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.
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
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 |
A
+ pH2AX | - pH3 = Clastogen |
---|---|
+ pH3 = Aneugen | |
- pH2AX | - pH3 = Cytotoxic/- ve |
+ pH3 = Cell cycle inhibitor/Aneugen | |
Threshold 1.5 |
B
-S9 | +S9 | Mechanism | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
pH2AX | pH3 | pH2AX | pH3 | ||||||||
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:
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
Learn more about toxicology in our popular Mechanisms of Drug-Induced Toxicity guide
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