2.1 Drugs and regents
Trichlorfon (Jiangshan pesticide and chemical Co., Ltd, Nantong, China), CAS name dimethyl 2,2,2-trichloro-1-hydroxyethylphosphonate (C4H8Cl3O4P), was purchased from the local pesticide market, containing 90% active ingredient. Different dilutions were prepared by adding distilled water. DDVP standard solution was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd at a concentration of 1.00 mg/ml. All other chemical reagents in the experiments were of analytical grade.
2.2 Experimental Design
Goldfish, Carassius auratus (weight 20.65 ± 2.18 g [mean ± SE], length 10.82 ± 0.41 cm) was purchase from local market in Nanjing. The purchased fish were fasted for three days to adapt to the new environment. They were fed with commercial food every day, and the food residues and feces were filtered out of the water to maintain water quality. Goldfish were domesticated for two weeks with the dechlorination of tap water from Nanjing city, China, and refreshed every 24 h.
After domestication, a preliminary experiment was conducted before the formal test to determine the lethal concentration (LC50) of trichlorfon exposure within 96 h. In brief, goldfish were exposed to a series of two-fold diluted trichlorfon concentrations at 3.6, 7.2, 14.4, 28.8, 57.6 and 115.2 mg L-1. The water was completely replaced every 24 h, at the meanwhile, the trichlorfon concentration remained constant during exposure period. And fish exposed at 30.5 mg /L had a 50% mortality rate at 96 h, hence, LC50/10 and LC50/5 mg L-1 was set as low-dose (LD)and high-dose (HD) for the final experience concentration.
The goldfish were randomly divided into three groups: control (CON), LD and HD, with twelve fish in each group. The experimental conditions and methods were strictly carried out according to the preliminary experiment. 50 ml of water was collected at different time points 0, 3, 6, 12 and 24 h, and stored at -80°C for backup, with three replicates at each time point. After 96 h exposure, soft tissues samples of the liver, kidney and brain were collected and immediately frozen in liquid nitrogen and then stored at −80 °C for further analysis. All animal studies strictly abide by the rules of the animal ethics committee of Nanjing University of Science and Technology.
2.3 Biochemical Assay
For biochemical assay, tail vein blood was collected and centrifuged at 3000 rpm for 10 min to obtain serum samples. Serum levels of blood urea nitrogen (BUN), methane dicarboxylic aldehyde (MDA) alanine aminotransferase (ALT), Superoxide Dismutase (SOD), Aspartate aminotransferase (AST), creatinine (CRE), and Superoxide Dismutase (SOD) were measured using commercially available kits (Nanjing Jiancheng Biotech Inc., China) according to the manufacturers’ instructions.
2.4 Histopathological and Eosin (H&E) Staining
For histopathological examination (Xu et al., 2017), the tissues of goldfish were cleaned with normal saline, fixed in 10% formaldehyde solution for 24 h and embedded in paraffin. Paraffin sections, 5 μm thick, were stained with hematoxylin and eosin (H&E), and observed under light microscopy (NIKON Eclipse Ci), photographed with the imaging system (NIKON digital sight DS-FI2).
2.5 Metabolite extraction from tissues
The tissue samples of the goldfish were washed by physiological saline to remove blood and tissue fluid, and then dried and weighed. Weighed tissue specimens were immersed in cold solvent (methanol : water 4:1, v/v), ground and homogenized on ice by a tissue homogenizer, and vortexed and mixed. Cold Chloroform and distilled water was added, and the mixture was mixed by vortexing for approximate 30 s (Tang et al., 2017). After homogenization, the mixture was centrifuging for 10 min at 12000 rpm and divided into two layers. The polar upper phase was transferred to a new micro-centrifugal tube; the organic solvent accounting for about half of the total volume was volatilized completely under the condition of nitrogen, the remaining liquid was placed in the ultra-low temperature refrigerator (-80℃), and then freeze-dried. The lyophilized samples were dissolved in 550 μl 99.8% D2O phosphate buffer (pH 7.0) containing Na2HPO4 and NaH2PO4, 0.05% (w/v) sodium 3-(trimethylsilyl) propionate-2,2,3,3-d4 (TSP)). After vortex and centrifugation, the supernatant was pipetted into standard NMR tube (5 mm) for 1H NMR tests.
2.6 1H NMR spectroscopy
1H NMR spectra were detected at 298 K using a Bruker Avance III 500 MHz NMR spectrometer (Bruker GmbH, Karlsruhe, Germany). D2O was used as a field frequency lock-solvent and TSP was used as chemical shift reference (1H, 0.00 ppm). Water suppression sequence (Carr-Purcell-Meiboom-Gill, CPMG), used for attenuating broad signals from macromolecules, was applied to obtain 1H NMR spectrum. The free induction decays (FIDs) were collected into 32K data points with a spectral width of 10,000 Hz and 64 scans.
2.7 1H NMR Data Processing and Analysis
Topspin 2.1 (Bruker GmbH, Karlsruhe, Germany) and MestReNova 8.0.1, (Mestrelab Research SL, Santiago de Compostela, Spain) software were used for phase correction and baseline adjustment of 1H NMR spectrum, and then further processing with R-package (http://cran.r-project.org/) (Ihaka and Gentleman, 1996). Each spectrum was binned using an adaptive, intelligent algorithm (De et al., 2008) between 0.65 and 9.4 ppm after removal of the residual water signals. spectrum was normalized using the total spectral area normalization so as to facilitate comparison between the samples (Craig et al., 2006).
A supervised orthogonal partial least-squares discriminant analysis (OPLS-DA) (Gu et al., 2011) were performed for multivariate statistical analysis, which can identify different groups and screen for effective discriminants. The validity of the OPLS-DA model was evaluated through permutation test to determine the level of significance of group separation, a model with p-values< 0.05 was regarded as significant. The scores plot showed the classification of groups, and the corresponding loadings plot showed discriminatory potential of some of the metabolites responsible for the separation of the two groups, a warm-colored signal indicated a more significant contribution to the class separation than a cold-colored signal (Lv et al., 2018). The fold change values were calculated with an associated P-values adjusted for multiple comparisons using the Benjamini & Hochberg method (Benjamini and Hochberg, 1995) with the scripts written in R language, as visualized in colored tables 1,2 and 3. The statistical significance of differences in metabolites between groups was evaluated by Student's t-test or Mann-Whitney test. Data were expressed as mean ± SD and P < 0.05 was considered statistically significant.
2.8 HPLC Analysis of water sample
Chromatographic analysis was performed on Shimadzu LC-20AT high performance liquid chromatography system with Inertsustain® C18 reversed phase column (4.6×250 mm,5 μm, GL Science, Tokyo, Japan) and a detection wavelength of 206 nm. The column temperature was maintained at 30 ℃, and the mobile phase consisted of solvent A (0.1% formic acid water) and solvent B (acetonitrile); the elution procedure used 40% acetonitrile water isocratic elution with a duration of 15 min. Flow rate was set at 1 ml/min with an injection volume of 20 µl.
2.9 HPLC-MS/MS data acquisition and analysis
Chromatographic analysis was performed on a SCIEX Exion LCTM ultra-high performance liquid chromatography (UHPLC) System with a Phenomenex Kinetex® Biphenyl C18 column (2. 6 μm, 100 x 2.1 mm). A gradient elution using 0.1% formic in water as solvent A and acetonitrile as solvent B was performed with a flow rate of 0.3 mL/min over 25 minutes. The gradient elution process was shown in Table 1. The column temperature was set at 40 ℃ and the inject volume was 2 μL.
Table 1 Summary of LC gradient used to analyze the samples
Time (minutes)
|
Composition of Gradient (B%)
|
0
|
5
|
4
|
24
|
14
|
52
|
16
|
70
|
18
|
95
|
22
|
10
|
22.1
|
5
|
25
|
5
|
A TripleTOF® 5600+ system with a DuoSpray Source (electrospray ionization) was used for data acquisition in positive mode over a mass range of 100 – 1250 m/z. Automated calibration was performed using an external calibrant delivery system (CDS) which infuses calibration solution prior to sample introduction. A TOF MS survey scan experiment (100 ms) incorporating IDA set to monitor ten candidate ions (total cycle time of 350 ms) was performed with a collision energy of 35 eV and a spread of ±15 eV. The optimal conditions were set as follows: Ion source gas 1 (GS1), 55 psi; Ion source gas 2 (GS2), 55 psi; curtain gas (CUR), 35 psi; spray voltage, 5.5 kV (positive ESI mode); source heater temperature, 550 °C. Data were processed using Analyst TF 1.8.1 software. For features of interest, the MS and MS/MS data were further evaluated using Formula Finder and the structural elucidation tool within SCIEX OS Software.