Nitroxide metabolism in the human keratinocyte cell line HaCaT.
Metabolism of different nitroxides with piperidine structure used as spin labels in electron spin resonance (ESR) studies in vitro and in vivo was investigated in human keratinocytes of the cell line HaCaT by GC and GC-MS technique combined with S-band ESR. Besides the well known reduction of the nitroxyl radicals to the ESR silent hydroxylamines as primary products our results indicate the formation of the corresponding secondary amines. These reductions are inhibited by the thiol blocking agent N-ethylmaleimide and by the strong inhibitors of the thioredoxin reductase (TR) 2-chloro-2,4-nitrobenzene and 2,6-dichloroindophenol. The competitive inhibitor TR inhibitor azelaic acid and the cytochrome P-450 inhibitor metyrapone lack any effects. The rates of reduction to the hydroxylamines and secondary amines were dependent on the lipid solubility of the nitroxides. Therefore, it can be assumed that the nitroxides must enter the cells for their bioreduction. The mostly discussed intracellular nitroxide reducing substances ascorbic acid and glutathione were unable to form the secondary amines. In conclusion, our results suggest that the secondary amine represents one of the major metabolites of nitroxides besides the hydroxylamine inside keratinocytes formed via the flavoenzyme thioredoxin reductase most probably. Further metabolic conversions were detected with 4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl and the benzoate of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl as substrates.
Kroll
C
C
Department of Pharmacy, Humboldt University of Berlin, Germany.
Langner
A
A
Borchert
H H
HH
D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003497:Cyclic N-Oxides / Q000378:metabolism / Q000633:toxicity; D004578:Electron Spin Resonance Spectroscopy; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D015394:Molecular Structure; D009589:Nitrogen Oxides / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*; D010880:Piperidines / Q000737:chemistry / Q000378:metabolism* / Q000633:toxicity*; D013113:Spin Labels; D013329:Structure-Activity Relationship
D003497:Cyclic N-Oxides; D009589:Nitrogen Oxides; D010880:Piperidines; D013113:Spin Labels; C015527:4-amino-TEMPO; C001803:tempol; C003959:TEMPO