7,12-Dimethylbenz[a]anthracene (DMBA)

In splenocytes, which is isolated from mice, lymphocyte function is completely suppressed by the treatment with 50 μM DMBA. The susceptibility of splenocytes isolated from mEH-null mice is lower than from wildtype mice on the immunosuppression activity of DMBA.^[1]

Spleens are harvested and placed in a sterile phosphatebuffered saline. Cell suspensions are prepared by gentle dissociation using glass slides. Debris is separated from cells by filtration and cell suspensions are centrifuged at 250 x g for 5 min. Cells are seeded in a volume of 100 µl in 96-well plates. Mitogens were added in a volume of 10 µl at final concentrations of 25 µg/ml for LPS and 5 µg/ml for PHA, and then the cells were incubated in 5% CO2 at 37°C for 3 days. The cells were harvested onto filter strips using a harvester and [3 H]-thymidine incorporation was determined by liquid scintillation. For mitogen-induced lymphocyte proliferation assays after DMBA treatment in vitro, cell preparations from untreated mice were simultaneously exposed to mitogen (LPS) and DMBA.

The metabolic activation of DMBA in the spleen plays a key role in the splenic immunotoxicity, mEH-null mice are more resistant to DMBA-induced cytotoxicity as well as immunosuppression.^[1]