γH2AX Double Strand DNA Damage Response and Genotoxicity Assay

Understand the potential genotoxicity of your compound in our γH2AX double strand DNA damage response assay.

Our γH2AX double strand DNA damage response assay is in our portfolio of genotoxicity services. Cyprotex deliver consistent, high quality data and can adapt protocols based on specific customer requirements.

Anti-γH2A.X (phospho S139) as an indicator of DNA double-strand breaks (DSBs) and genotoxicity

Histone H2A variant H2A.X, a component of the nucleosome core structure has a special role in DNA repair. Phosphorylation of H2A.X at residue Ser-139 (Anti-γH2A.X) by PI3K-like kinases, including ATM, ATR and DNA-PK, is an early cellular response to the generation of DNA double-strand breaks (DSBs)¹.
Detecting the formation of DSBs using the γH2A.X (Ser-139) has emerged as a highly specific and sensitive molecular marker for monitoring DNA damage¹.
DSBs form when both strands of the DNA double helix are broken, irrespective of how they are formed they are found to be highly toxic and can ultimately be fatal².
Cyprotex’s DNA Damage assay uses High Content Screening (HCS) to identify both DNA Damage and Cytotoxicity.

Taking into account the ability of the automated γH2AX assay to predict genotoxicity in vivo to the same accuracy as currently used in vitro assays, while its use of human metabolic competent cells, and its automated scoring, its limited use of test compound since small volumes are needed and its simple and rapid applicability to study large numbers of chemicals as it is amenable to robotised procedures, there are many arguments in favor of its usefulness as in vitro genotoxicity test. Especially its high sensitivity to detect DNA-reactive GTX compounds is a positive asset.

³Tsamou et al. (2012) Mutagenesis 27(6); 645-652

Protocol

Double strand DNA damage response assay

Cell Line	HepG2 (other cell types available on request)
Multiplexing	Combination with other mechanistic endpoints available on request Compatible with the in vitro HCS Micronucleus Test (MNT)
Analysis Platform	Cellomics ArrayScan^® VTI or XTI (Thermo Scientific)
Test Article Concentrations	8 point dose response curve with highest concentration based on cell loss or solubility limit (3 replicates per concentration)
Test Article Requirements	50 µL solution at 200x highest concentration or equivalent amount in solid
Time Points	In absence or presence of aroclor 1254 induced rat liver S9: 24 hr exposure time
Quality Controls	Negative control: 0.5% DMSO (vehicle) Positive controls: Cyclophosphamide (S9 positive control) and chlorambucil (positive control)
Data Delivery	Minimum effective concentration (MEC) and AC₅₀ value for each measured parameter (cell loss, nuclear morphology, DNA fragmentation and DNA damage)

Data

Data from Cyprotex's double strand DNA damage response assay

Figure 1
Representative HCS images for cells treated with (A) 200µM chlorambucil (positive control) (B) vehicle control in the absence of S9 fraction (C) 200µM cyclophosphamide (metabolizing system positive control) and (D) vehicle control in the presence of S9 fraction over a 24 hr period. Cell nuclei are stained blue (Hoechst) with pink staining observed in the nucleus of cells positive for γH2A.X (indicated by white triangles). Cellular filamentous actin is stained green (phalloidin).

Compound	MEC	AC₅₀	+ve/-ve	MEC	AC₅₀	+ve/-ve
	Minus Rat Liver S9			Plus Rat Liver S9
In vivo genotoxin
Benzo(a)pyrene	0.133	29.8	+	1.93	103	+
Chlorambucil	9.69	55.2	+	7.53	66.2	+
Cisplatin	0.356	8.64	+	0.589	11.7	+
Colchicine	0.03	>0.2	+	0.0239	0.194	+
Cyclophosphamide	NR	NR	-	11.3	253	+
Cytarabine	0.0175	0.964	+	0.0376	10.8	+
Etoposide	0.346	5.23	+	0.703	>10	+
Formaldehyde	145	>400	+	97.6	825	+
Griseofulvin	8.56	>300	+	12.5	>300	+
Hydroxyurea	274	442	+	301	506	+
Mitomycin C	0.0116	1.12	+	0.0788	2.35	+
Vinblastine	0.0054	>0.02	+	0.0091	>0.05	+
In vivo non-genotoxin
Cyclosporin A	NR	NR	-	NR	NR	-
Diclofenac	NR	NR	-	NR	NR	-
Acrylonitrile	NR	NR	-	NR	NR	-
Amoxicillin	NR	NR	-	NR	NR	-
Cefuroxime	NR	NR	-	NS	NS	-
Hydrocortisone	NR	NR	-	NR	NR	-
Lansoprazole	NR	NR	-	NR	NR	-
Nalidixic acid	NR	NR	-	NR	NR	-
Citalopram	NR	NR	-	NR	NR	-
Eugenol	NR	NR	-	NR	NR	-
Norfloxacin	NR	NR	-	NR	NR	-
Resorcinol	NR	NR	-	NR	NR	-

NR = no response NS = not significant

Table 1
γH2A.X data for 24 validation compounds categorized according to literature data^3-5.

HepG2 cells were treated for 24 hours with test compound in the absence and presence of aroclor 1254 induced rat liver S9. The compounds were analysed using Cellomics ArrayScan^® VTI or XTI (Thermo Scientific). Cyprotex data correlates well with literature in vivo data^3-5.

Figure 2
Graphical representation of γH2A.X for chlorambucil (positive control) and cyclophosphamide (positive control for metabolizing system). A: is in the absence of aroclor 1254 induced rat liver S9. B: is in the presence of aroclor 1254 induced rat liver S9. Red dashed line represents the vehicle control limits.

Chlorambucil causes a concentration dependent increase in γH2A.X compared to vehicle control treated cells in both the absence and the presence of metabolizing system (aroclor 1254 induced rat liver S9). No response was observed for cyclophosphamide in the absence of metabolizing system, however in the presence of the metabolizing system cyclophosphamide shows a concentration dependent increase in γH2A.X. Data represents mean of triplicate incubations ± standard deviation

References

¹Mah LJ et al., (2010) γH2A.X: a sensitive molecular marker of DNA damage and repair. Leukemia 24; 679 – 686
²Chapman JR et al., (2012) Playing the End Game: DNA Double-Strand Break Repair Pathway Choice. Molecular Cell 47(4); 497-10
³Tsamou M et al., (2012) Performance of in vitro γH2A.X assay in HepG2 cells to predict in vivo genotoxicity. Mutagenesis 27(6); 645-652
⁴Diaz D et al., (2007) Evaluation of an automated in vitro micronucleus assay in CHO-K1 cells. Mutation Research 630;1-13
⁵Hastwell PW et al., (2009) Analysis of 75 marketed pharmaceuticals using the GADD45a-GFP ‘GreenScreen HC’ genotoxicity assay. Mutagenesis, 24(5); 455-463