Characterization of estrogen and androgen activity of food contact materials by different in vitro bioassays (YES, YAS, ERα and AR CALUX) and chromatographic analysis (GC-MS, HPLC-MS).

Onno C Meijer,Johannes Mertl,Michael Pyerin,Johannes Bergmair,Angelika Grininger,Michael Washüttl,Veronica Osorio,Manfred Tacker,Christian Kirchnawy,Alexander Richter

doi:10.1371/journal.pone.0100952

Onno C Meijer, Johannes Mertl + Show 8 more

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https://doi.org/10.1371/journal.pone.0100952

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Abstract

Endocrine active substances (EAS) show structural similarities to natural hormones and are suspected to affect the human endocrine system by inducing hormone dependent effects. Recent studies with in vitro tests suggest that EAS can leach from packaging into food and may therefore pose a risk to human health. Sample migrates from food contact materials were tested for estrogen and androgen agonists and antagonists with different commonly used in vitro tests. Additionally, chemical trace analysis by GC-MS and HPLC-MS was used to identify potential hormone active substances in sample migrates. A GC-MS method to screen migrates for 29 known or potential endocrine active substances was established and validated. Samples were migrated according to EC 10/2011, concentrated by solid phase extraction and tested with estrogen and androgen responsive reporter gene assays based on yeast cells (YES and YAS) or human osteoblast cells (ERα and AR CALUX). A high level of agreement between the different bioassays could be observed by screening for estrogen agonists. Four out of 18 samples tested showed an estrogen activity in a similar range in both, YES and ERα CALUX. Two more samples tested positive in ERα CALUX due to the lower limits of detection in this assay. Androgen agonists could not be detected in any of the tested samples, neither with YAS nor with AR CALUX. When testing for antagonists, significant differences between yeast and human cell-based bioassays were noticed. Using YES and YAS many samples showed a strong antagonistic activity which was not observed using human cell-based CALUX assays. By GC-MS, some known or supposed EAS were identified in sample migrates that showed a biological activity in the in vitro tests. However, no firm conclusions about the sources of the observed hormone activity could be obtained from the chemical results.

Highlights

Food contact materials have been identified as a possible source of endocrine active substances (EAS) that may interfere with the human endocrine system [1]
AR CALUX reproducibility The relative standard deviation was 13% for a solvent blank spiked with 100 pmol/l DHT and 14% for a solvent blank spiked with 400 pmol/l DHT
An analysis of the same samples with the human cellbased ERa CALUX assay did not show any antiestrogenic effects, not even when the concentration of the dimethyl sulfoxide (DMSO) extract in the ERa CALUX was increased to 1% to ensure direct comparability with the yeast estrogen screen. These results clearly demonstrate that the observed suppression of hormone activity is specific to the yeast cell-based YES

Summary

Introduction

Food contact materials have been identified as a possible source of endocrine active substances (EAS) that may interfere with the human endocrine system [1]. EAS can act as endocrine disrupting chemicals (EDCs), that may alter the activity of natural steroid hormones by modifying their regulatory pathways, interacting with steroid receptors and antagonizing endogenous hormones, or mimicking steroid hormone dependent effects [2] Physiological processes such as reproduction, cell growth and regulation of glucose levels are generally regulated by steroid hormones and are vulnerable to EDC effects [3]. Many in vivo studies with mammals have linked an increased EDC intake to a variety of reproductive and physiological abnormalities such as reduced sperm quality in males, feminization of laboratory animals, obesity, or the development of breast, ovarian and prostate cancers [4,5,6,7] These studies have increased awareness and concern about possible health impairment in humans due to EDC-exposure, since humans have an endocrine system similar to other mammals [8]. Most of the in vitro assays are based on the activation of steroid hormone dependent receptors by EAS, followed by the activation and transcription of a reporter gene (e.g. firefly luciferase) [3,13]

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