Specific pathogen-free ICR mice were purchased from the Animal Experimental Research Center of Anhui Medical University (Anhui, China). Before the formal experiment, the mice were adaptively fed for 1 week. All mice had free access to food and water under a 12:12-h light-dark cycle at 22 ± 1 °C. Then, 7-week-old ICR female mice were mated with fertile 10 to 12-week-old ICR males (female: male = 2:1). A vaginal plug was detected at next day after mating (the presence of a vaginal plug is defined as gestational day 0, GD0). Pregnant mice were randomly divided into four groups (n = 6 per group) and exposed to the following interventions from GD4 to GD18: 1) control group (DEHP 0 mg/kg), mice treated with corn oil; 2) DEHP 500 mg/kg group, mice treated with DEHP at a dose of 500 mg/kg/day by gavage; 3) DEHP 1000 mg/kg group, mice treated with DEHP at a dose of 1,000 mg/kg/day by gavage; 4) DEHP + Fer-1 group, mice treated with 1,000 mg/kg/day of DEHP and 5 mg/kg/day of ferroptosis inhibitor ferrostatin-1 (Fer-1). Dosage information and design in this experiment were based on recent previous research (Li et al., 2012; Zhao et al., 2021). All mice were sacrificed by cervical dislocation under diethyl ether anesthesia on GD18. Schematic diagram of experimental design in mice is shown in Figure 1A. All animal experiments were carried out with the approval of the Experimental Animal Ethics Committee of Anhui Medical University (No: 20190302).

On GD18, the mice were euthanized. Blood samples were collected and centrifuged to separate the serum. Liver tissue was excised and weighed immediately and divided into two parts: one for histological assays by fixing in 4% paraformaldehyde and the other for western blotting by freezing in liquid nitrogen for 20 min. All blood and liver samples were stored at −80°C for further experiments.

DEHP, MEHP, and RSL3 were purchased from Aladdin (Shanghai, China). Fer-1 was purchased from Sigma‒Aldrich (Shanghai, China). Alanine aminotransferase (ALT), aspartate aminotransferase (AST), MDA, GSH detection kits and glucose kits (glucose oxidase method) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A hematoxylin and eosin (H&E) staining kit, BCA protein assay kit, lysis buffer and Reactive Oxygen Species Assay Kit were purchased from Beyotime Technology (Shanghai, China). CCK8 was purchased from Apex-Bio (Houston, United States). DMEM was purchased from Gibco (New York, United States). Antibodies for solute carrier family 7 member 11 (SLC7A11) and GPX4 were purchased from Abcam (MA, United States). All horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Zen Bio (Chengdu, China). TRIzol^® reagent was purchased from Invitrogen (San Giuliano Milanese, Italy), and the SYBR Green kit was purchased from Life Science Biotechnology (Beijing, China). Primers for detecting Fth1, Ftl, Gpx4, Lpcat3, Slc7a11, Ptgs2, Lpcat3, Nrf2, and β-actin were synthesized by Invitrogen (San Giuliano Milanese, Italy).

Serum AST and ALT levels were measured by using an automatic biochemistry analyzer (Hitachi Automatic analyzer, Japan).

Samples of liver tissue were collected and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned for H&E staining. The histopathological observations were performed using a Microscope (Tissue FAXS-S plus, TG, Austria). First, the hepatic lobule structure was found under a ×4 objective lens. Second, the size, morphological structure, and arrangement of hepatocytes were observed under ×20 and ×40 objective lenses.

Hepatic iron accumulation was assessed by Perl’s Prussian Blue (PPB) staining. Paraffin-embedded liver tissue sections were deparaffinized and rehydrated through graded alcohols in water. The paraffin sections were stained with Prussian blue (10 mg/ml) for 15 min, washed in running water for 1 min and counterstained with 0.5% aqueous neutral red solution for 1 min. Tissue cells showing bright blue dots in the cytoplasm were examined under a phase-contrast microscope. Subsequent dehydration steps used exact timings to maintain consistent counterstain intensity and distribution. Each slice was randomly selected, and the percentage of Prussian blue-stained area was measured by image analysis (Tissue FAXS-S plus, TG, Austria).

The liver ferrous iron (Fe²⁺) concentration was assessed with an Iron Assay Kit (Solarbio Life Science Institute, China). Liver tissue (0.1 g) was added to 1 ml of extracting solution for ice bath homogenization and centrifuged at 4,000 × g for 10 min at 4°C. The supernatant was collected for further testing according to the manufacturer’s instructions. The absorbance of the supernatant was measured on an EnSight multimode detection platform (EnSight, PE, United States) at a wavelength of 520 nm.

The concentration of GSH, GSH/GSSG ratio, and MDA in liver homogenized supernatant were measured to evaluate the antioxidant status and lipid peroxidation. The liver homogenate was prepared as previously described. The total protein concentration in the supernatant was also determined by the BCA assay. All operations were performed according to the manufacturer’s instructions.

HepG2 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, BI, Israel) and 1% penicillin‒streptomycin. The HepG2 cell line was maintained in a humidified incubator at 37 °C with 5% CO₂. The cells were then separated into five groups: control (DMSO), 100 μM (MEHP), 200 μM (MEHP), 1 μM RSL3 (GPX4 inhibitor), and 200 μM (MEHP)+RSL3. HepG2 cells were pretreated in serum-free medium for 6 h and then incubated with MEHP for 24 h. All reagents were dissolved in DMSO (Sigma, United States) before use. Cells were treated with DMSO in the control group.

To investigate the effect of MEHP and RSL3 on HepG2 cell viability, HepG2 cells were inoculated at a density of 10⁴ cells/well in 96-well plates and then incubated in cell culture media containing 100–500 μM MEHP concentrations for 24 h. After 24 h of incubation, cell proliferation was evaluated using the Cell Counting Kit-8 kit, following the manufacturer’s instructions. Cell viability% = (absorbance of experimental group - absorbance of blank)/(absorbance of control group - absorbance of blank) × 100%.

To measure glucose metabolism in HepG2 cells treated with MEHP. The glucose levels were measured by following the manufacturer’s instructions. We also analyzed cell viability to measure the glucose consumption ratio after MEHP treatment.

ROS levels in HepG2 cells were measured by the fluorescent probe DCFH-DA. HepG2 cells were inoculated in 6-well plates using a Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China) and subjected to various treatments for 24 h. The culture medium was switched to serum-free medium, and 10 mol/L dichloro-fluorescein diacetate was incubated for 30 min in the dark. Finally, flow cytometry and fluorescence microscopy were used to estimate the ROS level in the cells.

Total RNA was extracted from liver tissues using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions and standardized to 1 μg/μL. The purification of RNA was performed according to the ratio of absorbance at 260 nm and 280 nm. The reaction solution was configured according to the instructions mentioned in the Reverse Transcription Kit (Roche, Switzerland). Then, RT-PCR was performed with SYBR Green Master Mix (Roche, Switzerland). The relative concentration of mRNA levels was normalized by the comparative Ct (2^−ΔΔCt) with β-actin as an internal reference. The specific primer sequences applied in this study are presented in Table 1.

Total proteins were extracted from the liver and quantified using the BCA Protein Assay Kit (Beyotime Biotechnology, China). Proteins were separated by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes, blocked with skimmed milk powder, and incubated at 4 °C overnight with primary antibodies. The membranes were washed with Tris-buffered saline with Tween 20 (TBST) and incubated with secondary antibodies conjugated with HRP. Antibody-protein complexes were detected using an ECL Prime Western Blotting Detection Reagent (Advansta, California, USA) and visualized using a Tanon digital imaging system (Fine-do X6, Shanghai, China). β-actin was used as an internal control protein.

All independent experiments were repeated at least 3 times, and the data are expressed as the mean ± standard deviation (SD). Student’s t test or one-way analysis of variance (ANOVA) was used to compare significant differences among groups, and Dunnett’s test was used for multiple comparisons using GraphPad Prism eight software. p < 0.05 (*), p < 0.01 (**), and p < 0.001 (***) indicated a statistically significant difference.

The results of H&E staining, inflammatory cytokine levels, and liver function parameters are shown in Figure 1. H&E staining was performed to observe histopathological changes in the liver. Representative micrographs from H&E-stained liver sections revealed swollen hepatocytes, necrosis, inflammatory cell infiltration (red arrow), and hyperemia in the central vein (black arrow) in mice treated with 1,000 mg/kg DEHP (Figure 1B). The liver is the key target organ of DEHP exposure. AST is mainly found in the liver cytoplasm and hepatocyte mitochondria. ALT is mainly distributed in the liver cytoplasm. The levels of AST and ALT were significantly elevated (p < 0.01) in the 1,000 mg/kg DEHP group, which suggested damage to the liver parenchyma (Figures 1C,D). We found that the liver weight/body ratio was markedly increased (p < 0.001) in the DEHP-exposed group compared to the control group (Figure 1E), the expression of the inflammatory factor interleukin-1β (IL-1β) was significantly evaluated, and the level of the inflammatory factor interleukin-6 (IL-6) in liver tissue was much higher in the 1,000 mg/kg DEHP group (p < 0.05) (Figures 1F,G). From the above results, it has been demonstrated that gestational DEHP exposure induces liver injury in pregnant mice.

To explore the effects of DEHP exposure on iron metabolism in the livers of pregnant mice. Liver Fe²⁺ concentration and PPB staining were performed. The level of Fe²⁺ was significantly elevated, and PPB staining showed increasing iron accumulation in the liver tissue (red arrow) in the 1,000 mg/kg DEHP group (p < 0.01, Figures 2A–C). Moreover, the MDA content was significantly increased (p < 0.05) in the livers of mice in the 1,000 mg/kg DEHP-exposed group (Figure 2D). To assess hepatic redox homeostasis after DEHP exposure, GSH and the GSH/GSSG ratio were also measured. They were markedly decreased in the 1,000 mg/kg DEHP-exposed group compared to the control group (Figures 2E,F).

Iron metabolism-related genes were analyzed by qPCR. We found that the Ftl and Fth1 genes were highly expressed (p < 0.05, p < 0.01), and the Tfr1 gene was expressed at low levels in the 1,000 mg/kg DEHP-exposed group (p < 0.05, Figures 2G–I). Collectively, these results suggested that gestational DEHP exposure might alter iron metabolism in the liver and promote lipid peroxidation.

To further determine whether DEHP exposure could induce ferroptosis in mice livers, ferroptosis-related genes and proteins were detected. We found that the ferroptosis-related genes Gpx4, Slc7a11, and Nrf2 were significantly downregulated, and Ptgs2 and Lpcat3 were significantly highly expressed in the 1,000 mg/kg DEHP-exposed group (Figures 3A–E). Since Gpx4 is the key gene in ferroptosis, we further conducted western blotting and revealed that the protein levels of GPX4 and SLC7A11 were markedly downregulated (p < 0.05, Figures 3F–H). The above results indicated that gestational DEHP exposure may lead to iron overload and further induce ferroptosis in mouse livers.

To determine whether ferroptosis plays a potential role in DEHP-induced liver injury. Fer-1 was injected into mice before DEHP exposure. Compared with the 1,000 mg/kg DEHP-exposed group, the levels of AST and ALT and the organ coefficient of the liver were significantly decreased in the 1,000 mg/kg DEHP + Fer-1 group (p < 0.05, Figures 4A–C). The level of liver Fe²⁺ was not significantly changed compared to that in the DEHP-exposed group (Figure 4D). Furthermore, the infiltration of inflammatory cells and hyperemia in the central vein was diminished after Fer-1 treatment (Figure 4E). In addition, the level of MDA in the liver was decreased (p < 0.05) in the 1,000 mg/kg DEHP + Fer-1 group, and the levels of GSH and the GSH/GSSG ratio were not markedly increased (Figures 4F–H). Overall, the data further indicated that Fer-1 was able to alleviate liver injury in DEHP-exposed mice.

In addition, ferroptosis-related genes and proteins were detected in DEHP-exposed mouse livers after treatment with Fer-1. Compared with the 1,000 mg/kg DEHP-espoused group, the mRNA level of Slc7a11 was significantly increased and Ptgs2 was obviously decreased (p < 0.05), while the expression of Nrf2 and Gpx4 showed an increasing trend that was not significantly different in the DEHP + Fer-1 group (Figures 5A–D). In addition, the protein levels of SLC7A11 and GPX4 were markedly increased (p < 0.05) in the DEHP + Fer-1 group (Figures 5E–G). In conclusion, the results demonstrated that DEHP may promote ferroptosis through the SLC7A11/GPX4-related pathway in pregnant mice livers.

To further determine the role of ferroptosis in MEHP-induced hepatotoxicity, an in vitro study was conducted in HepG2 cells. RSL3 (a specific GPX4 inhibitor) was utilized to explore the potential molecular mechanism. We evaluated cell viability after treatment with MEHP and RSL3 (Figures 6A,B). In addition, we measured the glucose levels in the cell lines (Figures 6C,D), and the results suggested the occurrence of abnormal glucose metabolism in the MEHP-treated group. After evaluating the level of ROS, we found that the ROS level was higher in the MEHP + RSL3 group than in the RSL3-treated group (Figures 6E–H). Furthermore, we also discovered that the protein expression of GPX4, SLC7A11 and Nrf2 was significantly downregulated after treatment with MEHP + RSL3 (Figures 6I-L). These in vitro data support the idea that ferroptosis is involved in the molecular mechanism of MEHP-induced hepatotoxicity.