Co-exposure to Fluoride and Arsenic induces Endoplasmic Reticulum Stress in C2C12 Myoblasts and MC3T3 Preosteoblasts.

Abstract

Original abstract online at
https://www.sciencedirect.com/science/article/abs/pii/S0300483X26001733?via%3Dihub

Highlights

First report of fluoride and arsenic co-exposure effects in C2C12 myoblasts
Fluoride and arsenic co-exposure induces oxidative stress in C2C12 and MC3T3 cells
Fluoride and arsenic co-exposure induces ER stress in C2C12 and MC3T3 cells
C2C12 myoblasts are more susceptible to fluoride and arsenic co-exposure

Environmental exposure to fluoride and arsenic is a major public health concern in many regions worldwide. Although both toxicants are known to impair skeletal development, the cellular mechanisms underlying their combined effects on skeletal muscle and bone cells remain poorly understood. This study, investigated the impact of sodium fluoride (NaF) and arsenic trioxide (As2O3) co-exposure, for the first time, in C2C12 myoblasts (at 0.14 mM NaF + 2.4 µM As₂O₃) and MC3T3 preosteoblasts (at 0.64 mM NaF + 13 µM As₂O₃)

Co-exposure significantly reduced cell proliferation and decreased the expression of the lineage specific transcription factors such as MYOD and RUNX2 in C2C12 and MC3T3 cells, respectively. Both individual and combined treatments increased ROS production, with co-exposure producing the highest oxidative stress. Further, fluoride and arsenic co-exposure exerted its effects by inducing endoplasmic reticulum stress in both cell types which was witnessed by the activation of the unfolded protein response (UPR) pathways. Specifically, in C2C12 myoblasts, co-exposure predominantly activated the PERK–Atf4–Chop pathway while suppressing IRE1a–Xbp1 expression, indicating unresolved ER stress. In contrast, MC3T3 preosteoblasts activated both PERK–Atf4–Chop and IRE1a–Xbp1 pathways, suggesting engagement of adaptive ER stress mechanisms.

Collectively, these findings demonstrate that fluoride and arsenic co-exposure disrupts cellular homeostasis by inducing ER stress in both myoblasts and preosteoblasts. The differences in activation of different UPR pathways suggests C2C12 myoblasts to be more susceptible to fluoride and arsenic co-exposure compared to MC3T3 preosteoblasts.

Graphical abstract

Introduction

Two of the most common inorganic groundwater pollutants, fluoride and arsenic, are widespread contaminants and pose significant risks to public health (Appelo and Postma, 2004).

Fluoride is a highly electronegative and an extremely reactive compound, with its recommended levels in drinking water not exceeding 1.5 mg/L (WHO, 2011). However, in fluorosis endemic areas, it is reported that fluoride is present in high levels in water; its source being rocks and granulite and population residing around fluoride endemic areas worldwide suffer from bone and muscle weakness (Teotia et al., 1998). Specifically, chronic and acute exposure to fluoride causes dental mottling and skeletal fluorosis (Asawa et al., 2015, Kebede et al., 2016, Krishnamachari, 1986). Besides, gastrointestinal, renal, neuronal, liver and reproductive (Antonio et al., 2017, Pereira et al., 2013, Vale et al., 2019, Wang et al., 2017, Zhang et al., 2008) disorders are also caused by fluorosis. In addition to this, fluorosis causes musculoskeletal damage and destroys collagen structure in muscles, tendons, ligaments and bone.

Similarly, arsenic is a groundwater contaminant with its maximum permissible limits not exceeding 10 ug/L (WHO, 2011) and exposure to arsenic is established as a major public health concern all over the world (González-Martínez et al., 2024). Arsenic is also distributed in earth’s crust, rocks, soil, air, water, sea water and deep sea sand-bed (Guha Mazumder, 2003). Exposure to arsenic results in skin lesions, hepatic and renal disease, cardiovascular and cerebrovascular pathologies, type 2 diabetes, respiratory illnesses, neurobehavioral disorders in children and cancer in kidney, liver, lungs, urinary bladder and skin, haemolysis, hypertension and shock (Rahaman et al., 2021).

The gravity of the problem becomes even more serious when humans are co-exposed to both fluoride and arsenic. It is reported that the coexistence of fluoride and arsenic is seen in many areas of the world such as Asia (India, China, Japan, Korea and Pakistan), Africa (Ethiopia, Ghana, Nigeria and Tanzania) and Latin America (Argentina, Bolivia, Chile, Colombia, Peru and Mexico) (Kumar et al., 2020). Although the individual toxic effects of fluoride and arsenic is already established in the literature, a clear-cut understanding on their combined toxicity is still to be strongly established. Evidence thus far demonstrates that combined exposure to fluoride and arsenic can induce hepatotoxicity, nephrotoxicity, cardiotoxicity, neurotoxicity, and reproductive toxicity (González-Alfonso et al., 2023, González-Alfonso et al., 2025, Mukherjee et al., 2024, Tian et al., 2023a). However, there is still scarcity of data to provide their effects on the skeletal muscle and to a certain extent, the bone.

Skeletal muscles are the main protein reservoir in the body accounting for approximately 40% of the total body weight and are crucial for exercise and energy metabolism. Skeletal muscles are responsible for physical activities and also act as the main energy metabolism tissue and participate in the uptake, utilization, and storage of energy metabolism substrates such as glucose, lipids, and amino acids (Yin et al., 2021). On the other hand, bone is composed of cells (osteoblasts, osteocytes, and osteoclasts), fibres, and an extracellular matrix which all form the complex tissue and plays several important functions in the body (Šromová et al., 2023). Hence, any disturbance caused by extracellular triggers may affect the normal functioning of such crucial organs and be detrimental to overall human health and wellbeing.

To this end, we investigated the effects of fluoride and arsenic co-exposure on skeletal muscle and bone using in vitro models, namely the C2C12 skeletal muscle cell line (C2C12 myoblast) and the MC3T3 bone cell line (MC3T3 preosteoblasts). Importantly, this is the first report of fluoride and arsenic co-exposure effects on the skeletal muscle C2C12 myoblasts.

Section snippets

Cell culture

C2C12 myoblasts were cultured in DMEM supplemented with 10% Foetal Bovine Serum (FBS), 1% Glutamax and 1% Penicillin-Streptomycin (all from Gibco, Thermo Fisher Scientific, USA) at 37°C and 5% CO₂. MC3T3 preosteoblasts were cultured in alpha MEM supplemented with 10% FBS, 1% Glutamax, 1% Penicillin-Streptomycin at 37°C and 5% CO₂. Cells were trypsinized at 70%-80% confluency using 0.25% trypsin-EDTA (Thermo Fisher Scientific, USA).

MTT Assay

C2C12 myoblasts and MC3T3 preosteoblasts were screened for

Fluoride and arsenic co-exposure reduced cell proliferation of C2C12 myoblasts and MC3T3 preosteoblasts

C2C12 myoblasts and MC3T3 preosteoblasts were seeded in 96 well plates at a cell density of 3,000 cells per well and screened for cytotoxicity following exposure to sodium fluoride (NaF) and arsenic trioxide (As2O3), individually and in combination through MTT assay.

Based on the results obtained from the dose response curves IC30 values for C2C12 myoblasts were 3 mM for NaF (Fig. 1 a), 2 µM for As2O3 (Fig. 1 b), and 0.14 mM NaF + 2.4 µM As2O3 (Fig. 1 c) for the combined treatment. Similarly, for

Discussion

Fluoride and arsenic are two major groundwater pollutants that are distributed worldwide, posing a threat to the health of millions of people (Chouhan and Flora, 2010). Fluoride exposure can lead to dental fluorosis, skeletal fluorosis, and damage to other organs (Everett, 2011), while arsenic poisoning can cause damage to the skin, heart tissue and other organs (States et al., 2009). Despite extensive research on the individual toxicological effects of fluoride and arsenic, studies…

Conclusion

The present study highlights the effects of co-exposure to fluoride and arsenic for the very first time in the skeletal muscle C2C12 myoblasts and on bone MC3T3 preosteoblasts. The study findings suggests that fluoride and arsenic co-exposure induces the production of oxidative stress in both the cell lines. Additionally, co-exposure also hampers the expressions of MYOD and RUNX2 in myoblasts and preosteoblasts respectively. Most importantly, co-exposure causes endoplasmic reticulum stress in…

Uncited reference

(Zhang et al. (2015))

Funding

This work was funded by Yenepoya Deemed to be University Seed grant (YU/Seed grant/159-2024) awarded to the principal investigator SSP and co-principal investigator BB to carry out this research. DBK and JRP are also supported by University’s Junior Research Fellowship.

CRediT authorship contribution statement

Sudheer Shenoy P: Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Data curation, Conceptualization. Apoorva H. Nagendra: Writing – review & editing, Validation, Resources, Conceptualization. Bipasha Bose: Writing – review & editing, Visualization, Validation, Supervision, Project administration, Methodology, Funding acquisition, Formal analysis, Data curation. Deepika Bhat. K:

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors would like to acknowledge Yenepoya Research Centre, Yenepoya Deemed to be University for the infrastructure and core facility support for conducting this research. Additionally, we also acknowledge Freepik and Vectors Tank from www.flaticon.com for the muscle cell and bone cell images used in the graphical abstract of the manuscript.

Declaration of conflict of interest

The authors declare no conflicts of interest

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