Transfluthrin-based insecticide exposure: assessment of cognitive function and anxiety-like behavior in rats

Transfluthrin-based insecticide exposure: assessment of cognitive function and anxiety-like behavior in rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transfluthrin-based insecticide exposure: assessment of cognitive function and anxiety-like behavior in rats David Anuoluwapo Oyeniran, Tobiloba Samuel Olajide, Toheeb Olalekan Oyerinde, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9528142/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 18 You are reading this latest preprint version Abstract Background The central nervous system is susceptible to environmental toxicants, including synthetic pyrethroid insecticides, which are widely used for domestic and agricultural pest control. Transfluthrin, a fast-acting pyrethroid, is commonly incorporated into insecticide papers for indoor use. However, concerns regarding its potential neurotoxic effects persist. This study evaluated the impact of transfluthrin-based insecticide paper (TBIP) smoke on cognitive function and anxiety-like behavior in adult male Wistar rats. Methods Thirty Male rats were randomly assigned into three groups (n = 10 per group): Group A (control) was exposed to clean ambient air; Group B was exposed to smoke from 6 g of TBIP for 4 hours daily; and Group C was exposed to smoke from 12 g of TBIP for 8 hours daily, both for 4 weeks via whole-body inhalation. After exposure, behavioral assessments were performed using the Elevated Plus Maze (EPM) to assess anxiety-like behavior at week 4 and to evaluate learning and memory at weeks 1, 2, and 4. The Y-maze was similarly used to assess working memory at weeks 1, 2, and 4. At the end of the exposure period, rats were sacrificed, and their brains were harvested to isolate the hippocampi for biochemical analysis and histopathological investigation. Results Rats exposed to TBIP exhibited significant reductions in learning and memory performance, increased anxiety-like behaviors, reduced body/ brain weights, and altered oxidative parameters and AChE activity compared to the controls. Conclusion These findings suggest that prolonged exposure to transfluthrin-based insecticides can impair neurobehavioral function, possibly via oxidative stress, nitrosative stress, and disruption of cholinergic signaling pathways. These indicate the need for cautious use and regulatory review of household insecticides containing transfluthrin. transfluthrin pyrethroid insecticide cognitive function anxiety acetylcholinesterase brain Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION The brain coordinates a wide range of complex functions, including cognition, behavior, and physiological regulation, through the intricate activity of billions of neurons. These neurons communicate via synapses in complex networks that ensure the proper functioning of the nervous system. However, neuronal degeneration or imbalances in excitatory and inhibitory signals can disrupt these processes, contributing to the onset and progression of neurological and psychiatric disorders ( 1 ). Nigeria, like many other countries, is experiencing a growing mental health crisis. An estimated 20% of its population, around 40 million individuals, suffers from mental health conditions, including depression, anxiety, and other psychiatric disorders ( 2 – 4 ). Recent data show that approximately 7 million Nigerians live with depressive disorders, while 4.9 million are affected by anxiety disorders ( 5 ). In comparison, in the United States, approximately 6% of adults experienced moderate to severe levels of anxiety symptoms. In 2019, 7% were reported to have depressive symptoms, with higher prevalence rates among women than men ( 6 ). more so, in the United States, an estimated 57.8 million adults were reported to have some form of mental illness ( 7 ). These disorders significantly burden individuals and healthcare systems, underlining the urgency of identifying contributing factors, especially those related to environmental exposure. Anxiety disorders are characterized by symptoms such as restlessness, fatigue, irritability, and persistent fear or worry, which can severely impair daily functioning ( 8 – 10 ). Another major neurological condition is dementia, with Alzheimer’s disease (AD) being its most common form. AD typically begins with mild memory loss and gradually progresses to severe cognitive decline. The disease predominantly affects brain regions, including the hippocampus and amygdala, which are necessary for memory formation and emotional regulation ( 11 – 13 ). A critical pathological feature of AD is the degeneration of cholinergic neurons connecting the basal forebrain and hippocampus, which correlates strongly with memory impairment ( 14 – 17 ). Oxidative stress, neuroinflammation, and disruptions in lipid metabolism are key pathological processes underlying both neurodegenerative and mood disorders ( 18 – 20 ). The brain is particularly vulnerable to oxidative stress owing to its high lipid content, elevated oxygen consumption, and intense metabolic activity ( 18 ). These characteristics predispose neural tissues to lipid peroxidation, ultimately compromising membrane integrity and contributing to neuronal dysfunction ( 21 , 22 ). Disruption of the brain’s antioxidant defense system, including key enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx), can arise from excessive endogenous production of reactive oxygen species (ROS) or exposure to exogenous stressors that enhance ROS generation ( 23 , 24 ). Such imbalance is often reflected by increased levels of malondialdehyde (MDA), a well-established marker of lipid peroxidation ( 25 ). The detrimental effects of oxidative stress are further amplified in the presence of neuroinflammation. The bidirectional interaction between oxidative stress and inflammatory processes plays a critical role in both the maintenance of normal brain function and the pathogenesis of neurological disorders ( 26 ). Each process can potentiate the other, leading to progressive cellular damage and functional impairment ( 27 ). A growing body of evidence points to environmental toxicants such as pesticides, herbicides, and insecticides as major contributors to neuropsychiatric conditions ( 28 ). These chemicals can induce oxidative stress, provoke neuroinflammatory responses, and disrupt neuronal signaling pathways, thereby increasing the risk of conditions such as AD, anxiety, and depression ( 29 – 31 ). Transfluthrin, a widely used pyrethroid insecticide across Africa, has become a chemical of concern due to its extensive application in household pest control and agricultural practices ( 32 , 33 ). Emerging studies have reported its toxic effects on various organs and the immune system, highlighting its potential risks to human health and the environment ( 34 – 37 ). The neurotoxic effects of pyrethroid insecticides are primarily attributed to their interference with voltage-gated sodium channels, leading to prolonged depolarization and hyperexcitability of the nervous system ( 38 , 39 ). They can also modulate acetylcholinesterase (AChE) activity in the brain, often leading to reduced enzyme activity ( 40 ). Although organophosphates are well-known AChE inhibitors, studies have also demonstrated that certain pyrethroids can affect AChE activity in brain regions such as the cerebral cortex, cerebellum, and hippocampus, resulting in impaired neural function in these regions ( 40 , 41 ). They bind to these channels and prevent their normal inactivation, causing them to remain open longer than usual. This results in repetitive neuronal firing and sustained excitatory signaling ( 37 ). Additionally, pyrethroids have been shown to affect isoforms of voltage-sensitive calcium channels, further disrupting calcium homeostasis and impairing neurotransmitter release ( 37 , 42 , 43 ). Beyond ion channel interference, exposure to this type of insecticide is associated with the induction of oxidative stress, a state characterized by an imbalance between the production of ROS and the body’s antioxidant defenses. This oxidative burden can damage cellular components, including lipids, proteins, DNA, and neurons, thereby contributing to neurodegeneration ( 44 , 45 ). Moreover, they may trigger neuronal cell death through mechanisms such as excitotoxicity (excessive glutamate-mediated stimulation) and apoptosis (programmed cell death) ( 46 – 48 ). Emerging evidence also suggests that pyrethroids impair mitochondrial function, disrupting cellular energy production and increasing neuronal vulnerability to stress and injury ( 48 , 49 ). Despite advances in our understanding of pesticide-induced neurotoxicity, significant gaps remain, particularly regarding the behavioral and cognitive consequences of specific agents like transfluthrin. To address this, the present study aims to investigate the neurobehavioral and cognitive effects of exposure to smoke from transfluthrin-based insecticide paper (TBIP) in adult male Wistar rats, thereby contributing to a more comprehensive understanding of environmental risk factors in neurological health. MATERIALS AND METHODS. A total of thirty ( 30 ) male Wistar rats (8 to 10 weeks old) weighing 150–170 g were procured from a breeding stock at the University of Ibadan in Oyo State and were kept under standard laboratory conditions, with unrestricted access to water and food, and a consistent 12-hour daylight cycle. Rats were given a 2-week acclimation period to the laboratory environment prior to the onset of experiments. The insecticide used in this study was Rambo® (Bayer Cropscience), distributed by Gongoni Company Limited. It was procured from a reputable retail outlet in Ejigbo, Lagos State, Nigeria. The paper, which weighed 6g, comprised 0.45% transfluthrin, 2.5% essential oil, and 97.05% inert ingredients. The rats were divided into three groups (A - C) of 10 rats each. Group A was exposed to regular environmental air, Group B rats were exposed to smoke from 6g (burning for 4 hours), and Group C rats were exposed to smoke from 12g (burning for 8 hours) of TBIP via whole-body inhalation for 4 weeks. Dose and method of exposure were done according to the study by Oyeniran, Ojewale ( 35 ). Rats were exposed to transfluthrin smoke using a custom whole-body inhalation chamber (43 × 30 × 26 cm) made of transparent acrylic for observation and containment. A transfluthrin-impregnated paper was ignited in a connected sealed combustion unit, and the resulting smoke was channeled into the chamber via heat-resistant tubing. The chamber was equipped with small ventilation outlets to prevent oxygen depletion while still maintaining a sufficient concentration of smoke. Animals were closely monitored for signs of distress during and after exposure. After exposure, rats in Groups A, B, and C underwent behavioral assessments at the first, second, and fourth weeks. Spatial working memory was evaluated using the Y-maze, while the Elevated Plus Maze (EPM) was used to assess the learning and memory capacity. At the end of the fourth week, the rats were subjected to the EPM to evaluate anxiety-like behavior. After the 4-week exposure, the rats were weighed and euthanized following anesthesia with ketamine (80 mg/kg, i.p.) to induce deep unconsciousness, followed by cervical dislocation. The brains were then carefully excised, weighed, and processed for both biochemical assays and histopathological evaluation ( 35 , 50 ). Experimental procedures involving the animals and their care were conducted in conformity with the guiding principles for research involving animals as recommended by the Declaration of Helsinki and the Guiding Principles in the Use and Care of Animals ( 51 ). Likewise, all methods are reported in accordance with ARRIVE guidelines ( 52 ) BEHAVIORAL ANALYSIS Assessment of Anxiety-like behaviour using the EPM Test In this study, the EPM test for evaluating anxiety-like behavior was configured in a plus (+) shape and elevated 50 cm above the ground. It consisted of two open arms, each measuring 45 cm in length and 10 cm in width, and two enclosed arms of the same length and width, with enclosing walls 30 cm high, all extending from a central square platform (10 cm × 10 cm). To ensure acclimatization, rats were transferred to the testing room 30 minutes before the experiment and maintained under dim lighting. At the start of the test, each rat was positioned on the central platform, facing any of the open arms, and allowed to freely explore the maze for 5 minutes (300 seconds). After every trial, the maze was thoroughly sanitized using 70% ethanol to eliminate residual olfactory cues. Behavioral parameters recorded included: time spent in open arms (TSOA), time spent in closed arms (TSCA), number of open arm entries (NOAE), and number of closed arm entries (NCAE) ( 53 ). Assessment of Learning and Memory using the EPM Test Twenty-four hours before the test, each rat was placed at the distal end of an open arm of the EPM, facing away from the central platform. The time taken to move from the open arm to any of the enclosed arms (measured in seconds) was recorded as the transfer latency (TL) of learning. After the rat entered an enclosed arm, it was allowed to explore the maze for 30 seconds before being removed. The same protocol was performed after a 24-hour interval to assess memory retention. The time taken to enter an enclosed arm was recorded again as the transfer latency (TL) of memory ( 54 , 55 ). Behavioral assessments were conducted by a student who was blinded to the experimental group assignments, employing a single-blind approach to minimize observer bias. Assessment of spatial working memory using the Y-Maze The Y-maze was used to assess spontaneous alternation behavior as an index of spatial working memory, as well as general locomotor activity. The apparatus consisted of three identical arms (41 cm long, 15 cm high, and 5 cm wide) positioned at 120° angles from each other. Each rat was placed at the end of one arm and allowed to explore the maze freely. Arm entries were recorded when the animal’s tail completely entered an arm, and the sequence of entries was noted over a 5-minute session. Spontaneous alternation was defined as consecutive entries into all three arms without repetition (e.g., A, B, C). The number of actual alternations was determined from successive overlapping triplet sets of arm entries. The percentage alternation was calculated using the formula: [(number of actual alternations) / (total number of arm entries − 2)] × 100 ( 56 ). Determination of Lipid Peroxidation and Antioxidant Enzyme Activities A 10% (w/v) brain tissue homogenate (prefrontal cortex and hippocampus) was prepared in 0.9% sterile saline solution. The homogenate was centrifuged at 6000 rpm for 10 minutes at 4°C, and the resulting supernatant was collected for biochemical analyses. Catalase (CAT) activity was assayed according to the method of Aebi ( 57 ), which is based on the rate of decomposition of 1 µM hydrogen peroxide (H₂O₂), and the results were expressed as U/mg protein. Superoxide dismutase (SOD) activity was determined following the procedure of ( 58 ), while reduced glutathione (GSH) content was measured using the method described by Ellman et al. ( 58 ). Lipid peroxidation was evaluated by determining malondialdehyde (MDA) levels through the formation of a red-colored complex with thiobarbituric acid (TBA), and MDA concentrations were expressed as nmol/mg protein. All assays were performed in duplicate to ensure analytical reliability ( 59 ). Determination of Nitric Oxide (NO) Nitric oxide levels were determined by colorimetric quantification of nitrite in brain tissue homogenates using the Griess assay. The Griess reagent consisted of 1.5% sulphanilamide in 1 mol/L HCl containing 0.15% N-(1-naphthyl)ethylenediamine dihydrochloride. In this procedure, nitrates present in the samples were first reduced to nitrites, which then reacted with the Griess reagent to form a chromophoric azo compound exhibiting a purple coloration. The resulting absorbance was measured at 540 nm to determine the nitrite and, consequently, NO concentration ( 60 ). Evaluation of Acetylcholinesterase Activity Each brain region was homogenized in 50 mM Tris-HCl buffer (pH 7.4) containing 1.15% potassium chloride to prepare a 10% (w/v) tissue homogenate. The homogenate was then centrifuged at 10,000 × g for 10 minutes at 4°C, and the resulting supernatant was collected for the determination of acetylcholinesterase (AChE) activity. AChE activity in both the hippocampus and prefrontal cortex was measured using the method of ( 61 , 62 ) Ellman et al. (1961), as modified by ( 61 , 62 ), employing acetylthiocholine iodide as the substrate. In this assay, AChE catalyzes the hydrolysis of acetylthiocholine iodide to yield thiocholine and butyric acid. The liberated thiocholine reacts with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) to produce 5-thio-2-nitrobenzoic acid, a yellow-colored compound. The intensity of the color is measured spectrophotometrically at 412 nm and is proportional to the AChE activity. Histological Evaluation For histological analysis, fixed brain tissues in 10% neutral buffered formalin were grossly dissected to expose the regions of interest. The tissues were subsequently dehydrated through a graded ethanol series and cleared in xylene. They were then infiltrated and embedded in molten paraffin wax at 58°C. Paraffin blocks were prepared, and serial sections of 5 µm thickness were cut using a microtome. The sections were rehydrated and stained with hematoxylin and eosin (H&E). Thereafter, sections of the prefrontal cortex and hippocampus were examined under a light microscope at ×400 magnification ( 35 ). Statistical Analysis Data was analyzed statistically using analysis of variance (ANOVA), followed by Tukey post-hoc test using GraphPad Prism 8.0 software. Differences between means were considered significant at p < 0.05 when compared with the control, and results were expressed as Mean ± SEM. RESULTS Effects of smoke from TBIP on body and organ weight. The body weight of the experimental group was lower than that of the control group. At p < 0.05, group C showed a significant reduction in body weight compared with the control. The brain weights of rats in groups B and C were significantly lower than those in the control group. Additionally, the relative brain weight (brain weight/final body weight) of the high-exposure group was significantly lower than that of the control at p < 0.05 (Table 1 ). Table 1 Effect of smoke from TBIP on body and organ weight. Groups Control TBIP 6g TBIP 12g Initial body weight (g) 152 ± 7.68 140 ± 6.86 156 ± 4.57 final body weight (g) 205 ± 5.08 195 ± 9.12 184 ± 7.55 Body-weight difference (g) 53 ± 3.22 55 ± 4.98 28 ± 5.35* Brain weight (g) 1.57 ± 0.04 1.22 ± 0.06* 1.05 ± 0.03* Relative brain weight 0.0077 ± 0.01 0.0063 ± 0.02 0.0057 ± 0.01* * P < 0.05; significantly different from control. Values are expressed as mean ± SEM for n = 10 in each group. Effect of smoke from TBIP on anxiety-like behaviors in rats using the EPM test. Exposure to TBIP significantly reduced the time spent in the open arms (TSOA) and the number of open arm entries (NOAE) in the 6 g and 12 g groups compared to the control (p < 0.05). Conversely, the time spent in the closed arms (TSCA) was significantly increased in the treated groups, while the number of closed arm entries (NCAE) was significantly higher in the TBIP 12 g group compared to the control (p < 0.05; Fig. 1 ). Effect of Smoke From TBIP on Learning and Memory Using EPM. During the first week of the learning phase, rats exposed to TBIP 12 g exhibited a significantly longer transfer latency to the closed arm compared to the control group (p < 0.05). In weeks 2 and 4, both TBIP-treated groups (6 g and 12 g) showed prolonged learning times relative to the control (p < 0.05). A similar pattern was observed in the retention (memory) phase, where the TBIP 12 g group showed a significantly longer transfer latency in week 1. Both the TBIP 6 g and 12 g groups exhibited extended transfer latencies in weeks 2 and 4 compared to the control at p < 0.05 (Fig. 2 ). Effect of Smoke From TBIP on Memory Performance Using the Y-maze Rats in the control group consistently demonstrated good memory performance (spontaneous alternation) in the Y-maze test throughout the evaluation period. At week 1, rats exposed to 6 g of TBIP exhibited memory performance comparable to the control group (p < 0.05). However, by weeks 2 and 4, a significant decline in memory performance was observed in this group compared with the control. In contrast, rats exposed to the higher dose of TBIP (12 g) showed a significant reduction in memory performance at all three assessment time points (weeks 1, 2, and 4) when compared to the control group (p < 0.05), as shown in Fig. 3 . Effect of Smoke From TBIP on Oxidative Parameters in the prefrontal cortex and hippocampus The oxidative stress assessment revealed that MDA levels in both the prefrontal cortex and hippocampus were significantly elevated in rats exposed to 6 g and 12 g of TBIP compared to the control group at p < 0.05. On the other hand, the activities of antioxidant enzymes SOD, CAT, and GSH were markedly reduced at both doses in the prefrontal cortex and hippocampus. However, for GSH, a significant decrease was observed only at the higher dose (12 g) in both brain regions (Fig. 4 a and 4 b). Effect of Smoke from TBIP on NO Levels in the Prefrontal Cortex and Hippocampus of Rats : The results showed that NO levels in both the prefrontal cortex and hippocampus of rats exposed to TBIP were significantly higher compared to the control group, p < 0.05 (Fig. 5 ). Effect of TBIP smoke on Acetylcholinesterase levels The acetylcholinesterase enzyme activity in the prefrontal cortex was significantly reduced in the high dose group compared to the control, while the low dose was not significantly different from the control. However, in the hippocampus, it was found that both experimental groups had a significant acetylcholinesterase level compared to the control at p < 0.05 (Fig. 6 ). Histopathological Evaluation of the effect of smoke of TBIP on Prefrontal Cortex and Hippocampal Subregions Histological analysis revealed dose-dependent neurodegenerative changes in the prefrontal cortex and hippocampal subregions of TBIP-treated rats compared to controls. In the prefrontal cortex, the control group showed normal cortical architecture with intact pyramidal neurons, glial cells, and well-organized neurites. TBIP exposure induced progressive neuronal degeneration, cytoplasmic vacuolation, focal lesions, and disrupted neurite arrangement. These alterations were more severe in the 12 g group, showing extensive vacuolation and pyknotic neurons. CA1 and CA3 regions of the control sections displayed well-defined laminar organization with compact pyramidal neurons. TBIP 6 g caused a mild reduction in pyramidal layer thickness and moderate vacuolation, while the 12 g group exhibited pronounced disorganization, neuronal degeneration, loss of pyramidal cell density, and numerous pyknotic neurons within the tissue parenchyma. In the dentate gyrus, the control group exhibited intact molecular, granule, and polymorphic layers. However, TBIP-treated groups retained this general structure but showed neuronal degeneration and vacuolation, particularly in the subgranular zone, alongside cytoplasmic vacuolation in glial and interneuronal cells (Fig. 7 ). DISCUSSION The integrity of cognitive and emotional function depends on coordinated biochemical signaling, synaptic plasticity, and structural stability across key brain regions such as the hippocampus and prefrontal cortex ( 63 ). Disruption of these processes by environmental toxicants, such as pyrethroid-based insecticides, can lead to profound neurobehavioral and neurochemical alterations ( 64 ). In the present study, it was seen that exposure to smoke from TBIP resulted in significant impairments in learning and memory, increased anxiety-like behaviours, and reductions in body and brain weights. Alterations in oxidative and nitrosative stress markers, decreased AChE activity, and marked histopathological damage in the prefrontal cortex and hippocampus were also observed. The reduction in body weight observed, particularly in the group exposed to smoke from a higher dose (12 g) of transfluthrin paper, corroborates earlier findings reported by Kalra and Sangha ( 65 ). Grewal et al. ( 66 ) also reported similar weight loss following pyrethroid exposure. Studies involving mosquito vaporizer fumes containing 0.88% Transfluthrin ( 67 ) and repeated exposure to cypermethrin ( 66 ) have also documented decreased body weight, aligning with our findings. This effect is likely multifactorial, involving reduced food intake, gastrointestinal irritation, and impaired nutrient absorption ( 68 ). Additionally, systemic toxicity associated with pyrethroid exposure may directly impair metabolic processes, contributing to weight loss ( 35 ). The observed reduction in brain weight, both absolute and relative, aligns with previous reports indicating that pyrethroid groups of insecticides may reduce organ weights, including the brain, kidney, and testes ( 35 , 66 , 69 ). Oxidative stress-induced neuronal degeneration may further contribute to this reduction in brain mass ( 70 , 71 ), corroborating the neuronal depletion seen in the prefrontal and hippocampus of rat exposure to TBIP. A major finding of this study is the impairment in cognitive function, as evidenced by increased transfer latency in the elevated plus maze and reduced spontaneous alternation in the Y-maze. These findings indicate deficits in both learning acquisition and memory retention. The hippocampus and prefrontal cortex function as an integrated network in memory processing ( 72 ). The hippocampus is responsible for encoding and consolidating new information, while the prefrontal cortex is involved in higher-order processing, organization, and retrieval of memory ( 73 , 74 ). Disruption of this circuitry provides a plausible explanation for the observed cognitive deficits. Previous studies have shown that pyrethroid exposure impairs learning and memory in rats and mice, often in association with oxidative stress, dopaminergic dysfunction, and hippocampal damage ( 75 – 77 ). For instance, exposure to flumethrin has been shown to downregulate memory-related genes such as GluRA1, Nmdar1, and Tyr1, thereby impairing olfactory learning and memory ( 78 ). Similarly, chronic administration of deltamethrin has been associated with hippocampal endoplasmic reticulum stress, impaired neurogenesis, and learning deficits ( 79 ), which is consistent with the neuronal depletion and vacuolation observed in the dentate gyrus in the present study. Chlorpyrifos, permethrin, and cyfluthrin, similar to transfluthrin, have been reported to affect cell survival, permeability, and tight junction in an in-vitro model of the human blood-brain barrier (BBB) ( 80 ). Suggesting that the ability of transfluthrin to cross the BBB may have contributed to its effect on the prefrontal cortex and hippocampus, resulting in neurobehavioral deficits as seen in the rat exposure to TBIP. The present findings demonstrate that transfluthrin exposure induces pronounced anxiety-like behavior, as evidenced by reduced exploration of the open arms and a clear preference for the closed arms in the EPM. Consistent with this observation, transfluthrin-exposed rats in previous studies have similarly exhibited increased anxiety-like behavior, hyperactivity, and impairments in spatial learning and memory ( 81 ). Another study reported the anxiogenic potential of pyrethroids such as permethrin ( 82 ), and reduced exploratory behavior and altered locomotor indices in paradigms like negative geotaxis ( 82 ). More broadly, cypermethrin, recognized as an emerging neurotoxin, has been shown to induce a spectrum of behavioral and psychological disturbances, particularly following prolonged exposure ( 83 ). Together, these findings suggest that anxiety-like behavior represents a consistent neurobehavioral outcome of pyrethroid toxicity, such as cypermethrin, permethrin, and transfluthrin. This reinforces the vulnerability of neural circuits governing emotion and cognition to pyrethroid exposure ( 81 ). Previous studies using cypermethrin have reported a significant reduction in total distance traveled at higher doses (20 and 80 mg/kg). They also demonstrated that reductions in open-arm time and entries were significant, indicating anxiogenic effects ( 84 ). This aligns with the present findings, where the observed increase in closed-arm preference is more probably linked to anxiety. The observed behavioral alterations may be closely linked to neuroinflammatory processes within brain regions critical for emotional regulation, including the amygdala, hippocampus, and prefrontal cortex ( 75 , 76 , 85 ). Neuroinflammation, characterized by elevated levels of pro-inflammatory cytokines such as TNF-α and IL-1β, has been strongly implicated in both anxiety disorders and cognitive dysfunction ( 86 , 87 ). Supporting this, Gargouri et al.( 75 ) reported that bifenthrin exposure induced oxidative stress, neuroinflammation, and memory deficits in rats, thereby strengthening the association between oxidative damage, inflammatory responses, and behavioral impairments ( 85 ). These pathways may have likely contributed to the anxiety-like phenotype observed in the present study. The neurotoxic effects of pyrethroids, including transfluthrin, are largely mediated through their action on voltage-gated sodium channels. These compounds prolong the open state of sodium channels, thereby delaying channel inactivation and promoting sustained neuronal depolarization ( 81 , 88 ). This aberrant increase in neuronal excitability disrupts normal synaptic transmission and neural network stability, ultimately manifesting as behavioral abnormalities. In more severe cases, such prolonged depolarization can lead to conduction block and neuromuscular dysfunction due to impaired repolarization dynamics ( 84 , 89 ). Oxidative stress emerges as a central mechanism underlying the neurotoxic effects observed in this study. The marked elevation in MDA levels was accompanied by significant reductions in key antioxidant defenses, including SOD, CAT, and GSH, in both the prefrontal cortex and hippocampus. These findings clearly indicate a disruption of redox homeostasis. These findings are consistent with earlier reports showing increased lipid peroxidation and compromised antioxidant capacity in rat brain following pyrethroid exposure ( 90 ). A study on zebrafish found that transfluthrin and prallethrin-based insecticides cause deleterious effects on antioxidant systems ( 91 ). Given the brain’s high lipid composition and substantial oxygen demand, it is particularly susceptible to oxidative injury ( 92 ). Lipid peroxidation can destabilize neuronal membranes, impair ion gradients, and disrupt synaptic signaling, ultimately predisposing neurons to degeneration and cell death ( 92 , 93 ). The SOD and GPx are antioxidant enzymes ubiquitous in living organisms, acting as an endogenous defense against ROS ( 94 ). Muhammad et al. showed in their study that repeated oral administration of Bifenthrin for 14 days led to an increase in lipid peroxidation and a decrease in antioxidant enzymes in the Brain of Oryctologus cuniculus ( 95 ). The concurrent increase in MDA and depletion of endogenous antioxidant systems observed in this study strongly support oxidative stress as a key driver of transfluthrin neurotoxicity. Among the various mechanisms by which insecticides impair neural development and function, oxidative stress remains one of the most prominent ( 96 ). It contributes to cellular damage through the oxidation of lipids, proteins, and nucleic acids, processes that have been widely implicated in the pathogenesis of neurodegenerative and neurodevelopmental disorders ( 21 ). Supporting this, animal studies have consistently demonstrated that pyrethroids induce oxidative stress across multiple tissues ( 97 ). Exposure to Pyrethroid bifenthrin was found to induce neuronal damage, oxidative stress, and cause neuroinflammation in the hippocampus of rats, which may lead to cognitive and memory impairment ( 90 ). Similar to what was reported in our study, cypermethrin a well-studied pyrethroid, has been reported to exert its toxic effects through excessive generation of reactive oxygen species, significantly reducing antioxidant enzymes such as SOD, GST, GSH, CAT, glutathione reductase, and GPx, while simultaneously increasing lipid peroxidation in serum and tissues ( 98 , 99 ). In an experimental study, its exposure leads to mitochondrial dysfunction, characterized by impaired electron transport and increased ROS production, accompanied by elevated oxidative stress markers and diminished antioxidant enzyme activities ( 100 ) [16]. These alterations not only compromise cellular energy metabolism but also trigger downstream pathological events, including DNA damage and apoptosis ( 84 , 101 ). In addition to oxidative stress, nitrosative stress also appears to contribute to neuronal damage. The significant increase in NO levels observed in this study is indicative of enhanced reactive nitrogen species (RNS) production. This is consistent with a previous study ( 102 ). Elevated NO levels, particularly through inducible nitric oxide synthase (iNOS) activation, can lead to the formation of peroxynitrite, a highly reactive molecule that induces protein nitration, lipid peroxidation, and DNA damage ( 103 ). In line with our present result, oxidative/nitrosative stress in the hippocampus of rats treated with Pyrethroid bifenthrin, as shown by increased levels of MDA, protein carbonyls (PCO), and NO, and reduced levels of enzymatic and non-enzymatic antioxidants, was reported in a previous study ( 75 ). This oxidative/nitrosative stress cascade may have exacerbated neuronal injury and promoted neuroinflammation, further contributing to cognitive and behavioural impairments. In a human study, increased concentration of nitrite and nitrate in plasma, suggesting an increased production of NO in volunteers exposed to mosquito repellent pyrethroids (allethrin and prallethrin), was documented compared to controls ( 104 ). More so, supporting our finding, exposure to pyrethroids like deltamethrin and bifenthrin as been shown to result in an increase in NO in the prefrontal cortex and hippocampus, which acts as a mediator of neurotoxicity, oxidative stress, and neuroinflammation ( 90 , 105 ). Elevated NO levels, often resulting from the activation of inducible iNOS by pro-inflammatory microglia, contribute to neuronal damage, cognitive impairment, and long-term behavioral deficits, such as learning and memory dysfunction ( 106 , 107 ). This was similar to the finding in our study Alterations in cholinergic neurotransmission represent another critical mechanism underlying the observed deficits. The significant reduction in AChE activity in the prefrontal cortex and hippocampus suggests impaired regulation of acetylcholine (ACh) levels. ACh is a key neurotransmitter involved in learning, memory, and attention, and its activity is tightly regulated by AChE ( 108 , 109 ). Reduced AChE activity may result in excessive accumulation of ACh in the synaptic cleft, leading to prolonged activation of muscarinic and nicotinic receptors. This sustained stimulation can cause receptor desensitization, synaptic fatigue, and impaired neurotransmission [60–63]. Overactivation of M1 muscarinic receptors may disrupt intracellular signaling pathways, including the phosphoinositide cascade, thereby impairing long-term potentiation (LTP), a critical process for memory consolidation ( 110 , 111 ). Similarly, desensitization of nicotinic receptors can impair fast synaptic transmission, contributing to deficits in attention and learning. This may have resulted in the behaviour deficit observed in the EPM and Y-Maze in our present study. The prefrontal cortex (PFC) and hippocampus are distinct, interconnected brain regions that, while working together in memory processes, serve different primary functions ( 112 ). The hippocampus typically shows higher acetylcholinesterase (AChE) activity, as it is a major recipient of dense cholinergic innervation from the basal forebrain, which may have accounted for the difference in the AChE activities in both the prefrontal cortex and hippocampus region ( 113 ). Pyrethroids have also been reported to directly interact with AChE, potentially inhibiting its activity through binding to its hydrophobic and anionic sites [67]. Previous studies have reported a decrease in acetylcholinesterase (AChE) activity in the brain of the Wistar rat after exposure to transfluthrin ( 114 ). Similarly, another study also demonstrated a dose-dependent reduction in AChE activity following cypermethrin exposure, as well as decreased enzyme activity after short-term pyrethroid treatment ( 65 ). These findings are consistent with the present study and further support the role of cholinergic dysfunction in pyrethroid-induced neurotoxicity. Another study observed that there was an increase in plasma nitrite and nitrate, and decreased activity of acetyl cholinesterase (AChE) following allethrin and prallethrin exposure in human subjects ( 115 ). Inhibition of acetylcholinesterase (AChE) activity in target tissues is widely employed as a biomarker of pesticide intoxication ( 116 ). Several earlier studies have demonstrated a strong correlation between AChE inhibition in blood and that observed in target tissues, supporting the reliable indicators of tissue-specific neurotoxicity induced by insecticides ( 117 ). Moreover, cholinergic imbalance may indirectly affect other neurotransmitter systems, including dopaminergic and serotonergic pathways, which are critical for mood regulation and cognitive function ( 118 , 119 ). Disruption of dopamine signaling, particularly through D1 and D2 receptors, has been associated with anxiety and depressive-like behaviours ( 120 , 121 ), while altered serotonergic transmission in the amygdala and hippocampus may further exacerbate anxiety states ( 122 , 123 ) The histopathological findings observed in this study provide structural evidence supporting the biochemical and behavioural alterations. Neuronal degeneration, cytoplasmic vacuolation, pyknotic nuclei, and disrupted neuronal architecture were evident in both the prefrontal cortex and hippocampus, with greater severity at higher exposure levels. These findings are consistent with a study that reported neural degeneration, infiltration of lymphocytes, and pyknosis of the cytoplasm of neurons in brain tissues of rats exposed to transfluthrin ( 124 ). Another study reported that pyrethroids induce mitochondrial dysfunction, oxidative damage, and neuronal loss in the hippocampus and prefrontal cortex of rats ( 125 ). Another study reported that cerebral necrosis in the hippocampus and the striatum was caused by the pesticide ( 126 ). Degenerative changes with chromatolytic cells in the microarchitecture of the cerebellum, hippocampus, and prefrontal cortex have been documented following both low and high doses of exposure to permethrin ( 82 ). In the hippocampus, damage to the CA1 and CA3 regions, as well as the dentate gyrus, is particularly significant, as these areas are essential for memory encoding and consolidation ( 15 ). This was also reported in our findings. The observed degeneration in the subgranular zone suggests impaired neurogenesis, which may further contribute to the cognitive deficits seen in our behavioural evaluation. Previous studies have reported degenerative and chromatolytic changes in the prefrontal and hippocampus region of the brain of exposed rats, which were the consequence of neurochemical and oxidative disruptions ( 82 ). The compromised structural integrity of the prefrontal cortex and hippocampus is capable of affecting vital neurologic functions of these two regions, leading to behavioral deficits ( 82 ).The findings above correlated well with the imbalance in the redox system that followed transfluthrin toxicity. Oxidative stress has been suggested to be part of the pathophysiology of neurologic conditions associated with toxicity ( 127 , 128 ). Neurogenesis in the dentate gyrus is essential for hippocampal function, as these neurons integrate into existing circuits and support learning and memory ( 79 , 129 ). Moreover, impairments in adult neurogenesis have been shown to result in deficits in hippocampal-dependent learning and memory ( 130 ). Therefore, transfluthrin-induced disruption of hippocampal neurogenesis may also underlie the behavioral deficits observed in rats. CONCLUSION Overall, the findings of this study demonstrate that exposure to transfluthrin smoke induces significant neurobehavioral, biochemical, and structural alterations in the brain. The convergence of oxidative stress, nitrosative stress, cholinergic dysfunction, and histopathological damage provides a comprehensive mechanistic framework for understanding TBIP-induced neurotoxicity. These results are consistent with a growing body of evidence highlighting the adverse neurological effects of pyrethroid-based insecticides and emphasize the potential public health risks associated with their widespread use. Declarations Ethics declarations Experimental procedures involving the animals and their care were conducted in conformity with the guiding principles for research involving animals as recommended by the Declaration of Helsinki and the Guiding Principles in the Use and Care of Animals ( 51 ) and approved by the animal experiment ethics committee of the College of Medicine, Lagos State University. Consent for publication Not applicable Competing interests The authors declare no competing interests. Funding Not applicable Author Contribution D.A.O. conceptualized, designed the study, performed the experiment, and drafted the original manuscript; T.S.O. interpreted the results, prepared the figures, and revised the manuscript; T.O.O. collected and analyzed the data and contributed to its interpretation; O.K.I. conducted the literature search and contributed to manuscript review and editing; O.M.I. reviewed and edited the manuscript and approved the final version for publication. 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Noradrenergic and cholinergic systems take centre stage in neuropsychiatric diseases of ageing. Neurosci Biobehavioral Reviews. 2023;149. ZARRINDAST MR, Khakpai F. The modulatory role of dopamine in anxiety-like behavior. 2015. Hasbi A, Nguyen T, Rahal H, Manduca JD, Miksys S, Tyndale RF, et al. Sex difference in dopamine D1-D2 receptor complex expression and signaling affects depression-and anxiety-like behaviors. Biology sex differences. 2020;11(1):8. Karayol R, Medrihan L, Warner-Schmidt JL, Fait BW, Rao MN, Holzner EB, et al. Serotonin receptor 4 in the hippocampus modulates mood and anxiety. Mol Psychiatry. 2021;26(6):2334–49. Sidorova M, Kronenberg G, Matthes S, Petermann M, Hellweg R, Tuchina OP et al. Enduring Effects of Conditional Brain Serotonin Knockdown, Followed by Recovery, on Adult Rat Neurogenesis and Behavior. Cells. 2021;10. Sylvestar Darvin S, ASK S, Vincent S, Ignacimuthu. HISTOPATHOLOGY AND PROTEIN PROFILE CHANGES IN, WISTAR RATS EXPOSED TO TRANSFLUTHRIN. International Conference on Contemporary Resarch Trends in Diagnostics and Therapeutics; Anna University. Research Gate2015. Lombán IA, Torres BL, Guerra JEM, Caballero MM, Larrañaga MRM, Navarro AA et al. PYRETHROID INSECTICIDE LAMBDACYHALOTHRIN INDUCE OXIDATIVE STRESS AND MITOCHONDRIAL DAMAGE IN RAT HIPPOCAMPUS AND PREFRONTAL CORTEX. Revista de Toxicología. 2024;41(1). Gasmi S, Rouabhi R, Kebieche M, Boussekine S, Salmi A, Toualbia N, et al. Effects of Deltamethrin on striatum and hippocampus mitochondrial integrity and the protective role of Quercetin in rats. Environ Sci Pollut Res. 2017;24(19):16440–57. Dasgupta R, Jain N. Histopathological assessment of transfluthrin toxicity in rat liver and kidney tissues. J Environ Biol. 2024;45(1):45–52. Jha L, Transfluthrin. Unmasking The Hidden HealthRisks Of A Common Mosquito Repellent. nternational J Creative Res Thoughts (IJCRT). 2025;13(6). Kozareva DA, Cryan JF, Nolan YM. Born this way: Hippocampal neurogenesis across the lifespan. Aging Cell. 2019;18(5):e13007. Disouky A, Lazarov O. Adult hippocampal neurogenesis in Alzheimer's disease. Prog Mol Biol Transl Sci. 2021;177:137–56. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 14 May, 2026 Reviewers agreed at journal 14 May, 2026 Reviewers agreed at journal 12 May, 2026 Reviews received at journal 11 May, 2026 Reviewers agreed at journal 10 May, 2026 Reviews received at journal 09 May, 2026 Reviewers agreed at journal 09 May, 2026 Reviewers agreed at journal 09 May, 2026 Reviewers agreed at journal 09 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers invited by journal 07 May, 2026 Editor assigned by journal 07 May, 2026 Editor invited by journal 04 May, 2026 Submission checks completed at journal 02 May, 2026 First submitted to journal 02 May, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9528142","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":640710531,"identity":"ee9a6231-0b52-4192-a993-e664104b975b","order_by":0,"name":"David Anuoluwapo Oyeniran","email":"data:image/png;base64,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","orcid":"","institution":"Elizade University","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"Anuoluwapo","lastName":"Oyeniran","suffix":""},{"id":640710532,"identity":"e0b4ce79-8f34-4b95-acce-22ea6ccfd07a","order_by":1,"name":"Tobiloba Samuel Olajide","email":"","orcid":"","institution":"University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Tobiloba","middleName":"Samuel","lastName":"Olajide","suffix":""},{"id":640710533,"identity":"78950ebe-b3e8-4b37-8391-ad62cce84f6c","order_by":2,"name":"Toheeb Olalekan Oyerinde","email":"","orcid":"","institution":"University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Toheeb","middleName":"Olalekan","lastName":"Oyerinde","suffix":""},{"id":640710534,"identity":"7d3bc863-45dd-46eb-85ae-c038bd2575c3","order_by":3,"name":"Olayemi Kafilat Ijomone","email":"","orcid":"","institution":"University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Olayemi","middleName":"Kafilat","lastName":"Ijomone","suffix":""},{"id":640710535,"identity":"ea7b10ab-e715-4eda-a7d9-5fb7a7842c05","order_by":4,"name":"Omamuyovwi Meashack Ijomone","email":"","orcid":"","institution":"University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Omamuyovwi","middleName":"Meashack","lastName":"Ijomone","suffix":""}],"badges":[],"createdAt":"2026-04-25 20:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9528142/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9528142/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109452056,"identity":"46c8b047-d64a-4ab9-ba35-97663f10b19a","added_by":"auto","created_at":"2026-05-18 09:11:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":87051,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of smoke from TBIP exposure on anxiety-related behaviors in the EPM.\u003c/strong\u003e Parameters assessed include (A) time spent in the open arms (TSOA), (B) time spent in the closed arms (TSCA), (C) number of open arm entries (NOAE), and (D) number of closed arm entries (NCAE). Data are expressed as mean ± SEM (n = 10 per group). *P \u0026lt; 0.05 indicates a significant difference compared to control.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-9528142/v1/f49efa9cc9824d308d4c0229.png"},{"id":109452057,"identity":"38e45ea3-c3ef-4c5e-8fa8-0627436ce458","added_by":"auto","created_at":"2026-05-18 09:11:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62608,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eshows the effect of smoke from TBIP on (A) learning and (B) memory in rats \u003c/strong\u003e* P \u0026lt; 0.05; significantly different from control. Values are expressed as mean ± SEM for n=10 in each group.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-9528142/v1/95369fb0f2aa6ad326eb72e7.png"},{"id":109452100,"identity":"33954a29-8fa7-49c3-b7d7-6b59dab97dea","added_by":"auto","created_at":"2026-05-18 09:11:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":18263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of smoke from TBIP on memory performance in the Y-maze. \u003c/strong\u003e* P \u0026lt; 0.05; significantly different from control. Values are expressed as mean ± SEM for n=10 in each group.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-9528142/v1/1db9c75b9387873e4e2cef55.png"},{"id":109452071,"identity":"1030caea-777e-4a86-9857-19408462e9a6","added_by":"auto","created_at":"2026-05-18 09:11:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":154466,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea: shows the effect of smoke from TBIP on oxidative parameters in the hippocampus of rats. \u003c/strong\u003e* P \u0026lt; 0.05; significantly different from control. Values are expressed as mean ± SEM for n=10 in each group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb: shows the Effect of smoke from TBIP on oxidative parameters in the prefrontal cortex of rats\u003c/strong\u003e. * P \u0026lt; 0.05; significantly different from control. Values are expressed as mean ± SEM for n=10 in each group.\u003c/p\u003e","description":"","filename":"4a.png","url":"https://assets-eu.researchsquare.com/files/rs-9528142/v1/eeac964207ef691e7a658037.png"},{"id":109452149,"identity":"66f3ba1c-569f-48ab-ae19-22e4c41333a4","added_by":"auto","created_at":"2026-05-18 09:12:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":387855,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eshows the effect of smoke from TBIP on NO Levels. a) Prefrontal cortex, b) Hippocampus of rats\u003c/strong\u003e. * P \u0026lt; 0.05; significantly different from control. Values are expressed as mean ± SD for n=10 in each group.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-9528142/v1/1c7bea823ba7b17fe10f317c.png"},{"id":109452214,"identity":"5af95eff-d41d-4b63-8700-71de306b150c","added_by":"auto","created_at":"2026-05-18 09:12:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":61786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eshows the effect of smoke on TBIP on acetylcholinesterase activity a) Prefrontal cortex, b) Hippocampus.\u003c/strong\u003e * P \u0026lt; 0.05; significantly different from control. Values are expressed as mean ± SEM for n=10 in each group.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-9528142/v1/6337afb789fc054ca550f982.png"},{"id":109452172,"identity":"3ad86d77-6d64-4573-beda-2b7bfb7ef602","added_by":"auto","created_at":"2026-05-18 09:12:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1549747,"visible":true,"origin":"","legend":"\u003cp\u003eShows histological feature of prefrontal cortex and the hippocampus subregions; cortex showing normal pyramidal neurons (yellow arrowhead), glial cells (black arrowhead), and neurites (black asterisk) in control, with TBIP 6 g and 12 g groups exhibiting degeneration (red arrowhead), vacuolation (green arrowhead), lesions (white arrowhead), disorganized neurites (yellow asterisk), and pyknotic neurons (blue arrowhead). CA1 region of the hippocampus (H\u0026amp;E, ×400) showing molecular (red asterisk), pyramidal (yellow asterisk), and polymorphic (black asterisk) layers in control. TBIP 6 g shows reduced PCL thickness and vacuolated glial cells (yellow arrowhead), while 12 g shows disorganized PCL, degeneration (red arrowhead), vacuolation (yellow arrowhead), and pyknotic neurons (black arrowhead). CA3 region of the hippocampus (H\u0026amp;E, ×400) showing distinct molecular (red asterisk), pyramidal (yellow asterisk), and polymorphic (black asterisk) layers in control. TBIP 6 g and 12 g groups show degeneration and vacuolation (red arrowhead), with the 12 g group exhibiting disorganized pyramidal cells and numerous pyknotic neurons (black arrowhead). Dentate gyrus (H\u0026amp;E, ×400) showing molecular (red asterisk), granule (yellow asterisk), and polymorphic (black asterisk) layers in control. TBIP 6 g and 12 g groups retain the three-layered structure but show degeneration and vacuolation at the subgranular zone (black arrowhead), with cytoplasmic vacuolation in polymorphic (red arrowhead) and granular (yellow arrowhead) cells.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-9528142/v1/594b9a94368fa9f15439bf0b.png"},{"id":109452252,"identity":"935db505-c829-4237-b9b5-586d29854aa8","added_by":"auto","created_at":"2026-05-18 09:12:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2530494,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9528142/v1/29a0edb9-cabf-4682-a905-2ce77c57b30b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transfluthrin-based insecticide exposure: assessment of cognitive function and anxiety-like behavior in rats","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe brain coordinates a wide range of complex functions, including cognition, behavior, and physiological regulation, through the intricate activity of billions of neurons. These neurons communicate via synapses in complex networks that ensure the proper functioning of the nervous system. However, neuronal degeneration or imbalances in excitatory and inhibitory signals can disrupt these processes, contributing to the onset and progression of neurological and psychiatric disorders (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNigeria, like many other countries, is experiencing a growing mental health crisis. An estimated 20% of its population, around 40\u0026nbsp;million individuals, suffers from mental health conditions, including depression, anxiety, and other psychiatric disorders (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Recent data show that approximately 7\u0026nbsp;million Nigerians live with depressive disorders, while 4.9\u0026nbsp;million are affected by anxiety disorders (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In comparison, in the United States, approximately 6% of adults experienced moderate to severe levels of anxiety symptoms. In 2019, 7% were reported to have depressive symptoms, with higher prevalence rates among women than men (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). more so, in the United States, an estimated 57.8\u0026nbsp;million adults were reported to have some form of mental illness (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). These disorders significantly burden individuals and healthcare systems, underlining the urgency of identifying contributing factors, especially those related to environmental exposure.\u003c/p\u003e \u003cp\u003eAnxiety disorders are characterized by symptoms such as restlessness, fatigue, irritability, and persistent fear or worry, which can severely impair daily functioning (\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Another major neurological condition is dementia, with Alzheimer\u0026rsquo;s disease (AD) being its most common form. AD typically begins with mild memory loss and gradually progresses to severe cognitive decline. The disease predominantly affects brain regions, including the hippocampus and amygdala, which are necessary for memory formation and emotional regulation (\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). A critical pathological feature of AD is the degeneration of cholinergic neurons connecting the basal forebrain and hippocampus, which correlates strongly with memory impairment (\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Oxidative stress, neuroinflammation, and disruptions in lipid metabolism are key pathological processes underlying both neurodegenerative and mood disorders (\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The brain is particularly vulnerable to oxidative stress owing to its high lipid content, elevated oxygen consumption, and intense metabolic activity (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). These characteristics predispose neural tissues to lipid peroxidation, ultimately compromising membrane integrity and contributing to neuronal dysfunction (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Disruption of the brain\u0026rsquo;s antioxidant defense system, including key enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx), can arise from excessive endogenous production of reactive oxygen species (ROS) or exposure to exogenous stressors that enhance ROS generation (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Such imbalance is often reflected by increased levels of malondialdehyde (MDA), a well-established marker of lipid peroxidation (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe detrimental effects of oxidative stress are further amplified in the presence of neuroinflammation. The bidirectional interaction between oxidative stress and inflammatory processes plays a critical role in both the maintenance of normal brain function and the pathogenesis of neurological disorders (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Each process can potentiate the other, leading to progressive cellular damage and functional impairment (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eA growing body of evidence points to environmental toxicants such as pesticides, herbicides, and insecticides as major contributors to neuropsychiatric conditions (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). These chemicals can induce oxidative stress, provoke neuroinflammatory responses, and disrupt neuronal signaling pathways, thereby increasing the risk of conditions such as AD, anxiety, and depression (\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Transfluthrin, a widely used pyrethroid insecticide across Africa, has become a chemical of concern due to its extensive application in household pest control and agricultural practices (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Emerging studies have reported its toxic effects on various organs and the immune system, highlighting its potential risks to human health and the environment (\u003cspan additionalcitationids=\"CR35 CR36\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). The neurotoxic effects of pyrethroid insecticides are primarily attributed to their interference with voltage-gated sodium channels, leading to prolonged depolarization and hyperexcitability of the nervous system (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). They can also modulate acetylcholinesterase (AChE) activity in the brain, often leading to reduced enzyme activity (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Although organophosphates are well-known AChE inhibitors, studies have also demonstrated that certain pyrethroids can affect AChE activity in brain regions such as the cerebral cortex, cerebellum, and hippocampus, resulting in impaired neural function in these regions (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). They bind to these channels and prevent their normal inactivation, causing them to remain open longer than usual. This results in repetitive neuronal firing and sustained excitatory signaling (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Additionally, pyrethroids have been shown to affect isoforms of voltage-sensitive calcium channels, further disrupting calcium homeostasis and impairing neurotransmitter release (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Beyond ion channel interference, exposure to this type of insecticide is associated with the induction of oxidative stress, a state characterized by an imbalance between the production of ROS and the body\u0026rsquo;s antioxidant defenses. This oxidative burden can damage cellular components, including lipids, proteins, DNA, and neurons, thereby contributing to neurodegeneration (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Moreover, they may trigger neuronal cell death through mechanisms such as excitotoxicity (excessive glutamate-mediated stimulation) and apoptosis (programmed cell death) (\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Emerging evidence also suggests that pyrethroids impair mitochondrial function, disrupting cellular energy production and increasing neuronal vulnerability to stress and injury (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Despite advances in our understanding of pesticide-induced neurotoxicity, significant gaps remain, particularly regarding the behavioral and cognitive consequences of specific agents like transfluthrin. To address this, the present study aims to investigate the neurobehavioral and cognitive effects of exposure to smoke from transfluthrin-based insecticide paper (TBIP) in adult male Wistar rats, thereby contributing to a more comprehensive understanding of environmental risk factors in neurological health.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS.","content":"\u003cp\u003eA total of thirty (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e) male Wistar rats (8 to 10 weeks old) weighing 150\u0026ndash;170 g were procured from a breeding stock at the University of Ibadan in Oyo State and were kept under standard laboratory conditions, with unrestricted access to water and food, and a consistent 12-hour daylight cycle. Rats were given a 2-week acclimation period to the laboratory environment prior to the onset of experiments.\u003c/p\u003e \u003cp\u003eThe insecticide used in this study was Rambo\u0026reg; (Bayer Cropscience), distributed by Gongoni Company Limited. It was procured from a reputable retail outlet in Ejigbo, Lagos State, Nigeria. The paper, which weighed 6g, comprised 0.45% transfluthrin, 2.5% essential oil, and 97.05% inert ingredients. The rats were divided into three groups (A - C) of 10 rats each. Group A was exposed to regular environmental air, Group B rats were exposed to smoke from 6g (burning for 4 hours), and Group C rats were exposed to smoke from 12g (burning for 8 hours) of TBIP via whole-body inhalation for 4 weeks. Dose and method of exposure were done according to the study by Oyeniran, Ojewale (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Rats were exposed to transfluthrin smoke using a custom whole-body inhalation chamber (43 \u0026times; 30 \u0026times; 26 cm) made of transparent acrylic for observation and containment. A transfluthrin-impregnated paper was ignited in a connected sealed combustion unit, and the resulting smoke was channeled into the chamber via heat-resistant tubing. The chamber was equipped with small ventilation outlets to prevent oxygen depletion while still maintaining a sufficient concentration of smoke. Animals were closely monitored for signs of distress during and after exposure.\u003c/p\u003e \u003cp\u003eAfter exposure, rats in Groups A, B, and C underwent behavioral assessments at the first, second, and fourth weeks. Spatial working memory was evaluated using the Y-maze, while the Elevated Plus Maze (EPM) was used to assess the learning and memory capacity. At the end of the fourth week, the rats were subjected to the EPM to evaluate anxiety-like behavior. After the 4-week exposure, the rats were weighed and euthanized following anesthesia with ketamine (80 mg/kg, i.p.) to induce deep unconsciousness, followed by cervical dislocation. The brains were then carefully excised, weighed, and processed for both biochemical assays and histopathological evaluation (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Experimental procedures involving the animals and their care were conducted in conformity with the guiding principles for research involving animals as recommended by the Declaration of Helsinki and the Guiding Principles in the Use and Care of Animals (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Likewise, all methods are reported in accordance with ARRIVE guidelines (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e)\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eBEHAVIORAL ANALYSIS\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eAssessment of Anxiety-like behaviour using the EPM Test\u003c/h2\u003e \u003cp\u003eIn this study, the EPM test for evaluating anxiety-like behavior was configured in a plus (+) shape and elevated 50 cm above the ground. It consisted of two open arms, each measuring 45 cm in length and 10 cm in width, and two enclosed arms of the same length and width, with enclosing walls 30 cm high, all extending from a central square platform (10 cm \u0026times; 10 cm). To ensure acclimatization, rats were transferred to the testing room 30 minutes before the experiment and maintained under dim lighting. At the start of the test, each rat was positioned on the central platform, facing any of the open arms, and allowed to freely explore the maze for 5 minutes (300 seconds). After every trial, the maze was thoroughly sanitized using 70% ethanol to eliminate residual olfactory cues. Behavioral parameters recorded included: time spent in open arms (TSOA), time spent in closed arms (TSCA), number of open arm entries (NOAE), and number of closed arm entries (NCAE) (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eAssessment of Learning and Memory using the EPM Test\u003c/h3\u003e\n\u003cp\u003eTwenty-four hours before the test, each rat was placed at the distal end of an open arm of the EPM, facing away from the central platform. The time taken to move from the open arm to any of the enclosed arms (measured in seconds) was recorded as the transfer latency (TL) of learning. After the rat entered an enclosed arm, it was allowed to explore the maze for 30 seconds before being removed. The same protocol was performed after a 24-hour interval to assess memory retention. The time taken to enter an enclosed arm was recorded again as the transfer latency (TL) of memory (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). Behavioral assessments were conducted by a student who was blinded to the experimental group assignments, employing a single-blind approach to minimize observer bias.\u003c/p\u003e\n\u003ch3\u003eAssessment of spatial working memory using the Y-Maze\u003c/h3\u003e\n\u003cp\u003eThe Y-maze was used to assess spontaneous alternation behavior as an index of spatial working memory, as well as general locomotor activity. The apparatus consisted of three identical arms (41 cm long, 15 cm high, and 5 cm wide) positioned at 120\u0026deg; angles from each other. Each rat was placed at the end of one arm and allowed to explore the maze freely. Arm entries were recorded when the animal\u0026rsquo;s tail completely entered an arm, and the sequence of entries was noted over a 5-minute session. Spontaneous alternation was defined as consecutive entries into all three arms without repetition (e.g., A, B, C). The number of actual alternations was determined from successive overlapping triplet sets of arm entries. The percentage alternation was calculated using the formula: [(number of actual alternations) / (total number of arm entries\u0026thinsp;\u0026minus;\u0026thinsp;2)] \u0026times; 100 (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eDetermination of Lipid Peroxidation and Antioxidant Enzyme Activities\u003c/h3\u003e\n\u003cp\u003eA 10% (w/v) brain tissue homogenate (prefrontal cortex and hippocampus) was prepared in 0.9% sterile saline solution. The homogenate was centrifuged at 6000 rpm for 10 minutes at 4\u0026deg;C, and the resulting supernatant was collected for biochemical analyses. Catalase (CAT) activity was assayed according to the method of Aebi (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e), which is based on the rate of decomposition of 1 \u0026micro;M hydrogen peroxide (H₂O₂), and the results were expressed as U/mg protein. Superoxide dismutase (SOD) activity was determined following the procedure of (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e), while reduced glutathione (GSH) content was measured using the method described by Ellman et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Lipid peroxidation was evaluated by determining malondialdehyde (MDA) levels through the formation of a red-colored complex with thiobarbituric acid (TBA), and MDA concentrations were expressed as nmol/mg protein. All assays were performed in duplicate to ensure analytical reliability (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Nitric Oxide (NO)\u003c/h2\u003e \u003cp\u003eNitric oxide levels were determined by colorimetric quantification of nitrite in brain tissue homogenates using the Griess assay. The Griess reagent consisted of 1.5% sulphanilamide in 1 mol/L HCl containing 0.15% N-(1-naphthyl)ethylenediamine dihydrochloride. In this procedure, nitrates present in the samples were first reduced to nitrites, which then reacted with the Griess reagent to form a chromophoric azo compound exhibiting a purple coloration. The resulting absorbance was measured at 540 nm to determine the nitrite and, consequently, NO concentration (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEvaluation of Acetylcholinesterase Activity\u003c/h3\u003e\n\u003cp\u003eEach brain region was homogenized in 50 mM Tris-HCl buffer (pH 7.4) containing 1.15% potassium chloride to prepare a 10% (w/v) tissue homogenate. The homogenate was then centrifuged at 10,000 \u0026times; g for 10 minutes at 4\u0026deg;C, and the resulting supernatant was collected for the determination of acetylcholinesterase (AChE) activity. AChE activity in both the hippocampus and prefrontal cortex was measured using the method of (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e) Ellman et al. (1961), as modified by (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e), employing acetylthiocholine iodide as the substrate. In this assay, AChE catalyzes the hydrolysis of acetylthiocholine iodide to yield thiocholine and butyric acid. The liberated thiocholine reacts with 5,5\u0026prime;-dithiobis(2-nitrobenzoic acid) (DTNB) to produce 5-thio-2-nitrobenzoic acid, a yellow-colored compound. The intensity of the color is measured spectrophotometrically at 412 nm and is proportional to the AChE activity.\u003c/p\u003e\n\u003ch3\u003eHistological Evaluation\u003c/h3\u003e\n\u003cp\u003eFor histological analysis, fixed brain tissues in 10% neutral buffered formalin were grossly dissected to expose the regions of interest. The tissues were subsequently dehydrated through a graded ethanol series and cleared in xylene. They were then infiltrated and embedded in molten paraffin wax at 58\u0026deg;C. Paraffin blocks were prepared, and serial sections of 5 \u0026micro;m thickness were cut using a microtome. The sections were rehydrated and stained with hematoxylin and eosin (H\u0026amp;E). Thereafter, sections of the prefrontal cortex and hippocampus were examined under a light microscope at \u0026times;400 magnification (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData was analyzed statistically using analysis of variance (ANOVA), followed by Tukey post-hoc test using GraphPad Prism 8.0 software. Differences between means were considered significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 when compared with the control, and results were expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eEffects of smoke from TBIP on body and organ weight.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe body weight of the experimental group was lower than that of the control group. At p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, group C showed a significant reduction in body weight compared with the control. The brain weights of rats in groups B and C were significantly lower than those in the control group. Additionally, the relative brain weight (brain weight/final body weight) of the high-exposure group was significantly lower than that of the control at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEffect of smoke from TBIP on body and organ weight.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTBIP 6g\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTBIP 12g\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eInitial body weight (g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e152\u0026thinsp;\u0026plusmn;\u0026thinsp;7.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e140\u0026thinsp;\u0026plusmn;\u0026thinsp;6.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e156\u0026thinsp;\u0026plusmn;\u0026thinsp;4.57\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003efinal body weight (g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e205\u0026thinsp;\u0026plusmn;\u0026thinsp;5.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e195\u0026thinsp;\u0026plusmn;\u0026thinsp;9.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e184\u0026thinsp;\u0026plusmn;\u0026thinsp;7.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBody-weight difference (g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e55\u0026thinsp;\u0026plusmn;\u0026thinsp;4.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e28\u0026thinsp;\u0026plusmn;\u0026thinsp;5.35*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eBrain weight (g)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRelative brain weight\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.0077\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.0063\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.0057\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e* P\u0026thinsp;\u0026lt;\u0026thinsp;0.05; significantly different from control. Values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM for n\u0026thinsp;=\u0026thinsp;10 in each group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of smoke from TBIP on anxiety-like behaviors in rats using the EPM test.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eExposure to TBIP significantly reduced the time spent in the open arms (TSOA) and the number of open arm entries (NOAE) in the 6 g and 12 g groups compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, the time spent in the closed arms (TSCA) was significantly increased in the treated groups, while the number of closed arm entries (NCAE) was significantly higher in the TBIP 12 g group compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Smoke From TBIP on Learning and Memory Using EPM.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDuring the first week of the learning phase, rats exposed to TBIP 12 g exhibited a significantly longer transfer latency to the closed arm compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In weeks 2 and 4, both TBIP-treated groups (6 g and 12 g) showed prolonged learning times relative to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). A similar pattern was observed in the retention (memory) phase, where the TBIP 12 g group showed a significantly longer transfer latency in week 1. Both the TBIP 6 g and 12 g groups exhibited extended transfer latencies in weeks 2 and 4 compared to the control at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Smoke From TBIP on Memory Performance Using the Y-maze\u003c/h2\u003e \u003cp\u003eRats in the control group consistently demonstrated good memory performance (spontaneous alternation) in the Y-maze test throughout the evaluation period. At week 1, rats exposed to 6 g of TBIP exhibited memory performance comparable to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, by weeks 2 and 4, a significant decline in memory performance was observed in this group compared with the control. In contrast, rats exposed to the higher dose of TBIP (12 g) showed a significant reduction in memory performance at all three assessment time points (weeks 1, 2, and 4) when compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of Smoke From TBIP on Oxidative Parameters in the prefrontal cortex and hippocampus\u003c/h2\u003e \u003cp\u003eThe oxidative stress assessment revealed that MDA levels in both the prefrontal cortex and hippocampus were significantly elevated in rats exposed to 6 g and 12 g of TBIP compared to the control group at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. On the other hand, the activities of antioxidant enzymes SOD, CAT, and GSH were markedly reduced at both doses in the prefrontal cortex and hippocampus. However, for GSH, a significant decrease was observed only at the higher dose (12 g) in both brain regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEffect of Smoke from TBIP on NO Levels in the Prefrontal Cortex and Hippocampus of Rats\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThe results showed that NO levels in both the prefrontal cortex and hippocampus of rats exposed to TBIP were significantly higher compared to the control group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEffect of TBIP smoke on Acetylcholinesterase levels\u003c/h2\u003e \u003cp\u003eThe acetylcholinesterase enzyme activity in the prefrontal cortex was significantly reduced in the high dose group compared to the control, while the low dose was not significantly different from the control. However, in the hippocampus, it was found that both experimental groups had a significant acetylcholinesterase level compared to the control at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological Evaluation of the effect of smoke of TBIP on Prefrontal Cortex and Hippocampal Subregions\u003c/h2\u003e \u003cp\u003eHistological analysis revealed dose-dependent neurodegenerative changes in the prefrontal cortex and hippocampal subregions of TBIP-treated rats compared to controls. In the prefrontal cortex, the control group showed normal cortical architecture with intact pyramidal neurons, glial cells, and well-organized neurites. TBIP exposure induced progressive neuronal degeneration, cytoplasmic vacuolation, focal lesions, and disrupted neurite arrangement. These alterations were more severe in the 12 g group, showing extensive vacuolation and pyknotic neurons. CA1 and CA3 regions of the control sections displayed well-defined laminar organization with compact pyramidal neurons. TBIP 6 g caused a mild reduction in pyramidal layer thickness and moderate vacuolation, while the 12 g group exhibited pronounced disorganization, neuronal degeneration, loss of pyramidal cell density, and numerous pyknotic neurons within the tissue parenchyma. In the dentate gyrus, the control group exhibited intact molecular, granule, and polymorphic layers. However, TBIP-treated groups retained this general structure but showed neuronal degeneration and vacuolation, particularly in the subgranular zone, alongside cytoplasmic vacuolation in glial and interneuronal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe integrity of cognitive and emotional function depends on coordinated biochemical signaling, synaptic plasticity, and structural stability across key brain regions such as the hippocampus and prefrontal cortex (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Disruption of these processes by environmental toxicants, such as pyrethroid-based insecticides, can lead to profound neurobehavioral and neurochemical alterations (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, it was seen that exposure to smoke from TBIP resulted in significant impairments in learning and memory, increased anxiety-like behaviours, and reductions in body and brain weights. Alterations in oxidative and nitrosative stress markers, decreased AChE activity, and marked histopathological damage in the prefrontal cortex and hippocampus were also observed. The reduction in body weight observed, particularly in the group exposed to smoke from a higher dose (12 g) of transfluthrin paper, corroborates earlier findings reported by Kalra and Sangha (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). Grewal et al. (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e) also reported similar weight loss following pyrethroid exposure. Studies involving mosquito vaporizer fumes containing 0.88% Transfluthrin (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e) and repeated exposure to cypermethrin (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e) have also documented decreased body weight, aligning with our findings. This effect is likely multifactorial, involving reduced food intake, gastrointestinal irritation, and impaired nutrient absorption (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e). Additionally, systemic toxicity associated with pyrethroid exposure may directly impair metabolic processes, contributing to weight loss (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). The observed reduction in brain weight, both absolute and relative, aligns with previous reports indicating that pyrethroid groups of insecticides may reduce organ weights, including the brain, kidney, and testes (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). Oxidative stress-induced neuronal degeneration may further contribute to this reduction in brain mass (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e), corroborating the neuronal depletion seen in the prefrontal and hippocampus of rat exposure to TBIP.\u003c/p\u003e \u003cp\u003eA major finding of this study is the impairment in cognitive function, as evidenced by increased transfer latency in the elevated plus maze and reduced spontaneous alternation in the Y-maze. These findings indicate deficits in both learning acquisition and memory retention. The hippocampus and prefrontal cortex function as an integrated network in memory processing (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e). The hippocampus is responsible for encoding and consolidating new information, while the prefrontal cortex is involved in higher-order processing, organization, and retrieval of memory (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e). Disruption of this circuitry provides a plausible explanation for the observed cognitive deficits. Previous studies have shown that pyrethroid exposure impairs learning and memory in rats and mice, often in association with oxidative stress, dopaminergic dysfunction, and hippocampal damage (\u003cspan additionalcitationids=\"CR76\" citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). For instance, exposure to flumethrin has been shown to downregulate memory-related genes such as GluRA1, Nmdar1, and Tyr1, thereby impairing olfactory learning and memory (\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e). Similarly, chronic administration of deltamethrin has been associated with hippocampal endoplasmic reticulum stress, impaired neurogenesis, and learning deficits (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e), which is consistent with the neuronal depletion and vacuolation observed in the dentate gyrus in the present study. Chlorpyrifos, permethrin, and cyfluthrin, similar to transfluthrin, have been reported to affect cell survival, permeability, and tight junction in an in-vitro model of the human blood-brain barrier (BBB) (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e). Suggesting that the ability of transfluthrin to cross the BBB may have contributed to its effect on the prefrontal cortex and hippocampus, resulting in neurobehavioral deficits as seen in the rat exposure to TBIP.\u003c/p\u003e \u003cp\u003eThe present findings demonstrate that transfluthrin exposure induces pronounced anxiety-like behavior, as evidenced by reduced exploration of the open arms and a clear preference for the closed arms in the EPM. Consistent with this observation, transfluthrin-exposed rats in previous studies have similarly exhibited increased anxiety-like behavior, hyperactivity, and impairments in spatial learning and memory (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnother study reported the anxiogenic potential of pyrethroids such as permethrin (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e), and reduced exploratory behavior and altered locomotor indices in paradigms like negative geotaxis (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e). More broadly, cypermethrin, recognized as an emerging neurotoxin, has been shown to induce a spectrum of behavioral and psychological disturbances, particularly following prolonged exposure (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e). Together, these findings suggest that anxiety-like behavior represents a consistent neurobehavioral outcome of pyrethroid toxicity, such as cypermethrin, permethrin, and transfluthrin. This reinforces the vulnerability of neural circuits governing emotion and cognition to pyrethroid exposure (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e). Previous studies using cypermethrin have reported a significant reduction in total distance traveled at higher doses (20 and 80 mg/kg). They also demonstrated that reductions in open-arm time and entries were significant, indicating anxiogenic effects (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e). This aligns with the present findings, where the observed increase in closed-arm preference is more probably linked to anxiety. The observed behavioral alterations may be closely linked to neuroinflammatory processes within brain regions critical for emotional regulation, including the amygdala, hippocampus, and prefrontal cortex (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e). Neuroinflammation, characterized by elevated levels of pro-inflammatory cytokines such as TNF-α and IL-1β, has been strongly implicated in both anxiety disorders and cognitive dysfunction (\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e). Supporting this, Gargouri et al.(\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e) reported that bifenthrin exposure induced oxidative stress, neuroinflammation, and memory deficits in rats, thereby strengthening the association between oxidative damage, inflammatory responses, and behavioral impairments (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e). These pathways may have likely contributed to the anxiety-like phenotype observed in the present study. The neurotoxic effects of pyrethroids, including transfluthrin, are largely mediated through their action on voltage-gated sodium channels. These compounds prolong the open state of sodium channels, thereby delaying channel inactivation and promoting sustained neuronal depolarization (\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e). This aberrant increase in neuronal excitability disrupts normal synaptic transmission and neural network stability, ultimately manifesting as behavioral abnormalities. In more severe cases, such prolonged depolarization can lead to conduction block and neuromuscular dysfunction due to impaired repolarization dynamics (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOxidative stress emerges as a central mechanism underlying the neurotoxic effects observed in this study. The marked elevation in MDA levels was accompanied by significant reductions in key antioxidant defenses, including SOD, CAT, and GSH, in both the prefrontal cortex and hippocampus. These findings clearly indicate a disruption of redox homeostasis. These findings are consistent with earlier reports showing increased lipid peroxidation and compromised antioxidant capacity in rat brain following pyrethroid exposure (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e). A study on zebrafish found that transfluthrin and prallethrin-based insecticides cause deleterious effects on antioxidant systems (\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e). Given the brain\u0026rsquo;s high lipid composition and substantial oxygen demand, it is particularly susceptible to oxidative injury (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e). Lipid peroxidation can destabilize neuronal membranes, impair ion gradients, and disrupt synaptic signaling, ultimately predisposing neurons to degeneration and cell death (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e). The SOD and GPx are antioxidant enzymes ubiquitous in living organisms, acting as an endogenous defense against ROS (\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e). Muhammad et al. showed in their study that repeated oral administration of Bifenthrin for 14 days led to an increase in lipid peroxidation and a decrease in antioxidant enzymes in the Brain of \u003cem\u003eOryctologus cuniculus\u003c/em\u003e (\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e). The concurrent increase in MDA and depletion of endogenous antioxidant systems observed in this study strongly support oxidative stress as a key driver of transfluthrin neurotoxicity. Among the various mechanisms by which insecticides impair neural development and function, oxidative stress remains one of the most prominent (\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e). It contributes to cellular damage through the oxidation of lipids, proteins, and nucleic acids, processes that have been widely implicated in the pathogenesis of neurodegenerative and neurodevelopmental disorders (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Supporting this, animal studies have consistently demonstrated that pyrethroids induce oxidative stress across multiple tissues (\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e). Exposure to Pyrethroid bifenthrin was found to induce neuronal damage, oxidative stress, and cause neuroinflammation in the hippocampus of rats, which may lead to cognitive and memory impairment (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e). Similar to what was reported in our study, cypermethrin a well-studied pyrethroid, has been reported to exert its toxic effects through excessive generation of reactive oxygen species, significantly reducing antioxidant enzymes such as SOD, GST, GSH, CAT, glutathione reductase, and GPx, while simultaneously increasing lipid peroxidation in serum and tissues (\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e, \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e). In an experimental study, its exposure leads to mitochondrial dysfunction, characterized by impaired electron transport and increased ROS production, accompanied by elevated oxidative stress markers and diminished antioxidant enzyme activities (\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e) [16]. These alterations not only compromise cellular energy metabolism but also trigger downstream pathological events, including DNA damage and apoptosis (\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e, \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to oxidative stress, nitrosative stress also appears to contribute to neuronal damage. The significant increase in NO levels observed in this study is indicative of enhanced reactive nitrogen species (RNS) production. This is consistent with a previous study (\u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e). Elevated NO levels, particularly through inducible nitric oxide synthase (iNOS) activation, can lead to the formation of peroxynitrite, a highly reactive molecule that induces protein nitration, lipid peroxidation, and DNA damage (\u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e). In line with our present result, oxidative/nitrosative stress in the hippocampus of rats treated with Pyrethroid bifenthrin, as shown by increased levels of MDA, protein carbonyls (PCO), and NO, and reduced levels of enzymatic and non-enzymatic antioxidants, was reported in a previous study (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). This oxidative/nitrosative stress cascade may have exacerbated neuronal injury and promoted neuroinflammation, further contributing to cognitive and behavioural impairments. In a human study, increased concentration of nitrite and nitrate in plasma, suggesting an increased production of NO in volunteers exposed to mosquito repellent pyrethroids (allethrin and prallethrin), was documented compared to controls (\u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e104\u003c/span\u003e). More so, supporting our finding, exposure to pyrethroids like deltamethrin and bifenthrin as been shown to result in an increase in NO in the prefrontal cortex and hippocampus, which acts as a mediator of neurotoxicity, oxidative stress, and neuroinflammation (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e105\u003c/span\u003e). Elevated NO levels, often resulting from the activation of inducible iNOS by pro-inflammatory microglia, contribute to neuronal damage, cognitive impairment, and long-term behavioral deficits, such as learning and memory dysfunction (\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e, \u003cspan citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e). This was similar to the finding in our study\u003c/p\u003e \u003cp\u003eAlterations in cholinergic neurotransmission represent another critical mechanism underlying the observed deficits. The significant reduction in AChE activity in the prefrontal cortex and hippocampus suggests impaired regulation of acetylcholine (ACh) levels. ACh is a key neurotransmitter involved in learning, memory, and attention, and its activity is tightly regulated by AChE (\u003cspan citationid=\"CR108\" class=\"CitationRef\"\u003e108\u003c/span\u003e, \u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e). Reduced AChE activity may result in excessive accumulation of ACh in the synaptic cleft, leading to prolonged activation of muscarinic and nicotinic receptors. This sustained stimulation can cause receptor desensitization, synaptic fatigue, and impaired neurotransmission [60\u0026ndash;63]. Overactivation of M1 muscarinic receptors may disrupt intracellular signaling pathways, including the phosphoinositide cascade, thereby impairing long-term potentiation (LTP), a critical process for memory consolidation (\u003cspan citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e, \u003cspan citationid=\"CR111\" class=\"CitationRef\"\u003e111\u003c/span\u003e). Similarly, desensitization of nicotinic receptors can impair fast synaptic transmission, contributing to deficits in attention and learning. This may have resulted in the behaviour deficit observed in the EPM and Y-Maze in our present study. The prefrontal cortex (PFC) and hippocampus are distinct, interconnected brain regions that, while working together in memory processes, serve different primary functions (\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e). The hippocampus typically shows higher acetylcholinesterase (AChE) activity, as it is a major recipient of dense cholinergic innervation from the basal forebrain, which may have accounted for the difference in the AChE activities in both the prefrontal cortex and hippocampus region (\u003cspan citationid=\"CR113\" class=\"CitationRef\"\u003e113\u003c/span\u003e). Pyrethroids have also been reported to directly interact with AChE, potentially inhibiting its activity through binding to its hydrophobic and anionic sites [67]. Previous studies have reported a decrease in acetylcholinesterase (AChE) activity in the brain of the Wistar rat after exposure to transfluthrin (\u003cspan citationid=\"CR114\" class=\"CitationRef\"\u003e114\u003c/span\u003e). Similarly, another study also demonstrated a dose-dependent reduction in AChE activity following cypermethrin exposure, as well as decreased enzyme activity after short-term pyrethroid treatment (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). These findings are consistent with the present study and further support the role of cholinergic dysfunction in pyrethroid-induced neurotoxicity. Another study observed that there was an increase in plasma nitrite and nitrate, and decreased activity of acetyl cholinesterase (AChE) following allethrin and prallethrin exposure in human subjects (\u003cspan citationid=\"CR115\" class=\"CitationRef\"\u003e115\u003c/span\u003e). Inhibition of acetylcholinesterase (AChE) activity in target tissues is widely employed as a biomarker of pesticide intoxication (\u003cspan citationid=\"CR116\" class=\"CitationRef\"\u003e116\u003c/span\u003e). Several earlier studies have demonstrated a strong correlation between AChE inhibition in blood and that observed in target tissues, supporting the reliable indicators of tissue-specific neurotoxicity induced by insecticides (\u003cspan citationid=\"CR117\" class=\"CitationRef\"\u003e117\u003c/span\u003e). Moreover, cholinergic imbalance may indirectly affect other neurotransmitter systems, including dopaminergic and serotonergic pathways, which are critical for mood regulation and cognitive function (\u003cspan citationid=\"CR118\" class=\"CitationRef\"\u003e118\u003c/span\u003e, \u003cspan citationid=\"CR119\" class=\"CitationRef\"\u003e119\u003c/span\u003e). Disruption of dopamine signaling, particularly through D1 and D2 receptors, has been associated with anxiety and depressive-like behaviours (\u003cspan citationid=\"CR120\" class=\"CitationRef\"\u003e120\u003c/span\u003e, \u003cspan citationid=\"CR121\" class=\"CitationRef\"\u003e121\u003c/span\u003e), while altered serotonergic transmission in the amygdala and hippocampus may further exacerbate anxiety states (\u003cspan citationid=\"CR122\" class=\"CitationRef\"\u003e122\u003c/span\u003e, \u003cspan citationid=\"CR123\" class=\"CitationRef\"\u003e123\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe histopathological findings observed in this study provide structural evidence supporting the biochemical and behavioural alterations. Neuronal degeneration, cytoplasmic vacuolation, pyknotic nuclei, and disrupted neuronal architecture were evident in both the prefrontal cortex and hippocampus, with greater severity at higher exposure levels. These findings are consistent with a study that reported neural degeneration, infiltration of lymphocytes, and pyknosis of the cytoplasm of neurons in brain tissues of rats exposed to transfluthrin (\u003cspan citationid=\"CR124\" class=\"CitationRef\"\u003e124\u003c/span\u003e). Another study reported that pyrethroids induce mitochondrial dysfunction, oxidative damage, and neuronal loss in the hippocampus and prefrontal cortex of rats (\u003cspan citationid=\"CR125\" class=\"CitationRef\"\u003e125\u003c/span\u003e). Another study reported that cerebral necrosis in the hippocampus and the striatum was caused by the pesticide (\u003cspan citationid=\"CR126\" class=\"CitationRef\"\u003e126\u003c/span\u003e). Degenerative changes with chromatolytic cells in the microarchitecture of the cerebellum, hippocampus, and prefrontal cortex have been documented following both low and high doses of exposure to permethrin (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e). In the hippocampus, damage to the CA1 and CA3 regions, as well as the dentate gyrus, is particularly significant, as these areas are essential for memory encoding and consolidation (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). This was also reported in our findings. The observed degeneration in the subgranular zone suggests impaired neurogenesis, which may further contribute to the cognitive deficits seen in our behavioural evaluation. Previous studies have reported degenerative and chromatolytic changes in the prefrontal and hippocampus region of the brain of exposed rats, which were the consequence of neurochemical and oxidative disruptions (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e). The compromised structural integrity of the prefrontal cortex and hippocampus is capable of affecting vital neurologic functions of these two regions, leading to behavioral deficits (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e).The findings above correlated well with the imbalance in the redox system that followed transfluthrin toxicity. Oxidative stress has been suggested to be part of the pathophysiology of neurologic conditions associated with toxicity (\u003cspan citationid=\"CR127\" class=\"CitationRef\"\u003e127\u003c/span\u003e, \u003cspan citationid=\"CR128\" class=\"CitationRef\"\u003e128\u003c/span\u003e). Neurogenesis in the dentate gyrus is essential for hippocampal function, as these neurons integrate into existing circuits and support learning and memory (\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR129\" class=\"CitationRef\"\u003e129\u003c/span\u003e). Moreover, impairments in adult neurogenesis have been shown to result in deficits in hippocampal-dependent learning and memory (\u003cspan citationid=\"CR130\" class=\"CitationRef\"\u003e130\u003c/span\u003e). Therefore, transfluthrin-induced disruption of hippocampal neurogenesis may also underlie the behavioral deficits observed in rats.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eOverall, the findings of this study demonstrate that exposure to transfluthrin smoke induces significant neurobehavioral, biochemical, and structural alterations in the brain. The convergence of oxidative stress, nitrosative stress, cholinergic dysfunction, and histopathological damage provides a comprehensive mechanistic framework for understanding TBIP-induced neurotoxicity. These results are consistent with a growing body of evidence highlighting the adverse neurological effects of pyrethroid-based insecticides and emphasize the potential public health risks associated with their widespread use.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eEthics declarations\u003c/h2\u003e \u003cp\u003eExperimental procedures involving the animals and their care were conducted in conformity with the guiding principles for research involving animals as recommended by the Declaration of Helsinki and the Guiding Principles in the Use and Care of Animals (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e) and approved by the animal experiment ethics committee of the College of Medicine, Lagos State University.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eConsent for publication\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eD.A.O. conceptualized, designed the study, performed the experiment, and drafted the original manuscript; T.S.O. interpreted the results, prepared the figures, and revised the manuscript; T.O.O. collected and analyzed the data and contributed to its interpretation; O.K.I. conducted the literature search and contributed to manuscript review and editing; O.M.I. reviewed and edited the manuscript and approved the final version for publication.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to acknowledge the contributions of Mr. Oguntola Jamiu at the College of Medicine, Lagos State University, for his technical support.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data are incorporated within the manuscript and are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePaliwal R, Sulakhiya K, Paliwal SR, Singh V, Kenwat R, Paramanik D. 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Brain Sci. 2019;9(11):300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarker GR, Banks PJ, Scott H, Ralph GS, Mitrophanous KA, Wong L-F, et al. Separate elements of episodic memory subserved by distinct hippocampal\u0026ndash;prefrontal connections. Nat Neurosci. 2017;20(2):242\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeles-Grilo Ruivo LM, Baker KL, Conway MW, Kinsley PJ, Gilmour G, Phillips KG, et al. Coordinated Acetylcholine Release in Prefrontal Cortex and Hippocampus Is Associated with Arousal and Reward on Distinct Timescales. Cell Rep. 2017;18(4):905\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHazarika H, Krishnatreyya H, Tyagi V, Islam J, Gogoi N, Goyary D, et al. The fabrication and assessment of mosquito repellent cream for outdoor protection. Sci Rep. 2022;12(1):2180.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNarendra Maddu NG, Fareeda Begum S, Sreekanth B. Mosquito repellent pyrethroid induced biochemical and Biophysical changes in plasma and antioxidant status in human Male volunteers exposed to long term allethrin and Prallethrin inhalation. Int J Med Pharm Sci (IJMPS). 2014;4(6):17\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFajardo L, Ocampo P. Inhibition of acetylcholinesterase activities in whitegoby, Glossogobius giuris from the East Bay of Laguna Lake, Philippines. 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSramek JJ, Cutler NR. RBC cholinesterase inhibition: a useful surrogate marker for cholinesterase inhibitor activity in Alzheimer disease therapy? Alzheimer Dis Assoc Disord. 2000;14(4):216\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeif P. Does Dysregulation Of The Indirect Pathway Contribute To The Pathophysiology Of Catatonia Through Neurotransmitter Imbalance? 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Revista de Toxicolog\u0026iacute;a. 2024;41(1).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGasmi S, Rouabhi R, Kebieche M, Boussekine S, Salmi A, Toualbia N, et al. Effects of Deltamethrin on striatum and hippocampus mitochondrial integrity and the protective role of Quercetin in rats. Environ Sci Pollut Res. 2017;24(19):16440\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDasgupta R, Jain N. Histopathological assessment of transfluthrin toxicity in rat liver and kidney tissues. J Environ Biol. 2024;45(1):45\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJha L, Transfluthrin. Unmasking The Hidden HealthRisks Of A Common Mosquito Repellent. nternational J Creative Res Thoughts (IJCRT). 2025;13(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKozareva DA, Cryan JF, Nolan YM. Born this way: Hippocampal neurogenesis across the lifespan. Aging Cell. 2019;18(5):e13007.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDisouky A, Lazarov O. Adult hippocampal neurogenesis in Alzheimer's disease. Prog Mol Biol Transl Sci. 2021;177:137\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-neuroscience","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nros","sideBox":"Learn more about [BMC Neuroscience](http://bmcneurosci.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/nros/default.aspx","title":"BMC Neuroscience","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"transfluthrin, pyrethroid, insecticide, cognitive function, anxiety, acetylcholinesterase, brain","lastPublishedDoi":"10.21203/rs.3.rs-9528142/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9528142/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe central nervous system is susceptible to environmental toxicants, including synthetic pyrethroid insecticides, which are widely used for domestic and agricultural pest control. Transfluthrin, a fast-acting pyrethroid, is commonly incorporated into insecticide papers for indoor use. However, concerns regarding its potential neurotoxic effects persist. This study evaluated the impact of transfluthrin-based insecticide paper (TBIP) smoke on cognitive function and anxiety-like behavior in adult male Wistar rats.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThirty Male rats were randomly assigned into three groups (n\u0026thinsp;=\u0026thinsp;10 per group): Group A (control) was exposed to clean ambient air; Group B was exposed to smoke from 6 g of TBIP for 4 hours daily; and Group C was exposed to smoke from 12 g of TBIP for 8 hours daily, both for 4 weeks via whole-body inhalation. After exposure, behavioral assessments were performed using the Elevated Plus Maze (EPM) to assess anxiety-like behavior at week 4 and to evaluate learning and memory at weeks 1, 2, and 4. The Y-maze was similarly used to assess working memory at weeks 1, 2, and 4. At the end of the exposure period, rats were sacrificed, and their brains were harvested to isolate the hippocampi for biochemical analysis and histopathological investigation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eRats exposed to TBIP exhibited significant reductions in learning and memory performance, increased anxiety-like behaviors, reduced body/ brain weights, and altered oxidative parameters and AChE activity compared to the controls.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings suggest that prolonged exposure to transfluthrin-based insecticides can impair neurobehavioral function, possibly via oxidative stress, nitrosative stress, and disruption of cholinergic signaling pathways. These indicate the need for cautious use and regulatory review of household insecticides containing transfluthrin.\u003c/p\u003e","manuscriptTitle":"Transfluthrin-based insecticide exposure: assessment of cognitive function and anxiety-like behavior in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-18 09:09:52","doi":"10.21203/rs.3.rs-9528142/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision 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