Cell Transformation assay CTA (D. Ramos)

CTA: Cell Transformation Assay on BALB/c 3T3

David Ramos

Investigador CERETOX

[email protected]

JORNADA TOX®

1 de Febrero de 2013

Parc Científic de Barcelona

Principle of the test

Reference

OECD DRP 31

In vitro test:

Phenotypic alterations (characteristic of tumorigenic cells) in cultured cells

induced by carcinogens. These cells induce tumours in susceptible animals

(Berwald and Sachs, 1963, 1965)

Fast

Cost efficient

Initial screening for carcinogenic potential

3 types of CTA:

Syrian hamster emrbyo cell (SHE pH:6.7)

Syrian hamster emrbyo cell (SHE pH:7)

BALB/c 3T3

Materials

Experimental system:

Clone A31-1-1 derived from BALB/c 3T3

Transformation: associated with malignancy

Sensitive to tumour-promoting agents

Morphological changes related to neoplasia

immortals, autocrine GF, tumorigenecity…

Medium: DMEM+10% hiFBS+Ab

Solvent Control: Water, DMSO, acetone and ethanol < 5%, 0.2%, 0.5%, 0.1%

Positive Control: 1-stage : 5µg/mL MCA

2-stage : 0.2µg/mL MCA as initiator + TPA 0.2 µg/mL as tumour promoter

Ref. Kakunaga and Crow (1980)

MCA = 3-methylcholanthrene

TPA = 12-o-tetradecanoylphorbol-13-acetate

Experimental design: cytotoxicty test

Cells seeded at 100 cells/60-mm dish,

3 dishes per treatment, 24 h

Wide range: 5 concentrations

Exposure: 72 h + 7 day fresh medium

Fixed and stained with 20% Giemsa’s solution

Solvent control

Positive Control, DMSO 3%

Determined by colony-forming efficiency (CFE)

respect control

Colonies

Concentration Replica 1 Replica 2 Replica 3 Replica 4 CFE (%) Effect

NP 197 200 0 0 0 0 0,00 100,00

NP 197 66,66 3 5 6 14 5,69 94,31

NP 197 22,22 15 18 19 52 21,14 78,86

NP 197 7,4 27 37 33 97 39,43 60,57

NP 197 2,469 60 69 70 199 80,89 19,11

Solvent control 5% H2O 74 92 80 246 100,00 0,00

Positive Control 3% DMSO 30 35 41 106 43,09 56,91

Experimental design: transformation assay

5 concentrations:

High: 80-90% decrease in

CFE

3 intermediate doses

Low: NO effect in CFE

Non toxic substances:

Soluble: ≤ 5 mg/mL

Insoluble: ≤ 2-times

visible solubility

Cells seeded at 104cells/60-mm dish,

10 dishes/treatment, 4 mL culture medium, 24 h

1-stage CTA

Genotoxic substance 72 h

Medium x2 / week

during 3½ weeks

Medium x1 / week

during 2 weeks

2-stage CTA

Non-genotoxic susbtance 72h as

inductor

refilled with fresh

normal medium for 3 days

TPA as promotor x2/week

during 2 weeks

Mediumx2/ week during week

once a week

during the following 2 weeks

MCA 72h as inductor

Refill

Substance as promotor x2 / week

during 2 weeks

medium x2/ week during week

once a week

during the following 2 weeks

Fixed and stained with 20% aqueous Giemsa for scoring focus formation

Refilled with fresh medium for 3 days

Medium1x / week during 2 weeks

Medium x2 / week during 1 week

Results The average number of foci Type III per dish in a treatment group

(Foci type III: Spindle-shaped markedly basophilic cells. Piling-up and criss-crossing, clear and marked).

Statistical analysis of treatments relative to the solvent control

2-stage CTA MCA as inductor+TPA as promotor Foci Type III

Treatment Concentration(µg/ml) FD/TD* Foci III/dish

Solvent Control 10,00% 9/10 3,30

Substance 78,125 9/10 3,10

Substance 156,25 10/10 4,40

Substance 312,5 9/10 4,90

Substance 625 9/9 5,11

Substance 1250 9/10 3,50

Control MCA 0.2 10/10 52,00

FD/TD: Number of dishes with foci/total number of dishes examined

Foci Type III

0

10

20

30

40

50

60

10,00% 78,125 156,25 312,5 625 1250 0.2

Solvent Control

Substance Substance Substance Substance Substance Control MCA

Fo

ci

III/d

ish

Concentration(µg/ml)

Foci Type III

CERETOX Parc Científic de Barcelona - Edifici Cluster

c/Baldiri Reixac, 10-12

08028 Barcelona

T. 93 403 71 83

www.pcb.ub.cat/ceretox

[email protected]

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