Full length articleThe fungal neurotoxin penitrem A induces the production of reactive oxygen species in human neutrophils at submicromolar concentrations
Graphical abstract
Introduction
Penitrem A (Fig. 1) is a toxic secondary metabolite mainly produced by the fungal species Penicillium crustosum (Moldes-Anaya et al., 2011). Intoxication with the mycotoxin penitrem A in animals has been reported recurrently in the literature for decades (Arp and Richard, 1979, Boysen et al., 2002, Cysewski et al., 1975, Eriksen et al., 2009, Hocking et al., 1988, Lowes et al., 1992, Naudé et al., 2002, Richard et al., 1981, Schell, 2000, Tiwary et al., 2009, Walter, 2002, Young et al., 2003). A few cases of neurological disease in humans are also presumptively caused by penitrem A (Cole et al., 1983, Gordon et al., 1993, Lewis et al., 2005). The incidence of penitrem A-induced neuromycotoxicosis may be under-diagnosed, especially in humans, because of the lack of knowledge about penitrem A's deleterious effects and the difficult access to appropriate analytical tools for detection and determination of the neurotoxin.
The main route of penitrem A exposure is through consumption of contaminated food and/or feed in humans and animals (Eriksen et al., 2009). After ingestion, the toxin is rapidly absorbed into the bloodstream and distributed throughout the body (Moldes-Anaya et al., 2009). In particular, penitrem A crosses the blood-brain-barrier, where it exerts its toxicological effects (Moldes-Anaya et al., 2009). In animals, clinical signs of intoxication range from tremors, seizures and hyperthermia to ataxia and nystagmus, all signs of motor system dysfunction (Eriksen et al., 2013). In humans, hyperthermia, nausea and emesis, diplopia, severe tremors and bloody diarrhoea (Eriksen et al., 2013) have been observed. Existing evidence shows that the CNS is the primary target for penitrem A (Moldes-Anaya et al., 2011, Norris et al., 1980, Stern, 1971), however, the peripheral nervous system (PNS) may also be affected (Knaus et al., 1994, McLeay et al., 1999, Wilson et al., 1972). Penitrem A is able to impair GABAergic amino acid neurotransmission and selectively antagonise high-conductance Ca+2-activated potassium (BK) channels. Dysfunctional neurotransmission and inhibition of neuronal BK channels are probably involved in the neurotoxic actions observed in both humans and animals (Moldes-Anaya et al., 2011, Eriksen et al., 2009). In a previous study, we have shown that penitrem A induces the production of reactive oxygen species (ROS) in primary cerebellar granule neurons from rats (Berntsen et al., 2013), opening for a new mechanism of action for the neurotoxin. Neuropathology has in general been limited to the cerebellum (Cavanagh et al., 1998), but massive pathological changes have also been reported in other organ systems of the thoracic and peritoneal cavities (Hayes et al., 1976). Hayes et al. (1976) also reported a significant increase in neutrophil levels in blood, while Cavanagh et al. (1998) reported a selective increase in brain cerebellar perfusion and neutrophil infiltration after penitrem A exposure.
Among circulating immune cells, neutrophils are one of the initial participants in the body’s defence against external pathogens, and are involved in acute phases of inflammation. Neutrophils phagocyte, kill and digest invading pathogens by using proteinases, cytotoxic proteins, chelators and free radicals (Babior, 2000). Once in the blood, unstimulated neutrophils are mainly in free circulation. When needed, they exit the bloodstream and migrate actively towards the sites of inflammation, infiltrating the affected organs. Simultaneously, large oxygen consumption denoted as the respiratory burst takes place (Babior, 2000). During the respiratory burst NADPH oxidase catalyses the one-electron reaction of O2 to O2●− (Babior, 2004). O2●− is highly reactive and unstable and will dismutate to H2O2, which may subsequently generate ROS such as OH−, hydroxyl radical (●OH), singlet oxygen (1O2) and hypochlorous acid (HOCl). ROS are by-products of normal cellular metabolism, and cells possess several mechanisms to avoid damage induced by ROS and other free radicals. When generation of ROS exceed basal levels and the mechanisms of defence against ROS-induced injury are overwhelmed, oxidative stress occurs (Kehrer, 1993). Oxidative stress can cause cellular damage and subsequent cell death because ROS oxidise critical cellular components such as lipids, proteins and DNA (Kehrer, 1993). The activation of neutrophils may also stimulate ROS overproduction and cause inflammation and tissue injury.
The mycotoxin penitrem A recurrently causes intoxication in animals, and clinical signs of intoxication and even hospitalisation have also been reported in humans. In a previous study, we reported that penitrem A induced the production of ROS in rat cerebellar granule cells. The purpose of the present study was to examine the capability of penitrem A to induce the production of ROS in human neutrophil granulocytes, as well as to examine possible mechanisms involved in this induction. Human neutrophil granulocytes represent an easily available test system, relevant for its role in the body’s defence mechanism against external pathogens.
Section snippets
Chemicals and reagents
α-tocopherol (vitamin E), 5-amino-2,3-dihydro-1,4-phtalazindione (luminol; ≥ 97%), cyclosporine A (CsA; ≥ 98.5%), dimethyl sulfoxide (DMSO; ≥ 99.9%), N-ω-nitro-l-arginine methyl ester (L-NAME), methanol (MeOH; ≥ 99.9%) and penitrem A ≥ 95%, were supplied by Sigma-Aldrich (St. Louis, MO, USA). 2′,7′- dichlorodihydrofluorescein diacetat (DCFH-DA), Hanks’ Balanced Salt Solution (HBSS; 10x) and HEPES buffer, came from GIBCO/Invitrogen (Oslo, Norway). 1,2-bis(o-aminophenoxy)-ethane-N,N,N’N’-tetraacetic acid
Studies of penitrem A-induced ROS production in human neutrophil granulocytes
Preliminary studies revealed that penitrem A (10 μM) significantly increased the production of ROS (314% of DMSO control) measured by the DCF assay, while no increase in ROS production was measured using the luminol assay (Fig. 2). The polychlorinated biphenyl PCB 153 was included as a positive control. Based on these results, further studies of penitrem A-induced ROS production, such as concentration-response studies and mechanistic studies, were performed using the DCF assay. The absence of
Discussion
To date, there is no report on whether the mycotoxin penitrem A is able to affect the oxidative function of neutrophil granulocytes. The present study aimed to advance in this area by studying the effects of penitrem A on the production of ROS by neutrophils. We have found that penitrem A is able to induce a concentration-dependent boost of ROS production even at submicromolar levels without causing significant damage to the cells. Further we have also unravelled some of the mechanism that may
Conclusion
Our study has shown that penitrem A significantly increased the production of ROS by neutrophil granulocytes at low concentrations (0.25 μM), without causing damage to the cells. A concentration-response curve yielded an EC50 value of 3.8 μM. The maximal increase in ROS production was approximately 330% at a concentration of 12.5 μM. The mechanism of ROS formation seems to involve an activation of several MAPK signalling pathways as well as an increase in intracellular calcium.
We suggest that the
Conflict of interest statement
The authors declare that they have no conflict of interest.
Acknowledgements
The Research Council of Norway (grant NFR 173498) and internal funding from the Norwegian Veterinary Institute and the University of Oslo supported this study. The authors wish to thank the volunteers providing blood for the study, the biomedical laboratory scientists Hildegunn Dahl and Stine Martinsen for taking the blood samples as well as Kamilla G. Haugland for helping in the preparation of the graphical abstract.
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