Co-exposure to arsenic and fluoride to explore the interactive effect on oxidative stress and autophagy in myocardial tissue and cell.

Co-contamination of arsenic and fluoride is widely distributed in groundwater. However, little is known about the interactively influence of arsenic and fluoride, especially the combined mechanism in cardiotoxicity. Cellular and animal models exposure to arsenic and fluoride were established to assess the oxidative stress and autophagy mechanism of cardiotoxic damage using the factorial design, a widely used statistical method for assessing two factor interventions. In vivo, combined exposure to high arsenic (50 mg/L) and high fluoride (100 mg/L) induced myocardial injury. The damage is accompanied by accumulation of myocardial enzyme, mitochondrial disorder, and excessive oxidative stress. Further experiment identified that arsenic and fluoride induced the accumulation of autophagosome and increased expression level of autophagy related genes during the cardiotoxicity process. These findings were further demonstrated through the in vitro model of arsenic and fluoride-treated the H9c2 cells. Additionally, combined of arsenic-fluoride exposure possesses the interactively influence on oxidative stress and autophagy, contributing to the myocardial cell toxicity. In conclusion, our data suggest that oxidative stress and autophagy are involved in the process of cardiotoxic injury, and that these indicators showed interaction effect in response to the combined exposure of arsenic and fluoride.

Arsenic-fluoride co-exposure induced endoplasmic reticulum stress resulting in apoptosis in rat heart and H9c2 cells.

Exposure to arsenic (As) or fluoride (F) has been shown to cause cardiovascular disease (CVDs). However, evidence about the effects of co-exposure to As and F on myocardium and their mechanisms remain scarce. Our aim was to fill the gap by establishing rat and H9c2 cell exposure models. We determined the effects of As and/or F exposure on the survival rate, apoptosis rate, morphology and ultrastructure of H9c2 cells; in addition, we tested the related genes and proteins of endoplasmic reticulum stress (ERS) and apoptosis in H9c2 cells and rat heart tissues. The results showed that As and/or F exposure induced early apoptosis of H9c2 cells and caused endoplasmic reticulum expansion. Additionally, the mRNA and protein expression levels of GRP78, PERK and CHOP in H9c2 cells were higher in the exposure groups than in the control group, and could be inhibited by 4-PBA. Furthermore, we found that As and/or F exposure increased the expression level of GRP78 in rat heart tissues, but interestingly, the expression level of CHOP protein was increased in the F and As groups, while significantly decreased in the co-exposure group. Overall, our results suggested that ERS-induced apoptosis was involved in the damage of myocardium by As and/or F exposure. In addition, factorial analysis results showed that As and F mainly play antagonistic roles in inducing myocardial injury, initiating ERS and apoptosis after exposure.

Proteomics and transcriptomics jointly identify the key role of oxidative phosphorylation in fluoride-induced myocardial mitochondrial dysfunction in rats.

The regulation of mitochondrial function, which is dominated by oxidative phosphorylation (OXPHOs), is important in fluoride induced cardiovascular disease. Based on the previous study of fluoride-induced mitochondrial structure and membrane potential abnormalities, this study integrated ITRAQ protein quantification and RNA-Seq methods to analyze the sequencing data of rat myocardial tissue under fluoride exposure (0, 30, 60 and 90 mg/L). A total of 22 differentially expressed genes associated with the OXPHOs pathway were screened by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) co-enrichment analysis, and were localizated by Interaction Network and calculated inter-genes and inter-omics correlations by Pearson correlation. In general, fluoride exposure can down-regulate genes related OXPHOs, particularly affecting the assembly of the complex I including Ndufa10, resulting in abnormal mitochondrial ATP synthesis and reduced myocardial energy supply. Most importantly, this study shows that the enriched information from the proteomics can explain the change process of energy production, but the specific molecules involved in energy supply cannot be obtained via transcriptomics information alone. Based on the results of transcriptional and protein analysis, our findings contribute to an innovative understanding of the pathways and molecular changes of myocardial injury induced by fluorosis.

Co-exposure to inorganic arsenic and fluoride prominently disrupts gut microbiota equilibrium and induces adverse cardiovascular effects in offspring rats.

Co-exposure to inorganic arsenic (iAs) and fluoride (F-) and their collective actions on cardiovascular systems have been recognized as a global public health concern. Emerging studies suggest an association between the perturbation of gut bacterial microbiota and adverse cardiovascular effects (CVEs), both of which are the consequence of iAs and F- exposure in human and experimental animals. The aim of this study was to fill the gap of understanding the relationship among co-exposure to iAs and F-, gut microbiota perturbation, and adverse CVEs. We systematically assessed cardiac morphology and functions (blood pressure, echocardiogram, and electrocardiogram), and generated gut microbiota profiles using 16S rRNA gene sequencing on rats exposed to iAs (50 mg/L NaAsO2), F- (100 mg/L NaF) or combined iAs and F- (50 mg/L NaAsO2 + 100 mg/L NaF), in utero and during early postnatal periods (postnatal day 90). Correlation analysis was then performed to examine relationship between significantly altered microbiota and cardiac performance indices. Our results showed that co-exposure to iAs and F- resulted in more prominent effects in CVEs and perturbation of gut microbiota profiles, compared to iAs or F- treatment alone. Furthermore, nine bacterial genera (Adlercreutzia, Clostridium sensu stricto 1, Coprococcus 3, Romboutsia, [Bacteroides] Pectinophilus group, Lachnospiraceae NC2004 group, Desulfovibrio, and two unidentified genera in Muribaculaceae and Ruminococcaceae family), which differed significantly in relative abundance between control and iAs and F- co-exposure group, were strongly correlated with the higher risk of CVEs (correlation coefficient = 0.70-0.88, p < 0.05). Collectively, these results suggest that co-exposure to iAs and F- poses a higher risk of CVEs, and the part of the mode of action is potentially through inducing gut microbiota disruption, and the strong correlations between them indicate a high potential for the development of novel microbiome-based biomarkers of iAs and/or F- associated CVEs.

Fluoride Exposure and Blood Pressure: a Systematic Review and Meta-Analysis.

Fluoride exposure may cause changes in blood pressure, but this conclusion is controversial. Therefore, this meta-analysis aims to investigate the potential relationship between fluoride exposure and blood pressure or hypertension. PubMed, Web of Science, China National Knowledge Infrastructure (CNKI), WANFANG MED ONLINE, and Chinese Scientific Journals Full-Text Databases (VIP) were searched; in addition, two related studies were added manually. In total, 7 observational studies were identified, the pooled odds ratios (ORs) for hypertension between high and reference fluoride exposure groups were calculated, and the pooled standardized weighted mean difference (SMD) of systolic blood pressure (SBP) and diastolic blood pressure (DBP) was estimated using an inverse-variance weighted random-effects model; next, sensitivity analysis and subgroup analysis were used to assess potential sources of heterogeneity; furthermore, publication bias was assessed using the Begg and Egger test. In brief, there were no statistical differences between exposure groups and control groups in terms of blood pressure or hypertension when all included studies considered. However, subgroup analysis indicated that blood pressure will rise with the increase of fluoride exposure concentrations in endemic fluorosis areas. The corresponding pooled SMD estimates were 0.31 (95% CI 0.11, 0.51) and 0.27 (95% CI 0.11, 0.43) for SBP and DBP. Funnel plots suggested no asymmetry. Our findings support the possibility of a positive correlation between fluoride exposure and blood pressure in endemic fluorosis areas. Additional evidence is needed to assess the dose-response relationship between fluoride exposure and blood pressure.

ITRAQ-based proteomics reveals the potential mechanism of fluoride-induced myocardial contraction function damage.

Fluorosis is a worldwide public health problem, and its adverse effects on the heart have been confirmed by many studies. Abnormal myocardial contractions are often associated with impairment of cardiac function as a cause or consequence. We designed two-part experiments to search for biomarkers and clarify the underlying molecular mechanism of fluoride on myocardial contraction. First, we used Pressure-volume Loop analysis to evaluate changes in myocardial function indexes with multiple fluoride exposure levels in mice (0, 30, 70, and 150 mg/L) exposed for 4 weeks. The results showed that fluoride exposure affects the heart pump function and reduces cardiac contractility. Then, we established a rat model of fluoride exposure (0, 30, 60, and 90 mg/L) for 6 months to carry out proteomic analysis of fluoride-induced myocardial contractile injury. Hematoxylin-eosin (H&E) staining was used to determine the severity of myocardial injury, and myocardial tissue samples were submitted for isobaric tags for relative and absolute quantitation (ITRAQ) analysis. A total of 1607 proteins were successfully identified with 294 differentially expressed proteins (DEPs) in fluoride treated groups. According to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, 12 DEPs were confirmed to be involved in pathways related to myocardial contraction. Furthermore, we constructed a protein-protein interaction (PPI) network for these 12 core DEPs to illustrate the role and location of each DEP in the myocardial contraction pathway. The results of this study are helpful for identify a potential mechanism and biomarkers of fluoride-induced myocardial contraction function damage, moreover, which can provide a new insight into the heart toxicity of fluoride in animals at the proteomics level.

Deregulation of autophagy is involved in nephrotoxicity of arsenite and fluoride exposure during gestation to puberty in rat offspring.

Exposure to fluoride (F) or arsenite (As) through contaminated drinking water has been associated with chronic nephrotoxicity in humans. Autophagy is a regulated mechanism ubiquitous for the body in a toxic environment with F and As, but the underlying mechanisms of autophagy in the single or combined nephrotoxicity of F and As are unclear. In the present study, we established a rat model of prenatal and postnatal exposure to F and As with the aim of investigating the mechanism underlying nephrotoxicity of these pollutants in offspring. Rats were randomly divided into four groups that received NaF (100 mg/L), NaAsO2 (50 mg/L), or NaF (100 mg/L) with NaAsO2 (50 mg/L) in drinking water or clean water during pregnancy and lactation; after weaning, pups were exposed to the same treatment as their mothers until puberty. The results revealed that F and As exposure (alone or combined) led to significant increases of arsenic and fluoride levels in blood and bone, respectively. In this context, F and/or As disrupted histopathology and ultrastructure in the kidney, and also altered creatinine (CRE), urea nitrogen (BUN) and uric acid (UA) levels. Intriguingly, F and/or As uptake induced the formation of autophagosomes in kidney tissue and resulted in the upregulation of genes encoding autophagy-related proteins. Collectively, these results suggest that nephrotoxicity of F and As for offspring exposed to the pollutants from in utero to puberty is associated with deregulation of autophagy and there is an antagonism between F and As in the toxicity autophagy process.

Co-exposure to Arsenic-Fluoride Results in Endoplasmic Reticulum Stress-Induced Apoptosis Through the PERK Signaling Pathway in the Liver of Offspring Rats.

Arsenic and fluoride are two of the major groundwater pollutants. To better understand the liver damage induced during development, 24 male rats exposed to fluoride (F), arsenic (As), and their combination (As + F) from the prenatal stage to 90 days after birth were selected for analysis. Histopathological results showed vacuolar degeneration in the As and As + F groups. Compared to those in the control group, aspartate aminotransferase and alanine aminotransferase levels were significantly increased in the combined group. Catalase activity significantly decreased in the treatment groups compared to that in the controls, and the malondialdehyde content in the As and As + F groups was significantly higher than those in the control group. We further evaluated whether this damage is linked to endoplasmic reticulum stress and its related pathways. The mRNA expression levels of PERK, GRP78, EIF2α, ATF4, and CHOP as well as the protein levels of CHOP was significantly increased in the As + F group compared with the control group. These results demonstrate that As, F, and their combination could lead to liver function damage and reduce the antioxidant capacity of the liver to cause oxidative damage to tissues. Moreover, the combination of As and F triggers endoplasmic reticulum stress-induced apoptosis in liver cells by activating the PERK pathway in the unfolded protein response. As and F seem to have different independent effects, whereas their combination resulted in more severe effects overall.

Subchronic exposure to arsenite and fluoride from gestation to puberty induces oxidative stress and disrupts ultrastructure in the kidneys of rat offspring.

Underground drinking water is commonly contaminated with arsenite (As) and fluoride (F) associated with chronic kidney diseases in humans; however, the combined renal toxicity of these pollutants and the underlying mechanisms are still unclear. The aim of the present study was to investigate the interaction between As and F regarding toxic effects on the kidney of rat offspring exposed to pollutants during prenatal and postnatal development. Pregnant rats were randomly divided into four groups that received NaAsO2 (50 mg/L), NaF (100 mg/L), NaAsO2 (50 mg/L) and NaF (100 mg/L) in drinking water, or clean water, respectively, during gestation and lactation. After weaning, six male pups were randomly selected from each group and continued on the same treatment as their mothers for up to three months. The results revealed that subchronic exposure to high-dose As and/or F decreased the organ coefficient of the kidneys and disrupted kidney ultrastructure, moreover inhibited the activity of antioxidant enzymes and increased the generation of malondialdehyde in the kidney. As exposure alone or combined with F led to an upregulation of nuclear factor erythroid 2-related factor-2 (Nrf2) and its regulatory targets (Ho-1, Gclc, and Nqo1), whereas the effect of F alone was not significant. These results suggest that the renal toxicity of As and F is associated with the induction of mitochondrial damage and oxidative stress, and alters the expression of Nrf2 and its regulatory targets. Furthermore, variance analysis results showed that an interaction between As and F in the toxicity process.

Comparative Transcriptomics Reveals the Role of the Toll-Like Receptor Signaling Pathway in Fluoride-Induced Cardiotoxicity.

Many studies have shown that fluorosis due to long-term fluoride intake has damaging effects on the heart. However, the mechanisms underlying cardiac fluorosis have not been illuminated in detail. We performed high-throughput transcriptome sequencing (RNA-Seq) on rat cardiac tissue to explore the molecular effects of NaF exposure. In total, 372 and 254 differentially expressed genes (DEGs) were identified between a group given 30 mg/L NaF and control and between a group given 90 mg/L NaF and control, respectively. The transcript levels of most of these genes were significantly down-regulated and many were distributed in the Toll-like receptor signaling pathway. Transcriptome analysis revealed that herpes simplex infection, ECM-receptor interaction, influenza A, cytokine-cytokine receptor interaction, apoptosis, and Toll-like receptor signaling pathway were significantly affected. IL-6 and IL-10 may play a crucial role in the cardiac damage caused by NaF as external stimuli according to protein-protein interaction (PPI) network analysis. The results of qRT-PCR and Western blotting showed a marked decreased mRNA and protein levels of IL-1, IL-6, and IL-10 in the low concentration fluoride (LF) and high concentration fluoride (HF) groups, which was in agreement with RNA-Seq results. This is the first study to investigate NaF-induced cardiotoxicity at a transcriptome level.