Chemicals and reagents.
Perfluorooctanoic acid (PFOA, 99.2% pure), purchased from Fluka Sigma-Aldrich, USA, was dissolved in deionized water. SOD, MDA, GSH-PX biochemical parameter kits were purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Antibodies against FAS, FASL (mouse monoclonal), Bcl-2, Bax, and Caspase-3 were purchased from Boster Biological Technology (Pleasanton, CA, USA), and a ready-to-use SABC immunohistochemical kit was sourced from Boster Biological Technology Co Ltd (Wuhan, China).
Treatment of animals.
8-week-old female and male Kunming mice were purchased from Sibeifu Animals Biotech Co. Ltd. (Beijing, China), license number SCXK(Beijing) 2016-0002. They were given free access to mouse chow and water, with a 12 h light cycle from 7:00–19:00. After a week adaptation to their new environment, females in estrous were mated with males 1:1 overnight. And the next morning, if a vaginal plug was detected, that day was designated as gestational day 0 (GD 0). Sixty pregnant mice at GD 0 were randomly divided into six groups (n = 10). Group A as the control (PFOA 0 mg/kg bw), and the rest groups were given PFOA at various concentrations by oral gavage on GD 1–7. Group B (1 mg/kg bw), group C (5 mg/kg bw), group D (10 mg/kg bw), group E (20 mg/kg bw) and group F (40 mg/kg bw).
All mice were weighed on GD 9. Pregnant mice were sacrificed by cervical dislocation after collecting blood on GD9 (48 h after last treatment). Liver and uterus were removed and the absolute and relative weights measured. The left horn of the uterus and part of the liver were promptly fixed in Bouin's Fluid. 0.2 g liver tissue was ground into a 10% homogenate in 0.9% saline at 4℃. The homogenate was centrifuged at 3000 rpm for 15 min, and the supernatant was stored at -80℃. The experiment was conducted in the animal house of Hebei Agricultural University, and all experimental protocols were approved by the Animal Protection Committee of Hebei Agricultural University before the study began.
The liver and uterus were collected discarding adherent fat and fascia, rinsed in saline, then dried with filter paper. Absolute and relative liver and uterus weights were measured and the organ index calculated as follows; Organ index (%) = organ weight (g) / body weight (g) × 100%
Liver and uterine morphology.
Uterus and liver tissues were fixed in Bouin's solution for 48 h, dehydrated in graded ethanol, transparented in xylene, and embedded in paraphine. Then 5 µm thick serial sections were prepared. The sections were stained with hematoxylin-eosin (HE) and the morphology and pathological changes in the uterine and liver sections were observed under a microscope.
Detection of liver oxidation index.
MDA, SOD, and GSH-PX were detected via the thiobarbituric acid method (TBA), xanthine oxidase assay, and the dithiobis nitrobenzoic acid assay, respectively, using kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Measurements were conducted in accordance with the manufacturer’s instructions. Protein content of the liver homogenate was measured by spectrophotometer using the Coomassie brilliant blue G-250 method.
Immunohistochemical detection of FAS、FASL、Bax、Bcl-2、and Caspase-3 protein expression in the uterus.
The 5 µm uterus sections were mounted on polylysine treated glass slides, placed in an oven, and baked at 60℃ for 60 min to promote adherence. Sections were dewaxed in xylene for 10 min twice, then hydrated. 3% H2O2 was added for 8 min at room temperature to inactivate endogenous enzymes. the sections were rinsed three times in double distilled water before washing in 0.01M citrate buffer (pH6.0) for antigen retrieval. 5% BSA blocking buffer was added dropwise and the slides were incubated at room temperature for 20 min, then incubated with 1:100 dilution of rabbit anti rat antibody at 4℃ overnight. A negative control for each group, 0.01 mol/L PBS solution (pH7.4) without any antibody was used and processed in the same manner. The following day, all slides were removed the refrigerator, left to come up to room temperature, then washed in PBS for 3 x 2 min. Biotinylated goat anti-rabbit IgG was added to the slide and it was incubated at 37℃ for 20 min, washed in PBS for 3 x 2 min. SABC solution was then added for 20 min at 37℃ followed by washing in PBS for 4 x 5 min. Sections were visualized using a DAB kit, dehydrated by the conventional method, mounted by neutral balsam, and observed under a microscope.
Brown particles in the cell membrane or cytoplasm were regarded as positive in each slice, 10 fields (magnification 40x for the objective lens) were selected randomly, the number of positive cells in each field of view was analyzed in Image J software.
Polylysine-treated slides were hydrated in gradient alcohol and incubated with 20 µg/ml Proteinase K at 37 °C for 20 min, then washed twice with PBS. The TUNEL assay was conducted in accordance with the manufacturer’s instructions. The images were collected using a microscope, the positive cells were pooled from 5 slices in each group and 8 fields in each section. Each experiment consisted of 10 slices, two negative control sections, and one positive control section.
Statistical analyses were performed using one-way analysis of variance (ANOVA) using SPSS 19.0. Data were presented as means and standard errors (Mean ± SD). P < 0.05 shows the significant difference.