Protective Role of Antioxidants in Cadmium-induced Reproductive Toxicity

 
ZSC contains antioxidant secondary metabolites that have demonstrated potent protective bioactivity against oxidative stress-induced cellular damage. ZSC richness in flavonoids, saponins and polyphenols has been attributed to its its robust antioxidant properties and its specific protective effects against heavy metal toxicity. Plants secondary metabolites are multifaceted in their action, though the protective properties of ZSC is central to its antioxidants content scavenging the ROS. Studies have shown that ZSC extract significantly ameliorate oxidative stress markers and histopathological damage in the liver and kidneys of rats exposed to cadmium (Al-attar, 2011). Similarly, ZSC ameliorated lead-induced oxidative damage in rat tissues, suggesting its potential protective role against other heavy metals, including cadmium (Al-Jassabi et al., 2012). There are reports on its anti-inflammatory effects associated with heavy metal toxicity (Fan et al., 2023). Similarly, ZSC bioactive metabolites can modulate signaling pathways involved in apoptosis and cell survival, thereby preserving ovarian and uterine cellular integrity. For instance, flavonoids in ZSC have been shown to inhibit the activation of pro-apoptotic proteins and enhance the expression of anti-apoptotic proteins, protecting cells from oxidative stress-induced apoptosis (Hosseinzadeh et al., 2013).
 
Understanding the effects of cadmium on female reproductive health and identifying protective agents like ZSC is crucial for developing effective strategies to mitigate reproductive toxicity. This research aims to provide valuable insights into the protective role of Ziziphus spina-christi, contributing solution to the growing prevalence of infertility and reproductive diseases especially in area of high exposure to cadmium.
 

 
Samples from the leaves of ZSC were collected in Dammam region (26.43°N 50.1°E) during October to December 2023.  The samples were cleaned thoroughly and stored in a dark, sanitary environment until they were completely dried. Post drying, the leaves were ground into a fine powder. This powdered ZSC was then soaked in water for 24 hours and the resulting solution was filtered through Whatman paper. Following maceration, the extract was evaporated at temperatures between 50 and 60°C, after which it was frozen at -20°C until needed. The extract was prepared at a dosage concentration of 100 mg per kg of body weight dissolved in water (Ammari et al., 2024).

Experimental animals
 
The study was conducted on 24 healthy rats, equally divided into male and female groups (12 of each), with body weights ranging from 180 to 220 grams and ages between 12 and 14 weeks. These animals were obtained from the Zoology Department’s animal facility at the college Science, King Saud University (KSU). The rats were housed in a well-ventilated environment at a controlled temperature of 25±2°C, under a 12-hour light-dark cycle. They were provided with a standard diet and had access to water throughout the experiment. All procedures were carried out in compliance with the ethics committee guidelines and Institutional Animal Care standards at KSU (Approval no: KSU-SE-23-06). The rats were divided into four groups, each consisting of six animals (three males and three females). The control group received water, the second group was administered 100 mg of ZSC, the third group received cadmium at a dose of 1 mg/kg and the fourth group was treated with both ZSC and cadmium for a period of 21 days.
 
Oxidative stress examinations
 
After weighing the livers from each group of rats, the tissues were immediately homogenized in phosphate-buffered saline (PBS) with a pH above 7.4. The homogenates were then centrifuged at 5000 g for 15 minutes at 4°C. The supernatant was collected, with 10% of the total volume used for measuring oxidative stress markers, including glutathione (GSH) and malondialdehyde (MDA), along with other antioxidants present in the liver.
 
Statistical analysis
 
All statistical analyses for the experiment were performed using GraphPad Prism software (version 10.1.1). The Shapiro-Wilk test was employed to ensure normal distribution of the data. A two-way ANOVA was used to analyze animal body weights, considering both day and extract variables, followed by Tukey’s multiple comparison test. Hormone levels and reproductive organ weights were analyzed using a one-way ANOVA, also followed by Tukey’s test for post-hoc analysis. The results are expressed as mean values with standard deviations and a p-value of 0.05 or lower was considered statistically significant.
 

Oxidative stress plays a crucial role in the pathogenesis of various diseases. This is often exacerbated by toxic substances such as Cadmium (Cd). Antioxidant defenses, such as catalase, superoxide dismutase (SOD) and glutathione (GSH), counteract oxidative damage, while elevated levels of malondialdehyde (MDA) and cortisol indicate increased oxidative stress and physiological stress, respectively. This study investigates the impact of different treatments on antioxidant levels and stress markers in both male and female rats. The treatments include a control group, cadmium (Cd) exposure, ZSC administration and a combination of cadmium with ZSC. The study measures key parameters including catalase, superoxide dismutase (SOD), malondialdehyde (MDA), glutathione (GSH) and cortisol levels. In the examination of catalase activity, we observed a similar baseline catalase activity in both sexes in the control group. In Cadmium exposed group, a significantly reduced catalase activity was observed in both sexes while males show a slightly greater decrease. The ZSC treatment group demonstrated an increased catalase levels in both genders, while females experiencing a slightly higher improvement. Interestingly, the treatment of ZSC with cadmium exposed group restored catalase activity, while females show a stronger response (Fig 1). Catalase is crucial for detoxifying hydrogen peroxide, a significant decrease in catalase activity in rats exposed to cadmium has been reported by Choudhary and Singh, 2000. However, the reports of Saied et al., (2008) support our findings on ZSC potency to improve the catalase activity. The slight gender difference observed may be due to hormonal influences, as estrogen has been shown to upregulate antioxidant enzymes (Chainy and Sahoo, 2020).
 

 
On the other hand, Increased MDA level indicates lipid peroxidation and oxidative stress. Our findings are consistent with that of Stacey et al., (1980) on elevated MDA levels in cadmium-exposed rats. Our results show a similar baseline MDA level in the control group for both sexes while the Cadmium exposure caused a significant increase in MDA levels, while male rats demonstrate a slightly greater increase. However, ZSC treatments in both Cadmium unexposed and exposed groups show a reduced MDA levels in both sexes, with females showing a slightly and stronger response respectively (Fig 1). ZSC’s antioxidant properties due to its moderation of MDA levels and (Yossef et al., 2011). In addition, the slightly greater reduction in females could be linked to more effective lipid metabolism and antioxidant defense (Marra et al., 2002).
 
Furtherly, GSH is a critical antioxidant marker and its reduction by cadmium is well reported (Xu et al., 2003). Our studies show the baseline level for GSH is comparable in both sexes in the control group. Cadmium exposure caused a significant drop in GSH levels, with an increased response in males. ZSC treatment increased GSH levels, with females experiencing a slightly greater improvement. The treatment of cadmium exposed group with ZSC significantly restored GSH levels, with females showing a slightly better antioxidative response (Fig 1). The slightly better response in females might be due to enhanced GSH synthesis and recycling, influenced by hormonal differences (Chainy and Sahoo, 2020). The examination of cortisol also shows that both sexes had similar baseline cortisol levels in the control group. Cadmium exposure elevated cortisol levels, more significantly in males. ZSC treatment reduced cortisol levels in both genders, with females showing a marginally larger reduction. In correlation with other antioxidant markers testes, the treatment of Cadmium exposed group with ZSC significantly decreased cortisol levels, with females showing a stronger stress-reducing effect.
 
In general, our results demonstrate that ZSC exhibits a protective antioxidative effect against cadmium-induced toxicity in both male and female rats. In particular: Females consistently showed slightly better recovery in antioxidant enzyme activities and stress markers, suggesting potential gender differences in response to oxidative stress and antioxidative treatment. The significant restoration of catalase, SOD, GSH levels and the reduction in MDA and cortisol levels emphasize the importance of ZSC as a promising therapeutic agent to mitigate oxidative damage and reproductive toxicity caused by cadmium.
 

ZSC shows promise as a protective agent against cadmium induced oxidative stress. The slightly better response in females suggests potential gender-specific benefits warrants further investigation. In all, these findings support the therapeutic potential of ZSC in managing oxidative stress and its associated health effects.

The authors sincerely acknowledge the Researcher Support Project (RSP-2024R112) for funding this work at King Saud University, Riyadh, Saudi Arabia.

Funding
 
The authors sincerely acknowledge the Researcher Support Project (RSP-2024R112) for funding this work at King Saud University, Riyadh, Saudi Arabia.
 
Data availability
 
The data sets used in the current study are available from the corresponding author upon request.

The authors claim that there are no conflicting interests.