Fumonisin B2 Induces Mitochondrial Stress and Mitophagy in Human Embryonic Kidney (Hek293) Cells-A Preliminary Study.

Ubiquitous soil fungi parasitise agricultural commodities and produce mycotoxins. Fumonisin B2 (FB2), the structural analogue of the commonly studied Fumonisin B1 (FB1), is a neglected mycotoxin produced by several Fusarium species. Mycotoxins are known for inducing toxicity via mitochondrial stress alluding to mitochondrial degradation (mitophagy). These processes involve inter-related pathways that are regulated by proteins related to SIRT3 and Nrf2. This study aimed to investigate mitochondrial stress responses in human kidney (Hek293) cells exposed to FB2 for 24 h. Cell viability was assessed via the methylthiazol tetrazolium (MTT) assay, and the half-maximal inhibitory concentration (IC50 = 317.4 µmol/L) was estimated using statistical software. Reactive oxygen species (ROS; H2DCFDA), mitochondrial membrane depolarisation (JC1-mitoscreen) and adenosine triphosphate (ATP; luminometry) levels were evaluated to assess mitochondrial integrity. The relative expression of mitochondrial stress response proteins (SIRT3, pNrf2, LONP1, PINK1, p62 and HSP60) was determined by Western blot. Transcript levels of SIRT3, PINK1 and miR-27b were assessed using quantitative PCR (qPCR). FB2 reduced ATP production (p = 0.0040), increased mitochondrial stress marker HSP60 (p = 0.0140) and suppressed upregulation of mitochondrial stress response proteins SIRT3 (p = 0.0026) and LONP1 (p = 0.5934). FB2 promoted mitophagy via upregulation of pNrf2 (p = 0.0008), PINK1 (p = 0.0014) and p62 (p < 0.0001) protein expression. FB2 also suppressed miR-27b expression (p < 0.0001), further promoting the occurrence of mitophagy. Overall, the findings suggest that FB2 increases mitochondrial stress and promotes mitophagy in Hek293 cells.

Rooibos, a supportive role to play during the COVID-19 pandemic?

This article presents the potential health benefits of Rooibos to be considered a support during the COVID-19 pandemic. The recent pandemic of COVID-19 has led to severe morbidity and mortality. The highly infectious SARS-CoV-2 is known to prime a cytokine storm in patients and progression to acute lung injury/acute respiratory distress syndrome. Based on clinical features, the pathology of acute respiratory disorder induced by SARS-CoV-2 suggests that excessive inflammation, oxidative stress, and dysregulation of the renin angiotensin system are likely contributors to the COVID-19 disease. Rooibos, a well-known herbal tea, consumed for centuries, has displayed potent anti-inflammatory, antioxidant, redox modulating, anti-diabetic, anti-cancer, cardiometabolic support and organoprotective potential. This article describes how Rooibos can potentially play a supportive role by modulating the risk of some of the comorbidities associated with COVID-19 in order to promote general health during infections.

Fumonisin B1 -induced mitochondrial toxicity and hepatoprotective potential of rooibos: An update.

Fumonisins are a family of potentially carcinogenic mycotoxins produced by Fusarium verticillioides. Several fumonisins have been identified with fumonisin B1 (FB1 ) being the most toxic. The canonical mechanism of FB1 toxicity is centered on its structural resemblance with sphinganine and consequent competitive inhibition of ceramide synthase and disruption of lipidomic profiles. Recent and emerging evidence at the molecular level has identified the disruption of mitochondria and excessive generation of toxic reactive oxygen species (ROS) as alternative/additional mechanisms of toxicity. The understanding of how these pathways contribute to FB1 toxicity can lead to the identification of novel, effective approaches to protecting vulnerable populations. Natural compounds with antioxidant properties seem to protect against the induced toxic effects of FB1 . Rooibos (Aspalathus linearis), endemic to South Africa, has traditionally been used as a medicinal herbal tea with strong scientific evidence supporting its anecdotal claims. The unique composition of phytochemicals and combination of metabolic activators, adaptogens and antioxidants make rooibos an attractive yet underappreciated intervention for FB1 toxicoses. In the search for a means to address FB1 toxicoses as a food safety problem in developing countries, phytomedicine and traditional knowledge systems must play an integral part. This review aims to summarize the growing body of evidence succinctly, which highlights mitochondrial dysfunction as a secondary toxic effect responsible for the FB1 -induced generation of ROS. We further propose the potential of rooibos to combat this induced toxicity based on its integrated bioactive properties, as a socio-economically viable strategy to prevent and/or repair cellular damage caused by FB1 .

The neglected foodborne mycotoxin Fusaric acid induces bioenergetic adaptations by switching energy metabolism from mitochondrial processes to glycolysis in a human liver (HepG2) cell line.

Metabolic flexibility defines the capacity of cells to respond to changes in nutrient status. Mitochondria are important mediators of metabolic flexibility and dysfunction is associated with metabolic inflexibility and pathology. Foodborne toxins are often overlooked as potential factors contributing to metabolic toxicity. Fusaric acid (FA), a neglected mycotoxin, is known to disrupt mitochondrial function. The aim of this study was to investigate the molecular mechanisms underlying a metabolic switch in response to FA. This study investigated the effects of FA on energy homeostasis in cultured human liver (HepG2) cells. HepG2 cells poised to undergo oxidative and glycolytic metabolism were exposed to a range of FA concentrations (4, 63 and 250 µg/mL) for 6 h. We determined mitochondrial toxicity, acetyl CoA levels and cell viability using luminometric, fluorometric and spectrophotometric methods. Expression of metabolic proteins (PDK1, PKM2, phosphorylated-PDH E1α and HIF-1α) and mRNAs (HIF-1α, PKM2, LDHa and PDK1) were determined using western blot and qPCR respectively. Our data connects a constitutive expression of HIF-1α in response to FA, to the inhibition of pyruvate decarboxylation through up-regulation of PDK-1 and phosphorylation of Pyruvate Dehydrogenase E1α subunit. Moreover, we highlight the potential of FA to induce a glucose "addiction" and phenotype reminiscent of the Warburg effect. The findings provide novel insights into the impact of this neglected foodborne mycotoxin in the dysregulation of energy metabolism.

Fusaric acid induces NRF2 as a cytoprotective response to prevent NLRP3 activation in the liver derived HepG2 cell line.

Fusaric acid (FA) is a neglected fusarium mycotoxin despite its ubiquitous presence. FA is a niacin related compound and mediates toxicity via oxidative stress and mitochondrial dysfunction. The NLRP3 inflammasome is a multiprotein scaffold that plays a key role in IL-ß maturation. We investigated the effects of FA on IL-1ß processing, NRLP3 inflammasome priming and activation along with the potential of FA to initiate cytoprotective mechanisms using spectrophotometry, luminometry, qPCR and western blots in the HepG2 liver cell line. FA disrupted synthesis and maturation of IL-1ß by inhibiting NRLP3 priming and activation. Further experimentation revealed an up-regulation of NRF2 with concomitant elevation in the anti-oxidant enzyme SOD2 and autophagy markers suggesting that FA induces NRF2 cytoprotective programs in these cells. We conclude that FA attenuates inflammasome priming and activation and sheds light on the immunosuppressive potential of FA in liver cells.

Toxicity assessment of mycotoxins extracted from contaminated commercial dog pelleted feed on canine blood mononuclear cells.

Raw ingredients of pet food are often contaminated with mycotoxins. This is a serious health problem to pets and causes emotional and economical stress to the pet owners. The aim of this study was to determine the immunotoxicity of the most common mycotoxins (aflatoxin, fumonisin, ochratoxin A and zearalenone) by examining 20 samples of extruded dry dog food found on the South African market [10 samples from standard grocery store lines (SB), 10 from premium veterinarian lines (PB)]. Pelleted dog food was subjected to extraction protocols optimized for the above mentioned mycotoxins. Dog lymphocytes were treated with the extracts (24 h incubation and final concentration 40 µg/ml) to determine cell viability, mitochondrial function, oxidative stress, and markers of cell death using spectrophotometry, luminometry and flow cytometry. Malondialdehyde, a marker of oxidative stress showed no significant difference between SB and PB, however, GSH was significantly depleted in SB extract treatments. Markers of apoptosis (phosphatidylserine externalization) and necrosis (propidium iodide incorporation) were elevated in both food lines when compared to untreated control cells, interestingly SB extracts were significantly higher than PB. We also observed decreased ATP levels and increased mitochondrial depolarization in cells treated with both lines of feed with SB showing the greatest differences when compared to the control. This study provides evidence that irrespective of price, quality or marketing channels, pet foods present a high risk of mycotoxin contamination. Though in this study PB fared better than SB in regards to cell toxicity, there is a multitude of other factors that need to be studied which may have an influence on other negative outcomes.

Fusaric Acid Induces DNA Damage and Post-Translational Modifications of p53 in Human Hepatocellular Carcinoma (HepG2 ) Cells.

Fusaric acid (FA), a common fungal contaminant of maize, is known to mediate toxicity in plants and animals; however, its mechanism of action is unclear. p53 is a tumor suppressor protein that is activated in response to cellular stress. The function of p53 is regulated by post-translational modifications-ubiquitination, phosphorylation, and acetylation. This study investigated a possible mechanism of FA induced toxicity in the human hepatocellular carcinoma (HepG2 ) cell line. The effect of FA on DNA integrity and post-translational modifications of p53 were investigated. Methods included: (a) culture and treatment of HepG2 cells with FA (IC50 : 580.32 µM, 24 h); (b) comet assay (DNA damage); (c) Western blots (protein expression of p53, MDM2, p-Ser-15-p53, a-K382-p53, a-CBP (K1535)/p300 (K1499), HDAC1 and p-Ser-47-Sirt1); and (d) Hoechst 33342 assay (apoptosis analysis). FA caused DNA damage in HepG2 cells relative to the control (P < 0.0001). FA decreased the protein expression of p53 (0.24-fold, P = 0.0004) and increased the expression of p-Ser-15-p53 (12.74-fold, P = 0.0126) and a-K382-p53 (2.24-fold, P = 0.0096). This occurred despite the significant decrease in the histone acetyltransferase, a-CBP (K1535)/p300 (K1499) (0.42-fold, P = 0.0023) and increase in the histone deacetylase, p-Ser-47-Sirt1 (1.22-fold, P = 0.0020). The expression of MDM2, a negative regulator of p53, was elevated in the FA treatment compared to the control (1.83-fold, P < 0.0001). FA also inhibited cell proliferation and induced apoptosis in HepG2 cells as evidenced by the Hoechst assay. Together, these results indicate that FA is genotoxic and post-translationally modified p53 leading to HepG2 cell death. J. Cell. Biochem. 118: 3866-3874, 2017. © 2017 Wiley Periodicals, Inc.

Fusaric acid induces mitochondrial stress in human hepatocellular carcinoma (HepG2) cells.

Fusarium spp are common contaminants of maize and produce many mycotoxins, including the fusariotoxin fusaric acid (FA). FA is a niacin related compound, chelator of divalent cations, and mediates toxicity via oxidative stress and possible mitochondrial dysregulation. Sirtuin 3 (SIRT3) is a stress response deacetylase that maintains proper mitochondrial function. We investigated the effect of FA on SIRT3 and oxidative and mitochondrial stress pathways in the hepatocellular carcinoma (HepG2) cell line. We determined FA toxicity (24 h incubation; IC50 = 104 µg/ml) on mitochondrial output, cellular and mitochondrial stress responses, mitochondrial biogenesis and markers of cell death using spectrophotometry, luminometry, qPCR and western blots. FA caused a dose dependent decrease in metabolic activity along with significant depletion of intracellular ATP. FA induced a significant increase in lipid peroxidation, despite up-regulation of the antioxidant transcription factor, Nrf2. FA significantly decreased expression of SIRT3 mRNA with a concomitant decrease in protein expression. Lon protease was also significantly down-regulated. FA induced aberrant mitochondrial biogenesis as evidenced by significantly decreased protein expressions of: PGC-1α, p-CREB, NRF1 and HSP70. Finally, FA activated apoptosis as noted by the significantly increased activity of caspases 3/7 and also induced cellular necrosis. This study provides insight into the molecular mechanisms of FA (a neglected mycotoxin) induced hepatotoxicity.