2,3,7,8-Tetrachlorodibenzo-p-dioxin induces alterations in myogenic differentiation of C2C12 cells

Abstract

Dioxin-induced toxicities that affect the development of the motor system have been proposed since many years. However, cellular evidence and the molecular basis for the effects are limited. In this study, a cultured mouse myoblast cell line, C2C12, was utilized to examine the effects of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) on myogenic differentiation and expression of acetylcholines- terase (AChE), a neuromuscular transmission-related gene. The results showed that TCDD exposure at 10—10 M repressed the myotube formation of C2C12 cells by disturbing the fusion process and sup-
pressing the expression of myosin heavy chain, a myobute structural protein, and not by induction of cytotoxicity. Furthermore, TCDD dose dependently suppressed the transcriptional expression and enzymatic activity of AChE during the myogenic differentiation, particularly in the middle stage. How- ever, the administration of aryl hydrocarbon receptor antagonists, CH223191 and alpha-naphthoflavone, did not completely reverse the TCDD-induced downregulation of muscular AChE during myogenic dif- ferentiation. These findings suggest that low dose exposure to dioxin may result in disturbances of muscle differentiation and neuromuscular transmission.

1. Introduction

Dioxins have been linked to multiple intoxications including chloracne, immunotoxicity, neurotoxicity, hepatoxicity, reproduc- tive toxicity, and tumor development (Denison et al., 2011; Pohjanvirta and Tuomisto, 1994; van Leeuwen et al., 2000). In recent years, people have paid more attention to dioxins’ neuro- toxicities, especially the developmental toxicities in the nervous system. Significant negative associations between the mental developmental index and the dioxin level in maternal blood have been found among 6-month-old male infants in Sapporo cohort study (Kishi et al., 2013; Nakajima et al., 2006). Additionally, animal studies have also revealed that maternal exposures to 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) could disrupt the memory function of the offspring and alter the differentiation pattern of the neural progenitor cells in mice (Haijima et al., 2010; Mitsuhashi et al., 2010). Dioxins not only cause alterations in advanced brain functions (Michalek et al., 2001; Powers et al., 2005; Schantz and Bowman, 1989) but also in the development and function of the skeletal muscles. A recent epidemiological study showed that altered functions in motor domains of the nervous system occurred in 4-month-old infants living in dioxin-contaminated areas in Vietnam, whose function of object manipulation and movement of the limbs and torso were interfered by perinatal dioxin exposure (Tai et al., 2013). Furthermore, defects in the myogenesis of the palate has been proposed as one of the mechanisms for the for- mation of the cleft palate in prenatal TCDD-exposed mice (Yamada et al., 2014). Coletti et al. also reported that the differentiation of cells in both the myogenic cell line and the primary myogenic cell cultures was specifically impaired by exposure to commercial mixtures of polychlorobiphenyl (PCBs) congeners, in which dioxin-like PCBs were included (Coletti et al., 2001). Thus, dioxin exposure might have effects on the myogenic differentiation of the skeletal muscle cells, which deserves extensive investigations on the direct cellular evidence and underlining molecular basis.

Apart from muscle development, dioxin could affect various aspects of functions associated with locomotion, such as functions of the motor neurons, conduction of the motor nerves, and func- tions of the neuromuscular junction (NMJ) and muscle innervation. Decrease in motor nerve conduction velocity has been reported to occur in the peroneal nerve of 156 dioxin-exposed workers from a pesticide plant (Thomke et al., 1999). Consistent with this finding in human, electrophysiological studies showed a dose-dependent slowing down in the conduction velocity of motor and sensory parts of the sciatic nerve in adult male Han/Wistar rats exposed to TCDD (Grahmann et al., 1993). Apart from peripheral nerves, di- oxins may affect innervation of the muscles. Myalgia and myas- thenia were major complaints among dioxin-exposed chemists (Schecter and Ryan, 1992). Amyotrophy could be observed in muscular tissues of dioxin-exposed rats (Max and Silbergeld, 1987). However, whether the neuromuscular transmission can be affected by dioxin exposure is still devoid of solid evidence.

Acetylcholinesterase (AChE) is an enzyme that hydrolyzes the neurotransmitter acetylcholine into acetic acid and choline and plays a vital role in terminating the nervous impulse in the pe- ripheral and central cholinergic nervous systems. In the peripheral nervous system, AChE mainly exerts its function at NMJs, and the major functional asymmetric form of AChE is present at the basal lamina of the NMJs (Soreq and Seidman, 2001). Abnormal expres- sion levels or subcellular locations of AChE at NMJs may lead to abnormalities in neuromuscular transmission, which controls muscle contractions to maintain normal movement function including breathing (Massoulie and Millard, 2009). The expression of AChE at NMJs is tightly controlled during development and after maturation, in which both the presynaptic neurons and post- synaptic myotubes make contributions (Tsim et al., 2010). The regulations of AChE during the process of myogenesis and NMJ formation have been extensively studied (Gaspersic et al., 1999; Siow et al., 2002). The C2C12 mouse myoblast cell line is widely used as an in vitro model for the study of myogenesis (Katase et al., 2016; Nozaki et al., 2016; Siow et al., 2002). The differentiation profile of AChE during myogenesis has revealed that AChE expression is markedly increased from the myoblast to myotube stages of cultured C2C12 cells and getting prepared for the inner- vation process (Fuentes and Taylor, 1993; Siow et al., 2002). Recently, alterations in neuronal AChE expression have been studied in dioxin-treated neuroblastoma cells, in which dioxin exposure significantly suppressed the AChE activity through aryl hydrocarbon receptor (AhR)-mediated transcriptional down- regulation in the SK-N-SH cells (Xie et al., 2013). However, whether muscular AChE expression could be altered by dioxins remains unclear.

Given the aforementioned evidence, we hypothesized that dioxin may interfere the process of myogenesis and the expression of AChE during myogenic differentiation. Therefore, in this study, we sought to define alterations in myogenic differentiation of C2C12 cells and in the differentiation profile of AChE expression upon dioxin exposure during the time. Finally, the role of AhR in gene alterations was explored.

2. Materials and methods

2.1. Cell culture and differentiation

C2C12 murine cell line, obtained from the American Type Cul- ture Collection (Manassas, VA, USA), was maintained in a growth medium (GM), including Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Gaithersburg, MD, USA) supplemented with 20% fetal bovine serum (FBS) (Corning, New York, USA) and 1%
penicillinestreptomycin (P/S) (Gibco), and incubated at 37 ◦C in a water-saturated 5% CO2 incubator. To induce fusion of cultured myoblasts, cells were first allowed to grow in DMEM with 10% FBS until confluent. Then the medium was changed to a differentiation medium (DM) including DMEM with 2% heat-inactivated horse serum (HS) (Gibco) to induce the differentiation. The medium was changed every 24 h over the following 6 days.

2.2. Chemical treatment

C2C12 cells were seeded onto 6-well plates and cultured in the GM or DM. TCDD, the most potent congener of dioxins, was pur- chased from Wellington Laboratories, Inc. (Ontario, Canada). Two antagonists of the AhR-dependent pathway, CH223191 or alpha- naphthoflavone (ANF) (Zhao et al., 2010; Ramadass et al., 2003), were obtained from Sigma (St. Louis, MO, USA).C2C12 cells were continuously treated with TCDD during the myogenic differentiation. The dosing of C2C12 cells was conducted every day in which the DM containing TCDD or solvent control was replenished every 24 h from the first day of induction (day 0). To reveal the role of AhR, C2C12 cells were pretreated with CH223191 (10—6 M) or ANF (10—5 M) for 3 h before the treatment with TCDD (Zhao et al., 2010; Ramadass et al., 2003). The solvent dimethyl sulfoxide (DMSO) was present in all treatments at less than 0.1%.

2.3. Morphological analysis

The morphological change of the cells during myogenic differ- entiation was analyzed by hematoxylin and eosin (H&E) staining. C2C12 cells were seeded onto 6-well plates. Five different images were randomly captured per well under an inverted light micro- scope (CKX41, Olympus, Japan) with a digital camera (DS126311; Canon Inc., Taiwan). The myotube and nuclei numbers were counted by using Image-Pro Plus 6.0 from which the average numbers from the five images randomly captured per well were obtained. The fusion index was calculated as the ratio of nuclei incorporated into myotubes to the total number of nuclei in all images captured. The number of nuclei per myotube was deter- mined as the average number of the nuclei in the myotubes from the images captured per well. In this study, the myotubes with two or more nuclei were counted (Ge et al., 2014; Shafey et al., 2005).

2.4. Cell viability analysis

The cells were cultured in 6-well plates to induce differentiation and carry out TCDD treatment. Cell viability was determined using CellTiter-Glo® luminescent cell viability assay kit (Promega, Madi- son, WI, USA) according to the manufacturer’s instructions. The absorbance was measured with a GLOMAX™ Multi detection sys- tem (Promega).

2.5. Quantitative real-time PCR (qRT-PCR)

The total RNA was extracted using GeneJET RNA purification kit (Thermo Waltham, MA, USA). cDNA was prepared using 2 mg of total RNA and the RevertAid first strand cDNA synthesis kit (Thermo) according to the manufacturer’s instructions. Real-time PCR was performed on equal amounts of cDNA using GoTaq® qPCR master mix kit (Promega) according to the manufacturer’s instructions. The SYBR green signal was detected by a Roche 480 multiplex quantitative PCR system (Roche, Basel, Switzerland) and the DDCT method was used to quantify the relative mRNA expression levels (Winer et al., 1999).

We determined the gene expression of AChE T subunit (AChET), myosin heavy chain (MyHC), and cytochrome P4501A1 (CYP1A1) by qRT-PCR, and b-actin was used as an internal control for nor- malizations. The CT values of b-actin in all groups were within a range of 16.45 ± 0.54 (mean ± SD, n = 118), and the coefficient of variation was 3.30%. There was no significant change in the b-actin expression from all treated groups. The sequences of all primers used are shown in Table 1, and their amplification efficiencies were within 100%e110%.

2.6. Determination of AChE enzymatic activity

AChE enzymatic activity was determined by a modified method of Ellman (Ellman et al., 1961). Cells were collected, and total pro- teins were extracted using the high-salt lysis buffer (1 M NaCl and 80 mM disodium hydrogen phosphate, pH 7.4) that is supple- mented with 0.5% Triton X-100 and 2.5 mM benzamidine, a protease inhibitor, for 30 min at 4 ◦C. Ten mL cell lysates were added in 96-well plates and incubated with 0.1 mM tetraisopropylpyr-
ophosphoramide (iso-OMPA), an inhibitor of butyrylcholinesterase (BChE), and 0.5 mM 5,5′-dithiobis(2-nitrobenzenoic acid) (DTNB) for 30 min at room temperature to inhibit BChE activity and allow saturation of nonspecific reaction with DTNB. Acetylthiocholine iodide (0.625 mM) was subsequently added to initiate AChE- specific reaction. Readings at 405 nm were repeated at 2-min in- tervals for 50 min with a microplate spectrometer (TECAN Infinite F200 Pro; Ma€nnedorf, Switzerland). Protein concentration was measured according to the method of Bradford, (1976). The velocity of the reaction was calculated from the slope of the fitting line obtained from optical density (OD) change over time. Arbitrary units of enzymatic activity were expressed as velocity (mOD per minute) per microgram of protein. All reagents were obtained from Sigma.

2.7. Western blot analysis

Cells were washed with phosphate-buffered saline and ho- mogenized in lysis buffer. Equal amounts of total protein, 20 mg, were loaded on a 10% running gel and a 4% stacking gel of sodium dodecyl sulfate polyacrylamide gel. Proteins captured in the gel were transferred to a pure nitrocellulose blotting membrane (Pall, New York, USA). The membrane was blocked for 1 h in blocking buffer (LI-COR Biosciences, Lincoln, NE, USA). Membranes were
incubated overnight at 4 ◦C with primary antibodies. Primary antibody dilutions were 1:1000 for anti-MyHC (Santa Cruz Biotechnology (SC-20641), Santa Cruz, CA, USA) and 1:10000 for anti-b-actin (Sigma (A1978)). After intensive washing with a solu- tion composed of 20 mM Tris base, 137 mM NaCl, and 0.1% Tween 20 at pH 7.6, membranes were incubated with an appropriate secondary antibody (Goat anti-Rabbit IRDye 800CW and Goat anti- Mouse IRDye 680CW, LI-COR Biosciences) for 2e3 h. Antibody reactive bands were detected with an Odyssey® infrared imaging system (LI-COR Biosciences). Band intensities were quantified using Image Studio Lite Software (LI-COR Biosciences).

2.8. Statistical analysis

We performed statistical analyses using GraphPad Prism 5.0. Data are represented as mean ± SEM. Each experiment was per- formed in triplicate and repeated at least three times. The signifi- cance of difference was determined by one-way or two-way ANOVA. We considered p < 0.05 as statistically significant.

3. Results

3.1. TCDD disturbs myotube formation of C2C12 cells

Cytotoxicity of dioxin in cultured myoblasts and myotubes was revealed by the cell viability assay. Results showed that continuous exposures to a series of TCDD concentrations (10—12 M to 10—9 M) did not significantly change the cell viability of cultured myoblasts (collected on day 1; Fig. 1A) and myotubes (collected on day 6; Fig. 1B). This result suggested that TCDD exposure did not induce cytotoxicity in C2C12 cells in the present study.
We further investigated the effects of low concentration (10—10 M) of TCDD on myogenic differentiation. From Fig. 2A, myotube formation could be observed morphologically in all groups on day 3 and day 6 by H&E staining. A significant sup- pression of myotube formation was shown morphologically in TCDD-treated groups compared with DMSO controls (Fig. 2A). Quantitative analysis also showed that myotube formation started on day 3 and reached a maximum number on day 6 of differenti- ation, which is consistent with the literature (Fig. 2B; Siow et al., 2002). The TCDD exposure significantly reduced the myotube number on day 6 (Fig. 2B). This suppressive effect on myotube formation may have resulted from the disturbance of the fusion process. To examine the effect of dioxin on the fusion process, two widely used indexes, the fusion index and the nuclei number per myotube (Ge et al., 2014; Shafey et al., 2005), were utilized in this study. The results showed that TCDD significantly attenuated the fusion index on day 3 (~21% decrease from control) and day 6 (~22% decrease from control) (Fig. 3A) and the nuclei number per myo- tube on day 3 (~7% decrease from control) and day 6 (~13% decrease from control) (Fig. 3B). This result implicated that in the presence of TCDD, less number of myoblasts undergo fusion process and the myotubes formed possess less cell content.

Myotube formation was further examined at the molecular level. Effect of TCDD treatment on the expression of MyHC, a structural protein in myotubes, was studied. In line with the liter- ature, the expression of MyHC was increased along with the in- crease in myotube formation in the solvent control groups (Fig. 4; Hwang et al., 2015). However, after continuous TCDD (10—10 M) exposure, the expression of MyHC was significantly suppressed at the mRNA level (~35% decrease from control; Fig. 4A) and the protein level (~30% decrease from control; Fig. 4B) on day 6, which was in accordance with the suppressive effect found in the morphological study (Fig. 2).

3.2. TCDD suppresses AChE expression during the myogenic differentiation of C2C12 cells

Effects of dioxin on the enzymatic activity of AChE were examined during the myogenic differentiation of C2C12 cells. Consistent with the literature, in the solvent control groups, AChE activity was gradually increased from day 1 to day 6. After exposure to 10—10 M TCDD, AChE activity was significantly reduced by approximately 42% on day 3 and 28% on day 6 compared with the controls (Fig. 5A), although the increasing profile of AChE activity was maintained.

We further examined dioxin effect on the expression of AChET subunit, a major AChE transcript in muscles (Legay et al., 1995). The results showed that compared to the solvent control, the mRNA level was significantly decreased on day 3 (~49% decrease from control) and day 6 (~31% decrease from control) by dioxin exposure, which is consistent with the alterations in AChE activity (Fig. 5B). Because more obvious effects were found on day 3, we chose the third day of differentiation to reveal the dose response of AChE expression to TCDD exposure. A series of TCDD concentrations (3 × 10—12 M to 10—10 M) was employed, and significant alterations in the enzymatic activity and AChET mRNA expression occurred in all TCDD groups and 10—11 Me10—10 M TCDD groups, respectively (Fig. 6). The maximum suppressive effects were obtained in the 10—10 M group with ~50% of changes in either enzymatic activity or mRNA expression (Fig. 6). These results suggested that dioxin can transcriptionally suppress AChE expression during the myogenic differentiation of C2C12 cells.

3.3. AhR does not directly mediate the suppression of AChE by TCDD exposure during myogenic differentiation of C2C12 cells

Based on the mediating role of AhR in the alterations of dioxin- responsive genes (Denison et al., 2011; Sorg, 2014), we further explored the involvement of AhR in the present suppression of muscular AChE expression caused by TCDD exposure. Two AhR inhibitors, CH223191 and ANF, were administrated 3 h before the continuous exposure to TCDD from differentiation day 0 to day 3. CYP1A1 served as a positive control, which is a classical responsive gene upon AhR activation (Denison et al., 2011; Abel and Haarmann-Stemmann, 2010). We found that TCDD treatment induced dramatical upregulation of CYP1A1 expression, and pre- treatment with CH223191 or ANF could significantly block this increase. In the presence of CH223191 (10—6 M) or ANF (10—5 M), the expression of CYP1A1 reduced by ~80% and ~95% compared to that in TCDD treatment groups, respectively (Fig. 7A and D). However, with the pretreatment of CH223191, the suppressive ef- fects of TCDD on AChE activity and AChET mRNA expression were not changed significantly (Fig. 7B and C). Interestingly, unlike CH223191, the pretreatment of ANF slightly reversed the suppres- sions caused by TCDD exposure, although not back to the control levels (Fig. 7E and F). It was notable that CH223191 or ANF treat- ment alone significantly suppressed the AChE expression compared to that in solvent control groups (Fig. 7B, C, E and F).

4. Discussion

Emerging evidence has shown that perinatal exposure of dioxin may affect the development of the motor system. Nishijo et al. re- ported that oral administration of TCDD (0.1 mg/kg body weight) to pregnant rats from gestational day 9 to day 19 delayed the righting response of the offspring on inclination, which suggested that perinatal TCDD exposure interfered with the development of the motor system in offspring (Nishijo et al., 2007). Furthermore, the myogenic differentiation process has been proposed as a target cellular event of toxic pollutants. Chiu et al. reported that benzo(a) pyrene (BaP) and its main epoxide metabolite were capable of inhibiting myogenic differentiation of human skeletal muscle- derived progenitor cells (HSMPCs) (Chiu et al., 2014). Another report demonstrated that upon continuous treatment with PCBs, the myogenic differentiation of in vitro cell models was inhibited in a dose-dependent manner (Coletti et al., 2001). When muscle undergoes differentiation or regeneration after damage or exercise or during diseases, the skeletal muscle satellite cells will be acti- vated and lead to new myofiber formation through proliferation, differentiation, and fusion (Cancino et al., 2013; Chiu et al., 2014; Martinello et al., 2011). C2C12 cell line is an ideal cell model for studying the process of myogenesis, which can rapidly fuse into myotubes under low-serum condition (Katase et al., 2016; Ku and Park, 2013). In line with the literature, the C2C12 myoblasts could undergo normal fusion to form multinucleated myotubes from the first day till the sixth day of differentiation in the present study (Bajaj et al., 2011; Siow et al., 2002). The morphological indexes such as the fusion index, myotube number and nuclei per myotube, and the expression of the marker protein MyHC were all found to be increased during the C2C12 myogenic differentiation in the present study, which is consistent with the literature (Ge et al., 2014; Shafey et al., 2005; Hwang et al., 2015). By using this myogenic differentiation model, we focused on exploring the effect of dioxin on myogenesis. We showed direct evidence that TCDD exposure repressed the myogenic differentiation of cultured C2C12 cells by disturbing myotube formation and maturation, and not by inducing cytotoxicity. This finding indicated that one of the crucial events of the motor system development, myogenesis, may be affected by dioxin exposure at an environmental relevant con- centration (10—10 M) (Xie et al., 2013).

Apart from muscle development, previous studies proposed neuromuscular toxicities induced by dioxin in rat upon low-dose exposures at 2.2, 4.4, 6.6, and 8.8 mg/kg body weight (Grahmann et al., 1993). In the present study, we found that dioxin treatment at low concentrations could decrease the enzymatic activity of AChE during the myogenic differentiation of C2C12 cells. Because dioxin is well known to regulate gene expression at the transcrip- tional level, we hypothesized that dioxin might affect the expres- sion of AChE transcript. Moreover, similar to what we found for neuronal AChE (Xie et al., 2013), TCDD did not directly inhibit AChE activity in C2C12 cell lysate (Data not shown), which further sug- gested that the decrease in the enzymatic activity might result from the transcriptional suppression. We did find a significant decrease in the mRNA level of AChET subunit upon TCDD exposure at low concentrations (10—11e10—10 M). Absence of AChE can lead to marked neuromuscular alterations. Mouisel et al. found that mus- cle weight and maximal tetanic force were reduced in 1.5-month- old AChE-knockout mice as compared to wild-type mice after short periods (500 ms) of repetitive nerve stimulations (Mouisel et al., 2006). Additionally, exposure to monocrotophos, an organic phosphate pesticide, induced severe muscle weakness in rats as a result of the significant inhibition of muscular AChE activity (30%e 60%) (Raghupathy et al., 2010). Considering the crucial role of AChE at the NMJs, the present findings suggested possible interferences of dioxin with the neuromuscular transmission. On the other hand, abnormal muscle development or muscle damages may lead to alteration in muscular AChE expression (Michel et al., 1994; Guo et al., 2009). Therefore, we presumed that inhibition of myo- genesis by dioxin may lead to the alterations of AChE and subse- quently affect the normal neuromuscular transmission.

It is well-known that AhR, a transcription factor of bHLH (basic Helix-Loop-Helix)-PAS (Per-ARNT-Sim) family, can regulate a vari- ety of physiological and developmental processes (McIntosh et al., 2010; Sorg, 2014) and mediate decrease in the neuronal AChE ac- tivity through transcriptional downregulation (Xie et al., 2013). It also plays a crucial role in the effect of dioxins on metabolomics profile of muscle (Lin et al., 2011). Chiu et al. also demonstrated that AhR was involved in BaP, and its epoxide metabolite induced inhibition of the myogenic differentiation in HSMPCs (Chiu et al., 2014). However, in this study, dioxin-induced suppression of AChE activity and transcriptional expression could not be completely reversed by application of the AhR antagonists, although the antagonists effectively counteracted the induction of CYP1A1 caused by TCDD. Nevertheless, based on the slight reverse of AChE expression in the presence of ANF, the possible role of AhR in muscular AChE regulation could not be ruled out completely. However, treatment with another AhR agonist, b-naphthoflavone (BNF; at 10—7 ~ 10—6 M), did not significantly affect the expression of AChET mRNA and AChE activity and also the expression of CYP1A1 (data not shown). The distinct effects of BNF on the expression of muscular AChE and CYP1A1 from those of TCDD might be due to the ligand-specific variation in AhR functionality as proposed previously (Zhao et al., 2010). These pieces of evidence suggest that AhR may not be the major mediator in the dioxin- induced muscular AChE suppression. Furthermore, the reason for the suppressive effect of CH223191 or ANF on AChE expression needs further investigations to answer whether AhR could play a physiological role in maintaining the normal expression level of muscular AChE expression.

Regarding the possible mechanisms for TCDD-induced sup- pression of muscular AChE, we hypothesized that an endogenous regulatory mechanism for AChE expression during C2C12 myogenic differentiation may be involved. MyoD family consists of muscle- specific transcription factors that participate in the management of C2C12 differentiation including withdrawal from the cell cycle, the expression of myotube-specific genes, and cell fusion to form multinucleate myotubes (Hwang et al., 2015). Myogenin is one of the myogenic regulatory factors, which has been shown to partic- ipate in the early phase of myotube formation and the induction of AChE transcription during myogenic differentiation process (Angus et al., 2001). Furthermore, it has been reported that myogenin plays a role in the BaP-induced inhibition of HSMPC myogenic differen- tiation (Chiu et al., 2014). Thus, whether myoD or myogenin could mediate the AChE suppression by dioxin during the myogenic process is worthy of further investigations. This investigation might be helpful to explain the different role of AhR in dioxin-induced muscular AChE alteration from that of neuronal AChE (Xie et al., 2016; Xu et al., 2014).

5. Conclusion

We demonstrate for the first time that TCDD can inhibit the myogenic differentiation and the expression of AChE in mouse C2C12 cells. In the middle stage of the myogenic differentiation, TCDD significantly suppresses the transcriptional expression of AChE in a dose-dependent manner, which might consequently lead to a decrease in the enzymatic activity of AChE. However, AhR signaling pathway may not be the predominant mechanism for the downregulation of AChE by TCDD during C2C12 differentiation. These findings suggest that low-dose exposure to dioxin may result in disturbances of muscle differentiation and neuromuscular transmission.BAY 2416964 The actual roles of AhR in these effects need further investigations.