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[Preprint]. 2024 Mar 1:rs.3.rs-3995767. [Version 1] doi: 10.21203/rs.3.rs-3995767/v1

Fluoride-related changes in the fetal cord blood proteome; a pilot study

Sami T Tuomivaara 1, Dana E Goin 2, Susan J Fisher 3, Steven C Hall 4, Aras N Mattis 5, Pamela K Den Besten 6
PMCID: PMC10925477  PMID: 38464284

Abstract

Background:

Fluoride exposure during pregnancy has been associated with various effects on offspring, including changes in behavior and IQ. To provide clues to possible mechanisms by which fluoride affects human fetal development, we completed proteomic analyses of cord blood serum collected from second-trimester pregnant women residing in Northern California with either high or low fluoride exposure, as identified by maternal serum fluoride concentrations.

Objective:

To identify changes in cord blood proteins associated with maternal serum fluoride concentration in pregnant women living in Northern California.

Methods:

The proteomes of 19 archived second-trimester cord blood samples representing highest and lowest serum fluoride concentrations from a cohort of 48 women living in Northern California, previously analyzed for serum, urine and amniotic fluoride concentrations, were characterized by mass spectrometry. Proteins highly correlated to maternal serum fluoride concentrations were identified, and further compared in a group of samples from women with the highest serum fluoride to the group with the lowest maternal serum fluoride concentrations.

Results:

Nine cord blood proteins were significantly correlated with maternal serum fluoride concentrations. Six of these proteins, including apolipoprotein B-100, delta homolog 1, coagulation factor X, mimecan, plasma kallikrein, and vasorin, were significantly decreased in the cord blood from women with the highest serum fluoride levels.

Conclusion:

Changes in the relative amounts of second trimester cord blood proteins included proteins associated with the development of the fetal hematopoetic system.

INTRODUCTION

Tooth enamel fluorosis, a biomarker for fluoride exposure, has steadily increased in the US [1, 2] resulting in hypomineralization of tooth enamel and at higher exposures, increasing the risk for dental disease[3]. Increasing levels of fluorosis are of concern in view of recent human studies showing associations between fluoride exposure during either pregnancy or the first year of life, and impaired neurodevelopment of offspring [4–9].

Rodent models show fluoride-related effects on cell function and neurodevelopment [10, 11], associated with increased oxidative stress and inflammation [12–16]. However, the relevance of these studies to human health has been questioned because rodents require 5 to 10-fold higher levels of fluoride in drinking water to achieve plasma fluoride levels similar to those found in humans [17]. Mechanisms responsible for possible fluoride-related effects in humans remain poorly understood, though reports of age and sex related effect of fluoride on children, identified by analyses of NHANES data, suggest that fluoride can affect multiple cells and tissues[18–22].

In this study, we investigated possible effects of fluoride on the developing fetus by comparing the cord blood proteome of second-trimester pregnant women in Northern California [23]. Cord blood samples were obtained from a random cohort of 48 samples of healthy second trimester pregnant women that we had previously analyzed for serum, urine, and amniotic fluid fluoride concentrations. Urine fluoride concentrations of this cohort were similar to cohorts from larger retrospective studies of Candian [23] and Mexican women [5, 6] showing fluoride related change in offspring neurodevelopment.

METHODS

Study Samples

This study was approved by the University of California, San Francisco Committee on Human Research. Archived cord blood serum samples, from a cohort of 48 women [23], were selected for whole proteome analysis. Ten samples were from women who had the lowest serum fluoride, and 9 samples were from women with the highest serum fluoride in from this cohort. All women resided in communities with water fluoride levels ranging from 0.2 to 0.9 ppm. The community water fluoride concentration recommended by CDC was 1.0 ppm prior to 2015, and was subsequently change to 0.7 ppm fluoride in 2015, and therefore all were near or below the maxium recommended levels for artificial water fluoridation.

Proteomic analysis

The cord blood samples were doped with protease inhibitors, and after clearing and dilution, the 14 most abundant proteins, including serum albumins were removed using immobilized antibodies. Total protein concentration was determined with BCA protein assay. The samples were buffer exchanged into ammonium bicarbonate, denatured with urea, reduced and alkylated, and digested with trypsin. The resulting peptide mixtures were desalted and analyzed by liquid chromatography-tandem mass spectrometry. Protein abundance in a sample was inferred from the number of peptide identifications. To account for the analytical variability, relative peptide abundance was calculated for each protein in each sample by dividing the number of peptides identified from a given protein by the total number of peptides identified in that sample.

Statistical analyses

Demographics were compared by Kruskal-Wallis one-way analysis of variance test. Pearson’s correlation coefficients were calculated for the relative protein abundance. The samples were grouped relative to highest and lowest maternal serum fluoride concentrations and and significance (p-value) from paired Welch’s t-test were calculated. All data analyses were conducted in the R statistical computing environment (version 4.0 or newer).

RESULTS

Demographics of the study population showed community water fluoride levels at the time of sample collection from the 10 women with the lowest serum fluoride concentrations (0.0040 to 0.0073 ppm) ranged from 0.2 to 0.8 ppm. Community water levels at the time of sample collection, for 9 women with higher serum fluoride (0.0224 to 0.0593 ppm fluoride) ranged from 0.16 to 0.9 ppm. Community water fluoride concentrations for all subjects were near or below 1.0 ppm, which was the optimal water fluoride levels recommended by the Center for Disease Control (CDC) at the time of collection.

The group of women with higher serum fluoride levels, were further divided into middle range serum fluoride of 0.0224 to 0.0291 ppm fluoride, and high serum fluoride ranging from 0.0444 to 0.0593 ppm fluoride. There were no significant differences in maternal age, smoking status or ethnicity between the fluoride groups (Table 1). However, the higher community water fluoride levels were associated with higher serum fluoride concentrations, with significant differences between groups.

Table 1.

Demographics of study population (N = 19)

Low serum fluoride Middle serum fluoride High serum fluoride p-value
N 10 6 3
Mean (SD)
Serum fluoride (ppm) 0.006 (0.001) 0.025 (0.003) 0.052 (0.007) 0.00
Water fluoride (ppm) 0.36 (0.32) 0.59 (0.26) 0.80 (0.11) 0.05
Age 24.0 (3.9) 23.8 (3.5) 25.3 (4.6) 0.82
BMI 27.3 (5.8) 25.5 (3.4) 27.5 (3.5) 0.80
N (%)
Smoker 6 (60.0) 4 (66.7) 3 (100.0) 0.44
Race/ethnicity
White 5 (50.0) 3 (50.0) 2 (66.7) 0.88
Black 3 (30.0) 2 (33.3) 1 (33.3) 0.99
Latina 2 (20.0) 1 (16.7) 0 (0.0) 0.72
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Mass spectrometry analysis of the cord blood proteome of the 19 samples showed significant correlations (Pearson’s correlation coefficient greater or less than 0.5) of 9 proteins with maternal serum fluoride concentrations (see Fig. 1).

Figure 1.

Figure 1

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Plots showing fetal cord blood proteins significantly correlated to maternal serum fluoride concentrations. Relative amounts of protein per fraction for individual samples at each maternal serum fluoride concentration, are shown by the red dots. Standard deviations for low, medium and high maternal serum fluoride, are shown by bars.

Proteins identified as significantly correlated to maternal serum fluoride were grouped into low and higher serum fluoride concentration (combined medium and high serum fluoride) and relative protein concentrations were compared. Six of the 9 cord blood proteins were significantly different in the low as compared to higher fluoride group (p ≤ 0.05) (see Table 2). These cord blood proteins were all significantly reduced in the higher as compared to lower fluoride group.

Table 2.

Protein (gene) Pearson’s correlation coefficient
n = 19		 Higher (n = 9) vs low (n = 10) F
(paired Welsh’s t-test)		 Fetal Tissue Biological process
Apolipoprotein B-100 −0.532 p = 0.045* Placenta liver Transports lipids from mother to fetus[24]
Coagulation Factor X −0.571 p = 0.020* Liver uterine wall Coagulation and wound healing [25]
Mimecan −0.531 p = 0.024* Extracellular matrix of multiple types of cells and tissues Regulation of glucose metabolism; negatively correlates with fat and bone mass [26]
Plasma Kallikrein −0.554 p = 0.012* Blood vessel walls Coagulation and wound healing [27]
Protein delta homolog 1 −0.527 p = 0.010* Placenta Placental growth [28]
Vasorin −0.656 p = 0.031* Liver; vascular, and smooth muscle Blood vessel contraction [29]
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DISCUSSION

Fetal circulation occurs through the umbilical vein that carries maternal blood and nutrients filtered through the placenta to the fetus, and 2 umbilical arteries that transport blood to and from the placenta. The placenta forms a barrier to macromolecules, including most proteins, to cross from maternal to fetal circulation [30]. However, fluoride crosses the placenta [23, 31], and is also increased in the placenta relative to cord blood fluoride concentrations [32, 33]. Previous studies have shown that fetal cord blood fluoride concentrations reflect maternal blood fluoride [34], and therefore in this study we used maternal serum fluoride concentrations as a biomarker for fetal fluoride exposure [23].

Cord blood serum was collected between 2014 and 2016 from second trimester pregnant women residing in Northern California [23]. All women were healthy without known underlying medical conditions, and their community water fluoride concentrations were at or below optimal water fluoride concentrations, as recommended by the US Center for Disease Control (CDC) at the time of sample collection (1.0 ppm fluoride). The 9 cord blood proteins found to be significantly associated with the maternal serum fluoride concentration were not correlated with risk factors for pregnancy outcomes including maternal age [35, 36], smoking status [37, 38], BMI [39], and race/ethnicity.

These proteins included protein delta-like homolog 1 (DLK1), a transmembrane protein highly expressed by stromal cells of the placental villi [28] and by the fetal liver hepatocytes [40], which was negatively associated with maternal serum fluoride. DLK1 regulates glucose metabolism [41] and placental growth hormone [42], and is reduced in small for gestational age fetuses [28]. The placenta is a primary hematopoietic stem cell (HSC) niche during pregnancy, which is believed to seed the fetal liver [43, 44], and depletion of DLK1 from of human fetal liver hepatoblasts in culture reduces the percentage of mature hematopoietic cells [40].

DLK1 also regulates notch signaling and affects neurodevelopment [45]. In mice, DLK1 was shown to regulate hippocampal neurogenesis and cognition. Reduced DLK1 levels triggers cognitive abnormalities [46], suggesting the possibility that fluoride-related effects on fetal neurodevelopment may be associated with reductions in DLK1.

The fetal liver is the primary organ for erythropoiesis during development, functioning as a vascular connection between the developing placental vessels connecting the heart [47]. It does not perform the traditional digestive and filtration functions because nutrients are normally carried to the fetus from the mother via the placenta. Other proteins significantly downregulated relative to maternal serum fluoride are also associated with hematopoiesis, and include apolipoprotein B-100 (APOB), coagulation factor X, plasma kallikrein, and vasorin. APOB, which is synthesized by the placenta, is a likely pathway for lipid transfer required for fetal growth [24]. Vasorin is a transmembrane glycoprotein[48] with a possible role in the regulation of the glycogen-mediated mTOR-ULK1 signaling pathway in the liver[49], and mimecan, also known as osteoglycin which is a small leucine-rich proteoglycan (SLRPs) present in the extracellular matrix of multiple tissues, including adipose tissue.

The possibility that fluoride may affect the liver in humans is supported by NHANES survey data from 2013–2016 that shows higher water fluoride and plasma fluoride concentrations are associated with lower blood urea nitrogen among US adolescents. However, while the fetal hematopoietic system is separate from that of the maternal circulatory system, the placenta is of dual origin, comprised of both fetally and maternally derived cells and maternal inflammation can alter maternal–fetal interactions. Maternal immune activation during pregnancy, can affect the development of the fetal hematopoietic system [50], and therefore it possible that fluoride related effects to increase maternal inflammation may also have a role in fetal development. Analysis of available NHANES survey data (2013–2014 and 2015–2016) of children aged 6–19, shows plasma fluoride concentrations positively correlated with total white blood cell count, segmented neutrophils and monocytes when adjusted for age, gender, and BMI as predictors. While there is no available data linking second trimester maternal white blood cell counts to fluoride exposure, further studies to explore this possibility are warrented.

In summary, this is the first study to assess possible fluoride related changes associated with human fetal development at low level fluoride exposure. Our finding in this pilot study, that cord blood proteins associated with the development of the fetal hematopoietic system were significantly downregulated relative to maternal fluoride exposure, supports the possibility that fluoride related mechansims affecting fetal development and may be associated with the placenta and fetal hematopoesis during second trimester fetal development. The second trimester of fetal development is also critical for development of the prefrontal cortex and integration of control of attention, thought, emotions and actions [51]. Therefore, the identification of cord blood proteins altered by relative levels of maternal fluoride exposure may direct future studies of fluoride related mechanism that may affect fetal development, including neurodevelopment.

Acknowledgements:

We are grateful to Tracey J Woodruff, Director, Program on Reproductive Health and the Environment, University of California, San Francisco, for support and helpful discussions.

Funding

This work was supported by NIH P01ES022841, US EPA RD 83543301, and the Center for Children’s Oral Health Research (UCSF).

Funding Statement

This work was supported by NIH P01ES022841, US EPA RD 83543301, and the Center for Children’s Oral Health Research (UCSF).

Footnotes

Ethics approval and consent to participate.

This study was approved by the University of California, San Francisco Committee on Human Research.

Competing interests

The authors declare that they have no competing interests.

Contributor Information

Sami T. Tuomivaara, University of California, San Francisco San Francisco

Dana E. Goin, University of California, San Francisco San Francisco

Susan J. Fisher, University of California, San Francisco San Francisco

Steven C. Hall, University of California, San Francisco San Francisco

Aras N. Mattis, University of California, San Francisco San Francisco

Pamela K. Den Besten, University of California, San Francisco San Francisco

Availability of data and materials

The datasets generated and/or analyzed during the current study are not publicly available due to patient privacy protections, but are available from the corresponding author on reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The datasets generated and/or analyzed during the current study are not publicly available due to patient privacy protections, but are available from the corresponding author on reasonable request.

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