Metabolism of benzo[ a]pyrene by murine splenic cell types

Abstract

The objective of the present study was to determine which splenic cell type(s) of B6C3F1 mice was capable of metabolizing B(a)P. Separation of splenocytes based on density by centrifugation through discontinuous Percoll gradients along with immunomagnetic negative selection or antibody-mediated complement lysis was utilized to obtain highly enriched populations of splenocytes for B(a)P metabolism studies. Immunofluorescent cell staining in conjunction with flow cytometry and examination of Giemsa-stained cytospin cell preparations indicated that B- or T-cell populations of greater than 95% purity and an 80–90% pure population of splenic macrophages were routinely attained. Splenic cell populations were incubated with [ 3H]B(a)P for 24 hr. High-pressure liquid chromatography was used to separate and quantitate the B(a)P metabolites generated by the enriched splenic cell populations. The results of these studies demonstrate that the macrophage is the cell type responsible for the metabolism of B(a)P within the spleen. The major metabolites of B(a)P produced were as follows: an unidentified peak of polar metabolites containing polyhydroxylated metabolites, B(a)P-9,10-dihydroxy-9,10-dihydrodiol, and B(a)P-7,8-dihydroxy-7,8-dihydrodiol. Other splenic cell types examined, including B and T cells, polymorphonuclear cells, or the spleen capsule did not produce amounts of B(a)P metabolites significantly above background levels. Based on these findings, macrophages are the splenic cell types which metabolize B(a)P. As a result, macrophages may be the cell type targeted by B(a)P resulting in suppression of splenic humoral immune responses.