The effect of an organophosphorus pesticide, chlorpyrifos (from different local sources), on the testicular tissue in adult male albino rats
Article information
Abstract
Chlorpyrifos is a widely used organophosphorus pesticide for agricultural purposes to control early disease in crops. Acting as an endocrine disrupting agent for male reproductive systems which leads to reproductive toxicity. Accordingly, this study aimed to investigate the potential mechanisms by which three commercial formulations of chlorpyrifos interfere with androgen receptor function and expression in male rats. The research was conducted according to the ethical guidelines established by the institutional animal care and use committee (IACUC), and the protocol received approval from Cairo University- Faculty of Science under approval number (CU/IF/12/23). Twenty-four male Wistar rats were equally allocated to four groups. The control group, chlorpyrifos groups orally received (17.43, 23.43, 21.40 mg/kg) for 28 days (5 days /week). The serum testosterone hormone was estimated, and the testes were collected, and fixed in 10% buffered formalin for histopathological and immunohistochemical examination. Results indicated that chlorpyrifos formulations caused a marked decrease in testosterone levels and downregulation of androgen receptor expression. Moreover, a significant reduction in tubular diameter, lumen diameter, and thickness of germinal epithelial cells was detected along with the Jonson score. In summary, exposure to the three chlorpyrifos formulations resulted in notable alterations in testosterone levels, decreased expression of androgen receptors, and compromised spermatogenesis, culminating in testicular damage and male infertility. Of the formulations assessed, chlorpyrifos-W was identified as the most effective disruptor of androgen signalling, demonstrating higher toxicity compared to the other formulations.
Introduction
The prevalence of male infertility in both humans and animals has been attributed to a variety of toxins and pesticides present in the environment. Environmental or occupational exposure to pesticides triggers the increasing incidence of male infertility, which is considered a great public and economic concern [1-4].
Chlorpyrifos (CPF) is a non-systemic organophosphorus insecticide with the formula [0,0–diethyl 22 0- 3,5,6-trichloro-2-pyridyl thiophosphonate], widely used for pest control through contact, ingestion, and inhalation. It is effective against various types of insects, including Coleoptera, Diptera, Homoptera, and Lepidoptera. It can be applied to soil or foliage for different crops. Additionally, it is used in the management of domestic pests, mosquitoes, and within animal cages [5]. According to the World Health Organization [6], it is a class II pesticide that has a moderate level of harm. Furthermore, the toxicity of the substance occurs by irreversibly blocking the acetylcholinesterase enzyme, which leads to the accumulation of acetylcholine in the cholinergic synapse. This buildup can stimulate acetylcholine receptors, or it can activate non-cholinergic pathways such as oxidative stress [7,8]. CPF was introduced to the Egyptian market under more than ten trade names, while the most common trade names used are Pyripan, Chlorzan, and Pestban [9]. Endocrine disruptors (EDs) are chemicals that interfere with normal hormone regulation by affecting hormone receptors and enzymatic pathways for hormone biosynthesis, bioavailability, or metabolism [10]. Excessive production and release of EDs into the environment are considered critical reasons for disrupting the physiological function of reproduction [11]. Previous evidence demonstrated that CPF acts as an endocrine disruptor by changing sex and reproductive hormone levels or altering male reproductive abilities and the expression of some steroidogenic enzymes [8, 12-14].
Elsharkawy et al. [15] found that administering male rats with chlorpyrifos significantly decreased the number of spermatogenic cells and the mean number of distinct germ cell types in all stages. Moreover, alternative changes in the interstitial tissue include necrosis in some tubules associated with edema and a fall in the mean number and nuclear volume of Leydig cells. Also, a drop in testosterone levels was noted. These results are in conjunction with impairment in the testicular antioxidant system.
An administration of CPF induces the generation of free radicals, which imply oxidative stress, which is considered an important mechanism for testicular damage via inducing mitochondria dysfunction, mitochondria-dependent apoptosis, and downregulation of steroidogenic genes [15, 16]. Male fertility is assessed based on the quantity and quality of sperm, proper functioning of Leydig cells, and a balanced hormonal profile. Various factors might lead to male infertility, including hormonal disorders, sexually transmitted diseases, anatomical issues, genetic factors, and environmental and lifestyle factors [17-19]. Irrefutably, proper reproductive activities are needed for critical cellular mechanisms. Therefore, the disruption of these cellular mechanisms leads to infertility [11]. Therefore, the present study was conducted to assess the reproductive toxicity of three commercial local formulations of chlorpyrifos pesticide (CPF-H, CPF-KZ, CPF-W) in male albino rats throughout male sex hormone, androgen receptor expression and architecture of testicular tissue.
Materials and Methods
Tested Compounds
Three commercial formulations of chlorpyrifos with the active ingredient, 48% emulsifiable concentrate (an organophosphorus group), were obtained from different local sources and were used in the current study:
First: Trade name Pyripan (CPF-H), obtained from El-Helb company, Egypt.
Second: Trade name Chlorzan (CPF-KZ), obtained from Kafr El-Zyat, company, Egypt.
Three: Trade name pestban (CPF-W), obtained from Watania company, Egypt.
Experimental Animals
A total of Eighty-Four healthy adult male albino rats (Rattus norvegicus) weighing 160 ± 10 g were purchased by the Department of Mammalian Toxicology, Central Agricultural Pesticides Laboratory, Agricultural Research Center, Egypt. The animals were housed in plastic cages with sawdust bedding, humidity (50% ± 10), and temperature (23 ± 3℃). The lighting period was controlled at a 12-hr light/12-hr dark cycle. Rats were given a normal diet and water ad libitum throughout the study. The experimental protocol was approved by Cairo University- Faculty of Science (CU/IF/12/23).
Acute oral toxicity study
The present study was carried out to define the oral median lethal dose (LD50) of three commercial formulations of Chlorpyrifos from different local sources using Weil's method [20]. Four different ascending concentrations of each formulation were used [CPF-H (54.25, 65.10, 78.125, 93.75 mg/kg), CPF-KZ (78.125, 93.75, 112.5, 135 mg/kg), CPF-W (78.125, 93.75, 112.5, 135 mg/kg)]. Animals were observed for signs of toxicity during the first 24 hours after administration of three formulations, and mortalities were recorded throughout 14 days.
Sub-acute toxicity study
Experimental design
Following a two-week accommodation period, a total of twenty-four animals were randomly categorized into four equal groups, as described below: control group, orally given distilled water, and the second (CPF-H), third (CPF-KZ) and fourth (CPF-W) groups orally received 1/4LD50 ((17.43, 23.43 and 21.40 mg/kg b.wt) from three formulations (CPF-H, CPF-KZ, CPF-W), respectively) for 28 days (5 days/week). The animals were continuously monitored, and their weight was measured twice a week throughout the entire trial period. Based on this data, the dosage was changed as needed.
The sub-acute dose (representing ¼ LD50) for each commercial formulation was diluted in water and used for dosing throughout the experimental period (fresh preparation daily).
Collection of samples and processing of tissues
After the experiment's 28 days, blood samples were taken from fasted rats and left to clot at room temperature. The blood samples were then centrifuged at a speed of 3600 r.p.m for 20 min. The sera were separated and quickly frozen for hormone assay. Rat testes were dissected out, rinsed from blood in isotonic saline, and blotted dry with a piece of filter paper. Then, testes were weighed and fixed in 10% buffered formalin for histopathological and immunohistochemical examination.
Hormonal assay
Determination of Serum Total Testosterone Level
Serum total testosterone was determined according to the method of Auletta et al. [21] using a solid phase radioimmunoassay kit purchased from Diagnostic Products Corporation (USA).
Histological and immunohistochemical examinations
The testicular samples were subjected to a series of procedures, including washing, dehydration in increasing concentrations of ethanol, clearing in xylene, and ultimately embedding in pure paraffin wax. Serial sections of 5 μm thickness were cut with a rotary microtome and stained by hematoxylin and eosin stain. The photomicrographs were captured using AmScope light microscope Model Number, United States. At least two histopathologists independently reviewed the photomicrographs to minimize potential bias in the evaluation of the tissue samples. Tissue sections for staining and photomicrography were selected randomly to minimize selection bias. The stained sections were examined to evaluate tubular diameter, lumen diameter, and thickness of germinal epithelial cells in seminiferous tubules (30 sections/animal) [22] by ImageJ software (NIH Bethesda, USA). Moreover, the spermatogenesis was graded from 1 to 10 using Johnsen's mean testicular biopsy score [23] according to the following criteria: full spermatogenesis (10), many late spermatids with disorganized spermatogenesis (9), few spermatozoa (8), no spermatozoa, many early spermatids (7), few early spermatids, arrest of spermatogenesis at spermatid stage (6), no spermatids or spermatozoa with many spermatocytes (5), only a few spermatocytes, arrest of spermatogenesis at the primary spermatocyte stage (4), only spermatogonia (3), no germ cells, Sertoli cells only (2) and neither germ cells nor Sertoli cells, tubular sclerosis (1).
The immunohistochemical reaction of the androgen receptor was carried out by preserving the testicular sections (5 μm) in a 3% solution of hydrogen peroxide for 10 min to block the action of endogenous peroxidase. Continuously, the slides were incubated for 48 h at 4°C with Primary antibodies for androgen receptors (1:200 dilution). The primary and secondary antibodies used were purchased from [Manufacturer(s)], with the following lot/batch numbers. The slides were warmed in an incubator for 25 min at 37°C then they were washed in phosphate buffer saline three times for 2 min. The testes section was treated with a streptavidin-biotin peroxidase complex. The immunohistochemical color was obtained by adding the diaminobenzidine chromogen solution. The immunohistochemical expression of the androgen receptor was evaluated by measuring the intensity of the brown-colored stained area (25 measurements/group) using ImageJ software.
Statistical analysis
The data presented in this study were statistically evaluated by a computer program (SPSS. Version 15). All data were expressed as mean values ± Standard error (SE) for each group, and statistical analysis of differences between means was carried out using one-way analysis of variance (ANOVA). In case of a significant F-ratio, the post hoc Least Significant Difference (LSD) test for multiple comparisons was used to evaluate the statistical significance between groups at P<0.05 significance.
Results
Acute oral toxicity study
The calculated median lethal dose (LD50) of CPF formulations in male rats were CPF-H (71.13 mg/kg b.w), CPF-KZ (93.75 mg/kg b.w), CPF-W (85.58 mg/kg b.w.).
Testes weight
Oral administration of male albino rats with three formulations of chlorpyrifos (H, KZ, W) showed a significant decrease (F= 9.46) in testes weight in group two (CPF-H) by (7.43%) and four (CPF-W) by (17.57%) while a non-significant decrease in group three (CPF-KZ) (4.73%) when compared with the control group (Table 1).
Effects of Sub-acute exposure with chlorpyrifos for a 28-day day on testes weight, histomorphometry parameters and Jonson score.
Serum testosterone level
Concerning male sex hormones, the results revealed a marked decline (p 0.05) in serum testosterone level in all experimental treatments CPF-H, CPF-KZ, and CPF-W (F=88.72) by (58.78%), (90.76%), and (93.14%), respectively, when compared with the control group. Notably, a significant decrease was observed in the testosterone level in group four (CPF-W) compared to the two and third exposed groups, as shown in Figure 1.
Effect of chlorpyrifos formulations on serum testosterone level and androgen receptor expression immunoactivity in testicular tissue. Each column represents mean ± SE. The letters a, b, c, and d denote significant change versus Control, CPF-H, CPF-KZ, and CPF-W groups, respectively. Significant differences (P<0.05).
Immunohistochemical analysis
The staining intensity of AR in the control group is expressed by (4.379 ± 0.344) in the Leydig cells. Moreover, the expression of AR was reduced in the Leydig cells of rats treated with the three CPF formulations. Notably, significant decrease in the number of positive cells of androgen receptor (F=37.96) in all CPF-formulation groups with mean intensities of 2.158 ± 0.204 (CPF-H), 1.893 ± 0.159 (CPF-KZ) and 1.224 ± 0.109 (CPF-W), as compared with the control group. From our results, the CPF-W formulation revealed a marked decrease in androgen receptor expression when compared with the other two CPF formulations, CPF-H and CPF-KZ, as shown in Figure 1&2.
Photomicrograph of AR expression in interstitial cells (Leydig cells). Control (a). Chlorpyrifos formulations exhibited a significant decrease in positive cells of AR expression in Leydig cells (b (CPF-H), c (CPF-KZ), d (CPF-W)). Sclar bar 20μm. Photomicrographs were taken at 600 magnification.
Histopathological examination
Light microscopic analysis
The testes of control rats showed normal histological findings of seminiferous tubules and interstitial tissue. Each tubule was lined with stratified germinal epithelium. Healthy spermatogenic cells include spermatogonia, spermatid, and spermatozoa. Notice the presence of interstitial cells Leydig. In comparison, the treated groups with CPF formulations showed different degrees of testicular tissue damage that appeared as disorganized seminiferous tubules, shrinkage, and decreased lumen diameter, as well as a significant loss in the spermatogenic cell lineage. Remarkably, a set of seminiferous tubules in CPF-formulations groups have reduction and disruption of the germinal cells, which causes a decline in the height of germinal epithelial cells and irregular tubular outline.
Moreover, Absence of spermatozoa in some of the seminiferous tubule's lumen. Within the lumen of certain seminiferous tubules of CPF formulations, injured spermatogenic cells (Sloughed germ cells) were found falling. Concerning the inter-tubular connective tissue, a small number of interstitial cells were observed in CPF formulation groups, and there was an increase in interstitial width when compared with the control group (Figure 3).
photomicrographs of H&E-stained testicular section from control and CPF-treated groups. Control (a & b) showed normal histological structure of mature, active seminiferous tubule (ST) with complete spermatogenic series lined with stratified germinal epithelium (G), spermatogonia (bifid arrow), spermatid (hollow arrow), spermatozoa (S) and Leydig cells (arrow). CPF-formulations (CPF-H, CPF-KZ, CPF-W) (c-h) exhibited disorganized seminiferous tubules and a significant loss in the spermatogenic cell lineage (double head arrow). A set of seminiferous tubules were degenerated, and no spermatozoa in the seminiferous tubule's lumen. Within the lumen of certain seminiferous tubules, injured spermatogenic cells (Sloughed germ cells) were found falling (triangle). In the inter-tubular connective tissue, CPF formulations treated rats demonstrated fewer interstitial cells (arrow) and an increase in testicular inter-tubular space (*). Photomicrographs were taken at 240 (a, c, e, g) and 600 (b, d, f, h) magnification.
Histomorphological analysis and Johnson score
As Recorded in Table 1, the administration of three formulations of chlorpyrifos resulted in a significant decrease (F=22.28) in tubular diameter in formulated groups (CPF-H, CPF-KZ, CPF-W) by (10.81%, 17.77%, 24.49%), respectively. as compared with the control value. Moreover, luminal diameter was significantly reduced (F=130.39) in treated groups by (6.38% CPF-H, 12.62% CPF-KZ, 19.63% CPF-W), (p< 0.05) when compared with the control group. Notably, a marked decrease (96.37) in the thickness of germinal epithelia was recorded in all experimental treated groups by (23.31% H, 32.28% KZ, 38.45% W) as compared with the control group. Concerning the Johnson score, the results illustrated a significant decrease (F=19.84) by 20.08%, 27.60%, and 30.18% in the H, KZ, and W groups, respectively, when compared with the control group (Table 1). In addition, the morphometric analysis of testes in CPF formulation groups exhibited CPF-W as more effective than the other two formulations (CPF-H&CPF-KZ).
Discussion
Extensive use of pesticides under uncontrolled applications induces serious environmental pollution and health hazards for human and animal models, as well as disruption in the male reproductive system, which may affect male fertility.
The obtained results show a significantly decreased testes weight of chlorpyrifos formulation groups as compared with the control group. This reduction may be due to a diminished tubular size, which is confirmed by testis histopathological findings, spermatogenic arrest, and inhibition of steroid biosynthesis of Leydig cells [9, 16].
Testosterone is the principal androgenic hormone that is essential for the growth and function of testicular tissue, sexual differentiation, maturation, and development of male sex organs. A reduction in androgen bioavailability and production leads to a decrease in testes' weight and separation and disordered germ cells of seminiferous tubules. Finally, it may cause testicular dysfunction [24,25].
Our results are consistent with Hazarika et al. [26], who reported that an administration of CPF induced a marked reduction in sperm count along with a decline in serum testosterone level. This disruption may be due to the molecular interaction of CPF and some of its degradation products with androgen receptors. In this respect, the molecular docking of CPF with AR display of the binding interaction involved 23 AR residues while the native ligand involved 22 AR residues, in comparison between docking analysis for CPF and native androgen with AR, exhibited that 19 AR residues were common between CPF and native ligand. As well as there are four residues which are important for the interaction between CPF and androgen receptors. The work of Jung et. al. [27] reported that CPF is considered an AR antagonist thereby suppressing AR homo-dimerization in the cytosol depending on AR binding affinity. Lastly, it led to an endocrine-disrupting effect. Furthermore, the CPF bind with androgen receptors causing conformational changes in the AR, which lead to a downstream response of the androgen signaling pathway. So, CPF can act as a potent androgen disruptor and has a concurrent effect against the androgen ligand.
The reduction in testosterone level in our study may be due to the inhibitory effect of CPF by different mechanisms, such as a significant reduction in expression of some steroidogenic enzymes (3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase), downregulated expression of testicular StAR protein, a decline in sialic acid content in testicular tissue along with increase cholesterol level and diminished secretion of pituitary hormones (Luteinizing hormone (LH) and Follicle-stimulating hormone (FSH)) through inhibition of hypothalamo-hypophyseal axis [28]. The mode of action of CPF is crucial for the reduction of testosterone levels where CPF exposure inhibited acetylcholine esterase activity in the brain followed by disruption of neurotransmission in synthesis and/or release of gonadotropin (FSH&LH). This disruption blocked the testosterone biosynthesis pathway [29].
Evidence suggests that the administration of CPF in model animals induces the generation of free radicals, which triggers a decreased antioxidant defence system and induces DNA damage in different organs such as the liver, testes, brain, and kidney. Consequently, this oxidative stress plays an important role in inducing male reproductive toxicity, particularly alteration changes in testicular tissue [8, 9, 16, 30].
CPF administration excess ROS production which leads to altered permeability of the mitochondria membrane. This defect induces cytochrome c release in cytosol and activated caspase-3 which in turn initiates an apoptosis cascade and Bax to release apoptogenic proteins into the cytoplasm. On the contrary, a decline in antiapoptotic and antioxidant molecules (Bcl-2) which protect mitochondria membrane integrity [31-33]. This suggestion corroborates the work of Hassan et al. [14] who illustrated that CPF can induce cell death in testes and neurons thereby upregulation of caspase-3 expression, upregulation of Bax at the mRNA level and downregulation of Bcl-2. On the other hand, administration of CPF to male rats resulted in a significant MDA level and a decline in SOD and CAT activities in testicular tissue [34]. Likewise, excessive ROS accumulation after CPF exposure induces the hypothalamic-pituitary-adrenal axis to release cortisol thereby the crosstalk between HPG and HPA axes. The cortisol hurts LH secretion by the anterior pituitary. Subsequently, the reduction in testosterone production from Leydig cells [28]. The oxidative damage caused by CPF to Leydig cells results in a decreased number of Leydig cells where testosterone is synthesized. All the above agrees with previous studies which illustrated that CPF has anti-androgenic activity [12, 16, 35-37]. In our study, the reduction in interstitial cells and increase in interstitial space may be attributed to the decline of serum testosterone levels.
The androgen receptor is one of the important indicators for decreasing sperm count and increasing infertility [38]. However, the level of AR plays a critical role in the normal structure and function of testicular tissue where the interaction of androgen with androgen receptor regulates target gene expression, which is responsible for differentiation, development, and maintenance of male reproductive function [24].
Hazarika et al. [26] reported that CPF acts as a potent AR ligand that can interfere with the natural binding of endogenous androgen ligands, resulting in the dysfunction of AR signalling. Moreover, the metabolites of this pesticide can disrupt hemostasis and cause dangerous damage. The obtained results in our study revealed a marked reduction in the immune expression of AR in Leydig cells, which affects steroidogenic functions, causing the arrest of spermatogenesis at the round spermatid stage [39]. Jonson Score and morphometric analysis confirmed this observation. Lastly, the reduction in AR protein expression in treated rats with CPF formulations is associated with impairment of spermatogenesis.
Erthal et al. [40] clearly that malathion, an organophosphorus, induced downregulation of 17 β-HSD and androgen receptor expression genes in testicular tissue. The reduction in the number of Sertoli cells is in direct proportion to AR expression. In the same trend, the downregulation of the androgen receptor expression gene in our work may be due to the depletion in the number of Leydig cells which results from apoptosis in testicular tissue. These results were confirmed by [41] who reported that the two concentrations of CPF (5& 15 mg/kg) administrated to male rats for 25 days caused a decline in the number of Sertoli, Leydig, and germ cells.
According to growing data, subacute exposure to CPF formulation might have an antagonistic effect on androgen receptor expression due to chlorpyrifos's anti-androgenic properties. Moreover, the downregulation of androgen receptor expression in testes of chlorpyrifos formulation treatments correlated with a parallel decline in serum testosterone levels.
Previous studies revealed that CPF and its metabolites are harmful to animals. TCP (3, 5, 6-trichloro-2- pyridinol) is one of the important final metabolites of CPF and has the same structure of trichloropyridine structure as CPF. Moreover, it is more persistent than CPF in soil and aquatic systems, and TCP has an anti-androgenic effect and can participate in CPF-induced inhibition of steroid hormone receptor binding or related signalling pathways [42-45].
Gao et al. [46] point out that TCP contributes to CPF-induced paracrine functional damage in Sertoli cells. It also binds to the testosterone binding site of AR on Sertoli cells and inhibits the binding between testosterone and AR.
Reproductive organs have a high lipid content, so pesticides can be stored in them and cause testicular dysgenesis syndromes, such as atrophy of seminiferous tubules and germ cell degeneration [47].
Regarding histopathological and morphometric examination, our results, consistent with the previous studies, illustrated that organophosphorus pesticides can cross the blood barrier of testicular tissue, induce oxidative stress and lipid peroxidation, and cause damage to the testicular membranes, the degeneration of the spermatogenic epithelium and Leydig cells. This subsequently causes alteration changes in the structure of seminiferous tubules and results in disruption of spermatogenesis and reduction of sperm count [9, 35, 36, 48, 49].
Our results are by 16, 50. Both researchers revealed that CPF induced different degrees of testicular tissue damage that was observed as disruption of the germinal cells, a small number of sperm cells in the lumen, and degenerative changes in interstitial and Sertoli cells in treated groups.
In the same trend of result, Babazadeh and Najafi [51] reported that light microscope examination showed a marked decline in the number of Leydig cells, germinal epithelial height, tubular differentiation index, repopulation index, and spermiation index. These previous results lead to spermatogenesis arrest and cause male infertility. All the above results were confirmed by the decline of the Johnson score in CPF-treated rats, where the Johnson score is an indicator of the deficiency of spermatogonia, spermatocytes, spermatids, and spermatozoa in testicular tissue.
In connection with morphometrics, a marked decline in tubular diameter may be a result of germ cell layer disarrangement and decreases in the number of germinal cells [52]. As well as detached germ cells filling the tubular lumen, degeneration, necrosis of germinal cells, irregularity, and atrophy of seminiferous tubules lead to a significant decrease in luminal diameter and height of germinal epithelial. Moreover, the migration of sloughing of germ cells from disintegrated epithelium into the lumen caused a reduction in luminal diameter and a marked increase of intraepithelial empty spaces [53].
From the afore-mentioned results, Oral administration of three commercial formulations of chlorpyrifos to male rats induces male reproductive toxicity through the reduction of serum testosterone level, suppression of androgen receptor expression, and degeneration of seminiferous tubules by two different mechanisms. First, the endocrine disrupting properties of CPF, and second, the accumulation of CPF and its metabolites in testicular tissues, caused an elevation of ROS level, which leads to testicular oxidative injury after CPF formulation exposure. However, the degree of injury differs according to CPF formulations. Where the CPF-W formulation was more effective than the CPF-KZ formulation, which was more effective than the CPF-H formulation, this variation may be due to the manufacture of three formulations.
Conclusions
This study's results indicate that chlorpyrifos (CPF) formulations have considerable harmful effects on the male reproductive system. The effects encompass changes in testicular weight, disturbance of testicular histoarchitecture, and a reduction in male sex hormone levels, especially testosterone. Furthermore, we noted a downregulation of androgen receptor expression, suggesting that CPF disrupts androgen signalling pathways. These disruptions result in testicular injury and may cause male infertility following sub-acute exposure. Considering the possible reproductive toxicity of CPF, especially the stronger effects of the CPF-W formulation. These findings highlight the differential effects of chlorpyrifos products on the male reproductive system, with important implications for pesticide regulation and safety.
Notes
Conflict of interest
No potential conflict of interest was reported by the authors.
CRediT author statement
RAM: Conceptualization, Methodology, Software, Data curation, Writing- Original draft preparation, Validation, Visualization, Writing- Reviewing and Editing. HAA; Data curation, Formal analysis, Software, Investigation, Validation, Visualization, Writing–Original draft, Writing- Reviewing and Editing.