In vitro cytotoxicity of fungi spoiling maize silage

Penicillium roqueforti, Penicillium paneum, Monascus ruber, Alternaria tenuissima, Fusarium graminearum, Fusarium avenaceum, Byssochlamys nivea and Aspergillus fumigatus have previously been identified as major fungal contaminants of Danish maize silage. In the present study their metabolite production and in vitro cytotoxicity have been determined for fungal agar and silage extracts. All 8 fungal species significantly affected Caco-2 cell viability in the resazurin assay, with large variations for each species and growth medium. The 50% inhibition concentrations (IC50) of the major P. roqueforti metabolites roquefortine C (48 μg/mL), andrastin A (>50 μg/mL), mycophenolic acid (>100 μg/mL) and 1-hydroxyeremophil-7(11),9(10)-dien-8-one (>280 μg/mL) were high. Fractionating of agar extracts identified PR-toxin as an important cytotoxic P. roqueforti metabolite, also detectable in maize silage. The strongly cytotoxic B. nivea and P. paneum agar extracts contained patulin above the IC50 of 0.6 μg/mL, however inoculated onto maize silage B. nivea and P. paneum did not produce patulin (>371 μg/kg). Still B. nivea infected maize silage containing mycophenolic acid (∼50 mg/kg), byssochlamic acid and other metabolites, was cytotoxic. In contrast hot-spots of P. roqueforti, P. paneum, M. ruber and A. fumigatus were not more cytotoxic than uninoculated silage.