DNA damage in dermal fibroblasts and endothelial cells exposed to gamma rays treated by the combination of pentoxifyline and a-tocopherol.

C Laurent, J-P Pouget, P. Voisin, 5th Comet Assay Workshop, 29-30/08/2003, Aberdeen, Ecosse.

PURPOSE: This in vitro study aims to better understand the mechanisms of action of the combination of pentoxifylline (PTX) and α-tocopherol (αT) on DNA damage in primary cultures of dermal fibroblasts and endothelial cells exposed to gamma rays. PTX is a xanthine derivative known for its anti-inflammatory effects and αT has the highest antioxidant activity among the vitamin E compounds structures. When administered simultaneously, the two drugs showed their effectiveness in vivo by reducing the late damage observed after skin radiation exposure. However, the mechanisms of action of this combination are still not elucidated.
METHODS: Primary cultures of dermal fibroblasts and endothelial cells were irradiated at the plateau phase at the experimentally chosen dose of 10 Gy using a 137Cs irradiator. Fifteen minutes prior to irradiation, cells were incubated in presence of 500 µM PTX and/or 500 µM trolox, the water-soluble analogue of αT. Cell survey was measured by trypan blue exclusion test. ROS generation was estimated by DCFH oxidation and DNA damage were assessed by the comet and micronuclei assays. The antioxidant capacity of the drugs was also determined by ORAC method.
RESULTS: Addition of the drugs either separately or simultaneously led to an increase in endothelial cells and fibroblasts survival after radiation exposure. ORAC test showed PTX and trolox have an antioxidant capacity which is increased when the compounds are used simultaneously. Generation of ROS was mainly observed immediately after irradiation. In agreement with previous data, the use of PTX and trolox allowed to decrease this production either immediately for endothelial cells, or some minutes after radiation exposure for fibroblasts. The number of strand breaks and alkali-labile sites measured in the irradiated cells by the comet assay was increased in presence of the drugs and persisted for several hours, whereas micronuclei formation was not emphasized.

CONCLUSIONS: The combination of PTX and trolox leads to an increase in viability of irradiated dermal endothelial cells and fibroblasts. That could be explained by the ROS generation decrease associated to the high antioxidant capacity of these drugs. However, preliminary results indicate that, in contrast with micronuclei frequency, the number of DNA strand breaks measured by the comet assay was increased after radiation exposure in treated cells. Further research works are needed to solve this apparent discrepancy.