Division of Neonatology, Perrino Hospital, Brindisi, Italy;
Endocrine Disruptors and Human Health
G. Latini* ,1,2
, G. Knipp 3 , A. Mantovani
4 , M.L. Marcovecchio
5 , F. Chiarelli
5 and O. Söder
1 Division of Neonatology, Perrino Hospital, Brindisi, Italy;
2 Clinical Physiology Institute, National Research Council of
Italy (IFC-CNR); 3 Purdue University, Department of Industrial and Physical Pharmacy,575 Stadium Mall Dr., West
Lafayette, IN 47907; 4 Food and Veterinary Toxicology Unit, Dept. Veterinary Public Health and Food Safety, Istituto
Superiore di Sanità, Viale Regina Elena, 299 00161, Rome; 5 Department of Pediatrics, University of Chieti, via dei
Vestini 5, Chieti, Italy; 6 Department of Women’s and Children’s Health, Paediatric Endocrinology Unit, Karolinska
Institute and University Hospital, S-17176, Stockholm, Sweden
Abstract: Endocrine-disrupting chemicals (EDCs) are a group of diversely natural compounds or synthetic chemicals that
can interfere with the programming of normal endocrine-signalling pathways during pre- and neonatal life, thus leading to
adverse consequences later in life. In addition, early life exposure to EDCs may alter gene expression and consequently
transmit these effects to future generations.
Keywords: Endocrine-disruptors, environment, endocrine system, phthalates, pregnancy, neonate, fetal.
Endocrine-disrupting chemicals (EDCs) are a large and increasing group of diversely natural compounds or synthetic chemicals present in the environment that include persistent halogenated pollutants, such as polychlorinated biphenyls (PCBs), polybrominated diphenylethers (PBDEs) and me- tabolites, industrial compounds, such as bisphenol A (BPA), alkylphenols and phthalate acid esters, as well as pharmaceu- ticals, pesticides, such as chlorpyrifos, fungicides including vinclozalin and phytoestrogens.
Man-made EDCs range across all continents and oceans. EDCs, which are typically present as complex mixtures and not as single substances, may mimic, block or modulate the synthesis, release, transport, binding, metabolism and/or elimination of natural endogenous hormones in wild animals and humans . In particular, EDC may interfere with hor- monal signalling systems and alter feedback loops in the brain, pituitary, gonads, thyroid, and other components of the endocrine system.
Growing evidence shows that EDC may also modulate the activity/expression of steroidogenic enzymes and steroi- dogenic pathways [2-5].
In addition, EDC can also promote activation of meta- bolic sensors, such as the peroxisome proliferator-activated receptors (PPARs) . As a consequence, there is an increas- ing concern worldwide on the potential adverse effects of ED on human health, although their impact on human be- ings’ health is not yet clear.
However, endocrine signalling pathways play an impor- tant role during prenatal differentiation; thus, developing organisms may be particularly sensitive to ED effects. In
*Address correspondence to this author at the Division of Neonatology,
Ospedale A. Perrino, s.s. 7 per Mesagne, 72100 Brindisi, Italy;
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fact, scientific evidence indicate that exposure to ED during critical periods of development can induce permanent changes in several organs, including molecular alterations, although the consequences of this disruption may not appear until later [7-11]. The mechanisms by which ED exert their action remain largely unclear; however, many ways have been identified by which ED can affect signal transduction systems .
Early life exposures to EDCs may alter gene expression via non-genomic, epigenetic mechanisms, including DNA methylation and histone acetylation, thus interfering with the germ-line. By contaminating the environment with ED hu- man race might be permanently affecting the health of sub- sequent generations [13-15]. Within the broad ED topic we have focussed on specific issues, selected since they are highly relevant to the up-to-date assessment of potential hu- man health risks from ED exposure.
ED IN THE FOOD CHAIN: HOW THEY INTERACT
WITH NATURAL COMPOUNDS?
Diet is a significant source of exposure to ED for the general population, as well as a source of concern for con- sumers’ health. One major issue is the “cocktail” effect: one cannot rule out additivity of different ED present in whole diet at low level, but hitting the same targets, e.g. nuclear receptors . Furthermore, it is not just the daily dose that matters. Many ED can bioaccumulate in lipid fraction of tissues, originating a mixture “body burden” of contaminants of different origin that can include dioxins, polychlorinated biphenyls, chlorinated pesticides and their metabolites, as well as brominated flame retardants . Other compounds may also concentrate in food chains, thus adding to the over- all ED burden, e.g., organotins . However, the modern conception of food toxicology cannot consider diet just as an exposure source of external harmful substances. Contami- nants such as ED may interact with the same metabolic pathways as natural food components such as polyunsatu-
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rated fatty acids, trace elements, vitamins and other bioactive substances (e.g. polyphenols) that cannot be considered nu- trients as there is no recognized deficiency . Dietary hab- its are related to socioeconomic status, cultural and religious factors, individual choices (e.g. vegetarianism/veganism); and dietary habits themselves may have the most important impact on the intake of both nutrients and contaminants. For instance, greater exposure to persistent ED is associated with the high consumption of fatty foods of animal origin [20, 21]. Thus, for specific food commodities a balanced evalua- tion is needed about contaminant-associated risks and nutri- tional benefits. A relevant example is represented by salmon- ids and other seafood, a useful source of nutrients such as polyunsaturated fatty acids as well as a major source of ED and other bioaccumulating contaminants, such as meth- ylmercury. Evidence might justify recommendations to in- crease as well as to reduce fish consumption, quite an uneasy situation for risk managers: decreasing fish consumption (and its nutritional benefits) may not be necessary in Europe, but monitoring of contaminants in edible fish should be con- tinued, as well as the development of novel aquaculture feeds, less liable to contamination .
Most important, effects of contaminants and natural food components may interact on the same pathways and targets. The outcomes of interactions may be complex, depending on dose and targets; e.g., phytoestrogens can protect against some hormone-dependent cancers, as well as postmeno- pausal osteoporosis, but may also interfere with receptor- mediated signal transduction (e.g. by inhibiting protein kinase) and DNA replication . Up to date, scientific data available on interactions between xenobiotics and “natural” substances in food are still limited; below, some relevant examples are provided
Iodine and ED
Iodine is the main determinant of thyroid development and function; seafood and milk are the main dietary sources. Subclinical iodine deficiency is still a common problem in many areas, including Europe ; thyroid is also increas- ingly recognized as a major target for ED, including newly recognized ones, such as organpophopsphorus insecticides . Yet, only a few papers target low iodine status in rela- tion to susceptibility to xenobiotics. Somewhat unexpectedly phthalates, the widespread plasticizers known mainly as antiandrogens, can modulate basal iodide uptake mediated by the sodium/iodide symporter in thyroid follicular cells in vitro: the effect was not shared by all phthalates and was independent from cytotoxicity . Many phytoestrogens may interfere with iodination of thyroid hormones. Some (e.g., naringenin, and quercetin, which contain a resorcinol moiety) are direct and potent inhibitors of thyroid peroxi- dase, others (myricetin, naringin) show noncompetitive inhibition of tyrosine iodination with respect to iodine ion, whereas biochanin A may act as an alternate substrate for iodination . A Czech biomonitoring study in children also indicated an adverse effect of genistein on thyroid func- tion . The drinking-water contaminant perchlorate inhib- its thyroidal iodide uptake; however, iodine-deficient female rats were more resistent to the inhibition of iodine absorption from perchlorate exposure than normal rats . Thus, the
interaction between iodine and some thyroid-targeting ED may be less straightforward than expected.
Phytoestrogens and the “xeno”ED
Due to their pleomorphic biological effects, phytoestro- gens are a sort of “natural ED”, whose overall dietary intake of phytoestrogens may be significant also in Europe [23, 30, 31]. Flavonoids (daidzein, genistein, quercetin, and luteolin) can at least partly antagonize the proliferation-stimulating activity of synthetic estrogenic ED in estrogen-dependent MCF-7 human breast cancer cells: thee ED included anionic detergent by-products alkylphenols, plastic additive bisphe- nol A, and the PCB 4-dihydroxybiphenyl [32, 33]. These findings suggest that phytoestrogens can compete with es- trogenic ED on shared biological targets, thus exerting a pro- tective action . In other models no interaction was observed: genistein did not modulate the effects on human astroglial cells by two persistent ED, the polybrominated flame retardant PBDE-99 and the PCB mixture Aroclor 1254 . As it is sometimes the case, in vivo studies provide a more complex picture. Genistein and the estrogenic chlorinated insecticide methoxychlor had an additive impact on both immune function and immune functional development in rats; the developing thymus appeared as a sensitive target of combined exposure . In estrogen reporter (ERE-tK- Luciferase) male mice genistein modulated the actions of both estradiol and persistent ED in liver and testis with tis- sue-specific features: the antiestrogenic action of beta- hexachlorocyclohexane in the testis and o,p’-DDT in the liver was antagonized, whereas genistein had an additive effect with the ER agonist p,p’-DDT in the liver . Two predefined mixtures of phytoestrogens and synthesis ED were tested in the uterotrophic assay on prepubertal rats: the composition of each mixture (what chemicals and to what amount) was based on human exposure data. The phytoes- trogen mixture did elicit an uterotrophic response, whereas the synthetic one has no effect itself nor an additive effect with phytoestrogens, possibly because of exposure levels too low . The combined exposure to estrogenic and antian- drogenic ED is suggested as a potential risk to male repro- ductive development. Genistein and the antiandrogenic fun- gicide vinclozolin, alone or in combination, were investi- gated concerning the induction of hypospadias in mice: the incidences were 25%,, 42% and 41% for genistein, vinclo- zolin and combined treatment, respectively, indicating a less than additive effect . On the other hand, genistein, as well as the methyl donor folic acid, both antagonized the DNA hypomethylating effect of bisphenol A in mouse em- bryos . The available data indicate that interactions be- tween phytoestrogens and ED can be important, but cannot simply explained in terms of additivity or antagonism; in- deed, additivity and antagonism may vary, depending on the chemicals, endpoints and lifestages.
ED and Vitamin A Pathways
Retinoic acid is the internal form of vitamin A interacting with the nuclear receptors RAR and RXR, whose natural ligands are all-trans-retinoic acid and 9-cis-retinoic acid, respectively. Retinoic acid pathways cross-talk with those of the aryl hydrocarbon receptor (AhR), the direct cell target for dioxins and dioxin-like compounds . Dioxins are potent
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inducers of cytochrome P450 (CYP) 1A1, that in its turn may enhance the dioxin effects; the concurrent supplementa- tion of vitamin A inhibits CYP1A1 activity in dioxin- exposed mice, reducing liver damage as well as CYP1A1 and AhR mRNA expression . Mice lacking retinoid binding proteins were especially responsive to dioxin- induced liver retinoid depletion, intracellular retinoid bind- ing protein I being the main factor. RAR- and RXR- knockout mice were essentially sensitive as wild-type mice, with the exception of RXRbeta-/- mice which showed no decrease in hepatic Vitamin A concentration; this suggest a possible role of RXRbeta in dioxin-induced retinoid disrup- tion . Retinoid storage and metabolism were also dis- rupted in female rats of two strains with different dioxin sen- sitivity (Long-Evans and Han/Wistar) . Comparison of dioxin effects on liver retinyl palmitate in AhR+/- and AhR- /- mice support disruption of retinoid homeostasis as a pri- mary AhR- mediated mode of action of dioxin-like chemi- cals . Retinoid pathways can be a critical target also for polybrominated diphenyl ethers: in rats treated orally with pentaBDE-71, decrease of hepatic apolar retinoids was the most sensitive effect, together with reduced thyroid hormone . These studies might also hint to vitamin A deficiency as a susceptibility factor towards some persistent ED.
Although the portfolio of scientific evidence is still quite limited, several other examples can be retrieved from the Endocrine disrupting chemicals – Diet Interaction Database – EDID, the only dedicated database available on ED-nutrient interactions . One further instance is the general protec- tive action elicited by “antioxidant” vitamins C and E to- wards the effects of several EDs, including dioxin-like poly- chlorinated byphenyls (PCB) and phthalates; indeed, several ED-related modes of action seem to eventually lead to in- creased oxidative stress . Overall, new evidence on in- teractions between ED and natural food components may disclose new insights on food-related factors modulating vulnerability as well as on nutrient intake as support to risk prevention and/or risk reduction strategies.
EXPOSURE TO EDCS AND IMPACT ON THE FETO-
Maternal exposure to EDCs has been demonstrated to be a significant reason for increases in adverse pregnancy and fetal outcomes. The placenta protects and nourishes the fetus by regulating nutrient and xenobiotic homeostasis between the maternal and fetal compartments. As discussed below, xenobiotics that can affect this placental homeostatic control may lead to abnormal fetal development by altering fetal exposure to toxic compounds and/or nutrient homeostasis [65, 66, 69]. An important aspect of this review is to high- light some areas in which EDCs have drawn considerable attention due to the many potential fetotoxic effects, which may be caused upon in utero exposure. For example, recent evidence suggests a link between EDC exposure and the fetal origins of neurological impairment that cannot be ig- nored even though their mechanistic basis is not well under- stood [47-51]. EDCs are hypothesized to induce functional and/or structural changes in specific neuroendocrine path- way(s), effects being largely dependent upon the phase (ges- tational time) and level of exposure . In addition, the potential for additive or synergistic effects of low dose com-
binations of EDCs are not well established and require con- siderable investigation. . Moreover, the role of other factors including diet, exercise and genetics has not been well characterized, adding to the difficulty in delineating the role of EDCs on neurodevelopment. Finally, the pharma- cokinetic and pharmacodynamic relationships for EDCs dif- fer and there exists a potential for placental and fetal accu- mulation not accurately measured in maternal plasma [52, 54]. For example, a recent study revealed that when both newborns and adults are exposed to the same bisphenol A (BPA) levels, newborns retain up to 3 times more than adults .
BPA is an EDC due to its ability to interact with estrogen receptor (ER and ) isoforms, androgen receptors (AR), and possessing a high affinity for the estrogen related recep- tor (ERR ) during mammalian brain development . BPA eluted from polycarbonate drinking bottles was demon- strated to exert an estrogenic like neurotoxic effect in devel- oping cerebellar neurons . BPA has also been demon- strated to alter fetal neurodevelopment through thyroid hor- mone (TH) pathways, as recently in a TH-dependent den- dritic Purkinje cell development in a murine cerebellar cul- ture assay [51, 57].
BPA is metabolized into BPA glucuronide, which is hormonally inactive, and excreted via the urine with a half- life below 6 hours . Despite the rapid metabolism, the U.S. CDC have found BPA levels in 95% of all human urine samples tested, suggesting broad and continuous BPA expo- sure [59, 60]. Chronic exposure to low levels of BPA may still cause developmental toxicity due to the significant po- tential for bioaccumulation in the human placenta and fetus . For example, human BPA levels in the placenta and amniotic fluid were 5 folds higher at weeks 15 to 18 com- pared to maternal serum . Furthermore, fetal and perina- tal exposure to BPA has been linked to several neurodevel- opmental changes and disorders including autism and the related autism spectrum of disorders (ASD), schizophrenia, impaired neurotransmission, attention deficit and hyperactiv- ity disorders, and potentially sexual dimorphic related changes in brain structure and function, as recently reviewed by Brown .
Phthalates are a ubiquitous class of environmental terato- gens capable of exerting their toxic effects through several nuclear hormone receptors including the androgen receptor (AR) antagonism [52, 63], ER agonism [52, 64] and/or trans-activation of the peroxisome-proliferator activated receptors (PPAR) and isoforms, either directly or indi- rectly [65-69]. Phthalates are classified as peroxisome prolif- erator chemicals due to their effects on peroxisomal lipid metabolism . Moreover, they may also exert their neuro- toxic effects through altering zinc metabolism [71, 72] or by altering intracellular Ca2+ concentrations leading to the for- mation of reactive oxygen species potentially through a pro- tein kinase C mediated pathway . It has also been sug- gested that DEHP inhibits membrane Na+-K+ ATPase in the rat brain, a phenomena linked to several neurodegenerative and psychiatric disorders .
Phthalate reproductive toxicology research [63, 65-69] has been largely focused on di-(2-ethylhexyl)-phthalate
Endocrine Disruptors and Human Health Mini-Reviews in Medicinal Chemistry, 2010, Vol. 10, No. 9 849
(DEHP), an industrial plasticizer that is ubiquitously dis- persed in the environment. Human DEHP exposure most likely begins in the mother’s womb, where DEHP has been demonstrated to readily cross the placenta and accumulate in the fetus [67, 68]. DEHP mediated direct or indirect PPAR effects on placental essential fatty acid (EFA) homeostasis have also been of interest [64-69]. The fetus requires mater- nal dietary intake and placental transfer of EFAs to guide proper pregnancy outcomes and fetal development, e.g. neu- rodevelopment [75-78]. The placenta plays a fetoprotective role by accumulating EFAs from maternal circulation and directionally transporting them into fetal compartment [79, 80]. With regards to proper neurodevelopment, EFAs includ- ing docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) are known to play critical roles in mye- logenesis and serve as essential components in neurogenesis, thus making the fetoprotective role of the placenta essential for neurodevelopment [76-80]. EFA imbalances have been linked to several neurological disorders including autism and ASDs, bipolar disorder, and schizophrenia, suggesting that a proper EFA supply is required to protect the CNS develop- ment [81, 82].
PPAR and regulate the expression of several fatty acid transport conferring proteins and metabolizing enzymes that maintain essential fatty acid (EFA) homeostasis and can be trans-activated by DEHP and its metabolites, mono-(2- ethylhexyl)-phthalate (MEHP) and 2-ethylhexanoic acid (EHA) [65, 66, 83]. Recent studies revealed that DEHP and its metabolites MEHP and EHA, can significantly increase the expression of EFA homeostasis proteins, EFA and lipid accumulation in the lipid metabolome of HRP-1 in vitro rat placental cell line [65, 84]. It was also demonstrated that the resulting increase in the expression of fatty acid transport- conferring proteins in these cells also led to a significant increase in fatty acid and lipid accumulation in the cells . DEHP exposure has been revealed to alter the expression of EFA homeostasis proteins in the in vivo rat placenta . In this study, radiolabeled AA and DHA where administered to rat dams at gestational day (GD) 20 and the maternal, pla- cental and fetal disposition of the labeled EFAs were as- sessed . AA was significantly reduced in the maternal and fetal plasma, yet increased significantly in the placenta upon DEHP exposure. DHA levels significantly increased in the maternal plasma and decreased in the fetal plasma upon DEHP exposure contrasted to the control vehicle. DEHP exposure also elicited a statistically significant decrease of both AA and DHA in the developing fetal brain. Lipomic analysis also revealed that DEHP reduced fetal pup brain accumulation of several critical fatty acid and lipid classes including a significant decrease in sphingomyelin (SM) of 54% . DEHP exposure elicited a significant reduction of DHA in five lipid fractions (namely, cholesterol ester (CE), diacylglyceride, phosphatidylserine, lysophasphatidyl cho- line (LYPC) and SM), whereas AA was significantly de- creased CE and LYPC. SM and DHA levels are critical for proper brain development including neurogenesis and neu- ronal differentiation [76-78]. Moreover, the brain weight and active neuronogenesis rapidly increases from GD15 to term in the rat fetus . These results suggest that DEHP may adversely affect on fetal neurodevelopment.
Recently, it has been found that 2,3,7,8-tetrachlorodi- benzo-p-dioxin (TCDD) can reduce n-3 and n-6 EFAs in contrast to the control when administered to the cynomolgus macaque at GD15 or 20 and the brains were isolated at GD24-26 . Although the mechanism of action was not defined, improper neural tube closing and other neurodevel- opmental aberrations were observed and attributed to the improper EFA balance [86-89]. Interestingly, TCDD also has an estrogenic response, acting through ERs, potentially interacting with the aryl hydrocarbon receptor (AHR) in rats [90, 91].
In summary, several EDCs have been demonstrated to
exert their teratogenic effects on fetal neurodevelopment.
Considerable attention is necessary to elucidate the mecha-
nisms by which individual or multiple combinations of
The post Division of Neonatology, Perrino Hospital, Brindisi, Italy;