Issue Date: August 8, 2016
Making sure chemical toxicity tests don’t miss metabolites
Once hailed as a safe alternative to the persistent organochlorine insecticide DDT, methoxychlor was widely used for decades to fight flies, mosquitoes, roaches, and other household pests. It was also extensively applied to fruits and vegetables, grains, and livestock.
But after laboratory animal studies linked the substance to developmental and reproductive side effects—including miscarriages, reduced fertility, and small litter size—regulators took action. The European Union banned sales of methoxychlor in 2002 and the U.S. followed suit in 2003.
Without data from animal studies, regulators would have likely missed the adverse effects of methoxychlor. The insecticidal chemical itself is relatively benign. Once it enters the body, however, metabolic enzymes in the liver convert the chemical into 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE), a substance with estrogenic activity. This metabolite disrupts the body’s endocrine system.
Compounds such as methoxychlor present a major challenge for regulators who are hoping to reduce the number of animal studies needed to demonstrate the safety of chemicals. They pin big hopes on high-throughput human cell-based assays that would replace slow and costly in vivo animal studies for evaluating chemical toxicity. Standing in their way, however, is the fact that such cell-based assays can’t yet detect the effects of unrecognized metabolites.
“When a chemical comes into your body, the first thing your body will do is send it to the liver,” says Kevin Crofton, deputy director of the National Center for Computational Toxicology at the U.S. Environmental Protection Agency. “The liver is going to recognize it as foreign, and try to chew it up and spit it out.”
During that metabolic process in the liver, chemicals are often converted into less hazardous compounds. But sometimes, as in the case of methoxychlor, they are converted into more toxic compounds.
If scientists rely on an assay that doesn’t account for metabolism, “what if we put the chemical in there and there is no effect?” Crofton asks. Compounds such as methoxychlor, which are not themselves toxic but give rise to toxic metabolites, may appear safe in such an assay.
Several research groups are trying to solve this problem. They are developing tools that could be easily bolted onto existing human cell-based high-throughput assays to monitor effects from both parent chemicals and their metabolites.
EPA, along with the National Toxicology Program and the National Institutes of Health’s National Center for Advancing Translational Sciences, launched a challenge earlier this year to accelerate these efforts. Its ten semifinalists are now competing for five $100,000 awards, to be announced in December.
Many of the semifinalists are developing approaches that rely on primary human hepatocytes, or liver cells, to metabolize a chemical that is under study. Metabolites are then transferred to a high-throughput assay. In two cases, however, researchers are making use of new gene-editing tools to put genes that encode for metabolic enzymes into cells.
EPA scientists, meanwhile, are also developing solutions. “We want to retrofit and screen thousands and thousands of chemicals to determine whether or not metabolism changes the result,” Crofton says.
The agency is facing the daunting task of prioritizing which of the tens of thousands of chemicals in U.S. commerce should be assessed for health risks. Changes Congress made in June to the U.S. law that governs commercial chemicals, the Toxic Substances Control Act, set deadlines over the next few years for EPA to evaluate the safety of chemicals.
“We have a very diverse portfolio of bioassays. We’d like to be able to use a solution with as many of those assays as possible.”
—Keith Houck, research toxicologist, Environmental Protection Agency
EPA is also required to make decisions regarding the safety of new chemicals within 90 days. “If you have to rely on animal data, you won’t have it in time,” Crofton says. “But if we have this huge battery of in vitro data that is available to help decision makers, they may be able to make a better decision. We are never going to have in vivo data for tens of thousands of chemicals,” he adds.
Environmental regulators aren’t the only ones interested in high-throughput chemical toxicity screening. Drug companies have long been interested in the bioactivity of metabolites formed when pharmaceuticals are converted in the liver. Such reactions have huge implications for drug-drug interactions and often change the bioactivity of a drug. The bioactivity of many anticancer drugs, for example, increases after metabolism in the liver.
In many cases, drug companies are interested in learning which of the body’s metabolic pathways chemicals act through, Crofton says. In contrast, EPA is merely trying to determine whether a chemical’s bioactivity changes when it is metabolized. If the activity does change, EPA may want to go to the next step and try to identify the pathway and what enzymes are responsible, he notes.
Many of the technologies being cultivated for EPA’s challenge are simultaneously undergoing development for pharmaceutical applications. Drug companies could benefit from approaches that consider metabolism in the early stages of drug discovery, when they screen large chemical libraries seeking lead molecules to treat various diseases. Without considering metabolism during such screening, pharma researchers are likely to miss many potential drug candidates, says Keith Houck, a research toxicologist with EPA.
Large consumer product companies are also interested in how metabolism figures into the safety of chemicals in their products. Unilever, for example, has teamed up with EPA to determine if the company can use high-throughput in vitro approaches to evaluate the health risks of a few natural products found in some of its consumer products. Unilever has long been committed to reducing animal testing.
Despite widespread recognition of the need to incorporate metabolism into high-throughput cell-based chemical screening assays, no system has been shown to reproduce the metabolic effects of the liver itself, Houck says. The goal of EPA’s challenge is to identify approaches that are flexible enough to work with many different commercially available high-throughput assays.
“Metabolism occurs in multiple places in the body.”
—Chris Vulpe, professor, University of Florida
EPA has a program called ToxCast that aims to screen thousands of chemicals for biological activity using hundreds of high-throughput assays. These assays are commercially available and come in a range of formats and types, including both biochemical and cell-based. The data are integrated with existing in vivo animal data and structure-activity information to computationally predict the toxicity of chemicals.
It is critical that the tools being developed in EPA’s metabolism challenge be compatible with these ToxCast assays, Houck says. “We have a very diverse portfolio of bioassays. We’d like to be able to use a solution with as many of those assays as possible.”
The technologies being developed through the agency’s challenge use various approaches to accomplish that goal.
Several of the proposals use primary hepatocytes to mimic liver metabolism. In such approaches, the small molecule of interest is allowed to diffuse into the liver cell where it is metabolized. The metabolite exits the liver cell and is then placed in an existing assay test system.
One semifinalist in the challenge is planning to grow liver and brain tissue on a single three-dimensional culture plate. They hope the system will allow them to assess the neural toxicity of both the parent compound and any of its liver metabolites.
Other technologies in the challenge use magnetic beads to capture the S9 fraction, a mixture of liver enzymes including cytochrome P450s. These enzymes metabolize drugs and other chemicals the body is exposed to. Internally, EPA is developing a method to encapsulate the S9 fraction of metabolic liver enzymes using a polysaccharide called alginate. The alginate S9 mixture forms gel-like microspheres that are attached to the tips of prongs on a lid that fits over a standard multiwell plate.
“It is like having little pieces of liver on the end of these tips. You stick them down into each well,” Crofton explains. “When you put the chemical in the well, the chemical gets metabolized by the S9 fraction, and the metabolite comes out.”
EPA scientists and a couple of semifinalists are also working on ways to make cultured liver cells act more like liver tissue. “Some of these cells have more metabolizing capability than others,” says Crofton. Using gene insertion techniques, “we can make the cells express more metabolizing enzymes.”
“The fundamental problem with current in vitro toxicity testing is that you are using these immortalized cell lines,” says semifinalist Chris Vulpe, a professor at the University of Florida. The trick, he says, is making the cells express enzymes in the amounts normally found in the liver.
Using a modified version of the CRISPR gene-editing technique, Vulpe’s team is working to turn on and activate transcription of DNA that encodes cytochrome P450 enzymes. Eventually he hopes to move beyond liver to other tissues, including intestinal, kidney, and breast tissues. “Metabolism occurs in multiple places in the body,” Vulpe says.
Whether EPA’s challenge will ultimately lead to a reduction in animal testing remains to be seen. But some experts predict that such technologies could be used by pharmaceutical companies to help weed out toxic drug candidates before they are tested in animals.
“It will probably help in reducing animal models,” says Randy McClelland, chief executive officer of SciKon Innovation, a North Carolina-based company developing fluid culture systems with integrated channels. Although the company is not one of the semifinalists in EPA’s challenge, it is working with some of the semifinalists to develop solutions for the next stage of the competition.
Rusty Thomas, director of EPA’s National Center for Computational Toxicology, says he is impressed with the creativity and innovation of the proposals submitted for the challenge. “The next stage is going to be important for demonstrating whether those innovative ideas can be put into practice,” he says.
EPA expects the challenge to enter its final stage in early 2017.
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