Tributyltin and the Female Hypothalamic-Pituitary-Gonadal Disruption

Abstract

The hypothalamic-pituitary-gonadal (HPG) axis is the principal modulator of reproductive function. Proper control of this system relies on several hormonal pathways, which make the female reproductive components susceptible to disruption by endocrine-disrupting chemicals such as tributyltin (TBT). Here, we review the relevant research on the associations between TBT exposure and dysfunction of the female HPG axis components. Specifically, TBT reduced hypothalamic gonadotropin-releasing hormone (GnRH) expression and gonadotropin release, and impaired ovarian folliculogenesis, steroidogenesis, and ovulation, at least in part, by causing abnormal sensitivity to steroid feedback mechanisms and deleterious ovarian effects. This review covers studies using environmentally relevant doses of TBT in vitro (1 ng—20 ng/ml) and in vivo (10 ng—20 mg/kg) in mammals. The review also includes discussion of important gaps in the literature and suggests new avenue of research to evaluate the possible mechanisms underlying TBT-induced toxicity in the HPG axis. Overall, the evidence indicates that TBT exposure is associated with toxicity to the components of the female reproductive axis. Further studies are needed to better elucidate the mechanisms through which TBT impairs the ability of the HPG axis to control reproduction.

tributyltin, endocrine-disrupting chemicals, female, hypothalamic-pituitary-gonadal axis, ovary

Impact Statement

• The studies show that exposure to the pollutant tributyltin is linked with abnormal female reproductive function by altering GnRH expression, gonadotrophin release, ovarian folliculogenesis, steroidogenesis, and ovulation.

• The toxic effects of TBT on the hypothalamic-pituitary-gonadal axis may explain how TBT exposure reduces female fertility.

Tributyltin

Tributyltin (TBT) is an organometallic pollutant that belongs to the family of organotin compounds (Graceli et al., 2013; Hoch, 2001). Organotin compounds are synthetic chemical tetravalent derivatives of tin (IV), with a general formula of R(4-n)SnXn, where R represents organic substituents and X can be a halide, anion, or an organic group linked covalently through a heteroatom (O, N, S, Cl, etc.) (Hoch, 2001) (Figure  1). TBT is the most studied organotin. It has many industrial uses, such as in the manufacturing of antifouling paints, as a preservative in woods, papers, and textiles, a component in broad-spectrum biocides (agricultural fungicides, etc.), and a stabilizer in plastic production (Antizar-Ladislao, 2008; Fent, 1996). TBT easily enters the environment because of improper disposal of TBT and its metabolites, such as dibutyltin and monobutyltin. Accumulation of TBT in the environment is followed by accumulation in different levels of the food chain (Graceli et al., 2013; Podratz et al., 2020).

Figure 1.

Five of the most common tributyltin chemicals.

In the environment, TBT has a degradation half-life of days to months in water and up to several years in sediment (ECHA, 2004). Its bioaccumulation does not follow environmental equilibrium partitioning (Graceli et al., 2013; Meador, 2000). In the aquatic ecosystem, organisms can be exposed to TBT via the water column, sediments, and ingestion of contaminated food. In the terrestrial ecosystem, organisms may be exposed via TBT-contaminated sediments, and by ingestion of TBT-contaminated food or water (Antizar-Ladislao, 2008). Agroindustry progress beginning in the 1960s increased the use of TBT and TBT contamination in the environment across the world, leading to several deleterious effects of TBT exposure in numerous systems, predominantly in the sea ecosystem (Graceli et al., 2013; Podratz et al., 2012). For example, TBT acts as a potent molluscicide and indirectly leads to imposex development, which is an abnormal endocrine syndrome with the imposition of male sex characteristics in female mollusks (Fent, 1996). Due to the toxicological effects of TBT in target and nontarget species, the use of TBT-based antifouling paints was globally banned in 2008 by the International Convention on the Control of Harmful Antifouling Systems (Maciel et al., 2018). Despite some reduction in TBT pollution, this chemical is still detected in sea sediment (0.5–20,000 ng/g), seawater (0.1–281,8 ng/l), and seafood (0.15–19,757 ng/g), representing an environmental issue along coastal areas under the influence of maritime activities such as Europe (Furdek et al., 2012; Keithly et al., 1999), Asia (Cao et al., 2009; Chen et al., 2019; Garg et al., 2011), South Africa (van Gessellen et al., 2018), Oceania (Haynes and Loong, 2002; Keithly et al., 1999; Roach and Wilson, 2009), North America (Garcia-Romero et al., 1993; Keithly et al., 1999; Tallmon, 2012), and South America (Abreu et al., 2021; Batista-Andrade et al., 2018; Castro et al., 2018). Thus, residual TBT contamination is still an environmental and health concern (Figure  2).

Figure 2.

TBT levels were reported in sea ecosystems (sediment and water) and seafood on different continents. TBT, tributyltin; APP, antifouling paint particle.

For the general human population, the major route of exposure to TBT is ingestion through the consumption of contaminated water, beverages, and food, particularly seafood (Azenha and Vasconcelos, 2002; He et al., 2021). The European Food Safety Authority (EFSA) and the United States Environmental Protection Agency (US-EPA) established a human tolerable daily intake (TDI) of 250 and 300 ng/kg/day of TBT, respectively. The TDI was derived by applying a 100-fold safety factor to the mouse from the no-observed-adverse-effect levelof 25 μg/kg/d (EFSA, 2004; US-EPA, 1997; Vos et al., 1990). TBT (155 ng/ml) was detected in human blood from study participants in Michigan, USA (Kannan et al., 1999) and in the human placenta (0.32 ng/g) in Finland between 1997 and 1999 (Rantakokko et al., 2014). Thus, studies have shown that TBT levels in human tissue range between 0.01 and 85.0 ng/g (Kannan et al., 1996; Nielsen and Strand, 2002; Rantakokko et al., 2013; 2014).

The toxicologic consequences of TBT exposure are complex and involve many signaling pathways in various endocrine organs and species (de Araújo et al., 2018a; Graceli et al., 2020; He et al., 2021; Marques et al., 2018). TBT acts as an endocrine-disrupting chemical (EDC) and reproductive toxicant (de Araújo et al., 2018; Podratz et al., 2020; Sena et al., 2017). EDCs are defined by the Endocrine Society and the World Health Organization (WHO) as “an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations” (WHO, 2013; Zoeller et al., 2012). The structural similarities between EDCs and endogenous hormones lead to EDCs being able to interfere with the regular function of hormone receptors (Delfosse et al., 2015; Zoeller et al., 2012). EDCs can act as agonists or as antagonists in genomic and nongenomic nuclear receptor pathways, nonsteroidal receptors, and ion channels, leading to improper molecular signaling, abnormal growth, inflammation, enzyme activity, and transcriptional coactivation (Gore et al., 2015). Importantly, EDCs can interfere with endogenous hormone synthesis and degradation to alter hormone levels and action time. Recent studies have also identified that TBT can follow an epigenetic mode of action by altering genomic methylation and causing histone modifications, often leading to deleterious transgenerational consequences (Chamorro-Garcia et al., 2017).

Hypothalamic-Pituitary-Gonadal Axis

TBT exposure has been shown to interfere with the hypothalamic-pituitary-gonadal (HPG) axis (Sena et al., 2017). The HPG axis is the principal modulator of the mammalian reproductive system, remaining silent throughout childhood and resuming activity just before pubertal changes (Palmert and Boepple, 2001). In the hypothalamus, a complex neuronal network culminates in the release of gonadotropin-releasing hormone (GnRH), a neuropeptide that was identified as the master regulator of HPG axis (Abreu and Kaiser, 2016; Herbison, 2016; Roux et al., 1997). GnRH neurons originate from the olfactory placode and migrate to the hypothalamic regions that project their axons to the median eminence to release GnRH in a pulsatile manner. In females, GnRH is released into the hypophyseal portal circulation to stimulate the secretion of the gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the basophil cells of the anterior pituitary. The gonadotropins stimulate the release of sex steroids (androgens, estrogens, and progesterone), ovarian follicular development, production of oocytes, and ovulation. The sex steroids also exert either negative or positive feedback at the level of the hypothalamus to regulate the release of GnRH or at the anterior pituitary to control gonadotropin release (Abreu and Kaiser, 2016; Zuckerman, 1956) (Figure  3). In addition, serum gonadal sex steroids such as estrogen and progesterone are responsible for the development of female external genitalia, along with other physical changes (Abreu and Kaiser, 2016).

Figure 3.

Schematic representation of female HPG axis and TBT effects. Neuronal hypothalamic networking to control GnRH release in pituitary portal system to modulate gonadotrophin (LH and FSH) release to modulate ovarian folliculogenesis and steroidogenesis. Sexual steroids negative/positive feedback mechanisms to control hypothalamic GnRH and gonadotropins release. TBT targets (GnRH neurons, gonadodrophs, feedback mechanism, folliculogenesis, steroidogenesis, granulosa and theca cells, ovulation, and CL formation). ARC, arcuate nucleus; AVPV, anteroventral periventricular; FSH, follicle-stimulating hormone; GABA, gamma-aminobutyric acid; GnRH, gonadotropin-releasing hormone; KISS, kisspeptin; LH, luteinizing hormone; NPY, neuropeptide Y; + and −, stimulatory and inhibitory stimulus, respectively.

Kisspeptin (KISS) is an important neuropeptide that modulates GnRH (Dumalska et al., 2008; Lehman et al., 2013). KISS neurons project to GnRH neurons and are directly, but differentially responsive to estrogen (Lehman et al., 2013; Smith et al., 2005). In the last several years, a complex and finely regulated neural network overseeing GnRH activity has been progressively unveiled. Neurons producing KISS, neurokinin B, glutamate, gamma-aminobutyric acid, neuropeptide Y, and other neuropeptides have been identified as major regulators of GnRH secretion (Naulé et al., 2021). These neuronal systems undergo several morphological, molecular, and functional changes during development, particularly as puberty approaches (Herting and Sowell, 2017).

The ovaries are responsible for female gamete production and maturation, as well as the production and release of the sex steroid hormones that control sexual development and proper reproductive function (Puttabyatappa and Padmanabhan, 2018). The ovarian cortex is an outer part containing the ovarian follicles (the ovarian functional units) and is enclosed in a capsule called tunica albuginea. A fully developed follicular unit contains the female gamete (ovum), and the surrounding cells comprised of theca cell layers (the androgen producing cells) and granulosa cells (estrogen producing cells). The ovarian medulla is an inner part that contains vascular elements (blood and lymphatic vessels) (Puttabyatappa and Padmanabhan, 2018).

The bipotential primordial germ cells, which form the ova in the females, first mitotically divide to form oogonia, which undergo meiosis I to form the primary oocytes during fetal life (Hummitzsch et al., 2015). Primary oocytes stay arrested in the diplotene stage of meiosis until ovulation. The process of oocyte maturation is closely associated with follicular development. Upon the formation of primary oocytes, the oocytes are surrounded by a squamous cell layer called pregranulosa or follicular cells to form the primordial follicles (McGee and Hsueh, 2000). In mammals, the ovaries contain a finite set of primordial follicles at or around birth that constitutes the follicular pool or ovarian reserve. The ovarian reserve will become reduced throughout the lifespan of the female mammalian (gestational development, birth, puberty, reproductive life, and senescence) (Gougeon et al., 1995). This reduction has been linked with programmed cell death or apoptosis (Escobar et al., 2011). The stages of follicle growth (primordial, preantral, antral, and preovulatory) and their ovulation are dependent on the harmonious action of gonadotropin and sex steroids in the HPG axis (Liu and Hsueh, 1986). As detailed, below, this review highlights the effects of TBT exposure on various aspects of the HPG axis.

MATERIAL AND METHODS

Articles used in this current review were selected from the PubMed database without exclusion based on publication year of study or positive or negative findings. We utilized the following inclusion criteria: work evaluating direct TBT exposure effects on the female reproductive system (the HPG axis) using mammalian models in vitro and in vivo. We included searches on any TBT dose, time, and life stage of exposure. Only data that described the relationship between relevant TBT dose or exposure and effect were included in the review. Each original study was critically evaluated for appropriateness of the journal quality, indexing, model, the use of adequate controls (negative and positive), the range of doses tested, methodology, and statistical methods. In addition, data analyzing the combined effect of more than one type of EDC and TBT were not included. Conference proceedings and articles not written in English were excluded. To the extent possible, we made efforts to be consistent in the description, discussion, and integration of findings in the effects of TBT exposure on the female reproductive system.

TBT EFFECTS ON THE FEMALE REPRODUCTIVE AXIS

TBT exposure can interfere with the normal function of the female reproductive organs in the HPG axis, such as the hypothalamus, anterior pituitary, and ovary (de Araújo et al., 2018; 2018a; Marques et al., 2018) (Figure  3). An integrative and critical summary of studies demonstrating the direct effects of TBT on the female HPG axis is described below.

TBT Effects on the HPG Axis

A few studies reported that TBT exposure is associated with abnormalities in the HPG axis (Table  1). Our group reported that female rats exposed to 100 ng/kg/d TBT, a dose lower than the TDI, for 15 days had reduced hypothalamic GnRH mRNA expression and exogenous KISS responsiveness, decreased basal and surge LH levels, reduced exogenous GnRH responsiveness, as well as decreased expression of both estrogen receptor (ER) (ER-alpha and ER-beta) in the pituitary compared with controls. In addition, TBT exposure caused lower responsiveness to estrogen negative feedback, as evidenced by TBT-induced diminished GnRH mRNA expression and serum LH and FSH levels in ovariectomized rats, leading to reduced fertility (Sena et al., 2017). Further, Kariyazono et al. (2015) reported that exposure to TBT chloride (1, 5, and 10 mg/kg) from gestational day (GD)15 to 20 leads to increased beta-subunit of LH (LH-beta) mRNA expression in the fetal pituitary.

Table 1.

Summary of Direct TBT Effects Specifically on the Female Reproductive HP Axis

Model	Exposure Time/Form of Exposure	Dosage	Age at Analysis	Endpoint(s)	References
In vivo
Pregnant rat/fetuses	GD15/single dose by gavage	10 mg as Sn/kg/corn oil	GD20-fetuses	↑ LH-beta mRNA in pituitary	Kariyazono et al. (2015)
Adult rat	Gavage by 15 days	100 ng/kg/d	12-week-old	↓ Basal and surge serum LH ↓ GnRH mRNA in hypothalamus ↓ Responsiveness to GnRH and KISS ↑ Serum testosterone ↓ Serum estrogen ↓ ER-alpha protein in pituitary ↓ ER-beta protein in pituitary	Sena et al. (2017)
In vitro
Mouse hypothalamic GnRH neuronal (GT1-7) cell line	For 1 h		—	↑ Oxidative damage and apoptosis ↓ Cell viability ↑ Phosphorylation levels of Ask1/Mkk7/Jnk, Ap-1, c-Jun, and Cl-Casp3 ↑ Bax mRNA expression ↓ Bcl-2 mRNA expression	Li et al. (2021)

Abbreviations and symbols: ↑, increased; ↓, reduced; d, day; ER, estrogen receptor; GD, gestational day; GnRH, gonadotropin-releasing hormone; KISS, kisspeptin; LH, luteinizing hormone.

Collectively, these data suggest that TBT disrupts the proper functioning of the female HPG axis at least in part by causing abnormal KISS and GnRH action involved in the hypothalamic and pituitary control of reproduction (Sena et al., 2017).

Similarly, studies have reported that TBT exposure affects the HPG axis using in vitro models (Li et al., 2021; Mellon et al., 1990). TBT chloride exposure (1 mg/l) for 1 h increased oxidative damage and apoptosis in a GnRH-secreting neuronal cell line (GT1-7 cells) (Mellon et al., 1990). Specifically, TBT exposure increased the pro-apoptotic markers Annexin V, caspase-3 protein, Bax expression, and downregulated expression of the anti-apoptotic marker Bcl-2 (Li et al., 2021). TBT also activated the MAPK pathway by increasing levels of phosphorylated ASK1/MKK7/JNK, and AP-1 pathways (Li et al., 2021). In addition, estrogen exposure protected GT1-7 cells from TBT-induced apoptosis by normalizing the MAPK and AP-1 pathways (Li et al., 2021). Taken together, the current literature presents some evidence that TBT exposure interferes with both hypothalamic and pituitary function in vitro and in vivo (Table  1).

Despite the known deleterious effects of TBT on the hypothalamus and pituitary, only 2 studies have explored the effect of TBT directly at the level of the GnRH neurons, gonadotropin release, or LH-beta expression in female rats (Sena et al., 2017, Kariyazono et al., 2015). Although KISS acts as an important player to modulate GnRH release, it is unlikely to be the sole mediator of this process, as a complex neuronal network was reported to participate in GnRH neuron regulation (Naulé et al., 2021). Future studies need to clarify the effect of TBT on this neuronal network and determine how TBT affects GnRH pulsatility, the ability of gonadotroph cells to release LH and FSH, and the regulation of estrogen-positive feedback mechanisms. In addition, TBT is an obesogenic chemical that causes metabolic complications (obesity, hyperleptinemia, hypoadiponectinemia, insulin resistance, liver lipid accumulation, etc.), which occur with the TBT-induced HPG axis abnormalities (Sena et al., 2017). Studies should determine if these TBT-induced metabolic effects are causes and/or consequences of TBT-induced HPG abnormalities.

Several studies reported that TBT exposure impairs nuclear and membrane-associated receptors such as the retinoid X receptor (RXR), peroxisome proliferator-activated receptor (PPAR) gamma, angiotensin-II receptors, and ERs (Penza et al., 2011; Bertuloso et al., 2015; Ceotto Freitas-Lima et al., 2018). Despite these findings, only one study has explored the effect of TBT on ER signaling directly at the level of the pituitary and linked changes in ER signaling to female reproductive function (Sena et al., 2017). Although Sena et al. (2017) demonstrates that TBT exposure reduced ER-alpha and ER-beta protein expression in the pituitary, it did not change ER expression in the hypothalamus. However, Sena et al. (2017) did not address if the TBT-induced reduction in ERs in the pituitary is linked with low serum LH levels or changes in ER expression in gonadotrophs. Additionally, abnormalities in GnRH neuron responsiveness to estrogen negative feedback could be altered by TBT agonist actions in ER signaling in the HPG axis, but this has yet to be explored. Future studies need to clarify the pathways by which TBT impairs GnRH neurons, gonadotrophs, and their relationship in the control of the reproductive axis. In vitro protocols, such as 3D cell culture chips that can evaluate the GnRH cell-gonadotroph cell relationship in response to TBT and elucidating the role of their principal modulator players (eg, ERs, KISS) still need to be evaluated.

TBT Effects on the Ovary

TBT is known to adversely affect the ovary in female rodent models. Specifically, TBT disrupts ovarian germ cells, ovarian reserve, steroidogenesis, folliculogenesis, ovulation, and corpora lutea (CL) formation in mice and rats (Kishta et al., 2007; Podratz et al., 2012; Sena et al., 2017) (Table  2 and Figure  3). Kishta et al. (2007) reported that exposure to TBT (10 and 20 mg/kg) from GD0-19 or GD8-19 leads to gonocyte abnormalities such as lipid droplet accumulation, irregular and dilated endoplasmic reticulum, and reduced gonocyte number in female rat offspring. Female mice exposed in utero to TBT (10, or 100 µg/kg body weight-bw/d) from GD6 through the period of lactation had early puberty onset (determined by earlier vaginal opening and first vaginal estrus), impaired estrous cyclicity, abnormal ovarian morphology, a reduction in CL number, and an increase in the presence of cystic follicles when they reached adult age (Si et al., 2012).

Table 2

Summary of Direct TBT Effects Specifically on the Ovaries

Model	Exposure Time/Form of Exposure	Dosage	Age at Analysis	Endpoint(s)	References
In vivo
Mouse Kun Ming	GD6-lactation/gavage	10–100µg/kg bw/d	PND21- 49, 77, 112, 152	↑ Weight (trend) Impaired estrous cyclicity (prolonged D or E) ↓ CL (trend)	Si et al. (2012)
Mouse	PND49-56/gavage for 12 days	500 ng/kg/d	PND61–68	↓ Reserve (primordial and primary follicles) ↓ CL and antral follicles Irregularity in morphology ↑ Cystic and antral follicles ↓ Number of healthy follicles Impaired estrous cyclicity (more days in M-D) ↑ Inflammation (macrophage)	Sarmento et al. (2020)
Rat	GD0-19 or 8-19/gavage	10 and 20 mg/kg	GD20-fetuses	↑ Germ cells with pyknotic nuclei ↓ Germ cells/area ↑ Apoptotic cells	Kishta et al. (2007)
Rat	PND21/gavage for 7 days	1–10 mg/kg bw	PND28	↑ PPAR-gamma, Ap2, Cd36, Pepck, Tnf-alpha, resistin, and Tnfr1 mRNA expression ↑ Apoptotic follicles ↓ Weight	Lee et al. (2012)
Rat	PND85/gavage for 15 days	100 ng/kg/d	PND85–110	↑ Tin levels ↓ Weight and size Impaired estrous cyclicity (less days in P and persistent M-D) ↓ CL ↑ Fibrosis ↓ ER-alpha protein expression ↑ Apoptosis and proliferation	Sena et al. (2017)
Rat	PND85/gavage for 30 days	100 ng/kg/d	PND115	Impaired estrous cyclicity (more days in M-D) ↓ Weight ↑ CYP11A1 protein expression ↓ CYP19 mRNA expression ↓ CL ↑ Cystic and atretic follicles ↑ Apoptotic cells in antral follicles ↑ Pro-caspase 3 protein expression ↑ PPAR-gamma protein expression ↑ CEBPβ and Lipin-1 mRNA expression ↓ ER-alpha protein expression ↑ Lipid drops accumulation ↑ Cholesterol levels ↑ Inflammation (neutrophils) ↑ Oxidative stress ↑ Fibrosis	de Araújo et al. (2018)
Rat	PND1/gavage for 16 days	10 and 100 ng/kg/d	PND56	↓ CL and antral follicles ↑ Cystic and atretic follicles ↓ Size Impaired estrous cyclicity (more days in M)—100 ng ↑ SLC27A6, RXR-alpha, RXR-beta1, RXR-beta2, RXR-gamma, and PPAR-gamma protein expression—100 ng ↑ ADCY2, FSHR, CYP19, GATA4, and SET protein expression	Yang et al. (2019)
In vitro
Granulosa-like tumor (KGN) cell line	For 48, 72 h or 7 days	20 ng/ml		↓ CYP19 activity ↓ Estradiol production—7 days ↓ CYP19 mRNA—48 hs	Saitoh et al. (2001)
Bovine COCs and follicular GCs	For 4–24 h	50 pM, 100 pM, and 50 nM		↑ Progesterone—50 pM ↑ HSD mRNA expression—50 pM ↓ Estradiol—50 pM ↓ CYP19 mRNA expression—COC—50 pM ↑ Estradiol—CG—100 pM ↑ CYP19 mRNA expression—CG—50 pM ↑ FSH—CG—50 nM	Schoenfelder et al. (2003)
Primary theca cells of human, sheep, cow, pig, and mice	For 12–72 h	1 or 10 ng/ml		↑ Star mRNA expression—10 ng ↑ Srebf1 and Abca1 mRNA expression ↓ Cholesterol	Pu et al. (2019)

Abbreviations and symbols: ↑, increased; ↓, reduced; CL, corpora lutea; COC, cumulus oocyte complex; CYP19, aromatase; CYP11A1, cytochrome P450, family 11, subfamily A, polypeptide 1; d, day; D, diestrus; E, estrus; ER, estrogen receptor; FSH, follicle-stimulating hormone; FSHR, follicle-stimulating hormone receptor; GD, gestational day; GC, granulosa cell; HSD, hydroxysteroid dehydrogenase; M, metestrus; PND, postnatal day; PPAR-gamma, proliferator-activated receptor-gamma; RXR, retinoid X receptor.

Our group reported that TBT exposure (100 ng/kg/d for 15 days) caused abnormal ovarian morphology, including increased apoptosis and proliferation in theca and granulosa ovarian cells and an increase in collagen deposition, suggesting that TBT exposure causes ovarian fibrosis and leads to irregular ovarian function in adult female rats (Podratz et al., 2012; Sena et al., 2017). We also observed important changes in ovarian steroidogenic enzymes, with TBT-induced reductions in aromatase mRNA and increases in CYP11A1 protein expression (de Araújo et al., 2018). Additionally, we observed that TBT exposure caused abnormal ovarian fat tissue accumulation (adipogenesis), with increased cholesterol levels, lipid droplet accumulation, and increased expression of PPAR-gamma protein expression, and mRNA for C/EBP-beta, and Lipin-1. TBT exposure also caused ovarian cystic follicles, inflammation (neutrophils and macrophages presence), oxidative stress, fibrosis, and reduced ER-alpha protein expression (de Araújo et al., 2018).

Lee et al. (2012) also showed that TBT (1 mg/kg TBT for 7 days) altered expression of ovarian adipogenic genes such as PPAR-gamma and AP2 mRNA expression, leading to irregular ovarian function in 3-week-old female rats. Female rats exposed to TBT (10 and 100 ng/ kg/d) from postnatal day (PND)1–16 exhibited irregular estrous cyclicity, disturbed ovarian development (with fewer CLs and antral follicles and increased presence of atretic and cystic follicles), hyperandrogenism, high serum LH levels, and reduced levels of serum sex hormone-binding globulin. Further, TBT exposure increased the RXR/PPAR signaling pathway and other proteins associated with androgen syntheses, such as follicle-stimulating hormone receptor (FSHR), CYP19, and the transcription factor GATA-4 in rats (Yang et al., 2019). Collectively, our data, as well as data from other studies, suggest that TBT exposure leads to similar features of polycystic ovary syndrome, such as hyperandrogenism, cystic ovarian follicles, irregular estrous cyclicity, high LH levels, obesity, abnormal lipid profile, glucose metabolism, and insulin resistance (de Araújo et al., 2018; Podratz et al., 2012; Sena et al., 2017; Yang et al., 2019).

Recently, Sarmento et al. (2020) showed that TBT exposure (500 ng/kg/d TBT for 12 days) induces irregular estrous cyclicity and abnormal ovarian morphology, linked with a reduction in primordial and primary follicle numbers in adult female mice, suggesting that TBT exposure causes ovarian reserve depletion. In addition, TBT exposure caused a reduction in antral follicles, CLs, and total healthy ovarian follicles together with an increase in cystic follicles and serum testosterone levels as well as ovarian inflammation, as evidenced by the increased presence of macrophage and mast cells (Sarmento et al., 2020).

In addition, a few studies report TBT affects the ovary using in vitro models (Saitoh et al., 2001; Schoenfelder et al., 2003) (Table  2). In a human granulosa-like tumor cell line KGN, TBT exposure for 72 h at 20 ng/ml (∼6 nM) suppressed both aromatase activity and aromatase gene expression by 30% compared with control (Saitoh et al., 2001). In addition, exposure with more than 1000 ng/ml TBT was very toxic to the KGN cells and caused immediate cell death within 24 h, whereas 200 ng/ml was found to cause KGN apoptosis (Saitoh et al., 2001). Podratz et al. (2012) observed a dose-dependent mutagenic effect of TBT (2, 0.2, and 0.02 pg/ml for 3 h) using Chinese hamster ovary cells (CHO-K1). Interestingly, the doses of TBT in the study were nearly pharmacologically equivalent to the doses that reportedly induce imposex in female gastropods (Bryan et al., 1986). TBT exposure for 4, 12, and 24 h (50 pM) also increased progesterone levels, reduced testosterone and estrogen levels, reduced expression of aromatase CYP19 and 3 beta-HSD mRNA in bovine cumulus-oocyte complex cultures (Schoenfelder et al., 2003). In addition, 50 pM TBT, but not 100 pM TBT, blunted the LH-mediated estrogen production in follicular granulosa cell culture (Schoenfelder et al., 2003), showing the contradictory and complex effect of TBT on ovarian steroidogenesis.

Recently, Pu et al. (2019) evaluated the effects of TBT exposure on cholesterol trafficking, luteinization, and steroidogenesis in ovarian theca cells of 5 species (human, sheep, cow, pig, and mouse) using environmentally relevant doses of TBT (1 or 10 ng/ml) for 12, 24, 36, 48, or 72 h. TBT upregulated Star and Abca1 in ovine cells and SREBF1 mRNA in theca cells. TBT also reduced intracellular cholesterol and upregulated ABCA1 protein expression; however, it did not alter testosterone or progesterone production. RXR antagonist and RXR-alpha knockdown studies demonstrate that the effect of TBT is partially through RXR signaling. The effects of TBT on ABCA1 and STAR expression were recapitulated in all 5 species. TBT stimulated theca cell cholesterol extracellular efflux via the RXR pathway, triggering a compensatory upregulation of STAR that regulates cholesterol transfer into the mitochondria and SREBF1 for de novo cholesterol synthesis. Similar results were obtained in all 5 species evaluated and are supportive of TBT’s conserved mechanism of action across mammalian species. Collectively, the existing data demonstrate that exposure to TBT can lead to ovarian dysfunction using in vivo and in vitro models (Table  2). Future studies need to clarify the pathways by which TBT affects ovarian follicle growth, ovulation, CL formation, the steroidogenic relationship between theca and granulosa cells, and determine if TBT changes ovarian sensitivity to gonadotropin actions or ovarian ER signaling.

LIMITATIONS, STRENGTHS, AND FUTURE DIRECTIONS

This review relied on a search strategy designed to address the direct associations between TBT exposure and female reproductive dysfunction in the HPG axis. The current state of knowledge has some important limitations. First, few studies have integrated the direct effects of TBT exposure on the HPG axis. Second, this review did not include epidemiological studies as we did not find consistent data in the literature. The lack of human epidemiological data on TBT exposure and the causal link between TBT exposure and reproductive abnormalities development stands out as a limitation in the field.

The strengths of this review include the description of only the direct effects of TBT and its damaging actions on HPG axis function. Thus, we can focus solely on these data to elucidate what is needed to clarify the effects of TBT on the female HPG axis. This review also helps to manage and focus on future studies as we continue to establish the safe dose of TBT exposure.

Considering the toxicity data resulting from studies on TBT exposure in the female reproductive system, in conjunction with the limited data on the direct effects of TBT, it is critical for future studies to better delineate the results to be made available. These future studies need to encompass more results in the HPG axis, both in vitro and in vivo as what we have so far is very limited. More in vivo studies including species other than rodents could also help increase knowledge about TBT toxicity. Epidemiological studies using different types of samples and at different times (in pregnancy, after birth, preconception, etc.) will be relevant. Although TBT use in antifouling paints was globally banned in 2008 by the International Convention on the Control of Harmful Antifouling Systems, important residual contamination is still being observed worldwide. Thus, TBT environmental monitoring (water, sediment, and seafood) and translational evaluation will be relevant to clarify environmental and health concerns.

CONCLUSIONS

As this review illustrates, TBT is an environmental chemical that can act as an EDC through a variety of mechanisms. The diversity of pathways and precision of biological hormone actions in the reproductive system make the female reproductive system particularly susceptible to disruption by exogenous agents. In addition, the wide range of possible phenotypes and endpoints makes the integration of studies on TBT to understand mechanisms a difficult task. Evidence shows TBT affects the function of the female reproductive HPG axis. Specifically, TBT reduced hypothalamic GnRH expression (GnRH neuron as target) and release of pituitary LH (gonadotroph as target), leading to lower KISS and GnRH agonist responsiveness. TBT also caused abnormal HPG axis function linked with irregular ovarian steroidogenesis (steroidogenesis as target) and follicular development (folliculogenesis as target), lower ovarian reserve, CL number (ovulation and CL formation as target), abnormal estrogen and testosterone levels (steroidogenesis as target), atretic and cystic follicles, inflammation, oxidative stress, and fibrosis. All these changes maybe explain the TBT-induced reduction in fertility in the female rodent (Sena et al., 2017). Future interdisciplinary studies should recognize the prevalence of nonmonotonic dose-response curves and the importance of low-dose studies. In addition, mechanistic and epidemiological studies are needed on TBT and other chemical pollutants together suspected that have EDC actions.

FUNDING

Fundação de Amparo à Pesquisa e Inovação do Espírito Santo (FAPES) (572/2018 and 140/2020); the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (304724/2017); the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Code 00). The Biochemistry kit support from Bioclin-Quibasa Research Program (Bioclin Research Program).

DECLARATION OF CONFLICTING INTERESTS

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Author notes

Kayke L. Barbosa, Raquel S. Dettogni, and Charles S. da Costa contributed equally to this study.