Metabolism of carbon tetrachloride to trichloromethyl radical: An ESR and HPLC-EC study.
Extensive ESR spin-trapping studies with alpha-phenyl-N-tert-butylnitrone (PBN) have shown that carbon tetrachloride (CCl(4)) is metabolized to trichloromethyl radical ((*)CCl(3)). However, the ESR analysis of alpha-phenyl-N-tert-butylnitrone (PBN)-spin trapped (*)CCl(3) in biological systems appears to be complicated. It has been reported that after in vivo administration of PBN and CCl(4) to rats, most of the PBN-CCl(3) adduct collected in the bile was ESR silent, suggesting reduction of the nitroxide to its hydroxylamine form. The PBN-CCl(3) nitroxide was also shown to undergo a NADPH-dependent reduction in the presence of liver microsomes. Thus, it appears that the variability (or the absence) of the ESR signal of PBN-CCl(3) nitroxide in biological systems reflects, at least in part, the fluctuations in the equilibrium between the nitroxide and hydroxylamine forms of this adduct. To test this possibility, ESR and HPLC experiments with electrochemical detection (EC) were conducted for analysis of the major redox form of the PBN-CCl(3) adduct in vivo. Standard procedures for the in vitro preparation of both redox forms of PBN-CCl(3) and for their HPLC-EC analysis and electrochemical profiles were established. The intensity of the initially observed ESR spectrum of PBN-CCl(3) nitroxide of the liver extract from a CCl(4)- and PBN-treated rat was relatively constant; after an addition of K(3)[Fe(CN)(6)] to the extract, the intensity of the ESR spectrum increased by 1 order of magnitude, most likely due to the co-oxidation of ESR silent PBN-derived hydroxylamines. The addition of PBN-CCl(3) nitroxide to the liver homogenate resulted in the rapid loss of the ESR signal. The HPLC-EC analysis of the liver extract revealed that the in vivo spin trapping of (*)CCl(3) with PBN leads to a preferential formation of the ESR silent PBN-CCl(3) hydroxylamine. The predominant presence of the hydroxylamine derivative was also detected in the blood of a CCl(4)-treated rat. The results of this work are discussed in terms of combination of the ESR spin trapping and HPLC-EC techniques for the detection of ESR silent radical adducts in biological systems.
Stoyanovsky
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DA
Department of Biochemistry and Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029, USA.
Cederbaum
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AI
D000818:Animals; D001711:Biotransformation; D002251:Carbon Tetrachloride / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics; D002851:Chromatography, High Pressure Liquid; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals; D006898:Hydroxylamines / Q000737:chemistry; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley
D005609:Free Radicals; D006898:Hydroxylamines; D007202:Indicators and Reagents; C025280:trichloromethyl free radical; D002251:Carbon Tetrachloride