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Rev Fac Cien Med Univ Nac Cordoba
Rev Fac Cien Med Univ Nac Cordoba
Revista de la Facultad de Ciencias Médicas
0014-6722
1853-0605
Universidad Nacional de Córdoba Argentina postmaster@ucbscm.edu.ar, postmaster@bfcmcb.sld.ar, cielo@fcmcb.sld.ar, bfcmcb@biomed.uncor.ar

38941224
42505
10.31053/1853.0605.v81.n2.42505
Artículos Originales
Effect of training on bone quality in rats with insulin resistance induce by fluoride consumption
Efecto del ejercicio físico sobre la calidad ósea en ratas con resistencia a la insulina inducida por el consumo de fluoruroEfeito do exercício físico na qualidade óssea em ratos com resistência à insulina induzida pelo consumo de fluoretohttps://orcid.org/0000-0003-1114-319X
Lombarte Mercedes 1 mercedes_lombarte@yahoo.com.ar

https://orcid.org/0000-0003-3708-0756
Fina Brenda Lorena 2 brendafina@gmail.com

García Victoria Inés 1 garciavictoriaines@hotmail.com

https://orcid.org/0000-0002-1331-1591
Lupo Maela 3 maela_lupo@hotmail.com

https://orcid.org/0000-0002-3953-278X
Rigalli Alfredo 1 arigalli@conicet.gov.ar

1 Bone Biology Laboratory, School of Medicine, Rosario National University Argentina
2 Grupo de Descargas Eléctricas, Universidad Tecnológica Nacional Facultad Regional de Venado Tuerto Argentina
3 University Centre for Environmental Studies, School of Medicine, Rosario National University Argentina
28 6 2024
28 6 2024
81 2 270284
20 9 2023
02 5 2024
Copyright © Universidad Nacional de Córdoba
2024
Universidad Nacional de Córdoba
https://creativecommons.org/licenses/by-nc/4.0/ Este es un artículo de acceso abierto distribuido bajo los términos de la Licencia de Atribución Creative Commons, que permite el uso, distribución y reproducción sin restricciones en cualquier medio, siempre que el trabajo original esté debidamente citado.
https://revistas.unc.edu.ar/index.php/med/article/view/42505/45443
https://revistas.unc.edu.ar/index.php/med/article/view/42505/45440
Abstract

When large amounts of Fluoride are consumed produces insulin resistance, but exercise can reverse insulin resistance in rats, because of a high fluoride uptake by bone tissue. However, bone quality has not been studied in those experiments. Therefore, the aim of this work was to evaluate bone quality in rats treated with fluoride when performing exercise. Sprague-Dawley rats were divided into 3 groups (n=6 per group): Control (drinking water without fluoride), Fluoride (drinking water with fluoride 15 mg/L for 30 days) and Exercise (daily running on a treadmill and drinking water with fluoride 15 mg/L for 30 days). Then, bone mineral density, mechanical and histological properties and bone fluoride level were measured. No effect of treatment on any bone parameters were observed. These results indicate that exercise normalizes glucose metabolism in insulin-resistant rats by bone fluoride uptake; however, this increase in bone fluoride does not manifest in bone deterioration

Resumen

Cuando se consumen grandes cantidades de fluoruro se produce resistencia a la insulina, pero la realización de ejercicio puede revertir dicho efecto en ratas, debido a una alta captación de fluoruro por el tejido óseo. Sin embargo, la calidad ósea no ha sido estudiada. Por ello, el objetivo de este trabajo fue evaluar la calidad ósea en ratas tratadas con flúor que realizan ejercicio. Se trabajó con ratas Sprague-Dawley que se dividieron en 3 grupos (n=6 por grupo): Control (recibieron agua sin flúor), Flúor (recibieron agua con flúor 15 mg/L durante 30 días) y Ejercicio (realizaron ejercicio diariamente en cinta ergométrica y recibieron agua con fluoruro 15 mg/L por 30 días). Luego, se midieron la densidad mineral ósea, las propiedades biomecánicas e histológicas y el nivel de fluoruro óseo. No se observó ningún efecto del tratamiento sobre ningún parámetro óseo. Estos resultados indican que el ejercicio normaliza el metabolismo de la glucosa en ratas resistentes a la insulina mediante la captación ósea de fluoruro; sin embargo, este aumento del fluoruro óseo no se manifiesta en deterioro óseo.

Resumo

Quando grandes quantidades de flúor são consumidas, a resistência à insulina é produzida, mas a realização do exercício pode reverter esse efeito em ratos, devido a uma alta absorção de flúor pelo tecido ósseo. No entanto, a qualidade óssea não foi estudada. Por isso, o objetivo deste trabalho foi avaliar a qualidade óssea em ratos tratados com flúor que realiza exercício. Foi trabalhado com ratos Sprague-Dawley que foram divididos em 3 grupos (n=6 por grupo): Controle (recebeu água sem flúor), Flúor (recebeu água com flúor 15 mg/L durante 30 dias) e Ejercicio (realizou exercício diariamente em cinta ergométrica e recepção de água com flúor 15 mg/L por 30 dias). Luego, se mediu a densidade mineral ósea, as propriedades biomecânicas e histológicas e o nível de fluoruro óseo. Nenhum efeito do tratamento foi observado em nenhum parâmetro ósseo. Esses resultados indicam que o exercício normaliza o metabolismo da glicose em ratos resistentes à insulina por meio da captação óssea de fluoruro; Contudo, este aumento do fluoruro óseo não se manifesta na deterioração do óseo.

Palabras clave

ejercicio físico
flúor
huesos
resistencia a la insulina
Keywords

fluorine
bone and bones
insulin resistance
Palavras-chave

exercício físico
flúor
osso e ossos
resistência a insulina
==== Body
pmcCONCEPTOS CLAVE

Qué se sabe sobre el tema

El flúor es un elemento que se encuentran naturalmente en algunas fuentes de agua y cuando su ingesta es elevada tiene numerosas consecuencias para la salud. Afectando a huesos, dientes y también al sistema endócrino. Se sabe que la realización de ejercicio físico puede prevenir el efecto negativo de una ingesta moderada de flúor sobre la resistencia a la insulina.

Qué aporta este trabajo

Este trabajo muestra que la mayor captación ósea de flúor estimulada por el ejercicio no trae consecuencias negativas para el hueso pero si ayuda a revertir la resistencia a la insulina.

Divulgación

El Flúor comúnmente presente en las pastas dentales es un elemento que ayuda a prevenir las caries y se encuentra naturalmente en algunas fuentes de agua de consumo. Sin embargo, una ingesta elevada puede producir resistencia a la insulina. La realización de ejercicio físico puede revertir las consecuencias nocivas para la salud al estimular la captación de este elemento en los huesos sin causar daño en los mismos.

Introduction

Fluoride (F-) is a substance that has been used in the prevention and treatment of dental caries for many years. However, when large amounts are ingested, fluoride gives rise to a condition known as fluorosis 1 . Fluoride enters the body spontaneously or as a therapeutic resource. The main route of F- entry into the organism is drinking water. Several studies have documented the relationship between the concentration of F- in drinking water and fluorosis. Fluorosis is a public health problem that affect millions of people in the word 2 . For this reason, the World Health Organization (WHO) recommends a maximum concentration of 1.5 mg/L of F- in drinking water9.

Deleterious effects of F- are not limited to bones and teeth, several toxic effects on the endocrine system have been also reported 4 . The chronic ingestion of F- produces insulin resistance5-6, which was also observed in large parts of the world7-8 where F- content in drinking water is higher than the recommended by the WHO 9 . Conversely, physical activity improves the effect of insulin on target tissues 10 . Moreover, the performance of daily physical activity could reduce negative effects of chronic ingestion of F- on glucose homeostasis11-12. A decrease in insulin resistance was observed in rats that did physical activity and consumed water with 15 mg/L of F-. Normalization of insulin resistance by exercise could be the consequence of the decrease in plasma F- levels and increase in bone F- content due to stimulated bone formation by exercise 13 . A lower plasma F- level and higher bone F- content observed in trained fluoride-treated rats supports the previous hypothesis. On the other hand, harmful effects of F- on bone structure and quality have been reported when fluoride is consumed14,15. Therefore, the aim of this study was to evaluate bone quality and strength in rats that reversed fluoride-induced insulin resistance when performing exercise

Materials and methods

Experimental groups

Animals were kept in collective cages with water and balanced food (Gepsa, Pilar, Córdoba, Argentina) ad libitum, in a temperature-controlled environment of 23-25°C, with a 12h-12h light-dark cycle and filtered airflow. Experiments were conducted under international standard rules for animal care16 and has been approved by the Ethical Committee of the School of Medicine of Rosario National University (Nº 6739/2014, November 27 of 2014).

Eighteen 70-day-old female Sprague-Dawley rats (body weight 266.6±7.4g) were randomly assigned to treatments constituting three experimental groups (n=6 per group): Control (drinking water without F-), Fluoride (drinking water with F- 15 mg/L) and Exercise (daily running on a treadmill during 60 min at 2.25 m/min and drinking water with F- 15 mg/L)12. A group that realized exercise but drink water without fluoride was not include because the aim of this work is study the bone quality in rats that have insulin resistance consequence of drink water with fluoride. The concentration of 15 mg/L of F-was chosen to simulate the ingestion of artificially fluoridated water by humans, considering that rodents metabolize F faster than humans 17 . After 30 days of treatment, rats were euthanized by CO2 inhalation after isofluorane anesthesia 24 h after the last exercise session and with 12 h of fast. Femurs and tibias were obtained for mechanical testing, bone mineral density (BMD), histological analysis, and bone fluoride level measurements. Blood samples were obtained by heart puncture into heparinized tubes and plasma was processed for glucose, insulin, and fluoride measurement18.

Biochemistry measurement

Plasma glucose levels were measured with colorimetric technique using a spectrophotometer (Perkin Elmer lambda 11, Norwalk, CT. USA) and with a commercial kit (Wiener Laboratorios, Rosario, Argentina).

Plasma insulin levels were measured by RIA (radio inmuno assay) using a commercial kit (Ria kit Rat insulin, Millipore Corporation, Billerica, MA, USA) and with a solid scintillation counter Alfanuclear Cmos (Buenos Aires, Argentina).

Plasma glucose and insulin levels were used to calculate HOMA-IR index (homoeostasis model assessment - insulin resistance) according to:

HOMA-IR = fasting glucose level (mmol/L) x fasting insulin level (µUI/mL)/22.5

This index allows to evaluate insulin resistance, a high value of HOMA-IR index indicates insulin resistance.

Plasma fluoride levels were measured by direct potentiometry using an ion selective electrode ORION 94-09, Orion Research (MS, USA) after isothermal distillation; and for bone fluoride measurement, bone tissue was incinerated for 6 h at 550°C before distillation process12.

Mechanical testing

The cortical and trabecular bone strength of femurs was determined with a three-point bending test at midshaft, and a compression test in distal epiphysis19. The mechanical tests were performed on a mechanical testing machine designed by the engineering department of the Bone Biology Laboratory with a 300 N load cell with 0.01 N of discrimination and an accuracy of 10 µm in displacements. The support span in three-point bending test was 11 mm. The area of the circular compression platen was 7 mm2 and a 2.5 mm thickness transversal section of distal epiphysis of femurs was used. In both tests, the speed was 0.01 mm/s and was monitored on a computer. Load versus displacement plots were recorded by the software Biomedical Data Acquisition Suite 1.0, (Argentina, 2011) to determine bone properties. Ultimate load (N) was defined as the highest load and the fracture load (N) was recorded as the load when bone fractured. The stiffness (N/mm) was determined as the slope of the linear portion of the load versus displacement curve.

BMD measurement by X-ray absorptiometry

At the end of the experiment, bone mineral density (BMD, mg Ca/cm2) was measured by an Xray equipment (70 KV) simultaneously with an aluminium step wedge which was previously calibrated with known Ca concentrations18.

Measurements were done on digital images with ImageJ 1.40 software. The BMD was determined in the same places where biomechanical tests were performed. Trabecular BMD was measure in the epiphysis of femurs and cortical BMD were measure in diaphysis of femur. All measurements were blinded.

Bone Histomorphometry

The proximal epiphysis of the left tibiae was fixed in 10% phosphate buffered formaldehyde and decalcified in 10% EDTA before embedded in paraffin. Five-mm longitudinal sections were stained with hematoxylin & eosin. Bone histomorphometric analyses were performed in digital images obtained at a 40x magnification of proximal epiphysis. The following measurements were performed with ImageJ 1.40 software (NIH, Maryland, USA): (1) total tissue volume, TV (μm2); (2) trabecular bone volume, BV (μm2); and 3) trabecular bone surface, BS (μm). With these values, histomorphometrical variables were calculated: 1) bone volume, BV/ TV (%) = [BV100/TV]; (3) trabecular thickness, Tb.Th (μm) =[2/(BS/ BV)]; (4) trabecular number, Tb.N (1/mm) = [(BV/TV)/(Tb.Th)]; and (5) trabecular separation, Tb.Sp (μm)= [(1/Tb.N)-Tb.Th]. The 3D parameters from 2D measurements were assessed using standard stereology theory.

The analysis of trabecular interconnectivity was performed as previously published. The following parameters were measured: total number of nodes (Nd) and number of terminals (Tm). With these parameters, we proceeded to calculate an interconnectivity parameter known as node to termini ratio (R = Nd/Tm). The greater the value of R, the more connectivity the trabecular bone has 19 .

Statistical analysis

Results are shown as media ± Standard Error (SE). The comparisons between groups were made using one-way ANOVA, and LSD post-test. Differences were considered significant when p<0.05. Statistical analyses were performed with R 2.14.1 software.

Results

Insulin resistance increased after 30 days of fluoride consumption in the same way as previous work 12 (Figure 1).

Figure Nº 1 HOMA-IR of rats that received the different treatments. Data are shown as mean ± SE, Control, Fluoride and Exercise. Different letters indicates that the group are significantly different. p<0.05 one way ANOVA, and LSD post-test.

However, insulin sensitivity did not differ fluoride group might be due to lower plasma fluoride between Control and Exercise group. The decrease level (Figure 2) and higher bone fluoride level of HOMA-IR index in exercise group respect to. (Figure 3).

Figure Nº 2 Plasma fluoride levels of rats that received the different treatments. Data are shown as mean ± SE, Control (drinking water without F-), Fluoride (drinking water with F- 15 mg/L) and Exercise (daily running on a treadmill during 60 min at 2.25 m/min and drinking water with F- 15 mg/L). Different letters indicates that the group are significantly different. p<0.05 one way ANOVA, and LSD post-test.

Figure Nº3 Bone fluoride levels of rats that received the different treatments. Data are shown as mean ± SE, Control (drinking water without F-), Fluoride (drinking water with F- 15 mg/L) and Exercise (daily running on a treadmill during 60 min at 2.25 m/min and drinking water with F- 15 mg/L). Different letters indicates that the group are significantly different. p<0.05 oneway ANOVA, and LSD post-test.

Plasma fluoride level in fluoride group is two times higher than plasma fluoride level in exercise group; although the statistical test showed no significant difference, this difference may be biologically relevant and has been reported in previous work 12 . The lack of statistically significant differences may be the result of the great variation observed in the Control group. This variation could be the consequence of plasma fluoride level not being controlled by a homeostatic system and was reported in others works 20 .

Table 1 show the effect of treatments on bone properties. Bone quality was studied through the biomechanical properties of the bones. Both through two parameters, fracture strength and stiffness in two tests, the 3-point bending test which allows us to evaluate the properties of the diaphysis of a long bone which is mainly composed of cortical bone. In addition, the compression test of a slice of the proximal epiphysis of the femur was used to evaluate the biomechanical properties of the trabecular bone. In addition, the study was complemented with measurements of bone mineral density and a histomorphological analysis with parameters that evaluate the amount of trabecular bone (BV/TV, TbN, TbSp, TbTh) and a trabecular connectivity index (R). The variables were compared with a oneway ANOVA and F- consumption did not affect any of bone properties. These results indicate that the treatments did not modify the quality or quantity of bone.

Table Nº1 Effect of treatment on bone variables of rats. The table shows the mean SE of groups. Significant differences were not found. (One way ANOVA, p>0.05)

Variable/Factor	Control	Fluoride	Exercise	
Three-point test Ultimate load (N)	100.10 5.08	100.11 2.70	97.11 1.16	
Three points test Stiffness (N/mm)	238.98 27.08	259.87 7.05	271.73 34.3	
Compression test Ultimate load (N)	76.08 16.21	72.29 11.66	79.19 9.60	
Compression test Stiffness (N/mm)	422.72 55.45	440.06 87.92	334.37 55.64	
Cortical BMD (mg Ca/cm2)	16.8 0.69	14.3 0.42	15.9 1.33	
Trabecular BMD (mg Ca/cm2)	28.1 2.13	27.4 1.44	26.9 0.72	
BVTV	33.62 3.5	35.46 13.2	24.93 5.3	
TbN	7.66 0.4	10.24 4.4	9.60 4.07	
TbSp	0.082 0.004	0.064 0.03	0.085 0.04	
TbTh	0.044 0.005	0.042 0.03	0.028 0.005	
R	0.58 0.06	0.49 0.08	0.49 0.05	

The table shows the mean±SE of groups. Significant differences were not found. (One way ANOVA, p>0.05). BMD: bone mineral density, %BV/TV: bone volume, TbN: trabecular number, TbSp: trabecular separation, TbTh: trabecular thickness, R: interconnectivity parameter known as node to termini ratio.

Discussion and conclusions

Drinking water is the main source of fluoride; it contains varying amounts of fluoride depending on the region 21 . The WHO has set an upper limit value of 1.5 mg/L 3 . In large parts of the world, such as India, Iran, Pakistan, Tanzania, China, and the southwest of Buenos Aires and Santa Fe provinces of Argentina, the amount of fluoride in drinking water exceeds this limit 2 . The products used in the prevention of tooth decay constitute another major source of chronic ingestion of fluoride 22 .

The results presented in this paper come from rats that develop insulin resistance due to the ingestion of 15 mg/L F- in drinking water for 30 days 12 . Moreover, previous works demonstrated that 15 mg/L of F- for rats would be equivalent to 3 mg/L for humans because rats metabolize fluoride faster 16 . Consequently, the results found in this work could be extrapolated to people consuming water with 3 mg/L or more of fluoride, which consequently produces an average intake of 5-10 mg of F- per day.

Physical activity and chronic fluoride administration have antagonistic effects on insulin action4,10. Previous work had demonstrated that controlled exercise could be a tool to counteract insulin resistance induced by fluoride. However, normalization of the insulin resistance was accompanied by a high fluoride uptake by the bone 12 .

Physical exercise stimulates bone formation 13 which can be beneficial for bone, but fluoride have deleterious effects on it14,15. Therefore, in this work we evaluate bone properties of rats that received simultaneously F- in drinking water and daily physical training.

Bone biomechanical, histologic properties and BMD were studied, and no effect of treatment was found despite of the high content of F-. These results indicate that the increased uptake of F- by bone tissue in rats that perform exercise is not accompanied by loss of bone strength. Other works found decrease in trabecular bone area of the tibia, changes in bone metabolism markers, poorer connectivity, and less trabecular bone network in cancellous bone of rats treated with fluoride 15 . In this work we do not found change in bone properties. However, work that found effect in bone used high doses of fluoride 50mg/l 23 or administrated the fluoride by orogastric tube 15 . In previous work of our group 24 , we found that the bone effect of fluoride is different when fluoride is intake by orogastric tube or in drinking water. This may explain the difference between the results of this work and other studies. It is important to highlight that future studies that analyse longer periods of time are required to complete this study.

Exercise was effective in reduce the toxic effect of fluoride in brain 25 , liver 26 , kidney 27 and intestine 28 . In glucose homeostasis were found a reduction of plasma insulin level 12 , changes in protein expression in muscle and liver11,29. Moreover, changes induce for exercise in pharmacokinetics of fluoride were reported 30 . Exercise is a recommended practice for improve bone health 13 and reduce de insulin resistance 10 . Thus, an improvement in bone quality induced by exercise would be expected. Although this work does not report a bone benefit from exercise, other authors have found such an effect in mice treated with 100mg/l of fluoride 31 . It is important to note that no deleterious effects of fluoride ingestion were observed either. It is likely that the dose of fluoride used, or the exposure time was not sufficient to produce bone effects, although they were observed at the level of glucose homeostasis.

In summary, this work proposes that exercise is a useful mechanism for reduce the toxic effect of fluoride. Although the result of this work does not confirm this hypothesis other work found positive results11-12,25-28,31.

Physical activity is clearly not a solution for people who consume water with high concentrations of fluoride. The appropriate solution for these people is to change the source of drinking water, for instance having access to a reverse osmosis system supply or a new water source with an appropriate concentration of fluoride. However, without these possibilities, physical exercise would be very useful to reduce the toxic effects caused by fluoridated drinking water consumption.

Agradecimientos

We thank Ornela N Acciarri and Hilda S Moreno for technical assistance. We thank Wiener Lab for providing reactive kits.

Limitaciones de responsabilidad:

La responsabilidad del trabajo es exclusivamente de quienes colaboraron en la elaboración del mismo.

Conflicto de interés:

Ninguno.

Fuentes de apoyo:

This work was supported by Consejo Nacional de Investigaciones Científica y Técnicas, CONICET, (Grant PIP 122-200801-00462) and Agencia Nacional de Promoción Científica y Técnica, ANPCyT, (Grant PICT-PRH 50).

Originalidad:

Este artículo es original y no ha sido enviado para su publicación a otro medio de difusión científica en forma completa ni parcialmente.

Cesión de derechos:

Quienes participaron en la elaboración de este artículo, ceden los derechos de autor a la Universidad Nacional de Córdoba para publicar en la Revista de la Facultad de Ciencias Médicas de Córdoba y realizar las traducciones necesarias al idioma inglés.
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