Endocrine Disrupting Chemicals: Epigenetic Relevance and Mechanisms

Genistein contributes to protection against the development of cancer. Through the demethylation of genes p16, O-6-methylguanine-DNA methyltransferase (MGMT), GSTP1, retinoic acid receptor beta (RARB) and mutL homolog 1 (hMLH1), that exert tumour suppressor function, genistein inhibits angiogenesis and proliferation, two major cellular processes. Rajvir Dahiya and his colleagues have found that genistein affects various miRs, such as miR-1260b, causing histone modification and demethylation of DNA and, consequently, upregulating secreted frizzled related protein 1 (SFRP1) and SMAD4 in Pca cells ^{86, 87}.

Shahana Majid et al., demonstrated a new role for genistein in prostate cancer. BTG3 is a potential tumour suppressor gene that is downregulated in prostate malignancy. Genistein has been found to reactivate the hypermethylated BTG3 gene by the demethylation of its promoter. More specifically, it prompts the demethylation of CpGs, the decrease of DNMT, the inhibition of the activity of methyl-CpG binding domain protein 2 (MBD2) and subsequent induction of prior silenced BTG3 gene ^{88, 89}.

A possible mechanism that regulates gene transcription is the methylation of DNA and the acetylation of histones ^{90, 91}. Treatment with genistein prompts the hypomethylation of hTERT in the E2F-1 sites of recognition ⁸⁹. Studies have shown that telomerase activity can be suppressed by genistein as a result of transcriptional regulation of hTERT ⁴⁴. Moreover, hTERT promoter can be restructured in the presense of genistein in the chromatin level in MCF-7 cell lines ⁹².

The evidenced hypomethylation in the E2F-1 sites leads to increased attachment of the repressor to this binding site, suggesting a cooperation between genetics and epigenetics in the modulation of hTERT transcription when this phytoestrogen compound is present ⁸⁹.

Thus, genistein could be combined with an epigenetic modulator in order to reach its full epigenetic potential in controlling telomerase’s action in breast malignant cells ⁸⁹.

Neonatal exposure in DES/Genistein evokes an adult-onset modification in uterine epigenome. Nsbp1 contribution in chromatin restructure and transcriptional function accompanied with the evidence that Nsbp1 transcription is defined by an epigenetic mechanism mediated by estrogen signals, encourages the theory of Nsbp1 participation in tumorigenesis after neonatal exposure to DES/genistein ⁹³.

Normally, in mice without genistein-treatment, Nsbp1 expression is weak prior to puberty. Estrogenic hormones, more specifically estradiol, cause the silence of Nsbp1. However, neonatal exposure to genistein and the co-occurrence of ovarian hormones cause Nsbp1 to get expressed actively throughout life. Neonatal genistein/DES treatment is evidenced to result in promoter hypomethylation of Nsbp1. Consequently, through a mechanism that possibly prevents the silencing of Nsbp1, uterine cancer incidence could increase at some point afterwards ⁹³.

In 1938, a nonsteroidal estrogen, called DES, was synthesized in the United States to prevent miscarriage and other pregnancy complications. It was used as an estrogen for decades and its effects continue to be evident ⁹⁴. DES was administered to women all around the world in nonstandardized quantities and forms such as pills and liquid creams. Many women were unaware that they were taking DES in the U.S., Europe, and Australia ^{95, 96}. Although DES has not been prescribed for women during pregnancy in the USA for 40 years, adverse outcomes continue to occur in women exposed in utero. Given the above, continued monitoring for established and unexpected adverse outcomes seems prudent ⁹⁶.

DES was used until 1971- in which year the Herbst paper appeared in the medical literature and the U.S. Food and Drug Administration (FDA) issued a warning about the ³² relationship between the development of adenocarcinoma and the use of DES in pregnancy in adolescent girls and young women whose mothers had taken DES while they were pregnant. Moreover, DES had been widely used in agriculture to chemically castrate male chickens and increase weight gain in cattle. In addition, until the 1970s when the effects of DES on female offspring were reported, its use in the sheep and cattle industry was prominent. Thus, DES could be characterized as an environmental estrogen ⁹⁷.

Despite the information that DES was a human carcinogen operating through a previously unknown mechanism, it continued to be used in clinical trials. DES reduces testosterone levels facilitating the treatment of advanced prostate cancer. Moreover, it is used in the treatment of postmenopausal breast cancer ^{97, 98}.

As a xenoestrogen, DES has a similar receptor affinity to the naturally occurring estrogen (estradiol) activating the ER-alpha (ERa). It has a significant oral activity as an estrogenic substance and invokes a strong hormonal response equivalent to an injected dose of the steroidal estradiol-17β ⁹⁹. DES is a lipid soluble, well absorbable compound that reaches a peak concentration within 20–40 min following the oral administration ¹⁰⁰. DES contributes to the prevention of miscarriages as well as bleeding resulting from preterm labor and gestation ^{98, 101}. DES metabolism may proceed via two routes. One route can produce hormonally inactive metabolites such as β-di- enestrol. The other pathway could produce compounds, such as DES- epoxide, which still retain a considerable amount of estrogenic activity. Evidence for such a bilateral metabolism of certain substances has been documented ⁹⁹. If DES undergoes such metabolism, then large doses or repeated ingestion could produce compounds in the body that are hormonally active and/or carcinogenic. It has a half-life of 3–6 h and is primarily excreted in urine ¹⁰⁰.

In breast cancer, a miRNA gene, MIR-9-3, is usually hypermethylated. miR-9-3 is an apoptosis modulator. Consequently, the silencing of the gene through an epigenetic mechanism could lead to the proliferation of cancer cells ¹⁰². One study indicated that breast progenitor cells' exposure to DES could cause this epigenetic repression. DES downregulates the target miRNA, namely miR-9-3 through both genomic and nongenomic pathways resulting in mediation of gene transcription. Continuation of this signaling could leave a mark able to be inherited through remodeling of chromatin and subsequent DNA methylation in the promoter region of this miRNA ¹⁰².

Hoxa10 gene expression can be modified by deviant DNA methylation after in utero DES exposure. DES, through a molecular mechanism that includes gene methylation, induces developmental changes in the programming of the reproductive tract ¹⁰³. DNA methylation caused by persistent administration of DES in utero can lead to permanent changes in Hox genes expression. The mechanism of the above mentioned epigenetic effect includes DES-mediated up-regulation of DNMTs ¹⁰³.

There is the so called 'second hit model' which is concerned with the impact of neonatal DES exposure to uterine adenocarcinoma development in adulthood ¹⁰⁴. Under normal circumstances, Nsbp1 acts in the uterus only during the period of development. The first hit is neonatal exposure to DES which causes hypo or hypermethylation in a treatment-specific way that results in altered Nsbp1 expression in prepuberty. Prepubertal ovariectomy on mice has been found to lead to the establishment of these methylation patterns through aging. The secondary hit results in elevated Nsbp1 expression, not only before puberty, which is related to the development of uterine adenocarcinoma. This hit involves additional estradiol presence during puberty which causes Nsbp1 promoter to shift to hypomethylated condition in the models which were estrogen exposed immediately after birth. It seems that there are underlying epigenetic events that are yet to be revealed by future trigger points ¹⁰⁴.

Estradiol modulates the transcription of HOTAIR. This antisence transcript participates in silencing of genes and plays an important role in breast cancer. More specifically, it has been reported that in breast cancer cell lines the administration of BPA and DES prompts the expression of this long non-coding RNA. HOTAIR expression is upregulated in rat mammary glands upon treatment with BPA and DES, as well. According to Luciferase assay, the promoter EREs of HOTAIR (to a greater extent ERE2 and ERE3) are triggered by DES and BPA, suggesting their potential involvement in endocrine disruption mediated via BPA and DES. The attachment of the MLL family of histone methylases and ERs (MLL1 and MLL3) to HOTAIR promoter EREs induced by BPA and DES causes chromatin modification (H3K4-trimethylation, histone acetylation), which is crucial for gene activation. ERs knockdown leads to decreased DES and BPA mediated HOTAIR expression. In summary, it is demonstrated that HOTAIR expression is epigenetically induced by DES and BPA, without the presence of estrogen being necessary ^{104, 105}.

Bisphenols (BPs) constitute a broad group of chemicals. BPA (2,2-bis-(4-hydroxyphenyl)propane;) is largely used as a monomer in the production of polycarbonates (PC), of epoxy resins and as an additive in other polymeric materials ^{106, 107}. Each year the atmospheric burden of BPA is estimated to be 100 tons ^{108, 109}.

Epoxy resins mark the introduction of BPA to mainstream production. They were initially applied in food and drink industry to protect the products from the metal packaging since epoxy resins have been used as internal protective coatings in cans for food and beverages and in drinking water storage tanks. BPA is used to make polycarbonate plastics, such as baby bottles ^{108, 110}, and has plenty more applications in electronic devices and even in dentistry for sealants ^{108, 111, 112}. Increased exposure to BPA may be caused, inter alia, by environmental pollution e.g. dust, air, drinking water, surface water, wastewater, leachate from landfills ^{113, 114, 115, 116, 117, 118, 119}.

According to existing studies, the route of human exposure to polycarbonate plastics is through epoxy resins and other substances related to foods and drinks ^{116, 120, 121} Indeed, canned food is responsible for 10-40% of BPA intake as indicated by one risk assessment study ^{106, 121}. Following the worldwide intensive discussion triggered by toxicological findings, alternative options have been provided to replace this chemical. BPA substitutes, though, have been proved to induce delayed toxicity ¹⁰⁶. There is a huge amount of information about BPA biological effects. However, there have been discrepancies in outcomes among the various studies, so investigation of the issue is still in progress ¹²².

BPA is a chemical acting as a xenoestrogen ¹²³. It binds to ERs ¹²⁴ exhibiting a 10-fold higher affinity to ERb than to ERa ¹²³. The endocrine disrupting properties of BPA were discovered in pursuit of synthetic estrogens by Dodds et al. ¹²⁵. However, as indicated by the same authors, DES, which had already been synthesized, had a much stronger estrogenic potential than BPA which was defined as weak ¹⁰⁶. Despite its environmental tenuous estrogenic activity there is not a safety threshold preventing cellular response stimulation, even in BPA concentrations lower than the ones required so that attachment to the ERs is achieved ¹²⁶. Thus, classical ER-dependent nuclear pathways is only one of the ways through which BPA elicits its effects ¹²².

BPA applied in utero or neonatally affects body weight, increases susceptibility to prostate and breast cancer and prompts changes in the reproductive function. BPA affects, to a greater extent, epigenetic mechanisms in early development i.e., stem cell stage. There is evidence supporting the enhancement of yellow coat colour of viable yellow agouti (Avy) in mouse offspring, after maternal exposure to BPA ¹²⁷. The change in progeny coat color was induced by reduced methylation of CpGs (cytosine-guanine dinucleotide) in Agouti gene and, more specifically, upstream of this gene originating from an intracisternal A particle retrotransposon ¹²⁷.

PDE4D4, which plays a regulatory role in cyclic adenosine monophospate breakdown, was analyzed in detail to determine its epigenetic response to estrogen exposure neonatally. 5′-flanking CpG Island of PDE4D4 was the methylating target of BPA and estradiol ¹⁰⁷. This specific methylation complex is generally required for epigenetic modulation. However, of great significance is the fact that the relationship between methylation level and gene expression is inversely correlated in the tissues of prostate. Thus, the progressive hypermethylation of PDE4D4 promoter in physiological aging prostates results in decreased gene expression. Interestingly, animals exposed neonatally to estradiol or BPA maintain a hypomethylated status, thus engendering increased PDE4D4 expression throughout life. Cell line studies have confirmed the model of transcriptional modulation and methylation of PDE4D4 where NbE-1 cells, which normally originate from immortalized epithelial ones, show reduced expression of the gene when PDE4D4 is hypermethylated. On the other hand, AIT cells, which are tumorigenic and originate from dorsal prostate, exhibit hypomethylating status. To sum up, there is a possible contribution of prostate’s epigenome in epithelial cell conversion ¹⁰⁷.

PBDEs are widely used as additives to plastics, furniture, textiles, electric cable insulation, water and sewage pipes, office equipment and electronic devices and for fire retardancy reasons i.e., to prevent and retard the spread of fire ¹²⁸. Therefore, PBDEs move relatively easily from these items into the environment ¹²⁹. They can be found in house and office air and dust, in dryer lint, and in air intake filters ²⁷. Their presence has been evidenced in landfills, sewage treatment plants, in the manufacture and recycling procedures of PBDEs ^{130, 131}. Developing countries carry the heavy burden of e-waste recycling collected from industrialized countries ¹³². Human beings and wildlife have been widely affected from PBDE production. Infant and toddler bodies contain three to nine times more PBDEs compared to adults because of maternal milk consumption and dust inhalation. North America is heavily burdened with PBDEs compared to Asia and Europe. The most abundant congener are tetra-BDE or BDE-47, hexa-BDE or BDE-153, and deca-BDE or BDE-209. They can enter human bodies via many routes. Human beings can be exposed to PBDEs through ingestion, food chain or dust and inhalation ^{27, 133}.

Regarding their chemical structure, PBDEs have two phenyl rings linked by an ether bond. The position and number of the bromine atoms results in 209 possible compounds, referred to as PBDE congeners. Technical PBDE products are produced by brominating diphenyl ether when a catalyst is present ^{134, 135}.

In humans, exposure to PBDEs can lead to disturbances in thyroid hormones. The associations appear to be life stage and dose dependent. It has been suggested that PBDEs possibly exhibit hyperthyroid effects ¹³⁶. Nonetheless, among Canadian women aged 30–51 years, higher levels of exposure were associated with elevated prevalence of hypothyroidism ¹³⁷. According to a study among US women, those with the highest levels of exposure to PBDEs were more likely to report a previous diagnosis of thyroid disease. This association though, was stronger among postmenopausal women ¹³⁸. The mechanisms of PBDE toxicity are complex and have not been fully resolved. PBDEs’ toxic effects are possibly mediated through some nuclear hormone receptor pathways, in particular the pathways involving thyroid hormone receptor (TR), ER and aryl hydrocarbon receptor (AhR), AR and progesterone receptor (PR) ¹³⁹.

Darnerud and his colleagues have reported that PBDE-209 increases the incidence of hepatocellular carcinomas ⁹. Studies have indicated that PBDE-209 can affect global DNA methylation decreasing their level. Moreover, it may affect secondary messengers and cause oxidative stress. These actions are potential causal factors inducing PBDE-209 neurotoxicity. Researchers concerned with the interaction between discrete levels of PBDE-209 and hippocampal neurons have reported that rats exposed neonatally to PBDE-209 could differ in cell’s apoptotic rate and viability, oxidative stress, global methylation levels and molecular signaling and intracellular second messenger. More specifically, PBDE-209 could decrease global levels of gene DNA methylation, especially in the 10 and 30 g/ml groups. However global levels of gene DNA methylation were increased when the concentration of PBDE-209 was elevated ⁹. This result may be attributed to DNMT adaptability. Decreased global gene DNA methylation caused by PBDE-209 might be a cause of carcinomas ¹³⁸.

Tumor necrosis factor alpha (TNFa) is a gene involved in inflammations and its methylation status gets affected by exposure to environmental PBDEs, even perinatally ¹⁴⁰. Maternal exposure to PBDE47 results in CpG site promoter methylation of this proinflammatory gene, according to the evidence provided by the Boston Birth Cohort (BBC). Measurement of PBDE levels in a number of paired samples in maternal blood and cord blood at birth has demonstrated alteration in TNFα promoter methylation ¹⁴⁰. As indicated, immune system’s functions can also be affected by exposure to PBDE ^{141, 142, 143}. TNFa, a cytokine, originates from macrophages, fibroblasts, B and T cells and is involved in immune-related diseases ^{144, 145, 146} and cancer, as well ¹⁴⁷. PBDEs are a potential causal factor of ectopic TNFa gene expression in the progeny. TNFa promoter in girls is more susceptible to hypomethylation after maternal exposure to this endocrine disruptor. Thus, TNFa hypomethylated status is inversely correlated with cord blood TNFα protein quantities i.e., they increase. In total, there is evidence that the progeny’s immunological reaction could be epigenetically reprogrammed through in utero PBDE exposure.

Autism is a neurodevelopmental disorder which could be caused by a combination of external factors and genetic susceptibilities. Rett syndrome (RTT) shares autistic traits, and the MECP2 factor plays a regulatory role in the epigenome of this X-linked autism spectrum disorder (ASD) ¹⁴⁸. A pattern of ‘two hits’ was suggested to explore the interplay between a single -BDE-47 exposure and a specific gene-MECP2. MECP2 transcription leads to a protein that recognizes DNA methylation and is recruited to regulate neuronal function and maturation ^{149, 150, 151}. More specifically, mutations in MECP2 cause RTT ¹⁵². Daily perinatal exposure of mutant Mecp2308/1 dams to BDE-47 caused DNA hypomethylation in the progeny brain accompanied by decreased gregariousness following a motive that was independent from genotype. In addition, the exposure adversely affected the survival of the offspring and the knowledge acquisition process of fully developed females. Nonetheless, MECP2 mutation in the offspring of perinatally exposed dams led to a reversion of learning abilities which were accompanied by higher levels of DNMT3a. These results demonstrate a complex epigenetic and behavioral interaction ¹⁴⁸.

TCDD, a potent environmental toxicant, evokes adverse health effects in a pattern defined by the gender, the tissue, the life stage and the species. It also prompts modification in the transduction of estrogen signals. Adverse health effects include hepatotoxicity, teratogenicity, cancer, severe anorexia-like wasting. It could even lead to death ^{153, 154, 155, 156, 157}. Dioxin adverse health effects are induced by AHR ¹⁵⁸. TCDD has strictly non-commercial use, occuring as a contaminant in specific chlorophenoxy acid and chlorophenols herbicides ¹⁵⁹. Moreover, TCDD can originate from combustion incidences and, to a greater extent, it may be formed when specific metal catalysts, such as copper, are present ^{160, 161}. Some of the largest TCDD production facilities include waste disposal burning, manufacture of metals, and natural fuels or wood combustion ¹⁶².

Dioxins, like other chlorinated forms, accumulate in food source sequences (bioaccumulation) ¹⁶⁰. TCDD and structurally related halogenated aromatic compounds are industrial chemicals and combustion byproducts that have been identified in fish, wildlife, humans, and throughout the environment ¹⁶³. Because of the long biological half-life and the low water solubility of dioxins, food chain is susceptible to water derived accumulation of high quantities of TCDD ¹⁶⁴.

TCDD is degenaration resilient and lipophilic, and its properties make it able to accumulate in the adipose tissue, as well ¹¹. TCDD has both estrogen agonist and antagonist activity. Characteristically, in females it exerts AHR-dependent antiestrogenic effects in their reproductive tract, such as restrained estrogen-induced elevation in uterine weight, gene expression reactions and DNA composition ¹⁰. TCDD, also, promotes estrogen-like effects. More specifically, it increases the ER DNA-binding potential without the presence of estrogen in the uterus of the rodent being required ¹⁶⁵. Additionally, TCDD administration to MCF-7 exerts estrogen-like cell cycle phase conversion and impacts on mitogenic activity ¹⁰. TCDD also prompts tumors which are estrogen-dependent ¹⁶⁶ and enhances endometriosis in TCDD-charged women and model organisms ^{167, 168, 169, 170}.

TCDD-exposed preimplantation embryos were transferred to unexposed recipients (mice). On Embryonic Day 14, the aforementioned TCDD exposed fetuses gained less weight than the unexposed controls. The levels of expression of the imprinted genes Igf2 (insulin-like growth factor 2 gene) and H19 decreased after preimplantation embryos’ exposure to TCDD. It is evidenced that in the imprint region H19/Igf2, the methylation was increased in TCDD exposed fetuses and embryos compared to the controls. Moreover, TCDD caused an elevation in methyltransferase activity. Thus, exposure to the environmental toxicant TCDD at the preimplantation stage affects imprinted genes altering their DNA methylation state, influences their expression level in early development, and the altered methylation status is maintained throughout the fetal stage ¹⁷¹.

TCDD possibly mediates in the development of endometriosis. This endocrine disruptor could participate in transcription activities at variable levels, even in the epigenetic one ¹⁷². More specifically, it could act in an epigenetic fashion, modifying consequently the normal processes induced by AHR pathways. This could affect the reproductive machinery both by altering the homeostasis of the peritoneal microenvironment, and provoking progesterone resistance ¹⁷².

It has also been reported that progesterone sensitivity can be debilitated by TCDD as it prompts epigenetic alterations with the contribution of toxicant-exerted inflammatory processes ¹⁷³. The methylation level in PR gene of female mice uterine samples was analyzed. The results showed that PR was partially methylated in 60% of the F1 female extracted tissues (in utero exposure to TCDD). Similar tissues of controlled mice were largely unmethylated. F3 females had also methylated PR in 40% of these animals ¹⁷³. Thus, silencing PR with epigenetic mechanisms induced by TCDD exposure could be the potential causal factor in inheritable infertility phenotype as well as an associated risk factor of spontaneous preterm birth (sPTB) in mice ¹⁷⁴. There is an obvious correlation between hypermethylation reported in these specific mice and the infertility rates in F1 and F3 mice which had earlier been examined ¹⁷⁴.

PCBs were initially synthesized in the 1930s to be used as lubricants in capacitors and adaptors and, also, as pesticides, hydraulic fluids or plasticizers ^{178, 179}. PCB production has been forbidden since 1970 but they still are a crucial health risk factor for the public since they are still persistent and widely dispersed in the environment, as well as in the food chain ^{180, 181}. The main sources of dispersion are degenerating structures and waste disposals, as well as the simultaneous PCB production through industrial activities such as paint pigments ¹⁸².

So far, 209 possible congeners of PCBs with different biological activities have been recorded. These congeners could be categorized into dioxin- and non-dioxin-like, DL and NDL respectively, depending on their molecular structure. Both of these types are abundant in the environment. DL PCBs activate AHR by attaching to it, just like dioxins do, whereas the so called NDL PCBs elicit minor AHR activity ¹⁷⁸. Evidence indicates NDL PCBs dominance upon DL PCBs as an environmental burden and in human tissues ^{183, 184}. There is evidence about the involvement of the AHR ^{178, 185} as well as other receptors in supporting the role of PCBs as EDs and interfering with calcium homeostatic mechanisms ^{186, 187}. Several adverse health effects in both human beings and animals, such as birth weight reduction, disruption of reproductive system development, immune dysfunction, and altered brain development, have been correlated with exposure to PCBs during intrauterine life ^{188, 189, 190, 191, 192}.

PCBs are organic chlorine compounds, highly lipophilic and chemically stable. They undergo limited catabolism after absorption and accumulate in the liver and adipose tissues. In addition, PCBs and their metabolites are easily transferred to the fetus through the placenta ^{193, 194}. Thus, PCB exposure in gestation could be considered as inheritable ^{135, 195, 196, 197}. Thyroid effects may are congener- or metabolite-dependent ¹³⁵. PCBs can disturb the homeostatic control of the thyroid system through a number of levels, in a pattern that can be characterized as congener-dependent. Each PCB metabolite plays a distinct role ¹³⁵. There are studies suggesting that PCBs are inversely correlated to thyroid hormones amount. A few studies suggest a neutral relationship between thyroid hormones and increased plasma PCB with a non obvious interplay ¹⁹⁸. Other studies suggest that serum PCBs and thyroid hormone levels change in the same direction ^{199, 200}.

PCBs lead to toxicity in fetals when exposed prenatally and predispose children to poor health. When fetal cells get exposed to PCBs during the period of pregnancy they become susceptible to cytotoxic effects as well as genotoxic ones. Cell lines were examined at specific time points during a period of time, in order for global methylation levels and other cellular processes to be evaluated in response to PCB administration ²⁰¹. For 120 days, sheep embryonic fibroblasts (SEF) as well as amniocytes (SA) were exposed to limited quantities of PCBs. Fetal cells got hypermethylated by PCBs. The hypermethylation, though, was irreversible even when blocking the administration of Aroclor 1254 (A1254 is a commercial mixture of PCBs) to these cells. Global genome methylation in SEF in response to PCB treatment remained high even after discontinuation of PCB use in the 60th day, for up to one month. This indicates that fetal cell exposure to PCBs prompts irreversible epigenetic changes that could follow and affect growth process until adulthood ^{3, 28}.

PCBs can modify epigenetic mechanisms ^{3, 5}. Prenatal exposure to PCB compounds decreases DNMT expression and activity in the liver of the offspring ²⁰².

It has been reported that exposure to reconstituted PCB compounds during gestation prompts Jarid1b expression (a demethylase) as well as Sirtuin1 (SIRT1) (a deacetylase) ⁵. Thus, a decrease in their target protein forms in progeny’s liver is demonstrated. The animals exposed themselves exhibited a reduced AR gene expression ⁴.

PFOA is one of the principal PFCs that are widely used in industrial and everyday consumer products including textile coatings, flame retardants, surfactants, food packaging items and lubricants ²⁰⁷. Moreover, PFOA is derived from the degradation of different fluoropolymers and is not further degenerated from environmental and metabolic breakdown. The United States general population was found to have average serum PFOA concentrations approximately 4 ng/mL ²⁰⁸. In occupationally exposed populations, concentrations may range from 428 to 12,000 ng/mL ²⁰⁹. PFOA is a persistent pollutant, thus, it can gather in tissues via multiple exposure routes ³⁰. PFOA can be found in liver, milk and serum ^{210, 211, 212}. It is also likely to be detected in blood, primarily, and in the kidney and liver as well, according to studies on monkeys and rats. Liver is a significant target organ ^{213, 214, 215}. As PFOA is widely spread and chemically stable several concerns have been raised with regard to the recorded PFOA accumulation recorded in aquatic and terrestrial biota, human serum as well as its environmental persistence ^{216, 217, 218}.

Estrogenic signaling probably induces PFOA mediated tumor development in trouts in a peroxisome proliferation independent manner ²⁰⁸. PFOA can cause hepatocarcinogenesis postinitiation to the rainbow trout model, even though this model is peroxisome proliferation insensitive. The molecular profiling of trout liver showed estrogenic gene marks which exhibited an important relation with E2. This mechanism is a distinct one for PFOA mediated cancer development. Thus, tumorigenesis is not attributed to the PFOA’s role as a PPARα agonist or as PP ²⁰⁷.

There has been one epidemiological study evaluating associations between PFAAs and the methylation of DNA. Environmental exposures lead to aberrant methylation motives in circulating fetal DNA. Prenatal exposure to perfluoroalkyl chemicals, such as PFOA, is likely to induce global DNA hypomethylation in cord serum ²¹⁹. A study on newborns reported a negative association between PFOA concentrations in cord blood and total methylated cytosine in DNA isolated from cord serum while no such association was observed with PFOS.

PFOA prompts GSTP altered methylation. It is known that GSTP is hypomethylated in the disease-free tissue. On the other hand, it is transcriptionally silenced and hypermethylated in human liver and prostate cancer cells as well as in leukemia cells ^{220, 221, 222, 223}. Hypermethylation is also common in myelodysplastic syndromes ^{224, 225}. The necessary genes for xenobiotic detoxification in L02 cells have a lack of transcription even though they are derived from a normal cell lineage, similar to immortalized human hepatocyte IHH1 cells ^{30, 226}. In these L02 cells, however, an epigenetic level detoxification response was shown where GSTP promoter region methylation could be elevated from PFOA, whereas global DNA genome methylation remained unchanged ³⁰. The reports in gene-specific as well as global methylation in the specific study are contrasting ^{227, 228, 229}.

GSTP promoter contains two SP1 (specificity protein 1) TF binding recognition sites that are very important cis-elements as they are required for the basal activity of the gene ²³⁰. In the aforementioned L02 model, GSTP promoter CpGs were completely methylated with the exception of the seriate sites region from −152 until −124. Subsequent PFOA treatment prompts CpGs to be further methylated. SP1 binding can be perturbed by cytosine methylation at the recognition sites ^{220, 231}. This indicates that SP1 binding may have a protectful role for CpG sites preventing methylation processes during development ^{220, 232}. PFOA modified the status of methylation in these sites regardless of the protection provided to SP1.

DNMT3A is the potential basal contributor to GSTP excessive hypermethylation since DNMT3B and DNMT1 mRNA transcript levels have been found to be unchanged ^{233, 234}. Thus, it is suggested that PFOA may affect GSTP SP1 sites and through DNMT3A expression alter DNA methylation. However, further investigation should follow to determine this detoxification response pathway ³⁰.

As

As is tasteless, odorless, colorless and even at high concentrations, human exposure to it is not easily avoided ²³⁵. It is a widely distributed metalloid, occurring in rock, soil, water and air. As contamination of the environment from both natural and anthropogenic sources is a major environmental concern due to its persistence and carcinogenic effects on living organisms. Exposure of the general population occurs mainly through oral ingestion via water and food, while occupational exposure to As occurs in workers in the paint, ceramics, pesticide, insecticide, and wood preservatives industry ²³⁶. People can be exposed to it through ingestion of As while dermal As exposure is through contaminated soil. Another route of exposure is the inhalation of suspended arsenical dusts in the air ^{237, 238}. It can be consumed through vegetables and crops which have taken up the As from contaminated soil and irrigation water. As is all over the environment. Once it is present in the environment, it can easily spread from soil to water and vice versa and then to plants, from air to soil through dust and atmospheric deposition and from air to surface and via precipitation to groundwater ²³⁹.

The presence of As in surface soils is either natural or artificial. Anthropogenic activities e.g. mining and metallurgical activities affect the toxic heavy metal content of agricultural soils. Artificial presence refers to the introduction of As by herbicides and long term watering with As burdened irrigation waters. Natural presence refers to As already present within the soils ²⁴⁰.

As activates or inhibits responses, maintaining, probably, the same action mechanism. In low doses As effect is stimulatory. In rat hepatoma cells (EDR3) 0.05–1 μM (6–120 ppb) of As, with the mediation of glycocorticoid receptor (GR), was found to induce gene activation of both the endogenous tyrosine aminotransferase (TAT) and other reporter genes ^{241, 242}. At higher concentrations (1–3 μM), As effects became inhibitory for GR-mediated transcription. In EDR3 hepatoma cells, when As concentrations ranged from 0.045 to 2.7 μM, a similar biphasic response was observed for the mineralocorticoid receptor (MR), AR, and PR ²⁴³.

In the same way, in the NT2 human embryonic carcinoma cells and in the GH3 rat pituitary tumor cells a biphasic response was revealed similar to the one observed for steroid receptors, on genomic-mediated induction by the retinoic acid receptor RAR and the TR ²⁴⁴.

Breast cancer MCF-7 cell line was used to explore As impact on ER ²⁴². As levels (0.25–3 μM), which are normally non-cytotoxic for cells, led to an important inhibition of genomic activation mediated by estradiol, whereas low levels of exposure exhibited no induction effect ²⁴⁴. Similar results were also obtained in an in vivo study with administration of non-cytotoxic concentrations (1–50 μM/kg) of NaAsO2 to chicken embryos, where Davey et al. showed a significant inhibition of ER-dependent gene transcription of the 17b-estradiol (E2)-inducible vitellogenin II gene.

Concerning to estrogenic effect and inhibition of spermatogenesis, As inhibits estradiol binding, possibly by activating ERa through the construction of a high-affinity cluster with the receptor’s hormone binding site. It was shown that in utero As exposure leads to marked alterations in gene expression in fetal liver involving a complex interplay between steroid metabolism and estrogen signaling pathways ²⁴⁵. Overexpression of these estrogen-linked genes, such as X-inactive specific transcript (Xist), Anterior gradient protein 2 homolog (Agr2), Tffl, C-reactive protein (CRP)-ductin, ghrelin and obestatin prepropeptide (Ghrl), keratin 1-19 (Krt1-19), and cytochrome P450 (Cyp2a4,) are indicative of endocrine disruption effects of inorganic As at an early life stage ²⁴².

After reporting spermatogenic degeneration in animals treated with estradiol, Jana et al. (2006) ²⁴⁶ suggested that an estrogen-type mechanism of action may be responsible for As-related reproductive toxicity. The results were repeated in a study conducted on human cancer cells of breast (MCF-7) that were exposed to 0.1, 1, 5, or 10 μM of sodium arsenite ²⁴⁷. Interestingly enough, the arsenite-mediated effects included decreased expression of ERa and increased PR expression, like estradiol does. In addition, inhibition of arsenite estrogen-like activity was observed when an antiestrogen was present, suggesting that ERa is the target receptor. A marked effect was arsenite’s ability to activate ERa even at low concentrations (1 nM).

Ιn a study conducted by Jana et al., male Sprague Dawley rats were administered 5 mg/kg sodium arsenite in drinking water for 6 d/wk, for 4 wk. The findings showed an alteration in the reproductive activity of the treated animals and, more specifically, reduction in testicular mass and decrease in plasma concentrations of testosterone and gonadotropin ²⁴⁶. In vivo studies investigating the As effect on the male reproductive system reported sperm toxicity, inhibition of testicular androgenesis, and reduction in testicular and accessory sex organ weights. Sarkar et al. (2003) ²⁴⁸ administered sodium arsenite to male Wistar rats at variable doses and in the 5- and 6-mg/kg groups was evidenced important decreases in the internal reproductive organs’ weights, in the epididymal spermatozoa, and in concentrations of Follicle-stimulating hormone (FSH), Luteinizing hormone-LH, and testosterone. A similar study has reported a significant reduction in testicular weight, sperm count and motility, and testicular enzymatic activities ²⁴⁹. The aforementioned study of Jana et al. gave similar results ²⁴⁶.

PLA2G2C expression leads to a phospholipase, an enzyme participating in phospholipids’ hydrolysis and their conversion into lysophospholipids and fatty acids ³¹. These lipid mediators’ roles in inflammation, cell growth, and cell death, ²⁵⁰ make them potentially important for cancer progression ^{31, 251}. As is an established skin carcinogen and overexpression of Cyclooxygenase-2 (COX2) is associated with As exposure ^{252, 253, 254}. Interestingly enough, it has been shown that skin carcinogens induce A2 phospholipases (e.g., light ultraviolet B, phorbol ester) which eventually exert synthesis of prostaglandin via COX2 resulting in skin carcinogenesis ^{255, 256}.

The methylation levels seemed to increase at cg04605617 (chr1: 20,501,558) corresponding to higher As exposure. This locus of PLA2G2C is in its first exon. This locus have been mildly associated with elevated PLA2G2C gene expression. PLA2G2C locus methylation was the determining locus that was correlated with both blood and urinary As concentrations compared to other genes examined ³¹.

SQSTM1 is involved in a variety of diseases such as cancer, neurodegenerative diseases, insulin resistance and obesity ²⁵⁷. In vitro evidence indicates that As induces the factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway prompting, consequently, a skin carcinogenesis mechanism ^{258, 259}. Higher As administration has been correlated with reduced methylation at the locus cg01225779 in the SQSTM1 5´ untranslated region (UTR) ³¹. This is a locus of the SQSTM1 gene that has been involved in many diseases such as cancer, obesity, insulin resistance, and neurodegenerative diseases ²⁵⁷. SQSTM1 is translated to a protein that is ubiquitin binding and modulates the activity of kappa-B nuclear factor (NF-κB) signaling pathway ²⁵⁷.

SLC4A4 expression leads to a sodium bicarbonate cotransporter that participates in the modulation of bicarbonate absorption and secretion and intracellular pH as well. SLC4A4 is, also, associated with decreased body weight/size and abnormal ion homeostasis in mice. Gene mutations of SLC4A4 are related to hypertension ²⁶⁰, a health outcome that has been associated with exposure to As ²⁶¹. Higher As was associated with reduced levels of methylation at locus cg06121226. This locus is inside SLC4A4 body and was determinedly correlated with elevated SLC4A4 gene expression ³¹.

Serum immunoglobulins appear to be increased under the presence of As ²⁶², which may induce skin carcinogenesis ²⁶³. The heavy locus of immunoglobulin contains diversity (D), variable (V), constant (C), joining (J) immunoglobulin segments and lymphoma has been correlated with translocations in the specific region ²⁶⁴. Increased methylation at cg13651690 locus was observed in response to higher As exposure. This locus is inside Immunoglobulin heavy locus IGH body ³¹. Additionally, in response to water contamination with As, an important replication of the locus was demonstrated.

PAH are pervasive in our environment and are widely distributed all over atmosphere ²⁶⁵. PAHs is a group of compounds containing a high number of chemicals ubiquitously distributed. The physicochemical properties of PAHs make them highly mobile in the environment, allowing them to distribute across air, soil, and water bodies where their presence is ubiquitous ^{266, 267}. PAHs are genotoxicants and carcinogens and, therefore, pose a serious threat to the health and well-being of humans ²⁶⁸. Interestingly enough, PAHs have traditionally been examined for their genotoxic damage potential while their ability to cause epigenetic transformations has been widely ignored ²⁶⁹. PAHs are released into the environment from both natural and anthropogenic sources ²⁷⁰. Combustion of organic materials causes the widespread occurrence of PAHs. Incomplete burning of fuels, garbage, or other organic substances such as tobacco and plant material are some of PAHs sources ²⁷¹. Likewise, volcanic eruptions and conflagrations can equally contribute to environmental PAH burden ²⁷¹. A risk evaluation of the group of children is of high priority since they constitute a susceptible population, in part because they tend to have higher PAH exposure due to behavioral patterns such as hand-to-mouth behavior, time spent outside in contact with soil and dust, and higher inhalation rate per bodyweight unit ²⁷².

Regarding their chemical structure, PAHs consist of 2 to 7 fused aromatic rings ^{273, 274}. They are made of hydrogen and carbon formed into up to seven fused or condensed aromatic ring forms ²⁷⁵. PAHs' molecular weight is positively correlated with their carcinogenic potential and negatively correlated with their acute toxicity potency. The most potent PAH carcinogens have been identified to include benzo^aanthracene, benzo^apyrene (B^aP), and dibenz^ahanthracene ^{274, 276, 277}. PAHs are highly lipophilic and, therefore, miscible in organic solvents.

PAHs are considered as EDs because they mimick endogenous hormones, interfering consequently with the organisms’ homeostasis. Schraplau et al., 2015 ²⁷⁷ isolated hepatocytes from male Wistar rats exposed to B^aP for 72 h and examined them for thyroid hormone activity and related RNA expression profiles. It was demonstrated that B^aP reduces the metabolic rate by breaking down thyroid hormone. It is indicated that B^aP exposure inhibits steroidogenesis ²⁷⁸.

Moreover, PAHs are either estrogenic or anti-estrogenic depending on the types and locations of the samples. However, the mechanisms responsible for their activities still remain unclear. The analyses of specific PAHs lead to several possibilities such as a direct interaction between PAHs and ERs (estrogenic), AHR-mediated suppression of estrogenic activity (anti-estrogenic), and modulation of estrogen or other signaling pathways (estrogenic or anti-estrogenic) ²⁷⁹.

PAH exposure transplacentally can possibly cause methylation in ACSL3 5′CGI of umbilical cord’s blood cell ³². ACSL3 encodes enzymes fundamental in fatty acid metabolic activities ²⁸⁰. There is a lack of biomarkers enhancing the theory that asthma induction can be affected by transplacental exposure to PAHs. However, there is some preliminary evidence supporting this relationship ²⁸¹. Prenatal monitoring of exposure to airborne PAH was completed for each mother participating in the study in person. The gene ACSL3 showed the highest concordance between the methylation level and the level of its expression. Consequently, ACSL3 was further examined and a significant positive correlation between the methylation status and maternal exposure to airborne PAH was shown ³².

IFNγ is a well known asthma-related gene. The gene methylation status is affected in umbilical cord blood cell in response to maternal PAH exposures ³³. Interestingly enough, in vitro studies on BaP impact, a PAH prototype, on lung cancer and Jurkat T lines, showed induction of IFNγ promoter hypermethylation ²⁸² and decreased IFNγ expression. Cord blood cells' exposure to PAH also prompted the methylation of IFNγ promoter in the offspring of exposed women. An alteration in the methylation status in specific CpG sites of the IFNγ promoter sequence is a potential indicator of exposures to environmental pollutants ³³.

PAH exposure results in altered methylation of the Foxp3 promoter region ²⁸³ which was later associated with cellular functional changes, and an increase in total plasma immunoglobulin E (IgE) levels and impaired Treg function ²⁸⁴. Asthma is associated with impaired function of regulatory T cells (Tregs). These cells maintain tolerance in healthy individuals by exerting suppressive effects on a variety of immune cells involved in allergic disease progression, including activated T cells, eosinophils, basophils, antigen-presenting cells, and mast cells ²⁸⁵.

Anopheles mosquitoes are a public health hazard in tropical and subtropical areas since they can cause malaria ²⁹⁰. Thus, their eradication has been a main goal in these areas. Initially, DDT was used for insect elimination ²⁹¹. However, DDT's hazardous nature was demonstrated by its significant adverse effects in living organisms ^{292, 293}. Interestingly, DDT compound levels throughout the environment and DDT accumulation in living organisms follow a causal relationship that is, also, statistically significant ²⁹⁴.

DDT is an issue still relevant for many reasons. First of all, DDT was widely used in the past affecting persistently women’s health by increasing the risk of breast cancer, even in contemporary society as the generation of women born during this period of extreme DDT use is alive ²⁹⁵. It is worth noting that DDT still remains a controversial issue since it is still in use for malaria control in both Asia and Africa in accordance with WHO recommendations ^{296, 297}. Moreover, DDT is persistent in the natural environment so people worldwide continue to be exposed to it, as indicated by clinically relevant levels in animal tissues and soil ²⁹⁸. Africa, Mexico and China ^{299, 300} are the most susceptible regions since fundamental principles like disposal, storage, and safe use are not always applied, thus ³⁰¹, prompting human exposure and environmental contamination. Furthermore, we should bear in mind that DDT detrimental health effects are here to stay since climate change supports the expansion of malaria vectors ³⁰².

The increase in reports of abnormalities in male sex development in wildlife and humans coincided with the introduction of “estrogenic” chemicals, such as DDT, into the environment. This, probably, reflects the AR-mediated antiandrogenic function of DDT metabolite dichlorodiphenyldichloroethylene (p,p'-DDE) ^{302, 303}.

p,p'-DDE-induced sex development abnormalities in males may are AR-mediated. Although ER is considered to induce these phenotypic alterations, AR- mediated events are also consistent with inhibition. p,p'-DDE has a slight ability to attach to the ER, nonetheless it has been found to restrain androgen attachment to the AR, androgen induced transcription and androgen action in growth stages of male rats ³⁰³.

The male reproductive system is susceptible to the antagonistic effects of DDT metabolite as p,p'-DDE prompts inhibition of AR-androgen binding and subsequent inhibition of transcriptional activity. AR transcriptional activity in cell culture requires a lower concentration of p,p'-DDE compared to the levels that accumulate in the environment, and more specifically, in areas where DDT remains in use or is present in contaminated ecosystems. In the mid-1960s, period during which DDT was widely used, p,p'-DDE levels were found in tissues from stillborn infants suggesting a way of transplacental route. Indeed, in vitro rat model confirmed the risk of perinatal exposure of infants ^{304, 305}.

Along a period of a month, DDT was administered by gavage to male young rats at doses of 0, 0.006, 0.06, 0.6, 6, and 60 mg/kg/day. According to Shutoh Y et al., a low dose of DDT (0.06 mg/kg/day) is considered to be hormetic. It can induce transcriptional down-regulation and DNA hypomethylation in the young hypothalamus ³⁴.

Epigenetic mediated transgenerational inheritance caused by environmental factors, potentially contributes to obesity induction as well as other associated diseases ³⁰⁶. As shown in an in vivo study, exposure of rats to DDT can lead to obesity and altered DNA methylation up to F3 generation. More specifically, sperm DNA methylation was modified in Tubb3, Carm1m and Slc4a4 in a statistically significant relation to the control lineage. The epigenetic transmission was derived from both gender germlines ³⁰⁶.

DDT was inversely associated with global methylation levels in a study conducted on humans ²⁰⁶. Inverse correlations between percent 5-methylcytosine and many of the persistent organic pollutant (POP) concentrations were measured and linear regressions, adjusting for cigarette smoking and age, showed statistically significant inverse linear relationships mainly for the Alu assay for p,p′-DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane), p,p′-DDE (1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene😉, and other POPs and PCBs ²⁰⁶.

Vz

Fungicides have a variety of applications such as on landscapes, golf courses, industrial activities, lawn turf, and woody ornametals; thus, they can be readily spread to water supplies ³⁰⁷. Vz (RS)-3-(3,5-Dichlorophenyl)-5-methyl-5-vinyloxazolidine-2,4-dione is a systemic dicarboximide fungicide effective in the control of diseases by inhibiting spore germination caused by Botrytis cinerea, Sclerotinia sclerotiorum, and Moniliniam spp. Dicarboximide fungicides have been widely used in Europe to protect grapes, fruits, vegetables, hops, ornamental plants, and turf from fungi damage ³⁰⁸. In the United States, Vz is registered for use on several fruits and vegetables, ornamental plants, and turfgrass. Vz is not persistent in the field but is rather degraded to several metabolites in the soil and/or within the plants themselves ³⁰⁸. After application, DDTs are readily accessible to living organisms and untreated foods since their distribution is induced by volatilization and air circulation ³⁰⁹. Tap water cannot eliminate human exposure to DDT since it cannot remove it from produces ¹⁵⁸.

Vz is an environmental compound acting as an anti-androgen. Anti-androgens exhibit a mechanism which is determined by interaction with the hormone receptor ^{158, 310}. Two Vz metabolites can be totally attached to monkeys, rats and humans, as well as AR ³¹¹. The effects of antiandrogens to sexual development are serious and can even affect male reproductive system by inducing hypospadias, a situation where the urinary opening is not at the appropriate location of the penis ^{312, 313, 314, 315, 316}.

Vz and its active metabolites, M1 and M2, principally bind competitively to AR, thereby antagonizing the binding of natural androgens to the receptor ^{310, 317, 318}. Reduced anogenital distance, nipple retention, hypospadias, cryptorchidism, decreased sex accessory gland growth, induction of prostate inflammation and reduced sperm production at adulthood are abnormalities of androgen-regulated sexual differentiation in male offspring caused by administration of Vz to pregnant rats ^{318, 319, 320}.

Transgenerational inheritance of acquired traits can be derived either from a chromosomal or from an epigenetic modification. A brief prenatal exposure to vinclozolin or methoxychlor (which is an estrogenic chemical) during the sex determination stage has been found to cause adult decreased spermatogenic capacity and elevated infertility until up to the fourth generation. These effects were correlated with modified DNA methylation in germ line ³⁰⁸.

The transgenerational actions of Vz appear to involve an epigenetic (i.e. DNA methylation) reprogramming of the male germ line ³⁰⁸. A study in an outbred strain of mice also showed that gestational exposure to parental oral Vz produces a reduction in sperm count and sperm head abnormalities ³²¹. Furthermore, it has been suggested that the detrimental transgenerational Vz effects on the reproductive system of males are induced by imprinting defects ³²¹. More specifically, in vivo studies examined the methylation status of paternally and maternally imprinted genes after prenatal exposure to Vz. The results demonstrated altered DNA imprinting pattern in two paternal imprinted genes (Gtl2 and H19) and three maternal ones (Peg3, Peg1 and Snrpn) in the offsprings ³²¹.