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

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.

A rapid analytical method to quantify complex organohalogen contaminant mixtures in large samples of high lipid mammalian tissues.

In vitro investigations of the health impact of individual chemical compounds have traditionally been used in risk assessments. However, humans and wildlife are exposed to a plethora of potentially harmful chemicals, including organohalogen contaminants (OHCs). An alternative exposure approach to individual or simple mixtures of synthetic OHCs is to isolate the complex mixture present in free-ranging wildlife, often non-destructively sampled from lipid rich adipose. High concentration stock volumes required for in vitro investigations do, however, pose a great analytical challenge to extract sufficient amounts of complex OHC cocktails. Here we describe a novel method to easily, rapidly and efficiently extract an environmentally accumulated and therefore relevant contaminant cocktail from large (10-50 g) marine mammal blubber samples. We demonstrate that lipid freeze-filtration with acetonitrile removes up to 97% of blubber lipids, with minimal effect on the efficiency of OHC recovery. Sample extracts after freeze-filtration were further processed to remove residual trace lipids via high-pressure gel permeation chromatography and solid phase extraction. Average recoveries of OHCs from triplicate analysis of killer whale (Orcinus orca), polar bear (Ursus maritimus) and pilot whale (Globicephala spp.) blubber standard reference material (NIST SRM-1945) ranged from 68 to 80%, 54-92% and 58-145%, respectively, for 13C-enriched internal standards of six polychlorinated biphenyl congeners, 16 organochlorine pesticides and four brominated flame retardants. This approach to rapidly generate OHC mixtures shows great potential for experimental exposures using complex contaminant mixtures, research or monitoring driven contaminant quantification in biological samples, as well as the untargeted identification of emerging contaminants.

Biochemical and Transcriptomic Effects of Herring Gull Egg Extracts from Variably Contaminated Colonies of the Laurentian Great Lakes in Chicken Hepatocytes.

Determining the effects of complex mixtures of environmental contaminants poses many challenges within the field of ecotoxicology. In this study, graded concentrations of herring gull egg extracts, collected from five Great Lakes breeding colonies with variable burdens of organohalogen contaminants (OHCs), were administered to chicken embryonic hepatocytes to determine effects on 7-ethoxyresorufin-O-deethylase (EROD) activity, porphyrin accumulation, and mRNA expression. EROD activity and porphyrin accumulation permitted the ranking of colonies based on the efficacy of eliciting an aryl hydrocarbon receptor-mediated response. An avian ToxChip polymerase chain reaction (PCR) array provided more exhaustive coverage in terms of potential toxicity pathways being affected, including xenobiotic and lipid metabolism and the thyroid hormone pathway. Herring gull eggs from Channel Shelter Island (CHSH, Lake Huron) and Gull Island (GULL, Lake Michigan) had among the highest OHC burdens, and extracts elicited a biochemical and transcriptomic response greater than that of extracts from the other three, less polluted colonies. For example, EROD EC50 values and porphyrin ECthreshold values were lower for CHSH and GULL extracts than for the other colonies. Extracts from CHSH and GULL altered 15 and 13 of 27 genes on the PCR array compared to no more than eight genes for the less contaminated sites. The combination of a well-established avian in vitro assay, two well-characterized biochemical assays, and the avian ToxChip PCR array permitted the geographical discrimination of variably contaminated herring gull eggs from the Great Lakes. Such high-throughput assays show potential promise as cost-effective tools for determining toxic potencies of complex mixtures in the environment.