Application of in vitro bioassaysfor water quality monitoring

Dr.Ir. Harrie Besselink

This research has received funding from the European Union’s Seventh Framework Programme under the grant agreement no. 308339.

BioDetection Systems BV (BDS)Science Park 4061382 AD AmsterdamThe Netherlandsharrie.besselink@bds.nl

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Taken from Escher et al

Why use bioassays?

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• Focus on biological effects• Identity compound frequently unknown• Present as a complex mixture• Activities of individual compounds largely unknown• Sometimes at relatively low concentrations• Compounds can act in concert

• We cannot determine all compounds chemically• …. and if we could we would most of the time not know

their biological effect(s)

Why use bioassays?

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Why use bioassays?

• Generation of simple, rapid screening systems for monitoring and identifying complex mixture for various toxicological endpoints

• Widespread implementation • suitable for regulatory purposes• rapid availability• mechanistic base• cost effective• Transferable• high-throughput• OECD/ECVAM validated methods as anchors/quality control

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CALUX approach

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Nucleus

Ligandbinding

Cytosol

Transcription

ProteinsEnzymesLuciferase

Add substrate (luciferine)

Light

ChemicalResponsiveElement(CRE)

Ligand

Transport protein

Hsp

Chemical receptor

Principle CALUX bioassay

03.02.2015 7Hanahan and Weinberg 2000, Cell 100, 57-70

Signaling pathways: selection of assays

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Endpoint Mechanisme Actieve stoffen Assay

Nuclear receptor Estrogen receptor (alpha) (xeno)hormones, pharmaceuticals, pesticides, plasticizers

ER (alpha) CALUX

Androgen receptor (xeno)hormones, pharmaceuticals, pesticides, ? AR CALUX

Progesteron receptor (xeno)hormones, pharmaceuticals, pesticides, ? PR CALUX

Glucocorticoid receptor (xeno)hormones, pharmaceuticals, pesticides, ? GR CALUX

Thyroid receptor (beta) (xeno)hormones, pharmaceuticals, pesticides, ? TRbeta CALUX

RAR receptor (alpha) Pharmaceuticals, pesticides, ? RAR CALUX

Peroxisome proliferator-activated receptor alpha

Pharmaceuticals, pesticides, ? PPARalpha CALUX

Peroxisome proliferator-activated receptor gamma

Pharmaceuticals, pesticides, ? PPARgamma CALUX

Dioxin receptor PCBs, dioxines DR CALUX

Dioxin receptor PAHs PAH CALUX

Stress pathway p53 pathway Nitro-PAHs, pharmaceuticals, heavy metals, ? P53 CALUX

Nrf2 pathway Heavy metals, ? Nrf2 CALUX

Cytotoxicity ? Control CALUX

Latest countStable CALUX: n=28

CALUX bioassay panel

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ER AR PR TR GR RAR DR

KappaB cytox PPAR

• Endocrine

ER AR PR TR GR RAR DR

KappaB nrf2 p53 p21 cytox

PPAR

ER AR TR GR RAR DR

KappaB cytox PPAR

• DevelopmentER AR PR TR GR RAR DR

KappaB nrf2 p53 p21 cytox

PPAR

• Mutagen/carcinogen

ER AR PR GR RAR DR

KappaB nrf2 cytox PPAR

• Immuno

KappaB nrf2 p53 p21 cytox • Acute

Effect-related profiling by bioassays

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Fields of application

Bio-based economy

PharmaFood risk & benefit

Chemical safety

Environment

CALUX panel

Bio-based economy

PharmaFood risk & benefit

Chemical safety

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Dutch & Australian case studies

Scre

enin

g to

olbo

xSelection of relevant endpoints in water

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Dutch River water: Dommel highest ER CALUX and intersex in bream

Estradiol equivalents (pmolEEQ/l) Compartment n Range Median Industrial wastewater Effluent 3 0.2 – 9.5 0.9 Influent 5 5.8 – 560 317 Municipal wastewater Effluent 10 <LOD – 2.2 0.3 Untreated influent 13 2.4 - 275 27 Surface water Surface water 90 <LOD – 0.61 0.07 Polder ditches 11 0.003 – 0.74 0.03 Rainwater 3 0.01 – 0.22 0.13

Dutch National Water Research (LOES 2002):Industrial and Municipal Wastewater

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Rhine Monitoring (ER, AR, TR, GR):temporal effects

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• All types of endocrine activity present

• Identity of active compounds unknown

Monitoring wastewater / surface water

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Development and optimalisation of wastewater purification using CALUX bioassay

Pharmafilter: purification of wastewater for hosptials

Bioassay unit Test 1

(26 jan.) Test 2

(16 febr.) Test 3

(28 febr.) Test 1-3 filtrate

AR CALUX (ng DHT eq./l) 66 62 96 <0.31 Era CALUX GR CALUX PR CALUX

(ng 17βestradiol eq./l) (ng Dex. Eq./l) (ng Org2058 eq./l)

69 35

<1.3

52 2044 4.6

138 297 23

<0.04 <7.6 <1.3

Evaluatie rapport Pharmafilter;Full scale demonstratie in het Reinier de Graaf Gasthuis, Delft. Stowa rapport 29. 2012

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Thank you for your attention!