Investigations in this laboratory have demonstrated for the first time the feasibility of spin trapping free radicals in vivo produced during metabolic processes, using electron paramagnetic resonance spectroscopy (EPR). Production of .CCl3 and lipid radicals (L.) during CCl4 metabolism was demonstrated to occur in livers of intact animals, and the radicals were detected only in the endoplasmic reticulum (E.R.), which is the site of their production. This was done by administering the spin trapping agent to rats simultaneously with the CCl4.
The specific aims are to determine the effect of the dose size of CCl4 and of the amount of spin trapping agent [phenyl t-butyl nitrone (PBN), or trimethoxyphenyl t-butyl nitrone (MO)3PBN)] administered on the intensity and duration of .CCl3 and L. radical formation in the hepatic E.R. in vivo. This will be done by giving the spin trapping agent at various times after CCl4 administration and trapping radicals for a specified time before removal of livers and extraction of the trapped lipid radicals for EPR analyses. This will provide the opportunity to observe radical production at sequential windows of time, information needed to determine the relationship of L. production to the generation of CCl3. L. formation is the initiation step of lipid peroxidation, which is believed to be associated with the toxicity of CCl4. Since lipid peroxidation to CCl4 metabolism is inhibited in liver microsomes in vitro by prior treatment of animals with radical-scavenging antioxidants, we propose to investigate the influence of an effective scavenger, Alpha-tocopherol, on L. formation in vivo. Antioxidants provide partial protection against CCl4 toxicity, but is it not known if this protection may involve scavenging of radicals generated during CCl4 metabolism. On the other hand, CCl4 toxicity is enhanced by prior treatment of rats with phenobarbital (PB). We will investigate the influence of prior treatment of rats with BP on the production of .CCl3 and L. radical in vivo. It should be possible to assess if a relationship exists between radical formation and the known toxicity of CCl4 in these physiological states. Isolation of the lipids from the hepatic E.R. will be done to determine which lipid classes form the L. adducts. We will determine if production of the L. and .CCl3 radicals which occurs in microsomal systems metabolizing CCl4 in vitro shows similar behavior as production occurring in vivo. This will aid in assessing the validity of the in vitro studies on these radical events.
