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Updated: Aug 25, 2026

Flow Cytometric Measurement Of ROS Production In Macrophages In Response To FcγR Cross-linking
Published on: March 7, 2019
Azidothymidine promotes free radical generation by activated macrophages and hydrogen peroxide-iron-mediated
Andrei M Komarov1, Jonathon M Hall, William B Weglicki
1Department of Physiology and Experimental Medicine, The George Washington University Medical Center, 2300 Eye Street, NW, Ross Hall, Rm. 451A, Washington, DC 20037, USA. phyamk@gwumc.edu
Abstract:
Azidothymidine (AZT) and AZT monophosphate (AZT-MP) in concentrations as low as 10 and 50 microM, respectively, promote oxidation of chemically deacetylated 2',7'-dichlorodihydrofluorescein (DCDHF) to 2',7'-dichlorofluorescein (DCF) by rat peritoneal macrophages activated with latex. Cells were incubated with AZT and AZT-MP for 18 h, washed out from residual AZT or AZT-MP and activated with latex for 30 or 60 min in the presence of DCDHF. Latex-activated cells oxidize DCDHF extracellularly due to release of hydrogen peroxide and low-molecular iron complexes, which is verified using catalase, desferal and the peroxidase inhibitor sodium azide. AZT and AZT-MP increase DCDHF oxidation due to additional release of hydrogen peroxide as demonstrated by catalase inhibition of DCDHF oxidation and direct H(2)O(2) measurement. Thymidine and thymidine phosphates did not show any effect on macrophage activation. In separate experiments we evaluated the in vitro prooxidant activity of AZT, AZT-MP, AZT triphosphate (AZT-TP), AZT glucuronide (GAZT) and 3'-amino-3'-deoxythymidine (AMT) in a cell-free system using the hydrogen peroxide-iron-mediated oxidation of DCDHF. Under these conditions, AZT and AZT phosphates exhibit a prooxidant effect in concentrations as low as 100 microM. Furthermore, GAZT is a less effective prooxidant and AMT acts like an antioxidant. Thymidine did not show any effect.
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