Testing Endocrine Disruption in Biota Samples: A Method to Remove Interfering Lipids and Natural Hormones

Abstract

A cleanup method was developed to remove coextracted lipids and natural hormones from biota samples in order to test the endocrine-disrupting (ED) capacity of their extracts in in vitro bioassays. Unspiked and spiked fish tissues were cleaned with a combination of dialysis, gel permeation chromatography (GPC), and normal-phase liquid chromatography (NP-HPLC). The spiking mixture consisted of a broad range of environmental pollutants (endocrine disruptors and genotoxic compounds). Chemical recoveries of each test compound, and thyroid-hormone-like and (anti)androgenic activities of the cleaned extracts were investigated. Despite the chemical and toxicological complexity of the spiking mixture and the sequential sample treatment, chemical analysis revealed acceptable recoveries on average: 89 ± 8% after each cleanup step separately and 75 ± 3% after the whole extraction and cleanup procedure in the extracts. In addition, recovered activities in the bioassays were in good agreement with the spiking levels. The developed cleanup method proved to be capable of lipid and natural hormone removal from fish extracts, enabling the measurement of selected endocrine-hormone-like activities in T(4)*-TTR and AR-CALUX bioassays. The method can be used as a sample preparation method of biota samples for toxicity profiling and effect-directed analysis (EDA).

Novel cell-based assay for detection of thyroid receptor beta-interacting environmental contaminants

“…Several approaches and cell lines for detection of EDCs, including tests for thyroid hormone disruption (Fini et al 2007; Grimaldi et al 2015; Scholz et al 2013; Simon et al 2010; Steinberg 2013) have been proposed. However, alternative methods for fast and low-cost high-throughput detection of TR-interacting EDCs in the environment are still in a great demand.…”

“…It could be followed by chemical analyses and transcriptional studies using endogenous genes or luciferase based reporter constructs. TR beta CALUX reporter gene assay, for example, is a high-throughput screening method based on TRβ-mediated transcriptional response (Simon et al 2010; Steinberg 2013). Both, the TRβ CALUX and the translocation assay show activity in the picomolar and nanomolar range upon stimulation with T3.…”

Blood Plasma Sample Preparation Method for the Assessment of Thyroid Hormone-Disrupting Potency in Effect-Directed Analysis

“…Baker) at a flow-rate of 5 mL/min, as described before. 17 The 0À40 min (F 1 ) and 41À49 min (F 2 ) NP-HPLC fractions were collected, resulting in the fractions E 1 F 1 , E 1 F 2 , E 2 F 1 , and E 2 F 2 (Figure 1). In these fractions recoveries (chemical analysis) and competitive TTRbinding activities (T 4 *-TTR assay) were determined.…”

“…The presence of endogenous hormones in biota extracts may interfere with the measurement of endocrine disrupting compounds in in vitro bioassays. 17,19 Therefore, NP-HPLC fractionation was initially used to separate endogenous hormones from POPs in biota extracts as was previously done in studies on estrogens in blood matrices 20 and androgens and THs in fish tissues. 17 The NP-HPLC elution profile of the spiking compounds (data not shown) revealed that OH-PCBs eluted in the same subfraction (E 1 F 2 ) as the endogenous THs.…”

Coupling passive sampling with in vitro bioassays and chemical analysis to understand combined effects of bioaccumulative chemicals in blood of marine turtles

“…Facilitated transport of hydrophobic chemicals by biological matrices (e.g., proteins) into passive sampling devices has been observed in many studies (Oomen et al, 2000;Heringa et al, 2006;Kramer et al, 2007). The advantage of PDMS over solvents to extract chemicals is that charged molecules (e.g., proteins) do not diffuse into PDMS and only a small proportion of lipid does, which minimizes co-extraction of complex matrices, thus significantly reducing solvent use and cleanup efforts compared to conventional solvent extraction methods (Simon et al, 2010(Simon et al, , 2011Suzuki et al, 2011). The PDMS-based approach may also offer chemical extraction from biological samples to be performed immediately in situ or in vivo, thus circumventing the logistical need to transport and store samples (Allan et al, 2013).…”