18
Jul
Developmental Neurotoxic Effects of Widely Used Neonicotinoid Insecticide Underestimated by EPA
(Beyond Pesticides, July 18, 2024) A recent study in The Journal of Toxicological Sciences shows that a single dose of the neonicotinoid insecticide clothianidin (CLO) induces behavioral abnormalities, predominantly in female mice, throughout key stages of development. In testing mice at various ages, sex-specific changes were identified that highlight not only varied effects on males and females but also how pesticide exposure at a young age can cause lasting impacts throughout adulthood in mammalian species.Â
The researchers, at the Laboratory of Animal Reproduction and Development at Tohoku University in Japan, “utilized murine [mouse] models to compare the sex-specific differences in behavioral effects following CLO exposure at different developmental stages. [They] orally administered CLO to male and female mice as a single high-dose solution (80 mg/kg) during the postnatal period (2-week-old), adolescence (6-week-old), or maturity (10-week-old), and subsequently evaluated higher brain function.†Â
As the authors remark, “Most studies on the neurotoxicity of CLO have targeted only males, with limited insights regarding the neurodevelopmental toxicity in females. There are significant sex differences in brain development due to hormonal, genetic, epigenetic, and other sex-specific factors. Moreover, there are also a number of sex-based differences in the prevalence of developmental disorders, such as attention deficit hyperactivity disorder, and autism spectrum disorder, with susceptibility to neurological disorders being as much as 2- to 5-fold greater in one sex than in the other.†This study began with an aim to address the discrepancy in past clothianidin studies by testing exposure in both male and female mice at multiple stages in their lifecycle.Â
The researchers continue in saying, “Many reports have indicated varying behavioral toxicities due to chemical exposure depending on sex. Given these findings and the high susceptibility of the brain to chemical exposure during the developmental period, it is possible that CLO exposure during development may cause differences between males and females. Therefore, it is imperative to observe the toxic effects of CLO in females as well.â€Â
The study design includes a solution of methyl cellulose and acetone with dissolved clothianidin that was administered to mice “at two weeks of age (2w-CLO I), six weeks of age (6w-CLO I), and ten weeks of age (10w-CLO I), respectively. The 2w-CLO II, 6w-CLO II, and 10w-CLO II groups received the same treatments.†The control groups, called vehicle control groups I and II (VC I and VC II), also received the solution with methyl cellulose and acetone (known as the vehicle) without any neonicotinoid insecticide at 2, 6, and 10 weeks of age to establish a baseline. A behavioral battery test (BBT) was conducted at 3 months of age in group I and at 7 months of age in group II for both the control and treatment groups, which consisted of an open field test, light/dark transition test, and contextual/cued fear conditioning test.Â
Locomotor activity in the mice was measured in the open field test by the total distance traveled by the mice, total number of movement episodes, and the time spent at the center area during the test. In the light/dark transition test, the “total distance traveled in a light box, the latency to enter the light box for the first time, and the transition number between the light and dark boxes†was recorded. The contextual/cued fear conditioning test consisted of a series of experiments in which mice were placed into a chamber with stainless steel rods on the floor. These rods were utilized with tone-shock pairings to condition the mice and analyze their fear of the chamber with and without the tone that had been followed by a shock. This allowed the researchers to measure the freezing response (inability to move due to fear) of the mice under those conditions. Following behavioral testing, the brains of all mice were surgically removed for histological and immunohistochemical analysis.Â
After the single dose of clothianidin and before the behavioral tests began, “acute peripheral neurotoxicity in our mouse models, such as tremors in the limbs†was noted by the authors. Within the tests, behavioral changes are seen in exposed female mice more than the males. Anxiety-like behavior in these mice was first noted in the open field test. “The time spent at the center area by female mice of the 2w-CLO II group was significantly decreased… This result was attributed to the excessive anxiety of these mice after introduction to a novel environment,†the authors state. They continue in saying, “These results suggest that CLO administration during the early postnatal period induces anxiety-like behaviors only in female mice.â€Â
The results of the light/dark transition test show that the distance traveled in the light box by the female mice in the 6w-CLO I group significantly decreased and that female mice in 2w-CLO II group entered the chamber with a notable delay relative to female mice of the VC II group. The fear tests reveal that the contextual freezing rate significantly decreased in 6w-CLO II female mice and the cued freezing rate significantly decreased among 2w-CLO II and 10w-CLO II group female mice.Â
With the fear test results, the authors comment that this “suggests that CLO exposure during adolescence may lead to spatial memory abnormalities only in female mice. We noted similar responses during the cued fear test, as the freezing rate among female mice in the 2w-CLO II and 10w-CLO II groups significantly decreased. Moreover, the freezing rate of the 6w-CLO II female mice was lower than that of their VC II group counterparts… In contrast, the freezing rate response of male CLO II mice during the cued fear test, was comparable to that of control males. These results suggest that cued memory retrieval impairments occur only in the female CLO II groups.â€Â
These sex-specific behavioral changes highlight the importance of testing both males and females for effects of pesticide exposure, as reactions can vary greatly. The researchers also observed a reduction in astrocytes in the female mice of the 2w-CLO II group and 6w-CLO II group. “Astrocytes within the hippocampus undergo a rapid increase during early postnatal development, particularly around postnatal day 14. As such, the decrease in the number of astrocytes may have been influenced by CLO exposure during the early postnatal period,†the researchers postulate.Â
Astrocytes are a subtype of glial cells making up most cells in the human central nervous system, which raises alarm for pesticides like clothianidin that affect astrocyte numbers. “Astrocytes in the hippocampus are largely involved in mammalian behavior. Astrocytes actively contribute to information processing and the establishment of the blood-brain barrier. Several neurological disorders, including anxiety and fear-related disorders, are thought to be associated with astrocyte dysfunction,†the authors report. Sexual dimorphism, which shows variations in individuals of different sex in the same species, occurs in human brain composition with females having more glial cells than males and can help explain why astrocytes may be impacted more predominantly in females. Â
“The increased anxiety-related behaviors displayed by the 2w-CLO II group, together with the limited spatial associative memory capabilities of the 6w-CLO II group during behavioral tests, were consistent with the typical behavioral abnormalities observed when hippocampal astrocyte function is compromised,†the researchers find. “Therefore, the reduced number or reduced activity of astrocytes in the [hippocampus] of 2w-CLO II and 6w-CLO II adult female mice may impede normal information transmission, and could be related to these behavioral abnormalities.â€Â
Neonicotinoids, such as clothianidin, are widely used as agonists, which mimic the action of acetylcholine at the nicotinic acetylcholine receptor (nAChR), and lead to insect death. While insects are the intended targets of neonicotinoids, many organisms have nAChRs and “reports have indicated that neonicotinoids can cross the mammalian blood-brain barrier and demonstrate a higher affinity for mammalian nAChRs than originally thought,†the study authors share. Â
Clothianidin is commonly sprayed over forests and agricultural areas and is known for its neurotoxic effects on mammals. Since “CLO is used as a wood preservative, as well as in food crops, and given that multiple generations of people are exposed to CLO, understanding the differences in the effects of CLO on behavior based on developmental stages is essential,†the researchers emphasize. With brain plasticity differing greatly at different developmental stages, studies like these are crucial. Â
Prior research reveals that acute CLO exposure induces neurotoxicity as a behavioral abnormality, and this study highlights the behavioral differences between males and females after a single-dose CLO administration across multiple developmental stages. The authors conclude that, “The observed female-specific behavioral abnormalities underscore the importance of evaluating the effects of chemicals in both sexes, not just in males.†Beyond Pesticides has previously covered studies that show sex-specific effects, favoring both males and females, for a multitude of pesticides (see more here, here, here, and here). These impacts are trivialized by the U.S. Environmental Protection Agency (EPA) in current regulatory processes.Â
While there are a multitude of studies that pinpoint detrimental effects on many species after exposure to pesticides, there are data gaps in the studies analyzed in risk assessments. EPA underestimates the real-world threats from pesticides (such as neonicotinoid insecticides), ignores potential chemical interactions or synergistic effects, and has a lack of scientific integrity with an over-reliance on industry-funded science that contradicts peer-reviewed studies. Â
Within single species, there can be great variation between sexes and even time of exposure as shown in this study. Between species in the same family, there can be drastically different tolerance levels as well. Risk assessments only focus on a snapshot of data and do not look at the whole picture for how pesticides threaten the health of all organisms and the environment. This study, which adds further complexities to the research that needs to be considered by EPA when approving regulations for chemicals that cause unreasonable harm, supports those who advocate for a precautionary approach. Â
The only solution moving forward is to look at the overwhelming body of science that supports organic agriculture as the answer for sustainably producing food and managing land. Organic agriculture protects not only wildlife and the environment but is also of particular importance to farmworkers, consumers, and children. Children are even more susceptible to the effects of pesticide exposure because they have developing organ systems that are more vulnerable and less able to detoxify toxic chemicals. Â
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All unattributed positions and opinions in this piece are those of Beyond Pesticides. Â
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Source:Â
Kaku, K. et al. (2024) A single dose of clothianidin exposure induces varying sex-specific behavioral changes in adulthood depending on the developmental stage of its administration, The Journal of Toxicological Sciences. Available at: https://www.jstage.jst.go.jp/article/jts/49/7/49_301/_article.  Â
