Superfund Research Program
Mechanisms of Chlorpyrifos Developmental Neurotoxicity
Chlorpyrifos is the most commonly used insecticide in the United States - an estimated 20 to 25 million tons are applied annually. The organophosphate pesticide is sold in more than 800 different products, but is most commonly known as Dursban® (home and commercial use) and Lorsban® (agricultural use).
Domestic use accounts for nearly 50% of chlorpyrifos applications. People use it in and around their homes to control termites, lawn insects, ants, cockroaches, fleas, ticks, and mosquitoes. Farmers use chlorpyrifos on more than 50 types of crops including corn, apples, tomatoes, oranges, grapes, cotton, alfalfa, and berries. Chlorpyrifos is so popular in part because its chemical stability and persistence reduce the need for repeated application. However, this persistence also enhances the likelihood of prolonged exposure.
Like all organophosphates, chlorpyrifos kills insects and other animals by impacting the function of the nervous system. Chlorpyrifos inhibits the activity of acetylcholinesterase (AChE), an enzyme which breaks down the neurotransmitter acetylcholine. Without AChE, acetylcholine accumulates. As a result, a nerve that is firing will continue to fire - causing rapid twitching of involuntary muscles, convulsions, paralysis, and ultimately death.
In June, 2000, the U.S. Environmental Protection Agency (EPA) announced restrictions on chlorpyrifos use based on new scientific information indicating that the insecticide is more toxic to infants, children, and pregnant or nursing women than was previously understood. The regulatory modifications will remove chlorpyrifos from retail sale and restrict most residential and professional uses by the end of 2001. However, because the ban permits existing stocks to be sold to consumers and allows limited use by certified applicators, chlorpyrifos will continue to be used in large quantities. Equally important, its agricultural use will remain for the foreseeable future, and there are increasing problems concerning storage and disposal - chlorpyrifos is included as one of the pesticides designated for problems involving Superfund sites.
Scientists at Duke University are conducting extensive, multidisciplinary research on chlorpyrifos. Their work spans all levels of biological organization - from the cellular and molecular events underlying the toxic mechanisms, through the morphological assembly of the nervous system, to the eventual behavioral outcomes.
To identify the mechanisms by which chlorpyrifos disrupts brain development, the Duke scientists focused on the basic processes controlling cell division and differentiation. The researchers studied several test systems including cell-free systems (fractionated cell extracts that maintain biological function), neuronal cell cultures, and newborn rats treated with apparently subtoxic doses of chlorpyrifos. They identified three different types of effects:
- Interference with the replication of DNA required for cell division.
- Alterations in the expression and function of the specific molecules that control cell development (nuclear transcription factors). Specifically, researchers noted changes in two transcription families that are essential to cell replication and differentiation: AP1 and Sp1.
- The generation of reactive oxygen species, compounds that produce oxidative damage.
These findings are important because they indicate that the mechanisms of chlorpyrifos developmental neurotoxicity are clearly different from its "standard mechanism" of systemic toxicity - the inhibition of cholinesterase.
To identify which types of brain cells are targeted by the pesticide, the Duke scientists compared the effects of chlorpyrifos on neuronal cells, which receive and conduct electrical impulses, and glia cells, which are the supportive tissue of the brain, but which provide important guidance enabling the brain to "wire up" correctly during development. The Duke investigators found that glia cells are more sensitive to chlorpyrifos than neurons. Because glia cells develop much later than neurons, and are specifically involved in allowing neurons to make the proper connections to their targets, these findings indicate that the developing brain is likely to be vulnerable to chlorpyrifos exposure at least through early childhood.
When evaluating neurotoxicity, it is important to determine if adverse effects at the cellular level impact behavioral function. The Duke scientists conducted a series of standard behavioral tests with adolescent and adult rats following neonatal exposure to subtoxic doses of chlorpyrifos. They assessed activity, learning, and memory and noted both immediate and long-term gender-selective behavioral abnormalities, with males showing greater sensitivity to chlorpyrifos exposure in some tests. Females were not spared however - for some tests, their responses were "masculinized," that is, their behaviors resembled those of males instead of females.
Additionally, the Duke scientists have been successful in designing an invertebrate system for a rapid screening method to evaluate the developmental neurotoxicity of chlorpyrifos and related pesticides. They based the system on the knowledge that the chemicals which facilitate sea urchin nerve cell growth (neurotrophins) are the same compounds that have been identified as the targets of chlorpyrifos neurotoxicity during mammalian development. Therefore, processes that result in biochemical or behavioral effects in mammalian neurodevelopment will result in visible abnormalities in the developing sea urchin. Following exposure to chlorpyrifos, the researchers observed marked abnormalities in the sea urchins, appearing exactly at the developmental stage expected if the neurotrophins were impacted. This work represents a major step towards development of a rapid screening tool that can be used to predict eventual, adverse effects specific to neural development, but that does not require the use of vertebrate animals.
The findings of the Duke University studies represent significant progress towards a more complete understanding of the developmental toxicity of chlorpyrifos, and by implication, of the entire class of organophosphate pesticides. The identification of three mechanisms of chlorpyrifos neurotoxicity that do not involve inhibition of cholinesterase indicates that classic method used to predict organophosphate-induced toxicity (quantification of cholinesterase inhibition) is inadequate to predict the developmental neurotoxicity of chlorpyrifos. This highlights the importance of examining endpoints other than cholinesterase inhibition to evaluate pesticide toxicity and establish safe exposure levels for pregnant women and children.
The discovery that the period of vulnerability to chlorpyrifos exposure extends into early childhood has important implications, in terms of basic neurotoxicology as well as in regulatory issues. Chlorpyrifos is a semi-volatile compound; it can vaporize and be re-deposited on surfaces in treated rooms for weeks after application. As a result, it may adhere to objects such as children's toys that are brought into the room hours or days after the pesticide is applied. Young children with high frequency mouthing behavior may be at risk of acute exposure to chlorpyrifos residues. This research indicates that apparently non-toxic childhood exposure to pesticides, which can go undetected because of the lack of overt symptoms, may have long-term impacts on behavior, and emphasizes the necessity to conduct long-term evaluations to identify later-emerging behaviors.
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To learn more about this research, please refer to the following sources:
- Buznikov GA, Nikitina LA, Bezuglov VV, Lauder JM, Padilla S, Slotkin TA. 2001. An invertebrate model of the developmental neurotoxicity of insecticides: effects of chlorpyrifos and dieldrin in sea urchin embryos and larvae. Environ Health Perspect 109(7):651-661. PMID:11485862
- Garcia SJ, Seidler FJ, Crumpton TL, Slotkin TA. 2001. Does the developmental neurotoxicity of chlorpyrifos involve glial targets? Macromolecule synthesis, adenylyl cyclase signaling, nuclear transcription factors, and formation of reactive oxygen in C6 glioma cells. Brain Res 891(1-2):54-68. PMID:11164809
- Auman JT, Seidler FJ, Slotkin TA. 2000. Neonatal chlorpyrifos exposure targets multiple proteins governing the hepatic adenylyl cyclase signaling cascade: implications for neurotoxicity. Brain Res Dev Brain Res 121(1):19-27. PMID:10837889
- Crumpton TL, Seidler FJ, Slotkin TA. 2000. Developmental neurotoxicity of chlorpyrifos in vivo and in vitro: effects on nuclear transcription factors involved in cell replication and differentiation. Brain Res 857(1-2):87-98. PMID:10700556
- Crumpton TL, Seidler FJ, Slotkin TA. 2000. Is oxidative stress involved in the developmental neurotoxicity of chlorpyrifos?. Brain Res Dev Brain Res 121(2):189-195. PMID:10876031
- Dam K, Seidler FJ, Slotkin TA. 2000. Chlorpyrifos exposure during a critical neonatal period elicits gender-selective deficits in the development of coordination skills and locomotor activity. Brain Res Dev Brain Res 121(2):179-187. PMID:10876030
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