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Abstract

Different studies have suggested that fluoride can induce apoptosis in non-skeletal tissues, however, evidence from these experimental studies is still controversial. This meta-analysis aims to clarify the mechanism of fluoride-induced apoptosis in non-skeletal tissues of experimental animals. Primary studies which measured apoptosis were identified through exhaustive database searching in PubMed, Embase, Web of Science Core Collection, Scopus, and references of included studies. A random effects model with standardized mean difference (SMD) was used for meta-analyses. The heterogeneity of the studies was evaluated using Higgin’s I2 statistics. The risk of bias and publication bias were assessed using the SYRCLE’s risk of bias tool and Egger’s test, respectively. There was an increase in total apoptotic cells, and the expression of Bax, Bax/Bcl-2 ratio, caspase-3, caspase-8, caspase-9, Cyt c, and p53, and a decrease in the expression of Bcl-2 in the fluoride-treated groups as compared to the control groups. However, there was no evidence of a difference in the expression of APAF-1 in the two groups. The subgroup analysis highlighted the role of the intervention period in modification of the apoptotic effect of fluoride and that the susceptibility and tolerance of different animal species and tissues vary. Meta-regression analysis indicated that the studies’ effect size for total apoptotic cells was influenced by animal species and that of Bax by the sample source. The results of this meta-analysis revealed that fluoride causes apoptosis by up-regulating caspase-3, -8, and -9, Cyt c, p53, Bax, and down-regulating Bcl-2 with a concomitant up-regulation of the Bax/Bcl-2 ratio.

Excerpt:

1. Introduction

… Apoptosis is a highly regulated and programmed cell death characterized by specific biochemical and morphological features that culminate in cellular shrinkage to apoptotic bodies that are engulfed by neighboring macrophages []. This occurs without an accompanying inflammatory response. Characterized morphological changes accompanying apoptosis include cleavage of cytoskeletal filament fibers, cell cytoplasm, and nucleus condense, DNA fragmentation by the action of endonucleases, fragmentation of cell organelles (Golgi apparatus, endoplasmic reticulum, and mitochondria), and blistering of the cell plasma membrane. The cells become round and separate from neighboring cells []. Apoptosis plays a key role in the elimination of unnecessary or damaged cells and a variety of normal biological processes such as cell proliferation and differentiation, aging, and tissue homeostasis [,].
A growing body of literature has shown that apoptosis induced by oxidative stress plays a key role in the pathogenesis of fluorosis. Oxidative stress can be triggered by promoting reactive oxygen species (ROS) production and reducing antioxidant function []. Excess cellular levels of ROS cause damage to proteins, nucleic acids, lipids, membranes, and organelles, which can lead to the activation of cell death processes such as apoptosis [

Other molecular mechanisms underlying fluoride-induced apoptosis include disruption of mitochondria outer membrane and release of cytochrome c into the cytosol, which activates caspases-9 and -3 (intrinsic) apoptotic pathway, activation of the cell surface death receptors (extrinsic Fas/FasL-caspase-8 and -3 pathway), alterations in the ratio of anti-apoptotic-apoptotic Bcl-2 proteins, upregulation o f p53 expression, expression of apoptosis-related genes, endoplasmic reticulum stress and disturbances in protein synthesis []. Even though this subject has been thoroughly and extensively evaluated, studies employed different protocols and methods, different test groups and sizes, and different types of experimental animals which, among other factors, yielded conflicting findings. Bai et al. [] for example, reported an increase in the percentage of apoptotic renal cells in chicken whereas Campos-Pereira et al. [] reported no evident increase in positive terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) induced by fluoride in rat hepatocytes indicating the absence of apoptosis. In another study, fluoride exposure did not produce any signs of apoptosis evidenced by the lack of an increase in caspase-3 [

In contrast, several studies reported an increase in caspase activity after exposure to fluoride [20, 21, 22, 23]. It is therefore important to provide a systematic evaluation and meta-analysis of these studies to clarify the mechanism of fluoride-induced apoptosis in experimental animals….

4. Discussion

In this study, we presented the results of the first systematic review and meta-analysis aimed at elucidating whether fluoride causes apoptosis in non-skeletal tissues of experimental animals. The systematic review is based on 47 studies measuring 10 biomarkers of apoptosis identified systematically from four major scientific databases. Results from the meta-analysis reported here have shown that fluoride increased the total apoptotic cells, the level of Bax/Bcl-2 ratio, and the expression of Bax, caspase-3, -8, -9, Cyt c, and p53 and decrease the expression of Bcl-2. There was, however, no evidence of a difference in the expression of APAF-1 between the fluoride and control groups. All biomarkers showed high levels of heterogeneity except for Cyt c which had moderate heterogeneity. There was evidence of publication bias in studies measuring apoptotic cells, Bax, Bcl-2, caspase-3, and Cyt c. The sensitivity analysis for studies measuring total apoptotic cells and the expression of Bax, Bcl-2, Bax/Bcl-2 ratio, caspase-3, and p53 showed that differences in the biomarkers of apoptosis between animals treated with fluoride and the controls were not influenced by any single study, suggesting the robustness of the outcome of the meta-analysis. Our study also showed that the apoptotic effect of fluoride might be dependent on the intervention period. In addition, different animal species have different sensitivity and tolerance to fluoride. Our meta-analysis, therefore, provides a theoretical basis for the molecular mechanism of fluoride-induced toxicity in non-skeletal tissues of experimental animals.
The known molecular mechanisms underlying fluoride-induced apoptosis are different and varied. They include amongst others, disruption of outer mitochondria membrane, activation of caspases, alterations in the ratio of anti-apoptotic-apoptotic Bcl-2 proteins, upregulation of p53 expression, expression of apoptosis-related genes, and disturbances in protein synthesis [10,16]. Caspases are divided into initiator (?2, ?8, ?9, ?10) and effector (?3, ?6, ?7) caspases and function by a cascade, depending on whether the lethal stimuli are generated at the cell membrane, activating the extrinsic pathway (death receptor-mediated pathway) or intracellularly, activating the intrinsic pathway (mitochondria-mediated pathway) [12,16]. Most of the morphological changes associated with apoptosis can be attributed to the cleavage of initiator caspases at specific target sites which activate a set of effector caspases [ The disruption of the outer mitochondrial membrane by apoptotic stimuli initiates the mitochondrial pathway with the subsequent release of Cyt c from mitochondria to the cytosol. The release of Cyt c triggers apoptosome assembly from APAF-1, dATP (deoxyadenosine triphosphate), and procaspase-9, which in turn activates caspase-3 and -7, leading to oligonucleosomal DNA fragmentation [16,19]. The extrinsic pathway originates from the plasma membrane through the association of transmembrane death receptors TNF-? or Fas and their ligands inducing oligomerization of the receptor. This leads to receptor aggregation that binds adaptor molecules such as Fas-associated death domain (FADD) which then bind to initiator caspases prodomain e. g caspase-8 or -10 to promote their activation with subsequent stimulation of caspase-3. Caspase-3 cleaves one of its substrates, poly(ADP-ribose) polymerase (PARP), resulting in the degradation of nuclear DNA. Thus, intrinsic and extrinsic pathways converge at the activation of caspase-3 [16,68]. The ability of fluoride to trigger an intrinsic pathway has been demonstrated both in vitro and in vivo in many cell types. Song et al. [] demonstrated increased caspase-3 and -9 protein and mRNA relative expression in the liver of rats chronically exposed to fluoride. In other studies, an increase in cytosolic Cyt c [] and activation of caspase-3 was reported in HL-60 (human leukemia cell line) cells [. …
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