Endocrine disruptors are known to cause harmful effects to human through various exposure routes. These chemicals mainly appear to interfere with the endocrine or hormone systems. As importantly, numerous studies have demonstrated that the accumulation of endocrine disruptors can induce fatal disorders including obesity and cancer. Using diverse biological tools, the potential molecular mechanisms related with these diseases by exposure of endocrine disruptors. Recently, pathway analysis, a bioinformatics tool, is being widely used to predict the potential mechanism or biological network of certain chemicals. In this review, we initially summarize the major molecular mechanisms involved in the induction of the above mentioned diseases by endocrine disruptors. Additionally, we provide the potential markers and signaling mechanisms discovered via pathway analysis under exposure to representative endocrine disruptors, bisphenol, diethylhexylphthalate, and nonylphenol. The review emphasizes the importance of pathway analysis using bioinformatics to finding the specific mechanisms of toxic chemicals, including endocrine disruptors.

To analyze their different mechanisms, comprehensive analysis is required. As a typical comprehensive analysis in biology, pathway analysis can be efficient. Today, the quality and quantity of biological data are increasing. To process the large amount of data, a new field called bioinformatics has developed. Pathway analysis is one of bioinformatics tools whose goal is to identify the pathways significantly impacted. Pathway analysis has become the first choice for gaining insight into the underlying biology of genes and proteins that are differentially expressed. Through pathway analysis, researchers can find the direct interactions, find the shortest paths, functionally group pathways, find the shortest pathway between selected genes/proteins, and find Primary/Secondary/Tertiary relationships. Finally, it can infer molecular mechanisms.

Endocrine disruptors are chemicals that interfere with the hormone systems and produce adverse developmental, reproductive, neurological, and immunological effects in mammals. Endocrine disruptors can be found in many products including plastic bottles, metal food cans, detergents, flame retardants, food, toys, cosmetics, and pesticides. Although limited scientific information is available on the potential adverse human health effects, concern arises because endocrine disrupting chemicals presenting in the environment at very low levels have been shown to have adverse effects. Some research shows that these substances are also adversely affecting human health in similar ways, resulting in reduced fertility and increased progression of some diseases, including obesity, diabetes, endometriosis, and some cancers. These chemicals have also been referred to as endocrine modulators, environmental hormones, and endocrine active compounds. 1 Because the hazards of endocrine disruptors are well known, a more complete study of the molecular mechanism is needed.

With identifying evidence of the harmfulness of endocrine disruptors, their use is heavily restricted and the human body burden of the endocrine disruptors decline. The first step in reducing the body burden is eliminating or phasing out their production. The second step toward lowering human body burden is awareness of and labeling of foods that are likely to contain high amounts of endocrine disruptors. Endocrine disruptors were first discussed as a global issue at the ‘Rio summit’ in 1992. ‘Agenda 21’ was adopted at the summit and focuses on environmental safety management is of toxic and dangerous agents. Since that summit, endocrine disruptors have been regulated by various international organizations ( ). 11 BPA has been controlled by restricting policy in countries around the world. This policy applies to World Health Organization as well as individual countries. 12 The use of some phthalates has been restricted in the European Union (EU) since 1999 and in the United States since 2008. 13 , 14 NP also is prohibited in the EU, the USA, and in other countries. 7 , 15

Several environmental substances including heavy metals which seem to act as endocrine disruptors are reported. Numerous studies have demonstrated that tissues including kidney, liver and testis are sensitive to heavy metals toxicity. 8 Heavy metals are released into the environments from industrial and agricultural products. 9 Particularly, exposure through tobaccos is major source to human exposure with heavy metals. 10

The increase in household products containing pollutants and the decrease in building ventilation indoor air to become a significant source of endocrine disruptor exposure. 4 In addition, endocrine disruptors accumulate from a variety of routes in the body ( ). 5 Phthalates are easily released into the environment and it is known that exposure of phthalates in the air induce asthma in children. 6 NPs are produced industrially, naturally, and by the environmental degradation of alkylphenol ethoxylates. It originates principally from the degradation of NP ethoxylates which are widely used as industrial surfactants. 7

Food is the major route of exposure to endocrine disruptors ( ). According to an article reported by Schecter et al., 2 a total of 32 food samples from three major supermarket chains in Dallas were contaminated with polybrominated diphenyl esters (PBDEs). In this study, PBDEs are detected mainly in fish, meat, and dairy products. BPA exposure also occurs through diet, including contaminated food and water. 3

MOLECULAR MECHANISMS WITH ENDOCRINE DISRUPTORS

In general, endocrine disruptors are thought to affect an organism’s endocrine system. Additionally endocrine disruptors are known to affect other diseases such as cancer and obesity ( ).16–18 In the case of obesity, endocrine disruptors are called obesogens. This chapter deals with molecular mechanisms of endocrine disruptors already studied.

1. Inhibition of endocrine receptors Endocrine disruptors can affect every level of the endocrine system. First, they can disrupt the action of enzymes involved in steroidogenesis. These enzymes can be inhibited, as can the enzymes involved in metabolism of estrogens. For instance, some polychlorinated biphenyl (PCB) metabolites inhibit sulfotransferase, resulting in an increase of circulating estradiol.19 The transport of hormones is also targeted by certain compounds capable of interacting with the binding sites of sex hormone binding globulin, thus competing with endogenous estrogens.20 The most studied mode of action of endocrine disruptors is their ability to bind and activate endocrine receptors (ERs) in target tissue.16 However, it is of note that the two ERs mediate distinct biological effects in many tissues, such as the mammary glands, bone, brain, and vascular system in both males and females. Therefore, because ERα and ERβ show different tissue distribution and distinct physiological functions, endocrine disruptors could display agonist or antagonist activity in a tissue-selective manner or during development. Considering the significant differences in structural features and relative ligand binding affinity of the ER subtypes, endocrine disruptors can induce distinct conformational changes in the tertiary structure of the ERs, affecting the recruitment of cofactors differently. These interactions between ERs and coactivators/corepressors are critical steps in ER-mediated transcriptional regulation and consequently the modulation of the expression of ER-target genes. Moreover, the genistein effect is often tissue specific, depending on numerous factors such as the expression of specific cofactors, the ERα/ERβ ratio, and the level of expression of certain intracellular kinases, including cytoplasmic tyrosine kinases. Genistein has been reported to have both proliferative and anti-proliferative effects in cancer cells.21 Endocrine disruptors generally act in 100 to 1,000 folds greater concentrations than estradiol but can have additive or synergic effects with endogenous estradiol or when they are present in combination.22 Furthermore, the ability of some endocrine disruptors to act as agonists in certain tissues and as antagonists in the others leads to the development and use of selective ER modulators, in particular for anti-hormonal treatments, such as tamoxifen and raloxifene. Some endocrine disruptors can also affect the ER non-genomic pathways and induce an endocrine disruption.23 For instance, a study performed on structurally different endocrine disruptors showed that at high concentrations, BPA and diethylstilbestrol are able to activate ERs via the activation of mitogen-activated protein kinase and phosphotidyl inositol 3-kinase in breast cancer cells. In addition, the activation of protein kinase C (PKC) by some endocrine disruptors has been observed.24 Interestingly, PKC has been reported to modulate ERα transcriptional activity.25 Therefore, synergic or additive effects between these pathways to combine the activation of ER signaling could be possible. Cadmium is well known as a endocrine disruptor which affects the synthesis and/or regulation of several hormones.26,27 Indeed, cadmium affected progesterone synthesis in JC-410 porcine granulose cells and activated the ERα and/or mimic estrogen in different tissues (e.g., uterus and mammary gland) and breast cancer cell lines.28–30 Cadmium regulates androgen receptor gene expression and activity in LNCap cells, a hormone-dependent human prostate cancer cell line, and also mimics androgenic effects in rats and mice.31 In male rodents, it is well established that cadmium significantly alters the circulating levels of several hormones (e.g., testosterone, luteinizing hormone [LH], and follicle-stimulating hormone [FSH]).32 Moreover it decreased steroidogenic acute regulatory protein, LH receptor and cyclic adenosine monophosphate (cAMP) levels in the testis.33 Cadmium affected the circadian pattern release of noradrenaline, a regulator of hypothalamus hormone secretion, which resulted in changes in the daily pattern of plasma testosterone and LH levels.32 In addition, plasma levels of pituitary hormones (e.g., LH, FSH, prolactin, and adrenocorticotropic hormone) were modified after cadmium exposure.34

2. Obesity mechanism Endocrine disruptors play another role in obesity and the metabolic programming of obesity risk. Their action predicts the existence of chemical obesogen, molecules that inappropriately regulate lipid metabolism and adipogenesis to promote obesity. Although until now, data have been scant; some epidemiological and in vitro studies suggested a link between environmental chemical exposure and obesity.35 The endocrine disruptors inducing obesity are called obesogens and have been reviewed.35 Obesogens have been shown to target transcription regulators found in gene networks that function to control intracellular lipid homeostasis as well as proliferation and differentiation of adipocytes. The major group of regulators that is targeted is a group of nuclear hormone receptors known as peroxisome proliferator activated receptors (PPARα, δ, and γ). These hormone receptors sense a variety of metabolic ligands, including lipophilic hormones, dietary fatty acids, and their metabolites, and, depending on the levels of these ligands, control transcription of genes involved in balancing the changes in lipid balance in the body.36 In order to become active and properly function as both metabolic sensors and transcription regulators, the PPAR receptors must heterodimerize with another receptor known as the 9-cis retinoic acid receptor (RXR). The RXR receptor, itself, is the second major target of obesogens next to the PPAR receptors.35 The central regulator in this process is the PPARγ, which associates with the RXR receptors and binds DNA targets as a heterodimer to directly regulate the expression at the transcriptional level.37 PPARγ is considered to be the master regulator of adipogenesis and plays key roles in nearly all aspects of adipocyte biology.38 It was recently proposed that PPARγ may function in adipogenesis without the need to be activated by a ligand. When the ligand binding domain of PPARγ was mutated such that the receptor was unresponsive to known agonists, the ability of preadipocytes to differentiate into adipocytes in cell culture was unaffected.39 The most reasonable interpretation of these data is that either PPARγ can act as an unliganded transcription factor to mediate adipogenesis, or that an as yet unknown endogenous ligand is being produced in response to the induction cocktail. Several endocrine disruptors are known to affect PPARγ activity and induce adipogenesis. Notable among these are organotins such as tributyltin and triphenyltin and certain phthalates.40,41 Triorganotins and phthalates also have the ability to induce adipocyte differentiation in a variety of cell culture models.42,43 Other endocrine disruptors are known to promote adipogenesis, but probably do not act through PPARγ. These include BPA, organophosphate pesticides, monosodium glutamate, and PBDEs.44,45 PCBs bind the aryl hydrocarbon receptor in adipocytes and increase adipogenesis.46 BPA and alkylphenols stimulate adipogenesis in 3T3-L1 cells, and BPA diglycidyl ether was recently shown to induce adipogenesis in human and mouse bone marrow-derived mesenchymal stem cells.17 Although several endocrine disruptors are associated with adipogenesis and obesity in animal models, tributyltin is the only endocrine disruptor known to cause in utero effects on adipocytes via activation of PPARγ.47 Prenatal exposure to tributyltin in mice led to a substantial increase in the amount of triglycerides in newborn tissues which normally have little to no fat at all, although, the experiments did not distinguish whether more lipid was stored in existing cells, more cells were produced, or both.43 Other endocrine disruptors are likely to promote adipogenesis, in utero, although it is possible that this is secondary to broader metabolic imbalances. For instance, certain PCBs and PBDEs reduce thyroid function as does the antibacterial compound triclosan.48,49 The mechanisms of action are not completely certain, but possible modes include interference with thyroid hormone synthesis, transport, metabolism, or clearance.50