Inhibitory effects of NaF on mitochondrial energy generation in human platelets in vitro.

Abstract

Background: fluoride is a beneficial ion that has been used in various fields, from industrial products to therapeutics. However, due to its narrow therapeutic index, fluoride sometimes acts as a toxic agent at relatively higher concentrations in the human body. Based on the interest in genetic stability, its cytotoxic effects have been investigated mainly in nucleated, adherent cells, such as fibroblasts. However, the sensitivity of blood cells, especially anucleate platelets, to fluoride is poorly understood. To fill this gap in the literature, we investigated the effects of relatively low levels of fluoride on platelet energy metabolism, function, and viability.

Methods: Platelet-rich plasma (PRP) was prepared from 15 non-smoking healthy male adults (age: 28–63) and treated with NaF (0.5 or 1.0 mM) in microtubes for up to 3 days. Platelet function was evaluated based on aggregation and adhesion activities. Platelet energy metabolism was evaluated based on intracellular ATP levels, extracellular lactate levels, and respiration activities. The mitochondrial membrane potential (Em) and localization of reactive oxygen species (ROS) were visualized using cytochemical methods. Platelet viability was evaluated by cell counting and tetrazolium reduction.

Result: NaF (1 mM) significantly reduced platelet viability and inhibited functions. Behind these phenomena, NaF substantially decreased mitochondrial Em and increased ROS production along with significant decreases in oxygen consumption and ATP levels. Simultaneously, NaF increased the lactate levels. Although not statistically significant, similar effects were observed at 0.5 mM NaF.

Conclusion: At relatively low levels, NaF has the potential to attenuate platelet function probably primarily through the inhibition of mitochondrial energy generation. Cytotoxicity may be directly related to ROS production. These findings suggest that when used topically, for example, for caries prevention in the oral cavity, NaF could interfere with wound healing and tissue regeneration by endogenous and exogenously added platelets in the form of PRP.

Full-text article online at https://www.frontiersin.org/journals/toxicology/articles/10.3389/ftox.2024.1421184/full

Excerpt:

4 Discussion

The primary question in this study was whether non-mitogenic platelets are as sensitive to reduced viability as adherent mitogenic cells to low fluoride levels. This study demonstrated that approximately 1 mM is the border of fluoride-induced cytotoxicity in human platelets, essentially as shown in many other nucleated cell types regardless of their karyotypes (Helgeland and Leirskar, 1976; Hirano and Ando, 1997; Jeng et al., 1998; He et al., 2015). In nucleated cells that have the mitogenic capacity for cell proliferation, it has been ambiguously, but generally, thought that the cytotoxic action is due to the balance of the mitogenic potential and toxic effects. The present platelet’s similarity in cytotoxic fluoride levels suggests that fluoride cytotoxic action could always be expressed universally regardless of cell mitogenic potential.

The second question was, which is the major target of fluoride involved in energy depletion and dysfunction between glycolysis and mitochondrial OXPHOS? This study also demonstrated that the fluoride-induced functional inhibition occurred simultaneously with the inhibition of mitochondrial OXPHOS as monitored by mitochondrial Em, respiration activity, and ATP level. Fluoride is well known to inhibit glycolysis (Cimasoni, 1971; ten Cate and van Loveren, 1999; Qin et al., 2006; Dimeski et al., 2015) and is thus used for laboratory testing of blood samples. Considering the general concept regarding platelet energy metabolism that the glycolysis system functions as the main ATP generator regardless of their resting or activation states (ten Cate and van Loveren, 1999; Dimeski et al., 2015; DeFronzo et al., 2016; Ryoo and Kim, 2018), it was hypothesized that fluoride primarily inhibits glycolysis to decrease extracellular lactate levels. Against this expectation, fluoride significantly increased the extra-platelet lactate levels. From a biochemical point of view, the “unexpected” changes could be explained by the concept that pyruvate is produced as usual but not efficiently utilized in the following TCA cycle and consequently accumulated and released in the form of lactate (DeFronzo et al., 2016; Ryoo and Kim, 2018). In line with this scenario, the primary target of fluoride in platelets can be considered the mitochondrial energy generation system rather than the glycolysis system (Gambino, 2013). The schematic diagram showing this mechanism is shown in Figure 6A. Judging from the dissimilarity of concentration-effectiveness relationships between viability and other indexes, it seems more likely that the reduced viability may be directly caused by increased mitochondrial ROS production rather than energy depletion because ROS induces not only mitophagy but also nonspecific damage to cell phospholipid bilayers and the plasma membrane (Cordeiro, 2014). Further investigations are required to clarify this mechanism: however, our findings regarding the positive correlations between reduced energy metabolism and dysfunction would provide an opportunity to again investigate the balance between glycolysis and OXPHOS in platelets.

Figure 6. (A) Schematic diagram showing the proposed mechanisms of fluoride action on platelet viability, energy metabolism, and functions. Fluoride passes across the plasma membrane and primarily acts on mitochondria, resulting in decreased energy generation and increased ROS production. The former and the latter influence platelet functions and viability, respectively. (B) Representative clinical case of platelet exposure to high fluoride levels in the oral cavity. When wounds, such as sockets, are treated with platelet concentrates, varnishing of adjacent teeth with high fluoride levels has a high risk of impeding wound healing.

The technical limitations for the determination of mitochondrial Em should be briefly discussed here. There are several fluorescent dyes, such as JC-1, tetramethylrhodamine ethyl ester (TMRE), and tetramethylrhodamine methyl ester (TMRM), to determine mitochondrial Em (Laboratories, 2024). Each dye has both advantages and disadvantages. Resistance against photobleaching is the primary critical issue for these dyes, while the secondary issue is the unavailability of fixed cells. In clinical studies using limited volumes of precious human samples, it is important to use them efficiently by performing several independent experiments in parallel, without significant loss. In line with this policy, we chose MT-1, a fluorescent dye that can be applied to fixed cells and observed after fixation (Takeda and Dai, 2022; Ushiki et al., 2022).

Concerning the limitation of this study, we should carefully interpret the in-vitro cell-based data. The oral cavity is always covered by biofilm that is chronically formed on its various surfaces due to the presence of rich nutrients and other favorable environments (Ray and Pattnaik, 2024) and protects against invasion of fluoride as well as mucous barrier (Bierbaumer et al., 2018). However, the biofilm is frequently washed out and reformed by saliva, drinking, and eating, whereas it could be drastically changed by periodontal and peri-implant diseases and dental caries. Thus, the situation regarding the exposure of platelets to clinically applied fluoride could vary with individual oral health conditions. It should be noted that this study examined the effects of fluoride on platelets by simply focusing on the basic contact between platelets and fluoride ions.

Compared with other tissues or organs, the oral cavity has a higher probability of exposure to high levels of fluoride. In the case of mouth rinse, NaF levels in the solution usually range from 0.05% to 0.2%, which is converted to 11.9–47.6 mM (Shimizu et al., 2022). For varnish, higher levels of NaF (2.7–5.0%; 643.0–1190.8 mM) are conventionally applied to prevent dental caries under professional control (Baik et al., 2021) (Figure 6B). These concentrations were extraordinarily higher than those used in this study. Even though the high levels of fluoride coated on the tooth surface could gradually be washed out and diluted by food, drink, saliva, and mouth-washing, biologically significant fluoride levels are expected to remain for a while and influence surrounding tissues. Thus, in the presence of wounds, cysts, or inflamed pockets in the oral cavity, it is plausible that NaF mouth rinsing or coating damages infiltrating platelets, delaying hemostasis and subsequent wound healing or exacerbating inflammation. Similar possibilities are imaginable in the case of overlapping PRP therapy. Platelets externally applied in the form of PRP could also be disrupted by the topical application of fluoride (Figure 6B); therefore, PRP may lose its efficacy in wound healing and tissue regeneration.

In the last 3 decades, PRP has been widely used for tissue regeneration in various fields including dentistry (Kawase, 2015). PRP is often used for socket preservation and alveolar bone augmentation of the oral cavity. Thus, when the fluoride coating remains on the surface of adjacent teeth, PRP’s regenerative potential of PRP could be impeded because it is not solely due to its concentrated growth factors, but also due to platelet activity (Kawase, 2022). It has been scientifically proven that fluoride, whose concentrations vary with the method of application, is beneficial for maintaining oral health through the remineralization of enamel, inhibition of bacterial growth, prevention of tooth decay, and decrease in tooth sensitivity (Pitts et al., 2017). In addition, fluoride treatment is undoubtedly a highly cost-effective therapeutic option. Therefore, this study did not indicate the overall withdrawal of fluoride from the oral cavity. Instead, clinicians should pay more attention to such topical conditions and carefully use fluoride when wounds exist nearby or when PRP is applied nearby.