An in vitro and in vivo evaluation of the immuno and neurotoxicological effects of fusaric acid on altered protein kinase signalling cascades.
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Mycotoxins are naturally occurring toxins produced by moulds which contaminate numerous crops and foodstuffs including cereals, nuts and fruits. When consumed, mycotoxins pose a serious threat to both human and animal well-being, causing acute poisoning or chronic effects such as immune deficiency and cancer. Fusaric acid (FA), a ubiquitous mycotoxin produced by Fusarium species, is a frequent contaminate of staple grain-based foods, and is a specific inhibitor of dopamine-β-hydroxylase in human and animal hosts, thereby affecting the autonomic nervous system by causing significant alterations in catecholamine metabolism. The latter effect is believed to occur through the competitive binding of FA with tryptophan to albumin in circulation. Although several studies have reported the oral activity of FA in circulation and the nervous system, the immuno- and neurotoxic effects of FA are unknown. Therefore, in this study we aimed to investigate the immunotoxic and neurotoxic potential of FA, using in vitro human and in vivo murine models. On a daily basis, protein kinases are required to integrate developmental cues and environmental stimuli to decide cell fate (cell death or survival). Thus, alterations to mitogen-activated protein kinases (MAPKs) in the immunotoxicity of FA was assessed in vitro on healthy human peripheral blood mononuclear cells (PBMCs) and on the human acute monocytic leukemic (Thp-1) cell line at an acute exposure (1 day) (Chapter 2); with Thp-1 cells (IC50-107.7 μg/ml) showing a greater susceptibility to FA exposure than PBMCs (IC50-240.8 μg/ml). Notably, elevated stress-induced stimuli (increased oxidative stress and ATP depletion) activated the pro-apoptotic signalling of phosphorylatedextracellular signal-regulated kinase (p-ERK), resulting in initiation of intrinsic apoptosis evidenced by the decreased phosphorylation of B-cell lymphoma 2 (p-Bcl-2; an anti-apoptotic protein) and subsequent activation of caspase-9 and caspase-3/7 activities in Thp-1 cells. In contrast, a caspaseindependent (reduced caspase -8, -9 and -3/7 activities) form of cell death (paraptosis) was induced in PBMCs that was possibly mediated by ERK and c-Jun N-terminal kinase (JNK) in response to metabolic stress (decreased cellular ATP availability). The significant ATP depletion in immune cells then led to the assessment of the metabolic effects of FA in the brain; since the brain is exposed to the peripheral effects of FA and is a highly metabolic organ. Therefore, the next chapter (Chapter 3) investigated the neurometabolic effects of FA via the protein kinase B (Akt) and AMP-activated protein kinase (AMPK) signalling pathways (which are central regulators of cellular energy and metabolism) in C57BL/6 mice at acute (1 day) and prolonged exposure (10 days). Acute exposure to FA, augmented Akt signalling following the increased expressions of upstream regulators phosphatidylinositol 3-kinase (PI3K), mammalian target of rapamycin (mTOR) and p70 ribosomal S6 protein kinase (p70S6K). Activated Akt inhibited glycogen synthase kinase 3 (GSK3) activity with the simultaneous activation of AMPK, p53 phosphorylation and reduced glucose transporter (GLUT)-1 and -4 expression, potentially suppressing neuronal glucose entry. However, following prolonged exposure, FA dampened PI3K/Akt and AMPK signalling, but increased the expression of GLUT transporters (1 and 4) in mice brain. Despite the differential regulation of glucose receptors by the PI3K/Akt and AMPK pathways (at acute and prolonged exposures), neuronal ATP failed to rise despite the increased pyruvate dehydrogenase E1ß (PDHE1β) activity [a regulatory subunit of glycolysis and the tricarboxylic acid cycle (TCA) cycle] at both 1 and 10 days; suggesting that FA mediates ATP depletion independent of metabolic signalling. Given the evident neurometabolic disturbances mediated by FA and its importance as a risk factor for neurometabolic-related diseases, the next chapter (Chapter 4) investigated the neurotoxic potential of FA in C57/BL6 mice following acute (1 day) and prolonged exposures (10 days) and its influence on cyclic AMP (cAMP) response element binding (CREB) signalling, an essential transcription factor, commonly activated by MAPKs, that is responsible for the brain’s neuroprotective responses through the regulation of neurotrophic and metabolic signals in the brain. After an acute administration of FA, CREB signalling was enhanced with a simultaneous increase in brain derived neurotrophic factor (BDNF) expression; whilst FA suppressed CREB/BDNF signalling following a prolonged exposure. In contrast, protein expressions of MAPKs (ERK, JNK and p38) negatively correlated with CREB activity; inferring that FA induced MAPK-independent activation of CREB responses. Consistent with caspase activation in Thp-1 cells, FA increased caspase activities (8, -9 and -3/7) at 1- and 10 days postexposure, although to a lesser extent at a prolonged treatment. However, despite enhanced caspase activity, microanalysis of brain tissue showed no prominent histological markers of damage to extracellular tissue or neuronal cells. Although FA showed no significant neurotoxicity, alterations in glial cell density patterns at both acute and prolonged exposures were observed. Besides the neuroprotective roles of CREB/BDNF signalling in the brain, CREB and BDNF are also involved in memory development and psychiatric disorders. Therefore, although FA may have not been neurotoxic, dysregulation of CREB/BDNF signalling impacts normal brain functions which potentially plays a role in the development of neurological disorders with longer periods of exposure.Collectively, although FA demonstrated significant toxicity towards Thp-1 cells, FA did not display cytotoxicity to healthy immune and neuronal cells, suggesting that compromised cellular systems may be more vulnerable to the effects of FA. In addition, while FA did alter MAPK, PI3K/Akt, AMPK and CREB pathways, which are important regulators of cell survival/death and energy homeostasis, these pathways are also involved in several other fundamental cellular processes, including gene expression, cell differentiation, inflammation, and synaptic plasticity; thus, modifications to their activities could have severe outcomes in health and disease.