Dramatic Increase in Oxidative Stress in Carbon-Irradiated Normal Human Skin Fibroblasts

Carine Laurent,François Sichel,Alexandre Leduc,Virginie Prévost,Jean-Louis Lefaix,Ivannah Pottier

doi:10.1371/journal.pone.0085158

Abstract

Skin complications were recently reported after carbon-ion (C-ion) radiation therapy. Oxidative stress is considered an important pathway in the appearance of late skin reactions. We evaluated oxidative stress in normal human skin fibroblasts after carbon-ion vs. X-ray irradiation. Survival curves and radiobiological parameters were calculated. DNA damage was quantified, as were lipid peroxidation (LPO), protein carbonylation and antioxidant enzyme activities. Reduced and oxidized glutathione ratios (GSH/GSSG) were determined. Proinflammatory cytokine secretion in culture supernatants was evaluated. The relative biological effectiveness (RBE) of C-ions vs. X-rays was 4.8 at D0 (irradiation dose corresponding to a surviving fraction of 37%). Surviving fraction at 2 Gy (SF2) was 71.8% and 7.6% for X-rays and C-ions, respectively. Compared with X-rays, immediate DNA damage was increased less after C-ions, but a late increase was observed at D10% (irradiation dose corresponding to a surviving fraction of 10%). LPO products and protein carbonyls were only increased 24 hours after C-ions. After X-rays, superoxide dismutase (SOD) activity was strongly increased immediately and on day 14 at D0% (irradiation dose corresponding to a surviving fraction of around 0%), catalase activity was unchanged and glutathione peroxidase (GPx) activity was increased only on day 14. These activities were decreased after C-ions compared with X-rays. GSH/GSSG was unchanged after X-rays but was decreased immediately after C-ion irradiation before an increase from day 7. Secretion of IL-6 was increased at late times after X-ray irradiation. After C-ion irradiation, IL-6 concentration was increased on day 7 but was lower compared with X-rays at later times. C-ion effects on normal human skin fibroblasts seemed to be harmful in comparison with X-rays as they produce late DNA damage, LPO products and protein carbonyls, and as they decrease antioxidant defences. Mechanisms leading to this discrepancy between the two types of radiation should be investigated.

Highlights

Effects of conventional radiation therapy (RT) using low-LET X-rays on tumours and on normal tissues have been investigated for decades
The β value was 3-fold lower after C-ions compared with X-rays, with a value of 0.02 explaining the almost linear shapes of the C-ion survival curve
glutathione peroxidase (GPx) activity was unchanged after X-rays except for a slight significant increase at day 14, but was decreased after C-ions compared with X-rays, with an RBE10% of 0.59 at day 7 (Figure 4C)

Summary

Introduction

Effects of conventional radiation therapy (RT) using low-LET (linear energy transfer) X-rays on tumours and on normal tissues have been investigated for decades. Of 35 patients treated for unresectable bone and soft tissue sarcoma by a dose escalation protocol, 35 and 27 presented acute or late skin reactions, respectively, after exposure to doses ranging from 52.8 to 73.6 GyE in 16 fixed fractions. They were followed up from 29.5 to 71.7 months after C-ion RT. In vitro studies have demonstrated an active role of dermal fibroblasts and endothelial cells in response to irradiation by the use of an antiinflammatory and antioxidant treatments [5,6], which have proven very effective in patients presenting late skin complications [7]

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Results

Conclusion