Duluth, 19-21 May 2010 IDENTIFYING GENOTOXIC AND NON GENOTOXIC CARCINOGENS WITH CELL TRANSFORMATION...

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


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


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


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).


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

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 ……



Inhibition of DNA repairActivation of protooncogenesInactivation of tumor-suppressor genes of antimetastasis genes


Efficient controls at every steps,



Inhibition of DNA repairActivation of proto-oncogenesInactivation of tumor-suppressor genes of antimetastasis genes


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…)


- 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


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


Thank you

Duluth, 19-21 May 2010 IDENTIFYING GENOTOXIC AND NON GENOTOXIC CARCINOGENS WITH CELL TRANSFORMATION...

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