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. 2025 Jul 10;3(11):1353–1365. doi: 10.1021/envhealth.5c00156

Reproductive Toxicity and Transgenerational Effects of Anesthetic Etomidate in Zebrafish: PCOS-like Ovarian Changes, Oxidative Stress, and Metabolism Disruption

Xuewei Li 1, Xuhui Lin 2, Zile Zhuang 1, Yuxuan Luo 1, Yihan Li 1, Said Ahmed Isse 1, Zhenzhong Chen 3, Fanliang Meng 4, Qizhi Luo 1,*, Bofeng Zhu 1,*, Xuncai Chen 1,*
PMCID: PMC12645302  PMID: 41306307

Abstract

Etomidate (ETO), a benzodiazepine commonly used by pregnant women with anxiety disorders, has been increasingly detected in aquatic environments. However, its reproductive toxicity and intergenerational effects have not been adequately assessed. In this study, adult female fish were employed to investigate the reproductive toxicity after ETO exposure. After 4 weeks of exposure, zebrafish ovaries exhibited polycystic ovary syndrome (PCOS)-like changes, including polycystic ovaries, reduced spawning rates, and elevated androgen levels. Moreover, ETO was transmitted to offspring via maternal ovaries, significantly increasing larval mortality and deformity rates while reducing their locomotor activity. Transcriptomic and metabolomic profiling revealed oxidative stress responses, disrupted unsaturated fatty acid and glutathione metabolism, and damage to ovarian granulosa cells and the zona pellucida, as confirmed by reactive oxygen species (ROS) fluorescence staining. Combined multiomics analysis demonstrated that ETO exposure exacerbated ovarian oxidative stress, impaired the functional structure of ovaries, and disrupted follicular cell maturation via hyperactivation of the mechanistic target of the rapamycin complex 1 (mTORC1) pathway. These changes culminated in PCOS-like effects in zebrafish. This study provides critical insights into the mechanisms of ETO-induced reproductive toxicity and transgenerational impacts, offering a foundation for more comprehensive risk assessments and potential interventions.

Keywords: anesthetic etomidate, reproductive toxicity, PCOS-like ovarian changes, multiomics, oxidative stress, metabolism disruption

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1. Introduction

Anxiety and sleep disorders affect up to 15% of pregnant women, with 10–26% of those diagnosed being prescribed benzodiazepines. , In Norway, approximately 1.5% of all pregnant women receive benzodiazepine prescriptions. However, benzodiazepine use during pregnancy is not without risks. A large-scale Korean cohort study revealed that first-trimester exposure to benzodiazepines is associated with a slightly elevated risk of congenital malformations, particularly heart defects. Beyond their clinical use, benzodiazepines have become an environmental concern. A European analysis of surface water samples from 30 rivers across seven major basins found that 86% contained one or more benzodiazepines at nanogram-level concentrations. Studies, including those by Luykx, have shown that psychotropic drugs are inefficiently removed by wastewater treatment plants and that households are significant contributors to psychotropic contamination in surface water.

Among these benzodiazepines, etomidate (ETO), first synthesized in 1964, is widely used in anesthesia due to its favorable hemodynamic properties. However, in recent years, ETO has also been misused as a new psychoactive substance (NPS) due to its ability to induce dissociative states and altered consciousness, similar to ketamine and propofol. , Environmental monitoring data indicate widespread ETO contamination across multiple matrices. Wastewater treatment plants in Shanghai and Zunyi, China, exhibited ETO concentrations ranging from 2.4 to 35.4 ng/L and 1.7 to 29.3 ng/L, respectively. Human exposure assessments revealed elevated ETO levels in biological samples, with hair concentrations of 0.2–45 μg/g and urinary levels of 1.0–80 μg/L detected in e-cigarette users. Indoor air monitoring demonstrated significant aerosolization potential, showing particulate ETO concentrations reaching 750.70 ng/m3 during e-cigarette combustion. Occupational exposure was evidenced by hair samples from drug testing laboratory personnel containing up to 19.73 μg/g ETO. These findings collectively demonstrate substantial environmental persistence and human exposure pathways for ETO. Although some levels are significantly below thresholds that directly impact aquatic organisms, additive and sublethal effects cannot be ruled out.

Our previous research demonstrated that chronic exposure to ETO impairs short-term memory and increases aggression in zebrafish. Clinical studies have shown that misuse of ETO can result in dizziness, mental disorders, and even death. Long-term use can damage the endocrine system, liver, and nervous system, with neurotoxic effects including apnea or even death. In zebrafish larvae, acute ETO exposure has shown a dose-dependent reduction in spontaneous behavior. , In recent years, studies have demonstrated that benzodiazepines can enhance the activity of γ-aminobutyric acid A (GABA-A) receptors, affecting neuroendocrine regulation, which can lead to male sexual dysfunction and alterations in sex hormone levels. Despite extensive studies on the effects of ETO, the reproductive toxicity of ETO remains underexplored, raising concerns about its potential risks to reproductive health and fetal development.

In this study, we employed an integrated multiomics approach to investigate the reproductive toxicity and intergenerational effects of ETO exposure in female zebrafish. By analyzing histopathological changes, oxidative stress levels in parental ovaries, developmental parameters, and the motor abilities of their offspring, we identified hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) as a critical event driving polycystic-like ovarian changes. This study provides valuable insights into the molecular mechanisms of ETO-induced reproductive toxicity, contributing to a deeper understanding of the toxicities of ETOs and their environmental and clinical risks. Moreover, it highlights the need for heightened awareness of the aquatic environmental, iatrogenic, and occupational exposure risks associated with ETO.

2. Materials and Methods

2.1. Chemicals and Animals

ETO (CAS: 33125–97–2; 98%) and dimethyl sulfoxide (DMSO, CAS: 67–68–5; >99.9%) were purchased from TMRM Quality Inspection Technology Co., Ltd. (Beijing, China) and Solarbio (Beijing, China), respectively. Stock solutions were dissolved in DMSO, and fresh stock solutions were prepared weekly. All other chemicals were purchased from Macklin and were of analytical grade. Adult wild-type zebrafish (3 months old) were purchased from Hubei Chuangxin Biotechnology Co., Ltd. (Wuhan, China).

2.2. Fish Maintenance and Exposure

Female zebrafish were cultured at 28 ± 0.5 °C with a 14 h:10 h light–dark cycle in aerated water and exposed to etomidate for 4 weeks. Wild-type adult zebrafish were divided into 3 treatment groups, including control (0 μg/L), low-dose exposure to etomidate (20 μg/L), and high-dose exposure to etomidate (200 μg/L). The concentration of DMSO was kept at 0.001% (v/v) in all of the containers. The exposure was conducted in 5 L tanks, each containing 20 females in triplicates. The exposure solution was replaced daily with fresh stock solution. After 4 weeks of ETO exposure, zebrafish were euthanized by immersion in ice–water for 15 min and dissected, and samples were immediately frozen in liquid nitrogen and stored at −80 °C for further analysis.

2.3. Mating and Monitoring F1 Zebrafish

Prior to experimental initiation, sexually mature female zebrafish (3 months old) with confirmed reproductive competence underwent pairing selection and were acclimated in 5 L aquaria. At the conclusion of the one-week acclimation period, 5 females per group were randomly selected for mating trials to quantify baseline spawning output. During the subsequent four week ETO exposure phase, mating trials were conducted weekly on Monday evenings. Specifically, 5 ETO-exposed female zebrafish from each tank (3 replicate tanks per group; 5 females randomly sampled per tank, n = 15) were transferred with 2 healthy, uncontaminated male zebrafish into spawning tanks containing system water. The following day, the transparent baffle was removed to allow unrestricted mating for 1 h. Spawned eggs were quantified and washed three times with embryo medium. The number of hatched larvae from each treatment group was counted at 72 h postfertilization (hpf), and the hatching rate was calculated as the number of hatched (n = 15) larvae divided by the total number of eggs, expressed as a percentage. The hatching rate (n = 15), abnormalities (n ≥ 3, 30 larvae in one replicate), and mortality (n ≥ 3, 30 embryos or larvae in one replicate) of embryos and larvae were monitored at different time points.

2.4. Quantification of ETO in Zebrafish Tissues

ETO levels were quantified using liquid chromatography–mass spectrometry (LC-MS/MS) (Thermo Scientific, Waltham, USA), following the detection standard published by the Anti-Drug Information Center of the Ministry of Public Security of China. The specific procedures were as follows: Each ovarian tissue was regarded as one sample (n = 4). A 1 mL methanol solution was added to each sample, followed by grinding, shaking, ultrasonic treatment (10 min), and centrifugation (4 °C, 13,000 rpm, 15 min). The supernatant (400 μL) was mixed with 200 μL of 0.1% formic acid in water, and the mixture was transferred to an injection vial. ETO concentrations were determined using a SCIEX Triple Quad 5500 LC-MS/MS system (Thermo Scientific) with external standard calibration (A, 0.1% formic acid water; B, 0.1% formic acid acetonitrile; C-18 column). The specific method of the LC-MS instrument is described in the Supporting Information Section 1.

2.5. Coefficient of Condition (K) and the Gonado-Somatic Index (GSI)

Upon completion of the exposure period, female zebrafish were euthanized through 15 min of immersion in ice water. Following necropsy, body weight (BW) and body length (BL) were recorded to calculate the coefficient of condition (K) using the formula K = (BW/BL3) × 100 (n = 14). Ovaries were subsequently excised and weighed (GW) for determination of the gonado-somatic index (GSI) according to the equation GSI = (GW/BW) × 100 (n = 14).

2.6. HE Stains of Ovarian Histology

Ovaries (n = 3) were dissected and fixed in 4% paraformaldehyde for 24 h. After gradient dehydration (70%, 80%, 90%, 95%, and 100% ethanol), the samples were cleared in xylene and embedded in paraffin. Sections (3 μm) were cut using a Leica microtome (Weztlar, Germany), collected on slides, and stained with hematoxylin and eosin. The sections were examined under a Nikon ECLIPSE Ni Series microscope (Tokyo, Japan).

2.7. Enzyme-Linked Immunosorbent Assay (ELISA)

Gonads (n = 3) and brains (n = 3, 3 brains were pooled into one sample) were homogenized in PBS (pH = 7.4) and then centrifuged at 13,000 rpm for 10 min at 4 °C, and the supernatant was collected for analysis. Testosterone (T), 11-ketotestosterone (11-KT), estradiol (E2), anti-Müllerian hormone (AMH), and luteinizing hormone (LH) levels in tissue homogenates were measured using Zebrafish ELISA Kits (Dogesce, Beijing, China). The levels of phosphorylated mechanistic target of rapamycin (p-mTOR) and phosphorylated ribosomal protein S6 kinase (p-S6K) in tissue homogenates were measured using Zebrafish ELISA kits (Bioleaper, Shanghai, China).

2.8. Light/Dark Preference Behavior Analysis of F1 Zebrafish

At 120 hpf, zebrafish larvae with normal appearances (n = 24) were collected from each treatment group and transferred to 96-well plates containing 200 μL of culture solution. The larvae were acclimated to a ViewPoint Zebrabox testing system (ViewPoint Life Sciences, Lyon, France) for 15 min. Light/dark preference behavior was recorded over a 20 min period, alternating between the light and dark every 5 min.

2.9. Methods for Fluorescence Staining of ROS in Gonads

The gonads (n = 3) were fixed in 4% paraformaldehyde for 24 h, dehydrated in 30% sucrose solution, and sliced using a frozen microtome (Leica). Sections were fixed with pure methanol for 10 min, washed with PBS (3 × 10 min), and stained with the ROS-specific fluorescent dye dihydroethidium (Beyotime, Shanghai, China) at 37 °C for 30 min. The sections were washed with PBS and stained with DAPI (Thermo Fisher) for 10 min in the dark. The samples were sealed with an antifluorescence-quenching tablet and imaged using an inverted laser confocal microscope (FV3000, Olympus Corporation, Tokyo, Japan, 535 nm/610 nm).

2.10. Transcriptomic and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analyses

Ovarian (n = 3, 2 ovary were pooled into one sample) tissue from zebrafish was homogenized in 1 mL of TRIzol reagent (Thermo Fisher) for ribonucleic acid (RNA) extraction following the manufacturer’s instructions. RNA quality was verified using a NanoDrop spectrophotometer (Thermo Scientific). A total of 500 ng of RNA was used to construct a bulk RNA sequencing library. PolyA­(+) RNA was reverse transcribed using SuperScript II, and second-strand synthesis was performed using a template-switching reaction followed by 15 qRT-PCR cycles for cDNA (cDNA) amplification with DNA Polymerase I and RNase H. The cDNA was purified using the AMPure XP system and quantified using the Agilent High Sensitivity DNA Assay on a Bioanalyzer 2100 system (Agilent, California, USA). Sequencing was performed on the NovaSeq 6000 platform (Illumina). Differentially expressed genes (DEGs) were identified using DESeq2 with a threshold of p < 0.05 and an absolute fold change of ≥2. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were conducted on the DEGs for functional annotation.

For ovary tissue analysis, total RNA was extracted using the TRIzol reagent (Thermo Fisher). cDNA was synthesized by using a reverse transcriptase kit (Takara Biochemicals, Shiga, Japan). qRT-PCR was conducted in a SYBR Green system (Thermo Fisher). Gene expression levels were normalized to β-actin as a reference gene, and relative abundance was calculated using the [log2­(2–ΔΔCT)] method. Primer sequences used for qRT-PCR are listed in Table S1.

2.11. TMT-Based Quantitative Proteomic Analysis and Parallel Reaction Monitoring (PRM) Verification

For sample preparation and proteomic analysis, ovaries from two zebrafish were pooled as a single biological replicate (n = 3), homogenized in 200 μL of lysis buffer (2% SDS, 100 mmol/L DTT, 150 mmol/L Tris-HCl pH 8.0), and sequentially processed through vortexing, 95 °C boiling, ultrasonication, and centrifugation (12,000 × g, 10 min). Protein concentration was determined using a BCA assay (Bio-Rad, California, USA). Protein digestion was performed via the Filter-Aided Sample Preparation (FASP) method. TMT-labeled peptides were fractionated using a Waters XBridge BEH130 C-18 column (Massachusetts, USA, 3.5 μm, 2.1 × 150 mm) on an Agilent 1290 HPLC system. LC-MS/MS analysis was conducted on a Q Exactive HF-X mass spectrometer coupled to an Easy nLC 1200 system (Thermo Scientific), operating in data-dependent acquisition mode. Raw files were processed through Proteome Discoverer 2.4 with stringent filtering criteria: ≥6 amino acid residues per peptide, ≥2 unique peptides per protein, and a 1% false discovery rate (FDR) at both peptide and protein levels. Differentially expressed proteins (DEPs) were defined as those exhibiting >1.2-fold change (FC) with p < 0.05. Functional annotation was performed using GO/KEGG databases, while interaction networks were constructed via STRING and visualized in Gephi.

For PRM validation, protein extraction and tryptic digestion followed protocols identical to those of TMT experiments, with triplicate biological replicates. PRM targets were selected based on shotgun proteomics data, prioritizing peptides demonstrating symmetrical elution profiles and consistent identification across runs. Skyline 4.1 software facilitated peak integration and manual verification. Normalized peptide abundance (AUC/protein average) was used for quantitative comparisons. Both TMT and PRM analyses were performed by Shanghai Bioprofile Technology Co., Ltd. under standardized protocols.

2.12. Metabolomic Analysis

After 4 weeks ETO exposure, ovaries (n = 3) of zebrafish were collected from each group, homogenized in water (200 μL), methanol (400 μL), and acetonitrile (400 μL), vortexed for 30 s, subjected to ultrasound for 10 min (ice bath), and then frozen at 20 ° C for 1 h to precipitate proteins. The samples were centrifuged (13,000 r/min, 15 min, 4 °C), and the supernatant was evaporated in a lyophilizer (Tokyo Rikakikai Co., Ltd., Tokyo, Japan.). The dried extract was reconstituted in 100 μL of acetonitrile:water (1:1), and centrifuged again, 60 μL of supernatant was collected and transferred to a sample bottle. 10 μL of supernatant was extracted from each sample and blended into quality control (QC) samples. One QC sample was injected between every four samples for batch correction and assessment of signal stability. Two solvent blanks solutions (methanol) were injected between different groups to avoid cross-group contamination. Metabolites were identified using a Triple TOF X500R mass spectrometer (Thermo Scientific), with data analysis performed using MetDNA and MetaboAnalyst software (http://metdna.zhulab.cn) (https://www.metaboanalyst.ca).

2.13. Statistical Analysis

Experimental data are presented as mean ± standard error of the mean (SEM), with at least three replicates in all experiments. Graphs were generated using GraphPad Prism 9.5 and Origin 2024b. Statistical analysis was performed using one-way ANOVA followed by LSD or two-tailed Student’s t test, with significance indicated as *p < 0.05, **p < 0.01, and ***p < 0.001. Heatmaps were generated using the R package heatmap or GraphPad Prism 9.5, and bubble plots of KEGG and GO results were created with the R package ggplot2 and Bioinformatics (http://www.bioinformatics.com.cn/).

3. Results and Discussion

3.1. ETO Exposure Induces Symptoms Similar to Polycystic Ovary Syndrome (PCOS) in Zebrafish

After 4 weeks of ETO exposure, it was found that ETO accumulated in the ovaries of zebrafish. Examining the embryos spawned by the F0 female zebrafish that had been exposed to ETO showed that ETO could transfer to the offspring through the ovaries of F0 zebrafish (Table S2). Also, significant changes in oocyte development were observed in F0 zebrafish ovaries (Figure A). The proportion of immature oocytes (primary growth oocytes, PO) was significantly higher in the ETO-exposed group compared to the control, while the number of vitellogenic and postvitellogenic follicles (EV and LV) was markedly reduced (Figure B). Additionally, a significant increase in atretic follicles was observed in exposed group (Figure C). Similar to women with polycystic ovary syndrome (PCOS), who have approximately twice as many primary and secondary follicles compared to normal ovaries, these findings suggest that chronic ETO exposure induces polycystic ovarian changes in zebrafish. Histological analysis also revealed disrupted follicular architecture in mature oocytes of the ETO group, including irregular invaginations of the zona pellucida and degeneration or disappearance of the granulosa cell layers. Granulosa cells secrete factors such as estradiol (E2) and growth factors essential for follicular development. This indicates that after ETO exposure, it might not only cause damage to the ovarian granulosa cell layer but also impede follicular development by modifying the normal hormone levels (such as E2) necessary for ovarian development. As a consequence, it leads to polycystic-like alterations in the ovaries of zebrafish within the ETO group.

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Chronic exposure to ETO induces PCOS-trait ovarian phenotype and reproductive endocrine disorders. (A) Representative HE sections of the ovaries of the F0 zebrafish, #: the ovulated follicle, *: the atretic follicle, the black arrows for the granular cell layer, and the yellow for the zona pellucida. Scale bar: 100 μm. (B) The proportion of different follicular stages. (PO: primary growth oocyte, CO: cortical follicular oocyte, EV: vitellogenic oocyte, LV: mature oocyte). (C) The number of atretic follicles. (D) GSI and K. (E, F) The contents of T, 11-KT, E2, and AMH, respectively. (Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

We assessed the reproductive outcomes in ETO-exposed zebrafish. Fecundity was significantly reduced in the ETO group compared to controls (Figure A). Additionally, the condition factor (K) and gonadosomatic index (GSI) of the exposed zebrafish were significantly elevated (Figure D). The GSI is a recognized biomarker for reproductive studies in fish, reflecting gonadal growth under endocrine control. , Enlarged ovaries with polycystic and atretic follicles, as well as invaginated zona pellucida layers, further confirm the structural and functional abnormalities in the ovary of zebrafish induced by ETO exposure. Similarly, polycystic ovary syndrome (PCOS), a prevalent endocrine disorder, is characterized by hormonal imbalances, inhibited follicular maturation, formation of multiple small cysts due to follicular degeneration, and elevated levels of free testosterone. PCOS affects 10–13% of women of reproductive age, accounting for over 90% of adolescent and adult hyperandrogenism. It is associated with significant long-term health risks, including infertility, metabolic disorders, mental health challenges, and endometrial cancer. −

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Study of fetal and postnatal effect of F1 zebrafish after prenatal ETO exposure. (A) Spawning rate of the F0 zebrafish, survival rate, malformation rate, and heart rate of the offspring. (B) Representative malformations of the offspring, red arrows for pericardial edema, and blue for scoliosis. Scale bar: 1000 μm. (C) Top: Dot plot of swimming distance of F1 larvae in the light/dark preference experiment. Middle: Representative swimming trajectories of larva, red line for light time, and black line for dark one. Bottom: left: The average distance of larva moved during light times; right: The average distance of larva moved during dark times. (Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

The above results provide preliminary evidence that exposure to ETO induces symptoms similar to those of polycystic ovary syndrome (PCOS) in zebrafish.

3.2. Reproductive Endocrine Disorders in ETO Exposure Zebrafish

Hyperandrogenemia is a key clinical hallmark of polycystic ovary syndrome (PCOS). To investigate this, testosterone (T), 11-ketotestosterone (11-KT), and estradiol (E2) levels were measured using ELISA. The results showed a significant increase in both T and E2 levels in the ETO-exposed group compared to controls (Figure E, F). Elevated T levels are also known to promote adipogenesis, which may contribute to obesity, , and this also explains why the K and GSI of zebrafish in the ETO-exposed group are higher. Mechanistically, elevated T in women drives visceral fat deposition via androgen receptor-mediated inhibition of lipolysis and enhanced adipogenesis. Androstenedione is the primary precursor for the synthesis of both T and E2, produced in the ovaries and the adrenal cortex. In the ovary, the conversion of androstenedione to T occurs in theca cells via 17β-hydroxysteroid dehydrogenase (17βhsd), and T is subsequently converted into E2 in granulosa cells by the enzyme cytochrome P450 19A1 (cyp19a1). Previous studies have reported upregulation of steroidogenic enzymes in theca cells of women with PCOS. In our study, the expression of 17βhsd was significantly downregulated in the ovaries of ETO-exposed zebrafish compared to controls (Figure S1), suggesting that excess T may trigger negative feedback mechanisms in zebrafish.

Notably, the functional androgen in zebrafish, 11-KT, was significantly lower in the ETO-exposed group compared with the control group (Figure E). This may be due to decreased expression of cytochrome P450 11B (cyp11b). cyp11b is a key enzyme in the synthesis of 11-KT, which converts T to 11-KT. Exposure to ETO resulted in downregulation of cyp11b expression via qRT-PCR (Figure S1), which led to a blockage of the conversion of T to 11-KT as evidenced by a decrease in the synthesis of 11-KT and an accumulation of T, which explains the increase in the level of T and the decrease in the level of 11-KT. Meanwhile, the elevated E2 levels correspond with increased cyp19a1 expression.

Additionally, neuroendocrine dysregulation is known to contribute to enhanced androgen production in the ovary and plays a role in PCOS pathogenesis. ETO exposure led to an increase in LH content in zebrafish (Figure S2). Upregulation of lhr expression may exacerbate LH signaling, which is consistent with the measured LH levels. In contrast, downregulation of FSHR expression attenuates the FSH effects. This would result in an abnormally elevated LH/FSH ratio, which is typical of PCOS patients. Anti-Müllerian hormone (AMH) levels are 2- to 3-fold higher in women with PCOS and are used to evaluate ovarian reserve function. In our study, AMH levels were significantly elevated in ETO-exposed zebrafish compared to those in controls (Figure F). These hormonal alterations in ETO-exposed zebrafish mimic the endocrine disturbances observed in PCOS patients with upregulated E2 and T levels, leading to hyperandrogenism. This, combined with increased AMH, disrupts follicular growth and maturation, resulting in a similar pathophysiological profile to PCOS.

3.3. Prenatal ETO Exposure Exerts a Negative Effect against Their Offspring

Prenatal ETO exposure induces transgenerational epigenetic alterations, contributing to developmental toxicity in the offspring of zebrafish. Offspring from ETO-exposed F0 zebrafish exhibited a significantly reduced survival rate and an increased malformation rate compared to controls (Figure A), including scoliosis and pericardial edema in zebrafish (Figure B). Interestingly, no statistical difference in heart rate was observed between the offspring of ETO-exposed and control groups (Figure A), possibly due to etomidate’s minimal cardiovascular and respiratory depression effects.

Research by Baron-Cohen et al. links elevated prenatal androgen levels, such as T, to autism spectrum disorder (ASD) development. Similarly, Kosidou et al. found an association between maternal PCOS and an increased risk of autism in offspring. Consistent with these findings, our study demonstrates that larvae from ETO-exposed zebrafish exhibited reduced locomotion distance in both light and dark conditions during light/dark preference behavior analysis (Figure C). In addition, mass spectrometry analysis revealed the presence of ETO in the offspring embryos after maternal ETO exposure (Table S2). Our previous study on ETO revealed that ETO exposure induces behavioral abnormalities in zebrafish, including short-term memory impairment and increased aggression. This suggests that ETO exposure elevates ovarian T levels in the parental ovary of zebrafish while simultaneously transferring to offspring through ovarian damage, resulting in direct ETO exposure. These intertwined mechanisms collectively may alter fetal brain development, potentially contributing to neurodevelopmental disorders in offspring larvae.

In summary, the accumulation of ETO in ovaries not only disrupts normal functional structures such as ovarian granulosa cells and the zona pellucida via unknown mechanisms, thereby delaying oocyte development, but also transfers to offspring through damaged oocytes, leading to subsequent developmental impairments and reduced deteriorating activity. The transgenerational toxic effects of ETO are hypothesized to be mediated by oxidative stress. Excessive reactive oxygen species (ROS) accumulation or heightened degradation disrupts redox homeostasis, triggers oxidative stress, and impairs embryonic development through multiple pathways. Among these, DNA damage is a critical factor: low-level oxidative stress induces base oxidation and abasic sites, while more severe oxidative stress causes DNA strand breaks. These DNA alterations confer mutagenic risks to gametes, with nuclear and DNA modifications potentially resulting in offspring malformations and even mortality.

3.4. ETO Exposure Results in Altered Ovarian Transcriptomic Profiles in F0 Zebrafish

To elucidate the molecular mechanisms and pathways underlying PCOS induced by ETO, we conducted RNA sequencing on ovaries from the control and 200 μg/L ETO-exposure groups, followed by differential gene expression analysis. Hierarchical clustering of differentially expressed genes (DEGs) revealed consistent relationships among the six sequenced samples, grouped into nine distinct clusters (Figure A). We identified 427 DEGs, comprising 234 downregulated and 193 upregulated genes, in the 200 μg/L ETO-exposure zebrafish compared with controls (Figure B, C). To gain functional insights, gene enrichment analysis was performed on the DEGs using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). KEGG pathway analysis indicated that downregulated genes in the ETO-exposed group were associated with sphingolipid metabolism and proteasome pathways, while upregulated genes were linked to metabolic pathways, folate biosynthesis, and mucin-type O-glycan biosynthesis (Figure D). Previous studies have highlighted the central role of deregulated sphingolipid metabolism in metabolic disorders. Li et al. suggested that serum sphingolipids could serve as diagnostic biomarkers for PCOS subgroups. Furthermore, using the Gene Ontology (GO) database, we explored biological processes (BPs) associated with DEGs, identifying 10 enriched BP terms (Table S3). The key BP terms included regulation of transcription (DNA-templated), RNA biosynthetic process, aromatic compound biosynthesis, RNA metabolic process, cellular response to oxidative stress, and superoxide metabolic process. Our findings indicate that the most affected BPs were cellular response to oxidative stress and superoxide metabolic process, suggesting that ETO exposure induces ovarian oxidative stress.

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Chronic exposure to ETO induces alterations of ovarian transcriptomic profiles in F0 zebrafish. (A) The trend analysis of each cluster. (B) Volcano map based on DEGs. (C) Bar chart of DEGs due to ETO exposure. (D) Enrichment analysis of KEGG function based on DEGs.

3.5. ETO Exposure Results in Altered Ovarian Metabolomic Profiles in F0 Zebrafish

PCOS is a common endocrine disorder associated with both reproductive and metabolic dysfunctions. Genetic studies suggest a significant overlap between PCOS and metabolic disorders. , To understand how ETO exposure influences ovarian metabolic profiles, we performed untargeted metabolomics using LC/Q-TOF MS. Results revealed a clear distinction between the metabolomic profiles of the 200 μg/L ETO-treated group and the control group (Figure A, B). Fourteen differentially expressed metabolites (DEMs, p < 0.05 and AUC = 1) were identified, with seven upregulated and seven downregulated metabolites (Figure C,Figure S3). KEGG enrichment analysis showed that ETO exposure primarily affected pathways including purine metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis, phenylalanine metabolism, glutathione metabolism, biosynthesis of unsaturated fatty acids, and linoleic acid metabolism (Figure D). Similarly, a study by Dong et al. using UPLC-QTOF-MS revealed metabolic profiling differences between PCOS patients and controls, showing alterations in amino acid and nucleoside metabolism, glutathione metabolism, and lipid metabolism. , Linoleic acid (LA), a precursor of arachidonic acid and an omega-6 polyunsaturated fatty acid, plays a vital role in inflammation and steroid hormone regulation, processes linked to metabolic syndrome. , Excessive LA, commonly found in vegetable oils, has been associated with an increased risk of obesity and oxidative stress. Additionally, elevated glutathione levels observed in the glutathione metabolism pathway also hint at oxidative stress in the ovary after ETO exposure. As the most abundant cellular antioxidant, glutathione is crucial for neutralizing reactive oxygen species (ROS). In conclusion, ETO exposure significantly disrupts unsaturated fatty acid and glutathione metabolism in zebrafish ovaries, and these disruptions primarily induce oxidative stress, compromising the ovarian oxidation-antioxidant balance and contributing to ovarian damage.

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Chronic exposure to ETO induces metabolomic changes. (A) Heatmap of different expressed metabolisms between the control and ETO-treated (200 μg/L) group. (B) PCA diagram. (C) Interaction network of DEMs. (D) Enrichment analysis of KEGG function based on DEMs.

3.6. ETO Induces Ovarian Oxidative Stress via Elevating the Level of ROS

To verify the results of transcriptomics and metabolomics, we employed ROS-specific fluorescence staining to visualize the levels of ROS in the ovaries of zebrafish. Remarkably, the results were in accordance with those derived from the transcriptomic and metabolomics profiles. Specifically, in the zebrafish ovaries of the ETO exposure group, the content of ROS was significantly elevated (Figure A), which served as solid evidence that chronic exposure to ETO indeed induced oxidative stress in the zebrafish ovaries. Additionally, oxidative-stress-related gene expression was evaluated via qRT-PCR (Figure B). Nuclear factor E2-related factor 2 A (nrf2a), catalase (cat), and gpx1 (glutathione peroxidase 1) are all important regulators in oxidative stress. ETO exposure markedly downregulated gpx1 and nrf2a expression, indicating an impaired antioxidant system in ovary in zebrafish. nrf2a serves as the central regulator of cellular antioxidant responses. Under oxidative stress, nrf2a activates the expression of antioxidant enzymes such as cat and gpx1 to neutralize ROS and mitigate oxidative damage. Notably, prolonged or severe oxidative stress triggers a feedback mechanism that suppresses nrf2a expression to prevent excessive antioxidant activation. As a canonical nrf2a target gene, gpx1 expression reduction can mainly be attributed to nrf2a inhibition. In contrast, cat expression exhibits partial nrf2a independence. Consequently, although partial antioxidant compensation is achieved through catalysis, the suppressed nrf2a/gpx1 axis ultimately results in insufficient ROS clearance capacity. This mechanistic imbalance leads to the incomplete resolution of oxidative stress. These findings suggest that ETO exacerbates oxidative stress, damaging the ovaries. ,

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Compared with the control, chronic exposure to ETO induces oxidative stress in the ovary of zebrafish. (A) Fluorescence staining of ROS in the ovary. (B) The expression of mRNA in the ovaries compared with control. (Significance levels: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Oxidative stress is known to act as an ″initiator″ for oocyte aging and reproductive pathologies, causing follicular atresia, abnormal meiosis, reduced fertilization rates, delayed embryonic development, and PCOS. Ovarian follicular atresia, often linked to granulosa cell apoptosis, has been associated with ROS-induced oxidative stress, as observed in oxidant-treated mice. The ROS can cause severe damage to the oocytes by interfering with their normal physiological functions and cellular structures. Additionally, it triggers apoptosis in ovarian granulosa cells. Mechanistically, numerous studies have shown that ROS induce DNA breaks as well as reduce base oxidation and DNA repair in the GC, reducing the transfer of nutrients and survival factors to the oocyte, leading to apoptosis and initiating follicular atresia. The apoptosis process involves a series of complex molecular and cellular events, ultimately leading to disruption of normal cellular homeostasis within the ovaries. As a consequence of these adverse effects, the zebrafish ovaries undergo significant morphological and functional alterations, presenting a polycystic ovary-like appearance. This polycystic ovary-like change is not only a visible manifestation of the damage but also indicates a profound disturbance in the ovarian microenvironment.

Furthermore, the presence of excessive ROS and the resulting ovarian damage seem to play a role in promoting the transmission of ETO from maternal oocytes to the offspring. It is hypothesized that the damaged oocytes, which have been affected by ETO-induced oxidative stress, might act as carriers, facilitating the transfer of ETO to the next generation. This potential mechanism of transgenerational transmission of ETO through maternal oocytes is of great interest, as it may have far-reaching implications for understanding the long-term impact of ETO exposure on the reproductive system of zebrafish and potentially other organisms, highlighting the need for further in-depth investigations to elucidate the underlying molecular and cellular mechanisms involved.

3.7. ETO Exposure Results in Altered Ovarian Proteomic Profiles in F0 Zebrafish

To explore how ETO exposure affects ovarian proteins, we conducted a TMT-based quantitative proteomic analysis on ovaries from zebrafish exposed to 200 μg/L ETO and controls. A total of 210 differentially expressed proteins (DEPs) were identified, with 99 downregulated and 111 upregulated proteins (Figure A–C). KEGG pathway analysis revealed that downregulated proteins were primarily enriched in four pathways, while upregulated proteins were associated with linoleic acid metabolism, endocytosis, glycerophospholipid metabolism, ether lipid metabolism, base excision repair, and the mTOR signaling pathway (Figure D). Gene ontology (GO) analysis highlighted several biological processes (BP) linked to the DEPs, such as the phospholipid catabolic process, negative regulation of TORC1 signaling, COPII-coated vesicle budding, and TORC1-related signaling processes (Table S4). The mammalian target of rapamycin (mTOR) is a conserved serine/threonine kinase existing as two complexes, mTORC1 and mTORC2, which regulate numerous cellular functions, including cell proliferation, adhesion, metabolism, and invasion. , The activation or inactivation of mTORC1 depends on signals like nutrients, energy, growth factors, and hormones, including estradiol (E2). Dysregulation of mTOR signaling is implicated in conditions such as cancer, diabetes, neurodegenerative disorders, aging, and reproductive diseases.

6.

6

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Chronic exposure to ETO induces alterations of ovarian proteomic in F0 zebrafish. (A) The circular heatmap of different expressed proteins between the control and ETO-treated (200 μg/L) group. (B) Volcano map based on DEPs. (C) Pie chart of DEPs due to ETO exposure. (D, E) Enrichment analysis of KEGG function based on downregulated and upregulated DEPs, respectively. (F) Pathway of multiomics integrated analysis induced by ETO exposure. The blue font represents downregulation, and the red font represents upregulation.

In female reproduction, mTOR signaling is crucial for regulating ovarian reserve, follicle development, oocyte meiotic maturation, ovarian aging, and steroidogenesis in ovarian somatic cells. − For instance, E2 has been shown to activate mTORC1 signaling in vaginal epithelium. Inhibition of mTORC1 in rats through intracerebroventricular administration of rapamycin resulted in reduced LH and E2 levels, delayed vaginal opening, and ovarian and uterine atrophy. Recent studies have linked mTOR signaling to PCOS, ovarian cancer, and premature ovarian failure. , Kuang et al. reported elevated mTOR expression and phosphorylation in granulosa cells of PCOS patients compared to non-PCOS individuals. Inhibition of mTOR with small-molecule inhibitors has shown therapeutic potential for conditions like PCOS and premature ovarian failure while preserving ovarian function during chemotherapy. , The ovarian levels of the phosphorylated mechanistic target of rapamycin (p-mTOR) and the phosphorylated ribosomal protein S6 kinase (p-S6K) in zebrafish were quantified using ELISA kits. ETO exposure significantly increased the ovarian levels of both proteins (Figure S4). As critical regulators of mTOR signaling activity, p-mTOR represents the activated form of mTOR, while p-S6K serves as a direct downstream effector of mTORC1. The marked elevation in these phosphorylation events demonstrates overactivation of the mTORC1 signaling pathway.

In a nutshell, our findings suggest that excessive activation of the mTOR signaling pathway, particularly mTORC1, may contribute to ETO-induced PCOS. Further investigation of this pathway could elucidate the mechanisms underlying PCOS pathogenesis and progression, offering potential therapeutic targets for reproductive diseases.

3.8. Integrated Multiomics Analysis Reveals the Mechanisms of Reproductive Toxicity Induced by ETO Exposure in Zebrafish

Advances in multiomics technologies, including transcriptomics, proteomics, and metabolomics, have enabled detailed insights into disease mechanisms. In this study, we employed multiomics approaches to investigate the mechanisms underlying ETO-induced polycystic ovary syndrome (PCOS)-like lesions in zebrafish ovaries (Figure F). Chronic ETO exposure resulted in significant metabolic imbalances, excessive reactive oxygen species (ROS) production, and disrupted unsaturated fatty acid and glutathione metabolism profiles. Additionally, ovarian sex hormone levels (E2 and T) were significantly elevated, and the mTOR signaling pathway was hyperactivated. The mTOR pathway, comprising mTORC1 and mTORC2 complexes, regulates metabolism, immune responses, autophagy, and cellular proliferation. − Chronic ETO exposure led to ROS accumulation, which activates AMP-activated protein kinase (AMPK), which enhances the inhibition of mTORC1. Dysregulation of this pathway, associated with granulosa cell proliferation and steroidogenesis, contributes to ovarian dysfunction, follicular abnormalities, and PCOS progression. Furthermore, by integrating protein interaction data with transcriptomics, we identified three overlapping genes, namely, itln1, spa17, and tph1b, between the proteomic and transcriptomic data sets (Figure S5A, B). PRM-based quantification confirmed the downregulation of itln1 and spa17, consistent with the findings in PCOS (Table S5). Itln1 is implicated in insulin resistance, , while spa17 is associated with fertilization and ovarian pathologies. , Additionally, tph1b, crucial for serotonin synthesis, may link endocrine dysregulation and behavioral changes in PCOS.

These findings highlight the intricate molecular interactions caused by ETO exposure, particularly the role of oxidative stress and mTOR signaling in driving PCOS-like ovarian changes in zebrafish.

A limitation of this study is that the concentration of ETO exposure is higher than that in the environment. While this elevated exposure range enabled mechanistic investigation of toxicity pathways, it may reduce direct environmental extrapolation. Such high-dose approaches remain scientifically valid for identifying potential hazard thresholds and molecular mechanisms, although their predictive value for ecological risk assessment requires cautious interpretation. Our findings emphasize the need to investigate low-dose exposure to better characterize the environmental health risks of ETO. Future studies employing environmentally relevant concentrations will be critical for establishing accurate dose–response relationships and ecological safety thresholds.

4. Conclusions

This study utilized a combined multiomics analysis approach integrating transcriptomics, metabolomics, and proteomics to investigate the reproductive toxicity and transgenerational effects of chronic etomidate (ETO) exposure on zebrafish. Our findings reveal that ETO exposure delays the maturation of primary oocytes in maternal zebrafish, disrupts the normal functional structure of the ovaries, disturbs sex hormone levels, and diminishes reproductive capacity, manifesting in polycystic ovary syndrome (PCOS)-like ovaries. Additionally, ETO exposure can be transmitted to offspring through the maternal ovaries, leading to increased mortality and deformity rates and reduced locomotor activity in the larvae. Transcriptomic and metabolomic data indicate disruptions in unsaturated fatty acid (LA) and glutathione metabolism, suggesting oxidative stress. Further evidence of ovarian oxidative stress and damage induced by ETO was confirmed by ROS fluorescence staining. The multiomics analysis revealed that the overactivation of mTORC1 plays a key role in the development of polycystic ovaries in zebrafish. In summary, this study demonstrates that excessive ETO exposure can exacerbate ovarian damage, induce PCOS-like changes, and cause reproductive toxicity and transgenerational effects through mTORC1 hyperactivation and increased oxidative stress. These findings offer novel insights into the molecular mechanisms of ETO-induced reproductive toxicity and transgenerational effects and highlight the value of multiomics approaches in evaluating the impact of environmental toxicants on reproductive function and offspring development.

Supplementary Material

eh5c00156_si_001.pdf (407.9KB, pdf)

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Grants No.22206072 and 82293652) and the Science and Technology Program of Guangzhou (2024A04J2742).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/envhealth.5c00156.

  • LC-MS/MS operation conditions, mRNA expression in ovaries vs control, LH content, DEMs, p-mTOR and p-S6K contents, integrated transcriptomics and proteomics results, primers used in this study, ETO contents, GO data based on DEGs and DEPs, contents of the three proteins (PDF)

#.

X.Li. and X.Lin. contributed equally to this work.

The authors declare no competing financial interest.

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