Role of metabolism in cocaine-induced immunosuppression in splenocyte cultures from B6C3F1 female mice.

Cocaine has been reported to directly suppress the in vitro immune responses at very high concentrations. In the present study, the possible role of metabolism in cocaine-induced immunosuppression was investigated in splenocyte cultures isolated from B6C3F1 female mice. Since cocaine can be metabolized by both esterase and P-450 monooxygenase, we studied the direct effects of cocaine, benzoylecgonine and norcocaine on the in vitro T-dependent antibody response to SRBC. Direct exposure to cocaine only produced a modest (30%) but nonsignificant suppression of the antibody response, while benzoylecgonine, a primary product of metabolism by the esterase pathway, was devoid of activity. In contrast, direct exposure to norcocaine, the initial product of N-demethylation by the P-450 pathway, produced significant suppression at concentrations greater than or equal to 10 microM. Similar results were observed in studies measuring LPS and Con A mitogenicity. Furthermore, a significant suppression was observed when splenocytes were preincubated for 1 h with 1 mM cocaine in the presence of liver S-9 fractions isolated from phenobarbital-induced mice. Meanwhile, no suppression was obtained when splenocytes were preincubated in the presence of untreated S-9 fractions. To characterize the mechanism of our results, the capacity of both untreated and phenobarbital-induced microsomes to produce formaldehyde from cocaine was compared. The N-demethylation of cocaine was NADPH-dependent and phenobarbital-induced microsomes produced approx. 6-times higher amounts of formaldehyde, indicating a greater portion of cocaine could be metabolized through the P-450 pathway to its toxic metabolites. Finally, because benzoylecgonine shares with cocaine the presence of a methyl group on the tropane nitrogen, we also compared the ability of N-demethylation from cocaine and benzoylecgonine in mouse liver microsomes. Our results indicated that benzoylecgonine could not be demethylated as determined by a failure to generate any formaldehyde. These results offer further support that the N-demethylation pathway is a critical step to cause its immunotoxicity.