DIRECT OBSERVATION OF SPIN-TRAPPED CARBON-DIOXIDE RADICALS IN HEPATOCYTES EXPOSED TO CARBON-TETRACHLORIDE

Carbon dioxide radical adducts of the spin trapping agent, αphenyl N-t-butyl nitrone (PBN), have been observed to occur in the urine and bile of rats exposed to carbon tetrachloride as well as in perfusates of liver in which the perfusion medium contained carbon tetrachloride (Connor er al., J. Biol. Chem., 261, 4542, (1986)). The carbon dioxide adduct was proven to be derived from CCI, by use of 13-C-labelled compound. These adducts were not observed in the liver itself suggesting that they might be rapidly secreted from the liver. However, using isolated hepatocytes, we have demonstrated that the carbon dioxide radical adduct can be observed directly in the liver cells as it is formed. Since this water-soluble adduct cannot be extracted by non-aqueous solvents such as chloroform or toluene, its formation in liver in vivo or in perfused livers was not detected. Lowering the oxygen tension in the system diminished the intensity of production of the carbon dioxide adduct, consistent with the adduct being produced as a result of ·OOCCl3 generation. It is not clear the extent to which this adduct is formed as a result of the ·CO2 radical or is produced by metabolic oxidation of the trichloromethyl radical adduct of PBN per se to the carbon dioxide radical adduct. The intensity of the signal of the carbon dioxide radical adduct suggests that adduct conversion may be the route of formation since it seems unlikely that a sufficient amount of the halocarbon could be metabolized to ·COCl or ·CO2 radicals to generate a signal of the magnitude involved. The ·CO2 adduct is readily observed in intact hepatocytes, but the ·CCl3 adduct is not (although we know the ·CCl3 adduct has been produced in these cells), indicating that the ·CO2 adduct is present in considerable abundance compared to the ·CCl3 adduct. © 1990 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.