/MDA
MDA/LPO level increased over a time exposure of 7 and 14 days, against controls in a concentration-dependent manner (Figure 2). The highest MDA level was obtained in 75 mg kg-1 nano-ZnO treated groups after 14 days (143.37%, P< 0.05). A positive correlation was observed between nano-ZnO increasing concentrations and elevated MDA level (r = 0.92, P<0.01) after 14 day exposure to nanoparticles.
Figure 2. The level of malondialdehyde (MDA/LPO) in kidney of homing pigeon exposed to nano-ZnO after 7 and 14 days. a,b: data not sharing a common letter are significantly different (P<0.05) between treatments in the same exposure time.
CAT activity
In Figure 3, the CAT activity was shown to be elevated significantly in all treated groups (except in 30 mg kg-1 groups) in a concentration-dependent manner. The highest elevation was in 75 mg kg-1 groups after 14 days by 62.81 % (P<0.05). The correlation between nano-ZnO increasing level and elevated activity of CAT was r = 0.90 (P<0.05) following 14 day exposure to nano-ZnO.
Figure 3. The activity of catalase (CAT) in kidney of homing pigeons exposed to nano-ZnO after 7 and 14 days. a,b: data not sharing a common letter are significantly different (P<0.05) between treatments in the same exposure time.
Content of CP
The CP content was increased, in a concentration-dependent trend, with increases in nano-ZnO comparing to controls (Figure 4). The highest and significant CP value was obtained in 75 mg kg-1 nano-ZnO treated groups after 14 days (91.62 %, P<0.05). Here, a correlation was observed between increment in CP value and nano-ZnO concentrations was r = 0.89 (P<0.05).
Figure 4. Alterations in the carbolynated proteins (CP) content in kidney of homing pigeons exposed to nano-ZnO after 7 and 14 days. a,b: data not sharing a common letter are significantly different (P<0.05) between treatments in the same exposure time.
Value of TAC
The TAC values of kidney samples were found to be reduced upon increase in nano-ZnO concentrations after 7 and 14 days (Figure 5). The lowest TAC value was obtained in 75 mg kg-1 nano-ZnO treated group after 14 days by -40.02 % (P<0.05). A negative correlation between TAC value and increasing nano-ZnO concentrations was observed by r = -0.87 (P<0.05) following 14 day exposure.
Figure 5. Total antioxidant capacity (TAC) in kidney of homing pigeons exposed to nano-ZnO after 7 and 14 days. a,b: data not sharing a common letter are significantly different (P<0.05) between treatments in the same exposure time.
Blood plasma biochemistry
In Table 1, plasma urea and uric acid, and creatinine were increased non-significantly with the increase in the nano-ZnO concentrations after 7 and 14 days. The highest values for plasma urea, ureic acid, and creatinine were obtained following treatment with 75 mg kg-1 nano-ZnO after 14 days by 31.74 %, 12.63 %, and 15.83 %, respectively (P<0.05).
Table 1. Alterations in plasma urea, uric acid, and creatinine of homing pigeons exposed to nano-ZnO after 7 and 14 days. .
| Exposure time (days) | ||
nano-ZnO (mg/kg) |
Parameter |
7 |
14 |
0 | U | 0.61 ± 0.03 | 0.63 ± 0.04 |
UA | 281 ± 83 | 285 ± 79 | |
C | 21.32 ± 0.23 | 22.49 ± 0.29 | |
30 | U | 0.68 ± 0.02 | 0.72 ± 0.03 |
UA | 293 ± 81 | 295 ± 91 | |
C | 23.67 ± 0.26 | 24.98 ± 0.31 | |
50 | U | 0.70 ± 0.02 | 0.81 ± 0.05 |
UA | 301 ± 85 | 302 ± 87 | |
C | 24.332 ± 0.25 | 24.67 ± 0.21 | |
75 | U | 0.78 ± 0.04 | 0.83 ± 0.05 |
UA | 305 ± 81 | 321 ± 79 | |
C | 25.39 ± 0.34 | 26.05 0.38 | |
Units: U: urea (m mol/L), UA: uric acid (µ mol/L), C: creatinine (µ mol/L).
Histological examinations
Figure 6A shows normal architecture in parenchyma and well-arranged urinary tubules without any defects. Our results indicate that tissue damages in the pigeon kidney samples were only observed in pigeons treated with 75 mg kg-1 nano-ZnO after 14 day of exposure including aggregation of nano-ZnO in glomerulus and tubular necrosis lines (Figure 6B). These tissue changes, somewhere less, and somewhere more are observed in all specimens of homing compared to the control group where these changes were not present.
Figure 6. Histology of kidney of homing pigeons in control group (A, 10×) and treated with 75 mg kg-1 of nano-ZnO after 14 days (B 40×) (H&E). D: duct, N: nanoparticle aggregation in glomerulus, NL: tubular necrosis line.
DISCUSSION
The organisms are exposed to nano-ZnO through dermal, inhalational, and oral routes. Emerging reports have indicated various types of toxicity such as mitochondrial dysfunction and genotoxicity related to these NPs [1,17]. In contrary, antimicrobial, anti-inflammatory, and antioxidant properties of nano-ZnO have also been reported [17]. According to previous results, the accumulation of nano-ZnO is high in certain tissues including liver, kidneys, lung, brain, and spleen [5]. However, evidence regarding toxic effects of nano-ZnO on birds are still scanty and this study was performed to define the possible renal toxicity of nano-ZnO in pigeons (Columba livia f. urbana).
Content of LPO/MDA
It is known that generation of ROS and induction of OS has been defined to be the major contributing factor in nano-ZnO mediated toxicity [18]. In our study, elevated level of MDA reflects the induction of LPO process followed by oxidative damages in pigeon kidney samples. In agreement with our results, Heidai-Moghadam et al. (2019) revealed that OS was increased in the kidney by nano-ZnO through enhancing MDA content and reducing activities of SOD [19]. Exposure to nano-ZnO leads to ROS generation and altered antioxidant defense system in cells that interference with normal cellular processes [20]. MDA is a relatively reactive and toxic molecule which can rapidly interact with thiols and amino groups leading to formation of stable adduct proteins and cell inactivation, so if it is not removed, it may potentiate pathological processes [21]. In the previous work from our team, it was showed that nano-ZnO could affect the blood markers of pigeons, where oxidative damages may be the potential mechanism underlying this intoxication. We found that nano-ZnO has capable to augment the level of MDA in the blood plasma samples of homing pigeons in a dose-dependent manner following 14 day treatment with NPs [21].
|
CAT activity
According to what emerged from the results, increased CAT activity clearly indicates that nano-ZnO had potency to induce oxidative damages in the pigeon’s kidney. CAT is an antioxidant enzyme which decomposes H2O2 in the oxidative stress status, and protects the tissue from related oxidative damages [3]. CAT may detoxify nano-ZnO toxic effects in the treated pigeons. In Japanese quails, upon intoxication with nano-ZnO, the CAT activity was increased to eliminate the oxidative damages [22]. According to Mittler’s ROS theory, it can be assumed that at nano-ZnO exposure resulted in the production of excessive H2O2 and lipid radicals in pigeons and the CAT activity was increased to adapt to the oxidative status [23]. We, in the previous investigation, also demonstrated that nano-ZnO at similar doses caused an increase in CAT activity in the blood plasma samples of pigeons indicating propagation of oxidative damages in the blood compartments [21].
Content of CP
Here, we showed increased MDA and CP content in nano-ZnO treated kidneys in pigeons. The occurrence of CP has been considered as a hallmark of ROS-mediated protein oxidation in animal models exposed to certain pollutants. The MDA can react with the amino group of certain amino acids and form reactive carbonyl groups that cause protein oxidation leading to the cell inactivation [13]. Thus, any increase in MDA concentration may lead to protein damage likely occurs due to the elevated CP levels. Jenni-Eiermann et al. (2014) found lower concentrations of PC during the night in resting birds than in flying birds due to higher metabolic rate and ROS generation in the latter [24]. Meanwhile, an increase in LPO accompanied by enhancement of the CP content was detected in the muscle of pigeons living in areas with different levels of pollution [25].
Value of TAC
The TAC value refers to the antioxidative status of an organism, and includes the synergic and redox interactions between the different molecules present in the biological fluids [26]. To counteract the oxidative damages, the organism’s body is well-equipped with antioxidant defense mechanisms. Cellular non-enzymatic antioxidant defense potentially includes reduced glutathione (GSH), vitamins C and E, etc. Amongst, depletion of GSH by oxidants could be due to the direct conjugation of their metabolites with GSH leading to OS [3]. Our results indicate that TAC value in kidney samples of treated pigeons with nano-ZnO was decreased in a concentration-dependent manner which can be attributed to the nano-ZnO ability to lower the power of antioxidant defense systems. Hence, it can be concluded that in our study, induction of OS and excessive ROS production in pigeon’s body might be the reason for the antioxidant power depletion in the kidney samples under the stress of nano-ZnO toxicity. In agreement to this results, we earlier showed that antioxidative power of blood plasma samples of pigeons was depleted under the stress of nano-ZnO administration marked by reduced TAC value [21].
Blood plasma biochemistry
Blood biochemistry analysis is a common tool for the early diagnosis and correction of nutritional and metabolic disorders before the emergence of more serious symptoms. In our results, nano-ZnO administration caused an elevation in plasma urea, uric acid, and creatinine levels which proved to be non-significant increases. Plasma urea concentration has traditionally been considered as an inappropriate parameter to evaluate renal function in bird [27]. Plasma uric acid concentration has traditionally been considered as an inappropriate parameter to evaluate renal function in birds [27], and elevations are seen with severe renal diseases. Also, the elevated level of creatinine in blood is consequently a sign of impaired kidney function. Any reduction of glomerular filtration can lead to an increased plasma creatinine concentration [28]. Here, although their concentrations did increase in the response to nano-ZnO stress, but the concentrations did not exceed significantly border line for these variables. In other animals, administration of nano-ZnO caused a significant elevation in the blood urea, uric acid, and creatinine levels [19].
Histological examinations
The kidney is an organ highly vulnerable to damage caused by ROS, likely due to the abundance of long-chain polyunsaturated fatty acids in the composition of renal lipids [29]. Our results indicate different tissue damages including NP aggregation in glomerulus along with tubular necrosis lines in kidney samples of homing pigeons treated with higher concentrations of nano-ZnO 75 mg kg-1 for 14 days. An explanation for that would be induction of OS due to imbalance between abnormal generation of ROS production and antioxidant defense function in animal tissues. Free radicals can induce some protein modifications, i.e., unfolding or alteration of protein structure in cells [30], resulting in the tissue damages. In the most cases, ROS production leads to cytotoxicity and oxidative damages to macromolecules including lipid, protein, and DNA peroxidation. Besides, OS induced by many toxic compounds causes disruption of several major metabolic pathways within target organs such as kidneys [31]. In agreement with our results, AbdAlaziz and Albaker (2021) demonstrated that nano-ZnO treatments cause swelling, degeneration and necrosis of the renal epithelial cells, hemorrhage between the renal tubules and within the glomerulus, with infiltration of inflammatory cells upon examination of microscopic sections [32]. In regard to the role of nano-ZnO in nephrotoxicity in other animal models, Yan et al. (2012) reported that nano-ZnO could disturb the energy metabolism and cause mitochondria and cell membrane impairment in rat kidney leading to nephropathy [33]. Meanwhile, it was showed that nano-ZnO treatments cause infiltration of inflammatory cells around glomerular capillaries and degeneration of proximal and distal tubules in mice [34].
CONCLUSIONS
We demonstrated that the nano-ZnO has deleterious effects to pigeons at higher concentrations. Nano-ZnO affected kidney function and structure in homing pigeons through the induction of oxidative stress. However, further investigation regarding nano-ZnO stress in pigeons are required.
ACKNOWLEDGEMENTS
The authors kindly thank Shahrekord University, Shahrekord, Iran for providing the financial support to this study.
Ethical consideration
All experiments have been complied with the ARRIVE guidelines 2.0 and carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and also with Directive 2010/63/EU revising Directive 86/609/EEC on the protection of animals used for scientific purposes was adopted on 22 September 2010.
Conflict of interest
The authors declare that there is no conflict of interest.
Authors’contributions
MA designed and supervised study and drafted original manuscript. HRN performed experiments, analyzed data. All authors read and approved the final manuscript.
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