Earthworm cytochrome P450 determination and application as a biomarker for diagnosing PAH exposure

Abstract

We developed a new microsome purification method and used the Omura and Sato method to measure the total content of cytochrome (Cyt) P450 in earthworm (Eisenia fetida) microsomes. In method development, two different pretreatments, i.e. solubilization or manual separation were used to purify worm microsomes. Solubilization was more effective than manual separation and difference spectra showed a peak at 450 ± 1 nm in microsomes received solubilization pretreatment. We conducted a 48 h contact test by exposing worms on pyrene (Py) and benzo[a]pyrene (BaP) spiked filter paper. A dose-response relationship was established between total P450 content and the concentration of Py or BaP ranging from 10⁻⁶ mg mL⁻¹ to 10⁻² mg mL⁻¹. Results show that total Cyt P450 content in earthworms is a promising biomarker for diagnosing PAHs exposure at sublethal dose ranges.