The Role of Environmental Endocrine Disruptors on Leydig Cell Death and Senescen

Abstract

Environmental endocrine disruptors, as exogenous chemicals that interfere with hormonal behavior, are known to cause testicular Leydig cell death and senescence. The incidence of diseases of the male reproductive system has been increasing over the past half-century. Genetic defects alone cannot explain the rapid increase in incidence, and there is growing evidence that environmental factors or lifestyle changes are responsible for the high incidence in recent years. Testicular Leydig cells occupy an important role in the male reproductive system. In this study, we review the mechanisms by which environmental endocrine disruptors promote both death and senescence of testicular Leydig cells, refine the former into two programmed death modes, apoptosis, and autophagy, and further explore the interactions among them, thus summarizing the advances of the toxic effects of environmental endocrine disruptors on testicular Leydig cells, and expecting to provide a new therapeutic idea.

INTRODUCTION

Environmental endocrine disruptors (EDCs) are exogenous chemicals that can interfere with hormonal behavior and are widely found in daily life. It can bind to different receptors in the body and enhance or inhibit normal endocrine functions through a variety of mechanisms, which in turn cause a variety of diseases [1]. Leydig cells, located in the interstitial spaces between the seminiferous tubules, are the primary source of androgens.

Leydig cells play a key role in the regulation of spermatogenesis by influencing the production of growth factors and steroids and have been closely associated with many androgenic reproductive disorders [2]. In recent years, various studies have shown that EDCs promote apoptosis and senescence of testicular Leydig cells, which reduces testosterone production and produces androgenic toxicity. Apoptosis and autophagy are modes of programmed cell death; apoptosis maintains a precise dynamic balance between cell division and cell death and is a physiological protective mechanism that removes unwanted, damaged, or dangerous cells without harming surrounding cells or tissues, but inappropriate activation or inhibition of apoptosis can lead to a variety of diseases [3]; in normal cells, autophagy occurs continuously at a low rate to maintain the dynamic balance of the intracellular environment. When cells encounter special conditions, such as metabolic stress, autophagy occurs in large quantities [4]. Cellular senescence generally refers to cellular replicative senescence, and common hypotheses on the molecular mechanisms of cellular senescence include the “telomerase hypothesis” and the “oxidative stress hypothesis” [5].

By clarifying the specific mechanisms of EDC-induced Leydig cell death and senescence, this article aims to provide new insights for identifying drug targets and developing medications for related male reproductive system diseases.

MAIN BODY

1. Environmental endocrine disruptors cause Leydig cell death

1) Environmental endocrine disruptors promote apoptosis in testicular Leydig cells

Apoptosis is a novel form of cell death distinct from necrosis, in which the lysosomes within the dying cells remain intact. The apoptotic cells detach from the surrounding tissue and are eventually phagocytosed, without eliciting an inflammatory response in the body [6]. The apoptotic pathways of Leydig cells are mainly divided into the extrinsic pathway initiated by death receptors and the intrinsic pathway initiated by mitochondria. EDCs primarily induce Leydig cell apoptosis through the latter pathway [7]. EDCs can induce Leydig cell apoptosis through both caspase-dependent and caspase-independent pathways, and they are involved in the regulation of Leydig cell apoptosis.

(1) Environmental endocrine disruptors promote caspases-dependent apoptotic pathways

The discovery of caspases originated from studies on cell apoptosis in Caenorhabditis elegans, and they are a group of structurally similar proteases located in the cytoplasm [8]. Members of the caspase family can be divided into two categories: initiator caspases and effector caspases [9]. Initiator caspases, including caspase-2, -8, -9, -10, and -11, are responsible for cleaving the precursors of effector caspases. Effector caspases, including caspase-3, -6, and -7, are responsible for cleaving structural and regulatory proteins within the nucleus and cytoplasm.

Cadmium increases the expression of mitochondrial fission proteins DRP1 and FIS1 while decreasing the expression of mitochondrial fusion proteins OPA1 and MFN1. This results in the accumulation of mitochondrial superoxide, excessive mitochondrial fission, and the release of cytochrome c, ultimately leading to Leydig cell apoptosis. By targeting DRP1, the use of Mdivi-1 (an inhibitor of DRP1) can reduce mitochondrial fission, partially prevent the release of cytochrome c from mitochondria to the cytosol, and attenuate cell apoptosis [10]. Chlorella vulgaris is a species of green microalga. Via suppression of apoptotic-related genes including caspase3 and activating steroidogenic-related genes including StAR and HSD17β3 in the Leydig cells. C. vulgaris alleviates the adverse effects of cadmium (Cd) [11].

7,12-Dimethylbenz(a)anthracene (DMBA), a typical polycyclic aromatic hydrocarbon, is catalyzed by the CYP1B1 enzyme to produce its ultimate metabolite, DMBA-DE. Exposure to DMBA induces a significant increase in reactive oxygen species (ROS) generation, which initiates the depolarization of mitochondrial membrane potential (MMP) and enhances the activation of caspase-3. Subsequently, DMBA-DE binds to DNA, inducing p53 activation, which ultimately leads to apoptosis through caspase-3 activation [12].

Acrylamide (AA) is present in many cooked foods rich in carbohydrates, and glycidamide (GA) is the ultimate toxic metabolite of AA. Cyanidin-3-O-glucoside (C3G) exhibits a protective effect against AA- and GA-induced reproductive toxicity in Leydig cells. After exposure to AA (1.925 mM) and GA (0.872 mM), Leydig cells were treated with 10 to 50 µM C3G for 24 hours, resulting in up-regulated expression of Bcl-2 protein and down-regulated expression of pro-apoptotic protein Bax and cleaved caspase-3 [13].

(2) Environmental endocrine disruptors promote non-caspases-dependent apoptotic pathways

Plastic particle pollution poses a novel threat to both ecosystems and human health. Polystyrene nanoparticles (PS-NPs, with a size of 20 nm) can disrupt the integrity of the plasma membrane, as evidenced by increased lactate dehydrogenase release and depolarization of the cell membrane potential. Furthermore, PS-NPs affect the mitochondrial DNA copy number, collapsing the MMP and accompanying energy metabolism disturbances. PS-NPs induce apoptosis in Leydig cells and exert cytotoxic effects [14].

A cohort study presents novel data supporting the hypothesis that intrauterine paracetamol exposure during the masculinization programming window (8–14 weeks of gestation) may adversely affect male reproductive development, specifically shorter anogenital distance in male infants [15]. However, these findings need confirmation. One experiment simulated clinical medication and testicular development, constructing mouse models with varying doses and durations, and found dose-dependent inhibition of fetal testicular cell proliferation and increased apoptosis. Exposure to acetaminophen resulted in the activation of the Wnt signaling pathway and the inhibition of the Notch signaling pathway [16]. For more advancements, please refer to the comprehensive reviews [17,18,19].

Cannabidiol (CBD), one of the primary components extracted from the Cannabis sativa plant, is utilized as a prescription medication for treating seizures in numerous countries. When CBD concentrations are below 30 µM, it can reduce cell viability, induce G1 cell cycle arrest, and inhibit DNA synthesis. Exposure to high concentrations of CBD (≥50 µM) for 24 hours or to a lower concentration (20 µM) for 6 days can induce apoptosis in Leydig cells [16].

Copper oxide nanoparticles (CuONPs) are metallic multifunctional nanoparticles known for their excellent electrical conductivity, catalytic properties, and antibacterial characteristics. Exposure to CuONPs activates the ERK1/2 signaling pathway, which further promotes apoptosis and cell cycle arrest in TM3 Leydig cells, ultimately leading to Leydig cell injury and disorders in steroidogenesis [20]. Mangiferin (MF) protects against diethylhexyl phthalate (DEHP)-induced testicular dysfunction partly by inhibiting apoptosis. It modulated the apoptotic pathway by suppressing cytochrome C mRNA, Fas ligand, Bax, caspase-3 activity, and upregulating HSP70 expression [21].

(3) Involvement of environmental endocrine disruptors in the regulation of apoptosis

EDCs can induce Leydig cell apoptosis by activating p53, regulating gene expression, and increasing oxidative damage. P53 is a crucial tumor suppressor gene and a pro-apoptotic factor [22]. Normally, the activity of p53 in cells is maintained at a very low level. When cells encounter abnormal conditions, such as DNA damage or abnormal growth caused by overexpression of oncogenes, p53 is activated, leading to cell growth arrest by blocking the cell cycle and even triggering apoptosis.

The survival of Leydig cells depends on external signals, and the apoptotic program will be initiated if they lose survival signals or receive death signals. p53 is a crucial pro-apoptotic intracellular transcription factor, and the apoptotic signals received by mitochondria often originate from p53. Benzo[b]fluoranthene (BbF) activates p53 by inhibiting the Akt-Mdm2 signaling pathway, reducing the expression of p-Akt and Bcl-2, and increasing the in vivo and in vitro expression of Bax and cleaved caspase-3, further inducing apoptosis in Leydig cells [23].

EDCs can trigger Leydig cell apoptosis through gene regulation. Bisphenol A (BPA) is a significant environmental pollutant. With increasing exposure to BPA, the mRNA levels of TET1, Cav3.2, and Cav3.3 decrease significantly. Notably, TET1 significantly promotes the proliferation of TM3 Leydig cells and inhibits their apoptosis. TET1 enhances the expression of Cav3.3 through DNA hydroxymethylation regulation. This suggests that TET1 may serve as a potential epigenetic marker for the reproductive toxicity associated with BPA exposure [24].

EDCs can also elevate cellular apoptosis levels by increasing xidative damage. Cisplatin (CDDP), a chemotherapeutic drug used to treat many different types of tumors, significantly decreases total antioxidant capacity, superoxide dismutase (SOD), and glutathione (GSH) levels in mouse testes when administered. Additionally, it increases malondialdehyde (MDA) levels, leading to increased apoptosis of germ cells and an elevated Bax/Bcl-2 ratio in mouse testes [25].

However, it is noteworthy that apoptosis plays a crucial role in the development of the biological reproductive system. Phthalates, as plasticizers, are widely used in many consumer products, and dipentyl phthalate (DPeP) is one of them. By downregulating the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Insl3 in fetal Leydig cells, DPeP induces a reduction in cell apoptosis but leads to a decrease in the number of adult Leydig cells. Additionally, DPeP lowers SIRT1 and Bcl2 levels in the testes of adult rats [26]. In conclusion, DPeP adversely affects both fetal and adult Leydig cell development following in-utero exposure.

2) Environmental endocrine disruptors promote autophagy in testicular Leydig cells

Autophagy is a process by which Leydig cells degrade their substances through the fusion of lysosomes with double-membrane-encased cellular materials [27]. Normal Leydig cells need to constantly degrade dysfunctional or unnecessary cellular structures, such as various proteins, organelles, and cytosolic components, to maintain dynamic intracellular environmental balance. It is noteworthy that autophagy serves as a self-protection mechanism to promote Leydig cell survival. On the one hand, when Leydig cells face metabolic stress, such as a lack of nutritive or growth factors or exposure to hypoxic environments, they degrade their protein macromolecules or organelles to provide raw materials or ATP for cell survival [28]. On the other hand, autophagy possesses self-cleaning capabilities which is shown in Fig. 1. Unlike ubiquitin-mediated protein degradation pathways, autophagy can degrade not only misfolded protein polymers but also entire dysfunctional organelles such as mitochondria, peroxisomes, and Golgi apparatuses in Leydig cells [29].

Fig. 1. Environmental endocrine disruptors (EDCs)-induced Leydig cell death and senescence.

(1) The multiple pathways of environmental endocrine disruptors in inducing autophagy

In the normal state of cells, growth factors are present at normal concentrations and, upon binding to receptors on the cell surface, activate phosphatidylinositol kinase (PI3K), which subsequently activates AKT/PKB. This activation then proceeds through tuberous sclerosis complex-related proteins TSC1/2 and the G-protein Rheb to activate the protein kinase mammalian target of rapamycin (mTOR). mTOR is capable of inhibiting the kinase activity of Atg1, thereby suppressing the occurrence of autophagy [30]. Consequently, reducing the phosphorylation of PI3K can induce autophagy; upregulating proteins related to autophagosome formation can also induce autophagy.

The aforementioned BPA induces autophagy by decreasing the phosphorylation of AKT1 and mTOR [31]. 1,25-dihydroxyvitamin D3 (VD3) binds to the vitamin D receptor (VDR) to regulate male reproduction and mitigate BPA-induced Leydig cell injury by enhancing autophagy. Supplementing VD3 could eliminate the inhibition of BPA in VDR expression [32]. Lead, on the other hand, induces autophagy by upregulating autophagy-related proteins such as Beclin 1, Dynein, LC3-I, and LC3-II, while downregulating the mTOR proteins [33]. Furthermore, EDCs can also stimulate autophagy as a protective mechanism. Prenatal exposure to DEHP downregulates CYP11A1 and HSD3B2 through m6A epigenetic regulation and induces autophagic protection in adult Leydig cells as a response to DEHP exposure [34].

(2) The interplay between apoptosis and autophagy in Leydig cells

As mentioned above, autophagy is a form of cell death, but unlike apoptosis, it is more of a self-protection mechanism that promotes cell survival. Silica nanoparticles (SNPs) activate autophagy in Leydig cells by increasing the levels of BECLIN-1, ATG16L, and LC3-II; they promote apoptosis in Leydig cells by increasing the Bax/Bcl-2 ratio and activating caspase 8 and caspase 3 levels. Furthermore, autophagy reduces SNPs-induced cell apoptosis by regulating caspase 8 levels. The depletion of BECLIN-1 increases caspase 8 levels. In summary, these shreds of evidence demonstrate that SNPs activate BECLIN-1-mediated autophagy, which prevents SNPs-induced testicular toxicity by inhibiting caspase 8-mediated cell apoptosis in Leydig cells. However, the accumulation of SNPs in the testes ultimately leads to male reproductive toxicity [35]. This study suggests that autophagy can inhibit apoptosis in Leydig cells, a viewpoint that is supported by more research. Tributyltin chloride (TBTCL), a commonly used preservative, induces endoplasmic reticulum stress and inhibits autophagy flux, contributing to Leydig cell apoptosis and cell cycle arrest. This provides new insights into the mechanisms of EDC-induced testicular toxicity [36]. The EDC mentioned above, cadmium, also induces Leydig cell apoptosis by inhibiting mitochondrial autophagy, leading to the accumulation of mitochondrial damage [10].

2. Environmental endocrine disruptors promote senescence in testicular Leydig cells

Like the theory that intracellular telomere shortening leads to cellular senescence, the theory of oxidative damage is also one of the major theories explaining the mechanisms of senescence in Leydig cells [37]. This theory proposes that the aging phenomenon arises from the accumulation of damage caused by ROS produced during metabolic activities. Among the oxygen absorbed by organisms, 2% to 3% is converted into ROS, including superoxide anion, hydrogen peroxide, and hydroxyl radicals [38]. ROS exerts damaging effects on biomacromolecules such as proteins, lipids, and nucleic acids, and can also cause specific mutations in mitochondrial DNA. The specific molecular mechanisms supporting the theory of oxidative damage are as follows [5]: in normal young Leydig cells, the activation of cyclin-dependent kinases (CDK) leads to the phosphorylation of Rb protein, which separates from the transcription factor E2F. The released E2F activates the transcription of downstream genes, promoting the transition of cells from the G1 phase to the S phase and ensuring the normal progression of the cell cycle. As cells replicate, telomere shortening triggers the activation of intracellular DNA repair systems, including p53. In turn, p53 induces the expression of p21, which inactivates CDKs, thereby preventing the phosphorylation of Rb protein. Consequently, Rb cannot separate from E2F, keeping E2F in a continuously inactivated state and preventing the normal initiation of transcription of several key factors during the G1/S transition, leading to cell cycle arrest and cellular senescence in Leydig cells. In another signaling pathway, other signals such as oxidative damage can induce the expression of p16, also causing cell cycle arrest.

1) Environmental endocrine disruptors promote oxidative stress

The theory of oxidative damage is one of the major theories explaining the mechanisms of aging. Levels of GSH, catalase, SOD, and MDA are currently common indicators used to study oxidative stress [39]. 1-Nitropyrene (1-NP) is a representative nitro-polycyclic aromatic hydrocarbon found in diesel exhaust. In mice exposed to 1-NP, the level of MDA, a marker of oxidative stress in the testes, increased, while GSH decreased. The results indicate that long-term exposure to 1-NP can induce endoplasmic reticulum stress caused by ROS and disrupt steroidogenesis [40]. Rutin possesses a strong antioxidant capacity. Using an H₂O₂-induced Leydig cell model of oxidative damage, Rutin was found to reduce levels of ROS and MDA, while increasing levels of GSH and testosterone [41]. Icariin reversed the adverse effect of phthalate on Leydig cell proliferation, and decreased ROS levels [42]. More developed therapies are listed in Table 1 [10,11,13,21,25,33,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57]. Doxorubicin is an anticancer drug that can induce oxidative stress by downregulating several antioxidant enzymes, such as SOD, catalase, and glutathione peroxidase (GPx) [58]. Risperidone (RIS) is a commonly used drug throughout life for the treatment of schizophrenia. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and serum testosterone levels, as well as GSH, catalase, SOD, and MDA levels, were measured as indicators of oxidative stress. It was found that normal sperm morphology decreased in the RIS groups, and histopathological degeneration occurred in testicular tissue in a dose-dependent manner. The toxic effects of RIS targeted Leydig cells, which may be associated with impairment of the hypothalamic-pituitary-gonadal (HPG) axis. RIS induces reproductive toxicity in male rats by causing oxidative stress and disrupting hormonal regulation [39].

Table 1. Agents involved in reducing cellular apoptosis, autophagy, and oxidative stress mediated by environmental endocrine disruptors (EDCs).

EDC	Agent	Models	Drug administration regimen	Targeted cell	Mechanism	Effect	Reference
Cisplatin	Melatonin	Mice model In vivo	10 mg/kg/d body weight, i.p., 2 weeks	Leydig cells Spermatogonia Spermatocytes	MDA↓; TAC, GSH ↑; BAX/BCL-2↓	Reduction of oxidative stress, suppression of apoptosis	[25]
Cisplatin	Acacia hydaspica ethyl acetate extract	Rat model In vivo	400 mg/kg/d body weight, orally, 21 days	Germinal cells Sertoli cells Leydig cells	POD↑, SOD↑, CAT↑, QR↑	Augmentation of the antioxidants defense mechanism; reversal of fertility hormone levels; suppression of histomorphological alterations and DNA damages	[43]
Cisplatin	Gallic acid	Rat model In vivo	50 mg/kg/d body weight, i.g., 10 days	Spermatogonia Leydig cells Sertoli cells	CAT↑, GSH↑, SOD↑; Caspase-3↓, Bax↓, 8-OHdG↓	Reduction of oxidative stress, suppression of apoptosis and DNA damage, and restoration of structural and functional deterioration	[44]
Cisplatin	Tranilast	Rat model In vivo	100, 300 mg/kg/d body weight, orally, 7 days	-	Modulation of TRAIL/caspase‐8/cJNK signaling; MAD↓, TAC↑, GSH↑, SOD↑	Reduction of oxidative stress, modulation of proliferation, and suppression of apoptosis.	[45]
Cisplatin	Bilobetin	Rat model In vivo	6, 12 mg/kg/d body weight, orally, 10 days	Spermatogenic cells	Modulation of Nrf-2/Keap-1 Signaling, activation of Nrf-2, inhibition of Keap-1 P53↓, Cytochrome C↓, caspase-3↓; improved Ki67	Enhancement of antioxidant effects, reduction of apoptotic signals, amelioration of testicular injury, considerable boosting of serum testosterone levels, and restoration of relative testicular weight	[46]
Cisplatin	Amifostine and melatonin	Rat model In vivo	5 mg/kg/d body weight, 5 days, (melatonin); 400 mg/kg, i.p., on day 5 (Amifostine)	Germinal epithelium cells Leydig cells	MAD↓, GSH, SOD↑; Caspase-3↓, 8-OHdG↓	Reduction of oxidative stress, suppression of apoptosis	[47]
Cadmium	Artocarpus altilis (Moraceae)	Rat model In vivo	200 mg/kg/d body weight, p.o., 3 weeks	Spermatogonial cells Leydig cells Sperm cells	TBARS↓; DPPH ↓, OH radicals↓	Reduction of lipid peroxidation	[48]
Cadmium	Mdivi-1	TM3 Leydig cells line In vitro	0.1, 1 µM	Leydig cells	DRP1↓	Inhibition of mitochondrial fission	[10]
Cadmium	Glutamine	Rat model In vivo	1g/kg, p.o., 30 days	Leydig cells Sperm cells Sertoli cells	G6PD↑	Decrease in lipid peroxidation and impairment of NO-dependent endothelial function	[49]
Cadmium	Chlorella vulgaris	Rat model In vivo	500 mg/kg body weight, orally, 10 days	Sertoli cells Leydig cells	SOD↑, CAT↑, GSH↑ and MDA↓; caspase3↓ StAR↑, HSD17β3↑	Suppression of oxidative stress and modulation of spermatogenesis and steroidogenesis	[11]
Cadmium	D-Aspartate	Rat model In vivo	0.1 mM /day/g body weight, i.g., 15 days	Germ cells	Cyt c↓, caspase-3↓; SOD↑, CAT↑	Reduction of oxidative stress	[50]
Lead	Selenium	Hyline chickens model In vitro	0.5 µmol/L, Six times	Leydig cell	HSP27↓, HSP40↓, HSP60↓, HSP70↓, HSP90↓	Reduction of oxidative stress	[33]
Lead	Melatonin and vitamin C	Rat model In vivo	500 mg/kg/d body weight; 5 mg/kg/d (melatonin); i.p., 12 weeks	Leydig cells Spermatogenic cells	AOC↑, SOD↑, GSH↑, ZO-1↑, and NF-κB signaling↓	Reduction of oxidative stress	[51]
Tributyltin	Omega-3 fatty acid	Rat model In vivo	250 mg/kg/day body weight, orally, 8 weeks	Germinal epithelial cells Leydig cells Sperm cells Sertoli cells	Nitric oxide↑; inhibiting 5 alpha-reductase; CYP51↑, MAD↓, GPX↑	Improvement in spermatogenesis with elevated GnRH, FSH, LH, and testosterone levels; alleviation of ROS generation and DNA damage	[52]
Di(2-ethylhexyl) phthalate	Icariin	Mice modelv In vivo	50, 100, 150 mg/kg/d body weight, i.p., 28days	Leydig cells	ROS↓; Δψm levels↑	Prevention of ROS accumulation and promotion of testosterone secretion	[42]
Di(2-ethylhexyl) phthalate	Mangiferin	Rat model In vivo	20 mg/kg/day body weight, i.p., 7 days	Leydig cells Spermatogenic cells Sertoli cells	Nrf2↑, HO-1↑, Glutathione↑, TAC↑ Regulation of NF-κB/Cyt c/HSP70; Fas ligand↓, Bax↓, IHC↓; caspase-3↓; c-Kit signaling cascades↑; StAR↑	Reduction of oxidative stress, suppression of apoptosis	[21]
Bisphenol A	Resveratrol	Mice model In vivo	100 mg/kg/day body weight, orally, 56 days	Leydig cells Sperm cells Sertoli cells	MAD↓, SOD↑	Prevent testicular structural changes and sperm quality GnRH, FSH, LH↑ Testosterone↑	[53]
Bisphenol A	Curcumin and piperine	Gerbils model In vivo	100 mg/kg/d body weight (curcumin); 20 mg/kg/d body weight (piperine); i.g.; every two days, 29 weeks	Prostate epithelial cell	-	Weight loss, anti-inflammatory potential, and control of prostate epithelial cell homeostasis	[54]
Bisphenol A	Fenugreek seeds aqueous extract	Rat model In vivo	200 mg/kg/d body weight, orally, 8 weeks	Germ cell	ROS↓, LPO↓; caspase-3,9↓	Reduction of oxidative stress and apoptosis	[55]
Bisphenol A	Hydroethanolic Murraya koenigii leaves extract	Mice model In vivo	200 mg/kg/d body weight, orally, 8 weeks	Germ cell	ROS↓, LPO↓; Bcl↓; caspase-3,9↓	Reduction of oxidative stress and apoptosis	[56]
Bisphenol A	Carvacrol	Rat model In vivo	75 mg/kg body weight, orally, 28 days	Spermatogenic cells Leydig cells Sperm cells	CAT↑, GPX↑, GSH ↑; NF-κB↓, TNF-α↓ Caspase-3↓, Bcl-2↓, PCNA ↑	Improvement of sperm parameters, boosting of testosterone levels, reduction of oxidative stress, inflammation, and apoptosis, and promotion of proliferation	[57]
Acrylamide Glycidamide	Cyanidin-3-O-Glucoside	R2C Leydig cell line In vitro	0, 10, 25, 50, 100 µM	Leydig cell	-	Reduction of oxidative stress, mitochondrial membrane depolarization, and apoptosis; activation of steroidogenic enzymes	[13]

-: the related information is not mentioned in the current reference, and it is not clear.

2) The interplay between senescence and death in Leydig cells

EDCs can induce Leydig cell apoptosis through oxidative stress. T-2 toxin, an inevitable mycotoxin in food and feed products, induces oxidative stress characterized by increased levels of ROS and MDA, inhibited activities of catalase and SOD, accompanied by elevated caspase-9, 8, and 3 activities, and Leydig cell apoptosis [59]. As mentioned previously, AA is a common chemical produced during food processing and widely used in various industrial and laboratory processes. Exposure to AA and its metabolite GA resulted in decreased cell viability, increased excessive oxidative stress, and apoptosis in both cell types. Further in vitro experiments demonstrated that the reproductive toxicity of AA and GA is primarily due to oxidative stress-induced apoptosis [1].

EDCs can induce autophagy in Leydig cells through oxidative stress. Tri-ortho-cresyl phosphate (TOCP), which is widely used in industry as a plasticizer, plastic softener, and flame retardant, significantly induces oxidative stress in Leydig cells but does not affect apoptosis. TOCP markedly increases the levels of LC3-II, the ratio of LC3-II to LC3-I, and the contents of autophagy proteins Atg5 and Beclin 1. Transmission electron microscopy reveals an increase in autophagic vacuoles in the cytoplasm of the cells, indicating that TOCP can induce autophagy [60]. All relevant EDCs mentioned in the text are listed in Table 2 [10,12,13,14,15,20,23,25,26,33,35,39,60,61,62].

Table 2. The effects of different environmental endocrine disruptors (EDCs) on the death and senescence of Leydig cell.

Category		EDCs	Sources	Uses/exposures	Model	Dose	Route	Exposure period	Result	Reference
Heavy metal	-	Cadmium	-	Battery production, the plastics and coating, sectors, and metal smelting	Mice model In vivo	0.5, 1.0 and 2.0 mg/kg/day body weight	i.p.	4 weeks	Mitochondrial fission↑; cytochrome c↑; apoptosis↑	[10]
Heavy metal	-	Lead	-	Constantly accumulates in soil, water and air	Hyline chicken Leydig cell In vitro	20 µmol/L	-	24 h	Autophagy↑	[33]
Chemicals	-	Bisphenol A	-	Manufacture of epoxy resins and plastics	TM3 Leydig cell line In vitro	0, 20, 40, and 80 µM	-	72 h	Inhibition of cell vi- ability; mRNA TET1↓, mRNA Cav3.2↓, mRNA Cav3.3↓; apoptosis↑	[61]
Chemicals	Polycyclic aromatic	7,12-dimethylbenzanthracene	Incomplete combustion of organic materials	Cigarette smoke, barbecued food, and automobile exhaust	TM3 Leydig cell line In vitro	-	-	-	ROS↑; depolarization of mitochondrial membrane potential; caspase-3 activation; CYP1B1 activation	[12]
Chemicals	Polycyclic aromatic hydrocarbon	Benzo[b] fluoranthene	-	Air, water, soil, pavement and food	Mice model In vivo In vitro	0, 8, 20, and 50 mg/kg/day body weight	i.g.	35 days	p53-mediated mitochondrial apoptosis pathway activation; apoptosis↑	[23]
Chemicals	Organophospho- rous compound	Phosphate	-	Plasticizer, plastic softener, and flame-retardant	TM3 Leydig cell line In vitro	-	-	-	Oxidative stress↑; autophagy↑	[60]
Chemicals	Carcinogen	Acrylamide	Cooked carbohydraterich foods	Wastewater treatment, mining, textile, paper manufacturing, surface coating and cosmetics	R2C cell line In vitro	0, 0.5, 1, 2, 4, and 6 mM	-	24 h	ROS↑; mitochondrial membrane potential↓	[13]
Chemicals	Nanoparticles	Polystyrene nanoparticles	Macroscopic plastic objects breakdown (size 20 nm)	Freshwater, marine, terrestrial and atmospheric systems	TM3 Leydig cell line	50, 100 and 150 µg/mL	-	24 h	Mitochondrial impairment; cytomembrane destruction	[14]
Chemicals	Nanoparticles	Silica nanoparticles	Silicon dioxide	Agriculture, cosmetics, food additives, and biomedical applications such as drug delivering system	Mice model In vivo	25 mg/kg	i.v.	One time	BECLIN-1-mediated autophagy activation	[35]
Chemicals	Nanoparticles	Copper oxide nanoparticles	(size 50–100 nm)	Antibacterial materials, catalysts, superconducting materials and sensing materials	Mice model In vivo	10, 25 mg/kg/d	i.g.	2 weeks	ERK1/2 signaling pathway activation; apoptosis↑; cell cycle arrest	[20]
Chemicals	Phthalates	Dipentyl phthalate	-	Plasticizers	Rat model In vivo	10, 50, 100, and 500 mg/kg/day body weight	Orally	7 days	Lhcgr↓; Scarb1↓; Star↓; Cyp11a1↓; Hsd3b1↓; Cyp17a1↓; Hsd17b3↓; Insl3↓; apoptosis↑; SIRT1↓; BCL2↓	[26]
Pharmaceuticals	-	Cannabidiol	Extracted from the plant Cannabis sativa	Treatment for seizures	TM3 Leydig cells In vitro	50 and 20 µM	-	24 h (50 µM) 6 days (20 µM)	Mitochondria and lysosome dysfunction; oxidative stress↑; autophagy↑	[62]
Pharmaceuticals	-	Risperidone	-	Treatment for schizophrenia	Rat model In vivo	1.25, 2.5 and 3 mg/kg	Orally	28 days	Oxidative stress↑; hormonal regulation disruption	[39]
Pharmaceuticals	-	Acetaminophen	-	Antipyretic and analgesic drugs	Mice model In vivo In vitro	100, 200, and 400 mg/kg/day body weight	i.g.	Second/third trimester	Spermatogonial cell proliferation inhibition; apoptosis↑; Wnt/Notch signal pathway change	[15]
Pharmaceuticals	-	Cisplatin	-	Treatment for cancer	Mice model In vivo	mg/kg/day body weight	i.p.	One time	Oxidative stress↑	[25]
Biological	Fungus	T-2 toxin	Mycotoxin	Food products and feeds	TM3 Leydig cells In vitro	0, 2, 4, 6, 8, 10, 12 and 14 nM	-	24 h	Oxidative stress↑; apoptosis↑; caspase-9, 8, and 3 activities↑	[61]

-: the related information is not mentioned in the current reference, and it is not clear.

CONCLUSIONS

There is a significant association between EDCs and male reproductive system disorders. Mammalian male reproductive development encompasses a gonadal formation phase followed by a hormone-driven differentiation phase. Failures in these processes may result in Differences in Sex Development (DSD), which may encompass abnormalities of the male reproductive tract such as cryptorchidism, hypospadias, infertility, and testicular germ cell cancer. These disorders are also regarded as part of a testicular dysgenesis syndrome in males. While DSDs are primarily attributed to genetic abnormalities, the manifestation of testicular dysgenesis syndrome is frequently associated with environmental factors [63]. Many longitudinal studies have evaluated the link between pubertal timing and exposure to endocrine-disrupting chemicals during prenatal or pubertal periods [64,65]. What's more, the molecule linked to the proposed mechanism can be a biomarker for infertility and fertilization prognosis. Intracellular Ca²⁺ homeostasis is crucial for normal cellular functions, and its disruption can cause apoptosis. Cd increases the concentration of Ca²⁺ in Leydig cells by boosting phospholipase C activity, while PREP reduces cytosolic Ca²⁺ by modulating phosphatases. Cd regulates PREP expression and localization in testes, suggesting PREP as a potential fertility marker [66].

This article reviews EDCs toxic mechanisms on Leydig cells, including apoptosis, autophagy, and senescence. However, the articles proposed in this review were mainly studied using rodent models. Although, animal models are crucial for assessing human toxic mechanisms, considering challenges in exposure assessment and tissue sampling [67]. The limitations and potential confounding factors, which increase heterogeneity and complicate comparisons between studies, cannot be ignored. First off, animal studies often report findings based on higher exposures than humans face. For instance, low doses of phthalates showing anti-androgenic effects in animals are often 10 to 100 mg/kg/day, while human exposure is much lower [68]. To overcome this, studies have constructed models for human-relevant doses and found deregulated Leydig function and testicular toxicity in rats exposed to phthalate mixtures below threshold doses [69]. Apart from that, EDCs mixtures don't follow a classic dose-response model, necessitating discussions on bi-phasic effects and additive/synergistic effects in risk assessments. Bioaccumulation of EDCs exerts complex influences, and assessing EDC mixtures across a range of doses in multiple models is crucial. In addition, evaluating animal studies requires considering model differences (in vitro vs in vivo), exposure regimen, drug metabolism, and species variations [70]. Notably, testicular steroidogenesis regulation differs between humans (hCG-LH receptor-driven) and rodents (paracrine-driven) [71]. Moreover, maternal lifestyle and environmental factors affecting human EDCs exposure may differ fundamentally from experimental rodent conditions. Finally, due to method limitations, few studies exist on EDCs effects on male puberty, with more focus on fetal testis development. Regulatory bodies should also prioritize using human-relevant models to assess EDCs effects. Recently, experimental systems have expanded beyond traditional in vivo and in vitro methods to include in vitro culture, ex vivo tissue culture, xenograft models, and transplantation. See this review for details [63]. Studies have applied these methods, including culturing first-trimester human fetal testes and xenografting second-trimester testes into mice, to confirm the effects of acetaminophen and ibuprofen on fetal germ cell development [72]. Furthermore, with research technology advances, the direct effects of Valproic acid were examined using the validated first-trimester fetal testes organ culture system and the transcriptomic analysis [73]. Moreover, epidemiological studies and systematic reviews can provide insights into EDCs impacts on male reproductive disorders. More research based on human tissue and epidemiological studies is needed to verify animal experimental results.

Some confounding factors in animal studies have been discussed above. On top of that, bias from sample size, representativeness, interference from other EDCs, dose-response relationships, and recall error affect study credibility in the context of epidemiological studies. In studying the reproductive toxicity of EDCs, lifestyle factors cannot be neglected. Epidemiological investigations show increasing health risk behaviors like smoking, drug use, and alcohol in pregnant women and adolescents, linked to andrological disorders and impaired male fetal/puberty testicular development [74]. Besides, the existing research lacks longitudinal studies, and often quantifies EDCs levels only in mothers during pregnancy or children after birth, without direct mother-fetus exposure assessment. Chemical measurement is missing, presumed by occupational exposure [75]. Larger, carefully designed prospective studies are needed.

In this review, we proposed some drug development studies based on anti-EDCs-induced Leydig cell apoptosis, autophagy, and oxidative stress. Other relevant studies are listed in Table 1. Drawing from our understanding, we offer four perspectives on strategies for developing novel pharmaceuticals. To begin with, similarly to the need for adopting updated experimental methods in conjunction with epidemiological studies for toxicity assessment and underlying mechanism research, the development of new drugs also requires the updating of research methodologies and further validation through studies with evidence sourced from clinical trials and epidemiological studies. Secondly, new drug research should focus on Leydig cells' crucial role in EDC-induced male reproductive toxicity. Leydig cells synthesize testosterone, which is essential for male reproductive development and sperm vitality, their death or senescence mainly causes male reproductive damage [10]. Thirdly, many of the studies we reviewed solely focus on changes in overall oxidative stress indicators, and there is a need for more research to delve into specific mechanisms in order to identify potential drug targets. For example, a study found that Melatonin exerted its anti-apoptotic and anti-oxidative effects by activating MT1/MT2 and stimulating SIRT1/Nrf2 signaling to prevent the cisplatin-induced apoptosis of Leydig cells [13]. Additionally, using cutting-edge technologies can speed up target discovery. Computational toxicology models reveal BbF-induced male reproductive toxicity via Leydig cell apoptosis and the Akt/p53 pathway [23]. However, these predictions need experimental validation. Last but not least, current research is mostly limited to the fetal period. More studies should discuss the detrimental effects of EDs exposure and potential treatment strategies on male puberty and the transition age (defined as age 18–25 years).

The assessment of the toxicity of EDCs and their correlation with male reproductive disorders, the in-depth exploration of underlying mechanisms, the discovery of pharmacological targets, and the development of therapeutic agents are still in their preliminary stages. Hence, there may be a long way to go in this field of research. We hope that our insights into the role of EDCs on Leydig cell death and senescence, coupled with the preliminary drug development strategies we have proposed, will ignite further research endeavors and pave the way for significant and impactful discoveries in the future.