Cucumeropsis mannii seed oil protects against Bisphenol A‐induced testicular mitochondrial damages

Abstract There has been increasing search for the ameliorative properties of seed oils against toxicants. bisphenol A acts as an estrogenic endocrine‐disrupting chemical capable of causing male infertility. This study aimed to explore Cucumeropsis mannii seed oil effects against mitochondrial damage in rats using bisphenol A. Forty‐eight rats were randomly assigned to six groups (n = 6) of eight rats each and fed the same food and water for 6 weeks. The group A rats were given 1 mL olive oil, while the ones in group B were given bisphenol A at 100 mL/kg body weight via oral route. Group C received C. mannii seed oil 7.5 mL/kg body weight C. mannii seed oil, while group D, group E, and group F were pre‐administered bisphenol A at 100 mL/kg body weight, followed by treatment with C. mannii seed oil at 7.5, 5, and 2.5 mL/kg body weight, respectively. Antioxidant enzymes, glutathione, reactive oxygen species, testicular volume, malondialdehyde, body weight, and testicular studies were done using standard methods. The results of the bisphenol A‐administered group showed a significant decrease in the antioxidant enzymes, glutathione, body weight, and testicular volume with elevation in the levels of reactive oxygen species, malondialdehyde, and testicular indices. BPA + CMSO‐treated group showed a significant increase in GPx activity compared with BPA‐exposed rats. CMSO treatment significantly increased catalase activity in comparison with that of rats exposed to BPA. Remarkably, C. mannii seed oil and bisphenol A co‐administration significantly reversed the abnormalities observed in the dysregulated biochemical biomarkers. Our findings suggest that C. mannii seed oil has considerable antioxidant potential which can be explored in therapeutic development against systemic toxicity induced by exposure to bisphenol A. Cucumeropsis mannii seed oil protects against bisphenol A‐induced testicular mitochondria damages.


| INTRODUC TI ON
Infertility has long been a global health barrier to successful reproduction for couples seeking to have one or more children. Infertility affects millions of people of reproductive age all over the world. Data estimate that between 48 million couples and 186 million people suffer infertility worldwide (WHO, 2021). Approximately 90% of male infertility is caused by reduction in sperm count and/or sperm quality (Gurunath et al., 2011). Male infertility accounts for 20%-30% of all infertility cases worldwide (Gurunath et al., 2011;Mohapatra et al., 2010). In sub-Saharan Africa, male infertility is prevalent, ranging from 20% to 46%. Several factors contribute to sperm defects, including genetic abnormalities, environmental pollutants, smoking, hormone deficiency, and alcohol or cocaine use (Gurunath et al., 2011;Leslie et al., 2022). Recent data show that men are solely responsible for 20% of infertility cases worldwide and contribute to another 30%-40% of all cases of infertility (Leslie et al., 2022;WHO, 2021).
Bisphenol A (BPA) is among the most common environmental chemicals produced all around the world, accounting for approximately 6 billion pounds per year. Although it is used to produce epoxy resins and polycarbonate plastics, BPA is released from container walls by heat, thus penetrating the body system to exert genotoxic and cytotoxic effects (Awuchi & Awuchi, 2019;Dobrzyńska & Radzikowska, 2013;Geens et al., 2012). According to Sirasanagandla et al. (2022) and Mohapatra et al. (2010), BPA is an estrogenic endocrine-disrupting chemical capable of causing male infertility. Furthermore, a review by Ranjit et al. (2010) reported that BPA affects sperm production, lowers serum testosterone levels, and thins the seminiferous epithelium. Takahashi and Oishi (2003) also discovered that BPA affects the activities of testicular mitochondria. The interaction of BPA with androgen and estrogen receptors causes reproductive toxicity (Cimmino et al., 2020;Sirasanagandla et al., 2022). After high subcutaneous and oral dosing, BPA may also potentiate uterotrophic effects (Cimmino et al., 2020;Sirasanagandla et al., 2022). Because of its endocrine-disrupting properties, BPA can cause oxidative stress, resulting in the generation of reactive oxygen species such as superoxide radicals, hydroxyl radicals, and hydrogen peroxide (Awuchi & Awuchi, 2019;Gurunath et al., 2011).
Cucumeropsis mannii is indigenous to tropical Africa, especially in the West of the Great Rift Valley. It is cultivated for food and as a source of oil in this tropical region (Agu et al., 2022). In addition to being known as white-seed melon and Mann's cucumeropsis in English, it is also known as ahu-ilu in Igbo, agushi in Hausa, and elegushi in Yoruba (Koffi et al., 2008) in Nigeria. It has climbing vines that may reach a length of 4 m and are coated with stiffened hair-like structures. The roughly palmate or heart-shaped leaves may reach lengths of 12 cm and widths of 14 cm (Mbuli et al., 1983). It has tiny yellow petals that are around a centimeter long on both the male and female flowers. About 19 cm long and 8 cm broad, the fruit is egg-shaped or elongated oval in form. The fruit has a creamy texture and traces of green. The fruits and seeds can both be consumed and also employed for various industrial processes and products (Okwundu et al., 2021). In west African countries, various parts of the C. mannii plants have been utilized (Agu et al., 2022;Okwundu et al., 2021). Ghanaians use the juice from the flesh fruit concocted with various condiments and apply to navel of newly born infants to fast-track the curing progression till cord vestiges drip off; equally, in Gabon, steeped leaves have been in use as a purgative to constipated suckling babies (Agu et al., 2022). The Sierra Leonian cattle boys conventionally make use of the desiccated C. manni fruit shell as a horn warner (Awuchi, 2023;Leakey et al., 2022). The kernel of the seed contains semi-drying oils for making soap, cooking, lighting, livestock feed, and the fruit for preparing healing ointment (Koffi et al., 2008;Okwundu et al., 2021;Olarewaju et al., 2021).
Similarly, research has revealed that C. mannii seeds are a good source of essential amino acids, essential fatty acids, minerals, vitamins, polyphenols, and other important phytochemicals, which are nutraceuticals explorable in tackling various illnesses (Basharat et al., 2023;Kortse & Oladiran, 2013;Okwundu et al., 2021). For instance, the essential fatty acids such as oleic, linoleic, stearic acid, and omega-6 fatty acids which are the major components of the C. mannii seeds that have antimicrobial, antioxidant, and anti-inflammatory properties, explaining their therapeutic potentials against infections such as gastrointestinal tract infections, infertility, oxidative damage, etc. (Kortse & Oladiran, 2013;Nwozo et al., 2023). The characteristic phytochemical constituents reported present in C. mannii seed and its oil according to Badifu and Ogunsua (1990) were alkaloids, phenols, flavonoids, terpenes, and tannin, which are exogenous antioxidants. Agu et al. (2022) reported that Cucumeropsis mannii seed oil (CMSO) reversed altered testicular histology/biochemistry in male rats against toxicity induced by BPA. We considered that there is no limit to the exploration of plants' therapeutic potentials (Aja et al., 2014) and adopted the use of animal model to further enlist C. mannii seed oil potentials as an emerging therapeutic natural product. Therefore, the present study investigated the effects of C. mannii seed oil (CMSO) against mitochondrial damage induced by bisphenol A in male albino rats. The study demonstrated that CMSO is suitable as a nutritional and pharmacological alternative against infertility caused by exposure to toxicants such as BPA.

| Collections and authentication of plant materials
The C. mannii (Abakaliki wild type: Egusi) seed was obtained from Iboko market, Izzi Local Government Area, Ebonyi state, Nigeria.

| CMSO extraction
The seeds of C. mannii were peeled and ground using automated grinder. Seed oil was then locally extracted using mechanical pressing with the help of mortar and pestle (Ferrentino et al., 2020).
Water drops were introduced to improve oil release as the water helps in the rupturing of the cells, partly by binding to gum/mucilage (hydrocolloids; Dror et al., 2006), which are allowed to sediment. The extracts were stood for 2-3 days undisturbed to sediment and was then separated by decantation to recover oil with purer quality. The oil was then kept in clean bottles for use.

| Acute toxicity of CMSO
CMSO acute toxicity in Wistar Albino rats (male) was evaluated according to OECD (2008) as specified in the guideline No. 425. Acute toxicity was done using the test procedure of limit dose according to the guideline No. 425. For this experiment, 2 months old male albino Wistar rats were employed for the animal study and were acclimatized in the laboratory for 7 days before the experiment. Fifty mL/ kg of CSMO was orally given to a female rat after fasting overnight.
Thereafter, the rat was strictly monitored for any observed behavioral or physical change for initial 30 min after extract's administration, and then periodically observed for the next 24 h, with more attention within the first 4 h, and then monitored on daily basis for 14 days. Foods were given to the rats after 3-4 h administration of CMSO. After the first rat survival, the other four rats (male) were recruited, then fasted for 4 h. The rats subsequently received the same CMSO dose followed by same observation/monitoring, which continued for additional 14 days to watch for possible toxicity (Eleazu et al., 2021;Saleem, 2017;Tadesse et al., 2014). At 50 mL/kg limit test dose, there was no signs of gross behavioral or physical changes in the rats, such as motor activity, reduction in feeding, or hair erection during the 24 h and 14-day monitoring periods. Consequently, 5 mL/kg limit dose (10%) was chosen as intermediate/middle dose, 2.5 mL/kg (half of this) was chosen as lower dose, while 7.5 mL/kg (1.5 times the middle dose) was chosen as the higher dose according to the guideline of OECD (2008) as stated in the OECD guideline No. 425.

| Experimental rats
The animals used for the study were adult albino Wistar rats ob-

| Experimental design
Total of 48 male albino Wistar rats were recruited for the experiment and randomly grouped into six groups labeled group A, group B, group C, group D, group E, and group F, with each group having eight rats. Group A, group B, and C were used as control, while group D, group E, and F received the treatment ( Table 1).
CMSO and BPA administration was simultaneously given once per day by oral intubation for 6 weeks.

| Collection of tissue sample
The sacrificing of the rats was done under mild anesthesia by cervical dislocation. Scrotal sacs were dissected using a sharp blade to remove the right and left testicles (Garcia & Sajjad, 2022). Their volume, length, width morphology was studied for all the groups. The calculation of testicular volume and indices was done using: Where, L = length, D = width, and K = 0.9.
The supernatant was retained, while a fresh isolation medium was used to wash the pellet and then recover using the initial supernatant (Bello et al., 2021;Park & Pang, 2021). The centrifugation of the pooled fractions was done for 10 min at 500 g and supplemented with protease and phosphatase inhibitors. The obtained supernatant was centrifuged for 15 min at 5000 g with the aim obtaining the mitochondria pellet. The pure mitochondria were recovered using centrifugation at 4°C for 10 min at 12,000 g.

| Antioxidant Enzymes' measurement
GPx was determined using Flohe and Gunzler (1984) method. A suspension of fresh isolation of mitochondria was made in 1 mL of buffer (50 mM Tris-HCl, 0.22 mM NADPH (Sigma), 1 mM GSH (Sigma), 5 mM EDTA, and 0.4 U glutathione reductase (Sigma) (pH 7.6)). The initiation of the reaction was done by the addition of Spectrochem (tertiary butyl hydroperoxide) to 0.22 mM final concentration, and absorbance was read at 340 nm. The enzyme activity was calculated as nmol NADPH oxidized/min/mg protein using a molar extinction coefficient of 6.22 × 10 3 M −1 cm −1 .
The catalase activity assay was performed according to the method reported by Khodayar et al. (2018). In brief, suspensions of mitochondria were added to H 2 O 2 (0.01 M) and Tris-HCl (0.05 mM), mixed, and then incubated for 10 min. After which 4% ammonium molybdate was added. Absorbance was determined at 410 nm.
Superoxide dismutase was determined using Flohe and Otting (1984) method. Sonication was used to disrupt the 1 mg mitochondrial pellet suspension in 0.1 mM EDTA and 50 mM phosphate buffer (pH 7.8). After centrifugation, the supernatant was collected and analyzed for MnSOD activities. The mixture of the assay contains a sample, reaction buffer (0.5 M xanthine (Sigma), 0.1 mM NaOH (E. Merck), and 2 lM cytochrome c (SRL) in 0.1 mM EDTA and 50 mM phosphate buffer, at pH 7.8). The initiation of the reaction was done with the addition of 0.2 U/mL in 0.1 mM EDTA (xanthine oxidase (Sigma)). The absorbance change was read at 438 nm for 3 min.
Activities of SOD were determined as its inhibitory ability against 50% ferricytochrome c decrease and presented as U/min/mg protein.
The GSH level was determined according to the Umapathy et al.

| Statistical analysis
The statistics was done using GraphPad Prism 5.04 (GraphPad). The data were presented as mean ± standard deviation. ANOVA (one-way) with Tukey's test was done. Generally, the significance level was p < .05.

| RE SULTS
As shown in Figure 1, BPA induction caused a significant decrease in glutathione peroxidase activities in comparison with control rats. The group of rats treated with CMSO (BPA + CMSO) showed a significant increase in GPx activity when compared to that of BPAexposed animals. Figure 2 showed that SOD activity was significantly decreased on induction with BPA when compared to the normal control group.
Interestingly, CMSO treatment showed a significant increase in SOD activity when compared to that of BPA-exposed rats.
Catalase activity as shown in Figure 3 significantly decreased in group exposed to BPA in comparison with the normal control.
Conversely, CMSO treatment significantly increased catalase activity in comparison with that of rats exposed to BPA. Figure 4 revealed that GSH level was significantly decreased in rats exposed to BPA in comparison with the normal control.
However, CMSO treatment significantly increased and restored GSH levels when compared to BPA-exposed rats.
As shown in Figure 5, BPA administration significantly increased MDA level in the testicular mitochondria in comparison with the normal control. Interestingly, treatment with CMSO (BPA + CMSO) showed a significant decrease in the level of MDA when compared to the BPA group. Figure 6 revealed ROS level significantly increased in testicular mitochondria of BPA-treated rats over the normal control. Rats in BPA + CMSO-treated group had a significant reduction in ROS level when compared to the BPA-treated group.
As shown in Figure 7, a significant increase in testicular indices was observed in BPA-treated rats over the normal control.

Groups SOD (U/mg protein)
F I G U R E 3 CMSO effects on catalase activity in testicular mitochondria in testicular toxicity induced by BPA in the albino rats. Results are calculated as mean ± standard deviation (n = 6). Mean with different signs have significant difference (p < .05).

| DISCUSS ION
Bisphenol A, a well-established endocrine disruptor, has been shown to induce cell permeability and enzyme inactivation (Catala, 2009). Sakaue et al. (2007) reported that BPA caused a decrease in antioxidant enzymes (GPx, SOD, and CAT), sperm count, and motility, F I G U R E 4 CMSO effects on reduced glutathione level in testicular mitochondria in testicular toxicity induced by BPA in the albino rats. Results are calculated as mean ± standard deviation (n = 6). Mean with different signs have significant difference (p < .05).    Nwozo et al., 2023). However, co-administration of CMSO and BPA restored and significantly increased GSH levels to near normal, which may be due to the reducing properties and potential of CMSO.

Weight of Rats (g)
More so, our study showed a remarkable increase in testicular lipid peroxidation in the BPA-treated group. This result is consistent with the observation of Biedermann et al. (2010), who affirmed that BPA administration caused rapid degradation of lipids that ultimately led to the high level of MDA in the test sample, thus generating oxidative stress in testicular tissue. In furtherance to this, Li et al. (2012) reported that BPA declined the activity of the males' specific cytochrome P450 isoforms (testosterone, 2, and 6β hydroxylase), thus provoking ROS that impairs sperm function. Furthermore, the present study is consistent with the study of Kabuto et al. (2003), who reported that BPA administration significantly decreased the level of testosterone and progesterone and led to the accumulation of ROS.
The buildup of ROS that may have triggered the oxidative breakdown of lipids and resulted in the rise in MDA levels shown in the current study is one possible explanation. Free radicals may be able to steal electrons from the lipids in cell membranes as a result of these processes, damaging the cells. This process is carried out by a free radical chain reaction, which has the potential to further impact polyunsaturated fatty acids with many double bonds between methylene bridges (-CH 2 ) that contain reactive hydrogen atoms.
However, co-treatment of CMSO significantly decreased the level of ROS, which is most probably due to the therapeutic potential and reducing ability of CMSO or perhaps due to mitochondrial GSH's effects on the inner membrane permeability and sulphydryl group in the reduced state. Its metabolic implication is the impairment of the electron transport chain, thus inhibiting oxidative phosphorylation that may ultimately alter cellular metabolism and decrease sperm motility, thus resulting in infertility. This increase in ROS could result from the leakage of electrons at complex I and complex III from electron transport chains that lead to a partial reduction of oxygen and the formation of superoxide anions Novo & Parola, 2008). Collectively, both superoxide and hydrogen peroxide generated in this process is considered mitochondrial ROS (Turrens, 2003). During oxidative phosphorylation (OXPHOS), the electron transport chain inside the inner mitochondrial membrane generates most of the mitochondrial ROS (Awuchi & Twinomuhwezi, 2021;Nolfi-Donegan et al., 2020).
The rats' body weight and testes were both drastically reduced by BPA, while testicular indices were noticeably elevated. This result is consistent with observations made by Gurmeet et al. (2014), who found that rats exposed to BPA saw a rise in the testicular index and a concurrent decrease in testicular volume and body weight. Similar findings have been reported for BPA-exposed rats that had significantly lower testicular volume and body weight than their control counterparts. Numerous studies have demonstrated that following the treatment of BPA, the body weight and testicular volume of male rats significantly decreased, with a concomitant rise in testicular indices (Korkmaz et al., 2010;Nanjappa et al., 2012;Norazit et al., 2012).
However, the significant decrease in testicular volume and BW with a corresponding increase in testicular indices observed in this study may be caused by reduced gender hormones' bioavailability, pointing to the males' reproductive endocrine stipulation. The volume and weight of testis have direct relationship with the spermatogenic cells' mass. Consequently, reduced volume and mass of testis affect the activity of spermatogenesis with a decrease in germ cells. Additionally, it is pertinent to note that intoxication with BPA reduced the BW of the experimental rats by exerting damages on key molecules, including proteins of testis. However, co-treatment with CMSO significantly increased the BW and testicular volume with a concomitant decrease in testicular indices of male rats, which may be due to the therapeutic efficacy and protective potential of CMSO. As a result, our findings confirmed the previous observation by Anjum et al. (2011) that there was a significant decrease in body weight and testicular volume as well as an increase in testicular indices in BPA-exposed groups. However, pre-treatment of melatonin markedly increased testicular weight and volume, with a decrease in testicular indices.
Based on the findings from the present study, BPA exposure resulted in systematic impairments in the testicular mitochondria of male rats. It caused a dysregulation in the activities and levels of markers of oxidative damage investigated in this study. Prolonged exposure to BPA resulted in a drastic decrease in the body weight of rats, testicular volume, and a marked increase in testicular indices.
Interestingly, the administration of C. mannii seed oil (CMSO) significantly normalized these toxic effects of BPA in all cases of markers investigated. Therefore, CMSO is an emerging nutraceutical of importance against systematic toxicity induced by exposure to BPA.

| CON CLUS ION
This study explored the effects of C. mannii seed oil against mito-

ACK N OWLED G M ENTS
The authors acknowledge Ebonyi State University, Abakaliki, Nigeria, and Kampala International University, Kampala, Uganda for providing the facilities used in undertaking this study.

FU N D I N G I N FO R M ATI O N
No funding was obtained for this study.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there is no financial or non-financial conflict of interest in the study.

DATA AVA I L A B I L I T Y S TAT E M E N T
Additional data will be made available on request.

E TH I C A L A PPROVA L
In this study, we received ethical approval number EBSU/BCH/ ET/19/001 from the Ethical Committee of the Biochemistry Department, Ebonyi State University, Nigeria, and followed their recommendations as adopted from the International Standard Procedure for Experimental Animal Handling of the National Institute of Health (NIH), USA (NIH Publications No. 8023, revised in 1996).

I N FO R M E D CO N S E NT
Not applicable.

CO N S E NT FO R PU B LI C ATI O N
All the authors on this manuscript gave their consent to publish.