Are developing brains affected by commonly used chemicals?

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.