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Piacenza, 23 marzo 2015

Caratterizzazione degli effetti endocrini di contaminanti della filiera-agro-alimentare: dal progetto ReProTect a LIFE-EDESIA

Stefano Lorenzetti [email protected]

Dip.to Sanità Pubblica Veterinaria e Sicurezza Alimentare Istituto Superiore di Sanità – ISS INDICE

 PRESENTAZIONE Di cosa ci occupiamo e in che modo

 INTERFERENZA ENDOCRINA Definizioni Meccanismi e Modi di Azione Effetti avversi e Biomarcatori di effetto

 IL PROGETTO EUROPEO ReProTect (2006-10) Lo sviluppo di un metodo sperimentale in vitro alternativo alla sperimentazione animale: dalla tossicogenomica al «phenotypic anchoring»

 IL PROGETTO EUROPEO LIFE-EDESIA (2013-2016) Lo screening dell’Interferenza Endocrina mediante l’uso di biomarcatori di effetto in saggi funzionali INDICE

 PRESENTAZIONE Di cosa ci occupiamo e in che modo

 INTERFERENZA ENDOCRINA Definizioni Meccanismi e Modi di Azione Effetti avversi e Biomarcatori di effetto

 IL PROGETTO EUROPEO ReProTect (2006-10) Lo sviluppo di un metodo sperimentale in vitro alternativo alla sperimentazione animale: dalla tossicogenomica al «phenotypic anchoring»

 IL PROGETTO EUROPEO LIFE-EDESIA (2013-2016) Lo screening dell’Interferenza Endocrina mediante l’uso di biomarcatori di effetto in saggi funzionali Di cosa ci occupiamo

Analisi del rischio (risk analysis) Gestione del rischio (Risk management) VALUTAZIONE DEL RISCHIO Interferenti Endocrini (risk assessment) Comunicazione del rischio ( IE ) (Risk communication) o Identificazione degli eventi avversi (hazard identification) Endocrine Disruptors (EDCs) Caratterizzazione degli eventi avversi: meccanismi e relazione dose-risposta (hazard characterization)

Valutazione dell’esposizione (exposure assessment)

Caratterizzazione del rischio (risk characterization) Di cosa ci occupiamo

Contaminanti ambientali e della catena alimentare

 NUTRIENTI macro-nutrienti (carboidrati, proteine, lipidi) micro-nutrienti (vitamine, sali minerali) Interferenti Endocrini ( IE ) o  NON NUTRIENTI (poli)fenoli – plant bioactives Endocrine Disruptors (EDCs)  CONTAMINANTI pesticidi / biocidi, AMBIENTALI & plasticizzanti, … DEGLI ALIMENTI Di cosa ci occupiamo

Endocrine Active Substances / EASs: “a substance having the inherent ability to interact or interfere with one or more components of the endocrine system resulting in a biological effect, but need not necessarily cause adverse effects.” EFSA Journal 2013;11(3):3132

Interferenti Endocrini In other words, “Endocrine Disruptors/EDs are Endocrine Active ( IE ) Substances/EASs causing adverse effects mediated by endocrine o mechanisms” Endocrine Disruptors Rovida, De Angelis, Lorenzetti. ALTEX 30, 2/13 (EDCs) “An endocrine disrupter is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations.” WHO/IPCS 2002 STATE OF THE ART ASSESSMENT OF ENDOCRINE DISRUPTERS ec.europa.eu/environment/endocrine/.../summary_state_science.pdf In che modo ce ne occupiamo

Uso di strategie integrate per il principio di sostituzione

Endocrine Disruptors in silico/in vitro – Evaluation and Substitution for Industrial LIFE12 ENV/IT/000633 Interferenti Endocrini ( IE ) o Endocrine Disruptors (EDCs)  Ftalati, Bisfenoli, Parabeni In che modo ce ne occupiamo

Uso di strategie integrate per il principio di sostituzione

Endocrine Disruptors in silico/in vitro – Evaluation and Substitution for Industrial LIFE12 ENV/IT/000633 Interferenti Endocrini ( IE ) o IN SILICO TOXSCREEN . CHEMICO-PHYSICAL PROPERTIES . TOX PROPERTIES (e.g. cancerogenic, Endocrine Disruptors mutagenic, binding to Nuclear Receptors) (EDCs) . MOLECULAR DOCKING . QSAR  Ftalati, Bisfenoli, IN VITRO TOXSCREEN Parabeni Cell specific endpoint: Functional Assay – Phenotypic anchoring Molecular endpoint: Cell aspecific endpoint: . prostate: PSA secretion gene expression of Nuclear Cell Viability (MTS . trophoblast: bhCG secretion Receptors of interest (qPCR) assay) . liver: intracellular lipid accumulation and AFP secretion

Gene reporter assays: AR-, ER-, PPAR-gene reporter assays (OECD and/or IHCP-JRC guidelines and/or protocols under the validation programme) In che modo ce ne occupiamo

Uso di marcatori clinici in sperimentazione tossicologica (aspetti traslazionali) Which biomarkers are searched for ED-like activity ?

ligan d functional markers Interferenti Endocrini cell-specific, clinically relevant NR inhibitor endpoints ( IE ) y protein o N NR NR Endocrine Disruptors R binding dimerization (EDCs) changed NR translocation cell function cytoplasm protein

nucleus AAAAA mRNA

NR- NR target gene Part of a RE Mode of Action

transcriptional activation molecular markers: Part of a mRNA, miRNA, proteins, metabolites Mechanism of Action (incl. toxicogenomics) Adapted from Lorenzetti and Narciso, 2012 DOI: 10.1039/9781849735353 In che modo ce ne occupiamo

Uso di marcatori clinici in sperimentazione tossicologica (aspetti traslazionali) Which biomarkers are searched for EDC-like activity ?

ligan d functional markers A geneInterferenti reporter Endocriniassay or a cDNA cell-specific, clinically relevant NR inhibitor endpoints microarray (do IE not ) identify an y protein o endocrine disrupting property N NR NR R binding dimerization BUTEndocrine only a part Disruptors of a potential (EDCs) changed mechanism of action of a chemical NR translocation cell function WHOSE endocrine disruption cytoplasm protein nucleus AAAAA property (the adverse effect) mRNA NR- NR target gene Part of a remain to be assessed… by a RE Mode of Action functionally-relevant endpoint transcriptional activation molecular markers: Part of a mRNA, miRNA, proteins, metabolites Mechanism of Action (incl. toxicogenomics) Adapted from Lorenzetti and Narciso, 2012 DOI: 10.1039/9781849735353 In che modo ce ne occupiamo

SECREZIONE DELLA PSA come marcatore di attività biologica (1)

ESTRADIOLO GENISTEINA (GEN) (E2)

+ DHT 10nM + DHT 10nM

Results are expressed as mean ± SD of three independent experiments: * p <0,05,** p<0,01, ***p<0,001 vs

Smeriglio#, Trombetta#, Marcoccia#, et al. 2014, Intracellular Distribution and Biological Effects of Phytochemicals in a Sex Steroid-Sensitive Model of Human Prostate Adenocarcinoma. Anti-cancer Agents Med Chem., 14(10):1386 In che modo ce ne occupiamo

SECREZIONE DELLA PSA come marcatore di attività biologica (2)

FLAVONOLO FLAVONOLO APIGENINA (API) LUTEOLINA (LUT) API LUT

Free PSA Total PSA MTS assay Free PSA Total PSA MTS assay

3500 180 3500 180 160 2500 130 2500 140 120 100 1500 80 1500 80

cc 60 %Viability 500 30 500 40 %Viability

20 % PSA %PSA secretion -500 -20 % PSA Secretion -500 0

+ DHT 10nM + DHT 10nM

Results are expressed as mean ± SD of three independent experiments: * p <0,05,** p<0,01, ***p<0,001 vs Smeriglio#, Trombetta#, Marcoccia#, et al. 2014, Intracellular Distribution and Biological Effects of Phytochemicals in a Sex Steroid-Sensitive Model of Human Prostate Adenocarcinoma. Anti-cancer Agents Med Chem., 14(10):1386 In che modo ce ne occupiamo

SECREZIONE DELLA PSA come marcatore di attività biologica (3)

Localizzazione intracellulare Molecole Terreno (Medium) Frazione Frazione Frazione di coltura/M microsomale / FM citoplasmatica / C nucleare / N Molecole di riferimento DHT 44,66 47,96 1,95 5,01 E2 9,65 58,39 8,50 23,14 Polifenoli vs Polifenoli + Ormoni Steroidei APIGENINA (API) 22,60 61,57 7,16 8,21 API + 10nM DHT 12,46 42,92 17,95 26,29 API + 10nM E2 0,92 89,29 1,13 6,42 LUTEOLINA (LUT) 3,83 81,11 4,50 9,02 LUT + 10nM DHT 48,93 60,38 10,86 18,76 LUT + 10nM E2 5,63 82,39 3,77 8,80

SECREZIONE DELLA PSA come marcatore di attività biologica (4)

V. XANTHOPHOENICEUM FENILETANOIDE EXRACT (VCE) VERBASCOSIDE (VERB)

+ DHT 10nM + DHT 10nM 153.7 pM VERB

Marcoccia et al. 2014. Inhibition of the DHT-induced PSA secretion by Verbascum xanthophoeniceum and S. REPENS (SPO) Serenoa repens extracts in human LNCaP prostate epithelial cells. J. Ethnopharmacol., 155(1):616-25.

+ DHT 10nM INDICE

 PRESENTAZIONE Di cosa ci occupiamo e in che modo

 INTERFERENZA ENDOCRINA Definizioni Meccanismi e Modi di Azione Effetti avversi e Biomarcatori di effetto

 IL PROGETTO EUROPEO ReProTect (2006-10) Lo sviluppo di un metodo sperimentale in vitro alternativo alla sperimentazione animale: dalla tossicogenomica al «phenotypic anchoring»

 IL PROGETTO EUROPEO LIFE-EDESIA (2013-2016) Lo screening dell’Interferenza Endocrina mediante l’uso di biomarcatori di effetto in saggi funzionali WHERE WE STARTED FROM…

Endocrine Active Substance / EAS “a substance having the inherent ability to interact or interfere with one or more components of the endocrine system resulting in a biological effect, but need not necessarily cause adverse effects.” EFSA Journal 2013;11(3):3132

Endocrine Disruptor / ED “An endocrine disrupter is an exogenous substance or mixture that alters function(s) of the endocrine system and consequently causes adverse health effects in an intact organism, or its progeny, or (sub)populations.” WHO/IPCS 2002

In other words, … Endocrine Disruptors (EDs) (i.e., EASs causing adverse effects mediated by endocrine mechanisms) Rovida, De Angelis, Lorenzetti. ALTEX 30, 2/13 Endocrine activity… “endocrine activity as a collection of modes of action, potentially leading to adverse outcomes, rather than a (eco)toxicological hazard in itself.” EFSA Journal 2013;11(3):3132

… as a sum of different Mode of Actions “A change in morphology, physiology, growth, reproduction, development or lifespan of an organism which results in impairment of functional capacity or impairment of capacity to compensate for additional stress or increased susceptibility to the harmful effects of other environmental influences”. Federal Institute for Risk Assessement (BfR), Berlin workshop 2009”

In vitro only nuclear receptor binding & regulation of gene transcription define an endocrine activity ?

OECD 2002 … AND WHERE DO WE GO FROM HERE

In summary, currently available definitions of “endocrine disrupter” are either neutral in terms of specifying the toxicological relevance of the effects to be described, or they introduce the idea of adversity. The former is in danger of being insufficiently discriminatory, the latter shifts the problem to defining what adversity should mean in an endocrine context, which could be too restrictive and not inclusive enough. At the core of this dilemma is the fact that “endocrine disruption” cannot presently be anchored to specific assay outcomes in a straightforward way.

STATE OF THE ART ASSESSMENT OF ENDOCRINE DISRUPTERS ec.europa.eu/environment/endocrine/.../summary_state_science.pdf A TYPICAL SCHEME OF NR-MEDIATED SIGNALLING

ligand

NR inhibitory protein

NR NR binding NR dimerization

changed NR translocation cell function protein cytoplasm

nucleus AAAAA mRNA

NR-RE NR target gene

Adapted from Lorenzetti and Narciso, 2012 DOI: 10.1039/9781849735353 A TYPICAL SCHEME OF NR-MEDIATED SIGNALLING

How to search for «new» biomarkers of effect ?

ligand

NR functional markers inhibitory protein

NR NR binding NR dimerization

changed NR translocation cell function protein cytoplasm

nucleus AAAAA mRNA

NR-RE NR target gene Part of a Mode of Action

Part of a Mechanism of Action molecular markers

Adapted from Lorenzetti and Narciso, 2012 DOI: 10.1039/9781849735353 NRs do not transactivate alone

 Ligand-NR interaction is as important as the presence of dozens of transcriptional coregulators at the DNA binding site within the promoter of NR-target genes. Indeed, the NR-dependent trascriptional activation (or repression) of NR-target genes depends on specific co-activators (or co- repressors) present within the nucleus of a specific cell in a well determined very short-time period. A TYPICAL SCHEME OF NR-MEDIATED SIGNALLING

How to search for «new» biomarkers of effect ?

ligand

NR functional markers inhibitory protein

NR NR binding NR dimerization

changed NR translocation cell function protein cytoplasm

nucleus AAAAA mRNA

NR-RE NR target gene Part of a Mode of Action

Part of a Mechanism of Action. molecular markers toxicogenomics Adapted from Lorenzetti and Narciso, 2012 DOI: 10.1039/9781849735353 INDICE

 PRESENTAZIONE Di cosa ci occupiamo e in che modo

 INTERFERENZA ENDOCRINA Definizioni Meccanismi e Modi di Azione Effetti avversi e Biomarcatori di effetto

 IL PROGETTO EUROPEO ReProTect (2006-10) Lo sviluppo di un metodo sperimentale in vitro alternativo alla sperimentazione animale: dalla tossicogenomica al «phenotypic anchoring»

 IL PROGETTO EUROPEO LIFE-EDESIA (2013-2016) Lo screening dell’Interferenza Endocrina mediante l’uso di biomarcatori di effetto in saggi funzionali ReProTect

Thematic Priority Development of new in vitro tests to replace animal experimentation Project title Development of a novel approach in hazard and risk assessment of reproductive toxicity by a combination and application of in vitro, tissue and sensor technologies

WorkPackage 4 (WP4 coordinator: Alberto Mantovani) Strategies for in vitro test batteries in reproductive/developmental toxicity: a toxicogenomic approach involving expression / regulation of androgen receptor genes Reproductive toxicology as a frame to develop “a proof of concept”

The androgen receptor directly regulate development, maturation, functionality and homeostasis of the prostate gland and, in particular, of the prostate epithelium that secrete the prostatic fluid (1/3 in volume of the male ejaculate) an essential component to ensure male fertility. PROSTATE and HUMAN HEALTH: TDS as an example

Prostate epithelial cells (prostatic fluid)

Adapted from Skakkebaek NE et al., 2001, Human Reproduction 16: 972–8 Testicular Dysgenesis Syndrome (TDS): exposure in utero to environmental factors (anti- androgenic compounds) in Western Europe and USA nei paesi dell’Europa occidentale e negli USA, are responsible of male infertility and associated-diseases/malformations. LNCaP cells as an in vitro tool…

PSA secretion assay cDNA microarray (free PSA, upper panel; total PSA, bottom panel)

CTRL DHT MT 2OH LIN LNCaP, human epithelial cell line, 600,0 *** treated with androgen receptor /AR ***

agonist/antagonist: 500,0 g/L]

number of modulated genes m 400,0 Modulated UP-regulated DOWN-regulated 300,0 genes genes genes 200,0 DHT 2369 1390 979 100,0

MT 2600 1442 1158 [ concentration free PSA 0,0 1

2OH- 3995 2069 1926 CTRL DHT MT 2OH LIN 600,0 FTA

500,0 LIN 304 139 165 *** *** 400,0

g/L] ***

m [ 300,0 Phenotypic anchoring

200,0 concentration 100,0

0,0 PSA Total PSA Lorenzetti et al., unpublished data 1 LNCaP : SEX STEROID RECEPTOR STATUS

ERa, ERb

 In our experimental culture conditions, only ERb and ART877A are expressed ART877A whereas ERa is NOT expressed THUS allowing us to reduce the redundancy between

M AR and ERa signalling on common target MT

DHT genes CTRL Maranghi et al., 2007 FROM TOXICOGENOMICS TO PHENOTYPIC ANCHORING… Unsupervised clustering

Figure 1A MT 3 DHT 2 2OH-FTA 2 2OH-FTA 1 2OH-FTA 3 DHT 1 2OH-FTA 4 MT 4 DHT 4 MT 2 MT 1 DHT 3 LIN 1 Figura 1B Androgens UP-regulated DOWN-regulated Treatment LIN 3 modulated genes genes genes LIN 2

DHT 2369 1390 979

MT 2600 1442 1158

2OH-FTA 3995 2069 1926 Lorenzetti et al., unpublished data

LIN 304 139 165 PSA/KLK3 Figure 2C PSA secretion Figure 2D Free PSA Total PSA gene expression 600 600 90 * *** 500 500 75 *** *** *** *** 400 400 60 * *** 300 300 45 * 30

PSA (ug/L) PSA 200 200 (arbitrary units) (arbitrary

100 100 gene expression PSA 15

0 0 0 CTRL DHT MT 2OH-FTA LIN CTRL DHT MT 2OH-FTA LIN CTRL DHT MT 2OH-FTA LIN FROM TOXICOGENOMICS TO PHENOTYPIC ANCHORING… (2)

PSA secretion: time- and DHT dose-dependency Figure 1B Free PSA Total PSA

30 30 * * * * * * * * * * * * CTR * * * * 25 * * * * * 25 * * * * -9 * * * * * * 10 M * * * * * 20 20 10-8 M 15 15 * * * * * * * * * * * * * -7 * * * * * * * * * * 10 M

PSA (ug/ul) PSA 10 10 -6 * 10 M * * * * * * * * * 5 * * * * * 5 * * * * * 10-5 M 0 0 6 24 48 72 96 6 24 48 72 96 hours hours

PSA secretion PSA/KLK3 gene expression Figure 2C Figure 2D Free PSA Total PSA 600 600 90 * *** 500 500 75 *** *** *** *** 400 400 60 * *** 300 300 45 * 30

PSA (ug/L) PSA 200 200 (arbitrary units) (arbitrary

100 100 gene expression PSA 15

0 0 0 CTRL DHT MT 2OH-FTA LIN CTRL DHT MT 2OH-FTA LIN CTRL DHT MT 2OH-FTA LIN

Lorenzetti et al., unpublished data TAKE HOME MESSAGE

 Transactivation o gene reporter assay in in vitro models do not characterize an effect as adverse or not, but just indicate the ligand binding in that particular cellular environment

 Transactivation assay or, better, any gene expression profiling should be associated to a functional assay to search for a mode of action

 Molecule interactions and/or gene expression modulation are not toxicological endpoints

Assessment of adversity is not unique to endocrine related effects. Scientific criteria for assessment of adversity have not been generally defined. In general, but not always, transient, inconsistent and minor fluctuations at the biochemical and molecular level may be considered adaptive, i.e. non-adverse. Changes at the cell-, organ-, organism-, or (sub)population-level resulting in pathology or functional impairment in vivo, as well as altered timing of development, may be considered adverse. It is therefore difficult to propose ED- specific criteria for adversity and expert judgement in a weight-of-evidence approach is needed to assess substances for possible endocrine disrupting properties. EFSA Journal 2013;11(3):313 FROM PHENOTYPIC ANCHORING TO AN INDEPENDENT SCREENING TOOL…

Cell aspecific endpoint: Cell Viability (MTS ASSAY) Screening 10 chemicals in a double blind + feasibility study: Cell specific endpoint: Functional Assay . 2 confirmed as AR-interfering chemicals (PSA SECRETION) (vinclozolin and BPA) . 1 newly identified as androgen-like chemical HOW WE MOVED ON… (3)

Projects in progress: Italian Ministery of Health and Rovereto Town Council national projects

Plant GMO- Insecticides Drugs bioactives Plasticizers Herbicides Herbicides Fungicides & metabolites /Biocides Doping agents 5a-di-hydro- Glufosinate Chlorpyripho 17a-methyl Genistein Di-n-butyl phthalate Terbuthylazin Vinclozolin testosterone ammonium Vinclozolin M1 s testosterone (GEN) (DBP) e (TBT) (VIN) (DHT) (GA) (CPF) (MT) di-hydroxy- Di-(2- Tributylin Resveratrol Linuron flutamide (2OH- ethylexyl)phthalate Glyphosate Zineb Vinclozolin M2 oxide (RESV) (LIN) FTA) (DEHP) (TBTO) Spirodiclofe Apigenin Bisphenol A Ethylene Bicalutamide (BIC) Nitrofen Mancozeb n (API) (BPA) Thiourea (ETU) (SPI) Luteolin Permethrin 17b-estradiol (E2) Fenarimol (LUT) (PERM) Naringenin Carbendazi Lindane (NRG) m (gHCH) Quercetin (QRC) 2. In vitro:

1. In silico 2a Cytotoxicity (MTS assay) test 3. Data Analysis:

Molecular Docking 2b Protein (free and total PSA) secretion assay Integrated in silico – in vitro data analysis 2c Gene expression by qPCR of the 48 human Nuclear Receptors (hNRs) & target genes

2d Chemical Biodistribution The experimental model LNCaP as an in vitro model system of the (human) prostate epithelium (the tumour-developing cells): LNCaP: AR mutated (ART877A) in the Ligand Binding Domain LBD PCa: > 90% mutation in AR-LBD; about 50% are ART877A

Epithelial cell Basal cell layer Basement membrane This in vitro approach provides: Capillary walls Free PSA Protein-bound PSA 1. a preliminary in vitro screening method to detect chemicals affecting male It is originated from a metastasis of prostatic fertility in a prostate-mediated manner. adenocarcinoma (PCa) Tumorigenic in mice xenografts 2. an alternative in vitro method to Androgen Sensitive perform the principle of the 3Rs It expresses a mutated androgen receptor (ART877A), the (Replacement, Reduction and main one in human PCa A model of androgen-dependent PCa Refinement) for the use of animal PSA (Prostate-Specific Antigen) is secreted and AR- experimentation. modulated  Under proper growth condition, it expresses only the sex steroid receptor AR and ERβ and not ERα as it occurs in physiological condition

• Lorenzetti et al., 2011, Annals Ist Sup Sanità, 47(4):429-444 • Lorenzetti, Marcoccia, Narciso, Mantovani. 2010. Repr. Toxicol. 30(1):25-35 • Lorenzetti, Narciso, Marcoccia, Altieri. 2012. J. Biol. Res, 1(LXXXIV):36-41. • Smeriglio#, Trombetta#, Marcoccia# et al., Anticancer Agents Med Chem, (# equal contributors) • Marcoccia, et al., Genes Nutr, manuscript in preparation The performed in vitro activities Cell viability (MTS) assay - PSA secretion assay – Chemical intake & Intracellular distribution in LNCaP

Reference Chemicals Endocrine Active Substances (EAS) to be tested for their androgen-like activity MTS PSA Intracellular MTS PSA Intracellular Drugs , doping agents and vehicle Family Herbicides Family assay secretion distribution assay secretion distribution 5α-di-hydro-testosterone (DHT) steroid hormone ● ● ● glufosinate ammonium (GA) Phosphinic acid ● ● ● (androstanolone) Glyphosate (N-(phosphonomethyl)glycine) Fosfonoglicine ● ● ● 2OH-Flutamide (2OH-FTA) No steroid drug ● ● ● (GLYPH) (derived aa) bicalutamide no steroid drug ● ● ● Lindane organochlorine ● ● ●

E2 steroid hormone ● ● ● Terbuthylazine (TBT) Clorotriazine ● ● ●

MT doping agent ● ● ● Linuron (LIN) derived from urea ● ● ● MTS PSA Intracellular Fungicide and metabolites Family assay secretion distribution Plant-derived bioactives vinclozolin (VIN) ● ● ● MTS PSA Intracellular Metabolite M1 of the Vinclozin 2-[[(3,5- Polyphenols Family assay secretion distribution dichlorophenyl)-carbamoyl]oxy]- 2- ● ● ● methyl-3-buteonic acid dicarboximide Quercetin (QRC) flavonoid ● ● ● Metabolite M2 of the Vinclozolin 3,5- Naringenin (NRG) flavanone ● ● ● dichloro-2-hydroxy-2-methlybut-3- ● ● ● enanilide Apigenin (API) flavanone ● ● ● Fenarimol pyrimidine ● ● ● Zineb (Ethylene-1,2-bisdithiocarbamate Luteolin (LUT) flavanone ● ● ● ● ● ● de zinc) dithiocarbamates Resveratrol (RESV) flavonoid ● ● ● Mancozeb Tiocarbamide ● ● ● Metabolite of the Ethylene (thiourea) Genistein (GEN) isoflavone ● ● ● ● ● ● dithiocarbamate ethylenethiourea MTS PSA Intracellular Biocides Family assay secretion distribution Chlorpyriphos (CPF) organophosphates ● ● ● Permethrin pyrethroid ● ● ● Tributyltin oxide (TBTO) Organ-stannic ● ● ● MTS PSA Intracellular Plasticizers Family Vehicle assay secretion distribution bisphenol A (BPA) phenols ● ● ● MTS PSA Intracellular Substance Family bis(2-ethylhexyl) benzene-1,2- assay secretion distribution phthalate ● ● ● dicarboxylate (DEHP) DMSO solvent ● ● ● di-n-butyl-phthalate (DBP) phthalate ● ● ● FOOD: A SOURCE OF (NON) NUTRIENTS &… ENVIRONMENTAL CONTAMINANTS

 NUTRIENTS macro-nutrients (carbohydrates, proteins, lipids) micro-nutrients (vitamins, trace elements)

 NO NUTRIENTS (poli)phenols BIOACTIVE COMPOUNDS

 DIETARY AND pesticides, biocides, ENVIRONMENTAL plasticizers, … ENDOCRINE CONTAMINANTS DISRUPTORS (EDCs)

• Naringenin/NRG • Resveratrol/RESV

• Luteolin/LUT • Genistein/GEN

• Quercetin/QRC • Apigenin/API LNCaP - PSA secretion: screening plant extracts (1) LNCaP - PSA secretion: screening plant extracts (2)

Serenoa repens extracts are used as anti-inflammatory natural therapeutic drugs in BPH but it did not suppress PSA secretion (Habib et al., 2005) • SPO (Serenoa Repens – saw palmetto oil) • VCE (Verbascum xanthophoeniceum) • Verb (verbascoside)

153.7 pM Verb • For the first time, a S. repens extract (SPO) has been shown to inhibit DHT-induced PSA secretion • V. xanthophoeniceum extract (VCE) acted similarly to SPO but more efficiently • One purified component of VCE, the phenylethanoid glycoside verbascoside (Verb) is less efficient than its parental extract Conclusions

SCREENING OF PLANT EXTRACT ACTIVITIES  For the first time, a S. repens extract (SPO) has been shown to inhibit DHT-induced PSA secretion  V. xanthophoeniceum extract (VCE) has been shown to act similarly but more efficiently than SPO on DHT-induced PSA secretion  the phenylethanoid glycoside verbascoside (Verb), a VCE component, has been shown to have much less effect than its parental extract on DHT-induced PSA secretion INDICE

 PRESENTAZIONE Di cosa ci occupiamo e in che modo

 INTERFERENZA ENDOCRINA Definizioni Meccanismi e Modi di Azione Effetti avversi e Biomarcatori di effetto

 IL PROGETTO EUROPEO ReProTect (2006-10) Lo sviluppo di un metodo sperimentale in vitro alternativo alla sperimentazione animale: dalla tossicogenomica al «phenotypic anchoring»

 IL PROGETTO EUROPEO LIFE-EDESIA (2013-2016) Lo screening dell’Interferenza Endocrina mediante l’uso di biomarcatori di effetto in saggi funzionali LIFE-EDESIA project: Action B4

THE CORE OF ACTION B4 AT A GLANCE Cell specific endpoint: Functional Assay – Molecular endpoint: Phenotypic anchoring Cell aspecific endpoint: gene expression of . prostate: PSA secretion Cell Viability Nuclear Receptors of . trophoblast: bhCG secretion (MTS assay) interest . liver: intracellular lipid accumulation (qPCR) and AFP secretion

Gene reporter assays AR-, ER-, PPAR-gene reporter assays (OECD and/or IHCP-JRC guidelines and/or protocols under the validation programme) • • OECD guidelines for the testing of chemicals http://www.oecd.org/env/ehs/testing/oecdguidelinesforthetestingofchemicals.htm JRC-IHCP website http://ec.europa.eu/dgs/jrc/index.cfm : e.g., the “Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists” (OECD TG 455); the “BG1Luc Estrogen Receptor

Transactivation Test Method for Identifying Estrogen Receptor Agonists and Antagonists” (OECD TG 457). A gene reporter assays do not identify an endocrine disrupting property but only a part of a potential mechanism of action of a chemical whose endocrine disruption property remain to be assessed… ! : by a functionally-relevant endpoint (?) Lorenzetti S, Mantovani A. 2014. Reproductive and Developmental Toxicity Testing: issues for 3Rs implementation. In: Reducing, 41 Refining, and Replacing the Use of Animals in Toxicity Testing (Chapter 12, pp. 330-347), edited by Dave G Allen and Michael D Waters, RSC Publishing, Cambridge (UK); DOI:10.1039/9781849737920-00330. Confronto tra concetti base di tossicologia e interferenza endocrina

2013

2012

Lorenzetti, Marcoccia, Mantovani (2015), in press Come la tossicologia in vitro e i functional assays possono contribuire agli studi in vivo

Secrezione della PSA

TDS (Testicular Dysgenesis Syndrome)

AOP (Adverse Outcome Pathway)

Lorenzetti, Marcoccia, Mantovani (2015), in press LIFE-EDESIA: contatti

«COME TALK TO ME… All the things that we both might say” (P. Gabriel)

Stefano Lorenzetti [email protected]

Istituto Superiore di Sanità – ISS Dipartimento Sanità Pubblica Veterinaria e Sicurezza Alimentare Reparto Tossicologia Alimentare e Veterinaria Viale Regina Elena 299 – 00161 Roma (I) 0649902512

Peter Gabriel concert - Vienna, 3 ottobre 2013