Introduction
Endocrine-related disorders can raise globally through exposure to synthetic chemicals that mimic hormones and disturb the female reproductive system [1]. These chemicals may be found as nano-products in all sectors of life, including medicine, pharmaceuticals, food additives cosmetics, paint self-cleaning glass, pollution treatment [2], and electric devices [3]. Furthermore, nanoparticles can reach the ovaries and disrupt their normal functions by causing follicle damage, irreversible ovarian dysgenesis, or premature ovarian failure [4]. As a result, the possible hazardous effect of nanoparticles on ovaries has received a lot of attention [5]; however, the reproductive toxicity of nanomaterials has received little study [6].
Magnetite nanoparticles, like those naturally occurring on Earth as magnetite (Fe3O4) and maghemite (γ-Fe2O3), have inspired the development of synthetic magnetite nanoparticles )MNPs( for diverse biomedical applications [7]. While their potential benefits are vast, it's crucial to investigate their safety due to the limited knowledge of their cellular and molecular-level toxicity [8].
Recently, the deleterious influence of iron oxide nanoparticles on the development and function of endocrine and reproductive systems depends on their interactions with biological components, cellular absorption, and physicochemical properties [9]. In this respect, iron is one of the most important elements in life, as it is involved in the structure of nearly all of the body's cells. However, iron can accumulate inside the cells and induce oxidative stress via the formation of reactive oxygen species (ROS) and protein aggregation [10]. These ROS can dysregulate folliculogenesis, oocyte maturation, steroid synthesis, and cause tissue damage through lipid peroxidation [11]. This causes mitochondrial malfunction, oxidative phosphorylation in the inner mitochondrial membrane, and apoptosis-related gene overexpression [12].
The usage of flavonoids as an antioxidant against oxidative damage is an important way to maintain reproductive health. Quercetin (QUE) is a flavonoid found in grains, vegetables, and fruits [13]. It has unique antioxidant properties through its ability to scavenge radicals, regulate cellular antioxidant enzymes, chelate transition metals, and inhibit lipid peroxidation [14]. Moreover, QUE has been proposed as a potential therapeutic agent for many pathologies, including viral infection [15], inflammation/allergy [16], hypertension, and atherosclerosis [17]. Furthermore, recent studies have reported that QUE has beneficial effects on ovarian cancer [18] and ovarian oxidative damage [19].
Nowadays, reports about the negative effects of iron oxide nanoparticles are contradictory, and additional research is still needed to assess their possible reproductive toxicity. Moreover, the possible protective efficacy of QUE on MNPs–induced ovarian toxicity is still unknown. Therefore, this study aimed to examine the potential protective effect of QUE against MNPs-induced ovarian toxicity and steroidogenesis disruption in albino rats, with a mechanistic focus on the role of the oxidative stress-mediated upregulation of apoptotic gene expressions.
Materials and Methods
Chemicals used
MNPs (≥ 95 %) were purchased from Nano Gate Company (Egypt). The nanosize of MNPs was confirmed by a high-resolution transmission electron microscope (HR-TEM, Tecnai G20, FEI). Before administration, MNPs were suspended in distilled water and sonicated for 20 minutes. QUE (≥ 95 %) and other analytical grade chemicals were purchased from Sigma Chemical Company (USA).
Animals
Forty female rats (Rattus norvegicus), three months old, weighing 180-200 g were collected from the National Organization for Drug Control & Research (Egypt). The rats were housed in stainless steel cages at a regulated temperature (25 ± 5 °C), with 12:12 hour light-dark cycles. They had unrestricted access to rat stander's feed as well as clean tap water. This protocol (MLS/D/07/21) has been approved by Al Aqsa University in line with the Guidelines on the use and care of laboratory animals.
Experimental design
In this study, rats were screened to estimate the estrous cycle via vaginal smear examination [20], and rats that displayed a normal estrous cycle were used. Rats were divided equally into four groups, where the control group received distilled water (1 ml/kg body weight) and the QUE group received 100 mg/kg body weight of QUE (1 ml/kg body weight) via gavage [21]. The MNPs group intraperitoneally received 50 mg/kg/day of Fe3O4 nanoparticles in 1 ml/kg body weight [22]. The MNPs+QUE group was intubated with the same dose of QUE 2 hours before MNPs intoxication for four weeks. A 28-day exposure spans approximately 6 estrous cycles, capturing various stages of follicle development, ovulation, and corpus luteum formation, allowing for a comprehensive evaluation of potential effects on different ovarian functions.
Animals were anesthetized with sodium pentobarbital (200 mg/kg) after four weeks and blood samples were collected in a serum separator tube. After clotting, it was centrifuged (1000 x g, 15 minutes) to separate the serum, which was aliquoted and stored at -20 °C for hormonal testing. The anterior wall of the abdomen was removed with a transverse incision. The left ovarian tissues were excised, washed, dried, and preserved in neutral-buffered formalin for histological examination. The right ovarian tissues were homogenized in saline (1:10, w/v), centrifuged at 10,000 x g for 15 minutes at 4 °C, and the clear supernatant was removed and used for biochemical parameter testing. The other ovarian tissues were stored at -80 °C for the reverse transcription-polymerase chain reaction (RT-PCR).
Histopathological study
The ovarian tissues were fixed and hydrated in a graded series of ethanol before being cleaned with xylene and embedded in paraffin blocks. Each tissue block was sectioned into 5 µ m thick sections, stained with hematoxylin and eosin [23]. The histomorphological analysis was done by a light microscope (20 sections/animal) and scored by imaging software (ImagePro Plus 5.1, Media Cybernetics, USA).
Assessment of oxidative stress parameters
The malondialdehyde (MDA) level was determined at 534 nm using the method of Satoh [24]. The levels of reduced glutathione (GSH) were determined using the method described by Ellman [25], whereas the enzymatic activity of superoxide dismutase (SOD) was estimated using the method described by Marklund and Marklund [26].
Steroid hormones analysis
The levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), estrogen (ER), and progesterone (PR) in serum were estimated according to the manufacturer's instructions of rat ELISA kits (MyBioSource, Inc., San Diego, USA).
Determination of Bax and caspase-3 by RT-PCR
The RNeasy Mini Kit (catalog no.74104, Qiagen, Germany) was used to extract total RNA. The Revert Aid Reverse Transcriptase (catalog no. K1622, Thermo Fisher) generated the appropriate cDNA. With a total reaction volume of 25 μl (12.5μl 2× SYBR Green PCR Master Mix, 1μl from each primer direction, 2μl cDNA, and 8.5μl of RNase-free water), RT-PCR was done using the Quantitect SYBR Green PCR kit (catalog no. 204141, Qiagen, Germany). The PCR cycles consisted of denaturation at 94 °C for 15 seconds, annealing for 30 seconds at the appropriate annealing temperature corresponding to each specific gene primer (Table 1, Metabion, Germany), and extension at 72 °C for 30 seconds for 40 cycles. To calculate relative expression, the threshold cycle (Stratagene MX3005P software, Agilent Technologies, GmbH) of the examined genes was compared to β-actin [27].
In silico docking analysis
The crystal structure of FSH (8I2G) LH (7FIH), ER (5KRI), and PR (3D90) hormones in complex with their receptors besides caspase-3 (6CKZ) in complex with its inhibitor was downloaded from the protein data bank (PDB) database. The Discovery Studio software (version v21.1.0.20298) was used for protein preparation by removing water, ligands, heteroatoms, duplicate, and unnecessary chains, then polar hydrogen and charges were added. The QUE conformer (CID 5280343) was downloaded from the PubChem database to optimize its geometry by Open Babel [28]. The in silico docking study was done using AutoDock Vina software [29], and obtained conformers were analyzed using Discovery Studio (version v21.1.0.20298). For method validation, all original or co-crystalized ligands were extracted from their corresponding receptors and then re-docked with the same binding pocket of their receptors [30].
Statistical analysis
The IBM SPSS software package (SPSS, version 25, IBM, Chicago, IL, USA) was used for statistical analysis. Data were initially submitted to the exploratory analysis of normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test). The data are presented as the mean ± the standard error of the mean (SEM). A one-way analysis of variance (ANOVA) followed by Turkey's honestly significant difference (HSD) test was used to determine the differences between groups at probability (p) ≤ 0.05.
Results
MNPs HR-TEM analysis
The size of MNPs ranged from 7.92 to 15.90 nm (Figure 1), which enclosed within a homogeneous nanosize range and confirmed the manufacturer's specifications (< 50 nm).
Follicular development
Ovaries from all experimental groups showed all stages of folliculogenesis (Table 2). The number of primordial, primary, secondary, and Graafian follicles did not differ significantly (p > 0.05) between the experimental groups. As compared with control a non-significant (p > 0.05) decrease in primordial, primary, secondary, and Graafian follicles was observed in MNPs (by 13.70, 12.90, 29.27, 21.053 %), MNPs + QUE (by 8.22, 6.45, 24.39, 15.79 %) and also in QUE group (by 4.11, 9.68, 21.95, 10.53 %, respectively).
Follicular development
Ovaries from all experimental groups showed all stages of folliculogenesis (Table 2). The number of primordial, primary, secondary, and Graafian follicles did not differ significantly (p > 0.05) between the experimental groups. As compared with control a non-significant (p > 0.05) decrease in primordial, primary, secondary, and Graafian follicles was observed in MNPs (by 13.70, 12.90, 29.27, 21.053 %), MNPs + QUE (by 8.22, 6.45, 24.39, 15.79 %) and also in QUE group (by 4.11, 9.68, 21.95, 10.53 %, respectively).
Histopathological observations
The ovary from the control (Figure 2A) and QUE (Figure 2B) groups showed normal stroma (S) and developing secondary follicle (SF) with normal histological appearance of granulosa cells, and theca cells. The atretic follicles (AF) with darkly stained nuclei and a secondary follicle (SF) with disordered follicular granulosa cells were noted in MNPs group (Figure 2C). Furthermore, the MNPs+QUE group (Figure 2D) showed a secondary follicle (SF) with good characteristic features surrounded by theca cells and still has some pyknotic nuclei and atretic follicles (AF) was noted.
Hormonal profile
In comparison with the control group; serum FSH (46.04 %), LH (38.8 %), E2 (49.4 %), and PR (41.3 %) levels were significantly reduced (p < 0.05) after the intoxication with MNPs. Remarkably, QUE has normalized the level of LH (p > 0.05) in the combined group, but the FSH (17.03 % p), E2 (33.93 %,), and PR (23.19 %) hormones levels (p < 0.05) did not normalize (Table 3).
Biochemical analysis
Compared with the control, the MDA level in ovarian tissues of the MNPs intoxicated group was significantly elevated (p < 0.05) by 69.54 %, while the levels of SOD (32.9 %) and GSH (29.95 %) were remarkably decreased (p < 0.05). Compared with the MNPs group, the levels of MDA (by 19.54 %, p > 0.05), SOD (by 3.68 % p > 0.05), and GSH (by 17.05 % p < 0.05) were markedly alleviated in the MNPs + QUE group. Moreover, the level of MDA and SOD activity reached the control value after QUE co-treatment, but GSH did not return to this value (Table 4).
Each value represented the means ± SEM. Tukey's honestly significant difference (p ≤0.05) test was applied after a oneway analysis of variance (ANOVA). The differences between the groups within the same row were represented by means with different letters.
Relative quantity of Bax and caspase-3
Bax and caspase-3 gene expression in the ovarian tissues of MNPs were significantly (p < 0.05) increased by 7.2- fold and 11.7-fold, respectively as compared with the control level (Figure 3). In the MNPs+QUE group, the expression of Bax (3.49-fold) and caspase-3 (8.23-fold) genes were significantly (p˂0.05) downregulated, however; QUE could not recover the gene expression of Bax and caspase-3 to the normal expression (Figure 3).
Docking analysis
Method validation
The root means square deviation (RMSD) between the docked and experimental co-crystalized ligands revealed that the first poses of the docked co-crystalized ligands nearly superimposes (RMSD < 1) with the experimental ones (Figure 4). Thus, the docking method was reasonably accurate and reproducible.
Docking simulation of QUE with caspase-3 binding pocket (6CKZ)
As shown in Figure (5), QUE docked inside a 6CKZ binding pocket where the interaction occurred at binding energy of -7.2 kcal/mol (Figure 5A) where five conventional hydrogen bonds formed with Gly122, His121, Ser120, Gln161, and Arg207 with bond length 2.37, 1.85, 2.32, 2.21, and 3.09Å, respectively (Figure 5B). Moreover, QUE formed a hydrophobic interaction with Arg207 residue inside the caspase-3 enzyme active site. Other observed interaction involving Pi-donor hydrogen bonds and Pi-Sulfur interaction with Cys163, Pi-donor hydrogen with His121, and Pi-Cation interactions with Arg207 besides van der Waals interaction with Ala162, Glu123, Ser205, and Trp206 (Figure 5B).
Docking simulation of QUE with FSH receptor (8I2G)
QUE docked inside the 8I2G binding pocket where the interaction occurred at a binding energy of -8.7 kcal/mol with three conventional hydrogen bonds formed with Pro519, Ala607, and Ile588 with bond lengths 2.24, 2.60, and 2.26 Å, respectively. Moreover, QUE formed good hydrophobic interactions through its carbon-hydrogen skeleton with Met520, Ala595, Ala592, Ala607, and Ile588 residues inside the receptor binding pocket. Furthermore, QUE becomes close to Phe353, Ile522, Ser596, Val531, L518, Leu610, Pro587, Phe591, and Ile602 across van der Waals interaction (Figure 6B) besides close vicinity of His615 (Figure 6A).
Docking simulation of QUE with LH receptor (7FIH)
The 7FIH-QUE complex formed at binding free energy of -8.4 kcal/mol (Figure 7. A) where QUE formed three hydrogen bonds inside the binding pocket of 7FIH protein receptor through its hydroxyl groups as hydrogen bond donor with oxygens of Glu451 (2.55 Å) and Val447 (2.98 Å), and through its oxygen as hydrogen bond acceptor with Ser450 (2.80 Å). Regarding the hydrophobic interactions, it was formed with Ala589, Met517 134, Pro516, and Val447 amino acid residues inside the receptor active site. Van der Waals interactions formed with Ala592, Leu532, Ile528, Asn535, Ile531, Thr446, Tyr612, Ile585, Leu608, and Ser604 (Figure 7B) besides close vicinity of Lys595 (Figure 7A).
Docking simulation of QUE with ER receptor (5KRI)
QUE docked inside the 5KRI binding pocket where the interaction occurred at a binding energy of -8.3 kcal/mol (Figure 8A) and two conventional hydrogen bonds formed with Arg394 and Glu353 with bond lengths 1.92 and 2.02Å, respectively. Moreover, QUE formed good hydrophobic interactions through its carbon-hydrogen skeleton with Ile424, Met421, Pro404, Leu391, Ala350, and Leu387 residues inside the receptor binding pocket. Moreover, QUE becomes close to Phe425, Leu428, Met388, Leu349, Leu384, Leu525, His524, and Gly521 across van der Waals interaction (Figure 8B).
Docking simulation of QUE with PR receptor (3D90)
The 3D90-QUE complex formed at binding free energy of -9.2 kcal/mol (Figure 9A) where three conventional hydrogen bonds formed with Met759, Gln725, and Leu718 with bond lengths 2.12, 2.24, and 2.94Å, respectively (Figure 9B). Moreover, QUE formed hydrophobic interaction with Phe778 and Leu718 residues inside the PR active site. Other observed interaction involving Pi-Sulfur interaction with Met759 besides close vicinity to Leu763, Val760, Leu797, Phe794, Tyr890, Leu715, Cys891, Phe905, Asn719, Gly722, Trp755, Leu721, and Arg766 (Figure 9B).
Discussion
Recently, there has been an increased concern about the toxicity of nanoparticles as they can pass the bloodstream to deposit on tissues and cause adverse events [31]. In this respect, MNPs may accumulate in the ovaries and induce structural and functional changes, however; their adverse mechanistic effects on reproductive performance are still unknown. Therefore, the current work evaluated the potential protective roles of QUE against the toxic effects of MNPs on rats' ovaries, with a mechanistic focus on hormone dysregulation, oxidative damage, and apoptosis.
The growing follicles are extremely sensitive to hazardous substances, these negative consequences could arise once MNPs cross the blood-follicle barrier to enter the ovary via the bloodstream [32]. Former investigations have found that metal-based nanoparticles produce deformities in the histology of laboratory animals' reproductive organs, causing a distraction in reproductive cell synthesis [33,34]. Moreover, nanoparticles can arrive at the ovaries via the circulatory system and aggregate in granulosa and theca cells, causing ovum dysplasia, as well as disrupting their normal function, especially their key role in hormone production [35]. Therefore, the serum FSH, LH, E2, and PR levels were lowered following MNPs intoxication. Our data suggested that MNPs may interfere with the function of antral follicles by causing genotoxicity [34] and/or changing the activity of enzymes that produce E2 and steroid hormones [36]. Similarly, steroidogenic enzymes in the corpora luteal may be disrupted, resulting in insufficient PR and E2 levels [37]. Moreover, the hypothalamus-pituitary–ovarian axis may be disturbed by a lack of ovarian-derived steroid hormones, resulting in LH and FSH suppression [38].
Recently, FSH and LH enhance ovarian activity via binding with G-protein-coupled receptors on the surface of target cells, granulosa cells, where the binding specificity is governed by critical interaction sites involving both common and hormone-specific subunits [39]. Those hormones undergo a coordinated conformational change upon binding, which changes bulging loops in their receptor [40]. Therefore, this specific binding can be interrupted by interacting with MNPs that may interfere with the normal folding and conformations of proteins, especially those nanoparticles that have highly toxic properties as they can move deep inside the cells and elicit a purge of ROS [41].
Our results have confirmed the oxidative injury induced by MNPs by increasing the level of MDA and declining the content of GSH besides SOD activity in the ovarian tissues. These findings suggested that the variability of the oxidantantioxidant system could be a key mechanism by which MNPs promote cellular damage. This could be attributable to the liberation of free iron from uncoated particles inside the tissue, which can produce ROS [42] to interact with biological components and increase the level of MDA as a subsequent result [43]. Moreover, MDA level is adversely associated with LH concentrations as an increase in oxidative stress can distribute LH levels, which promotes pathophysiology in female infertility, principally via fluctuations in the gonadotrophin hormones [34]. Furthermore, the declined GSH content may result from its free radical scavenging properties to protect ovarian cells from MNPs-induced oxidative stress [44]. As a result, the depletion of GSH concentration, which is maintained intracellularly through the dependent action of its related enzymes, leads to an antioxidant enzyme activity reserve [45]. Therefore, the decreased SOD activity in ovarian tissue could be due to an excess of oxy-radical resulting from MNPs poisoning [43], which is immediately transformed to H2O2 by SOD activity and subsequently eliminated as water by catalase enzyme [46].
Our findings proposed that MNPs could stimulate ROS production, which disrupts the redox status and initiates the mitochondrial route of apoptosis via upregulation of the Bax and caspase-3 gene expression in ovarian tissues [47]. This pathway is primarily regulated by the Bcl-2 family of proteins and activated via ROS production [48]. In this pathway, excessive ROS may distribute the mitochondrial outer membrane which initiates the interaction between an activated Bax with other Bcl-2 and causes apoptosis by releasing cytochrome-c from mitochondria to the cytoplasm [49]. The cytoplasmic cytochrome-c can activate caspase-9, which stimulates the effector caspase-3 [50] to initiate an apoptotic signaling cascade and produce DNA damage [51]. Our study suggested that MNPs might slightly interact with necessary transcription factor sequences on Bax and caspase-3 genes. This close adherence with the ability for ROS production at these sites may become a message for the activation of redox-sensitive transcription factors [52], that can positively regulate these apoptotic enhancer genes and increase their expression levels.
Remarkably, co-treatment with QUE revealed beneficial improvements in the levels of females' fertility hormones and ovarian histology. These improvements may result from the phytoestrogenic activity of QUE which regulates the binding of E2 with E2-α and β receptors, resulting in enhancements of ovarian folliculogenesis by increasing the number of healthful follicles and hormone production [53]. This hypothesis has been confirmed by Amevor et al [54], who reported that QUE could regulate the secretion and concentration of reproductive hormones and their receptors, particularly E2. It's also thought that QUE can control the specific regulatory enzymes that switch steroid hormone creation and metabolism [55], like FSH, LH, E2, and PR. Therefore, QUE can ameliorate the infertility action of MNPs by regulating reproductive system functions such as folliculogenesis, and oocyte maturation, and it may be useful in the treatment of reproductive disorders such as endometriosis, and hormonal disorders [56].
Our in silico study showed that QUE interacted with amino acid residues such as Met520 and Val53 that can activate FSH receptors. This interaction gives a medium activity to the receptor and may protect this residue from oxidation by ROS [57]. Correspondingly, QUE could interact with the main residues responsible for the activation of the LH receptor as Met517, Ala589, Ser604, and Lys595 [39] as well as the main residues responsible for the activation of the E2 receptor as Met421, Phe425, and Ala350 [58]. Also, PR can be activated by QUE by interacting with Met759 and close to Met909 via the allosteric agonist [59].
We also observed that QUE can reduce ovarian oxidative injury in the MNPs-intoxicated rats by decreasing MDA levels and increasing GSH content as well as the enzymatic activity of SOD. These improvements may be related to the scavenging activity of QUE and metal-chelating abilities that hinder ROS-caused oxidative destruction and hormonal disturbances [53]. This scavenging characteristic has resulted from the catechol and hydroxyl groups [22] that may give QUE the ability to decrease ROS production and hinder lipid peroxidation by reducing the level of MDA and enhancing the biosynthesis of GSH. Furthermore, QUE could lower oxidative stress via transcriptional activation of nuclear factor erythroid 2–related factor 2 (Nrf2), which control many antioxidant genes, including catalase, SOD, glutathione peroxidase 2, heme-oxygenase-1 (HO-1), and thioredoxin [60].
Subsequently, the regulation of cellular antioxidant hemostasis by QUE could prevent oxidative injuries and histopathological alterations that hinder the release of cytochrome c from mitochondria by decreasing the expression of apoptosis-related genes (Bax and caspase-3). Another possible anti-apoptotic efficacy of QUE is increasing the expression of the anti-apoptotic protein (Bcl-2), which downregulates the expression of apoptosis-related genes (Bax and caspase-3) and prevents the apoptosis cascade [54]. Moreover, the activation of sirtuins by QUE may prevent Bax-dependent apoptosis and eliminate many pro-apoptotic transcription factors [61]. Remarkably, our docking study showed that QUE could interact as an inhibitor at a high affinity with the binding pocket of caspase-3, especially with His121 and Cys163, which are the main residues of the enzyme catalytic activity [62] as mentioned on the UniProt database (P42574).
Conclusions
Our data represent the amelioration efficiency of QUE in the MNPs-impaired female reproductive system, oxidative injury, and mitochondrial apoptotic pathway. These efforts may be elicited by improving the endogenous antioxidant status followed by reducing ovarian apoptosis to restore hormonal homeostasis. Based on our findings, women should avoid exposure to nanoparticles due to their toxic effects on the reproductive system. Moreover, further research is needed to confirm the possible signaling pathway that would explain the effects of QUE on the endocrine system that regulates folliculogenesis.
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