Abstract-Relative potency (REP) estimates are widely used to characterize and compare the potency of a wide variety of samples analyzed using in vitro bioassays. Relative potency estimates are generally calculated as a simple ratio: the EC50 of a wellcharacterized standard divided by the EC50 of a sample. Such estimates are valid only when the dose-response curves for the sample and standard are parallel and exhibit the same maximum achievable response (efficacy). These conditions are often either violated or cannot be demonstrated. As a result, there is a need to calculate and present REPs in a manner that addresses the potential uncertainties caused by violation of the assumptions of parallelism and equal efficacy. Multiple point estimates, over the range of responses from EC20 to EC80, can be used to derive relative potency ranges (REP 20-80 range). The width of a REP range is directly proportional to the degree of deviation from parallelism between sample and standard dose-response curves. Thus, REP 20-80 ranges both test the assumption of parallelism and characterize the amount of uncertainty in an REP estimate resulting from deviation from parallelism. Although uncertainties due to unequal efficacy cannot be easily characterized mathematically, a systematic method for evaluating sample efficacy has been developed into a framework to guide the derivation and application of REP estimates based on in vitro bioassay results. Use of the systematic framework and REP 20-80 ranges was illustrated using three sample data sets. It is hoped that the framework and discussion presented will facilitate the use of bioassay-derived REP estimates to characterize samples of both known and unknown composition without ignoring the assumptions underlying REP estimation.
Polychlorinated naphthalenes (PCNs) are ubiquitous environmental pollutants that are structurally similar to other polychlorinated diaromatic hydrocarbons (PCDHs), such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs). Despite being ubiquitous, much less is known about the fate, transport, and biological effects of individual PCN congeners than other PCDHs. The purpose of the current study was to utilize an in vitro assay (H4IIE-luc) to determine potencies relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 20 individual PCNs (from 75 possible congeners) and 6 Halowax mixtures. H4IIE rat hepatoma cells (H4IIE-Luc), which are stably transfected with an Ah receptor (AhR)-controlled luciferase reporter gene construct, respond specifically to AhR agonists and are thus a reasonable measure of AhR-mediated, or dioxin-like, activity. The most potent congeners were 1,2,3,4,6,7-hexa-CN (PCN 66), 1,2,3,5,6,7-hexa-CN (PCN 67), and 1,2,3,4,5,6,7-hepta-CN (PCN 73), with relative potencies as compared to TCDD of 0.004, 0.001, and 0.001, respectively. Significant structure−activity relationships were observed. For example, lateral substitution is an important determinant of AhR-mediated activity, but not sufficient, as illustrated by the inactivity of 2,3,6,7-tetra-CN to elicit AhR-mediated activity. Relative potencies of the Halowax mixtures with AhR-mediated activity were 0.0089, 0.000038, and 0.0000018 for 1051, 1014, and 1013, respectively. The relative potencies derived from this study were applied to literature-derived data on concentrations of PCN congeners in environmental mixtures to assess the potential contribution of PCNs to total TCDD equivalents (TEQs) in environmentally weathered complex mixtures.
In vitro cell bioassays are useful techniques for the determination of receptor-mediated activities in environmental samples containing complex mixtures of contaminants. The cell bioassays determine contamination by pollutants that act through specific modes of action. This article presents strategies for the evaluation of aryl hydrocarbon receptor (AhR)-(hereafter referred as dioxin-like) or estrogen receptor (ER)-mediated activities of potential endocrine disrupting compounds (EDCs) in complex environmental mixtures. Extracts from various types of environmental or food matrices can be tested by this technique to evaluate their 2,3,7,8-tetrachlorodibenzop-dioxin equivalents (TCDD-EQs) or estrogenic equivalents (E(2)-EQs) and to identify contaminated samples that need further investigation using resource-intensive instrumental analyses. Fractionation of sample extracts exhibiting significant activities, and subsequent reanalysis with the bioassays can identify important classes of contaminants that are responsible for the observed activity. Effect-directed chemical analysis is performed only for the active fractions to determine the responsible compounds. Mass-balance estimates of all major compounds contributing to the observed effects can be calculated to determine if all of the activity has been identified, and to assess the potential for interactions such as synergism or antagonism among contaminants present in the complex mixtures. The bioassay approach is an efficient (fast and cost effective) screening system to identify the samples of interest and to provide basic information for further analysis and risk evaluation.
In 1990, a portion of the Kalamazoo River in Michigan, USA, was designated a Superfund site because of the presence of polychlorinated biphenyls (PCBs) in the sediment and floodplain soils. During a four-year period from 2000 to 2003, several avian species were monitored for reproductive effects and concentrations of PCBs in tissues attributed to food chain transfer from contaminated sediments. The tree swallow (Tachycineta bicolor) was chosen as a model receptor for contamination of passerine species. A top-down methodology was used to evaluate the bioaccumulation of PCBs, including non-ortho and mono-ortho congeners, in tree swallow eggs, nestlings, and adults at the Kalamazoo River area of concern (KRAOC) and at an upstream reference site. Generally, a sixfold difference in tissue concentrations of total PCBs was observed between the two sites with concentrations in eggs and nestlings at the KRAOC ranging from 0.95 to 15 microg PCB/g wet weight. Concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQsWHO Avian) for PCBs, based on bird-specific World Health Organization toxic equivalence factors, were 10- to 30-fold greater in the KRAOC than at the reference location. Egg and nestling TEQsWHO-Avian ranged from 0.21 to 2.4 ng TEQ/g wet weight at the KRAOC. Hazard quotients calculated from literature-derived toxicity reference values were below 1.0 at both the target and the reference site based on the no-observed-adverse-effect level and the lowest-observed-adverse-effect level.
Approximately 125 km of the Kalamazoo River, located in southwestern Michigan (USA), are designated as a Superfund site, with polychlorinated biphenyls (PCBs) as the contaminant of concern. Mink (Mustela vison) are a naturally occurring predator in this area and also a species of concern because of their known sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally similar compounds, such as PCBs. Four of nine mink trapped from the Kalamazoo River area of concern (KRAOC) exhibited histological evidence of a jaw lesion previously identified in ranch mink. The jaw lesion, hyperplasia of squamous epithelium in the mandible and maxilla, is known to be caused by 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and TCDD. Mink trapped from an upstream reference area (Fort Custer Recreation Area [FCRA]) did not exhibit the lesion. Mean concentrations of total PCBs were 2.8 and 2.3 mg/kg wet weight in the livers of mink from the KRAOC and FCRA, respectively, and TCDD toxic equivalent (TEQ) concentrations were 0.30 and 0.11 microg/kg wet weight, respectively. Significant correlations were found between the severity of the lesion and the hepatic concentrations of total PCBs and TEQs. To our knowledge, this is the first published report of the lesion occurring in wild mink.
A series of field studies was conducted to determine the bioaccumulation of polychlorinated biphenyl (PCB) congeners in the terrestrial food web of the Kalamazoo River flood plain. Samples included colocated soils, native plants likely to be consumed by wildlife, several taxa of terrestrial invertebrates, small mammals, passerine bird eggs, nestlings, and adults, and great horned owl plasma and eggs. Mean concentrations of total PCBs in samples from the former Trowbridge impoundment were 6.5 mg/kg dry weight for soils and 0.023, 0.13, 1.3, 1.3, 1.6, and 8.2 mg/kg wet weight for plants, small herbivorous mammals, depurated earthworms, shrews, great horned owl eggs, and house wren eggs, respectively. Historical data from the Kalamazoo River have reported Aroclor-equivalent total PCB concentrations in the terrestrial food web; however, the degree of environmental weathering of the parent PCB mixtures was unknown. In this study, earthworms and composite samples of coleoptera exhibited PCB congener patterns that were similar to patterns in colocated soils. However, in plants, less chlorinated PCBs (e.g., mono-, di-, tri-, and tetrachlorinated biphenyls) were predominant, and in small mammals, there was a notable enrichment of PCBs 153, 180, 138, 118, and 99. In general, concentrations of PCBs were lower in most biota than in soil from the Kalamazoo River Area of Concern (KRAOC) although there was a modest biomagnification of PCBs from lower trophic level biota to highertrophic levels. As a consequence of environmental weathering of PCBs in the terrestrial food web of the KRAOC, the relative potency of the PCBs (expressed as mg TEQs/kg PCBs) decreased from soil to most biota. While there was a general trend, as expected, in which concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs) increased with total PCBs, this relationship was rather poor (R2 = 0.13). Taken together, these data suggest that the differential accumulation of PCB congeners in the terrestrial food web can be explained by congener-specific differences in bioavailability from soil, exposure pathways, and metabolic potential of each of the food web components.
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