Most of the tens of thousands of chemicals in use today, including many pesticides and industrial chemicals, have not been tested for their ability to alter how the nervous system develops. The lack of such neurotoxicity data makes it impossible to investigate what role chemicals in the environment are playing in neurodevelopmental disorders.
The need for data is growing as autism, attention deficit hyperactivity disorder, dyslexia, obsessive-compulsive disorder, Tourette’s syndrome, and other neurodevelopmental disorders in children are on the rise worldwide (Pediatrics 2019, DOI: 10.1542/peds.2019-0811; LancetNeurol. 2014, DOI: 10.1016/S1474-4422(13)70278-3; Eur. Child Adolesc. Psychiatry 2014, DOI: 10.1007/s00787-014-0553-8). Genetics, better awareness, and different diagnostic criteria cannot explain the increased prevalence of such disorders over the past 2 decades, raising questions of whether exposure to chemicals in the womb and during early development is a factor.
Lead, methylmercury, polychlorinated biphenyls, and organophosphates are known neurodevelopmental toxicants, but other chemicals may also harm the developing brain. The challenge is testing large numbers of chemicals rapidly.
A transatlantic partnership between researchers at the US Environmental Protection Agency and scientists in Germany funded by the European Food Safety Authority (EFSA) could help speed up the process. The effort, which began about 15 years ago, has now led to the development of more than a dozen in vitro developmental neurotoxicity tests that provide answers quickly and reduce the need for time-consuming animal testing.
The suite of assays uses various types of neural cells. The tests probe the effects of chemicals on key cellular events in the development of the nervous system, including growth and division of neural stem cells, called neuroprogenitors; cell migration and differentiation; growth of cell-signaling structures; formation of junctions called synapses between neurons; formation of networks of neural cells; and cell death.
“All of these are things that have to happen for the nervous system to develop,” says Timothy Shafer, a research toxicologist at the EPA’s Office of Research and Development who helped develop some of the assays. “They take place at different times and in different regions, but if a chemical disrupts any of these events, then it has the potential for disrupting the development of the nervous system,” he says.
The limitation of using cellular assays is that any one test on its own is unlikely to detect all chemicals that are developmentally neurotoxic. “A chemical may disrupt development of the nervous system through proliferation but not have any effect on differentiation,” Shafer says. “Or a chemical may affect network formation but not proliferation.” Running large numbers of chemicals through a battery of multiple assays that target different cellular events increases the chances of finding those that pose the most risk.
Most of the assays rely on an automated fluorescence microscope to examine how chemicals affect key neurodevelopmental processes at the cellular level. Others take advantage of microelectrode arrays to examine how neurons function and form networks. To increase throughput, researchers perform the assays in multiwell plates that facilitate testing multiple chemicals at different concentrations.
The microelectrode array uses a specialized 48-well plate that acts like a computer chip. The center of each well has 16 microelectrodes, each about 50 µm in diameter. The plate is connected to a computer so that researchers can measure the electrical activity on each electrode. “You can culture any kind of an electrically active tissue over these electrodes, and that allows you to measure the spontaneous activity of that electrically active tissue,” such as the firing of a neuron, Shafer says.
Researchers involved in the international effort are developing a guidance document that should help researchers and regulators “interpret the data that comes out of this in vitro battery,” says Ellen Fritsche, head of a molecular toxicology group at the Leibniz Research Institute for Environmental Medicine in Düsseldorf, Germany, who developed several of the assays. The scientists expect to finalize the guidance, which is being coordinated by the Organisation for Economic Co-operation and Development (OECD), next year.
In 2018, EFSA funded Fritsche and Marcel Leist, a toxicologist at the University of Konstanz, in Germany, to run numerous chemicals through the suite of in vitro assays to see how they performed. Deciding which chemicals to test was a difficult and important decision, Fritsche says. The German researchers collaborated with the US EPA’s Shafer to determine which chemicals made the most sense.
The researchers selected 121 compounds, including metals, pharmaceuticals, and pesticides from different classes. They selected chemicals that in vivo and epidemiology studies have shown to be developmentally neurotoxic in humans or animals, as well as some that are considered to be not developmentally neurotoxic.
Fritsche and Leist ran the chemicals through various assays that each use a different cell type. The assays developed in Fritsche’s lab rely on clusters of neuroprogenitor cells called neurospheres. Neuroprogenitor cells cannot divide indefinitely as stem cells do, but they can become neurons and different types of glial cells, which help protect neurons, Fritsche says. These neuroprogenitor cells undergo development in multiwell plates just as they do in the brain, she says.
Separately, Leist and colleagues developed assays that focus on the development of the peripheral nervous system, which connects the brain and spinal cord to the rest of the body. Those tests use neural crest cells, which are precursor cells for the peripheral nervous system, the intestinal nervous system, and cartilage and bones in the face and skull.
And EPA researchers are running the same 121 compounds through the assays that they developed. Those tests include cell proliferation and death in human neuroprogenitor cells, the outgrowth of signaling structures called neurites in human and rat neurons, and the formation of synapses in rat neurons. They are also testing network formation in rat neurons using microelectrode arrays.
The data that have been generated so far indicate that the in vitro battery of tests the teams have assembled does a good job of predicting which chemicals affect neurodevelopmental processes, Fritsche says. The next step is to close some gaps, mostly related to glial cell proliferation and maturation, she notes. In addition to protecting neurons, glial cells form myelin, an insulating layer that helps transmit electrical impulses along neurons. Fritsche and Leist are hoping to develop those assays quickly so they can complement the other in vitro tests.
In the US, regulators are using the organophosphate pesticides as a case study to build confidence in the in vitro methods. The chemicals have a robust set of existing toxicological data, says Anna Lowit, senior science adviser in the EPA’s Office of Pesticide Programs. For most of the organophosphate pesticides, there are epidemiology studies in humans and many studies in animals, she says. Some of the organophosphates, such as chlorpyrifos, also have a lot of in vitro mechanistic information, she notes.
The EPA’s goal is to learn how to bring the in vitro methods into the regulatory program, Lowit says. As part of its regular process for registering pesticides, which typically involves reassessing them every 15 years, the EPA is reevaluating 22 organophosphate pesticides. “The risk assessments and risk management decisions are still being worked on,” Lowit says. Data from the suite of in vitro tests could be used as part of those analyses, but the EPA is waiting for a panel of advisers to weigh in on the readiness of such data. The panel met in September and is expected to release its report later this month.
One of the big challenges with building confidence in the in vitro assays is that even when in vivo data exist, “sometimes the animal data are not very reliable, or they are very difficult to interpret,” Leist says. Additionally, “the functions we can interrogate with a live animal are very different from the functions we can interrogate with cell assays,” Leist says. The live animal tests typically examine whether exposure to a chemical changes behavior, such as avoidance of bright light. It is difficult to connect that behavior to an effect on one of the key cellular processes, such as cell migration or synapse formation, he says.
1. Proliferation: Neural stem cells, also called neuroprogenitor cells, replicate and produce cells that turn into neurons (pink) and glial cells (yellow). Glial cells protect neurons and form myelin, an insulating layer that helps transmit electrical impulses along neurons.
2. Apoptosis: Neuroprogenitor cells produce more neural cells than needed. Unnecessary cells die.
3. Differentiation and migration: Neural stem cells change from one type to another and move to different locations.
4. Neurite outgrowth: Neurons create structures called neurites that make up the wiring of the nervous system.
5. Synaptogenesis: Junctions form that allow electrical or chemical signaling between neurons.
6. Maturation: Glial cells called oligodendrocytes become fully functional and able to coat neurons with myelin.
7. Neural network formation: Neurons form interconnected circuits that carry out specific functions.
“The really powerful learning phase will start when we have compounds with established in vitro activity patterns, which we can use to compare unknown compounds,” Leist says. “Then we compare within one system.”
“The goal was not necessarily to replace animal models,” says Rusty Thomas, director of the EPA Office of Research and Development’s Center for Computational Toxicology and Exposure. “It was to evaluate the safety—the potential developmental neurotoxicity—of these chemicals in a better, more robust way by evaluating these biological processes that they may impact,” he says.
The EPA could use the in vitro assays to screen large numbers of chemicals for their potential to harm the developing nervous system. Chemicals that are identified as potentially problematic could then be tested in living animals, such as zebrafish and possibly rats.
Unike rats, zebrafish develop really fast, says Stephanie Padilla, a research toxicologist at the EPA who is leading an effort to use zebrafish to detect chemicals that are developmental neurotoxicants. Within 6 days of fertilization, a zebrafish egg develops into a swimming fish that can find food and escape predators, she says.
The researchers expose developing zebrafish to a chemical and determine whether the substance affects the overall development of the fish in terms of how the fish looks and behaves. For example, when they exposed developing zebrafish to lead, the fish ended up with smaller brains with fewer synapses than the brains of unexposed zebrafish, Padilla says. With respect to behavior, the researchers monitored the movement of the fish in response to light and dark. Zebrafish exposed to lead moved less in the light than unexposed zebrafish.
The advantage of using an animal model over cell-based methods is that all the different systems that talk to one another are in place. Particularly important is that “all the pathways, the whole blueprint for development of the nervous system, is very similar between all vertebrates,” Padilla says. “So doing research on zebrafish isn’t that crazy if you are trying to predict what is happening to humans.”
In Europe, “our vision would be different,” Leist says. There, researchers are focused on substituting animal studies with nonanimal tests, he says. In humans, chemicals may affect cognition, intelligence, language, social interactions, and attention span, Leist says. “These are all things that are really difficult to assess in animals or even impossible.”
Cell-based in vitro assays can’t directly assess those effects either. But “if a cell doesn’t grow neurites, or if it doesn’t migrate when it should normally migrate, I feel it is easier to interpret that this is really an adverse effect,” Leist says. “If a rat likes to sit in the light, I find it a bit more difficult to interpret.”
The OECD guidance document will be essential for getting regulators around the world to accept the use of in vitro developmental neurotoxicity data in regulatory decisions. Without the OECD guidance, which determines validity in the regulatory context, industry will be hesitant to conduct the tests, Leist says. But with such guidance, “it is not such a big regulatory step to say data that have been produced according to this guidance would be accepted for certain purposes,” he says. Many avenues exist to get such data into regulatory decision-making “without tumbling the whole system.”