Discussion

The current investigation used a catechin supplement with cadmium as an experimental model to evaluate antioxidant properties against induced oxidative stress. The study aimed to test whether catechin could improve haematological and biochemical alterations caused by cadmium-induced oxidative stress.

The endogenous antioxidant system comprises enzymes such as superoxide dismutase, catalase, and glutathione peroxidase, as well as non-enzymatic molecules. These antioxidants target specific free radicals and operate in different ways to mitigate their harmful effects, helping to maintain the balance between oxidative reactions and antioxidant activity. Antioxidants prevent chain reactions initiated by free radicals by stabilising these reactive molecules. They donate electrons to free radical molecules that possess unpaired electrons, thereby blocking their high reactivity.

Intra- and extracellular physiological metabolic processes, such as cell respiration, are mediated by free radicals. However, certain circumstances, including diseases and toxicities, generate excess amounts of free radicals.^{(Reference Tijjani, Olatunde, Zangoma, Egbuna, Danyaro, Abdulkarim, Mahmoud, Muhammad, Egbuna, Găman and Jeevanandam29)} The body’s internal antioxidant system manages these excess free radicals to prevent their harmful effects.

Oxidative stress, also known as oxidative overload, occurs when there is an imbalance between oxidation reactions and antioxidant activities. This imbalance is referred to as redox status. This imbalance reduces the effectiveness of the body’s natural antioxidant system at managing the harmful effects of ROS. Disruptions to this balance caused by disease conditions or environmental factors are considered triggers of oxidative stress.^{(Reference Fazelian, Moradi, Agah, Hoseini, Heydari, Morvaridzadeh, Omidi, Pizarro, Ghafouri and Heshmati30)}

Oxidative stress is characterised by the depletion of antioxidant molecules and plays a critical role in tissue damage and disease development. It is characterised by the excessive production of ROS, including free radicals such as hydroxyl and superoxide radicals, as well as non-radical compounds such as hydrogen peroxide.^{(Reference Staudacher, Trujillo, Diederichs, Dick and Radi31)}

Free radicals have a hazardous impact when the antioxidant system is not efficient enough to counteract them. These unstable molecules can oxidise other cellular biomolecules, resulting in oxidative stress. This stress can damage cellular proteins and DNA, potentially leading to cell death. One significant outcome of this oxidative damage is lipid peroxidation, which primarily affects cell membranes.

Excessive generation of ROS is triggered by cadmium toxicity, which eventually leads to oxidative stress and significant damage to the liver and kidneys.^{(Reference Li, Tan, Wang, Zhang, Lao and Wong32)}

Cadmium is toxic and leads to the oxidation of cell membrane lipids, significantly reducing adenosine triphospahte (ATP) production and glutathione levels in the mitochondria. Cadmium toxicity also disrupts the function of antioxidant enzymes, increasing oxidative stress. Ultimately, cadmium toxicity triggers apoptosis by activating caspases.^{(Reference Pham-Huy, He and Pham-Huy33)}

Reliable markers used to assess antioxidant status in the present study include total thiols, glutathione, superoxide dismutase, glutathione peroxidase, catalase, and total antioxidant capacity (TAC), all of which were found to be significantly decreased in rats exposed to cadmium. These decreases suggest that excessive oxidative stress negatively impacted these antioxidants. It is proposed that ROS generated during this process caused the oxidation of these antioxidant molecules, leading to altered functions and reduced capacity to combat oxidative damage. Certain endogenous antioxidants, including glutathione and catalase, directly eliminate free radicals.

When antioxidants become oxidised and inactivated, free radicals increase, leading to heightened oxidative stress. Catalase breaks down hydrogen peroxide (H₂O₂), a compound strongly linked to lipid peroxidation. Reduced catalase activity allows hydrogen peroxide to exert powerful oxidative effects, particularly in rats. These effects contribute to the Fenton reaction, which produces highly damaging hydroxyl radicals (OH).^{(Reference Feng, Chen and Lin34)}

The present results showed a significant increase in malondialdehyde (MDA) levels in cadmium-intoxicated rats, which could be attributed to lipid peroxidation of cell membranes. MDA is a biomarker of lipid peroxidation resulting from oxidative damage.^{(Reference Nichole, Ying, Marian and Fereidoon35)}

The current biochemical profile indicates that cadmium toxicity substantially affects the body’s natural antioxidant system and alters the blood’s biochemical profile. This has led to a depletion of antioxidant molecules, decreased overall antioxidant capacity, and disrupted the natural balance between oxidants and antioxidants. When the body’s natural antioxidant system is compromised, introducing external sources of antioxidants becomes essential to restore this balance. Antioxidants in supplement form are expected to protect biological systems from oxidative damage by quickly eliminating excess free radicals and enhancing the body’s antioxidant system. Supplemented and endogenous antioxidants collaborate to neutralise and eliminate free radicals. It is worth mentioning that the byproducts resulting from the reaction of antioxidant molecules with free radicals can eliminate additional radicals, thus enhancing the overall antioxidant effectiveness.^{(Reference Nichole, Ying, Marian and Fereidoon35,Reference Feng, Ding, Lin and Chen36)}

Serum enzymes (ALT, AST, and ALP) were significantly elevated in cadmium-exposed rats, indicating liver damage induced by cadmium. This increase may be attributed to lysosomal damage caused by lipid peroxidation, which releases enzymes into the bloodstream and leads to higher enzyme levels.

The levels of these serum enzymes, which are linked to hepatic function, were not elevated in rats administered catechins compared to the control group. This excludes the possibility of a hepatotoxic effect from the catechin dose used. Furthermore, cadmium exposure resulted in increased blood urea and creatinine levels, suggesting kidney damage in the affected rats. Blood urea nitrogen (BUN), urea, and creatinine levels were not elevated in the catechin-administered rats compared to the control group. This excludes any potential nephrotoxic effects of the utilised catechin dose.

Catechins are not synthesised in the human body, but their bioactivities make them representative of exogenous antioxidants, which enhance the activity of the antioxidant system and combat oxidative stress. Previous studies have examined catechin’s potential to combat oxidative stress and related damage while maintaining redox status.^{(Reference Huang, Deng, Xie, Shi and Huang37)}

The potent antioxidant properties of catechins are mainly related to the hydroxyl groups in their chemical structure. The location and number of these groups on the aromatic ring of epigallocatechin gallate (EGCG) explain its greater antioxidant efficacy compared to other catechins.^{(Reference Patani, Hardingham and Liddelow38–Reference Della Via, Alvarez, Basting and Saad40)} ECGC’s ability to combat ROS is attributed to the structure of the D- and B-rings.^{(Reference Mokra, Joskova and Mokry41,Reference Zhang, Mao, Cui, Zhang, Zhao and Tang42)} Catechins’ antioxidant properties are explained by multiple mechanistic pathways, including the scavenging of free radicals (such as peroxyl radicals, superoxide anions, nitric oxide, peroxynitrite, and hydrogen peroxide), the neutralisation of radicals, and the inhibition of lipid peroxidation.^{(Reference Rana, Samtiya, Dhewa, Mishra and Aluko43,Reference Haddad, Mohammed, Gopalan, Ayoub, Nasim and Assi44)} Two mechanisms explain the antioxidant activities of catechins: the hydrogen atom transfer mechanism and the single-electron transfer mechanism. In the first mechanism, free radicals receive a hydrogen atom from antioxidants and become more stable products. In the second mechanism, antioxidants donate a single electron to free radicals, which then become stabilised.^{(Reference Zwolak45,Reference Nikoo, Regenstein and Ahmadi Gavlighi46)} Furthermore, ECGC modulates signalling pathways (NF-κB, PKC, MAPK, Nrf2, and PI3K/Akt), which are disrupted by oxidative stress. In this way, ECGC enables the antioxidant system to eliminate ROS. Catechins, especially ECGC, increase the activities of SOD, catalase, and GPX through the inhibitory modulation of Nrf2, which downregulates the expression of antioxidant enzymes.^{(Reference Olson, Briggs, Devireddy, Iovino, Skora and Whelan47,Reference Talebi, Talebi, Farkhondeh, Mishra, Ilgün and Samarghandian48)}

Catechins, especially epigallocatechin gallate (EGCG), increase the activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) by inhibiting the modulation of nuclear factor erythroid 2-related factor 2 (Nrf2), which downregulates the expression of antioxidant enzymes. ECGC has been demonstrated to ameliorate excess NO levels, which, under conditions of oxidative stress, can induce RN (reactive nitrogen species) generation in the form of peroxynitrite.^{(Reference Zwolak45,Reference Nikoo, Regenstein and Ahmadi Gavlighi46)}

ECGC can also inhibit xanthine oxidase activity, thereby mitigating increasing levels of ROS.^{(Reference Kurutas49,Reference Hossen, Kaiqi, Hua, Junsong, Mingquan and Yanping50)}

Significantly decreased blood cadmium levels in rats previously intoxicated with cadmium and then treated with catechin are attributed to cadmium chelation. Flavonoids possess the property to chelate metals to form metal complexes, and this help counter the activity of the oxidising agents through inhibiting the liberation of free iron that contributes to Fenton reaction.^{(Reference Chen, Wu, Yang, Chang, Sheu and Liu51,Reference Wang and Chen52)} ECGC can chelate transition metals^{(Reference James, Wang and Wang53)}, and this metal-chelating property is related to the ortho-3,4-dihydroxy and 4-keto,3-hydroxyl moieties.^{(Reference Zhang, Mao, Cui, Zhang, Zhao and Tang42)} The metal chelating efficacy of catechin was found to be comparable to that of potent flavonoids such as quercetin.^{(Reference Kostyuk, Potapovich, Vladykovskaya, Korkina and Afanas’ev54)}

Undoubtedly, reducing cadmium levels in the blood significantly limits the initiative step of oxidative stress and contributes to the recovery of the endogenous antioxidant system.

The present findings align with previous studies that concluded catechins enhance the antioxidant system. As demonstrated in this study, epigallocatechin gallate (ECGC) has the potential to significantly increase the expression of antioxidant enzymes, which are reliable biomarkers of antioxidant activity.^{(Reference Mandel, Amit, Reznichenko, Weinreb and Youdim55)}

Additionally, the present results provide evidence that epigallocatechin gallate (EGCG) effectively inhibits lipid peroxidation, as indicated by the significant decrease in malondialdehyde (MDA) levels, a marker of lipid peroxidation.^{(Reference Bawono, Khairinisa, Jiranusornkul and Levita56,Reference Wu, Liao, Zhu, Huang, Chen and Huang57)}

In brief, the present experimental work demonstrated significant increases in antioxidant markers, including total thiols, glutathione, catalase, glutathione peroxidase, superoxide dismutase, and total antioxidant capacity, in rats exposed to cadmium and administered ECGC catechin. These results support the positive impact of ECGC on the expression of antioxidant molecules and highlight its role in enhancing endogenous antioxidant capacity.

Haematological and biochemical assays conducted on rats exposed to cadmium and treated with ECGC exhibited improved parameters that were restored to control levels. These results may support the hypothesis that catechins have a synergistic antioxidant role in reducing oxidative stress induced by cadmium toxicity.

The results clearly demonstrate ECGC’s antioxidant properties in reducing oxidative stress and damage caused by cadmium toxicity. However, further research is necessary to fully understand the molecular mechanisms by which this potent catechin exerts its antioxidant effects in the context of heavy metal toxicity. In other words, while these results demonstrate the effectiveness of ECGC as an antioxidant, additional research is necessary to elucidate the detailed molecular mechanisms underlying these properties.