Metabolic radiolabeling: experimental tool or Trojan horse? (35)S-Methionine induces DNA fragmentation and p53-dependent ROS production.
Despite the general assumption that widely used radiolabeled metabolites such as [(35)S]methionine and (3)H-thymidine do not adversely affect or perturb cell function, we and others have shown that such low-energy beta-emitters can cause cell cycle arrest and apoptosis of proliferating cells. The goal of the present study was to elucidate the targets and mechanisms of [(35)S]methionine-induced cellular toxicity. Comet analyses (single-cell electrophoresis) demonstrated dose-dependent DNA fragmentation in rabbit smooth muscle cells within a time frame (1-4 h) well within that of most radiolabeling protocols, whereas fluorescence analyses using a peroxide/hydroperoxide-sensitive dye revealed production of reactive oxygen species (ROS). Although ROS generation was inhibitable by antioxidants, DNA fragmentation was not inhibited and was in fact observed even under hypoxic conditions, suggesting that beta-radiation-induced DNA damage can occur independently of ROS formation. Studies with p53(+/+) and p53(-/-) human colorectal carcinoma cells further demonstrated the dissociation of early DNA damage from ROS formation in that both cell types exhibited DNA fragmentation in response to radiolabeling whereas only the p53(+/+) cells exhibited significant increases in ROS formation, which occurred well after significant DNA damage was observed. These findings demonstrate that metabolically incorporated low-energy beta-emitters such as [(35)S]methionine and (3)H-thymidine can induce DNA damage, thereby initiating cellular responses leading to cell cycle arrest or apoptosis. The results of this study require a reevaluation using low-energy beta-emitters to follow not only experimental protocols in vivo processes, but also acceptable exposure levels of these genotoxic compounds in the workplace and environment.
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Department of Biochemistry and Molecular Biology, The George Washington University, School of Medicine and Health Sciences, Washington, DC 20037, USA. bcmvwh@gwumc.edu
Heikka
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Dieffenbach
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D000818:Animals; D015687:Cell Hypoxia; D002478:Cells, Cultured; D053938:DNA Fragmentation / Q000187:drug effects / Q000528:radiation effects*; D004307:Dose-Response Relationship, Radiation; D008715:Methionine / Q000494:pharmacology*; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013462:Sulfur Radioisotopes / Q000494:pharmacology*; D013936:Thymidine / Q000494:pharmacology; D013997:Time Factors; D014316:Tritium / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53
D017382:Reactive Oxygen Species; D013462:Sulfur Radioisotopes; D016159:Tumor Suppressor Protein p53; D014316:Tritium; D008715:Methionine; D013936:Thymidine