Foodborne Cereulide Causes Beta-Cell Dysfunction and Apoptosis
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Foodborne Cereulide Causes Beta-Cell Dysfunction and Apoptosis Roman Vangoitsenhoven1, Dieter Rondas1, Inne Cre`vecoeur1, Wannes D’Hertog1, Pieter Baatsen2, Matilde Masini3, Mirjana Andjelkovic4, Joris Van Loco4, Christophe Matthys1, Chantal Mathieu1, Lut Overbergh1, Bart Van der Schueren1* 1 Laboratory for Clinical and Experimental Medicine and Endocrinology, KU Leuven, Leuven, Belgium, 2 EM Facility, VIB Bio Imaging Core and VIB department for the Biology of Disease, KU Leuven, Leuven, Belgium, 3 Department of Translational Research and of New Surgical and Medical Technologies, University of Pisa, Pisa, Italy, 4 Food, Medicines, and Consumer Safety, Scientific Institute of Public Health, Brussels, Belgium Abstract Aims/Hypothesis: To study the effects of cereulide, a food toxin often found at low concentrations in take-away meals, on beta-cell survival and function. Methods: Cell death was quantified by Hoechst/Propidium Iodide in mouse (MIN6) and rat (INS-1E) beta-cell lines, whole mouse islets and control cell lines (HepG2 and COS-1). Beta-cell function was studied by glucose-stimulated insulin secretion (GSIS). Mechanisms of toxicity were evaluated in MIN6 cells by mRNA profiling, electron microscopy and mitochondrial function tests. Results: 24 h exposure to 5 ng/ml cereulide rendered almost all MIN6, INS-1E and pancreatic islets apoptotic, whereas cell death did not increase in the control cell lines. In MIN6 cells and murine islets, GSIS capacity was lost following 24 h exposure to 0.5 ng/ml cereulide (P,0.05). Cereulide exposure induced markers of mitochondrial stress including Puma (p53 up-regulated modulator of apoptosis, P,0.05) and general pro-apoptotic signals as Chop (CCAAT/-enhancer-binding protein homologous protein). Mitochondria appeared swollen upon transmission electron microscopy, basal respiration rate was reduced by 52% (P,0.05) and reactive oxygen species increased by more than twofold (P,0.05) following 24 h exposure to 0.25 and 0.50 ng/ml cereulide, respectively. Conclusions/Interpretation: Cereulide causes apoptotic beta-cell death at low concentrations and impairs beta-cell function at even lower concentrations, with mitochondrial dysfunction underlying these defects. Thus, exposure to cereulide even at concentrations too low to cause systemic effects appears deleterious to the beta-cell. Citation: Vangoitsenhoven R, Rondas D, Cre`vecoeur I, D’Hertog W, Baatsen P, et al. (2014) Foodborne Cereulide Causes Beta-Cell Dysfunction and Apoptosis. PLoS ONE 9(8): e104866. doi:10.1371/journal.pone.0104866 Editor: Jianhua Zhang, University of Alabama at Birmingham, United States of America Received April 7, 2014; Accepted July 14, 2014; Published August 13, 2014 Copyright: ß 2014 Vangoitsenhoven et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by grants from the Flemish Research Foundation (G.0857.13, www.fwo.be) and the University of Leuven (GOA 13/006 and IOF KP/12012, www.kuleuven.be/research/funding). C. Mathieu has a clinical research fellowship and W. D’Hertog a postdoctoral fellowship from the Flemish Research Foundation (FWO). I. Cre`vecoeur has a grant from the Agency for Innovation of Science and Technology (IWT, 121086, www.IWT.be). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction ionophore, and is structurally very similar to the well-known valinomycin. Cereulide has been shown to uncouple oxidative The prevalence of type 1 and type 2 diabetes is rising and this phosphorylation by permeabilizing the mitochondrial membrane increase is believed to be related to environmental factors. Next to [3]. As a result, cereulide inhibits cell proliferation, RNA synthesis sedentary lifestyle and western style diet, it is known that certain and motility in different cell types [4–6]. As mitochondrial ATP environmental polluents, such as polychlorinated biphenyls, production is a key element for glucose-stimulated insulin xenoestrogens or cadmium can cause beta-cell dysfunction and production, cereulide’s mitochondriotoxic properties might be ultimately cell death, both key traits of the pathophysiology of specifically harmful for beta-cell function and survival. Exposure of diabetes [1,2] . porcine pancreatic islets to 1 ng/ml of cereulide, decreased insulin Thus, foodborne toxins, ubiquitous in this era of prepackaged content and increased necrotic cell death within 2 days [7]. and take-away meals, might contribute to the current rise in Cereulide is highly resistant to heat, acidity and proteolysis, diabetes prevalence. Here, we propose cereulide, a toxin produced which renders it difficult to eradicate from the food chain and by certain strains of Bacillus cereus, as a putative culprit. It is a prevents its neutralization in the digestive tract. Moreover, the cyclic dodecadepsipeptide (1.2 kDa), that acts as a potassium highly lipophilic character of cereulide (log Kow 5.96) suggests PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e104866 Cereulide Causes Beta-Cell Dysfunction and Apoptosis possible bioaccumulation in human tissues which could further Pancreatic islet isolation increase the potential health hazard [8,9]. Whole mouse pancreatic islets were isolated from 2–3 weeks old Cereulide is well known to cause acute and possibly fatal emetic C57Bl/6J mice (Charles River Laboratories, Brussels, Belgium) as food poisoning, with an estimated lethal dose of 8 mg/kg body described before [23]. Islets were handpicked under a stereomi- weight [10]. Most of the cases of acute toxicity involved the liver croscope, and cultured in RPMI 1640 supplemented with 10% (fulminant hepatic failure) and cereulide has been detected in the heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 mg/ gut, peritoneum and spleen of intoxicated patients [11]. Shiota et ml streptomycin, 2 mM GlutaMAX (all from Invitrogen-Gibco). al. have been able to detect 4 ng/mL cereulide in plasma of 2 Principles of laboratory animal care (NIH publication no. 85–23, children presenting with emetic food poisoning [12], which revised 1985) were followed and all animal experimental supports the hypothesis that cereulide enters both the portal and procedures were approved by the Ethics Committee of the systemic circulation. University of Leuven (permit number P056/2013). Animals were Chronic exposure to low concentrations of cereulide might have sacrificed by carbon dioxide gas, and all efforts were made to a more devious effect. The exact prevalence of cereulide toxin in minimize suffering. food remains uncertain, mainly because a full diagnostic workup is seldom performed in self-limiting gastro-intestinal syndromes, and Cell death assay a simple and cheap diagnostic test is not available. Almost all of The percentage of living, apoptotic, and necrotic cells was the available data are derived from dramatic - and sometimes fatal assessed by microscopic cell counting. MIN6, INS-1E, HepG2 and – cases of acute food poisoning. The prevalence of the Bacillus COS-1 cells were cultured in 96-well plates. After 24 and 72 h of cereus infections (all strains combined) is rising throughout Europe exposure to cereulide concentrations ranging from 0.05 ng/ml to [13]. Agata et al. report concentrations of 0.01 to 1.28 mg/g in 13 5 ng/ml, cells were incubated for 15 min with propidium iodide out of 14 food samples, and others have measured concentrations (PI, 20 mg/mL, Invitrogen) and Hoechst HO342 (4 mg/mL, up to 13.2 ng/g food [14,15]. Very recently, Messelha¨usser and Invitrogen) at 37uC. Whole mouse islet were exposed to cereulide colleagues, investigated 4300 food samples linked to foodborne concentrations ranging from 0.05 ng/ml to 5 ng/ml, for 24 hours. illness over a 7 year period in Bavaria, and confirmed that At least 500 cells, or 20 cultured islets, were evaluated in each cereulide is not only present in cooked rice dishes and pasta, but experimental condition by two researchers, one of them unaware also in infant meals, cauliflower and milk [16]. Our group has of the sample identity, on an inverted fluorescent microscope shown that 7.4% of rice dishes randomly collected from Asian (Nikon, Brussels, Belgium). restaurants in Belgium, contained low concentrations of cereulide (4 ng/g food) upon sampling and this prevalence increases to Caspase 3/7 activity 12.9% when food is exposed to temperature abuse conditions MIN6 cells were plated in a 96-well plate and exposed for 24 h (25uC) [17]. Thus, although large scale prevalence data for the cereulide toxin itself are lacking, repeated exposures to nanogram to cereulide. Luminescence was measured on a Victor Wallac levels of cereulide through food are likely. Up until now, little is workstation (Perkin Elmer, Wallac, Finland), using the Caspase known about the effects of exposure of beta-cells to low Glo 3/7 assay (Promega, Madison, WI), according to the concentrations of the toxin. In this study, we set out to investigate manufacturer’s instructions.