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HCS Genotoxicity: pH3 and pH2AX Assay

Cyprotex have released a pH3 and pH2AX assay, designed to detect the potential of genotoxicity and aid the elucidation of the mechanisms involved. 

Cyprotex deliver consistent, high quality data with the flexibility to adopt protocols based on specific customer requirements. 

Assessment of genotoxicity using pH3 and pH2AX biomarkers

  • High Content Screening uses automated fluorescence microscopy to measure indicators of cellular health and quantify biomarkers. 

  • Clastogens and aneugens are key classes of genotoxic agents. Clastogens directly damage DNA, resulting in double-stranded DNA breaks. Aneugens produce numerical chromosome aberrations, formally known as aneuploidy (the result of "lagging" chromosomes). 

  • Phospho-histone 3 (pH3) is a marker of mitosis and cell cycle arrest during the G2/M phase. Aneugens have been shown to increase levels of pH31. Phospho-histone 2AX (pH2AX) is a marker for double-stranded DNA breaks, indicating direct DNA damage caused by clastogens and some aneugens. 

  • HepG2 cells were selected due to their wild type p53 expression, shown to be important for accurate genotoxicity prediction2

Aneugenic compounds induced either an increase or a decrease in p-H3 depending on their mode of action. Clastogens induced γH2AX, and cytotoxic compounds generated a marked decrease in these two biomarkers.

1Khoury L, Zalko D and Audebert M (2016) Arch Toxicol 90(8); 1983-1995


HCS genotoxicity: pH3 and pH2AX assay protocol

Cell Line HepG2 cells
Time Point 24 hours
HCS Analysis Platform  Cellomics ArrayScan® VTI (Thermo Scientific)
Metabolizing System With or without S9 fraction
Test Article Requirements 5-10 mg solid or equivalent solution depending upon top concentration required (200x to maintain 0.5% vehicle).
Assay Controls Chlorpromazine and colchicine (+S9)
Colchicine and methyl methanesulfonate (-S9)
Endpoints Cell count
Nuclear size
DNA structure
pH3 level
pH2AX level
Genotoxicity category


Data from the pH3 and pH2AX assay

Figure 1:
Representative HCS images of pH3 and pH2AX immunocytochemistry. 

HepG2 cells were treated with colchicine and etoposide for 24 hours prior to staining for pH3 (green) and pH2AX (red). Hoechst 33342 (blue) provides a cell count and identifies changes in DNA structure associated with cytotoxicity. Colchicine, an aneugen, demonstrated a dose dependent increase in pH3 staining. The clastogen etoposide shows an increase in γH2AX in a dose dependent fashion.


+ pH2AX - pH3 = Clastogen
+ pH3 = Aneugen
- pH2AX - pH3 = Cytotoxic/- ve
+ pH3 = Cell cycle inhibitor/Aneugen
Threshold 1.5  


vinblastine Aneugen NR NR <0.004 2.28 0.0193 1.6 <0.004 2 Cell cycle inh/Aneugen Aneugen
colchicine 0.022 1.47 0.0112 4.2 0.0592 1.39 0.00889 1.93 Cell cycle inh/Aneugen Cell cycle inh/Aneugen
paclitaxel 0.325 1.3 0.006 3.91 0.189 1.41 0.0253 3.95 Cell cycle inh/Aneugen Aneugen
carbendazim NR NR 1.56 2.74 0.0272 3.23 0.0136 1.34 Cell cycle inh/Aneugen Clastogen
griseofulvin 17.2 1.41 4.54 3.2 26.1 1.74 8.81 2.68 Cell cycle inh/Aneugen Aneugen
methyl methanesulfonate Clastogen 124 2.53 NR NR 41.1 3.49 264 1.34 Clastogen Clastogen
etoposide 0.141 1.63 NR NR 0.288 1.61 NR NR Clastogen Clastogen
4-nitroquinoline N-oxide 0.277 6.18 1.76 -0.553 0.727 6.97 NR NR Clastogen Clastogen
chlorambucil 2.12 3.05 NR NR 4.15 7.01 185 1.31 Clastogen Clastogen
cyclophosphamide NR NR NR NR 17.6 1.86 166 1.2 Cytotoxic/- ve* Clastogen
araC 0.029 3.95 NR NR 0.0272 3.23 0.0245 1.32 Clastogen Clastogen
7, 12-dimethylbenz[a]anthracene <0.08 1.57 NR NR 1.55 1.81 NR NR Clastogen Clastogen
chlorpromazine Cytotoxic NR NR NR NR NR NR NR NR Cytotoxic/- ve Cytotoxic/- ve
CCCP 1.22 1.43 NR NR 6.66 1.3 7.01 1.33 Cytotoxic/- ve Cytotoxic/- ve
staurosporine 0.179 1.27 <0.012 -0.257 2.73 1.27 0.203 -0.655 Cytotoxic/- ve Cytotoxic/- ve


Table 1:
pH3 and pH2AX data for a set of 15 compounds incubated over 24 hr with HepG2 cell in the presence and absence of S9.

HepG2 monolayers were treated for 24 hours with and without S9 fraction. Mechanisms were predicted with criteria set out in A), green boxes show correctly predicted compounds. B) The positive threshold was set at 1.5 fold increase compared to vehicle only controls. Orange boxes are above the threshold, blue boxes are below the threshold. *Cyclophosphamide is metabolically activated.


1 Khoury L et al. (2016) Complementarity of phosphorylated histones H2AX and H3 quantification in different cell lines for genotoxicity screening. Arch Toxicol 90; 1983-1995

2 Kumari R et al., (2014) p53 regulation upon genotoxic stress: intricacies and complexities. Mol Cell Oncol 1(3); DOI: 10.4161/23723548.2014.969653 

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