Methods Experimental animal Adult earthworms E. fetida (Savigny, 1826) were collected from Vermiculture Research Station, DS College (Dr BRA University), Aligarh, India, and were assimilated in an experimental chamber without light, at low temperature (approximately 24°C), and kept in earthworm beddings. The worms were acclimated for 2 weeks before cell collection following Brousseau et al.[27] with regular feeding. Extrusion of coelomocytes Earthworm coelomocytes were collected

using a non-invasive method following [28–30]. Briefly, each worm was rinsed in cold water and placed on a paper towel. One fourth of the posterior part was massaged to expel the content of the lower gut. Then, each worm was placed AMN-107 for 3 min in a 15-ml polypropylene tube containing 30 ml of cold extrusion medium [Nacl C646 clinical trial (71.2 mM), EDTA

P505-15 disodium salt (6.7 mM), GGE (50.4 mM), ethanol (2% v/v) and a supplement of antibiotic and antimycotic agents: penicillin G sodium salt (100 U/ml), streptomycin sulphate (100 μg/ml), amphotericin B (25 mg/ml)]. Ethanol (5%) was added to the extrusion medium immediately before cell extrusion. After 3 min, the worm was removed and the volume was made up to 12 ml by adding ice-cold Ca-free Luria Broth Agar Media containing 1.5 mM NaCl, 4.8 mM KCl, 1.1 mM MgSO4 · 7H2O, 0.45 M KH2PO4, 0.3 mM Na2PO4 · H2O and 4.2 mM NaHCO3 adjusted to pH 7.3 and osmolarity adjusted to 300 mosM [27]. Finally, the cells were re-suspended in Ca-LBSS (containing 3.8 mM

CaCl2) and loaded in a culture plate with Methane monooxygenase Dulbecco’s Modified Eagle Medium (DMEM) supplement with foetal bovine serum. The selected choloragocytes were subjected to subculturing. Viability determination The cell viability was determined by both trypan blue staining and flow cytometry. In this case, 5 μl of a 1 mg/l propidium iodide solution was added to 500 μl of cell suspension and the fluorescence measured in FL3. Exposure of ZnO NPs Chloragocytes were seeded into a 96-well plate at 5 × 105 cells/ml and treated with ZnO NPs (for 3, 6, 12, 24 and 48 h) of diameters 100 and 50 nm (0.5, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/l). ZnO NPs were purchased from Sigma-Aldrich (St. Louis, MO, USA), and their morphology and size were examined by transmission electron microscopy (TEM) at The Energy Research Institute, New Delhi, India. DNA damage analysis The Comet assay was performed as described by Singh et al.[31]. Ethidium bromide-stained nuclei were examined with a fluorescent microscope (Leica Microsystems, Wetzlar, Germany). Images were analyzed with the software CASP according to the method of Collins et al.[32] (Figure 1). Figure 1 DNA damage of coelomocytes (A) in the control and (B) after exposure to 100-nm NPs (3 mg/l). Statistical analysis Results are the means of three replicates. Two-way analysis of variance (ANOVA) was performed by using the SPSS 10.5 software.