Evaluation of the reliability of human teeth matrix used as a biomarker for fluoride environmental pollution

Journal Pre-proof Evaluation of the reliability of human teeth matrix used as a biomarker for fluoride environmental pollution Azza Ben Said Cheima Telmoudi Kaouthar Louati Faten Telmoudi Dorra Amira Mohamed Hsairi Abderrazek Hedhili

PII:

S0003-4509(19)30194-4

DOI:

https://doi.org/doi:10.1016/j.pharma.2019.10.006

Reference:

PHARMA 637

To appear in:

Annales Pharmaceutiques Franc¸aises

Received Date:

22 July 2019

Accepted Date:

23 October 2019

Please cite this article as: Said AB, Telmoudi C, Louati K, Telmoudi F, Amira D, Hsairi M, Hedhili A, Evaluation of the reliability of human teeth matrix used as a biomarker for fluoride environmental pollution, Annales Pharmaceutiques Franc¸aises (2019), doi: https://doi.org/10.1016/j.pharma.2019.10.006

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Evaluation of the reliability of human teeth matrix used as a biomarker for fluoride environmental pollution. Évaluation de la fiabilité de la matrice des dents humaines utilisées comme biomarqueur de la pollution environnementale au fluorure. Azza Ben Saida,b*, Cheima Telmoudib, Kaouthar Louatib, Faten Telmoudic, Dorra Amirad, Mohamed Hsairie, Abderrazek Hedhilid a

Centralized cytotoxic preparation unit, SALEH AZAIZ Institute, Boulevard of April 9th 1938,

1006, Tunis, Tunisia. University of pharmacy, Road Avicenne 5000 Monastir, Tunisia.

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Private medical analysis laboratory, Fatma residence in block F 1st floor, avenue Dar Fadhal El

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2036, Tunis, Tunisia. d

Toxicology Laboratory and Toxicology-Environment Research Laboratory LR12SP07 at El

Manar University, Tunis, Tunisia.

Department of statistics and medical informatics, SALEH AZAIZ Institute, Boulevard of April

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9th 1938, 1006, Tunis, Tunisia.

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* Corresponding author: Azza Ben Said

Tel: + 216 94 519 968

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ABSTRACT

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E-mail address: [email protected]

Environmental contamination by heavy metals has been a matter of great concern in many countries for several decades. Human exposure to such elements may cause adverse health effects and young children are particularly at risk. Many matrixes have been used for determination of metal exposure levels. Hard tissues such as teeth and bones have some advantages compared to other matrix. Dental tissues are considered to be suitable for long-term metal exposure due to their stability, resistance to physical and chemical degradation and their good preservation over time. Several studies have analyzed the teeth of animals for assessment the relationship between increased fluoride exposure and dental fluorosis, however few studies have been conducted on human teeth. Thus, the aim of the present study was to assess the reliability of human teeth matrix used as a biomarker for fluoride environmental pollution in TUNISIA, and to evaluate the relationship with place of residence, age, dental caries and sex.

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Teeth samples (n=123) were collected from individuals living in Gafsa (fluoride-polluted area which inhabitants are to a great extent exposed to heavy metals) and Tunis (non polluted area). Samples were analyzed using a potentiometric method. The fluoride concentration was found to be significantly higher in teeth from Gafsa samples than those from Tunis. Their control levels were respectively 6793.1 µg/g and 1068.8 µg/g. The results indicate that there is a clear relation between fluoride concentration and residence of living. An increased level of dental fluorosis in fluoridated communities has been used to evaluate historical chronic exposure to fluoride in these communities, despite constant fluoride levels in the drinking-water.

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The fluoride concentration was also observed to be significantly increased in polluted area with age and in carious teeth whereas, no significant difference was observed for sex. Our study confirms well that human teeth used as a bioindicator for environmental pollution provide good chronological information on exposure, and highlighted the risks incurred by

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consumers living in such polluted area.

Keywords: Dental fluorosis; environment; fluoride; biomarker; teeth; risk assessment.

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RESUME :

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La contamination de l’environnement par des métaux lourds est un sujet de grande préoccupation dans de nombreux pays depuis plusieurs décennies. L'exposition humaine à ces

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tels éléments peut avoir des effets néfastes sur la santé et les jeunes enfants sont particulièrement à risque. De nombreuses matrices ont été utilisées pour déterminer le niveau d'exposition aux métaux. Les tissus durs tels que les dents et les os présentent certains avantages par rapport aux autres matrices. Les tissus dentaires sont considérés comme convenables à une exposition prolongée aux métaux en raison de leur stabilité, résistance à la dégradation physique et chimique et de leur bonne conservation dans le temps. Plusieurs études ont analysé les dents d'animaux pour évaluer la relation entre l'exposition accrue au fluorure et la fluorose dentaire, mais peu d'études ont été menées sur les dents humaines. La présente étude vise donc à évaluer la fiabilité de la matrice des dents humaines utilisées comme biomarqueur de la pollution environnementale au fluorure en TUNISIE, et à évaluer la relation entre le lieu de résidence, l'âge, la carie dentaire et le sexe. Des échantillons de dents (n = 123) ont été collectés chez des personnes vivant à Gafsa (zone polluée par le fluorure dont les habitants sont en grande partie exposés aux métaux lourds) et à Tunis (zone non polluée). Les échantillons ont été analysés en utilisant une méthode potentiométrique. La concentration de fluorure s'est révélée significativement plus élevée dans les dents des échantillons de Gafsa que ceux de Tunis. Leurs dosages de contrôle étaient respectivement de 2 Page 2 of 26

6793,1 µg/g et de 1068,8 µg/g. Les résultats indiquent qu'il existe une relation claire entre la concentration de fluorure et le lieu de résidence. Une augmentation du niveau de fluorose dentaire dans les communautés fluorées a été utilisée pour évaluer l’historique d’exposition chronique aux fluorures dans ces communautés, malgré des niveaux constants dans l'eau de boisson. On a également observé que la concentration en fluorures a augmenté de manière significative dans la zone polluée avec l’âge et le statut carieux, alors aucune différence significative n’était observée avec le sexe. Notre étude confirme bien que les dents humaines utilisées comme bioindicateur de la pollution environnementale fournissent de bonnes informations chronologiques sur l'exposition et mettent en évidence les risques encourus par les consommateurs vivant dans une telle zone

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polluée. ABBREVIATION F-ISE: Fluoride ion selective electrodes

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KEYWORDS:

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Fluorose dentaire, environnement, fluorure, biomarqueur, dents, évaluation des risques.

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GPC: Gafsa Phosphate Company GMB: Gafsa mining basin TCG: Tunisian Chemical Group

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TISAB: Total Ionic Strength Adjustment Buffer

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INTRODUCTION Environmental contamination from heavy metals has been a matter of great concern in many countries for several decades. Human exposure to such elements may cause adverse health effects and young children are particularly at risk [1]. Our organism absorbs toxic elements. Their presence in the environment is a result of their natural existence or connected to industrial activities. Toxic metals taken up by the body are spread to different tissues and organs. Hard tissues, which form our bones and teeth, accumulate most heavy metals absorbed by the body [1]. Fluorine is the most reactive of all elements and widespread in the natural environment in the

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form of fluoride ion (F-) at varied concentration in rocks, soil, water, air, plants and animals [24], with higher concentrations in areas where there are recent/past pyroclastic activities or geologic uplift. Fluorides are also widely used in many industrial processes. The systemic human fluoride exposure can occur when drinking water, eating foods containing fluoride or

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by inhalation [5, 6].

During exposure, all calcified tissues incorporate fluoride during the development of their

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mineral phases. Since fluoride is an avid mineralized tissue seeker, approximately 99% of its amount retained in the body is found in bones and teeth [7]. It binds calcium and replaces the

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hydroxyl groups in the mineral part of the bone, which is mostly hydroxyapatite. Skeletal accumulation of fluoride can therefore be regarded as a mechanism of detoxification [7]. This accumulation reflects the long-term impact of exposure to fluorides. Depending on the

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levels of exposure, fluoride has both beneficial and toxic effect on human health [8, 9]. The presence of fluoride in drinking water is known to reduce dental cavities among consumers. Although, fluoride deficiency is one of the factors contributing to the development of dental caries, increased fluoride intake does, however, not only affect amelogenesis but also dentinogenesis [10] causing dental fluorosis, also known as mottled enamel, various bone disorders (skeletal fluorosis) [7, 10-11] and in extreme cases carcinogenesis [6, 10-12]. The risk of fluorosis extends across the entire dentition during the period of tooth development and is associated with the cumulative dose of fluoride over this whole time period. Due to the above mentioned disorders by high concentrations of fluoride, careful control of levels is so required. The presence of fluoride in industrial effluents must be monitored because many plants are quite sensitive to fluoride while others are fluoride accumulators [13]. Animals from areas contaminated by industrial emissions containing fluoride may also accumulate it predominantly in hard tissues. So, in industrialized countries, a major concern should be taken

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concerning the environment contamination and for workers who are daily exposed to heavy metals [6, 10, 14]. The most important biological matrix used for determination of metal exposure levels are blood, urine, hair and saliva [15-16]. Nevertheless, and because of their nature, urine and blood data provide information on recent exposure (approximately 30 days). Information concerning environmental contamination and chronic incorporation were possible by the use of indicators as contaminant dumps through long periods of time [17]. Hard tissues such as teeth and bones have some advantages compared to other matrix. Teeth are considered to be suitable bioindicator since they are easily obtained than bones, presenting

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the same structure of bone and relatively stable. They provide a permanent cumulative and sound record of chronic and/or recent environmental exposure to heavy metals [18]. Several studies have analyzed the teeth of animals for assessment the relationship between increased fluoride exposure and dental fluorosis. Teeth samples were often collected from fox

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and deer species (roe deer and red deer), living in regions with high fluoride levels as biomarkers of fluoride pollution [6, 14, 19-20]. However few studies have been conducted on

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human teeth [21-24]. Thus, the aim of the present study was to assess the reliability of human teeth matrix used as a biomarker for fluoride environmental pollution in TUNISIA, and to

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evaluate the relationship with place of residence, age, dental caries and sex. MATERIALS AND METHODS

A cross-sectional study over a period of 6 months was conducted to evaluate the different

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factors that may influence the accumulation of fluoride in teeth. 1. Collection of samples

Actually, teeth fulfill nearly all of our investigation needs since: (1) they are easily obtainable than bones; (2) presenting the same structure and relatively stable, therefore, they have the same metal affinity, and (3) their remodeling is slow and, as a consequence, the contaminants clearance is much smaller towards other organs. Thus, teeth allow for an exposure longstanding record.

A total of one hundred and twenty three teeth were collected randomly from various patients (57 men, 66 women) aged between 6 to 60 years old (mean age 26.9±17.7 years). For statistical analysis, the samples were divided into 2 age groups: 6-20 years and 21-60 years. All teeth were extracted by dentist between 2013 and 2014. Age, sex, caries status (carious or non carious) and place of residence were determined for each donor. In order to evaluate the reliability of human teeth matrix used as a biomarker for fluoride environmental pollution, zone of residence history questionnaires assessing previous exposure 6 Page 6 of 26

to pollution during life were conducted, so that only subjects born and living in the two areas: Gafsa and Tunis town were included in this study, even for the older of them. Each patient from Gafsa twon

had specified his living in or outside the GMB. Collection of permanent teeth was not so easy to obtain from donors, and the extraction of teeth was in most cases justified by previous associated pathologies such as caries and dental fluorosis in order to more emphasize the relation between fluoride concentrations and the development of such pathologies, and to evaluate historical chronic exposure to fluoride in high polluted

communities. 2. Description of the selected regions

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Gafsa is located in the southwestern of Tunisia. It is one of the most important phosphate producers in the world (more than 10 million tons/year) which operates seven open-pit quarries and one underground mine distributed within about 1.250 km2 areas around the mining basin including Mdhilla, Metlaoui, Moulareb and Redeyef cities (fig.2) [25, 26].

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The exploitation sites (extraction and enrichment) of Gafsa phosphate company (GPC) are grouped, according to their geographical location, in five production centers located within a

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radius of 50 km around the town of Metlaoui (main mining town). The open-air deposits are at a distance of four kilometers from the town of Metlaoui and Moulares and ten kilometers from

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the towns of Rdeyef and Mdhilla. The crude phosphates extracted from the deposits are transferred to the laundries, which are units for washing and phosphates enrichment. There are eight laundries in the study area, five of which are located in the mining towns [26].

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Another unit, dedicated to the enrichment and chemical treatment of phosphate, named Tunisian Chemical Group (TCG), which is located 3 km to the north. It's the city of Mdhilla. Thus, GPC dominates throughout the mining territory and TCG is only near the region of Mdhilla. From this, the Gafsa mining basin (GMB) is a highly industrialized area exposed to severe atmospheric fluoride pollution, and its inhabitants are to a great extent exposed to heavy metals [26, 27].

Tunis, the capital city, located on northeast coast of Tunisia (in the Maghreb region of North Africa) is not exposed to elevated fluoride deposition. Gafsa area (exposed region) is located at 350 Km from Tunis capital (the controlled region). Only patients born and living in these two areas were included in our study. 3. Chemical reagents and standards: Fluoride stock solutions (1000 µg/g) were prepared from sodium fluoride. Nitric acid at 1% was used for glassware washing process.

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The only reagent to be prepared is TISAB (Total Ionic Strengh Adjustment Buffer) which is a buffer solution that maintains pH within 5 and 6 during assays. Besides it gives a high total ionic strength, stabilizing the solution and avoiding ionic interference by polyvalent cations such as Al(III), Fe(III) and Si(IV), which are able to complex or precipitate with fluoride and reduce the free fluoride concentration in the solution. TISAB contains 58 g of sodium chloride, 57 mL of glacial acetic acid, 4 g of 1,2cyclohexanediamine N,N,N’,N’-tetra-acetic acid (CDTA) and approximately 150 mL of 6 mol L−1 NaOH in a volume of 1000 mL. Using a combined glass electrode, the pH is adjusted between 5 and 5.5.

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4. Preparation of equipments All containers were washed after each use and allowed to be soaked overnight in nitric acid solution, then rinsed the next day with distilled water and dried in an oven. 5. Sample preparation

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After extraction, each tooth was rinsed with bidistilled water to remove organic materials deposited on the teeth (blood, saliva and dental tissue) and then stored in tightly sealed Teflon

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vessels. To facilitate the pulverizing process, each tooth had been previously fractured into small pieces, dried at 105°C for 10 minutes and then pulverized with mortar and pestle. Thus,

6. Sample analysis

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the fine obtained powder was stored in desiccators (in a hermetically sealed glass vial).

We prepare four standard solutions at 100 µg/g, 10 µg/g, 1 µg/g and 0.1 µg/g with appropriate

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serial dilution from the stock solution at 1000 µg/g. Pipette 25 ml of each of the standard solutions; add 25 ml of TISAB in clean, dry beakers. For sample solution: The powder was again dried at 105°C for 4 hours immediately prior to being dissolved. A 25 ml of TISAB was added to 0.5 g of sample accurately weighted into a vial of 100 ml. The mixture was boiled thoroughly for 2 minutes. Adjust the resulting solution to 50 ml with distilled water.

Fluoride ion selective electrodes (F-ISE) (Jenway) were adopted as method for fluoride determination. Fluoride concentrations are expressed as µg/g. Immerse the fluoride electrodes in each of the solutions and measure the potential in millivolt using a potentiometer. Draw the calibration line of the potential as a function of the logarithm of fluoride concentration (E against log [F-]) ions and determine the concentration of the unknown solutions. The method for the quantitative determination of fluoride levels was linear in the concentration range between 1.9 and 19 103 ppm. The detection limit reached 0.63 ppm. 8 Page 8 of 26

7. Statistical analysis The data were statistically analyzed using the SPSS statistical version 16.0 software. Categorial data were described by proportions’ calculation while mean, standard devitation (SD) and median were calculated for continuous variables. Student t-test was used to compare means of groups (in case of a group samples size greater than 30) and Kruskal wallis test was applied in the remaining situations. Tables and charts were prepared using Excel and Graph Box. All data were analyzed to compare differences between groups by calculating means and SD. Only p values less than 0.05 were considered significant.

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RESULTS The overall statistics (standard deviations, medians, maximum and minimum levels) for dental fluoride concentration associated with sex, age, and carious status whatever was the place of residence were summarized in table 1.

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A general description of the main population characteristic for each place of residence was presented in table 2.

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Fluoride concentration in teeth was ranged between 251.5 µg/g to 23761 µg/g. 1. Residence of living

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As illustrated in table 1 and Fig.1, fluoride levels in teeth samples from Gafsa were found to be significantly higher than those in Tunis: mean levels are 6793.1 and 1068.8 µg/g respectively. The comparison of fluoride levels between mining and non mining area does not show any

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significant difference (P> 0.05) (table 3). 2. Sex status

Women present approximately the same fluorine concentration as men (4044.6 µg/g and 3950.1 µg/g respectively (table 1)). The results of the present study have statistically showed a nonsignificant difference regarding fluorine concentration between men and women in the two areas (Table 2).

3. Age factor Fluoride concentration in teeth was significantly in linear increase with age (P<0.05) (Fig.2). Fig.3 and table 2 show that in Tunis, controlled area, fluoride level was not different regardless the age (P>0.05), however, it was significantly higher in patients aged between 21 and 60 years in the exposed area (P = 0.006). 4. Caries status

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No relationship could be established between fluoride levels and caries status of studied person (P>0.05) (table 1). In Tunis, fluoride level was higher in non-carious teeth, while in GAFSA exposed area, it was slightly higher in carious teeth than those non carious (P>0.05) (table 2). DISCUSSION 1. Distribution of fluoride levels in tooth Fluoride is distributed in different dental tissues such as enamel, dentine, and cementum. Since each extracted tooth was totally pulverized, so our results have been obtained on fluoride content found in both enamel and dentin. Following literature, various fluoride levels were found in the same tooth. The highest concentrations

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in enamel are found at the surface and can be around 2000mg/kg in persons residing in nonfluoridated regions (representing a 6% replacement of OH- by F- in hydroxyapatite) and 3000mg/kg (8% replacement of OH- by F- ) in fluoridated areas. These concentrations fall sharply after the outermost 10-20 µm of enamel to levels ranging from hundreds of mg/kg to

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around 50 mg/kg, depending on whether the area is fluoridated or not. Beyond this depth the fluoride content remains fairly constant up to the enamel-dentine junction, in contrast with bone

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and dentine where accumulation of fluoride continues throughout life. Once human enamel is fully formed, its fluoride concentration can only be permanently altered at subsurface regions,

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as a result of trauma caused, for example, by caries, erosion, or abrasion. In the outermost surface layers, however, fluoride concentrations are independent of de- and remineralisation processes and can increase due to diffusion of fluoride from the oral environment which can

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vary according to exposure to ingested fluoride sources, therapeutic agents, saliva and dental plaque. In contrast to enamel, dentine continues to accumulate fluoride throughout life. The fluoride content of dentine is derived entirely by the systemic route. Within the dentine, the fluoride concentration is generally higher than that of the majority of enamel and tends to increase gradually from the enamel-dentine junction to the dentine pulpal surface. This may provide some benefit in combination with additional sources of fluoride, since a higher fluoride concentration is required in dentine to inhibit demineralisation and enhance remineralisation, compared with enamel [28].

Another study conducted by Yoshikazu K. had showed that although the fluoride concentration

in the enamel could have been changed due to its post-eruptive environment, the coronal dentine takes up fluoride from the pulp until about the age of 50 and the root dentine and the cementum take up fluoride from both the pulp and the periodontal ligament all during the life of the tooth [29]. 2. Effect of life residence on fluoride concentration and dental fluorosis 10 Page 10 of 26

2.1 Effect on fluoride concentration Our results reveal a significantly higher fluoride concentration in teeth from GMB than those from Tunis, the controlled area (table 2). Their control levels were respectively 6793.1 µg/g and 1068.8 µg/g. This high level is related to the fact that this region is more exposed to

environmental heavy metals. Indeed, the exposition to natural phosphate is considered as the primary source of fluoride pollution. Gafsa region is subdivided into mining and non-mining areas. All mining towns are located around phosphate extraction and processing points. The main four mining towns are: Metlaoui, Rdeyef, Moulares and Mdhilla [26].

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Since the end of the 19th century, the mining area has specialized in the single-activity extraction and enrichment of phosphate for reasons of economic profits. This production is assured by GPC since 1893 which exploits seven open-pit quarries and one underground mine scattered within about 1250 km2 areas around the GMB [26].

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The mining industry is a polluting activity because of the destruction of the ecosystems and the landscape, whether during the opening and the quarrying operations or at the level of the

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discharges of the sterile materials and open sludge [26].

The phosphate wastes of muddy nature are rejected by the laundries directly in the wadis,

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polluting them. The settling ponds, created in 1990, serve to prevent the spread of pollutants in nature by simple storage. These settling ponds are more or less impervious to the permeability and pollution of neighboring regions. Since the year 1985, underground mines were abandoned

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and replaced by open pit mines. Unfortunately, these mines present until nowadays the risk of collapse and landslide.

So the extraction of phosphates seriously pollutes the region. The area has suffered an intense degradation of soil resources, vegetation cover and an advanced deterioration of agricultural. In order to give off the superficial rock layers and access to the layers of deep phosphate ore, the GPC uses dynamite, without respecting the standards of use that limit the amount of dynamite per surface and therefore the amount of dust in suspension. Important quantities of pollutants from industrial activities, landfills and from the trucks of a private transportation company replacing the wagons for the transport of phosphate until the treatment factories are emitted as particles in the atmosphere that becomes filled with phosphate particles and heavy metals such as cadmium and uranium. A gray dust covers the plants, the streets, the roofs of houses located several miles away. So, Gafsa region is all contaminated in both mining and non-mining regions (table 3).

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Millions of tons of sludge are continued to be dumped in the wadis of the region. These sludges are loaded with heavy metals (cadmium, nickel, copper, zinc, chromium, etc.) and results in clogging and asphyxiation of soil, changing their permeability characteristics giving them a cracked appearance with disappearance of the vegetation cover [26]. Soils closed to abandoned mines and even very remote were found to be highly contaminated with heavy metals. The stagnancy of industrial dust has covered them with white layers [27, 30], like the soil at the Mdhilla Laundries which is marked by a blackish to greyish pollutant cover [26]. The physico-chemical characterization of soils has shown that fluoride levels are significantly more important than in water, due to the accumulation by years of fluoride dust

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from phosphoric origin [26]. These high levels require careful control because excessive concentrations of soil by metallic trace elements are toxic, influence the uptake of these elements by plants which are quite sensitive to fluoride [13], as well as to animals which have no other source of food than the

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forage species available in these area, accumulating fluoride predominantly in hard tissues [6, 14-15, 19-20]. Health problems in livestock sector were reported (falling teeth, skeletal

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disorders and decreased milk production) [26]. The direct contact of mammals with the muddy discharge zones has weakened the tones and affected the legs due to the high level of the toxicity

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rate [26]. Meat from farmed animals constitutes a likely channel for human transmission of heavy metals [26]. So, pollution caused by the GPC activities reverberates on agricultural products, therefore on the quality of food products intended for human consumption [31].

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The inhalation and ingestion of fluoride loaded particles in ambient air is one of the important factors for accumulation of fluoride and dental fluorosis. In GMB, foul smells coming from the GCT are toxic gaseous emissions that contain mainly cadmium, sulfur oxide and Fluorine following investigations. To these odors, small particles are present from the terrarium of phosphogypsum on the roofs of housing. Their impact has reached neighboring communities even outside the GMB (table 3) [26].

Drinking water is also considered to be an important element contributing to fluoride accumulation in teeth. Several studies from Mexico, India, Argentina, Korea, America, China, had indicated that drinking water containing higher concentration of fluoride is the greatest source of fluoride human environmental exposure [24-27, 30-31]. These high levels are related to the local environment. Real data were obtained from sources used for consumption in Tunisia. In Gafsa, fluoride levels are much higher than the allowed limit in Tunisia drinking water (1.5 mg/L) and those from international levels. Concentration of fluoride was found to be 2.22 mg/l in Gafsa and 0.43 mg/l in Tunis [32]. Thus, children living 12 Page 12 of 26

in Gafsa have higher fluoride exposed levels than fluoride toxicity reference value following Health Canada [33], and they are particularly at risk for developing dental fluorosis as demonstrated by many studies [24, 34], which the severity is dependent upon fluoride dose, the timing and duration of fluoride exposure according to Richter H, et al [14]. Despite the low levels of fluoride in feed waters, the majority of the wells exploiting the superficial aquifer exceed largely the standards of potability, and since there is no control, groundwater must be so used with caution in agriculture. Many countries through the world have a serious problem of fluoride contamination in drinking water. Levels at 0.3-7.0 mg/mL had lead to chronic fluoride toxicities in villagers and animals

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of Udaipur district (INDIA) [24]. So, the high concentration of fluoride in Gafsa town is attributed to the GPC activities contaminating soil, vegetation, air, groundwater resources, and altering ecosystems in many area of this region.

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Human exposure to such elements may cause adverse health effects and young children are particularly at risk [1]. Many diseases can be developed such as pneumonia, asthma and

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respiratory effects by the inhalation of atmospheric pollution; cancers associated with radioactive elements contained in phosphate ores; fluorosis due to fluorine presence in the dust

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and the reserves of contaminated water at a rate three to four times higher than the limit threshold recommended by the WHO [35]. 97% of investigations have stated that there is at least one family member who suffers from a disease caused by the negative impacts of mining

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pollution. From these adverse health effects, the inhabitants of the Gafsa town are well recognized by their teeth mainly fluorosis, silicosis, reneal stones,etc [26]. Our results are in conformity to some studies described in literature. In Maracoo, a research has highlighted that soils close to mining sites and even very remote are highly contaminated with heavy metals: Pb, Zn, Cu and Cd [31].

Another research conducted in the Gulf of Gabes (Southeast of Tunisia) [36], had shown that high fluoride pollution was detected in the surrounding soils of the coastal superphosphate industries. The study was conducted in vicinity of factories analyzing plant functional traits combined with plant fluoride accumulation and soil metal concentrations. Authors had also compared the fluoride level in human teeth from a phosphatic fertilizer plant environment and no exposed area. They had reported higher fluoride levels at 248-8891 µg/g of dried weighted powder in teeth samples from the exposed area [24]. 2.2 Effect on dental fluorosis

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The benefits of fluoride in the prevention and control of dental caries have been accepted for many years. However, alongside these benefits it is recognized that the ingestion of fluoride during the period of tooth development increases the risk of developing dental fluorosis, a developmental defect seen as hypomineralization of the enamel [37]. Above 0.1 mg fluorine/kg/day, fluorosis develops dependent on dose. It is due to an overdose of Fluor, for several months or years, occurring during the period of mineralization of the teeth. The complete development of the enamel crystals is disturbed by the excess of fluorine leading to a porous fluorotic tissue. If the damage is important, the porous enamel is likely to incorporate any colored exogenous element and generate coloration of the teeth (going from

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the simple white spot to a brown tablecloth). The severity of alterations is multifactorial and depends on the ingested dose, the moment of exposure (enamel formation phase), the duration of impregnation or accumulation, and the misunderstanding of the various sources of fluoride intake that may increase risk of fluorosis

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such as water fluoridation, fluorides in foods and beverages, infant formulas with and without fluoridated water, fluoride dentifrices, mouthrinses, and gels [38, 39].

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In the majority of cases, the repercussion of fluorosis is mainly aesthetic. We must therefore be particularly vigilant for children aged 0-4 years, in the mineralization period of the crowns of

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the incisors, especially at this age and until around the age of 6 years, a large amount of toothpaste is ingested involuntarily. The limit dose not to be exceeded to avoid any risk of fluorosis is 0.05 mg/day per kg of body weight, all intakes combined, without exceeding 1 mg/

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day (WHO data) [38].

In our study, populations with low or moderate exposure to fluoride through optimally fluoridated community water supplies, fluorosis was presented as diffuse white lines or opacities of the enamel surface as a result of an increase in the porosity of the fluorotic enamel. However, in Gafsa populations exposed to higher levels of fluoride for example, high levels of fluoride in groundwater used for cooking and drinking; fluorosis manifests more severe hypomineralization with pitting and loss of the surface enamel. Our results are in conformity to researches described in literature. In Maracoo, a study consisted in hydrochemical knowledge of the existing aquifer system near to mines and phosphates operating factors (Khouribga Province, Morocco) [40], has highlighted the groundwater pollution by fluoride from phosphate origin, and explained the development of fluorosis in these regions, despite the low levels of fluoride in feed waters because the majority of their rural population use groundwater extracted from traditional wells close to their "Douar" as their only alimentary source. 14 Page 14 of 26

Another study conducted in Chiang Mai, Thailand had shown that consumption of drinking water with fluoride content >0.9 ppm and use of cooking water with fluoride content >1.6 ppm were associated with an increased risk of aesthetically significant dental fluorosis [37]. Moller IJ and Polson S, had reported that several hundreds of children living within 1-1.5 km area of the mine were affected by dental fluorosis due to the dust pollution from phosphate mines [41]. Death C, et al., had demonstrated that dental lesions were positively associated with bone fluoride accumulation in multiple species of marsupial in high fluoride industrial area [42]. 3. Effect of age on fluoride concentration

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The difference of fluoride concentration between the two range of age (6-20 years) and (21-60 years) was statistically significant (P <0.05) (table 1). Our results show that concentration increases with age, in accordance with data from literature [4, 10, 14, 43]. Many studies had examined bones and teeth of wild and domesticated animals for indirect assessment of the

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degree of environmental contamination with fluoride. They had shown that the concentrations of fluoride in mammals generally increase with age as a result of bioaccumulation. In the case

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of people and other long-lived mammals, including ungulates and carnivores, the concentration of fluoride clearly increases with their age [4, 43].

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Akumagai A, et al., had shown that there is an accumulation of metals in teeth by age explained by a long-term exposure to a polluted environment [44]. Also in the red fox, there is a clear relationship between age and the fluoride concentration in

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bone tissues. Authors had demonstrated the statistically significant correlation coefficient (r) concerning the studied mandibles [4, 43]. This increased level of fluorine by age can be explained by the fact that F- anion substitutes calcium sites in the hyroxyapatite crystals of dentin, since the main component of hard tissues of the body, including teeth, is hydroxyapatite. Under physiological conditions, during the process of mineralization, various elements can be included in the hydroxyapatite structure. The elemental composition of the hydroxyapatite during teeth changes is a long-term process, which means that the metals that are included in its structure are released only to a small extent. Metals absorbed into the body and deposited in the teeth tissue remain there throughout lifetime. Thus, in conditions of occupational or environmental exposure, the concentration of toxic elements in teeth increases with age. This feature determines the usefulness of teeth when assessing a long-term exposure. 4. Effect of sex on fluoride concentration

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No relationships could be established between the fluoride levels in exfoliate human teeth and the sex of subjects. Similar conclusions were reached by studies in India [24]. However, it may be a slight difference. This may reflect a difference in terms of food preferences, hormonal metabolism, tobacco or other physiological influences. In literature, a study had demonstrated that fluoride concentration of erupted enamel from woman teeth was significantly higher than that of man, suggesting that this might be due to a longer period of pre-eruptive maturation in man teeth [25]. 5. Effect of fluoride concentration on carious status The effects of fluoride on human health may be whether beneficial or harmful depending on

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the exposed dose. At low concentration, fluoride is used for dental caries and osteoporosis prevention at the dose of 0.5 - 12.0 mg/day and 20 -150 mg/day respectively [45]. On the other hand, at high concentration, fluoride will be harmful to health [10-12].

Millions populations are exposed to toxic effects in areas of endemic fluorosis specified with

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high fluorine content in the natural drinking water (21-80 mg/day) [5, 8]. Numerous studies have demonstrated that low concentrations of fluoride ion in drinking water (<0.5 mg/L) were

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associated with increased dental caries whereas contents of 0.5-1.5 mg/L are protective against the disease [32, 46]. The minimum concentration of fluoride required in drinking-water is

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approximately 0.5 mg/L [47].

However, exposure to higher concentrations via drinking water (>1.5 mg/L), some teas, and fluoride contaminated salt lead to carious teeth and can cause deformities of the hard tissues,

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namely dental and skeleteal fluorosis [1, 17].

The protective power of fluorine was explained, on the one hand, to balance maintenance between demineralization and remineralization, and on the other hand to metabolism inhibition of cariogenic bacteria. So, fluoride is widely used in dental products, and water fluoridation programs have been implemented by many countries as a routine measure of public health in order to help reduce dental cavities among the population [32, 48]. Our results have showed that fluorine concentration in carious teeth is higher than those noncarious, in accordance to data in literature which indicate that fluorine, when presented at high concentration in teeth is associated with fluorosis [10]. Following our study, we can explain that the high fluorine concentration in carious teeth is associated to an increased accumulation of fluorine in the dentine part of tooth and so its possible role in predisposing or causing dental caries. Dental enamel consists of 96% of mineral matter. The remaining 4% is water and organic matter. The mineral matter consists mainly of calcium hydroxyapatite crystals which chemical 16 Page 16 of 26

formula is Ca10 (PO4)6 (OH)2. The highly mineral composition of the enamel gives it the advantage to be extremely hard and also vulnerable to a demineralization process usually presented as caries [49]. The main phosphate mineral found in phosphate rocks in Gafsa is carbonate fluorapatite. This mineral differs from fluorapatite and hydroxyapatite by the strong substitution in its PO4 network with CO3 essentially and Ca by Mg, Na, Sr and other rare earth elements and trace elements such as Cd, Zn, Cu, U, Th [50]. For Gafsa polluted area, the high rates of fluoride and fluorosis observed in most of our samples are linked to an increased prevalence of dental caries. Two explanations are proposed: in severe

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forms of fluorosis, post-eruptive loss of teeth external enamel leads to wells formation that food debris can stagnate in these areas and contribute to carious lesion susceptibility. A second hypothesis is the hypomineralization under dental surface which may be more causal to carious lesion risk [51].

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Our case in Tunis, non-carious status may be explained that fluoride at low concentrations provides protection against dental caries, especially in children. This protective effect increases

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with concentration up to about 2 mg of fluoride per liter of drinking-water. CONCLUSION

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Environmental contamination from heavy metals has been a matter of great concern in many countries for several decades. Human exposure to such elements may cause adverse health effects and young children are particularly at risk.

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Fluoride are the most reactive of all elements, widely used in many industrial processes that are found with higher concentrations in areas with environmental pollution. The systemic human fluoride exposure can occur when drinking water, eating foods containing fluoride or by inhalation. During exposure, all calcified tissues incorporate fluoride during the development of their mineral phases. Since fluoride is an avid mineralized tissue seeker, approximately 99% of its amount retained in the body is found in bones and teeth. Human excessive systemic exposure can lead to disturbances of bone homeostasis and enamel development. The risk of fluorosis extends across the entire dentition during the period of tooth development and is associated with the cumulative dose of fluoride over this whole time period. Teeth are easily available biological materials than bones and good markers for exposure to environmental pollution since they provide a permanent cumulative and relatively stable record. In our study, we have assessed the reliability of human teeth matrix for fluoride environmental pollution, and the relationship between fluoride level with place of residence, age, sex, and carious status. The region is the most important factor influencing metal concentrations that 17 Page 17 of 26

fluoride levels were found significantly higher in teeth from Gafsa samples than those from Tunis. This high concentration in Gafsa town is attributed to the GPC activities contaminating soil, vegetation, air, groundwater resources, and altering ecosystems in many areas even outside the GMB. An increased level of fluorosis in fluoridated communities has been used to evaluate chronic historical exposure to fluoride in these communities, despite constant fluoride levels in the drinking-water. Also, fluoride concentration was observed to be significantly increased in polluted area with age and in carious teeth whereas, no significant difference was observed for sex. Our study has well confirmed that human teeth used as a bioindicator for environmental

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pollution provide good chronological information on exposure. Our research has highlighted the risks incurred by consumers living in such exposed area. Remediation measures must so be taken to immobilize pollutants and limit their transport to the environment. However, for many Tunisians, the GPC must continue to produce without being prevented by protest movements

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because phosphate is a national wealth, which accounts for 10% of exports.

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Conflict of Interest None of the authors of this paper has financial or personal relationship with other people

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or organisations that could inappropriately influence or bias the content of the paper.

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Acknowledgement The authors want to thank the Toxicology-Environment Research Laboratory at El Manar University, Tunis, TUNISIA for its constant support and encouragement providing necessary

facilities to carry out this work. The authors thank also the dentists who took part in this

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study.

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Formatting of funding sources This research did not receive any specific grant from funding agencies in the public,

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commercial, or not-for-profit sectors.

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List of figure captions Fig.1: Distribution of dental fluoride level by region. Fig.1: Répartition du taux de fluorure dentaire par region. Fig.2: Distribution of dental fluoride level by age. The results concern all subjects, whatever was the place of residence. Fig.2: Répartition du taux de fluorure dentaire par âge. Les résultats concernent tous les sujets, quel que soit le lieu de résidence. Fig.3: Distribution of dental fluoride level by age following the place of residence.

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Fig.3: Répartition du taux de fluorure dentaire par âge suivant le lieu de résidence.

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