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Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Polyethylene Microplastics Induced Hepatic Oxidative Stress and Early Non-alcoholic Fatty Liver Disease in Wistar Rats

D
Diwakar Maurya1,2
A
Atul Katarkar1
P
Pankaj M. Kulurkar1,2
S
Shilpa A. Deshpande3
K
Kannan Krishnamurthi1,2
S
Saravanadevi Sivanesan1,2,*
1Waste and Chemical Toxicity Assessment, CSIR-National Environmental Engineering Research Institute, Nagpur-440 020, Maharashtra, India.
2Academy of Scientific and Innovative Research, Ghaziabad-201 002, Uttar Pradesh, India.
3Priyadarshini J.L. College of Pharmacy Electronic Zone Building, MIDC, Nagpur-440 016, Maharashtra, India.

ABSTRACT

Background: Polyethylene microplastics (PE-MPs) are persistent environmental pollutants found extensively in various ecosystems. However, their specific impact on liver function remains inadequately investigated.

Methods: Adult male wistar rats were orally exposed to PE-MPs (1-10 µm) at doses of 0.1, 1 and 5 mg/kg/day over 28 days. Attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) and fluorescence microscopy identified accumulations of PE-MPs. Liver tissues were examined histologically using Haematoxylin and Eosin staining. Oxidative stress was measured by measuring malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH) levels. Transcriptomic analysis was performed to detect differentially expressed genes (DEGs). Functional enrichment analyses were conducted using gene ontology (GO) and kyoto encyclopaedia of genes and genomes (KEGG) pathways. Key genes were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Result: Accumulation of PE-MPs was detected in liver tissue. Histological evaluation revealed dose-dependent liver injury, including inflammatory infiltration and periportal fibrosis. Elevated MDA levels indicated increased oxidative stress via lipid peroxidation (LPO). qRT-PCR investigation confirmed the upregulation of mitochondrial dysfunction-related genes (NDUFC, UQCRH, MT-CO2), pro-inflammatory genes (TNF-α, CXCL1, IL-1β) and fibrosis markers (IL-6, Col1A1, α-SMA). Transcriptomic analysis identified 189 DEGs linked with oxidative stress, inflammation and lipid metabolism pathways. Altogether, PE-MPs exposure induces oxidative stress, mitochondrial dysfunction, inflammation and fibrosis, contributing to non-alcoholic fatty liver disease (NAFLD) like hepatic alterations. This highlights the potential risk of PE-MPs as emerging environmental threats to liver health.

INTRODUCTION

Globally, the issue of microplastic (MP) contamination is becoming more and more problematic. By 2050, global plastic production is predicted to reach 33 billion tons, a sharp rise from 1.5 million tons in 1950 and 400 million tons in 2020 (Lei et al., 2024). MPs are composed of multiple polymer categories, specifically polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC) and polystyrene (PS). MPs are accumulated from birth and continue to accumulate throughout life (Braun et al., 2021). Faecal samples from adults and newborns contained MPs (Wen et al., 2022). Furthermore, both humans and animals have demonstrated that microplastics disrupt the intestinal barrier, enter the bloodstream and accumulate in organs, including the gastrointestinal tract, kidneys, liver and brain (Deng et al., 2017a).
       
The liver is an essential organ accountable for detoxification and metabolism, making it a primary target for microplastic (MP) accumulation. MPs can cause liver damage, disrupt digestive functions, impair bile acid secretion and interfere with metabolic processes, leading to serious health concerns (Hou et al., 2024). Recent studies have documented hepatotoxicity and disruptions in lipid metabolism associated with MPs, induced oxidative stress and inflammation (Chiang et al., 2024). Humans consume MPs every day through food, drink and the air. We previously reported that MPs were present in bottled drinking water, with adults consuming around 883 and children consuming 553 particles per capita per day, respectively (Patil et al., 2024). The incidence of MPs in the airborne was also reported by our group earlier (Narmadha et al., 2020). The assessment of MP-induced hepatotoxicity has been investigated, with particular emphasis on PS-MPs (Hamza et al., 2023). However, the specific impact of PE-MPs on liver toxicity is highly elusive. In this study, we evaluate the impact of oral doses of PE-MPs administered to wistar rats (0.1, 1 and 5 mg/kg/day) serving as the independent variable on dependent variables such as oxidative stress biomarkers, histopathological changes (inflammation, fibrosis) and differential gene expression profiles in expose liver tissue. Therefore, the main objective of present study to identify accumulation of PE-MPs in liver tissue, potential dose-dependent biological responses, mechanistic pathways contributing to liver dysfunction and NAFLD-like alterations.

MATERIALS AND METHODS

Microplastic (MP) particles
 
MPs Nanochemazone supplied the High-Density Polyethylene (HDPE)-MPs that had diameters between 1 to10 µm (CAS No 9002-88-4, Canada).
 
Rats and experimental protocols
 
The study was conducted at Priyadarshini J.L. College of Pharmacy, Nagpur, under ethical approval protocol number PJLCP/2021-2022IAEC/36, dated November 29, 2021, granted by the Institutional Animal Ethics Committee of Priyadarshini J.L. College of Pharmacy. 24 male Wistar rats were chosen to avoid variability caused by estrous cycle. Rats were housed in a 12-hour light/dark cycle, at a temperature of 22±3oC and with humidity levels ranging from 55% to 65%. Animals were randomly divided in to four groups (6 animal in each group, as per IAEC and CPCSEA guidelines to minimize animal use): Group 1 (control) received corn oil along with food and water free from microplastics, groups 2 received 0.1 mg/kg/day, group 3 received 1 mg/kg/day and group 4 received 5 mg/kg/day dosage of PE-MPs per day for 28 days. The dose range (0.1, 1 and 5 mg/kg/day) and 28-day exposure period were selected based on prior microplastic toxicology studies and OECD guidelines to represent environmentally relevant, moderate and high exposures and to allow for detection of early hepatic alterations (Lu et al., 2018b; Patil et al., 2024; Djouina et al., 2023). On day 29, the rats were anesthetized and subsequently euthanized, organ removed and kept at -80oC for further analysis.
 
MP accumulation in liver tissue digest
 
Preserved organs (0.1 g) were digested following the given protocol (Deng et al., 2017b). Nile Red (lipophilic dye that binds to plastic polymers) staining was used to evaluate PE-MPs in liver tissue digest. Using glass microfiber filter paper (Whatman, Cat. No. 1822-047), the liver tissue digest was vacuum-filtered. Each filter paper was stained with 2-3 drops of Nile Red dye (HI-MEDIA, TC707) and incubated for 10 minutes at 60oC to dry. Fluorescence imaging of the filter paper was performed using an Olympus BX51 microscope to determine the presence of the accumulated PE-MPs. Subsequently, Liver tissue digests were filtered and analyzed using ATR-FTIR analysis. PE-MPs showed characteristic peaks: CH2 stretching at 2913 and 2847 cm-1, in-plane bending at 1462 and 1377 cm-1 and CH2 rocking at 717 cm-1 (Narmadha  et al., 2020).
 
Direct visualization of MP in the liver
 
The PHAD method was employed to directly visualize PE-MPs in the liver sections (Marinho and Hanscheid, 2023).
 
Biochemical analyses of liver sample
 
A 10% homogenate was made from rat liver samples in phosphate buffer saline (PBS) and the supernatant was extracted by centrifuging the samples at 2665g for five minutes at 4oC (Lohiya et al., 2017). Commercially available kits were employed to detect oxidative stress biomarkers following the manufacturer’s guidelines such as reactive oxygen species (ROS), superoxide dismutase (SOD; HIMEDIA), malondialdehyde (MDA, HIMEDIA), catalase (CAT), glutathione S-transferase (GST, HIMEDIA) and total antioxidant capacity (TAC; HIMEDIA). All assays were carried out in triplicate.
 
Histopathology in liver tissue
 
Liver tissues from control and treated groups were fixed in formalin and sectioned using a microtome. Hematoxylin and eosin (H and E) staining was performed on 5 mm-thick sections for histopathological characterisation (Deng et al., 2017b). Brightfield images were taken with Olympus BX51 microscope (Ravikumar et al., 2022). Leukocyte infiltration and fibrotic score were analysed with Image J software.
 
qRT-qPCR analysis
 
Total RNA was extracted using TRIzol reagents (Ambion, Cat. No. 15596018). Reverse transcription was performed using a cDNA synthesis kit (Applied Biosystems, Cat. No. 4368814). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Wang et al., 2023) served as the housekeeping gene and the primer sequences are accessible in Table 1.

Table 1: RT-qPCR primer sequence.


 
Transcriptome investigation and differential expression genes expression
 
Liver samples were from control and higher doses (5 mg/kg/day) group was analysed for multiplex transcriptome sequencing. The total RNA was isolated, purified using TRIzol method and Nucleospin RNA kit. KAPA HyperPrep kit was used for synthesis, amplification and purification of cDNA. The cDNA library was measured using a bioanalyzer and transcriptome sequencing was achieved on the Illumina NovaSeq 6000 platform. All differentially expressed genes were plotted using Enhanced Volcano and a Venn diagram showed the distinct and frequently expressed transcripts. Using ClusterProfiler, detailed GO tree diagrams and quality visuals were created and GO and pathway enrichment analyses revealed key biological mechanisms (Love, 2014).
 
Statistical analysis
 
The data were expressed as mean ± standard deviation (SD). Student t-test (a non-parametric) was performed using Prism 8 (Graph pad software). P-value <0.05 was considered statistically significant. 

RESULTS AND DISCUSSION

PE-MPs accumulation in liver tissues
 
PE-MPs were detected in the livers of the experimental groups, whereas no such accumulation was observed in the control group. Nile red staining revealed the presence of PE-MPs in liver tissue sections (Fig 1A). A clear progressive accumulation of PE-MPs with a concentration-dependent pattern was observed across the experimental groups (Fig 1B), consistent with previous findings on MP biodistribution in mammalian organs (Deng et al., 2017a). Additionally, ATR-FTIR analysis of the liver tissue extracts further confirmed the occurrence of PE-MPs (Fig 1C). The liver sections were examined using the PHAD method showed the occurrence of PE-MPs in the liver tissues of exposed rats. These observations confirmed an increase in hepatic deposition of PE-MPs in a dose-dependent manner (Fig 1D-E), suggesting a proportional relationship between exposure level and bioaccumulation. These results not only confirm hepatic uptake of ingested PE-MPs but also align with earlier studies on tissue retention and distribution of MPs, reinforcing the liver’s role as a primary accumulation and detoxification site.

Fig 1: Accumulation of PE-MPs in rat liver tissue.


  
Histopathological analysis of liver tissues
 
PE-MPs accumulation in hepatic tissue induced a gradual increase in multifocal centrilobular necrosis and inflammation with increase in dosage of PE-MPs. Compared to the controls, livers from PE-MPs-treated rats also showed notable Leukocyte infiltration and periportal fibrosis (Fig 2A). These histological changes were supported by quantitative morphometric data, which demonstrated significant increases in neutrophil infiltration and fibrotic scores with escalating PE-MP dosages (Fig 2B). This aligns with earlier observations reported by (Deng et al., 2017a) where fluorescent polystyrene microplastics (PS-MPs) were shown to accumulate in mouse liver and induce oxidative damage and inflammation, laying a foundational understanding of microplastic-induced tissue toxicity. The dose-dependent progression of fibrosis and inflammation observed in our study is consistent with earlier work by (Lu et al., 2018b) who demonstrated that PS-MP exposure in mice disrupted hepatic lipid metabolism and promoted hepatic steatosis and inflammation. Similarly, (Zhao et al., 2021) found that chronic PS-MP ingestion triggered immune cell activation and natural killer cell infiltration in the liver, contributing to fibrosis via immunopathological pathways.

Fig 2: (A) Representative images of H and E-stained liver sections from rats exposed for 28 days. Control group shows normal histopathology of liver but experimental group shows notable gradual increase in neutrophil infiltration and periportal fibrosis with increasing dosages. Upper panel, 10X Scale bar, 50 mm and lower panel with inset 40X Scale bar, 10 mm. n= 6 rat/group; 2 section/slide. (B) Quantification of neutrophil infiltration in liver sections of rats treated with increasing doses of PE-MPs (0.1, 1 and 5 mg/kg/day) for 28 days. (B) Fibrotic score expressed as percentage area affected in liver tissue, demonstrating a progressive increase in fibrosis severity with higher PE-MP exposure. Data are expressed as mean ± SD. rat n = 6 per group.


 
Evaluation of oxidative stress by biochemical assay
 
Liver tissue extracts from rats were analyzed for ROS levels following PE-MP exposure. No significant changes in ROS levels were detected across any of the exposed doses after 28 days (p>0.05) (Fig 3A), possibly due to the inherently unstable nature of ROS (Andrés Juan  et al., 2021). The PE-MP-exposed groups exhibited significantly higher MDA levels, reflecting enhanced LPO compared to the control group (p<0.05) (Fig 3B). Dose-dependent increase GST activity was showed on PE-MP exposure (p<0.05) (Fig 3C). Furthermore, significant increases in SOD, catalase CAT and total antioxidant capacity were observed in high exposure group (5 mg/kg/day) (Fig 3D-F). At low doses of experiments groups (0.1 mg/kg/day and 1 mg/kg/day), no notable changes were detected in CAT, SOD, or total antioxidant levels (Fig 3D-F). The increase in MDA levels confirms ROS involvement. Djouina et al. (2023) have shown elevated levels of MDA in mice exposed to PE-MPs, which is further responsible for aggravating liver dysfunction (Djouina et al., 2023). These data corroborate earlier reports by Lu et al., (2018c) and Zhao et al., (2021) who documented similar upregulation of antioxidant defenses in response to polystyrene microplastic (PS-MP) exposure, suggesting a conserved cellular adaptive mechanism across microplastic types.

Fig 3: Evaluation of oxidative stress and anti-oxidant markers.


 
Transcriptomic analysis, GO and KEGG pathway analysis of DEGs
 
Fan et al., 2022 reported 293 upregulated and 351 downregulated genes in mice livers after 20 weeks of PS-MPs ingestion. Wang et al., (2022) observed 69 DEGs gene (low-exposure dose) and 178 (high-exposer dose), with a mix of upregulated and downregulated genes. To explore transcriptional alterations, transcriptome sequencing was performed on liver tissues. A higher dose 5 mg/kg/day PE-MPs were chosen to investigate the molecular mechanisms of liver toxicity following 4 weeks of exposure. The analysis identified 162 differentially expressed genes (DEGs) compared to the control group, comprising 59 down regulated and 103 upregulated genes (|log2FC| > 0), as depicted in the volcano plot (Fig 4A). A heat map of the top 50 DEGs further illustrates the gene expression changes in 5 mg/kg/day group (Fig 4B). Transcriptomic data, validated by five randomly chosen DEGs, selected for quantitative PCR (qPCR) which includes three down regulated genes (CCNB1, CCNA2 and AUNIP) and two upregulated genes (LCN2 and RPL12). The qPCR results corroborated the sequencing findings, supporting the accuracy and reliability of the transcriptomic analysis (Fig 4C-D). KEGG pathway analysis of DEGs showed alterations in lipid metabolism pathways, including prolactin signaling, alcoholic liver disease, PPAR signaling, NAFLD, retinol metabolism and drug metabolism. Gene ontology (GO) annotation further highlighted enrichment in pathways related to cell cycle suppression and negative regulation of apoptosis. Additionally, GO analysis pointed to mitochondrial involvement, with enrichment in oxidative stress-related processes, including mitochondrial transport chains and electron transport functions. These findings suggest that PE-MP exposure disrupts mitochondrial function and lipid metabolism as part of the liver’s adaptive response. Among the top 50 DEGs, several key genes, such as RGD1565355, Hsd17b13, Lpin1, (CD36-like), Car3, Spc25, Xbp1, Pdk4 and Sgms2, were associated with lipid metabolism processing pathways implicated in NAFLD. Analysis of transcriptomic profiles alongside KEGG pathway mapping indicated NAFLD pathway activation in PE-MP-treated liver samples. Given that LPO emerged as a major contributor to liver injury, we further validated its upstream and downstream effects.

Fig 4: Transcriptomic analysis.



Activation of the NAFLD pathway
 
Exposure to 5 mg/kg/day PE-MPs led to transcriptomic changes affecting lipid metabolic processes and NAFLD-associated pathways, with evident mitochondrial participation. We suggest that mitochondrial impairment drives LPO, leading to inflammatory responses, neutrophil infiltration and fibrosis characteristic of NAFLD. To verify this observation, qPCR analysis showed a significant upregulation of mitochondrial dysfunction-related genes UQCRH, NDUFC and MT-CO2 in 5 mg/kg/day group compared to controls (Fig 5A). qPCR validation showed a surge in mRNA levels of IL-1β, CXCL1 and TNF-α key markers of neutrophil infiltration and inflammation involved in NAFLD in the high exposure 5 mg/kg/day PE-MPs group (Fig 5B). Additionally, fibrosis-related genes (Liu et al., 2021) IL-6, α-SMA and Col1A1, were also significantly upregulated (Fig 5C). Elevated expression of CXCL1 facilitates the transition from hepatic steatosis to steatohepatitis by increasing oxidative stress and promoting neutrophil infiltration. IL-1β expression is crucial in the transition from steatosis to NASH and fibrosis, mediated by NLRP3 inflammasome pathway. Experimental data suggest that MPs, including PE-MPs, stimulate pro-inflammatory cytokines expression such as IL-1β, TNF-α and CXCL1, thereby aggravating hepatic inflammation and promoting NAFLD (Musso et al., 2018). These transcriptomic and qPCR findings are consistent with prior studies using other microplastic types such as PS-MPs, which have similarly demonstrated disruption of mitochondrial bioenergetics, inflammation and fibrogenesis in murine models (Fan et al., 2022; Wang et al., 2022).

Fig 5: NAFLD pathway activation leads to neutrophil infiltration, inflammation and liver fibrosis.

CONCLUSION

In conclusion, this study demonstrates that PE-MPs have hepatotoxic effects in the Wistar rat models. The comprehensive analysis includes PE-MPs accumulation, biochemical profiling, histopathological examination and NAFLD pathway evaluation. We sense a clear association between PE-MPs exposure and liver toxicity. These findings suggest that PE-MP accumulation induces LPO, leading to mitochondrial dysfunction, inflammation and fibrosis, thereby activating the NAFLD pathway. Although this research primarily focuses on the hepatotoxicity and NAFLD pathway, future studies should investigate PE-MPs toxicity in other organs.

CONFLICT OF INTEREST

All authors declared that there is no conflict of interest.

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    Full Research Article

    Polyethylene Microplastics Induced Hepatic Oxidative Stress and Early Non-alcoholic Fatty Liver Disease in Wistar Rats

    D
    Diwakar Maurya1,2
    A
    Atul Katarkar1
    P
    Pankaj M. Kulurkar1,2
    S
    Shilpa A. Deshpande3
    K
    Kannan Krishnamurthi1,2
    S
    Saravanadevi Sivanesan1,2,*
    1Waste and Chemical Toxicity Assessment, CSIR-National Environmental Engineering Research Institute, Nagpur-440 020, Maharashtra, India.
    2Academy of Scientific and Innovative Research, Ghaziabad-201 002, Uttar Pradesh, India.
    3Priyadarshini J.L. College of Pharmacy Electronic Zone Building, MIDC, Nagpur-440 016, Maharashtra, India.

    ABSTRACT

    Background: Polyethylene microplastics (PE-MPs) are persistent environmental pollutants found extensively in various ecosystems. However, their specific impact on liver function remains inadequately investigated.

    Methods: Adult male wistar rats were orally exposed to PE-MPs (1-10 µm) at doses of 0.1, 1 and 5 mg/kg/day over 28 days. Attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR) and fluorescence microscopy identified accumulations of PE-MPs. Liver tissues were examined histologically using Haematoxylin and Eosin staining. Oxidative stress was measured by measuring malondialdehyde (MDA), superoxide dismutase (SOD) and reduced glutathione (GSH) levels. Transcriptomic analysis was performed to detect differentially expressed genes (DEGs). Functional enrichment analyses were conducted using gene ontology (GO) and kyoto encyclopaedia of genes and genomes (KEGG) pathways. Key genes were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR).

    Result: Accumulation of PE-MPs was detected in liver tissue. Histological evaluation revealed dose-dependent liver injury, including inflammatory infiltration and periportal fibrosis. Elevated MDA levels indicated increased oxidative stress via lipid peroxidation (LPO). qRT-PCR investigation confirmed the upregulation of mitochondrial dysfunction-related genes (NDUFC, UQCRH, MT-CO2), pro-inflammatory genes (TNF-α, CXCL1, IL-1β) and fibrosis markers (IL-6, Col1A1, α-SMA). Transcriptomic analysis identified 189 DEGs linked with oxidative stress, inflammation and lipid metabolism pathways. Altogether, PE-MPs exposure induces oxidative stress, mitochondrial dysfunction, inflammation and fibrosis, contributing to non-alcoholic fatty liver disease (NAFLD) like hepatic alterations. This highlights the potential risk of PE-MPs as emerging environmental threats to liver health.

    INTRODUCTION

    Globally, the issue of microplastic (MP) contamination is becoming more and more problematic. By 2050, global plastic production is predicted to reach 33 billion tons, a sharp rise from 1.5 million tons in 1950 and 400 million tons in 2020 (Lei et al., 2024). MPs are composed of multiple polymer categories, specifically polyethylene terephthalate (PET), polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC) and polystyrene (PS). MPs are accumulated from birth and continue to accumulate throughout life (Braun et al., 2021). Faecal samples from adults and newborns contained MPs (Wen et al., 2022). Furthermore, both humans and animals have demonstrated that microplastics disrupt the intestinal barrier, enter the bloodstream and accumulate in organs, including the gastrointestinal tract, kidneys, liver and brain (Deng et al., 2017a).
           
    The liver is an essential organ accountable for detoxification and metabolism, making it a primary target for microplastic (MP) accumulation. MPs can cause liver damage, disrupt digestive functions, impair bile acid secretion and interfere with metabolic processes, leading to serious health concerns (Hou et al., 2024). Recent studies have documented hepatotoxicity and disruptions in lipid metabolism associated with MPs, induced oxidative stress and inflammation (Chiang et al., 2024). Humans consume MPs every day through food, drink and the air. We previously reported that MPs were present in bottled drinking water, with adults consuming around 883 and children consuming 553 particles per capita per day, respectively (Patil et al., 2024). The incidence of MPs in the airborne was also reported by our group earlier (Narmadha et al., 2020). The assessment of MP-induced hepatotoxicity has been investigated, with particular emphasis on PS-MPs (Hamza et al., 2023). However, the specific impact of PE-MPs on liver toxicity is highly elusive. In this study, we evaluate the impact of oral doses of PE-MPs administered to wistar rats (0.1, 1 and 5 mg/kg/day) serving as the independent variable on dependent variables such as oxidative stress biomarkers, histopathological changes (inflammation, fibrosis) and differential gene expression profiles in expose liver tissue. Therefore, the main objective of present study to identify accumulation of PE-MPs in liver tissue, potential dose-dependent biological responses, mechanistic pathways contributing to liver dysfunction and NAFLD-like alterations.

    MATERIALS AND METHODS

    Microplastic (MP) particles
     
    MPs Nanochemazone supplied the High-Density Polyethylene (HDPE)-MPs that had diameters between 1 to10 µm (CAS No 9002-88-4, Canada).
     
    Rats and experimental protocols
     
    The study was conducted at Priyadarshini J.L. College of Pharmacy, Nagpur, under ethical approval protocol number PJLCP/2021-2022IAEC/36, dated November 29, 2021, granted by the Institutional Animal Ethics Committee of Priyadarshini J.L. College of Pharmacy. 24 male Wistar rats were chosen to avoid variability caused by estrous cycle. Rats were housed in a 12-hour light/dark cycle, at a temperature of 22±3oC and with humidity levels ranging from 55% to 65%. Animals were randomly divided in to four groups (6 animal in each group, as per IAEC and CPCSEA guidelines to minimize animal use): Group 1 (control) received corn oil along with food and water free from microplastics, groups 2 received 0.1 mg/kg/day, group 3 received 1 mg/kg/day and group 4 received 5 mg/kg/day dosage of PE-MPs per day for 28 days. The dose range (0.1, 1 and 5 mg/kg/day) and 28-day exposure period were selected based on prior microplastic toxicology studies and OECD guidelines to represent environmentally relevant, moderate and high exposures and to allow for detection of early hepatic alterations (Lu et al., 2018b; Patil et al., 2024; Djouina et al., 2023). On day 29, the rats were anesthetized and subsequently euthanized, organ removed and kept at -80oC for further analysis.
     
    MP accumulation in liver tissue digest
     
    Preserved organs (0.1 g) were digested following the given protocol (Deng et al., 2017b). Nile Red (lipophilic dye that binds to plastic polymers) staining was used to evaluate PE-MPs in liver tissue digest. Using glass microfiber filter paper (Whatman, Cat. No. 1822-047), the liver tissue digest was vacuum-filtered. Each filter paper was stained with 2-3 drops of Nile Red dye (HI-MEDIA, TC707) and incubated for 10 minutes at 60oC to dry. Fluorescence imaging of the filter paper was performed using an Olympus BX51 microscope to determine the presence of the accumulated PE-MPs. Subsequently, Liver tissue digests were filtered and analyzed using ATR-FTIR analysis. PE-MPs showed characteristic peaks: CH2 stretching at 2913 and 2847 cm-1, in-plane bending at 1462 and 1377 cm-1 and CH2 rocking at 717 cm-1 (Narmadha  et al., 2020).
     
    Direct visualization of MP in the liver
     
    The PHAD method was employed to directly visualize PE-MPs in the liver sections (Marinho and Hanscheid, 2023).
     
    Biochemical analyses of liver sample
     
    A 10% homogenate was made from rat liver samples in phosphate buffer saline (PBS) and the supernatant was extracted by centrifuging the samples at 2665g for five minutes at 4oC (Lohiya et al., 2017). Commercially available kits were employed to detect oxidative stress biomarkers following the manufacturer’s guidelines such as reactive oxygen species (ROS), superoxide dismutase (SOD; HIMEDIA), malondialdehyde (MDA, HIMEDIA), catalase (CAT), glutathione S-transferase (GST, HIMEDIA) and total antioxidant capacity (TAC; HIMEDIA). All assays were carried out in triplicate.
     
    Histopathology in liver tissue
     
    Liver tissues from control and treated groups were fixed in formalin and sectioned using a microtome. Hematoxylin and eosin (H and E) staining was performed on 5 mm-thick sections for histopathological characterisation (Deng et al., 2017b). Brightfield images were taken with Olympus BX51 microscope (Ravikumar et al., 2022). Leukocyte infiltration and fibrotic score were analysed with Image J software.
     
    qRT-qPCR analysis
     
    Total RNA was extracted using TRIzol reagents (Ambion, Cat. No. 15596018). Reverse transcription was performed using a cDNA synthesis kit (Applied Biosystems, Cat. No. 4368814). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Wang et al., 2023) served as the housekeeping gene and the primer sequences are accessible in Table 1.

    Table 1: RT-qPCR primer sequence.


     
    Transcriptome investigation and differential expression genes expression
     
    Liver samples were from control and higher doses (5 mg/kg/day) group was analysed for multiplex transcriptome sequencing. The total RNA was isolated, purified using TRIzol method and Nucleospin RNA kit. KAPA HyperPrep kit was used for synthesis, amplification and purification of cDNA. The cDNA library was measured using a bioanalyzer and transcriptome sequencing was achieved on the Illumina NovaSeq 6000 platform. All differentially expressed genes were plotted using Enhanced Volcano and a Venn diagram showed the distinct and frequently expressed transcripts. Using ClusterProfiler, detailed GO tree diagrams and quality visuals were created and GO and pathway enrichment analyses revealed key biological mechanisms (Love, 2014).
     
    Statistical analysis
     
    The data were expressed as mean ± standard deviation (SD). Student t-test (a non-parametric) was performed using Prism 8 (Graph pad software). P-value <0.05 was considered statistically significant. 

    RESULTS AND DISCUSSION

    PE-MPs accumulation in liver tissues
     
    PE-MPs were detected in the livers of the experimental groups, whereas no such accumulation was observed in the control group. Nile red staining revealed the presence of PE-MPs in liver tissue sections (Fig 1A). A clear progressive accumulation of PE-MPs with a concentration-dependent pattern was observed across the experimental groups (Fig 1B), consistent with previous findings on MP biodistribution in mammalian organs (Deng et al., 2017a). Additionally, ATR-FTIR analysis of the liver tissue extracts further confirmed the occurrence of PE-MPs (Fig 1C). The liver sections were examined using the PHAD method showed the occurrence of PE-MPs in the liver tissues of exposed rats. These observations confirmed an increase in hepatic deposition of PE-MPs in a dose-dependent manner (Fig 1D-E), suggesting a proportional relationship between exposure level and bioaccumulation. These results not only confirm hepatic uptake of ingested PE-MPs but also align with earlier studies on tissue retention and distribution of MPs, reinforcing the liver’s role as a primary accumulation and detoxification site.

    Fig 1: Accumulation of PE-MPs in rat liver tissue.


      
    Histopathological analysis of liver tissues
     
    PE-MPs accumulation in hepatic tissue induced a gradual increase in multifocal centrilobular necrosis and inflammation with increase in dosage of PE-MPs. Compared to the controls, livers from PE-MPs-treated rats also showed notable Leukocyte infiltration and periportal fibrosis (Fig 2A). These histological changes were supported by quantitative morphometric data, which demonstrated significant increases in neutrophil infiltration and fibrotic scores with escalating PE-MP dosages (Fig 2B). This aligns with earlier observations reported by (Deng et al., 2017a) where fluorescent polystyrene microplastics (PS-MPs) were shown to accumulate in mouse liver and induce oxidative damage and inflammation, laying a foundational understanding of microplastic-induced tissue toxicity. The dose-dependent progression of fibrosis and inflammation observed in our study is consistent with earlier work by (Lu et al., 2018b) who demonstrated that PS-MP exposure in mice disrupted hepatic lipid metabolism and promoted hepatic steatosis and inflammation. Similarly, (Zhao et al., 2021) found that chronic PS-MP ingestion triggered immune cell activation and natural killer cell infiltration in the liver, contributing to fibrosis via immunopathological pathways.

    Fig 2: (A) Representative images of H and E-stained liver sections from rats exposed for 28 days. Control group shows normal histopathology of liver but experimental group shows notable gradual increase in neutrophil infiltration and periportal fibrosis with increasing dosages. Upper panel, 10X Scale bar, 50 mm and lower panel with inset 40X Scale bar, 10 mm. n= 6 rat/group; 2 section/slide. (B) Quantification of neutrophil infiltration in liver sections of rats treated with increasing doses of PE-MPs (0.1, 1 and 5 mg/kg/day) for 28 days. (B) Fibrotic score expressed as percentage area affected in liver tissue, demonstrating a progressive increase in fibrosis severity with higher PE-MP exposure. Data are expressed as mean ± SD. rat n = 6 per group.


     
    Evaluation of oxidative stress by biochemical assay
     
    Liver tissue extracts from rats were analyzed for ROS levels following PE-MP exposure. No significant changes in ROS levels were detected across any of the exposed doses after 28 days (p>0.05) (Fig 3A), possibly due to the inherently unstable nature of ROS (Andrés Juan  et al., 2021). The PE-MP-exposed groups exhibited significantly higher MDA levels, reflecting enhanced LPO compared to the control group (p<0.05) (Fig 3B). Dose-dependent increase GST activity was showed on PE-MP exposure (p<0.05) (Fig 3C). Furthermore, significant increases in SOD, catalase CAT and total antioxidant capacity were observed in high exposure group (5 mg/kg/day) (Fig 3D-F). At low doses of experiments groups (0.1 mg/kg/day and 1 mg/kg/day), no notable changes were detected in CAT, SOD, or total antioxidant levels (Fig 3D-F). The increase in MDA levels confirms ROS involvement. Djouina et al. (2023) have shown elevated levels of MDA in mice exposed to PE-MPs, which is further responsible for aggravating liver dysfunction (Djouina et al., 2023). These data corroborate earlier reports by Lu et al., (2018c) and Zhao et al., (2021) who documented similar upregulation of antioxidant defenses in response to polystyrene microplastic (PS-MP) exposure, suggesting a conserved cellular adaptive mechanism across microplastic types.

    Fig 3: Evaluation of oxidative stress and anti-oxidant markers.


     
    Transcriptomic analysis, GO and KEGG pathway analysis of DEGs
     
    Fan et al., 2022 reported 293 upregulated and 351 downregulated genes in mice livers after 20 weeks of PS-MPs ingestion. Wang et al., (2022) observed 69 DEGs gene (low-exposure dose) and 178 (high-exposer dose), with a mix of upregulated and downregulated genes. To explore transcriptional alterations, transcriptome sequencing was performed on liver tissues. A higher dose 5 mg/kg/day PE-MPs were chosen to investigate the molecular mechanisms of liver toxicity following 4 weeks of exposure. The analysis identified 162 differentially expressed genes (DEGs) compared to the control group, comprising 59 down regulated and 103 upregulated genes (|log2FC| > 0), as depicted in the volcano plot (Fig 4A). A heat map of the top 50 DEGs further illustrates the gene expression changes in 5 mg/kg/day group (Fig 4B). Transcriptomic data, validated by five randomly chosen DEGs, selected for quantitative PCR (qPCR) which includes three down regulated genes (CCNB1, CCNA2 and AUNIP) and two upregulated genes (LCN2 and RPL12). The qPCR results corroborated the sequencing findings, supporting the accuracy and reliability of the transcriptomic analysis (Fig 4C-D). KEGG pathway analysis of DEGs showed alterations in lipid metabolism pathways, including prolactin signaling, alcoholic liver disease, PPAR signaling, NAFLD, retinol metabolism and drug metabolism. Gene ontology (GO) annotation further highlighted enrichment in pathways related to cell cycle suppression and negative regulation of apoptosis. Additionally, GO analysis pointed to mitochondrial involvement, with enrichment in oxidative stress-related processes, including mitochondrial transport chains and electron transport functions. These findings suggest that PE-MP exposure disrupts mitochondrial function and lipid metabolism as part of the liver’s adaptive response. Among the top 50 DEGs, several key genes, such as RGD1565355, Hsd17b13, Lpin1, (CD36-like), Car3, Spc25, Xbp1, Pdk4 and Sgms2, were associated with lipid metabolism processing pathways implicated in NAFLD. Analysis of transcriptomic profiles alongside KEGG pathway mapping indicated NAFLD pathway activation in PE-MP-treated liver samples. Given that LPO emerged as a major contributor to liver injury, we further validated its upstream and downstream effects.

    Fig 4: Transcriptomic analysis.



    Activation of the NAFLD pathway
     
    Exposure to 5 mg/kg/day PE-MPs led to transcriptomic changes affecting lipid metabolic processes and NAFLD-associated pathways, with evident mitochondrial participation. We suggest that mitochondrial impairment drives LPO, leading to inflammatory responses, neutrophil infiltration and fibrosis characteristic of NAFLD. To verify this observation, qPCR analysis showed a significant upregulation of mitochondrial dysfunction-related genes UQCRH, NDUFC and MT-CO2 in 5 mg/kg/day group compared to controls (Fig 5A). qPCR validation showed a surge in mRNA levels of IL-1β, CXCL1 and TNF-α key markers of neutrophil infiltration and inflammation involved in NAFLD in the high exposure 5 mg/kg/day PE-MPs group (Fig 5B). Additionally, fibrosis-related genes (Liu et al., 2021) IL-6, α-SMA and Col1A1, were also significantly upregulated (Fig 5C). Elevated expression of CXCL1 facilitates the transition from hepatic steatosis to steatohepatitis by increasing oxidative stress and promoting neutrophil infiltration. IL-1β expression is crucial in the transition from steatosis to NASH and fibrosis, mediated by NLRP3 inflammasome pathway. Experimental data suggest that MPs, including PE-MPs, stimulate pro-inflammatory cytokines expression such as IL-1β, TNF-α and CXCL1, thereby aggravating hepatic inflammation and promoting NAFLD (Musso et al., 2018). These transcriptomic and qPCR findings are consistent with prior studies using other microplastic types such as PS-MPs, which have similarly demonstrated disruption of mitochondrial bioenergetics, inflammation and fibrogenesis in murine models (Fan et al., 2022; Wang et al., 2022).

    Fig 5: NAFLD pathway activation leads to neutrophil infiltration, inflammation and liver fibrosis.

    CONCLUSION

    In conclusion, this study demonstrates that PE-MPs have hepatotoxic effects in the Wistar rat models. The comprehensive analysis includes PE-MPs accumulation, biochemical profiling, histopathological examination and NAFLD pathway evaluation. We sense a clear association between PE-MPs exposure and liver toxicity. These findings suggest that PE-MP accumulation induces LPO, leading to mitochondrial dysfunction, inflammation and fibrosis, thereby activating the NAFLD pathway. Although this research primarily focuses on the hepatotoxicity and NAFLD pathway, future studies should investigate PE-MPs toxicity in other organs.

    CONFLICT OF INTEREST

    All authors declared that there is no conflict of interest.

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