An approach to understanding toxicity induction by filamentous fungi on human cell lines
Egbuta, Mary Augustina
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Filamentous fungi occur widely in different parts of the environment including water, soil and air. Their occurrence in the environment especially in large amounts and under certain conditions pose dangerous health risks to humans, especially immunocompromised individuals as a result of the compounds they produce during metabolism. In this regard, filamentous fungi are associated with a range of diseases including invasive and superficial infections. In this study, species from the genera Aspergillus, Fusarium and Penicillium were used to investigate their combined toxic effects when exposed to two human cell lines (hepatocytes and renal epithelial cells “in vitro”). Reference isolates used were: Aspergillus niger (A. niger), Aspergillus flavus (A. flavus), Fusarium oxysporum (F. oxysporum), Fusarium verticillioides (F. verticillioides), Penicillium chrysogenum (P. chrysogenum) and Penicillium expansum (P. expansum) isolated from maize samples. Isolates were cultured on Malt Extract agar/broth and Potato dextrose agar/broth at three incubation periods (4, 9 and 14 days). Isolates were identified following deoxyribonucleotide acid (DNA) extraction, amplification and sequencing of amplified products. Fungal species were further screened for mycotoxin production after different incubation periods by high performance liquid chromatography (HPLC) analysis using specific mycotoxin standards. Production of mycotoxins varied among isolates with F. verticilliodes and F. oxysporum producing more mycotoxins compared to other species. Aspergillus flavus produced aflatoxins (AFs) at different stages of growth up to 11.6μg/g at 9 days whereas, A. niger produced ochratoxin A (OTA) between 8.63*10⁻⁶ and 5.8*10⁻⁴μg/g at 4 and 14 days of growth respectively. Production of fumonisin B₁ (FB₁), deoxynivalenol (DON) and nivalenol (NIV) by Fusarium species was up to 114.6μg/g at 4 days, 0.15μg/g at 14 days and 1035.27μg/g at 4 days respectively. One characteristic of toxicity induction by microorganisms on both human and animal cells is the reduction of cell viability of the latter. A resazurin salt assay test was conducted in order to determine this effect. Human hepatocytes and renal epithelial cells were exposed to individual filamentous fungi species and their combinations for 24, 48 and 7h and cell viability determined by the ability of the cells to reduce resazurin to resofurin. Individual filamentous fungi and their combinations were able to induce a reduction in cell viability of the human cell lines at 72h of exposure with initial increase in cell proliferation at 24 and 48h. After incubation for up to 72h, there was reduction of cell viability down to 39.9 and 35.6% for hepatocytes and renal epithelial cells respectively. Filamentous fungi combinations, especially combinations of A. niger species and others had more deleterious cell viability reduction compared to individual species. Cytokine secretion/production is one of the means through which the human system combats infections. Although these cytokines contribute to protecting the human system from infections, an imbalance in their secretion could help in promoting inflammation upon infection. To investigate the induction of cytokine production by the hepatocytes upon exposure to individual filamentous fungi species and their combinations, cytometric Bead Array (CBA) of Th1 and Th2 human cytokines were determined. The cells were exposed to fungal isolates individually and in combination for 3, 6, 12 and 24h and cytokine expression measured using an Accuri C6 flow cytometer. Cytokine expression was measured for some of the cells exposed to A. flavus, F. verticillioides, F. oxysporum, P. chrysogenum and P. expansum with the production of interleukin 2 (IL-2), interleukin 4 (IL-4) and interferon gamma (IFN-γ). Fungi combinations containing F. verticillioides and F. oxysporum induced secretion of five cytokines; IL-2, IL-4, IFN- γ, Tumour necrosis factor (TNF) and interleukin 10 (IL-10) up to 2.940pg/ml, 3.693pg/ml, 4.720pg/ml, 2.093pg/ml and 0.623pg/ml. This study has been able to fill the knowledge gap in terms of synergistic action of some filamentous fungal species when exposed to certain cells in the human system. Furthermore, the production of DON by F. oxysporum in this study is a novel finding which has not been documented. The significance of this study is that the continuous exposure of humans to co-occurring filamentous fungi can be deleterious resulting in abnormal cell multiplication and reduction in cell viability as well as organ shut down.