Effect of Cypermethrin Exposure during Gestation on the Learning and Memory Ability of Pups

Synthetic pyrethroids are more prominently used insecticides in agricultural areas due to their great effectiveness and minimal toxicity to mammals. Two forms of synthetic pyrethroids exist, distinguished by changes in their target location and chemical structure. Type I pyrethroids also known as non-cyano pyrethroids, alter sodium channels in neuronal membranes and cause them to open for comparatively brief amounts of time. The alpha-cyano group found in type II pyrethroids causes a greater delay in sodium channel inactivation, which prolongs the depolarization of the neuronal membrane (Hocine et al., 2016). Type I pyrethroids generally cause tremors, while Type II pyrethroids containing the α-cyano group cause salivation/choreoathetosis syndrome (Morgan et al., 2018). Cypermethrin is a member of type II Synthetic Pyrethroids.It is commonly used to kill agriculture and domestic pests (Carriquiriborde et al., 2007). It is neurotoxic and targets the central nervous system of mammals and insects (Ray, 2001). Cypermethrin has become one of the most used insecticides since its introduction in developing countries such as Egypt, Saudi Arabia, Nigeria, the Caribbean and India (Madu, 2015). Due to this, there is an increasing risk of contamination of food with insecticides and contaminated food may harm humans and domestic animals.Despite their low toxicity, persistent use of insecticides may be dangerous to mammals (Usmani and Knowles, 2001). Rats exposed to type II acute pyrethroid poisoning orally or intravenously exhibit salivation, increased jaw opening, rolling gait due to increased extensor tone in hind limbs, tremors and impaired coordination followed by tonic seizures and death. There is evidence that type II pyrethroid exposure causes memory impairments in rats (Nieradko-Iwanicka and Borzêcki, 2008). There is ample evidence of pyrethroid exposure in children, newborns and pregnant women (Berkowitz et et al ., 2003). Pyrethroid exposure raises the risk of fetal toxicity and embryonic resorption (Joya and Sangha, 2016).The mother’s cypermethrin exposure had an impact on the body and organ weight of the offspring (Huang and Li, 2014). Increments in pre and post-implantation losses, reduction in the implantation sites, corpus luteal and early mortality rate in pyrethroids-exposed rats were also noted (Ahmad, 2010). Research has demonstrated that mothers who were engaged in farming practices either before or during the first three months of gestation have a greater chance of having children with nervous system defects, mouth clefts, or numerous malformations (Ramon-Yusuf et al. 2017). Cypermethrin can pass through the placenta and impact physiological processes linked to the development of the fetus’s nervous system (Dewailly et al., 2014) and fetal weight (Madu, 2015). Pesticide exposure is linked to diseases that manifest later in life (such as neurodegenerative disorders) as well as ailments that begin in childhood. In the previous study, it was reported that the herbicide atrazine toxicity also leads to memory impairment (Rastegar-Moghaddam et al., 2018). Cyfluthrin exposure can cause nerve damage, leading to impairments in learning, memory, spatial exploration and autonomic motor function (Xie et al., 2023). Acute and chronic exposure to chlorpyrifos causes cognitive impairment in animal models (Yan et al., 2012). Learning and memory impairment in rats were studied by different scientists on different pesticide exposure using different behaviour tests but very few studies were conducted to find out the effect of synthetic pyrethroids exposure on the learning and memory of rats. Thus, the current study was created to evaluate the spatial learning and memory of pups whose mothers were treated with CYP during gestation. The cognitive ability of pups was studied using the MWM task.

Name of the institute
 
The research was carried out at the Zoology Department, Maharshi Dayanand University, Rohtak.
 
Research period
 
1.5 to 2 years.
 
Chemicals
 
Cypermethrin (25 % EC), was acquired from the pesticide shop in Rohtak. Following the previously published studies (Sangha et al., 2013) with a few minor alterations, the doses for the current investigation were selected. As a result, the doses of 25 mg/kg, 50 mg/kg and 75 mg/kg body weight of rats were chosen as the low, medium and high doses, respectively.
 
Animals
 
From the disease-free small animal house at the Lala Lajpat Rai University of Veterinary and Animal Science in Hisar, Haryana, twenty-four fertile male and female Wistar albino rats (3 to 4 months old) were purchased.  They were kept in plastic polypropylene cages under control conditions of temperature (25^oC), humidity, proper ventilation and a 12-hour light/dark cycle. Animals were allowed to feed on standard laboratory food pellets and ad libitum to drinking water. The Institutional Animal Ethical Committee of M.D.U. Rohtak has approved the protocol used in this study. Animals were acclimatized for one week before the conduction of the experiment.
 
Mating
 
Oestrus female and fertile male rats were placed in one cage in the ratio of (2:1) for the mating. The vagina of the female was checked daily for the vaginal plug. The presence of spermatozoa in the females’ vaginal fluid confirmed that they had successfully mated. Vaginal plug presence is regarded as gestational day 0 (GD0) and daily gain in weight and belly size is further confirmation of pregnancy.
 
Experimental design
 
Pregnant female rats were randomly assigned to four different groups, each with 4 rats. Group I served as a control group, receiving no cypermethrin exposure. The experimental groups were designated as Groups II, III and IV.Pregnant females of the experimental groups received CYP orally at 25 mg/kg, 50 mg/kg and 75 mg/kg body weight (BW) from gestation day 6 till parturition. Doses were freshly prepared daily, dissolved in water and then given according to the body weight of the rats. Behavioural activity, food consumption, water intakeand body weight were closely monitored throughout the prenatal stage. After parturition, the growth and development of pups were studied for one month. Parameters such as litter weight, litter size, opening of eyes and the arrival of fur in control and treated groups were recorded. The MWM was used to examine the cognitive ability of the pups after a month.
 
Behavioral test
 
Learning and memory of control and CYP-treated animals were studied through the Morris water maze (MWM) followed by Vorchees and Williams, (2006) with slight modifications. Five different tests were performed by the pups in MWM such as spatial acquisition test, reversal spatial acquisition test, discrimination learning test, latent learning test and trial-dependent working and memory test. All MWM test was performed by the pups of control and cypermethrin-treated female rats after one month of birth to check the effect of CYP on the learning and memory of pups.
 
Statistical analysis
 
The data was shown as mean±SE. Tukey’s post hoc comparisons test and one-way analysis of variance (ANOVA) were utilized to assess the statistical significance of variations in the mean values of the experimental and control groups. At p≤0.05, p≤0.01, p≤0.001 and p≤0.0001, the difference was statistically significant.

Changes in body weight and behavior of pregnant females during the gestation period
 
Following CYP treatment during the gestation period, no evident adverse symptoms were observed in the lower dose (25 mg) group females. However, mild nausea, irritability and salivation were observed during the last days of gestation. In the 50 mg and 75 mg groups, pregnant females showed less intake of food and water, diarrhoea, respiratory distress and a tilted neck. The observed signs of toxicity in the current study show similarity with the results of previous study (Madu, 2015). Similar results were also noted in the study of Ramon-Yusuf et al. (2017) where rats were treated in combination with cypermethrin and dimethoate.
       
Pregnant females of the control groups showed a significant increase in weight during the gestation period throughout the experiment. However, CYP-treated pregnant females of all experimental groups showed less gain in body weight during the gestation period than control group. 75 mg CYP treated group showed a lesser gain in weight as compared to the lower dose treated group (Fig 1). Mortality was also observed in the 75 mg dose group. Joya and Sangha (2016) also reported less gain in body weight in pregnant females treated with 0.1 mg/kg of deltamethrin. Similar results were noted in the study of Madu (2015).


 
Growth and developmental study of pups
 
Control group pups showed a significant increase in their body weight and length. At post-natal day (PND) 5, 20, 25 and 30, the higher dose (75 mg CYP) exposed group exhibited a significant (p<0.05) reduction in their body weight from the control group (Fig 2A). 50 mg and 75 mg CYP exposed group denoted a significant (p<0.05) decrease in their body length from the control group (Fig 2B). CYP treated group showed a lesser gain in their body weight and length. The growth and developmental findings of the current investigation were consistent with the results of previous studies (Joya and Sangha, 2016; Madu, 2015; Savithri et al., 2014). The body fur arrival time and opening of the eyes were found slightly delayed in the treated group as compared to the control group. 75 mg CYP treated group pups’eye-opening and fur arrival time was also found significantly different (p<0.01) from the control and 25 mg CYP group (Fig 2C). A significant delay in developmental landmarks after deltamethrin exposure was also noted in previous studies (Joya and Sangha, 2016; Lazarini, 2001). The growth of 75 mg CYP-treated female pups was comparatively less than the lower dose-treated groups. Body texture, size and behavioral activity were also different in the CYP-exposed group compared to the control group. Mortality was also reported in the experimental group’s pups, this parameter was recorded as high in the 75 mg CYP-treated group and at some extant in the 50 mg group. The observed mortality of the offspring was found to be similar to the findings of (Savithri et al., 2014).


 
Learning and memory study using MWM
 
From the results of the MWM task, it was observed that the performance of the control group pups was better than CYP-treated group pups.
       
In the spatial acquisition test, less time was taken by the control group pups to reach the platform as compared to CYP-treated group pups (Fig 3A). Reference memory data of the spatial test showed that the control group pups spent more time in the platform area than the CYP-treated group pups which means that the control group pups remember the position of the platform than the CYP-treated group pups (Fig 3B).


       
Similarly, in reversal spatial tasks better performance was noticed in the case of the control group than in CYP-treated groups (Fig 4A). In the case of experimental groups, 25 mg CYP group performs far better than the 50 and 75 mg group pups.Reference memory data also proved the same thing that controls and 25 mg group pups were able to memorize the position of the platform than the 50 and 75 mg CYP group pups (Fig 4B).


       
In the discrimination learning test, pups have to differentiate between true (static) and false (floating) goals. From the data, it was observed that the control group pups chose a static platform over a floating platform in most trials but CYP-treated pups chose more floating trials as compared to the control group pups (Fig 5C). On 1st day of the trial, all group pups take more time to reach the goal but on the last or 5th day of the trial control and 25 mg group pups take lesser time to reach the goal than 50 mg and 75 mg group pups (Fig 5A). Reference memory data also revealed that control group pups also spent more time in the static platform quadrant than experimental group pups (Fig 5B).


       
In the latent learning task, pups were placed on the platform for 5-10 seconds before each trial so that pups remember the location of the platform. However, it was observed that the 50 mg and 75 mg group pups performed poorly even after placing the pups on the platform before starting the trials. The performance of the control and 25 mg group was comparable in this task (Fig 6A). Reference memory data explained that the control group pups remember both the platform position (SW as well as NE) because control pups spent approximately equal time in both directions but CYP-treated pups significantly (p<0.001) spent more time in the NE direction than SW as in the last 2 days of task platform was located in NE direction so CYP treated group pups able to build up only short memory than control group pups. Time spent by the treatment group in the SW quadrant showed a highly significant (p <0.0001) difference from the control group pups (Fig 6B).


       
In trial-dependent working and spatial learning and memory tasks, the control group demonstrated superior performance. The 25 mg group’s performance was likewise similar to that of control group but 50 mg and 75 mg treated groups performed poorly in this task. They take more time to reach the successive goal than the control and 25 mg CYP group (Fig 7). From the data of Morris water maze task, it was observed that CYP treatment affects the learning and memory of pups. Terry et al., (2003) and Yan et al., (2012) also discovered that rats’ performance on a spatial learning task was compromised following the treatment to Chlorpyrifos. A mixture of endosulfan and methyl parathion exposure leads to increased latency in searching the platform by the rats (Castillo et al., 2002). In the previous studies, it is clearly stated that CYP exposure leads to nervous toxicity caused by oxidative stress and the generation of free radicals which harms the brain’s cellular structure. So, the hippocampus is the crucial component of the brain that plays a vital role in learning and memory and injury to the hippocampus leads to the destruction of the memory. The impairment in learning and memory tasks of the treated group could be due to impairment in the hippocampus region of the central nervous system (Lu et al., 2021). Rastegar-Moghaddam et al. (2018) was also in agreement with our study by stating that atrazine-treated groups spent more time reaching the platform and also remained for less time in the target quadrant as compared to the control group. Similar results were also observed in the study of Zhang et al., (2019) where propamocarb fungicide-treated mice spent less time in the target zone and had poor memory. According to Nishiyori et al., (2014) a lady who attempted suicide after ingesting pesticides had anxiety and short-term memory problems, which is consistent with our findings. Additionally, rats exposed to permethrin demonstrated a marked decline in their short-term memory and spatial orientation (Nasuti et al., 2003). Beta-cyfluthrin was found to impair both motor coordination and spatial memory (Syed et al., 2016). According to the current research, animals’ ability to learn and remember things is adversely affected by CYP exposure, which is consistent with findings by Gargouri et al., (2018). Ozdemir et al., (2014) found that offspring exposed to clothianidin exhibit low performance in the MWM task, which is in line with the current investigation. The prenatal exposure to melamine also damaged the cognitive abilities of rats. Similar results were also reported in the previous studies (Ghasemnejad-Berenji et al., 2021; Maodaa et al., 2024; Murshed et al., 2023; Wasnik et al., 2023). Xie et al., (2023) validated the current investigation by exposing the rats to three different doses (6.25 mg, 12.5 mg and 25 mg/kg) of cyfluthrin and observed a reduction in the spatial learning and memory abilities of rats. According to Imam et al., (2018), rats exposed to dichlorvos and chlorpyrifos for 14 days may have neurocognitive impairments and exhibit more anxiety-like behaviors.

The current investigation concluded that prenatal exposure to CYP adversely affects the developmental parameters of the neonates.Body texture, weight, length, fur arrival, eye-opening time and behavioral activity of pups were also affected due to cypermethrin. CYP exposure impaired spatial learning and memory of pups. Exposure to CYP has a deleterious effect on the brain development of both adults and pups.

The authors are highly thankful to the Department of Zoology, M.D.U. Rohtak and the Council for Scientific and Industrial Research (CSIR) for their support to carry out the research work.

The authors declare that they have no conflict of interest.