The antioxidant effects of coenzyme Q10 on albino rat testicular toxicity and apoptosis triggered by bisphenol A

Polycarbonate plastics for packaging and epoxy resins are both made with the industrial chemical bisphenol A (BPA). This investigation looked at the histological structure, antioxidant enzymes, and albino rats’ testis to determine how coenzyme Q10 (CoQ10) impacts BPA toxicity. For the experiments, three sets of 18 male adult rats were created: group 1 received no therapy, group 2 acquired BPA, and group 3 got the daily BPA treatment accompanied by coenzyme Q10, 1 h apart. The experimental period ran for 14 days. The biochemical biomarkers catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) were altered as a result of BPA exposure. The testicular histological architecture, which is made up of apoptosis, was also exaggerated. Furthermore, rats given BPA and CoQ10 treatment may experience a diminution in these negative BPA effects. These protective properties of CoQ10 may be correlated with the ability to eliminate oxidizing substances that can harm living species. The outcomes might support the hypothesis that CoQ10 prevented oxidative damage and boosted rats’ stress responses when BPA was introduced. Thus, by shielding mammals from oxidative stress, CoQ10 aids in the growth and development of the animals. BPA is extremely hazardous to humans and can persist in tissues. Human reproductive functions are a worry due to human exposure to BPA, especially for occupational workers who are typically exposed to higher doses of BPA. As a result, in order to reduce the health risks, BPA usage must be minimized across a diverse range of industries, and improper plastic container handling must be prohibited. By giving CoQ10 to patients, BPA’s harmful effects on reproductive structures and functions may be avoided.


Introduction
BPA is a short-lived anthropopic compound that has been widely utilized in plastic over the past few decades for a variety of purposes, including healthcare facilities, hygiene items, household products, and food containers. By interacting with hormone signaling factors, the prevalent endocrine-disrupting chemical (EDC) BPA has the power to change metabolic and reproductive processes. In fact, BPA has already been discovered in domestic dust, consumer items, water, soil, nutrients, and air, in addition to being found in biological matrices (Bilal et al. 2019;Chang et al. 2019;Mohammed et al. 2020).
BPA exposure has been linked to detrimental health effects in both humans and animals. Along with the endocrine and reproductive systems, it also has an impact on the immune and central nervous systems through a number of pathways, including oxidative stress (Gassman 2017). Research on lab animals is providing more and more proof that these non-persistent chemical influences reproductive function, even at very low exposure levels (Vandenberg et al. 2019). This is crucial since BPA exposure has the potential to be chronic, effectively equating it with a persistent substance.
BPA also affects two steroidogenic enzymes that reduce the activity of the aromatase enzyme. Aromatase is often located in the brain, Leydig cells, and adipose connective tissue as well as is crucial for the production of steroid hormones. Androgens can permanently change into estrogens owing to it. According to earlier studies, testicular mitochondrial enzyme levels were decreased in mice exposed to BPA (Anjum et al. 2011).
Multiple experimental models link oxidative stress and related indicators to BPA toxicity and humans (Gassman 2017). There is substantial evidence that the decline in antioxidant defense and/or reactive oxygen species (ROS) generation, which alters the oxidative balance throughout the cell, including in the mitochondria, has a significant impact on BPA organ damage (Gassman 2017;Grewal et al. 2021).
BPA in rat liver cells has been demonstrated to facilitate transformation of xanthine dehydrogenase into xanthine oxidase, which elevates the levels of ROS (Sakuma et al. 2010). BPA's damaging effects on human health may be mediated by Production of ROS and DNA oxidative damage (Gurmeet et al. 2014). Apoptosis, also known as programmed cell death, is a common physiological and pathological occurrence. It is engaged in the process of spermatogenesis, in which the removal of defective sperm cells to produce convenient sperm quality and quantity is known as apoptosis of germ cells. Numerous factors, including a deficiency of hormones, radiation exposure, heat, and toxicants, have an impact on the apoptosis of germ cells (Li et al. 2009).
The health and defense processes of organs and body tissues are crucially dependent on antioxidant compounds. The human body produces antioxidants in a range of ways to protect against oxidation and nitrosation damage. Frequently, extensive or abnormal apoptosis of germ cells results in oligospermia and azospermia (Kurutas 2016).
There is evidence that a potent antioxidant called CoQ10 can lessen the harm caused by several contaminants (Arany et al. 2017) and influences the fertility positively (Ben-Meir et al. 2015). It was discovered that CoQ10 therapy was beneficial in correcting oocyte quality and quantity decline with age as well as the oocyte mitochondrial disease linked to obesity reversed by the administration of CoQ10. But despite the fact that various research on in vitro fertilized women show a link between raised CoQ10 levels and enhanced fertility other individuals have been unable to locate an effective (Ben-Meir et al. 2015;Bentov et al. 2014;Gat et al. 2016;Xu et al. 2018).
This study looked at both topics: tracking the effects of BPA and investigating CoQ10's protective effects over adult albino rats testis. According to the research, adding such a natural antioxidant to one's diet may be a costefficient and limited strategy to lessen the effect that BPA exposure has on fertility.

Animals and treatments
The King Khalid University Ethics Committee gave the study, its prior approval before it could be carried out (Abha, Saudi Arabia). In the current investigation, 18 healthy, mature male albino rats (Rattus norvegicus) (weighing roughly 250-300 g, 50-60 days old) were employed. King Khalid University's Department of Laboratory Animals provided the rats (Abha, Saudi Arabia). The animals were kept in 12-h light/dark cycles with a temperature of 24 °C in polycarbonate rat cages, as is normal practice in laboratories. Ad libitum supplies of food and liquid were made available. The 14-day treatment regimen was administered (Takahashi &Oishi 2003). Three sets of six rats each were created randomly.
Group 1: acted as a control and was given corn oil at a dose of 2 ml/kg body weight (BW) every day. Group 2: was administered 100 mg/kg BW of BPA diluted in 2 ml/kg BW of corn oil once daily. Group 3: received BPA and CoQ10 doses of 100 mg/ kg BW dissolved in 1 ml/kg BW corn oil and 10 mg/kg BW diluted in 1 ml/kg BW corn oil every day, 1 h apart.
The chosen volume of corn oil in the current investigation was 2 ml/kg, which was how BPA was introduced orally by Wei et al. (2011).

Chemicals and preparation
Bisphenol A (BPA): (2,2 bis-4-hydroxyl phenyl propane) was acquired through Sigma Chemicals Co. (≥ 99% purity; Sigma 239,658, St Louis, MO, USA). In water, BPA was dissolved and given through oral use to the experimental animals at a concentration of 100 mg/kg bw/day. CoQ10 (Kaneka; ubiquinol ≥ 96%, ubiquinone ≥ 2%) (≥ 98% purity; Sigma C9538) supplied to the treated animals at a rate of 10 mg/kg body weight per day for 4 weeks after being blended with 1 ml/kg BW of corn oil. Corn Oil was used as a vehicle for BPA and CoQ10.

Evaluation of CAT and SOD activities and serum MDA levels
SOD experiments were conducted since using the xanthine: xanthine oxidase system as a superoxide generator prevented the reduction of nitro blue tetrazolium. Spectrophotometer tuned at 560 nm; the proportion of SOD activity in serum was determined and measured in units per mg of protein. Plasma CAT activity was measured and reported as units/g Hb by monitoring the oxidation of H 2 O 2 . The fundamental idea behind the Ohkawa et al. (1979) method, which was employed to gauge the rates of lipid peroxidation, is a spectrophotometric measurement of the color that MDA produces when thiobarbituric acid interacts with it. At 532 nm; the serum's absorbance was measured and represented as nanomoles/mg of protein (Tekin &Seven 2022).

Histopathological studies
After being given ether anesthesia, the rats were sacrificed. Testicular specimens were preserved in Blouin's solution after being dissected and inspected microscopic histologically. Following fixation, to prepare for 3-5-µm-thick sections, after embedding the testis tissue in paraffin, blocks were created. Hematoxylin and eosin was used to stain the sections after they had been deparaffinized and rehydrated for inspection under a light microscope (Abu-Abed &Brinkmann 2020).

Ultrastructural preparations
All animal groups' testis were sectioned into minor pieces, each measuring around 1 mm 3 , and then quickly fixed at 4 °C for 18-24 h in freshly prepared 3% glutaraldehyde. The specimens were washed in phosphate buffer (pH 7.4) before being post-fixed for an hour at 4 °C in isotonic 1% osmium tetroxide. Toluidine blue was used to stain semi-thin sections to pinpoint the area of interest before utilizing glass knives on an ultra-microtome to generate ultrathin sections. Following uranyl acetate and lead citrate staining, sections were looked at a JEM-1011 transmission electron microscope from Joel Ltd. In Japan, which was run at 80 kV in the faculty of medicine at KKU (Eid et al. 2021).

Statistical analysis
The Statistical Package for the Social Sciences (SPSS) software was used to tabulate and analyze the data using statistical package version 16. (SPSS, Inc., USA). The mean and standard deviation (X ± SD) of quantitative data were used to express them. Following a one-way analysis of variance, a post hoc Tukey test was used to assess the significance of differences between groups p < 0.05 which was considered statistically significant.

Biochemical results
The CoQ10 supplementation to BPA group considerably decreased the elevated MDA ( Fig. 1), this verified CoQ10's antioxidant properties. In addition, Figs. 2 and 3 demonstrate that the BPA group's CAT and SOD activity were significantly lower when compared to control. The BPA group's CAT and SOD activity considerably enhanced when CoQ10 was given to them.

Group I
Histological analysis of the testis from this group revealed seminiferous tubules that range in shape from spherical to oval and lined by multiple layers of germinal epithelium and disconnected by a minimal range of interstitial tissue holding Leydig cells. This germinal epithelium had pyramidal Sertoli cells and oval, darkly stained spermatogonia adjacent to the basement membrane. The largest spermatocytes found inside the seminiferous tubules were primary spermatocytes. Small cells called spermatids were arranged in 2-4 rows and near together to the lumen. Moreover, the lumen of the majority of the tubules contained eosinophilic threads that represented the sperm tails. Additionally, spindle-shaped myoid cells sitting on the basement membrane's outer surface were found ( Fig. 4A; Table 1).

Group II
Multiple vacuoles, considerable disruption of the basement membrane, degeneration of the lining epithelial cells, irregularly shaped basement membrane, and architectural loss were all visible in the seminiferous tubules of the BPA group ( Fig. 4B; Table 1).

Group III
Comparing the histological results of the BPA group to CoQ10 plus BPA treated group, these changes were considerably reversed ( Fig. 4C; Table 1).

Groups I
A seminiferous tubule encircled by basement membrane and lined with spermatogonia, primary spermatocytes with nuclei and mitochondria, and Sertoli cells with nuclei and mitochondria were all visible upon ultrathin sections of the testes from this group of rats. Spermatids were composed of large, rounded euchromatic nuclei, a pronounced Golgi apparatus, an acrosomal cap on one side of the nucleus, and mitochondria arranged peripherally. Centrioles were visible in several cross sections of sperm. Endothelial cells ringed the typical basement membrane in a cross-section of a blood capillary, myoid cells, and Leydig interstitial cells were observed (Fig. 5A, B, C, and D; Table 1).

Fig. 1
CoQ10 protects rats from BPA-induced oxidative stress biomarkers. At the end of the study, the values of MDA in serum levels were assessed in the three groups of rats included in this study: control, BPA, and CoQ10 + BPA. The findings are the mean (± SD) f; n = 6. Experiments were carried out in triplicate. *p < 0.05 in comparison to control group, ***p < 0.05 in contras Fig. 2 CoQ10 protects rats from BPA-induced oxidative stress biomarkers. At the end of the study, the values of CAT in serum levels were assessed in the three groups of rats included in this study: control, BPA, and CoQ10 + BPA. The findings are the mean (± SD) f; n = 6. Experiments were carried out in triplicate. *p < 0.05 in comparison to control group, ***p < 0.05 in contras

Group II
Cell-damaging effects of BPA were seen in these samples' ultrathin sections. Damaged primary spermatocytes with pleomorphic nuclei and mitochondria, disintegrated spermatogonia, injured Sertoli cells with mitochondria, and lipid droplets lined a seminiferous tubule. A myoid cell with a nucleus encircled the tubules in an atypical manner. Numerous vacuoles and lysosomes, a prominent Golgi apparatus, and disrupted mitochondria were all present in apoptotic spermatids. Cross sections of sperm revealed damaged cytoplasmic inclusions, including vacuoles and a substantial percentage of lipid droplets. In the injured interstitial region, the damaged blood capillary basement membranes had an irregular shape and were surrounded by pleomorphic endothelial cells (Fig. 6A, B, C, and D; Table 1).

Group III
The components of the rat testis of this group underwent a noticeable improvement. Germinal epithelium-covered seminiferous tubules demonstrated a return to normal architecture. Spermatogonia, primary spermatocytes, and Sertoli cells with mitochondria and nuclei are all observable intact. Tubules were encircled by basement membrane and myoid cells with typical nuclei. Spermatids had a large, rounded Fig. 3 CoQ10 protects rats from BPA-induced oxidative stress biomarkers. At the end of the study, the values of SOD in serum levels were assessed in the three groups of rats included in this study: control, BPA, and CoQ10 + BPA. The findings are the mean (± SD) f; n = 6. Experiments were carried out in triplicate. *p < 0.05 in comparison to control group, ***p < 0.05 in contras The interstitial spaces in-between the tubules contain interstitial cell of Leydig (L) and vascular capillaries (C). B Damaged seminiferous tubules with disorganized germinal epithelium, abnormal spermatogenic cells, and irregular outline basement membrane (Bm) as well as multiple vacuoles (V) are observed. C Seminiferous tubules with germinal epithelium showing recovery toward normal architecture in the bisphenol-A plus ubiquinone (CoQ10) treated group euchromatic nucleus, a prominent Golgi apparatus and the acrosomal cap on one side of the nucleus, and mitochondria that were arranged peripherally. Numerous sperm cross sections containing centrioles were visible. A cross section of a blood capillary revealed intact basement membrane surrounded by endothelial cells with nuclei in the interstitial area, which contained Leydig cells (Fig. 7A, B, C, and D; Table 1).

Discussion
Male infertility is a worldwide issue that affects about 4% to 12% of men; it accounts for about 20 to 70% of cases globally, with the highest incidence in Africa and Eastern Europe (Agarwal et al. 2015). There is mounting evidence that exposure to endocrine-disrupting chemicals like BPA is one of the factors that contribute to infertility and male reproductive problems (Cariati et al. 2019;Siracusa et al. 2018). In business, BPA is commonly used, especially in the production of polycarbonate plastics and food packaging, because it is strong and stable at high temperatures. PA is regarded as one of the substances used most frequently in industrially produced chemicals worldwide (Bosch et al. 2016). It contains a tiny (228 Da) particle, is white in color, is a solid particle at room temperature, and smells of phenol. It has also been demonstrated that BPA has antagonistic effects on androgenic receptors, which are regarded to be the main regulators of androgen cell signaling. In particular, spermatogenesis, one of the most important processes, depends on androgen receptors for the growth and function of the male reproductive system (Bin-Meferij &El-Kott 2015, Siracusa et al. 2018).
The BPA treated group's light microscopic findings revealed disorganization, disrupted seminiferous tubules, damaged spermatogonia containing vacuoles, Sertoli cells, and both primary and secondary spermatocytes. The interstitium contained the pleomorphic spermatids, aberrant interstitial cells, myoid cells, and deformed blood capillaries. One of the detrimental effects of BPA on male fertility that have been noted in earlier studies is damage to testicular histoarchitecture, defective spermatogenesis as well as decreased LH and testosterone (Chianese et al. 2018;Ma et al. 2018). Findings point to a significant relationship between the paternal urine BPA content and the high prevalence of infertility treatment failure in the couple (Mínguez-Alarcón et al. 2021). DNA damage has been linked to an increased risk of infertility in elderly (Das et al. 2013;Rosiak-Gill et al. 2019), and the reduction in sexual and reproductive hormones (Belloc et al. 2014, Eisenberg &Meldrum 2017. The level of the hormone testosterone may be lowered due to interstitial cells that manufacture testosterone is dispersed throughout the connective tissue in between the coiled somniferous tubules (Akingbemi et al. 2004). An ultrastructural study of Sertoli cells revealed larger intracellular vacuoles, electron-dense bodies, expanded mitochondria, and cell debris, which supported these modifications (Gurmeet et al. 2014).
Seminiferous tubules from male BPA treated rats were examined under an electron microscope, and some of the lining cells had severe cytoplasmic vacuolation (Gurmeet ). These could be clarified by an ionic and osmotic imbalance that inhibits water from entering cells and causes cellular vacuolization; this is recognized as a type of cell ageing. Additionally, the expansion of the smooth endoplasmic reticulum, which may be a sign of alterations in cellular permeability, is possibly what causes the vacuolization of germinal and sustentacular cells. Additionally, it appeared from the ultrastructural data that several germinal cells in each rat exposed to BPA had undergone apoptosis. BPA caused degenerative alterations to the basement membrane, which preserves the testicular tissues' structural and functional integrity (Liu et al. 2013). It is possible that BPA-induced lipid peroxidation and a drop in testosterone levels are what caused the ultrastructural abnormalities in the rat testis given BPA treatment. Where as in the seminiferous tubules, testosterone is necessary for attachment of germ cell generations. As a result, low levels of testicular testosterone may cause germ cells disconnection from the seminiferous epithelium, which would then trigger to their death, that lead to male infertility (Blanco-Rodríguez &Martínez-García 1998). Numerous lines of research examine the protective role of natural and synthetic antioxidants in preventing the harmful effects of xenobiotic chemicals on the male reproductive system. As an endocrine disruptor, the tested substance caused oxidative stress in the testicles, liver, and kidney (Alshehri et al. 2021;Bindhumol et al. 2003;Rezvanfar et al. 2013). It is recognized that excessive ROS created in mitochondria and microcosms can cause oxidative stress, which can disrupt proteins, lipids, and nucleic acids. Numerous diseases, such as cancer, infertility, and neurological conditions, might result from this , Kabuto et al. 2004, Shati &El-Kott 2021.
According to the biochemical results, rats exposed to BPA exhibited significant changes in their enzyme activity, including an increase in MDA and a decrease in CAT and SOD. Data showed that BPA increased oxidative stress, altered levels of SOD and CAT, and induced aberrant interactions with radical oxygen in a range of biological systems (Aitken & Roman 2008). Decreases in lipid peroxidation products like MDA can therefore cause antioxidant enzymes to become inhibited (Aitken &Roman 2008, Khalaf et al. 2022, Obata &Kubota 2000. Rong et al. (Rong et al. 2021) claimed that selenoprotein iodothyronine deiodinase upregulation results in high levels of ROS generation and altered antioxidant enzymes (CAT, SOD) performance. According to Tiwari &Vantage (Tiwari &Vanage 2017), the decline of numerous antioxidant enzymes, including SOD, CAT, and non-enzymatic reduced glutathione, is associated with a decline in sperm quality. Khalaf et al. (2019) showed that the dams of offspring testis had been subjected to BPA had higher levels of H 2 O 2 and lipid peroxidation, and antioxidant defense systemsdepletion. BPA affected the morphology of the seminiferous tubule epithelium during the beginning of puberty, impairing sperm production and gonadotropin discharges. It also increased the ROS formation and decreased CAT and SOD antioxidant activity (Ullah et al. 2019). Adult rats exposed to BPA had dose-dependently higher ROS levels, and the loss of essential proteins insulin receptor substrate-2 and glucose transporter-8 in the testicles-respectively necessary for spermatogenesis and testicular energy metabolism-proved that metabolic dysfunction and oxidative stress were linked to reproductive system dysfunction (D'Cruz et al. 2012). Adult rats' epididymis, testicles, and immunological cells (lymphocytes and bone marrow) all showed a considerable rise in lipid peroxidation as a result of BPA exposure.
Previous studies have shown that BPA has detrimental effects on the male genital system; 15 days of oral exposure resulted in a reduction in the number of spermatocytes, Leydig cells, serum testosterone, and the testis' overall antioxidant capacity (Yousaf et al. 2016). Wang et al. (2014) found that BPA administration decreased sperm production while increasing caspase-9 and -3 activities and TUNELpositive cells. Antioxidant capabilities are a feature of CoQ10 and its effects on lipid peroxidation, antioxidant defenses that had been compromised, and arsenic-induced rise in caspase-3 levels were all dramatically reduced (Fouad et al. 2011). Based on the findings of cited studies, BPA and CoQ10 dosages and treatment durations were chosen (Wang et al. 2014;Yousaf et al. 2016).
Comparing the groups who received BPA with CoQ10 to those that received BPA alone, the data obtained showed a noticeably improved testicular structure. The outcomes also demonstrated that CoQ10 treatment reduced MDA while increasing CAT and SOD activity. A considerable reduction in testicular MDA levels was seen after treatment with CoQ10 in comparison to the BPA group and an increase in testicular SOD. These results are in line with the preceding research (Saleh et al. 2017). According to Saleh et al. (2017), CoQ10 supplementation dramatically reduced ROS levels. CoQ10 is a powerful antioxidant. The by-products of lipid peroxidation during free radical reactions are scavenged by CoQ10 (Sohet et al. 2009) and prevents the production of ROS (Iqbal et al. 2022;Sohet et al. 2009;Tsuneki et al. 2007). An important cellular antioxidant is CoQ10 with the ability to reduce inflammation and scavenge free radicals Lamia et al. 2021). Following exercise training, CoQ10 can modify the nuclear factor's production erythroid 2-related factor 2 (Nrf2), supporting its function in inflammation and antioxidant defense (Pala et al. 2016).

Study limitation
Despite these findings, there are many limitations in this research. Our data is still observational, which is essential. Furthermore, based on these findings, it is currently unclear to pinpoint the upstream mechanism by which CoQ10 protects the testicular parenchyma. As a result, concentrating more on these systems utilizing animals or cells will be more beneficial. The CoQ10 dose employed was based on a previously tested testicular parenchyma protective dose in the toxicity of BPA. However, more research employing a dose-response curve is strongly advised. Identifying alternative pathways that regulate inflammation and oxidative stress could also be a promising area for future research. Lastly, this study focused solely on CoQ10's ability to protect against BPA induced testicular toxicity. However, examining this effect testicular structure and function, as well as all assessed markers at different time intervals, could be more informative. Moreover, more research into the therapeutic effects of different portions of the reproductive organ specimens on rats with pre-existing toxicity will add to our understanding of this impact and the mechanisms of action of this antioxidant.

Conclusion
The findings might support the hypothesis that CoQ10 mitigated BPA-induced stress response and exacerbated oxidative stress in rats through lowering oxidant impacts. Therefore, by eliminating free radicals and reactive oxygen species from the body, CoQ10 promotes development and healthy growth in mammals.