Long-term exposure to polychlorinated biphenyl 126 induces liver fibrosis and upregulates miR-155 and miR-34a in C57BL/6 mice

Fernanda Torres Quitete; Ananda Vitória Silva Teixeira; Thamara Cherem Peixoto; Bruna Cadete Martins; Geórgia Correa Atella; Angela de Castro Resende; Daniela de Barros Mucci; Fabiane Martins; Julio Beltrame Daleprane

doi:10.1371/journal.pone.0308334

. 2024 Aug 12;19(8):e0308334. doi: 10.1371/journal.pone.0308334

Long-term exposure to polychlorinated biphenyl 126 induces liver fibrosis and upregulates miR-155 and miR-34a in C57BL/6 mice

Fernanda Torres Quitete ^1,^#, Ananda Vitória Silva Teixeira ^1,^#, Thamara Cherem Peixoto ¹, Bruna Cadete Martins ¹, Geórgia Correa Atella ², Angela de Castro Resende ³, Daniela de Barros Mucci ¹, Fabiane Martins ^1,⁴, Julio Beltrame Daleprane ^1,^*

Editor: Partha Mukhopadhyay⁵

PMCID: PMC11318903 PMID: 39133714

Abstract

Environmental pollutants, including polychlorinated biphenyls (PCBs), act as endocrine disruptors and impair various physiological processes. PCB 126 is associated with steatohepatitis, fibrosis, cirrhosis, and other hepatic injuries. These disorders can be regulated by microRNAs (miRNAs). Therefore, this study aimed to investigate the role of miRNAs in non-alcoholic fatty liver disease associated with exposure to PCB 126. Adult male C57BL/6 mice were exposed to PCB 126 (5 μmol/kg of body weight) for 10 weeks. The PCB group showed lipid accumulation in the liver in the presence of macro- and microvesicular steatosis and fibrosis with increased inflammatory and profibrotic gene expression, consistent with non-alcoholic steatohepatitis (NASH). PCB exposure also upregulated miR-155 and miR-34a, which induce the expression of proinflammatory cytokines and inflammation in the liver and reduce the expression of peroxisome proliferator-activated receptor α, which, in turn, impairs lipid oxidation and hepatic steatosis. Therefore, the present study showed that PCB 126 induced NASH via potential mechanisms involving miR-155 and miR-34a, which may contribute to the development of new diagnostic markers and therapeutic strategies.

Introduction

Environmental factors such as persistent organic pollutants have been highlighted as endocrine disruptors capable of interfering with physiological processes. Exposure to these pollutants, even at low doses, is associated with obesity and non-alcoholic fatty liver disease (NAFLD) [1–4]. Polychlorinated biphenyls (PCBs) are widely used in industrial processes, and despite their banned production, are still present in the environment because of their high thermodynamic stability and resistance to biodegradation [2, 5, 6]. PCBs, including their more toxic congener PCB 126, are present in the diet [7] and are associated with obesity, NAFLD, insulin resistance, diabetes, oxidative stress, and metabolic syndrome [1–3, 8–12]. Their mechanism of action involves the aryl hydrocarbon receptor (AhR), which regulates the transcription of several genes involved in NAFLD progression [2, 11]. These findings are concerning as the NHANES 2003–2004 showed that 100% of the adult population had detectable serum levels of PCBs [5]. Additionally, a 2010 study involving an American population estimated that the daily intake of PCBs was approximately 30 ng/day [13].

PCBs are associated with inflammation, accumulation of triglycerides in the liver, steatohepatitis, fibrosis, cirrhosis, and hepatocarcinoma [1–3, 8–12]. These disturbances are associated with various molecular markers, such as microRNAs (miRNAs), clarifying their participation in the onset and progression of various pathological processes, either as diagnostic molecular markers or potential therapeutic targets [14–17]. NAFLD is strongly associated with miR-155, miR-34a, and miR-122, as these miRNAs possibly regulate different processes of lipid metabolism and are involved in the inflammatory process characteristic of NAFLD [14–16, 18]. miR-122 downregulation and miR-34a upregulation are consistently found in individuals with NAFLD [16]. In contrast, miR-34a and miR-155 are associated with obesity, accompanied by the accumulation of triglycerides in the liver, as they target the mRNA of molecules such as sirtuin 1, which is reduced in individuals with NAFLD and acts by regulating energy homeostasis through the modulation of transcription factors [14–17].

Although several studies have shown an association between miRNAs and the onset of NAFLD [14–17], to date, no study has investigated the association between the expression patterns of these miRNAs and exposure to PCBs. Therefore, the present study aimed to investigate the participation of miRNAs in the development of NAFLD associated with exposure to PCB 126 to clarify the molecular mechanisms by which these disorders occur, providing candidate diagnostic markers and new therapeutic strategies.

Material and methods

Animals and experimental groups

Male C57BL/6 mice aged 3 months old were maintained on a 12 h/12 h dark/light cycle with controlled humidity (60 ± 10%) and temperature (21 ± 2°C) and free access to food and water. This study was approved by the Animal Ethics Committee of the State University of Rio de Janeiro (protocol number CEUA/013/2019) in accordance with the ARRIVE guidelines and was performed in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Mice were randomly assigned to two groups (n = 10 for each group) according to treatment with PCB 126 (5 μmol/kg of body weight, diluted in corn oil) [19, 20] or vehicle (corn oil), which was administered through intragastric gavage biweekly at weeks 2, 4, 6, and 8 of the study (Fig 1A).

Fig 1 — (A) Overview of the experimental approach: C57BL/6 male mice were allocated to receive vehicle–control group or PCB 126 (5 μM/kg) biweekly for 10 weeks. At the end of 10 weeks, mice were euthanized and blood and liver were collected for further analyses. (B) Average food intake per animal per day. (C) Body mass during 10 weeks of experiment; values expressed as mean ± standard deviation (n = 10 per group).

All animals received a normocaloric and normolipidemic standard diet (14%, 10%, and 76% of energy as protein, fat, and carbohydrates, respectively; total energy, 15 kJ/g). The diet was manufactured by PragSolucoes (Jaú, São Paulo, Brazil) and was in agreement with the recommendations of the American Institute of Nutrition (AIN 93M) [21].

Food intake and body mass were measured weekly. At the end of the experiment (10 weeks), the animals were fasted for 6 h and then deeply anesthetized with an intraperitoneal injection of sodium thiopental [60 mg/kg body mass, associated with 2% lidocaine (10 mg)] to withdraw blood using a heparinized syringe by cardiac puncture. Blood samples were centrifuged at 3000 × g for 15 min at 4°C to obtain the plasma. The samples were stored individually at −20°C until the analytical assays. In addition, livers were carefully dissected, weighed, frozen in liquid nitrogen, and stored at −80°C or fixed in formalin for subsequent analyses and histology, respectively.

Plasma analyses

Total cholesterol, triglyceride, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and fasting glucose concentrations in plasma samples were evaluated using commercial colorimetric kits (Bioclin, Belo Horizonte, Brazil). Insulin (#EZRMI-13-K, Millipore, MO), interleukin 6 (IL-6; #BMS603-2, Invitrogen, CA, USA), and tumor necrosis factor-α (TNF-α; #88-7324-88 Invitrogen, CA, USA) levels in plasma were determined using enzyme-linked immunosorbent assay (ELISA) with commercially available kits.

Histological and stereological analysis of the liver

After being fixed in formalin, hepatic tissue was placed in Paraplast Plus (Sigma-Aldrich, St Louis, MO, USA). Subsequently, 5-μm sections were placed on slides and stained with hematoxylin-eosin and Sirius red. The slides were analyzed and images were captured randomly (JPG format, 36-bit color, 1360 × 1024 pixels) under a light microscope (Olympus BX51 with DP71 digital camera; Olympus Optical, Tokyo, Japan). The volume density (Vv) of hepatocytes was determined using the STEPanizer software version 1.8 via 16-point tests. The results were calculated by dividing the sum of the points found by the sum of the total points of the system and were expressed as percentages [22, 23]. Alternatively, to evaluate interstitial fibrosis, the slides were stained with Sirius red solution for 1 h and counterstained with hematoxylin [24, 25]. Furthermore, the extent of fibrosis was quantitatively measured and characterized as the proportion of tissue area positively stained with Picrosirius Red relative to the total liver tissue area, as described previously [22, 26].

Determination of hepatic cholesterol, triglyceride, and hydroxyproline contents

Liver samples (50 mg) were homogenized in 1 mL isopropanol (Vetec, Rio de Janeiro, Brazil) and centrifuged (50 x g/10 min/4°C). Total cholesterol and triglyceride levels in the supernatant were measured using a colorimetric method with a commercial kit (Bioclin, Belo Horizonte, Brazil). Hydroxyproline levels were quantified using a colorimetric method (catalog # MAK008; Sigma-Aldrich, St. Louis, MO, USA) and expressed as micrograms of hydroxyproline per milligram of liver tissue.

Determination of liver fatty acid composition

Total lipids from the liver were extracted as described by Bligh and Dyer [24], with modifications. After incubation in a chloroform-methanol-water solution (2:1:0.8, v/v), 10 mg of liver sample was centrifuged (1500 × g for 20 min at 4°C). Chloroform was added to the supernatant. After centrifugation (1500 × g for 20 min), the organic phase was removed and dried under a nitrogen stream. The extracted lipids were analyzed using thin-layer chromatography (TLC) for neutral lipids on a DC Silica gel 60 plate (Merck Millipore, HE, Germany) [27]. After the run, the plates were submerged for 10 s in Charring solution (3% CuSO₄ and 8% H₃PO₄ (v/v)), dried, and heated to 110°C for 10 min. The TLC plates were analyzed using densitometry (Image Master software from Total Lab, Auckland, New Zealand). The samples were also analyzed using gas chromatography–mass spectrometry (GC–MS) [28]. Lipid samples were dissolved in 1% sulfuric acid in methanol. The GC-MS analysis was performed on a Shimadzu GCMS-QP 2010 Plus system using an HP Ultra 2 column (5% phenyl methylpolysiloxane; Agilent Technologies, 25 m × 0.20 mm × 0.33 μm). The injector temperature was set to 250°C. The column temperature was programmed from 40 to 160°C at 30°C/min, 160–233°C at 1°C/min, 233–300°C at 30°C/min, and then held at 300°C for 10 min. Electron ionization (EI-70 eV) was performed using a quadrupole mass analyzer operating at scans from 40 to 440 amu. The interface was set at 240°C and the ion source at 240°C. Lipid components were identified by comparing their mass spectra to those of the NIST05 MS library contained in the mass spectrometer. Retention indices were used to confirm the identity of the peaks in the chromatogram using the Supelco 37 Component FAME Mix certified reference material (Sigma-Aldrich, St. Louis, MO, USA).

Total RNA and miRNA extraction and real-time reverse transcription polymerase chain reaction (RT-qPCR)

Total RNA and miRNA were extracted from the liver under RNase-free conditions using RNAzol RT (RN 190) reagent (Molecular Research Center, Cincinnati, OH, USA). Total RNA and miRNA were quantified using a NanoVue Plus Spectrophotometer (GE Healthcare, Buckinghamshire, UK). cDNA was prepared from total RNA using a High-Capacity cDNA Reverse Transcription Kit (catalog #:4368814) (Applied Biosystems, Foster City, CA, USA) and from miRNA using a TaqMan MicroRNA Reverse Transcription Kit (catalog #:4366597) (Applied Biosystems, Foster City, CA, USA). The mRNA levels of Ahr (assay ID: Mm00478932_m1), Mcp1 (assay ID: Mm00656886_m1), Cxcl1 (assay ID: Mm04207460_m1), Il-6 (assay ID: Mm00446190_m1), Tnfa (assay ID: Mm00443258_m1), Tgfβ1 (assay ID: Mm03024091_m1), Smad3 (assay ID: Mm03024086_m1), Collagen1α (assay ID: Mm01309565_m1), Cxcl9 (assay ID: Mm00434946_m1), Caspase3 (assay ID: Mm01195085_m1), Sirt1 (assay ID: Mm01168521_m1), and Ppara (assay ID: Mm00440939_m1) and the miRNA levels of miR-155 (assay ID: 002571), miR-122 (assay ID: 002245), and miR-34a (assay ID: 00426) in the liver were determined. The mRNA and miRNA levels of the markers cited above were measured using TaqMan Fast Advanced Master Mix (catalog #4444963) (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. RT-qPCR was performed in triplicate for each sample using an Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Oligonucleotide primers and probes were prepared by Applied Biosystems (Foster City, CA, USA). Co-amplification of mouse Gapdh mRNA (assay ID: Mm99999915_g1) and U6 snRNA (assay ID: 001973), with various internal controls, was performed for all samples. The results were normalized to GAPDH mRNA and U6 snRNA levels using the 2^-ΔΔCT method. This method can be used to calculate relative changes in gene expression, as determined by real-time quantitative PCR [29].

Western blotting analysis

Hepatic protein extraction involved liver homogenization with buffer and protease inhibitors, followed by centrifugation [25]. Supernatants were collected and equal protein amounts were suspended in SDS-buffer, heated, and separated via SDS-PAGE. Electrophoresis used 30 μg protein aliquots, transferred to nitrocellulose membranes. Membranes were blocked with TBS-T solution containing 5% albumin and incubated overnight with cleaved caspase-3 (Cell Signaling, Massachusetts, USA), caspase 3, SMAD 6 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody. After washing, membranes were incubated with secondary antibodies, washed again, and protein expression was detected using ECL kit (GE Healthcare, Madison, WI) and ChemiDoc Resolutions System. Quantitative analysis was performed using Image Pro Plus software v 7.01.

Statistical analysis

The results are shown as the means ± standard deviation. GraphPad Prism 10.2 (GraphPad Software, La Jolla, CA, USA) was used for statistical analyses and graphics. All data were tested for normal distribution and homogeneity of variance using Bartlett’s test. Experimental data were analyzed using Student’s t-test, and differences were considered significant at p < 0.05.

Results

Food intake, body mass, and plasma analysis

No differences were observed in average daily food intake (Fig 1B) or body mass (Fig 1C) between the groups during the 10-week experimental period. The PCB group showed higher plasma triglyceride, glucose, and insulin levels (+68%, +68%, and +53%, respectively; p < 0.001; Table 1) than the C group. Plasma markers of hepatic function were also evaluated, and the PCB group showed increased levels of ALT and AST (4.9- and 1.7-fold increase, respectively; p < 0.001; Table 1) compared to the C group. In addition, the PCB group showed higher levels of the proinflammatory cytokines IL-6 and TNF-α (+63% and +43%, respectively; p < 0.001, Table 1) than the C group. Surprisingly, the plasma cholesterol concentration was lower in the PCB group than in the C group (-15%; p < 0.05, Table 1).

Table 1. Biochemical parameters in plasma.

Plasma analysis	C	PCB
ALT (U/L)	9.6 ± 4.1	56.9^*** ± 16.1
AST (U/L)	70.4 ± 2.9	191.8^*** ± 3.1
Cholesterol (mmol/L)	1.45 ± 0.2	1.23^* ± 0.2
Triglycerides (mmol/L)	0.37 ± 0.0	0.62^*** ± 0.1
Glucose (mmol/L)	6.50 ± 1.2	10.9^*** ± 1.2
Insulin (pmol/L)	93.3 ± 1.6	142.8^*** ± 2.6
IL-6 (pg/ml)	45.4 ± 3.3	73.8^*** ± 3.8
TNF-α (pg/ml)	14.0 ± 1.5	20.0^*** ± 1.1

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C–Control group; PCB–PCB 126 group.

Values expressed as mean ± SD (n = 10).

* p value < 0.05;

** p value < 0.01;

***p value < 0.001.

Liver analysis

Histological and stereological analyses were performed on the liver samples, and the stained sections showed microvesicular and macrovesicular steatosis of small and large lipid droplets dispersed in the livers of animals exposed to PCB (Fig 2A). Vv [steatosis, liver] hepatic steatosis was higher in the PCB group than in the C group (3.5-fold increase; p < 0.001, Fig 2B). Consistent with this result, the PCB group showed higher hepatic triglyceride (+98%; p < 0.001, Fig 2C) and cholesterol (+40%; p < 0.001, Fig 2D) contents than the C group. PCB serves as a classical ligand for the aryl hydrocarbon receptor (AhR), and the activation of this transcription factor is associated with the exacerbation of hepatic steatosis. As anticipated, its expression was elevated in the PCB group compared to that in the control group (2.5-fold increase, p < 0.0001; Fig 2E). In addition to macro- and microsteatosis in the liver, inflammatory infiltrates were observed on histological slides. The expression levels of Mcp1 (3-fold increase; p < 0.0001, Fig 2F) and Cxcl1 (2-fold increase; p < 0.001, Fig 2G) were higher in the PCB group than in the control group. Consistent with systemic inflammation findings, the hepatic expression of Il-6 (0.5-fold increase, p < 0.01, Fig 2H), Tnf-α (2-fold increase, p < 0.01, Fig 2I).

Fig 2 — (A) Stereology of the liver; (B) Vv steatosis (%) of mouse liver; (C) hepatic triglyceride content; (D) hepatic cholesterol content; (E) aryl hydrocarbon receptor (AhR) gene expression in the liver; gene expression of inflammation-related genes: (F) *Mcp1*, (G) *Cxcl1*, (H) *Il-6* and (I) *Tnf-α*. C: Control group; PCB: PCB 126 group. Values are expressed as mean ± standard deviation (n = 10 per group for hepatic lipid content; n = 5 per group for histological and stereological analysis; n = 8 per group for RT-PCR). ** p value < 0.01; *** p value < 0.001; ****p value < 0.0001.

Furthermore, the PCB group exhibited fibrotic areas, suggesting potential progression from hepatic steatosis to non-alcoholic steatohepatitis (NASH) (Fig 3A). To confirm the presence of fibrosis, we quantified the Sirius red staining area, and the PCB group showed a 4.5-fold increase in Sirius red staining than the control group (Fig 3B, p < 0.0001). Hydroxyproline quantification was 6 times higher in the PCB group than in the control group (Fig 3C, p < 0.0001). Genes involved in the development of fibrosis, namely Tgfβ1 (3.5-fold increase, p < 0.0001, Fig 3D), Smad3 (4-fold increase, p < 0.0001, Fig 3E), Collagen1α (3.4-fold increase, p < 0.0001, Fig 3F), and Cxcl9 (3-fold increase, p < 0.0001, Fig 3G), were also upregulated in the PCB group compared to the control group. Hepatic fibrosis, which may result from steatosis, is exacerbated by the death of hepatocytes. The PCB group exhibited increased Caspase 3 gene expression (2-fold increase; Fig 3H) and increased cleaved caspase 3 protein expression (Fig 3I and 3J; p<0.001). Protein level of SMAD 6 was up-regulated in PCB group (Fig 3K and 3L; p < 0.001).

Fig 3 — (A) Picrosirius red staining; (B) % Picrosirius Red area; (C) hydroxyproline content in the liver; profibrogenic gene expression: (D) *Tgfβ1*, (E) *Smad3*, (F) *Collagen1α*, (G) *Cxcl9*, and cell death-related gene: (H) *Caspase 3*. Protein Levels of cleaved caspase 3 (I and J) and SMAD 6 (K and L) were assed. C: Control group; PCB: PCB 126 group. Values are expressed as mean ± standard deviation (n = 8). ** p value < 0.01; *** p value < 0.001.

The fatty acid profile was also evaluated in liver samples, and no differences were observed in the amounts of saturated, monounsaturated, and polyunsaturated fatty acids between groups or in the amounts of n-6 and n-3 essential fatty acids (Table 2).

Table 2. Fatty acids profile in liver samples.

Fatty acids	C	PCB
Lauric acid (C12:0)	0.326	0.144
Tridecanoic acid (C13:0)	0.000	0.000
Myristic acid (C14:0)	0.609	0.591
Pentadecanoic acid (C15:0)	0.190	0.145
Palmitic acid (C16:0)	17.998	17.298
Margaric acid (C17:0)	0.265	0.193
Stearic acid (C18:0)	6.582	6.704
Arachidonic acid (C20:0)	0.271	0.228
Behenic acid (C22:0)	0.211	0.190
Tricosanoic acid (C23:0)	0.000	0.069
Lignoceric acid (C24:0)	0.298	0.284
Myristoleic acid (C14:1)	0.000	0.044
Palmitoleic acid (C16:1)	3.234	4.330
Cis-10-heptadecenoic acid (C17:1)	0.201	0.156
Oleic acid (C18:1n9c)	21.117	19.585
Elaidic acid (C18:1n9t)	4.632	6.512
Cis-11-eicosenoic acid (C20:1n9)	1.220	1.229
Erucate acid (C22:1n13)	0.438	0.286
Nervonic acid (C24:1n9)	0.394	0.334
Gamma-linolenic acid (C18:3n6)	0.237	0.164
Linoleic acid (C18:2n6c)	16.964	21.517
Arachidonic acid (C20:4n6)	9.172	6.429
cis-5.8.11.14.17-Eicosapentaenoic acid (C20:5n3)	0.405	0.308
Dihomo-alpha-linolenic acid (C20:3n6)	1.826	2.323
cis-11.14-eicosadienoic acid (C20:2)	0.799	1.097
cis-4,7,10,13,16-docosapentaenoic acid (C22:5n6)	0.774	0.399
cis-4.7.10.13.16.19-Docosahexaenoic acid (C22:6n3)	9.442	6.632
7,10,13,16-Docosatetraenoic acid (C22:4n6)	0.984	0.799
Docosapentaenoic acid (C22:5n3)	1.425	1.856
Cis-13,16-docosadienoic acid (C22:2)	0.000	0.144
Saturated fatty acids (%)	26.75	25.85
Monounsaturated fatty acids (%)	31.24	32.48
Polyunsaturated fatty acids (%)	42.03	41.67
Total (%)	100	100

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C–Control group; PCB–PCB 126 group.

Values expressed as percentage (%).

In addition, SIRT1 and PPAR-α, biomarkers associated with steatosis development, were assessed in the liver. No differences were observed in Sirt1 mRNA expression (Fig 4A), whereas the mRNA expression of Ppara decreased in the PCB group compared to the C group (-63%; p < 0.05, Fig 4B). Furthermore, miRNAs associated with NAFLD were evaluated in the liver, and no differences were observed in the expression of miR-122 (Fig 4C); however, the expression of miR-34a (1.7-fold-increase; p < 0.05, Fig 4D) and miRNA-155 (1-fold increase, p < 0.001, Fig 2E) were elevated in animals exposed to PCB when compared with the C group.

Fig 4 — (A) Sirtuin-1 (*Sirt1*) gene expression in the liver. (B) Peroxisome proliferator-activated receptor alpha (*Ppar-α*) gene expression in the liver. (C) *miR-122* expression in the liver (D) *miR-34a* expression in the liver. (E) *miR-155* expression in the liver. C: Control group; PCB: PCB 126 group. Values are expressed as mean ± standard deviation (n = 8). * p value < 0.05; *** p value <0.001.

Discussion

Organic pollutants, particularly PCBs, have been identified as important factors that promote health problems. In this context, PCB 126 is associated with the development of obesity, inflammatory processes, and NAFLD [1, 2, 4]. The identification of molecular mechanisms and biomarkers that regulate the development of these disorders, such as miRNAs, is of great importance to provide a better understanding of the participation of these compounds in the current health panorama of the population and to guide public policies in the care of exposure to these substances.

The present study evaluated the impact of chronic exposure to PCB 126 for 10 weeks in mice, and, at the end of treatment, no differences were observed in food intake or body mass between the groups. However, the PCB group showed altered biochemical parameters in plasma, such as an increase in markers of hepatic function, ALT and AST, increased levels of triglycerides, glucose, and insulin, and lower levels of cholesterol, indicating that, although this group did not present an altered body composition, exposure to PCB 126 disturbed some metabolic processes. An altered profile of glucose metabolism with elevated plasma glucose concentrations was observed in mice exposed to different types of PCB, including PCB 126 [30, 31]. This hyperglycemic condition possibly results from insulin resistance in the liver, muscle, and adipose tissue through mechanisms involving TNF-α which, at higher levels, impair the insulin signaling cascade and glucose uptake by the GLUT4 transporter in insulin-dependent tissues [30, 32]. Although not the focus of the present study, these results are similar to those observed here, showing that animals exposed to PCB 126 present with impaired glycemic homeostasis.

In addition, the PCB group showed a proinflammatory profile in plasma, with elevated levels of IL-6 and TNF-α. Exposure to PCB 126 induces the expression of inflammatory cytokines, including TNFα and IL-1β, possibly promoting the polarization to the proinflammatory M1 phenotype in a human monocyte cell culture. In addition, an increase in monocyte chemoattractant protein-1 (MCP-1) in PCB 126-activated macrophages suggests the induction of chemokines regulating immune cell recruitment and infiltration of monocytes/macrophages [33]. Additionally, the gene expression of major chemokines for different immune cell infiltrations was higher in the livers of the PCB group, suggesting chemotaxis of the inflammatory cells.

To confirm that PCB 126 promotes NAFLD, liver samples were evaluated and the PCB group showed lipid accumulation in the presence of macro- and microvesicular steatosis and increased hepatic cholesterol and triglyceride contents. Consistent with steatohepatitis, the PCB group exhibited fibrosis and inflammatory infiltration of the liver. PCB 126 interferes with metabolic pathways in the liver and there is an association between this pollutant and the molecular development of NAFLD. Exposure to PCB 126 increases lipid accumulation in hepatocytes and triglyceride concentrations in the liver of rats through mechanisms involving microsomal triglyceride transfer protein and diacylglycerol O-acyltransferase 2 (DGAT-2), which are important molecules in the hepatic synthesis and export of triacylglycerides [1].

Administration of PCB 126 to mice resulted in notable histological damage and vacuolar degeneration in the liver, with the accumulation of lipid droplets, increased inflammation, and collagen accumulation, showing liver fibrosis [34]. Furthermore, similar to the results of the present study, liver injury was confirmed when serum AST and ALT levels increased [34]. These results, which are in agreement with the data presented in this study, demonstrate that PCB 126 induces liver inflammation, fibrosis, and injury in mice.

All the effects described for PCB 126 appear to occur through the AhR, a receptor in the liver to which PCB binds and regulates the transcription of several genes involved in NAFLD development [2, 11]. In the present study, Ahr gene expression increased in the liver of the group exposed to PCB 126, suggesting that this pollutant may promote its effects through this receptor. PCB-126 is a potent endocrine disruptor that interferes with thyroid and steroid hormone receptors, affecting liver lipid metabolism. This exposure may increase cholesterol synthesis or hinder its elimination by altering liver metabolic pathways and enzyme activities like HMG-CoA reductase. PCB-126 also induces oxidative stress and inflammation in the liver, which can damage cells and trigger a defensive increase in cholesterol synthesis. Additionally, it impacts the function and expression of lipid transport proteins, such as LDL and HDL, affecting cholesterol transport and removal [35, 36].

Furthermore, the expression of Tnfa and Il6 was evaluated in the liver, and PCB 126 was found to positively regulate the expression of these cytokines, showing that these animals presented increased inflammation in the liver tissue. This is consistent with the histological and stereological analyses, where lipid accumulation, inflammatory infiltration, and fibrosis were verified, showing the development of NASH. These results corroborate previous studies that showed that PCB 126 promotes an inflammatory state in the liver with an increase in proinflammatory cytokines such as TNF-α, IL-1β, IL-6, MCP-1, C-C Motif Chemokine Ligand 2 (Ccl2), Ccl3, and Ccl5, contributing to the development of liver injury [33, 34].

In the present study, genes involved in lipid metabolism, such as Sirt1 and Ppara, were evaluated in the livers of the animals. SIRT1, a protein deacetylase dependent on NAD+, plays a pivotal role in maintaining the energy balance within the body. Specifically, hepatic SIRT1 governs lipid equilibrium by enhancing the function of PPARα, a nuclear receptor crucial for adapting to fasting and starvation. When SIRT1 is deficient in liver cells, it undermines PPARα activity, diminishing fatty acid oxidation. This disruption contributes to the onset of hepatic steatosis and inflammation, particularly in the context of a high-fat diet. [37]. In the present study, PCB 126 did not alter the expression of Sirt1 but reduced the expression of Ppara. It is important to highlight that the present study evaluated the gene expression of Sirt1. Although no changes were observed in the PCB group, the hypothesis that the protein content or enzymatic activity may be reduced and promote the reduction of gene expression of Ppara cannot be ruled out. In this context, activation of PPARα leads to increased expression of several target genes involved in fatty acid oxidation, and lack of PPARα, in a mouse model, promotes an excessive accumulation of triglycerides in the liver [38, 39].

Accumulation of triglycerides in the liver tissue, inflammation, steatohepatitis, liver fibrosis, and cirrhosis are associated with microRNAs, suggesting their participation in the development and progression of NAFLD [14–17, 40]. The present study evaluated the expression of miRNAs that have already been associated with these disorders, such as miR-155, miR-122, and miR-34a [14–17], to clarify the possible contribution of these markers in regulating the changes observed in the livers of animals exposed to PCB 126. The mechanism by which PCB-126 (polychlorinated biphenyl-126) regulates miR-155 and miR-34a expression is closely tied to its interaction with the aryl hydrocarbon receptor (AhR) signaling pathway [41]. PCB-126 is a potent ligand for AhR, a transcription factor that is activated upon binding with various ligands, including environmental pollutants like dioxins and PCBs. When PCB-126 binds to AHR, it triggers the translocation of the AhR-ligand complex into the nucleus [20, 42]. Once inside the nucleus, the AhR-ligand complex can bind to specific DNA sequences known as xenobiotic response elements (XREs) located in the promoter regions of various genes, including those encoding for miRNAs like miR-155 and miR-34a. This binding can either upregulate or downregulate the transcription of these miRNAs, depending on the context and the specific interactions at the gene promoters. miR-155 and miR-34a are microRNAs that play crucial roles in various biological processes, including inflammation, cell cycle regulation, and apoptosis [33, 36, 43].

The regulation of these miRNAs by AhR signaling can influence these processes, potentially leading to various biological effects. The alteration of miR-155 and miR-34a expression by PCB-126 through AhR signaling can have significant pathophysiological implications [3]. For example, dysregulation of these miRNAs may contribute to the development of diseases like cancer, where they are known to play roles in regulating apoptosis, proliferation, and the immune response. In summary, PCB-126 regulates miR-155 and miR-34a expression primarily through its interaction with AhR, leading to changes in gene transcription that can have broad biological and health implications. This pathway is a critical aspect of understanding the toxicological effects of PCBs and their impact on cellular and molecular processes [20, 43–45].

Liver expression of miR-155 increased in the PCB group, and this miRNA plays a role in the regulation of Kupffer cells (KCs) and is involved in the inflammatory processes in NAFLD [40]. miR-155 is highly expressed in total liver, hepatocytes, and KCs of a mice model of liver disease, and this upregulation contributes to TNF-α production [46]. Furthermore, miR-155 is upregulated in patients with NASH, possibly representing a specific biomarker for this pathological condition [40]. This finding is corroborated by the data presented here, which showed that the animals exposed to PCB 126 developed NASH.

Additionally, increased expression of miR-34a was observed despite the unaltered expression of miR-122 in the liver of the PCB group. miR-34a is upregulated in the liver of patients and rodents with NASH, and its contribution to the development of this disorder appears to occur through mechanisms involving PPARα and SIRT1, which are specific targets of mir-34a [40, 44, 45, 47, 48]. In this context, the upregulation of miR-34a results in the downregulation of hepatic PPARα and SIRT1, which results in reduced fatty acid oxidation and the development of steatosis [44]. These data are in agreement with the results observed in the present study because upregulation of miR-34a and reduced Ppara expression were observed in the PCB group. However, an unchanged expression of Sirt1 suggests that PCB 126 promoted the development of NASH, at least in part, through mechanisms involving reduced fatty acid oxidation in the liver owing to reduced PPARα expression, possibly induced by miR-34a upregulation.

In summary, the present study showed that PCB 126 induced NASH through increased inflammatory processes, impairment of lipid oxidation, and development of hepatic steatosis associated with increased expression of proinflammatory cytokines, reduced expression of PPARα, and upregulation of miR-155 and miR-34a in the liver. To our knowledge, this is the first study to demonstrate the upregulation of miR-155 and miR-34a induced by PCB 126 and their possible contribution to the development of NAFLD. These findings may contribute to the development of novel diagnostic markers and therapeutic strategies.

Supporting information

S1 Data. Data from experiments.

(XLSX)

pone.0308334.s001.xlsx^{(3.1MB, xlsx)}

Data Availability

All relevant data are within the manuscript and its Supporting information files.

Funding Statement

This research was supported by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro-FAPERJ (grant numbers E-26/211.193/202, E-26/201.234/2022, and E-26/210.332/2022) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-CAPES, 001.

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PLoS One. doi: 10.1371/journal.pone.0308334.r001

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PONE-D-24-04954Long-term polychlorinated biphenyl 126 exposure induces liver fibrosis by upregulation of miR-155 and miR-34a in C57BL/6 micePLOS ONE

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Reviewer #1: The present study "Long-term polychlorinated biphenyl 126 exposure induces liver fibrosis by upregulation of miR-155 and miR-34a in C57BL/6 mice" analyzed some mechanism of fatty liver disease induced by PCB126. Here are some major concerns:

1. microRNA is not epigenetic marker. The authors need to rewrite the manuscript with accurate concepts.

2. Fibrosis is not an indicator of NASH. If the authors want to prove NASH, infiltration of multiple types of immune cells needs to be experimentally confirmed. Also, the authors need to use experiments to prove hepatocyte death.

3. What is the mechanism to regulate miR-155 and miR-34a expression? How is it related to AHR signal?

4. Which cells in the liver express AHR, miR-155 and miR-34a?

5. The authors need to use molecular biology methods to prove whether TNF-alpha and IL-6 are direct targets of miR-155. And whether PPARalpha is a direct target of miR-34a.

6. To prove miR-155 and miR-34a participate in the pathogenesis, the authors need to use molecular methods to interfere with the expression of these two microRNAs in the liver and repeat the animal experiment, in comparison to the unrelated interference treatment.

7. Fibrosis is only supported by Sirus Red staining, which is too limited. The authors need to perform hydroxyproline analysis, collagen quantification, fibrosis related gene expression to confirm fibrosis.

8. What is the mechanism for cholesterol elevation in PCB treated mice?

Reviewer #2: The authors tested a commonly used pollutant, PCB-126, and observed liver fibrosis and steatosis in mice, as has been shown before. Nevertheless, they also showed increased expression of some miRNAs related to lipid metabolism.

Overall, the results seem concise and nicely presented, and the text is well written, although there are some typos and some mistakes that could need to be revised. Some points of the text are redundant as well, but I’ll bring it along to my revision. Regarding Picrosirius Red, did you make any kind of measurement of the area affected? I would recommend analyzing the area with ImageJ, which is available for free, and several macros could help with the analysis. This could be enriching for the data you show.

The discussion section feels hard to follow. Try to reorganize it to group the results by “themes”. E.g., the Second paragraph (starting in line 245) could come after the third one. Also, this second paragraph gets very redundant with the information that’s about to be presented. Please, reorganize, and check and rewrite what is needed.

Statistical-wise, was there any normality test used? It is important to determine if the data was properly analyzed by the Student’s T-test. Also, something that could add to your conclusions, is correlation. Use simple linear correlation to compare the miRNA you measured with other results you observed. E.g., in Figure 3, you add two correlation analysis between IL-6 and TNF-α by the expression of miR-155, and in Figure 4, the same thing with PPAR-α and miR-34a. Other possibilities are evaluating the correlation between the results you got in the stereology of the liver and the expression of these two miR-34a. I understand that, although correlations could be misleading, once you already are exposing the relation in the discussion section that would be justified. Feel free to try other correlations that could work well and present some of them. This could reinforce your results.

- Line 76: …to date, no study…

- Lines 91 to 97: Information is shown twice. Make the last sentences of the paragraph more complete and you can’t need the subsequent paragraphs.

- Line 100: You can use graphical accents on the words that have it originally, as in São Paulo.

- Line 104: Which drug did you use for euthanasia, pentobarbital or thiopental? Also, use the right symbols {[()]}

- Line 122: Reference 20 doesn’t make sense with what is in the text. It’s about chow, not histology.

- Lines 170 to 186: Gene names and transcripts are two different things. The gene for TNF-α is Tnfa, and, as this is a rodent gene, the first letter is capitalized and subsequent in lowercase, and italics. For it to be clearer, you can add the proper name of the genes in the parenthesis, before the assay ID. Please, be aware of this and fix it along with the text.

- Line 186: Use the proper reference for Livak and Schimittgen.

- Paragraph starting in line 221: Text is hard to follow. Please, rewrite it to present the data with better flow.

- Line 282: Reference of the “other study” should come by the end of the first sentence.

- Line 342: Please, double-check the verbal tense of the verb induce here.

- Line 346: relation instead of relationship.

**********

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Reviewer #2: No

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PLoS One. 2024 Aug 12;19(8):e0308334. doi: 10.1371/journal.pone.0308334.r002

Author response to Decision Letter 0

10 May 2024

Dear reviewers,

We greatly appreciate your contribution to improving our article. We have addressed each question point by point and hope that all raised concerns have been adequately answered.

We have worked diligently, returning to the laboratory bench to conduct additional experiments aimed at addressing and working through all the issues raised.

We are available for further clarification if needed.

Sincerely,

Julio Daleprane

RESPONSE TO REVIWERS

1. microRNA is not epigenetic marker. The authors need to rewrite the manuscript with accurate concepts.

A: We conducted a thorough reading of the entire manuscript and reformulated the sentences.

A: In response to the reviewer's query regarding our methods to elucidate the development of steatosis into fibrosis, we conducted gene expression assays for markers of different cell types. These included MCP1 for macrophages, CXCL1, and IL-8—key chemokines for neutrophil chemotaxis. Notably, CXCL1 was found to significantly elevate neutrophil infiltration (Figure 2). Additionally, beyond the fibrosis previously demonstrated by picrosirius red staining, we quantified the area occupied by the staining, measured tissue hydroxyproline concentrations, and assessed the expression of pro-fibrotic genes such as TGF-beta1, Smad3, Collagen1, and CXCL9. Cell death was evaluated by assessing Caspase3 expression in hepatic tissue (Figure 3).

Figure 2.

Figure 3.

3. What is the mechanism to regulate miR-155 and miR-34a expression? How is it related to AHR signal?

A: The mechanism by which PCB-126 (polychlorinated biphenyl-126) regulates miR-155 and miR-34a expression is closely tied to its interaction with the aryl hydrocarbon receptor (AhR) signaling pathway. PCB-126 is a potent ligand for AhR, a transcription factor that is activated upon binding with various ligands, including environmental pollutants like dioxins and PCBs. When PCB-126 binds to AHR, it triggers the translocation of the AhR-ligand complex into the nucleus.

Once inside the nucleus, the AhR-ligand complex can bind to specific DNA sequences known as xenobiotic response elements (XREs) located in the promoter regions of various genes, including those encoding for miRNAs like miR-155 and miR-34a. This binding can either upregulate or downregulate the transcription of these miRNAs, depending on the context and the specific interactions at the gene promoters. miR-155 and miR-34a are microRNAs that play crucial roles in various biological processes, including inflammation, cell cycle regulation, and apoptosis. The regulation of these miRNAs by AhR signaling can influence these processes, potentially leading to various biological effects. The alteration of miR-155 and miR-34a expression by PCB-126 through AhR signaling can have significant pathophysiological implications. For example, dysregulation of these miRNAs may contribute to the development of diseases like cancer, where they are known to play roles in regulating apoptosis, proliferation, and the immune response. In summary, PCB-126 regulates miR-155 and miR-34a expression primarily through its interaction with AhR, leading to changes in gene transcription that can have broad biological and health implications. This pathway is a critical aspect of understanding the toxicological effects of PCBs and their impact on cellular and molecular processes.

4. Which cells in the liver express AHR, miR-155 and miR-34a?

A: As described by Yan et al (2019), AhR is expressed in hepatic stellate cells, hepatocytes and Kupffer cells [1]. It has been described that miR-155 is highly expressed in hepatocytes and Kupffer cells, being involved in inflammatory processes that control innate and adaptive immunity in nonalcoholic fatty liver disease, in addition to an important role in the initial hepatic lipid accumulation and modulating lipid metabolism [2]. miR-34a is expressed in hepatocytes and hepatic stellate cells and was associated to induction of hepatocytic apoptosis, regulation of lipid metabolism and stimulation of hepatic stellate cell activation and fibrosis [3].

[1] Yan J, Tung HC, Li S, Niu Y, Garbacz WG, Lu P, Bi Y, Li Y, He J, Xu M, Ren S, Monga SP, Schwabe RF, Yang D, Xie W. Aryl Hydrocarbon Receptor Signaling Prevents Activation of Hepatic Stellate Cells and Liver Fibrogenesis in Mice. Gastroenterology. 2019 Sep;157(3):793-806.e14. doi: 10.1053/j.gastro.2019.05.066. Epub 2019 Jun 3.

[2] Dongiovanni P, Meroni M, Longo M, Fargion S, Fracanzani AL. miRNA Signature in NAFLD: A Turning Point for a Non-Invasive Diagnosis. Int J Mol Sci. 2018 Dec 10;19(12):3966. doi: 10.3390/ijms19123966.

[3] Hochreuter MY, Dall M, Treebak JT, Barrès R. MicroRNAs in non-alcoholic fatty liver disease: Progress and perspectives. Mol Metab. 2022 Nov;65:101581. doi: 10.1016/j.molmet.2022.101581.

5. The authors need to use molecular biology methods to prove whether TNF-alpha and IL-6 are direct targets of miR-155. And whether PPARalpha is a direct target of miR-34a.

A: Unfortunately, it was not possible to perform molecular biology analyzes to prove such associations, but we revised and rewrote the discussion section so that a direct relationship between these markers is not suggested.

“However, these associations have already been well described in previous studies. Tili et al (2007) suggest that miR-155 may act directly or indirectly to increase the rate of translation of TNF- transcripts, possibly through their redistribution across the cytoplasm and/or enhancement of TNF- transcript stability. Furthermore, they showed that miR-155 targets transcripts encoding proteins, such as IKKε (IκB kinase ε), FADD (Fas-associated death domain protein) and Ripk1 (receptor (TNFR superfamily)-interacting serine-threonine kinase 1) whose ultimate function results in the activation of the LPS/TNF- pathway, while it increases TNF- production [1]. In addition, other study described that miR-155 is a master regulator of inflammation, enhancing the translation of TNFα during innate immune responses by Toll-like receptor ligands [2].

Thus it has already been described that the overexpression of miR-155 promoted activation of NF-κB, through mechanisms involving the downregulation of SOCS1, and elevating the production of proinflammatory cytokines, such TNF-α and IL-6 [3, 4]

Concerning to PPARalpha and its relation to mir-34a, previous studies have already reported that the upregulation of miR-34a resulted in the downregulation of hepatic PPARα and SIRT1 that are the direct targets of miR-34a and that silencing of miR-34a restored the expression of SIRT1 and PPARα, resulting in activation of PPARα downstream genes, such as AMPK and Hydroxymethylglutaryl-CoA Reductase (HMGCR) [2, 5].”

[1] Tili E, Michaille JJ, Cimino A, Costinean S, Dumitru CD, Adair B, Fabbri M, Alder H, Liu CG, Calin GA, Croce CM. Modulation of miR-155 and miR-125b levels following lipopolysaccharide/TNF-alpha stimulation and their possible roles in regulating the response to endotoxin shock. J Immunol. 2007 Oct 15;179(8):5082-9. doi: 10.4049/jimmunol.179.8.5082.

[3] Chen C, Luo F, Liu X, Lu L, Xu H, Yang Q, Xue J, Shi L, Li J, Zhang A, Liu Q. NF-kB-regulated exosomal miR-155 promotes the inflammation associated with arsenite carcinogenesis. Cancer Lett. 2017 Mar 1;388:21-33. doi: 10.1016/j.canlet.2016.11.027.

[4] Tan L, Jiang W, Lu A, Cai H, Kong L. miR-155 Aggravates Liver Ischemia/reperfusion Injury by Suppressing SOCS1 in Mice. Transplant Proc. 2018 Dec;50(10):3831-3839. doi: 10.1016/j.transproceed.2018.08.060.

[5] Ding J, Li M, Wan X, Jin X, Chen S, Yu C, Li Y. Effect of miR-34a in regulating steatosis by targeting PPARα expression in nonalcoholic fatty liver disease. Sci Rep. 2015 Sep 2;5:13729. doi: 10.1038/srep13729.

A: Unfortunately, we were unable to perform these experiments, but we revised and rewrote the discussion section to try to minimize this direct association of miRs with the pathogenesis of NASH.

A: We performed additional experiments as suggested (Fig 3).

8. What is the mechanism for cholesterol elevation in PCB treated mice?

A: PCB-126 is known to be a potent endocrine disruptor. It can interfere with thyroid and steroid hormone receptors, thereby affecting lipid metabolism in the liver. Changes in hormone levels may result in increased cholesterol synthesis or reduced elimination. Simultaneously, exposure to PCB-126 can alter liver metabolic pathways, increasing cholesterol biosynthesis and/or decreasing its conversion into bile acids. This may be caused by changes in the expression or activity of key enzymes such as HMG-CoA reductase, which is crucial in the cholesterol biosynthetic pathway. Additionally, PCB-126 can induce oxidative stress and inflammation in the liver. Oxidative stress may damage liver cells, while inflammation can trigger a lipid response that includes increased cholesterol synthesis as a defense mechanism. Finally, PCB-126 may affect the function and expression of lipid transporter proteins, such as low-density (LDL) and high-density (HDL) lipoproteins, impacting cholesterol transport and removal from the liver. To improve the quality of the work, we have included the information in the text. [1,2]

1. Mohammadparast-Tabas P, Arab-Zozani M, Naseri K, Darroudi M, Aramjoo H, Ahmadian H, et al. Polychlorinated biphenyls and thyroid function: a scoping review. Rev Environ Health. 2023. doi:10.1515/reveh-2022-0156

2. Yang Y, Mei G, Yang L, Luo T, Wu R, Peng S, et al. PCB126 impairs human sperm functions by affecting post-translational modifications and mitochondrial functions. Chemosphere. 2024;346: 140532. doi:10.1016/j.chemosphere.2023.140532

1.Regarding Picrosirius Red, did you make any kind of measurement of the area affected? I would recommend analyzing the area with ImageJ, which is available for free, and several macros could help with the analysis. This could be enriching for the data you show.

A: We performed additional experiments as suggested (Fig 3).

2.The discussion section feels hard to follow. Try to reorganize it to group the results by “themes”. E.g., the Second paragraph (starting in line 245) could come after the third one. Also, this second paragraph gets very redundant with the information that’s about to be presented. Please, reorganize, and check and rewrite what is needed.

A: The discussion section was reviewed and we reorganize and rewrite some paragraphs to make it easier to understand.

3.Statistical-wise, was there any normality test used? It is important to determine if the data was properly analyzed by the Student’s T-test. Also, something that could add to your conclusions, is correlation. Use simple linear correlation to compare the miRNA you measured with other results you observed. E.g., in Figure 3, you add two correlation analysis between IL-6 and TNF-α by the expression of miR-155, and in Figure 4, the same thing with PPAR-α and miR-34a. Other possibilities are evaluating the correlation between the results you got in the stereology of the liver and the expression of these two miR-34a. I understand that, although correlations could be misleading, once you already are exposing the relation in the discussion section that would be justified. Feel free to try other correlations that could work well and present some of them. This could reinforce your results.

A: Dear Reviewer, Thank you for the opportunity to clarify the conduct of our data analyses. To carry out the statistical analyses, we initially performed a normality test on all data, as now described in the methodology section. Subsequent statistical analyses were then performed. We attempted to conduct a correlation test with the studied variables; however, unfortunately, the sample size was too small to establish this relationship. As a result, we changed how we present the results, and no longer claim causality between miRNAs and other inflammatory and fibrotic indicators. We now merely discuss these in light of existing data in the literature.

4.Line 76: …to date, no study…

A: Done.

5.Lines 91 to 97: Information is shown twice. Make the last sentences of the paragraph more complete and you can’t need the subsequent paragraphs.

A: We rewrite the sentence.

6.Line 100: You can use graphical accents on the words that have it originally, as in São Paulo.

A: Done.

7. Line 104: Which drug did you use for euthanasia, pentobarbital or thiopental? Also, use the right symbols {[()]}

A: Sorry for this mistake. The drug used was sodium thiopental and the sentence has already been corrected.

8. Line 122: Reference 20 doesn’t make sense with what is in the text. It’s about chow, not histology.

A:It has been corrected.

9. Lines 170 to 186: Gene names and transcripts are two different things. The gene for TNF-α is Tnfa, and, as this is a rodent gene, the first letter is capitalized and subsequent in lowercase, and italics. For it to be clearer, you can add the proper name of the genes in the parenthesis, before the assay ID. Please, be aware of this and fix it along with the text.

A: We correct this throughout the text.

10. Line 186: Use the proper reference for Livak and Schimittgen.

A: Done.

11. Paragraph starting in line 221: Text is hard to follow. Please, rewrite it to present the data with better flow.

A: We rewrote the paragraph to improve understanding of the data presented.

12. Line 282: Reference of the “other study” should come by the end of the first sentence.

A: The reference was included at the end of the sentence.

13. Line 342: Please, double-check the verbal tense of the verb induce here.

A: Done.

14. Line 346: relation instead of relationship.

A: Done.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0308334.s002.docx^{(1.4MB, docx)}

PLoS One. doi: 10.1371/journal.pone.0308334.r003

Decision Letter 1

Partha Mukhopadhyay

16 Jun 2024

PONE-D-24-04954R1Long-term exposure to polychlorinated biphenyl 126 induces liver fibrosis and upregulates miR-155 and miR-34a in C57BL/6 micePLOS ONE

Dear Dr. Daleprane,

Your manuscript was reviewed by same experts and one of them raised few comments , which must be addressed.

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: The current version of "Long-term exposure to polychlorinated biphenyl 126 induces liver fibrosis and upregulates miR-155 and miR-34a in C57BL/6 mice" made significant improvement compared to the original submission. Here are some more major and minor concerns:

Major concern

1. Caspase-3 expression cannot indicate apoptosis. The authors should either perform western blot to evaluate cleaved caspase-3, or IHC for TUNEL in histological samples. If using western blot to detect cleaved caspase-3, total or full-length caspase-3 protein level should also be shown still.

Minor concern

1. IL-8 is typically not considered present in murine. Human IL-8 equivalent in mice is CXCL1, which the authors have already examined. IL-8 measurement can be deleted.

2. miR-155 expression level should be moved to figure 4 from figure 2

3. The authors answered the potential mechanism linking AHR to miR-155 and miR-34a regulation in the rebuttal letter. Please include this information to the discussion section with proper citations.

Reviewer #2: I am satisfied with the alterations the authors made to the manuscript. I recommend its publication.

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

**********

PLoS One. 2024 Aug 12;19(8):e0308334. doi: 10.1371/journal.pone.0308334.r004

Author response to Decision Letter 1

19 Jun 2024

Dear Reviewer,

We would like to express our gratitude for the thorough review and valuable feedback on our manuscript. We have carefully considered each of your comments and have made the necessary revisions accordingly. Below, we address each of your concerns in detail:

Major Concern

1. Caspase-3 Expression:

We acknowledge that caspase-3 expression alone cannot definitively indicate apoptosis. In response to your suggestion, we have performed a Western blot analysis to evaluate cleaved caspase-3. Additionally, we have included the total or full-length caspase-3 protein level in our results. The updated data can be found in the revised manuscript (Figure 3I and 3J). We believe this provides a more comprehensive evaluation of apoptosis in our samples.

Minor Concerns:

1. IL-8 Measurement:

We appreciate your observation regarding IL-8. As suggested, we have removed the IL-8 measurement from our manuscript, considering that IL-8 is not typically present in murine models and we have already examined the murine equivalent, CXCL1. The manuscript has been updated accordingly to reflect this change.

2. miR-155 Expression Level:

We have moved the miR-155 expression data from Figure 2 to Figure 4 as requested. This rearrangement aligns better with the overall flow of our results and discussion.

3. Mechanism Linking AHR to miR-155 and miR-34a Regulation:

We have incorporated the information regarding the potential mechanism linking AHR to the regulation of miR-155 and miR-34a into the discussion section of our manuscript. Proper citations have been added to support this information. We believe this addition strengthens the discussion and provides a clearer understanding of the mechanisms involved.

We sincerely thank you again for your constructive feedback, which has significantly improved the quality of our manuscript. We hope that the revised version meets your expectations and addresses all your concerns satisfactorily.

Thank you for your time and consideration.

Best regards,

Julio Daleprane

Attachment

Submitted filename: Response to Reviewers.docx

pone.0308334.s003.docx^{(13.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0308334.r005

Decision Letter 2

Partha Mukhopadhyay

23 Jul 2024

Long-term exposure to polychlorinated biphenyl 126 induces liver fibrosis and upregulates miR-155 and miR-34a in C57BL/6 mice

PONE-D-24-04954R2

Dear Dr. Daleprane,

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Section Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: (No Response)

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: (No Response)

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: (No Response)

**********

6. Review Comments to the Author

Reviewer #1: (No Response)

**********

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Reviewer #1: No

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PLoS One. doi: 10.1371/journal.pone.0308334.r006

Acceptance letter

Partha Mukhopadhyay

1 Aug 2024

PONE-D-24-04954R2

PLOS ONE

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Data. Data from experiments.

(XLSX)

pone.0308334.s001.xlsx^{(3.1MB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0308334.s002.docx^{(1.4MB, docx)}

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Submitted filename: Response to Reviewers.docx

pone.0308334.s003.docx^{(13.9KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting information files.

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PERMALINK

Long-term exposure to polychlorinated biphenyl 126 induces liver fibrosis and upregulates miR-155 and miR-34a in C57BL/6 mice

Fernanda Torres Quitete

Ananda Vitória Silva Teixeira

Thamara Cherem Peixoto

Bruna Cadete Martins

Geórgia Correa Atella

Angela de Castro Resende

Daniela de Barros Mucci

Fabiane Martins

Julio Beltrame Daleprane

Roles

Abstract

Introduction

Material and methods

Animals and experimental groups

Fig 1. Characterization of the experimental model.

Plasma analyses

Histological and stereological analysis of the liver

Determination of hepatic cholesterol, triglyceride, and hydroxyproline contents

Determination of liver fatty acid composition

Total RNA and miRNA extraction and real-time reverse transcription polymerase chain reaction (RT-qPCR)

Western blotting analysis

Statistical analysis

Results

Food intake, body mass, and plasma analysis

Table 1. Biochemical parameters in plasma.

Liver analysis

Fig 2. Effects of PCB 126 on cellular, morphological, and molecular features of the liver after 10 weeks of exposure.

Fig 3. Histological and molecular parameters associated with liver fibrosis after chronic PCB 126 exposure.

Table 2. Fatty acids profile in liver samples.

Fig 4. Molecular parameters of lipid metabolism in the liver after chronic PCB 126 exposure.

Discussion

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Partha Mukhopadhyay

Roles

Author response to Decision Letter 0

Decision Letter 1

Partha Mukhopadhyay

Roles

Author response to Decision Letter 1

Decision Letter 2

Partha Mukhopadhyay

Roles

Acceptance letter

Partha Mukhopadhyay

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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