Paternal Zearalenone Exposure Is Associated with Hepatic Dysfunction in F1 Offspring: Insights from Proteomic Analysis

TL;DR

Paternal exposure to zearalenone causes liver problems in offspring, with proteomic analysis revealing immune and inflammatory pathway disruptions.

Contribution

This study reveals novel intergenerational effects of paternal zearalenone exposure on offspring liver health and immune pathways.

Findings

F1 offspring of ZEN-exposed fathers developed hepatic steatosis and oxidative stress.

Proteomic analysis showed dysregulation of NF-κB and chemokine signaling pathways in F1 offspring livers.

Reduced MHC-I and increased MHC-II levels suggest altered immune responses in affected offspring.

Abstract

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin that adversely affects directly exposed individuals, yet the intergenerational consequences of paternal ZEN exposure remain poorly understood. In this study, we investigated the impact of paternal ZEN exposure on hepatic outcomes in F1 offspring, with a focus on the underlying molecular mechanisms. Kunming male mice (F0) were fed a ZEN-supplemented diet (10 mg/kg bw/day) for 5 weeks. Their F1 offspring developed hepatic steatosis, elevated oxidative stress, and a chronic inflammatory state. Proteomic analysis of F1 livers revealed significant dysregulation of immune and inflammatory pathways, including NF-κB and chemokine signaling, with reduced MHC-I and increased MHC-II levels. These findings provide mechanistic insight into how paternal ZEN exposure disrupts hepatic immune-metabolic homeostasis in F1 offspring, highlighting a critical and understudied pathway in intergenerational toxicology.

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Full text

1. Introduction

Environmental endocrine-disrupting compounds (EDCs) are increasingly recognized for their role in hormonal imbalances and reproductive disorders [1]. Among these, Zearalenone (ZEN) is a stable, nonsteroidal estrogenic mycotoxin produced by Fusarium fungi, which frequently contaminates cereal crops worldwide [2,3]. ZEN exhibits structural similarities to natural estrogens and binds to estrogen receptors. These interactions can activate estrogen response elements and lead to reproductive toxicity [4,5]. In males, prolonged exposure to ZEN disrupts hormonal homeostasis, impairs Leydig cell autophagy, and reduces sperm count [6,7]. In females, it is associated with menstrual irregularities and infertility [8,9]. Beyond reproductive toxicity, ZEN has been reported to exert neurotoxic, hepatotoxic, immunotoxic, and genotoxic effects, highlighting the importance of investigating its systemic and long-term biological risks.

The maximum residue limits (MRLs) for ZEN in human food and animal feed have been established by regulatory agencies worldwide. The European Union has set MRLs for ZEN at 20 μg/kg in processed cereal-based foods for infants and 100 μg/kg in unprocessed cereals [10,11]. In China, the national standard (GB 2761-2017) limits ZEN to 60 μg/kg in wheat and corn [12]. These regulatory thresholds underscore the public health concern surrounding ZEN contamination.

Recent studies indicate that EDCs such as ZEN can induce heritable effects by compromising germ cell integrity and altering epigenetic marks, thereby disrupting cellular homeostasis in subsequent generations [13,14]. Such alterations have been linked to reproductive, metabolic, and immune dysfunction in offspring [15,16]. While most studies have focused on maternal exposure and transplacental effects [17], the consequences of paternal ZEN exposure remain poorly defined, despite growing recognition of paternal epigenetic inheritance.

Notably, the potential intergenerational hepatic consequences of paternal ZEN exposure are unknown. To address this knowledge gap, the present study investigates the impact of paternal ZEN exposure on hepatic health in unexposed F1 offspring, with a focus on oxidative stress, lipid metabolism, and immune-related molecular pathways. Using an integrated approach such as histology, biochemistry, and proteomic profiling, we characterized the resulting phenotype and elucidated underlying molecular mechanisms. This work provides novel insight into a paternal route of intergenerational toxicity and its mechanisms.

2. Results

2.1. Effect of ZEN Exposure on the Body Weight, Organ Indices, and Sperm Quality in F0 Male Mice

To assess the impact of ZEN exposure, we analyzed body weight, organ indices, and sperm quality in F0 male mice. ZEN exposure in F0 male mice significantly reduced body weight, epididymis index, total sperm count, and sperm viability compared to the control group (Figure 1). These findings suggest that ZEN harms male reproductive health, which could have an impact on the subsequent F1 generation.

2.2. Effect of Paternal ZEN Exposure on Reproductive Performance

The reproductive performance of F0 male mice exposed to ZEN was evaluated by examining litter characteristics of their F1 offspring (Figure 2). The average birth weight of pups was significantly reduced in the ZEN group compared to controls, whereas alive litter size and sex ratio were not affected. These results indicate that paternal ZEN exposure impairs early offspring growth, as reflected by reduced birth weight.

2.3. Effect of Paternal ZEN Exposure on Body Weight and Organ Indices in F1 Offspring

The effects of paternal ZEN exposure on F1 offspring growth and organ development were evaluated at 4 and 8 weeks of age (Figure 3). At 8 weeks, there was a notable sexual dimorphism in body weight: The F1 male offspring of males exposed to ZEN exhibited substantial increase (p < 0.05) compared to their respective controls, while female offspring showed a significant decrease (p < 0.01) (Figure 3A,E).

Concurrently, the liver index was significantly elevated in both male and female F1 offspring from the ZEN group at 8 weeks compared to controls (Figure 3B,F), suggesting liver enlargement. Alterations in kidney and spleen indices were less pronounced (Figure 3C,D,G,H). Given the consistent and significant impact on liver mass, subsequent analyses focused on characterizing this hepatic phenotype.

In addition, the adverse effects of paternal mice consuming ZEN on the reproductive organs of the F1 generation were also noted, as shown in Figure S1. Because of significant differences in these phenomena between the genders, we did not pursue this phenomenon further in subsequent studies.

2.4. Effect of Paternal ZEN Exposure on the Serum Biochemical Parameters in F1 Offspring

Paternal ZEN exposure induced sex-specific alterations in serum markers of liver function and lipid metabolism in F1 offspring (Figure 4). In males, offspring of ZEN-exposed male mice exhibited a significant decrease in serum AST and a trend toward reduced ALT, along with a significant reduction in HDL-C, compared to those in the control group. In contrast, female F1 offspring of ZEN-exposed male mice exhibited a significant increase in serum AST activity and a trend toward elevated ALT. Levels of LDL-C, triglycerides (TG), and total cholesterol (TC) were not significantly altered in either sex. These results demonstrate that paternal ZEN exposure dysregulates hepatic function and lipid homeostasis in a sex-dependent manner.

2.5. Effect of Paternal ZEN Exposure on the Hepatic Biochemistry and Lipid Accumulation in F1 Offspring

Analysis of hepatic biochemistry revealed significant alterations in F1 offspring (Figure 5A–D). A distinct pattern was observed for liver transaminases: female F1 offspring of male mice from the ZEN group exhibited substantial reductions in both ALT (p < 0.01) and AST (p < 0.001) compared to those of control females. In contrast, changes in male F1 offspring were less uniform.

Concurrently, hepatic lipid content was significantly elevated. Levels of total cholesterol (TC) and triglycerides (TG) were markedly increased in both male and female F1 offspring of male mice exposed to the ZEN group relative to their respective controls (Figure 5C,D).

This biochemical evidence of hepatic lipid overload was corroborated by histology. Oil Red O staining confirmed pronounced lipid droplet accumulation in the livers of F1 offspring of male mice consuming ZEN compared to those of mice consuming a control diet (Figure 5E), indicating clear hepatic steatosis.

2.6. Effect of Paternal ZEN Exposure on the Hepatic Gene Expression in F1 Offspring

Transcriptional profiling of F1 livers revealed significant alterations in key metabolic and inflammatory pathways (Figure 6). Expression of lipid synthesis genes (ACLY, SCD1, FASN) was downregulated, while lipid degradation genes (ATGL, HSL) were upregulated in both male and female offspring of male mice exposed to the ZEN diet compared to controls (Figure 6A–E).

Concurrently, a pro-inflammatory transcriptional shift was evident. Expression of the pro-inflammatory cytokine TNF-α was markedly increased, whereas anti-inflammatory genes (IL-10, TGF-βR2) were decreased in F1 offspring of ZEN-exposed male mice (Figure 6F–H). Furthermore, key regulators of the antioxidant response, Nrf2 and Keap1, were significantly downregulated (Figure 6I,J). Collectively, these gene expression changes indicate a state of enhanced hepatic lipolysis, inflammation, and oxidative stress in F1 offspring of male mice consuming the ZEN diet, aligning with the observed biochemical and histological signs of liver dysfunction.

2.7. Proteomic Analysis of F1 Offspring Liver Tissues

To gain a systems-level understanding of the hepatic impact, label-free quantitative proteomics was performed on F1 liver tissues. Principal component analysis (PCA) revealed clear separation between the ZEN and control groups in both male and female offspring, confirming that paternal exposure induces significant alterations in the hepatic proteome (Figure 7A,B).

Differential expression analysis identified 727 DEPs in females (229 upregulated, 498 downregulated) and 359 DEPs in males (196 upregulated, 163 downregulated) (Figure 7C). Intersection analysis revealed 74 DEPs common to both sexes (Figure 7D), representing a core set of proteins consistently altered by paternal ZEN exposure. Hierarchical clustering of these 74 common DEPs showed robust intra-group consistency and clear inter-group separation (Figure 7E).

Functional enrichment analysis of the 74 common DEPs highlighted a pronounced immune-centric signature. Gene Ontology (GO) terms were strongly associated with immune cell migration and defense responses (e.g., leukocyte chemotaxis, T cell migration) (Figure 8A). KEGG pathway analysis further demonstrated significant enrichment of key inflammatory and immune signaling pathways, including NOD-like receptor signaling, NF-κB signaling, and chemokine signaling (Figure 8B).

Notably, analysis of specific immune regulators revealed a coordinated shift in antigen presentation machinery: MHC class I proteins were downregulated, while MHC class II proteins were upregulated in F1 offspring from the ZEN group (Figure 8C,D).

Pathway enrichment related to antigen presentation and hepatic immune imbalance (Figure S2) supports the notion that this MHC imbalance contributes to hepatic immune dysregulation. Collectively, the proteomic data revealed a state of hepatic immune dysregulation and chronic inflammation in F1 offspring, characterized by altered immune signaling and antigen presentation, which provides a molecular basis for the observed metabolic and inflammatory phenotype.

2.8. Validation of Proteomics Results by RT-qPCR

To validate the proteomic findings, mRNA expression levels of two representative MHC-I genes (H2Q1 and H2Q6) were quantified using RT-qPCR (Figure 9). In male F1 offspring of ZEN-exposed male mice, H2Q1 was decreased significantly, and H2Q6 showed a decreasing trend compared to the controls. In female offspring of male mice consuming ZEN, H2Q1 exhibited a decreasing trend, and H2Q6 was significantly reduced as compared to those of male mice consuming the control diet. The consistency between these transcriptional changes and the corresponding proteomic data confirms the reliability of the proteomic dataset and substantiates that the identified protein alterations reflect genuine biological changes induced by paternal ZEN exposure.

3. Discussion

Zearalenone (ZEN) is a potent estrogenic mycotoxin whose reproductive toxicity is well-documented, and most of the research has predominantly focused on maternal or direct exposure pathways [18]. This study challenges that paradigm by demonstrating that paternal ZEN exposure is sufficient to impair F0 reproductive health and induce a significant, sex-specific hepatic phenotype in their unexposed F1 offspring.

The main mechanism of ZEN toxicity is its action as a potent xenoestrogen with strong affinity for estrogen receptors (ERα and ERβ) [7,19]. This ER-mediated disruption is the most probable main cause of the reported intergenerational effects [20,21]. In F0 males, ZEN exposure significantly impaired reproductive parameters, including reduced body weight, epididymis index, sperm count, and viability. These findings align with evidence that ZEN disrupts the hypothalamic–pituitary–gonadal (HPG) axis, compromises testosterone synthesis, and induces testicular oxidative stress and apoptosis [22,23,24]. Crucially, this ER-mediated impairment of paternal gonadal activity and germ cell health may have long-term effects on subsequent generations. Consistent with this, F1 offspring of F0 male exposed to the ZEN diet exhibited reduced birth weight, aligning with studies showing parental exposure to endocrine-disrupting chemicals can influence offspring developmental progress [16,25].

The transmission of hepatic dysfunction from ZEN-exposed male mice to their unexposed offspring is consistent with the growing concept of paternal epigenetic inheritance [16]. However, it is critical to emphasize that this mechanism remains hypothetical and speculative, as epigenetic markers were not directly measured in the present study. We hypothesize that ZEN-induced oxidative stress in paternal testes could potentially alter the sperm epigenome (e.g., DNA methylation, histone modifications, or small RNA profiles), thereby reprogramming hepatic developmental pathways in F1 offspring. This proposed mechanism is presented as a testable framework for future research and requires direct experimental validation through sperm epigenetic profiling.

A major finding was the significant sexual dimorphism in the F1 outcomes. Females exhibited reduced body weight and elevated serum ALT and AST, while males showed increased body weight and decreased hepatic transaminases. These variations most likely arise from the interaction between inherited factors and sex-specific hormonal environments. The reduction in ALT and AST in males, despite clear hepatic steatosis and inflammation, may indicate a distinct phase of hepatic adaptation or dysfunction, a phenomenon noted in metabolic disorders [26]. These opposing phenotypes highlight sex as a critical biological variable in intergenerational toxicology.

Paternal ZEN exposure triggered an integrated hepatic pathophysiology in F1 offspring. We observed significant metabolic dysregulation, evidenced by elevated serum triglycerides and cholesterol, and a compensatory transcriptional shift in lipid-handling genes. This metabolic state was linked to a condition of chronic oxidative stress and inflammation, characterized by impaired Nrf2-Keap1 antioxidant signaling and elevated pro-inflammatory cytokines (TNF-α). These pathways are established consequences of sustained ER disruption and create a vicious cycle that promotes further lipid accumulation and injury [27,28].

An apparent paradox emerged in our transcriptional data: hepatic steatosis and elevated triglyceride levels occurred alongside downregulation of lipogenic genes (ACLY, SCD1, FASN) and upregulation of lipolytic genes (ATGL, HSL). We interpret this pattern as a compensatory response to pre-existing hepatic lipid accumulation, rather than a primary driver of steatosis. The liver likely attempts to counteract lipid overload by suppressing de novo lipogenesis and enhancing lipolysis, but these transcriptional adaptations are insufficient to resolve the established steatotic phenotype.

Notably, male F1 offspring exhibited a significant reduction in serum HDL-C levels. This finding suggests that hepatic lipid accumulation may result, at least in part, from disrupted lipid transport and export mechanisms. Impaired reverse cholesterol transport or reduced VLDL secretion could contribute to intrahepatic lipid retention. Alternative mechanisms warranting future investigation include increased hepatic fatty acid uptake (e.g., via CD36), impaired mitochondrial β-oxidation, and altered lipoprotein metabolism. Resolving this metabolic paradox will require direct functional assays of hepatic lipid flux. Such disturbances align with effects reported from other parental exposures to endocrine disruptors, highlighting a shared paradigm [13,29].

Our proteomic analysis further provided a systems-level validation of this pathophysiology, revealing significant enrichment of pathways central to hepatic immune regulation and inflammation, including NF-κB signaling, chemokine activity, and NOD-like receptor pathways. This dysregulation explains the sustained inflammatory milieu and suggests a mechanism for macrophage/Kupffer cell activation. Dysregulated chemokine signaling and NF-κB activation have been reported to drive oxidative stress and tissue remodeling [30,31,32], consistent with the histopathological and metabolic changes observed in the liver of F1 offspring.

Furthermore, altered MHC I/II expression indicates broader immunological dysregulation. This shift in antigen presentation machinery more likely reflects immune-metabolic reprogramming associated with hepatic lipid overload and chronic inflammation [33]. Thus, these findings provide context rather than definitive proof of such pathology in this model. The concordance between proteomic findings and RT-qPCR validation of selected DEPs strengthens the reliability of the observed molecular alterations.

The phenotypic changes observed in F1 offspring represent a pharmacological/toxicological model effect. The ZEN dose employed in this study (10 mg/kg bw/day) was selected from prior subchronic toxicity studies to elicit a clear phenotypic response for hypothesis generation and proof-of-concept. This dose exceeds typical environmental exposure levels and regulatory maximum residue limits (MRLs) by several orders of magnitude and is not intended to model human dietary intake. Establishing dose–response relationships and a no-observed-adverse-effect level (NOAEL) requires dedicated future studies.

Taken together, our findings suggest that paternal ZEN exposure is associated with impaired male reproductive health and altered hepatic physiology in F1 offspring (Figure 10). This work establishes the paternal exposure pathway as a critical determinant of intergenerational metabolic health. Future studies, such as dose–response modeling, multi-omics validation, direct epigenetic profiling, and comprehensive immune characterization, are needed to fully define the mechanistic basis and long-term consequences of paternal ZEN exposure.

4. Materials and Methods

4.1. Materials and Chemicals

ZEN (ZEN, purity ≥ 98%) was purchased from Qingdao Pribolab Engineering Co., Ltd. (Qingdao, China). The rodent feed was obtained from Beijing HFK 105 Bioscience Co., Ltd. (Beijing, China). All other chemicals were of analytical grade.

4.2. Animals and Experimental Design

All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of China Agricultural University (Approval No.: AW21111202-1-5) and were conducted in strict accordance with the EU Directive 2010/63, ARRIVE guidelines, and institutional animal welfare standards to ensure humane treatment and minimize stress in both parental (F0) and offspring (F1) mice.

Twenty specific pathogen-free (SPF) male Kunming mice (5 weeks old) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China). Mice were housed under controlled conditions (temperature: 22 ± 2 °C; relative humidity: 50–60%; 12 h light/12 h dark cycle) with ad libitum access to water and nutritionally complete rodent breeding feed.

After a one-week acclimatization period, mice were randomly assigned to control and ZEN treatment groups (n = 10 per group). The ZEN-supplemented diet was prepared by thoroughly mixing crystalline ZEN into a powdered standard diet. The concentration of ZEN in the feed was calculated to achieve a target daily intake of 10 mg ZEN per kg body weight, based on standard daily feed consumption values for mice this age and strain. The ZEN group received a ZEN-supplemented diet (10 mg/kg body weight) for 5 weeks, while the control group received a standard diet without ZEN. Feed intake was monitored throughout the exposure period to confirm consistent dosing.

The dose was selected based on prior subchronic toxicity studies in mice [6,7] and was chosen to elicit a clear phenotypic response for hypothesis generation. This dose exceeds typical environmental exposure levels and is not intended to model human dietary intake but rather to establish proof-of-concept for paternal intergenerational effects. Establishing a no-observed-adverse-effect level (NOAEL) and dose–response relationship will require future dedicated studies. Sample size was determined based on previous power calculations (power = 0.8, α = 0.05).

At 11 weeks of age, each male mouse was mated with two 9-week-old female mice. After the mating period, all F0 male mice were euthanized by CO_2_ inhalation in accordance with AVMA guidelines. Blood samples were collected via the retro-orbital sinus, and tissues were immediately frozen in liquid nitrogen for subsequent analysis.

Pregnant females were housed individually (one mouse per cage) for 3 weeks to give birth to the pups. The litter size and birth weights of the F1 pups were recorded. The female mice were allowed to wean their F1 pups for 3 weeks. Subsequently, the F1 male and female mice were raised separately until they were 8 weeks old.

Four experimental F1 groups were established: Control-Male, Control-Female, ZEN-Male, and ZEN-Female (n = 10 replicates/group). For each litter born to an F0 dam, two same-sex F1 pups with body weights closest to the litter median were selected and maintained. At 4 weeks of age, one pup from each replicate was randomly selected, euthanized, and sampled. The remaining littermate was then sampled at 8 weeks of age. Therefore, n = 10 biological replicates per group. All the F1 pups were given the same diet without ZEN.

At 4 and 8 weeks, one mouse per replicate was euthanized. Blood and tissues were collected, weighed, and stored at −80 °C. The experimental design is summarized in Figure 11.

4.3. Sperm Quality

4.3.1. Sperm Viability

One epididymis was minced in 1 mL pre-warmed saline (37 °C). After 15 min, 10 µL of suspension was placed on a hemocytometer and examined under a microscope (Olympus CX23). Active sperm were recorded as Grade IV according to WHO criteria. Sperm viability (%) was calculated as:

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4.3.2. Sperm Count

Semen samples were transferred to a counting chamber. The sperm count was calculated as:

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4.4. Serum Biochemical Indicators

The serum concentrations of alanine aminotransferase (ALT, Cat# C009-2-1), aspartate aminotransferase (AST, Cat# C010-2-1), high-density lipoprotein cholesterol (HDL-C, Cat# A112-1-1), low-density lipoprotein cholesterol (LDL-C, Cat# A113-1-1), total cholesterol (TC, Cat# A111-1-1), and triglycerides (TG, Cat# A110-1-1) were measured using commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. All assays were performed in biological duplicates, and each measurement was performed in technical triplicates.

4.5. Liver Histology

Frozen liver sections (8 µm) were air-dried, fixed in 10% formalin (10 min), rinsed with distilled water, and immersed in 60% isopropanol (30 s). Sections were stained with 0.5% Oil Red O in 60% isopropanol (15 min), mounted, and scanned (3D HISTECH Panoramic MIDI). Lipid accumulation was quantified with Case Viewer software (v2.43).

4.6. RT-qPCR of Liver Metabolism and Inflammation-Related Gene

Total RNA was extracted from F1 liver tissue using the Eastep^®^ Super Total RNA Extraction Kit (Cat# 15596018; Promega, Beijing, China). RNA concentration and purity were measured with a NanoPhotometer^®^ N60 (IMPLEN, Munich, Germany). cDNA was synthesized using the TRUE script RT Kit with gDNA eraser (Cat# PC5402; Aidlab Biotechnologies, Beijing, China).

Two-step RT-qPCR was conducted using SYBR Green qPCR Mix (Cat# PC3302; Aidlab Biotechnologies, Beijing, China) on a Bio-Rad CFX Connect™ system (Bio-Rad, Hercules, CA, USA). Primers were designed using NCBI sequences and synthesized by Sango Biotech (Shanghai, China). Primer efficiency was validated by standard curve analysis, and amplicon specificity was confirmed by melt curve analysis. GAPDH was used as the reference gene; its expression stability was confirmed across all experimental groups. Relative expression was calculated using the 2^−ΔΔCt^ method [34]. Primer sequences, product sizes, and accession numbers are listed in Table 1.

4.7. Proteomics Analysis

A total of 10 liver samples were collected per experimental group. To ensure tech-nical consistency and reduce variability, these 10 samples were randomly paired to generate five independent replicates (n = 5 per group) for label-free proteomic analysis. Liver proteins were extracted using RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA), and concentration was determined using a BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). Samples were analyzed individually by LC-MS/MS on an Orbitrap Fusion Lumos system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a C18 column (75 μm × 25 cm, 2 μm particle size, Thermo Fisher Scientific, Waltham, MA, USA). Chromatographic separation used a 120 min linear gradient from 2% to 35% mobile phase B (0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min.

Raw data were processed using MaxQuant (v2.1.0.0) against the UniProt mouse database. A total of 3492 proteins were identified at a false discovery rate (FDR) < 0.01, at both peptide and protein levels prior to filtering. All samples were analyzed in a single batch; therefore, batch effect correction was not applicable. Local normalization [35] was applied to all protein abundance measurements obtained from label-free proteomics. Inter group intensity normalization was not applied. Differentially expressed proteins (DEPs) were defined as those with |log_2_ fold-change| ≥ 0.263 (corresponding to a 1.2-fold change) and adjusted p-value (FDR) < 0.05. Principal component analysis (PCA) was performed to assess global proteomic variation, and no significant batch effects were detected.

4.8. Statistical Analysis

Data were analyzed in GraphPad Prism (v8.0.1). For F1 offspring, the two same-sex littermates from each cage were averaged to produce a single biological replicate, yielding n = 10 true biological replicates per experimental group. Two-way ANOVA was used to assess the effects of paternal exposure and sex, followed by Tukey’s post hoc test for multiple comparisons. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was set at * p < 0.05. p-values between 0.05 and 0.10 were considered non-significant and are described as numerical trends without inferential claims. For proteomic data, multiple testing correction was applied using the Benjamini–Hochberg procedure (FDR < 0.05).

5. Conclusions

In conclusion, paternal ZEN exposure was associated with altered hepatic immune regulation in F1 offspring, characterized by reduced MHC-I and increased MHC-II expression, along with enrichment of inflammatory signaling pathways. These immune-related proteomic changes likely contribute to the observed sexual dimorphism in growth, hepatic steatosis, chronic inflammation, and immune dysregulation. The integrated data support a model wherein initial estrogen receptor-mediated disruption in the male mice contributes to dysregulated hepatic immune-metabolic disorders in offspring. While the intergenerational transmission of these effects is consistent with potential epigenetic involvement, this mechanism remains hypothetical and requires direct experimental validation. This work establishes the paternal exposure pathway as a critical determinant of intergenerational health, expanding the scope of ZEN toxicity beyond traditional maternal-focused paradigms.