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 trichloromethyl radical (.CCl3). However, the ESR analysis of (alpha)-phenyl-N-tert-butylnitrone (PBN)-spin trapped .CCl3 in biological systems appears to be complicated. It has been reported that after in vivo administration of PBN and CC14 to rats, most of the PBN-CC13 adduct collected in the bile was ESR silent, suggesting reduction of the nitroxide to its hydroxylamine form. The PBN-CC13 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-CC13 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-CC13 adduct in vivo. Standard procedures for the in vitro preparation of both redox forms of PBN-CC13 and for their HPLC-EC analysis and electrochemical profiles were established. The intensity of the initially observed ESR spectrum of PBN-CC13 nitroxide of the liver extract from a CC14- and PBN-treated rat was relatively constant; after an addition of K3[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- CC13 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 •CC13 with PBN leads to a preferential 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 combination of the ESR spin trapping and HPLC-EC techniques for the detection of ESR silent radical adducts in biological systems.