ROS - MolBiol.ru

Oxygen and its Radicals
Cytomic Analysis of Reactive Oxygen Species
José-Enrique O’Connor
Laboratory of Cytomics
Centro de Investigación Príncipe Felipe - University of Valencia
Valencia, SPAIN
Oxygen and its Radicals
1. Oxygen and Reactive Oxygen Species (ROS)
2. Oxidative Stress: Causes and consequences
3. Analysis of oxidative stress and ROS
4. Cytomics of ROS:
a) ROS in leukocyte functions
b) ROS in platelet signalling
c) ROS in bacteria
d) ROS and in vitro toxicology
LEO the lion says GER!
LEO the lion says GER!
Loss of Electrons is Oxidation,
Gain of Electrons is Reduction
Oxygen and its Radicals
1. Oxygen and Reactive Oxygen Species (ROS)
2. Oxidative Stress: Causes and consequences
3. Analysis of oxidative stress and ROS
4. Cytomics of ROS:
a)ROS in leukocyte functions
b) ROS in platelet signalling
c) ROS in Bacteria
d) ROS and in vitro toxicology
Large amounts of reactive
oxygen in aerobic organisms
are generated by respiring
mitochondria
MITOCHONDRIAL RESPIRATION GENERATES
REACTIVE OXYGEN SPECIES (ROS)
Singlet
Oxygen
O12
H2O
hn
e-
O2
+ H+
O-2
Molecular
Oxygen
Superoxide
Radical
(Triplet)
(HO2)
e+ H+
e-
O=2
Peroxide
(H2O2)
+ H+
OH·
Hydroxyl
Radical
e+ H+
H2O
NON-MITOCHONDRIAL GENERATION OF ROS
Other enzymes producing superoxide
are
xanthine
oxidase,
NADPH
oxidases and cytochromes P450.
Hydrogen peroxide is produced by
several
enzymes
including
monoxygenases and oxidases.
ROS INTERPLAY (OR A ROS IS A ROS IS A ROS…)
Fenton Reaction:
Hydroxyl free radicals (·OH) are produced when H2O2 and a
transition metal react together:
Fe2+ + H2O2 ↔ Fe3+ + ·OH + OH-
Ferrous ion is regenerated by superoxide:
O2·- + Fe3+ ↔ O2 + Fe2+
Haber-Weiss Reaction:
O2·- + H2O2 ↔ O2 + ·OH + OHTransition metals act as catalysts for ·OH generation
Oxygen and its Radicals
1. Oxygen and Reactive Oxygen Species (ROS)
2. Oxidative Stress: Causes and consequences
3. Analysis of oxidative stress and ROS
4. Cytomics of ROS:
a)ROS in leukocyte functions
b) ROS in platelet signalling
c) ROS in bacteria
d) ROS and in vitro toxicology
REDOX METABOLISM AND OXIDATIVE STRESS
All forms of life maintain a reducing environment within their
cells. The cellular redox environment is preserved by
enzymes that maintain the reduced state through a
constant input of metabolic energy.
Disturbances in this normal redox state can cause
oxidative stress, with toxic effects through the production
of peroxides and free radicals that damage all components
of the cell, including proteins, lipids, and DNA.
Production of ROS
Oxidative Stress
PROOXIDANTS AND ANTIOXIDANTS
Cellular oxidants
Endogenous
Exogenous
Source
Metabolism
Redox cicling compounds
Peroxides
Metals (Fenton reaction)
Radiation
Oxidative Species
O2-. H2O2 .OH .NO .OCl
O2
-.
H2O2
.OH
.OH
Cellular antioxidants
Enzymatic
Superoxide dismutase
Catalase
Glutathione reductase
Glutaredoxin
Thioredoxin
Non-Enzymatic
Vitamin E
Glutathione
Vitamin C
OXIDATIVE STRESS
Oxidative stress is caused by an imbalance between the
production of reactive oxygen and the ability of a biological
system to readily detoxify the reactive intermediates or to
repair the resulting damage.
OXIDATIVE STRESS AND TISSUE DAMAGE
POSITIVE EFFECTS OF ROS
However, reactive oxygen species can be beneficial, as they
are used by the immune system as a way to attack and kill
pathogens and as a form of cell signaling in many cell types.
POSITIVE EFFECTS OF ROS: IMMUNE SYSTEM
The immune system uses the lethal effects of oxidants as a
central part of its mechanism of killing pathogens; with
activated phagocytes producing both ROS and reactive
nitrogen species.
These include superoxide (•O2-), nitric oxide (•NO) and their
particularly reactive product, peroxynitrite (OONO-)
POSITIVE EFFECTS OF ROS : CELL SIGNALLING
Stimulation with most agonists
(thrombin, collagen, etc)
ROS and platelet
activation
+
+
PI3-kinase
NADPH oxidase
.
O2
Superoxide
Decrease bioactivity of NO
Adhesion
Aggregation
Oxygen and its Radicals
1. Oxygen and Reactive Oxygen Species (ROS)
2. Oxidative Stress: Causes and consequences
3. Analysis of oxidative stress and ROS
4. Cytomics of ROS:
a)ROS in leukocyte functions
b) ROS in platelet signalling
c) ROS in bacteria
d) ROS and in vitro toxicology
METHODS FOR DETECTION OF ROS
DIRECT METHODS FOR DETECTION OF ROS
Complex spectroscopy techniques:
• Electron Spin Resonance
• Nuclear Magnetic Resonance
• X-ray Photoelectron Spectroscopy
• Absorption Spectroscopy
DIRECT METHODS FOR DETECTION OF ROS
Biochemical methodology:
• Free radical markers: Stable, specific or non-specific
derivates of physiological substances can be measured
• Lipid peroxidation products (isoprostanes, TBARS)
• Amino acid oxidation products (meta-tyrosine, ortho-tyrosine,
hydroxy-Leu, dityrosine etc.)
• Peptide oxidation products (oxidized glutathione)
• Measurement of the decrease of antioxidants (TAS, reduced
glutathione)
DIRECT METHODS FOR DETECTION OF ROS
Oxidative stress may be quantified by following
the reaction of ROS with fluorogenic substrates:
• Flow cytometry
• Confocal microscopy
• High-content analysis by bioimaging
CYTOMIC ANALYSIS OF ROS
ROS in leukocyte
functions
ROS in platelet
signalling
ROS and bacterial
cytomics
ROS and in vitro
toxicology
CYTOMIC ANALYSIS OF ROS
Oxidative stress may be analysed directly by interaction of
ROS, oxidized molecules or antioxidants with fluorescent
probes or fluorogenic substrates
FLUORESCENT PROBES FOR OXIDATIVE STRESS
• ROS (fluorogenic substrates):
• DHDCF, DHRH123, Hydroethidine, MitoSox Red
• Glutathione and SH- groups:
• Monobromobimane, Monochlorobimane, o-PTA
• Chloromethyl fluorescein
• Mercury Orange
• Peroxidized lipids:
• cis-Parinaric acid, Bodipy 665
• Oxidized bases in DNA:
• Antibody anti-8-oxodGuanine-[FITC, PE]
GREEN FLUORESCENT PROBES FOR ROS
• Dihydrodichloro fluorescein:
• Peroxidase substrate: Detection of H2O2 and MPO activity
• Oxidative burst and leukocyte activation
• Analysis of Oxidative stress
• Dihydro rhodamine 123:
• Peroxidase substrate: Detection of H2O2/OH·, peroxynitrite
• Oxidative burst and leukocyte activation
• Analysis of Oxidative stress
• Sensitive to peroxynitrite: Analysis of nitrosative stress
ORANGE/RED FLUORESCENT PROBES FOR ROS
• Dihydro ethidium (Hydroethidine):
• Superoxide-sensitive probe: Detection of O2· anion
• Oxidative burst and leukocyte activation
• Analysis of Oxidative stress
• MitoSOX Red mitochondrial superoxide indicator:
• MitoSOX Red reagent is live-cell permeant and is rapidly and
selectively targeted to the mitochondria.
• Once in the mitochondria, MitoSOX Red is oxidized by superoxide
and exhibits bright red fluorescence upon binding to nucleic acids.
• MitoSOX Red reagent is readily oxidized by superoxide but not by
other ROS- or reactive nitrogen species (RNS)–generating systems.
• Oxidation of the probe is prevented by superoxide dismutase.
PROBLEMS USING FLUOROGENIC PROBES FOR THE
ANALYSIS OF ROS AND OXIDATIVE STRESS
• Probe oxidation may not be dependent of a single ROS / RNS
• Probe oxidation may require enzymes or metal ions.
• Peroxydases or cytochrome C may oxidize directly probes
• Probe oxidation may generate ROS and interfere with ROS
metabolism
• The oxidized products may be not retained within the cell (e.g.,
DCF leakage in apoptosis or membrane damage)
• Some probes reveal indirectly ROS and require parallel
measurements (e.g. DHRH123 and Dy)
PROBLEMS USING FLUOROGENIC PROBES FOR THE
ANALYSIS OF ROS AND OXIDATIVE STRESS
DiHydroDiChloroFluorescein [DHDCF]
+ tert-Butyl Hydroperoxide
DHDCF [F.A.U]
CONTROL
PROPIDIUM IODIDE
PROBLEMSUSING FLUOROGENIC PROBES FOR THE
ANALYSIS OF ROS AND OXIDATIVE STRESS
MITOSOX
+ Plumbagin
MITOSOX [F.A.U]
CONTROL
PROPIDIUM IODIDE
PROBLEMS USING FLUOROGENIC PROBES FOR THE
ANALYSIS OF ROS AND OXIDATIVE STRESS
USE OF APPROPRIATE CONTROLS IS IMPORTANT:
• Prooxidants
• Free radical scavengers
• Metal chelators
• Soluble antioxidants
• Membrane antioxidants
• Enzyme inhibitors
• Genetically modified organisms
Oxygen and its Radicals
1. Oxygen and Reactive Oxygen Species (ROS)
2. Oxidative Stress: Causes and consequences
3. Analysis of oxidative stress and ROS
4. Cytomics of ROS:
a) ROS in leukocyte functions
b) ROS in platelet signalling
c) ROS in bacterial function
d) ROS and in vitro toxicology
CYTOMIC ANALYSIS OF ROS
ROS in leukocyte
functions
ROS in platelet
signalling
ROS in bacteria
ROS and in vitro
toxicology
ROS IN LEUKOCYTE FUNCTIONS
The immune system uses the lethal effects of oxidants by
making production of oxidizing species a central part of its
mechanism of killing pathogens; with activated phagocytes
producing both ROS and reactive nitrogen species.
These include superoxide (•O2-), nitric oxide (•NO) and
their particularly reactive product, peroxynitrite (OONO-)
Although the use of these highly reactive compounds
causes damage to host tissues, the non-specificity of
these oxidants is an advantage since they will damage
almost every part of their target cell.
This prevents a pathogen from escaping this part of
immune response by mutation of a single molecular target.
ROS IN LEUKOCYTE FUNCTIONS
The Oxygen-Dependent Microbiocidal System
Granulocytes and other phagocytic cells possess a
membrane NADPH oxidase, which takes reducing
equivalents from the hexose monophosphate shunt and
transfers these to molecular oxygen to produce superoxide
and other active oxygen species. A further myeloperoxidase
converts peroxide produced in this system to microbiocidal
products, probably including hypochlorite.
ROS IN LEUKOCYTE FUNCTIONS
Missing in X-CGD
phox
gp91
p22
p47
PO4
_
}
O2 .
H 2 O2
phagocytic vesicle
p40
phox
phox
phox
phox
p67
rac2
FLOW CYTOMETRIC ANALYSIS OF ROS IN LEUKOCYTES
DCDHF
DHRH123
DHE
Phagocytic Vacuole
NADPH Oxidase
NADPH
O2
NADP
SOD
DCF
O2H2O2
HE
O2H2O2
DCF
OH-
FROM John Paul Robinson, The University of Purdue
FLOW CYTOMETRIC ANALYSIS OF ROS IN LEUKOCYTES
Plasmid pMEK91 GFP
FLOW CYTOMETRIC ANALYSIS OF ROS IN LEUKOCYTES
whole blood
100 mL aliquots
Incubation with ROS or NO donors:
• Prooxidants: H2O2, tBOOH, paraquat
• Chemotactic stimuli: LPS
• Microorganisms: Zymosan, E.coli
• Drugs: Anaesthetics, Muscle relaxants, Plant extracts
• NONOates: DETA-NO
• Inducers of NO synthase: LPS+Cytokines
Incubation (10 min/37ºC) with:
• CD45-PC5 (5 mL)
• DHRH123 (1 mM)
• HE (1 mM)
FLOW CYTOMETRIC ANALYSIS OF ROS IN LEUKOCYTES
WBC
RBC
PEROXIDATIVE
PMN
SUPEROXIDE
PMN
Lymphocytes
Monocytes
Lymphocytes
SUPEROXIDE
Monocytes
PEROXIDATIVE
POLYCHROMATIC ANALYSIS OF ROS IN LEUKOCYTES
BODIPY 665
HE / TMRM
630/30
DET 10
(FL8)
DET 5
(FL3)
780/40
670/30
613/20
630/30
7-AAD
670/20
MONOCHLOROBIMANE
DET 6
(FL4)
DET 8
(FL6)
740LP
CD45-PC7
DET 7
(FL5)
450/65
DET 4
(FL2)
580/30
DET 3
(FL1)
530/40
HE / TMRM
DCFH-DA
DET 9
(FL7)
488/10
488 nm
351 nm
DAF-FM
DHR123
CMF
DET 2
(SSC)
635 nm
MoFlo FILTER SETTINGS
POLYCHROMATIC ANALYSIS OF ROS IN LEUKOCYTES
1. Phenotype-based selection of leukocytes:
•
CD45 PC7
2. Functional analysis:
•
Mitochondrial membrane potential:
•
•
•
Reactive oxygen species (ROS):
•
Dihydrodichlorofluorescein (DCFH)
•
Hydroethidine (HE)
Peroxidized lipids:
•
•
Tetramethylrhodamine (TMRM)
BODIPY 665
Glutathione:
•
Monochlorobimane (MCB)
•
Chloromethyl fluorescein (CMF)
POLYCHROMATIC ANALYSIS OF ROS IN LEUKOCYTES
1. Phenotype-base selection of leukocytes:
•
100 μl of EDTA-anticoagulated whole blood + 10 μl of CD45 PC7
•
Incubate for 15 minutes at room temperature in the dark
2. Staining of whole blood samples for functional analysis:
•
•
•
•
Dilute immunostained whole blood:1/20 in PBS
•
Stain with two different protocols:
Tube 1:
•
Dihydrodichlorofluorescein (DCFH) 5 μg/ml
•
Tetramethylrhodamine (TMRM) 60 nM
•
Monochlorobimane (MCB) 40 μM
•
BODIPY 665 20 ng/ml
Tube 2:
•
Chloromethyl fluorescein (CMF) 0.5 μM
•
Hydroethidine (HE) 5 μg/ml
•
BODIPY 665 20 ng/ml
Incubate for 30 minutes at 37ºC in the dark with shaking
CYTOMIC ANALYSIS OF ROS
ROS in leukocyte
functions
ROS in platelet
signalling
ROS in bacteria
ROS and in vitro
toxicology
ROS and platelet activation
Stimulation of platelets with most of agonists
(thrombin, collagen, others)
ROS generation
Antioxidants have antiplatelet effects
• Flavonoids
• Vitamin C
• Vitamin E
• Other...
Mechanisms of platelet activation
Platelet agonists
+
+
PI3-kinase
NADPH oxidase
.
O2
Superoxide
Decrease bioactivity of NO
Adhesion
Aggregation
FLOW CYTOMETRIC ANALYSIS OF ROS IN PLATELETS
A
PRP
1,75
Collagen
(mg/ml)
Collagen (g/m L)
1,5
10
7.5
5
2.5
1
1,25
1
Fluorescence ratio (DHR123)
2
2
Fluorescence ratio (DHR123)
B
WB
1,75
Collagen
(mg/ml)
Collagen (g/m L)
1,5
10
7.5
5
2.5
1
1,25
1
0,75
0,75
0
50
100
150
Time (sec)
200
250
A
0
50
100
150
Time (sec)
Effect of collagen on ROS generation in platelets
200
250
B
CYTOMIC ANALYSIS OF ROS
ROS in leukocyte
functions
ROS in platelet
signalling
ROS in bacteria
ROS and in vitro
toxicology
CYTOMIC ANALYSIS OF ROS IN BACTERIA
A prokaryote is not a small eukaryote!!
(Harald Steen)
CYTOMIC ANALYSIS OF ROS IN BACTERIA
• Flow cytometric functional assays in live bacteria
are limited in part by the cell wall, which impairs
penetration of vital dyes and imposes a need for
permeabilization procedures.
• These manipulations are time consuming, may
affect
cell
physiology,
aggregation or lysis.
and
provoke
cell
CYTOMIC ANALYSIS OF ROS IN BACTERIA
A SUGGESTION FROM ORLANDO FURIOSO
Prese nuovo consiglio, e fu il migliore,
Di vincer con altre arme il mostro crudo:
Sbabarbagliar lo vuol con lo splendore
Ch’era incantato nel coperto scudo.
CYTOMIC ANALYSIS OF ROS IN BACTERIA
• Escherichia coli strains which have been already assessed
in assays of mutagenicity (E. coli WP2 tester strains)
• Genetically modified WP2 strains deficient in some key
genes involved in the control of specific pathways in the
antioxidant defence
• Functional flow cytometry to detect and quantify
intracellular generation of reactive oxygen and nitrogen
species (ROS and NOS) and antioxidant products
CYTOMIC ANALYSIS OF ROS IN BACTERIA
G. Herrera et al.:
Cytometry 49: 62-69 (2002)
Current Protocols in Cytometry 11.16.1-11.16.9 (2003)
Lettere GIC 13: 23-28 (2004)
CYTOMIC ANALYSIS OF ROS IN BACTERIA
• Escherichia coli B WP2 strains have been used for
mutagenic assays because they possess an altered cellwall lipopolysaccharide that leads to increased membrane
permeability.
CYTOMIC ANALYSIS OF ROS IN BACTERIA
H2O2
O2.-
Fe+2
Cu+2 .OH
OxyR
RpoS
ahpCF
katG
katE
H2O+ 1/2 O2
GSH
GSSG
H2O2
SoxR/SoxS
sodA
sodB
sodC
gorA
GSSG
O2 -
GSH
Mechanism of OxyR activation by H2O2
Reduced OxyR
Oxydized OxyR
CYTOMIC ANALYSIS OF ROS IN BACTERIA
CYTOMIC ANALYSIS OF ROS-INDUCED DNA DAMAGE IN BACTERIA
• Oxidative damage to DNA is repaired by several enzymes
that belong to the base excision repair (BER) pathway
• Several mechanisms in E. coli limit mutagenesis due to the
relatively abundant oxidative lesion 8-oxo-7,8 dihydroguanine
(8-oxoG):
• MutM: at the sites of 8-oxoG formation
• MutT:
hydrolizes
8-oxoGTP
and
prevents
8-oxoG
incorporation into DNA
• MutY: repairs post-replicative adenines mispaired with 8-
oxoG lesions
• Similar pathways occur in human cells
CYTOMIC ANALYSIS OF ROS
ROS in leukocyte
functions
ROS in platelet
signalling
ROS and bacterial
gene engineering
ROS and in vitro
toxicology
Prediction of human toxicity by in vitro methods
Optimisation and Pre-validation of an In Vitro Test
Strategy for Predicting Human Acute Toxicity
The Integrated Project A-Cute-Tox
Integration of in vivo data and in vitro cytotoxicity with
additional information on biokinetics, metabolism and target
organ toxicity (neuro-, nephro- and hepatotoxicity).
WP 1 Generation of “high quality” in vivo d
Cytomics in WP 4
and establishment of a depository list of m
compounds
• Sources of in vivo data
• Data mining
• Prioritisation/selection of chemicals
• Acquisition, depository and distribution of referen
PARTNER 11
(CIEMAT)
50
75
100
125
A
25
WP 4
0
PARTNERS
11 + 9
% of inhibition of platelet activation
CFU-GM Assay
CFU-Meg Assay
-4
-3
-2
-1
0
1
New cell systems
log [cyclosporin]/mM
Platelet Inhibition
•Whole blood assay
•CFU-GM
New endpoints
•cytomic analysis
•Adaptable to robotic screening
platform
PARTNER 21
(VUB)
W
•
•
•
•
•
Cytokine Secretion Assay/ELISA
W
PARTNERS
21 + 9
PARTNER 9
(UVEG)
Cytomic Panel for Cytotoxicity Screening
Multiplex 11-Cytokine Assay
Cytomic Panel for Oxidative Stress Screening
The role of Cytomics in A-Cute-Tox
• Multiplexed assay of cytokine secretion by human neo-hepatocytes: WP8
• Cytomic prediction of cholestasis and steatosis: WP7.3
MULTIPARAMETRIC ASSESSMENT OF EARLY CYTOTOXICITY
Ca++
Mitochondrial membrane
potential
H2O2
DNA damage
ATP
Plasma membrane
potential
Superoxide
OH·
Intracellular lipid
accumulation
Plasma membrane
permeability
Lipid peroxidation
H2O2
OH·
The role of Cytomics in A-Cute-Tox
Human cell lines:
• SH-SY5Y Neuroblastoma
• HepG2 Hepatoma
• A.704 Renal Adenocarcinoma
Miniaturized 96-well format cytomic assays:
• Cytomic Panel for Cytotoxicity Screening
• Cytomic Panel for Oxidative Stress Screening
Flow Cytometry
High-Content Analysis by Bioimaging
The role of Cytomics in A-Cute-Tox
CPCS includes FCM assays of:
• Viability (Propidium Iodide)
• Intracellular Ca2+ (Fluo-4 AM)
• Mitochondrial membrane potential (TMRM)
• Plasma membrane potential (DiBAC)
CPOSS includes assays of:
• Superoxide anion (MitoSox Red) by FCM
• Peroxidative activity (DHDCF-DA) by FCM
• HCA assay of oxidative damage to:
• Genomic DNA (nuclear anti 8-oxo-dG)
• Mitochondrial DNA (cellular anti-8-oxo-dG)
The role of Cytomics in A-Cute-Tox
Other cytomic assays used and not shown here:
• Multiplexed assay of cytokine secretion by PBMC cultures and by whole
blood cultures
• Multiplexed assay of cytokine secretion by cultures of monocyte-derived
human neo-hepatocytes
• Assay of fibrinogen-receptor conformational change in whole blood
platelets activated with thrombin
• Kinetic assay of NBD-bile acid uptake by fresh isolated rat hepatocytes
(Cholestasis alert assay)
• FCM and HCA assay of exogenous lipid accumulation by HepG2 cells
(Steatosis alert assay)
ASSAY PERFORMANCE AND PREDICTIVITY
• Assays are well reproducible, with typical
CVs lower than 15%
• Refflect early changes in live cells and are
not estimations of cell death
• Allow to calculate IC50/EC50 values
Range-finding experiment
Flow cytometric assay of viability
with propidium iodide
Functional experiment:
High-Content Analysis assay for
oxidative stress in attached cells
fixed after sublethal exposure
Raw data:
Algorithm-based quantitative
analysis of bioimage stacks
(Developer Toolbox)
Calculation of IC50 and derivation of
IC20 (Phototox software)
Functional experiment:
Flow cytometric panel of assays for
cytotoxicity or oxidative stress in live
cells in sublethal exposure
Calculation of EC50 (Excel) and IC50
(Phototox)
HEP G2.MITOSOX.P.FENOL
y = 0,5033Ln(x) + 0,5799
R2 = 0,7925
y = 0,4765Ln(x) + 0,5716
R2 = 0,7924
y = 0,4899Ln(x) + 0,5769
R2 = 0,7923
2,50
RMFI +
Raw data:
Quantitative analysis (MFI) of FCS
files (MXP, CXP, Expo32 Software)
[TOXIC] LOG
2,00
RMFI -
1,50
RMFI
1,00
Logarítmica (RMFI +)
0,50
Logarítmica (RMFI -)
Logarítmica (RMFI)
0,00
0
2
4
6
8
10
RMFI
12
14
16
18
20
ASSAY PERFORMANCE
DIAZEPAM
CONTROL
Target segmentation
Nuclei
CONTROL
BHP
DAPI
Channel
Defining the Measures
Cells
NUCLEI
- Area
- Mean FITC Intensity
NUCLEI + GRANULES
NUCLEI + CELLS
- Granule Area
- Mean FITC Intensity
- Mean FITC Intensity
of Cytoplasm
of granules
- Ratio Nuc/Cytoplasm
- Count Granules in
Nuclei
Granules
FITC
Channel
ASSAY PERFORMANCE AND PREDICTIVITY
To assess the predictive
value of in vitro assays, IC50
or EC50 are correlated (R2)
with human and rat in vivo
LC50 or LD50 log values.
Our assays correlate better with the human in vivo data (Log EC50/IC50 M
vs Log LC50 M) than with rodent in vivo data (Log LD50 M) or in vitro
standard assay (Neutral Red Uptake in 3T3 cells)
ASSAY PERFORMANCE AND PREDICTIVITY
How does perform our
“competitor” in vitro assay
3T3 NRU?
• 3T3 NRU vs rat LD50: R2 = 0.54
• 3T3 NRU vs human LC50: R2 = 0.56
• rat LD50 vs human LC50: R2 = 0.50
Our assays correlate better with the human in vivo data (Log EC50 M vs
Log LD50 M) than does the in vitro standard assay (Neutral Red Uptake in
3T3 cells)
DATA MINING AND TOXIC CLASSIFICATION
WP4 has uploaded 2175 entries in the project shared database (AcutoxBase)
2175 / 9984 ~ 21.78% of all entries
*
*
*
DATA MINING AND TOXIC CLASSIFICATION
DATA MINING AND TOXIC CLASSIFICATION
DATA MINING AND TOXIC CLASSIFICATION
All the in vivo LC50/LD50 and in vitro IC50/EC50 obtained
are log transformed and standardized for cluster analysis
and hierarchization using Cluster and Treeview software,
both available at http://rana.lbl.gov/EisenSofware.htm.
The results are then clustered according to the toxic classes
of the Globally Harmonised System of Classification and
Labelling of Chemicals (GHS), based on rat LD50 values,
and to the reported human LC50 values (as from
Acutoxbase).
DATA MINING AND TOXIC CLASSIFICATION
http://www.osha.gov/dsg/hazcom/ghs.html
DATA MINING AND TOXIC CLASSIFICATION
I
II
III
IV
V
Our assays (based on human cells) do not separate perfectly
compounds belonging to the mid-toxic GHS classes (based on rat data)
but identify compounds labelled as non-toxic by GHS and may provide
alerts for specific toxicity.
DATA MINING AND TOXIC CLASSIFICATION
I
II
III
IV
V
As expected, there is no perfect match between compound clustering
according to their GHS classes (rat in vivo toxicity), to human in vivo
toxicity or in vitro toxicity (NRU assay in mouse 3T3 fibroblasts)
DATA MINING AND TOXIC CLASSIFICATION
I
II
III
IV
V
Compound cluster according to Cytomic Panel of Cytotoxicity
Screening refflects much better the classification according to human in
vivo toxicity (LD50)
DATA MINING AND TOXIC CLASSIFICATION
0.719
0.89
0.83
I
II
III
IV
V
Compound cluster according to Cytomic Panel of Oxidative Stress
Screening refflects much better the classification according to human in
vivo toxicity (LD50)
Oxygen? "I rarely use it myself, sir. It promotes rust."
www.uv.es/cytomics
• A-Cute-Tox: Optimization and pre-validation of an in vitro test strategy for predicting acute human toxicity.
• Predictomics: Short-term in vitro assays for long-term toxicity.
• Development of a cytomic platform of high-content miniaturized assays for the detection of in vitro cytotoxicity and
prediction of acute human toxicity of drugs and xenobiotics.
• Improvement of the translational prediction to humans of the non-clinical safety assays.