1) Lorenzetti et al., 2010. Reprod Toxicol, 30:25-35; Mørck et al., 2010. Reprod Toxicol, 30:131-7; Kwiecińska et al., 2011. Pharmacol Rep, 63:1195-202; Lorenzetti et al., 2011. Annals Ist Super Sanità, 47:429-44.

2) Kawai et al., 2001. Hepatology, 33:676-691; Fujimura et al., 2009. J Appl Toxicol, 29:356-363.

3) Lorenzetti, Mantovani. 2014. Reproductive and Developmental Toxicity Testing: Issues for 3Rs Implementation, pp.330-47. In: Reducing, Refining and Replacing the Use of Animals in Toxicity Testing, Eds. D.

Allen, MD Waters, RCS Publishing DOI:10.1039/9781849737920-00330; Tsai et al., 2014. Vitam Horm, 94:427-35. http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/test-submission/tests-submitted-to-eurl-ecvam;

http://www.oecd-ilibrary.org/environment/test-no-455-performance-based-test-guideline-for-stably-transfected-transactivation-in-vitro-assays-to-detect-estrogen-receptor-agonists_9789264185388-en.

Lorenzetti S 1,#,

Basile L 2, Benfenati E 3, Fiorino F 4, Lauthier J 2, Marcoccia D 1, Perissutti E 4, Roncaglione A 3, Mantovani A 1

1 Istituto Superiore di Sanità (ISS)-National Health Institute, Food and Veterinary Toxicology Unit, Dept. Food Safety and Veterinary Public Health, viale Regina Elena 299, 00161 Rome, Italy2 EtnaLead srl, via San Nullo 5/i, 95123 Catania, Italy

2 Istituto Ricerche Farmacologiche Mario Negri (IRFMN), via La Masa, 20156 Milan, Italy4 Università degli Studi di Napoli Federico II, via D. Montesano 49, 80131 Napoli, Italy

Functional biomarkers as toxicological endpoints

in searching for plasticizers' alternatives

Abstract. One of the goals of the LIFE-EDESIA project is to apply the substitution principle to Endocrine Disruptor Compounds (EDCs) of "equivalent concern" on the basis of

both endocrine disruption effects and the potential exposure of general population to Substances of Very High Concern (SVHC) such as phthalates and bisphenols. Hence, a new, robust and

cost-effective in silico/in vitro approach to evaluate suitable chemicals for replacing EDC of equivalent concern has been proposed making use of i) chemical selection through an in silico

toxicological process to identify potential substitutive chemicals, both on the basis of the state of the art and applying available in silico methods (molecular docking, QSAR); and ii) a

subsequent in vitro evaluation of toxicological and ED effects making use of human-derived cell lines such as fetal hepatocytes (HuH6), throphoblasts (BeWo) and prostate epithelium

(LNCaP) representative of EDC-target tissues. As specific toxicological and EDC screening endpoints we will make use of well established clinical and functional biomarker as biomarkers of

effect: a-fetoprotein (AFP) secretion and lipid accumulation (steatosis) in HuH6, bhCG secretion in BeWo and Prostate-Specific Antigen (PSA) secretion in LNCaP will be monitored by

ELISA, fluorimetric and colorimetric assays in order to compare the ED effects of reference plasticizers (e.g. BPA, DEHP) versus the in silico selected alternative chemicals. Finally, it will

be performed a comparison of the chemical effects between the proposed cell-based bioassays and the chemicals' ability to transactivate their nuclear receptor mediators by ER-, AR- and

PPAR-reporter assays.

Endocrine Disruptors in silico/in vitro - Evaluation and Substitution for Industrial Applications (LIFE-EDESIA)

References.

The in silico – in vitro approach of the LIFE-EDESIA project (Implementing Actions B1-B5).

LNCAP

(PROSTATE EPITHELIUM)

BEWO

(TROPHOBLAST)

HUH6

(FETALHEPATOCYTE)

HUMAN CELL LINES 1

(and represented target tissues)

FUNCTIONAL BIOMARKERS 1,2

(based on human clinical biomarkers)

PSA secretion (free and total PSA)

by a dual-label time-resolved fluoroimmunoassay, DELFIA: free PSA(europium-labeled antibodies/615nm) and total PSA (samarium-labeledantibodies/642 nm).

bhCG secretion

by a solid phase enzyme-linked immunosorbent assay (bHCG ELISA)based on the sandwich principle.

AFP secretion and hepatosteatosis

by a solid phase enzyme-linked immunosorbent assay (AFP ELISA) anda steatosis colorimetric assay kit, respectively.

Cell specific endpoint:

Functional Assay –

Phenotypic anchoring

prostate: PSA secretion

trophoblast: bhCG secretion

liver: AFP secretion

and/or intracellular lipid

accumulation

Molecular endpoint:

gene expression

of Nuclear

Receptors of

interest

Cell aspecific

endpoint:

Cytotoxicity

Gene reporter assays

AR-, ER-, PPAR-gene reporter assays(OECD and/or IHCP-JRC guidelines and/or protocols under the validation programme)

THE IN VITRO CORE AT A GLANCE

GENE REPORTER ASSAYS 3

(on main targeted Nuclear Receptors)

THE IN SILICO TOXSCREEN CORE

CHEMICO-PHYSICAL PROPERTIES

TOX PROPERTIES (e.g. cancerogenic, mutagenic, binding to Nuclear Receptors)

MOLECULAR DOCKING

QSAR

CYTOTOXICITY 1

Cell viability (MTS) assay

GENE EXPRESSION

qPCR of the Nuclear Receptors of interest

AR-BASEDDetection of (anti-) androgenic activity ofchemicals (i.e. JRC-ECVAM TM2010-07under validation; TM2010-02/PALM underperformance evaluation)

ER-BASEDDetection of (anti-) estrogenic activity ofchemicals (i.e. JRC-ECVAM TM2009-02/MELN under validation; TM2009-11under performance evaluation; or OECDTG-455 7)

PPAR-BASEDDetection of (anti-) PPAR activity by arecombinant PPRE-driven luciferaseassay

Acknowledgements. Contacts.

life.edesia@iss.ithttp://www.iss.it/life

http://www.iss.it/life/index.php?lang=2 stefano.lorenzetti@iss.it #

https://www.facebook.com/pages/Life-Edesia/180734252116032?ref=stream

LIFE-EDESIA is supported by the LIFE+ programme (LIFE12 ENV/IT/000633).