Inhibition of Aflatoxin Production by Paraquat and External Superoxide Dismutase in Aspergillus flavus.

Tomohiro Furukawa,Shohei Sakuda

doi:10.3390/toxins11020107

Abstract

Aflatoxin contamination of crops is a worldwide problem, and elucidation of the regulatory mechanism of aflatoxin production, for example relative to the oxidative–antioxidative system, is needed. Studies have shown that oxidative stress induced by reactive oxygen species promotes aflatoxin production. However, superoxide has been suggested to have the opposite effect. Here, we investigated the effects of the superoxide generator, paraquat, and externally added superoxide dismutase (SOD) on aflatoxin production in Aspergillus flavus. Paraquat with an IC50 value of 54.9 µM inhibited aflatoxin production without affecting fungal growth. It increased cytosolic and mitochondrial superoxide levels and downregulated the transcription of aflatoxin biosynthetic cluster genes, including aflR, a key regulatory protein. The addition of bovine Cu/ZnSOD to the culture medium suppressed the paraquat-induced increase in superoxide levels, but it did not fully restore paraquat-inhibited aflatoxin production because bovine Cu/ZnSOD with an IC50 value of 17.9 µg/mL itself inhibited aflatoxin production. Externally added bovine Cu/ZnSOD increased the SOD activity in fungal cell extracts and upregulated the transcription of genes encoding Cu/ZnSOD and alcohol dehydrogenase. These results suggest that intracellular accumulation of superoxide impairs aflatoxin production by downregulating aflR expression, and that externally added Cu/ZnSOD also suppresses aflatoxin production by a mechanism other than canonical superoxide elimination activity.

Highlights

Aflatoxins are potent carcinogenic toxins produced mainly by Aspergillus flavus and Aspergillus parasiticus, which infect agricultural crops, including corn and peanut
When A. flavus IFM 47798 was incubated for 48 h at 28 ◦ C in potato dextrose broth (PDB) liquid medium, about 1–2 ppm aflatoxin B1 was detected in the culture broth
As the fungal mycelial dry weight was not changed significantly by 500 μM paraquat, the inhibitory activity of this superoxide generator was specific to aflatoxin B1 production

Summary

Introduction

Aflatoxins are potent carcinogenic toxins produced mainly by Aspergillus flavus and Aspergillus parasiticus, which infect agricultural crops, including corn and peanut. The enzymatic genes responsible for aflatoxin biosynthesis are located in a cluster in the genomes of A. flavus and A. parasiticus. The transcription of genes encoding aflatoxin biosynthetic enzymes is positively regulated by the transcription factor AflR, whose gene is in the same cluster [8]. Environmental factors such as light and pH, trophic factors such as carbon and nitrogen sources, and several transcription factors recognizing these cues have been found to affect aflatoxin production, but the regulatory mechanisms of these factors leading to aflatoxin production have not been clarified in detail [9,10,11,12]

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Results

Conclusion