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 (CCl4) is metabolized to trichlor omethyl radical ((CCl3)-C-.). However, the ESR analysis of alpha-phenyl-N-t ert-butylnitrone (PBN)-spin trapped (CCl3)-C-. in biological systems appear s to be complicated. It; has been reported that after in vivo administratio n of PBN and CCl4 to rats, most of the PBN-CCl3 adduct collected in the bil e was ESR silent, suggesting reduction of the nitroxide to its hydroxylamin e form. The PBN-CCl3 nitroxide was also shown to undergo a NADPH-dependent reduction in the presence of liver microsomes. Thus, it appears that the va riability (or the absence) of the ESR signal of PBN-CCl3 nitroxide in biolo gical systems reflects, at least in part, the fluctuations in the equilibri um between the nitroxide and hydroxylamine forms of this adduct. To test th is possibility, ESR and HPLC experiments with electrochemical detection (EC ) were conducted for analysis of the major redox form of the PBN-CCl3 adduc t in vivo. Standard procedures for the in vitro preparation of both redox f orms of PBN-CCl3 and for their HPLC-EC analysis and electrochemical profile s were established. The intensity of the initially observed ESR spectrum of PBN-CCl3 nitroxide of the liver extract from a CCl4- and PEN-treated rat w as 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 PEN-derived hydroxylamines. Th e addition of PBN-CCl3 nitroxide to the liver homogenate resulted in the ra pid loss of the ESR signal. The HPLC-EC analysis of the liver extract revea led that the in vivo spin trapping of (CCl3)-C-. with PEN leads to a prefer ential formation of the ESR silent PBN-CCl3 hydroxylamine. The predominant presence of the hydroxylamine derivative was also detected in the blood of a CCl4-treated rat. The results of this work are discussed in terms of comb ination of the ESR spin trapping and HPLC-EC techniques for the detection o f ESR silent radical adducts in biological systems.