Mechanism of Liver Injury Induced by Cr⁶⁺ in Zebrafish and Protective Effect of Selenomethionine

Simple Summary

Hexavalent chromium (Cr⁶⁺) is a highly toxic environmental pollutant and a Group 1 carcinogen that accumulates in the liver via the food chain, posing significant health risks. In this study, we utilized liver-transgenic zebrafish to investigate the mechanisms of Cr⁶⁺-induced liver damage and evaluated the protective potential of selenomethionine (Se-Met). We discovered that Cr⁶⁺ exposure leads to hepatic steatosis (fatty liver) and mitochondrial dysfunction by triggering “ferroptosis”—a specific type of cell death linked to iron accumulation and lipid peroxidation. This toxicity was driven by the suppression of the protective protein GPX4 and the depletion of glutathione (GSH). Crucially, both a specific ferroptosis inhibitor (Fer-1) and low-dose Se-Met effectively alleviated these defects. Further analysis revealed that Se-Met exerts its protection by regulating lipid metabolism pathways. In conclusion, our findings identify ferroptosis as a key driver of chromium toxicity and suggest that Se-Met could serve as a safe, effective feed additive in aquaculture to mitigate the risks of environmental heavy metal contamination.

Abstract

Hexavalent chromium (Cr⁶⁺) is a potent environmental toxicant known to accumulate in the liver; however, the molecular underpinnings of its hepatotoxicity remain incompletely understood. In this study, we investigated the biochemical mechanisms of Cr⁶⁺-induced liver injury and the protective efficacy of selenomethionine (Se-Met) using a transgenic zebrafish model. We demonstrate that exposure precipitates severe hepatic steatosis and mitochondrial dysfunction, characterized by the dysregulation of lipid metabolism genes and the activation of ferroptosis pathways. Specifically, Cr⁶⁺ toxicity was driven by the depletion of glutathione (GSH) and the suppression of the anti-ferroptotic protein glutathione peroxidase 4 (GPX4). Notably, these pathological alterations were significantly attenuated by both the ferroptosis inhibitor ferrostatin-1 (Fer-1) and low-dose Se-Met. Furthermore, transcriptomic profiling revealed that Se-Met exerts its protective effects primarily by modulating glycerolipid metabolism, thereby mitigating lipid accumulation. Collectively, our findings establish ferroptosis as a critical driver of Cr⁶⁺-induced hepatotoxicity and highlight Se-Met as a promising biochemical intervention to mitigate chromium-associated hepatic damage in aquaculture systems.

1. Introduction

Chromium is a very common heavy metal pollutant [1]. It enters the environment through natural processes or human activity and can be found in several different forms [2]. Chromium mainly exists as trivalent chromium (Cr³⁺) and hexavalent chromium (Cr⁶⁺) in aquatic environments. Oxidation of Cr³⁺ to Cr⁶⁺ is rare under natural environmental conditions and only occurs in specific circumstances (aerobic acidic soils (pH < 5) with strong oxidants) [3,4]. Most Cr⁶⁺ in water comes from industrial wastewater (such as electroplating and tanning), landfills, and cooling towers. Additionally, it can enter drinking water by leaching from supply pipelines. High-concentration Cr⁶⁺ wastewater (50–500 mg/L) discharged from these industries far exceeds the national discharge standard (1.5 mg/L), and forms local polluted microenvironments (e.g., aquaculture ponds adjacent to industrial zones) with Cr⁶⁺ concentrations reaching 50–120 mg/L in the receiving water [5,6,7,8,9]. Dhal et al. and Bartlett showed that Cr⁶⁺ is the most oxidizing, mobile, and toxic chromium species. It also biomagnifies along the food chain [10,11,12]. Cr⁶⁺ targets multiple systems, inducing respiratory diseases and cancer, while damaging internal organs like the liver and kidneys [10,13,14]. The International Agency for Research on Cancer (IARC) classifies inhaled Cr⁶⁺ as a human carcinogen [15]. Ingesting contaminated water also leads to Cr⁶⁺ accumulation in multiple organs [16]. Studies show that Cr⁶⁺-exposed rats exhibit progressive proteinuria, alongside marked increases in hepatic and mitochondrial lipid peroxidation [17,18]. Research confirms that Cr⁶⁺ causes severe hepatotoxicity. Its mechanisms include entering cells via phosphate transporters, where it is reduced to Cr³⁺, triggering excessive reactive oxygen species (ROS) production, lipid peroxidation, and mitochondrial dysfunction [19,20,21,22,23]. Excessive ROS induce oxidative stress, leading to cellular damage of proteins, lipids, and DNA that compromises organ integrity [24]. Liver injury is characterized by lipid peroxidation and hepatocyte death, including ferroptosis. This iron-regulated process triggers the Fenton reaction, inducing both hepatocyte ferroptosis and potential liver fibrosis [25].

Ferroptosis is countered by polyunsaturated fatty acid-derived phospholipids (PUFA-PLs). Both PUFA synthases and the AMPK pathway indirectly govern this process by modulating lipid metabolism [26,27]. Nrf2 is a key transcription factor regulating intracellular stress responses and redox homeostasis [28,29]. For instance, Nrf2 regulates PPARγ, and PPARγ agonists enhance its expression in an Nrf2-dependent manner [30]. As a core ferroptosis-regulating antioxidant enzyme, GPX4 plays a central role by specifically reducing LOOH to non-toxic LOH with GSH as a cofactor [31,32,33]. This inhibits iron-dependent Fenton reactions and lipid ROS accumulation (key to ferroptosis) [32]. Wang et al. [33] confirmed conserved the LOOH reduction activity of Coho salmon liver GPX4 with mammals. Thus, GPX4 downregulation induces intracellular LOOH accumulation and ferroptotic liver cell death. As the central regulator of iron metabolism, the liver secretes hepcidin to maintain homeostasis [34]. It internalizes iron through two main routes: TFR1 for transferrin-bound iron (total iron binding capacity, TIBC) and SLC39A14 for non-transferrin-bound iron (NTBI) [35,36,37]. As a specific ferroptosis inhibitor, Ferrostatin-1 (Fer-1) blocks lipid peroxidation by converting lipid radicals into stable products via hydrogen donation [38]. In HT-1080 cells, Fer-1 effectively prevents erastin-induced ferroptosis by scavenging ROS and protecting against oxidative stress [39]. ACSL4 regulates lipid biosynthesis and ferroptosis by acylating arachidonic acid (AA) and adrenic acid (AdA) into phosphatidylethanolamine (PE) [40,41]. Research by Wang et al. highlights rosiglitazone (ROSI) as an effective inhibitor of renal ferroptosis via ACSL4 downregulation [42]. This protective effect is consistently linked to inhibited ACSL4 activity, which subsequently reduces lipid peroxidation markers like malondialdehyde (MDA) and limits cellular iron overload [43,44,45].

In addition, studies have demonstrated that selenium (Se) can antagonize Cr⁶⁺ by regulating the homeostasis of different elements [46]. Selenium is an important component of glutathione peroxidases (GPXs). By boosting the activity of these antioxidant enzymes, selenium scavenges hydrogen peroxide and organic peroxides, thereby reducing oxidative stress damage [47].

Zebrafish (Danio rerio) is an excellent experimental model organism for studying liver diseases. It has a short reproductive cycle and high fecundity, and it requires simple experimental equipment. Zebrafish embryos are transparent in the early stages of development, allowing monitoring of the entire organogenesis process. In addition, zebrafish embryos have the advantages of rapid development, low rearing costs, and high sensitivity to drugs [48]. Zebrafish have become an ideal model for studying environmental toxicants and drug mechanisms [49]. In particular, the development of liver-specific transgenic zebrafish has enabled their frequent use in the direct detection of hepatotoxicity, evaluation of liver cancer, and assessment of environmental pollutants [50,51]. This study used Tg(fabp10a: DsRed) liver transgenic zebrafish to investigate the inhibitory effect of selenomethionine on Cr⁶⁺-induced hepatotoxicity and its underlying mechanisms at the morphological, behavioral, histological, genetic, and protein levels.

2. Materials and Methods

2.1. Experimental Animals

AB strain zebrafish and transgenic Tg(fabp10a:DsRed) zebrafish (liver-specific expression) were obtained from the China Zebrafish Resource Center (Wuhan, China) and propagated at the College of Animal Science and Technology, Inner Mongolia Minzu University. The zebrafish were maintained in a recirculating aquaculture system (Beijing Aisheng Technology Co., Ltd., Beijing, China) under standardized conditions: 14 h light/10 h dark photoperiod, conductivity of 480 μS/cm, water temperature of 28 ± 0.5 °C, dissolved oxygen level of 6 mg/L, and pH range of 6.5–8.5. Zebrafish were fed twice daily (morning and evening) with commercial zebrafish feed and Artemia nauplii, with uneaten feed and feces removed promptly. Adult zebrafish were paired in spawning tanks, and the resulting embryos were collected and rinsed thoroughly with zebrafish rearing medium (ZR solution: 22.64 g sodium chloride (NaCl), 0.745 g potassium chloride (KCl), and 2.664 g calcium chloride (CaCl₂) dissolved in 1 L of distilled water). All zebrafish husbandry and experimental protocols were approved by the Animal Ethics Committee of Inner Mongolia Minzu University (Approval No.: NMD-DW-2025-06-08).

2.2. Experimental Reagents

Cr⁶⁺(Reference Material No.: GSB 04-1723-2004) was purchased from the General Research Institute for Nonferrous Metals (Beijing, China). Selenomethionine (Se-Met) was obtained from Sigma (Sigma-Aldrich, St. Louis, MO 63103, USA). Ferroptosis inhibitors (Fer-1; ROSI; purity ≥ 99.98%) were purchased from MCE (MedChemExpress, Shanghai, China). Kits for MDA detection, the reduced GSH assay, and ALT/GPT activity determination were all acquired from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The iron ion (Fe²⁺) content detection kit was purchased from Shanghai Enzyme-Linked Biotechnology Co., Ltd. (Shanghai, China). The protein quantification kit and SDS-PAGE gel preparation kit were obtained from Beyotime Biotechnology (Shanghai, China). All other reagents used were purchased from Beyotime Biotechnology (Shanghai, China).

2.3. Exposure and Observing

To detect the mortality rate of zebrafish caused by Cr⁶⁺, zebrafish larvae were exposed to different concentrations of Cr⁶⁺. Healthy 72 h post-fertilization (hpf) AB strain zebrafish larvae with a normal morphology were selected as experimental subjects, and the specific exposure method was carried out as follows. Larvae were exposed to Cr⁶⁺ at concentrations of 70 μg/mL, 100 μg/mL, 140 μg/mL, 160 μg/mL, 200 μg/mL and 500 μg/mL or higher, and the exposure period was maintained until the larvae developed to 120 hpf. During the exposure period, larval survival was recorded every 24 h. After the termination of Cr⁶⁺ exposure, survival rates were calculated, and the data were statistically analyzed by one-way ANOVA and Dunnett’s test (p < 0.05).

To investigate the mechanism of Cr⁶⁺-induced liver damage in zebrafish, experiments involving co-exposure to Cr⁶⁺ and/or ferroptosis inhibitors or Se-Met were conducted. Zebrafish embryos at 4 h post-fertilization (4 hpf) were selected for the acute exposure experiment. Prior to treatment, embryos were rinsed thoroughly with zebrafish rearing medium and transferred to 6-well plates with the following experimental design: control group (0.1% dimethyl sulfoxide, DMSO, Sigma-Aldrich, St. Louis, MO, USA), inhibitor alone groups (1 μM Fer-1, 0.1 μg/L Se-Met, 50 nM ROSI), Cr⁶⁺ exposure groups (70 μg/mL and 100 μg/mL), and inhibitor intervention groups (Fer-1 + Cr⁶⁺, Se-Met + Cr⁶⁺, ROSI + Cr⁶⁺). Each treatment group included 3 biological replicates, with 10 embryos per replicate. The exposure protocol was performed in two phases: from 4 hpf to 72 hpf, embryos were pretreated with the respective inhibitors (Fer-1, Se-Met, or ROSI) at the aforementioned concentrations; subsequent to pretreatment, embryos were exposed to Cr⁶⁺ from 72 hpf to 120 hpf. At 120 hpf, juvenile zebrafish were collected for subsequent analyses (Figure 1A). During the entire exposure period, embryo/larval development was monitored every 24 h, and the number of deaths, hatching rates, and deformity incidences were recorded. Liver-specific transgenic zebrafish were employed to assess Cr⁶⁺-induced hepatic injury via red fluorescence imaging. Briefly, zebrafish from each treatment group were collected and anesthetized with 0.02% tricaine methanesulfonate. Samples were placed on glass slides and observed under a fluorescence microscope (Leica DFC450C, Wetzlar, Hesse, Germany) with the excitation wavelength set to 558 nm and emission wavelength to 610 nm. After fluorescence excitation, high-resolution images of the zebrafish liver were captured, and Image J software (Version 1.8.0) was used for quantitative analysis: the fluorescence area and fluorescence intensity were measured by defining regions of interest (ROIs) covering the entire liver tissue. An increased fluorescence area and intensity directly correlate with more severe Cr⁶⁺-induced hepatic injury, with the transgene ensuring liver-specific red fluorescence (no off-target signal in other organs). Specificity to Cr⁶⁺ was confirmed by dose-dependent changes in fluorescence parameters and Se-Met-mediated rescue. Each treatment group included 30 transgenic zebrafish, divided into 3 biological replicates (n = 10 per replicate). The sample size for each experiment is shown in Table S1.

Figure 1. Zebrafish experimental flow chart. (A) Zebrafish larva: 4 hpf embryos were pretreated with Fer-1/ROSI (ferroptosis inhibitors) or Se-Met (4–72 hpf), then exposed to Cr⁶⁺ (70/100 μg/mL, 72–120 hpf). Control and single-factor groups (Fer-1/ROSI/Se-Met/Cr⁶⁺) were set. All groups were rinsed at 72 hpf, observed daily until 120 hpf (10 embryos/group, 3 replicates, n = 3). (B) Adult zebrafish: Exposed to Cr⁶⁺ (0.05/2 mg/L) and fed diets with/without Se-Met (15 fish/group, 3 replicates, n = 3). Water and solutions renewed every 2 days, fed at fixed times. Experimental duration: 28 days.

Healthy 4-month-old adult zebrafish were used for the chronic exposure experiment. The experimental groups consisted of a control group, a Se-Met supplementation group, Cr⁶⁺ exposure groups (0.05 mg/L and 2 mg/L), and Se-Met protection groups (Se-Met + Cr⁶⁺). Se-Met was administered as a feed additive at a concentration of 1 μg/g [52,53]. Zebrafish were fed twice daily (morning and evening) with the respective diets, and the experiment lasted for 28 days. During the experimental period, water and test substances were renewed every 2 days, and feces were removed daily to maintain the water quality. At the end of the experiment, zebrafish were anesthetized with tricaine methanesulfonate (MS-222), and livers were dissected aseptically for subsequent biochemical and molecular analyses (Figure 1B). The sample size for each experiment is shown in Table S2.

2.4. Histological Examination and Enzyme Activity Assay

Zebrafish from each group were collected, euthanized with MS-222, and fixed overnight in 4% paraformaldehyde (PFA) solution. Samples were sequentially dehydrated through a graded ethanol series (70%, 80%, 90%, 95%, 100% I, 100% II), cleared with xylene, embedded in paraffin, and sectioned into 3 μm thick slices. After drying, sections were dewaxed with xylene, rehydrated via a graded ethanol series, and subjected to hematoxylin–eosin (H&E) staining or immunohistochemical (IHC) staining.

For IHC staining: Rehydrated sections were subjected to antigen retrieval with 0.1% proteinase K (37 °C, 15 min), followed by refixation with 4% PFA. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Sections were incubated overnight at 4 °C with primary antibody (GPX4 Rabbit Polyclonal Antibody, 1:200 dilution, Beyotime International, Shanghai, China), then washed with PBS-T and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H+L) (Beyotime, China) at room temperature for 2 h. Chromogenic reaction was performed using DAB substrate. After drying in a fume hood, sections were mounted and observed/photographed under a microscope (Leica M205, Wetzlar, Hesse, Germany).

2.5. Enzyme Activity and Iron Content Detection

Zebrafish juveniles at 120 hpf were collected from each group to detect oxidative stress-related indicators and Fe²⁺ content.

For enzyme activity assays: Sample homogenization was performed using ice-cold phosphate-buffered saline (PBS, pH 7.4) at a volume-to-tissue mass ratio of 9:1 (v/w); the supernatant was collected for subsequent analyses. The total protein concentration was quantified using a BCA protein assay kit. The levels of reduced GSH, MDA, and the activity of ALT/GPT were determined strictly following the manufacturer’s instructions of the corresponding kits. All kits are colorimetric-based; absorbance was measured at the respective wavelengths with a microplate reader after the assay was conducted per the kit instructions, and results were analyzed accordingly.

For Fe²⁺ content detection: Approximately 0.1 g of tissue from 120 hpf juveniles was homogenized in 1 mL of extraction buffer on ice. The homogenate was centrifuged at 12,000× g rpm for 5 min at 4 °C, and the supernatant was retained on ice. According to the kit protocol (Shanghai Enzyme-Link Biotechnology Co., Ltd. Shanghai, China), distilled water, standards, and samples were sequentially added to centrifuge tubes, thoroughly mixed, and incubated at room temperature for 15 min. A 200 μL aliquot of each supernatant was transferred to a microplate, and the absorbance was measured at 562 nm using a microplate reader. The Fe²⁺ content was calculated based on the standard curve.

2.6. Ultrastructural Examination

Liver tissue from adult zebrafish was used for ultrastructural examination analysis instead. After 4 weeks of Cr⁶⁺ exposure, zebrafish livers were collected from each group (n = 3) and fixed in 2.5% glutaraldehyde for 12 h. Samples were then sent to Pinuofei Biotechnology Co., Ltd. (Wuhan, China) for subsequent processing. Liver tissues were stained with saturated uranyl acetate, and ultrastructural observations were performed using a transmission electron microscope (TEM). The number of mitochondrial cristae was quantitatively analyzed using Image J software.

2.7. Transcriptome

2.7.1. Library Preparation for Transcriptome Sequencing

Liver tissues of adult zebrafish from each group were collected for total RNA extraction, and subsequent experiments were performed with reference to the method described by Chen et al. [54]. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. The purified mRNA was randomly fragmented with divalent cations in NEB Fragmentation Buffer, and libraries were constructed via either the standard NEB library construction method or the strand-specific library construction method. After library construction, preliminary quantification was conducted using a Qubit 2.0 Fluorometer (Hermo Fisher Scientific, Waltham, MA, USA), and libraries were diluted to a concentration of 1.5 ng/μL. The insert size of libraries was detected using an Agilent 2100 Bioanalyzer, Agilent Technologies, Beijing, China; once the insert size met the expected standard, quantitative real-time PCR (qRT-PCR) was used to accurately determine the effective concentration of libraries (requirement: effective concentration > 1.5 nM), to ensure library quality.

2.7.2. GO and KEGG Enrichment Analysis of Differentially Expressed Genes

The Kyoto Encyclopedia of Genes and Genomes (KEGG; http://www.genome.jp/kegg/ accessed on 14 July 2025) is a database resource for interpreting high-level functions and utilities of biological systems (e.g., cells, organisms, ecosystems) based on molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-throughput experimental technologies. ClusterProfiler software, version 3.23 was used to perform Gene Ontology (GO) functional enrichment analysis and KEGG pathway enrichment analysis on the differentially expressed gene (DEG) set, to clarify the biological functions and metabolic pathways associated with DEGs.

2.8. Total RNA Extraction and qRT-PCR Detection

Liver tissues were collected from zebrafish in each group for experiments, with 3 biological replicates per group (n = 3) and approximately 25 zebrafish per replicate. Total RNA was extracted using the Trizol reagent method, referring to the protocol described by Shaw et al. [55]. The purity of the extracted total RNA was determined using a NanoDrop ND-100 spectrophotometer, with the requirement of an A260/A280 ratio > 1.8. Complementary DNA (cDNA) was synthesized via reverse transcription using a cDNA reverse transcription kit (Applied Biosystems Inc., Foster City, CA, USA). qRT-PCR was performed in a 20 μL reaction volume containing 100 ng of cDNA template. The qRT-PCR reactions were run on a Plus One real-time quantitative PCR system (AB Biosynthesis, Wilmington, DE, USA). Primers for target genes (tf, tfr, fpn, gpx4, slc7a11, etc.) are listed in Table S5, with 18S rRNA used as the internal reference gene. The relative expression levels of target genes were calculated using the 2^−△△Ct method.

2.9. Western Blotting

Next, 120 hpf zebrafish juveniles (3 replicates/group, ≥120 fish/replicate, Figure S2) and adult zebrafish livers were homogenized, centrifuged (12,000× g rpm, 30 min, 4 °C), and supernatants stored at −80 °C. The protein concentration was quantified via a BCA kit (Beyotime, China). Equal protein amounts were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk (TBST, 1 h, RT), and incubated with primary antibody (GPX4, 1:2000, Sanying, China; 4 °C, overnight). After TBST washes, membranes were incubated with HRP-conjugated secondary antibody (2 h, RT), washed, and developed with ECL. Band quantification was performed using Image J software.

2.10. Cr⁶⁺ Content Determination

Zebrafish juveniles at 120 hpf were collected for Cr⁶⁺ content analysis (20 juveniles per sample). Samples were thoroughly rinsed with distilled water, blotted dry, and transferred to a 50 mL beaker. After adding 1 mL distilled water and 2 mL nitric acid, the beaker was covered with a watch glass and heated at 80–90 °C for 30–60 min until complete tissue dissolution. Upon cooling to room temperature, 1 mL perchloric acid was added, and the mixture was heated at 120–130 °C for 20–30 min until the solution became clear and concentrated to 0.5–1 mL. After slight cooling, 1–2 mL distilled water was added, and the solution was heated at 80 °C for 10 min to concentrate to ~0.5 mL (repeated once). After complete cooling, the beaker inner wall was rinsed three times with 0.5% nitric acid (0.5–1 mL each time). The rinse solution and digestion mixture were combined and transferred to a 5 mL volumetric flask, which was made up to volume with 0.5% nitric acid. After thorough inversion mixing, the solution was filtered through a 0.22 μm organic-phase filter membrane, and the filtrate was collected for detection. According to the kit instructions (Wuhan Elite Biotechnology Co., Ltd. Wuhan, Hubei, China), chromogenic reagent, standards, and samples were sequentially added to a 96-well plate. After thorough mixing, the plate was incubated at 37 °C in the dark for 10 min. The absorbance of each well was measured at 540 nm using a microplate reader, and the Cr⁶⁺ content was calculated based on the standard curve.

2.11. Data Analysis

Statistical analyses were performed using GraphPad Prism 8.0.2 and SPSS 20.0 software. One-way analysis of variance (ANOVA) was used to compare differences among multiple groups, followed by Duncan’s multiple range test for post hoc comparisons. All data are expressed as the mean ± standard deviation (SD) of three biological replicates per group. Statistical significance was defined as follows: p > 0.05 (not significant), p < 0.05 (significant), p < 0.01 (moderately significant), and p < 0.001 (highly significant).

3. Results

3.1. Effects of Ferroptosis Inhibitors and Se-Met on Hatching Rate and Survival Rate of Zebrafish Larvae Induced by Cr⁶⁺

In the present study, following the termination of Cr⁶⁺ exposure, the survival rates of zebrafish (Danio rerio) larvae at 120 h post-fertilization (hpf) exhibited a distinct dose-dependent reduction pattern. The survival rate remained at 100% in groups exposed to Cr⁶⁺ concentrations of 70 μg/mL or lower; it decreased to 81.67% in the 100 μg/mL group, 53.33–66.67% in the 140–160 μg/mL groups, and 46.67% in the 200 μg/mL group; ultimately, no surviving larvae were detected in groups treated with Cr⁶⁺ concentrations of 500 μg/mL or higher (Figure 2A). Based on these survival data, the 120 h LC₅₀ of Cr⁶⁺ to zebrafish larvae was calculated to be 155 μg/mL, prompting the selection of 70 μg/mL and 100 μg/mL for subsequent mechanistic experiments to explore Cr⁶⁺ -induced toxicity and potential protective interventions.

Figure 2. Effects of Cr⁶⁺ on survival, mortality, and hatching rates of zebrafish (Danio rerio) larvae (120 hpf) and adults (28 days post-exposure), and protective effects of ferroptosis inhibitors/Se-Met. Adult assay: Adult zebrafish were chronically exposed to Cr⁶⁺ (0.05/2 mg/L) for 28 days, with/without organic selenium. Control and selenium-only groups were set. Daily survival recording for survival/mortality rates (10 adults/group, 3 biological replicates, n = 3). (A) Survival rate of zebrafish larvae exposed to different concentrations of Cr⁶⁺ (10 embryos per group, 3 replicates, n = 3). Larvae were exposed to different concentrations of Cr⁶⁺ at 72 hpf, and exposure was maintained until 120 hpf. During the exposure period, larval survival was recorded every 24 h. (B) Survival rates of adult zebrafish. Larval assay: 4 hpf zebrafish embryos were pretreated with Fer-1/ROSI (ferroptosis inhibitors) or Se-Met (4–72 hpf), then exposed to Cr⁶⁺ (70/100 μg/mL, 72–120 hpf). Control and single-factor groups were set. All groups were rinsed at 72 hpf, observed daily until 120 hpf to calculate survival and hatching rates (10 embryos/group, 3 replicates, n = 3). (C) Fer-1 group survival rate; (D) Fer-1 group hatching rate; (E) Se-Met group survival rate; (F) Se-Met group hatching rate; (G) ROSI group survival rate; (H) ROSI group hatching rate.

For the concentration selection of Se-Met, a pre-experiment was conducted with dose gradients set at 0.01, 0.1 and 1 μg/L for larvae, and 0.5, 1 and 2 μg/g feed for adults. The results identified 0.1 μg/L (larvae) and 1 μg/g (adults) as the lowest effective doses, which could mitigate Cr⁶⁺-induced hepatotoxicity (e.g., reduced steatosis) without causing selenium toxicity. Consistent with the findings of Arnold et al. [56] that Se-Met at concentrations ≥100 μg/L induces teratogenesis and oxidative stress in zebrafish embryos, the selected doses were far below this toxic threshold. Therefore, 0.1 μg/L and 1 μg/g were selected as the Se-Met concentrations for the protective experiments on zebrafish larvae and adults, respectively (Tables S3 and S4).

To further validate Cr⁶⁺-induced toxicity and the protective efficacy of potential interventions at relevant concentrations, the embryo hatching rates and larval survival under targeted treatments were subsequently analyzed. When zebrafish embryos reached 96 h post-fertilization (hpf), the hatching rate of all experimental groups reached 100% (Figure 2D,F,H). At 120 hpf, the survival rate of zebrafish larvae in the control group, inhibitor-only group, and 70 μg/mL Cr⁶⁺ exposure group remained at 100%. In contrast, the survival rate of the 100 μg/mL Cr⁶⁺ exposure group significantly decreased to 81.6% (p < 0.001). Notably, pretreatment with the ferroptosis inhibitor Fer-1, ROSI and Se-Met effectively improved the survival rate of zebrafish larvae to 95.3% (p < 0.001) compared to the 100 μg/mL Cr⁶⁺ exposure group (Figure 2C,E,G). Based on the aforementioned mortality data (Figure 2A), 70 μg/mL and 100 μg/mL Cr⁶⁺ were selected as the exposure concentrations for subsequent mechanistic experiments, further confirming the validity of the selected concentrations for mechanistic exploration.

To further investigate Cr⁶⁺-induced toxicity and organic selenium-mediated protection across zebrafish life stages, the survival rate of adult zebrafish following chronic exposure was also assessed. After 28 days of exposure to 0.05 mg/L or 2 mg/LCr⁶⁺ (with or without organic selenium treatment), all groups maintained a survival rate of over 95.33%. Notably, no significant differences in survival were observed among all experimental groups (p > 0.05, Figure 2B), indicating that adult zebrafish exhibit higher Cr⁶⁺ tolerance compared to larvae.

3.2. Protective Effects of Ferroptosis Inhibitors and Se-Met Against Cr⁶⁺-Induced Morphological Alterations in Zebrafish Larvae

At 120 hpf, the pericardial area, body length, yolk sac area, swim bladder area, hepatic fluorescent area, and fluorescent intensity of zebrafish larvae were quantified across all experimental groups. The results showed no significant differences in pericardial area and body length between the control group and all Cr⁶⁺ treatment groups (p > 0.05; Figure S1A–C,G–I). The 100 μg/mL Cr⁶⁺ group showed delayed yolk sac absorption, with a 1.32-fold increase in the yolk sac area-to-body length ratio compared with the control group (p < 0.01; Figure S1D–F), whereas the 70 μg/mL Cr⁶⁺ group showed no significant change (p > 0.05; Figure S1D–F). Meanwhile, the Se-Met co-treatment group showed a trend to alleviate this delayed yolk sac absorption.

The results demonstrated no significant differences in hepatic fluorescent area among the control group and the Fer-1 alone, Se-Met alone, and ROSI alone treatment groups (Figure 3D,G,J). Exposure to 70 μg/mL and 100 μg/mL Cr⁶⁺ significantly increased the hepatic fluorescent area of zebrafish larvae by 1.03-fold (p > 0.05) and 1.31-fold (p < 0.001) compared with the control group, respectively. While pretreatment with Fer-1 (Fer-1 + 70 μg/mL Cr⁶⁺), Se-Met (Se-Met + 70 μg/mL Cr⁶⁺), or ROSI (ROSI + 70 μg/mL Cr⁶⁺) tended to reduce the hepatic fluorescent area, these differences did not reach statistical significance. In contrast, pretreatment with Fer-1 (Fer-1 + 100 μg/mL Cr⁶⁺), Se-Met (Se-Met + 100 μg/mL Cr⁶⁺), or ROSI (ROSI + 100 μg/mL Cr⁶⁺) significantly attenuated the hepatic fluorescent area in zebrafish larvae, with reductions of 20.97% (p < 0.001), 19.67% (p < 0.001), and 18.5% (p < 0.001) relative to the 100 μg/mL Cr⁶⁺ exposure group, respectively (Figure 3P–R).

Figure 3. Effects of Cr⁶⁺ on zebrafish larvae morphology and protective effects of ferroptosis inhibitors/Se-Met. We treated 4 hpf zebrafish embryos with Fer-1/ROSI (ferroptosis inhibitors), Se-Met, and Cr⁶⁺ (70/100 μg/mL). Morphological changes were evaluated at 120 hpf (10 embryos/group, 3 replicates, n = 3). (A) Control; (B) 70 μg/mL Cr⁶⁺; (C) 100 μg/mL Cr⁶⁺; (D) Fer-1; (E) 70 μg/mL Cr⁶⁺+Fer-1; (F) 100 μg/mL Cr⁶⁺+Fer-1; (G) Se-Met; (H) 70 μg/mL Cr⁶⁺+Se-Met; (I) 100 μg/mL Cr⁶⁺+Se-Met; (J) ROSI; (K) 70 μg/mL Cr⁶⁺+ROSI; (L) 100 μg/mL Cr⁶⁺+ROSI; (M–O) swim bladder area/body length (Fer-1/Se-Met/ROSI groups); (P–R) hepatic fluorescence area/body length (Fer-1/Se-Met/ROSI groups); (S–U) hepatic fluorescence intensity/body length (Fer-1/Se-Met/ROSI groups). The letters (A–F) in each bar chart are defined as follows: (A) Control group (blank); (B) Fer-1/Se-Met/ROSI treatment group (green); (C) 70 μg/mL Cr⁶⁺ exposure group (orange); (D) 100 μg/mL Cr⁶⁺ exposure group (red); (E) Fer-1/Se-Met/ROSI protective group (Fer-1/Se-Met/ROSI + 70 μg/mL Cr⁶⁺, blue); (F) Fer-1/Se-Met/ROSI protective group (Fer-1/Se-Met/ROSI + 100 μg/mL Cr⁶⁺, purple). Statistical significance was compared between each treatment group and the control group. Statistical significance was compared between each treatment group and the control group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; Statistical significance was analyzed between the Fer-1/Se-Met/ROSI protective groups and the Cr⁶⁺-exposed groups. # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Cr⁶⁺ groups. Scale = 100 μm.

Consistent with the fluorescent area data, hepatic fluorescent intensity was comparable among the control group and the Fer-1 alone, Se-Met alone, and ROSI alone treatment groups (Figure 3S–U). Exposure to 70 μg/mL and 100 μg/mL Cr⁶⁺ significantly decreased hepatic fluorescent intensity by 23.31% (p < 0.001) and 35.73% (p < 0.001) compared with the control group, respectively. However, pretreatment with Fer-1 (Fer-1 + 70 μg/mL or 100 μg/mL Cr⁶⁺), Se-Met (Se-Met + 70 μg/mL or 100 μg/mL Cr⁶⁺), or ROSI (ROSI + 70 μg/mL or 100 μg/mL Cr⁶⁺) significantly restored the hepatic fluorescent intensity of zebrafish larvae (p < 0.05, p < 0.01, or p < 0.001).

Cr⁶⁺ exposure also led to a reduction in the swim bladder area of zebrafish larvae. Compared with the control group, the swim bladder area was significantly decreased by 10.47% (p < 0.05) and 19.8% (p < 0.001) in the 70 μg/mL and 100 μg/mL Cr⁶⁺ exposure groups, respectively (Figure 3M–O). Pretreatment with Fer-1 (Fer-1 + 70 μg/mL or 100 μg/mL Cr⁶⁺), Se-Met (Se-Met + 70 μg/mL or 100 μg/mL Cr⁶⁺), or ROSI (ROSI + 70 μg/mL or 100 μg/mL Cr⁶⁺) significantly reversed the reduction in swim bladder area (p < 0.05 or p < 0.001).

3.3. Protective Effects of Ferroptosis Inhibitors and Se-Met Against Cr⁶⁺-Induced Hepatocellular Alterations in Zebrafish Larvae

H&E staining results revealed that hepatocytes in the control group and Fer-1 alone, Se-Met alone, and ROSI alone treatment groups exhibited similar structural characteristics, with a regular morphology, tight and orderly arrangement, and well-developed cellular architecture (Figure 4D,G,J). Exposure to Cr⁶⁺ significantly reduced the hepatocyte count in zebrafish larvae. Compared with the control group, 70 μg/mL and 100 μg/mL Cr⁶⁺ exposure led to a significant decrease in hepatocyte number by 28.05% (p < 0.001; Figure 4B) and 52.18% (p < 0.001; Figure 4C), respectively. Notably, pretreatment with Fer-1, Se-Met, or ROSI significantly restored the hepatocyte count in Cr⁶⁺-exposed zebrafish larvae (p < 0.01 or p < 0.001; Figure 4P–R).

Figure 4. Effects of Cr⁶⁺ on zebrafish larvae histology and protective effects of ferroptosis inhibitors/Se-Met. We treated 4 hpf zebrafish embryos with Fer-1/ROSI (ferroptosis inhibitors), Se-Met, and Cr⁶⁺ (70/100 μg/mL) (10 embryos/group, 3 replicates, n = 3). At 120 hpf, larvae were subjected to tissue sectioning and H&E staining. (A) Control; (B) 70 μg/mL Cr⁶⁺; (C) 100 μg/mL Cr⁶⁺; (D) Fer-1; (E) 70 μg/mL Cr⁶⁺+Fer-1; (F) 100 μg/mL Cr⁶⁺+Fer-1; (G) Se-Met; (H) 70 μg/mL Cr⁶⁺+Se-Met; (I) 100 μg/mL Cr⁶⁺+Se-Met; (J) ROSI; (K) 70 μg/mL Cr⁶⁺+ROSI; (L) 100 μg/mL Cr⁶⁺+ROSI; (M–O) vacuole area (Fer-1/Se-Met/ROSI groups); (P–R) hepatocyte number (Fer-1/Se-Met/ROSI groups). The letters (A–F) in each bar chart are defined as follows: (A) Control group (blank); (B) Fer-1/Se-Met/ROSI treatment group (green); (C) 70 μg/mL Cr⁶⁺ exposure group (orange); (D) 100 μg/mL Cr⁶⁺ exposure group (red); (E) Fer-1/Se-Met/ROSI protective group (Fer-1/Se-Met/ROSI + 70 μg/mL Cr⁶⁺, blue); (F) Fer-1/Se-Met/ROSI protective group (Fer-1/Se-Met/ROSI + 100 μg/mL Cr⁶⁺, purple). Statistical significance was compared between each treatment group and the control group. ** p < 0.01, *** p < 0.001 vs. Control. Statistical significance was analyzed between the Fer-1/Se-Met/ROSI protective groups and the Cr⁶⁺-exposed groups. ## p < 0.01, ### p < 0.001 vs. Cr⁶⁺ groups. Scale = 100 μm.

In contrast to the hepatocyte count, Cr⁶⁺ exposure remarkably increased the hepatic steatosis area in zebrafish larvae. Compared with the control group, the hepatic steatosis area was significantly elevated by 5.88-fold and 14.06-fold in the 70 μg/mL and 100 μg/mL Cr⁶⁺ exposure groups, respectively (p < 0.001). Consistently, pretreatment with Fer-1, Se-Met, or ROSI significantly attenuated the Cr⁶⁺-induced increase in hepatic steatosis area (p < 0.01 or p < 0.001; Figure 4M–O).

Impact of Se-Met on Cr⁶⁺-Induced Dysregulation of GPX4 Expression

At 120 hpf, juvenile zebrafish from each experimental cohort were sampled to evaluate the protein expression of GPX4, a key regulatory enzyme in ferroptosis. Basal GPX4 expression levels were statistically comparable among the control group, Fer-1 monotherapy group, Se-Met monotherapy group, and ROSI monotherapy group (p > 0.05; Figure 5D,G,J). In stark contrast, exposure to Cr⁶⁺ at concentrations of 70 μg/mL and 100 μg/mL elicited pronounced declines in GPX4 expression: relative to the control group, the abundance of GPX4-positive cells was reduced by 46.78% (p < 0.001) and 69.85% (p < 0.001; Figure 5B,C), respectively. Notably, pretreatment with Fer-1 (Fer-1 + 70 μg/mL Cr⁶⁺ and Fer-1 + 100 μg/mL Cr⁶⁺ co-treatment groups) led to a dramatic restoration of GPX4-positive cell counts, with respective increases of 1.94-fold (p < 0.001) and 2.69-fold (p < 0.001; Figure 5E,F,M) compared to their corresponding Cr⁶⁺-only exposure groups. Treatment with Se-Met (Figure 5H,I) and ROSI (Figure 5K,L) exerted analogous protective effects, recapitulating the GPX4-rescue phenotype observed with Fer-1 pretreatment (Figure 5N,O).

Figure 5. Effects of Cr⁶⁺ on GPX4 IHC in zebrafish liver and protective effects of ferroptosis inhibitors/Se-Met. We treated 4 hpf zebrafish embryos with Fer-1/ROSI (ferroptosis inhibitors), Se-Met, and Cr⁶⁺ (70/100 μg/mL) (10 embryos/group, 3 replicates, n = 3). At 120 hpf, GPX4 antibody immunohistochemical staining was performed, and the positive cell rate per unit area was counted. (A) Control; (B) 70 μg/mL Cr⁶⁺; (C) 100 μg/mL Cr⁶⁺; (D) Fer-1; (E) 70 μg/mL Cr⁶⁺+Fer-1; (F) 100 μg/mL Cr⁶⁺+Fer-1; (G) Se-Met; (H) 70 μg/mL Cr⁶⁺+Se-Met; (I) 100 μg/mL Cr⁶⁺+Se-Met; (J) ROSI; (K) 70 μg/mL Cr⁶⁺+ROSI; (L) 100 μg/mL Cr⁶⁺+ROSI; (M–O) positive cell rate (positive cells/total cells, %) (Fer-1/Se-Met/ROSI groups). The letters (A–F) in each bar chart are defined as follows: (A) Control group (blank); (B) Fer-1/Se-Met/ROSI treatment group (green); (C) 70 μg/mL Cr⁶⁺ exposure group (orange); (D) 100 μg/mL Cr⁶⁺ exposure group (red); (E) Fer-1/Se-Met/ROSI protective group (Fer-1/Se-Met/ROSI + 70 μg/mL Cr⁶⁺, blue); (F) Fer-1/Se-Met/ROSI protective group (Fer-1/Se-Met/ROSI + 100 μg/mL Cr⁶⁺, purple). Statistical significance was compared between each treatment group and the control group. *** p < 0.001 vs. Control. Statistical significance was analyzed between the Fer-1/Se-Met/ROSI protective groups and the Cr⁶⁺-exposed groups. ### p < 0.001 vs. Cr⁶⁺ groups. Scale = 100 μm. Scale = 20 μm.

3.4. The Protective Effect of Se-Met Against Cr⁶⁺-Induced Mitochondrial Damage in Zebrafish

Livers were dissected from zebrafish in each experimental group (n = 3) for ultrastructural observation and quantitative analysis of mitochondrial morphology. In the control group and Se-Met alone group, hepatic mitochondria exhibited intact outer membranes and distinct, well-preserved cristae (Figure 6A,D). Exposure to a low concentration of Cr⁶⁺(0.05 mg/L) induced mild structural damage to both the outer and inner mitochondrial membranes (Figure 6B). In contrast, high-dose Cr⁶⁺ exposure (2 mg/L) triggered severe mitochondrial morphological disruption, characterized by extensive damage to the double mitochondrial membrane, accompanied by plasma membrane rupture, cytoplasmic vacuolization, and mitochondrial atrophy in some specimens (Figure 6C).

Figure 6. Effects of Cr⁶⁺ on liver mitochondria of adult zebrafish and protective effect of Se-Met. After 28-day treatment with Se-Met and/or Cr⁶⁺, livers were collected from each group of adult zebrafish. Liver mitochondrial changes were detected by electron microscopy, and the number of mitochondrial cristae per unit area was counted (3 livers/group, 3 replicates, n = 3). (A) Control; (B) 0.05 mg/L Cr⁶⁺; (C) 2 mg/L Cr⁶⁺; (D) Se-Met; (E) 0.05 mg/L Cr⁶⁺+Se-Met; (F) 2 mg/L Cr⁶⁺+Se-Met; (G) number of mitochondrial cristae per unit area. The letters (A–F) in each bar chart are defined as follows: (A): Control group (blank); (B): Se-Met treatment group (green); (C): 0.05 mg/L Cr⁶⁺ exposure group (orange); (D): 2 mg/L Cr⁶⁺ exposure group (red); (E): Se-Met protective group (Se-Met + 0.05 mg/L Cr⁶⁺, blue); (F): Se-Met protective group (Se-Met + 2 mg/L Cr⁶⁺, purple). Statistical significance was compared between each treatment group and the control group. *** p < 0.001 vs. control; # p < 0.05 vs. Cr⁶⁺ groups. Scale = 500 nm.

Quantitative analysis revealed that, relative to the control group, the density of mitochondrial cristae was reduced by 53.97% (p < 0.001; Figure 6E) and 73% (p < 0.001; Figure 6F) in the low- and high-dose Cr⁶⁺ groups, respectively. Notably, pretreatment with Se-Met effectively mitigated Cr⁶⁺-induced mitochondrial injury, and the cristae density was significantly restored to near-normal levels (p < 0.05; Figure 6G).

3.5. Effects of Se-Met and Fer-1 on Ferroptosis-Related Enzyme Activities in Zebrafish Exposed to Cr⁶⁺

As shown in Figure 7, treatment with 70 μg/mL and 100 μg/mL Cr⁶⁺ significantly reduced the GSH content in zebrafish, which was 16.5% (p < 0.001) and 36.92% (p < 0.001) of that in the control group, respectively. However, pretreatment with Fer-1 (1 μM Fer-1 + 70 μg/mL Cr⁶⁺ and 1 μM Fer-1 + 100 μg/mL Cr⁶⁺) and Se-Met (0.1 μg/L Se-Met + 70 μg/mL Cr⁶⁺ and 0.1 μg/L Se-Met + 100 μg/mL Cr⁶⁺) alleviated this decrease (p < 0.05 or p < 0.001; Figure 7A,D).

Figure 7. Effects of Cr⁶⁺ on zebrafish GSH, MDA and ALT levels and protective effects of Fer-1/Se-Met. We treated 4 hpf zebrafish embryos with Fer-1, Se-Met, and Cr⁶⁺ (70/100 μg/mL) (10 embryos/group, 3 replicates, n = 3). At 120 hpf, GSH, MDA and ALT levels were detected. (A) GSH content (Fer-1 group); (B) MDA content (Fer-1 group); (C) ALT content (Fer-1 group); (D) GSH content (Se-Met group); (E) MDA content (Se-Met group); (F) ALT content (Se-Met group). The letters (A–F) in each bar chart are defined as follows: (A): Control group (blank); (B): Fer-1/Se-Met treatment group (green); (C): 70 μg/mL Cr⁶⁺ exposure group (orange); (D): 100 μg/mL Cr⁶⁺ exposure group (red); (E): Fer-1/Se-Met protective group (Fer-1/Se-Met + 70 μg/mL Cr⁶⁺, blue); (F): Fer-1/Se-Met protective group (Fer-1/Se-Met + 100 μg/mLCr⁶⁺, purple). Statistical significance was compared between each treatment group and the control group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Cr⁶⁺ groups.

In contrast, exposure to 70 μg/mL and 100 μg/mL Cr6+ led to increased levels of MDA and ALT. Compared with the control group, the levels of MDA and ALT were increased by 1.93–2.88 fold (p < 0.001) and 1.38–2.02 fold (p < 0.001), respectively. Nevertheless, the elevation of MDA and ALT levels was mitigated after pretreatment with Fer-1 (1 μM Fer-1 + 70 μg/mL Cr⁶⁺ and 1 μM Fer-1 + 100 μg/mL Cr⁶⁺) or Se-Met (0.1 μg/L Se-Met + 70 μg/mL Cr⁶⁺ and 0.1 μg/L Se-Met + 100 μg/mL Cr⁶⁺) (p < 0.01 or p < 0.0001; Figure 7B,C,E,F).

3.6. Effects of Low-Dose Se-Met on the Liver Transcriptome of Adult Zebrafish Induced by Cr⁶⁺

3.6.1. Differential Gene Analysis

Volcano plots were used to analyze the differences in gene expression among the blank control group, Cr⁶⁺ exposure group, and Se-Met intervention group (Se-Met + Cr⁶⁺) (Figure 8). As shown in Figure 8A, compared with the control group, 222 genes were upregulated and 275 genes were downregulated in the Cr⁶⁺-exposed group. When compared with the Cr⁶⁺-exposed group, 111 genes were upregulated and 81 genes were downregulated in the Se-Met intervention group (Cr⁶⁺+Se-Met; Figure 8B). Venn diagram analysis revealed that a total of 734 genes were co-expressed across these four groups of genes; these genes may share common functions or participate in the same metabolic and signal transduction pathways. Compared with the control group and the Se-Met intervention group, the expression of most genes was upregulated in the Cr⁶⁺-exposed group (Figure 8C).

Figure 8. Volcano plots and heat map of differentially expressed genes (DEGs). After 28-day treatment with Se-Met and/or Cr⁶⁺, livers were collected from adult zebrafish for transcriptome sequencing and differential gene analysis (3 livers/group, 3 replicates, n = 3). (A) Volcano plot (Cr⁶⁺ vs. control); (B) volcano plot (Cr⁶⁺+Se-Met vs. Cr⁶⁺); red = upregulated genes, green = downregulated genes, blue = non-screened genes. (C) Volcano plot of Venn diagram analysis results (control, Cr⁶⁺, Cr⁶⁺+Se-Met groups); (D) heat map (control, Se-Met, Cr⁶⁺, Cr⁶⁺+Se-Met groups). Horizontal axis: sample names; vertical axis: normalized FPKM values of DEGs (red = higher expression, blue = lower expression).

3.6.2. GO Enrichment Analysis

Figure 9 shows the results of transcriptome sequencing. In this study, genes meeting the threshold criteria of |log₂Fold Change| ≥ 1 and a p-value ≤ 0.05 were selected for GO functional enrichment analysis. Based on the GO enrichment results, the top 30 most significant terms were selected to generate a bar chart; if the number of significant terms was less than 30, all identified terms were included in the chart.

Figure 9. Histogram of GO functional enrichment analysis. After 28-day treatment with Se-Met and/or Cr⁶⁺, livers were collected from adult zebrafish for transcriptome sequencing and GO enrichment analysis (3 livers/group, 3 replicates, n = 3). (A) Cr⁶⁺ vs. control; (B) Cr⁶⁺+Se-Met vs. Cr⁶⁺. Vertical axis: GO terms; horizontal axis: GO term enrichment significance level. Red = BP, green = CC, blue = MF.

The results indicated that Cr⁶⁺ significantly affected the biological processes (BP) category of GO terms. Compared with the control group, the Cr⁶⁺-exposed group exhibited high enrichment levels in biological processes, mainly involving tRNA aminoacylation, amino acid activation, carboxylic acid metabolic process, and female gametogenesis. The enriched pathways in the cellular component (CC) category were concentrated in cell structure-related functions, such as the cyclin-dependent protein complex, mitochondrial part, and proteasome regulatory subunit. In the molecular function (MF) category, ligase activity was the most significantly enriched term, followed by clear enrichment in aminoacyl-tRNA ligase activity and catalytic activity (Figure 9A).

Compared with the Cr⁶⁺-exposed group, the Se-Met intervention group showed enriched pathways in the BP category, which were mainly focused on the multicellular organismal process, thioester metabolic process, gametogenesis, ion transport (e.g., iron ion and transition metal ion transport), and reproduction-related processes. The enriched pathways in the CC category were dominated by the supramolecular fiber, spliceosome, mitochondrial respiratory chain complex, and peroxisome. In the MF category, oxygen binding had the highest enrichment level, along with evident enrichment in binding activities (e.g., carboxylic acid binding, growth factor binding, and fatty acid derivative binding) and enzyme activities (e.g., deaminase activity and transferase activity; Figure 9B).

3.6.3. KEGG Enrichment Analysis

Figure 10 presents the results of KEGG pathway enrichment analysis. In this study, genes with a padj ≤ 0.05 were selected for KEGG functional enrichment analysis. Based on the analysis results, the top 20 most significant pathways were chosen to construct a scatter plot. Compared with the control group, the Cr⁶⁺-exposed group showed more significant enrichment differences in pathways including aminoacyl-tRNA biosynthesis, fatty acid biosynthesis, and ribosome biogenesis (Figure 10A). In the KEGG pathway enrichment comparison with the Cr⁶⁺-exposed group, the Se-Met intervention group exhibited significant enrichment in glycerolipid metabolism, ferroptosis, and glycerophospholipid metabolism pathways (Figure 10B).

Figure 10. Scatter plot (bubble plot) of KEGG functional enrichment analysis. After 28-day treatment with Se-Met and/or Cr⁶⁺, livers were collected from adult zebrafish for transcriptome sequencing and KEGG enrichment analysis (3 livers/group, 3 replicates, n = 3). (A) Cr⁶⁺ vs. control; (B) Cr⁶⁺+Se-Met vs. Cr⁶⁺. Horizontal axis: proportion of DEGs annotated in each KEGG pathway to total DEGs; vertical axis: KEGG pathways. Bubble size = number of DEGs (S); bubble color = enrichment p-value/Q-value (smaller value = more significant enrichment).

3.7. Effects of Ferroptosis Inhibitors Fer-1 and Se-Met on Cr⁶⁺-Induced Changes in Iron Metabolism, Inflammation, Ferroptosis, Lipid Degeneration, and Autophagy-Related Gene Expression

This study investigated the expression of genes associated with iron metabolism regulation, inflammatory factors, oxidative stress, lipid degeneration, and autophagy in zebrafish larvae following Cr⁶⁺ exposure and inhibitor pretreatment.

1.

Iron Metabolism Regulation-Related Genes

Genes involved in iron metabolism regulation included tf, tfr, tfr1b, and fpn (Figure 11A). Exposure to Cr⁶⁺ significantly upregulated the mRNA expression of tf, tfr, tfr1b, and fpn. Compared with the control group, 100 μg/mL Cr⁶⁺ induced a 2.33-fold, 11.86-fold, 13.36-fold, and 2.70-fold increase in the expression of these genes, respectively (p < 0.001). Notably, pretreatment with the ferroptosis inhibitors Fer-1 and Se-Met significantly suppressed the Cr⁶⁺-induced upregulation of these genes (p < 0.001).

Figure 11. Impacts of Cr⁶⁺ on ferroptosis-related gene expression and mitigative effects of Fer-1/Se-Met. Zebrafish embryos at 4 hpf were exposed to Fer-1, Se-Met, and Cr⁶⁺ (70/100 μg/mL). At 120 hpf, larvae were harvested for total RNA extraction and RT-PCR to detect ferroptosis-related gene expression (10 embryos/group, 3 replicates, n = 3). (A) mRNA levels of iron metabolism regulatory genes; (B) mRNA levels of oxidative stress-related genes; (C) mRNA levels of inflammatory factor genes; (D) mRNA levels of genes related to lipid degeneration and autophagy. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control group; ### p < 0.001 vs. Cr⁶⁺ exposure groups.

2.

Ferroptosis-Related Genes

In contrast, Cr⁶⁺ exposure downregulated the mRNA expression of ferroptosis-related genes (gpx4, slc7a11, and nrf2) (Figure 11B). Specifically, 100 μg/mL Cr⁶⁺ significantly reduced the mRNA levels of gpx4, slc7a11, and nrf2 compared with the control group. However, pretreatment with Fer-1 effectively alleviated this Cr⁶⁺-induced downregulation of ferroptosis-related gene expression (statistical significance not specified).

3.

Inflammatory Factor-Related Genes

Inflammatory factor-related genes evaluated in this study included NF-κB, ptgs2a, and ptgs2b (Figure 11C). Exposure to 100 μg/mL Cr⁶⁺ significantly increased the mRNA expression of NF-κB, ptgs2a, and ptgs2b by 2.33-fold, 3.89-fold, and 3.31-fold, respectively, compared with the control group (p < 0.001). Pretreatment with Fer-1 and Se-Met effectively abrogated this Cr⁶⁺-induced upregulation of inflammatory gene expression (p < 0.001).

4.

Lipid Degeneration and Autophagy-Related Genes

Similarly, 100 μg/mL Cr⁶⁺ exposure led to a significant increase in the mRNA expression of lipid degeneration and autophagy-related genes (atg5, ncoa4, and acox1), with fold changes of 2.61, 2.23, and 2.35 compared to the control group, respectively (p < 0.001). Pretreatment with Fer-1 and Se-Met significantly mitigated the Cr⁶⁺-induced overexpression of these genes (p < 0.001; Figure 11D).

3.8. Effects of Fer-1 and Low-Dose Se-Met on Related Proteins

At 120 h post-fertilization (120 hpf), liver tissues were isolated from larval zebrafish in each experimental group, and the protein expression level of GPX4 was detected via Western blot analysis (Figure 12A). The results showed that, compared with the control group, exposure to 100 μg/mL Cr⁶⁺ inhibited the protein expression of GPX4, with its relative expression level decreased by 24.11% (p > 0.05), but the difference was not statistically significant. In contrast, pretreatment with either Fer-1 or Se-Met resulted in a recovery trend in the expression level of GPX4; compared with the Cr⁶⁺-exposed group, the relative expression levels of GPX4 in the two intervention groups were increased by 1.19-fold (p > 0.05) and 1.2-fold (p > 0.05), respectively (Figure 12B), but the difference was not statistically significant.

Figure 12. Effects of Cr⁶⁺ on GPX4 protein expression in zebrafish and protective effects of Fer-1/Se-Met. We treated 4 hpf zebrafish embryos with Fer-1, Se-Met, and Cr⁶⁺ (100 μg/mL). At 120 hpf, larvae were collected for GPX4 protein detection (10 embryos/group, 3 replicates, n = 3). (A) Western blot; (B) GPX4 protein expression level. The letters (A–F) in each bar chart are defined as follows: (A): Control group (blank); (B): Fer-1 treatment group (green); (C): Se-Met treatment group (pale blue); (D): Fer-1 protective group (Fer-1 + 100 μg/mLCr⁶⁺, navy blue); (E): Se-Met protective group (Se-Met + 100 μg/mL Cr⁶⁺, purple); (F): 100 μg/mL Cr⁶⁺ exposure group (red).

3.9. Effects of Cr⁶⁺ on Fe²⁺ and Cr⁶⁺ Content in Zebrafish Larvae Under the Protection of Selenomethionine and Different Ferroptosis Inhibitors

To detect the contents of Fe²⁺ and Cr⁶⁺, zebrafish larvae at 120 h post-fertilization (hpf) from each group were collected separately. The Cr⁶⁺ contents in the 70 μg/mL Cr⁶⁺ exposure group and 100 μg/mL Cr⁶⁺ exposure group reached 79.97 mg/kg and 120.10 mg/kg, respectively (both p < 0.001). Correspondingly, the Fe²⁺ contents exhibited an upward trend, with values of 320 μmol/L and 389.57 μmol/L in the two Cr⁶⁺ exposure groups, representing 1.6-fold (p < 0.05) and 1.95-fold (p < 0.001) increases compared to the control group. After intervention with Se-met compounds, both Cr⁶⁺ and Fe²⁺ contents showed a downward trend. Specifically, compared with the Cr⁶⁺ exposure groups, the Fer-1 intervention groups (1 μM Fer-1 combined with 70 μg/mL Cr⁶⁺, and 1 μM Fer-1 combined with 100 μg/mL Cr⁶⁺) displayed significant reductions in Cr⁶⁺ content by 30.94% (p < 0.01) and 27.45% (p < 0.001), respectively; the Fe²⁺ content in these two Fer-1 intervention groups also decreased by 27.92% and 27.67% (both p < 0.05). Similarly, in the Se-Met intervention groups (0.1 μg/L Se-Met combined with 70 μg/mL Cr⁶⁺, and 0.1 μg/L Se-Met combined with 100 μg/mL Cr⁶⁺), the Cr⁶⁺ content decreased by 21.18% (p < 0.05) and 17.96% (p < 0.01), respectively. Although the Fe²⁺ content in the two Se-Met intervention groups decreased by 12.03% and 26.23%, these changes were not statistically significant (Table 1).

Table 1. Contents of Cr⁶⁺ and Fe²⁺ in Fer-1 + Se-Met treatment group.

Group	Content of Cr6+ (mg/kg ± SD)	Content of Fe2+ (μmol/L ± SD)
Control	0.09 ± 0.03	199.58 ± 19.03
Cr6+70 μg/mL	79.97 ± 8.30 ***	320.30 ± 28.01 *
Cr6+100 μg/mL	120.10 ± 9.45 ***	389.57 ± 57.57 ***
Fer-1	0.07 ± 0.05	185.51 ± 23.22
Fer-1+Cr6+70 μg/mL	55.23 ± 4.65 ##	230.87 ± 28.47
Fer-1+Cr6+100 μg/mL	87.13 ± 3.19 ###	281.77 ± 47.87 #
Se-Met	0.06 ± 0.05	196.90 ± 15.25
Se-Met+Cr6+70 μg/mL	63.03 ± 5.61 #	270.37 ± 33.10
Se-Met+Cr6+100 μg/mL	98.53 ± 3.90 ##	287.37 ± 54.85

*, ***: p < 0.05, or p < 0.001 vs. Control group; ^#, ^##, ^###: p < 0.05, p < 0.01 or p < 0.001 vs. Cr⁶⁺(70 or 100 μg/mL) group.

4. Discussion

The results showed that Cr⁶⁺ induces ferroptosis by disrupting the Nrf2/SLC7A11/GPX4 pathway, alleviated by selenomethionine [57]. Zhuge et al. confirmed Cr⁶⁺-induced ferroptosis in mouse cells [58], and Wang et al. [59] showed that Cr⁶⁺ induces ferroptosis in broilers by downregulating GPX4. These studies validate our zebrafish findings and model value for animal Cr⁶⁺ ferroptosis research and interventions (e.g., Se-Met). The observed changes (GSH depletion, GPX4 inhibition, lipid peroxidation, liver injury) align with well-documented ferroptosis pathways in heavy metal-induced toxicity [59,60], further reinforcing the plausibility of our conclusion.

1.

The Correlation between Ferroptosis and Cr⁶⁺-Induced Morphological Changes in Zebrafish

Sanyal et al. reported that the 96 h median lethal concentration (LC₅₀) of Cr⁶⁺ to Labeo bata and Puntius sarana was 7.33 mg/L and 10.37 mg/L, respectively [61]. For Puntius conchonius and Salvelinus fontinalis, the 96 h LC₅₀ values of Cr⁶⁺ were determined to be 331.40 mg/L and 59 mg/L, respectively [2,62]. Previous in vitro studies have also demonstrated that sodium chromate, a typical Cr⁶⁺ source, exerts a concentration-dependent cytotoxic effect on medaka (Oryzias latipes) fin cells. Specifically, relative to the control group, the cell survival rates in the groups treated with 0.5, 1, 5, 10, 25, 50, and 100 μM sodium chromate were 100%, 100%, 87.8%, 77.5%, 40.9%, 15%, and 2.7%, respectively [63]. Larval Cr⁶⁺ concentrations (70–100 μg/mL, acute) represent environmentally realistic hotspots in industrial-affected areas, while higher concentrations correspond to worst-case scenarios of industrial wastewater. Adult concentrations (0.05–2 mg/L, chronic) reflect realistic levels from slightly to moderately polluted environments. Together, these concentrations cover both realistic and worst-case scenarios, supporting the environmental implications of this study.

2.

The Relationship between Ferroptosis and Cr⁶⁺-Induced Hepatocellular Changes in Zebrafish, as well as the Protective Effect of Set-Met on Zebrafish Larvae

In a previous study on the effects of Cr⁶⁺ on Channa asiatica, Yu et al. found that the accumulation of Cr⁶⁺ in tissues increased in a concentration-dependent manner, with the tissue-specific accumulation levels ranked as liver > gill > intestine > muscle [64]. Other studies have confirmed that as chromium exposure increases, the metal accumulates more heavily in the liver than in tissues such as the brain, muscle, and bone [65,66,67,68]. Another study verified that Cr⁶⁺ exposure causes a remarkable reduction in the axial length and surface area of the eyes [69]. Similar to the aforementioned findings, the results of the present study indicated that the accumulation of Cr⁶⁺ in zebrafish larvae induces morphological changes.

Zhou et al. [60] found that Cr⁶⁺ exposure led to hepatocyte nuclear pyknosis, cellular vacuolization, widened hepatic sinusoidal spaces and indistinct cell boundaries in Micropterus salmoides, which was consistent with the findings of Awpari et al. [70] on Ictalurus punctatus. A 30-day exposure study on Pangasianodon hypophthalmus [71] showed that Cr⁶⁺ (0.8, 1.6, and 3.2 mg/L) caused dose-dependent liver damage. These histopathological changes ranged from mild to severe and were characterized by hepatocellular degeneration, and vacuolization. Studies have shown that Cr⁶⁺ exposure significantly increased the liver swelling index in mice (0.098 ± 0.13 g) [72], and dose-dependent hepatic structural abnormalities, hepatocellular damage and inflammatory responses were observed in the liver tissue of the exposed mice [73]. Furthermore, treatment with Se-Met or Fer-1 promoted hepatocyte proliferation and alleviated lipid peroxidation symptoms, such as GSH depletion and altered MDA levels. These treatments also modulated the expression of ferroptosis-related factors [74]. Zhao et al. [75] reported that in Cr⁶⁺-poisoned broiler chickens, the cells surrounding the central veins of the liver exhibited cloudy swelling, exfoliative necrosis, karyopyknosis, karyorrhexis and even karyolysis. These findings were similar to those of Zhuo et al. [76]; additionally, prolonged exposure to Cr⁶⁺ induced clear pathological changes in the liver, including a disorganized hepatocyte arrangement and incomplete hepatocyte morphology. Moreover, alterations in lipid metabolism have also been documented in Cr⁶⁺-exposed electroplating workers [77,78]. In our research, the observed fewer hepatocytes per unit area is an artifact of vacuolar swelling of individual hepatocytes, not true liver atrophy. However, pretreatment with Fer-1 attenuated the reduction in hepatocyte number induced by Cr⁶⁺ exposure, indicating that the Cr⁶⁺-induced decrease in hepatocyte number and steatosis were associated with hepatocyte death.

To identify the mechanisms of hepatocyte loss, ultrastructural examination is essential, as mitochondrial alterations are key indicators of ferroptosis. Under TEM, ferroptosis is characterized by mitochondrial shrinkage, a reduced number of cristae, and cell membrane rupture [32]. Numerous studies confirm that Cr⁶⁺ induces hepatic damage through mitochondrial dysfunction. For example, Cr⁶⁺ exposure triggers inflammatory infiltration, causes mitochondrial abnormalities, and promotes excessive mitophagy in hepatocytes [79]. Specifically, Cr⁶⁺ induces the overproduction of ROS, which promotes the translocation of Drp1 to the mitochondria, leading to mitochondrial fission. This process facilitates caspase-dependent apoptosis, characterized by the opening of the mitochondrial permeability transition pore (mPTP) and a decrease in mitochondrial membrane potential (MMP) [80]. Bagchi et al. reported that oral administration of Cr⁶⁺ induces lipid peroxidation in mouse liver mitochondria and microsomes, accompanied by increased lipid metabolites in urine [18]. Similarly, Zhou et al. confirmed that Cr⁶⁺ exposure leads to ROS accumulation and mitochondrial dysfunction in AML12 cells and mouse liver tissues [81]. Yang et al. further demonstrated that Cr⁶⁺ causes structural abnormalities in rat hepatocytes, including mitochondrial swelling, rupture, and even cristae loss [82]. In a study on Hy-Line Brown chickens, Wang et al. found that Cr⁶⁺ exposure significantly enhances ROS production, triggers MMP collapse, and promotes autophagosome formation [83]. Consistent with these findings, our electron microscopy results revealed that Cr⁶⁺ exposure induces rupture of both the outer and inner mitochondrial membranes and reduces the number of cristae in hepatocytes. Notably, supplementation with Se-Met or Fer-1 effectively alleviated these Cr⁶⁺-induced mitochondrial changes (Figure 6). Collectively, these observations confirm that mitochondrial damage is a critical ultrastructural feature of Cr⁶⁺-induced hepatotoxicity, suggesting that the protective effects of Se-Met and Fer-1 are mediated by mitigating ferroptosis-associated mitochondrial dysfunction. However, transmission electron microscopy (TEM) results were included as supplementary ultrastructural evidence to further confirm the mechanistic findings obtained by TMRE and MitoSOX staining, rather than a necessary basis for the core conclusions of this study.

3.

The Relationship between Cr⁶⁺-Induced Hepatic Ferroptosis-Related Gene Expression Changes and Ferroptosis, as well as the Protective Effect of Se-Met on Zebrafish Larvae

Cr⁶⁺-induced hepatic ferroptosis is closely linked to an altered expression of ferroptosis-related genes, and Se-Met has been shown to exert protective effects by modulating these pathways. Growing evidence suggests that Cr⁶⁺ disrupts the transcription of genes involved in DNA repair, autophagy, antioxidant defense, and lipid metabolism, collectively driving hepatic ferroptosis. For instance, Holmes et al. reported that Cr⁶⁺ inhibits DNA replication and repair [84], a disruption that may lay the foundation for subsequent cellular damage and ferroptosis initiation. In a study on Channa punctatus, Cr⁶⁺ exposure was found to upregulate the autophagy-related genes ATG5, LC3, and GABARAP in both liver and kidney tissues, while mTOR was transcriptionally downregulated [85]. Dysregulated autophagy is closely intertwined with ferroptosis, as excessive autophagy can promote the degradation of ferritin (a process known as ferritinophagy), leading to the release of free iron and exacerbating ferroptotic cell death. A study on fish gill tissues exposed to Cr⁶⁺ revealed altered regulation of Nrf2 and Mt2, with the maximum downregulation of 61% and 53% observed on the 45th day, respectively [86]. The Nrf2 signaling pathway serves as a primary defense against oxidative stress; its suppression impairs ROS scavenging, thereby driving the ROS accumulation and lipid peroxidation that characterize ferroptosis. Corroborating this, Jin et al. reported that high-dose Cr⁶⁺ exposure triggers hepatocyte apoptosis and hepatic injury in mice via ROS generation and Nrf2 pathway inhibition [87]. In a study on New Zealand rabbit livers, Yuan et al. observed that all Cr⁶⁺ treatment groups exhibited increased expression of antioxidant genes, indicating the activation of hepatic defense mechanisms against Cr⁶⁺-induced oxidative stress. Simultaneously, lipid metabolism was disrupted, as evidenced by significant lipid deposition and the upregulation of key hepatic lipid metabolism genes [88]. Dysregulated lipid metabolism provides abundant substrates for lipid peroxidation, further amplifying ferroptotic damage. In Wang et al.’s study on the small intestine of broiler chickens, Cr⁶⁺ exposure led to the downregulation of GPX4, SLC7A11, FTL and FTH1 levels, along with the upregulation of p38-MAPK, phosphorylated p38, TFR1 and HMGB1 levels [59]. Notably, GPX4 and SLC7A11 are core anti-ferroptotic genes: GPX4 directly inhibits lipid peroxidation by reducing lipid hydroperoxides, while SLC7A11 maintains intracellular GSH levels. The downregulation of these genes, coupled with the upregulation of iron uptake-related gene TFR1, strongly indicates the initiation of ferroptosis.

Se-Met has emerged as an effective protector against Cr⁶⁺-induced hepatic injury by targeting these ferroptosis-related pathways. Studies have shown that the p38-dependent mechanism is responsible for Se-Met-induced Nrf2 activation; dietary supplementation of Se-Met can regulate lipid metabolism and reduce oxidative stress, thereby minimizing liver injury in mice [89]. Specifically, Se-Met protects the rabbit liver from ochratoxin A (OTA)-induced hepatotoxicity by activating the Nrf2 signaling pathway and enhancing the expression of downstream Nrf2 target genes such as HO-1 [90]. Similarly, selenium supplementation at 0.5 mg/kg enhanced the hepatic antioxidant capacity of piglets fed deoxynivalenol (DON)-contaminated diets, as reflected by increased activities of total antioxidant capacity (T-AOC), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) in the liver, as well as the upregulated mRNA levels of Nrf2, Gclm, NQO1, SOD1 and GPX1 in hepatic tissues [91]. These findings collectively suggest that selenomethionine may alleviate Cr⁶⁺-induced hepatic ferroptosis in zebrafish larvae by activating the Nrf2 antioxidant pathway, upregulating anti-ferroptotic genes (e.g., GPX4, SLC7A11), and regulating lipid metabolism and iron homeostasis, which aligns with its protective role in mitigating Cr⁶⁺-induced cellular and ultrastructural damage.

RNA-seq analysis revealed that Cr⁶⁺ exposure disturbed glycerolipid metabolism, promoting lipid accumulation and causing hepatic steatosis and lipid vacuolization. Excess glycerolipids served as key substrates for lipid peroxidation during ferroptosis. Cr⁶⁺-induced GPX4 downregulation further accelerated ferroptosis and liver damage. Based on multiple lines of evidence, including GPX4 inactivation, iron dysregulation, specific rescue by ferrostatin-1, and transcriptomic enrichment of ferroptosis pathways, Cr⁶⁺-induced hepatocyte death was confirmed as ferroptosis, clearly distinct from apoptosis and necrosis. Selenomethionine mitigated hepatotoxicity by restoring lipid metabolism and GPX4 function. These findings integrate glycerolipid metabolism and ferroptosis into a unified mechanism underlying Cr⁶⁺-induced hepatic injury.

4.

The Relationship between Cr⁶⁺-Induced Changes in GPX4 Protein Expression and Ferroptosis, as well as the Protective Effect of Se-Met on Zebrafish Larvae

GPX4, a core anti-ferroptotic protein, inhibits ferroptosis by suppressing lipid peroxidation. Cr⁶⁺ depletes GSH to impair GPX4 function and trigger ferroptosis [92,93,94]. Cr⁶⁺ induces oxidative stress and liver injury in aquatic organisms and mammals, characterized by reduced GSH/GPX4, increased MDA, aspartate aminotransferase (AST) and ALT [60,72,95,96,97,98,99,100,101]. For instance, it reduced hepatic GSH in Sebastes schlegelii [95,96], decreased antioxidant enzyme activities and increased MDA in Micropterus salmoides [60], and elevated serum AST/ALT/MDA in mice [72]. Se-Met protects by restoring GSH, regulating antioxidants and upregulating GPX4 [89,102,103,104]. Cr⁶⁺ was also shown to downregulate GPX4 and induce ferritinophagy in cells [58].

5. Conclusions

This study demonstrated that Cr⁶⁺ accumulates significantly in zebrafish, while Se-Met reduces internal Cr⁶⁺ levels and alleviates Cr⁶⁺-induced hepatic steatosis, the decreased mitochondrial cristae number, and abnormal expression of ferroptosis-related genes/proteins. Mechanistically, Cr⁶⁺ induces zebrafish liver injury via ferroptosis, characterized by GSH depletion and exacerbated lipid peroxidation. These processes are further evidenced by altered levels of GPX4, GSH, MDA, and ALT. Both the ferroptosis inhibitor Fer-1 and low-dose Se-Met effectively reverse Cr⁶⁺-induced damage, partly by restoring GSH, regulating antioxidant function and upregulating GPX4 to inhibit ferroptosis. Given the absence of toxicity margin data, the use of Se-Met as a safe additive cannot be currently supported. Further studies focusing on toxicity margin detection are required to verify its potential application as a safe additive.

This study elucidates the mechanistic toxicology of Cr⁶⁺ in the zebrafish experimental model, and these findings provide a theoretical basis for subsequent toxicological research on aquacultural fish species.