OXIDATIVE STRESS AND
IN VITRO CELL TRANSFORMATION
MAIRE Marie-Aline
2nd McKim Workshop on Reducing Data Redundancy in Cancer assessment
Baltimore, 8-10 may 2012
►



Cell transformation
Definition
Characteristics of transformed cells
Mechanisms of cell transformation
►



Oxidative stress in carcinogenesis
Definition
Sources of ROS
Effect of ROS on macromolecules, modulation of gene expression
►



Oxidative stress and cell transformation
Acrylonitrile
Chromium
Fibres and particles (e.g. silica)
Cell transformation
► multistage process that results in the conversion of normal cells into
fully malignant cells after exposure to carcinogen substances
Cell transformation assays :
•
•
•
ECVAM validation (2005-2009)
OECD recommendation (2007)
OECD TG (april 2012)
•
•
primary cells (Syrian hamster embryo), cell lines (Balb3T3, C3H101/2)
similar to that observed in vivo for both genotoxic and non-genotoxic
carcinogens
surrogate in vitro model for carcinogenesis in vivo
mechanisms of carcinogenesis
•
•
SHE cells
Balb3T3
Mechanisms of cell transformation
► Genotoxic and non-genotoxic mechanisms
•
disturbance in signal transduction
•
imbalance of cell proliferation/apoptosis
•
modulation of gene expression (cell cycle control, proliferation and differentiation)
•
alteration of DNA repair
•
oxidative stress
•
histone desacetylation, DNA methylation
•
inflammation
•
changes in intercellular communication
•
immunosuppression
► Several studies have involved oxidative stress in cell transformation
Oxidative stress
►Oxidative stress
 physiological, endogenous and exogenous processes that directly / indirectly affect the oxidant and
antioxidant balance
 occurs in cells or tissues when the concentration of reactive oxygen species (ROS) generated
exceeds the antioxidant capacity of the cells
 cellular oxidative damage
►Involved in aging process & and in the pathogenesis of a number of diseases :
 cardiovascular, metabolic, inflammatory and neurodegenerative diseases, cancers
►Cancers
 oxidative damage can participate in all stages of carcinogenesis process
 prooxidant state observed in human cancers / tumor cells
ROS : reactive oxygen species
►Key players in oxidative stress
 radicals, ions, molecules with a single unpaired electron in their outermost shell of electron
 highly reactive
► Free oxygen radicals :
► Non radical ROS :
•
•
•
•
•
•
•
•
•
•
•
•
•
superoxide (O2•-)
hydroxyl radical (•OH)
nitrite oxide (NO•)
organic radical (R•)
peroxyl radical (ROO•)
alkoxyl radical (RO•)….
hydrogen peroxide (H2O2)
singlet oxygen (1O2), ozone (O3)
organic hydroperoxides (ROOH)
hypochloride (HOCl)
peroxinitrite (ONO-)
dinitrogen dioxide (N2O2)…
highly reactive lipid- or carbohydratederived carbonyl compounds
► H2O2, O2•-, •OH = most studied ROS in carcinogenesis
Sources of ROS
Endogenous sources
peroxisomes,
mitochondria,
inflammatory cells,
P450 metabolism,
NADPH oxidase,
Lipoxygenase…
► e.g. Peroxisomes (organelle, consumption of O2)
 ROS production via peroxisomal oxidase (acyl coA and xanthine oxidase)
 Peroxisome proliferator increase the number and size of peroxisomes
Ex : hypolipidemic drugs, phthalates, halogenated coumpounds…
 Link between peroxisome proliferation and hepatocarcinogenesis
► e.g. Inflammatory cells (neutrophils, eosinophils, macrophages)
 Activation (endo / exogenous stimuli) leads to a respiratory burst :
• increase in oxygen uptake
• generation of ROS through NADPH oxidase
Phagocyte-derived ROS may be involved in the development of cancers
Sources of ROS
Endogenous sources
Exogenous sources
peroxisomes,
mitochondria,
inflammatory cells,
P450 metabolism,
NADPH oxidase,
Lipoxygenase…
Ionizing radiations, UV
Environmental toxicants
Chemotherapeutic agents
…
The majority of environmental, occupational and industrial
chemicals are able to generate free radical species primarily
or through their metabolic activation
Antioxidant defenses
ROS
Antioxidants
► Enzymatic
e.g. superoxide dismutase (SOD), catalase,
glutathione peroxidase (GP), peroxiredoxin, …
► Non-enzymatic
e.g. Glutathione (GSH), flavonoids (EGCG), carotinoids, vitamins (E, C), pyruvate,
urate, plant-derived antioxidants, metallothioneins, …
Effect of ROS on macromolecules
ROS
Antioxidants
Enzymatic
(SOS, CAT, GSH perox…)
Non-enzymatic
(VitE, GSH, VitC…)
Oxidative damage
Proteins
Lipids
ADN, ARN
Altered gene expression
Signaling
pathways
DNA
methylation
…
Effect of ROS on macromolecules
DNA
PROTEINS
DNA damage
Formation of carbonyl derivatives
single or double strand
breakage, base modifications,
deoxyribose and phosphate
modifications, DNA cross-link,
DNA adducts (e.g. 8OHdG)
Direct oxidation of amino acid chains
Oxidation-induced peptide cleavage
Modification of structure and
function of the membrane
cell death, mutation,
induction of transcription,
induction of signaling
pathways, genomic
instability
Changes to receptor proteins and
gap junction proteins
Stimulation or inhibition of
enzymes activity
LIPIDS
Lipid peroxidation
Lipid peroxyl radical
Lipid hydroxyperoxide
Modification
Reactive
of membrane
aldehydes
structure &
formation
function
(MDA, 4-HNE…)
Loss of cell
homeostasis
Alteration of gene expression by ROS
ROS
Antioxidants
Enzymatic
(SOS, CAT, GSHperox…)
Non-enzymatic
(VitE, GSH, VitC…)
Oxidative damage
Proteins
Lipids
ADN, ARN
Altered gene expression
Signaling
pathways
DNA
methylation
…
Effect of ROS on signaling pathways
ROS are integrated in signaling pathways
Homeostasis
Normal
growth and
metabolism
Response to oxidative stress
Low or transient level of ROS
High level of ROS
Regulation of cellular migration, proliferation,
survival, death response
Apoptosis and
necrosis
• alteration of expression of proto-oncogenes / growth factors
• activation of transcription factors (Nfr2, AP-1, Nf-kb, HIF-1, p53, Foxo…)
• activation of protein kinase pathways (MAPKs, Akt, PKC…)
dysregulation of these pathways =
potential mechanisms of ROS-induced carcinogenesis
Signaling pathways : e.g. Nrf2 and NFkB
Nfr2
• protective enzymes : xenobiotic detoxification, antioxidative response, proteome maintenance
• low level / loss of activity :  ROS production, DNA damage, predisposition to tumorigenesis
Effects of ROS on DNA methylation
Hydroxyl radical OH•
DNA damage
modified DNA structure
reduction or inhibition of
the methyl-accepting ability
base modifications, deletions, strand breakage,
chromosomal rearrangement …
8-OHdG, 8-hydroxyguanine, O6- methylguanine…
DNA methyltransferases
(DNMTs)
HYPOMETHYLATION
 transcriptional level, aberrant genes expression, activation of
proto-oncogenes, genomic instability
Oxidative stress and carcinogenesis
ROS
Antioxidants
Enzymatic
(SOS, CAT, GSHperox…)
Non-enzymatic
(VitE, GSH, VitC…)
Oxidative damage
Lipids, ADN, ARN, Proteins
Altered gene expression
Signaling pathways, DNA methylation…
CARCINOGENESIS
Oxidative stress and carcinogenesis
 most of these compounds induce cell transformation
(Klaunig et al., 2011)
Oxidative stress and cell transformation
1. Acrylonitrile
2. Chromium
3. Fibres and particles (e.g. Silica)
Example : Acrylonitrile
► Acrylonitrile (ACN) :
 intermediate used in manufacture of acrylic fibres, plastics, synthetic rubbers and resins
 exposition : manufacturing process, end-product usage, cigarette smoke, drinking water…
► IARC 2B
 genotoxicity equivocal in vitro and in vivo (Whysner et al., 1998)
 absence of ACN-DNA-adducts in brain (major target organ) :
mechanisms of carcinogenesis others than ACN-DNA reactivity (epigenetic mechanisms)
 mechanisms of carcinogenesis associated with oxidative stress (Jiang et al., 1998 ; Kamendulis et
al., 1999)
Acrylonitrile : oxidative stress
Acrylonitrile
Conjugaison with the cellular
antioxidant GSH
(major route of detoxification of ACN)
Metabolism of ACN via P450
Cyanide compounds
(reactive epoxide
cyanide ethylene oxide)
ROS by-products via
P450 2E1 and through
futile cycling
Early depletion of GSH
Contribution to an overall
decrease in antioxidants
Inhibition of the mitochondrial respiratory chain
Inhibition of antioxidant enzymes activity
(e.g. catalase)
Oxidative stress
Acrylonitrile : SHE cell transformation
► Acrylonitrile induces cell transformation for treatment >24 h:
►Decrease of ACN-cell transformation by antioxidants :
• Vitamin E (a-tocopherol)
• EGCG (-)-epigallocathecin-3-gallate (green tea flavonoid)
(adapted from Zhang et al., 2000 ; 2002)
Acrylonitrile : formation of 8OHdG
Treatment duration
24 hours
2 days
3 days
7 days
ROS
8OHdG
TF
+
+
+
+
++
+++
+
++
++
+++
• Increase of ROS and 8OHdG
• Not statistically increase of 8OHdG at 24h
• Consistent with the failure of 24h ACN treament to
induce TF
• Co-treatment with antioxydants result in a
decrease in 8OHdG formation
Antioxidants inhibit both TF and oxidized DNA damage
(adapted from Zhang et al., 2000)
Acrylonitrile : effect on oxidant/antioxidants
► Early and temporal depletion of enzymatic and non enzymatic antioxidant
 GSH, catalase, superoxide dismutase
► Activation of oxidant enzyme
 xanthine oxidase
Effects of ACN on catalase and xanthine
oxidase can be cancelled by inhibition
of P450 activity.
P450 metabolism required for
ACN-induced oxidative stress
and cell transformation ?
(adapted from Zhang et al., 2002)
Acrylonitrile : P450 metabolism & TF
► P450 metabolism is required for acrylonitrile effects :
 Co-treatment with 1-aminobenzotriazole (ABT) (nonspecific suicidal inhibitor of P450)
(adapted from Zhang et al., 2002 ; Kamendulis et al., 2002)
Acrylonitrile : conclusion
► Involvement of oxidative stress in cell transformation induced by ACN :
• Cell transformation is correlated with the formation of ROS and the increase of 8OHdG
• Cell transformation can be inhibited by antioxidants (EGCG and vitamin E)
• ROS induced by ACN may regulate expression of antioxidant defenses (Cat, SOD,
GSH) and oxidant enzyme (xanthine oxidase)
• Production of ROS requires oxidative metabolism and/or a metabolite of ACN
Oxidative stress in cell transformation
1. Acrylonitrile
2. Chromium
3. Fibres and particles (e.g. Silica)
Chromium
► Metalloid compounds :
 toxic metals : As, Cd, Ni, Pb, Cr...
 known to induce adverse effects on humans & promote carcinogenesis
 toxicological properties partly related to generation of ROS
e.g. direct mitochondrial respiration damage, ROS production via Fenton reaction, lipid
peroxidation, depletion of antioxydants…
► Chromium compounds :
 associated with malignant disease (e.g. lung cancer)
 exposure : welding, tanneries, chromium plating, exhaust from cars, cigarette smoke...
 Cr(VI) compounds : human carcinogen IARC 1
 Cr(III) : IARC 3, but key role in carcinogenesis induced by Cr(VI)
Chromium (VI) SHE cell transformation
► Hexavalent chromium compounds :
 Zn, Ca, Sr, Pb, Ba
 induce dose-dependant cell transformation
► in vivo validation of transformed SHE cells
 MT colonies checked for colony formation in soft agar and
injected back in newborn hamsters
strontium chromate
calcium chromate
(Elias et al., 1989)
SHE morphologically transformed colonies induced
by chromium compounds acquire tumorigenic
potential in time
Chromium (VI) compounds
► Potential carcinogenic mechanisms of chromium compounds
Cr(III)
CrSO42-
Reduction Cr(VI) → Cr(III)
Cr-DNA adducts
(e.g. Cr-Asc, Cr-GSH…)
Fenton-like reaction :
generation of hydroxyl
radicals
Oxidative stress may be involved in carcinogenesis and cell transformation
induced by Cr(VI)
(adapted from Henkler et al., 2010)
Oxidative stress in cell transformation
1. Acrylonitrile
2. Chromium
3. Fibres and particles (e.g. Silica)
Cell transformation induced by fibres & particles
► CTA recommended as alternative methods for evaluation of carcinogenicity of solid material
(Fubini et al., 1998 - ECVAM workshop 1998)
Fibres & particles
►Factors involved in the toxicity / carcinogenicity :
 chemical composition, structure, type
 dimension, diameter, length (phagocytosis)
 surface reactivity
• ability to generate ROS
• oxidative damage
Diatomaceous earth
► Genotoxicity :
 weak mutagenicity, aneugen and clastogen effects
 oxidative DNA damage
Quartz & Zeolite
►e.g. Silica :
 crystalline silica (quartz, cristobalite, tridymite…) : IARC 1
 amourphous silica (diatomaceous earth, calcined DE…) : IARC 3
Asbestos
ROS induced by silica
Main sources of ROS generated by silica
Inflammation
Cell-generated ROS
Neutrophils & Alveolar macrophages
•
•
•
•
•
•
Particle-generated
free radicals and ROS
Silica-based surface radicals
SiO•, SiO2, SiO3•, Si+ O2•-
Iron site active at the surface
Cells damage & lung injury
Membrane damage through lipid peroxidation : ↑of permeability, perturbation of intracellular homeostasis
Activation of cell signaling pathways (MAPK/ERK kinase…)
Activation of transcription factors (NFkB, AP-1, Nfr2…)
Increased expression of inflammatory cytokines (TNFa, IL-1…)
Apoptosis induction :
mitochondrial dysfunction
increased gene expression of death receptors & ligands (TNFa, FasL…)
Cell transformation induced by silica
► Treatments surface influence cell transformation of silica :
Treatment
Cell transfo.
Diatomaceous earth (DE)
natural DE acid-washed then calcined
+++
Quartz - (Min-U-Sil 5)
natural
+++
Quartz - HF
hydrofluoric acid
the outmost layers was dissolved
++
Quartz - Fe
enriched in iron
iron was deposited on the surface
++
Quartz - dfx
deferoxamine mesylate
iron was extracted by chelator
+
► Relationship between cell transformation, surface treatment and ROS generation :
*
DE
Min
HF
dfx
(Fubini et al., 2001)
*(30.4 µg/cm²)
Oxidative stress in silica-induced TF
► Co-treatments with antioxidants decrease cell transformation :
mannitol : quencher of HO• , catalase : dismutation of H2O2, SOD : dismutation of O2-•
• surface radicals react with H2O and/or H2O2 to form HO•
• catalase can inhibit this reaction (not SOD)
• consistent with the larger efficiency of catalase in inhibiting TF
(adapted from Fubini et al., 2001)
Conclusion : Silica
► Ability of surface to generate ROS :
relevant role in cell transformation, inhibition with antioxidant enzymes
similar results obtained with refractory ceramic fibers (Elias et al., 2002)
► Role of iron present at the surface in cell transformation :
• Small iron contamination present at the surface :
– increase biological reactivity of particles
iron chelation but also iron coating decrease cell transformation
• Large iron contamination :
– decrease some adverse effects
– reduce cellular uptake
– decrease attachment to the cell surface and/or internalization of iron-coated particles
→ decrease of cell transformation
Conclusion : CTA / oxidative stress
► Relevance of CTA :
 CTA provide evidence for several stages in neoplastic progression (long term)
 accurate and comprehensive recapitulation of the in vivo neoplastic process
 clarify carcinogenecity in case of ambiguous genotoxicity
 may be indicative of mode of action of substances
(duration treatment, sequential treatment, co-treatment…)
► Mutagenicity / genotoxicity assays in case of oxidative stress :
 indicative of mainly single changes
 in case of reparation of oxidative DNA damage : escape to the genotoxicity screening
 some events not directly related to DNA damage may occur before reparation :
•
activation by ROS of signaling pathways / epigenetic mechanisms
•
contribution to growth and neoplastic transformation
Thank you for your attention
Characteristics of transformed cells
► a block in cellular differentiation
 visualised as morphological transformation in the SHE CTA
► the acquisition of immortality expressed by :
 unlimited lifespan, aneuploid karyotype, genetic instability
► the acquisition of tumourigenicity closely associated with :
 in vitro phenotypes of foci formation, autocrine factor production, anchorage independent
growth in semi solid agar
► malignant growth when transformed cells are injected back into a suitable host
(LeBoeuf et al., 1999)
DNMTs
Me
CpG islands in promoter region
(Methyl CpG binding domain
MBP, Methyl CpG binding
protein)
maintenance : inheritance of gene silencing
de novo methylation : increase promoter methylation status
HYPERMETHYLATION :
Block access to the transcriptional machinery of
the promoter
 Gene silencing : tumor suppressor
HYPOMETHYLATION :
 Gene activation : oncogenes
Transcriptional activity
regulated by the chromatin’s
acetylation status :
Histones
acetylation/déacetylation
aberrant gene expression
associated with malignant
transformation
involved in the
acquisition of
transformed
phenotype
CARCINOGENESIS
Endogenous sources of ROS
PEROXISOMES
MITOCHONDRIA
INFLAMMATORY CELLS
Organelles, consomption of O2
Mitochondrial electron transport chain
Activation
(endo / exogenous stimuli) of
neutrophils, eosinophils,
macrophages
Production of ROS involves
peroxisomal oxidase (acyl coA
and xanthine oxidase)
Peroxisome proliferator increase
the number and size of
peroxisomes
Ex : hypolipidemic drugs,
phthalates, ester, halogenated
coumpounds…
Link between peroxisome
proliferation - induced ROS
and liver tumorigenesis
Complex I : NADH-ubiquinone
oxidoreductase
Complex III : ubiquinol cytochrome c
oxidoreductase
Undergo to a respiratory burst :
– increase in oxygen uptake
ROS-generated at complex III
regulate hypoxic activation of
HIFs (hypoxia inducible
factors), transcription factors,
involved in cell proliferation
and angiogenesis
– generation of ROS through
NADPH oxidase
Phagocyte-derived ROS may
be involved in the development
of cancers
Origin of ROS in silica cell transformation
► Nature and origin of ROS implied in silica cell transformation :
 Clear-cut relationship between HO• release and cell transformation
 Antioxidants decrease cell transformation :
−
mannitol : quencher of HO•
−
catalase : dismutation of H2O2
−
SOD : dismutation of O2-•
Involvement of HO•, H2O2, O2-•
in cell damages
► Iron yield of HO• radical via the Fenton reaction :
 Aqueous suspensions of quartz generate : H2O2, HO•, H2O2, O2
 Fenton reaction (ferrous ions impurity at the surface)
Fe2+ + H2O2 → Fe3+ + OH- + HO•
Origin of ROS in silica cell transformation
► Iron yield of HO• radical via the Haber-Weiss cycle
 In the presence of reductants (metabolites such as ascorbate, cysteine, gluthatione)
 O2-• as intermediate
Fe3+ + reductant (n) → Fe2+ + reductant (n-1)
(n= redox state of the reductant molecule)
Any scavengers of HO•, H2O2, O2-•
mannitol, catalase, SOD
inhibit the overall reaction
 this way has already been hypothesized for asbestos fibres
Origin of ROS in silica cell transformation
► Parallel free-radical mechanisms not involving iron and O2•-:
Surface radicals
SiO•, SiO2, SiO3•, Si+ O2•-
 surface radicals react with H2O and/or H2O2 to form HO•
Catalase (not SOD)
inhibit this reaction
 consistent with the larger efficiency of catalase in inhibiting cell transformation
Metal mixture Balb3T3 cell transformation
► e.g. metal mixture (As, Cd, Pb)
 As, Cd : IARC 1 and Pb : IARC 2B
 Induced cell transformation in Balb3T3 system
► Oxidative damage markers related with cell transformation of metal mixture
↑ ROS and ↓ cell viability : significantly correlated with the ↑ of cell transformation
(Silva-Aguilar et al., 2011)
Metal mixture Balb3T3 cell transformation
► Metal mixture induced cell transformation
as both initiator and promoter
initiation phase
promotion phase
► Co-treatment with antioxidant N-acetylcysteine*
Decreased TF if metals are initiator-only
Abolished TF if metals are promoter
Metal mixture : 2 µM As, 2µM Cd, 5µM Pb ; TPA : 0.1 µg/mL ; MNNG : 0.5 µg/mL
(Silva-Aguilar et al., 2011)
* Cysteine donor, promotes the reduction of glutathione (GSH)
Metal mixture Balb3T3 cell transformation
ROS
Initiation phase
Metals
Promotion phase
MNNG/Metals
day 1
day 4
day 7
day 11
day 16
day 21
↑
↑
genotoxicity
↑
↑
↓
LPx
catalase
↑
↑
↑
↑
↑
SOD
TAC
↑
↑
↑
↑
Metal mixture Balb3T3 cell transformation
Role of oxidative stress in cell transformation induced by Metal mixture
Initiation phase
macromolecules damage (DNA, lipids)
genotoxicity
• TF partly inhibited by antioxydant
• non-oxidative stress mechanisms
may be involded
Promotion phase
↑ of oxidative stress markers (ROS, LPx)
↑ antioxidant response (Cat, SOD)
↓ cell viability
• TF abolished by antioxidants
• ROS produced during this stage are related to ROS-tumor promotion
Metals carcinogenic effects linked to oxidative stress could be more related with
promotion stage than initiation stage
Oxidative stress : Arsenic / Cadmium