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

Protocol

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

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

		-S9				+S9				Mechanism
		pH2AX		pH3		pH2AX		pH3		Mechanism
Compound	Mechanism	MEC	Max response	MEC	Max response	MEC	Max response	MEC	Max response	-S9	+S9
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.

References

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