Sex-specific expression of the human NAFLD-NASH transcriptional signatures in the liver of medaka with a history of ancestral bisphenol A exposure

The progression of fatty liver disease to non-alcoholic steatohepatitis (NASH) is a leading cause of death in humans. Lifestyles and environmental chemical exposures can increase the susceptibility of humans to NASH. In humans, the presence of bisphenol A (BPA) in urine is associated with fatty liver disease, but whether ancestral BPA exposure leads to the activation of human NAFLD-NASH-associated genes in the unexposed descendants is unclear. In this study, using medaka fish as an animal model for human NAFLD, we investigated the transcriptional signatures of human NAFLD-NASH and their associated roles in the pathogenesis of the liver of fish who were not directly exposed but their ancestors were exposed to BPA during embryonic and perinatal development three generations prior. Comparison of bulk RNA-Seq data of the liver in BPA lineage male and female medaka with publicly available human NAFLD-NASH patient data revealed transgenerational alterations in the transcriptional signature of human NAFLD-NASH in medaka liver. Twenty percent of differentially expressed genes (DEGs) were upregulated in both human NAFLD patients and medaka. Specifically in females, among the total shared DEGs in the liver of BPA lineage fish and NAFLD patient groups, 27.69% DEGs were downregulated and 20% DEGs were upregulated. Off all DEGs, 52.31% DEGs were found in ancestral BPA-lineage females, suggesting that NAFLD in females shared majority of human NAFLD gene networks. Pathway analysis revealed beta-oxidation, lipoprotein metabolism, and HDL/LDL-mediated transport processes linked to downregulated DEGs in BPA lineage males and females. In contrast, the expression of genes encoding lipogenesis-related proteins was significantly elevated in the liver of BPA lineage females only. BPA lineage females exhibiting activation of myc, atf4, xbp1, stat4, and cancerous pathways, as well as inactivation of igf1, suggest their possible association with an advanced NAFLD phenotype. The present results suggest that gene networks involved in the progression of human NAFLD and the transgenerational NAFLD in medaka are conserved and that medaka can be an excellent animal model to understand the development and progression of liver disease and environmental influences in the liver.


Introduction
Nonalcoholic fatty liver disease (NAFLD) is a leading liver disease in Western countries, and its incidence is on the rise worldwide 1 .According to the "two-hit hypothesis," steatosis or NAFLD is caused by more than 5% accumulation of fat in hepatocytes in the first hit 2 .
Consequently, excess fat accumulation causes the second hit, which involves oxidative stress, pro-inflammatory cytokines synthesis, and ultimately, apoptosis 3 .Underdiagnosed and untreated NAFLD can lead to necro-inflammatory changes in the liver, which is known as non-alcoholic steatohepatitis (NASH) 4 .In NASH, liver stellate cells (HSCs) release collagen type I and III and fibronectin, which promote fibrosis through chronic liver inflammation 5 .As the disease progresses, ballooning degeneration, lobular inflammation, collagen fiber deposition, and cell death eventually cause fibrosis and cirrhosis, which require liver transplantation 6,7 .Several factors contribute to the pathogenesis of NAFLD.Unlike the 'two-hit hypothesis,' the 'multiple hit' concept addresses the interaction of multiple factors contributing to the pathogenesis of NAFLD.These include genetics 8 , environment 9 , lifestyle 10 , insulin resistance 11 , adipocyte differentiation 12 , and the intestinal microbiota 13 .Among the several factors contributing to NAFLD, environmental chemicals play a major role in triggering the pathogenesis of NAFLD 14- 16 .
As the most widely produced and prevalent environmental chemical worldwide, bisphenol A (BPA) is a potent endocrine disruptor 17 .Because of its ubiquity in nature and consumer goods, humans and non-human organisms are exposed to this chemical at early developmental stages, leaving behind long-lasting health effects in present and future generations [18][19][20][21] .Several epidemiological studies have found a link between BPA exposure and metabolic disorders, including cardiovascular disease, diabetes, and liver disease [22][23][24] .There is a positive correlation between BPA levels in urine and obesity, insulin resistance, ALT levels, and hepatic steatosis index levels (HSI) [25][26][27] .Both in vitro and in vivo studies showed that BPA increases de novo fatty acid synthesis by HepG2 cells and enhances hepatic triglyceride (TG) synthesis in mice [28][29][30] .BPA promotes excessive reactive oxygen species (ROS) production to advance liver pathogenesis by promoting mitochondrial dysfunction and lipoperoxidation in hepatocytes [31][32][33] .
Biologically, sex remains an important variable in the pathogenesis of metabolic disorders 34,35 .
Evidence suggests that direct BPA exposure can promote sex-specific hepatic dysfunction 36,37 .
By establishing the heritable epigenetic memories in germ cells, BPA not only causes adverse health outcomes in the immediate generation exposed to it but also promotes transgenerational health outcomes in unexposed generations [38][39][40][41][42] .We and others have shown that medaka fish can be an animal model for studying human NALFD 43,44 .Medaka provides additional benefits to understanding the heritability of this disease phenotype across generations.We have reported the heritability of NAFLD up to five generations and sex-specific NAFLD-NASH phenotypes in medaka fish caused by ancestral exposure to BPA 44 .It is unclear whether the transcriptional signatures of medaka NAFLD-NASH phenotypes are similar to human NAFLD-NASH patients.
. CC-BY 4.0 International license available under a was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.It  To compare transcriptional signatures associated with NAFLD between human NALFD and BPA-induced NAFLD in medaka fish, we analyzed bulk RNA seq data obtained from the liver of male and female medaka fish that developed sex-specific NAFLD phenotypes and compared them with publicly available human NAFLD and NASH transcriptional signatures.Using a system biology approach, we identified gene sets (biomarkers), transcription factor-driven pathways, and sex-specific up-and downregulated genes and their pathways in fish and human NAFLD-NASH.

Animal care and ancestral BPA exposure, BPA lineage maintenance, and sample collection
As a relevant animal model for human NAFLD, the present study used medaka fish, which shows liver phenotypes comparable to those of humans 44,45 .A four-month-old medaka from the BPA and control lineage that developed transgenerational NAFLD-NASH phenotypes was used to determine the transcriptional landscape and NAFLD-NASH genes profile.The maintenance, procedure for exposure, and euthanization were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of North Carolina Greensboro as previously published 44 .In the laboratory, medaka fish were reared in 20 L glass aquariums on a 14 hour:10 hour light cycle, recirculated water with 25% water exchanged every four hours at 26 ± 1 °C, and fed Otohime granular food and brine shrimp (Artemia nauplii) twice daily.Every exposure group had three biological replicate tanks, and within each biological replicate group, embryos were collected from three to five separate breeding pairs.To generate fish with transgenerational liver phenotypes, The third generation (F2) was used after initial BPA exposure at the first generation (F0).Bisphenol A exposure can affect metabolic and reproductive health of almost all vertebrates at various concentrations 46,22,[47][48][49] .The concentration of BPA (10 μg/L), which is relevant to the environmental levels in many regions of the world, was used as a test concentration 50,51 .BPA exposure solutions were prepared and measured using mass spectrometry as described elsewhere 52,53 .The exposure of BPA started after eight hours post-fertilization stage (hpf) and continued until day fifteen after fertilization (dpf).The BPA exposure occurred only at the first generation (F0) for first 15 days of life and never thereafter.Fish were reared in clean water with no further exposure until F2 generation.
The exposure window of the first 15 days includes a critical period of sex determination 54 and liver differentiation 55 in medaka but excludes the embryonic stem cell differentiation phase in medaka 56 .The uptake of BPA was 20 pg/mg egg/day 44 , which is less than the daily intake of BPA by humans 57 .The measured concentration of BPA was within <10% of the calculated concentration throughout the experiment.The evidence that embryonic BPA exposure (10 μg/L) at F0 generation (ancestors) leads to NAFLD, fertilization defects, and increased embryo mortality in subsequent unexposed generations has been previously demonstrated by our group 44,58,59 .At 120 days of age, six pairs of fish from the initial (F0) generation were bred to produce F1 offspring.The same mating methodology was used to produce the next F2 generation (the third generation, first transgenerational).Six males and nine females were used from each lineage (control and BPA).The experimental fish were euthanized with MS-222 (250 mg/L) at the age of four months, and the liver samples were collected from the fish.

RNA-seq library preparation, RNA sequencing, and data analysis
The liver of control and BPA lineage fish (9 females and 6 males) were used for total RNA extraction by using Quick RNA/DNA Miniprep Plus Kit (#D-7003, Zymo Research, CA, USA) according to the manufacturer's protocol as previously described 60 .RNA quality was tested by bleach gel electrophoresis 61 , and the quantity was determined by Nanodrop 2000 and Qubit (Thermofisher, Waltham, MA).The RNA of the liver from three fish was pooled to make one biological replicate per group for RNA sequencing.Transcriptome libraries were prepared using NEBNext Ultra II RNA Kit and manufacturer's protocol.The libraries were sequenced on Illumina Novaseq 6000 (Novogene Corporation, CA, U.S.A.) using a 150 bp paired-end sequencing strategy (short-reads), producing 20-40 million reads per biological replicate.
Bioinformatics analysis was performed by using the Longleaf clusters of Supercomputing Facility of the University of North Carolina Chapel Hill.The reads were first preprocessed with Fastp 0.23.2 62 , an ultra-fast all-in-one FASTQ preprocessor, which performs quality control, adapter trimming, quality filtering, per-read quality pruning, and many other operations with a single scan of the FASTQ data.The processed reads were then mapped to the medaka genome (Oryzias_latipes.ASM223467v1) using STAR 2.7.7a 63 .Finally, DESeq2 v1.34.0 was used to do the differential expression analysis 64 .Pathway and network analysis done by using Cytoscape 65 Shiney GO 66 , and R package 67 .

Comparative analysis of gene sets: NAFLD/NASH caused by ancestral BPA exposure vs publicly available NAFLD/NASH datasets
A set of DEGs established by direct BPA exposure in mice was obtained from the previously published publicly available data set (NCBI accession number PRJNA529277) 68 .
Human patient datasets were collected by compiling data set of GSE89632, GSE99010, GSE52748 and followed by previously published database 69 and GSE48452 70 .Overlapping DEGs between BPA lineage fish and human patient group were selected and illustrated by using VENNY (http://bioinfogp.cnb.csic.es/tools/venny/index.html).Throughout comparisons, expression data from BPA lineage livers were compared with those from controls, unless specifically mentioned.The transcriptome database has been submitted to NCBI as GSE252744 (token# oruzwusypfezxmp )

Results
RNA seq revealed sex-specific transcriptional alterations and potential biomarkers of transgenerational NAFLD induced by ancestral BPA exposure A bulk RNA sequencing was performed to identify transcriptional alterations in the liver.
Global transcriptome screening identified 11,928 and 16,826 differentially expressed transcripts in the liver of BPA lineage males and females compared to the control group.Based on the volcano plot, the livers of the BPA lineage males (Supplementary Figure 1A) displayed a significantly decreased number of unique DEGs than the livers of the BPA lineage females (Supplementary Figure 1B).Using strict selection criteria (5<log2FC< -4 and for females and 2.5 <log2FC< -2 for males), mRNA biomarkers associated with the transgenerational NAFLD phenotype were identified in males (Figure 1A) and females (Figure 1B) of the BPA lineage.As compared to the control group (Figure 1A), elovl5, igfbp1, tlr5, hck, and apcs were significantly upregulated, and elovl1, fbxo4, invs, prkaca, and pheta2 were significantly downregulated in the liver of the BPA lineage males.The top 10 up-and down-regulated genes associated with direct exposure to BPA in the male liver were provided in Supplementary Table 1A and B 71 .In the female liver, vtg3, fabp7, cacna2d4, esr1, aldh18a1, mttp, fas, and pycr1 were significantly upregulated while pparα, acox3, cdlk2, enpep, trak1 and ugt3a1 were significantly downregulated in the BPA lineage than the control lineage (Figure 1B).A list of significantly upand down-regulated DEGs in females due to direct BPA exposure was provided in Supplementary Table 1C and D 71 .As the metabolic pathways are common among medaka, mice, and humans 43 , the DEGs (biomarkers) induced by BPA exposure (direct, intragenerational) in the mouse liver were compared between the two vertebrate species-mice and medaka.Results suggest that transgenerational liver disease biomarkers are significantly different from those caused by direct exposure to BPA.

Transcription factors-mediated cancerous pathways were found in the livers from BPA lineage females
To gain insight into how transcription factors play a role in BPA-induced transgenerational NAFLD, we examined the expression of several transcription factors and their roles in triggering disease-specific pathways in the liver of BPA lineage medaka.In the livers of BPA lineage male, srf, sall1, rad21, elf1, mef2c were collectively formed transcription factors (TFs) network controlling expression of genes associated with NAFLD phenotype (Supplementary Figure 2A and Supplementary Table 2).TF networks and their expression profiles in the liver of BPA lineage females were shown in Supplementary Figure 2B and heatmaps (Figure 1C), respectively.In the liver of the BPA lineage females, transcription factors, mainly xbp1, myc, atf4, stat 4, and stat 6 were upregulated, whereas hnf4a, ppargc1a, klf5, sp1, and vdr were downregulated (Figure 1C).The number of disease-specific TFs found in the livers of BPA lineage females was significantly increased compared to the livers of BPA lineage males.Transcription factors found in the female livers of the BPA lineage were associated with transcriptional dysregulation in cancer, estrogen signaling pathways, Jak-STAT signaling pathways, Th1 and Th2 cell differentiation, and TGF beta signaling pathways in cancer (Figure 1D).Ingenuity pathway analysis (IPA) determined 50 cancerous genes (Supplementary Table 3) expressed in the female livers of the BPA lineage compared to the control lineage.Estrogen receptor-mediated (esr1 and esr2) activation of myc, rara, and ccnd1 and downstream activation of the cross-linking network were provided in Figure 1E and Supplementary Figure 3.

Both male and female livers of the BPA lineage expressed upregulated genes related to the innate immune pathway, LDL/HDL-mediated lipid transport, and downregulated genes related to lipid digestion
To determine the up and downregulated DEGs and their associated pathways contributing to the NAFLD pathogenesis, significantly altered genes in the liver of BPA lineage were screened by considering significance criteria |log 2 FC| > 0.5, and FDR< 0.1.In total, there were 115 and 1012 significantly upregulated genes and 387 and 702 significantly downregulated genes in the liver of BPA lineage males (Figure 2A) and females (Figure 2B), respectively.Out of all upregulated genes, BPA lineage had 11 common in both sexes, 104 male-specific, and 1001 female-specific DEGs (Figure 2A and Supplementary Table 4A).Out of all downregulated genes, 85 were common in both sexes, 302 were male-specific, and 617 were female-specific (Figure 2B and Supplementary Table 4B).As fatty liver disease phenotypes developed in both males and females from BPA lineages, we explored pathways triggered by up-and downregulated genes common in both sexes.According to KEGG analysis, commonly upregulated genes were enriched in chemokine and chemokine receptor activation, triggering innate immunity (Figure 2C), suggesting immunogenic pathways being impacted.In addition, downregulated genes common in males and females indicated impact in lipid digestion, mobilization and transport, lipoprotein metabolism, HDL/LDL-mediated lipid transport, and bile acid synthesis (Figure 2D).Supplementary Tables 5 and 6 demonstrated the enrichment of total pathways associated with DEGs in the BPA lineage males and females.The present results indicated that up-and downregulated genes in both sexes of BPA lineage fish contributed to dysregulation of immunogenic and lipid clearance pathways in the liver.

Sexually dimorphic expression of genes associated with fat metabolism and immunogenic genes in the BPA lineage fish.
To ascertain the role of fat metabolizing genes in unequal and sexually dimorphic fat deposition in the liver of the BPA lineage fish, we screened for the expression pattern of DEGs associated with cholesterol metabolism, fatty acid transport, lipogenesis, and lipolysis.Heatmap was used to demonstrate the expression pattern of fat metabolizing genes in the liver of BPA lineage males (Figure 3A) and females (Figure 3B).The expression of genes encoding enzymes for the lipogenic cycle, mainly sqle, mttp, cers5, and aco1 was significantly downregulated in the liver of the BPA lineage males compared to the control females (Figure 3A).However, lipogenic genes such as scd, mttp, and abcg1 were significantly upregulated in the liver of BPA lineage females compared to controls, except for pnpla3 (Figure 3B).This suggested that lipogenesis was elevated in females' livers than males of the BPA lineage.In contrast, genes encoding enzymes controlling the lipolytic cycle were significantly downregulated in both males and females of the BPA lineage.Downregulated lipolytic genes included acadm, cpt1ab, and pparδ in the males and pparα, pparδ, cyp 450, and crot in the females, suggesting that the lipolytic cycle was disrupted in the liver of BPA lineage fish.The mRNAs for srebf1, srebf2, pparγ, and pparggc1a were significantly decreased in the liver of BPA lineage females compared to the control females.Genes associated with fatty acid transport mechanisms, such as osbp, cetp, lrpap1, and slc27a1 in the male liver and apoab and fabp3 in the BPA lineage female livers, were significantly downregulated, suggesting restricted fat release from the liver in the BPA lineage fish.In contrast to aberrant expression of fat transport genes, the receptor protein CD36, which controls the intake of extrahepatic fat granules, was significantly increased in the liver of BPA lineage females compared to control females.The receptor protein cd36, which controls the intake of extrahepatic fat granules, was significantly upregulated in the liver of BPA lineage females, suggesting an increased intake of fatty acids rather than an effective fat clearance process.In the liver of both BPA lineage males and females, the DEGs associated with cholesterol metabolism were downregulated, but soat2 was upregulated in the liver of the BPA lineage males compared to control males.BPA lineage males showed downregulation of acot7, elovl6, and elovl5, while elovl1 was upregulated.The expression of tlr5, tnfaip3, cxcl12, hla-dqa1, rsl24d1, and cxcr4 were considerably increased in the liver of BPA lineage males compared to control males (Figure 3A).cxcr4, cxcl12 were upregulated and irf3, caspase3, caspase8, ap1m2, il10, tlr8 and irak1bp1 were upregulated in the liver of BPA lineage females compared to controls (Figure 3B).

The liver of BPA lineage females showed abnormal expression of genes associated with estrogen signaling, epigenetic processes, aberrant cell cycle regulation, activation of kinases, and cytokines
In the BPA lineage females, estrogen signaling pathway genes such as adcy9, cacna1s, cacna1d, and cacng8 were downregulated except for cacna2d4 (Figure 3B).In addition, genes encoding for estrogen-dependent receptors, including esrp1, esr1, cav2, and cavin1b, were significantly upregulated, whereas esr2 and esrp2 were downregulated (Figure 4A).A list of genes involved in estrogen receptor-mediated activation of downstream genes is shown in Supplementary Figure 3.To explore the involvement of epigenetic processes in disease phenotype, we examined genes encoding enzymes involved in epigenetic modification in the livers of the BPA lineage females, which showed increased NAFLD phenotype.The genes related to DNA demethylation, mainly tet1, tet2, and tet3, were significantly downregulated, and all subtypes of dnmts, pcna, and uhrf1 were significantly upregulated in BPA lineage females than control females (Figure 4B).The genes encoding histone deacetylation, mainly hdac1, hdac5, hdac7b, hdac11, hdac4 were significantly downregulated, but hdac9b, hdac8, hdac3, hdac12, hdac6, and sirt were upregulated (Figure 4B).This suggested that epigenetic dysregulation in the liver of BPA lineage females due to ancestral BPA exposure.The genes encoding kinases, including map3k21, ripk2, mapk1, and mtor were differentially expressed (Figure 4C), and cytokine genes were significantly upregulated in the liver of BPA lineage females than control livers (Figure 4D).The cell cycle regulation genes wee1, aruka, rrm2, and rrm2 were significantly upregulated in the liver of BPA-lineage females compared to control females.The cell cycle regulation genes wee1, aruka, rrm2, yme1|1 and rrm2 were significantly upregulated in BPA-lineage female livers compared to control female livers (Figure 4E) and these genes were associated with G1/S, G2/M transition, APC/C mediated degradation of cell cycle protein, DNA replication (Supplementary Figure 4A).

Impact on ER and mitochondrial genes in developing oxidative stress response in the liver of BPA lineage females
When compared between females in the BPA and control lineages, endoplasmic reticulum-mediated stress response genes, mainly Xbp1, hspa5, hyou1, atf4, atf6, and ddit3 were significantly upregulated in the liver of the BPA lineage females (Figure 4F).Among all ER stress genes, atf4, hspa5, ddit3, and hyou1 were upstream hub genes that control many downstream genes, such as mapk14, jun, ankrd1, and fgf19 (Supplementary Figure 4B).These genes are associated with the production of ROS, unfolded protein, and ER-mediated stress (Supplementary Figure 4B).To investigate mitochondrial gene expression associated with energy metabolism, we found a total of 25 DEGs linked to the electron transport chain (ETC).Among the genes encoding protein complex in ETC, cox6b2, cox7a2, nufa-3,-4,-5,-8, nduf-1,-2,-3,-6,-9 were significantly upregulated, while cox5a was only downregulated in BPA lineage females, suggesting dysregulation of mitochondrial energy metabolism pathway ( Figure 4G and Supplementary Figure 5 and 6).The genes encoding nuclear receptors were significantly downregulated except esr1 (Figure 4G).Furthermore, oxidative stress-induced genes, mainly gpx7, gpx4, and gpx3 were highly upregulated in BPA lineage females (Figure 3B).The present results indicated activation of oxidative stress in the liver of BPA lineage females, possibly mediated by mitochondrial and endoplasmic reticulum-mediated stress.

DEG-mediated pathways, Gene Ontology (GO), and IPA analysis revealed disease-related pathways triggered in females from the BPA lineage
In the BPA lineage males, upregulated genes in the DEG list were enriched in Th1 and Th2 cell differentiation, cysteine and methionine metabolism, the intestinal immune network for IgA production, and thiamine metabolism pathways (Figure 5A), suggesting dysregulation of immunogenic response.As revealed by the KEGG pathway analysis, male DEGs were associated with metabolic pathways, protein processing in the endoplasmic reticulum, cholesterol metabolism, and PPAR signaling (Supplementary Figure 7A).In the BPA lineage females, upregulated DEGs were typically associated with cell cycle and checkpoint, p53-independent DNA damage, unfolded protein response by activation of XBP1, respiratory electron transport, and apoptosis regulation pathways, suggesting cellular stress resulting in the development of severe phenotypic traits in females from the BPA lineage (Figure 5B and Supplementary Figure 7B).Downregulated DEGs in the males from the BPA lineages were mainly linked to fatty acid metabolism, steroid biosynthesis, fatty acid elongation, and metabolic pathways (Figure 5C).In contrast, beta-oxidation, PPARα regulation, lipid and lipoprotein metabolism, gluconeogenesis, and transport of fatty acid pathways were associated with downregulated DEGs, indicating perturbed lipolysis and fatty acid clearance mechanism in the liver of BPA lineage females (Figure 5D).In total, 15 pathways common to both males and females, 2 pathways specific to males, and 198 pathways specific to females were found to be activated in the liver of BPA lineage fish, suggesting sex-specific associations of disease pathways in liver disease development (Supplementary Figure 7C).Metabolic pathways, cholesterol metabolism, fatty acid elongation, and PPAR signaling pathways were found to be mutually triggered due to ancestral BPA exposure regardless of sex (Supplementary Figure 7C).According to KEGG pathway analysis, NAFLD, cytokine-cytokine receptor interaction, leukocyte migration, antigen processing and presentation, Jak-STAT signaling and MAPK, AMPK, Insulin, thyroid hormone signaling pathways were found to be associated with up-and downregulated DEGs, respectively in the liver of BPA lineage females compared to control females (Supplementary Figure 8).
Next, we performed gene ontology analysis with top-ranked significant DEGs in males and females from the BPA lineage.In the liver, Gene Ontology Enrichment (GEO) analysis revealed a total of 12 (male) and 94 (female) molecular functions (MF), 85 (male) and 678 (female) biological processes (BP), and 23 (male) and 183 (female) cellular components (CC) in BPA lineage fish (Supplementary Figure 9 and 10).In the liver of BPA lineage males, cholesterol metabolic and biosynthetic process (GO:0016126 and GO:0008203) fatty acid synthase and elongase activity (GO:0004312 and GO:0009922) were significantly enriched.
Cellular protein modification and metabolism (GO:0006464 and GO:0044267), ribosome biogenesis (GO:0042254), kinase activity (GO:0016301), mitochondrial membrane (GO:0005743 and GO:0031966), focal adhesion (GO:0005925) were detected in the liver of BPA lineage females.The results of IPA pointed out disease-specific canonical pathways, such as liver hyperplasia, hepatocellular carcinoma, liver steatosis, and liver necrosis in the liver of BPA lineage females (Supplementary Figure 11).Adipogenesis, triacylglycerol degradation, and retinol biosynthesis were found in the liver of BPA lineage males (Supplementary Figure 12), whereas liver necrosis, and hepatocellular carcinoma were found in the liver of BPA lineage females as per IPA.Upstream regulators hnf4a, tp53, and xbp1 expressed in the liver of the BPA lineage females (Supplementary Figure 12).

BPA exposure across humans, mice, and medaka
To investigate liver-specific pathways associated with direct and indirect BPA exposure, a comparison of KEGG pathway analyses was performed, as shown in the Venn diagram.The results showed that directly exposed mice and indirectly exposed medaka shared eight common pathways induced by BPA (Figure 6A).In males, metabolic pathways, cholesterol metabolism, PPAR signaling pathway, cholesterol metabolism, lysosome, and carbon metabolism pathways were common in databases obtained from direct and indirect BPA exposure.In females, KEGG pathway analysis (Figure 6B) found directly exposed mice and indirectly exposed medaka shared ninety-two common pathways induced by BPA.Common pathways in females represented oxidative phosphorylation, metabolic pathways, ribosomes, chemical carcinogenesis, and proteasomes in the liver.However, MTOR, AMPK signaling, insulin resistance, nonalcoholic fatty liver disease (NAFLD), and pathways in cancer were uniquely triggered in the female liver due to ancestral BPA exposure.Results indicated that in females, similar pathways were affected by the direct and indirect exposure to BPA.Compared to BPA lineage males and females, human and mouse transcription factors associated with steatosis were found most commonly in the liver of BPA lineage females (Supplementary Table 7), suggesting disease-specific transcription factors in humans and mice were significantly expressed in the female liver of BPA lineage.

BPA lineage.
Ancestral exposure led to NAFLD-NASH phenotypes in the liver of BPA lineage females 44 .
Here we screened for NAFLD-NASH signature genes in the livers of BPA lineage females and compared them with gene signatures associated with NAFLD-NASH in human patients.The BPA lineage females and human NAFLD patients shared 88 DEGs (Figure 7A).Additionally, 20% of DEGs were mutually upregulated between medaka and human NAFLD patients (Figure 7B).Among the total shared DEGs, 27.69% of DEGs were mutually downregulated.The total mutual DEGs triggered in the female liver of BPA lineage and human NAFLD patients were illustrated in Figure 7C.A total of 52.31% of DEGs expressed in BPA lineage females were expressed in human patients (Figure 7B).The commonly expressed DEGs in medaka and human NAFLD patients were found to be associated with immune response, cholesterol and lipid metabolic process (Figure 7D).The common downregulated genes were abcb11, aldh6a1, igf1, cyp1a1, and apof.Among the shared upregulated genes, atf3, cxcr4, plin1, gins1, and alpk2 were common in BPA-lineage females and human patients (Supplementary Table 8).In BPA lineage females, gene signaling networks identified hsp90ab1, myc, ctnb1, tp53, and mitf as potential upstream genes involved in human NAFLD (Supplementary Figure 13).In total, 14 pathways associated with NAFLD pathogenesis were found to be common between BPA lineage females and human NAFLD patients (Figure 7E).Among the common pathways, PPAR signaling and metabolic pathways were highly significant in human NAFLD patients (Figure 7F) and BPA lineage females (Figure 7G), respectively.Genes associated with gluconeogenesis, lipid metabolism, mitochondria, ceramide metabolism, insulin pathway, inflammatory response, cell adhesion, coagulation, cytokines controlling liver fat, and advanced liver disease in humans were comparable to liver genes of BPA lineage females suggesting an association of similar DEGs in NAFLD pathogenesis (Supplementary Table 9).In addition, 39 common DEGs were detected in patients with NASH and BPA lineage females, and only 15 DEGs showed ancestral BPA-specific expression (Figure 7H).Among common NASH-specific DEGs, igf1 was found to be an upstream regulator connected to downstream genes, including hnf1a, cyp7a1, mmp13, fbn1, and linked to adipogenesis in the liver (Supplementary Figure 14).The synthesis of extremely longchain fatty acyl CoAs, fatty acid triacylglycerol and ketone body metabolism, bile acid, and salt metabolism were found to be linked to the common NASH genes network (Supplementary Figure 15).

Discussion
BPA can alter metabolic health in humans and non-human organisms; however, excluding BPA from consumer goods may not indicate environmental safety.BPA exposure may still cause transgenerational liver disease in descendants who will not experience BPA exposure in the future.This study demonstrates ancestral BPA-specific transcriptional signatures linked to transgenerational NAFLD in the third generation (F2) induced by ancestral embryonic BPA exposure at the first generation (F0).We found that ancestral BPA exposure led to the development of the NAFLD phenotype and the activation of different disease pathways in its progression to NASH by activating genes found in human NAFLD-NASH patients.
Interestingly, the pathways linked with BPA exposure-induced liver disease caused by direct BPA exposure differed from those associated with ancestral BPA exposure, suggesting the involvement of novel mechanisms in developing liver diseases in unexposed descendants of exposed ancestors.
Global transcriptomic alterations were determined in the liver of both males and females, but female livers showed massive transcriptional alterations associated with DEGs and disease pathways.Several biomarkers, mainly bend7, aldh18a1, pycr2, and cacna2d4 identified in the liver of BPA-lineage females, have been described as "implicated in the activation of cancerous pathways" [72][73][74][75][76] .Interestingly, biomarkers linked with direct and ancestral BPA exposure were substantially different, indicating the involvement of novel pathways in the pathogenesis of BPA-induced transgenerational NAFLD.
Transcription factors, primarily sp1, elf1, klf5, and srebf2 were found in male livers, whereas upregulated myc, xbp1, atf4, stat6, and downregulated hnf4a, ppargc1a, and ctnnb1 were found in female livers of BPA lineage.Elevated levels of xbp1 and atf4 are linked to inflammation and fibrosis in humans and mice that are undergoing NAFLD 77,78 .Thus, overexpression of xbp1 in the liver of BPA lineage females could have triggered lipogenesis and a UPR-driven fibrogenic cascade 79,80 .Mutant mice for atf4 exhibited recovery in liver steatosis phenotype by AMPK-dependent inhibition of fatty acid synthase 81 .Elevated levels of ATF4 found in NASH patients and depletion of ATF4 displayed a protective role in advanced liver phenotype 81 .A previous study found that the activation of myc, one of the most commonly activated oncogenes, is a critical driver of liver carcinogenesis both in vitro and in vivo 82 .Yang et al. showed an activation of hnf4a, resulting in nuclear transcription factor-mediated attenuation of liver fibrosis 83 .As a result of ancestral BPA exposure, the downregulation of hnf4a and overexpression of xbp1 and atf4 could have induced liver fibrosis and progressed the NAFLD to NASH phenotype in medaka.It is worth mentioning that BPA induced equivalent expression of hnf4, xbp1, and atf4 in the livers of both immediately [84][85][86] and ancestrally exposed generations.The role of transcription factors activated in the female liver was associated with transcriptional misregulation in cancer, estrogen signaling route, Jak-STAT signaling, and TGF beta signaling system, according to enrichment analysis, and most of the pathways triggered were associated with the advanced stage of liver disease 87,88 .
In addition to a few common DEGs, we found more sex-specific DEGs between males and females of BPA lineage than in control.DEGs that were identified to be commonly elevated in both males and females were connected to chemokine and innate immune activation.
Interestingly, the same BPA lineage (F2 generation) exhibited transgenerational gut dysbiosis 89 , indicating a possible relationship between gut dysbiosis and activation of immunogenic response in the liver of BPA lineage fish, which could have enhanced the inflammation.Mutually expressed downregulated genes in both males and females of the BPA lineage were enriched in LDL and HDL-mediated lipid transport, lipid digestion, and synthesis of bile acids.Studies showed that low-density apolipoprotein receptor (LDLR) and high-density apolipoprotein receptor (HDLR) play an essential role in the primary route of circulatory lipid clearance in liver cells 90,91 .Choi et al. demonstrated that knockout (Ldlr −/− ) mice were impaired in apoB and apoE-containing lipoprotein clearance and induced steatosis and inflammation in liver 92,93 .
Together with published literature, downregulated genes associated with lipoprotein metabolism could have perturbed the release of fat droplets from the liver in the BPA lineage fish and promoted NAFLD.However, how the heritable epigenetic signature in sperm epigenome controls the differential expression of liver genes and how it is linked to pathogenesis need to be determined in future studies.
As fat-metabolizing genes play a primary role in lipid metabolism of the liver, we screened genes associated with cholesterol metabolism, fatty acid transport, lipogenic, and lipolytic genes in both male and female livers of BPA lineage.We found that most lipogenic genes were downregulated in males, whereas lipogenic genes such as scd, mttp, and abcg1 were dramatically elevated in female BPA livers, but pnpla3 was downregulated.According to the literature, direct BPA exposure increased adipogenesis in the liver by increasing expression of SCD and ABCG1 in vivo 84,94 , and ABCG1 was linked to steatohepatitis 95 .SCD1 gene knock-out mouse study showed a decrease in the lipogenic cycle and fat accumulation in the liver 96,97 .This suggested that activation of lipogenic genes in the female liver could contribute to abnormal fat accumulation.In contrast, genes linked with the lipolytic cycle in the liver, acadm, cpt1ab, and pparδ were downregulated in males, whereas cyp450, pparδ, pparα, and crot were downregulated in females of the BPA lineage.Interesting findings included the up-and downregulation of cpt in the male and female BPA lineages compared to controls, respectively.
Literature suggested that downregulation of pparα displayed severe hepatosteatosis 98 , and depletion of pparδ exhibited increased hepatic expression of inflammatory cytokines TNFα and IL1β and fibrosis in response 99 .Additionally, genes that control beta-oxidation and lipid metabolism, including cpt1b, srebf1, srebf2, pparγ, and pparggc1a were downregulated in liver of BPA lineage females.Previous studies showed that the upregulation of CPT genes determines the activation of beta-oxidation pathways 100 and direct exposure to BPA decreased CPT expression in a previous study 101 .Thus, an increased expression of cpt1b gene in the liver of BPA-exposed females was unique, indicating an enhanced mitochondrial lipid catabolism pathway.However, upregulation of cpt1b in the liver of BPA lineage females could have triggered the apoptotic mechanism by elevating CD4 + T 102 .Ke et al. showed that direct exposure to BPA decreased the DNA methylation levels of Srebf1 and Srebf2 that increased with lipid synthesis 103 .An ancestral BPA exposure led to downregulated srebf1 and srebf2 in the liver of BPA lineage females which might suppress sterol biosynthesis 104 .Instead of de novo fat synthesis in the liver of BPA lineage females, upregulation of gene encoding fat translocase protein in hepatocytes, cd36, could have induced the uptake of extrahepatic fat through the portal and central circulation [105][106][107] .Decreased cholesterol biosynthesis in the liver of the BPA lineage fish could have impaired steroidogenic hormonal pathways, leading to reproductive disorders found in the BPA lineage.As a result, dysregulation of fat metabolism could have contributed to abnormal fat accumulation and reduced sterol biosynthesis, leading to reproductive impairment in BPA lineage fish.Future research must explore the epigenetic control mechanisms involved in the transgenerational dysregulation of fat-metabolizing genes in the liver.
Carbohydrate digestion, Th1 and Th2 cell differentiation, cysteine and methionine metabolism were enriched in the liver of BPA lineage males.Pacana et al. showed that multiple alterations in methionine metabolism trigger NAFLD pathogenesis 108 .Increased hepatic production of Th1-related cytokines IFN, IL-12, and TNF triggered hepatitis in cholinedeficient-diet-fed steatotic mice 109 , as well as elevation of genes toward a Th1 cell differentiation when comparing NASH patients to those without NASH 110 .Transgenerational gut dysbiosis in the same male/ female fish from BPA lineage could have activated Th2 cell activation that could interfere with IL-4/STAT6 axis on insulin sensitivity, leading to the inhibition of PPARa, lipolytic gene 111 .Aberrant methionine metabolism and disruption in the lipolytic cycle in the BPA lineage males could have contributed to enhancing fatty acid elongation process and sterol biosynthesis 112 .In the female liver, pathways to P53-dependent DNA damage response, activation of NF-kappa B cells, activation of chaperone genes by XBP1, and dysregulation in the cell cycle were mainly enriched with upregulated DEGs in the BPA lineage females.Several studies showed that direct exposure to BPA induces DNA damage 111 , promotes cell proliferation in c-Myc-dependent manner in estrogen receptor (ER)-α-negative mammary cells 113 , increases the generation of reactive oxygen species (ROS) in the liver 114 , and induces the formation of DNA adducts 115 .Additionally, abnormal fat accumulation in BPA lineage females could enhance oxidative stress response and apoptotic mechanism by activating caspase and Gpx 116,117 .
Importantly, the signaling pathway activated by ancestral BPA exposure in the female liver, including disruption of ER homeostasis and the unfolded protein response (UPR), has been linked to lipid biosynthesis, insulin action, inflammation, and apoptosis [118][119][120] .Oxidative phosphorylation, chemical carcinogenesis, and metabolic pathways were mutually triggered in direct and indirect BPA exposure but in the liver of BPA lineage females.However, pathway enrichment analysis uncovered unique pathways such as AMPK signaling activation (43 genes, FDR -2.92E-06), MTOR signaling (48 genes, FDR -4.56E-05), Rap1 signaling (61 genes, FDR-4.04E-05),pathways in cancer (125 genes, FDR-0.000102067)only active in the female liver of the BPA lineage that has been associated to cancer [121][122][123][124] .Taken together, ancestral BPA exposure could have triggered harmful pathways, resulting in a severe liver phenotype.
We found 88 common DEGs associated with NAFLD in BPA lineage females and human NAFLD patients.Among the shared downregulated genes, a study showed that significantly decreased Cyp1a1 expression increased cholesterol synthesis and reduced intrahepatic lipid accumulation 126 .Other studies found that abcb11 knockout mice exhibited impaired mitochondrial fatty acid β-oxidation that might exacerbate cholestatic liver damage 127 .
The decreased expression of igf1 was associated with histologic severity of NAFLD 128 .
Overexpression of atf3-induced hepatic stellate cell activation was found in both in vivo and in vitro studies, and knockdown of atf3 alleviated advanced liver disease 129 .Compared with wildtype mice, mice lacking or overexpressing hepatic ATF3 exhibit decreased or increased RIPK3 expression in severe hepatic steatosis and necroptosis after partial hepatectomy.Based on live cell imaging experiments, atf3 induces necroptosis instead of apoptosis in cultured hepatocytes 130 .Upregulation of fabp5 was associated with the activation of hepatocellular carcinoma 131 .In the advanced stage of liver disease, cxcr4 expression was found to be increased 132 and is associated with hepatocellular carcinoma 133 .Overexpression of plin1 increases lipid metabolism and also enhances triglyceride levels 134 .In total, we found 39 genes common between NASH patients and livers of BPA lineage females, and 15 DEGs showed unique ancestral BPA exposure-specific expression patterns.Among common genes, cnga1, cox7b, igfbp2 were upregulated and cacna1h, cyp7a1, fat1,kntc1,lyz,me1,pcsk5, and pla2g7 were downregulated showing ancestral BPA specific expression patterns.However, the downregulation of igfbp2 was associated with fatty liver 135 , the upregulation of cox7b is linked to the disruption of mitochondrial electron transport change 136 , and the downregulation of cyp7a1 was associated with the synthesis of cholesterol 137 .Among NASH-associated DEGs, igf1 was highly downregulated in the liver of BPA lineage female fish compared to human patients.
Literature suggested that impairment of IGF1 synthesis results in a worsening state of insulin resistance 138 , and reduced IGF1 expression caused fibrosis 139 , suggesting igf1 could have enhanced NASH pathogenesis in the liver of BPA lineage females, a master regulator.

Conclusion
This study aimed to examine the global changes in transcriptional networks and their connection to the pathogenesis of NALFD and NASH in the grandchildren generation affected by ancestors' exposure to BPA.In the livers of the BPA lineage females, abnormal cytokine and kinase gene activation that might set off multiple disease pathways were found.Our results indicated that ancestral BPA exposure leads to the activation of several DEGs associated with human NAFLD-NASH patients with alteration of LDL/HDL-mediated lipid transport, metabolism of lipoprotein and lipids, p53-dependent DNA damage response, AMPK, mTOR, and cancerous pathways.Comprehensive transcriptional network analysis identified probable mechanisms of fat accumulation, oxidative stress response mediated by mitochondria and ER, and liver fibrosis in female livers caused by the impact of ancestral BPA exposure.

Figure 1 .
Figure 1.Liver histology of male and female liver of BPA lineage.(A) Control male (B) BPA lineage male and (C) Control female, (D) BPA lineage male.RNA seq determined the top ten up and down regulated genes in (E) male liver and (F) female liver of BPA lineage compared to control.(G) Transcription factors found in the female liver of BPA lineage.(H) Transcription factors drive pathways in the liver of BPA lineage females.(I) Estrogen receptor-mediated gene activation in the liver of BPA lineage female.

Figure 2 .
Figure 2. Comparison of upregulated DEGs in the liver of BPA lineage males and females (A) and enriched pathways of upregulated DEGs exhibited (B).Comparison of down-regulated DEGs in males and females (C) and associated pathways (D).

Figure 3 .
Figure 3. Heat maps showing fat metabolism genes, including cholesterol metabolism, lipolysis, lipogenesis, and fatty acid transport, were displayed with sex-specific expression patterns.Differentially expressed genes in the male liver (A) and female liver (B) are associated with fat metabolism are shown.Color codes indicate treatments and significantly affected pathways.

Figure 5 .
Figure5.The upregulated genes in the liver of BPA lineage male (A) showed activation of immunogenic pathways while the liver of BPA lineage female (B) showed enrichment of unfolded protein, P53 DNA damage response, and activation of chaperon genes.The downregulated genes in the liver of BPA lineage male (C) showed enrichment in steroid biosynthesis, fatty acid elongation but lipoprotein metabolism, lipid digestion, phospholipid metabolic pathways were enriched in the liver of BPA lineage female (D).

Figure 6 .
Figure 6.Comparative analysis of pathways triggered due to direct exposure (mice) and indirect exposure (medaka) of BPA exposure in males (A) and females (B) across the species.

Figure 7 .
Figure 7. Comparative analysis of DEGS found in human NAFLD and NASH patients and BPA lineage liver of males and females.(A) Barplot showing common DEGs in human NAFLD genes and the liver of BPA lineage female and male.(B) Percentage of mutual up and downregulated ancestral BPA exposure-specific DEGs in the female liver.(C) Gene signaling network of common DEGs in human NAFLD patients and females' liver of BPA lineage.(D) Comparison of pathways in human NAFLD patients and BPA lineage females.(E) Total common pathway enrichment in human NAFLD (E) and medaka female (F).(G) Total common NASH-specific genes in human patients and BPA lineage females.(H) Gene signaling network of common NASH genes in BPA lineages.

Figure 1 .Figure 2 .
Figure 1.Liver histology of male and female liver of BPA lineage.(A) Control male (B) BPA lineage male and (C) Control female, (D) BPA lineage male.RNA seq determined the top ten up and downregulated genes in (E) male liver and (F) female liver of BPA lineage compared to control.(G) Transcription factors found in the female liver of BPA lineage.(H) Transcription factors drive pathways in the liver of BPA lineage females.(I) Estrogen receptor-mediated gene activation in the liver of BPA lineage female. log2FC

Figure 3 .Figure 4 :Figure 5 .
Figure 3. Heat maps showing fat metabolism genes, including cholesterol metabolism, lipolysis, lipogenesis, and fatty acid transport, were displayed with sex-specific expression patterns.Differentially expressed genes in the male liver (A) and female liver (B) are associated with fat metabolism are shown.Color codes indicate treatments and significantly affected pathways.
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