Endocrine Active Substances:: understanding MoAs for risk assessment Alberto Mantovani Stefano Lorenzetti, Cinzia La Rocca, Laura Narciso, Sabrina Tait Food and Veterinary Toxicology Unit Dept Food Safety and Veterinary Public Health Istituto Superiore di Sanità - Roma alberto.mantovani@iss.it ISS website on endocrine disrupters http://www.iss.it/inte
Let's start from the EU definition of ED An E(ndocrine) D(isrupter) elicits adverse effects by interfering with the endocrine system In a whole, intact organism Or its progeny Or in a subpopulation NB: Endocrine Active Substances (see EFSA, 2010) An up-to-date food safety (e.g., risk-benefit assessment) needs to consider also substances that are essential or potentially beneficial to endocrine function, at least at certain dose levels (iodine, selenium, phytoestrogens)
Definitions have consequences Effects have to be adverse = overcoming cell/tissue /organism homeostatic checks'n'balances Endocrine mode of action is critical = endocrine-related effects at LOEL, at least in vulnerable sex and/or lifestage Whole organism level = a network is affected, e.g., action on gland leads to multiple impacts on organs/tissues, depending on age/gender targets (e.g., nuclear receptors) widespread in several (potentially sensitive) tissues e.g. thyroid Foetus/newborn: brain matiuation/growth Female in fertile age: reproductive competence, pregnancy loss Adult: goiter
Definitions have consequences (2) Subpopulation/progeny = different sensitivity (= different NOELs) based on “ endocrine status” = sex/lifestage Scientific evidence supports that major endpoints are related to programming = developmental exposure/delayed effects on target organ function/maturation (e.g., reproductive dysfunction seen from puberty) Some compounds may show ED activities only upon developmental exposure , e.g., the well known neurotoxicant (cholinesterase inhibitor) chlorpyriphos Thyroid effects in adult mice exposed in utero to dose levels devoid of cholinesterase inhibition activities ( De Angelis, Tassinari et al., Toxicol Sci, 2009 )
Endocrine disruption is a (group of) mode(s) of action which impact on risk assessment MoA = from molecular (mechanism) through to cell/tissue and organ/organism level (ED affects a signalling network) Is not a phenmenological effect (endpoint) per se Thus, 1) Endocrinology (e.g., colon is amongst the tissues with highest expression of ERbeta) should lead to have some more imagination pointing also to targets other than reproductive system/thyroid e.g., hypothalamus/pituitary, bone, metabolic syndrome some examples from our non-conventional studies
Neuroendocrine programming Developmental exposure to chlorpyriphos (not an ED till recently) alters the expression of specific neurohypophyseal hormones (oxytocin, vasopressin; not prolactin: pregnancy, blood pressure, social behavio r) in the mouse hypothalamus at dose levels not altering brain cholinesterase with marked gender differences ( Tait et al., Env Health Perspect 2009 ) Bone highly modulated by steroid ( osteoporosis: estrogen-modulated disease) Prepubertal exposure of rats to Semicarbazide (babyfood contaminant) = impaired growth of surface cartilage, together with a ED pattern: thyroid/ ovary, delayed female and accelerated male puberty, altered sex steroid balance. In vitro: antagonism with N-methyl-d-aspartate (NMDA) receptors ( Maranghi et al., 2010, Chem Biol Interact ) Is this an ED MoA ?
Metabolic Programming Prenatal exposure to diethyl-2-hexyl-phthalate (DEHP; agonist of PPARs, PXR, CoAR ) at doses corresponding to NOEL/LOEL = altered liver maturation, with altered glycogen/lipid metabolism (Maranghi, Lorenzetti et al., Reprod Toxicol, 2010) Liver is a potential (and overlooked) ED target: hormone catabolism, rich in nuclear receptors, concentrates xenobiotics PPARs, PXR, CoAR = regulation of first steps of steroid biosynthesis pathways Should we look more closely to adipocyte differentiation//proliferation ? Insulin-regulated pathways (LXR) ?
ED MoA 1)” Traditional” MoAs e.g., enzyme inhibitors (azole fungicides, steroid biosynthesis and in particular aromatase inhibitors) Also “antinutrient” activities (e.g., inhibition of iodine uptake/orgaification in thyroid, like glucosinolates in brassica vegetables) (contaminants interacting with nutrient metabolism: EDID database at www.iss.it/inte, Baldi & Mantovani, 2012) Extremely important, but rather straightforward Hazard characterization is related to the NOAEL/NOAEC for enzyme inhibition in the most vulnerable sex/lifestage
ED MoA 2) The problematic MoA: interaction with nuclear receptors Again, pesticides provide some good examples, like the AR antagonists: dicarboximide fungicides ( vinclozolin, procymidone) and, more ambiguous, the herbicide linuron Why problematic ? a) NR are differentially expressed a) in tissues, b) depending on gender, c) across lifestages b) NR cross-talk (e.g., ER beta-ER alpha), thus non-hormonal NR (RARs, RXR, LXR..) may impact on hormone homeostasis c) NR have physiological agonists, thus in vitro/mechanistic assays should consider ED action in the absence and in the presence of physiological agonists which may, e.g., result in agonism and antagonism, respectively)
ED MoA: Nuclear Receptor interaction May point to unusual Dose-Response (qualitatively different effects at different exposure levels: e.g., agonism at low and antagonism at high doses) ( Mind such D/R are rather expected with pharmacological agents, With nutrients: deficiency/requirement/excess) In my opinion NR interaction point especially to a largely different sensitivity across lifestages, and to the protection of the most susceptible lifestage/target Mind : some hormonal NR are somewhat overlooked: PR, TRs (peripheral effects of thyroid hormones)
TWO DANGERS : Sticking to current toxicity guidelines, looking for the “perfect” GLP study…and showing skepticism towards scientific evidence (may have some use as antibody towards over-fashionable topics, but it’s not the right way to proceed) OR DROWNING INTO COMPLEXITY ( we cannot conclude. .)
DON’T DROWN - Remember the RA principles applied to ED - Exploits the impressive knowledge gained by the basic and clinical endocrinology - Use omics to identify patterns - Chemicals are thousands (new ones are coming: think about nanomaterials?) Mechanisms are many Adverse outcome pathways are several (but limited) in number - Think phenomenological : you need to have an evidence of adversity, a mechanism should produce an event
EXAMPLE 1 (Lorenzetti et al., 2010) - Chemicals relevant to AR-regulated pathways - A cell model of a critical AR target: prostate - A clinically relevant marker : prostate-specific antigen (PSA) secretion - Specific effect: only relevant at non-cytotoxic concentration - 3 patterns identified (pattern parameters: magnitude of effect, concentration- response, relationship with cell proliferation, previous knowledge on AR/ER interaction) Androgens Antiandrogens ( both AR antagonists and post-receptor inhibitor: DBP) ER-binder
EXAMPLE 2 (Tait et al., 2010) Omics to identify patterns indicative of MoA to understand mixtures Hypothesis : additive action of substances with same MoA Mixture effects are highly relevant to some exposure patterns, like the concurrent body burden of different, low-level, lipophyllic ED PCB: transcriptome of PCB congeners at real-life levels in a potentially vulnerable model (human fetal penile cells) Three distinct and restricted patterns of gene modulation in the absence of cytoxicity identifying three potentially dose-additive mixtures Dioxin-like (cell-cell communication) Mix1 “estrogenic” (smooth muscle function) Mx3 “persistent” (steroid and lipid synthesis)
ED are a separate “niche ” where strange things do occur (U-shaped curves, low-dose effects) ?
Rather ED are a fisrst-line section of Toxicological RASS Traslation of molecular mechanisms into phenomena Gender and developmental windows issues Targets overlooked in current testing (body composition, diabetes, neuro-immune-endocrine interfaces) Interaction with advances in medical sciences to understand human relevance Cost-effective, predictive tools for hazard chacterization may support tackling RA issues such as mixture effects and biomarker development
Translational Prevention (from bench to risk assessment) That’s all Folks…
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