Materials
Chlorpyrifos (purity > 95 %) was obtained from Shuangma Chemical Co. Ltd. (Jiangsu, China). RPMI-1640, dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Aldrich Co. (St Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Life Technologies (Carlsbad, CA, USA). Heat-inactivated fetal bovine serum (FBS) was obtained from Chuanye Biotechnology, Ltd (Tianjin, China). Bladder cancer cell line T24, colon cancer cell line HT-29 and lymphocyte cell line Jurkat were purchased from Cell Center of Chinese Academy of Medical Sciences (Beijing, China).
Animals and treatment
Forty 6-8-week old male Wistar rats with body weight of 150–220 g were obtained from Laboratory Animal Technology Company (Beijing, China). The animals were maintained at dark/light cycle of 12 h, 22 ± 2 °C and 55 ± 10 % humidity. The rats were kept individually with free access to feed and water. Based on data from previous studies that the acute oral half-lethal doses (LD50) of chlorpyrifos were 163 mg/kg for male rat [14], we chose the doses of 1/125, 1/50, and 1/20 LD50 of chlorpyrifos as low dose (1.30 mg/kg body weight), medium dose (3.26 mg/kg body weight), and high dose (8.15 mg/kg) for the pesticide treatment groups in this study. The low dose of chlorpyrifos (1.30 mg/kg body weight) is close to the level of possible occupational exposure [15]. After 7 days of acclimatization, the animals were divided into three chlorpyrifos treatment groups (low dose: 1.30 mg/kg, medium dose: 3.26 mg/kg, and high dose: 8.15 mg/kg) and one vehicle control group with 10 rats in each group. The pesticide was dissolved in corn oil (0.5 ml/kg body weight) before being orally administered daily. The control group received an equivalent volume of corn oil daily. All of the animals were treated for consecutive 90 days to mimic the chronic exposure to pesticides in humans. At the end of the experiment, the samples of urine, feces, and blood were collected once from each of the animals (Fig. 1).
All animal procedures were performed in accordance with current China legislation and approved by the Animal and Medical Ethics Committee, which is affiliated to the Institute of Zoology, Chinese Academy of Sciences.
Preparation of body fluids samples
Ten milliliters (10 mL) of the collected 24-hour urine samples were processed with protein precipitation; the supernatant was lyophilized and suspended in 1 mL of phosphate buffer solution (PBS). The solution was sterilized by filtration with 0.22-µm filters, and then diluted ten times by complete cell culture medium to make a stock solution, which was further diluted to treat cells to determine the dose-response effect of urine extract on cell viability.
Feces collected from animals were suspended in 0.1 M cold PBS (1:5, w/v) and then incubated with PBS buffer overnight with stirring. The solution was centrifuged at 3, 000 × g for 30 min at 4 °C and then the supernatant was centrifuged at 15, 000 × g for 30 min at 4 °C. The supernatant was sterilized by filtration with 0.22 μm filters and diluted ten times with cell culture medium, which was further diluted to treat cells to determine the dose-response effect of feces extract on cell viability.
Serum collected from the animals was heated at 56 °C for 30 min to inactivate complement proteins and potential mycoplasma, and then the serum was diluted ten times in complete cell culture medium containing 10 % FBS to treat cells to determine the dose-response effect of the serum on cell viability.
Cell culture and treatment
The cells were cultured with DMEM (for T24 and HT-29 cell lines) or RPMI-1640 (for Jurkat cell line) plus 10 % FBS complemented with 100 U/mL penicillin and 100 µg/mL streptomycin. The cells were maintained at 37 °C in a humidified atmosphere containing 5 % CO2.
MTT staining
MTT staining was carried out according to the reference [16]. Briefly, T24 and HT-29 cells were seeded in 96-well plates with 1 × 104 cells/well. After 24 h, the urine stock solutions were added to T24 cells with different dilutions. Feces extracts were added to HT-29 cells with different dilutions. The corresponding urine and feces extracts from the vehicle-treated rats were also used to treat the cells, which served as vehicle controls. Culture medium was used as the negative control. After cells were treated for different time periods, 20 µl of MTT (5 mg/ml) was added to each well. Four hours later, the cell culture medium was discarded and 150 µl of DMSO was added to each well. OD570 values were measured by using Bio-Rad Benchmark microplate reader. Each treatment was performed in triplicate and each experiment was repeated at least three times.
To eliminate possible non-specific interference from the samples, we determined the absorbance of the culture medium sparked with the prepared urine stock solution and feces extract samples under the cell-free condition (all other operations are the same as the MTT assay except for in the absence of cells). The result showed that no difference of the OD570 values was found between the culture medium group (blank) and the spiked medium group.
Trypan blue staining
Jurkat cells were seeded in 12-well plate with 1 × 105 cells/well. After 12 h, the cells were treated with 300 µl of the 10-fold diluted serum from the rats treated with 1.30 mg/kg, 3.26 mg/kg, or 8.15 mg/kg chlorpyrifos, respectively. After cultured for different time periods, cells were stained by trypan blue and the live cell number in each well was counted. Serum samples from the corn oil-treated rats were used as the vehicle control and 10 % FBS in regular cell culture medium was used as the negative control. Each treatment was performed in triplicate and each experiment was repeated at least three times.
Determination of chlorpyrifos and TCP levels in the serum and urine
The assay of chlorpyrifos and its metabolite 3, 4, 5-trichloropyrindinol (TCP) was carried out according to the method of the previous study [17]. Briefly, 200 µl of the serum or urine were added to 600 µl of acetic ether and vortexed for 1 min. The solution was centrifuged at 1, 000 × g for 20 min and then the supernatants were collected, dried under nitrogen and then dissolved in 150 µl of methanol. The solution was centrifuged at 15, 000 × g for 20 min and then the supernatants were transferred into the vials. The samples were analyzed by the Agilent 1100 series HPLC system (Agilent Co., USA).
Statistics
All data are presented as mean ± Standard Error of the Mean (SEM). Statistical differences between treated and control groups were evaluated by GraphPad Prism 8.0 (SanDiego, CA, USA). The one-way Analysis of Variance (ANOVA) test followed by the post hoc Dunnett’s test was performed to access significant differences among all the groups. The difference was considered significant if the p-value was less than 0.05.