Grantee Research Project Results
2004 Progress Report: Comparative In Vitro Immunotoxicology of Organochlorine Mixtures: Validation for Use in Risk Assessment
EPA Grant Number: R829361Title: Comparative In Vitro Immunotoxicology of Organochlorine Mixtures: Validation for Use in Risk Assessment
Investigators: DeGuise, Sylvain
Institution: University of Connecticut
EPA Project Officer: Hahn, Intaek
Project Period: October 1, 2001 through September 30, 2004 (Extended to September 30, 2005)
Project Period Covered by this Report: October 1, 2003 through September 30, 2004
Project Amount: $666,649
RFA: Complex Chemical Mixtures (2000) RFA Text | Recipients Lists
Research Category: Hazardous Waste/Remediation , Land and Waste Management , Safer Chemicals
Objective:
The objectives of this research project are to:
- (1) determine the effects of in vitro exposure to mixtures of organochlorines (OCs) on peripheral blood neutrophil phagocytosis, splenocyte proliferation, and natural killer (NK) cell activity using B6C3F1 mice, the common model in immunotoxicology;
- compare the effects of in vitro exposure to mixtures of OCs on immune functions of different species of concern, including humans and marine mammals, using B6C3F1 mice as a reference species;
- validate in mice the significance of in vitro exposure to determine immunotoxicity compared to traditional in vivo exposures;
- and investigate the use of monoclonal and polyclonal antibodies to rapidly assess the exposure directly at the level of the individual target cell using flow cytometry, which will allow for the investigation of the association between exposure and effects at the cell level.
Progress Summary:
Recent research in our laboratory has used the in vitro exposure model to quantify the immunotoxic potential of polychlorinated biphenyls (PCBs) and dioxins, individually and in mixtures, in marine mammals, humans, and mice (Levin, et al., 2004; Levin, et al., 2005a; Levin, et al., 2005b; Mori, et al., 2005). Some of those results will be discussed below.
First, and maybe most importantly, there were marked differences between species in the susceptibility to the immunotoxic potential of PCBs and dioxins, individually and in mixtures (Figure 1).
Figure 1. The Immunomodulatory Effects of OCs, Individually or in Mixtures, Were Assessed on Con A-Induced T Lymphocyte Proliferation in Mice and Different Species of Marine Mammals Upon Experimental Exposure to Mixtures of Non-Coplanar PCB Congeners (PCB 138, 153, and 180, 5 ppm each), a Coplanar PCB Congener (PCB 169, 5 ppm), or TCDD (0.05 ppb), the Model Compound for Coplanar PCB Congeners. Results are expressed as the percentage increase or reduction compared to unexposed control when the differences were statistically significant (p<0.05) Exposure to organochlorines resulted in either increases or decreases in T lymphocyte proliferation, with marked differences between species. Data slightly modified from Mori, et al. (2005).
Another important finding is that different immune functions are not equally affected within a species and across species by a given chemical or mixture of chemicals. This is illustrated by the clear differences in the immunomodulatory effects of PCBs and dioxins, individually or in mixtures, on T lymphocytes (Figure 1) and neutrophil phagocytosis (Figure 2). Even for a given function, there were differences between the effects in different cell types, as illustrated by the differences in the effects on neutrophil and monocyte phagocytosis (Figure 3). The magnitude of the reduction in phagocytosis was much more marked in human monocytes than in human neutrophils, and PCB 169 had a significant effect on monocyte but not on neutrophil phagocytosis.
Figure 2. The Immunomodulatory Effects of OCs, Individually or in Mixtures, Were Assessed on Neutrophil Phagocytosis in Mice and Different Species of Marine Mammals Upon Experimental Exposure to Mixtures of Non-Coplanar PCB Congeners (PCB 138, 153, and 180, 5 ppm each), a Coplanar PCB Congener (PCB 169, 5 ppm), or TCDD (0.05 ppb), the Model Compound for Coplanar PCB Congeners. Results are expressed as the percentage increase or reduction compared to unexposed control when the differences were statistically significant (p<0.05) Exposure to organochlorines resulted in either increases or decreases in neutrophil phagocytosis, with marked differences between species. Data slightly modified from Levin, et al. (2005a).
Figure 3. Modulation of Human Neutrophil (upper panel) and Monocyte (lower panel) Phagocytosis (% of unexposed DMSO vehicle control) Upon a 3-Hour Exposure to Increasing Concentrations of Individual OCs. *p<0.05, significantly different from unexposed control (N=6). The concentrations for TCDD were 0, 0.05, 0.10, 0.15, 0.20, and 0.25 ppb. Data from Levin, et al. (2005b).
Effects were not only observed in marine mammals and mice, but also in human cells (Figure 3). The relationships between species were quantified based on the similarities and differences in their response to the immunomodulatory potential of mixtures of PCBs and dioxin, using the chemicals listed in Figures 1 and 2, and an example is provided for monocyte phagocytosis (Figure 4). The lack of clustering according to the phylogenetic relationships between species (for example, the fact that the cetaceans do not all group together) highlights the fact that phylogeny could not predict toxicity. In fact, our results show that sea otters represent the species whose response to the immunotoxic effects of PCBs and dioxin mixtures most resembles that of humans.
Figure 4. Dendrogram Representing Quantitative Clustering by Species Based on Similarities and Differences in Modulation of Monocyte Phagocytosis Compared to the Unexposed Control Cells. This dendrogram clearly documents that phylogeny could not predict toxicity. Data from Levin, et al. (2005b).
Taken together, our data reflect the inappropriateness of using the mouse model to predict effects in other species and predict the risk associated with exposure to chemicals if effects in other species are different. Not only are there differences in the magnitude of changes (or sensitivity) between species, but there appears to be differences in the mechanisms by which the immunotoxicity is manifested, as exemplified by the fact that non-coplanar PCBs 138, 153, and 180 suppressed phagocytosis in humans (Levin, et al., 2005b; Figure 3), bottlenose dolphins, and beluga whales (Levin, et al., 2004), but not in mice (Levin, et al., 2005a; Figure 2).
Our recent experience has also led us to investigate the effects of chemicals in mixtures, using different methods. Correlation analyses were used to test whether the effects of mixtures could be predicted from those of its components (Figure 5). Three outcomes were found using this approach:
- the lack of significant correlation;
- a significant negative correlation, which suggested that the effects of a mixture could not be determined by that of its components and that there are complex interactions (possibly synergistic or antagonistic);
- and a significant positive correlation, which suggested that the effects of a mixture could be predicted from those of its components with varying levels of confidence (depending on the strength of the correlation, defined by the R2) and precision (how close to 1 is the slope).
Figure 5. Correlation Analysis Between the Response Calculated as the Sum of the Effects of Individual Congeners Compared to Control and Measured (observed) Reduction in Neutrophil Phagocytosis for all Individual Congeners and Mixtures Described in Figure 1 and 2. The dotted line (if present) represents a theoretical perfect correlation resulting from a purely additive interaction (slope of 1 and R2=1). For Pacific white-sided dolphins (upper panel), the measured reduction in phagocytosis could be predicted by the simple addition of the effects of the components of the mixtures. The negative slope of the significant correlation in Northern fur seals (lower panel) was not biologically relevant and could not allow for the prediction of the effects of a mixture from that of its components. Data from Levin, et al. (2005a).
A second method used in our lab to evaluate the contribution of different chemicals to the effects of mixtures is to perform regression analysis using the data from all the mixtures tested, as well as individual chemicals (Figures 1 and 2), for a given function in a given species to assess the overall relative contribution of each chemical (independent variable) in explaining the variability of the dependent variable (the immune function measurement). Using such a method (Table 1), we demonstrated that most of the effects of PCBs and dioxin on neutrophil phagocytosis were mediated by the non-coplanar PCBs 138, 153, and 180, and not by the coplanar PCB 169 or dioxin (except for Steller sea lions).
Table 1. Forward Stepwise Regression Equations To Explain Changes in Neutrophil Phagocytosis (∆ P) in Different Species of Marine Mammals and Mice. Data from Levin, et al. (2005a).
Finally, the different congeners and mixtures used also could be quantitatively clustered, based on the similarities and differences of the immunotoxic effects they induced in different species. For example, the PCBs and dioxin mixtures listed above (Figures 1 and 2) were clustered based on their effects on neutrophil phagocytosis in marine mammals and mice (Figure 6). This grouping clearly separated the mixtures that contained none or one of the non-coplanar congeners 138, 153, or 180 (upper branch) from those that contained two or three of the non-coplanar congeners (lower branch).
Overall, the suite of data presented here demonstrates the ability of this laboratory to use an in vitro approach to demonstrate important concepts in immunotoxicology, including the ability to determine, quantify, and compare the relative immunotoxicity of chemicals in different species and to quantify interactions of chemicals in mixtures. Our results also have demonstrated the inadequacies of rodent models to predict toxic effects in different species, such as marine mammals and humans. This information will be vital to more precise and relevant assessment of the hazardous potential of chemicals in different species, one of the most important steps in risk assessment.
Figure 6. Dendrograms Representing Clustering by Species and Mixtures of OCs Based on Modulation of Neutrophil Phagocytosis Compared to the Unexposed Control Cells. M.m.-B6C3F1 mouse; T.t.-bottlenose dolphin; D.l.-beluga whale; L.o.-Pacific white-sided dolphin; O.o.-killer whale; C.c.-Commerson’s dolphin; C.u.-Northern fur seal; E.l.-sea otter; P.v.-harbor seal; E.j.-Steller’s sea lion. Brighter shades of green indicate greater reduction of phagocytosis compared to control and brighter shades of red indicate greater enhancement of phagocytosis compared to control. Data from Levin, et al. (2005a).
Future Activities:
We will complete our database and publish results for the effects of exposure to PCBs and dioxin on phenotyping of human leucocytes, LPS-induced B lymphocyte proliferation, and neutrophil respiratory burst in human and marine mammal cells, which will complete objectives 1 and 2. With regards to objective 3, we have performed in vivo exposures and await the results of analysis of tissue samples for concentrations of PCBs to complete this project. Objective 4 will not be completed successfully, as attempts at labeling cells with antibodies to PCBs did not result in significant labeling above control.
Journal Articles on this Report : 6 Displayed | Download in RIS Format
Other project views: | All 56 publications | 12 publications in selected types | All 8 journal articles |
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Type | Citation | ||
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Levin M, Morsey B, Mori C, DeGuise S. Specific non-coplanar PCB-mediated modulation of bottlenose dolphin and beluga whale phagocytosis upon in vitro exposure. Journal of Toxicology and Environmental Health-Part A-Current Issues 2004;67(19):1517-1535. |
R829361 (2004) R829361 (Final) |
not available |
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Levin M, DeGuise S, Ross PS. Association between lymphocyte proliferation and polychlorinated biphenyls in free-ranging harbor seal (Phoca vitulina) pups from British Columbia, Canada. Environmental Toxicology and Chemistry 2005;24(5):1247-1252. |
R829361 (2004) |
not available |
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Levin M, Morsey B, De Guise S. Calcium modulation as a possible mechanism for PCB-induced modulation of phagocytosis in marine mammals. Toxicology (submitted, 2005). |
R829361 (2004) |
not available |
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Levin M, Morsey B, Mori C, Nambiar PR, DeGuise S. Non-coplanar PCB-mediated modulation of human leukocyte phagocytosis: A new mechanism for immunotoxicity. Journal of Toxicology and Environmental Health-Part A-Current Issues 2005;68(22):1977-1993. |
R829361 (2004) R829361 (Final) |
not available |
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Levin M, Morsey B, Mori C, Nambiar PR, De Guise S. PCBs and TCDD, alone and in mixtures, modulate marine mammal but not B6C3F1 mouse leukocyte phagocytosis. Journal of Toxicology and Environmental Health-Part A 2005;68(8):635-656. |
R829361 (2004) R829361 (Final) |
not available |
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Mori C, Morsey B, Levin M, Nambiar PR, De Guise S. Immunomodulatory effects of in vitro exposure to organochlorines on T-cell proliferation in marine mammals and mice. Journal of Toxicology and Environmental Health-Part A 2006;69(4):283-302. |
R829361 (2004) R829361 (Final) |
not available |
Supplemental Keywords:
PCBs, dioxin, immunotoxicity, marine mammals, human, health risk assessment, chemical mixtures, animal bioassays, biodegradation, chemical exposure, immunotoxicology, organochloride mixtures, environmental chemicals,, RFA, Scientific Discipline, Health, Waste, Environmental Chemistry, Health Risk Assessment, Chemistry, chemical mixtures, Risk Assessments, Hazardous Waste, Ecology and Ecosystems, Hazardous, complex mixtures, organochlorine mixtures, chemical exposure, risk assessment, fate and transport , biodegradation, in vitro immunotoxicology, PCBs, exposure, environmental transport and fate, PCB, human exposure, hazardous chemicals, marine mammals, immunotoxicology, animal bioassays, contaminated soils, environmental chemicals, exposure assessmentProgress 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.