CYP enzymes have an important role
CYP450 Cell Cycle Compound Library synthesis have an important role in microsomal oxidase function across a broad range of taxa (Pakharukova et al., 2012). They are involved in metabolism and detoxification of both internally generated compounds and xenobiotics as the phase I metabolizing enzymes and play an important role in maintaining internal homeostasis (Anzenbacher, 2001). In some cases, however, the metabolites of CYP action on xenobiotics such as benzo(a)pyrene are also toxic or carcinogenic (Minacapelli et al., 2013). In the CYP super family, CYP1A2, CYP2E1 and CYP3A4 have significant roles in drug metabolism in humans and other mammals (Snyder, 2000, Huang and Yang, 2001). They are the three most studied sub-enzymes in the P450 super family, accounting for approximately 13%, 7% and 50% respectively, of the total CYP450 protein content in human liver and are found in similar abundance in many other mammals (Schumacher and Jose, 2012). Because earthworms are able to adapt to a wide range of polluted soil environments, live in direct contact with, and ingest the various pollutants, it can be hypothesized that their body tissues require a high detoxification capacity compared with many other organisms and that these three CYP enzymes would be involved in such a detoxification processes.
The expression of CYP1A2, CYP2E1 and CYP3A4 proteins has been reported for many animal species (Czerwinski et al., 2015). However, very few reports are available on CYP enzymes in earthworms. Song et al. (2015). reported using CYP3A4 enzymatic activity as the biomarker for exposure to pyrethyroids, but without quantification of CYP protein expression. Saint-Denis et al. (1999) found that methoxyresorufin-O-deethylase (CYP1A2) in the earthworm E. fetida exposed to benzo(a)pyrene was induced at low concentrations (0.05 and 1 mg kg−1 soil) but inhibited at high concentrations (100 and 1000 mg kg−1 soil). At the time of writing we are unaware of any studies reporting specifically on the expression of 2E1 in earthworms. Current knowledge of CYP450 enzymatic activity in earthworms is largely derived indirectly from detoxification studies with specific compounds likely to accumulate chemical residues in the environment (Song et al., 2015, Berghout et al., 1991, Achazi et al., 1997, Li et al., 2007). Given the increasing human impact on the environment, the importance of CYP450 enzymes for detoxification and the role of earthworms in maintaining soil health, it is important to have more specific information about CYP450 protein expression and regulation in earthworms.
In this study we investigated three earthworm species commonly found in the Shanghai area; the epigeic Amynthas carnosus, the endogeic Metaphire guillelmi and the widely studied composting earthworm Eisenia fetida. The expression of CYP1A2, CYP2E1 and CYP3A4 proteins was investigated at different developmental stages and in different body locations (anterior and posterior body regions as well as relative quantification between earthworm body wall, reproductive, and intestinal tissues), in each of the 3 species studied.
Materials and methods To obtain earthworm specimens, we identified 5 sites in Shanghai which were previously farmed (Soil physical and chemical properties: pH:7.5, EC 129.5 μS cm−1, organic matter: 32.1 g kg−1, total P: 0.958 g kg−1, total N: 1.85 g kg−1, total K: 2.53 g kg−1), but where farming ceased more than five years ago, and chose the two most frequently encountered species: A. carnosus and M. guillelmi as the test species. E. fetida, is the most widely used species for commercial breeding and ecotoxicity testing and so this species was also selected for inclusion in this study and purchased from a local commercial supplier. The specimens collected included adults, sub-adults and juveniles. The worms were depurated by placing on filter paper soaked with isotonic salt solution at 22 ± 1 °C for 2 d prior to the experiments (Wang et al., 2010). All subsequent operations were conducted at temperatures below 4 °C.