Mechanistic Insights into Fluoride-Induced Male Reproductive Injury: The Role of Nrf2/FSP1 Pathway-Mediated Ferroptosis.

Introduction

In soil, livestock and poultry farming, feed processing, slaughter, and packaging, toxic elements such as fluoride can enter and accumulate in animal-derived foods through various pathways, posing a serious risk to humans and animals [1]. Environmental fluoride pollution has intensified in parallel with rapid industrialization, and the impact of fluoride exposure on human health has become a major public health concern. Numerous studies have reported the harm fluoride exerts on multiple tissues and organs, encompassing pathological changes in bone tissue, neurotoxicity, and disruption of reproductive and immune processes [2,3]. However, evidence concerning the effects of fluoride exposure on the reproductive system and the underlying mechanisms remains limited. Studies have shown that the reproductive toxicity of fluoride exhibits sex-specific mechanisms, and differences in hormone levels, physiology, and gene expression between men and women can affect their response to environmental toxins [4]. Notably, the global decline in sperm count and the recognized association between male reproductive health and systemic diseases have drawn unprecedented attention to male reproductive health and have positioned this field as an emerging priority in environmental health research [5]. Supporting this concern, recent large-animal studies have demonstrated that the male reproductive system is highly responsive to external modulators; dietary supplementation with plant-derived essential oils significantly improved sperm concentration, motility, viability, and testicular development in Bunaji bulls, underscoring the need to elucidate mechanisms of reproductive toxicity from environmental toxicants such as fluoride [6]. In this context, conducting in-depth research on the specific mechanism by which fluoride exposure affects the male reproductive system is of both urgent and critical importance. This study focuses on the mechanism of male reproductive injury, aiming to provide supplementary evidence specifically from the male perspective for the sex-specific study of fluoride reproductive toxicity. Previous studies have demonstrated that fluoride poses a reproductive toxicity risk to male reproductive health. Zhang et al. found through in vitro and in vivo studies that sodium fluoride (NaF) treatment disrupted the blood-testis barrier in experimental mice, increased the rate of sperm deformities, reduced sperm motility, and caused reproductive damage through endoplasmic reticulum stress, oxidative stress, and other pathways [7]. Moreover, fluoride has been shown to interfere with reproductive hormone levels in the adult hypothalamic-pituitary-gonadal axis [8,9]. Current research has revealed that fluoride exposure can induce oxidative stress, cell apoptosis, pyroptosis, and autophagy in rat testicular tissue, resulting in male reproductive damage [[10], [11], [12], [13], [14]]. Recently, studies have found that environmental chemicals can induce ferroptosis by triggering oxidative and antioxidant imbalances in tissues. For instance, polystyrene microplastics have been shown to damage mitochondrial structure and function, leading to lipid peroxidation, redox imbalance, and ultimately ferroptosis in chicken spleen [15]. These findings provide new insights for the study of fluoride-induced reproductive damage mechanisms.

Ferroptosis is a mode of cell death driven by iron-dependent lipid peroxidation and is characterized by iron overload and subsequent lipid peroxidation. Morphologically, it is characterized by a decrease in mitochondrial membrane density, a reduction or disappearance of mitochondrial cristae, and rupture of the outer mitochondrial membrane [16,17]. Ferroptosis has been implicated in the onset and progression of various pathological conditions and diseases, including drug resistance in cancer treatment and brain injury [18]. Importantly, ferroptosis is increasingly recognized as a key mediator of reproductive system damage. Specifically, acute ferroptosis has been shown to induce oxidative damage to sperm DNA and testicular oxidative stress, leading to male reproductive dysfunction [19]. Moreover, exposure to environmental agents such as phthalates and arsenic has been shown to induce testicular ferroptosis, resulting in testicular tissue damage and spermatogenic dysfunction [[20], [21], [22]]. Notably, treatment with NaF elevates lipid peroxidation in the rat testes, and the level of malondialdehyde (MDA), an end product of lipid peroxidation, in the testicular tissue of rats exposed to fluoride is significantly higher than that of the control group [23]. Aldehydes such as MDA are regarded as markers of the “executive” stage of ferroptosis. Therefore, ferroptosis may play a role in the initiation and development of fluorosis [24]. Thus, fluoride may induce male reproductive system damage via the ferroptosis pathway. However, little evidence is available on whether fluoride exerts reproductive toxicity by inducing ferroptosis.

Acyl-CoA synthase long-chain family member 4 (ACSL4) promotes lipid peroxide generation, acts as an executor of ferroptosis, and serves as a ferroptosis biomarker [25,26]. In contrast, ferroptosis suppressor protein 1 (FSP1), also known as apoptosis – inducing factor mitochondria – associated 2 (AIFM2), functions as a potent inhibitor of ferroptosis. It acts through the FSP1-coenzyme Q₁₀ (CoQ₁₀)-NADPH axis [27,28]. It is noteworthy that evidence indicates that FSP1 is a transcriptional target of nuclear factor-erythroid 2 related factor 2 (Nrf2), and the Nrf2/FSP1 signaling pathway may mediate the onset of cellular ferroptosis [29,30]. Furthermore, Parlak et al. found that excessive fluoride exposure modulated Nrf2 expression and reduced its levels in rat testes [31]. Ommati et al. found that fluoride exposure inhibited the Nrf2/HO-1/NQO1 antioxidant pathway in testicular tissue and Leydig cells, leading to oxidative stress, mitochondrial damage, apoptosis, and autophagy [32]. These findings reveal the key role of Nrf2 in fluoride-induced male reproductive damage, but there is limited direct evidence supporting a role for Nrf2/FSP1 in ferroptosis in fluoride-induced male reproductive toxicity.

To this end, our study initially employed bioinformatics approaches to identify evidence supporting that fluoride induces damage to the male reproductive system via the ferroptosis pathway. Subsequently, we established a NaF-treated rat model to evaluate the effects of NaF treatment on reproductive function, the Nrf2/FSP1 pathway, and the expression of ferroptosis-related molecules in male Sprague-Dawley (SD) rats. Ultimately, we constructed an in vitro fluoride-exposed cell model to confirm that the Nrf2/FSP1 pathway mediates ferroptosis-induced reproductive damage.