Exploring the Impacts of Polyethylene Microplastics on Rat Liver

doi:10.21203/rs.3.rs-5196445/v1

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Exploring the Impacts of Polyethylene Microplastics on Rat Liver

https://doi.org/10.21203/rs.3.rs-5196445/v1

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The widespread presence of microplastics (MPs) has raised significant concerns due to their adverse impacts on organisms, public health, and ecological safety. Although hepatotoxic consequences of exposure to polystyrene microplastics (PS-MPs) have been studied recently, the potential effects of long-term accumulation of polyethylene microplastics (PE-MPs) in the liver remain unclear. In this study, we developed a rat model (Wistar) with doses of 0.1, 1, and 5 mg/kg of PE-MPs (with sizes ranging from 1–10 µM) over 4 weeks. As confirmed by FT-IR and fluorescence microscopy, PE-MPs exposure did not significantly affect body weight but led to dose-dependent accumulation in liver tissues. Histopathological assessment revealed signs of liver injury, accompanied by a significant dose-dependent increase in lipid peroxidation (LPO) in liver tissue extracts. Furthermore, transcriptomic profiling of the liver exposed to PE-MPs resulted in differentially expressed genes enriched in pathways linked to mitochondrial dysfunction, lipid and fatty acid metabolism, and non-alcoholic fatty liver disease (NAFLD). PE-MPs-induced LPO activates NAFLD pathways, which were further validated at the transcriptional level by involving genes affecting neutrophil infiltration, inflammation, and fibrosis. Thus, targeting the LPO pathway could serve as a potential avenue for intervention in PE-MPs-mediated liver toxicity.

MPs

Polyethylene Microplastics

FTIR

Histopathology

Lipid Peroxidation

Transcriptome-Seq

NAFLD

Microplastic pollution is a growing concern worldwide. Plastic production has already reached 1.5 to 400 million tons from 1950 to 2020 and is projected to be doubled by 2050 (Lim 2021). Microplastics (MPs) are composed of polymers like polystyrene (PS), polypropylene (PP), polyvinyl chloride (PVC), polyethylene terephthalate (PET), and polyethylene (PE), (Wang et al. 2020). Humans are exposed to MPs daily through drinking water, food, and air. Almost 883 and 553 particles/capita/day are consumed by adults and children, respectively (Mohamed Nor et al. 2021). Accumulation of MPs starts from birth and continues throughout life. MPs were identified in the stool of a newborn (Braun et al. 2021), infant and adult faecal samples. Interestingly, The concentration of MPs in infant stool was significantly higher than in adult stool (Liu et al. 2023), which is quite an alarming situation. Moreover, data from animal and human studies have shown that MPs compromise intestinal barrier integrity, enter into the bloodstream (Leslie et al. 2022), (Sun et al. 2022), and accumulate in different organs such as the liver, brain, gut, and kidney (Deng et al. 2017), (Mu et al. 2022). Prolonged inhalation of MPs can lead to lung damage and respiratory dysfunction (Cao et al. 2023a). MPs can cross the blood-brain barrier, potentially entering the brain and causing neurotoxicity (Bellettato and Scarpa 2018).

The liver is the largest metabolic organ, which plays an important role in detoxification and is the main target of MPs bioaccumulation. However, liver toxicity by MPs is still undervalued. MPs have been shown to be a useful model particle for researching the biological impacts and accumulation of MPs in living organisms (Sadri and Thompson 2014) (Yu et al. 2018). MPs (< 5 mm) can accumulate in the liver of zebrafish and cause oxidative stress. Moreover, MPs < 0.5 µm in Ctenopharyngodon idella induce severe hepatic congestion and considerable oxidative stress at high dosages (500 µg/L). Recently, hepatotoxicity and lipid metabolism disturbances have been well documented following ingestion of MPs along with induced oxidative stress as well as inflammation (Cheng et al. 2022), (Huang et al. 2022), (Lu et al. 2018), (Luo et al. 2022), (Wen et al. 2022), (Zheng et al. 2021), (Zhao et al. 2021).

Acute liver injury can result from medications and environmental insults. The environment has a larger concentration of small particle MPs, which makes it challenging to detect them at the lower size (Lu et al. 2018), (Roch et al. 2019), (Sjollema et al. 2016). Apart from that, plastic material type in disease pathophysiology is gaining more attention. Most of the studies have used polystyrene microplastic (PS-MP) to understand the pathophysiology or mechanisms of liver disease and animal model development. However, to our knowledge, to date, little information is available on PE-MPs induced liver toxicity and how it differentially contributes to liver disease development (Sheng et al. 2021). PE-MPs are commonly used plastics which contribute to 26% of global plastics (Plastics Europe 2023). PE-MPs are abundant among MPs and have the potential health risk depending upon size and concentration due to low biodegradability, high durability, and high thermostability. PE-MPs are mostly profuse in drinking water bottles and food, but little is known regarding their effects on the mammalian liver (Mortensen et al. 2021).

In the present study, we exposed the Wistar rat model to PE-MPs for 28 days (OECD 407). We hypothesized that the consumption of PE-MPs may accumulate in the liver and cause liver damage. The main objectives of our study were to investigate (i) accumulation and characterization of PE-MPs in the liver and associated hepatotoxicity, (ii) possible oxidative stress that leads to PE-MPs induced hepatotoxicity, and (iii) transcriptomic analysis to understand the molecular pathways associated with the liver toxicity. In the present study, we uncover a direct link between PE-MPs exposure and initiation of Non-alcoholic fatty liver disease (NAFLD) mediated by lipid peroxidation (LPO).

2.1. Particles

The High-Density Polyethylene (PE) microplastics (MPs) ranging in diameters from 1 to 10 µm were obtained from Nanochemazone (CAS No 9002-88-4, Canada). These MPs were then dispersed onto a glass microfiber filter (Whatman) (Catalog No.1822-047) before staining them with Nile red (HI-MEDIA, Catalog No. TC707). Subsequently, the samples were examined using a stereomicroscope and imaged with a fluorescence microscope (Olympus BX51). The morphology and size of the MPs were verified by a Scanning Electron Microscope (SEM, Tescan VEGA 3 XMU), while the particle size was determined using a particle size analyzer (Microtrac). Furthermore, Fourier transform infrared spectroscopy (ATR-FTIR, Bruker-Vertex 70) was employed to confirm the composition of the MPs.

2.2. Mice and experimental protocols

Twenty-four male Wistar Rat rats, which had reached sexual maturity, were housed and maintained in steel cages at the Animal House. The research has been ethically approved by the Institutional Animal Ethics Committee (IAEC), and approval details can be provided upon request. The rats were subjected to a normal lighting schedule with a light (12-hour)/dark (12-hour) cycle, temperature 22 ± 3ºC, and relative humidity of 55%-65%. The rats were provided with food pellets containing free soy, alfalfa, 40–50% carbohydrate, protein, and 4–7% adipose tissue twice daily, and had access to tap water ad libitum via polysulfone bottles. Animal care and treatment strictly adhered to the guidelines outlined in the Organization for Economic Cooperation and Development OECD-407 (2008). A cohort of sexually mature male Wistar rats was subjected to random allocation into four distinct groups, each comprising six individuals (n = 6/group). The group 1 control is administered with vehicle corn oil and maintained with microplastic-free water and food. Conversely, Groups 2 to 4 were subjected to escalating doses of high-density polyethylene microplastics (PE-MPs) at 0.1, 1, and 5 mg/kg body weight, respectively. These microplastics were suspended in corn oil and administered via oral gavage for a duration of 28 days. The selection of polyethylene microplastic doses adhered to the guidelines outlined in the Organisation for Economic Co-operation and Development (OECD) Protocol 407, specifically designed for evaluating the effects of various compounds with anonymized characteristics (OECD, 2008). On the 29th day, the rats underwent anaesthesia induced by diethyl ether and were subsequently euthanized through decapitation. Tissue samples, including liver, kidney, and gut tissues, were meticulously extracted and promptly preserved at -80°C in a deep freezer for subsequent and comprehensive investigative studies.

2.3. Histopathology

Liver tissues of control and treatments groups (0.1,1, and 5 mg/kg) were fixed in 10% formalin, processed using an automated tissue processor, and 5 mm sections were stained with H&E (haematoxylin and eosin) for histopathological examination (Deng et al. 2017).

2.4. MPs accumulation in tissue samples

Before processing the samples, ethanol was used to clean all working surfaces to avoid contamination altogether. Additionally, distilled water was used to rinse every piece of glassware three times. Throughout the entire analysis process, a laboratory blank was processed to ensure quality control and assurance, indicating minimal contamination of MPs particles from containers or airborne sources.

The lyophilized dry tissues weighing 0.1g underwent digestion in 1ml of 65% V/V nitric acid (HNO₃) at 70˚C for two hours, followed by digestion in 30% V/V hydrogen peroxide (H₂O₂) at 85˚C for two hours. All samples underwent vacuum filtration. The final 10 ml digested solution was diluted with deionized water and vacuum-filtered using a cellulose nitrate membrane. Subsequently, the filter paper was treated with Nile red (HI-MEDIA, TC707), and fluorescent Images were captured using a fluorescent microscope (Olympus BX51).

Direct visualization of MPs in tissue sections was performed by using the Projected Hot Air Deparaffinization (PHAD) method (Marinho and Hanscheid, 2023), which helps to efficiently remove the paraffin from the tissue sections. In short, histopathological slides containing liver tissue sections were positioned in front of a hair dryer. The hair dryer was activated at the specified settings and left operational for a duration of 20 minutes. Subsequently, the slide was washed with distilled water for 5 minutes upon completion of the drying period, followed by staining with Nile-red dye for 15 minutes. Once the staining process is completed, the slide is rinsed by gently introducing tap water to the edge of the slide, allowing it to flow over the sample until no further dye is observed. To decolorize the tissue sample, a 0.5% acid-alcohol solution (0.5 ml of HCL in 100 ml of 70% ethanol) is applied dropwise until no additional dye is released. Subsequently, the slide is retained with Nile-red dye, air-dried, and prepared for fluorescence microscopy observation.

2.5. Biochemical analyses

Liver samples were weighed and homogenized in Phosphate Buffer Saline (PBS) to create a 10% homogenate, and supernatant was obtained by centrifugation at 4°C for 5 minutes at 5000 rpm. The biomarkers and protein levels in the supernatant were determined according to the manufacturer protocol including malondialdehyde (MDA) assay kit (HIMEDIA, CCK023-100), reactive oxygen species (ROS) Assay Kit (HIMEDIA, CCK078-100), total antioxidant capacity (HIMEDIA, CCK071-100), glutathione S-Transferase (GST) (HIMEDIA, CCK028-100), superoxide dismutase (SOD) (HIMEDIA TC693) and catalase (CAT) assay kit (HIMEDIA TC693). All the biomarker levels in cells subjected to various concentrations of MPs (control, 0.1, 1, and 5 mg/kg) were determined using an absorbance reader, and each assay was performed in triplicate.

2.6. Total RNA extraction and cDNA preparation

Total RNA extraction was done by using the TRIzol reagent (Ambion 15596018) following the manufacturer’s protocol from the rat’s liver tissue. The purity of isolated RNA was analyzed with a nanophotometer (spectrophotometer) and 1% agarose gel electrophoresis. The reverse transcription of RNA was performed by using a kit (Applied Biosystems, 4368814) as per manufacturer protocol and stored at -20˚C. The primers listed in Supplementary Table 1 were used in the present study. RT-PCR of given genes was performed with the (Applied Biosystem Quant-3, UK) as per manufacturer instruction (Applied Biosystem, 4367659). The gene expression level was normalized by using the 2^−ΔΔCT method (Livak and Schmittgen 2001). Internal reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for relative fold change expression calculation.

2.7. Transcriptome Analysis and Differential Gene Expression

The transcriptome sequencing analysis was carried out using Illumina NovaSeq 6000. The total RNA isolated using the TRIzol method was purified by a Nucleospin RNA kit, followed by quantification. Nanodrop 2000, and the integrity and RNA quality were performed via 1% agarose gel electrophoresis. The quality assessment of samples was conducted using FastQC, followed by adapter trimming with the fastp tool to remove adapters from the 5’ to 3’ end. This tool, known for its swift data pre-processing capabilities, performs quality control, adapter trimming, filtering, and read pruning, supporting multi-threading. Data quality was evaluated using Phred or Q scores, represented as Qphred=-10log10(e), with "e" indicating the sequence error rate. Salmon, a rapid transcript quantification tool, aligned RNA-Seq read to the Rattus norvegicus reference (mRatBN7.2) and generated raw counts for subsequent analysis. DESeq2 package in R facilitated normalization, visualization, and differential expression analysis, prioritizing genes based on |log2FC|>0. Enhanced Volcano plotted all differentially expressed genes, while unique and commonly expressed transcripts were depicted in a Venn diagram. Gene ontology analysis and pathway enrichment provided insights into biological mechanisms, with ClusterProfiler generating high-quality images and GO tree lists (Love et al. 2014),(Ewels et al. 2016),(Chen et al. 2018),(Patro et al. 2017),(Wu et al. 2021).

2.8 Data analysis

The data was shown as mean ± SD. Statistical analysis was carried out using Prism 8 (GraphPad Software). The one-sample t-test and two-tailed unpaired t-test were used to determine the statistical significance between the experimental and control groups. P values < 0.05 were considered statistically significant.

3.1 Characterization of PE-MPs, animal treatment, and histopathological examination of liver toxicity

MPs were visualized on Whatman paper using bright field microscope (Figure. 1A) and capability to stain with Nile red was confirmed with a fluorescent microscope (Figure. 1A). The particle size was observed in the range between 1–10 µm with two major peaks, one at 1µm and other at 10µm (Figure. 1B). FTIR spectrum of PE-MPs showed that the central band of PE-MPs had a peak at ν_as and ν_s (2913 cm^− 1 and 2847 cm^− 1) due to symmetric and asymmetric CH_2, bending, stretching deformation corresponding to the polyethylene, δs and δas in-plane C-H (1462 and 1377 cm^− 1) and δ rocking C-H (-CH₂-) n ≥ 6 (717 cm^− 1) (Figure. 1C). SEM analysis further confirmed the PE-MPs used in the study were spherical in shape within the size range < 10 µm (Figure. 1D). As shown in schematics (Figure. 1E), the rats were fed with varying doses (Treatment group, 0.1. 1 and 5 mg/kg) of PE-MPs daily and control group with vehicle. Following 28 days of exposure, all the animals remained alive. There were no noticeable variations in daily food intake observed between the groups exposed to microplastics and the control groups. There were no significant differences in body weight between the treatment and control groups were observed. Histopathological examination was carried out to investigate the impact of PE-MPs on the liver. The liver of the rats treated with PE-MPs showed signs of neutrophil infiltrations compared to the control group. Rat treated 0.1 mg/kg dose showed considerable multifocal centrilobular necrosis along with inflammation on histology. The rat treated with medium-dose (1 mg/kg) showed mild periportal fibrosis. The rat treated with high-dose (5 mg/kg) showed moderate to severe multifocal necrosis accompanied by inflammation and inflammatory cell infiltration in the periportal, peribiliary, and other areas along with fibrosis (Figure. 1E).

3.2. Accumulation of PE-MPs in liver tissues

We hypothesized that PE-MPs accumulation is the key determinant of rat liver toxicity. To make this evident, we validated the accumulation of PE-MPs using key experimental methods. By using fluorescence microscopy, tissue liver extracts were stained with Nile red and dried on Watman paper, followed by microscopy showing explicit PE-MPs with red color fluorescence. whereas in the control group, no MPs were observed (Figure. 2A). The dose-dependent increase in the PE-MPs accumulation was quite evident from this experiment (Figure. 2B). The same liver tissue extract was further subjected to FTIR analysis, which confirmed PE-MPs accumulation (Figure. 2C) in the tissues. To get direct evidence of PE-MPs accumulation, the PHAD method followed by Nile red tissue staining was adopted. Fascinatingly, clear accumulations of PE-MPs were observed in treated groups but not in the control group (Figure. 2E). This further reconfirmed the dose-dependent accumulation of PE-MPs in rat liver (Figure. 2E).

3.3. Evaluation of oxidative stress and anti-oxidant markers

Rat liver tissue extract was subjected to ROS level detection; we did not observe a significant change in the level of ROS in any of the exposed doses of PE-MPs (Figure. 3A) after 28 days. But surprisingly, we confirmed the markedly elevated levels of lipid peroxidation measured as malondialdehyde (MDA) level in the PE-MPs exposed group compared to the control group in dose-dependent manner (Figure. 3B). Moreover, GST level was significant increased with increasing dosage of PE-MPs (Figure. 3C). A significant increase in superoxide dismutase (SOD), catalase (CAT) and total antioxidant (TTA) activity was showed only in the 5 mg/kg of PE-MPs dose group compared to the control group (Figure. 3D-F). Whereas at lower doses (0.1 and 1 mg/kg), no significant changes in the levels of SOD, CAT, and total antioxidants were observed. However, a sudden spike in SOD and CAT at high concentrations of PE-MPs may be a feedback response by the hepatic cell damage caused by LPO or free radicals released during lipid peroxidase chain reaction to protect hepatic cells from further damage.

3.4. Transcriptomic analysis results

PE-MPs induced LPO may disrupt the gene expression profile of rat liver and may lead to severe liver pathophysiology. To uncover the changes at the transcriptional level, transcriptome sequencing of liver tissue was performed. A dose of 5mg/kg of PE-MPs was selected in order to elucidate the underlying mechanism of hepatotoxicity at the transcriptional level after 4-weeks of exposure. The 5mg/kg-dose had 162 genes that were significantly different from the control, including 103 upregulated genes and 59 downregulated genes (log2FC > 2) as shown in the volcano graph (Figure. 4A, Supplementary Data 1) The heat map clustered for the top 50 genes were altered in the liver of rat in 5mg/kg group compared control groups (Figure. 4B). To verify the accuracy of transcriptome sequencing, five DEGs were randomly selected for qRT-PCR validation. These genes included three down-regulated genes (AUNIP, CCNB1, and CCNA2) and two up-regulated genes (RPL12 and LNC2) that were changed in 5mg/kg groups compared to the control group. These findings suggest that the outcomes of the transcriptome analysis and the RT-qPCR study supported the reliability of the transcriptome analysis (Figure. 4C-D).

3.5. Gene ontology and KEGG pathway analysis of differentially expressed genes

Gene ontology (GO) analysis was performed using the genes that were upregulated and downregulated DEGs in 5mg/kg dose compared to the control group for functional analysis. GO related to liver function and lipid metabolism were significantly enriched in PE-MPs expose group such as Prolactin signaling pathways, alcohol liver diseases, PPAR signaling pathways, drug metabolism, NAFLD and retinol metabolism (Figure. 5A). GO annotation analysis were further shown to have significant impact on cell cycle suppression and negative regulation on apoptosis (Figure. 5B). ROS generation mainly take place at mitochondria and endoplasmic reticulum, GO analysis clearly indicate the enrichment of mitochondrial transport chains which mainly contribute to generation of free radicals (Figure. 5C). Moreover, GO annotation analysis at biological process shown to have enrichment towards lipid metabolism and fatty acid metabolism, at cellular level enrichment of cytoplasmic changes mainly involving the mitochondria, mitochondria inner membrane and endoplasmic reticulum, at molecular level the enrichment of oxidoreductase and electron transport function was observed (Figure. 5D). Overall, this analysis suggested that the involvement of mitochondrial and lipid metabolic changes in adaptation to PE-MPs exposure. We observed Hsd17b13, Lpin1, RGD1565355, Spc25, Pdk4, Car3, Xbp1, and Sgms2 genes among the list of top 50 genes which established the link between lipid metabolism and the NAFLD pathway. The overlap between the genes enriched for the NAFLD pathway in liver tissue exposed to PE-MPs comes out from transcriptomic analysis with the KEGG database to confirm activation of the NAFLD pathway (Figure. 5E). Since, in the present study, we observed the LPO was the main risk factor behind the liver injury, we performed the validation of downstream and upstream effects as per directed in Figure. 5E.

3.6. NAFLD pathway activation leads to neutrophil infiltration, inflammation, and liver fibrosis

In transcriptomic analysis, the NAFLD pathway was enriched in the PE-MP exposed group (dose 5mg/kg) along with biological processes pointing towards the lipid and fatty acid metabolism with the involvement of mitochondria. We hypothesized that mitochondrial impairment leads to lipid peroxidation and activates NAFLD pathways involving neutrophil infiltration, inflammation, and liver fibrosis. To validate this, we performed the qRT-PCR analysis of the genes of mitochondrial dysfunction, such as UQCRH, MT-CO2, and NDUFC, which are associated with lipid oxidation. A significant increase in mRNA level of UQCRH, MT-CO2, and NDUFC was observed in the 5mg/kg dose group compared to normal (Figure. 6A). The markers for neutrophil infiltration (CXCL1) and proinflammatory cytokine (TNF-α and IL-1β) was further validated with qRT-PCR which are involved in NAFLD. A significant increase in mRNA levels of CXCL1, TNF-α, and IL-1β was observed in the 5mg/kg dose group compared to the control (Figure. 6B). Lastly, the fibrosis marker (Liu et al. 2021) Col1A1, IL-6, and α-SMA at mRNA level were significantly increased in the 5mg/kg dose group compared to normal (Figure. 6C). Overall, accumulation of PE-MPs in rat liver tissue triggers lipid peroxidation, initiating NAFLD. This is confirmed by the upregulation of markers associated with mitochondrial dysfunction, neutrophil infiltration, proinflammatory cytokines, and fibrosis, as depicted in the schema (Figure. 6D).

The liver is the primary target organ for MPs toxicity. MPs are transported to the liver through systemic circulation (Meng et al. 2022). The primary features of MPs-induced liver toxicity include oxidative stress, inflammation, and metabolic irregularities. In this study, rats were orally administered PE-MPs ranging from 1–10 µm in size via gavage at doses of 0.1, 1, and 5 mg/kg over a 4-week period. Biochemical and fluorescence analyses of liver tissue indicate a noteworthy accumulation of PE-MPs in the liver in a dose-dependent manner. We adopted the PHAD method for direct visualization of PE-MPs in the liver section, to avoid the melting of PE-MPs from xylene and efficient removal of paraffin from the section (Figure. 2D) (Marinho and Hanscheid 2023). Histopathologically, a dose-dependent increase in liver toxicity was observed. The rat treated with PE-MPs 5mg/kg dose showed higher levels of liver injury, neutrophil infiltration, inflammation, hepatocyte vacuolation, and dilated intercellular space compared to control.

Oxidative stress is the primary mechanism of MPs toxicity. SOD, CAT, and total TAA are crucial antioxidant enzymes that play a vital role in degrading ROS to mitigate cellular damage. They constitute essential components of the in vivo antioxidant defense mechanism (Auguet et al. 2022). Exposure to MPs induces excessive ROS production in the body and triggers activation of the antioxidant defense system (Prata et al. 2020). Wan et al. studied PS-MPs' impact on antioxidant enzymes in zebrafish larvae. They found elevated MDA levels and reduced activities of SOD, CAT, and TAA (Wan et al. 2019). Deng et al. (ref) built a mouse model to study PS-MPs exposure. They found that SOD activities increased, while CAT activity decreased (Deng et al. 2017). Yang et al., in a juvenile red crucian carp model, observed MP exposure increased MDA levels and an increase in antioxidant enzyme activity, which agrees with the present study (Yang et al. 2019). Zou et al. found that in the PS-MPs group, the activities of SOD, CAT, and TAA were notably reduced, while the MDA content was significantly elevated (Zou et al. 2023). Most of the study is in agreement with the present study in terms of the increase in MDA level. The fluctuations in antioxidant enzyme levels in response to MPs exposure are multifaceted. Factors such as MPs' size, type, concentration, and duration in contact with organisms and tissues under examination may contribute to these variations. However, the underlying mechanism behind these fluctuations remains elusive (de Sá et al. 2018). In the present study, an increase in GST level with an increase in PE-MP exposure further suggests that GST activity may protect the liver hepatocyte apoptosis against increased LPO. Despite observed upregulation of antioxidant levels (SOD, CAT, and TAA) but concomitant liver injury further suggests that PE-MPs induced LPO plays a pivotal role in liver damage and override the protective effects of antioxidants. One key observation of this study suggests that PE-MPs play a limited role in triggering ROS compared to PS-MPs (Das 2023). However, both types of MPs ultimately contribute to a shared stress response pathway, particularly LPO.

A previous study by Wang et al. demonstrated significant alterations in over 8000 genes in the liver of Gobiocypris rarus following 28 days of MPs exposure (Wang et al. 2022). Similarly, Fan et al. found upregulation of 293 genes and downregulation of 351 genes in mouse liver following 20 weeks of MPs ingestion (Fan et al. 2022). Wang et al. identified significant alterations in hepatic gene expression. In the low-dose group, 69 genes were affected, with 30 upregulated and 39 downregulated. In contrast, mice exposed to high-dose MPs showed changes in expression of 178 genes, with 72 were upregulated and 106 were downregulated (Wang et al. 2022). In the present study, following a 4-week exposure to PE-MPs, transcriptomic analysis revealed DEGs, with 103 genes upregulated and 59 genes downregulated in the rat liver. These DEGs exhibited close enrichment of NAFLD.

Recently, in human clinical samples, concentrations of MPs in cirrhotic liver tissues were observed which further confirms the capability of MP to cause diseases (Horvatits et al. 2022). NAFLD, comprising 25% of liver diseases, involves lipid accumulation, oxidative stress, neutrophil infiltration, inflammation, and fibrosis. ROS is mainly produced in the mitochondria and endoplasmic reticulum which instigate the LPO chain reaction. Our transcriptomic analysis revealed upregulation of Uqrch, Mt-co2, and Ndufc genes, which play key roles in the oxidative chain reaction. Validation of those genes in rat liver tissue samples exposed to PE-MPs indicates a connection to mitochondrial dysfunction that may amplify lipid peroxidation. Lipotoxicity is a characteristic of NAFLD accelerated by neutrophil infiltration, inflammation, and fibrosis (Figure. 5E) (Musso et al. 2018).

CXCL1 is a major chemoattractant for neutrophils. Its expression in the liver of NAFLD patients is well-documented. Elevated hepatic CXCL1 mRNA levels, dependent on toll-like receptor 4-MyD88 signaling, lead to neutrophil infiltration, exacerbating both hepatic inflammation and fibrosis. Moreover, CXCL1 overexpression mediated by adenovirus was enough to drive the progression from steatosis to steatohepatitis by instigating hepatic neutrophil infiltration and oxidative stress. These findings emphasize the significance of CXCL1-mediated neutrophil recruitment in NAFLD development (Bertola et al. 2010), (Nagata et al. 2022). In the current study, we validated the CXCL1 expression since we histopathologically observed the neutrophile infiltration.

Liver tissue inflammation is a hallmark feature of NAFLD progression. IL-1β, a pro-inflammatory cytokine, plays a crucial role in NAFLD development and progression, from liver steatosis to NASH and fibrosis. It requires proteolytic cleavage to become bioactive. One prominent activation mechanism in NAFLD is the classical NLRP3 inflammasome pathway, widely studied for its role in metabolic disturbances (Mirea et al. 2018), (Wree et al. 2014). TNF-α is a key inflammatory regulator by activating the NF-κB pathway. This activation triggers downstream signaling cascades, leading to nuclear translocation of NF-κB. Subsequently, NF-κB promotes the transcription of inflammation-associated proteins like TNF-α, IL-6, perpetuating a vicious cycle of inflammation (Schuster et al. 2018), (Zhang et al. 2017). Recently, Gasper et al. investigated the inflammatory response triggered by PS-MPs in the livers of both young and old mice. They noted a rise in the mRNA expression of the inflammatory cytokine TNF-α in PS-MP-exposed mice, regardless of age, compared to control groups (Gaspar et al. 2023). Coa et al. showed PE-MPs trigger activation of the NLRP3 inflammasome and increase expression of IL-1β in carp gills (Cao et al. 2023b). J Woo et al. showed inflammatory cytokines and chemokines such as TNF-α, IL-1β, IL-6, and CXCL1in the BALF of Polyvinyl microplastic exposed mice increased in a dose-dependent manner, and histopathological examination revealed Polyvinyl microplastic exposed mice showed inflammatory cell infiltration and contributed to neutrophilic lung inflammation (Woo et al. 2023).

Liver fibrosis is a pathological process that leads to abnormal deposition of extracellular matrix (ECM) in the liver. Shen et al. investigated the mitochondrial and nuclear damage caused by PS-MPs activating the cGAS/STING signaling pathway, which initiated the nuclear translocation of NFκB and upregulated pro-inflammatory cytokines expression of TNF-α and IL-1β, thus facilitating liver fibrosis (Shen et al. 2022). In the present study, we validate the neutrophile infiltration and inflammation marker (CXCL1, TNF-α, IL-1β) and fibrosis marker (Col1a1, α-sma, and IL-6) to establish the PE-MPs involvement in progressive NAFLD (Figure. 6).

The present study explored liver oxidative stress and transcriptome responses in rats affected by PE-MPs exposure. Although we observed PE-MPs accumulation in liver tissue, the study had some limitations. It remains unclear which specific cells, such as Kupffer cells, hepatocytes, cholangiocytes, or immune cells, are involved in the oxidative stress response. However, employing high-end microscopy in future studies could clarify the specific accumulation sites. PE-MPs induce lipid peroxidation and activate the NAFLD pathway, resulting in prolonged symptomatic effects such as neutrophil infiltration, inflammation, and fibrosis. Addressing lipid peroxidation pathways could serve as a therapeutic strategy for managing NAFLD induced by PE-MPs exposure. This approach may involve lifestyle modifications such as dietary changes and exercise, along with the use of antioxidants or pharmaceuticals targeting lipid peroxidation pathways.

Ethical approval: Ethical permission taken from the IAEC was obtained on 29/11/2021, and project proposal no. PJLCP/2021-22/IAEC/36

Consent to participate: Informed consent was obtained from all individual participants included in the study

Consent for publication: Consent to publish has been received from all participants.

Author contribution: Diwakar Maurya (carried out Animal experiments, transcriptome studies Data analysis, Data Compilation, Bioinformatics analysis, Manuscript writing); Atul Katarkar (Bioinformatic analysis Visualization, Writing - review & editing); Pankaj M. Kulurkar (carried out animal experiments and histopathology ); Shilpa A. Deshpande (provided with animal house facilities and guided in-vivo toxicity studies); K. Krishnamurthi (Guided Biochemical studies, Review & editing.); S. Saravanadevi (Conceptualization, methodology, Validation, formal analysis, investigation, data curation, Writing - review & editing, Resources, and Correspondence)

Funding: This is the Ph.D. work's publication; no external financial support was taken for this research.

Competing interests: The authors declare no competing interests.

Data availability: The data supporting this study's findings are available from the corresponding author upon reasonable request.

Acknowledgments

Diwakar Maurya acknowledges the Council of Scientific and Industrial Research (CSIR), New Delhi, and the Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India, for fellowship. Atul Katarkar is supported by the DBT-Ramalingaswami Re-Entry fellowship (Ref.No.BT/RLF/Re-entry/14/2021). KRC No.: CSIR-NEERI/KRC/2024MAY/WCTA/1

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Exploring the Impacts of Polyethylene Microplastics on Rat Liver

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Abstract

Figures

1. Introduction

2. Material and methods

2.1. Particles

2.2. Mice and experimental protocols

2.3. Histopathology

2.4. MPs accumulation in tissue samples

2.5. Biochemical analyses

2.6. Total RNA extraction and cDNA preparation

2.7. Transcriptome Analysis and Differential Gene Expression

2.8 Data analysis

3. Results

3.1 Characterization of PE-MPs, animal treatment, and histopathological examination of liver toxicity

3.2. Accumulation of PE-MPs in liver tissues

3.3. Evaluation of oxidative stress and anti-oxidant markers

3.4. Transcriptomic analysis results

3.5. Gene ontology and KEGG pathway analysis of differentially expressed genes

3.6. NAFLD pathway activation leads to neutrophil infiltration, inflammation, and liver fibrosis

4. Discussion

5. Conclusion

Declarations

References

Supplementary Files

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