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Duluth, 19-21 May 2010
IDENTIFYING GENOTOXIC AND NON GENOTOXIC CARCINOGENS
WITH CELL TRANSFORMATION ASSAYS
P. VASSEUR, M.A. MAIRE, C. RAST, S. ALEXANDRE, H. BESSI.
University of Metz, CNRS , France
Context, Definitions, History of CTA
SHE cells
Protocol
Mechanisms
The Balb 3T3, C3H10T1/2 cell lines
Performances of cell transformation assays (CTA)
Analyses SHE results
Conclusion
Duluth, 19-21 May 2010
Context
Long term rodent carcinogenicity assay : expensive, time consuming → not required for the evaluation of chemicals (EU), except the genotoxic ones produced at high tonnage
Short term in vitro and in vivo genotoxicity assays have been usedas surrogates to predict carcinogenicity.Yet,a number of chemical carcinogens to humans and mammals are negative in genotoxicity assays, but positive in cell transformation assays (CTA)
In 2007, OECD has recommended the development of guidelines for cell transformation assays for in vitro detection of chemical carcinogens
Duluth, 19-21 May 2010
OECD Environment, Health and Safety publications Series on Testing and Assessment N°31, 2006.
Detailed Review Paper (DRP 31) on Cell Transformation Assays for Detection of Chemical Carcinogens.
Environment Directorate, November 28, 2006, 170 p.
http://www.olis.oecd.org/olis/2007doc.nsf/LinkTo/NT00002F0A/$FILE/JT03230941.PDF
http:www.oecd.org/document/12/0,2340,en_2649_34377_1898188_1_1_1_1,00.html
Duluth, 19-21 May 2010
Cell Transformation
Cell transformation is the induction of phenotypic alterations in cultured cells that are characteristics of tumorigenic cells.
These phenotypic alterations can be induced by exposing mammalian cells to carcinogens.
Transformed cells that have acquired the characteristics of malignant cells have the ability to induce tumors in susceptible animals (Berwald and Sachs, 1963, 1965).
Duluth, 19-21 May 2010
Primary orsecondary SHE cells +BaP or 3MC
thensubcultured Foci of
rapidly
dividing cells
Subcutaneous Injection intonewborn hamster
TumoursTumours
Earle (1943) : Morphological changes in cell culture were associated with the oncogenicity of these cells in vivo Berwald and Sachs, 1963, 1965
MULTISTAGE TRANSFORMATION OF SYRIAN HAMSTER EMBRYO (SHE) CELLS
BY CHEMICAL CARCINOGENS
Further development and validation of SHE assay
DiPaolo et al., 1969
Pienta et al., 1977
Barrett et al., 1979
Newbold et al., 1982
Chouroulinkov & Lasne, 1976 ,…
demonstrated the ability of chemical carcinogens
from different chemical classes to induce
morphological transformation (MT) in vitro
Morphological transformation (MT)
= Changes exhibited by transformed cells related to neoplasia and associated with behaviour and growth control modifications :
. alteration of cell morphology
. disorganized pattern of colony growth
. acquisition of anchorage-independent growth (Combes et al., 1999)
Later on, transformed cells become able to :
. grow in semi-solid agar . produce autocrine growth factors . evolve to tumorigenicity when injected into appropriate hosts . divide indefinitely (immortalized), which is associated with other alterations like aneuploïd karyotype and altered genetic stability.
• Changes in - cytoskeleton - morphology of cells
& colonies
Characteristic phenotype of transformed cells :
normal transformed
Phenotypic changes / SHE CTA
- a random growth pattern of spindle shaped cells,- a piling up of cells in a criss-cross pattern (a loss of growth inhibition and of cell-cell orientation at confluency)
Normal SHE colony
Morphologically transformed SHE colony
Morphologically transformed SHE colony
Chemical carcinogens classified in two groups
. Genotoxic carcinogens
able to initiate cells to carcinogenesis through direct interaction with DNA,resulting in DNA damages and/or structural/numerical chromosomal aberrations which can be detected by genotoxicity tests.
. Non-genotoxic carcinogens
carcinogenic agents devoid of direct interaction with DNA.The indirect modifications to DNA structure, amount or function may inducealtered gene expression and/or signal transduction.
Generally, non-genotoxic carcinogens refer to carcinogens negative in genotoxicity assays performed to measure endpoints such as gene mutations and chromosomal damages (chromosomal aberrations, micronuclei).
The multistage process of carcinogenesis in vivo
Initiation Promotion Progression
Normal Foci Benign lesion Malignant lesion
Initiation Promotion Progression Invasion
MutagensVirusesRadiations
Factors of Cell growth, division Transcription
1. Genotoxicity 2. Epigenetic events ……
MutagensVirusesRadiations
Factors of Cell growth, division Transcription
Inhibition of DNA repairActivation of protooncogenesInactivation of tumor-suppressor genes of antimetastasis genes
1. Genotoxicity 2. Epigenetic events ……
Efficient controls at every steps,
MutagensVirusesRadiations
Factors of Cell growth, division Transcription
Inhibition of DNA repairActivation of proto-oncogenesInactivation of tumor-suppressor genes of antimetastasis genes
1. Genotoxicity 2. Epigenetic events ……
Efficient controls at every steps,
BUT, IF INACTIVATED
Histone desacetylationHypo/hypermethylation
Inhibition of DNA repairActivation of proto-oncogenesInactivation of - tumor-suppressor genes of - antimetastasis genes
1. Epigenetic events 2. Genotoxicity ……
Acetylation
Histone acetyl transferase
(HAT) open chromatin transcription, gene activation
Desacetylation Histone desacetylase (HDAC) + Methylation Histone methyl transferase + HMT
Methyl Binding Proteins (MBP)
compacted chromatin blocage transcription gene inactivation tumor suppressors genes p53, p16
Acetylation
Histones H3, H4
Desacetylation
Hypoacetylation H4observed in early stepsof carcinogenesis
Silencing of tumor suppressor genes
Hypermethylation H3 H4,Overexpression HMTin a variety ofneoplasia
DNA hypermethylationof promoter sequences→ transcriptional silencing
Histone acetylases
Histone desacetylase
Histone methyl transferases
active, open chromatinnormal state
Methylation
DNA
H3, H4
DNA methyl transferases
methionineSAM
Hypomethylation also tumorigenic
Iacobuzio-Donahue, Ann Rev Pathol. Mech. Dis.2009.
Acetylation
Histones H3, H4
Desacetylation
Hypoacetylation H4observed in early stepsof carcinogenesis
Silencing of tumor suppressor genes
Hypermethylation H3 H4,Overexpression HMTin a variety ofneoplasia
DNA hypermethylationof promoter sequences→ transcriptional silencing
Histone acetylases
Histone desacetylase
Histone methyl transferases
active, open chromatinnormal state
Methylation
DNA
H3, H4
DNA methyl transferases
methionineSAM
Nickel
Hypomethylation also tumorigenic
Arsenic, Alcohol CadmiumEffect dose dependent
Preferential binding to methylated CpG sites PAH (tobacco smoke)AFB1 (Herceg, Mutagenesis, 2007)
(Sutherland et al. Ann NY Acad Sc, 2003)
The MT phenotype of colonies expresses changes in the expression of genes involved in cell cycle control,
proliferation and differentiation.
resulting from genotoxicity and non-genotoxic mechanisms
leading to : - alteration of DNA repair - disturbance in signal transduction - histone desacetylation, DNA hypermethylation & hypomethylation - modulation of gene expression → disturbance of cell cycle control, proliferation and differentiation (Alexandre et al., 2003) Histone desacetylation, DNA hypermethylation & hypomethylation - oxidative stress (Jiung et al., 1999, Zhang et al. 2000) inflammation - imbalance of cell proliferation/apoptosis - changes in intercellular communication (Cruciani et al. 1997) - telomerase activation …. - immunosuppression
Disturbance in signal transduction from cell environment nucleus
cascade phosphorylation / dephosphorylation
transient activation of a number of intermediates
Non lipophilic Membrane growth + receptor factor
Syntheses, replication,
mitosis
Signal transductionkinase cascade
Lipophilic hormonesNuclear receptors
Nuclear transcription factors
Disturbance in signal transduction from cell environment nucleus
Growth + Receptor factor
Syntheses, replication, mitosis
A signal transduction pathway may be disrupted, activated or blocked, by analogs that substitute or interfere with some intermediates or the receptor itself.
An activation may be permanent, instead of transient, leading to a sustained response ( ex : cell cycle dysregulation, increased rate of mitosis)
cell response
Signal
Kinase C Inactiveprotein
Phosphorylated protein
activeCa++
Phorbol ester TPA
Phorbol ester
The tumor promoter TPA 12-O-Tetradecanoylphorbol-13-acetatesubstitutes to diacylglycerol (DG)and activates the PKC pathway
DG
COH
Oxidative stress is involved in acrylonitrile (ACN)-inducedmorphological transformationin SHE cells
0
0,5
1
1,5
2
2,5
ACN 0 25 50 75 0 25 50 75 µM
-tocopherol 5 5 5 5 µM
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
8-oxodGuo in SHE cells
ConVit. E
ACN
Vit. E
Zhang et al., 2000. Carcinogenesis 21, 727-733.
% M
T c
olo
nie
s
CONTROL APOPTOTIC
DEHP, Di-(2-ethylhexyl)phthalate, a non genotoxic carcinogen induces SHE cell transformation at doses inhibiting apoptosis (50 µM) in serum-deprived cells
DEHP 10 µM DEHP 50 µM
Maire et al., 2005. Toxicol Lett, 158, 237-245
Consequence : survival of abnormal cells
Mechanisms :
Surexpression of the antiapoptotic gene bcl-2
Repression of the protooncogene c-myc
DEHP inhibits apoptosis via surexpression of bcl-2 (antiapoptotic)
→ change in bax/bcl-2 ratio
Bcl-2 (26 kDa)
bcl-2 (500 pb)
and represses the protooncogene c-myc expression
(Maire et al., 2005, Toxicol Lett,158, 237-245)
Protocol of the SHE assay
Obtention of SHE cells (feeder and target cells)
Hamster 5000 radsembryos primary feeder cells13 days culturesGestation non target cells differentiated cells
storage- 196°C
SHE cells tested at clonal density
150 cells / dish 25-45 colonies / dish
7 days
of exposure
Fixation, colorationscoring
Cloning : 1 cell → 1 colony of hundred(s) cells )Cloning efficiency (> 20%) : 150 cells → 25-45 colonies
Normal colony Transformed
colony
Scoring of coded plates, under stereomicroscope
*Preliminary experiment for dose-range finding
*Definitive test
- 5 dose levels, vehicle control and positive control (BaP)
- Cytotoxicity evaluated by clonal efficiency
- Nb target cells adjusted in order to obtain 20-45 colonies/dish
- 40 dishes per concentration (or 10/conc. x 4 experiments)
- Transformation frequency and cloning efficiency established from 1000 scored colonies per concentration
- Statistical analyses
- Criteria of acceptance fulfilled
Experimental design (continued…)
Experimental design (continued…)
- statistical analysis for comparison between vehicle control and concentration level (Fisher’s exact test or 2) and positive dose-response trend (Cochran-Armitage test) - positive response is declared when :
. 2 positive (successive) concentrations, at least . or one positive concentration plus positive trend
-criteria for acceptance fulfilled . 20% cloning efficiency in controls . Nb transformed colonies in the range 25-45/dish
Experimental design (continued…)
Test medium : DMEM (without phenol red) with fetal calf serum (12-15%), 10% CO2
pH / exposure physiological pH : 7.0 - 7.35 exposure 7 days or
LeBoeuf’s modification (1986) pH : 6.7 exposure to the tested chemical 24 h or 7 days
* Balb 3T3, clone A31 Kakunaga, 1972 Yamasaki, 1985
* C3H10T ½ Chen and Heidelberger, 1969, Reznikoff et al., 1973
Development of cell transformation assays (CTA) on mouse established cell lines
In parallel to SHE cell MT assay ,
Cell Transformation Assays (CTA)
SHE
• diploïd, normal cells• metabolically competent• secondary cultures• low level of spontaneous
transformation• short term (7 days) exposure• mimics the first stages of
the neoplastic transformation
• Balb 3T3, C3H10T1/2
• aneuploïd cell lines• limited metabolic ability • infinite life span• high level of spontaneous
transformation• long term (> 4 weeks)• mimic the late stages of the
neoplastic process
The multistage process of carcinogenesis in vivo (a)
(a)
Initiation Promotion Progression
Normal Foci Benign lesion Malignant lesion
SHE Balb/c 3T3, C3H 10T1/2
From Combes et al., 1999. ATLA 27, 745-767.
Non-transformed Transformed
Morphologically transformed and non-transformed foci of BALB/c 3T3 cells (foci induced by 1 µg/ml 3-methylcholanthrene)
Photo Dr H Yamasaki.
Type III
Photo Dr J. Landolph.
Morphologically transformed and non-transformed foci of C3H 10T1/2 cells (treatment with 1µg/ml 3-methylcholanthrene for 24h)
Type I normal
Type II
PERFORMANCES OF THE CELL TRANSFORMATION ASSAYS
- OECD -
Comparison with commonly used short-term genotoxicity tests for assessing carcinogenic potential
Salmonella (Ames) test (mutagenesis assay)Mouse lymphoma L5178Y cell mutagenesis assayHPRT mutagenesis assayIn vitro chromosomal aberrationsIn vivo chromosomal aberrationsIn vivo micronucleus test
Data set
Data banks
- IARC, NTP, GENETOX, CCRIS, CPDB/Gold and Zeiger (1997)
- Heidelberger et al. (1983), Matthews et al.(1993), Leboeuf et al. (1996) and many other published articles …
Nb chemicals
SHE : 264
BALB: 186
C3H : 141
Organic
203
165
121
Inorganic
61
21
20
RodentCarcinogens
191
127
117
Non Carcinogens
73
59
24
SHE results on 64 metals and inorganic compounds
Asbestosis, ceramic fibres, cadmium, nickel, chromium compounds, …
SHE
Results
Rodent
carcinogen
Rodent
non carcinogen
+ 50 6 56
-, ?, eq 3 5 8
53 11 64
Elias et al. Carcinogenesis, 10-11, 2043-2052, 1989 ; Elias et al.,Toxicology In Vitro, 14, 409-422, 2000; Elias et al., J. Toxicol Environ Health, 65, 2007-2027, 2002; Elias et al, Ann. Occup. Hyg., 46, 53-57, 2002. …
Comparison with rodent carcinogenicity
Definitions
In vitro Carcinogen Non-carcinogen
+ a b
- c d
Concordance = % agreement with in vivo exp. (a+b)/(a+b+c+d)*100Sensitivity = % carcinogens that are positive (a/a+c)*100Specificity = % noncarcinogens that are negative (d/b+d)*100Positive Pred.= % positive calls that are carcinogens (a/a+b)*100Negative Pred.=% negative calls that are noncarcinogens (d/c+d)*100
False negative = c/ a+cFalse positive = b/ b+d
In vivo
Performance of CTA relative to rodent bioassay
Concordance
Sensitivity
Specificity
False negative
False positive
(Inconclusive
Not included)
BALB
149
68%
75%
53%
25%
47%
(28%)
C3H
96
73%
72%
80%
28%
20%
(30%)
SHE
264
86%
91%
74%
9%
26%
(10%)
n =
Performance of CTA relative to rodent bioassay
Concordance
Sensitivity
Specificity
False negative
False positive
(Inconclusive
Not included)
BALB
149
68%
75%
53%
25%
47%
(28%)
C3H
96
73%
72%
80%
28%
20%
(30%)
SHEpH 7.0204
85%
92%
66%
8%
34%
(12%)
n =
SHEpH 6.7
88
74
66
85
33
15
(2)
Carcinogens CTA positive
Direct alkylating agents lactones, epoxides aldehydes alkylsulfonates Indirect acting alkylating agents N-nitroso compounds Halogenated aliphatic hydrocarbons
Indirect acting, DNA covalent binding, Intercalating agents, Polycyclic aromatic hydrocarbons Aromatic amines, nitroarenes mycotoxins
Genotoxic Non genotoxic
Steroïds
Phthlates & HPP (fibrates) (SHE)
Polyhalogenated biphenyls
Halogenated aryl (insecticides)
PCDD
Biotoxins, cyanotoxins
Tumor promoters (TPA, okadaïc acid…)
False negatives in CTA
SHE
AnilineAnthraquinoneArochlor 1254 DDTEthinyl estradiolEthyl alcohold-LimoneneMetaproterenolMethylcarbamateNitrilotriacetate NTA5-nitro-o-toluidinePyridineTetrahydrofuranTEHP tris(2ethylhexyl) phosphate
BALB 3T3
2-AminoanthraceneChlorinated aliphatic hydro mono, di, tetra, hexachloroethane
Clofibrate1,2-epoxybutaneEthinyl estradiold-LimoneneMonuron2-4-DinitrotoluenePhthlates Butylbenzyl phthlate, DEHPProcarbazineTEHP
C3H
Inorganics lead acetate potassium dichromate nickel chloride sodium arsenateOrganics BrdU Phenobarbital Propyleneimine Styrene ThioacetamideDivergent responses Diethylstilbestrol DEHP Hexamethy phosphoramide 5-nitro-o-toluidine
Many known or suspected aneugens induce CT in SHE cells
AcrylamideAsbestosBenzeneBenomylCadmium chlorideChloral hydrateColcemidDESEconazole nitrateGriseofulvineHydroquinonePyrimethamineVincristine
SHE
+++++++++++++
In vitroABS
++
+/-
+/-+
+
++
In vivoMN
+
+
-/?+/?++
?+
RodentCarcinog
+++-++-+-++--
Performances of short-term genotoxicity tests on the chemicals of the data set
Concordance
Sensitivity
Specificity
False negative
False positive
(Inconclusive
Not included)
MLA170SHE
74%
86%
34%
14%
66%
29%
In vivo MN158
56%
57%
52%
43%
48%
31%
In vitroABS184
64%
65%
63%
35%
37%
15%
Ames252
51%
37%
81%
63%
19%
21%
n =
Non genotoxic (S. typhi) carcinogens SHE positive
Ames negativeAcetamideAcrylamideActinomycine DAmitroleAuramineBenzeneBrdUButylhydroxytolueneButylbenzylphthlateCatecholChlordaneChlorothalonilCinnamyl anthranilateClofibrateCyclosporineDecabromodiphenyloxideDieldrinDiethanolamineDEHP
DiethylstilbestrolDiethylthioureaDimethylhydrazineEstradiolEthionineEthylbenzeneEGBE Butyl glycolHexachlorobutadieneHexamethylphosphoramideHydroquinoneMethylpyrilene, HClMethyl eugenolMethylclofenapateMezereinMonuronN-nitroso ethylanilineOkadaïc acidOxymetholoneProcarbazine, HClProgesterone
ReserpineSafroleSulfamethoxazoleTPA phorbol esterThioureaTrichlorophenolTEHP tris(2ethylhexyl) phosphate
Wyeth 14043 (HPP)
One embryo 20-50 testsOne female (m ≈5-8 embryos) : 160-400 tests Renewal of target cells every year
Yet, cells and kits are now available and provided by some companies
Training necessary
6-8 weeks required for a confirmed result
Quite performant as alternative to rodent carcinogenicity assays.CTAs, in vitro assays necessary for non genotoxic carcinogens !
The use of toxicogenomics & proteomics will help in mechanistics for a better knowledge of :
. the link between gene expression, cytoskeleton alterations, neoplastic cell transformation,
. the pattern(s) of genomic changes common to some categories of non genotoxic carcinogens
. a base set of gene expression changes (if existing) typical of non genotoxic carcinogens
Duluth, 19-21 May 2010
Thank you