Bioactivation and covalent binding of halothane in vitro: Studies with [³H]- and [¹⁴C]halothane

To determine if the hydrogen atom of halothane (CF₃CHBrCl) is retained on the reactive intermediates that covalently bind to microsomal lipids and protein [³H]halothane and [¹⁴C]halothane were incubated with rat hepatic microsomes and a NADPH generating system. Both [³H]- and [¹⁴C]halothane were bioactivated and bound to a greater degree when incubations were performed in a N₂ atmosphere rather than an O₂ atmosphere. In fact, covalent binding of ³H-equivalents did not occur under an O₂ atmosphere. Binding of [³H]- and [¹⁴C] halothane equivalents was significantly enhanced when hepatic microsomes from phenobarbital- or Aroclor 1254-treated rats were used in the incubations. Omission of NADPH or incubation with CO was inhibitory to the binding of both [³H]- and [¹⁴C]halothane. The apparent kinetic constants for binding of halothane equivalents, Km and Vmax indicate a significantly higher Km but lower Vmax for the formation and/or binding of ³H-binding equivalents. The results indicate that halothane is primarily bioactivated under conditions that promote its reductive metabolism and that this reductive metabolism does not involve cleavage of the carbon-hydrogen bond of halothane. Differences in binding under N₂ and O₂ as well as between [³H]- and [¹⁴C]halothane suggest that multiple reactive intermediates may form during the biotransformation of halothane.