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Quercetin Abates Aluminum Trioxide Nanoparticles and Lead Acetate Induced Altered Sperm Quality, Testicular Oxidative Damage, and Sexual Hormones Disruption in Male Rats

This study examined the effects of exposure to lead acetate (PbAc) and/or aluminum trioxide nanoparticles (Al2O3NPs) on testicular function. Additionally, the probable reproprotective effects of quercetin (QTN) against Al2O3NPs and PbAc co-exposure in male Sprague Dawely rats were assessed. Al2O3NPs (100 mg/kg b.wt.), PbAc (50 mg/kg b.wt.), and QTN (20 mg/kg b.wt.) were orally administered for 60 days. Then, spermiogram, histopathological examinations of the testis and accessory glands, and immunohistochemical detection of androgen receptors (AR) and tumor necrotic factor alpha (TNF-α) were achieved. Moreover, serum levels of male sex hormones and testicular levels of antioxidant indices were estimated. The results showed that Al2O3NPs and/or PbAc caused significant sperm abnormalities, testicular oxidative stress, and histopathological changes. Furthermore, serum testosterone, LH, and FSH levels significantly decreased, while estradiol levels significantly increased. The Al2O3NPs and/or PbAc co-exposed group had more obvious disturbances. Furthermore, QTN co-administration significantly reversed the Al2O3NPs and PbAc-induced testicular histopathological alterations, reduced antioxidant defenses, and altered AR and TNF-α immune expression in testicular tissues. Conclusively, Al2O3NPs and/or PbAc evoked testicular dysfunction by inducing oxidative injury and inflammation. However, QTN oral dosing effectively mitigated the negative effects of Al2O3NPs and PbAc by suppressing oxidative stress and inflammation and improving the antioxidant defense system.


Introduction
In recent years, the usage of engineered nanomaterials for various biomedical and industrial purposes has significantly increased, raising multiple worries concerning harmful consequences on animal and human health [1]. In particular, the toxicological effects of inorganic nanoparticles (NPs) on reproductive organs have lately come to light [2]. Numerous studies have demonstrated that several NPs such as silver (Ag NPs), titanium dioxide (TiO 2 NPs), and aluminum trioxide (Al 2 O 3 NPs) can alter the integrity of the bloodtestis barriers (BTB) [3][4][5]. The BTB is a strong structure consisting of tight junctions, gap junctions, and adhesion junctions between adjacent Sertoli cells of the testes close to the base of the seminiferous tubule [6]. It functions as a suitable microenvironment for spermatogenesis and prevents cytotoxic substances from entering the seminiferous tubules, preventing foreign substances' effect on spermatogenic cells [7]. The underlying

PbAc, Al 2 O 3 NPs, and QTN Dose Selection
The PbAc dose was selected from several previous studies that reported that this dose causes male reproductive impairment [61][62][63]. However, the effect of the tested PbAc dose at co-exposure with Al 2 O 3 NPs has not yet been assessed. Additionally, in this study, the oral administration of Pb at a dose of 50 mg/kg b.wt. was used to reveal environmental mimicked Pb exposure. Oral exposure is the common direct route of environmental Pb exposure [64]. Furthermore, Pb exposure resulted in several pathological conditions in the populations exposed to it, even at a low level [65,66]. Thus, regardless of the higher amount of Pb exposure, the cumulative dose of Pb and the individual's susceptibility are strongly associated with health concerns [67,68].
Due to the limited amount of literature on the toxicity of Al 2 O 3 NPs, particularly reproductive toxicity [69], the selected dose of 100 mg/kg b.wt. Al 2 O 3 NPs have been reported to induce oxidative stress, inflammation, apoptosis, and DNA damage in various organs, including the liver, kidney, and brain [57,70,71]. However, the impact of Al 2 O 3 NPs on testis at the earlier dose has not yet been investigated.
The QTN dose (20 mg/kg b.wt.) has been reported to be efficient in mitigating male reproductive toxicity from exposure to various environmental pollutants and drugs [72][73][74].

Blood Sampling Collection
At the end of the trial (day 60), the rats were fasted for 12 to reduce variability in investigatory parameters, particularly body weight change. Then, rats were weighed and anesthetized with an intramuscular injection of a mixture of ketamine hydrochloride (50 mg/kg b.wt.) and xylazine (5 mg/kg b.wt.). Blood was drawn from each rat from the retro-orbital venous plexus by a well-sterilized glass capillary tube. The blood sample was collected into a centrifuge tube without K 2 EDTA for serum collection for hormonal analyses. All rats were decapitated, the testes were quickly dissected, connective tissues and fat were removed, and weighed.

Testicular Tissues Weights and Sampling
The absolute weight of the testicles was determined using a sensitive weighing balance (Radwag, Model AS220/C/2, Clarkson Laboratory and Supply Inc., Chula Vista, CA, USA). While the following formula determined the relative testicular weight: Relative testis weight = Testis weight/Body weight × 100. Each rat's left testis was used to measure AL and Pb content. Parts of the testes were rinsed with sterile saline and fixed in 10% buffered formalin for histopathological and immunohistochemical studies of AR and TNF-α. Right testicular samples (0.5 g) were homogenized in 5 mL phosphate buffer (pH 7.4) using an electrical homogenizer and then stored on ice. Supernatants from testicular homogenates were separated using a centrifuge set at 1200× g for 20 min at 4 • C, then frozen at −80 • C for later use in measuring antioxidant and oxidative stress biomarkers.

Semen Assessment
The cauda epididymidis from one testis was excised and cut with a sterilized scissor in a petri dish where the spermatozoa were dispersed in 2 mL prewarmed physiological saline solution at 37 • C to assess individual sperm motility [75]. A drop of the epididymal suspension was transferred into a clean glass slide pre-heated at 37 • C and covered with a pre-heated glass cover slide at 37 • C before being examined under a light microscope at high power (400×) magnification. Numerous microscopical fields were investigated to analysis about 200 sperms in these microscopic fields within 2-4 min after their extraction from the epididymis. A subjective scoring between 0 and 100 percent was used to assess the motile sperm cell percentage [76]. A hemocytometer was used to determine the total number of spermatozoa/mL [77]. Each rat's epididymal fluid was then mixed with the same volume of eosin-nigrosin stain. It spread onto slides that had been prepared to remove grease and debris to identify the morphological abnormalities of sperms under the microscope at a magnification of ×1000 [78]. The results were expressed as percentages.

Analysis of Al and Pb Residues
The liver samples were digested in the microwaves with 1 mL of 30% hydrogen peroxide and 8 mL of nitric acid. Then, the contents of Al and Pb were determined by an inductively coupled plasma-Optical Emission Spectrometer (ICP-OES, model 5100, Agilent, Santa Clara, CA, USA) with Synchronous Vertical Dual View (SVDV). Each measurement series' intensity was calibrated using a blank and at least three Merck Company standards (Darmstadt, Germany). External reference standards from Merck and standard reference material for trace elements in a quality control sample from the National Institute of Standards and Technology (NIST) were used to verify the instrument readings and ensure the accuracy and precision of the metal measurements.

Hormones Measurements
Serum samples were analyzed for hormone concentrations using enzyme-linked immunosorbent assay (ELISA) kits specific to rats, per the manufacturer's instructions. Cusabio Biotech Company provided rat testosterone and estradiol assay kits (Wuhan, China). Kamiya Biomedical Company provided rat follicle-stimulating hormone (FSH) and luteinizing hormone (LH) ELISA kits (Seattle, WA, USA) according to Zirkin and Chen [79]' method.

Oxidative Stress Biomarkers Analysis
Antioxidant enzyme activities and lipid peroxidation level (malondialdehyde (MDA) activity) were determined in testicular tissue homogenate. Commercial ELISA kits (Cusabio Biotech Co., Ltd., Wuhan, China) were used to measure glutathione peroxidase-like activity (GPx) because of the probable measurement of peroxiredoxin enzymatic activity and superoxide dismutase (SOD) [80,81]. The colorimetric assay of Ohkawa et al. [82] was used to determine MDA concentration.

Histopathological Evaluation
Tissue specimens from the right testis, prostate gland, and seminal vesicle were collected from all experimental groups and prepared according to Suvarna et al. [83]. They were fixed, dehydrated with ascending concentrations of ethyl alcohol, cleared in xylene, embedded, and blocked in paraffin. The sections were stained with Hematoxylin and Eosin (H&E) before being examined randomly with a light microscope.
2.11. Immunohistochemistry of AR and TNF-α Testicular tissue sections were dewaxed, rehydrated, and autoclaved in 10 Mm citrate buffer for 10 min at 120 • C, as described by Banchroft et al. [84]. Washing with PBS then 0.3% H 2 O 2 in methanol was applied for 15 m to block endogenous peroxidase. The slides were then blocked by adding a blocking buffer and incubated for 30 min at room temperature after being washed in PBS. According to the avidin-biotin-peroxidase complex protocol of Hsu et al. [85], the primary AR (rabbit monoclonal anti-androgen receptor antibody [ER179(2)]-ChIP Grade (ab108341) primary antibody, Abcam, USA) and TNF-α (Mouse anti-TNF-α antibody-(SC-52746) Santa Crus Biotechnology, Inc., Dallas, TX, USA) antibody were diluted by PBS and incubated/30 m, then washed with PBS three times/3 m each. The slides were treated with a biotinylated polyvalent secondary antibody and then coincubated for 30 min, followed by washing three times/3 m with buffer. Metal Enhanced (3,3'-Diaminobenzidine) DAB substrate (Abcam, Boston, MA, USA) working solution was applied to the tissue, incubated for 10 min, and then washed off twice/three minutes later with buffer to visualize the reaction.

Statistical Analysis
By SPSS version 14 (SPSS, Chicago, IL, USA), the data were displayed as mean ± standard error (SE). To evaluate the differences between the groups, we ran a one-way analysis of variance (ANOVA) and then used Tukey's Post-Hoc test for post hoc pairwise comparisons. When the p value is <0.05, the results show statistical significance. The computer program Graphpad (ISI Software, Philadelphia, PA, USA) was used. Table 1 showed no significant differences in the initial body weights among experimental groups. QTN administration alone revealed a significant (p < 0.05) increase (9.04%) in final body weight with a non-significant change in absolute and relative testicular weight relative to the control group.

Effects on Body WEIGHT Changes and Testicular Weight
The individual administration of Al 2 O 3 NP s or PbAc showed a significant (p < 0.05) decrease in final body weight by 7.58% and 19.28%, respectively, compared to the control group. Additionally, the PbAc-exposed group showed a significant decline in body weight gain by 75.24% relative to the control group. Moreover, the Al 2 O 3 NP s + PbAc group showed a significant (p < 0.05) reduction in final body weight and weight gain by 17.02% and 71.36%, respectively, compared to the control group. In contrast, the reduction in body weight and weight gain change was significantly (p < 0.05) minimized in Al 2 O 3 NP s + PbAc + QTN to be 7.98% and 18.44% lower than the control group ( Table 1).
The absolute and relative testicular weights were significantly (p < 0.05) reduced in the Al 2 O 3 NP s group (31.03% and 25.61%, respectively) and the PbAc group (40.89% and 26.83%, respectively). Of note, the highest reduction in absolute and relative testicular weights was recorded in Al 2 O 3 NP s + PbAc group to be 45.81% and 36.59%, respectively, lower than the control group. On the contrary, co-treatment with QTN in the Al 2 O 3 NP s + PbAc + QTN group improved the reduction in absolute and relative testicular weights to 5.91% and 2.44%, respectively, lower than the control group (Table 1).

Effects on Semen Quality
As demonstrated in Table 1, rats orally given QTN displayed a significant increase (p < 0.05) in sperm counts (9.33%) and sperm motility (6.38%) with a non-significant change in the percent of sperm abnormalities when compared to the control group. In comparison with the control groups, Al 2 O 3 NP s , PbAc, and Al 2 O 3 NP s + PbAc groups showed a significant reduction (p < 0.05) in the percentage of motile spermatozoa (43.61%, 62.41%, 81.20%, respectively) and sperm cell counts (17.77%, 27.56%, 46.27%, respectively) compared to control groups. The most reduction was observed in Al 2 O 3 NP s + PbAc co-exposed group when compared with individual exposure to Al 2 O 3 NPs or PbAc. A significant (p < 0.05) increase in abnormal sperm percentage was observed when Al 2 O 3 NP s (143.1%) or PbAc (160.08%) were administered alone and in combination (200.12%) compared to the control group. The combined Al 2 O 3 NP s + PbAc group revealed a greater elevation in abnormal sperm percentage when compared to values of both Al 2 O 3 NP s and PbAc groups (p < 0.05) ( Table 1 and Figure 1). On the other hand, co-administration of QTN with Al2O3NPs and PbAc significantly (p < 0.05) alleviated the decreases in spermatozoa motility (28.58%) and sperm cells concentrations (7.11%) and significantly dropped the abnormal spermatozoa percentage (95.08%) relative to control group.

Effects on Reproductive Hormones
Individual administration of QTN resulted in a significant (p < 0.05) decline in estra- On the other hand, co-administration of QTN with Al 2 O 3 NP s and PbAc significantly (p < 0.05) alleviated the decreases in spermatozoa motility (28.58%) and sperm cells concentrations (7.11%) and significantly dropped the abnormal spermatozoa percentage (95.08%) relative to control group.

Effects on Reproductive Hormones
Individual administration of QTN resulted in a significant (p < 0.05) decline in estradiol hormone (38.09%) and a significant increase in testosterone (68.75%), and FSH (33.25%), compared to the control group (Table 2). Instead, serum testosterone levels reduced significantly (p < 0.05) in Al 2 O 3 NP s + PbAc groups to be 93.75% lower than in the control group. Oppositely, there was a significant (p < 0.05) increase in serum testosterone concentration in the Al 2 O 3 NP s + PbAc + QTN group, compared to the Al 2 O 3 NP s + PbAc groups to the level non-significant from the control group. There was a decreased but non-significant serum FSH concentration in the rats individually exposed to Al 2 O 3 NP s (27.30%) or PbAc (27.30%). In comparison, the reduction in FSH level was significant (p < 0.05) (50.37%) in Al 2 O 3 NP s and PbAc co-exposed rats compared to the control ones. Nevertheless, the QTN co-administration in PbAc and Al 2 O 3 NP s co-exposed rats significantly (p < 0.05) minimized the reduction in FSH concentration to 19.85% lower than the control values ( Table 2).
Serum LH concentration significantly (p < 0.05) decreased in PbAc (46.8%) and Al 2 O 3 NP s + PbAc (52%) groups compared to the control group. Of note, the maximum significant (p < 0.05) reduction was observed in serum LH hormone level in the group administered Al 2 O 3 NP s + PbAc together. On the contrary, there was a significant (p < 0.05) increase in serum LH concentration in the Al 2 O 3 NP s + PbAc + QTN group compared to the Al 2 O 3 NP s + PbAc co-exposed group and restored to a non-significant level compared to the control value (Table 2). Table 2 displayed a significant (p < 0.05) increase in serum estradiol concentration in Al 2 O 3 NP s (24.37%), PbAc (31.49%), and Al 2 O 3 NP s + PbAc (51.04%) groups, compared to the control group. Oppositely, there was a significant (p < 0.05) decrease in serum estradiol concentration in the Al 2 O 3 NP s + PbAc + QTN group compared to the Al 2 O 3 NP s + PbAc group and very nearly to the control group.

Effects on Testicular Oxidative Stress Biomarkers
Regarding SOD, GPx-like activity, and MDA concentrations in testes, there were significant (p < 0.05) rises in SOD (65.77%) and GPx-like activity (42.28%) in the QTN group but a significant (p < 0.05) decrease (19.48 %) in testicular MDA level as compared to the control group. In comparison with the control groups, Al 2 O 3 NP s , PbAc, and Al 2 O 3 NP s + PbAc groups demonstrated a significant (p < 0.05) reduction in SOD by 29.70%, 61.19%, and 65.08%, respectively, compared to the control group. Additionally, testicular GPx-like activity level significantly (p < 0.05) decreased in the Al 2 O 3 NP s (19.29%), PbAc (20.47%), and Al 2 O 3 NP s + PbAc (44.68%) groups compared to the control group (Table 2). However, both SOD (5.16%) and GPx-like activity (5.52%) concentrations in testicular homogenates significantly (p < 0.05) in the Al 2 O 3 NP s + PbAc + QTN group relative to the control group.

Changes in Testicular Content of Al and Pb
Al 2 O 3 NP s exposure in Al 2 O 3 NP s , PbAc, or Al 2 O 3 NP s + PbAc group showed significant (p < 0.05) elevations in testicular Al level in comparison to control groups with the maximum elevation in Al 2 O 3 NP s + PbAc co-exposed group ( Table 2). On the contrary, co-administration of QTN for Al 2 O 3 NP s and PbAc co-intoxicated male rats resulted in a significant (p < 0.05) reduction in testicular Al residues compared with Al 2 O 3 NP s and PbAc co-exposed rats. Concerning testicular Pb residues, rats exposed to PbAc and/or Al 2 O 3 NP s for 60 consecutive days showed a significant (p < 0.05) accumulation in Pb relative to the control groups (Table 2). Moreover, Pb residue significantly increased in Al 2 O 3 NP s + PbAc co-exposed rats compared to the group individually administered PbAc. Oppositely, the co-administration of QTN for PbAc and Al 2 O 3 NP s co-intoxicated male rats revealed a significant (p < 0.05) decrease in testicular Pb residue compared with rats co-administered PbAc and Al 2 O 3 NP s .

Testis
Testis of the control group revealed normal structure. The active mature, functioning seminiferous tubules are lined with complete spermatogenic series and Sertoli cells. Spermatogonia, primary spermatocytes, spermatids, and mature spermatozoa were regularly arranged on the basement membrane. Interstitial spaces were filled with Leydig cells ( Figure 2A1,A2). Testis of corn oil and QTN-treated groups were similar to the control group's structure ( Figure 2B,C).
In addition, different pathological changes appeared in tissue sections from Al 2 O 3 NP s and/or PbAc groups. There was spermatogenesis distortion with loss of germinal layers in most tubules. Prominent tubular atrophy, irregular outlines, detachment, and basement membrane thickening were seen. Necrosis with darkly stained pyknotic nuclei, vacuolations, and mild Necrosis of Sertoli cells were noted. Giant spermatids were observed in some tubules. Interstitial edema was noted. Interstitial and sub-capsular blood vessels showed significant congestion, thickening in the wall with vaculations, and vasculitis. Capsule exhibited thickening, edema, and inflammatory cell infiltrations ( Figure 2D1-D3,E1-E3). These alterations were increased in severity in Al 2 O 3 NP s with the PbAc-treated group ( Figure 2F1-F3). On the other side, Al 2 O 3 NP s + PbAc + QTN-treated group showed apparently normal structure with active spermatogenesis compared with the control group. Few tubules showed mild epithelial vacuolations and edema in some sections ( Figure 2G).

Testis
Testis of the control group revealed normal structure. The active mature, functioning seminiferous tubules are lined with complete spermatogenic series and Sertoli cells. Spermatogonia, primary spermatocytes, spermatids, and mature spermatozoa were regularly arranged on the basement membrane. Interstitial spaces were filled with Leydig cells ( Figure  2A1,A2). Testis of corn oil and QTN-treated groups were similar to the control group's structure ( Figure 2B,C).

Prostate Glands
The histological picture of the prostate gland of the control group showed normal aggregations of tubuloalveolar acini with secretion inside their lumen and enclosed within an outer capsule. The acini lined with simple columnar or cuboidal epithelium and embedded in the fibromuscular stroma contained blood vessels ( Figure 3A). Prostate glands of corn oil and QTN groups were similar to the control group, as illustrated in Figure 3B,C.   The prostate gland of Al 2 O 3 NP s , PbAc, and co-exposure groups exhibited destruction of some acini and decreasing acinar size compared to the control group. Others had vacuolated flattened epithelium with epithelial hyperplasia and marked papillary projections. There were little vacuolated secretions in the lumen. Interstitial tissues showed congested blood vessels, edema, and monocellular inflammatory cells. The fibro-muscular layer was thicker than the control group ( Figure 3D,E1,E2,F1-F3). QTN could improve the observed changes caused by Al 2 O 3 NP s and PbAc accompanied by secretion inside the lumen. There were mild papillary projections, edema, and congestion in a few tissue sections ( Figure 3G).

Seminal Vesicles
As illustrated in Figure 4A-C, seminal vesicles of control, corn oil, and QTN-treated groups showed that the normal branched convoluted mucosal folds surrounded by smooth muscle and the lumen filled with secretion. Pseudostratified columnar epithelium with highly vesicular nuclei and foamy cytoplasm lined the folds. A well-developed fibromuscular capsule surrounded the gland from which trabeculae passed through them. Mild blood vessels congestion was observed in a few sections of corn oil and QTN-treated groups.
Antioxidants 2022, 11, x FOR PEER REVIEW 13 of 26 observed changes caused by Al2O3NPs and PbAc accompanied by secretion inside the lumen. There were mild papillary projections, edema, and congestion in a few tissue sections ( Figure 3G).

Seminal Vesicles
As illustrated in Figure 4A-C, seminal vesicles of control, corn oil, and QTN-treated groups showed that the normal branched convoluted mucosal folds surrounded by smooth muscle and the lumen filled with secretion. Pseudostratified columnar epithelium with highly vesicular nuclei and foamy cytoplasm lined the folds. A well-developed fibromuscular capsule surrounded the gland from which trabeculae passed through them. Mild blood vessels congestion was observed in a few sections of corn oil and QTN-treated groups.

Immunohistochemistry Analysis
As shown in Figure 5, the testis of control (A1,A2), corn oil (B1,B2), and QTN (C1,C2) rats showed normal AR expression. It stained interstitial cells with brown coloration and a lack of immune reaction in seminiferous tubules. On the contrary, it was observed that there was a noticeable reduction in the AR expression in the testis of Al 2 O 3 NP s and/or PbActreated groups ( Figure 5D1,D2,E1,E2,F1,F2). In contrast, the AR expression in Al 2 O 3 NP s + PbAc + QTN-treated groups was improved to the extent like that in the testis of the control group ( Figure 5G1,G2).

Immunohistochemistry Analysis
As shown in Figure 5, the testis of control (A1,A2), corn oil (B1,B2), and QTN (C1,C2) rats showed normal AR expression. It stained interstitial cells with brown coloration and a lack of immune reaction in seminiferous tubules. On the contrary, it was observed that there was a noticeable reduction in the AR expression in the testis of Al2O3NPs and/or PbAc-treated groups (Fig. 5D1,D2,E1,E2,F1 and F2). In contrast, the AR expression in Al2O3NPs +PbAc+ QTN-treated groups was improved to the extent like that in the testis of the control group ( Figure 5G1,G2).  (E1,E2) Testis of PbAc-exposed group showing weak androgen receptor expression. (F1,F2) Testis of Al 2 O 3 NPs and PbAc co-exposed group showing weak androgen receptor expression. (G1,G2) Testis of Al 2 O 3 NPs and PbAc co-exposed and QTN-treated group showing strong androgen receptor expression in the interstitial cells (Androgen receptor immunostain, ×100 for (A1-G1) and ×400 (A2-G2)). Yellow arrows denoted AR-positive immune stained cells.

Discussion
Body weight changes are considered one of the sensitive indicators of the adverse effects of toxic chemicals [86]. In the current experiment, PbAc-exposed groups (Pb and PbAc + Al2O3NPs) showed a significant reduction in their body weight compared to the control group. The body weight reduction associated with PbAc exposure could result from Pb-induced reduced food intake, increased catabolic state, and disturbed nutrient metabolism caused by changing zinc-dependent enzymes [87]. On the other hand, the reduction in the body weight gain was not significantly different in the Al2O3NPs-exposed

Discussion
Body weight changes are considered one of the sensitive indicators of the adverse effects of toxic chemicals [86]. In the current experiment, PbAc-exposed groups (Pb and PbAc + Al 2 O 3 NPs) showed a significant reduction in their body weight compared to the control group. The body weight reduction associated with PbAc exposure could result from Pb-induced reduced food intake, increased catabolic state, and disturbed nutrient metabolism caused by changing zinc-dependent enzymes [87]. On the other hand, the reduction in the body weight gain was not significantly different in the Al 2 O 3 NP s -exposed group compared to the control group. Comparably, other NPs have not evoked significant changes in body weight gains, despite other negative effects, even at large doses up to 1 g/kg b.wt. [88,89]. Of note, an obvious restoration of the body weight was recorded in the PbAc + Al 2 O 3 NPs + QTN-treated group. Comparably, QTN reduced the weight loss induced by other NPs such as iron oxide NPs [90]. In this regard, QTN supplementation has been reported to increase the skeletal muscles' insulin-stimulated glucose uptake and antioxidant capacity, positively reflecting on health and body weight [91]. QTN's antioxidant capacity has been related to its chemical structure, particularly the presence and position of the catechol group in the B ring and the hydroxyl (-OH) group, which control the redox mechanism and boost free radical scavenging ability [92]. High oxidative stress has been shown to impair mitochondrial production in skeletal muscle [93] and to influence the rate of cellular adenosine triphosphate synthesis [94].
The current study revealed a significant reduction in the testis weight and gonadosomatic index in Al 2 O 3 NP s , PbAc, and Al 2 O 3 NP s + PbAc exposed groups. The substantial decrease in the weight of the testis after Al 2 O 3 NP s and/or PbAc exposure was accompanied by alterations in their histology, such as tubular atrophy, detachment, and Sertoli cell necrosis. Comparable reductions in the testis weight and gonadosomatic index have previously been reported following PbAc [95] and Al 2 O 3 NP s [16] exposure in rats. Various reasons might be responsible for the earlier reductions, including spermatogenesis inhibition and reduction in germ, spermatid cells, and testosterone levels [16,96]. On the contrary, the QTN co-treatment significantly restored the testis weight and gonadosomatic index in the Al 2 O 3 NP s + PbAc + QTN group. Consistent with the earlier findings, QTN restored the reduction in testis weight resulting from exposure to other NPs such as TiO 2 NPs [97]. As revealed by the histopathological findings, the QTN-associated reinstatement of the testicular architecture could be responsible for the restoration of the testis weight.
The quality of sperm is the most important predictor of male fertility [98]. In the current study, the exposure to Al 2 O 3 NP s and/or PbAc decreased sperm count and sperm motility while it increased the abnormal sperm count. Comparable impaired sperm parameters were observed in rats exposed to Al 2 O 3 NP s [5] or PbAc [38,99]. The reduction in sperm motility was most likely caused by the influence of Al 2 O 3 NP s on mitochondrial function. In this regard, in an in vitro study using C18-4 cells, Braydich-Stolle et al. [100] reported that Al 2 O 3 NP s could cross the membrane and connect to mitochondria and sperm acrosomes. On the other hand, Leydig cells, which secrete testosterone, are considered the primary target for Pb, which could explain the depleted population of spermatogonia, spermatocytes, and spermatids [101]. Additionally, Pb has been reported to adversely affect the male accessory glands [102], which is apparent in the histopathological findings, with their important role in sperm physiology [103]. Of note, compared to individual exposures, the toxic effects of Al 2 O 3 NP s and PbAc resulted in a more dramatic reduction in sperm count, viability, and increased sperm abnormalities suggesting their synergistic interaction. On the contrary, QTN oral dosing significantly repaired the sperm-impaired parameters resulted from Al 2 O 3 NP s and PbAc co-exposure. Previous studies proposed that the positive effects of QTN on sperm characters could be partly related to its antioxidant activity [104,105]. Additionally, Taepongsorat et al. [106] suggested that QTN might indirectly affect sperm quality via the stimulation of the sex organs (testis and accessory sex organs) at the cellular and organ levels. In line with the earlier suggestion, the histopathological findings revealed the significant restoration of the testis, seminal vesicles, and prostate architecture in Al 2 O 3 NP s + PbAc + QTN-treated rats.
Male sex hormones are key in male fertility as they affect sperm production and health [107]. Herein, the single exposure Al 2 O 3 NP s and PbAc significantly increased the estradiol level and decreased testosterone, FSH, and LH but without significant difference compared to the control group. However, the co-exposure to Al 2 O 3 NP s and PbAc significantly increased serum estradiol while it significantly decreased serum testosterone, FSH, and LH compared to the control group. Comparable disturbance in the male sex hormone balance was earlier reported with exposure to Al 2 O 3 NP s [16] or PbAc [108]. Given this, Lan and Yang [8] reported that NPs exposure could cause inflammation and affect Leydig cells, lowering testosterone serum levels and compromising the BTB. Despite the scarce information on the effect of Al 2 O 3 NPs on the pituitary gland and hypothalamus, other NPs such as Ag NPs [109] and TiO 2 NPs [110] have been reported to alter HPG. Moreover, Pb toxicity in rats has been reported to cause a deformity in the pituitary gland and hypothalamus, thus affecting LH secretion [111,112]. On the contrary, QTN oral dosing in Al 2 O 3 NP s and PbAc co-exposed rats significantly corrected the male hormones balance. These findings could be related to the earlier reported ability of QTN to correct the defects in HPG axis imbalance [113]. Moreover, Sharma et al. [114] reported that the QTN-induced increase in antioxidant defense of Sertoli and Leydig cells is highly associated with increased testosterone, FSH, and LH levels. The same authors proposed that QTN may also protect the pituitary gland and subsequently enhance the level of FSH and LH.
Testicular oxidative stress has been known as a chief feature in male infertility [115]. Herein, Al 2 O 3 NP s or PbAc exposure provoked an obvious testicular oxidative stress in terms of reduced antioxidants (SOD and GPx-like activity) and increased lipid peroxidation (MDA) levels. Moreover, co-exposure to Al 2 O 3 NP s and PbAc resulted in a more dramatic decline in SOD and GPx-like activity and an increase in MDA, indicating a synergistic toxic effect of concurrent exposure rather than exposure alone. Similarly, Yousef et al. [17] verified the depletion of testicular antioxidants, including GPx-like activity, glutathione S-transferases (GST), catalase (CAT), SOD, and reduced glutathione (GSH) in rats that orally received 70 mg Al 2 O 3 NP s /kg b.wt. for 75 days. Moreover, Algefare et al. [103] demonstrated that PbAc decreased antioxidant enzyme activity and increased MDA levels in the testes of rats intraperitoneally injected 20 mg PbAc/kg b.wt. for 4 weeks. In this regard, Pb, like most divalent metals, is bound in tissues by ionic or coordination bonds and commonly bound to small peptides, cysteine, methionine, selenomethionine, and enzymes affecting their activity [101]. Also, Pb has been reported to induce free radical damage by two distinct pathways, the release of hydrogen peroxides, and singlet oxygen, reflected in increased MDA levels as the lipid peroxidation end product, and the direct exhaustion of antioxidant reserves [116]. Several studies also verified that Al 2 O 3 NPs are involved in the ROS generation, thereby leading to a decrease in the antioxidant enzyme activities [117,118]. On the contrary, QTN treatment significantly re-established the depleted antioxidant enzymes and repressed the incremented MDA resulted from Al 2 O 3 NP s and PbAc co-exposure. Several earlier studies confirmed the potent antioxidant effect of QTN [105,119]. In this respect, Tvrdá et al. [120] verified that QTN has a strong scavenging capacity for ROS in favor of the catechol moiety and free hydroxyl groups in its structure, guards sperm from ROS, and retains the male germ cells function. Moreover, QTN has been found to directly eliminate ROS and hydroxyl radicals and renovate endogenous redox homeostasis through increasing glutathione levels and eliminating free radical enzyme systems [121].
Regarding Al accumulation in testicular tissues, the current findings revealed that the control, corn oil, and QTN groups initially had minimal amounts of Al in their testicular tissue. In line with these findings, in the previous study by HM O et al. [122], minimal amounts of Al have been found in various organs, including the control group's liver, kidney, and spleen. The wide distribution of Al in the environment because of its diverse uses, especially in Al containers, utensils, and water purifiers, could be responsible for its presence in the groups that have not received it as oral dosing [123]. Of note, the PbAc group had significant concentrations of testicular Al compared to the control group. The PbAc exposure had been known to cause renal impairment [124], which might affect the excretion of Al and lead to its accumulation in the tissue. Earlier reports confirmed that impaired renal function considerably increases Al accumulation risk [125][126][127]. In the Al 2 O 3 NP s and Al 2 O 3 NP s + PbAc-exposed rats, significant concentrations of Al were found in their testicular tissues, with the maximum amount in the Al 2 O 3 NP s + PbAc co-exposed ones. In this regard, Al 2 O 3 NP s have the potential to cross intestinal barriers and enter the bloodstream. This transport procedure involves binding blood ligands and delivering them via receptor-mediated phagocytosis, endocytosis, and pinocytosis [128]. Moreover, Al 2 O 3 NPs have been reported to penetrate the BTB and injure the testicular biological membranes [5,17,18]. Additionally, Yousef et al. [11] have reported that the accumulation of Al 2 O 3 NP s in the hippocampus of rats affected mitochondrial membrane function and lipoprotein integrity. Interestingly, a relation between dysfunctions of the brain and testis has been evident in recent studies [129,130]. Of note, the marked accumulation of Al in testicular tissues of the Al 2 O 3 NP s + PbAc-co-exposed group could be related partly to the Pb-induced-impaired excretion of Al [125][126][127]. Additionally, NPs have been known for their large surface area that allows heavy metals to adsorb [131]. The heavy metals can enter the organism as a free ion and/or NPs-heavy metals complex, and NPs can act as carriers for their transport within the organism [132]. The earlier fact could elucidate the presence of minimal Pb in Al 2 O 3 NP s -exposed rats and significant Pb concentrations in Al 2 O 3 NP s + PbAc co-exposed rats. Moreover, some earlier reports confirmed that Pb can across BTB [133][134][135].
On the contrary, Pb and AL residues significantly decreased in the testicular tissue of Al 2 O 3 NP s + PbAc + QTN-treated rats compared to those co-exposed to Al 2 O 3 NP s + PbAc. It is proposed that QTN can reduce the Al and Pb concentrations in the testicular tissue by forming complexes. In this regard, it has been reported that the catechol function site in the QTN structure has the highest complexation power toward Pb [136]. Furthermore, QTN has been proposed as a potential metal chelator. The hydroxyl and carbonyl groups in the C ring of QTN are reported to be the chief metal complexing domains interacting with metal [137]. Moreover, QTN has been reported to efficiently protect the BTB integrity [138], which may partly explain its role in reducing Pb and Al accumulation in the testis.
The testis is a sex gland that produces sperm and androgens as part of the HPG axis [139]. Seminal vesicles and prostate glands are important male accessory sex glands that play an important role in sperm motility, capacitation, and survival [140,141]. The present study revealed that the testis, seminal vesicle, and prostate gland of Al 2 O 3 NPs and/or PbAc-exposed rat showed various pathological perturbations. Consistent with these findings, Al 2 O 3 NP and PbAc induced comparable testicular lesions in the studies of Hamdi [5] and Abdrabou et al. [142], respectively. Additionally, PbAc adversely impacted the histological architecture of seminal vesicles and prostate in the earlier studies of Aldaddou et al. [143] and Dhurvey et al. [102]. Moreover, other NPs such as TiO 2 NPs [144] and zinc oxide NPs [122] caused hyperplasia, congestion, and desquamation of the prostate's epithelial and lining cells, as well as congestion in the seminal vesicle. The earlier effects of Al 2 O 3 NP and PbAc could be highly linked to their oxidative stress-inducing effects. In contrast, the testis, seminal vesicle, and prostate gland histoarchitecture were significantly restored in Al 2 O 3 NP and PbAc co-exposed rats with QTN oral dosing. QTN antioxidant efficacy could be responsible for reestablishment of testis and male accessory gland function and architecture. In addition, QTN therapy decreased the levels of the oxidative stress metabolite F2-isoprostane and prostaglandin E2 in expressed prostatic secretions (EPS) while increasing the levels of prostatic β-endorphins [145][146][147]. Moreover, in a recent study, QTN protected against chronic histologic prostatitis in rats [148].
The present study revealed that the individual and combined administration of Al 2 O 3 NP s and PbAc reduced the immune expression of AR compared to the control group. The reduced testosterone content in the testis of Al 2 O 3 NPs and PbAc-exposed groups could be responsible for their reduced AR immunoexpression because of their positive correlation. Comparably, in silica NP-exposed mice, serum testosterone levels were considerably reduced parallel with decreased mRNA expression of AR and genes regulating testosterone synthesis, resulting in spermatogenesis malfunction [36]. Furthermore, Huang et al. [149] demonstrated that PbAc induced a dose-dependent reduction in AR expression in the spermatogenic cells, Sertoli cells, and Leydig cells. Given this, Pb is a known endocrine disruptor with estrogenic properties, and it may be responsible for antagonizing AR expression in male Wistar rats [42,150,151]. The decrease in testicular AR expression observed in PbAc-exposed rats may also be explained by a reduction in serum testosterone levels [42]. Nevertheless, QTN mitigated AR immune expression in the Al 2 O 3 NP s + PbAc + QTN-treated group, which may be linked to its antioxidant properties. Additionally, treating primary cultures of Leydig cells by QTN resulted in a dose-dependent upregulation of AR [152]. This might be because QTN promotes steroidogenesis in Leydig cells [153]. Moreover, as testosterone is known to regulate AR expression through 5α-reductase, the QTN-induced increase in AR immunoexpression possibly related to the aromatase-inhibiting property of QTN preventing the conversion of androgen into estrogen [154]. Overall, the considerable enhancement of semen quality, hormonal balance, and testicular oxidative status in the QTN-treated groups could be linked to its chelating activity, powerful antioxidant properties, and efficacy in balancing the HPG axis.
Pro-inflammatory cytokines like TNF-α cytokines in the testis may have certain physiological functions. However, when these cytokines are higher than normal, they harm sperm [155]. In the current experiment, the TNF-α level immune expression was significantly increased in the testicular tissue sections of Al 2 O 3 NP s , PbAc, and Al 2 O 3 NP s + PbAc-exposed rats compared to the control group. Comparably, El-Khadragy et al. [156] reported an elevation in the proinflammatory cytokine TNF-α in the testis of rats after intraperitoneal Pb exposure (20 mg/kg b.wt.) for 7 days. Additionally, the pro-inflammatory effects of NPs have been recently documented [157,158] and correlated with the binding of proteins to NP surfaces. For instance, the adsorption of TNF-α on carbon black, TiO 2 NPs, and aluminum oxide hydroxide (AlOOH) NP has been reported [159]. Additionally, exposure of rats to Al 2 O 3 NP s daily for 75 days resulted in a significant elevation of testicular levels of TNF-α [16]. As, the inflammatory reactions within the testis are strongly connected with oxidative stress [160], the Al 2 O 3 NP s and PbAc-induced oxidative stress could be responsible for the increase in TNF-α immunoexpression. On the contrary, QTN coadministration suppressed TNF-α immune expression in the Al 2 O 3 NP s + PbAc + QTNtreated group, which reflects its anti-inflammatory effect [161]. In this regard, the study of Nair et al. [162] showed that a possible mechanism of QTN-mediated suppression of TNF-α expression is mediated in downregulating NF-κβ1 gene expression.

Conclusions
The results showed negative effects of Al 2 O 3 NPs and PbAc alone or in combination on the testicular function and architecture. Various pathways, including the induction of oxidative stress, lipid peroxidation, disruption of the HPG axis and sex hormone imbalance, spermiogram issues, and histopathological changes, have been implicated in Al 2 O 3 NPs and PbAc reprotoxic effect. Enhancing TNF-α and suppressing AR immune expression in testicular tissue was another possible mechanism. Of note, the combination of Al 2 O 3 NPs and PbAc had greater reproductive toxicity than either one alone. However, further studies are warranted on Al 2 O 3 NPs and PbAc co-exposure outcomes at lower concentrations for longer durations. The current investigation also verified the potential therapeutic benefits of QTN against Al 2 O 3 NPs and PbAc-induced altered reproductive parameters in experimental rats, which were thought to be caused by its anti-oxidative anti-inflammatory properties, and TNF-α and AR modulation. Funding: This research was funded by Cairo University in a project entitled "Assessment of the risk hazards of co-exposure to nanomaterials and environmental contaminants with mitigation strategies using natural products" (Cairo university projects-12-2021).