Infrared microspectroscopy study of gamma-irradiated and H2O2-treated human cells.

Purpose: Fourier transform infrared microspectroscopy (FT-IRM), which allows simultaneous detection of biochemical changes in the various cellular compartments, was used as a new analytical tool to study early radiation- and oxidation-induced cellular damage at the molecular level in single human cells.

Materials And Methods: HaCaT keratinocytes were given a single dose of 6 Gy (137Cs) or 650 microM H2O2, neither of which is cytotoxic (neutral red assay) but both of which result in less than 10% clonogenic survival, and deposited on zinc sulphur (ZnS) windows for infra-red (IR) spectra acquisition, immediately and 2 h after treatment. DNA damage was assessed by comet assays in alkaline conditions.

Results: Comet assays showed that the yield of DNA damage was higher after H2O2 treatment than after gamma-irradiation. The comparison between spectra of irradiated and H2O2-treated cells showed common changes, but H2O2 treatment presented a broader spectrum of cellular oxidation than ionizing radiation. The bands characteristic of deoxyribose/ribose in nucleic acids centered at 966 and 997 cm(-1), the bands characteristic of nucleic acid bases centered at 1572, 1599, and 1691 cm(-1), as well as the bands characteristic of ordered secondary structure of DNA centered at 1713-1716 cm(-1), were changed in absorbance, sometimes accompanied by a shift. The bands characteristic of proteins centered at 1515, 1530, 1544 and 1640 cm(-1) were changed in absorbance indicating a decrease in secondary structure of proteins. Moreover, the absorbance of the bands at 1515 and 1630 cm(-1) was correlated the yield of reactive oxygen species. Two hours after both treatments most changes were persistent, suggesting either irreversible or not easily repaired damage or persistent oxidative stress.

Conclusion: As we previously demonstrated in radiation-induced apoptosis studies, these results show that FT-IRM, in correlation with other cellular biology techniques, might be useful for assessing immediate radiation- and oxidative-induced damage to nucleic acids and proteins in single human cells.