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Mixed Messages
Department of Pathology and Laboratory Medicine, Brown University, Providence, RI 02912
1 To whom correspondence should be addressed. Fax: (401)863-9008. E-mail: kim_boekelheide{at}brown.edu.
Received May 7, 2007; accepted May 8, 2007
Key Words: mixtures; endocrine disruptors; developmental toxicity; prenatal; reproductive tract; male; testis.
Our understanding of mixed exposures is still very much in its infancy, even though the vast majority of environmental exposures are mixed exposures. The basic reason for this lack of understanding is the huge investment needed to describe the dose-response behavior of the single components before a mixture can be interpreted as the net result of their interactions.
An alternative approach to understanding mixed exposures is simply to treat the mixture as the exposure and evaluate the effects of the mixture as a whole without trying to parse out the contributions of the components. This approach is certainly useful for relatively stable formulations produced in large amounts; you might think of gasoline as an example of such a product, but I bet that the differences among crude oil sources and refineries make the final product that we call "gasoline" quite variable. So, the fundamental problem with this approach is the lack of utility of the data beyond the narrow scope of the formulation itself. And since environmental exposures are an infinite set of mixed contaminants of varying quantities, their effects cannot be understood by the toxicity testing of each possible mixture taken one at a time. The one-at-a-time approach is clearly not the way to go, so we are back to developing a "toxicological science of mixtures."
DEVELOPING THE TOXICOLOGICAL SCIENCE OF MIXTURES IS NOT GOING TO BE EASY OR FAST OR CHEAP
The United States Environmental Protection Agency (USEPA) has described principles and procedures for how to conduct risk assessments of mixed exposures (USEPA, 2000
). This large document (209 pages) spells out some of the conceptual underpinnings and possible approaches. One key concept is the necessity to determine whether the components of a mixtures share "toxicologic similarity." Toxicologic similarity means sharing the same mode of action, defined as "a series of key events and processes starting with the interaction of an agent with a cell and proceeding through operational and anatomical changes causing disease formation" which, in some cases, "may be relaxed to require that these chemicals act only on the same target organ" (USEPA, 2000, pp. 10). WOW! This limited description is all that we get as an explanation of the biology that underlies toxicologic similarity—the implication being that we have a lot of work to do!
Another key concept is an assumption of "additivity," used "when the effect of the combination of chemicals can be estimated directly from the sum of the scaled exposure levels (dose addition) or of the response (response addition) of the individual components" (USEPA, 2000, pp. 10). Dose addition makes the assumption that the mixed chemicals share a similar mode of action, similar absorption, distribution, metabolism, and excretion, and similarly shaped dose-response curves. Response addition makes no such assumptions and is in practice applied to low risk estimates resulting from linear extrapolations of high-dose exposures, primarily in the risk assessment of mixtures of carcinogens.
DEVELOPING THE TOXICOLOGICAL SCIENCE OF MIXTURES IS NOT GOING TO BE EASY OR FAST OR CHEAP ... BUT YOU HAVE TO START SOMEWHERE
In a recently published series of technical reports and articles, the National Toxicology Program (NTP) examined the carcinogenic effects of exposures to binary and tertiary mixtures of dioxin-like compounds (for a representative publication, see Walker et al., 2005). This body of work is an amazing tour de force that took a decade to complete and required the deep pockets of the NTP to conduct. The bottom-line conclusion is that for carcinogenic end points, dose additivity successfully predicts the mixed behavior of the dioxin-like compounds when the doses are appropriately scaled by their respective toxic equivalency factors. This is great stuff and very reassuring. However, the examination of carcinogenic end points of similarly acting chemicals is likely the easy pickings in our attempts to understand how mixed contaminants work together to produce adverse outcomes. Cancer is a relatively simple end point because the characteristics of this end point do not change with dose, only the incidence.
And now to the work of Howdeshell et al. (2007) appearing in this issue. For many years, the Gray laboratory has studied the effects of endocrine disrupting chemicals on testicular development in the rat. This has been a concerted and focused effort that has led to the characterization of developmental events in the male that depend upon the synthesis of testosterone and insl3 by the fetal testis and downstream signaling responses to these hormones. Interference with fetal testicular hormone synthesis or androgen-dependent signaling events results in a suite of abnormalities, including epididymal agenesis, cryptorchidism, hypospadias, nipple and areola retention, small testes, impaired spermatogenesis, altered anogenital distance, delayed preputial separation, and Leydig cell hyperplasia. These abnormalities arise because of a failure to adequately trigger the requisite hormone-dependent events in a multistep pathway beginning with insl3 and testosterone synthesis within the fetal testis and proceeding to gubernacular maturation and androgen receptor activation in various end organs.
In Howdeshell et al. (2007), the effects of single and combined exposures to two phthalates, di-(n-butyl) phthalate and di(2-ethylhexyl) phthalate, were examined. The hypothesis being tested was that these two chemicals with the same molecular mechanism of action but different active metabolites would produce dose-additive adverse effects on the various end points in their model, including biomarkers of fetal testicular steroidogenesis, androgen-dependent organ weights, and reproductive malformations. Examining a remarkable plethora of outcomes, the predicted dose additivity of the mixed exposure held true, planting a stake in the ground for the development of the toxicological science of mixtures as applied to endocrine disruptors and hormone-dependent signaling pathways. The dose-response behaviors of endocrine disruptor action on hormone-dependent signaling pathways are fraught with controversy (U-shaped curves, anyone?), so the ability to predict outcomes in this setting is important.
More recent work from the Gray laboratory, presented in abstract form at the 46th Annual Meeting of the Society of Toxicology (Gray et al., 2007), takes this mixtures paradigm to another level. In this new work, pregnant rat dams were exposed to mixtures of phthalates (suppressors of testosterone synthesis within the fetal testis) and androgen receptor antagonists (acting at the end organs of this signaling pathway). The exposures were orchestrated so that any agent alone had very limited effects while the collective exposure robustly induced hypospadias and epididymal agenesis in the developing males. Overall, the chemicals clearly acted with dose additivity, not response additivity. These effects were induced by chemicals acting by different molecular mechanisms within different organ systems with different absorption, distribution, metabolism, excretion patterns, and differently shaped dose-response curves. By all of our familiar criteria, these chemicals are not toxicologically similar and do not share a mode of action as defined by the USEPA; and yet they can act together to inhibit this developmentally sensitive signaling pathway. Stay tuned for the upcoming publication of this exciting work!
The examples highlighted above illustrate both the progress that we are making to develop a toxicological science of mixtures and the limitations of our current approach. The toxicological science of mixtures is "big science" requiring lots of resources over a considerable period of time. The initial phases of any "big science" project are highly descriptive; it takes a lot of background knowledge to design a mixtures experiment that makes sense. The examples above are of intramural science performed within large federal agencies (USEPA and NTP/National Institute of Environmental Health Sciences [NIEHS]). The usual National Institutes of Health extramural grant system, largely focused on R01-type projects, is not designed to support this kind of work because by its very nature big science requires a long-term commitment and has a long prodromal descriptive phase. There are a few ways in which mixtures science is supported in the extramural community, notably by the NIEHS Superfund Basic Research Program, but overall this important area of exposure biology has not attracted the attention and support it needs and deserves. While we should celebrate the achievements in this emerging field noted above, we need to engage extramural researchers in this area, invest more in its development, and plan coherently because developing the toxicological science of mixtures is not going to be easy or fast or cheap ... but real-life exposures are almost always mixed exposures.
ACKNOWLEDGMENTS
Supported in part by National Institutes of Environmental Health Sciences grant P42 ES013660.
REFERENCES
Gray LE, Furr J, Wilson VS, Hochkiss AK, Howdeshell K, Rider C. Cumulative reproductive effects of in utero administration of mixtures of antiandrogens in male SD rats: Synergy or additivity? Toxicologist (2007) 96:385.
Howdeshell KL, Furr J, Lambright CR, Rider CV, Wilson VS, Gray LE. Cumulative effects of dibutyl phthalate and diethylhexyl phthalate on male rat reproductive tract development: Altered fetal steroid hormones and genes. Toxicol. Sci. (2007) 99(1):190–202.
USEPA. In: Supplementary Guidance for Conducting Health Risk Assessment of Chemical Mixtures (2000) Washington, DC: U.S. Environmental Protection Agency, Risk Assessment Forum, Office of Research and Development.
Walker NJ, Crockett PW, Nyska A, Brix AE, Jokinen MP, Sells DM, Hailey JR, Easterling M, Haseman JK, Yin M, et al. Dose-additive carcinogenicity of a defined mixture of dioxin-like compounds. Environ. Health Perspect. (2005) 113:43–48.[Web of Science][Medline]
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