| Due to high molluscicidal efficacy and low toxicity to humans and animals, niclosamide has been recommended by the World Health Organization(WHO) as the only chemical used for large-scale snail control and it is currently the most widely used molluscicide in China, which has play a vital role in schistosomiasis control. However, the agent exhibits a strong acute toxicity to aquatic animals including fish at a dose for snail control. During mollusciciding, niclosamide may enter into rivers, lakes, streams, which may lead to death of fish, shrimp and crab, thereby resulting in an economic loss and affecting breeding industry development. Lowering the toxicity of niclosamide to aquatic animals is therefore an important issue to be solved in schistosomiasis control program. To solve the problem of niclosamide toxicity to aquatic animals, the mechanisms and target of niclosamide toxicity to aquatic animals should be clarified first, which may provide experimental evidence for developing novel molluscicides with low toxicity to aquatic animals. The use of living fish to investigate the mechanisms underlying niclosamide toxicity is time-consuming and costly, and the drug degradation by fish themselves may result in a poor repeatability of the experiment results. The toxicologic testing using fish cell lines instead of living fish may allow direct drug-cell interaction, and have a short period of time and low cost. Fish cell lines have shown a high homogeneity, and genetic similarity, which may avoid the effect of various systems, resulting in a high repeatability. In addition, fish cells can be used for simultaneous detection of multiple drugs at multiple concentrations, thereby acquiring exact toxicological mechanisms. In the current study, zebrafish caudal fin AB.9 and African green monkey kidney Vero cell lines were used to investigate the cytotoxicity of niclosamide, so as to provide a novel approach for exploring the mechanism of niclosamide toxicity.1 AB.9 and Vero cell culture and stroage1.1 Cell cultureAB.9 cells were incubated at 28°C for 24 h, and prepared into cell suspension by gently pipetting. A half of the cell suspension was transferred to a new flask, and incubated at 28°C for a further 24 h. Cell growing to 80% confluence were used for passage, and cells at a log-growth phase were used for toxicological testing. Microscopy revealed that AB.9 cells were adherent to flask wall following 4 to 6 h of culture, and the cells spread completed, appearing an irregular shape.Vero cells were incubated at 37°C containing 5% CO2 for 24 h, and prepared into cell suspension by gently pipetting. A half of the cell suspension was transferred to a new flask, and incubated at 37°C containing 5% CO2. Cell growing to 80% confluence were used for passage, and cells at a log-growth phase were used for toxicological testing. Microscopy revealed that Vero cells were adherent to flask wall following 4 to 6 h of culture, and the cells spread completed, appearing an irregular shape.Both AB.9 and Vero cells were not polluted, and revived cryopreserved cell lines grew well, which can be stored and passaged, suggesting successful conservation of the cell lines.1.2 Plotting of cell growth curveThe AB.9 and Vero cells at 3 passages were used. When growing to 80% confluence, cells were prepared into cell suspensions, and seeded onto 96-well plates. The optical was determined using MTT assay at various time points, and cells were counted. The growth curve for AB.9 and Vero cells was plotted, and the optimum inoculation condition for these two cell lines was estimated.After AB.9 cells were inoculated into 96-well plates at a density of 3 × 104 cells per well, the cells were dormant after 0 to 24 h, at log-growth phase after 24 to 48 h, and then proceeded to the plateau phase. After Vero cells were inoculated into 96-well plates at a density of 3 × 104 cells per well, the cells were dormant after 0 to 24 h, at log-growth phase after 24 to 48 h, and then proceeded to the plateau phase. The highest cell viability was observed at log growth phase. Therefore, the optimum condition for use of AB.9 cells to detect niclosamide toxicity was inoculation of AB.9 cells in 96-well plates at a density of(3 to 6) × 104 cells per well, or to 6-well plates at a density of 5× 105 cells per well for 24 to 48 h, while the optimum condition for use of Vero cells to detect niclosamide toxicity was inoculation of Vero cells in 96-well plates at a density of(3 to 6) × 103 cells per well, or to 6-well plates at a density of 5× 104 cells per well for 24 to 48 h2 Toxicity of niclosamide to AB.9 and Vero cell2.1 Toxicity of niclosamide to AB.9 cellsAB.9 cell suspensions were prepared, and were seeded onto 6-well plates at a live cell density of 5× 105 cells per well, followed by incubation at 28°C for 24 h. The original culture medium was removed, and cells were washed three times with PBS. The media containing niclosamide solutions at concentrations of 6, 2, 0.667, 0.222, 0.07, 0.025, 0.008 mg/L were formulated, while serum-free RMPI 1640 medium containing 1% DMSO served as a solvent control, serum-free RMPI 1640 medium served as a negative control, and fresh 10% fetal bovine serum served as a blank control. The effect of niclosamide on AB.9 cell morphology was evaluated under a microscope, and the dose-response relationship between niclosamide and cytotoxicity to AB.9 cells was evaluated.Following 24 h incubation in the media containing niclosamide, the intracellular space of AB.9 cells gradually enlarged with the increase of niclosamide concentration, and the cell morphology became irregular and shrunk to round. Following treatment with niclosamide at a concentration of > 2 mg/L, all cells almost became round, and most cells appeared a vacuole-like shape, with cell debris seen. Niclosamide was toxic to AB.9 cells, and the cytotoxicity increased with drug concentration, with a maximum tolerance concentration of 2 mg/L.2.2 Toxicity of niclosamide to Vero cellsVero cell suspensions were prepared, and were seeded onto 6-well plates at a live cell density of 5× 104 cells per well, followed by incubation at 37°C containing 5% CO2 for 24 h. The original culture medium was removed, and cells were washed three times with PBS. The media containing niclosamide solutions at concentrations of 20, 5, 1.25, 0.313, 0.078, 0.02, and 0.01 mg/L were formulated, while serum-free RMPI 1640 medium containing 1% DMSO served as a solvent control, serum-free RMPI 1640 medium served as a negative control, and fresh 10% fetal bovine serum served as a blank control. The effect of niclosamide on Vero cell morphology was evaluated under a microscope, and the dose-response relationship between niclosamide and cytotoxicity to Vero cells was evaluated.Following 24 h incubation in the media containing niclosamide, the morphology of Vero cells did not alter with the change in the concentration of niclosamide in relative to negative controls, demonstrating no apparent cytotoxicity of niclosamide to Vero cells.2.3 Detection of cytotoxicity of niclosamide to AB.9 cells using MTT assayAB.9 cells were seeded onto two 96-well plates at a live cell density of 5×104 cells per well. After adherence to plate wall, the cells were given treatment with niclosamide solutions at concentrations of 6, 2, 0.667, 0.222, 0.07, 0.025, 0.008 mg/L, respectively, while serum-free RMPI 1640 medium containing 1% DMSO served as a solvent control, serum-free RMPI 1640 medium served as a negative control, and fresh 10% fetal bovine serum served as a blank control. After culture for 24 and 48 h, the absorbance value was measured at 490 nm. The time- and dose-specific effect of niclosamide on the survival of AB.9 cells was evaluated, and dose-response relationship was estimated.The survival of AB.9 cells treated with niclosamide at concentrations of 0.07 mg/L or less for 24 and 48 h was greater than that observed in blank controls, and the cell proliferation increased with the drug concentration. If AB.9 cells were treated with niclosamide at concentrations of > 0.07 mg/L, a reduced survival was seen in treated cells as compared to blank controls. The 24 and 48 h niclosamide IC50 values for AB.9 cells were 0.485 and 0.358 mg/L(R2 = 0.953 and 0.995). The cytotoxicity of niclosamide to AB.9 cells gradually increased, from stimulation of growth to inhibition of growth, followed by cell death. Niclosamide has an acute toxicity on AB.9 cells, and the fatal threshold concentration of niclosamide for fish cells and fish was comparable. It is therefore considered that use of AB.9 cell to determine the cytotoxicity of niclosamide may serve as a novel approach for detection of drug toxicity.2.4 Detection of cytotoxicity of niclosamide to Vero cells using MTT assayVero cells were seeded onto two 96-well plates at a live cell density of 5× 103 cells per well. After adherence to plate wall, the cells were given treatment with niclosamide solutions at concentrations of 20, 5, 1.25, 0.313, 0.078, 0.02 and 0.01 mg/L, respectively, while serum-free RMPI 1640 medium containing 1% DMSO served as a solvent control, serum-free RMPI 1640 medium served as a negative control, and fresh 10% fetal bovine serum served as a blank control. After culture for 24 and 48 h, the absorbance value was measured at 490 nm. The time- and dose-specific effect of niclosamide on the survival of AB.9 cells was evaluated, and dose-response relationship was estimated.The survival of Vero cells treated with niclosamide for 24 and 48 h was not significantly different from that observed in blank controls, and no obvious alteration was detected in the survival of Vero cells treated with 20 mg/L niclosamide. In addition, niclosamide showed no remarkable effect on the growth of Vero cells.2.5 Detection of cytotoxicity of niclosamide to AB.9 cells using SRB assayAB.9 cells were seeded onto two 96-well plates at a live cell density of 5× 104 cells per well. After adherence to plate wall, the cells were given treatment with niclosamide solutions at concentrations of 6, 2, 0.667, 0.222, 0.07, 0.025, 0.008 mg/L, respectively, while serum-free RMPI 1640 medium containing 1% DMSO served as a solvent control, serum-free RMPI 1640 medium served as a negative control, and fresh 10% fetal bovine serum served as a blank control. After culture for 24 and 48 h, the absorbance value was measured at 540 nm. The time- and dose-specific effect of niclosamide on the survival of AB.9 cells was evaluated, and dose-response relationship was estimated.The survival of AB.9 cells treated with niclosamide at concentrations of 0.07 mg/L or less for 24 and 48 h was greater than that observed in blank controls, and the cell proliferation increased with the drug concentration. If AB.9 cells were treated with niclosamide at concentrations of > 0.07 mg/L, a reduced survival was seen in treated cells as compared to blank controls. The 24 and 48 h niclosamide IC50 values for AB.9 cells were 0.298 and 0.318 mg/L(R2 = 0.985 and 0.968). The cytotoxicity of niclosamide to AB.9 cells gradually increased, from stimulation of growth to inhibition of growth, followed by cell death. Niclosamide has an acute toxicity on AB.9 cells, and the fatal threshold concentration of niclosamide for fish cells and fish was comparable. It is therefore considered that use of AB.9 cell to determine the cytotoxicity of niclosamide may serve as a novel approach for detection of drug toxicity.2.6 Detection of cytotoxicity of niclosamide to Vero cells using SRB assayVero cells were seeded onto two 96-well plates at a live cell density of 5× 103 cells per well. After adherence to plate wall, the cells were given treatment with niclosamide solutions at concentrations of 20, 5, 1.25, 0.313, 0.078, 0.02 and 0.01 mg/L, respectively, while serum-free RMPI 1640 medium containing 1% DMSO served as a solvent control, serum-free RMPI 1640 medium served as a negative control, and fresh 10% fetal bovine serum served as a blank control. After culture for 24 and 48 h, the absorbance value was measured at 540 nm. The time- and dose-specific effect of niclosamide on the survival of Vero cells was evaluated, and dose-response relationship was estimated.The survival of Vero cells treated with niclosamide for 24 and 48 h was not significantly different from that observed in blank controls, and no obvious alteration was detected in the survival of Vero cells treated with 20 mg/L niclosamide. In addition, niclosamide showed no remarkable effect on the growth of cells.3 Detection of AB.9 cell death of niclosamide3.1 Detection of AB.9 apoptosis using Annexin V/PIAB.9 cells were seeded onto two 6-well plates at a live cell density of 5× 105 cells per well. After adherence to plate wall, the cells were given treatment with niclosamide solutions at concentrations of 0.07, 0.2, 0,3, 0.4, 0.5 and 0.6 mg/L, respectively, while serum-free RMPI 1640 medium containing 1% DMSO served as a solvent control, serum-free RMPI 1640 medium served as a negative control, fresh 10% fetal bovine serum served as a blank control, and serum-free RMPI 160 medium containing apoptosis-inducing agents served as a positive control. After culture at 28°C for 24 h, cells were stained with Annexin V/PI, and the fluorescence for Annexin V, PI and Annexin V/PI was detected using flow cytometry. Cell apoptosis was estimated.Flow cytometry revealed the number of survival AB.9 cells treated with niclosamide at a concentration of > 0.07 mg/L for 24 h gradually decreased with the increase of drug concentration, and the percentage of necrotic cells gradually increased, while no obvious alteration was seen in the apoptotic rate of cells. The results demonstrate that niclosamide may cause necrosis of fish cells, and the target site may locate in the pathway trigger cell necrosis or proteins of respiratory chain in mitochondria.3.2 Detection of Vero apoptosis using Annexin V/PIVero cells were seeded onto two 6-well plates at a live cell density of 5× 104 cells per well. After adherence to plate wall, the cells were given treatment with niclosamide solutionsat concentrations of 5 and 20 mg/L, respectively, while serum-free RMPI 1640 medium containing 1% DMSO served as a solvent control, serum-free RMPI 1640 medium served as a negative control, fresh 10% fetal bovine serum served as a blank control, and serum-free RMPI 160 medium containing apoptosis-inducing agents served as a positive control. After culture at 28°C for 24 h, cells were stained with Annexin V/PI, and the fluorescence for Annexin V, PI and Annexin V/PI was detected using flow cytometry. Cell apoptosis was estimated.Flow cytometry revealed the necrotic rate and apoptotic rate of Vero cells treated with 5 and 20 mg/L niclosamide were not significantly different from those observed in negative controls(P > 0.05), indicating that niclosamide has no obvious effect on Vero cell death.4. Effect of niclosamide on cell proteome of AB.9AB.9 cells were treated with were treated with 0.5 and 0.07 mg/L niclosamide, while serum-free RMPI 1640 medium containing 1% DMSO served as a solvent control, serum-free RMPI 1640 medium served as a negative control, and fresh 10% fetal bovine serum served as a blank control. Following 24 h culture, cells were lysed, and centrifuged, and the supernatant was cell lysis solution with soluble protein. Approximately 500 μg soluble protein was collected, and the nucleic acids and lipids were removed to yield purified protein. The protein concentration was determined using Bradford method. The protein samples were dissolved in loading buffer, and then used for isoelectric focusing. Then, SDS-PAGE was performed, and the gels were stained with coomassie brilliant blue. Images were captured with a Gel Doc 2000 gel imager and processed using PDQuest 8.0 gel analysis software. Differentially expressed protein spots were screened, and cut. The protein was extracted from the gels, and identified using time-of-flight mass spectrometry(TOF-MS) in combination with bioinformatics.The protein spots on the electrophoresis profiles of protein isolated from AB.9 cells treated with different concentrations of niclosamide were analyzed using PDQuest8.0 electrophoresis analysis software, and each protein sample was checked with triple parallel electrophoreses. The repeatability was 77% to 100% for each protein profile, which meets the requirements for comparing the difference among samples. There were 193 ± 44,115 ± 22,102 ± 18.7,83 ± 25 protein spots detected in the blank control group, solvent control group, 0.07 mg/L niclosamide treatment group and 0.5 mg/L niclosamide treatment group, respectively, with the molecular weights of 10 to 120 k Da for most proteins, and 5 to 8 isoelectric points seen. There were 26 differentially expressed protein spots between 0.07 mg/L niclosamide treatment group and the blank control group, including 2 protein spots with up-regulated expression, 12 protein spots only detected in the blank control group and 12 protein spots only detected in the 0.07 mg/L niclosamide treatment group. There were 34 differentially expressed protein spots between 0.5 mg/L niclosamide treatment group and the blank control group, including 3 protein spots with up-regulated expression, 5 protein spots with down-regulated expression, 13 protein spots only detected in the blank control group and 13 protein spots only detected in the 0.5 mg/L niclosamide treatment group. TOF-MS identified 7 proteins, including DNA-dirceted RNA polymerase Ⅲsubunit RPC2, ch211-69g19.2 protein, 88517 protein, PREDICTED: insulin receptor substrate 2, M-phase phosphoprotein 6, novel protein, and fructose-bisphosphate aldolase C-B, which were associated with signaling transduction, cell division, cell growth, and intracellular glycolytic pathway. Further studies are required to investigate the exact functions and roles of these proteins identified. |
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