Method for Determining CYP2C9 Activity in Earthworms and its Responses to Benzo[a] pyrene or Pyrene in Soil

In this study, high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) for determining CYP2C9 activity in the earthworm Eisenia fetida using diclofenac as the substrate is developed and validated for the first time. The high sensitivity, selectivity, and excellent accuracy and precision of this approach permit the use of very low incubation concentrations of microsome protein (10-100 mu g mL(-1)). Using this method, CYP2C9 activity in earthworms in response to increasing doses of benzo[a]pyrene (B[a]P) or pyrene (0.12, 0.24, 0.48, and 0.96 mg kg-1) in soil is determined. The results suggest that the enzyme CYP2C9 did exist in earthworms and show that CYP2C9 activity responded to B[a]P or pyrene highly different. A significant enhancement of CYP2C9 activity is observed at low doses (0.12-0.24 mg kg(-1)) of B[a]P at days 3 and 14, while a clear inhibition is observed at high doses (0.48-0.96 mg kg(-1)) of B[a]P at day 14. The responses of CYP2C9 activity to pyrene are bell shaped, independent of exposure duration, and not inhibited during the entire experiment. In contrast, at the highest dose of pyrene, CYP2C9 activity reaches its minimal value, which is insignificantly different from the control. It is thus concluded that the CYP2C9 activity of earthworms can be used as a biomarker for monitoring B[a](P-) or pyrene-contaminated soil.