NCM460D cell line was purchased from INCELL Corporation (San Antonio, TX). NCM460D is a non-transformed human colonic epithelial cell line originally derived from normal colon tissue of a 68-year-old male donor. The cells were selected for stable in vitro growth and were not infected, immortalized, or genetically modified, preserving a normal genotype. Phenotypically, NCM460D cells express key intestinal epithelial markers, including cytokeratins, villin, colonic epithelial antigens, and a subset of cells exhibit mucin production, consistent with differentiated epithelial features (9). NCM460D cells are non-tumorigenic and grown in INCELL's enriched M3:10^™ medium (INCELL Corp.) to maintain their physiological phenotype, supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific, Waltham, MA), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco), at 37˚C in a humidified atmosphere containing 5% CO₂.

PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl) was purchased from Sigma-Aldrich (St. Louis, MO) and dissolved in dimethyl sulfoxide (DMSO) to generate a 10 mM stock solution. Working concentrations were freshly prepared by diluting the stock in M3:10^™ complete medium, ensuring the final DMSO concentration did not exceed 0.1% (v/v) in any treatment. Cells were seeded at 2x10⁴ cells/well in 96-well plates for viability assays or 2x10⁵ cells/well in 6-well plates for RNA extraction. For the cell viability assay, cells were treated with 0.5, 5, 50, or 200 µM PCB153, or an equivalent volume of DMSO (vehicle control), for 6, 24, or 48 h. For transcriptomic analysis and RT-qPCR validation, cells were exposed to PCB153 (0, 5, or 50 µM) or DMSO for 24 h under identical culture conditions in a 37˚C humidified atmosphere containing 5% CO₂.

The genotoxicity of PCB153 on intestinal epithelial cells was determined by PrestoBlue^TM HS Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA). Briefly, NCM460D cells (2x10⁴ cells/well) were seeded into a 96-well plate and treated with PCB153 at final concentrations of 0, 0.5, 5, 50 and 200 µM, with DMSO serving as the vehicle control. Treatments were performed for 6, 24, and 48 h. After treatment, PrestoBlue reagent was added directly to the culture medium as 1/10th of the total well volume and incubated for 1 h in a cell culture incubator at 37˚C, protected from direct light. Absorbance was measured at 570 nm, using 600 nm as a reference wavelength. Cell viability was calculated as the ratio of OD570/600.

NCM460D cells (2 x 10⁵ cells/well in 6-well plate) were treated with PCB153 (0, 5, or 50 µM) for 24 h, with DMSO serving as the vehicle control. Total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA) according to the manufactory protocol. RNA purity and concentration were assessed using the NanoDrop spectrophotometer (Agilent Technologies, Santa Clara, CA). Total 1 µg of RNA was reverse-transcribed to cDNA using high-capacity cDNA reverse transcription kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA). Reverse transcription was performed at 25˚C for 10 min, followed by 37˚C for 120 min, 85˚C for 5 min, and then held at 4˚C. Quantitative PCR was performed with SYBR Green master mix (Applied Biosystems) on a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). The thermocycling program consisted of an initial denaturation at 95˚C for 10 min, followed by 40 cycles of 95˚C for 15 sec and 60˚C for 60 sec. The relative expression of target gene was analyzed by the 2^-ΔΔCq method (10), normalizing each target to GAPDH and expressing data as fold-change relative to the solvent control. Primer sequences of MT1G, MT2A, LGR5, DACT1, LEF1 and GAPDH are provided in Table SI.

NCM460D cells (2x10⁵ cells/well in 6-well plate) were treated with PCB153 (5, or 50 µM) for 24 h, with DMSO serving as the vehicle control. Total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA) as previously described and purified by RNA Clean & Concentrator kit (Zymo Research, Irvine, CA) according to the manufactory protocol. RNA integrity and quantity were further assessed using Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) before the library construction for RNA sequencing. Total RNA was processed for Poly(A) mRNA isolation and preparation of cDNA libraries using NEBNext Ultra II RNA Library Prep Kit (New England Biolabs, Ipswich, MA) and barcoded with NEBNext Multiplex Oligos for Illumina (NEB), all according to the manufacturer's manuals. Library concentration was measured by Agilent Bioanalyzer (Agilent Technologies) and 2.6x10^-10 moles desired loading of final library were subjected to 100 bp pair-end sequenced (PE100) on the Illumina NovaSeqX platform at the University of Chicago Genomics Core Facility (Chicago, IL). RNA-Seq raw data has been uploaded to the database at the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/bioproject/) under BioProject accession number PRJNA1285092.

For transcriptomic analysis, we performed two independent biological experiments, each containing duplicate cultures per treatment condition (0, 5, and 50 µM PCB153). To minimize technical variation and maximize consistency across experiments, the duplicate cultures within each treatment group were pooled prior to RNA extraction, resulting in two biological replicates per treatment group used for RNA-seq. The output FASTQ sequences were aligned to reference human genome annotation (GENCODE version 48) using STAR software (version 2.7) (11). The aligned sequences were further sorted with Samtools (version 1.18) (12). Counting reads in features with htseq-count (version 2.0.2) (13). The count matrix was then normalized and differentially expressed were identified (adjusted P-value <0.05) with the DESeq2 package (version 1.46) (14) in R software (version 4.4). Gene Ontology (GO) was identified by clusterProfiler (version 4.14) (15) to discover the functional enrichment of pathways comparing experimental groups. ShinyGO 0.82 was used to visualize the pathway networks (16).

All statistical analyses were performed using GraphPad Prism (version 10.5). For cell viability assays (n=6) and RT-qPCR (n=3) experiments, differences among treatment groups were analyzed using one-way ANOVA, followed by Tukey's post hoc test for multiple comparisons. Data are presented as mean ± standard deviation (SD). A P-value of P<0.05 was considered statistically significant. Significance levels are indicated as ^*P<0.05, ^**P<0.01, ^***P<0.001, ^****P<0.0001.

To evaluate the cellular consequences of PCB153 exposure in a physiologically relevant intestinal model, we first established an in vitro system using the non-tumorigenic human intestinal epithelial cell line NCM460D. This line is derived from normal colonic mucosa and retains key features of differentiated intestinal epithelial cells, including expression of cytokeratins, villin, and other colonic epithelial markers, without exogenous genetic modifications (9). Due to these characteristics, NCM460D cells provide an appropriate model for examining transcriptomic disruptions in normal IECs.

To determine suitable exposure conditions for transcriptomic profiling, we next assessed the effects of PCB153 on NCM460D cell viability across a range of concentrations (0.5, 5, 50, and 200 µM) and exposure times (6, 24, and 48 h) (Fig. 1). This analysis was conducted to identify non-cytotoxic conditions appropriate for detection of meaningful alterations by RNA-seq, rather than to define a full cytotoxicity curve. At 6 h, cells exposed to 0.5-50 µM PCB153 showed little change in viability, indicating that this short exposure primarily captures immediate early-response genes and is not ideal for broader transcriptional profiling (Fig. 1A). In contrast, 200 µM PCB153 caused acute viability loss at this early time point, making it unsuitable for mechanistic studies (Fig. 1A). Cells were maintained >90% viability when exposed with both 5 and 50 µM PCB153 at 24 h, providing sufficient time for primary transcriptomic responses to develop while avoiding overt cytotoxicity (Fig. 1B). By comparison, 0.5 µM PCB153 produced minimal biological impact on NCM460D cells and was unlikely to yield detectable transcriptional changes (Fig. 1A-C). However, subtle but statistically significant reductions in cell viability emerged at 48 h, even at 5 and 50 µM intermediate doses, indicating that longer exposures may introduce nonspecific cytotoxic effects (Fig. 1C). Together, these findings identify 5 and 50 µM PCB153 for 24 h as optimal exposure conditions that balance IEC cellular integrity with meaningful transcriptional alterations while minimizing confounding cytotoxic effects and thus were selected for subsequent RNA-seq experiments.

Although recent studies have identified various adverse effects of PCB153 exposure, significant knowledge gaps remain regarding its impact and underlying mechanisms of cytotoxicity in normal intestinal epithelial cells. In addition, no studies have systematically investigated the dose-dependent effects of PCB153 exposure on normal IECs. To address these gaps, this study aimed to characterize the transcriptional responses and signaling pathways affected by PCB153 in human normal IECs using RNA-seq analysis. Principal component analysis (PCA) revealed a significant transcriptomic shift between control and PCB153-treated cells. More importantly, the transcriptomic changes followed a dose-dependent pattern in IECs exposed to PCB153 (Fig. 2A). Further analysis of differentially expressed genes (DEGs), using an adjusted P-value cutoff of <0.05, identified 329 upregulated and 246 downregulated genes in IECs treated with 5 µM of PCB153 compared to controls (Fig. 2B). Moreover, exposure to 50 µM of PCB153 resulted in 4,006 upregulated and 3,070 downregulated genes (Fig. 2C). Among these significantly differentially expressed genes, 136 were commonly downregulated, and 181 were commonly upregulated in both the 5 and 50 µM PCB153 treatment groups compared to controls (Fig. 2D). These results suggest that acute PCB153 exposure significantly alters the transcriptome of IECs, with higher doses exerting more pronounced effects than lower doses. The higher PCB153 exposure resulted in thousands of gene expression changes, indicating a dose-dependent impact on the IEC transcriptome. To validate the RNA-seq results, RT-qPCR was performed for several representative differentially expressed genes, including MT1G, MT2A, LGR5, DACT1, and LEF1. The RT-qPCR results confirmed a strong induction of MT1G and MT2A, and a marked suppression of LGR5, DACT1, and LEF1 following PCB153 exposure, consistent with the RNA-seq data (Fig. 2E). Taken together, these findings suggest that PCB153 exposure may profoundly influence IEC cellular functions and biological processes.

Through DEG analysis, we identified a core set of 317 genes (136 downregulated and 181 upregulated) shared between two doses of PCB153 exposure. Pathway analysis of this core set revealed significant alterations in several key biological processes. GO terms analysis of the 181 upregulated genes indicated significant enrichment in pathways related to mineral absorption, ribosome function, coronavirus disease, and axon guidance (Fig. 3A and B). The upregulation of mineral absorption pathways suggests that PCB153 disrupts metal homeostasis, particularly affecting zinc (Zn) and copper (Cu) transport. Metallothionein (MT) family genes (MT1E, MT1F, MT1G and MT2A), regulate essential metal ions (17,18), dramatically induced, suggesting an adaptive response to PCB153-induced metal transporter disruption, potentially leading to increased cellular stress due to metal accumulation or dysregulation of Zn/Cu levels. Additionally, the increased ribosome activity observed following PCB153 exposure suggests enhanced protein synthesis, which may burden cellular machinery, triggering ER stress, misfolded proteins, and conditions favoring tumorigenesis (19,20). The upregulation of coronavirus disease pathway (Path:hsa05171, aggregates 232 pathway genes) likely reflects the activation of a general ‘viral response-related pathways’ or inflammatory stress response, suggesting PCB153 exposure alters immune responses in the IECs, potentially increasing susceptibility to viral infections and chronic inflammation. Taken together, network analysis further revealed a potential connection between ribosome and coronavirus disease pathways in response to PCB153, suggesting that ribosome dysregulation may exacerbate gut inflammation during infections (Fig. 3B). Interestingly, PCB153 exposure also disrupted axon guidance signaling, suggesting potential alterations in gut-brain axis communication. Axon guidance genes, such as NTN4 [a member of the netrin family (21)], regulate neuronal connectivity, and their dysregulation may impair gut-brain signaling, potentially affecting gut motility and neurological function.

On the other hand, PCB153 exposure led to the downregulation of 136 core genes, which were significantly enriched in pathways related to DNA replication, ABC transporters, digestive secretions (gastric acid, pancreatic, and salivary), cGMP-PKG signaling, cell cycle, estrogen signaling, and Wnt signaling (Fig. 3C and D). Among these, the suppression of DNA replication and cell cycle genes suggests reduced IEC proliferation. Network analysis highlighted a core connection between gastric acid secretion, pancreatic secretion, and salivary secretion, indicating that PCB153 may significantly disrupt digestive processes (Fig. 3D). The downregulation of these pathways may impair protein, fat, and carbohydrate digestion, leading to malabsorption disorders (22). Furthermore, the cGMP-PKG signaling pathway is also connected to the digestive secretion function, which plays a role in intestinal motility and cellular homeostasis (23,24), was also downregulated (Fig. 3C and D). The suppression of ABC transporters (e.g., ABCA1, ABCG1) impairs cholesterol efflux, leading to lipid accumulation and oxidative stress (25). Similarly, downregulation of estrogen signaling in IECs may contribute to the disruptions in intestinal lipid homeostasis. Since PCBs are lipophilic and accumulate in lipid membranes, the inhibition of ABC transporters may increase intracellular PCB retention, exacerbating cellular toxicity and bioaccumulation. Most importantly, PCB153 exposure significantly disrupted the Wnt signaling pathway, which is critical for intestinal stem cell renewal and epithelial integrity (26,27) (Fig. 3C). Notably, due to the downregulation of WNT signaling, LGR5, a stemness-associated molecule, was significantly downregulated (Fig. 2D), suggesting impaired intestinal regeneration and homeostasis. Additionally, TCF4 and LEF1, two major Wnt transcription factors essential for IEC proliferation, ranked among the top 20 downregulated genes (Fig. 2D). Their suppression directly contributes the disrupts of WNT signaling linking to IECs renewal and regeneration, highlighting the detrimental impact of PCB153 on intestinal epithelial integrity.

Taken together, PCB153 exposure profoundly alters multiple essential signaling pathways in normal IECs, disrupting cell proliferation, lipid metabolism, immune responses, and gut-brain axis communication. These findings suggest that PCB153 exposure not only impairs gut homeostasis but may also contribute to tumorigenic potential and long-term metabolic dysfunction.

In this study, we further evaluated the dose-dependent effects of PCB153 on cell fate and signaling in IECs. We found that the 5 µM of PCB153 exposure resulted in the upregulation of the same cellular pathways as identified with the common core upregulated gene set, suggesting the lower dose of PCB153 exposure can initiate the cellular changes of mineral absorption, ribosome function, coronavirus disease, and axon guidance (Fig. 4A). We found that relatively low PCB153 exposure in IECs led to the downregulation in DNA replication, mismatch repair, homologous recombination, ABC transporters, cell cycle regulation, and cGMP-PKG signaling, effects that would be predicted to result in severe gut dysfunction (Fig. 4B). Impaired DNA repair and cell cycle arrest increase genomic instability, raising the risk of mutations and colorectal cancer (28,29). Downregulated ABC transporters reduce toxin efflux, suggesting that PCB153 exposure could augment further cellular PCB153 retention. Additionally, weakened intestinal regeneration can impair the barrier integrity and contribute to chronic inflammation and disrupted homeostasis. These changes collectively are predicted to compromise gut health, increasing susceptibility to disease, oxidative stress, and metabolic imbalances.

On the other hand, 50 µM of PCB153 exposure in IECs resulted in dramatical impairments of multiple signaling pathways. Using an FDR <0.05 as the significance cutoff, a total of 111 pathways were significantly upregulated and 68 were downregulated. The top 20 pathways are shown in Fig. 4C and D. To enhance interpretability, the enriched pathways were consolidated into functional modules based on shared biological relevance. Upregulated pathways were grouped into four major categories: ‘Immune & Host-Defense’, ‘Proteostasis & Translation’, ‘Proliferation/Oncogenic’, and ‘Others’. Downregulated pathways were classified into ‘Core Energy & Lipid Metabolism’, ‘Genome Maintenance & Proliferation’, ‘Neurodegeneration & Proteostasis Stress’, and ‘Transport/Signaling & Global’ modules. This modular grouping highlights the extensive remodeling of immune, metabolic, and stress-related processes in intestinal epithelial cells following high-dose PCB153 exposure.

To provide deeper biological context, we next analyzed the pathways driving these functional modules to delineate the specific cellular processes affected by PCB153 exposure. Exposure to 50 µM PCB153 in IECs induces significant cellular stress, inflammation, and cancer-related responses. Upregulation of mitophagy, autophagy, and protein processing in the ER suggests heightened stress adaptation mechanisms, while activation of TNF and NF-kB signaling points to chronic inflammation and immune dysregulation (30). Increased expression of cancer-related pathways, including bladder and pancreatic cancer, as well as cell cycle dysregulation and senescence, implies a potential pro-tumorigenic environment. Additionally, pathways linked to bacterial and viral infections (Shigellosis, Salmonella, E. coli, and Papillomavirus) are upregulated, suggesting gut barrier dysfunction and increased pathogen susceptibility (31). Thus, these alterations indicate that PCB153 compromises intestinal homeostasis, promotes inflammatory and oncogenic signaling, and weakens immune defenses, increasing the risk of gut-related diseases and malignancies. Conversely, we found that exposure to 50 µM of PCB153 in IECs intensively downregulates key metabolic, mitochondrial, and detoxification pathways, effects likely leading to impaired cellular function. Suppression of steroid and terpenoid biosynthesis, fatty acid metabolism, and amino acid degradation (valine, leucine, lysine) suggests disrupted lipid and energy homeostasis, which may contribute to metabolic disorders. Downregulation of oxidative phosphorylation and thermogenesis points to mitochondrial dysfunction and reduced energy production, potentially compromising cellular survival (32). Inhibition of chemical carcinogenesis (ROS detoxification) and carbon metabolism indicates reduced detoxification capacity, likely rendering PCB153-exposed cells more vulnerable to oxidative stress and secondary toxic insults. Furthermore, repression of pathways related to neurodegenerative diseases (Alzheimer's, Parkinson's, Huntington's, and ALS) raises concerns about systemic neurotoxic effects of PCB153 exposure. Collectively, these alterations suggest that high dose of PCB153 disrupts cellular metabolism, mitochondrial function, and detoxification mechanisms, potentially contributing to epithelial cell dysfunction, metabolic disorders, and increased disease susceptibility.

Taken together, both concentrations of PCB153 disrupt IEC homeostasis. The exposure to PCB153 exhibits dose-dependent transcriptomic shifts in IECs. The higher dose of PCB153 (50 µM) leads to a broader range of cellular effects, exacerbating cellular stress, metabolic disruption, and cancer-related signaling compared to lower doses (5 µM). These findings highlight the potential role of PCB153 in promoting epithelial cell dysfunction, immune dysregulation, and disease susceptibility.