Whole cell-ELISA to measure the gammaH2AX response of six aneugens and eight DNA-damaging chemicals

Abstract

The phosphorylated form of the histone protein H2AX (gammaH2AX) plays a central role in sensing and repairing DNA damage and is a sensitive marker for DNA double-strand breaks (DSB). Although a wide range of genotoxic agents that do not initiate DSB induce gammaH2AX, the range of chemicals that cause H2AX phosphorylation is not clear. We designed a novel, whole cell enzyme-linked immunosorbent assay (cell-ELISA) that can accurately quantify gammaH2AX levels and identify chemical compounds that induce gammaH2AX formation; our novel assay is more convenient than microscopic examination of gammaH2AX foci or flow cytometry. We measured gammaH2AX levels in CHL, CHO and V79 cells exposed to DNA-damaging, non-genotoxic and aneugenic chemicals using the cell-ELISA assay. The cell-ELISA results for the DNA-damaging compounds (methyl methanesulfonate, N-ethyl-N'-nitro-N-nitrosoguanidine, mitomycin C, cisplatin, irinotecan, etoposide, methotrexate and 5-fluorouracil) assayed showed that there was a concentration-dependent increase in gammaH2AX, which was 1.5-fold greater than the negative control; the only exception was a negative response of CHO cells to 5-fluorouracil. None of the 10 non-genotoxic compounds assayed showed similar increases in gammaH2AX and all exhibited concentration-dependent growth inhibition of the cells. The highest levels of gammaH2AX found from treatment with aneugens (vincristine, colcemid, paclitaxel, griseofulvin, 17-allylaminogeldanamycin and CH3310395), which are compounds that cause spindle dysfunction and have no genotoxic activity in the Ames test, were 1.5-fold lower than the negative control. In contrast, mitomycin C and etoposide, which both have aneugenic and DNA-damaging activities, induced a positive response. None of the aneugens caused an increase in gammaH2AX at concentrations that induce micronuclei. The chemical classes that show positive results in the cell-ELISA are different from those that are positive in the Ames or in vitro micronucleus test. By using the cell-ELISA for the level of gammaH2AX, we were able to distinguish DNA-damaging agents from non-genotoxic compounds or aneugens.