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•
 Luděk Bláha, PřF MU, RECETOX
•www.recetox.cz
•BIOMARKERS AND TOXICITY MECHANISMS
•01 - INTRODUCTION
•
OPVK_MU_stred_2

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Course summary
•1) Introduction
• - Intro and overview of the mechanisms beyond the toxicity
• (with special respect to environmental contaminants)
• - Intro and concept of biomarkers
•
•2) Details on selected important toxicity mechanisms
• - Membrane toxicity, enzyme inhibitions, oxidative stress, genotoxicity, Nuclear Receptors (AhR,
ER, AR ….) etc.
• - Methods to determine toxicity mechanism
•
•3) Biomarkers
• - What it is and how to find (identify) suitable biomarker(s)?
• - The overview of the most important biomarker classes
• - Methods of biomarker assessment
•

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The importance of understanding
to toxicity mechanisms

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1962
•© Patuxent Wildlife
Refuge, MA, USA
•http://www2.ucsc.edu/scpbrg/

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•In vivo: shell thinning
•In situ: bioaccumulation
-> bird population decline
•Bitman et al. Science 1970, 168(3931): 594
•
•
•Biochemistry discovered in 1970s:
Bird carbonate dehydratase

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•Thalidomide
http://pubs.acs.org/cen/img/83/i25/8325thalidomide.gif
•Originally marketed in 1957 as sedative / hypnotic
–also curing anxiety, gastritis, tension
–against nausea and morning sickness of pregnant
•TERATOGENICITY à Develoment of phocomelia = limb malformations
 (10 000 children worldwide / 40% survived)
•
http://blogs.smithsonianmag.com/smartnews/files/2012/09/thalidomide-image1.jpg
http://stealthepidemic.com/wp-content/uploads/2014/06/ThalidomideTiming-for-Sophomore.png
http://blogs.smithsonianmag.com/smartnews/files/2012/09/thalidomide-image1.jpg
•Currently still in use - completely different targets
: anticancer (multiple myeloma), antileprosis, immunosupression

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•Thalidomide
http://pubs.acs.org/cen/img/83/i25/8325thalidomide.gif
http://upload.wikimedia.org/wikipedia/commons/1/11/Chronology-TLP.png

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•Thalidomide
•... mechanisms of action
http://pubs.acs.org/cen/img/83/i25/8325thalidomide.gif
http://www.springerimages.com/img/Images/Springer/JOU=10238/VOL=2007.7/ISU=3/ART=2007_134/MediaObje
cts/MEDIUM_10238_2007_Article_134_Fig1.jpg
•(2) Teratogenicity
•(3) Anticancer
http://yamaguchi.bio.titech.ac.jp/english/images/thalidomide_en.jpg
•(1) Sedative effects
•... mechanism unknown

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MECHANISMS of chronic toxicity
•Various chronic effects have uniform biochemical basis
–
–
–
–
–
–principle studies with mechanistically based in vitro techniques
–estimation of in vitro effects of individual compounds
•HORMONE
•
•
•
•Biochemical
•effects
•TOXIN
•
•
•
•In vivo
•effects
•RECEPTOR
Understanding MoA ... may predict higher-level effects
•
•
•Organism
•
•Population & beyond

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Basics and keywords
from toxicology

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Toxicity - concept
•
•Escher, B. I., Behra, R., Eggen, R. I. L., Fent, K. (1997), "Molecular mechanisms in
ecotoxicology:
an interplay between environmental chemistry and biology", Chimia, 51, 915-921.
•MoA = mode of action

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From mechanisms (or modes of action) to biomarkers
-Chemical enters organism
+ may be metabolized/detoxified, transported, released ...
-Chemical reacts with target (e.g. DNA) and changes a specific nucleotide (e.g. G à de-oxo-G)
-
-Elevated de-oxo-G in blood
à Toxicokinetics
-
-
à Toxicodynamics
= toxicity mechanisms (MoA) and following toxic effects (e.g. mutation, cancer ...)
à (Selective) biochemical marker (biomarker)
= information about exposure and/or effect

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•
•Paracelsus (1493 - 1541)
Toxicity – the cause-effect paradigm
•‘What is there which is not a poison?
•
lAll things are poison and nothing without poison.
lSolely the dose determines that a thing is not a poison.
l
lToxicology – the science of doses
2

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What processes are beyond toxicokinetics?
•ADME
caption
•Toxicokinetics ...
•... EXPOSURE phase à Determines the final dose

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Toxicokinetics in fish


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ToxicoDYNAMICS
http://4.bp.blogspot.com/-MebbujGDXi0/UAu26D7WxII/AAAAAAAAACk/StePoxIb3Go/s1600/2.png Dynamic
simulation of processes causing toxicity and their grouping into toxicokinetics and toxicodynamics,
illustrated on the example of the aquatic invertebrate Gammarus pulex.
•MoA
•
•
•... and measurable
EFFECTS
•TARGETS = macromolecules
•(DNA/RNA, proteins, membrane lipids)

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What is toxicity? What are the types of effects?
•Toxicity
–degree to which a substance (at certain dose) can damage an organism
•
•Exposure & toxicity
–acute (immediate, high doses, days)
–chronic (sublethal / low doses, long-term)
•
•Effect & toxicity
–lethal (acute)
•mortality – definitive endpoint /  high doses
•easy to determine (single endpoint – death)
–nonlethal, sublethal (chronic)
•endocrine disruption, reproduction toxicity, immunotoxicity, tumor induction etc.
•difficult to determine (multiple endpoints)
•more specific – low concentrations / longer exposures
•often reflected by specific biochemical changes (biomarkers)
•
•Systems and organ & toxicity
–Systemic lethal toxicity
–Organ-specific toxicity (neurotoxicity, hepatotoxicity, nefrotoxicity ...)
–Developmental toxicity
–Reproduction toxicity
–

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Principles of toxicity testing
1)Define and know biological target (molecule, cell, organism, population) and its properties
2)
•2) Define and know chemical and its properties
•
•3) Define exposure of biological system to a chemical
-variable concentrations
-defined or variable duration (time)
-conditions (T, pH, life stage ....)
-
•4) Assess effects, i.e. Changes in measurable parameter in relationship to variable doses
•
•5) Dose-response evaluation & estimation of the toxicity value (i.e. concentration or dose):
• LDx, ICx, ECx, LOEC/LOEL, MIC ...
•
•

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•
•
auto0 AcuteToxScheme_RM HaF-adult arenic~4 Daphnia Dania pict
•Cu addition
•Effect concentrations expressed in total/dissolved Cu
•
Effect assessment - procedure

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How to study (chronic) toxicity ?
•In vitro studies (biochemical mechanisms)
–+ easy to perform, short-term - ecotoxicological relevancy
–+ highly controlled conditions - mostly with vertebrate cells
–+ lower amounts of chemicals needed
(new cmpnds screening)
–
•In vivo biotest testing
–+ unique whole organisms - only few (ecologically
–+ controlled conditions   nonrelevant) organisms used
–+ better ecological interpretation - mostly ACUTE assays
– - chronic: long exposures
•
•Field and in situ observations, epidemiological studies

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http://3.bp.blogspot.com/-dokK9_P3A4I/UZ38uN8AoUI/AAAAAAAAAJc/d1N8vDjsmAw/s1600/what+is+a+chemical+
reactions.jpg
http://upyourtalent.com/wp-content/uploads/2012/04/white-rat.jpg
https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcS52E4zw_1IWgj6a-zMCfagRjY0GxrrXUj7RPtc7h6W2mD
_trk2 http://www.lfu.bayern.de/analytik_stoffe/biol_analytik_toxizitaetstests/pic/159498_gr.jpg
•Organism
•Chemical
•Adverse Effects
•Death
•Altered Reproduction
•Inhibition of Growth
•
•Tumorigenicity
•Skin irritation
•…
•+
•Traditionally – Evaluation of adverse effects using the whole organism models
•REGULATORY FOCUS
(APICAL ENDPOINTS)
Výsledek obrázku pro government icon
Hazard assessment

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http://3.bp.blogspot.com/-dokK9_P3A4I/UZ38uN8AoUI/AAAAAAAAAJc/d1N8vDjsmAw/s1600/what+is+a+chemical+
reactions.jpg
http://upyourtalent.com/wp-content/uploads/2012/04/white-rat.jpg
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_trk2 http://www.lfu.bayern.de/analytik_stoffe/biol_analytik_toxizitaetstests/pic/159498_gr.jpg
•Organism
•Chemical
•Adverse Effects
Death
•Inhibition of Growth
•Altered Reproduction
•Tumor
•Skin irritation
•…
•+
•Traditionally – Evaluation of adverse effects using the whole organism models
http://www.tdi.ox.ac.uk/_asset/image/tdi-barcoded-plates.jpeg/fit/300/300/FF0000
http://wallpoper.com/images/00/27/10/25/rainbows-vials_00271025.jpg
•+
http://ehp.niehs.nih.gov/wp-content/uploads/118/4/ehp.0901392.g001.png
•10^4 Chemicals
•HTS
High-Throughput-Screening
•Chemical-biological interactions,
•Mechanistic Toxicological Data
Hazard assessment
•New – Ex vivo / in vitro / In chemico / In silico Methods

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Mode of Action (omics) toxicity testing
http://www.omics.jp/about/img/omics_01.jpg
And other „omes“
•Lipidome
•Microbiome …

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http://systemsbiology.usm.edu/SystemsBiologyCenter/EL_ERDC/ERDC_img/Earthworm-Toxicogenomics.jpg
Omes is not only for humans …

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MoA and omics are supported by strategic documents
& organizations
Toxicity Testing in the 21st Century: A Vision and a Strategy
US National Academies of Sciences
http://www.nap.edu/catalog/11970.html
https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcTAlr7MmMMxBP1Gffwd_0Gk4X2faiMuSVai0zxTF1WtiV1
DANMq
https://encrypted-tbn0.gstatic.com/images?q=tbn:ANd9GcQyQs5sVs1TzKletUOC20iLNg93Liu4X5Q_XF_I6rQWsL4
04Y83

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reactions.jpg
http://upyourtalent.com/wp-content/uploads/2012/04/white-rat.jpg
https://encrypted-tbn3.gstatic.com/images?q=tbn:ANd9GcS52E4zw_1IWgj6a-zMCfagRjY0GxrrXUj7RPtc7h6W2mD
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•Organism
•Chemical
•Adverse Effects
Death
•Inhibition of Growth
•Altered Reproduction
•Tumor
•Skin irritation
•…
•+
•Traditionally – Evaluation of adverse effects using the whole organism models
http://www.tdi.ox.ac.uk/_asset/image/tdi-barcoded-plates.jpeg/fit/300/300/FF0000
http://wallpoper.com/images/00/27/10/25/rainbows-vials_00271025.jpg
•+
http://ehp.niehs.nih.gov/wp-content/uploads/118/4/ehp.0901392.g001.png
•10^4 Chemicals
•HTS
High-Throughput-Screening
•Chemical-biological interactions,
•Mechanistic Toxicological Data
Hazard assessment
•New – Ex vivo / in vitro / In chemico / In silico Methods
•
•Key task/question:
•How to link MECHANISTIC INFORMATION with APICAL ENDPOINTS ?

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Adverse Outcome Pathways
•The EXISTING KNOWLEDGE is used to link the two anchor points: Molecular Initiating Event (MIE) and
Adverse Outcome (AO)
via a series of intermediate steps: Key Events
Chemical
Macro-Molecular Interaction
Cellular Response
Organ Response
Organism
Response
Population Response
Molecular Initiating
Event
Key
Event 1
Key
Event 2
Adverse Outcome
Tissue Effect
Key
Event 3
Chemical Property
Receptor/Ligand
Interactions
DNA Binding
Protein Oxidation
Gene Activation
Protein Production
Altered Signaling
Altered Physiology
Disrupted Homeostasis
Altered Development / Function
Lethality
Impaired Development
Impaired Reproduction
Altered Sex Ratio
Extinction
•Adverse Outcome Pathway
•Toxicity Pathway
•Mode of Action
•In silico, In chemico, In Vitro, Ex vivo
•In vivo
•Ankley, G. T., R. S. Bennett, et al. (2010). "Adverse outcome pathways: a conceptual framework to
support ecotoxicology research and risk assessment." Environmental Toxicology and Chemistry 29(3):
730-741.

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Concept of “Adverse Outcome Pathway” (AOP)
http://www.ufz.de/export/data/1/60568_Bionalytical%20Tools.jpg
•Mechanisms
or modes of action      à    à Effects

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AOP = Global strategy with support from OECD, EU, USA
•http://www.oecd.org/chemicalsafety/testing/projects-adverse-outcome-pathways.htm

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•http://aopkb.org/
•Key documents
•
•OECD Guidance document and a template for developing and assessing adverse outcome pathways
(Series No. 184, Series on Testing and Assessment)
•
•Handbook for AOP developers

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AOP Wiki
•https://aopkb.org/aopwiki/index.php/Main_Page
•Wiki-based platform for development of AOPs
•Only members of an OECD AOP development project can create / edit AOPs
•
•
https://aopkb.org/aopwiki/images/thumb/c/c9/FiveLogos.gif/780px-FiveLogos.gif

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•What AOPs are now
•in AOP Wiki (July 2017) ?
 OECD Endorsed (WNT and TFHA)
6
•Alkylation of DNA in male pre-meiotic germ cells leading to heritable mutations
•Aromatase inhibition leading to reproductive dysfunction
 EAGMST Approved
1
•Androgen receptor agonism leading to reproductive dysfunction
•
 EAGMST Under Review & for comments
18
 EAGMST Under Development
84
 SAAOP AOP Under Development
130+
•OECD Extended Advisory Group on Molecular Screening and Toxicogenomics (EAG MST)
•The Working Group of the National Coordinators of the Test Guidelines Programme (WNT)
•https://aopwiki.org/aops
https://aopkb.org/aopwiki/images/thumb/c/c9/FiveLogos.gif/780px-FiveLogos.gif

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AOP Example: MIE aromatase inhibition
•Environmental Toxicology and Chemistry, Vol. 30, No. 1, pp. 64–76, 2011

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•Aromatase inhibition leading to reproductive dysfunction (in fish)
•https://aopwiki.org/wiki/index.php/Aop:25
•

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AOP Example from RECETOX:
Modulation of RAR/RXR à developmental toxicity in fish
•Jonáš et al. 2014 Aquatic Toxicology
•http://dx.doi.org/10.1016/j.aquatox.2014.06.022
Concentration-response curves of the retinoid-like activity (expressed as % of ...
•Activation of RAR/RXR
•in P19/A15 cells by atRA and
cyanobacterial metabolites
Comparison of phenotypes of control and zebrafish embryos exposed to ...
•ZF exposed to ATRA and cyanobacterial  (120 hpf) - Control (A), exudates of C. raciborskii 3.3×
(B) and 10× (C), M. aeruginosa 10× (D) and D. quadricaudatus 17× (E). ATRA 4 μg/L (13.3 nM) (F),
12 μg/L (40 nM) ((G) and (H)), 36 μg/L (I) and 108 μg/L (J).
•atRA
•other RAs in cyanos

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•Kidd, K.A. et al. 2007. Collapse of a fish population following exposure to a synthetic estrogen.
Proceedings of the National Academy of Sciences 104(21):8897-8901
•Controls        +Ethinylestradiol
•5 ng/L (!)
•7 years
http://upload.wikimedia.org/wikipedia/commons/8/8d/Ethinylestradiol-2D-skeletal.png

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Keywords to remember and understand
•What is meant by the “mechanism of action” (or “mode of action”) in toxicology? •Why is it
necessary to understand MoAs? What is the AOP concept?
•What is toxicokinetics? What is ADME?
•What is toxicodynamics?
•What is the relationship between the exposure and the effect?
•What are the different types of toxicity?
•How can the (toxic) effect be measured / assessed?
•What types of “bioassays” are available to study toxicity and/or MoA?
•How is the result (i.e. „toxicity“) described in numbers?
•