Cytotoxic and genotoxic potencies of single and combined spore extracts of airborne OTA-producing and OTA-non-producing Aspergilli in Human lung A549 cells

Abstract

Aspergillus sclerotiorum (AS) is a well-known producer of ochratoxin A (OTA) while Aspergillus pseudoglaucus (AP) produces a wide range of extrolites with poorly investigated toxicity. These species are frequently co-occur in grain mill aeromycota. The aim of this study was to determine OTA levels in spore extracts using HPLC and immunoaffinity columns, and to examine the cytotoxicity of pure OTA, OTA-positive (AS-OTA(+)) and OTA-negative (AS-OTA(-)) spore extracts, as well as of AP spore extract, on human lung adenocarcinoma cells A549, individually and in combination, using a colorimetric MTT test (540nm). To establish which type of cell death predominated after treatments, a quantitative fluorescent assay with ethidium bromide and acridine orange was used, and the level of primary DNA damage in A549 cells was evaluated using the alkaline comet assay. OTA was detected in spore extracts (0.3-28µg/mL) of 3/6 of the AS strains, while none of the tested AP strains were able to produce OTA. Taking into account the maximum detected concentration of OTA in the spores, the daily intake of OTA by inhalation was calculated to be 1ng/kg body weight (b.w.), which is below the tolerable daily intake for OTA (17ng/kg b.w.). Using the MTT test, the following IC50 values were obtained: single OTA (53μg/mL); AS-OTA(+) (mass concentration 934μg/mL corresponds to 10.5μg/mL of OTA in spore extract); and 2126μg/mL for AP. The highest applied concentration of AS-OTA(-) spore extract (4940μg/mL) decreased cell viability by 30% and IC50 for the extract could not be determined. Single OTA and AS-OTA(+) and combinations (AP+AS-OTA(+) and AP+AS-OTA(-)) in subtoxic concentrations provoked significant primary DNA damage, apoptosis, and to a lesser extent, necrosis in A549 cells. Mixture of AP+AS-OTA(+) and AP+AS-OTA(-) in subtoxic concentrations showed dominant additive interactions. Despite the low calculated daily intake of OTA by inhalation, our results suggest that chronic exposure to high levels of OTA-producing airborne fungi in combination with other more or less toxic moulds pose a significant threat to human health due to their possible additive and/or synergistic interactions.

Keywords: A. pseudoglaucus; A. sclerotiorum; Cytotoxicity; Genotoxicity; Ochratoxin A; Spore extracts.