DEHP (C24H38O4, CAS: 117-81-7, purity 99.0%) was purchased from the National Institute of Standards and Technology. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), blood glucose (GLU), urea nitrogen (BUN), creatinine (CREA), total cholesterol (TC), triglycerides (TG) reagent kits were obtained from Maccura Biotechnology Co., Ltd. Total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and glutathione (GSH) contents were measured by using assay kits (Jiancheng Haihao Biotechnology Co., Ltd.). Serum transthyretin protein (TTR; cat. no. GD-S0712-A), TRH (cat. no. GD-S2054-A) and thyroid-stimulating antibody (TSAb; cat. no. GD-S1768-A) levels were measured using ELISA kits (Shanghai Guduo Biological Technology Co. Ltd.). Serum T4, T3, free triiodothyronine (FT3), FT4 and TSH levels were measured using radioimmunoassay kits (Beijing North Institute of Biotechnology Co., Ltd.). Anti-NIS polyclonal (cat. no. ab83816), anti-TSHr polyclonal (cat. no. ab202960), anti-thyroglobulin (Tg) monoclonal (cat. no. ab156008), TPO (cat. no. ab203057), anti-GAPDH (cat. no. ab8245) and anti-α tubulin (cat. no. ab7291) antibodies were obtained from Abcam. TTF-1 (D2E8 rabbit mAb cat. no. 12373) and PAX-8 (D2S2I rabbit mAb cat. no. 59019) were purchased from Cell Signaling Technology, Inc. TSH-β antibody (cat. no. MAB4507) was purchased from R&D Systems Inc. Alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (cat. no. ZB-2301), AP-conjugated rabbit anti-goat IgG (cat. no. ZB-2306) and AP-conjugated rabbit anti-mouse IgG (cat. no. ZB-2305) secondary antibody were purchased from ZSGB-BIO.

A total of 40 healthy 2-week-old male Wistar rats (70±10 g) were purchased from Vital River Laboratory Animal Technology Co. Ltd. All animals were housed under a constant temperature (23±1°C) and humidity (50–60%) and a 12-h light/dark cycle. All rats were provided with distilled water and standard AIN-93M diet ad libitum. Rats were randomly assigned to experimental groups (n=10): A control group, a 150 mg/kg/day DEHP group [~ five times the no-observed-adverse-effect level (NOAEL)], 300 mg/kg/day DEHP group (~10 times the NOAEL) and a 600 mg/kg/day DEHP group (~20 times the NOAEL). The NOAEL was obtained from a 104-week study on the chronic toxicity of DEHP in rats using a previously described method (41). Based on the investigation of DEPH properties in different experiments, a concentration of 600 mg/kg/day, which is 1/40 of the half lethal dose of DEHP for rats, was used in subsequent experiments (42). DEHP was administered to rats via gavage and control animals received peanut oil without phthalate. DEHP was intragastrically administered to rats daily for 90 consecutive days. All protocols were performed in accordance with the Regulations of the Ethical Committee for Research on Laboratory Animals, as assessed and approved by the Ministry of Health of China and the Institute of Zoology Animal and Medical Ethics Committee of Harbin Medical University. Subsequent experiments were conducted under the strict principles of Good Laboratory Practice to ensure good quality in vivo toxicology studies.

The body weight of the rats was measured weekly. On day 89, the rats were transferred to the metabolic cage, and urine samples were collected for 24 h. Rats were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (35 mg/kg). Euthanasia was performed by abdominal aortic bleeding. Blood samples (~5 ml/per rat) were collected from the abdominal aorta. Thyroid, pituitary, hypothalamus, liver, kidney and testis tissue samples were obtained and frozen in liquid nitrogen, and then were transferred to a −80°C refrigerator for storage until analysis. Serum biochemical indicators were detected using an automatic biochemical analyzer (Hitachi High-Tech Corporation) according to the manufacturer's instructions. Measurements of ALT, AST, TC and TG were performed according to the manufacturer's protocol.

The content of oxidation products and the activity of antioxidant enzymes are indicators of the level of oxidation and therefore the degree of oxidative damage (43). Antioxidant enzyme activities of SOD and CAT and antioxidant levels of MDA, GSH and T-AOC in serum were measured according to the manufacturer's protocols.

Serum T3, T4, FT3, FT4 and TSH levels were obtained using radioimmunoassay kits (Beijing North Institute Of Biotechnology Co., Ltd.) according to the instructions supplied with the Automatic Gamma Counter (Wallac Wizard-2 2470 Automatic Gamma Counter; PerkinElmer, Inc.).

Serum TTR, TRH and TSAb were measured according to the ELISA kit instructions. A BioTek 3 MFD instrument (BioTek Instruments, Inc.) was used to repeatedly measure standards solution and samples.

The urine iodine (UI) determination method was performed according to the principle of Ascerium-Cerium Catalytic Spectrophotometric Determination of Urine Iodine (WS/T 107–2006) and the analysis standard of iodine in urine (GBW09109h; National Institute of Standard Substance, Beijing, China) was based on the reference analysis provided by the National Iodine Deficiency Reference Laboratory (44,45). A TU-1901 double beam ultraviolet visible-visible spectrophotometer (Beijing Puxi General Instrument Co., Ltd.) was used to determine the iodine content in standard and diluted samples.

The thyroid, liver, kidney and testes samples from the four groups were fixed in 10% paraformaldehyde for 12 h, dehydrated and trimmed and embedded in paraffin blocks at room temperature. Multiple 4-µm thick sections were deparaffinized with xylene, stained with hematoxylin and eosin (H&E;1:5) for 5 min at 25°C and representative samples were analyzed by light microscopy (magnification, ×200). The number of follicular epithelial cells and the diameter of the follicular cavity were measured using Image-Pro Plus 6.0 image analysis software (Media Cybernetics, Inc.) to quantitatively assess histological changes in glands.

Total RNA of tissues was extracted using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) and treated with genomic DNA wiper. cDNA was synthesized from 1 g total RNA (65°C for 5 min and rapid cooling on ice) using ReverTra Ace qPCR RT kit (cat. no. FSQ-201; Toyobo Life Science). RT was performed at 37°C for 15 min and 98°C for 5 min. RT-qPCR was performed using THUNDERBIRD SYBR^® qPCR Mix with 50X ROX reference dye (cat. no. QPS-201T; Toyobo Life Science) according to the manufacturer's instructions. Expression levels were measured for NIS, PAX-8, Tg, TSHr, TTF-1, TTF-2, PDS, TPO, TSH-β, TRHr, D1, D2 and D3. The relative expression levels of target genes were calculated using 2^–ΔΔCq method (46). The primer sequences (Table I) were designed according to the cDNA sequence from GenBank. All reactions were run in triplicate. A melting curve was generated during amplification to verify the absence of primer dimers or incorrectly paired products. All primers were synthesized by Sangon Biotech, Co., Ltd.

Samples were processed to determine tissue (thyroid, liver, kidney and testis) protein concentrations as previously described (47). The protein concentrations were measured by the Bicinchoninic Acid Protein Assay kit (Beyotime Institute of Biotechnology.). Equal amounts of protein (20 µl per lane) were separated by electrophoresis on a 10% SDS polyacrylamide gel under reducing conditions and the separated proteins were transferred onto a polyvinylidene fluoride membrane. The polyvinylidene fluoride membranes were then blocked with 1% bovine serum albumin (cat. no. V900933; Sigma-Aldrich; Merck KGaA) in TRIS-buffered saline with 0.05% Tween-20 (TBST) at room temperature for 1 h, then incubated with NIS (1:1,000 dilution), TSHr (1:1,000 dilution), Tg (1:1,000 dilution), TPO (1:1,000 dilution), TTF-1 (1:1,000 dilution), PAX-8 (1:1,000 dilution), TSH-β (1:500 dilution) and GAPDH (1:2,000 dilution) overnight at 4°C. The next day, membranes were rinsed three times (10 min each) with 1% TBST and incubated with AP-conjugated goat anti-rabbit IgG secondary antibody (1:1,000 dilution), AP-conjugated rabbit anti-goat IgG (1:1,000 dilution) or AP-conjugated rabbit anti-mouse IgG (1:1,000 dilution) for 1–2 h at room temperature. After washing six times in TBST buffer, a chemiluminescence detection system (Tanon Science and Technology Co., Ltd.) was used to detect the resultant signals. The individual protein bands were quantified by using ImageJ software (v1.50; National Institutes of Health). Each western blot analysis was repeated ≥3 times.

Each experiment was performed ≥3 times. Statistical analysis was performed using SPSS version 20.0 (IBM Corp.). All data are expressed as the mean ± standard error of the mean. Differences among groups were analyzed using one-way analysis of variance (ANOVA), followed by Bonferroni's test. Graphs presenting the results were produced using GraphPad Prism 5.0 (GraphPad Software, Inc.) and P<0.05 was considered to indicate a statistically significant difference.

No rats succumbed during the treatments and rats in each group gained body mass normally over time. There was no difference in the physiological parameters of the rats in each group compared with time-matched control groups at all time-points (Fig. 1).

To validate the role of DEHP in organs, relative organ weight [(organ weight/body weight) × 10,000] were calculated (Fig. 2). Relative organ weight for thyroid, epididymis and testicular fat did not significantly differ among treatment and control groups (P<0.05). However, relative organ weights for liver, kidney were increased in DEHP treatment groups compared with the control group; relative organ weights for testis were decreased in DEHP 600 mg/kg/day treatment group compared with the control group.

To further clarify the potential effects of DEHP-induced oxidative stress, several oxidative stress-associated parameters were measured. The findings demonstrated that MDA levels were significantly increased following DEHP treatment, while the activities of T-AOC, SOD, CAT and GSH in DEHP-exposed rats were decreased compared with the control group (Fig. 3).

The effects of exposure to DEHP on biochemical indices are presented in Fig. 4. The results of routine blood and urine metabolic indicators ALT, AST, TG and UI revealed similarities among the four groups. Unexpectedly, compared with the 300 mg/kg/day dose group, there were significant differences in TC levels after treatment with 150 and 600 mg/kg/day.

As revealed in Fig. 5, no significant difference was observed in levels of THs (FT4, TRH, TTR and TSAb) in DEHP treatment compared with the control group (P>0.05). No changes were observed with 150 mg/kg/day DEHP (a no effect level) and that for FT3 and T4, the effects were not dose-dependent. However, levels of T4 and FT3 in the 300 mg/kg/day dose group were significantly decreased compared with that of the control group (P<0.05). Levels of T3 and TSH in the 600 mg/kg/day dose group were also significantly different compared with the control group (P<0.05).

Analysis of histological changes revealed altered TH levels following DEHP exposure. H&E staining demonstrated that the follicular epithelium was cube-shaped in the control group, colloids were uniform, most of the thyroid gland was normal, only some epithelium was shed and inflammatory cell infiltration was evident (Fig. 6A). In the low-dose group, the follicular epithelium was also cube-shaped, part of the epithelial cytoplasm was loose and epithelial shedding was evident (Fig. 6B). In the medium dose group, part of the follicular epithelium exhibited signs of necrosis, follicular collapse was observed along with inflammatory cell infiltration and residual follicular epithelial vacuolar degeneration was apparent. In addition, the cytoplasm appeared foamy and vacuolated and enlarged follicular epithelial cells were noticeable (Fig. 6C). In the 600 mg/kg/day dose group, most of the thyroid follicles were collapsed and disappeared, inflammatory cell infiltration was advanced, part of the filter follicle epithelium was shed and follicle epithelium vacuolar degeneration was even more pronounced. Additionally, the nucleus was deformed while the nuclear membrane was shrunken and chromatin was aggregated. The expansion of the rough endoplasmic reticulum was also evident, along with the appearance of vacuoles (Fig. 6D).

In Fig. 6E the liver tissue of the control group is presented. In the 150 mg/kg/day dose group, liver lobular structure is clear, hepatocytes are arranged neatly, but partly bulked (Fig. 6F). In 300 mg/kg/day dose, a small amount of fatty liver cells and punctate necrosis of the hepatocytes occasionally occurred (Fig. 6G). In the 600 mg/kg/day dose group, focal necrosis of hepatocytes, vacuum, hepatic sinusoidal dilation, obvious hydrodegeneration and occasional apoptotic hepatocytes were observed (Fig. 6H).

Histological studies of the kidneys in the control group revealed normal glomeruli and tubules (Fig. 6I). In the 150 mg/kg/day group, the glomeruli were contracted slightly and the tubular epithelial cells expanded (Fig. 6J). The glomerular epithelium of the 300 and 600 mg/kg/day group swelled, some of the renal tubules disappeared and protein components were visible in the tubes (Fig. 6K and L).

In the 0 and 150 mg/kg/day DEHP-induced group, the testes tissue was well organized with an intact epithelium (Fig. 6M and N). Exposure to DEHP at 300 and 600 mg/kg/day caused the seminiferous tubules at all levels of the seminiferous epithelium to be disorderly arranged with few layers, disintegration of germinal epithelial and reduction of round spermatozoa in the seminiferous tubules (Fig. 6O and P).

Results for thyroid gene expression are presented in Fig. 7. The mRNA levels of PDS, NIS, PAX-8, TPO, TTF-1 and Tg were significantly increased compared with the control group after DEHP treatment. mRNA levels of TSHr, D1 and TTF-2 significantly decreased compared with the control following DEHP treatment (P<0.05). Exposure to DEHP caused an initial decrease in mRNA expression of pituitary TSH-β and significant differences between 150 and 600 mg/kg/day groups (P<0.05) and 150 and 300 mg/kg/day groups (P<0.05). The mRNA levels of hypothalamus TRHr were increased after DEHP treatment, with significant differences between 600 mg/kg/day and the control group (P<0.01) and between 150 and 600 mg/kg/day groups (P<0.01).

Compared with that of the control group, the protein levels of NIS, PAX-8, Tg, TTF-1 and TPO were significantly increased after DEHP treatment (Fig. 8). TSHr protein levels significantly decreased compared with that of the control group following DEHP treatment (P<0.05). The protein levels of pituitary TSH-β were increased after DEHP treatment and significant differences between 150 and 600 mg/kg/day groups (P<0.05) and between 300 and 600 mg/kg/day groups (P<0.05) were observed.