Grantee Research Project Results
Final Report: Mechanistic Evaluation of the Toxicity of Chemical Mixtures
EPA Grant Number: R829358Title: Mechanistic Evaluation of the Toxicity of Chemical Mixtures
Investigators: LeBlanc, Gerald A.
Institution: North Carolina State University
EPA Project Officer: Hahn, Intaek
Project Period: September 24, 2001 through September 23, 2006
Project Amount: $465,281
RFA: Complex Chemical Mixtures (2000) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Environmental Justice , Safer Chemicals , Land and Waste Management
Objective:
Evaluating the toxicity of complex chemical mixtures is one of the major challenges facing modern toxicology. The virtually infinite number of chemical combinations that constitute environmentally relevant mixtures renders standard approaches for the toxicity characterization of chemicals obsolete. The overall objective of this research project was to test the hypothesis that the toxicity of complex chemical mixtures can be satisfactorily estimated by understanding the mechanism of toxicity of the individual constituents and utilizing algorithms that define interactions based on these mechanisms. Experiments were performed using an invertebrate model, Daphnia magna, because both acute and life-cycle toxicity evaluations can be performed with this species with reasonable cost, space, and time consideration. The following three objectives were established to test the hypothesis:
1. The concentration-response relationship for each of the model chemicals to be used to develop the mixtures modeling approach would be characterized.
2. The algorithm to be used to assess the toxicity of chemicals mixtures would be developed and experimentally tested using the data from individual chemicals generated under Objective 1.
3. The approach would be expanded to assess the chronic toxicity of environmentally relevant chemical mixtures.
All objectives were met. In addition, an interactive Web site was created with which users can assess the toxicity of chemical mixtures of interest.
Summary/Accomplishments (Outputs/Outcomes):
Integrated Addition and Interaction Model Development
Concentration Addition. With the Integrated Addition and Interaction (IAI) model, chemicals having the same mechanism of action are assigned to a common cassette (i.e., grouping). Toxicity associated with the cassette is then calculated using a concentration addition approach. The combined toxicity of chemicals within the same cassette is computed using the following equation:
where R is the response of the organisms to the chemical mixture within the cassette, Ci is the concentration of chemical i in the mixture, EC50i is the concentration of chemical i that causes a 50 percent response, and ρ´ is the average power (Hill slope) associated with all chemicals in the cassette.
Response Addition. The concept of response addition is used to compute the joint toxicity associated with the different chemical cassettes within a mixture. The response addition model is used because each cassette is assumed to represent a different mechanism of action. The response addition model can be depicted as:
where R represents the response to the mixture and Ri is the response to chemicals in cassette i.
The above two equations were integrated to determine the response associated with individual cassettes within a mixture and to sum these responses associated with the cassettes to yield the total response to the chemical mixture:
Chemical Interactions
The ability of one chemical in the mixture to modify the effective concentration of another was defined by coefficients of interactions or K-functions. K-functions were described by experimentally deriving the effect of concentrations of effector chemical on the EC50 values for the affected chemical. K-functions were derived for specific concentrations of effector chemical using the following equation:
where EC50a is the concentration of affected chemical that immobilizes 50 percent of the exposed animals and EC50a/e is the EC50 of the affected chemical when exposure occurred in the presence of a defined concentration of the effector chemical. These K-functions were then plotted against the concentrations of effector chemical from which they were derived. The logistic equation that defined this relationship was used to calculate K-functions for any concentration of effector chemical when modeling mixture toxicity. K-functions were integrated into this model to describe toxicokinetic interactions between chemicals:
where ki represents a function describing the extent to which the effector chemical present in the mixture at a defined concentration alters the active concentration of chemical i (the affected chemical). This application of the K-function is specific for toxicokinetic interactions whereby the effector chemical modifies the active concentration of the affected chemical. K-functions should have similar application in describing toxicodynamic interactions where the effector chemical affects the activity of the affected chemical; however, evaluation of toxicokinetic interactions was beyond the purview of the program.
Model Assessment
Two chemical mixtures were used to assess the accuracy of the IAI model. The first was a quaternary mixture of two organophosphate compounds, malathion and parathion, and two chlorophenols, 2-chlorophenol and 4-chlorophenol. These chemicals allowed for assessment of the integration of a concentration-addition and a response-addition model into a single algorithm. The model consisted of two cassettes (organophosphate cassette and chlorophenol cassette) that were each evaluated using concentration addition. The joint toxicity of the two cassettes then was assessed according to response-addition. The toxicity of 30 combinations of the four compounds was experimentally determined and compared to model predictions based on concentration addition alone, response addition along, and integrated addition. The integrated addition model provided the best estimate of the toxicity of the 30 combinations, demonstrating the utility of this approach over conventional approaches.
The second mixture consisted of malathion, parathion, and piperonyl butoxide. This ternary mixture allowed for integrated assessment of concentration addition (organophosphates), response addition (organophosphate cassette and piperonyl butoxide cassette), and toxicokinetic interaction (piperonyl butoxide is a potent inhibitor of the metabolic activation of the organophosphates). Thirty combinations of the three compounds were modeled and experimentally measured. Model predictions were highly consistent with experimental measurement (r2 = 0.72).
In combinations, these results demonstrate that the IAI model is superior to standard mixtures modeling approaches and holds promise as a means of assessing the combined toxicity of chemical mixtures in which some constituents share the same mechanisms of action, some have different mechanisms of action, and some chemicals modify the toxicity of other constituents.
Acute toxicity (immobilization after 48 hours exposure) was used as the toxicity endpoint in all initial assessments. Next, the utility of the IAI model to assess chronic toxicity of environmentally relevant chemical mixtures was evaluated. Two separate studies were undertaken. One consisted of an evaluation of the effects of a mixture of nine chemicals shown to be relatively common in surface waters of the United States (bisphenol A, caffeine, carbaryl, chlorpyrifos, N, N-diethyl-m-toluamide, diazinon, 1,4-dichlorobenzene, fluoranthene, 4-nonylphenol). The second study consisted of an evaluation of the chronic toxicity of a mixture of four polycyclic aromatic hydrocarbons (pyrene, phenanthrene, fluoranthene, and naphthalene). Endpoints of toxicity used in these experiments were reduced lifespan (first study), reduced growth rate (first and second study), and reduced fecundity (first study) over 21 days exposure.
Toxicity of the first mixture was modeled at various constituent combinations at which the ratio of the chemicals within the mixture was maintained at that reported for median detected environmental levels. Toxicity of the mixture was then determined experimentally and compared to model predictions. The model accurately predicted the most sensitive endpoint, as well as the lowest toxic effect level of the mixture. Results demonstrated that, for this mixture of chemicals, toxicity was not influenced significantly by interactions among the chemicals, and a single constituent dominated toxicity. According to model predictions and experimental results, the median detected environmental concentrations of chemicals constituting this mixture provided no margin of safety.
The toxicity of the polyaromatic hydrocarbon (PAH) mixture was modeled for concentrations of the materials reported to occur in the environment and on equitoxic concentrations. The effects of over 140 combinations of four mixture formulations on the growth rate of daphnids were experimentally determined and compared to model predictions. The IAI model tended to over predict the joint toxicity of these PAH mixtures when a common mode of action was assumed, and all chemicals were placed in the same cassette. Toxicity of the PAH mixture was well represented when each PAH was placed in its own cassette. Mixtures at environmentally relevant concentrations were both predicted and experimentally demonstrated to have no effect on daphnid growth rates. Results indicated that PAHs elicit toxicity to daphnids by multiple mechanisms and demonstrate an appropriate modeling approach to assess the toxicity of these mixtures.
Finally, we demonstrated that the IAI model could be used to establish the mechanism of action of chemicals. We observed that on treating daphnids with the terpenoid hormone methyl farnesoate or its synthetic analogs (pyriproxyfen, fenoxycarb, methoprene) the hemoglobin 2 gene was induced resulting in significantly elevated hemoglobin levels. A putative response element on the hemoglobin 2 gene was identified that may mediate this hormonal activity as was a 52 kD protein that binds to the response element following hormone treatment. Together, these results identified a regulatory pathway responsible for controlling hemoglobin levels that is distinct from the well characterized hypoxia/hypoxia inducible factor regulatory pathway.
We observed that the herbicide atrazine induced the hemoglobin 2 gene resulting in elevated circulating hemoglobin levels. The IAI model was used to establish whether this induction was mediated by the hormonal regulatory pathway. The joint action of hemoglobin and pyriproxyfen was modeled under the assumption that both chemicals shared the same mechanism of action (i.e., both chemicals were placed in the same cassette) and, alternatively, under the assumption that the chemicals induced hemoglobin by different mechanisms of action (i.e., each chemical assigned to different cassettes). Model results were compared to measured effects of the chemical combinations on hemoglobin 2 mRNA levels. Hemoglobin induction by the mixtures was accurately predicted when different mechanisms of action were assumed and not when a common mechanism of action was assumed. These results demonstrated that atrazine does not induce hemoglobin via the hormonal regulatory pathway and demonstrated the strength of the modeling approach in identifying mechanisms of action, which is critical to accurate mixtures toxicity modeling.
A free-access Web site has been created with which users can apply the IAI model to their own assessments of the toxicity of chemical mixtures (http://wang.tox.ncsu.edu/model5/ Exit ). The Web Site, called CATAM—A Computational Approach to the Toxicity Assessment of Mixtures—consists of a description of the model framework, a dictionary of terms used in the model, a tutorial, frequently asked questions, and two calculators (a quick calculator and a definitive calculator) with which users can enter data on individual chemicals and calculate the estimated hazard of the mixture. Monthly usage of this site by individuals outside of the principal investigator’s (PI) laboratory has increased by 325 percent since its inception. The site, along with publications from this program, has stimulated several collaborations between the PI and investigators, who are researching the toxicity of chemical mixtures.
Journal Articles on this Report : 11 Displayed | Download in RIS Format
| Other project views: | All 29 publications | 21 publications in selected types | All 17 journal articles |
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Gorr TA, Rider CV, Wang HY, Olmstead AW, LeBlanc GA. A candidate juvenoid hormone receptor cis-element in the Daphnia magna hb2 hemoglobin gene promoter. Molecular and Cellular Endocrinology 2006;247(1-2):91-102. |
R829358 (Final) R831300 (2005) R831300 (2006) R831300 (Final) R832739 (2008) |
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LeBlanc GA, Olmstead AW. Evaluating the toxicity of chemical mixtures. Environmental Health Perspectives 2004;112(13):A729-A730. |
R829358 (Final) |
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LeBlanc GA, Wang G. Chemical mixtures: greater-than-additive effects? Environmental Health Perspectives 2006;114(9):A517-A518. |
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Mu XY, LeBlanc GA. Synergistic interaction of endocrine-disrupting chemicals: model development using an ecdysone receptor antagonist and a hormone synthesis inhibitor. Environmental Toxicology and Chemistry 2004;23(4):1085-1091. |
R829358 (2003) R829358 (2004) R829358 (Final) R826129 (Final) |
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Mu XY, LeBlanc GA. Cross communication between signaling pathways: juvenoid hormones modulate ecdysteroid activity in a crustacean. Journal of Experimental Zoology Part A–Comparative Experimental Biology 2004;301A(10):793-801. |
R829358 (2004) R829358 (Final) R826129 (Final) R831300 (2004) |
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Mu XY, Rider CV, Hwang GS, Hoy H, LeBlanc GA. Covert signal disruption: anti-ecdysteroidal activity of bisphenol A involves cross talk between signaling pathways. Environmental Toxicology and Chemistry 2005;24(1):146-152. |
R829358 (2004) R829358 (Final) R831300 (2004) R831300 (Final) R832739 (2008) |
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Olmstead AW, LeBlanc GA. Toxicity assessment of environmentally relevant pollutant mixtures using a heuristic model. Integrated Environmental Assessment and Management 2005;1(2):114-122. |
R829358 (Final) |
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Olmstead AW, LeBlanc GA. Joint action of polycyclic aromatic hydrocarbons: predictive modeling of sublethal toxicity. Aquatic Toxicology 2005;75(3):253-262. |
R829358 (Final) |
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Rider CV, Gorr TA, Olmstead AW, Wasilak BA, LeBlanc GA. Stress signaling: coregulation of hemoglobin and male sex determination through a terpenoid signaling pathway in a crustacean. Journal of Experimental Biology 2005;208(Pt 1):15-23. |
R829358 (Final) R831300 (2004) R831300 (2006) R831300 (Final) R832739 (2008) |
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Rider CV, LeBlanc GA. An integrated addition and interaction model for assessing toxicity of chemical mixtures. Toxicological Sciences 2005;87(2):520-528. |
R829358 (Final) |
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Rider CV, LeBlanc GA. Atrazine stimulates hemoglobin accumulation in Daphnia magna: is it hormonal or hypoxic? Toxicological Sciences 2006;93(2):443-449. |
R829358 (Final) R831300 (Final) |
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Supplemental Keywords:
hazard assessment, narcotics, computational toxicology, synergy, antagonism, toxicity, Daphnia magna, surface waters, chemical mixtures, analytical models, biodegradation, chemical kinetics, complex mixtures, complex toxic chemical mixtures, contaminant transport models, contaminated sediments, environmental transport and fate, fate and transport, fate and transport , hazardous chemicals, hazardous organic substances, mechanistic research,, RFA, Ecosystem Protection/Environmental Exposure & Risk, Scientific Discipline, Waste, Hazardous, Fate & Transport, chemical mixtures, Environmental Chemistry, Hazardous Waste, Ecology and Ecosystems, environmental transport and fate, hazardous organic substances, toxicity testing, contaminated sediments, complex toxic chemical mixtures, mechanisitic research, analytical models, fate and transport , biodegradation, complex mixtures, fate and transport, chemical kinetics, contaminant transport modelsRelevant Websites:
http://www.tox.ncsu.edu/faculty/leblanc/ Exit
http://wang.tox.ncsu.edu/model5/ Exit
http://www.ncsu.edu/news/press_releases/05_01/021.htm Exit
http://jeb.biologists.org/cgi/content/full/208/1/i-a Exit
http://www.cals.ncsu.edu/agcomm/magazine/spring05/sex.htm Exit
Progress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.