The body wall intestine seminal receptacle
The body wall, intestine, seminal receptacle and seminal vesicle in the adult worms were also isolated and homogenized to make microsomal fractions to investigate the relative expression of CYP1A2, CYP2E1 and CYP3A4 in these different organs. In order to get sufficient mass, about 20 individual adult worms were dissected. There were three replicates for each treatment. The clitellum of E. fetida (0.497 g ± 0.046) is located at body segments xxv–xxxiii, and the gizzard, seminal receptacles and seminal vesicles are located posterior to the clitellum. The clitella of A. carnosus (1.563 g ± 0.035) and M. guillelmi (1.604 g ± 0.022) are located at segments xiv–xvi, and the gizzard, seminal receptacles, seminal vesicles, testes, prostate and PF 04418948 are located posterior to the clitellum (Chen, 1959). There are two pairs of seminal receptacles in E. fetida located at segments ix–x or x-xi and four pairs of seminal vesicles located at ix–xii. There are three or four pairs of receptacles in A. carnosus located at segments v/vi–viii/ix, three pairs of seminal receptacles in M. guillelmi located at segments vi/vii–viii/ix, and three or four pairs of seminal vesicles in A. carnosus and M. guillelmi located at segments v/vi–viii/ix (Chen, 1956, Chen, 1959).
Microsomal fractions of earthworm tissue were prepared as per a previous study with modifications (Zhang et al., 2006). Briefly, the earthworm tissue samples transferred into cold 0.15 mol l−1 KCl solution and were cut into shreds. The remaining solid tissue homogenized for 30 min at 8000 r.min−1. The supernatant centrifuged at 14,700 r.min−1 for 30 min and then the supernatant ultracentrifuged at 46,500 r.min−1 for 90 min. After dissolving precipitation, the mixed liquor was centrifugal at 38,000 r.min−1 for 45 min at 4 °C. Finally, the supernatant was collected and stored at −80 °C. The protein levels of CYP1A2, CYP2E1 and CYP3A4 in the microsomes were measured by Western blot. Rabbit polyclonal antibody anti-human, rat, mouse CYP 1A2, CYP2E1 and CYP3A4; mouse monoclonal anti-β-actin antibody; horseradish peroxidase labelled goat anti-rabbit IgG, goat anti-mouse IgG were used. The relative fluorescence intensity (RFI) of the three CYPs compared to β-actin were used to assess relative protein expression level. Statistical analyses were performed with SPSS Software Version 17.0 (SPSS Inc, Chicago, USA) and the data were expressed as the mean ± SD.
Discussion The results indicated that the main detoxification processes for xenobiotics in M. guillelmi are likely to occur in the posterior portion of the body, and that the anterior parts would have less detoxification functionality. Conversely, a majority of detoxification capacity in E. fetida and A. carnosus appears to be located in the anterior portion of the body. The anterior part includes the body wall and a range of interior organs and tissue types, whereas the posterior section comprises mainly the body wall, with muscle, intestine and circulatory vessel tissues. It is possible that the differential CYP expression could relate either to differences in internal tissue function, or to differing external challenges; such as where one species is litter dwelling and where another species is deep burrowing. Possible xenobiotic challenges may come from pollutants in litter or soil or from soil chemical reactions, for example changes to soil chemistry under reducing conditions when soil has lower oxygen status, either at greater depth or when wetter. E. fetida and A. carnosus also differed from M. guillelmi in having more CYP3A4 in the intestine than the latter species (Fig. 3), and this difference in CYP protein expression presumably also relates to habitat or dietary preference. It is known that M. guillelmi, lives in deeper soil layers, whereas the other two earthworm species live on the surface or in the upper layer of the soil (Chen, 1956) so these behavioural differences may be linked to the CYP expression differences noted above.