Fisetin modulates fluoride induced osteochondral toxicity in zebrafish larvae.

Introduction

Fluoride (F^–) is an essential trace element known for its dual role in skeletal health. At optimal levels, it supports bone and dental integrity, but chronic exposure to elevated concentrations can lead to skeletal fluorosis, a condition characterized by impaired bone remodeling, joint stiffness, and structural deformities (Everett, 2011; Johnston and Strobel, 2020). Excess F^– disrupts redox balance, induces oxidative stress, interferes with osteogenic signaling, and impairs matrix mineralization, ultimately weakening bone strength and architecture (Qiao et al., 2021).

Although the toxic effects of F^– on the skeletal system have been widely studied, the underlying mechanisms remain incompletely understood, particularly at low or subclinical exposure levels. Current evidence indicates that high F^– concentrations disturb the balance between osteoblast and osteoclast activity, leading to altered bone turnover (Jiang et al., 2020). F^– accumulation in bone is dose-dependent. Bone ash F^– concentrations below 1000 mg/kg typically show no noticeable skeletal changes, while those between 1000 and 6000 mg/kg may remain asymptomatic. Concentrations above 7000 mg/kg are frequently linked to skeletal deformities and pathological lesions (Choubisa, 2024).

Emerging studies suggest that even moderate F^– exposure can delay bone formation by interfering with signaling pathways and matrix protein expression (Pei et al., 2017; Yao et al., 2019). However, data on F^– toxicity during early skeletal development are limited. Most findings rely on rodent models, where their in utero development restricts real-time observation. In this context, zebrafish have gained attention as a promising alternative model. Their small size, high fecundity, transparent embryos, and external fertilization allow for non-invasive live imaging of skeletal development (Laizé et al., 2014). These features make zebrafish ideal for evaluating both pro-osteogenic and osteotoxic effects of environmental toxicants. Our previous study (Ottappilakkil and Perumal, 2025) demonstrated the biphasic effect of F^– exposure on zebrafish skeletal development, where low concentration (25 ppm) promoted osteogenesis and mineral deposition, while higher dose (50 ppm) impaired bone formation, reduced mineralization, and induced skeletal deformities. However, it is well recognized that such F^– induced osteogenic stimulation does not necessarily equate to improved bone health. Instead, the newly formed bone matrix often lacks appropriate structural organization and mechanical strength, rendering it more fragile despite increased mineral content. Thus, even the osteogenic dose of 25 ppm can be regarded as toxic in terms of bone quality (Everett, 2011). These findings confirm the biphasic role of F^– in bone biology and reinforce the utility of the zebrafish model in dissecting dose-dependent skeletal outcomes.

One of the central mechanisms implicated in F^– toxicity is oxidative stress. Studies across in vivo (Shanmugam et al., 2016; Ameeramja and Perumal, 2017; Luo et al., 2017; Ameeramja and Perumal, 2018; Sun et al., 2018), in vitro (Ameeramja et al., 2016; Li et al., 2017), and epidemiological settings (Shivarajashankara et al., 2001; Melila et al., 2019; Tao et al., 2022) have shown that excessive F^– intake disrupts the oxidant-antioxidant balance by increasing the production of reactive oxygen and nitrogen species (ROS and RNS) (Luo et al., 2017; Sun et al., 2018; Lu et al., 2017; Babu et al., 2022; Ottappilakkil et al., 2023a). F^– induced oxidative stress impairs antioxidant systems, such as catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH), while simultaneously promoting lipid peroxidation (LPO) and cellular damage (Cao et al., 2020).

Earlier research has increasingly focused on the use of natural antioxidants to counter F^– toxicity. Phytochemicals with strong free radical-scavenging activity can reduce ROS accumulation, inhibit pro-apoptotic pathways, and help restore redox homeostasis. Compounds like caffeic acid (Kanagaraj et al., 2015), curcumin (Fujiwara et al., 2021; Xu et al., 2024), quercetin (Nageshwar et al., 2018; Oyagbemi et al., 2018; Javanbakht et al., 2025), epigallocatechin gallate (Shanmugam et al., 2016; Xu et al., 2024), and protocatechuic acid derivatives (Ameeramja and Perumal, 2017; Ameeramja and Perumal, 2018) have shown protective effects in F^– exposed models. These phytochemicals offer potential dual benefits by limiting oxidative damage and reducing F^– retention in biological systems.

Fisetin (3,7,3′,4′-tetrahydroxyflavone, Fis) is a naturally occurring flavonol found in strawberries, apples, onions, and cucumbers (Arai et al., 2000; Pal et al., 2016; Sundarraj et al., 2018). Due to its potent antioxidant properties, Fis has been investigated for its therapeutic potential in conditions involving oxidative stress. It modulates key signaling pathways, including those regulating protein and lipid kinases, thereby enhancing cellular resilience against damage. Fis has also demonstrated bone-protective effects by suppressing the receptor activator of nuclear factor kappa B ligand (RANKL) induced osteoclast differentiation through inhibition of nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) signaling (Choi et al., 2012; Sakai et al., 2013). It disrupts actin ring formation and reduces the expression of carbonic anhydrase II and B3 integrin, both crucial for osteoclast maturation and bone resorption (Kim et al., 2014; Aditama et al., 2015).

At the same time, Fis promotes osteoblast differentiation and mineralization by upregulating the runt-related transcription factor 2 (RUNX2) and modulating osteogenic pathways, including PI3K-AKT and GSK-3B-mediated B-catenin signaling (Léotoing et al., 2014; Molagoda et al., 2021; Xu et al., 2021). These findings suggest that Fis may serve as a dual-action modulator of bone remodeling.

Although Fis has been widely studied for its protective effects against various xenobiotic-induced toxicities, its role in mitigating F^– toxicity, especially under mono-exposure conditions and during early bone development, remains largely unexplored. Our preliminary screening revealed that Fis interacts with F^–, indicating a possible binding or chelation mechanism that may further enhance its protective role (Ottappilakkil et al., 2023b).

Thus, in this study, we aimed to investigate the protective role of Fis against the toxic effects of F^– on early skeletal development in the zebrafish larval model. Each Fis and F^– exposures were designed in such a way as to eliminate the confounding effects of co-exposures and focus solely on independently induced outcomes. To simulate a preventive strategy, Fis was administered as a 24 h pretreatment before F^– exposure. A low concentration of F^– (25 ppm) was used for a brief window from 4 to 7 days post fertilization (dpf), aligning with the critical period of skeletal development. This approach allowed us to assess whether Fis could aid in resisting F^– toxicity, offering clearer insight into its potential as a protective agent.