Endocrine Toxicity of Micro- and Nanoplastics, and Advances in Detection Techniques for Human Tissues: A Comprehensive Review

Abstract

Background: Plastic pollution driven by human activities has become a critical global issue for human health. A growing literature demonstrates that micro- and nanoplastics (MNPs) contain endocrine-disrupting chemicals (EDCs) and other harmful compounds that enter the body easily, acting as agonists or antagonists for a wide range of hormonal receptors, and promoting endocrine toxicity. Endocrine disruption induced by MNPs occurs through the aberrant activation/inhibition of different signaling pathways that in addition to directly interfering with hormonal balances, trigger apoptosis, oxidative stress, and inflammation in endocrine cells. However, to date, the molecular mechanisms of these contaminants remain not completely elucidated. Furthermore, given the unanimous consensus on the negative impact of MNPs on human health, several methodologies have been developed to detect MNPs and contaminants not only in the environment but also in biological fluids and human tissues. Results: This review comprehensively summarizes the emerging experimental and clinical evidence explaining the mechanisms underlying the toxicity related to chronic plastic pollution in relation to the endocrine system. In addition, the review illustrates the new methodological approaches to detect MNPs in human biological samples, highlighting that employing complementary methods enables the precise characterization and quantification of MNPs. Conclusions: Future studies employing experimental, epidemiological, epigenetic, and multi-omics approaches are essential for understanding the short and long-term effects of MNPs on endocrine glands and developing effective strategies to mitigate their impact on human health.

1. Introduction

Microplastic and nanoplastic (MNP) pollution has emerged as a significant threat to human health. The major sources of plastic pollution include industrial emissions and agricultural activities, which release waste into the environment. Plastics degrade over time into smaller particles: microplastics (MPs, 1 μm to 5 mm) and nanoplastics (NPs, <1 μm) [1]. MPs originate from primary sources, intentionally produced during manufacturing, and secondary sources, formed through the environmental degradation of larger plastics via chemical, physical, and biological processes such as photodegradation, oxidation, and hydrolysis [2]. MNPs release numerous additives and chemicals, including alkylphenols, polybrominated diphenyl ethers (PBDEs), phthalates, dioxins, bisphenols, and heavy metals [3,4,5]. These substances are classified as endocrine-disrupting chemicals (EDCs) that impair hormone receptor function, posing severe risks to human health [6]. Such substances are commonly used as additives in the manufacturing of plastics to enhance specific properties, such as plasticity or flame resistance. MNPs containing this class of compounds can significantly enhance the bioavailability and therefore toxicity when ingested and they enter the digestive tract. The small size of these plastics and their high surface area may facilitate the transportation and absorption of these compounds, potentially posing health risks [7,8]. Human exposure to MNPs occurs primarily through contaminated food, especially seafood, with additional pathways including water consumption, air inhalation, and skin contact [9]. MNPs mainly enter the human body through biological membranes, the blood–brain barrier, and the digestive and respiratory systems. The sources include consuming food or liquids stored in plastic containers, inhaling airborne particles, and direct dermal exposure. Once in the digestive tract, MNPs can be transported via the bloodstream, potentially impacting various organs and contributing to chronic diseases, including cancer [10,11]. MPs and NPs act as carriers for harmful chemicals, including EDCs, which leach into liquids and disrupt endocrine function. Their toxicity is size dependent, with smaller particles having greater absorption and surface area, leading to higher EDC release. Many EDCs mimic hormone structures, interfering with endocrine signaling [11]. In 2009, Diamanti-Kandarakis et al. provided a comprehensive review of how EDCs affect endocrine systems, including male and female reproductive health, breast and prostate cancer, thyroid function, neuroendocrine regulation, and metabolic processes. These chemicals can exert their effects by mimicking or blocking hormone receptors, altering gene expression, and interfering with the critical developmental processes. The authors described multiple mechanisms of action, including interactions with nuclear hormone receptors (e.g., estrogen receptors [ERs], androgen receptors [ARs], progesterone, and thyroid receptors), as well as non-nuclear steroid hormone receptors (e.g., membrane ERs) and nonsteroidal receptors (e.g., neurotransmitter receptors). Importantly, they emphasized that the exposure to EDCs during fetal or infant development may lead to latent effects, which could manifest later in adulthood or even during aging [12]. Additionally, Zoeller et al. (2012) described how EDCs can exhibit non-traditional dose-response curves, similar to certain hormones. Zoeller et al. explained this concept in detail, highlighting that when hormone receptors are overstimulated (binding saturation), receptor downregulation occurs, leading to decreased cellular sensitivity to the hormone [13]. This phenomenon, known as high-dose inhibition, results in a paradoxical effect where low doses of a hormone or EDC stimulate a response, whereas higher doses suppress it. This leads to an inverted U-shaped dose-response curve, a form of non-monotonic dose response, which EDCs have been shown to replicate [12,13]. This has significant implications for risk assessments. A high dose of a chemical does not necessarily predict its effects at low doses because chemicals with non-monotonic dose responses can trigger multiple interactions at high doses that obscure the key effects observed at lower doses. Additionally, since endogenous hormones are naturally present at low levels in the body, even small fluctuations caused by EDCs can have significant biological consequences. These considerations challenge the traditional assumption of a safe threshold for EDC exposure. Studies in animals, epidemiological research, and theoretical models of hormone action suggest that EDCs can exert effects even at extremely low levels, supporting the idea that there is no threshold dose for these chemicals [14,15,16]. The lack of a threshold is particularly concerning because humans are continuously exposed to low doses of EDCs. Therefore, risk assessments should adopt a no-threshold approach, assuming that even minimal exposure may be harmful to human health [13].

More recently, La Merrill et al. (2020) proposed a framework for identifying and evaluating EDC hazards based on the ten key characteristics (KCs) of endocrine disruptors. These KCs include interactions with hormone receptors, the disruption of hormone synthesis, interference with hormone metabolism and transport, and epigenetic modifications. Applying this structured approach enhances the identification of potential EDCs beyond the traditional toxicological methods, improving risk assessment and regulatory decision making [17].

Given the evidence of the detrimental effects of MNP pollution on human health, it is crucial to assess the presence of MNPs in the human body through biological samples such as feces, sputum, and placenta. Over time, several technologies have been developed to detect qualitatively and quantitatively the MNPs in human tissues, such as Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and Pyrolysis–Gas chromatography/mass spectrometry (Pyrolysis-GC-MS).

This review aims to provide a comprehensive update on the molecular mechanisms driving the detrimental effects of MNPs on the endocrine system. Furthermore, the review focuses on the current methodologies to detect qualitatively and quantitatively the MNPs in endocrine systems, highlighting both their advantages and limits.

2. MNPs and the Reproductive System

Due to their small size, MNPs can accumulate in the cells of the reproductive system, altering the cellular morphology and disrupting physiological functions, ultimately impairing the fertility in both males and females with prolonged exposure. Numerous in vitro and in vivo studies have explored these effects [18,19,20]. Recent studies demonstrated that male mice exposed to MNPs exhibited reduced sperm counts and decreased spermatogenic cells [21]. Jin et al. reported that the long-term exposure to polystyrene microplastics (PS-MPs) in male mice induces testicular histological changes, abnormal spermatogenesis, and impaired hormone secretion. They found that PS-MPs disrupt the hypothalamic–pituitary–gonadal (HPG) axis, leading to decreased serum levels of testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). Specifically, PS-MPs downregulate the LH-mediated LHR/cAMP/PKA/StAR pathway, which is essential for testosterone synthesis [21]. Hou et al. observed significant reductions in the sperm count in mice administered daily doses of PS-MPs at 0.01 mg, 0.1 mg, and 1 mg. Additionally, PS-MPs were shown to activate pro-inflammatory pathways, such as the NF-κB pathway, increasing TNF-α and inflammatory cytokine expression, including IL-1β and IL-6. These findings are consistent with other mouse models, highlighting NF-κB as a critical regulator of PS-MP-induced testicular inflammation [19].

PS-MPs have been linked to the nuclear erythroid 2-related factor 2 (Nrf2) pathway. Nrf2, a key transcription factor involved in the antioxidant defense system, is negatively regulated by Kelch-like ECH-associated protein 1 (Keap-1). The dysregulation of this pathway suggests potential oxidative stress mechanisms underlying the toxic effects of PS-MPs [22,23]. Evidence indicates that the exposure to PS-MPs promotes oxidative stress, by affecting the p38 MAPK pathway and depleting the nuclear Nrf2 pathway, resulting in a reduced sperm quantity and quality in male mice [24]. In Balb/c mice, PS-MP exposure generates reactive oxygen species (ROS) and enhances the phosphorylation of p38 and JNK MAPK in the testes. Moreover, microplastics are known to promote apoptosis in sperm cells, leading to impaired spermatogenesis and diminished sperm quality. Notably, inhibiting NF-κB signaling has been shown to significantly reduce PS-MP-induced apoptosis [19]. Recently, epigenetic and protein modifications have emerged as critical factors in this context. Epigenetic changes can disrupt testosterone homeostasis through mechanisms such as non-coding RNA modulation, histone modification, and ubiquitination. A recent in vivo study examined the effects of beta-cypermethrin (β-CYP), a widely used pyrethroid pesticide, on testicular dysfunction via alterations to the MAPK pathway, DNA methyltransferases (DNMTs), and miRNA activity. The study revealed that β-CYP inactivates the JNK and p38/MAPK pathways, leading to the increased expression of miR-140-5p, a microRNA that directly targets the transcription factor SF-1, a regulator of steroidogenic proteins. This cascade results in decreased testosterone levels [25]. A more recent study investigated how polyethylene microplastics (PE-MPs) induce oxidative stress through protein modifications and miRNA regulation, ultimately reducing the testosterone production in mice. The findings showed that PE-MPs promote ubiquitin-mediated degradation and miR-425-3p regulation, which together decrease the glutathione peroxidase 1 (GPX1) levels, a protein critical for protecting cells against oxidative stress. This reduction triggers endoplasmic reticulum stress, activating metabolic enzymes that accelerate testosterone metabolism and further lower the testosterone levels [26].

Similar to studies on the male reproductive system, research using animal models has investigated the effects of MNPs on the female reproductive system. Some studies report that MNPs interfere with the reproductive physiology by altering the structural integrity of the uterus and ovaries. These changes can impair the embryo implantation in the uterus and disrupt the ovarian functions, including egg production and ovulation [27,28]. In an in vivo study, exposure to 0.5 μm microplastics was found to alter the hormone levels in female rats, specifically decreasing the anti-Müllerian hormone levels. This exposure activated the Wnt/β-Catenin signaling pathway, promoting oxidative stress and ovarian cell apoptosis, which reduced the ovarian reserve capacity [20]. MNPs exert their effects on the female reproductive system by triggering various signaling pathways. For example, exposure to MNPs increases the ROS accumulation, which negatively affects the reproductive functions [29]. During oxidative stress, the Nrf2/ARE signaling pathway plays a protective role in the female reproductive system. Nrf2 dissociates from its inhibitor, Keap1, in the cytoplasm and translocates to the nucleus, where it binds to the Antioxidant Response Element (ARE) to promote antioxidant enzyme expression [30]. However, evidence suggests that elevated MNP concentrations may inhibit the Nrf2 pathway despite an initial increase in the Nrf2 expression in ovarian granulosa cells. This inhibition leads to decreased levels of antioxidant enzymes, heightened oxidative stress, and increased ROS accumulation in the ovaries. ROS accumulation also activates the Wnt/β-Catenin signaling pathway, inducing ovarian fibrosis. This fibrosis is characterized by the increased expression of transforming growth factor-β (TGF-β), α-smooth muscle actin (α-SMA), and other fibrosis-related proteins [20,30,31]. These findings underscore the detrimental impact of MNPs on female reproductive health.

The NOD-like receptor protein 3 (NLRP3) is a protein complex that plays a critical role in the defense against various damage stimuli. It activates apoptotic and inflammatory responses by converting pro-caspase-1 into caspase-1 and pro-interleukin-1β (pro-IL-1β) and pro-interleukin-18 (pro-IL-18) into their active forms, interleukin-1β (IL-1β) and interleukin-18 (IL-18), respectively [32,33]. Recent studies have shown that MNP exposure can activate the NLRP3/caspase-1 pathway through oxidative stress, leading to a reduction in the ovarian reserve in animal models [34]. In addition, the Toll-like receptor 4 (TLR4)/NADPH oxidase 2 (NOX2) pathway plays a central role in oxidative stress and reactive oxygen species (ROS) production. TLR4 activation stimulates NOX2, a key enzyme in ROS generation. Overactivation of the TLR4/NOX2 signaling pathway by MNPs leads to excessive ROS production, resulting in cellular damage. In female rats, the MNP-induced activation of this pathway promotes oxidative stress and inflammation, driving the accumulation of fibronectin and collagen in uterine tissue. This excessive accumulation disrupts the uterine structure and function, contributing to the development of uterine fibrosis [35].

MNP and EDC exposure is linked to a reduced sperm quality and androgen levels in males [36,37], while in females, it contributes to ovarian dysfunction, hormonal imbalances, and pregnancy complications [30,37]. Despite the strong evidence for phthalates and BPA, further research is needed to clarify the long-term reproductive effects.

Overall, MNP and EDC exposure induces significantly adverse effects on human male and female reproductive systems through mechanisms involving oxidative stress, apoptosis, and the activation of various signaling pathways, potentially impacting the health of future generations (Figure 1). While the precise mechanisms of MNP toxicity remain unclear, further research is essential to elucidate their effects on reproductive health and the underlying molecular pathways.

3. MNPs and the Adrenal Gland

The adrenal glands play a pivotal role in maintaining physiological homeostasis via the secretion of steroid hormones. While the detrimental effects of MNPs on various organs are well documented, their specific impact on the adrenal glands has received limited attention. MNPs can act as hydrophobic surfaces that adsorb endocrine-disrupting chemicals (EDCs), facilitating their entry into organisms and their passage across epithelial barriers [11]. The adrenal glands are particularly susceptible to EDCs due to their structural and functional characteristics. The exposure to EDCs has been shown to activate the hypothalamic–pituitary–adrenal (HPA) axis, leading to elevated adrenocorticotropic hormone (ACTH) levels. This hormonal surge results in structural and functional changes in the adrenal glands. Additionally, EDC exposure disrupts the key steroidogenic enzymes in the adrenal cortex, such as aromatase, 5-reductase, CYP11A1, and CYP11B1, all crucial for steroidogenesis [38,39]. Animal studies have demonstrated that the exposure to 4-Bromodiphenyl ether (BDE-3), a flame retardant, can inhibit the phosphorylation processes in transcription factors and enzymes such as the cAMP response element-binding protein (CREB), AMP-activated protein kinase (AMPK), and c-Jun N-terminal kinase (JNK) [40]. An in vitro study using H295A cells, a fetal adrenal cortical cell model, revealed that Bisphenol A (BPA) activates estrogen receptor β (ERβ) through the Sonic Hedgehog (Shh) pathway. This activation promotes cell proliferation by directly inducing the transcription of the proliferation-related factors, such as cyclin D1 [41,42]. Redox homeostasis is essential for adrenal metabolism due to the high levels of lipid metabolism and the generation of reactive oxygen species (ROS) during steroidogenic reactions. This balance is maintained by non-enzymatic mechanisms, such as ascorbic acid—found in high concentrations in the adrenal cortex—and enzymatic systems like glutathione peroxidase and thioredoxin. However, EDC exposure can stimulate ROS production, impairing the activity of antioxidant enzymes [43]. A recent study conducted by Farag et al. highlighted the adreno-toxic effects of polyethylene microplastics (PE-MPs) in animal models. The authors observed significant histological changes, including mitochondrial damage and structural alterations in the adrenal cortical cells. PE-MP exposure also led to the depletion of antioxidant enzymes such as glutathione (GSH) and superoxide dismutase (SOD), coupled with an increase in malondialdehyde (MDA), a lipid peroxidation marker indicative of membrane damage. In the presence of oxidative stress, PE-MPs activated the signaling pathways related to inflammation and apoptosis, as evidenced by the increased expression of IL-1β, NF-κB, caspase-3, and Bax [39]. Interestingly, melatonin treatment in mice provided protective effects against the oxidative, inflammatory, and apoptotic damage caused by PE-MPs, suggesting its potential as a therapeutic agent against microplastic-induced endocrine toxicity [39,43].

MNP and EDC exposure can disrupt adrenal function by altering hormone synthesis, inducing oxidative stress, and promoting inflammation and apoptosis [44,45]. BPA has been linked to a higher incidence of nonfunctional adrenal incidentalomas (NFAIs) and cortisol dysregulation, while phthalates may affect adrenal androgens and cortisol levels [45]. Further research is needed to clarify the molecular mechanisms and long-term health impacts of MNP-induced adrenal toxicity.

In conclusion, MNP- and EDC-induced disruptions may lead to endocrine dysfunction through altered hormone synthesis, oxidative stress, inflammation, and apoptosis (Figure 1). Further research is essential to elucidate the molecular mechanisms of MNP-induced adrenal toxicity and evaluate their long-term effects on human health.

4. MNPs and the Thyroid and Parathyroid Glands

Thyroid hormones play a pivotal role in regulating essential physiological processes, including metabolism, growth, and development [46]. Consequently, thyroid dysfunction can have far-reaching effects on overall health. Chronic exposure to MNPs has been shown to adversely affect thyroid function. Various additives and pollutants released by MNPs, such as polybrominated diphenyl ethers (PBDEs), bisphenol A (BPA), and phthalates, have been identified as thyroid-disrupting chemicals (TDCs) [47,48,49]. These TDCs enter the body primarily through the gastrointestinal tract, interfering with the biochemical pathways of triiodothyronine (T3) and thyroxine (T4) and disrupting thyroid hormone production and metabolism [48]. Thyroid dysfunction can also negatively impact other organs, including the central nervous system, especially during the early developmental stages [50]. BPA impacts thyroid gland function through multiple mechanisms, including the modulation of transcriptional activity at thyroid receptors and competition with hormonal transport proteins, leading to antagonistic effects [51]. Animal studies have examined the molecular impact of BPA and bisphenol S (BPS) on thyroid function. In 2018, the research investigated the interactions of BPA and BPS with paired box 8 (Pax8) and thyroid transcription factor 1 (TTF1) using in silico analyses. These regulators are critical for thyroid organogenesis, thyrocyte differentiation, and the biosynthesis of thyroid hormones such as thyroglobulin (TG), thyroperoxidase (TPO), and the sodium/iodide symporter (NIS). The findings demonstrated that the exposure to BPA and BPS significantly disrupts thyroid function [52]. A similar study by Zhang et al. confirmed the thyroid-disrupting effects of BPS and bisphenol F (BPF) through in vivo and in vitro models. This research utilized competitive fluorescence binding and molecular docking assays, showing that, like BPA, the exposure to BPS and BPF dysregulates the thyroid hormone (T3 and T4) and thyroid-stimulating hormone (TSH) levels. These disruptions alter the regulation of the genes associated with the hypothalamic–pituitary–thyroid (HPT) axis, ultimately leading to thyroid toxicity [53]. Phthalates and their derivatives can inhibit thyroid hormone synthesis by impairing NIS function, leading to reduced serum levels of T3 and T4, along with elevated compensatory TSH levels [50,54]. PBDEs, on the other hand, act as competitors for thyroid hormone transport proteins such as transthyretin. They also affect the HPT axis and alter hormone metabolism, further reducing the circulating levels of T3 and T4 [55].

Among TDCs, perfluoroalkyl and polyfluoroalkyl substances (PFAS) deserve particular attention. Commonly used in food packaging, PFAS can persist in the human body for 3–5 years, contributing to thyroid gland dysfunction. An experimental study has demonstrated that PFAS exposure increases the risk of reduced thyroid hormone levels in animal models [56]. A recent in vitro study reported that PFAS exerts adverse effects on thyroid-specific gene expression by interfering with the activation of the TSH/TSH receptor (TSHR) pathway in thyroid cells. The study revealed that PFAS impacts TSHR transcription, thereby inhibiting the cAMP signaling pathway and resulting in the dysregulation of thyroid hormone transcription [56]. An interesting recent study on mouse models has been conducted to evaluate the early effects of MNPs in the thyroid and parathyroid of mice after their intratracheal instillation and intragastric infusion for short-time exposure. The authors observed that MNPs induced dysfunction in the thyroid and parathyroid glands through dietary and respiratory routes. Additionally, transcriptomic analysis revealed that thyroglobulin synthesis was disrupted by MNPs, which interfered with the expression of the Pax8 and CREB genes. These genes play crucial roles in thyroid growth, differentiation, and thyroid hormone biosynthesis. Furthermore, MNPs exert harmful effects on parathyroid cells by modulating the expression of the transcription factor MafB, which is essential for parathyroid gland development and directly regulates the levels of parathyroid hormone (PTH) and cyclin D2. Moreover, MNPs affect the IP3R protein, which is involved in the signaling pathway that regulates intracellular and extracellular Ca²⁺ concentrations and normal cellular functionality, exerting an indirect influence on the PTH secretion from the parathyroid cells [57]. Finally, recent in vivo studies demonstrated that the exposure to PE-MP promoted a significant reduction in TSH and free-triiodothyronine levels, suggesting that PE-MP suppresses TSH secretion. The authors hypothesize that this suppression may result from different factors, such as negative feedback mechanisms, direct inhibition, hypothyroidism, and overall thyroid dysfunction, indicating that microplastics may adversely affect thyroid function [58]. Moreover, thyroid dysfunction can contribute to increased oxidative stress, often linked to mitochondrial dysfunction, which arises from an imbalance in thyroid gland function (hyperthyroidism or hypothyroidism). Furthermore, the authors demonstrated that naringin, a natural flavonoid with anti-inflammatory, anti-apoptotic, and antioxidant properties, can counteract the endocrine-disrupting effects and oxidative stress induced by PE-MP exposure, indicating that this natural compound can mitigate the harmful effects of microplastics [58].

Human studies have demonstrated that thyroid dysfunction is associated with EDCs. Specifically, a negative correlation has been observed between urinary phthalate levels and free/total T4, while a positive correlation has been found with serum TSH levels [59,60]. Similarly, BPA exposure may either increase or decrease serum T4 levels in humans, indicating a complex and potentially bidirectional effect on thyroid function [61]. In contrast, research on the parathyroid gland remains scarce. The few available human studies mainly focus on a limited class of EDCs, particularly derivatives associated with parathyroid tumors [62]. While human studies provide evidence linking EDC exposure to thyroid dysfunction, findings on the parathyroid gland are limited.

In conclusion, human studies provide evidence linking EDC exposure to thyroid dysfunction, though the effects appear complex and potentially bidirectional. In contrast, the data on EDC-related parathyroid disruption remain limited and primarily focus on the specific chemical derivatives associated with parathyroid tumors. Emerging research highlights the potential of natural compounds such as naringin to counteract oxidative stress and the endocrine-disrupting effects induced by MNPs, pointing toward a promising therapeutic strategy. Nevertheless, further studies are needed to clarify the molecular mechanisms by which microplastics disrupt thyroid and parathyroid function and evaluate natural bioactive compounds’ efficacy in mitigating these adverse effects.

5. MNPs and Adipose Tissue: ECD’s Obesogenic Effects

The endocrine system plays a crucial role in regulating the metabolism of fats, carbohydrates, and proteins, which provide the energy required for the body’s functional activities. The primary energy reservoir in the body is the adipocytes within adipose tissue. Due to its ability to produce hormones, adipose tissue is now recognized as an endocrine organ. However, alterations in hormone secretion by adipose tissue can disrupt the metabolic processes leading to abnormal fat deposition and contributing to the development of obesity [63]. Emerging evidence from the recent literature highlights that some EDCs, called obesogenic EDCs, can disrupt metabolic processes and adipocyte regulation, leading to an imbalance in body weight control and contributing to the onset of obesity [64,65]. EDCs impact various endocrine-regulated metabolic processes, including lipid and glucose metabolism and insulin signaling pathways [64,66]. These compounds have been shown to promote adipogenesis in cellular models, leading to fat cell formation and contributing to obesity and fat accumulation in both animal studies and humans [67]. At the molecular level, obesogenic EDCs can induce low-grade systemic inflammation, contributing to fat deposition and insulin resistance. Additionally, they can activate oxidative stress mechanisms and cause mitochondrial dysfunction. Several EDCs have been observed to interfere with adipogenesis by targeting the activation of peroxisome proliferator-activated receptors (PPARs), particularly PPARγ. Notably, phthalates act as selective activators of PPARγ, promoting the differentiation of 3T3-L1 preadipocytes into mature adipocytes. This effect is believed to be mediated by phthalate metabolites, which activate PPARγ and contribute to weight gain [68]. Recent investigations have explored the mechanisms of BPA during the differentiation of human preadipocytes into adipocytes, focusing on its interactions with PPARγ. Human preadipocyte cell lines exposed to BPA and its substitutes revealed that all the tested bisphenols decreased adipogenesis during differentiation. This inhibition was associated with the promotion of an inflammatory state, marked by the dysregulation of adiponectin and the chemokine monocyte chemoattractant protein-1 (MCP1), also known as CC-chemokine ligand 2 (CCL2). MCP1 plays a key role in mediating the macrophage infiltration into adipose tissue, further exacerbating inflammation. Additionally, BPA and its substitutes significantly downregulated insulin sensitivity, as indicated by reduced pAKT/AKT ratios upon insulin stimulation. This effect, combined with the inhibition of PPARγ activity, led to adipose tissue hypertrophy. These changes collectively contribute to an increased risk of developing metabolic syndrome, including insulin resistance [69]. Several in vitro studies have shown that the exposure to BPA leads to increased levels of interleukin (IL)-6 and interferon (IFN)-γ. Prolonged exposure further upregulates the mRNA expression of IL6, IL1-α, IFN-γ, and MCP1. Notably, this upregulated expression is suppressed following the removal of BPA [70].

Interestingly, EDCs can activate the various signaling pathways associated with the inflammation mechanisms, such as the mitogen-activated protein kinase (MAPK) pathway, in response to stress signals and cytokine secretion. JNK and p38 play critical roles in promoting inflammation within adipose tissue [71]. Additionally, TLR4 can recognize ligands such as lipopolysaccharides and saturated fatty acids, triggering the activation of NF-κB and MAPK signaling pathways, thereby amplifying the inflammatory response [72]. Oxidative stress and mitochondrial dysfunction can significantly impact adipose tissue, often as a consequence of inflammatory states. These processes contribute to an altered adipocyte function, the increased secretion of pro-inflammatory cytokines, and the perpetuation of metabolic disturbances, such as insulin resistance and impaired energy homeostasis.

In 2018, a study revealed that oxidative stress is specifically localized within the mitochondria in an in vitro model. This mitochondrial oxidative stress was shown to impair the insulin-dependent translocation of glucose transporter 4 (GLUT4) to the plasma membrane, leading to a reduction in glucose uptake [73]. Therefore, there is an oxidative stress impact also in the liver. Hepatic insulin resistance leads to a state of low-grade inflammation, characterized by the release of pro-inflammatory cytokines and the activation of oxidative stress pathways. This, in turn, triggers the release of IL-1β and IL-18, promoting the formation of the NLRP3 inflammasome. This inflammasome plays a key role in recognizing cellular stress signals and contributing to chronic inflammation and metabolic dysregulation. Additionally, insulin resistance can activate the IKKβ/NF-κB signaling pathway, which disrupts the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1). The resulting dysfunction affects the downstream processes, including the translocation and activity of GLUT4, which is essential for glucose uptake [72,74]. Obesogenic EDCs may influence epigenetic pathways, inducing epigenetic modifications that contribute to an increased predisposition to obesity. These compounds appear to act on the DNA methylation mechanisms by altering the activity of DNA methyltransferases and their cofactors. Additionally, they affect histone modifications and the expression of microRNAs (miRNAs), which are crucial regulators of gene expression [75]. The prolonged exposure to low doses of BPA has been shown to influence adipogenesis by reducing the methylation of the PPARγ promoter in preadipocyte cell lines [76]. Additionally, studies using in vitro models have demonstrated that butyl benzyl phthalate (BBP) increases the expression of miR-34a-5p, a microRNA implicated in adipogenesis, obesity, and the epigenetic regulation of insulin signaling [77].

The exposure to endocrine-disrupting chemicals (EDCs), such as BPA, BPF, BPS, and phthalates, has been associated with metabolic disorders, including obesity, diabetes, dyslipidemia, and hypertension, with potential sex-specific effects [78,79] [Kim, 2019, Liu, 2019]. However, inconsistencies in the study designs and exposure assessments highlight the need for standardized human biomonitoring programs for improved risk assessment [80] [Haverinen, 2021].

Despite the growing evidence, the research on the metabolic effects of EDCs in humans remains inconsistent due to the differences in study designs, exposure assessment methods, and population variability. Further well-conducted, harmonized human biomonitoring programs are essential for better risk assessment and policy actions.

In conclusion, the literature data suggest that EDCs significantly affect adipose tissue by disrupting its metabolic functions, promoting inflammation and oxidative stress, and activating various signaling pathways (Figure 1). These alterations contribute to adipose tissue dysfunction, reduced insulin sensitivity, the elevated secretion of pro-inflammatory cytokines, and imbalances in energy homeostasis. Continued investigation into these mechanisms is critical for developing targeted interventions and improving human health outcomes.

6. Epigenetic Consequences of Endocrine Disruption Plasticity

Endocrine system plasticity refers to its ability to adapt dynamically to physiological and environmental changes, ensuring homeostasis and optimal function throughout life. This plasticity is shaped by multiple factors, including the exposure duration, individual susceptibility, compensatory mechanisms, epigenetic modifications, and environmental influences. In this context, we can introduce the concept of Endocrine Disruptor Plasticity (EDP), which refers to the dynamic and adaptative nature of endocrine disruption in response to MNPs and their associated EDCs. The plasticity framework is particularly relevant for understanding the interindividual variability in the endocrine disruption outcomes. The genetic predisposition, life stage, and cumulative exposure history can all influence how endocrine systems respond to MNP-associated EDCs. Moreover, epigenetic alterations, such as DNA methylation and histone modifications, and non-coding RNAs may play a role in shaping long-term endocrine responses and transgenerational effects. Emerging evidence highlights the significant role of epigenetic modifications in response to the exposure to MNP and EDCs. While DNA sequence mutations are well-established contributors to various diseases, some abnormalities arise from molecular mechanisms unrelated to the traditional genetics or direct DNA mutations. These mechanisms involve the molecular environment surrounding DNA, which regulates genome activity independently of its sequence [81]. These heritable, mitotically stable modifications are classified as epigenetic processes [82]. While EDCs do not directly modify DNA sequences, they can induce alterations in DNA methylation patterns, histone modifications, and microRNA (miRNA) regulation, causing transcriptional changes associated with various diseases [83]. In 2017, Menezo et al. described the transgenerational effects of EDCs on endocrine and metabolic function, mainly through sperm epigenetic changes, including DNA methylation modifications. These alterations can influence gene expression, genomic imprinting, and genomic stability [84]. A notable example of epigenetic inheritance was demonstrated by Manikkam et al., who investigated the impact of the ancestral exposure to plastic-derived EDCs, such as bisphenol-A (BPA), bis(2-ethylhexyl) phthalate (DEHP), and dibutyl phthalate (DBP). Pregnant F0-generation rats were exposed to these chemicals during embryonic gonadal sex determination (days 8–14), and the disease phenotypes were analyzed in the F1 and F3 generations. The study reported a significant increase in reproductive disorders, obesity, and pubertal abnormalities in the F3 generation, with testicular and ovarian diseases, as well as metabolic dysfunction, manifesting in a transgenerational manner. The epigenetic analysis of sperm identified 197 differential DNA methylation regions (DMRs), suggesting that germline epimutations mediate these effects. The altered sperm epigenome undergoes permanent reprogramming, resisting DNA methylation reprogramming after fertilization. This results in the transmission of epigenetic modifications across generations, ultimately leading to changes in the epigenomes and transcriptomes of somatic cells and tissues, contributing to disease development [85]. This study strongly underscores the potential for environmental toxicants to induce inheritable health consequences, raising concerns about the widespread human exposure to plastics and phthalates [86]. Non-coding RNAs, like microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), play a crucial role in gene expression regulation. Tricotteaux-Zarqaoui et al. described how EDCs influence epigenetic modifications, leading to long-term reproductive health consequences. Studies on diethylstilbestrol (DES) and BPA have demonstrated that these compounds induce sex-specific DNA methylation changes. In women, reproductive tract abnormalities have been associated with alterations in the DNA methylation of estrogen-responsive genes, along with histone modifications following DES exposure [87]. Furthermore, BPA-induced alterations in DNA methylation impair folliculogenesis and reduce the oocyte quality, with these changes persisting into adulthood and contributing to long-term fertility issues [88]. To track these epigenetic modifications, the authors highlight the importance of advanced monitoring techniques, such as bisulfite sequencing and chromatin immunoprecipitation assays. Additionally, RNA sequencing and microarray analysis can be used to assess the expression of non-coding RNAs affected by EDC exposure [87,89]. The potential interventions to counteract the adverse effects of EDCs include dietary modifications and pharmacological approaches targeting DNA methylation. DNA methyltransferase inhibitors and histone deacetylase inhibitors have been proposed as possible therapeutic strategies [90].

In conclusion, EDP provides a novel perspective to understand the dynamic and adaptative nature of the endocrine system in response to environmental stressors, particularly the EDCs associated with MNPs. While some species may adapt to hormone disruptions, these changes could reduce fertility or affect metabolism, leading to long-term challenges. To better understand and mitigate the effects of EDC exposure, future research should integrate multi-omics approaches (genomics, transcriptomics, proteomics, and metabolomics), epigenetic studies, and biomonitoring strategies to identify the early biomarkers of disruption. The advances in detection technologies and regulatory measures are critical to monitoring EDC prevalence in biological and environmental systems. Additionally, targeted public health interventions are needed to reduce the exposure in vulnerable populations. Understanding the full scope of EDC-induced endocrine plasticity will be essential for developing effective strategies to safeguard human and ecological health.

7. Emerging Techniques to Efficaciously Detect the MNPs in Human Fluids and Tissues

The identification and the qualitative and quantitative analysis of MNPs involve methodologies that are strictly dependent on the matrix in which MNPs are contained, i.e., environmental or biological. The analysis of floating MNPs in seawater or freshwater requires a collection with a manta thrown from a boat. Then, a physical separation of MNPs from particles having a biological or inorganic nature is carried out via microscopic observation [91]. When MNPs have to be extracted from sediments collected from the environment, the separation is generally carried out by employing density separation using fluids with crescent densities [92]. Moving to biological matrices, the identification and analysis of MNPs pose an issue related to the separation methods to adopt and to the amount of available sample. There is an extensive literature documenting the identification of microplastics in human fluids and tissues; MNPs have been found in biological fluids and tissues such as saliva, blood vessels, and lungs [93]. In these cases, the microplastic analysis process includes a sampling phase, where the sample size depends on the availability of the fluid or tissue, especially in the case of a biopsy where the availability is indeed limited. Once the sample is available, the separation methods consist of the digestion of the biological phase. This can be carried out via treatments with bases such as KOH or NaOH [94,95,96]; acids, such as HNO₃, H₂O₂, and ZnCl₂; and proteases, if the biological matrix contains a high protein fraction [94,95,97,98]. After the digestion samples are washed and filtered with plastic-free filters, they are ready to be analyzed. The potential MNPs can be identified using reflected light optical microscopy [97] and scanning electron microscopy (SEM-EDS) [98], the latter offering a higher magnification and resolution which is useful for the identification of smaller MNPs. These techniques allow for the identification of the shape and size of the particles, but only through a chemical–molecular analysis, can it be established whether the particle is an MNP. The most commonly used techniques are Raman spectroscopy [96] and Fourier transform infrared spectroscopy (FT-IR) (including micro-FTIR and LDIR) [98,99]. Both techniques operate on the principle of interaction between electromagnetic radiation and the vibrations of the functional groups of molecules. As the dimensions of MNPs decrease and the complexity of the matrix increases, the aforementioned techniques may become insufficient to yield reliable results, because of the relatively low repeatability, reproducibility, and sensitivity of the procedures.

Besides spectroscopic techniques, the use of Pyrolysis–Gas chromatography–mass spectrometry (Py-GC-MS) is emerging for the identification and quantification of MNPs in environmental [100] and biological samples [99,101]. The analysis consists of the heating of the sample containing MNPs at high temperatures (550–700 °C) in an oxygen-free environment, which leads to the chemical transformation of the plastics (decomposition) into smaller volatile and semi-volatile molecules characteristic of each polymer. Gas chromatography (GC) and mass spectrometry (MS) separate and identify the resulting pyrolysis products. This technique can have a greater sensitivity with respect to spectroscopic methods, and after a calibration procedure, a quantitative assessment can be carried out, providing a mass/mass or mass/volume concentration, instead of particles/mass or particles/volume concentration that can be obtained by using counting techniques (optical/electron microscopy). However, Py-GC-MS does not provide information about the shape and size of MNPs; this information can only be obtained through microscopic observation.

Since the “background” environmental concentration of microplastics is at a level that can be often easily measured, and sometimes it is comparable to the amount of MNPs that is expected in some matrices, there is an emerging issue related to the MNPs’ environmental contamination for samples undergoing analysis. This contamination can be also due to the tools used for the analysis, and this is more critical as the expected concentration and sizes of MNPs decrease. To mitigate these risks, it is essential to use plastic-free tools and operate in clean rooms and laminar flow hoods. Despite all the measures that can be adopted, environmental contamination cannot be excluded. For this reason, a critical point of the analysis of MNPs in biological samples is the quality assurance/quality control (QA/QC), which is essential for accurate and reliable analyses [102]. The analytical technique employed for MNP analysis must be validated to confirm its accuracy, precision, and sensitivity. This validation should involve assessing the key parameters, including the linearity, limit of detection (LOD), and limit of quantitation (LOQ). Quality assurance and quality control measures, such as procedural blank samples and spiked samples, should be implemented to track the performance of the analytical method and ensure that there is no contamination. In the literature, not all researchers report the preparation and use of blanks in their laboratory processes. Blanks are conducted by replicating the laboratory experiment process to detect potential contaminants [103]. For example, a blank solution without any matrix should be processed alongside the digestion of samples [72,104]. During physical characterization using a stereomicroscope, an additional set of either dry or wet filter paper blanks should be positioned near the work area until the procedure is complete [105]. These blanks are then examined for atmospheric microplastics that could contaminate the samples during processing. Implementing these practices will yield more reliable and accurate results.

In conclusion, the identification and analysis of micro- and nanoplastics (MNPs) in biological matrices require meticulous methodologies. Utilizing complementary techniques, such as microscopic, spectroscopic, and pyrolysis methods, enables the precise characterization and quantification of MNPs. However, several challenges persist. One major limitation is the complexity of biological matrices and the lack of standardized methods, which can lead to inconsistencies and hinder data comparability across studies. Contamination poses another significant issue; both environmental background contamination and contamination from analytical tools can compromise the accuracy of the results. To address these challenges, it is crucial to implement stringent cleanroom protocols and utilize non-plastic tools. The regular use of blanks and control samples can further help identify and mitigate the contamination sources. Effective separation techniques, thorough quality assurance and quality control (QA/QC) measures, and robust analytical validation are essential to minimize the contamination risks and ensure reliable results. Although several organizations conduct interlaboratory tests for the determination of MNPs, there is a lack of such tests on biological-like matrices. This type of activity would help improve the reliability of the methods used to analyze these materials.

8. Concluding Remarks

The current evidence reported in the literature suggests that MNPs can adversely impact endocrine systems by triggering oxidative stress, inflammation, and apoptosis. MNPs, acting as EDCs, have also been reported to interfere with hormone production, regulation, and transport. By functioning as antagonists or agonists of hormone action, they can cause dysfunction in the related endocrine systems. In this context, growing evidence on the endocrine-disrupting effects of MNPs highlights the need for extensive research to address their impact on public health. Future research will address the study of the interconnection between MNPs and the associated chemicals that disrupt endocrine systems. This includes their interaction with post-transcriptional regulators like microRNA (miRNA) and epigenetic modifications. These emerging studies underscore the importance of expanding our understanding of the molecular and post-transcriptional effects that MNPs can have on endocrine systems. Additionally, epidemiological studies are likely to reveal stronger correlations between MNP exposure and endocrine-related diseases. In this context, new approaches, such as multi-omics studies, may help to better interpret the potential links between miRNA regulation and MNP exposure, ultimately improving the predictive models of toxicity to mitigate their impact on endocrine systems. Finally, an important topic concerns the analytical techniques currently available for the qualitative and quantitative detection of environmental contaminants in biological fluids and human tissues. Despite the advancements in this field, some methodological gaps persist. Therefore, further research is essential to advance the development of highly sensitive and specific analytical techniques, also allowing the collection of large-scale data for a better understanding of the impact of pollution on human health and, most importantly, for the early diagnosis of damage.

This complex interplay of environmental exposure, internal bioavailability, molecular disruption, and systemic outcomes is summarized in the conceptual framework proposed herein, which serves as a visual synthesis of the current knowledge and future research directions (Figure 2). Moreover, adopting a systemic level of analysis, ranging from individual behaviors and local waste management to national regulations and global agreements, provides a holistic perspective crucial for understanding and mitigating the impact of MNPs across the biological, societal, and ecological dimensions. Collectively, this knowledge will ultimately inform policies, therapeutics, and preventive measures to safeguard endocrine health globally.