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.