Cyclic depsipeptide mycotoxin exposure may cause human endocrine disruption: Evidence from OECD in vitro stably transfected transcriptional activation assays

Highlights

• The potential endocrine disrupting effects of ENN A1, and ENN B1 are assessed.

• ER and AR agonistic/antagonistic effects are detected by OECD TGs.

• ENN A1, and ENN B1 were determined to be ERα/AR antagonist.

• ENN A1, and ENN B1 bound to bound to ERα, and AR.

• Then, ENN A1, and ENN B1 inhibited the dimerization of ERα, and AR.

Abstract

The presence of cyclic depsipeptide mycotoxins in foods and feedstuffs could potentially cause endocrine disrupting effects on humans and wildlife by their inhibition of active steroidogenesis. Therefore, we attempted to assess the human estrogen receptor (ER) and androgen receptor (AR) agonistic/antagonistic effects of representative cyclic depsipeptide mycotoxins, enniatin A1 (ENN A1), and enniatin B1 (ENN B1), by OECD Performand Based Test Guideline (PBTG) No.455, VM7Luc ER transcriptional activation (TA) assay and OECD TG No. 458, 22Rv1/MMTV_GR-KO AR TA assay. No tested cyclic depsipeptide mycotoxins were found to be ER and AR agonists in VM7Luc ER TA and 22Rv1/MMTV_GR-KO AR TA assays. On the other hand, ENN A1, and ENN B1 exhibited the ER and AR antagonistic effects with IC₃₀ and IC₅₀ values in both TA assays. These two cyclic depsipeptide mycotoxins, which were determined as ER and AR antagonists by two in vitro assays, bound to ERα, and AR. Then ENN A1, and ENN B1 inhibited the dimerization of ERα, and AR. These results, for the first time indicated that ENN A1, and ENN B1 could have potential endocrine disrupting effects mediated by interaction of ERα and AR using international standard testing methods to determine the potential endocrine disrupting chemical.

Introduction

Various fungi, including Penicillium, Fusarium, Aspergillus and Alternaria are capable of producing mycotoxins, which are chemical hazards of increasing concern in food products due to a wide range of food types where they can be found [[1], [2], [3], [4]]. Mycotoxin contamination of foods and feeds may be a serious concern for both human and animal health because of their adverse effects [5,6]. Fusarium strains produce secondary metabolites, including trichothecenes, zearalenone, fusaproliferin, moniliformin, and fumonisins, many of which have exhibited adverse health effects, including neurological and reproductive disorders [[7], [8], [9]]. Enniatins (ENNs), which are regarded as emerging groups of Fusarium mycotoxin, are produced by various Fusarium strains via nonribosomal peptide synthetase systems [10]. These emerging mycotoxins have cyclic depsipeptide structures that consist of alternating residues of d-2-hydroxyisovaleric acid and branched-chain N-methyl-l-amino acids linked by peptide and ester bonds; the ionophoric properties that arise from their molecular structure are related to their biological activities [11]. ENNs were the most prevalent contaminants in various geographical regions and were commonly found to contaminate cereals and other feedstuffs [8,[12], [13], [14], [15]]. ENN B induces apoptotic cell death by mitochondrial modifications and cell cycle disruption [[16], [17], [18], [19]]. The European Food Safety Authority (EFSA) reported that the mean chronic exposure to the sum of ENN A, A1, B, and B1 ranged from 0.42 to 1.82 μg/kg b.w. per day and the 95th percentile exposure ranged from 0.91 to 3.28 μg/kg b.w. per day [20]. However, the Panel on Contaminants in the Food Chain (CONTAM) concluded that there was insufficient data to establish the health-based guidance values like a TDI or/and an acute reference dose (ARfD) for ENNs [20]. Although the intrinsic toxicity of cyclic depsipeptide mycotoxins seem to be lower than those of other mycotoxins from Fusarium strains, synergistic effects with other mycotoxins may play a role in the increased adverse effect to humans and animals [8,20,21].

Recently, it has been published that many fungal metabolites, such as ochratoxin A, patulin, alternariol, and zearalenone, present in food and feedstuffs may act as potential endocrine disruptors [[22], [23], [24], [25], [26]]. Cyclic depsipeptide mycotoxins have been found to inhibit a regulator of cholesteryl ester formation in the adrenal gland, acyl-CoA: cholesterol acyltransferase (ACAT) [27,28]. This inhibition of ACAT activity by ENNs exposure indicates that they could be potential endocrine disruptors because steroidogenic factor-1 dependent up-regulation of ACAT is important for maintaining readily available cholesterol esters, needed at times of active steroidogenesis [29]. Based on this hypothesis, Kalayou et al. (2015) and Fernández-Blanco et al. (2016) confirmed the endocrine-disrupting effects of beauvericin (BEA) and ENN B by in vitro bioassay [29,30]. However, the potential endocrine disrupting effects of ENNs have not been assessed by international standardized test guidelines, which have been established by the OECD for detection of endocrine-disrupting chemicals. The OECD suggested the five levels conceptual framework to assess the endocrine disrupting effects of chemicals, and second level in a conceptual framework outlined the in vitro testing methods to prove the endocrine processes affected induced by chemicals [31]. The OECD recommended that it is important to screen various chemicals for their potential endocrine-disrupting effects by in vitro assays before hazard assessment by in vivo studies for clarification of their safety [31].

In this study, we tried to assess the human estrogen receptor (ER) and human androgen receptor (AR) agonistic/antagonistic effects of ENN A1, and ENN B1 by OECD Performance Based Test Guideline (PBTG) No.455 [32], VM7Luc ER transcriptional activation (TA) assay, and TG No. 458, 22Rv1/MMTV_GR-KO AR TA assay [33].