engleski

Toxic effects of single and combined aflatoxin B1, ochratoxin A, and sterigmatocystin on human keratinocyte HaCaT cells

Aflatoxin B1 (AFB1), ochratoxin A (OTA) and sterigmatocystin (STC) are mycotoxins produced by filamentous fungi of the genus Aspergillus which exert various toxic properties in vivo and in vitro. The superficial layer of the skin (epidermis) is the most exposed part of the human body, and it's composed mostly of keratinocytes which have an important immune role by excreting various cytokines and could be subjected to harmful chemicals, e.g. the carcinogenic effect of mycotoxins such as AFB1. The purpose of this study was to investigate the cytotoxic, proinflammatory and genotoxic effects of single AFB1, OTA and STC, and their binary combinations on human keratinocyte HaCaT cells. Cells were exposed to single and combined mycotoxins for 24 h. Cytotoxicity was determined by MTT reduction assay. Excretion of cytokines IL- 1β, IL-6, IL-8, TNFα and IL-10 was measured by enzyme-linked immunosorbent assay (ELISA) in cell supernatant. The level of DNA damage was determined by alkaline comet assay. According to concentrations that inhibit metabolic activity of HaCaT cells by 50% (IC50), cytotoxic potencies of mycotoxins were as follows: STC (IC50= 44.42 μM ) > OTA (IC50= 98.93 μM), while AFB1 at 50 μM reduced cell viability by 40% and increase to 150 μM did not provoke further decrease of cell viability. AFB1+OTA and OTA+STC applied in subcytotoxic concentrations showed additive and synergistic effects while AFB1+STC exerted dominant antagonism. ELISA showed that untreated HaCaT cells do not produce IL-1β, TNFα and IL-10, while IL-6 and IL-8 were produced in concentrations of 507 pg/mL and 547 pg/mL, respectively. All three mycotoxins provoked the increase of IL-6 and IL-8 by 123-297% and 131- 228%, respectively. The binary mycotoxin combinations showed an antagonistic effect on cytokine excretion. The alkaline comet assay showed that all mycotoxins individually and in binary combinations provoke DNA fragmentation by significant increase of the tail intensity (1.68- 5.57%) as compared to untreated cells (0.57%). The effect of binary combinations on DNA fragmentation were additive (AFB1+OTA) ; additive or antagonistic (AFB1+STC) ; and dominantly antagonistic (OTA+STC). In conclusion individual mycotoxins AFB1, OTA and STC and their binary mixtures evoke cytotoxic, genotoxic and proinflammatory effects in HaCaT cells. Taking into account result of combined treatments, cytotoxic potency of particular binary mixtures may overcome genotoxic and proinflammatory action.

Combined toxicity; sterigmatocystin; aflatoxin; ochratoxin A; keratinocytest

nije evidentirano