Impaired neurogenesis induced by fluoride via the Notch1 signaling and effects of carvacrol intervention.

Abstract

Highlights

IQ was associated with fluoride exposure and circulating Notch1 levels in boys.
Neurological functions were impaired in NaF-treated rats.
Neurogenesis was inhibited in NaF-treated rats with neurological deficits.
NaF-induced Notch1 signaling was responsible for the inhibition of neurogenesis.
CAR significantly alleviated NaF neurotoxicity via Notch1 signaling.

The negative regulation on neurogenesis has been implicated in fluoride neurotoxicity, while the evidence is limited. To explore whether fluoride interferes with neurogenesis via the Notch1 signaling and the potential alleviation effects of carvacrol (CAR), we conducted in vivo and in vitro experiments, as well as epidemiological analyses in this study. The results showed that urinary fluoride levels and circulating Notch1 levels were associated with IQ levels in boys. NaF-treated rats had fewer neurons, lower densities of dendritic spines, depressed neurogenesis, and impaired learning and memory abilities. In vitro experiments using undifferentiated PC12 cells mimicking neurogenesis revealed that NaF suppressed differentiation and neurite outgrowth. Besides, Notch1 signaling activation was detected in vivo and in vitro. The latter was confirmed using an in vitro model supplemented with DAPT, a potent Notch1 inhibitor. Furthermore, CAR supplementation negatively regulated NICD1 and Hes1 expressions and promoted hippocampal neurogenesis, thereby improving neurological functions in NaF-treated rats. These findings indicated that the inhibition of neurogenesis in hippocampi induced by fluoride via Notch1 signaling activation may be one of the underlying mechanisms of its neurotoxicity, and that CAR significantly alleviated the neurotoxicity of NaF via the Notch1 signaling.

Graphical abstract

Excerpts:

2.2. Epidemiological study

In 2017, a cross-sectional study was conducted in a primary school located in the fluorosis area of Tongxu County, Henan Province. Children were included after excluding those who were non-local residents or had other fluoride exposure history, had recently take medications, had a history of brain injury or neurological disorders, or whose mothers had a history of adverse exposures (such as smoking or alcohol consumption) during pregnancy. Furthermore, to be consistent with the male SD rats in the animal model in terms of sex, a simple random sampling method was used to randomly select the required number of boys (n = 198) from the eligible boys. The sample size calculation and randomization design are shown in the supplementary files. The general information was collected by the face-to-face questionnaire survey. Physical examinations were performed for height and weight values, used for calculating body mass index (BMI). Samples of morning urine were collected. Urinary creatinine concentrations and urinary fluoride (UF) levels were evaluated by the picric acid method and the fluoride ion selective electrode method, respectively. Since multipotent progenitor/stem cells are present in human peripheral blood, and they can differentiate into neurons, glial cells, and other kinds of cells (Cesselli et al., 2009). Fasting peripheral venous blood samples were also collected, and the nucleated cell population was isolated. The BCA method was used to evaluate protein concentrations. The Notch1 levels in the peripheral blood of boys and rats, and in the hippocampi of rats were all estimated by the ELISA method. Then relative levels of Notch1 were calculated. Boys’ IQ levels were assessed by the Combined Raven’s Test. Informed consents were signed by all participants and their guardians, and the study design was approved by the Ethics Committee of Zhengzhou University (ZZUIRB, 2017-018).

… Results

3.1. Association among circulating Notch1 levels, fluoride exposure and IQ levels in boys

After dividing the boys into two groups based on the national standard (WS/T 256–2005) (Table S1), the levels of UF, urinary creatinine, and Notch1 were higher in boys with UF > 1.4 mg/L than those with lower UF concentrations (P < 0.05 for all). However, boys with higher UF levels got lower IQ score (P = 0.022). Additionally, the circulating Notch1 level was associated with the Notch1 levels in the hippocampi of rats (r = 0.865, P < 0.001) (Fig. 1 A), indicating that the circulating Notch1 level was a good surrogate marker for the Notch1 levels in the hippocampi. Among 198 boys aged 7–12 years, the level of UF was negatively associated with the IQ level of boys (B = -3.727, P = 0.003) after adjusting for potential confounders (Fig. 1 B). A positive association between the level of UF and Notch1 was observed, which was marginally significant (B = 8.729, 0.05<P < 0.1). Besides, a negative association between the level of Notch1 and IQ was observed, which was also marginally significant (B = -0.038, 0.05<P < 0.1) (Fig. 1 D). Mediation analysis showed that the circulating Notch1 level did not mediate the association between the level of UF and IQ (Table S2).

4. Discussion

… In the present study, fluoride exposure was negatively associated with boys’ IQ, and the spatial learning and memory abilities were significantly decreased in rats of each NaF group. All these pieces of evidence support the neurotoxicity of fluoride…

Original article online at https://www.sciencedirect.com/science/article/abs/pii/S0269749124010856?via%3Dihub