ToxCast on Target: In Vitro Assays and Computer Modeling Show Promise for Screening Chemicals

Government agencies rely on toxicity data to help make key regulatory decisions on pesticides and other chemicals in the environment. But thousands of chemicals, including many currently in commerce, have yet to be tested for potential toxicity and impact on human diseases. Such testing can be time-consuming and expensive; a full set of regulatory tests for a single chemical may use thousands of animals and cost millions of dollars. To help address this large backlog of untested chemicals, the U.S. Environmental Protection Agency (EPA) recently completed the first phase of its large-scale ToxCast™ program, which looks at the potential for combining faster and less expensive in vitro assays with computer modeling to screen and prioritize chemicals for human toxicity [EHP 118:485–493; Judson et al.]. This approach could both reduce the need for animal testing and speed up the regulatory process. 
 
The ToxCast program evaluates the use of in vitro assays for understanding the types of molecular and pathway perturbations caused by chemical exposures and computer-based prioritization models for in vivo toxicity. In phase I, researchers chose 309 chemicals for which toxicity data were already available. Using 467 in vitro assays across 9 technologies, including high-throughput cell-free assays and cell-based assays in multiple human primary cells and cell lines, they investigated a broad spectrum of chemical activities at the molecular and pathway levels. Matching these in vitro results with existing data helped researchers build initial prioritization models for predicting toxicity of similar but untested chemicals. 
 
This process also provided information on underlying mechanisms of toxicity, which are difficult to investigate directly using animal models. Using human cells or human cell constituents allowed researchers to measure the effects of chemicals on toxicity pathways that may be relevant to human disease. Based on the phase I examples, the ToxCast researchers feel confident that in vitro high-throughput data can help predict mechanisms of action for many other well-studied chemicals and indicate which other biological pathways may also be activated. This will lay the groundwork for screening untested chemicals and provide vital guidance for future testing. 
 
The authors hope molecular and computational models will help better guide targeted testing of environmental contaminants but caution that building this new paradigm will itself take time and require input from multiple government organizations. They are launching a second phase of ToxCast to expand on and further confirm that in vitro testing can help predict human toxicity. Phase II could be completed over the next several years.

Human exposure to phthalates is ubiquitous due to widespread commercial use. Although the compounds are reported to be rap idly metabolized, concentrations in the body appear to remain fairly stable due to ongoing exposure. The United States and Europe have banned some phthalates from consumer products primarily on the basis of reproductive toxicity data. However, not all phthalates are regulated; meanwhile, research indicates toxicity may extend to other endocrine targets such as the thyroid gland, which is critical for proper neurodevelopment. A new study now reports an association between prenatal exposure to certain phthalates and adverse effects on test scores used to evaluate children's behavior and executive functioning [EHP 118:565-571; Engel et al.].
The prospective study was based on a multi ethnic cohort of 404 women recruited dur ing their first pregnancy for the Mount Sinai Children's Environmental Health Study between 1998 and 2002. Each woman completed medi cal, sociodemographic, and lifestyle question naires and provided a urine sample between 25 and 40 weeks of pregnancy, which was used to measure phthalate metabolites. Metabolites were grouped according to their molecular weights.
When their children were approximately 4.5-5.5, 6-6.5, and 7-9 years old, 188 of the women completed the Behavior Rating Inventory of Executive Functioning (BRIEF) and the Behaviors Assessment System for Children Parent Rating Scales (BASCPRS), standardized forms used in clinical and research assessments of children's executive functioning and behav ior. Executive functions encompass planning to achieve goals, controlling attention and emotion, inhibiting inappropriate behaviors, and extrapolating from life experi ences. Problematic behaviors assessed included hyper activity, aggres sion, poor conduct, and issues with anxiety, attention, and adaptability.
Highmolecularweight phthalates-like those found in medical tubing and vinyl floor and wall coverings-were not associated with altered scores derived from the parentreport forms aside from a small association with reduced emotional control. However, lowmolecular weight phthalates-like those found in personal care products such as perfume, shampoo, cosmetics, and nail polish-were significantly associated with increased scores for aggression, attention and conduct problems, and depression.
The BASCPRS includes a scale to help researchers weed out inaccurate assessments. The higher the resulting F-score, the more likely the assessment is to reflect an excessively negative evaluation of the child, a failure to follow instructions, random responding, or difficulty reading. When parentreport forms were restricted to those with F-scores of 0 or 1 (leaving 161 children in the analyses), most associations remained strong for boys but not girls. The sole exception was conduct problems, which remained significant for both girls and boys. The behavioral problems assessed in this study are relevant to conditions such as oppositional defi ant disorder, conduct disorder, and attention defi cit/hyperactivity disorder. Diagnosing these condi tions requires extensive testing beyond the scope of this study. Furthermore, this study cannot confirm that phthalate exposure caused these problems via altered thyroid function-or any other mechanism. However, thyroidrelated phthalate toxicity makes a connection biologically plausible and underscores an urgent need to further investigate the effects of phthalates on neurodevelopment.
Julia R. Barrett, MS, ELS, a Madison, WI-based science writer and editor, has written for EHP since 1996. She is a member of the National Association of Science Writers and the Board of Editors in the Life Sciences.

ToxCast on Target
In Vitro Assays and Computer Modeling Show Promise for Screening Chemicals Government agencies rely on toxicity data to help make key regula tory decisions on pesticides and other chemicals in the environment. But thousands of chemicals, including many currently in commerce, have yet to be tested for potential toxicity and impact on human diseases. Such testing can be timeconsuming and expensive; a full set of regulatory tests for a single chemical may use thousands of animals and cost millions of dollars. To help address this large backlog of untested chemicals, the U.S. Environmental Protection Agency (EPA) recently completed the first phase of its largescale ToxCast™ program, which looks at the potential for combining faster and less expensive in vitro assays with computer modeling to screen and prioritize chemicals for human toxicity [EHP 118:485-493; Judson et al.]. This approach could both reduce the need for animal testing and speed up the regulatory process.
The ToxCast program evaluates the use of in vitro assays for understanding the types of molecular and pathway perturbations caused by chemical exposures and computerbased prioritization models for in vivo toxicity. In phase I, researchers chose 309 chemicals for which toxicity data were already available. Using 467 in vitro assays across 9 technologies, including highthroughput cellfree assays and cellbased assays in multiple human primary cells and cell lines, they investigated a broad spectrum of chemical activities at the molecular and pathway levels. Matching these in vitro results with existing data helped researchers build initial prioritization models for predicting toxicity of similar but untested chemicals.
This process also provided information on underlying mecha nisms of toxicity, which are difficult to investigate directly using animal models. Using human cells or human cell constituents allowed researchers to measure the effects of chemicals on toxicity pathways that may be relevant to human disease. Based on the phase I examples, the ToxCast researchers feel confident that in vitro highthroughput data can help predict mechanisms of action for many other well studied chemicals and indicate which other biological pathways may also be activated. This will lay the groundwork for screening untested chemicals and provide vital guidance for future testing.
The authors hope molecular and computational models will help better guide targeted testing of environmental contaminants but cau tion that building this new paradigm will itself take time and require input from multiple government organizations. They are launching a second phase of ToxCast to expand on and further confirm that in vitro testing can help predict human toxicity. Phase II could be completed over the next several years.
Rebecca Clay Haynes has written for EHP since 1993. Her work has also appeared on National Public Radio and in the Christian Science Monitor and The Environmental Forum. In addition, she is the author of two children's science books related to astronomy and space exploration.
Low-molecular-weight phthalates are found in many personal care products.

Persistent Organic Pollutants May Promote Insulin Resistance Syndrome
Animal studies indicate some persistent organic pollutants (POPs) may be endocrine disruptors and suggest similar health risks for humans. Recent studies have further suggested an association between exposure to POPs and the prevalence of type 2 diabetes. Now an experimental animal study reports evidence of a causal link between POP exposure and insulin resistance syndrome, a cluster of metabolic disorders-including type 2 diabetes-that are marked by sustained high blood sugar [EHP 118:465-471; Ruzzin et al.].
POPs accumulate in fatty tissue, where they can remain for years because they are not easily broken down. Fatty fish are a potential source of POP exposure in many human populations. However, n3 polyunsaturated fatty acids in fish oil may have beneficial health effects, possibly including protective effects on insulin resistance, that could counterbalance any adverse effects of POPs in fatty fish.
In the current study, rats were exposed for 28 days to highfat diets that contained either crude fish oil (from farmed Atlantic salmon) or fish oil that was refined to remove POPs. As expected, the crude fish oil contained much higher levels of POPs than the refined oil. Gene expression profile comparisons of the livers of the 2 treatment groups showed that POP exposure disrupted lipid homeostasis.
Rats on the unrefined fish oil diet gained more weight overall and had increased triacylglycerol, diacylglycerol, and total choles terol levels compared with rats fed refined fish oil. They also showed impaired insulin action in response to the highfat diet, whereas the highfat diet did not seem to cause insulin resistance in the rats fed refined fish oil. Further analysis revealed a reduction in the ability of insulin to stimulate glucose uptake in adipocytes treated with a mixture of POPs that was comparable to the mixture of chemicals in crude fish oil. Adipocyte responses to insulin varied with exposures to different mixtures of individual POPs.
The authors conclude that dietary exposure to POPs may be a risk factor for insulin resistance and associated metabolic disorders. Furthermore, the metabolic effects of POP exposures exacerbated deleterious effects of a highfat diet on rats and appeared to negate protective effects of n3 polyunsaturated fatty acids on insulin resistance.
Tanya Tillett, MA, of Durham, NC, is a staff writer/editor for EHP. She has been on the EHP staff since 2000 and has represented the journal at national and international conferences.

PBDEs Alter Development of Human Brain Cells
A new laboratory study demonstrating that polybrominated diphenyl ethers (PBDEs) can alter human fetal brain cells may explain at least in part the neurotoxicity recently documented in epidemiologic stud ies of young children exposed to PBDEs and previously shown in animal models [EHP 118:572-578; Schreiber et al.]. The new study, the first to examine PBDE neurotoxicity in a human cell-based sys tem, links the brain cell alterations to endocrine disruption.
A wealth of data demonstrates that babies can be exposed to signifi cant amounts of PBDE flame retardants both in the womb and through breastfeeding. Although all 3 PBDE formulations-penta, octa, and deca-are banned in Europe, and the penta and octa formulations were discontinued in the United States (deca also is banned in some states), PBDEs may still be used in some new U.S. products and in wares manufactured elsewhere. They also are found in a wide variety of older plastic consumer goods that remain in use in many homes, businesses, and automobiles. The PBDEs are known to migrate into indoor dust, posing a particularly high exposure risk to infants and toddlers because of their characteristic handtomouth behavior.
To investigate how PBDEs may impact the developing fetal brain, the team of scientists employed a method for evaluating human devel opmental neurotoxicity they had recently developed [EHP 117:1131[EHP 117: -1138 as an alternative to animal testing. This method uses primary fetal human neural progenitor cells (hNPCs) cultured to produce complex 3dimensional cellular systems called neurospheres. Neurospheres undergo the same basic processes that occur during the early stages of normal human brain development: cell proliferation, differentiation, and migration. Tests conducted with neurospheres may help identify exogenous substances that disturb these basic processes in vivo.
The researchers focused on 2 of the PBDE compounds that accu mulate the most in humans, BDE47 and BDE99. At concentrations below levels that cause cell death, they found these compounds could reduce the migration of the hNPCs-which suggests the possi bility of adverse effects on brain development-and the effects increased with higher PBDE concentrations. At the highest tested concentra tion (10 μM), BDE47 decreased the distance the cells migrated by more than 25% compared with unexposed cells, whereas the same concentration of BDE99 decreased the distance by more than 30%. Additional testing established that both compounds also interfered with the differentiation of immature progenitor cells into neurons and oligodendrocytes.
Further tests suggested the PBDE compounds affected cell migra tion and differentiation by interfering with thyroid hormone signaling, an endocrinedisrupting effect that could be associated with addi tional impacts throughout a person's life. Followup work to determine whether PBDEs cause the same effects in rodent neurospheres would facilitate extrapolation from animals to humans, the authors say.
Other work has shown the tendency of PBDEs to accumulate in brain and neuronal cells, and the researchers used radiolabeled BDE47 to measure an accumulation in test cells of approximately 60fold. Considered with available human exposure data, these data suggest current PBDE exposure levels are likely to be of concern for human health, the authors write. Two recent studies have linked PBDEs with subtle changes in childrens' IQs and behavior [EHP 117:1953[EHP 117: -1958 and EHP doi:10.1289/ehp.0901340], and the authors say additional studies are needed to assess these associations.
Kellyn S. Betts has written about environmental contaminants, hazards, and technology for solving environmental problems for publications including EHP and Environmental Science & Technology for more than a dozen years.

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This fluorescence micrograph of differentiated neural progenitor cells shows neurons in green, glial cells in red, and cell nuclei in deep blue (the turquoise results from the overlay of the blue and green channels).