Humans are frequently exposed to pesticides either directly, as workers in agricultural and industrial environments, or indirectly, via the consumption of food (vegetables, fruits, and cereals) and contaminated water. Many of these toxic chemical products may disrupt human reproduction by interfering with the endocrine function either by mimicking, modulating, or blocking the synthesis and metabolism of reproductive hormones such as estrogen and progesterone in women and testosterone in men.
High levels of pesticide exposure may induce toxicity in testicular tissues, thus disrupting testosterone synthesis and sperm production and/or quality and ultimately could reduce male fertility. Consequently, it is not surprising that the number of scientific studies on the reprotoxicity of pesticides has increased exponentially over the past 20 years, particularly in industrial-agricultural countries [1
Bifenthrin (BF) (2-methylbiphenyl-3-ylmethyl-(z
)-(1RS)-cis-3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropane carboxylate) is a type I synthetic pyrethroid used extensively against a broad spectrum of insect pests in agriculture and horticulture. As a potential food contaminant, BF can be commonly ingested by humans. Many recent experimental studies have demonstrated that BF is genotoxic, carcinogenic, neurotoxic and exhibits immunosuppressive and reprotoxic effects in a range of mammalian species and even in humans [2
]. In addition, BF has been reported to induce oxidative stress in human erythrocytes as well as mouse hepatic cells [4
] and to induce apoptosis in murine macrophages [3
] and human hepatocarcinoma cells. However, scientific knowledge on the mechanism by which BF induces reprotoxicity in in vivo models is currently limited to two main reports [5
Based on these studies, it has been proposed that the toxic effects of BF on reprotoxicity can occur through the production of high levels of mitochondrial reactive oxygen species (ROS), causing oxidative damage to various cellular components [4
]. Abnormal ROS production can induce detrimental chemical and structural modifications to sperm nuclear DNA as well as protein damage in sperm plasma and mitochondrial membranes [7
]. Mammalian sperm plasma membranes are extremely susceptible to lipid peroxidation induced by ROS because they have a high content of polyunsaturated fatty acids and insufficient antioxidant defense mechanisms [1
]. Therefore, the body and specially the testis tissues need additional strategies to defend against excessive ROS production induced by BF exposure. Nowadays, there is an increased demand for using dietary supplements in the prevention and treatment of ROS-related diseases.
Microalgae have attracted attention in the food industry and the principal genera used for functional foods are Chlorella
, and Spirulina
]. Spirulina platensis
(SP) is a unicellular cyanobacterium with a special composition of nutritional and bioactive substances (proteins, vitamins, minerals, pigments and phenolic acids, etc.) that are potential sources for a large range of medical applications [11
]. This cyanobacterium contains highly potent naturally antioxidant and free radical scavenging agents such as phycocyanin and beta-carotene [12
] that are well-known to protect against various diseases, such as renal failure and cancers [13
]. Therefore, it is not surprising that SP is generating growing interest in the scientific community because of its remarkable multi-organ protection property against many environmental toxic chemicals and heavy metal–induced toxic assaults [15
The aims of the present study were to (i) assess the reprotoxicity of BF in adult male mice using multilevel evaluations of testicular tissue including histology, oxidative enzyme activity monitoring and alterations of microRNA (miRNA) and mRNA expression levels and (ii) investigate the possible protective role of SP against the reprotoxicity effect induced by BF in this animal model of pathology. We provide a complete series of solid evidence that SP can be considered as a palliative treatment to protect against infertility in populations heavily exposed to pesticides.
2. Materials and Methods
2.1. Chemicals and Reagents
SP was isolated, identified and produced as described previously by Ben Amor et al. [11
]. All chemicals and reagents were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). The testosterone ELISA kit was purchased from BioVendor R&D (Brno, Czech Republic).
2.2. Evaluation of Physicochemical, Nutritional and Microbiological Qualities, and Antioxidant Activity of the Isolated SP
The physicochemical characteristics (pH, proteins, lipids, carbohydrates, sugars, fibers, minerals, etc.) and pigment content (chlorophylls, carotenoids, phycocyanins) of the SP dry powder were determined as described by Barkallah et al. [16
]. Concentrations of calcium, magnesium, potassium, sodium and iron were measured by atomic absorption spectroscopy (JY 38 S; Horiba, Montpellier, France). The fatty acid methyl esters (FAMEs) of total lipids were obtained by adding 500 µL of KOH (1N)–CH3
OH (2N) to the extracted lipids followed by a heating step of 10 min at 40 °C and the addition of 500 µL of n-hexane to the reaction mixture. The FAMEs in supernatants were then analyzed using gas chromatography (GC, Shimadzu GC-17A, Shimadzu Scientific Instruments, Columbia, MD, USA) and identified by comparison of their retention times with respect to pure standards of FAMEs purchased from Sigma and analyzed under the same conditions. FAMEs were quantified according to their percentage area, obtained by integration of the peaks. Vitamins were measured using the HPLC system method according to the Association of Official Analytical Chemists [17
]. The antioxidant activity of SP was evaluated according to the method described by Bersuder et al. [18
] using 1,1-diphenyl-2-picryl-hydrazil (DPPH) as indicator. Total viable bacterial count, mesophilic bacteria, yeasts and molds, and coliform group were enumerated (CFU g−1
) using the standard microbiological methods for the analysis of food [19
spp., and Staphylococcus aureus
were detected according to the methods established by Barkallah et al. [16
]. Metals in SP were determined using inductively coupled plasma-atomic emission spectrometry (JY 38 S; Horiba, Montpellier, France).
2.3. Animal Care
Approximately eight-week-old white Swiss male mice weighing 29 ± 3 g were obtained from provided by the Centre of Veterinary Research of Sfax, Tunisia. Experimental animal procedures were performed according to the recommendations of the European convention for the protection of vertebrate animals and in accordance with the Council Directive no. 2010/63/EU. Animals were kept in an air-conditioned room (22 ± 3 °C) with a relative humidity of approximately 40% and housed in stainless steel cages and subjected to a normal photoperiod (12 h dark/12 h light) before the beginning of the experiment. They were allowed free access to diet and water for one week during their acclimatization period and were daily observed to detect any source of suffering or abnormal behavior.
2.4. Experimental Protocol
After one week of acclimatization, the mice were randomly divided into four different groups of eight animals each. The first group of animals was administered physiological saline buffer (0.9% salt solution) and was used as a negative control group (C). The second group (BF) of animals was administered by oral gavage with BF at a dose of 5 mg/kg body weight on a daily basis during a period of 35 days. The third group (SP) of animals was also administered by oral gavage with SP but at a dose of 500 mg/kg on a daily basis over 35 days. The fourth group (SP + BF) of animals was administered with SP at a dose of 500 mg/kg/day, 2 h before BF administration at the same dose regimen as the second group of animals. These doses of BF and SP were selected based on our preliminary experiments (data not shown).
At the end of the treatment period, the animals of the different groups were weighed and sacrificed by cervical decapitation to avoid animal stress. The collected blood samples were left to clot at room temperature and then centrifuged at 3000 rpm for 15 min. Sera were then collected and stored at −20 °C for further biochemical analysis. The testes and epididymis were quickly excised from each animal, rinsed in ice-cold physiological saline buffer, and weighed to calculate the ratio of the organ weight to the body weight (%). Representative samples of testes were collected and fixed in 10% formalin solution for histological analysis. Other testes samples and the tails of the epididymis were used immediately for the analysis of different molecular and biochemical parameters and to study sperm parameters.
2.5. Sperm Collection and Analysis
Epididymal spermatozoa were collected by cutting the caudal region of the right epididymis into small pieces of approximately 5 mm and then incubated in 2 mL of pre-warmed physiological buffered saline (PBS) at 37 °C for 10 min to allow sperms to swim out. After a centrifugation step at 1600× g
for 15 min, supernatants were collected to evaluate the cell concentration, motility, viability and morphology of sperms by histological examinations. Briefly, sperm motility was analyzed microscopically by determining the number of all progressive spermatozoa from the total spermatozoa population. The final data were expressed as the percentage of sperm cells in each motility group as previously described by Kvist and Björndahl [20
Sperm viability was defined as the percentage of normal cells, according to the procedure described in the World Health Organization Manual [21
]. The study was assessed using the one-step eosin-nigrosin staining technique.
To evaluate the various abnormalities of spermatozoa, sperm suspension was stained with 0.2% final volume of eosin on slides before histological classification as previously described by Wyrobek and Bruce [22
2.6. Determination of Oxidative Stress Markers
Lipid peroxidation in the testicular tissue was estimated by measurement of malondialdehyde (MDA) content [23
]. Protein carbonyl contents were measured by using the spectrophotometric method [24
]. Testicular reactive oxygen species (ROS) levels were measured using DCF-DA (2′,7′-dichlorofluorescein diacetate) as fluorescent probe [25
]. The results were expressed as mean fluorescence intensity compared with values of the control group normalized to 100%. Nitric oxide production was determined based on the Griess reaction [26
]. The results were expressed as nmol/ 100 mg protein.
2.7. Determination of Non-enzymatic and Enzymatic Antioxidants in Testicular Tissues
The reduced glutathione (GSH) levels in testicular tissues were determined with Ellman’s reagent (DTNB; 5, 5-dithiobis-2-nitrobenzoic acid) as probe to quantify the total GSH content measured at 412 nm [27
]. The result was then expressed as µg mg−1
of tissue. Catalase (CAT) activity was assayed by the decomposition of hydrogen peroxide (H2
]. The enzyme activity was calculated as µmol H2
consumed/min/mg protein. Total superoxide dismutase (SOD) activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) [29
]. Results were then expressed as enzyme unit activity mg−1
protein. Glutathione peroxidase activity (GPx) was measured as previously described by Flohe and Gunzler [30
]. The final results were then expressed in terms of nmol GSH oxidized/min/mg protein.
2.8. Estimation of Testosterone in Sera
The testosterone level in mice sera was quantified using the Mouse Rat Testosterone ELISA Kit (BioVendor, Asheville, NC, USA) according to the manufacturer’s instructions.
2.9. Analysis of DNA Fragmentation
Testicular DNA samples of normal and experimental mice were isolated as previously described [31
2.10. Analysis of Gene Expression by Quantitative RT-PCR (qRT-PCR)
Extraction of total RNA from frozen testicular tissues was performed using the mirVana microRNA isolation kit with phenol according to the manufacturer’s instructions (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RNA integrity was assessed with the BioAnalyzer 2100 (Agilent technologies, Santa Clara, CA, USA). Samples with an RNA integrity number (RIN) superior or equal to 8 were considered for further analysis. The reverse transcription step was performed with the PrimeScript RT reagent Kit with gDNA eraser kit for the mRNA analysis while the TaqMan®
Advanced miRNA cDNA Synthesis Kit was used for the miRNA analysis (Applied Biosystems company, Thermo Fisher Scientific, Inc., Waltham, MA, USA). PCR products were generated from 100 ng of cDNA template using the QuantiFast SYBR Green PCR Master mix (Qiagen, Germantown, MD, USA) with specific forward and reverse primers to detect the expression of mRNAs or with a mixture of specific forward primers complementary to each miRNA mature sequence of interest used in combination with the universal qPCR reverse primer provided by the NCode VILO miRNA cDNA synthesis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The PCR primers used in this study are shown in Table S1
. All reactions were performed in triplicate on a Lightcycler®
480 Instrument II (Roche, Basel, Switzerland). Optimal q-PCR parameters were one cycle of 95 °C for 2 min followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. A melting curve analysis was performed using the following cycling parameters: 60 °C for 30 s and 5 °C temperature changes to the end temperature of 95 °C. For all samples, the mRNA expression level was normalized to the housekeeping beta-actin gene and to the snU6 level for quantification of mature miRNA. Finally, the relative expression levels of mRNA and miRNA were calculated using the standard 2−ΔΔCt
2.11. Protein Quantification
Total protein concentration was measured using pure bovine serum albumin (BSA) as standard [32
2.12. Testicular Histopathology
For the histological study, mice testes were removed and fixed in 10% buffered formalin. After routine paraffin processing, embedded testicular tissue samples were sectioned at 5 mm thickness using a Reichter 2040 Microtome (Medical Equipment Source, LLC; PA, USA) and stained with hematoxylin and eosin (H-E). The prepared sections were examined with a Leica® microscope fitted with a Sony® digital camera to capture images for the histological evaluation of testicular tissue alterations.
2.13. Statistical Analysis
All analytical determinations were performed at least in triplicate and values were expressed as the mean ± standard error of mean (SEM). One-way ANOVA and Tukey’s post-hoc multiple comparison tests were used to compare results with significant differences (p < 0.05). GraphPad Prism 6.0 for Windows (Graph Pad Software, San Diego, CA, USA) was used to perform all statistical analyses.
This study aimed to investigate the reproductive toxicity effect of BF in adult male mice using a multi-scale level of evaluation, starting from histological analysis of testicular tissues, biochemical measurement of oxidative stress and apoptosis markers to the quantification of the expression of targeted genes and miRNAs known to participate in spermatogenesis. The objective was to collect sufficient robust experimental evidence to gain insight into the underlying mechanisms of testicular toxicity induced by this pesticide. We hypothesized that the administration of SP extract, a microalga previously described as beneficial for other pathologies [13
], might also protect mice from the testicular toxicity induced by BF. The results presented here provide, for the first time to our knowledge, solid experimental evidence that the compounds in SP extract exercise a pleiotropic of defense mechanisms by almost completely reversing all the altered markers of testicular toxicity induced by BF. Regarding the urgent need for palliative treatments against the toxic effects of pesticides in general, overall, our study highlights the potential of SP extract as a palliative treatment to treat male fertility in countries exposed to BF.
We first found that the administration of BF for 35 days at a dose of 5 mg/kg bw significantly decreased the body weight of the animals with a pronounced effect on the weight of reproductive organs (testes and epididymis). We demonstrated that the reduction in testicular mass is correlated with a drastic decrease in epididymal sperm count, vitality, and motility, while the histological analysis of tissues revealed degeneration of the seminiferous epithelium, deterioration of germ cells, and shrinkage of seminiferous tubules.
The mechanism by which BF induces such drastic testes alterations is partially known and understood. Only two reports, both from the same group, have investigated this point—first in male offspring [5
] and second in adult mice [6
]. Results from these studies indicate that BF exposure leads to the overproduction of ROS, oxidative DNA damage, apoptosis of testicular cells, and change in intra-testicular testosterone production.
In our study, we confirm that BF exercises pleiotropic deleterious effects in testicular tissues, causing a reduction in sperm production. It is reasonable to speculate that lipid peroxidation as well as ROS induction in response to BF might be the primary source of tissue damage as reported elsewhere in other organs [4
]. In the testes, excessive ROS can have serious deleterious effects on many cellular components by reacting with polyunsaturated fatty acids to form lipid peroxides [33
]. Accumulation of the end products of lipid peroxidation may contribute to a decrease in testicular tissue integrity resulting in a loss of testes weight, apoptosis [4
], DNA fragmentation [34
], and reduction in the production of testicular androgens as evidenced in our study. We demonstrated that MDA, PCO, and NO levels in the testis were significantly increased in BF intoxicated mice, which might also contribute to testes alteration. We also observed depleted contents of GSH and down-regulated enzymatic activities of the redox system (SOD and CAT) in the testis, which reflects the inability of the antioxidant defense to manage the high levels of ROS produced by BF exposure. Consequently, the overproduction of ROS was accompanied by a significant change in the expression of mitochondrial apoptosis-related genes, p53 gene, and subsequent DNA fragmentation [34
]. These data are in line with those of other studies that have reported a change in p53 expression in murine organs after different synthetic pyrethroid treatments [35
]. We furthermore observed a drastic change in the transcription status of key genes related to testosterone synthesis such as P450scc
. Taken all together, these alterations explain well the reduction in sex hormones production, particularly testosterone, and decrease in sperm production, viability and motility that are the main causes of BF-induced infertility.
We then pursued this analysis further and investigated the hypothesis that BF might also alter the expression of specific miRNAs known to be crucial for spermatogenesis [36
]. We focused our attention on some miRNAs identified as biomarkers of male infertility such as miRNA-17, miRNA-34c, miRNA-34b, miRNA-449a and miRNA-449c [36
]. We found that the expression of miRNA-17, -34c, -34b, -449a, and miRNA-449c was significantly downregulated in BF-treated animals, while the expression of miRNA-122 was upregulated. Our results are in agreement with those of Hayashi and collaborators [37
], who showed that the disruption of miRNA-17 in the testes of adult mice resulted in severe testicular atrophy, empty seminiferous tubules, and depressed sperm production. This miRNA is also required for mouse primordial germ cell development and spermatogenesis [36
]. The observed downregulation of miRNA34b/c and miRNA-449 in the BF-treated mice is also in accordance with the results of Wu et al. [36
] who reported that reductions in these two miRNAs are causally associated with reduced fertility. Furthermore, Yuan et al. [38
] reported that miRNA-34bc/449-deficiency impairs both meiosis and the final stages of spermatozoa maturation. In contrast, the over-expression of miR-122a is reported [39
] to induce mitotic arrest and apoptosis of spermatocytes, which is also in agreement with the histopathology results of this study. Therefore, our study indicates that the deregulation in the expression of miRNAs might contribute as a consequence or a cause to the reproductive toxicity effect of bifenthrin in male mice. This deserves deeper investigation, notably to establish their diagnosis values.
We then investigated whether SP, known to exercise a beneficial effect in other pathologies [13
], might also provide positive outcomes as a protective and anti-infertility agent. The palliative effects of SP on body weight gain could be due to the fortification of the body by important nutrients such as proteins, vitamins, minerals and amino acids brought by SP. Interestingly, the administration of SP prior to BF significantly retrieved the testosterone level and the expression levels of genes related to cholesterol transport and testosterone synthesis. Our findings are in agreement with those of other authors, who showed that SP intake can effectively recover cadmium- and streptozotocin-induced deregulation of gene expression in the early and final steps of the steroidogenic pathway [40
]. The pre-treatment of mice with SP positively improved sperm quality parameters, as manifested by an increase in sperm motility and count. This improvement may be due to the richness of SP in zinc, which improves the activity of alkaline phosphatase enzyme in sperm [41
]. The pathological lesions induced by BF were also remarkably reduced by the pre-treatment with SP. The role of SP in modulating these testicular alterations may account for the antioxidant vitamin compounds of SP such as α-tochopherol (vitamin E) or ascorbic acid (vitamin C), which might improve testicular structure and function. Vitamin E could inhibit the peroxidation of cell membrane lipids by trapping lipid peroxyl (LOO−
) and many other radicals to help in counteracting oxidative damage and maintaining the levels of GSH and ascorbic acid in damaged tissues [42
]. The SP antioxidant effect has been linked to other bioactive molecules such as C-phycocyanin, β-carotene and chlorophylls [12
]. These pigments exert their antioxidant activities by increasing the endogenous antioxidant enzymes and scavenging a variety of reactive species such as superoxide and hydrogen peroxide radicals [15
]. SP also contains selenium, which is involved in the formation of glutathione peroxidase and other compounds such as selenocycteine and selenoglutathione that are known to counteract the toxic effects of pyrethroids [45
]. The ameliorative effect of SP was also accompanied by anti-inflammatory activity attested by a reduction in the expression of TNF-α and the production of NO in the testicular tissues of intoxicated mice. Polysaccharides of SP could also inhibit the expression of TNF-α, IL-1β, and COX-2 [46
], as we demonstrated in our study. A similar anti-inflammatory activity of SP was previously documented [47
]. Furthermore, SP counteracted the BF induced apoptotic events by attenuating the expression of the pro-apoptotic genes and ROS scavenging. A similar antiapoptotic effect of SP was previously documented [49
]. SP also regulated the expression of spermatogenesis and apoptosis miRNAs. This finding is in agreement with the results of Śmieszek and collaborators [50
], who showed that SP influenced the expression of apoptosis-related miRNAs and mRNA in Caco-2 cancer cells line.