Photon Factory Activity Report 2014 #32 (2015) B

BL-27A/2012G746

Membrane lipid peroxidation in lung cancer cells after monoenergetic X-ray
irradiation at the K-shell photoabsorption peak of phosphorus
Hiroshi Maezawa1,* Hiroko P. Indo2, Noriko Usami1, Hideyuki J. Majima2 and Katsumi Kobayashi1
1
Photon Factory, Tsukuba 305-0801, Japan2
Department of Oncology, Kagoshima University Graduate School of Medical and Dental Sciences,
Kagoshima 890-8544 Japan
1 Introduction
The mean energy deposition (MED) in a small volumeby photoninduced K-shell absorption followed by Augercascades in an appropriate atom may be sufficient tocause ionizing radiation damage due to moderateradiation clusters. According to calculations by Humm(1984), the MED after phosphorus K-absorption in a
volume with a 2 nm diameter is about 150 eV.
Phosphorus atoms are important constituents of DNA
and of the membrane lipid bilayer. In our previous work,
we showed that phosphorus K-absorption could
efficiently induce lethality of mammalian cells andinactivation of yeast cells, Escherichia coli andbacteriophages. Only a few studies have investigated themembrane damage induced by phosphorus K-absorption.
Indo et al. demonstrated that the formation of intracellular
membrane lipid peroxidation products, such as 4hydroxy-2-nonenal (HNE)-modified proteins, by X-rays(120kVp) correlated with the production of mitochondrial
reactive oxygen species
The purpose of this study is to experimentally verify
whether membrane lipid peroxidation is enhanced in cells
by Auger cascades after K-shell photoionization ofphosphorus.
2 Experiment
One day before irradiation, lung cancer cells wereplated on glass bottom dishes in DMEM supplemented
with 10% fetal bovine serum.
Monoenergetic X-ray irradiation was performed using a
Si crystal monochromater at BL-27A. Three energies
were chosen for irradiation: 2.147, 2.153 (phosphorus Kshell resonance absorption peak, P-K peak), and 2.160
keV. To assess the production of membrane lipid
peroxide, cells were exposed to 0.516 C/kg for eachenergy radiation.
After irradiation, the cells were incubated at 37 °C for 2
hours and subsequently immunofluorescently labeledwith anti-4-HNE mouse monoclonal antibody and AlexaFluor 488 goat anti-mouse IgG(H+L) conjugate.
Fluorescence images were obtained using a confocal laser
scanning unit, inverted microscope (20x objective lens),and color chilled 3CCD camera. The intensity of the laser
beam was set at a constant level to allow quantitative
comparison of the fluorescence for the different samples.
IPLab Spectrum software was used to obtain the values of
the average fluorescence intensity per cell.

3 Results and Discussion
The formation of intracellular HNE-modified proteins,observed in green, represents lipid peroxidation in A549cells (Fig. 1). All three tested irradiation energies led to asignificant increase in immunofluorescent intensity ascompared to that for the control. The fluorescent intensity
measured after irradiation at 2.153 keV was higher thanthat for 2.146 and 2.160 keV irradiation (P < 0.01).
Reproducibility of these results was confirmed throughthree experiments. The increment (21%) in relative
fluorescent intensity after 2.153 keV radiation was about
2 times as high as that after radiation at the other twoenergies. Therefore, these results indicate that 2.153 keV(P-K peak) radiation induced the peroxidation ofmembrane phospholipids more efficiently than P-K offpeak radiation. We suggest that the energy deposition in
the vicinity of phosphorus in the Auger cascade produced
efficient reactive oxygen species (e.g., superoxide,hydrogen peroxide) in mitochondria.

Fig. 1: Intracellular HNE generation in A549 cells
following irradiation. Representative images ofimmunofluorescencestaining(green)andthequantification of the fluorescence intensity are shown forirradiated and non-irradiated control cells. Bar represents
the mean ± s.d. (n>100);**: P <0.01; Scheffe’s F test.
References[1] J. L. Humm, KFA Report, 100 (1984).
[2] H. Maezawa et al., Acta Oncologica, 35, 889 (1996).
[3] H. P. Indo et al., Free Radical research, 46, 1029,(2012).
* mae_525rb@yahoo.co.jp