Genotoxicity screening using BlueScreen HC™ and · PDF file · 2014-03-18MMS:...
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www.citoxlab.com Genotoxicity screening using BlueScreen HC™ and BlueScreen HC S9™ assays: a validation study Thierry Orsière, Nick Pearson, Mylène Valin, Guillaume Sire, Séverine Sarlang, Jean-Jacques Legrand CiToxLAB France, Evreux. Introduction BlueScreen HC™ and HC S9™ are commercially available kits able to detect any class of genotoxic damage. The assay is based on a human lymphoblastoid TK6 cell line, GLuc-T01, which has been geneti- cally modified by the incorporation of a reporter system Gaussia Luciferase (GLuc) in association to the GADD45α gene which mediates the adaptive response to genotoxic stress. Cells exposed to a genotoxic compound increase the expression of the GADD45α reporter, which is quantified by the detection of luminescence. Simultaneously, cytotoxicity is evaluated by the determination of the cell density in compound treated wells compared to control wells. Abs: Absorbance BaP: Benzo[a]pyrene BS-HC-AM: BlueScreen HC-Assay Medium CPA: Cyclophosphamide DMBA: Dimethylbenz[a]anthracene ENU: N-ethyl-N-nitrosourea Flu: Fluorescence GADD45α gene: gene encoded the Growth Arrest and DNA-Damage- inducible protein GADD45 alpha GLuc: Gaussia luciferase HC: Human cells Lum: Luminescence MMS: Methyl Methane Sulfonate 4-NQO: 4 nitroquinoline 1-oxide SEM: Standard error of the mean VC: Vehicle control ABBREVIATIONS Materials and Methods BlueScreen HC and HC S9 kits were purchased from Gentronix (references: BlueScreen HC-20 and BlueScreen HCS9-20). Outline assay protocol: • Cell line: GLuc-T01 • Cell concentration at the beginning of treatment : 2 x 10 6 cells/ml • Controls: - Sterility control - Absorbance and/or fluorescence controls: compounds, culture medium with or without S9 • Negative controls (VC): vehicle treated wells (16 wells/plate) • Positive controls (4 wells/concentration/plate): - 4-NQO (0.125 and 0.5 μg/mL) for BlueScreen HC™ - CPA (5 and 25 μg/mL) for BlueScreen HC S9™ • Treatment and recovery periods: - 48 h + 0 h for BlueScreen HC™ - 3 h + 45 h for BlueScreen HC S9™ • Cell density evaluated by absorbance for BlueScreen HC™ and by relative DNA content measured by fluorescence after incorporation of a DNA stain solution for BlueScreen HC S9™ (Mean Abs. of Duplicate Test Wells – Mean Abs. of the Media Control) RCD = x 100 (Mean Abs. of the VC – Mean Abs. of the Media Control) (Mean Flu. of Duplicate Test Wells – Mean Flu. of the Media Control) RCD = x 100 (Mean Flu. of the VC – Mean Flu. of the Media Control) Relative cell density for BlueScreen HC: Relative cell density for BlueScreen HC S9: • Genotoxicity (i.e. induction of GLuc) evaluated by luminescence (Lum. of the Test Well – Lum. of the Media Control) Brightness = (Abs. of the Test Well – Abs. of the Media Control) Averaged Duplicate Test Well Brightness Luminescence Induction = Average Brightness of VC Wells Luminescence induction for BlueScreen HC: Luminescence Induction is calculated as follows (Lum. of the Test Well – Lum. of the Media Control) Brightness = (Flu. of the Test Well – Flu. of the Media Control) Luminescence induction for BlueScreen HC S9: Averaged Duplicate Test Well Brightness Luminescence Induction = Average Brightness of VC Wells Luminescence Induction is calculated as follows • Four compounds per 96-well plate • Eight 2-fold serial dilutions per compound (in duplicate) with a maximum tested final concentration of 500 μg/mL 1 2 3 4 5 6 7 8 9 10 11 12 A B C D E F G H Example of microplate layout for BlueScreen HC Test item 1 Test item 3 Diluent Positive control 4-NQO LOW No cells Test item 2 Test item 4 BC-HC-AM (sterility control) Positive control 4-NQO HIGH Cells GLuc-TO1 Acceptance criteria An assay is considered valid if the following criteria are met: - The luminescence induction ratio between treated and vehicle wells for each positive control (4-NQO or CPA) is higher for the high concentration than for the low concentration - The luminescence induction ratio between treated and vehicle wells exceeds 2 for each positive control at the high concentration Evaluation criteria (to be applied to positive controls and compounds) The graphical data for each compound is assessed for the relevance of a positive result. The genotoxicity is not evaluated for compound concentrations which induce a decrease in cell density greater than 70%. A compound is considered as genotoxic or progenotoxic when the ratio of lumines- cence between treated and vehicle controls wells increases with doses and exceeds 1.8 for BlueScreen HC or 1.5-fold for BlueScreen HC S9. A compound is considered as equivocal if a single concentration induces a luminescence ratio greater than 1.8 for BlueScreen HC or 1.5-fold for BlueScreen HC S9. Othe- rwise a compound is considered as non-genotoxic or non-progenotoxic. For each compound, the Lowest Effective Concentration (LEC) for cytotoxicity and genotoxicity are determined in μg/mL. VALIDATION STUDY Six known non-genotoxic, genotoxic or pro-genotoxic compounds were tested three times: • non-genotoxic compounds (tested with BlueScreen HC and HC S9) - Phenformin hydrochloride (up to 500 μg/mL) - D-mannitol (up to 500 μg/mL) • direct genotoxic compounds (tested with BlueScreen HC) - MMS (up to 62.5 μg/mL) - ENU (up to 500 μg/mL) • pro-genotoxic compounds (tested with BlueScreen HC S9) - 7, 12 DMBA (up to 31.25 μg/mL) - BaP (up to 62.5 μg/mL) Validation study criteria The following criteria were predefined for validating the assay at CIT: • all genotoxic and pro-genotoxic compounds are found positive in BlueScreen HC and BlueScreen HC S9, respectively, • all known non-genotoxic compounds are found negative in both BlueScreen HC and BlueScreen HC S9, • inter-laboratory reproducibility was assessed by comparison of CIT genotoxicity LEC results against Gentronix results. Results and discussion Positive control results met the above mentioned acceptance criteria in each assay. For each compound, averaged data of the three independent experiments are graphically presented in Figures 1 (BlueScreen HC) and 2 (BlueScreen HC S9). Phenformin-HCl and D mannitol were classified as negative for genotoxicity at concentrations up to 500 μg/mL (Fig 1A, B and Fig 2A, B). Methylmethane sulfonate and N-Nitroso-N-ethylurea were classified as positive for genotoxicity in BlueScreen HC assay with LEC equal to 15.6 and 62.5 μg/mL, respectively (Fig 1C, 1D). Benzo[a]pyrene and 7,12-dimethylbenzo[a]anthracene were classified as positive for genotoxicity in BlueScreen HC S9 assay with LEC equal to 7.81 and 1.95 μg/mL, respectively (Fig 2C, 2D). Fig.1. BlueScreen HC results [cytotoxicity (% relative cell density) and genotoxicity (luminescence induction) ± SEM) Fig.2. BlueScreen HC S9 results [Cytotoxicity (% relative DNA content) and Genotoxi- city (luminescence induction) ± SEM) Conclusion and discussion The BlueScreen HC™ and HC S9™ assays are considered as validated at CIT as these following criteria were met: - all tested genotoxic and pro-genotoxic compounds were found positive in BlueScreen HC and BlueS- creen HC S9, respectively. - all tested known non-genotoxic compounds were found negative. - inter-laboratory reproducibility was found satisfactory as all LEC for genotoxic or progenotoxic compounds were found at CIT within a 2-fold dilution interval of the LEC determined by Gentronix (data not shown). Fig.1 A: Phenformin hydrochloride 140.0 3.0 2.0 0.0 1.0 0.5 1.5 2.5 120.0 100.0 0.0 20.0 40.0 60.0 80.0 Blank 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 % Relative cell density Compound concentrations ( g/ml) Luminescence induction % Relative cell density Luminescence induction Fig.2 A: Phenformin hydrochloride % Relative DNA content Compound concentrations ( g/ml) Luminescence induction Blank 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 3.0 2.0 0.0 1.0 0.5 1.5 2.5 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 % Relative DNA content Luminescence induction Fig.1 B: D-mannitol % Relative cell density Compound concentrations ( g/ml) Luminescence induction 3.0 2.0 0.0 1.0 0.5 1.5 2.5 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 160.0 180.0 Blank 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 % Relative cell density Luminescence induction Fig.2 B: D-mannitol % Relative DNA content Compound concentrations ( g/ml) Luminescence induction 3.0 2.0 0.0 1.0 0.5 1.5 2.5 Blank 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 0.0 20.0 40.0 60.0 80.0 100.0 120.0 140.0 % Relative DNA content Luminescence induction Fig.1 C: MMS % Relative cell density Compound concentrations ( g/ml) Luminescence induction 3.0 2.0 0.0 1.0 0.5 1.5 2.5 Blank 0.49 0.98 1.95 3.91 7.82 15.63 31.26 62.52 120.0 100.0 0.0 20.0 40.0 60.0 80.0 % Relative cell density Luminescence induction Fig.2 C: BaP % Relative DNA content Compound concentrations ( g/ml) Luminescence induction 3.0 2.0 0.0 1.0 0.5 1.5 2.5 Blank 0.49 0.98 1.95 3.91 7.82 15.63 31.26 62.52 120.0 100.0 0.0 20.0 40.0 60.0 80.0 % Relative DNA content Luminescence induction % Relative cell density Compound concentrations ( g/ml) Luminescence induction 3.0 2.0 0.0 1.0 0.5 1.5 2.5 Blank 3.91 7.81 15.63 31.25 62.50 125.00 250.00 500.00 140.0 120.0 100.0 0.0 20.0 40.0 60.0 80.0 % Relative cell density Luminescence induction Fig.1 D: ENU % Relative DNA content Compound concentrations ( g/ml) Luminescence induction 120.0 100.0 0.0 20.0 40.0 60.0 80.0 3.0 2.0 0.0 1.0 0.5 1.5 2.5 Blank 0.24 0.49 0.98 1.95 3.91 7.81 15.62 31.24 % Relative DNA content Luminescence induction Fig.2 D: 7,12 DMBA
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Transcript of Genotoxicity screening using BlueScreen HC™ and · PDF file · 2014-03-18MMS:...
www.citoxlab.com
Genotoxicity screening using BlueScreen HC™ and BlueScreen HC S9™ assays: a validation studyThierry Orsière, Nick Pearson, Mylène Valin, Guillaume Sire, Séverine Sarlang, Jean-Jacques LegrandCiToxLAB France, Evreux.
IntroductionBlueScreen HC™ and HC S9™ are commercially available kits able to detect any class of genotoxic damage. The assay is based on a human lymphoblastoid TK6 cell line, GLuc-T01, which has been geneti-cally modifi ed by the incorporation of a reporter system Gaussia Luciferase (GLuc) in association to the GADD45α gene which mediates the adaptive response to genotoxic stress. Cells exposed to a genotoxic compound increase the expression of the GADD45α reporter, which is quantifi ed by the detection of luminescence.Simultaneously, cytotoxicity is evaluated by the determination of the cell density in compound treated wells compared to control wells.
Abs: AbsorbanceBaP: Benzo[a]pyreneBS-HC-AM: BlueScreen HC-Assay MediumCPA: CyclophosphamideDMBA: Dimethylbenz[a]anthracene
ENU: N-ethyl-N-nitrosoureaFlu: FluorescenceGADD45α gene: gene encoded the Growth Arrest and DNA-Damage-inducible protein GADD45 alpha GLuc: Gaussia luciferase
HC: Human cellsLum: LuminescenceMMS: Methyl Methane Sulfonate4-NQO: 4 nitroquinoline 1-oxideSEM: Standard error of the meanVC: Vehicle control
ABBREVIATIONS
Materials and MethodsBlueScreen HC and HC S9 kits were purchased from Gentronix (references: BlueScreen HC-20 and BlueScreen HCS9-20). Outline assay protocol:
• Cell line: GLuc-T01• Cell concentration at the beginning of treatment : 2 x 106 cells/ml• Controls:
- Sterility control - Absorbance and/or fl uorescence controls: compounds, culture medium with or without S9
• Negative controls (VC): vehicle treated wells (16 wells/plate)• Positive controls (4 wells/concentration/plate):
- 4-NQO (0.125 and 0.5 μg/mL) for BlueScreen HC™ - CPA (5 and 25 μg/mL) for BlueScreen HC S9™
• Treatment and recovery periods:- 48 h + 0 h for BlueScreen HC™ - 3 h + 45 h for BlueScreen HC S9™
• Cell density evaluated by absorbance for BlueScreen HC™ and by relative DNA content measured by fl uorescence after incorporation of a DNA stain solution for BlueScreen HC S9™
(Mean Abs. of Duplicate Test Wells – Mean Abs. of the Media Control)RCD = x 100
(Mean Abs. of the VC – Mean Abs. of the Media Control)
(Mean Flu. of Duplicate Test Wells – Mean Flu. of the Media Control)RCD = x 100
(Mean Flu. of the VC – Mean Flu. of the Media Control)
Relative cell density for BlueScreen HC:
Relative cell density for BlueScreen HC S9:
• Genotoxicity (i.e. induction of GLuc) evaluated by luminescence
(Lum. of the Test Well – Lum. of the Media Control)Brightness =
(Abs. of the Test Well – Abs. of the Media Control)
Averaged Duplicate Test Well BrightnessLuminescence Induction =
Average Brightness of VC Wells
Luminescence induction for BlueScreen HC:
Luminescence Induction is calculated as follows
(Lum. of the Test Well – Lum. of the Media Control)Brightness =
(Flu. of the Test Well – Flu. of the Media Control)
Luminescence induction for BlueScreen HC S9:
Averaged Duplicate Test Well BrightnessLuminescence Induction =
Average Brightness of VC Wells
Luminescence Induction is calculated as follows
• Four compounds per 96-well plate• Eight 2-fold serial dilutions per compound (in duplicate) with a maximum tested fi nal concentrationof 500 μg/mL
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
Example of microplate layout for BlueScreen HC Test item 1 Test item 3 Diluent Positive control 4-NQO LOW No cells Test item 2 Test item 4 BC-HC-AM (sterility control) Positive control 4-NQO HIGH Cells GLuc-TO1
Acceptance criteria
An assay is considered valid if the following criteria are met:- The luminescence induction ratio between treated and vehicle wells for each positive control (4-NQO or CPA) is higher for the high concentration than for the low concentration- The luminescence induction ratio between treated and vehicle wells exceeds 2 for each positive control at the high concentration
Evaluation criteria (to be applied to positive controls and compounds)
The graphical data for each compound is assessed for the relevance of a positive result.The genotoxicity is not evaluated for compound concentrations which induce a decrease in cell density greater than 70%. A compound is considered as genotoxic or progenotoxic when the ratio of lumines-cence between treated and vehicle controls wells increases with doses and exceeds 1.8 for BlueScreen HC or 1.5-fold for BlueScreen HC S9. A compound is considered as equivocal if a single concentration induces a luminescence ratio greater than 1.8 for BlueScreen HC or 1.5-fold for BlueScreen HC S9. Othe-rwise a compound is considered as non-genotoxic or non-progenotoxic. For each compound, the Lowest Effective Concentration (LEC) for cytotoxicity and genotoxicity are determined in μg/mL.
VALIDATION STUDY
Six known non-genotoxic, genotoxic or pro-genotoxic compounds were tested three times:• non-genotoxic compounds (tested with BlueScreen HC and HC S9)
- Phenformin hydrochloride (up to 500 μg/mL)- D-mannitol (up to 500 μg/mL)
• direct genotoxic compounds (tested with BlueScreen HC)- MMS (up to 62.5 μg/mL)- ENU (up to 500 μg/mL)
• pro-genotoxic compounds (tested with BlueScreen HC S9)- 7, 12 DMBA (up to 31.25 μg/mL)- BaP (up to 62.5 μg/mL)
Validation study criteriaThe following criteria were predefi ned for validating the assay at CIT:• all genotoxic and pro-genotoxic compounds are found positive in BlueScreen HC and BlueScreen HC S9, respectively,• all known non-genotoxic compounds are found negative in both BlueScreen HC and BlueScreen HC S9,• inter-laboratory reproducibility was assessed by comparison of CIT genotoxicity LEC results against Gentronix results.
Results and discussion Positive control results met the above mentioned acceptance criteria in each assay.For each compound, averaged data of the three independent experiments are graphically presented in Figures 1 (BlueScreen HC) and 2 (BlueScreen HC S9). Phenformin-HCl and D mannitol were classifi ed as negative for genotoxicity at concentrations up to 500 μg/mL (Fig 1A, B and Fig 2A, B). Methylmethane sulfonate and N-Nitroso-N-ethylurea were classifi ed as positive for genotoxicity in BlueScreen HC assay with LEC equal to 15.6 and 62.5 μg/mL, respectively (Fig 1C, 1D).Benzo[a]pyrene and 7,12-dimethylbenzo[a]anthracene were classifi ed as positive for genotoxicity in BlueScreen HC S9 assay with LEC equal to 7.81 and 1.95 μg/mL, respectively (Fig 2C, 2D).
Fig.1. BlueScreen HC results [cytotoxicity (% relative cell density) and genotoxicity (luminescence induction) ± SEM)
Fig.2. BlueScreen HC S9 results [Cytotoxicity (% relative DNA content) and Genotoxi-city (luminescence induction) ± SEM)
Conclusion and discussionThe BlueScreen HC™ and HC S9™ assays are considered as validated at CIT as these following criteria were met:- all tested genotoxic and pro-genotoxic compounds were found positive in BlueScreen HC and BlueS-creen HC S9, respectively.- all tested known non-genotoxic compounds were found negative.- inter-laboratory reproducibility was found satisfactory as all LEC for genotoxic or progenotoxic compounds were found at CIT within a 2-fold dilution interval of the LEC determined by Gentronix (data not shown).
Fig.1 A: Phenformin hydrochloride
140.0 3.0
2.0
0.0
1.0
0.5
1.5
2.5120.0
100.0
0.0
20.0
40.0
60.0
80.0
Blank
3.91
7.81
15.63
31.25
62.50
125.0
0
250.0
0
500.0
0
% R
elat
ive
cell
dens
ity
Compound concentrations ( g/ml)
Lum
ines
cenc
e in
duct
ion
% Relative cell density
Luminescence induction
Fig.2 A: Phenformin hydrochloride
% R
elat
ive
DN
A c
onte
nt
Compound concentrations ( g/ml)
Lum
ines
cenc
e in
duct
ion
Blank
3.91
7.81
15.63
31.25
62.50
125.0
0
250.0
0
500.0
0
3.0
2.0
0.0
1.0
0.5
1.5
2.5
0.0
20.0
40.0
60.0
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120.0
140.0
% Relative DNA content
Luminescence induction
Fig.1 B: D-mannitol
% R
elat
ive
cell
dens
ity
Compound concentrations ( g/ml)
Lum
ines
cenc
e in
duct
ion
3.0
2.0
0.0
1.0
0.5
1.5
2.5
0.0
20.0
40.0
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80.0
100.0
120.0
140.0
160.0
180.0
Blank
3.91
7.81
15.63
31.25
62.50
125.0
0
250.0
0
500.0
0
% Relative cell density
Luminescence induction
Fig.2 B: D-mannitol
% R
elat
ive
DN
A c
onte
nt
Compound concentrations ( g/ml)
Lum
ines
cenc
e in
duct
ion
3.0
2.0
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1.0
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Blank
3.91
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15.63
31.25
62.50
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0
250.0
0
500.0
00.0
20.0
40.0
60.0
80.0
100.0
120.0
140.0% Relative DNA content
Luminescence induction
Fig.1 C: MMS
% R
elat
ive
cell
dens
ity
Compound concentrations ( g/ml)
Lum
ines
cenc
e in
duct
ion
3.0
2.0
0.0
1.0
0.5
1.5
2.5
Blank
0.490.98
1.95
3.917.8
215
.6331.2
662.5
2
120.0
100.0
0.0
20.0
40.0
60.0
80.0
% Relative cell density
Luminescence induction
Fig.2 C: BaP
% R
elat
ive
DN
A c
onte
nt
Compound concentrations ( g/ml)
Lum
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e in
duct
ion
3.0
2.0
0.0
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Blank
0.490.98
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3.91
7.82
15.63
31.26
62.52
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100.0
0.0
20.0
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% Relative DNA content
Luminescence induction
% R
elat
ive
cell
dens
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Compound concentrations ( g/ml)
Lum
ines
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e in
duct
ion
3.0
2.0
0.0
1.0
0.5
1.5
2.5
Blank
3.917.8
115
.6331.2
562.5
0
125.00
250.00
500.00
140.0
120.0
100.0
0.0
20.0
40.0
60.0
80.0
% Relative cell density
Luminescence induction
Fig.1 D: ENU
% R
elat
ive
DN
A c
onte
nt
Compound concentrations ( g/ml)
Lum
ines
cenc
e in
duct
ion
120.0
100.0
0.0
20.0
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60.0
80.0
3.0
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1.0
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Blank
0.24 0.490.98
1.95
3.91
7.81
15.62
31.24
% Relative DNA content
Luminescence induction
Fig.2 D: 7,12 DMBA
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