Endocrine-disrupting chemicals (EDCs) are a heterogeneous group of substances that are able to interfere with the hormonal-signaling pathways and alter metabolic and reproductive functions. Among EDCs, bisphenol A (BPA) is a non-persistent anthropic compound widely employed in the last decades as a component of plastic for multiple applications (e.g., food packaging, personal care, household products, and medical devices) so it is now environmentally ubiquitous. Indeed, BPA has been detected in the atmosphere, soil, and aquatic environments, as well as in foodstuff, house dust, consumer products, and biotic matrices [1
Exposure to BPA is associated with deleterious health effects for animals and humans and affects not only endocrine and reproductive organs but also immune and central nervous systems through several mechanisms, including oxidative stress [3
]. Growing evidence from research on laboratory animals shows that this non-persistent compound alters male and female reproductive function even at extremely low exposure levels [4
]. This is relevant because BPA exposure may be chronic, making it functionally equivalent to a persistent compound.
The mechanisms underlying BPA toxicity may be related to its chemical properties in the body, including interactions with hormone receptors (i.e., estrogen receptors) in target cells [5
]. As a weak estrogen receptor ligand, BPA binds to the classical nuclear receptors with different affinity, for example, estrogen receptor (ER)α less than ERβ (about 10,000 times lower than 17-β-estradiol), and its potency of action becomes higher when the estradiol level is at very low concentrations [6
]. BPA also binds many other membranes and nuclear receptors, activating them at concentrations lower than those required to activate ERs [7
]. In particular, BPA binds to the membrane-bound form of estrogen receptor (mER)α and G protein-coupled receptor (GPR) 30, inducing non-genomic effects. GPR30 has been detected in several cell types, and it is involved in cell proliferation and apoptosis. Indeed, seminoma proliferation is induced by a low concentration of BPA through a GPR30 non-genomic activation [8
]. BPA also binds to another estrogen-related receptor (ERR)γ, an orphan nuclear receptor highly expressed in the fetal brain and placenta by which BPA influences fetal tissue development [9
Widespread contamination and dietary ingestion have led to BPA exposure in the general population, as evidenced by the scientific literature on BPA occurrence in human tissues and body fluids (i.e., urine, serum, plasma, saliva, breast milk, semen, follicular fluids, and adipose tissues) [10
A recent epidemiological study has hypothesized that higher urinary BPA concentrations in humans might negatively impact female reproduction [11
]. Moreover, in men, urinary BPA levels have been correlated to abnormal semen parameters [12
], and BPA-exposed individuals also showed reduced libido and erectile ejaculatory difficulties. The overall BPA effects on male reproduction appear to be more harmful if exposure occurs in utero [7
Given the challenges in both human exposure assessment and tissue sample collection, animal models are important for assessing the underlying toxic mechanisms observed in humans. First, differently from humans, rodents allow histological evaluation of various tissues, as well as for tissue- and cellular-specific molecular analysis. Second, rodent models provide excellent opportunities for sex-specific evaluations. In the experimental design, important factors must be considered, including the examined tissue, the structural, functional, and epigenetic differences between gender and species, the exposure time, and chemical dose. Third, despite the limitations of clinical studies, many animal studies have evidenced that BPA affects several reproductive endpoints.
BPA toxicity is associated with oxidative stress and related markers in several experimental models [3
] and humans [14
]. Mounting evidence shows that the production of reactive oxygen species (ROS) and/or decreased capacity of antioxidant defense significantly contribute to BPA organ toxicity, altering the oxidative balance in the mitochondria and generally in the cell [3
This review offers new insights and up-to-date literature survey on these issues to identify oxidative stress as the leitmotif underlying BPA’s mechanism of action with pleiotropic outcomes on reproduction. Besides, the review also examines the effect of antioxidant substances on the BPA-induced toxicity and analyzes their possible efficacy as a therapeutic strategy to limit the damage on female and male reproductive organs and their functions.
2. Oxidative Stress as a Mark of BPA Toxicity
Oxidative stress is a key component of inflammatory reactions and has been implicated in aging, cardio-metabolic, and immune diseases, neuronal degeneration, and the development and progression of cancer, with different and not completely understood mechanisms [16
]. The regulation of redox balance is essential to maintain cellular homeostasis, development growth, and survival. Indeed, physiological cellular metabolism generates ROS (i.e., superoxide anions, peroxides, and hydroxyl radicals) involved in redox balance, a well-orchestrated process. ROS comprise not only radical and nonradical oxygen derivatives but also nitrogen-containing compounds defined as reactive nitrogen species (i.e., nitric oxide and peroxynitrite). However, few data have related BPA toxicity to nitrosative species, in particular in the reproductive system. Redox balance is coordinated by numerous cellular components to avoid excess ROS and to prevent related deleterious effects (mutations, unchecked cell growth, and insensitivity to cell death signals).
As a metabolic and endocrine-disrupting chemical, BPA can impair oxidative homeostasis via direct or indirect mechanisms, including the increase of oxidative mediators and reduction of antioxidant enzymes, determining mitochondrial dysfunction, alteration in cell signaling pathways, and induction of apoptosis [17
]. BPA induces oxidative stress by decreasing antioxidant enzymes (superoxide dismutase (SOD), catalase, glutathione reductase (GR), and glutathione peroxidase (GSH-Px)) and increasing hydrogen peroxide and lipid peroxidation in the liver and epididymal sperm of rats; it can also alter organogenesis of the kidney, brain, and testis in mice [19
The ROS increase induced by BPA has been reported in several cell types with concentrations ranging from nanomolar to micromolar [3
]. Differences in ROS generation, duration, and cytotoxicity appear mainly based on the concentration of BPA, on cellular backgrounds, exposure time, and sensor fluorescent reagents [3
BPA has been recently associated with inflammation markers in addition to oxidative stress in humans [24
]. Previously, Yang et al. [14
] conducted a cross-sectional study on men and premenopausal/postmenopausal women; the authors observed that urinary BPA levels were positively associated to urinary oxidative stress and blood inflammatory biomarkers (alteration in white blood cell count and C-reactive protein) in postmenopausal women, evidencing that BPA effects depend on the gender and hormonal status. These authors assumed that the gender-related effect of BPA toxicity might be due to the estrogen level and receptor occupancy. As is known, the ER expression is gender and age-related [25
]. In postmenopausal women, the hormone level is very low, allowing a more extensive binding of xenoestrogen BPA to ER, triggering noxious cellular responses associated with oxidative stress and inflammation.
A strong relationship between oxidative stress and inflammation has been documented; these reactions are inextricably linked to cellular processes, and they may be both causes and/or consequences of cell damage. The ROS and nitrogen species generated by immune cells, as macrophages and neutrophils, contribute to the establishment of chronic inflammation. Malondialdehyde (MDA), an indicator of lipid peroxidation, and 8-hydroxy-deoxyguanosine (8-OHdG), a marker of DNA oxidation, have been widely considered as potential biomarkers for oxidative stress [26
] and several reports show their increase in BPA-exposed organisms [28
The ROS-induced DNA base modification has been shown to induce the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway of inflammation. Many other redox-sensitive signal transduction pathways like c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase and transcription factor AP-1 also participate and sustain the vicious cycle between inflammation and oxidative stress [29
The oxidative stress induced by BPA has been associated with human B-cells cytotoxicity [30
] and to the impairment of immune response in vitro in murine macrophages [31
], as well as in animal models [32
]. BPA exposure also exacerbates the expression of proinflammatory cytokines, as well as interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α), in many tissues and organs (i.e., serum, colon, liver) [17
] and the hydrogen peroxide species and ROS induced by BPA activates NF-κB [22
]. Furthermore, BPA-induced oxidative stress plays an important role in the activation of NOD-like receptor protein 3 (NLRP3) inflammasome with inflammatory co-stimulus (i.e., nitric oxide) in adipocyte cells [33
] or the liver of BPA-exposed obese mice (our unpublished results, presented as oral communication to the international congress of the British Pharmacological Society (2019), Edinburgh, UK).
Behind lipid peroxidation and inflammation reactions, the mechanisms of oxidative stress induced by BPA also include mitochondrial dysfunction. Low doses of BPA impair mitochondrial function in the liver [15
], leading to hepatic toxicity. Moreover, this pollutant induces ATP depletion, the release of cytochrome c, loss of mitochondrial mass and membrane potential, and alterations in the expression of genes involved in mitochondrial activity and metabolism [34
As is known, the excess of oxidative stress is involved in the neurodegeneration of many brain disorders [35
]. Tavakkoli et al. [18
] evidenced the neurotoxicity of BPA on the alteration of the expression of functional proteins influenced by the oxidative status that is related to neurological disorders as well as schizophrenia, depression, epilepsy, and brain tumors.
Evidence suggests a gender difference in responsiveness to oxidative stress [36
]. Females are less vulnerable to the protective effects of estrogen related to the hormone’s ability to act as an electron-donating antioxidant [37
]. Moreover, in females, mitochondria have higher levels of reduced glutathione than those of males, and the oxidative damage to mitochondrial DNA is lower in females than in males, due to higher expression and activities of manganese SOD and GSH-Px [36
]. Sex differences in oxidative stress depend not only on the higher antioxidant defense in females but also on the increased ROS generation in males [39
]. Indeed, some studies have been reported a gender-dependent NADPH oxidase activity, resulting in the excessive formation of reactive intermediates in male mice [40
]. Based on the intriguing literature concerning this issue, it will be relevant to carry out comparative studies on gender-related effects of BPA on oxidative damage.
4. Oxidative Stress-Induced BPA Toxicity on Male and Female Reproduction
Between 2000 and 2010, a lot of contradictory literature data on the effect of BPA on male fertility were collected, but many other recent reports have consistently established the adverse effects of BPA on male and female reproductive functions [13
In particular, in males, BPA impairs spermatogenesis and sperm quality, as evidenced by trans-generational studies; in females, it alters ovary, embryo development, and egg cell quality [94
]. Moreover, BPA impairs the reproductive function in offspring due to its capability to cross the blood–brain barrier [96
], in addition to its direct effects on reproductive organs.
The alterations of reproductive functions have irreversible consequences on adult fertility, mainly if these damages occur during the development of the reproductive organ in fetal life [97
]. In particular, oxidative stress induced by several EDCs, including BPA, is linked to male infertility [98
]. Many preclinical findings have shown the impact of prenatal exposure of BPA on male spermatogenesis, via several mechanisms of action. Quan et al. [99
] demonstrated that oral BPA exposure in dams (gestational days (GD)) [14
] provokes an increased ROS production, the activation of the apoptotic pathway in the testis of male offspring, confirmed by histopathological changes. BPA exposure in CD-1 dams (GD 7-14) also led to the alteration of sperm quality and motility in adult male mice, with changes in oxidative balance [100
]. Furthermore, in a murine model of chronic BPA exposure of dams, during all gestational period, and continuing the oral administration to male pups until to sexual maturation, damaged spermatogenesis was observed in progenies [101
]. This impairment was associated with decreased antioxidant defense and reduced expression of sirtuin 1, a key sensor of ROS production.
Very recently, data have confirmed that male pups exposed early to BPA produced oxidative stress in the testicular tissue [72
]. In this comparative study (among prenatal, perinatal, and postnatal BPA exposure), it was demonstrated that BPA-related epigenetic modifications occur independently from the time-exposure. Nevertheless, the magnitude of the BPA toxic effect on oxidative balance was time-exposure dependent, reaching a maximum effect during perinatal exposure (from pregnancy to lactation) [72
BPA exposure in adult rats increased ROS amount in a dose-dependent manner and oxidative stress was associated to metabolic impairment (hyperglycemia and hyperinsulinemia) and reproductive damage, as demonstrated by testicular reduction of insulin receptor substrate-2 and glucose transporter-8, two key proteins involved in testicular energy metabolism and spermatogenesis [102
]. In epididymis, testis and immune cells (i.e., lymphocytes and bone marrow) of adult rats, BPA provoked the significant increase of lipid peroxidation [103
] that is correlated to the impairment of sperm quality [104
] and the reduction of several antioxidant enzymes such as SOD, catalase, and non-enzymatic reduced glutathione [103
]. Recently, Khalaf et al. [105
] also reported an increased level of H2
, lipid peroxidation, and depletion of the antioxidant defense systems in the testis of offspring from dams exposed to BPA. At the early stage of puberty, BPA impaired sperm production and gonadotropin secretions and altered seminiferous tubule epithelium morphology, increasing ROS production and reducing the antioxidant activity of catalase and SOD enzymes [106
In testis, maintained alteration of the redox balance leads to ROS overproduction and the impairment of the antioxidant defense, resulting in increased oxidative stress that is the major cause of sperm dysfunction and male infertility [98
]. It is important to clarify that the clinical studies clearly addressed BPA reproductive toxicity to oxidative stress [107
]. However, considering the importance of oxidative balance in sperm physiology, oxidative stress could be the main detrimental mechanism underpinning BPA toxicity on the male reproductive system.
In seminiferous tubules, BPA reduced the activity of antioxidant enzymes and increased that of myeloperoxidase (MPO), substantiating inflammation and testicular dysfunction linked to high ROS production due to the peroxidation of spermatogenic cell membranes. These concurrent adverse effects negatively impact the mitotic and meiotic process of cells during spermatogenesis (i.e., spermatogonia, spermatocytes, spermatids, and spermatozoa) that result in an impaired quality and quantity of spermatozoa within seminiferous tubules.
Clinical findings have confirmed preclinical data: higher concentrations of BPA in the maternal placenta were associated with an increased risk of urogenital complications in children (i.e., cryptorchidism and hypospadias) [108
]. A cohort study showed a positive correlation between BPA maternal exposure and oxidative stress markers in the blood sample of neonates [109
]; notably, oxidative stress is a crucial driver of pregnancy complications [110
Moreover, a relationship between high blood/urinary BPA levels and anomalous semen parameters in men occupationally exposed has been reported, which also showed reduced libido and ejaculatory and erectile dysfunction (reviewed in Manfo et al. [13
]). Accordingly, Wang et al. [17
] found a positive correlation between BPA concentrations and oxidative markers in the urinary sample of adult men collected from days 0 to 90, a time window consistent to that of the spermatogenesis process.
A growing line of evidence indicates that perturbations induced by BPA and its substitutes can also affect female fertility leading to the alteration of oocyte meiotic maturation [111
]. Specifically, BPA has detrimental effects on oocyte progression and quality, chromosome segregation, increasing oxidative damage, along with oocyte apoptosis demonstrated both in vitro and in vivo studies [73
]. Indeed, oxidative stress, as well as DNA and epigenetic modifications, plays a crucial role in oocyte maturation and interferes with apoptosis, contributing to the alteration of germ cell nest breakdown and reproductive function [113
]. BPA can impair fertility, disrupting the physiological germ cell nest breakdown, as shown by the multiple transgenerational toxicities after in utero exposure in mice [114
]. This process is mainly controlled by the decrease of estrogens at the birth of pups and is also linked to the onset of apoptosis [115
]. During this critical stage, the exposure at different doses of BPA in mice blocks the natural apoptosis required for releasing oocyte from their germ cell nests [116
] and induces an alteration of key ovarian genes involved in the regulation of oxidative stress [114
Any error in oocyte maturation triggers other injuries, including infertility, pregnancy loss, and birth defects, and BPA negatively impacts all these disorders of female reproductive physiology by epigenetic mechanisms [117
Gupta et al. [118
] reported that BPA decreased the amplitude and frequency of spontaneous uterine contractions in isolated uterus, showing the estrous phase by involving nitric oxide release and nitrergic mechanisms. Moreover, BPA exposure in female rats induced the inhibition of steroidogenic enzymes, decreased level of estrogen, and eNOS overexpression in the ovary [119
Some epidemiological studies show that EDCs exposure can affect pregnant women [120
]; particularly, in placental mammals, BPA causes an unsuccessful pregnancy and the alteration of several placental molecular endpoints [23
], including hormone-related mRNA expression, micro-RNA expression, and DNA methylation [122
]. Other studies highlight the sex-specific relationship between BPA exposure and placental outcomes (reviewed in Strakovsky and Schantz [23
]) because of the double origin and development of the placenta (maternal and fetal tissues) and the following sexually dimorphic responses to ED [123
]. Consistently, it has been demonstrated that in CD-1 mice, daily exposure to BPA led to the reduction of placental mRNA expression of nuclear receptors disrupting the physiological function of the placenta [124
]. Very recently, Song et al. [125
] showed that in sheep, the exposure to BPA in the gestational period causes low birth weight mainly due to the placental dysfunction that differs between early and mid-gestation with stage-specific epigenetic alterations. In particular, BPA induces oxidative stress (i.e., nitrotyrosine formation) and inflammation (IL-1β), as well as lipotoxicity and modification of placental steroid milieu, in the later gestational period.
BPA toxicity on female reproduction also involves the occurrence of endometriosis, miscarriage, and abnormalities of uterus morphology [94
]. The common thread among these disorders is the increased oxidative stress [126
], along with the activation of inflammatory signaling. Indeed, Cho et al. [22
] have demonstrated the capability of BPA to induce oxidative stress and inflammatory mediators in human endometrial cells. Interestingly, these authors found a decrease in antioxidant enzymes in endometrial cells after BPA exposure, probably due to the excessive production of ROS related to BPA metabolization that exceeds the intracellular antioxidant defense capacity. Moreover, pregnant BALB/c mice exposed to different BPA doses revealed profound changes in endometrium morphology, characterized by hypertrophy and the formation of endometriosis-like structures in comparison to the not-treated mice [127
BPA has become a target of intense research based on its metabolic and endocrine interference and its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer. This review provides a framework for understanding how the induction of oxidative stress may contribute to the pleiotropic reproductive adverse effects observed after BPA exposure. The numerous examined studies show that reproductive organs in males are more vulnerable to BPA exposure than those in females. Evidence suggests that males may be more susceptible for several reasons (i) gender differences in glutathione levels and their relevance for detoxification process (lesser glutathione availability in males); (ii) greater sulfate-based detoxification capacity in females; (iii) greater inflammatory response in male reproductive organs; (iv) reduced vulnerability to oxidative stress in female organs.
Perinatal and developmental exposure to BPA can induce oxidative stress and lipid peroxidation; these effects are conserved across animal species and humans and can disrupt metabolic and endocrine physiological functions in the reproductive system. Often, oxidative stress appears as the primary abnormality in an organ; then, inflammation will develop and further exacerbate oxidative stress and vice versa.
Despite differences among doses, duration, model systems, and measured outcomes, growing and compelling evidence reports that wide variety of doses or concentrations of BPA (in vivo and in vitro or human exposure) promote ROS generation and alteration of redox balance, inducing mitochondrial dysfunction and the modulation of cell signaling pathways related to oxidative stress. The induction of oxidative stress by BPA can act in a dependent- or independent-manner to its endocrine and metabolic disrupting properties that may induce marked reproductive effects during prenatal, perinatal, and postnatal exposure or in adulthood. At the same time, the effect of BPA can be particularly severe when the exposure is associated with other risk factors, such as poor diet, metabolic impairment, and concurrent diseases.
To date, different antioxidant approaches have been identified to counteract BPA-induced damage in male and female reproductive systems. These agents improve male and female fertility, reducing multiple oxidative stress markers, lipid peroxidation, or DNA damage and restoring the antioxidant defense. However, new experimental and clinical studies are warranted to establish the specific molecular mechanisms underlying the protective effect of these antioxidant substances on oxidative stress and inflammation responsible for systemic and organ-specific toxicity of this ubiquitous xenobiotic. Further studies on antioxidants are needed to better define their usefulness in several gender-dependent reproductive dysfunctions and to establish their optimal dosage and scheme of treatment for counteracting BPA toxicity.