Date post: | 11-Jun-2015 |
Category: |
Documents |
Upload: | la-unidad-de-toxicologia-experimental-y-ecotoxicologia-utox-pcb |
View: | 256 times |
Download: | 0 times |
CTA: Cell Transformation Assay on BALB/c 3T3
David Ramos
Investigador CERETOX
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
http://goo.gl/VBsu7
http://goo.gl/BGLwt