A Framework for Cumulative Risk Assessment: Exploring the Carcinogenic Effects of Chemical Mixtures.

Humans are commonly exposed to mixtures of chemicals in their environments, rather than to one compound at a time. Accordingly, researchers have long been interested in the complex task of studying how mixtures affect disease end points.1 Cynthia Rider and colleagues recently proposed in Environmental Health Perspectives2 a research program on mixtures and cancer based on the key characteristics of carcinogenic chemicals.3

“We wanted to apply what we have learned from studying outcomes with shorter exposure periods to a more complex scenario,” says Rider, a toxicologist at the National Institute of Environmental Health Sciences and first author of the new paper. “Because cancer results from the cumulative impact of multiple low-dose hits in several pathways during a long period of latency, the analysis of mixture effects is especially challenging.”

Although people are exposed to many chemicals over their lifetimes—in food, water, personal care products, air, and other media—toxicology has historically studied agents individually. The authors of a new commentary recommend strategies for studying exposures to chemical mixtures. Image: © iStock/NoSystem Images.

Based on literature reviews and expert workshops, the team, organized by Martyn Smith at the University of California, Berkeley, proposed to anchor mixture analyses to a single carcinogen with a known effect. This approach allows researchers to use in vitro or in vivo assays to test specific hypotheses about changes in the dose–response curve when an organism is exposed to other chemicals in combination with the known carcinogen.

The proposal’s central tenet is that cancer results from a sequence of exposures to several carcinogens that affect the initiation, promotion, or progression of a slowly unfolding disease process.4,5 Thus, an appropriate research plan should build on both the key characteristics of carcinogenic chemicals and the hallmarks of cancer. “Key characteristics” refers to qualities or capabilities of a carcinogen that can lead to cancer, such as the ability to induce DNA damage or chronic inflammation, cause genomic instability, or suppress the immune system.3 “Hallmarks” refers to properties of cancer cells that are acquired during tumor formation, such as the ability to proliferate limitlessly and control formation of new blood vessels.6

The authors of the new paper proposed three strategies for studying how mixtures affect cancer outcomes. The first, a chemical screening, complements the other two approaches but may also stand alone. This approach involves mining existing data sets to select chemicals with some known link to cancer.

The second strategy is a transgenic model-based approach, for which the authors propose the rasH2 mouse 26-week bioassay. Animal models allow researchers to include nonchemical stressors, such as a high-fat diet, and better mimic the complexity of human cancers. “The critical piece for mixture studies is knowing what to expect,” says Rider. “The rasH2 mouse is [a Food and Drug Administration]-approved model with rich historical data for multiple cancers that provides expected joint effects as a benchmark for observed data.”

The third strategy, a disease-centered approach, may employ either an animal or in vitro organoid model. Organoid models, which may also be used in the screening approach, are well suited for testing how chemical mixtures affect cancer-relevant pathways or biomarkers.7,8

The authors also recommend experimental designs. For studying poorly understood cancers, they suggest starting with single-compound testing in an in vivo or in vitro screen, followed by high-throughput testing of multiple ratios of two chemicals along a single predicted response plane.9 For mixtures of more than two substances, the authors recommend the fixed ratio ray design,10 which focuses on a single fixed ratio of chemicals.

Alan Boobis, an emeritus professor of toxicology at Imperial College London, applauds the authors for addressing a topic whose importance has long been recognized by researchers and funding agencies. He agrees with anchoring mixture tests to a known carcinogen but would not limit animal models to the rasH2 just because it has extensive historical data.

“We have seen fantastic advances in systems biology and high-throughput screening technology,” says Boobis, who was not involved in the project. “I consider the rasH2 model a rather blunt instrument that may not provide sufficient power to detect low-dose effects and would prefer newer models that are better suited for proof-of-principle studies.”

For Susan Tilton, an associate professor of environmental and molecular toxicology at Oregon State University, the rasH2 model is a reasonable starting point that can be followed up with other models, perhaps designed for nonchemical stressors. “Evaluating mixtures is a challenging task,” she says. “To tackle this complex problem, leveraging a data-rich environment of existing models and toxicity information for individual chemicals is important.” Tilton also was not involved in the project.

Andreas Kortenkamp, a professor of human molecular toxicology at Brunel University London, hopes that carcinogenic effects of mixtures can eventually be predicted by modeling approaches alone, without further experiments. Kortenkamp, who also was not involved in the project, says, “This would be particularly attractive for risk assessment by regulatory agencies.”