A rapid method of preparing complex organohalogen extracts from avian eggs: Applications to in vitro toxicogenomics screening

Abstract

Double‐crested cormorants are piscivorous birds that breed in variably contaminated colonies across the Laurentian Great Lakes of North America. Collection and preparation of environmentally relevant extracts from eggs that contain variable concentrations of organohalogen contaminants represents a minimally invasive approach to characterize potential effects of exposure using in vitro bioassays. In the present study, a rapid, efficient lipid freeze‐filtration extraction method was used to prepare extracts from double‐crested cormorant eggs collected from 5 breeding colonies that had variable organohalogen contaminant burdens. Extracts, solubilized in dimethyl sulfoxide, were administered to chicken embryonic hepatocytes (CEHs) to determine effects on cell viability, 7‐ethoxyresorufin‐O‐deethylase (EROD) activity, and messenger RNA expression using a chicken ToxChip polymerase chain reaction (PCR) array. The EROD median effect concentration (EC50) values were lower for extracts with greater organohalogen contaminant burdens and thus permitted an initial ranking of colonies based on the efficacy of eliciting an aryl hydrocarbon receptor–mediated response. The ToxChip PCR array data provided a more exhaustive, pathway‐based evaluation of extract effects; variability in the transcriptomic profiles was associated with organohalogen contaminant burdens. For example, extracts from Mud Island (Detroit River, MI, USA) had among the highest organohalogen contaminant burdens and elicited a greater biochemical (EROD EC50 = 0.005) and transcriptomic response (22/43 genes altered on the array) in CEHs compared with the least contaminated site, which was Mandarte Island (BC, Canada; EROD EC50 = 0.172; 8/43 genes altered). Avian eggs represent a useful biomonitoring tool for determining complex mixture effects, and the combination of a rapid extraction method, an in vitro bioassay, and targeted endpoint evaluation (biochemical and transcriptomic) shows great promise as an environmental effects monitoring approach. Environ Toxicol Chem 2019;38:811–819. © 2019 Crown in the right of Canada. Published by Wiley Periodicals Inc. on behalf of SETAC.

Ecotoxicology assessments in avian species using cell-based models: A review

“…Those extracts induced EROD activity in embryonic chicken cells (CEHs) in a concentration-dependent manner with the expected additional decrease in activity seen in previous studies. 18,31 This is due to the “competitive inhibition of the reaction by the inducer.” 32 The most contaminated sites showed EC 50 values lower than the previous study, ranging from 0.003 to 0.007.…”

“…Similarly, another combination of embryonic hepatocytes and ToxChip study tested a new method for organohalogen extractions from contaminated DCCO eggs collected from the Great Lakes of North America region. 32 Lipid freeze filtration combined with high pressure-gel permeation chromatography allowed a sample to be collected and subjected to solid-phase extraction then prepared for hepatocyte exposures. The highest concentration extract from all five sites eggs was collected from reduced cell viability (neat concentration), and eggs from three sites also demonstrated reduced viability at 0.3 dilution.…”

Extracts from Dated Lake Sediment Cores in the Athabasca Oil Sands Region Alter Ethoxyresorufin‐O‐deethylase Activity and Gene Expression in Avian Hepatocytes

“…Activity of EROD is a well‐established biomarker of aryl hydrocarbon receptor (AhR) pathway perturbation following exposure to planar halogenated chemicals, PACs, and other complex environmental mixtures in fish, amphibians, and avian species (Smits et al 2000; Whyte et al 2000; Gauthier et al 2004; Colavecchia et al 2007; Crump et al 2015). In particular, measuring EROD activity in CEHs has been an effective screening approach for a wide range of complex mixtures to date (see Crump et al 2015, 2017, 2019; Mundy et al 2019). Hersikorn and Smits (2011) reported higher EROD activity in wood frog tadpoles raised within young (<7 yr old) wetlands formed from reclaimed oil sands process–affected materials.…”

“…A concordant decrease in expression was observed in CEHs exposed to delayed petcoke, but not fluid petcoke (Crump et al 2017), with the former having greater PAC concentrations, much like the impacted versus reference lake cores in the present study. In addition, CYP7B1 was down‐regulated in CEHs exposed to herring gull and double crested cormorant egg extracts, but only for the sites with the highest organohalogen contaminant egg burdens (Crump et al 2015, 2019).…”

“…2004; Colavecchia et al 2007;Crump et al 2015). In particular, measuring EROD activity in CEHs has been an effective screening approach for a wide range of complex mixtures to date (see Crump et al 2015Crump et al , 2017Crump et al , 2019Mundy et al 2019). Hersikorn and Smits (2011) reported higher EROD activity in wood frog tadpoles raised within young (<7 yr old) wetlands formed from reclaimed oil sands process-affected materials.…”