Sex-Specific Cypermethrin Induced Hippocampal Neurotoxicity Is Via the Modulation of Signaling Molecules for Antioxidant Defense, Membrane Integrity, Apoptosis and GABAergic Integrity

doi:10.21203/rs.3.rs-4974116/v1

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Sex-Specific Cypermethrin Induced Hippocampal Neurotoxicity Is Via the Modulation of Signaling Molecules for Antioxidant Defense, Membrane Integrity, Apoptosis and GABAergic Integrity

https://doi.org/10.21203/rs.3.rs-4974116/v1

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Pesticides, despite their intended use against pests, can have detrimental effects on non-target organisms, including humans. Its main route of exposure in urban areas is through dietary intake of fruits and vegetables. Oxidative stress, apoptosis and compromise to GABAergic interneurons’ integrity can be attributed to cypermethrin-induced neurotoxicity. The hippocampus, a vital region for memory and learning is particularly susceptible to the neurological toxicity caused by cypermethrin. Sixty adult male and female rats were grouped into control and cypermethrin-treatment groups. The treatment groups received oral dosages of 6.25mg/kg and 12.5mg/kg respectively for fourteen consecutive days. The daily weight was noted. The study was carried out on the hippocampal CA1 and CA3 regions of the rats using biochemical markers: GnRH, Na+/K + ATPase, COX-2, PG-E2 and immunohistochemical markers: Nrf2, CC3, BCL-2, parvalbumin and H&E. Cypermethrin caused a compensatory increase in body weight of the low cypermethrin group and decrease in body weight due to increased dose. Cypermethrin toxicity caused brain weight decrease which was seen more prominently in female high cypermethrin group. Using the biochemical markers, cypermethrin caused neuroinflammation and disrupted the normal functioning of the reproductive hormones and cell membrane; it was more prominent in females. Using the immunohistochemical markers, cypermethrin induced oxidative stress, apoptosis and compromise to the GABAergic interneuron integrity. The female rats expressed higher neuroprotection which can be attributed to estrogen and its signaling pathways.

The findings of the study shows that there are dose and sex-specific mechanisms may be involved in cypermethrin neurotoxicity, highlighting the importance of considering sex differences in toxicity studies.

Cypermethrin

Oxidative stress

apoptosis

GABAergic interneurons

hippocampal CA1 and CA3

Pesticides plays an important role in agricultural products as modern agricultural production is dependent on large quantities of chemical pesticide [1, 2]. Around the world, more than 1,000 pesticides are used to prevent pests from destroying or harming food [3]. While pesticides are advantageous when it comes to crop production, their persistent nature and bio-magnification can have major negative effects when used excessively [4]. Pyrethroids have been shown to cause neurotoxicity by different mechanisms, such as inflammation, oxidative stress, death of neuronal cells, and dysfunction of the mitochondria [5]. Exposure to cypermethrin causes changes in neurotransmitter levels, specifically GABA, as well as motor impairments and convulsions [6]. In Nigeria, many households involved in agriculture rely on inorganic fertilizers, with the common ones being: Atrazine, Chlorpyrifos, and cypermethrin [7].

Cypermethrin, a type II synthetic pyrethroid is among the most frequently detected pesticides in food with the main route of pesticide exposure in urban areas being through dietary intake of fruits and vegetables [8]. It acts on the nervous system and disturbs neuronal function by modulating sodium channels [9]. Cypermethrin can cause hepatotoxicity, nephrotoxicity and neurotoxicity 10. Cypermethrin is associated with oxidative stress as shown by increased oxidative stress markers on exposure to cypermethrin [11]. The generation of reactive oxygen species which leads to oxidative stress can result in cellular dysfunction and apoptosis [12]. Cypermethrin has also been shown to cause neurotoxicity by modulating the level of Gamma-aminobutyric acid, GABA [4]. Compared to other brain regions, the hippocampal region is more susceptible to pesticide-induced neurotoxicity, exhibiting more profound changes in cellular bioenergetics and elevated oxidative stress [13]. According to recent studies, the blood-brain barrier of the hippocampus may be more delicate than that of other brain regions and more vulnerable to disruptions in the transport processes that maintain homeostasis [14]. The effect of cypermethrin is dose-dependent, duration-dependent and based on sex [10].

`Sex is a determining factor and biological variable in susceptibility to environmental toxicants like pesticides [15]. There is debate over whether or not steroids contribute to sex differences in resilience or toxicity; the key, though, is understanding the mechanisms that mediate the protective versus toxic effects 15. Studies on experimental animals have shown that androgens can increase neurotoxicity following an insult, but the same androgens can also be neuroprotective [15]. For instance, the hormone estradiol has long-lasting effects on brain development that can vary from sex differences to a widespread trophic and neuroprotective effect. Given this information, this research proposes to determine the sex differences in the neurotoxicity caused by cypermethrin, focusing on oxidative stress, apoptosis, and GABAergic interneurons in the Hippocampus.

Ethical Approval

This research was approved by the University of Ilorin ethical review committee (UERC) (UIL/UERC/11/46KA072), following the recommendation of the College of Health Sciences ethical review committee, in compliance with the Institutional Animal Care and Use Committee (IACUC).

Animals and experimental design

Forty-eight (48) adult male and female Wistar rats with an average weight of 180 ± 200 g were obtained from the University of Ilorin Biological Garden, Ilorin. They were housed in cages and fed with standard laboratory diet and water ad libitum, in the animal holding unit of the Faculty of Basic Medical Sciences, College of Health Sciences, University of Ilorin, Ilorin. The rats were exposed to a 12 h light/dark cycle at room temperature for 7 days before the commencement of the experiments. All rats were handled in accordance with the standard guide for the care and use of laboratory animals.

Treatment plan

The animals were randomly divided into 3 groups of males (n=8) and female (n=8) rats as described below:

Control - received Corn oil (1 ml/kg.bw orally) daily

Low CP - received oral ingestion of CP (6.25 mg/kg.bw) daily

High CP - received oral ingestion of CP (12.5 mg/kg.bw) daily

Animals were treated for a period of fourteen (14) consecutive days

Daily Body Weight Measurements

The body weight of the rats was measured and recorded on the first day of the arrival of the rats into the animal housing facility of the college of health sciences, university of Ilorin before the commencement of acclimatization. After the administration began, their weights were measured and recorded daily throughout the duration of the experiment. Their brain weight was also measured and recorded immediately after removal. A weighing scale was used to measure the weight.

Animal sacrifice

24 hours after the last exposure, the rats were euthanized with intramuscular ketamine (10 mg/kg). The brains of rats designated for biochemical evaluations were removed and weighed. Hippocampal tissues (from Bregma –10 mm to –15 mm) of five brains from each group was grossed out, dipped in 30% sucrose solution for further processing.

Animals designated for immunohistochemistry study were perfused transcardially with normal saline followed by 10% buffered formal saline. The brains were excised and stored in specimen bottles containing 10% buffered formal saline.

Biochemical Analysis:

Hippocampal tissues were homogenized in 30% sucrose and centrifuged at 2500 rpm for 10 minutes. The supernatant was then collected in tubes for the analysis of the biomarkers bellow.

All biochemical markers where assayed with markers’ specific and highly sensitive kits: Prostaglndin-E2 (PG-E2), GnRH, COX-2,

The procedure for all the assays is summarized below with modifications following the specific instructions on the kits:

Gonadotropin Releasing Hormone (GnRH) Analysis

The brain tissues were homogenized to prepare them for GnRH analysis, a similar process to that of the Interleukin-6. After homogenization, the concentration of GnRH in the brain tissue homogenates was measured using a commercially available ELISA kit, following the manufacturer’s protocol. All reagents, samples, and standards were brought to room temperature before use. The GnRH standard was reconstituted and serial dilutions were prepared to generate a standard curve. The wells of a 96-well microplate were coated with anti-GnRH capture antibody and incubated overnight at 4°C. After washing the plate to remove unbound antibodies, the wells were blocked with a blocking buffer (typically containing bovine serum albumin) for 1 hour at room temperature to prevent non-specific binding. 100 µL of standards, controls, and tissue homogenate samples were added to the appropriate wells. The plate was incubated for 2 hours at room temperature, allowing GnRH present in the samples to bind to the capture antibodies. After washing the plate to remove unbound materials, a biotinylated anti-GnRH detection antibody was added to each well. The plate was incubated for 1 hour at room temperature. Following another wash step, a streptavidin-HRP conjugate was added to the wells and incubated for 30 minutes at room temperature. This step facilitates the binding of the HRP enzyme to the biotinylated detection antibody. After washing the plate to remove any unbound enzyme conjugate, a tetramethylbenzidine (TMB) substrate solution was added. The plate was incubated in the dark for 15-30 minutes at room temperature. The substrate reacts with HRP to produce a colorimetric signal. The reaction was stopped by adding a stop solution (typically 1N H2SO4), changing the color from blue to yellow. The optical density (OD) of each well was measured using a microplate reader at a wavelength of 450 nm, with a reference wavelength of 570 nm. A standard curve was generated by plotting the OD values of the GnRH standards against their known concentrations. A four-parameter logistic (4PL) curve-fitting model was used for accurate quantitation. The concentration of GnRH in the brain tissue homogenates was interpolated from the standard curve. The results were expressed in pg/mg of tissue protein, calculated by normalizing the GnRH concentrations to the total protein content in each homogenate, determined using BCA assay.

Sodium-Potassium Adenosine Triphosphatase (Na⁺/K⁺-ATPase) Analysis

The brain tissues were homogenized to prepare them for Na⁺/K⁺-ATPase activity analysis. A homogenization buffer containing 250 mM sucrose, 10 mM Tris-HCl (pH 7.4), and 1 mM EDTA was prepared to preserve the integrity of the membrane-bound enzymes during homogenization. Approximately 100 mg of brain tissue was placed in a homogenization tube with 1 mL of homogenization buffer. The tissue was homogenized using a mechanical homogenizer until a uniform suspension was obtained. The homogenate was then centrifuged at 10,000 x g for 15 minutes at 4°C. The supernatant containing the soluble proteins was carefully collected and stored at -20°C until further analysis. The activity of Na⁺/K⁺-ATPase in the brain tissue homogenates was measured using a colorimetric assay based on the release of inorganic phosphate (Pi) during the ATP hydrolysis reaction. A buffer containing 50 mM Tris-HCl (pH 7.4), 130 mM NaCl, 20 mM KCl, and 3 mM MgCl2 was prepared. ATP solution was prepared at a final concentration of 3 mM. Aliquots of the brain homogenate (50 µL) were added to microcentrifuge tubes. The reaction was initiated by adding 200 µL of ATPase assay buffer and 50 µL of ATP solution to the homogenate. The tubes were incubated at 37°C for 30 minutes. The reaction was stopped by adding 200 µL of ice-cold 10% trichloroacetic acid (TCA) to each tube. The samples were then centrifuged at 1,500 x g for 10 minutes to remove precipitated proteins. The Pi released during the ATP hydrolysis was measured using a colorimetric assay. To the supernatant (500 µL), 1 mL of freshly prepared ammonium molybdate reagent (containing 10 mM ammonium molybdate in 1 N H2SO4) was added, followed by 100 µL of reducing agent (containing 1.5% ascorbic acid). The mixture was incubated at room temperature for 10 minutes to allow color development. The absorbance of the blue complex formed was measured at 700 nm using a spectrophotometer. The amount of Pi released was quantified by interpolating the absorbance values from a standard curve generated using known concentrations of Pi. The Na⁺/K⁺-ATPase activity was expressed as µmol Pi released per mg of protein per hour (µmol Pi/mg protein/h). The total protein content in each homogenate was determined using BCA assay, and the enzyme activity was normalized to the protein concentration

Prostglandin-E2 (PG-E2) Assay

After harvesting the brain, the brain was dissected to get the hippocampal region. The hippocampus was homogenized in a lysis buffer. The PG-E2 levels were analyzed using ELIZA with a monoclonal anti-Prostaglandin E2 antibody against PG-2, which was performed in triplicate and validated using quality control measures to ensure accuracy and reliability. PG-E2 was extracted from the samples using solid-phase extraction. The data was analyzed using calibration curves to calculate the concentration of PG-E2 in the samples.

PGE2 levels in the brain tissues were quantified using a PGE2 EIA kit (Enzo Life Sciences). Standards were in the range of 2500–39.1 pg/ml. Briefly, the brain tissue was homogenized in 200 μl of homogenization buffer (0.1 m phosphate buffer, pH 7.4, containing 1 mm EDTA and 10 μm indomethacin), mixed with 1 ml of 100% ethanol, and kept on ice for 30 min. Samples were centrifuged at 3000 rpm for 10 min at 4°C and transferred to an empty tube, and the ethanol was evaporated under a constant stream of nitrogen. Samples were then resuspended in 250 μl of assay buffer, vortexed, and then allowed to rest for 5 min. This was repeated twice more. PGE2 levels were then measured according to manufacturer's instructions. Sample PGE2 concentrations were then normalized to the wet weight of the piece of tissue analyzed.

COX-2 (Cyclooxygenase-2)

COX-2 (Cyclooxygenase-2) is an enzyme involved in the inflammatory response and is often used as a biochemical marker in various contexts, particularly in cancer research and inflammatory diseases. Here’s a summary of the process of using COX-2 as a biochemical marker:

Hematoxylin & Eosin Staining

Tissue sections were stained with H&E to express general morphology. Slides were laid on 60-degree warm hot plate for few seconds and submerged in in xylene for 5mins, twice for deparaffinization. They were dipped in decreasing grades of ethanol (100%, 90%, and 70% respectively) for 5 mins each for rehydration, followed by a 5 min incubation in Hematoxylin solution for nuclear staining. The slides were washed with running water for 5 mins, dipped in 1% HCl in 70% alcohol for few seconds and dipped in ammonia water and then in ionizing water for varying period. Sections were counterstained in eosin solution for 10 mins, washed in water for 3 mins, then dehydrated in ascending grades of alcohol (70%, 90%, 100%) for 3 mins each, and cleared with two changes of xylene for 5 min each, before cover slipping.

Immunohistochemistry

To demonstrate the expressions of glia activation, oxidative stress and apoptosis associated proteins and inhibitory GABAergic interneurons in the hippocampal CA1 and CA3 sections following exposures CPM, the remaining 3 rats from each group were perfused with 10% buffered formalin, their brains removed and processed for immunohistochemical staining with anti-NRF2, anti-Caspase 3, anti-BCL2 and anti-PV respectively in the hippocampus.

Briefly, sections of paraffin embedded hippocampal tissues were deparaffinized with xylene, rehydrated through descending grades of ethanol (100%, 95%, 70% ethanol), and treated for protein un-masking in citrate-based solution (heat-mediated antigen retrieval), pH 6.0 (Vector^®, Burlingame, CA, USA) for 30 minis in a steamer. They were washed in PBS for 2 mins, treated with 0.3 % hydrogen peroxide solution in PBS for 10 mins to achieve endogenous peroxidase blocking, washed again in PBS for 2 mins and then incubated in 2.5% normal horse serum for 20 mins for protein blocking. The ssections were then incubated for 2 hours at room temperature in primary antibodies: Rabbit polyclonal to Nrf2 (Abcam, USA, ab31163) at 1:100, Rabbit polyclonal Cleaved Caspase-3 (Cell Signalling, Asp175) at 1:200, Goat polyclonal Iba-1 (Abcam, USA, ab5076) at 1:250, Goat polyclonal BCL2 (Santa Cruz, USA, 2E1: sc-33673) at 1:150 and Rabbit polyclonal PV antibody (Novus Biologicals, USA, NB120-11427) at 1:1000, followed by 5 minutes wash in PBS. Sections were incubated in ImmPRESS™ HRP Anti-Rabbit, anti-Goat or anti-Mouse IgG (Peroxidase) Polymer Reagent, made in horse (Vector^® Labs, USA), for 30 mins. They were washed in PBS for 5 mins twice, DAB Peroxidase (HRP) Substrate Kit (Vector^® Labs, USA) was added to develop color, rinsed well in tap water, and then counter-stained in hematoxylin. Lastly, the sections were dehydrated through ascending grades of ethanol (70%, 95%, 100%), cleared in Xylene, mount with Permount (Fischer Scientific, USA), and reaction sites were visualized as a brown staining.

Microscopy and Photomicrography

The sections were finally examined under an AmScope 40X-2500X LED Lab Compound Microscope and photographed using the AmScope 5.0 MP USB Still Photo & Live Video Microscope Imager Digital Camera 5MP, manufactured by iSCOPE corp., USA. The images were captured at 100X magnifications as indicated in micrographs and exported to JPEG image format.

Image Analysis

The images were analyzed using image J software. Manual cell counting and staining intensity were done using this software.

Cell Counting

Using image J, images were opened and the grid size (30000-50000 pixel²) was chosen for comfortable counting of X400 magnified images. The multipoint tool in the tool bar is selected. Counting was done manually by identifying and clicking on the specific cell types to count. The total count was exported to excel for further analysis.

Staining Intensity

Image is opened using the Image J software and converted to RGB gray scale stack which splits image into three channels/ images (Red, Green, blue). “Threshold” option was selected from the image tool in the tool bar, best separation image was selected and the image was threshold to adequately pick areas of staining. The images were quantified using mean gray value. The areas of staining interest were analyzed and result was then exported to excel for further analysis.

Statistical Analysis

The result was expressed as mean with standard deviation. Statistical analysis was performed using two-way analysis of variance (ANOVA) using Graph Pad prism 8.0 software. The difference was considered as statistically significant when p= ≤ 0.05.

High Dose, Not Low Dose of Cypermethrin Caused Weight Loss Comparably in Both Sexes

In both male and female rats exposed to cypermethrin, an appreciable weight gain in male (6.8%), and female (29.9%) was recorded in the low dose exposed rats, while the exposure to high dose cypermethrin resulted in a marked weight loss in both male (16.2%) and female (0.8%) compared to the control rats. The observed weight loss where more apparent in the male rats compared to the female (Fig. 1A). A higher brain weight (4.9%) and relative brain weight (18%) was recorded in the male rats exposed to low dose cypermethrin, while exposure to high dose caused both reduced brain weight (2.6%) and relative brain weight (11.5%) respectively (Fig. 1B and C).

Figure 1: Sowing Body weight changes (A), Brain weight (B) and Relative Brain weight (C) of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Dose And Sex-Specific Effects of Cypermethrin on Sex, Inflammatory and Membrane Biomarkers in Brains

Compared to the control, there was a 16.4% decrease and 14.3% increase in GnRH levels in the brain of low dose and high dose cypermethrin exposed rats respectively. On the other hand, low dose cypermethrin caused an increase in the GnRH concentration (14.4%), which is comparably higher than the 4.7% increase in GnRH level in the brain of the exposed rats (Fig. 2A).

In contrast to the GnRH concentrations, cypermethrin exposure caused a dose-dependent depletion in the concentrations of Na+/K+ ATPase (P=0.0178) in the female rats, with the most effect recorded in the brain of the high dose exposed rats (Fig. 2B). In contrast to the effects of cypermethrin on the Na+/K+ ATPase in the female rats’ brain, there was no obvious changes observed in the male rats (Fig. 2B).

On PGE-2 concentrations in the brains of the exposed rats, there was a dose-dependent statistical reduction in the males (P=0.0295 and P=0.0008 respectively) translating to 49.2% and 76.7% depletions, and in the females (P=0.0110 and P=<0.0001 respectively) translating to 49.2% and 76.7% depletions (Fig 2C). In comparison to the Na+/K+ ATPase concentrations following cypermethrin exposure, there was a dose-dependent depletion (5.6% and 10.1% respectively) in the concentrations of COX-2 in the female rats, with the most effect recorded in the brain of the high dose (P= 0.0141) exposed rats (Fig. 2D). In contrast to the effects in the female rats’ brain, there was no obvious changes observed in COX-2 concentrations in the male rats (Fig. 2D)

Figure 2: Graphical representation of GnRH (A), Na+/K+ ATPase (B), PGE-2 (C) and COX-2 concentrations in the brains of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Neuronal Integrities in The Hippocampal CA1 and CA3 Regions of Cypermethrin Exposed Rats

Compared to the male control, there was a decrease in number of neural cells in the hippocampal CA1 of the cypermethrin exposed rats. There was a dose-dependent 27.3 and 46.5% decrease in the mean number of neural cells in the hippocampal CA1 of the low dose (27.3) (P= <0.0001) and high dose (46.5) (P= <0.0001) respectively. Comparably in the female rats, there was an observable loss of neuronal cells in the CA1 region following low dose (13.5%) (P=0.0009) and high dose (18.1%) (P=<0.0001) cypermethrin exposures (Fig. 3A and B).

The effects of cypermethrin on the integrities of CA3 neurons is comparable to what is reported in the CA1 region. High dose cypermethrin caused a significant 19.6% loss mean number of neural cells (P= 0.0347) in the hippocampal CA3 of the male rats, and a 18.70% significant loss in the mean number of neural cells in the female rats (P= 0.0090) (Fig. 4A and B).

Figure 3: (A) Image representation (B) Graphical representations of the distribution of H&E in the hippocampal CA1 of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Figure 4: (A) Image representation (B) Graphical representations of the distribution of H&E in the hippocampal CA3 of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Cypermethrin Induced Oxidative Stress on The Hippocampal CA1 and CA3 Via Nuclear- Factor Erythroid 2- Related Factor 2 Modulation

In males, there is a statistical difference between CG and LCG (P= <0.0001), CG and HCG (P= 0.0159), and LCG and HCG (P= 0.0063). In females, there is a statistical difference between the CG and LCG (P= <0.0001), CG and HCG (P=<0.0001), and LCG and HCG (P= 0.0010).

Compared to the male CG, there was a decrease in the expression of Nrf2 in the LCG and HCG. There was a 64.7% and 35.1% decrease in the expression of Nrf2 in the hippocampal CA1 of the LCG and HCG respectively. Compared to the female CG, there was a substantial increase in the expression of Nrf2 in the LCG and HCG. There was a 132.7% and 72.8% increase in the expression of Nrf2 in the hippocampal CA1 of the LCG and HCG respectively. The female LCG had a higher level of Nrf2 expression amongst the cypermethrin-treatment groups. (Fig. 5)

In the CA3, there is no statistical sex difference but there is statistical difference amongst the groups. In males, there is statistical difference between the CG and LCG (P= 0.0083). In females, there is statistical difference between the CG and LCG (P= 0.0260).

Compared to the male CG, there was a decrease in the expression of Nrf2 in the LCG and HCG respectively. There was a 41.3% and 40.7% decrease in the expression of Nrf2 in the hippocampal CA3 of the LCG and HCG respectively. Compared to the female CG, there was an increase in the expression of Nrf2 in the LCG and HCG respectively. There was a 42.30% and 12.6% increase in the expression of Nrf2 in the hippocampal CA3 of the LCG and HCG respectively. The female LCG had a higher level of Nrf2 expression amongst the cypermethrin-treatment groups. (Fig. 6)

Figure 5: (A) Image representation (B) Graphical representations of the expression of Nrf2 in the hippocampal CA1 of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Figure 6: (A) Image representation (B) Graphical representations of the expression of Nrf2 in the hippocampal CA3 of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Cypemethrin Induced Apoptosis on The Hippocampal CA1 and CA3 Using Cleaved Capsace 3

There is no statistical sex difference. There is a statistical difference among the female groups. In females, there is a statistical difference between the CG and LCG (P= 0.0002), CG and HCG (P= <0.0001).

Compared to the male CG, there was an increase and slight decrease in the level of apoptosis in the LCG and HCG respectively. There was a 26% increase and <1% decrease in the level of apoptosis in the hippocampal CA1 of the LCG and HCG respectively Compared to the female CG, there was an increase in the level of apoptosis in the LCG and HCG respectively. There was 80.40% and 86.90% increase in the level of apoptosis in the hippocampal CA1 of the LCG and HCG respectively. The female HCG had a higher level of apoptosis amongst the cypermethrin-treatment groups. (Fig. 7)

Figure 7: (A) Image representation (B) Graphical representations of CC3 positive cells in the hippocampal CA1of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Expression Of Cleaved Caspase Three (CC3) In the Hippocampal Ca3 of Exposed Animals.

There is no sex-statistical difference and there is no statistical difference amongst the groups.

Compared to the male CG, there was an increase and slight increase in the level of apoptosis in the LCG and HCG respectively. There was a 24.4% and 5% increase in the level of apoptosis in the hippocampal CA3 of the LCG and HCG respectively compared to the female CG, there was an increase in the level of apoptosis in the LCG and HCG respectively. There was 43.5% and 47.3% increase in the level of apoptosis in the hippocampal CA3 of the LCG and HCG respectively. The male LCG had a higher level of apoptosis amongst the cypermethrin-treatment groups. (Fig 8)

Figure 8: (A) Image representation (B) Graphical representations of CC3 positive cells in the hippocampal CA3 of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Expression Of B-Cell Lymphoma - 2 (Bcl-2) In the Hippocampal CA1 of Exposed Animals.

There is no statistical sex difference but there is statistical difference amongst the groups. In males, there is statistical difference between the CG and LCG (P= 0.0003), CG and LCG (P= <0.0001).

Compared to the male CG, there was a decrease in the expression of BCL-2 in the LCG and HCG. There was a 35.6% and 33.9% decrease in the expression of BCL-2 in the hippocampal CA1 of the LCG and HCG respectively. Compared to the female CG, there was an increase in the expression of BCL-2 in the LCG and HCG. There was a 14.2% and 5.8% increase in the expression of BCL-2 in the hippocampal CA1 of the LCG and HCG respectively. (Fig. 9)

Fig 9: (A) Image representation (B) Graphical representations of the expression of BCL-2 in the hippocampal CA1 of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Expression Of B-Cell Lymphoma - 2 (Bcl-2) In the Hippocampal CA3 Of Exposed Animals.

There is statistical sex difference (P= 0.0072). There is no statistical difference among the groups.

Compared to the male CG, there was a progressive decrease in the expression of BCL-2 in the LCG and HCG. There was a 21% and 28.9% decrease in the expression of BCL-2 in the hippocampal CA3 of the LCG and HCG respectively. Compared to the female CG, there was an increase in the expression of BCL-2 in the LCG and HCG. There was a 30% and 24.2% increase in the expression of BCL-2 in the hippocampal CA3 of the LCG and HCG respectively. (Fig. 10)

Fig 10: (A) Image representation (B) Graphical representations of the expression of BCL-2 in the hippocampal CA3 of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Expression Of Parvalbumin in The Hippocampal CA1 of Exposed Animals.

There is statistical sex difference (P= <0.0001). There is statistical difference among the groups. In males, there is statistical difference between the CG and HCG (P= <0.0001), LCG and HCG (P= <0.0001). In females, there is statistical difference between the CG and LCG (P= 0.0041) CG and HCG (P=0.0091), LCG and HCG (P= 0.0001).

Compared to the male CG, there was an increase in the expression of parvalbumin in the LCG and HCG. There was a 9.2% and 72.4% increase in the expression of parvalbumin in the hippocampal CA1 of the LCG and HCG. Compared to the female CG, there was an increase and decrease in the number of parvalbumin positive cells in the hippocampal CA1 of the LCG and HCG respectively. There was a 15% increase and 22.7% decrease in the expression of parvalbumin in the hippocampal CA1 of the LCG and HCG respectively. (Fig 11)

Figure 11: (A) Image representation (B) Graphical representations of the expression of parvalbumin in the hippocampal CA1of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

Expression Of Parvalbumin in The Hippocampal CA3 of Exposed Rats.

There is no statistical sex difference. There is statistical difference among the groups. In males, there is statistical difference between the CG and HCG (P= 0.0048), LCG and HCG (P= 0.0141). In females, there is statistical difference between the CG and LCG (P= 0.0423) (Fig. 12B).

Compared to the male CG, there was an increase in the number of parvalbumin positive cells in the hippocampal CA3 of the LCG and HCG. There was a 3.8% and 53.4% increase in the expression of parvalbumin in the hippocampal CA3 of the LCG and HCG respectively. Compared to the female CG, there was an increase in the number of parvalbumin positive cells in the hippocampal CA3 of the LCG and HCG. There was a 32.2% and 20% increase in the expression of parvalbumin in the hippocampal CA3 of the LCG and HCG respectively (Fig 12A&B).

Figure 12: (A) Image representation (B) Graphical representations of the expression of parvalbumin in the hippocampal CA3 of male and female rats exposed to corn oil, low dose of cypermethrin and high dose of cypermethrin

High dose, not low dose of cypermethrin caused weight loss comparably in both sexes

Male rats subjected to low doses of cypermethrin showed an increase in body weight, which can be explained by the body's compensating response or an adaptive response to the initial stress the pesticide caused [16-18]. However, the observed reduction in body weight of the male LCG could be attributed to cypermethrin cumulative harmful effects on several organs, resulting in metabolic disruptions and total weight loss [19]. The decrease in body weight in the cypermethrin treated groups could be as a result of low food intake, diarrhea and food avoidance [20]. The reduction in body mass suggests that cypermethrin causes significant toxicity, which could be brought on by the loss of protein, fat, and other macromolecules [21].

The changes in body weight is dose-dependent [10,22]. Shuklan et al. showed a decrease in the body weight of the cypermethrin exposed which was caused by a decrease in food consumption especially in the high dose treated groups. In the research they carried out the females were more affected, as they recorded a death from the female high dose treatment group though the dose and duration used in their study was much higher and longer than the one used in this study. The result from this research shows an increase in the body weight of both cypermethrin-treated groups in the female rats compared to control. The increased weight gain in females could be due to cypermethrin’s induced toxicity on the endocrine system, causing reduction in metabolism and fat storage. The body weight of the female HCG group decreased compared to the female LCG group. The body weight changes of HCG in both the male and female rats showed a decrease compare to the LCG groups. This shows that an increase in dosage would result in a decrease in the body weight, hence the effect of cypermethrin on body weight is dose dependent.

Male rats treated to low doses of cypermethrin showed an initial rise in brain weight, which may be caused by the activation of antioxidant processes as a defense strategy against the pesticide's harmful effects. It could also be attributed to edema or inflammation, as cypermethrin is known to induce oxidative stress and neuroinflammation [6,9,11]. The increase in brain weight in the LCG male could also be attributed to up regulation of glial cells, such as astrocytes and microglia which can contribute to edema and weight gain. Astrocytes become activated and undergo morphological changes, including increased cellular proliferation and hypertrophy [17]. This increase in cell mass and size in the brain regions could contribute to the weight increase observed in the brain. The neurotoxic effects of pyrethroids seen in the HCG male may be due to neuronal loss or apoptosis, as studies shows that cypermethrin leads to neuronal loss [23]. It could also be the down regulation of BDNF which is important in neuron survival and growth [24,25].

The female more sensitive to the neurotoxicity of cypermethrin has showed by the decrease in brain weight. The higher brain weight seen in the HCG group may be due to compensatory mechanisms, such has up-regulation of antioxidant enzymes and neurotrophic factors which can mitigate the neurotoxic effects of cypermethrin. The changes in brain weight are inconsistent in works of literature, as some studies record an increase in brain weight on exposure to pyrethroids in the course of the experiments [16,17] and some a decrease in brain weight [21,26]. This research shows that the female brains were more affected by the neurotoxicity. The change in brain weight is both dose-dependent and sex-dependent.

Effect Of Cypermethrin on Sex-Specific Reproductive Hormones

The effect of cypermethrin on GnRH level in the brain is sex and dose-dependent. Cypermethrin is known as an endocrine-disrupting chemical [27-29]. Low doses of cypermethrin may disrupt the normal functioning of the GnRH neurons leading to decreased secretion of the hormone, which can be seen in the male rats. An increase in GnRH level in the high dose could be due to the body’s means of compensating for the neurotoxic effect of cypermethrin hence an increase in the level of GnRH. In the females, the increase in the GnRH level of the low dose could be attributed to the lipophilic nature of cypermethrin, allowing it to cross the blood-brain barrier, have access to the hypothalamus and stimulate GnRH release. The increase in the GnRH level of female can also be attributed to inflammation caused by cypermethrin [30]. Pyrethroids are endocrine disruptors that may have an impact on the hypothalamic-pituitary-gonadal axis [29]. This can have effect on the GnRH level through a negative feedback mechanism. Studies show that cypermethrin can cause reproductive toxicity due to its anti-androgenic properties [31], thereby limiting the production of sex hormones and stimulating the production of GnRH to produce LH and FSH to suffice for the deficit. Studies show that the sex hormones have neuroprotective properties [32,33], which will cause more production of GnRH.

Effect Of Cypermethrin on Membrane Integrities

Cypermethrin is known to affect neuronal function by altering ion channel activity leading to an increase in Na+ influx and K+ efflux, affecting homeostasis [34]. To counteract this, the neurons may up-regulate Na+/K+ ATPase activity in response to the change in homeostasis. In male rats, the increase in Na+/K+ ATPase could also be a response to protect cells against cypermethrin-induced oxidative stress and apoptosis, as studies show that Na+/K+ ATPase plays a role in regulating antioxidant defenses and preventing cell death [35,36]. The progressive decrease in the level of Na+/K+ ATPase activity in the female rats can be attributed to oestrogen. Cypermethrin exposure has been shown to affect oestrogen levels and disrupt oestrogen-mediated signaling pathways, which may contribute to the observed decrease in Na/K+ ATPase activity. The alteration in oestrogen levels can impair the neurons’ ability to maintain ionic balance. The progressive decrease in Na+/K+ ATPase activity with increasing cypermethrin suggests a dose-dependent disruption of neuronal function. This may lead to impaired synaptic transmission, neuronal excitability and neurodegeneration. The decrease in Na+/K+ ATPase activity in females could also be related to increased apoptosis and neuronal damage caused by cypermethrin [9]. The sex differences in this response may be attributed to differences in hormone regulation, neuronal structure and function between the males and females. Females are more susceptible to changes in Na+/K+ATPase activity caused by cypermethrin induction.

Cypermethrin Induced Neuroinflammation by Modulating PGE-COX-2 Pathway

The toxicity of pyrethroids causes the expression of pro inflammatory markers [37]. The progressive decrease of PG-E2 levels in both male and female rats shows a disruption in the prostaglandin-mediated signaling pathway. PG-E2 inhibits proinflammatory cytokines production thereby reducing neuroinflammation. In males, the slight increase of COX-2 in the low dose male represents a compensatory mechanism to maintain PG-E2 production. However, this increase is insufficient enough to counteract the decrease in COX-2 level due to cypermethrin’s direct inhibition of PG-E2 activity or expression. The compensatory mechanism can also be seen in the male high-dose group, which shows a higher level of PG-E2. The decrease in the COX-2 level in females can be attributed to hormonal regulation as oestrogen is shown to have a modulatory effect on the expression of PGE-2 and COX-2 expression. The decrease in COX-2 level further causes the decrease in the production of PG-E2 seen in the female rats. This agrees with other studies that shows that female rats are more susceptible to neuroinflammation. A highly oxidative environment can result from increased NADPH oxidase expression and activity, which can be induced by neuroinflammation [38,39]. The neuroinflammatory effect of cypermethrin is dose dependent and also sex dependent

Cellular Morphology in The Hippocampal CA1 and CA3 On Exposure to Cypermethrin

The progressive decrease in the number of neural cells in the hippocampal CA1 and CA3 of male rats is due to the neurotoxicity induced by cypermethrin [40,41]. This neurotoxicity leads to apoptosis, thereby reducing the number of viable neural cells. In females, there was an increase in the low dose due to the neuro-protection of the female hormones. There was also a decrease in the high dose of the females meaning that the toxicity effect of cypermethrin is dose-dependent in this case. There was a significant decrease in the neural cells in the high-dose group of cypermethrin-treated rats. This shows that the male rats are more prone to the neurotoxic effect of cypermethrin compared to the male rats. Loss of neural cells in the hippocampus can cause neurodegeneration, memory and cognitive impairment, neuroplasticity compromise, increased vulnerability to disease, and cellular stress in the brain.

Cypemethrin Induced Apoptosis on The Hippocampal CA1 and CA3 By Modulating Cleaved Capsace 3 And B-Cell Lymphoma - 2 (Bcl-2) Expressions

Cypermethrin can reduce the viability of cells and cause loss of neuronal cells [23,40,41]. The female rats were more susceptible to apoptosis with high expression of pro-apoptotic and anti-apoptotic marker. Due to pronounced cypermethrin-induced apoptosis, there was an up regulation of anti-apoptotic marker. The male rats were more affected at a lower dose, as seen by higher expression of pro apoptotic marker. The exposure to cypermethrin results in the inhibition of the Nrf2/ARE signaling pathway. This inhibition leads to the down-regulation of Nrf2 as well as its downstream genes. Studies show that apoptosis in the brain is induced by oxidative stress [42]. Consequently, this down-regulation ultimately results in oxidative damage and apoptosis in the neurons [23].

Cypermethrin Induced Oxidative Stress on The Hippocampal CA1 and CA3 Via Nuclear- Factor Erythroid 2- Related Factor 2 Modulation

The brain, in comparison to other organs in the body, is thought to be more susceptible to oxidative stress due to its high oxygen consumption, high polyunsaturated fatty acid (PUFA) content, and low levels of antioxidant enzymes [26]. The neurotoxicity caused by cypermethrin is linked to oxidative stress, decreased activity of antioxidant enzymes, and elevated indicators of inflammation [9].

The gender differences in the expression of NRF2 may be related to hormonal and genetic differences between males and females. Female rats have higher level of oestrogen which has antioxidant properties and can protect from oxidative stress [43,44]. Studies show that oestrogen and estrogenic compounds has neuroprotective capacity [45]. Estrogen upregulates neuroprotective pathway and can down regulates proinflammatory pathways. Estrogen has been shown to decrease pro inflammatory pathways, oxidative stress and apoptosis, and protect the mitochondria, which is a main source for the generation of ROS [15]. The decrease in Nrf2 levels in males is consistent with previous studies that shows that pyrethroid pesticides like cypermethrin can induce oxidative stress. This result could be dose-dependent: higher doses of cypermethrin may lead to pronounce decrease in Nrf2 levels. This could indicate oxidative stress and potential neurotoxicity in the hippocampus of male rats. Though in this study there was an up regulation of Nrf2 in the HCG female which could be a compensatory mechanism due to cypermethrin oxidative stress. This study also shows reduced Nrf2 expression in the HCG female compared to the LCG female, which can be attributed by the neurotoxicity of cypermethrin at high doses.

Effect Of Cypermethrin on GABAergic Interneurons Integrities In The Hippocampal CA1 And CA3

The increase in the expression of parvalbumin in the male rats signifies increased inhibitory neurotransmitter, GABA. Cypermethrin has been shown to modulate the Na+ channel gate, leading to hyper depolarization and delayed closure of Na+ voltage gates leading to hyper-excitability [46,47]. The increase in parvalbumin expression suggests toxicity due to prolonged opening of sodium channel gates. GABA is released to compensate for the excess excitatory effect caused by cypermethrin [48,49]. Cypermethrin may lead to the up regulation of parvalbumin in males, potentially as a compensatory mechanism to counteract the toxic effect of cypermethrin. The release of GABA in males can be said to be dose-dependent, as there was a more pronounced increase in the HCG. The expression of parvalbumin in the hippocampal CA1 female LCG group is increased compared to female HCG due to increased toxicity by cypermethrin in high dose. In the hippocampal CA3 of the female HCG group, there is an increase in the parvalbumin expression. Studies show that the hippocampal CA1 has been traditionally known to be more susceptible to insult and toxicity than the hippocampal CA3 [50]. Prolonged excitability in the hippocampal CA1 and CA3 it could cause impaired synaptic plasticity, neurodegeneration, impaired neurogenesis, disrupted neural functions.

Cypermethrin’s neurotoxic effect on hippocampal CA1 and CA3 is sex and dose dependent. The variation in its effect can be largely attributed to differences in gene and hormonal regulation.

Consent to Publication

All authors give consent to the publication of all the data, including micrographs from microscopic images, and graphs of quantitative data in the manuscript.

Competing Interests

The authors declare no competing interest.

Data Availability statement

The authors declare that the data supporting the findings of this study are available within the paper. Should raw data files be needed in another format, they are available from the corresponding author.

Funding

Not applicable

Acknowledgement

The authors will like to acknowledge the contributions of Ms. Ibrahim Zainab, for some her technical supports during the conduct of the experiments.

Author information

¹Neuroscience Unit, Department of Anatomy, College of Health Sciences, University of Ilorin, Ilorin 240003, Nigeria;

²Department of Psychiatry, University of Maryland School of Medicine, Baltimore, MD, United State of America;

³The Neuro- Lab, Department of Human Anatomy, School of Health and Health Technology, Federal University of Technology Akure, Akure 340252, Nigeria

Hedlund, J., Longo, S. B., & York, R. (2019). Agriculture , Pesticide Use , and Economic Development : A Global Examination ( 1990 – 2014 ). 0(0), 1–26. https://doi.org/10.1111/ruso.12303
Tudi, M., Ruan, H. D., Wang, L., Lyu, J., Sadler, R., Connell, D., & Chu, C. (2021). Agriculture Development , Pesticide Application and Its Impact on the Environment. 1–23.
WHO. (2022). Pesticde residues in Food. September.
Sharma, A., Kumar, V., Shahzad, B., Tanveer, M., Sidhu, G. P. S., Handa, N., Kohli, S. K., Yadav, P., Bali, A. S., Parihar, R. D., Dar, O. I., Singh, K., Jasrotia, S., Bakshi, P., Ramakrishnan, M., Kumar, S., Bhardwaj, R., & Thukral, A. K. (2019). Worldwide pesticide usage and its impacts on ecosystem. SN Applied Sciences, 1(11), 1–16. https://doi.org/10.1007/s42452-019-1485-1
Mohammadi, H., Ghassemi-barghi, N., Malakshah, O., & Ashari, S. (2019). Pyrethroid exposure and neurotoxicity : a mechanistic approach. 48, 74–89. https://doi.org/10.2478/aiht-2019-70-3263
Ali, H. F. H., El-Sayed, N. M., khodeer, D. M., Ahmed, A. A. M., Hanna, P. A., & Moustafa, Y. M. A. (2020). Nano selenium ameliorates oxidative stress and inflammatory response associated with cypermethrin-induced neurotoxicity in rats. Ecotoxicology and Environmental Safety, 195(March), 1–7. https://doi.org/10.1016/j.ecoenv.2020.110479
Udoh, G. D., & Gibbs, J. L. (2022). Commentary: Highlighting the need for pesticides safety training in Nigeria: A survey of farm households in Rivers State. Frontiers in Public Health, 10. https://doi.org/10.3389/fpubh.2022.988855
Tang, W., Wang, D., Wang, J., Wu, Z., Li, L., Huang, M., Xu, S., & Yan, D. (2018). Pyrethroid pesticide residues in the global environment: An overview. Chemosphere, 191(308), 990–1007. https://doi.org/10.1016/j.chemosphere.2017.10.115
Ashafaq, M., Hussain, S., Alshahrani, S., Siddiqui, R., Alam, M. I., Elhassan Taha, M. M., Almoshari, Y., Alqahtani, S. S., Jali, A. M., & Aljohani, H. M. (2023). Neuroprotective Effects of Nano-Curcumin against Cypermethrin Associated Oxidative Stress and Up-Regulation of Apoptotic and Inflammatory Gene Expression in Rat Brains. Antioxidants, 12(3), 1–14. https://doi.org/10.3390/antiox12030644
Shuklan, P., Raj, A., Chauhan, K., Madan, P., & Rani, S. (2023). Systematic Toxicity of Cypermethrin and Alterations in Behavior of Albino Rats. https://doi.org/10.1021/acsomega.3c00817
Afolabi, O. K., Aderibigbe, F. A., Folarin, D. T., Arinola, A., & Wusu, A. D. (2019). Oxidative stress and inflammation following sub-lethal oral exposure of cypermethrin in rats: mitigating potential of epicatechin. Heliyon, 5(8), e02274. https://doi.org/10.1016/j.heliyon.2019.e02274
Imam, A. L., Okesina, A. A., Sulamon, F. A., Imam, A., Ibiyeye, R. Y., Oyewole, L. A., Biliaminu, S. A., Shehu, M., Abdulhameed, A. O., Omoola, O. O., & Ajao, S. M. (n.d.). Orally administered Thymoquinone mitigates cypermethrin-induced dentate gyrus oxidative stress , preventing GABAergic interneuron degeneration and memory impairment in rats via the Nrf2 / ARE pathway . 1–20.
Elmorsy, E., Al-Ghafari, A., Al Doghaither, H., Salama, M., & Carter, W. G. (2022). An Investigation of the Neurotoxic Effects of Malathion, Chlorpyrifos, and Paraquat to Different Brain Regions. Brain Sciences, 12(8). https://doi.org/10.3390/brainsci12080975
Davidson, T. L., & Stevenson, R. J. (2024). Vulnerability of the Hippocampus to Insults: Links to Blood–Brain Barrier Dysfunction. International Journal of Molecular Sciences, 25(4). https://doi.org/10.3390/ijms25041991
Torres-Rojas, C., & Jones, B. C. (2018). Sex differences in neurotoxicogenetics. In Frontiers in Genetics (Vol. 9, Issue JUN). Frontiers Media S.A. https://doi.org/10.3389/fgene.2018.00196
Kašuba, V., Tariba Lovaković, B., Lucić Vrdoljak, A., Katić, A., Kopjar, N., Micek, V., Milić, M., Pizent, A., Želježić, D., & Žunec, S. (2022). Evaluation of Toxic Effects Induced by Sub-Acute Exposure to Low Doses of α-Cypermethrin in Adult Male Rats. Toxics, 10(12). https://doi.org/10.3390/toxics10120717
Omotoso, G., Oloyede, O., Lawal, S., Gbadamosi, I., Mutholib, N., Abdulsalam, F., Bature, A., Babalola, A., Ayeni, B., & Amedu, N. (2020). Permethrin exposure affects neurobehavior and cellular characterization in rats’ brain. Environmental Analysis Health and Toxicology, 35(4), 1–13. https://doi.org/10.5620/eaht.2020022
Zain, M. H., & Aitte, S. A. (2023). Histopathological changes in the kidney tissues of white Swiss mice (Mus musculus L.) exposed to alpha-cypermethrin. Journal of Nephropathology, 12(4). https://doi.org/10.34172/jnp.2022.17303
Seven, B., Yalçin, E., & Acar, A. (2022). Investigation of cypermethrin toxicity in Swiss albino mice with physiological , genetic and biochemical approaches. Scientific Reports, 1–15. https://doi.org/10.1038/s41598-022-15800-8
Abdel-Razik, R. K., Mosallam, E. M., Hamed, N. A., Badawy, M. E. I., & Abo-El-Saad, M. M. (2021). Testicular deficiency associated with exposure to cypermethrin, imidacloprid, and chlorpyrifos in adult rats. Environmental Toxicology and Pharmacology, 87(February), 103724. https://doi.org/10.1016/j.etap.2021.103724
Mahat, S., Jha, C. B., Shrestha, S., & Koirala, S. (2020). Effect of vitamin-E on cypermethrin induced toxicity in cerebral cortex of wistar albino rats: A histological study. Journal of Karnali Academy of Health Sciences, 3(1), 1–10. https://doi.org/10.3126/jkahs.v3i1.28457
Hadi, A. A., & Alwan, S. F. (2021). Physiology and Histopathology View project Cypermethrin Effects on The Weights of Body, Livers, Kidneys and Lungs in Rats View project. July.
Zhou, L., Chang, J., Zhou, M., Xiao, M., & Tan, H. (2019). Cypermethrin induces cell injury in primary cortical neurons of C57BL/6 mice by inhibiting Nrf2/ARE signaling pathway. Nan Fang Yi Ke Da Xue Xue Bao / Journal of Southern Medical University, 39(12), 1469–1475. https://doi.org/10.12122/j.issn.1673-4254.2019.12.11
Brigadski, T., & Leßmann, V. (2020). The physiology of regulated BDNF release. In Cell and Tissue Research (Vol. 382, Issue 1). Cell and Tissue Research. https://doi.org/10.1007/s00441-020-03253-2
Talbot, H., Saada, S., Barthout, E., Gallet, P. F., Gachard, N., Abraham, J., Jaccard, A., Troutaud, D., Lalloué, F., Naves, T., Fauchais, A. L., & Jauberteau, M. O. (2020). BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells. Scientific Reports, 10(1), 1–9. https://doi.org/10.1038/s41598-020-69307-1
Singh, R., Firdous, S., & Sharma, P. (2014). Neurotoxic effect of cypermethrin and protective role of resveratrol in Wistar rats. International Journal of Nutrition, Pharmacology, Neurological Diseases, 4(2), 104. https://doi.org/10.4103/2231-0738.129598
Darbre, P. D. (2018). Overview of air pollution and endocrine disorders. International Journal of General Medicine, 11, 191–207. https://doi.org/10.2147/IJGM.S102230
Graziosi, A., Sita, G., Corrieri, C., Angelini, S., d’Emmanuele di Villa Bianca, R., Mitidieri, E., Sorrentino, R., Hrelia, P., & Morroni, F. (2022). Effects of Subtoxic Concentrations of Atrazine, Cypermethrin, and Vinclozolin on microRNA-Mediated PI3K/Akt/mTOR Signaling in SH-SY5Y Cells. International Journal of Molecular Sciences, 23(23). https://doi.org/10.3390/ijms232314538
Ye, X., & Liu, J. (2019). Effects of pyrethroid insecticides on hypothalamic-pituitary-gonadal axis: A reproductive health perspective. Environmental Pollution, 245, 590–599. https://doi.org/10.1016/j.envpol.2018.11.031
Barabás, K., Szabó-Meleg, E., & Ábrahám, I. M. (2020). Effect of inflammation on female gonadotropin-releasing hormone (GnRH) neurons: Mechanisms and consequences. International Journal of Molecular Sciences, 21(2). https://doi.org/10.3390/ijms21020529
Liu, C., Qu, J., Wu, M., Huang, X., & Li, L. (2021). Cypermethrin triggers YY1-mediated testosterone biosynthesis suppression. Ecotoxicology and Environmental Safety, 225, 112792. https://doi.org/10.1016/j.ecoenv.2021.112792
Azcoitia, I., Barreto, G. E., & Garcia-Segura, L. M. (2019). Molecular mechanisms and cellular events involved in the neuroprotective actions of estradiol. Analysis of sex differences. Frontiers in Neuroendocrinology, 55(September), 100787. https://doi.org/10.1016/j.yfrne.2019.100787
Katragadda, V., Adem, M., Mohammad, R. A., Sri Bhasyam, S., & Battini, K. (2021). Testosterone recuperates deteriorated male fertility in cypermethrin intoxicated rats. Toxicological Research, 37(1), 125–134. https://doi.org/10.1007/s43188-020-00046-1
Gupta, P., Mahapatra, A., Suman, A., & Singh, R. K. (2023). In silico and in vivo assessment of developmental toxicity, oxidative stress response & Na+/K+-ATPase activity in zebrafish embryos exposed to cypermethrin. Ecotoxicology and Environmental Safety, 251(January), 114547. https://doi.org/10.1016/j.ecoenv.2023.114547
de Melo, A. D., Freire, V. A. F., Diogo, Í. L., Santos, H. de L., Barbosa, L. A., & de Carvalho, L. E. D. (2023). Antioxidant Therapy Reduces Oxidative Stress, Restores Na,K-ATPase Function and Induces Neuroprotection in Rodent Models of Seizure and Epilepsy: A Systematic Review and Meta-Analysis. Antioxidants, 12(7). https://doi.org/10.3390/antiox12071397
Huang, S., Dong, W., Lin, X., & Bian, J. (2024). Na+/K+-ATPase: Ion pump, signal transducer, or cytoprotective protein, and novel biological functions. Neural Regeneration Research, 19(12), 2684–2697. https://doi.org/10.4103/NRR.NRR-D-23-01175
Gargouri, B., Yousif, N. M., Attaai, A., Bouchard, M., Chtourou, Y., Fiebich, B. L., & Fetoui, H. (2018). Pyrethroid bifenthrin induces oxidative stress, neuroinflammation, and neuronal damage, associated with cognitive and memory impairment in murine hippocampus. Neurochemistry International. https://doi.org/10.1016/j.neuint.2018.08.004
Cicala, C., & Morello, S. (2023). Signaling Pathways in Inflammation and Its Resolution: New Insights and Therapeutic Challenges. International Journal of Molecular Sciences, 24(13), 10–13. https://doi.org/10.3390/ijms241311055
Teleanu, D. M., Niculescu, A. G., Lungu, I. I., Radu, C. I., Vladâcenco, O., Roza, E., Costăchescu, B., Grumezescu, A. M., & Teleanu, R. I. (2022). An Overview of Oxidative Stress, Neuroinflammation and Neurodegenerative Diseases. International Journal of Molecular Sciences, 23(11). https://doi.org/10.3390/ijms23115938
Li, J., & Bi, H. (2021). The effect and mechanism of cypermethrin- induced hippocampal neurotoxicity as determined by network pharmacology analysis and experimental validation. Bioengineered, 12(2), 9279–9289. https://doi.org/10.1080/21655979.2021.2000106
Özdemir, S., Altun, S., Özkaraca, M., Ghosi, A., Toraman, E., & Arslan, H. (2018). Cypermethrin, chlorpyrifos, deltamethrin, and imidacloprid exposure up-regulates the mRNA and protein levels of bdnf and c-fos in the brain of adult zebrafish (Danio rerio). Chemosphere, 203, 318–326. https://doi.org/10.1016/j.chemosphere.2018.03.190
Paravani, E. V., Simoniello, M. F., Poletta, G. L., Zolessi, F. R., & Casco, V. H. (2018). Cypermethrin: Oxidative stress and genotoxicity in retinal cells of the adult zebrafish. Mutation Research - Genetic Toxicology and Environmental Mutagenesis, 826, 25–32. https://doi.org/10.1016/j.mrgentox.2017.12.010
Borrás, C., Ferrando, M., Inglés, M., Gambini, J., Lopez-Grueso, R., Edo, R., Mas-Bargues, C., Pellicer, A., & Viña, J. (2021). Estrogen Replacement Therapy Induces Antioxidant and Longevity-Related Genes in Women after Medically Induced Menopause. Oxidative Medicine and Cellular Longevity, 2021. https://doi.org/10.1155/2021/8101615
Tsialtas, I., Georgantopoulos, A., Karipidou, M. E., Kalousi, F. D., Karra, A. G., Leonidas, D. D., & Psarra, A. M. G. (2021). Anti-apoptotic and antioxidant activities of the mitochondrial estrogen receptor beta in n2a neuroblastoma cells. International Journal of Molecular Sciences, 22(14). https://doi.org/10.3390/ijms22147620
Shvetcov, A., Ruitenberg, M. J., Delerue, F., Gold, W. A., Brown, D. A., & Finney, C. A. (2023). Neuroscience and Biobehavioral Reviews The neuroprotective effects of estrogen and estrogenic compounds in spinal cord injury. Neuroscience and Biobehavioral Reviews, 146(January), 105074. https://doi.org/10.1016/j.neubiorev.2023.105074
Ali, H. F. H., El-sayed, N. M., Dina, M., Ahmed, A. A. M., & Hanna, P. A. (2019). RECORDS OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES Cellular Mechanism involved in cypermethrin induced neurotoxicity.
Lin, M. H., Lin, J. F., Yu, M. C., Wu, S. N., Wu, C. L., & Cho, H. Y. (2022). Characterization in Potent Modulation on Voltage-Gated Na+ Current Exerted by Deltamethrin, a Pyrethroid Insecticide. International Journal of Molecular Sciences, 23(23), 1–19. https://doi.org/10.3390/ijms232314733
Bryson, A., Hatch, R. J., Zandt, B. J., Rossert, C., Berkovic, S. F., Reid, C. A., Grayden, D. B., Hill, S. L., & Petrou, S. (2020). GABA-mediated tonic inhibition differentially modulates gain in functional subtypes of cortical interneurons. Proceedings of the National Academy of Sciences of the United States of America, 117(6), 3192–3202. https://doi.org/10.1073/pnas.1906369117
Kilb, W., & Kirischuk, S. (2022). GABA Release from Astrocytes in Health and Disease. International Journal of Molecular Sciences, 23(24). https://doi.org/10.3390/ijms232415859
Einenkel, A. M., & Salameh, A. (2024). Selective vulnerability of hippocampal CA1 and CA3 pyramidal cells: What are possible pathomechanisms and should more attention be paid to the CA3 region in future studies? Journal of Neuroscience Research, 102(1), 1–38. https://doi.org/10.1002/jnr.25276

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Sex-Specific Cypermethrin Induced Hippocampal Neurotoxicity Is Via the Modulation of Signaling Molecules for Antioxidant Defense, Membrane Integrity, Apoptosis and GABAergic Integrity

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Abstract

Figures

Introduction

Materials And Methods

Daily Body Weight Measurements

Biochemical Analysis:

Hippocampal tissues were homogenized in 30% sucrose and centrifuged at 2500 rpm for 10 minutes. The supernatant was then collected in tubes for the analysis of the biomarkers bellow.

All biochemical markers where assayed with markers’ specific and highly sensitive kits: Prostaglndin-E2 (PG-E2), GnRH, COX-2,

Hematoxylin & Eosin Staining

Immunohistochemistry

Microscopy and Photomicrography

Image Analysis

Cell Counting

Statistical Analysis

Results

High Dose, Not Low Dose of Cypermethrin Caused Weight Loss Comparably in Both Sexes

Dose And Sex-Specific Effects of Cypermethrin on Sex, Inflammatory and Membrane Biomarkers in Brains

Neuronal Integrities in The Hippocampal CA1 and CA3 Regions of Cypermethrin Exposed Rats

Cypermethrin Induced Oxidative Stress on The Hippocampal CA1 and CA3 Via Nuclear- Factor Erythroid 2- Related Factor 2 Modulation

Cypemethrin Induced Apoptosis on The Hippocampal CA1 and CA3 Using Cleaved Capsace 3

Expression Of Cleaved Caspase Three (CC3) In the Hippocampal Ca3 of Exposed Animals.

Expression Of B-Cell Lymphoma - 2 (Bcl-2) In the Hippocampal CA1 of Exposed Animals.

Expression Of B-Cell Lymphoma - 2 (Bcl-2) In the Hippocampal CA3 Of Exposed Animals.

Expression Of Parvalbumin in The Hippocampal CA1 of Exposed Animals.

Expression Of Parvalbumin in The Hippocampal CA3 of Exposed Rats.

Discussion

High dose, not low dose of cypermethrin caused weight loss comparably in both sexes

Effect Of Cypermethrin on Sex-Specific Reproductive Hormones

Effect Of Cypermethrin on Membrane Integrities

Cypermethrin Induced Neuroinflammation by Modulating PGE-COX-2 Pathway

Cellular Morphology in The Hippocampal CA1 and CA3 On Exposure to Cypermethrin

Cypemethrin Induced Apoptosis on The Hippocampal CA1 and CA3 By Modulating Cleaved Capsace 3 And B-Cell Lymphoma - 2 (Bcl-2) Expressions

Cypermethrin Induced Oxidative Stress on The Hippocampal CA1 and CA3 Via Nuclear- Factor Erythroid 2- Related Factor 2 Modulation

Effect Of Cypermethrin on GABAergic Interneurons Integrities In The Hippocampal CA1 And CA3

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1