8-hydroxy-2--deoxyguanosine and Leukemia-L5178

Depletion of glutathione (GSH) in cells exposed to certain xenobiotics has been proposed to result in oxidative stress, which could lead to damage of cellular macromolecules such as proteins, lipids, and DNA. Diethyl maleate (DEM) is known to conjugate with GSH and rapidly lower cellular GSH levels. The objective of this study was to investigate the influence of DEM-induced GSH depletion on various genotoxicity and gene expression end points in mouse lymphoma L5178Y (TK(+/-)) cell cultures. Cells were exposed to DEM for 4 h at concentrations of 0, 6.7, 13.5, 26.9, 53.8, 107.6, 215.3, and 430.6 µg/mL (0.039-2.5 mM). Genotoxicity was evaluated by examining the induction of in vitro micronuclei (20 h post-treatment) and DNA strand breaks as measured by comet (immediately following treatment), and correlating these observations to cellular GSH levels. In the current study, GSH was decreased more than 50% at the lowest test concentration (6.7 µg/mL) and more than 95% at ≥ 107.6 µg/mL. A significant increase in micronuclei and DNA strand breaks was observed at concentrations of ≥ 26.9 µg/mL. Gene expression of seven apoptosis and oxidative-stress related genes showed significant alterations in only three genes only at the highest test concentration. Quantifiable levels of 8-OH-dG (≥ 2 adducts per 1 × 10(8) NT) were not detected at any treatment concentration. These results demonstrate an association between DEM-induced genotoxicity and GSH depletion in mouse lymphoma L5178Y (TK(+/-)) cells, but not with other oxidative markers.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Apoptosis; Biomarkers; Cell Line, Tumor; Cell Survival; Comet Assay; Deoxyguanosine; DNA Adducts; DNA Damage; Dose-Response Relationship, Drug; Gene Expression; Glutathione; Leukemia L5178; Maleates; Mice; Micronuclei, Chromosome-Defective; Micronucleus Tests; Mutagens; Oxidative Stress; Reactive Oxygen Species

We compared three 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyls (PROXYLs): carbamoyl-, methoxycarbonyl-, and hydroxymethyl-PROXYL (CM-, MC-, and HM-PROXYL, respectively) with respect to radioprotection, prevention of DNA damage, and in vivo distribution in mice. The PROXYLs provided protection to C3H mice against lethal X-irradiation (8 Gy) with the following order of magnitude, HM- > CM- approximately MC-PROXYL. In contrast, radioprotection at the cellular level assessed by the colony formation of leukemia cell line L5178Y showed no difference among them. The degree of protection from X ray-induced oxidation of DNA bases measured by the formation of 8-hydroxydeoxyguanosine in salmon DNA and the cleavage of DNA measured by electrophoresis of plasmid pBR322 DNA did not differ among the PROXYLs. Redox potentials were also similar for each. However, the blood concentration of the PROXYLs injected ip into the mice showed different maximum concentrations (HM- > CM- approximately MC-PROXYL), although all reached a maximum at around 5-10 min and gradually decreased thereafter. Their concentration in bone marrow showed a similar pattern, suggesting that the difference in in vivo radioprotection among the three PROXYLs is due to the difference in their distribution to bone marrow. In general, the radioprotection provided by stable nitroxides is affected not only by redox potential and reactivity in vitro but also by pharmacokinetics.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Body Temperature; Bone Marrow; Cell Line, Tumor; Cyclic N-Oxides; Deoxyguanosine; DNA; DNA Damage; Heart Rate; Leukemia L5178; Male; Mice; Oxidation-Reduction; Peroxides; Pyrrolidines; Radiation-Protective Agents; Salmon; Whole-Body Irradiation