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CTA: Cell Transformation Assayon BALB/c 3T3David RamosInvestigador CERETOXdramos@pcb.ub.cat                  JORNADA TOX® ...
Principle of the test    Reference    OECD DRP 31   In vitro test:   Phenotypic alterations (characteristic of tumorigen...
Materials Experimental system:    Clone A31-1-1 derived from BALB/c 3T3    Transformation: associated with malignancy  ...
Experimental design: cytotoxicty test Cells seeded at 100 cells/60-mm dish,  3 dishes per treatment, 24 h Wide range: 5 ...
Experimental design: transformation assay                                            Cells seeded at 104cells/60-mm dish,...
Results The average number of foci Type III per dish in a treatment group      (Foci type III: Spindle-shaped markedly ba...
CERETOXParc Científic de Barcelona - Edifici Cluster          c/Baldiri Reixac, 10-12              08028 Barcelona        ...
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Cell Transformation assay CTA (D. Ramos)

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Transcript of "Cell Transformation assay CTA (D. Ramos)"

  1. 1. CTA: Cell Transformation Assayon BALB/c 3T3David RamosInvestigador CERETOXdramos@pcb.ub.cat JORNADA TOX® 1 de Febrero de 2013 Parc Científic de Barcelona
  2. 2. 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
  3. 3. 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 promoterRef. Kakunaga and Crow (1980)MCA = 3-methylcholanthreneTPA = 12-o-tetradecanoylphorbol-13-acetate
  4. 4. 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 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 19,11Solvent control 5% H2O 74 92 80 246 100,00 0,00Positive Control 3% DMSO 30 35 41 106 43,09 56,91 Positive Control, DMSO 3%
  5. 5. Experimental design: transformation assay Cells seeded at 104cells/60-mm dish, 5 concentrations: 10 dishes/treatment, 4 mL culture medium, 24 h  High: 80-90% decrease in CFE 1-stage CTA 2-stage CTA  3 intermediate doses Non-genotoxic Genotoxic substance  Low: NO effect in CFE 72 h susbtance 72h as MCA 72h as inductor inductor  Non toxic substances: Medium x2 / week refilled with fresh normal medium for 3 days medium for 3 days Refilled with fresh Refill  Soluble: ≤ 5 mg/mL during 3½ weeks  Insoluble: ≤ 2-times Medium x1 / week TPA as promotor Substance as x2/week promotor x2 / week visible solubility during 2 weeks during 2 weeks during 2 weeks Medium x2 / weekmedium 1 week Mediumx2/ week during x2/ week during week during week once a week once a week during the following week Medium1x / 2 during 2 following during the weeks 2 weeks weeks Fixed and stained with 20% aqueous Giemsa for scoring focus formation
  6. 6. 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 60 Treatment Concentration(µg/ml) FD/TD* Foci III/dish Foci Type III 50 Foci III/dish Solvent Control 10,00% 9/10 3,30 40 Foci Type III 30 Substance 78,125 9/10 3,10 20 Substance 156,25 10/10 4,40 10 0 Substance 312,5 9/10 4,90 10,00% 78,125 156,25 312,5 625 1250 0.2 Substance 625 9/9 5,11 Solvent Substance Substance Substance Substance Substance Control MCA Control Substance 1250 9/10 3,50 Concentration(µg/ml) Control MCA 0.2 10/10 52,00FD/TD: Number of dishes with foci/total number of dishes examined
  7. 7. CERETOXParc Científic de Barcelona - Edifici Cluster c/Baldiri Reixac, 10-12 08028 Barcelona T. 93 403 71 83 www.pcb.ub.cat/ceretox ceretox@pcb.ub.cat http://goo.gl/BGLwt http://goo.gl/VBsu7
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