Abstract
Polychlorinated biphenyls (PCBs) are persistent organic pollutants (POPs) and are associated with thyroid diseases. Our previous study reported that 2,3’,4,4’,5-Pentachlorobiphenyl (PCB118) could induce thyroid dysfunction and the rat thyroid tissues exhibit abnormal mitochondrial ultrastructure. However, the more specific effects of PCB118 on mitochondria and the relationship between mitochondria and thyroid dysfunction remain unclear. In this study, Wistar rats were injected with PCB118 intraperitoneally at 0, 10, 100, and 1000 μg/kg/d for 13 weeks and FRTL-5 rat thyroid cells were treated with PCB118 (0, 0.25, 2.5, and 25 nM) for 24 hr, which did not influence the general conditions of rats and FRTL-5 cells viability. The detection of serum levels of thyroid hormones (THs) and the expression of sodium/iodide symporter (NIS) protein demonstrated that thyroid function was impaired after PCB118 exposure. Transmission electron microscopy showed mitochondrial damage in the thyroids of PCB118-treated rats. Biological processes analysis revealed that differentially expressed mRNAs in thyroid tissues induced by PCB118 were enriched in reactive oxygen species (ROS) metabolic process, hydrogen peroxide metabolic process, and hydrogen peroxide catabolic process. Moreover, mRNA expression of mitochondrial respiratory chain genes NDUFB3, UQCRC2, COX17, ATP5I and ATP5E decreased in PCB118-treated groups. In vivo and in vitro data showed that ROS production increased significantly after PCB118 exposure, accompanied by increased levels of phospho-c-Jun N-terminal kinase (P-JNK). Taken together, these results suggest that PCB118 could damage mitochondria by increasing oxidative stress and PCB118-induced thyroid dysfunction may be related to ROS-dependent activation of the JNK pathway.
INTRODUCTION
Polychlorinated biphenyls (PCBs) are a group of synthetic organochlorine compounds used in numerous industrial and consumer applications since they are very resistant to extreme temperature and pressure. Despite the ban on their production since the 1970s, PCBs show significant bioaccumulation in most compartments of the ecosystem and human tissues due to high chemical stability, resistance to degradation, and pronounced lipophilicity (Nichols et al., 2007; Pelletier et al., 2003).
PCBs cause adverse effects on nervous, reproductive, immune and endocrine systems (Coulter et al., 2019; Gaum et al., 2019; Spector et al., 2014). The thyroid is considered a potential target for endocrine toxicity of PCBs (Béchaux et al., 2014). It has been reported that PCBs interfere with thyroidal-related gene expression and thyroid function (Duntas and Stathatos, 2015; Katarzyńska et al., 2015). Our earlier studies showed that 2,3’,4,4’,5-Pentachlorobiphenyl (PCB118) could damage thyroid structure, interfere with THs and decrease the expression of sodium/iodide symporter (NIS) and thyroglobulin (TG) in human and animal thyrocytes (Guo et al., 2015; Tang et al., 2013; Xu et al., 2016).
Mitochondria are intracellular organelles which play a significant role in various processes such as energy metabolism, calcium homeostasis, the generation of free radicals, and cell survival and death (Kim et al., 2008). Mitochondrial dysfunction is implicated in a broad variety of pathological processes. Some studies have shown that mitochondrial structure and function abnormalities are involved in the occurrence and development of thyroid diseases (Cho et al., 2018; El-Sayed and Ibrahim, 2020).
In previous investigations, we have observed that rat thyroid tissues exhibited abnormal mitochondrial ultrastructure after exposure to PCB118. However, the more specific effects on thyroid mitochondria and the underlying mechanism by which mitochondria abnormality contributes to PCB-induced thyroid dysfunction are still unclear. This study aimed to elucidate the effects of PCB118 on thyroid mitochondria and to characterize the potential role of mitochondria involved in the thyroid dysfunction.
MATERIALS AND METHODS
Chemicals
PCB118 (CAS no.31508-00-6) was purchased from AccuStandard (USA).
Animal model
A total of 28 male Wistar rats of specific pathogen-free grade at age 6–8 weeks old were provided by Vital River (Beijing, China). The rats were randomly divided into four groups (0, 10, 100 and 1000 μg/kg/d), depending on the dosage of PCBs, and the animal models were established as previously reported (Xu et al., 2016). At the end of the experiments, the rats were euthanized by intraperitoneal injection of 2% sodium pentobarbital. Blood was drawn from the abdominal aorta, and serum was separated by centrifugation at 3,000 rpm for 10 min. The thyroid gland was stored at −80°C or fixed in 4% paraformaldehyde, 2.5% glutaraldehyde. The rat experiments were approved by the ethics committee of Nanjing Medical University (Approval No. IACUC-1705028-1).
Cell culture and treatment
FRTL-5 rat thyroid cells were provided by Dr Zheng Xuqin (Endocrine Department, The First Affiliated Hospital of Nanjing Medical University). FRTL-5 cells were cultured and treated with low concentrations of PCB118 (0.25, 2.5 and 25 nM) as previously described, which did not influence cell viability and apoptosis at these concentrations (Yang et al., 2015).
Enzyme-linked immunoabsorbent assay (ELISA)
The levels of serum free thyroxine (FT4), free triiodothyronine (FT3) and thyroid stimulating hormone (TSH) were determined using ELISA kits (Future Industrial, China) according to the manufacturer’s instructions.
Histology
Thyroids were fixed in 4% paraformaldehyde for 48 hr, and paraffin-embedded thyroids sections were stained with hematoxylin and eosin (HE) and observed under light microscope.
Transmission electron microscopy (TEM)
Thyroid specimens processing was carried out as described by Zhou et al. (2019). The ultrastructural features of thyroid tissues were examined under TEM (JEOL, Japan).
Gene Ontology (GO) functional enrichment analysis
The GO functional enrichment analysis was conducted by the R package clusterProfiler (v4.0.5) (Yu et al., 2012). The cutoff of the GO terms is adjusted P-value less than 0.05. The color indicaiting the P-value, size of bubble indicating the gene number belong to this term and the x axis represent the ratio of the differentially expressed genes with the genes in the whole genome belonging to this term.
Quantitative real-time PCR
Total RNA from thyrocyte was extracted with RNAiso Plus (Takara, Japan), and reverse transcribed with PrimeScript RT Master Mix Kit (Takara). Quantitative RT-PCR was performed using the SYBR-Green PCR kit on StepOnePlus system (Applied Biosystems, USA). The primer sequences are listed in Supplementary Table S1. Relative gene expression levels were calculated using the 2−ΔΔCT method.
Western blotting
Total protein was extracted from FRTL-5 cells or rat thyroid tissues using a RIPA lysis buffer. Antibodies against GAPDH, JNK and phospho-JNK (P-JNK) were purchased from Cell Signaling Technology (USA). Mouse anti-NIS antibody was obtained from Santa Cruz Biotech (USA). The protein bands were qualified with GAPDH as loading control using Image-J software (National Institute of Health, USA).
ROS staining
ROS levels in thyroid tissues were detected by the dihydroethidium (DHE) staining (Bestbio, China). The ROS levels in FRTL-5 cells were detected by reactive oxygen species assay kit (Yeasen, China). The images were observed under fluorescence microscope (Olympus, Japan). ROS levels were quantified using Image-J.
Statistical analysis
All results from at least triplicate experiments were shown as mean ± standard error of the mean (SEM), and analyzed by one-way ANOVA analysis of variance. Statistical analysis was carried out using GraphPad Prism 6 software, and statistical significance was defined as P < 0.05.
RESULTS
Effects of PCB118 on general condition of Wistar rats and FRTL-5 cell viability
In present study, the rats in each group showed no clinical symptoms of toxicity or behavioral disturbances after PCB118 exposure, and there were also no significant changes in body weight between PCB118-exposed groups and control group. In addition, at concentrations of 0.25 ~ 25 nM, PCB118 did not significantly affect FRTL-5 cell viability and apoptosis as determined in previous assays (Yang et al., 2015).
Effects of PCB118 on thyroid function
The serum concentrations of THs are summarized in Fig. 1A. Compared to the control group, the levels of serum FT3 and FT4 slightly decreased in the 10 μg/kg/d group, but significantly decreased in the 100 and 1000 μg/kg/d groups (P < 0.01). TSH level increased in PCB118-treated groups in a dose-dependent manner, and significant difference was observed in the 100 and 1000 μg/kg/d groups (P < 0.01) compared to the controls.
We further detected the expression of NIS protein, which is involved in TH synthesis. In thyroid tissues, NIS protein level decreased in PCB118-treated groups in a dose-dependent manner, especially in the 100 and 1000 μg/kg/d groups (P < 0.01) (Fig. 1B). In FRTL-5 cells, NIS protein level showed a dose-dependent decrease in the 0.25 and 2.5 nM groups, while significant decrease was found in the 25 nM group (P < 0.05) (Fig. 1C).
Histological alteration in thyroid tissues after PCB118 exposure
Compared to the control group (Fig. 2A), we found structural damages in the PCB118-treated groups. While inflammatory cell infiltration was low in the 10 μg/kg/day group (Fig. 2B), massive inflammatory cell infiltration, smaller follicular cavities, and disorderly arranged thyroid follicular epithelial were observed in the 100 and 1000 μg/kg/d groups (Fig. 2C and D).
TEM revealed ultrastructure disruption of thyroid in all PCB118-treated rats. The internal structure of thyrocytes was unclear, rough endoplasmic reticulum was distended and mitochondria were swollen (Fig. 3). Furthermore, dose-dependent changes in mitochondrial morphology were observed. Compared with the control group, mitochondrial cristae structure was blurred in the 10 μg/kg/d group (Fig. 4A and B). In the 100 and 1000 μg/kg/d groups (Fig. 4C and D), mitochondrial cristae were fractured, and even some mitochondria showed vacuolization.
The results GO enrichment analysis
GO enrichment revealed that differentially expressed mRNAs in thyroid tissues induced by PCB118 were enriched in biological processes such as reactive oxygen species (ROS) metabolic process, hydrogen peroxide metabolic process, and hydrogen peroxide catabolic process (Fig. 5). These biological processes are related to mitochondrial function (Liu et al., 2012b). To some extent, the results showed that PCB118 has effects on mitochondria in rat thyroid tissues.
Altered expression of mitochondrial respiratory chain genes in PCB118-treated rats and FRTL-5 cells
To determine whether the mitochondria in thyrocytes were affected by PCB118, the expression of mitochondria injury-related genes NDUFB3, UQCRC2, COX17, ATP5I and ATP5E in thyroid tissues was analyzed by qRT-PCR (Fig. 6). Compared with the control group, the expression of NDUFB3 mRNA was significantly reduced in PCB118-treated rats (P < 0.05). UQCRC2, COX17 and ATP5I mRNA expression showed a downtrend in the 10 μg/kg/d group, and significantly decreased in the 100 and 1000 μg/kg/d groups (P < 0.05). The expression of ATP5E mRNA was reduced significantly in all PCB118-treated rats (P < 0.05).
The expression of UQCRC2 and ATP5E mRNA was reduced significantly in all PCB118-treated FRTL-5 cells (Fig. 7) (P < 0.05). NDUFB3 mRNA expression decreased in a dose-dependent manner, and significantly decreased in the 2.5 and 25 nM groups (P < 0.05). The expression of COX17 mRNA decreased in a dose-dependent manner, but there was no statistical difference between the groups (P > 0.05).
Effects of PCB118 on oxidative stress in rat thyroids and FRTL-5 cells
As oxidative stress always occurs when mitochondria are impaired, we next performed DHE staining to analyze the level of ROS in thyroid tissues. Compared with the control group, the level of ROS in the thyroid increased significantly in the 100 and 1000 μg/kg/d groups (P < 0.05) (Fig. 8). Similarly, in PCB118-treated FRTL-5 cells, the level of ROS increased in a dose-dependent manner at the range of 0.25, 2.5 and 25 nM, and significantly increased in the 2.5 and 25 nM groups (P < 0.05) (Fig. 9).
PCB118 activated JNK in rat thyroids and FRTL-5 cells
To investigate the connection between mitochondrial damage and thyroid dysfunction, we detected the activation of JNK and found that PCB118 exposure significantly promoted JNK phosphorylation (P-JNK) in rat thyroids, especially in the 100 and 1000 μg/kg/d groups (P < 0.05), while there were no differences in total levels of JNK between the control and PCB118 groups (Fig. 10). Similar results were found in in vitro analysis, the expression of P-JNK protein significantly increased in all PCB118-treated groups (P < 0.05), and there was no significant change in total JNK protein level among all the groups (Fig. 11).
DISCUSSION
PCBs are universal organic pollutants and cause multiple toxic effects in the endocrine system (Maqbool et al., 2016). Among the congeners of PCBs, PCB118 is most closely related to thyroid dysfunction (Bloom et al., 2003). Our earlier studies revealed that PCB118 decreased serum levels of THs in animal models (Tang et al., 2013), and PCB118 could induce thyrocyte dysfunction and cause significant decrease of NIS in animal and human thyrocytes (Guo et al., 2015; Tang et al., 2013; Yang et al., 2015). In addition, our previous findings suggested that PCB118 could promote thyroid autophagy formation and cause the abnormalities in thyroidal structure. In this study, we also observed abnormalities in mitochondrial structure of rat thyroidal cells after PCB118 exposure. Mitochondria are crucial organelles which participate in the generation of adenosine triphosphate, metabolic regulation, the generation of reactive oxygen species, the maintenance of intracellular calcium homeostasis and the regulation of apoptosis. In recent years, mitochondria have received extensive attention due to their involvement in a broad range of pathological processes. However, the more specific effects of PCB118 on mitochondria and whether mitochondria are involved in PCB118-induced thyroid dysfunction are still unclear.
Studies have shown that chronic PCB118 exposure is a significant risk for thyroid dysfunction. In the study, we found that serum levels of THs decreased after PCB118 exposure in rats, consistent with such previous studies (Boas et al., 2006; Kato et al., 2004). As is well known, NIS is a transmembrane glycoprotein, and NIS-mediated iodide uptake plays a key role as the first step in TH biosynthesis (Mohammed et al., 2020). Decreased levels of NIS lead to the reduction of TH synthesis and secretion (Leung and Braverman, 2014). The present study indicated that PCB118 could reduce the expression of NIS protein in thyroid tissues and FRTL-5 cells. These data suggest that chronic PCB118 exposure causes thyroid dysfunction.
In this study, TEM showed that the mitochondrial ultrastructure of thyroid tissues was damaged in all PCB118-exposed groups, and the damage of mitochondria was aggravated in a dose-dependent manner. In addition, the functional enrichment analysis showed that differentially expressed mRNAs were enriched in reactive oxygen species (ROS) metabolic process, hydrogen peroxide metabolic process and hydrogen peroxide catabolic process, which are related to mitochondrial function. These results afforded preliminary proof that exposure to PCB118 could cause damage to thyroid mitochondria and induce oxidative stress.
The mitochondrial respiratory chain is located in the inner membrane of the mitochondria and includes complexes I-V, which constitute electron transport chains and are critical for mitochondrial metabolism (Liskova et al., 2021). NDUFB3 is a component of complex I, and the abnormality of NDUFB3 could lead to mitochondrial disorder (Hakim et al., 2019). UQCRC2 is one of the subunits of complex III and is a key regulator of oxidative stress. It was reported that deficiency of UQCRC2 leads to increased mitochondrial ROS production in airway epithelial cells (Aguilera-Aguirre et al., 2009). COX17 is essential for the assembly of cytochrome c oxidase. Mitochondria of COX17-downregulated HEK293 cell lines showed ultrastructural changes, including cristae reduction and mitochondrial swelling (Vanišová et al., 2019). Xia et al. found that the expression of ATP synthase gene ATP5E and ATP5I decreased in prenatal PFOS-exposed weaned rat heart (Xia et al., 2011). All these genes are essential for maintaining normal function of the mitochondria. qRT-PCR results showed the downregulation of NDUFB3, UQCRC2, COX17, ATP5E and ATP5I in thyroid tissues and FRTL-5 cells after PCB118 exposure, and this phenomenon was directly related to mitochondrial damage. The mechanism of mitochondrial damage caused by PCB118 has not yet been reported. Most of the toxicological studies on PCBs focused on the damage of mitochondrial ultrastructure and the occurrence of oxidative stress. Regarding the mechanism of mitochondrial damage, multiple studies highlight the importance of Ca2+ signaling in mitochondria. Enhanced store-operated calcium entry (SOCE) contributed to mitochondrial dysfunction (Rodríguez et al., 2022; Shanmughapriya et al., 2015). In our previous studies, we have found PCB118 increased intracellular calcium levels in a time- and dose-dependent manner. The channel protein mediating SOCE was also upregulated by PCB118 (Wang et al., 2020). In addition, we demonstrated that PCB118 upregulated the cytochrome P450 1A1 (CYP1A1) mRNA expression (Xu et al., 2016). It has been reported that mitochondrial activity and oxidative stress functions are influenced by CYP1A1 overexpression (Zhou et al., 2017). However, the above mechanisms are our speculation and more details on the underlying mechanism require further exploration.
Under normal conditions, the mitochondria are the sites of oxidative phosphorylation and energy generation, and are essential for maintaining redox balance and cellular homeostasis. While under stress conditions, mitochondria are destroyed and generate excessive levels of ROS, causing oxidative stress (Ansari et al., 2020; Bhatti et al., 2017; Schieber and Chandel, 2014). In an in vivo study, we found that ROS levels increased significantly in thyroid tissues after PCB118 exposure, and the same results were observed as well as in vitro. The above data supported that PCB118 significantly induced oxidative stress in rat thyroid cells.
ROS play a pivotal role in genomic damage, accelerating telomere shortening and mediating inflammation (Møller et al., 2014; Parrinello et al., 2003; von Zglinicki, 2002). Dysfunctional mitochondria increase ROS generation and it has been reported that ROS could promote the activation of the stress-activated JNK kinase (McCubrey et al., 2006; Schieber and Chandel, 2014; Shi et al., 2014). JNK is an important mediator of inflammation, and the JNK pathway plays an important role in THs homeostasis (Liu et al., 2012a). Previously, we found that PCB118 could induce thyroid dysfunction by activating the JNK pathway (Xu et al., 2016). Similarly, we observed that the levels of P-JNK protein increased significantly in vivo and in vitro after PCB118 exposure in this study, consistent with the changes of ROS. Based on these results, we speculate that JNK pathway is activated by ROS generated from dysfunctional mitochondria.
In conclusion, we demonstrated that chronic low-dose exposure to PCB118 could induce thyroid dysfunction and cause mitochondrial damage. PCB118-induced thyroid dysfunction may be related to ROS-dependent activation of the JNK pathway.
ACKNOWLEDGMENTS
This study was supported by the National Natural Science Foundation of China (No. 81670724, 82071582).
Conflict of interest
The authors declare that there is no conflict of interest.
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