Mechanisms Underlying Endothelin-1 Level Elevations Caused by Excessive Fluoride Exposure
Cellular Physiology and Biochemistry
Objective: To explore the mechanisms underlying endothelin-1 (ET-1) elevations induced by excessive fluoride exposure. Methods: We measured serum and bone fluoride ion content and plasma ET-1 levels and compared these parameters among different groups in an animal model. We also observed morphological changes in the aorta and endothelium of rabbits. In cell experiments, human umbilical vein endothelial cells (HUVECs) were treated with varying concentrations of NaF for 24h, with or without 10 µM
... U0126 pretreatment for 1 h. ET-1 levels in culture fluid and intracellular reactive oxygen species (ROS) levels, as well as ET1 gene, endothelin-converting enzyme-1 (ECE-1), extracellular signal-regulating kinase 1/2 (ERK1/2), pERK1/2 expression levels and RAS activation were measured and compared among the groups. Results: Plasma ET-1 levels of rabbits increased significantly in fluorinated groups compared with those in the control group. The rabbit thoracic aortas became slightly hardened in fluorinated groups compared with those in the control group, and some vacuoles were present in the endothelial cell cytoplasm of the rabbits in fluorinated groups. In our cell experiments, ET1 gene and ECE-1 expression levels in HUVECs and ET-1 expression levels in the cell culture supernatants increased significantly in some experimental groups compared with those in the control group. These trends paralleled the changes in intracellular ROS levels, RAS activation, and the pERK1/2-to-ERK1/2 ratio. After U0126 was added, ECE-1 expression and ET-1 levels decreased significantly. Conclusion: Excessive fluoride exposure leads to characteristic endothelial damage (vacuoles), thoracic aorta hardening, and plasma ET-1 level elevations in rabbits. In addition, the ROS-RAS-MEK1/2-pERK1/2/ERK1/2 pathway plays a crucial-and at least partial-role in ET-1 over-expression, which is promoted by excessive fluoride exposure. L. Sun and Y. Gao contributed equally to this work. Introduction Endemic fluorosis is a worldwide disease that seriously harms the health of residents in some endemic areas of China, India, and Africa. The disease manifests as dental and skeletal fluorosis, which affect young children and adults, respectively. In addition to causing skeletal and dental damage, excessive exposure to fluoride can exert other pathophysiologic effects. Specifically, excessive exposure to fluoride can cause metabolic, structural, and functional damage to the nervous system , kidneys, liver [2, 3], and cardiovascular system    and can reduce the intellectual capacity of children [7, 8] . In the past, the non-bone-related damage caused by fluorosis has often been overlooked because the onset of symptoms resulting from this damage is slow, and the injuries inflicted by this damage are nonspecific. In recent years, public concern regarding the damage to the cardiovascular system caused by excessive fluoride exposure has increased, particularly concern regarding the atherosclerosis and hypertension caused by excessive fluoride exposure. Our team found that excessive fluoride intake from drinking water is not only closely associated with essential hypertension  but also is associated with atherosclerosis . We also found that high fluoride exposure increases plasma endothelin-1 (ET-1) levels in subjects living in endemic fluoride areas  . As the most potent vasoconstrictive agent , ET-1 enhances vasoconstriction and exacerbates the processes of hypertension and atherosclerosis by aggravating cell hyperplasia and vascular smooth muscle cell migration      . ET-1 was originally isolated from porcine aortic endothelial cells and is predominantly secreted by endothelial cells. Mature ET-1 production is mainly controlled by endothelin-converting enzyme 1 (ECE-1)  . The results of ECE gene knockout studies suggest that ECE-1 is the major functional ECE in vivo, although additional ECE isoforms have been identified in animals and humans  . Therefore, ECE-1 is the limiting enzyme in ET-1 synthesis  . Some factors known to promote ET-1 production include insulin, angiotensin II, inflammatory mediators, oxidative stress and vascular shear stress  . Similarly, a signaling pathway also appears to be involved in ET-1 synthesis. Previous studies have reported that the extracellular signal-regulating kinase 1/2 (ERK1/2) signaling pathway plays a key role in ET-1 synthesis and secretion. ERK1/2 phosphorylation activates multiple transcription factors to not only enhance ET-1 production but also promote smooth muscle contraction, which is mediated by 20] . We inferred from our previous results that ET-1 plays a vital role in the development of hypertension and atherosclerosis caused by excessive fluoride exposure. However, the specific mechanism by which excessive fluoride exposure increases plasma ET-1 levels remains unclear. Therefore, the aim of this study was to explore the exact mechanism by which excessive fluoride exposure enhances plasma ET-1 levels. Materials and Methods Animals and treatment Twenty-four healthy, clean and male Japanese big-eared rabbits (2400-2600g) were selected and housed in an air-conditioned room with 45%-65% humidity and a temperature of 20±2°C. After a week of adaptive feeding, the rabbits were randomly assigned to three groups of eight animals each, according to their initial weights. Based on an earlier results of our team, the highly fluoridated water contained 100 mg fluoride ion/L, and the slightly fluoridated water contained 50 mg fluoride ion/L. Different groups of rabbits were given basic feed and drank tap water or fluoridated water (50 or 100 mg fluoride ion/L; the water was fluoridated with sodium fluoride). The experimental design of the study is presented in Table 1 . The feed and assigned drinking water were available at all times throughout the experiment. The rabbits were exposed to fluoride for 5 months and then sacrificed under chloral hydrate anesthesia. All the experiments were approved by the Animal Ethics Committee of Harbin Medical University. Measurement of intracellular reactive oxygen species (ROS) levels HUVECs were treated with different concentrations of fluoride ion for 24h. The cells were then collected and resuspended in dilute 2'7'-dichlorofluorescein diacetate solution at a density 1-20×10 6 cells/mL. The mixture was incubated at 37 °C for 20 min and then washed three times with serum-free culture medium. The cells were subsequently suspended in 0.5 mL of serum-free culture medium, and their fluorescence intensity was measured with a flow cytometer (Becton, Dickinson and Company, New York, USA). Statistical analysis All data were recorded with EpiData 3.02 and analyzed with SPSS 17.0 software (International Business Machines Corporation, Armonk, New York, USA). The results are presented as means ± SEM. Comparisons of ECE-1 expression and ET-1 (1.2 μg/mL) levels with and without U0126 pretreatment were made with Student's t test. All other indices were compared among all the experimental groups with one-way ANOVA. Dunnett's T3 and the least significant difference tests were used for all two-group comparisons. All tests were two-tailed, and P<0.05 was considered statistically significant. levels were noted among all groups (F=77.06, P<0.001). ET-1 levels in the high-dose group were significantly higher than those in all other groups (P<0.001), and ET-1 levels in the moderate-dose group were significantly higher than those in control and low-dose groups (P moderate vs control < 0.001, P moderate vs low = 0.001). However, there was no significant difference in ET-1 levels between low-dose and control groups (P =0.535). ECE-1 expression levels of HUVECs increased significantly in three dose groups ECE-1 expression levels in HUVECs of three dose groups (n=3) also increased gradually as fluoride ion concentrations increased, and significant differences in ECE-1 expression Fig. 4 . Intracellular ROS levels, RAS activation levels, and pERK1/2-to-ERK1/2 ratios in HUVECs of different groups and ECE-1 expression levels in HUVECs and ET-1 levels in cell culture supernatants after treatment with 1.2 μg/mL fluoride ion with or without U0126 pretreatment. Parts A, B, C, D, and E show the positive control, control, low-, moderate-and high-dose groups, respectively. Part F shows a comparison of the intracellular ROS levels in different groups. Part G shows the RAS activation levels in different groups. Parts H and I together show the expression levels of pERK1/2 and ERK1/2 and the ratios of pERK1/2 to ERK1/2 in HUVECs of different groups. The ratios of pERK1/2 to ERK1/2 were calculated from the expression levels of these proteins. *P < 0.01 compared with the control group. Parts J and K together show expression levels of ECE-1 in HUVECs of two different groups. The ratios of ECE-1/GAPDH were calculated based on the expression levels of these proteins. Part L shows the levels of ET-1 in cell supernatants of two different groups. *P < 0.01 compared with the 1.2 μg/mL group. with that in the control group. Pretreatment with U0126 clearly abolished ET-1 and ECE-1 over-expression in fluoride-treated HUVECs, implying that MEK1/2 activation is required in this process. All these results suggested that the RAS-MEK1/2-ERK1/2 signaling pathway plays a key role in fluoride-induced ET-1 over-expression in HUVECs.