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.

Toxicity, bioaccumulation and metabolism of pesticides in the earthworm

“…128) The concentrationdependent convex profiles (induction at lower concentrations but insignificant change or inhibition at higher ones) were observed for CYP content or EROD activity when the earthworm was exposed to BaP (E. fetida), 129) heavy metals (E. fetida) 116) and chlorpyrifos (Ap. caliginosa).…”

“…117,122,124,125) However, the difference spectrum of the reduced CO-treated microsomal fraction showed the peak at 420 nm instead of the 450 nm peak typical for CYPs, most likely due to the interference by giant hemoglobin characteristic to the earthworms. 114,122,123,129) The presence of CYP in the microsomal fraction was spectrophotometrically supported after removal of the hemoglobin through successive chromatographic purifications. 114,123,124) The estimated CYP content in E. fetida, 114) L. terrestris 123,124) and Dendrobaena veneta 130) was 0.008-0.16 nmol/mg protein, depending on the purification level and the experimental conditions in the enzyme kinetic assays.…”

1H NMR-based metabolomic observation of a two-phased toxic mode of action in Eisenia fetida after sub-lethal phenanthrene exposure

“…Similar concentration-dependent behaviour was also observed in E. fetida and other earthworm species exposed to PAHs where the concentration of cytochrome (Cyt) P450 was being monitored. [52][53][54] Exposure to xenobiotics results in an increased expression of Cyt P450 enzymes, allowing the monitoring of total Cyt P450 content to be a biomarker for exposure of xenobiotics to organisms. [22,53,55] PAHs have generally been shown to induce the Cyt P4501a isoenzyme in many species.…”

“…[52][53][54] Exposure to xenobiotics results in an increased expression of Cyt P450 enzymes, allowing the monitoring of total Cyt P450 content to be a biomarker for exposure of xenobiotics to organisms. [22,53,55] PAHs have generally been shown to induce the Cyt P4501a isoenzyme in many species. [22,[55][56][57] Zhang et al [53] investigated the concentration-dependent behaviour of E. fetida to pyrene (Py) and benzo[a]pyrene (BaP) by monitoring the total Cyt P450 content.…”

“…[22,53,55] PAHs have generally been shown to induce the Cyt P4501a isoenzyme in many species. [22,[55][56][57] Zhang et al [53] investigated the concentration-dependent behaviour of E. fetida to pyrene (Py) and benzo[a]pyrene (BaP) by monitoring the total Cyt P450 content. Total Cyt P450 content did not display any consistent correlation with PAH exposure and it was concluded that this observation was made because at certain concentrations, PAHs act as inducers of specific Cyt P450 isoenzymes while being an inhibitor for others.…”