Reproductive Toxicity of Aluminum Oxide Nanoparticles and Zinc Oxide Nanoparticles in Male Rats

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INTRODUCTION
Among various nanoparticles (NPs), aluminum oxide nanoparticles (Al 2 O 3 NPs) are one of the most important because of its promising technological applications.It is currently one of the two US market leaders for nanosized materials [1].
Based on these extensive commercial applications, they have drawn considerable attention as potential environmental pollutants [2].The genotoxicity observed with Al 2 O 3 NPs may be due to pro-inflammatory effects through a reactive oxygen species (ROS)-mediated mechanism [3].Prabhakar et al. [4] demonstrated significant increase in myocardial malondialdehyde with significant reduction in myocardial SOD and CAT enzymes activities and GSH concentration in nano-alumina intoxicated rats suggesting enhanced oxidative DOI: https://doi.org/10.35702/nano.10003stress state in myocardium.Some articles reported micronuclei increase [5], DNA damage [5] and chromosomal aberrations [6] caused by AlCl 3 NPs.Also, zinc oxide nanoparticles (ZnONPs) are among the metal oxide nanoparticles and have received considerable attention.This is due to their low production cost, ability to form diverse structures and various biological applications including drug delivery, bio-imaging probes, cancer treatment, antibacterial and immunomodulatory agent [7].Previous studies have shown the cytotoxic and pro-inflammatory effects of ZnONPs in different types of cells [8].In vitro and in vivo studies have shown that ZnO nanoparticles caused membrane damage, inflammation, DNA damage, apoptosis and complex cell-cell and cell-matrix interactions and changes in some hormones in mammalian cells [9].Adverse effects of ZnONPs can be mainly attributed to the generation of ROS leading to membrane damage, directly or indirectly [10].There is scarce information about the possible reproductive toxicity of the aluminum oxide nanoparticles (Al 2 O 3 NPs) and zinc oxide nanoparticles (ZnONPs) alone or in combination despite of their current use in novel technology.Therefore, the potential risks on reproductive health should be investigated, especially in those who are occupationally exposed to Al 2 O 3 NPs and ZnONPs.To achieve this aim, we determined semen characteristics, DNA fragmentation, mitochondrial transcription factor A gene, uncoupling protein 2 gene, p53, cytokines, oxidative stress and sex hormones and testes histopathology in male rats.

Tested compounds and doses
Al 2 O 3 NPs nanopowder (about 50 nm particle size) and ZnONPs nanopowder (about 100 nm particle size), were purchased from Sigma Aldrich Chemical Company (St. Louis, MO, USA).The dose of aluminum oxide nanoparticles was 70 mg/kg BW (aqueous suspension) and was chosen according to [11].The dose of ZnONPs was 100 mg/kg BW (aqueous suspension) was chosen [12].

Animals and experimental groups
Forty Wistar male albino rats 4-5 months age and weighing 160-170g were used in the present study.Animals were housed in a stainless steel wire cages and given food and water ad libitum.Animals were maintained in a controlled atmosphere, a temperature of 25 ± 5 °C and 50-70% humidity.
After two weeks of acclimation, animals were divided into four equal groups as follows: a control group and 3 treated groups; group 2, 3 and 4 which were orally treated with Al 2 O 3 NPs (70 mg/kg BW), ZnONPs (100 mg/kg) and Al 2 O 3 NPs plus ZnONPs, respectively.Rats were orally administered their respective doses daily for 75 consecutive days.

Blood samples collection and tissue preparations
At the end of the 75 th day of the experimental period, all animals of each group were anaesthetized with diethyl ether and sacrificed.Blood samples were collected by cardiac puncture from anaesthetized rats in test tubes containing heparin as an anticoagulant.Blood samples were centrifuged at 860 ×g for 20 min for the separation of plasma.Plasma was kept at -80 °C until analyses of the tested parameters.
Testes were immediately removed, washed using chilled saline solution (0.9%), and removed the adhering fat and connective tissues.testes were minced and homogenized (10%, w/v), separately, in ice-cold sucrose buffer (0.25 M) in a Potter-Elvehjem type homogenizer.The homogenates were centrifuged at 10,000 ×g for 20 min at 4 °C, to pellet the cell debris and the supernatant was stored at -80 °C for the determination of tested parameters.

Body and organs weights
Initial and final body weights of male rats were recorded and subsequently body weight gain (g/75 days) was calculated.
The reproductive sex organs (testes, prostates and epididymis) were immediately removed, washed using chilled saline solution (0.9%), removed the adhering fat and connective tissues, dried on tissue papers and weighed.

Quantitative analysis of testicular gene expression of mitochondrial transcription factor A (mtTFA) and uncoupling protein 2 (UCP2) using quantitative real time reverse transcriptase-polymerase chain reaction (qRT-PCR)
Total RNA was isolated from testicular tissues using GF-1 Total RNA Extraction Kit (Vivantis, Malaysia) ViP rime PLUS One step Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) Green Master Mix (Vivantis, Malaysia) was used for the relative quantitative determination of the gene expression of mtTFA [13] and UCP2 [14] at mRNA level using GAPDH as an internal reference gene for validation of the extraction procedure and calculation of relative expression.DOI: https://doi.org/10.35702/nano.10003

Assay of DNA fragmentation as a marker of cell death
Total DNA in the tissues was isolated using DNeasy kit (Qiagen, Germany) and the concentration and purity DNA were assessed using Nanodrop2000@ (Thermo Fisher Scientific, USA).Then DNA fragmentation, as a marker of cell death, was assayed [15].

ELISA measurements
Tumor suppressor gene p53, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were assayed using Enzymelinked Immunosorbent Assay (ELISA) kitsin testes tissue homogenates according to the manufacturer instructions using the serial standard of each parameter.

Markers of oxidative stress
The process of lipid peroxidation resulted in the end product of malondialdehyde (MDA).The MDA was determined as thiobarbituric acid-reactive substances (TBARS) assay in which MDA was heated with thiobarbituric acid (TBA) at a low pH to produce a pink chromogen with a maximum absorbance at 532 nm.The level of TBARS was calculated from a standard curve constructed using serial concentration of tetramethoxypropane (TMP) [16].The Griess reaction was used to determine the concentration of nitrite and nitrate as nitric oxide end products (NOx) in the deproteinized samples.
The Griess reaction was supplemented with the reduction of nitrate to nitrite by NADPH-dependent nitrate reductase.
The assay procedure consisted of two steps: the first required the diazotization of sulphanilic acid with nitrite ions followed by the second step of coupling this product with a diamine, resulting in a measurable pink metabolite 540 nm.The level of NOx was determined from the slope of the standard curve constructed using serial concentration of sodium nitrite.
The total antioxidant capacity (TAC) and the activities of superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and catalase (CAT) in the tissue homogenates were measured using colorimetric kits (Biodiagnostic, Egypt) according to the manufacturer instructions and using specific standard for each parameter.
Reduced glutathione content was assayed after protein precipitation using a metaphosphoric acid reagent.The assay was based on the oxidation of GSH by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to yield GSSG and 5-thio-2nitrobenzoic acid (TNB).The rate of TNB formation was assayed at 412 nm and was proportional to the GSH present in the sample.The rate of formation of TNB was monitored by recording the change in the absorbance.This was found to be 412 nm per minute (ΔA/min).The total glutathione content in the samples was determined from a GSH standard curve and the results were subsequently expressed as nmol/g tissue by dividing the concentration of glutathione in the sample by the weight (in grams) of tissue used to prepare the sample [17].

Semen characteristics
The left epididymis was excised and placed in a warmed petri dish contain 0.2 ml of calcium and magnesium free Hank's solution at 37°C.The tissue was minced with scalpels for approximately 1 min and placed in a 37°C incubator for 15 min prior to determining sperm motility.The suspension was stirred, one drop was placed on a warmed microscope slide, and a 22x22 mm coverslip was placed over the droplet.At least 10 microscopic fields were observed at 400 x magnification using a phase-contrast microscope, and the percentage of motile sperm was recorded [19,20].The coverslip was removed and the spermatozoa suspension was allowed to dry in air.The sample was stained with 1% eosin Y/5% nigrosin and examined at 400 x for morphological abnormalities.Three hundred spermatozoa from different fields were examined for each sample as described previously [19,20].
The right epididymis and specimens of right testis were frozen immediately after weighing.After thawing at room temperature, the whole epididymis and specimens of testis were homogenized in 0.5 ml of a solution of 0.9% NaCl containing 2% of Triton X-100.Ten strokes of a manual glass homogenizer were used for each sample.The testis and epididymis homogenates were diluted with 1.5 ml of the homogenization solution and spermatozoa were counted at 400 x magnification using a Neubauerhemocytometer.Three counts per sample were averaged [20].Assessment of sperm count and abnormal spermatozoa was performed using an eosin-nigrosine blue staining mixture as described by Blom [21].

Determination of reproductive and thyroid hormones
Testosterone was determined by using Enzyme-linked Immunosorbent Assay (ELISA) kit for the quantitative measurement of testosterone in plasma (DRG international co., U.S.A).Follicle stimulating hormone (FSH) was assayed was assayed by using ELISA kit in vitro quantitative detection of rat in plasma (Biovision CO., U.S.A).

Histological section preparation of testes
Testes was obtained from rats, and immediately fixed in 10% formalin, and then treated with a conventional grade of alcohol and xylol, embedded in paraffin and sectioned at 4-6 μm thickness.The sections were stained with Haematoxylin and Eosin (H&E) stains and photographed on the PC screen using a light microscope (Olympus BH-2; Japan) with a digital color camera attachment (Sanyo VVC-6975 P; Japan) and dial indicator for studying the histopathological changes [22].

Statistical analysis
Results are reported as means ± SE.Statistical analysis for all studied parameters was performed using the general linear model (GLM) produced by Statistical Analysis Systems Institute [23].Duncan's New Multiple Range Test was used to test the significance of the differences between means [24].Values of p<0.05 were considered statistically significant.

Initial and final body and sex organ weights
Data showed that there is no significant difference between control and all treated groups in the initial body weight.

Testicular gene expression of mitochondrial transcription factor-A (mtTFA) and uncoupling protein 2
Data showed suppression of the testicular gene expression of mitochondrial transcription factor-A (mtTFA) in testes of rats treated with aluminum oxide nanoparticles (Al 2 O 3 NPs), zinc oxide nanoparticles (ZnONPs) and their combination by about 50%, 45% and 59% of control value, respectively (Table 2).
The testicular gene expression of uncoupling protein 2 (UCP2) showed significant induction in the rats treated with Al 2 O 3 NPs, ZnONPs and their combination by about 193%, 130% and 317% of control value, respectively (Table 2).The treatment with the combination of Al 2 O 3 NPs plus ZnONPs showed the highest induction (more 3 folds than the control level).

DNA Fragmentation
The agarose gel electrophoresis of testicular genomic DNA showed very low or undetectable DNA laddering (DNA fragmentation) in the testes of the control rats (Fig. 1).
The DNA intact band appears to be condensed near the application point with no DNA smearing suggesting no DNA fragmentation.On the other hand, the ZnO and Al 2 O 3 nanoparticles-treatment alone or in combination resulted in massive DNA fragmentations which appear as DNA laddering (smearing).

Tissues levels of p53, TNF-α and IL-6
The rats treated with Al 2 O 3 NPs and ZnONPs alone or in combination showed significantly higher testes level of p53, TNF-α and IL-6 compared to control group.In addition, treatment with Al 2 O 3 NPs and ZnONPs in combination group showed significant increase in the level of p53 , TNF-α and IL-6 compared to Al 2 O 3 NPs and ZnONPs groups (Table 3).

Semen characteristics and sex hormones
Data showed that Al 2 O 3 NPs, ZnONPs and their combination caused significant reduction in sperm motility and sperm count, and significant induction in abnormal sperm compared to the control group (Table 4).Data showed significant decrease in the levels of testosterone and thyroid-Stimulating hormone (TSH), and significant increase in follicle stimulating hormone (FSH), luteinizing hormone (LH), tri-Iodothyronine (T3) and Thyroxin (T4) levels in rats treated with Al 2 O 3 NPs, ZnONPs and their combination compared to the control group (Table 5).

Antioxidant enzymes and free radicals
Treatment with Al 2 O 3 NPs, ZnONPs and their combination caused significant decrease in the activities of plasma and testes GPX, GST, CAT, SOD, GSH and TAC, and significant increase in TBARS and NO compared to control group.The effect of the combination of Al 2 O 3 NPs plus ZnONPs was more toxic than each other (Tables 6-9).

Activities of steroidogenic enzymes
Data showed that treatment with Al 2 O 3 NPs, ZnONPs and their combination caused significant (P<0.05)increase in steroidogenic enzymes 17β-hydroxysteroid dehydrogenase (17β-HSD) activity and significant decrease in 17-ketosteroid reductase (17-KSR) comparison with the control group (Table 10).mtTFA is expressed up to the late spermatocyte and early spermatid stage [27].Therefore, it is important to determine mtTFA in the present study to know if the tested nanoparticles have effects on mitochondrial transcription factor A (mtTFA), as a transcription factor is a multi-functional protein plays an important role in the maintenance of mitochondrial DNA (mtDNA) integrity, replication and transcription [28].
The obtained data showed that Al 2 O 3 NPs, ZnONPs and their combination caused significant decline in mtTFA by 50%, 45% and 59%, respectively (Table 2) and hence affect the mitochondrial DNA (mtDNA) integrity, replication and transcription [28].In line with this assumption it was documented that, ZnO nanoparticles induce mitochondrial dysfunction, morphological modification, and apoptosis [29].
In the present study, the enhanced expression of UCP2 in testicular tissues of rats treated with nanoparticles may facilitate the proton leak that may act as a possible control mechanism for mitochondrial ROS production in the testis, with a mild uncoupling leading to a reduction in mitochondrial membrane potential, which in turn reduces ROS production.
However this defense mechanism may have a serious metabolic consequence as the protein leak decrease the metabolic efficiency of the mitochondria through uncoupling the oxidation from phosphorylation leading to decreased ATP production.The later effect may participate directly and/or indirectly in the apoptotic and necrotic cell death.In line with    the present study, it was documented that, ZnO nanoparticle had a strong deleterious effect of mitochondrial function [30].The mechanism by which nanoparticles induce the gene expression of UCP2 is unclear however we can postulate that it may a direct consequence of oxidative stress and increased free radicals and lipid peroxide which found in the present data (Tables 6-9).
Testicular genomic DNA showed very low or undetectable DNA laddering (DNA fragmentation) in the testes of the control rats (Figure 1).and interliukin-6 (IL-6) of male rats treated with aluminum oxide nanoparticles, zinc oxide nanoparticles and their combination.Park et al. [32] demonstrated that Al 2 O 3 NPs for caused significant increase in inflammatory cytokines IL-6.Also, Rodrigo et al. [33] reported that aluminum nanoparticles have been shown to initiate inflammatory events in macrophages, including secretion of pro-inflammatory cytokines.
The present study showed that exposure to nanoparticles caused deteriorations in semen characteristics and imbalance in the reproductive hormones (Testosterone, FSH, LH) and thyroid hormones (TSH, T3 and T4) and this may be due to the increase the levels of free radicals and nitric oxide in testes and the reduction in the antioxidants (Tables 6-9).Dadong et al. [34] demonstrated that ZnO nanoparticles could decrease mitochondrial membrane potential, increase the production of oxygen free radicals (ROS) and lead to the over-expression of genes that activate caspase 12 in RGC-5 cells.Because the increase of oxygen free radicals lead to the production of oxidative stress in endoplasmic reticulum membrane and induce apoptosis and necrosis by expression of caspase 12 and finally cause cell death.ROS and membrane lipoperoxidation of Sertoli cells and the damage in Sertoli and leydig cells lead to imbalance in FSH, LH and test hormones.Naher et al. [35] showed an association with low antioxidant level in the semen and bad quality of spermatozoa of the infertile subjects.Also, decreased antioxidant level in the seminal fluid has a negative impact on semen quality.In addition, Aitken et al. [36] reported that infertile males who that produce high levels of ROS have a five-fold less chance of initiating a pregnancy than infertile males who produce low levels of ROS.The increase in TBARS can bring negative effects on motility, sperm-oocyte fusion, and induces midpiece abnormalities [37].
Shirvani et al. [38] reported that ZnO nanoparticles have impact on Sertoli cells, occurrence of vacuolation phenomenon, apoptosis and reduction in various cell lines including spermatogonia, round and elongated spermatids.
According to Bockelheide et al. [39], the presence of vacuoles

Table 10 :
Figure 5a & b which showed that testis of treated rats showed many severely distorted seminiferous tubules with disruption and irregularity of their basement membrane.Other tubules are illustrating focal losses of the germinal epithelium and the disturbed organization of the spermatogenic cells Notice the wide interstitial spaces in between some tubules while others are showing obliterated spaces containing few darkly stained Leydig cells.

Figure 1 : 7 DOI:
Figure 1: Stained agarose gel of testicular genomic DNA demonstrating apoptotic and necrotic cell deaths induced nanoparticles.lane 1 and 2: control, lane 3 and 4: ZnO nanoparticles treated rats, lane 5 and 6: Al 2 O 3 nanoparticles treated rats, lanes 7 and 8: ZnO plus Al 2 O 3 treated rats.DISCUSSIONFernandez et al.[25] found that determination of body weight supplies significant information on possible implications for the health of the organism and the toxicity of a compound.Also, the determination of absolute and relative weights of testes, epididymis, seminal vesicle and prostate can be utilized in the evaluation of risks from adverse effects on male reproductive apparatus.Body and organs weights are sensitive indicators of toxic chemicals and this is in agreement with the current study showed that exposure to Al 2 O 3 NPs, ZnONPs and their combination caused alterations in the body and sex organs weights.The effects of these nanoparticles may be referring to their toxicity on body and sex organs weights.The present study showed deterioration in sperm

Figure 2 A
Figure 2 A, B: Light micrographs of testis of control group which showed regular cross sections of seminiferous tubules (T) with clusters of interstitial cells of Leydig (L) in between.The tubules are lined by regular stratified layers of spermatogenic cells with different stages of spermatogenesis, including spermatogonia (Sg), spermatocytes (Sp) and spermatids (Sd).Spermatozoa (Sz) are seen filling the Lumina of the tubules.Sertoli cells (St) are seen resting on the basement membrane (B).

Figure 4 A
Figure 4 A, B: Light micrographs of testis of rats treated with ZnONPs which showed seminiferous tubules with irregular shapes and wide empty lumina (R).Decrease height of the lining epithelium and many spermatogenic cells with pyknotic nuclei are noticed (Y).The Leydig cells are almost absent with wide interstitial spaces (G).

Figure 5 A
Figure 5 A, B, C: Light micrographs of testis of the combination of Al2O3NPs with ZnO3NPs which showed many severely distorted seminiferous tubules (M) with disruption and irregularity of their basement membrane (W).Other tubules are illustrating focal losses of the germinal epithelium and the disturbed organization of the spermatogenic cells (P) Notice the wide interstitial spaces in between some tubules (G) while others are showing obliterated spaces containing few darkly stained Leydig cells (Z).

Figure 3 A 8 DOI:
Figure 3 A, B: Light micrographs of testis of rates treated with Al2O3NPs which showed distorted seminiferous tubules with loosely arranged detached spermatogenic cells (R), Most of the spermatogenic cells are with pyknotic nuclei (X) and falling into the seminiferous tubules lumina.Notice the wide interstitial spaces in between the tubules containing few darkly stained Leydig cells (G).
in the cytoplasm of Sertoli cells indicate direct harm of nanoparticles on these cells, such that one of the obvious tissue damage signs can be the presence of vacuoles in Sertoli cells cytoplasm and between cell lines of seminiferous tubules.Yoshida et al. [40] found degeneration in Leydig cell and damage in the seminiferous tubules due to exposure to nanoparticles.Increase in the activity of testicular 17β-HSD and decrease in the activity of 17-KSR in the present study may lead to decrease of testosterone which may explain the reduction of serum testosterone levels.One of the indicators of the chemical toxicity on reproductive system is the decreased level of testosterone [40].CONCLUSION In conclusion, the obtained results revealed adverse effects of Al2O3NPs and ZnONPs alone and or their combination on the testicular architecture and caused fertility problems through different pathways including; changing gene expression of the proteins involved in the mitochondrial biogenesis and function (mtTFA and UCP2), induction of oxidative stress, lipid peroxidation, nitric oxide production, apoptotic and necrotic cell death pathways in the testicular tissues.Also, induction of inflammatory pathway through enhancing production of Tumor necrosis factor α and interleukin-6, disturbing production of tumor suppressor p53, sex hormones imbalance, and histopathological changes.Also, the present study showed that the reproductive toxicity of the combination of Al2O3NPs plus ZnONPs was more pronounced than each one.

Table 1 :
The body and organs weights of male rats treated with aluminum oxide nanoparticles (Al 2 O 3 NPs), zinc oxide nanoparticles (ZnONPs) and their combination.

Table 2 :
The effect of aluminum oxide nanoparticles, zinc oxide nanoparticles and their combination on the testicular gene expression of mitochondrial transcription factor-A (mtTFA) and and uncoupling protein2(UCP2).

Experimental groups Control Al 2 O 3 NPs ZnONPs Al 2 O 3 NPs + ZnONPs
Data presented as Mean±SE.For each parameter, the groups with different superscript letters are significantly differ, p<0.05.(Numbers between parentheses represent the percentage change from control value).

Table 4 :
Semen characteristics of male rats treated with aluminum oxide nanoparticles, zinc oxide nanoparticles and their combination.

Table 5 :
Testosterone a Data presented as Mean±SE.For each parameter, the groups with different superscript letters are significantly differ, p<0.05.

Table 6 :
Plasma antioxidant enzymes of male rats treated with aluminum oxide nanoparticles (Al 2 O 3 NPs), zinc oxide nanoparticles (ZnONPs) and their combination.

Table 7 :
Plasma reduced glutathione and free radicals of male rats treated with aluminum oxide nanoparticles (Al 2 O 3 NPs), zinc oxide nanoparticles (ZnONPs) and their combination.

Table 8 :
Testes glutathione peroxidase, glutathione S-transferase, catalase, superoxide dismutase and total antioxidant capacity of male rats treated with aluminum oxide nanoparticles (Al 2 O 3 NPs), zinc oxide nanoparticles (ZnONPs) and their combination.

Table 9 :
Testes reduced glutathione and free radicals of male rats treated with aluminum oxide nanoparticles (Al 2 O 3 NPs), zinc oxide nanoparticles (ZnONPs) and their combination.