Assessment of the radioprotective effects of amifostine on human lymphocytes irradiated in vitro by gamma-rays using cytokinesis-blocked micronucleus assay

  Background: A radioprotective effect of amifostine as well as its ability to modulate the level of spontaneous and gamma-irradiation-induced genetic changes on human peripheral blood lymphocytes has been investigated . Amifostine, known as a potent radical scavenger, has been introduced as the most effective radioprotector, yet it is not completely approved for the clinical use. However, further in vitro and clinical studies are needed to clarify its mechanisms of action.   Materials and Methods : Whole blood samples from healthy donors were exposed to various doses of gamma-rays. Lymphocytes in cultures were treated with amifostine at different concentrations (2, 4 and 6 mM) in the presence or in the absence of 1 IU/ml alkaline phosphatase before or after gamma-irradiation. Standard procedure for the cytokinesis-block micronucleus (CBMN) assay was used to assess the effect of amifostine on radiation induced micronucleus in binucleate lymphocytes.     Results : Irradiated blood samples showed an increase in the total number of micronuclei (MN) significantly different from controls (p<0.05). However, pre-treatment of lymphocytes with amifostine in the presence of alkaline phosphatase, 15 minutes before irradiation, led to a significant decrease in the frequencies of MN and cells with more than one MN (p<0.05). Amifostine, in its own, produced little or no protection. However, the addition of amifostine with alkaline phosphatase to the cell cultures 15 minutes after irradiation produced substantial radioprotection significantly different from the frequencies of MN induced by radiation alone (p<0.05).   Conclusion : Results clearly indicated that gamma-rays induced MN in lymphocytes in a dose dependent manner. The highest protective effect was achieved when amifostine was phosphorilated by alkaline phosphatase and present before irradiation in the cellular environment, indicating its radical scavenging mechanism of radioprotection. Since the administration of amifostime after irradiation also led to a considerable decrease in the frequency of radiation induced MN, which might be possible for other mechanisms such as induction of cell cycle delay and hence influencing DNA repair, are involved in radioprotection by amifostine.