A new and rapid bioassay for the detection of gliotoxin and related epipolythiodioxopiperazines produced by fungi.
Gliotoxin is an immunosuppressive cytotoxin produced by numerous environmental or pathogenic fungal species. For this reason, it is one of the mycotoxins which must be systematically searched for in samples for biological control. In this study, a new, rapid and sensitive method for detecting gliotoxin has been developed. This bioassay is based on the induction of morphological changes in cultured cells (human KB cell line) by gliotoxin. Interpretation of the assay can be carried out after 1 h of incubation, either by direct microscopic observation, or with an automated microplate-reader at 630 nm. The limit of detection is 18-20 ng of gliotoxin in the well, depending on the used observation method. A high degree of specificity of the detection is brought about by the ability of the reducing reactant dithiothreitol to inhibit the biological activities of epipolythiodioxopiperazines (ETPs), such as gliotoxin, by reducing their polysulfide bridge. The bioassay allows a rapid primary screening of samples and a semi-quantitative evaluation of the gliotoxin concentration in extracts. The method has been used to study the gliotoxin production by different fungal strains, allowing to highlight 3 strains of Aspergillus fumigatus producing gliotoxin in various extracts.
Grovel
Olivier
O
S.M.A.B., Université de Nantes, Pôle Mer et Littoral - Faculté de Pharmacie, BP53508- 44035 Nantes cedex 01, France. olivier.grovel@univ-nantes.fr
Kerzaon
Isabelle
I
Petit
Karina
K
Robiou Du Pont
Thibaut
T
Pouchus
Yves-François
YF
D001232:Aspergillus fumigatus / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods; D004229:Dithiothreitol / Q000494:pharmacology; D005912:Gliotoxin / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D006801:Humans; D007624:KB Cells / Q000166:cytology / Q000187:drug effects*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D005912:Gliotoxin; D004229:Dithiothreitol