Effect of fluoride-induced testicular alteration in rats fed a high-fat diet
Article information
Abstract
Previous research on the well-known environmental pollutant fluoride has demonstrated that fluoride exposure can lead to oxidative stress-related male infertility. Obesity is another public health issue that has a detrimental impact on male fertility. Previously, findings on fluoride toxicity in high-fat diet (HFD) conditions associated with oxidative stress have been evidenced. This study aimed to evaluate the impact of subchronic fluoride exposure (5 mg/kg) plus a HFD on testicular alteration in Wistar rats. Animals were divided into four groups (control, HFD, fluoride, and fluoride 5 mg/kg plus HFD). The HFD contained a 50% kcal increase in fat (saturated fat), after 90 days of co-exposure to fluoride plus HFD, the animals showed a significant decrease in the adiposity index. The co-exposed group showed oxidative damage assessed through decreased glutathione (GSH) concentration (p < 0.0001), increased concentrations of malondialdehyde (MDA) (p < 0.0001), and the oxidation of proteins (p < 0.0001) vs the control group. Finally, testicular histology exhibited a reduction in spermatogonia and spermatocytes. The results of the study indicate that under these conditions, subchronic co-exposure to fluoride under HFD conditions could protect against the accumulation of epididymal fat, however, oxidative alteration at the testicular level is maintained.
Introduction
Fluoride is ubiquitous in some geological settings and can accumulate to unsafe concentrations in groundwater due to geochemical processes. It is common knowledge that aquifers in igneous, volcanic, and geothermally active rocks contain naturally high fluoride concentrations [1] Fluoride concentrations depend on geographic area and range between 0.32 - 15.36 ppm in Asia [2]; 0.1 - 11.4 ppm in the Middle East [3]; 1.69 - 3.52 ppm in Southeast Europe [4]. Fluoride is widely known for its benefits to oral health [5]. However, since it has been demonstrated that a high quantity of fluoride in water may constitute a risk to human health, the World Health Organization (WHO) has advised against exceeding 1.5 ppm of fluoride in drinking water [6]. The demonstrated negative effects of fluoride exposure on health include female and male reproductive alterations [7, 8], kidney toxicity [9], neurotoxicity [10], and endemic dental and skeletal fluorosis [1]. As a result of its detrimental effects on human health, the fluoride concentration in groundwater is currently recognized as a significant concern. The population at risk of fluoride in drinking water at concentrations greater than 1.5 ppm is 63 to 330 million people [11]. Fluoride exposure is associated with elevated levels of phospholipids, total cholesterol, and triglycerides in the blood, according to previous research with experimental animals [12]. Recent studies using multi-omics analyses demonstrated that miRNAs induce hepatic lipid and glucose metabolism disorders due to fluoride exposure in mice [13]. Another study further suggests that exacerbation of obesity in mice exposed to fluoride in combination with a high-fat diet causes a leaky gut and modifies the microbiota, particularly the growth of Erysipelatoclostridium ramosum [14].
Regarding reproduction, numerous studies indicate that fluoride exposure negatively impacts male reproduction on multiple levels. In the testes, it modifies tissue architecture, the presence of cell apoptosis, the cell cycle, thereby altering the spermatogenesis process [15]. Moreover, multiple studies have demonstrated that fluoride exposure impairs sperm functions - including morphology, motility, maturation, acrosome reaction, capacitation, hyperactivation, and chemotaxis - and alters the gene expression profile, resulting in decreased fertility [16–19]. In parallel with the global decline in male infertility, obesity - a public health disease characterized by a high body mass index, excessive fat accumulation in adipose tissue, etc. has spread widely in recent decades across both developed and developing nations due to unhealthy changes in nutritional composition, excessive calorie intake, and a lack of exercise [20]. The obesity epidemic has reached pandemic proportions on a global scale. The prevalence of obesity has increased across all age groups, regardless of geography, ethnicity, social class, or geographic region [21]. In addition, alteration of various sperm quality parameters (such as sperm concentration, motility, viability, morphology, DNA integrity, and mitochondrial function), endocrine changes, systemic and reproductive system inflammation, and oxidative stress increase male infertility rates [22–24].
Currently, worldwide, a large population is exposed to unsafe concentrations of fluoride through drinking water, and, at the same time, the obesity epidemic has reached pandemic proportions, as a consequence of a high-fat diet (HFD) consumed by the population. Environmental fluoride exposure and an HFD are considered stress factors that cause oxidative response [25, 26], but their combined effect on reproduction has not been evaluated. The goal of this study is to evaluate the effect of an HFD on fluoride-induced testicular toxicity in male rat.
Materials and Methods
Chemicals
Buffer-phosphate saline (PBS), Bouin solution, bovine serum albumin (BSA), 2-4-dinitrophenylhydrazine (DNPH), 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), guanidine, ethyl acetate, ethanol, hydrochloric acid (HCl), sodium fluoride (NaF), guanidine, thiobarbituric acid (TBA), TISAB II were from Sigma Chemical Co (St. Louis, MO). Trichloroacetic acid (TCA) was from J.T. Baker (Phillipsburg, NJ).
Animals and Experimental Design
Wistar rats were purchased from the Bioterium of the Autonomous University of Hidalgo (Hidalgo, Mexico) under the guidelines of the Institutional Ethics Animal Care and Use Committee (CIECUAL), which had already approved the experimental techniques. The animals were housed under standard conditions, which included a 12h/24h light/dark cycle, a constant temperature of 22 °C, and a humidity level of 50 %. All animals had access to water and food. A standard laboratory rodent diet (Formulab Chows 5008, Purina Mills, St Louis, MO) was given to control groups, while 50 % of the calories given to HFD-groups [27]. The formulab Chows 5008 food contains 3.23 Kcal/g of metabolizable energy (3.1 % simple carbohydrates, 8.1 % lipids, 23.6 % proteins, 3.3 % fiber, 6.1 % minerals). The HFD was prepared as follows: 730 g of previously ground rodent food, 200 g of lard, and 100.9 g of egg white were weighed and mixed. 300 mL of deionized water was added to make 1 kg of food, then baked at 180 °C for 20 minutes.
Thirty-six rats were randomly distributed into four experimental groups: control group, HFD group, 5 mg/kg fluoride exposure group, and HFD plus 5 mg/kg fluoride exposure group. The control and fluoride-exposed group consumed rodent food. In rodents, fluoride is distributed and eliminated more rapidly than in humans; doses up to 5 times higher have been suggested in rodents to achieve comparable serum levels in the exposed population [28]. The dose used is equivalent to 42 ppm of fluoride/day, therefore, it is within the range of environmental exposures in the human population. This dose has been previously reported to be associated with environmental exposure level of fluoride for humans and also represents toxicologically significant to male reproduction in rats [16, 19, 29]. Animals were administered daily (in the morning, at 9:00 am), through by gavage at a subchronic dose of fluoride equivalent to 5 mg/kg or deionized water for the control group. Body weight was recorded during treatment and urinary fluoride concentration was measured on days 0, 30, 45, 60, 75, and 90. After 90 days of treatment, rats were sacrificed and the amount of body fat (visceral, retroperitoneal, and epididymal) was recorded to determine the adiposity index. Testicles were immediately removed and homogenized in PBS, for determinations of stress and oxidative damage a) glutathione (GSH), b) malondialdehyde (MDA), and c) protein carbonyls (PC). Also, the morphological changes of the testes were evaluated through histological analysis.
Glutathione concentration (GSH)
The procedure previously described by Moron et al. [30] was used to assess GSH in testicular tissue. The production of a yellow colour following adding 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) to substances containing sulfhydryl groups served as the basis for the assay. 250 mL of homogenates and 250 mL of 10 % trichloroacetic acid (TCA) were combined and vortexed. After being centrifuged at 2500 g for 10 min, the upper layers were removed and combined with reaction solutions containing 2 mL of PBS and 500 μL of 4 % DTNB reagent. A plate reader was used to measure the absorbance at 412 nm after 10 minutes. GSH concentrations were expressed in μmol per gram of protein.
Protein carbonylation (PC)
Protein carbonyl concentration was measured in testicular tissue homogenates using 2-4-dinitrophenylhydrazine (DNPH), according to a previously described method [31]. In 1.5 mL tubes, 100 μL of testicular homogenate was deposited, and 500 μL of 10 mM DNPH was added. The mixture was kept at room temperature with constant stirring and protected from light. The hydrazones produced were then precipitated using 500 μL of 20 % TCA, after which the samples were centrifuged for 10 minutes at 9000 rpm. The pellet was washed three times with 1:1 ethyl acetate/ethanol before being solubilized in 1 mL of 6 mol L-1 guanidine and centrifuged at 9000 rpm for 10 minutes after being incubated at 37 °C for 15 minutes. absorbance was measured at 361 nm and the data were reported in nmol/mg of protein, using a molar absorption coefficient of 22,000 M-1 cm-1. Protein concentration was measured using a standard curve with BSA.
Lipid peroxidation
Malondialdehyde concentration was determined following the procedure described by Buege and Aust [32]. We added 2 mL of the TBARS reagent (15 % TCA and 0.375 % TBA diluted in HCl) to 100 μL of the testicular homogenate. The sample was boiled for 45 minutes, then placed in an ice bath before centrifuging at 4000 rpm for 10 minutes. The supernatant absorbance was measured at 532 nm and compared to a blank containing 100 μL of PBS with 2 mL of TBARS reagent. The data were reported in μmol of MDA/g of tissue and were computed using a molar extinction coefficient of 1.56 105 M-1 cm1.
Fluoride urinary concentration
Urine samples were taken from the animals exposed to fluoride (both with and without the HFD diet) at 0 days, 30 days, and 90 days. Animals were kept in metabolic cages during the urine collection process, and urine was collected for 24 h to measure fluoride content by a potentiometric technique using an ion-selective electrode (Orion 9609). Urine and TISAB II (1:1) were combined for analysis. The electrode was calibrated between 0.01 and 10 parts per million (ppm) [33]. The reading was duplicated everywhere.
Histology analysis
The testes were dissected and placed in Bouin's fixative, dehydrated in progressively higher concentrations of alcohol, cleaned with xylene, and then embedded in paraffin wax. The paraffin-fixed tissue blocks were then cut into 5 mm sections and stained with hematoxylin and eosin. The testes of four rats per group were then examined under a light microscope and an HD HD camera (Olympus Corp., Center Valley, PA, USA) was attached. Twenty seminiferous tubules from each animal were examined as part of a histological examination that assessed spermatogenesis.
Statistical analysis
Statistical analyses were performed in GraphPad Prism Version 8 (GraphPad Software, San Diego, CA). A one-way ANOVA was followed by a Bonferroni correction to evaluate pairwise differences. A p-value < 0.05 was considered significant.
Results
After 90 days of co-exposure, the body fat content was recorded at different levels (Table 1). As expected, animals in the HFD group the amount of retroperitoneal, epididymal fat and in the adiposity, index were significantly higher (2.61-fold) in the HFD group vs control group (p < 0.0001). Unexpectedly, the accumulation of epididymal and retroperitoneal fat were significantly lower (1.5-fold and 1.84-fold respectively) in the co-exposed group to fluoride plus HFD vs HFD-only group (p < 0.0001); this finding was accompanied by a significant decrease in the weight of the animals co-exposed to fluoride and HFD (Figure 1). In the current study, we quantified fluoride concentration in urine at different exposure times. We observed a similar fluctuation in fluoride excretion, in the 46 days of exposure. On day 60, fluoride excretion increased significantly (p < 0.001), and on day 75, it decreased significantly (p < 0.001) vs fluoride-exposed group (Figure 2).
Effect of fluoride plus HFD co-exposure on fat corporal.
Body weight of male rats exposed to fluoride with HFD diet. Results were expressed as mean ± SEM (n = 6), and the one-way ANOVA statistical test was performed with its Bonferroni post-test; a vs Control p < 0.001; b vs HFD p < 0.001.
Urinary fluoride concentration during exposed for all groups. Control group, HFD group, Fluoride 5 mg/kg group, Fluoride 5 mg/kg plus HFD. Values are mean ± SD (n = 6 per group), and the two-way ANOVA statistical test was performed with its Bonferroni post-test; a vs Control p < 0.001, b vs HFD p < 0.001, c vs Fluoride p < 0.05.
To assess oxidative stress on fluoride co-exposure plus HFD, we evaluated GSH, MDA, and PC concentration as of stress and damage markers in testes. As shown in Table 2, the co-exposed group (fluoride plus HFD), glutathione levels decreased significantly (2.8-fold) vs control group (p < 0.0001) and 2.4-fold vs HFD group (p < 0.0001). We also examined lipid peroxidation and PC as a marker of oxidative damage. Lipid peroxidation increased significantly in testicles of animals co-exposed to fluoride plus HFD (5.1-fold, p < 0.0001) vs control group, with no significant differences when compared to the fluoride-exposed and HFD groups. In addition, the level of lipid peroxidation increased in testicular tissue (4.4-fold) in the group exposed to fluoride (p < 0.0001) and (6.1-fold) in the HFD group (p < 0.0001) vs control group. Finally, PC increased significantly in the testes of animals co-exposed to fluoride plus HFD 3.3-fold (p < 0.0001) vs control group, 3.1-fold vs fluoride-exposed group (p < 0.0001), and 2.1-fold compared to the HFD group (p < 0.0001).
Effect of co-exposure of fluoride plus HFD on concentrations of markers of stress and oxidative damage in testicular tissue.
As shown in Figure 3, the control-group testicles showed typical testicular and regular architecture morphology, including the outermost layer of Sertoli cells and spermatogonia, the middle layer of spermatocytes, and the innermost layer of spermatozoa, which are signs of normal spermatogenesis. However, we observed a reduction in the size and number of the seminiferous tubules and irregular tubules, as well as decreased spermatogonia and spermatocytes in the testes of rats co-exposed to fluoride plus HFD.
Testicular histology in rats exposed to fluoride plus HFD and control groups. Normal histology in control group, histology alterations in Fluoride 5 mg/kg plus HFD. Circles shows disruption of spermatogenesis. HE stain, light microscope × 400.
Discussion
We used an in vivo model to evaluate the effect of HFD on reproductive toxicity induced by subchronic fluoride exposure. This study showed that animals co-exposed to fluoride plus HFD showed a significant decrease in corporal fat accumulation. As previously indicated in the Methodology section, the experimental animals were co-exposed to fluoride plus HFD at the same time. Fluoride exposure has been demonstrated to affect lipid metabolism, promoting dyslipidemia and exacerbating obesity in previous studies in animal models [12–14, 34, 35], and it has been suggested that fluoride exposure may affect the accumulation of body fat in the population, particularly in the context of HFD [36]. However, it has also been shown that the effect of fluoride on lipid metabolism and novo lipogenesis depends on the duration of exposure and type of HFD. Advances in understanding a mechanism to explain how fluoride interferes in lipid metabolism, which explains the decrease in fat index in animals when an HFD is combined with fluoride is a decrease in de novo lipogenesis that increases the hepatic steatosis[37]. However, because fluoride increases the expression of GRP78, a chaperone that regulates endoplasmic reticulum homeostasis and inhibits the apolipoprotein ApoE, [38] cholesterol transport decreases, reducing cholesterol and LDL levels[37]. A limitation of our study is that we did not evaluate histology in other organs including the liver and lipoproteins.
No other study has evaluated the kinetics of fluoride elimination in the co-exposure fluoride plus HFD; in our study, we observed a fluctuation in fluoride elimination during the 90 days of exposure. This fluctuation could influence the availability of fluoride in soft tissues and, consequently, its toxicity. In this study, we observed that from day 60 on, there is a tendency to decrease fluoride excretion, which would consequently increase its accumulation in tissues (hard and soft). Additional research is require to further investigate fluoride accumulation in an HFD.
To our knowledge, this is the first study to highlight the effect of fluoride plus HFD co-exposure on the testes. In this study, we evaluate oxidative stress, which refers as an imbalance between the generation of reactive oxygen species and the antioxidant defense system [39]. We observed a significant decrease in GSH concentration and a significant increase in MDA and PC concentrations in testes from animals exposed to fluoride plus HFD. These results correspond with previous studies that show that oxidative stress is one of the mechanisms underlying the reproductive toxicity of both fluoride [29, 40] and obesity and their related negative effects on the male reproductive system [24, 41]. Therefore, co-exposure of fluoride plus HFD causes oxidative stress and testicular alteration that could affect reproductive capacity. Recently, a study showed that reducing epididymal fat improves lipo-testicular toxicity and the blood-testicular barrier [42]. In this study, co-exposure of fluoride plus HFD significantly decreased testicular fat suggesting that the observed oxidative stress effect is mainly caused by fluoride exposure. Authors previously suggested that oxidative stress could play a significant role in changes in lipid metabolism; fluoride exposure with an HFD induces oxidative stress through an increase in GRP78, ERp29, an endoplasmic reticulum stress-inducible protein and superoxide dismutase-2 (SOD2), which in turn, due to a reduction in ApoE, decreases acetyl-CoA precursors that lead to a decrease in the formation of hepatic triglycerides [26]. Notably, chronic fluoride exposure elevates the expression of peroxisomal and mitochondrial proteins that are involved in fatty acid metabolism, thereby promoting their beta-oxidation-mediated catabolism [38]. Fluoride accumulation in testes induced oxidative stress by increasing oxidative status and a decrease in SOD1 activity and DNA damage in spermatozoa, considered dose-dependent markers of fluoride toxicity [43]. The findings of this study suggest considerable implications for populations exposed to these dietary conditions, showing how diet and exposure time influence fluoride toxicity.
In addition, fluoride exposure has been linked to pathological disorders and infertility in testicular tissue, as well as histological abnormalities in the testes of experimental animals [19, 44, 45], implicating lipid peroxidation, inflammation, and apoptosis as mechanisms of toxicity [46–49]. Another mechanism of fluoride toxicity in the testes is the induction of autophagy by fluoride exposure. This cell survival mechanism is activated under stressful conditions by triggering apoptosis in Sertoli cells [15]. According to a previous study, an unrestored arrest at the haploid stage of spermatogenesis may be caused by fluoride exposure, presumably through gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) and related regulatory proteins [50].
Conclusions
Subchronic exposure to fluoride under HFD conditions causes a decrease in epididymal fat, however, the oxidative damage induced by fluoride remains in the testes of experimental animals. This finding might reflect the various mechanisms activated by fluoride, so further studies are needed to understand the molecular mechanisms of fluoride exposure and HFD on testicular toxicity.
Acknowledgements
Authors thank to Álvaro Rubén Hernández-Cruz for helping with histological analysis. We also thank CONAHCYT for Itziar Hernández-Martínez maintenance scholarship.~
Notes
None to declare.
CRediT author statement
MSG: Conceptualization, Investigation, Methodology; Supervision, Writing- Reviewing and Editing; IHM: Conceptualization, Investigation, Methodology, Software; EOMS: Visualization, Investigation; KFFE: Investigation, Methodology; JAIV: Conceptualization, Investigation, Methodology, Supervision, Writing- Reviewing and Editing.