We initially determined that direct addition of cocaine to splenocytes from female B6C3F1 mice only produced nonsignificant suppression of the in vitro T-dependent antibody (Ab) response to SRBC, and only at the highest concentrations tested (21% and 41% at 10-5M and 10-4M, respectively). These results are not consistent with a direct immunomodulation by cocaine, because these concentrations are much greater than reasonable blood levels in cocaine users. The purpose of this study was to examine possible indirect effects of cocaine on the immune system, with an emphasis on a role by reactive metabolites. Cocaine is metabolized to norcocaine and, ultimately to more reactive intermediates, via a minor metabolic pathway mediated by the cytochrome P-450 system. The generation of these metabolites is associated with hepatotoxicity, which is both sex- and strain-dependent, and which can be increased following the administration of a P-450 inducer like phenobarbital (Pb). Subchronic (14 day) exposure in female B6C3F1 mice produced a significant decrease in the Ab response to SRBC only at the highest dose tested (80 mg/kg); lower doses were without effect, even though mice were obviously affected (i.e., female B6 mice were hyperactive when exposed to cocaine at 40 and 60 mg/kg/day). In contrast, a significant decrease of 50% was observed in female DBA/2 mice exposed to 60 mg/kg/day in male B6C3F1 mice exposed to 40 mg/kg/day. Pretreatment with Pb markedly increased the suppression in female B6C3F1 mice, with 30 mg/kg producing a decrease of ~30% and 60 mg/kg producing a significant decrease of >70%. Additional evidence has been derived from in vitro studies using the Ab response to SRBC by splenocytes from female B6 mice: (1) direct addition of norcocaine was 10-fold more suppressive than cocaine (decreases of 21% and 53% were noted at 10-6 M and 10-5 M, respectively); and (2) concentrations of cocaine, which were inactive when added to splenocytes alone, suppressed the Ab response when preincubated with an S-9 liver homogenate from Pb-induced female B6 mice, or when added to a co-culture system (primary hepatocyte-splenocyte from female B6 mice), which was developed and optimized by this laboratory to study the immunological effects of reactive intermediates. These preliminary results suggest that cocaine immunomodulation may be mediated, at least in part, through the generation of P-450-dependent reactive metabolites. The primary objective of this investigation will be to confirm a role by metabolism in cocaine's effects on immunocompetence. The studies will include a determination of a possible mechanism of action at a biochemical level - i.e., depletion of glutathione. A secondary objective will be to characterize the profile of activity of cocaine using optimal conditions for immunosuppression (i.e., as established with the Ab response to SRBC) and a number of parameters reflecting several aspects of immune function. These studies will provide some indication of the mechanism of cocaine's action at the cellular level by describing if it selectively affects a given cell type or functional endpoint.
