Abstract:
Stenocarpella maydis (previously known as Diplodia maydis) is one of the most
prevalent ear and stalk rot pathogens of maize globally, causing reductions of grain
quality and yield. Ingestion of S. maydis infected maize causes diplodiosis, a
neuromycotoxicosis of cattle and sheep. To date, diplodiatoxin, dipmatol, diplonine and
chaetoglobosins (K, L, M and O) have been isolated from S. maydis infected cultures. The mechanism of action of these metabolites is poorly understood and consequently, it
is not known whether these metabolites play a role in the aetiology of diplodiosis.
The toxicity and mechanism of action of the three S. maydis metabolites (i.e.
diplodiatoxin, dipmatol and diplonine) were evaluated for the first time in this study
using in vitro cell cultures, namely the mouse neuroblastoma (Neuro-2a), Chinese
hamster ovary (CHO-K1) and Mardin-Darby bovine kidney (MDBK) cells.
The cytotoxicity was firstly evaluated after exposure of the three cell lines to various
concentrations of diplodiatoxin, dipmatol and diplonine for 24, 48 and 72 h. The
xCELLigence, tetrazolium 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) and protein measurement assays were used. To better understand the
mechanism(s) involved in the observed cytotoxicity, necrotic and apoptotic cell death
pathways were investigated. The lactate dehydrogenase (LDH) and propidium iodide (PI)
flow cytometry assays were used to assess necrotic changes, and the caspase-3/7 and
Annexin V-FITC flow cytometry assays were used for apoptosis. Finally, transmission
electron microscopy (TEM) was used to evaluate the subcellular changes induced by the
S. maydis toxins in vitro.
Results obtained in this study indicate that both diplodiatoxin and dipmatol induced a
concentration-dependent cytotoxicity in Neuro-2a, CHO-K1 and MDBK cells and also
affected the activity of the mitochondrial succinate dehydrogenase enzyme. Conversely,
diplonine was not cytotoxic at comparable concentrations. Diplodiatoxin and dipmatol, at high concentrations, exerted their toxicity via the necrotic and the caspase-dependent
apoptotic cell death pathways. Mitochondrial damage, cytoplasmic vacuolation and
nuclear fragmentation were the major subcellular changes induced by diplodiatoxin and
dipmatol. Except for the elongation of mitochondria, no major subcellular changes were
observed following exposure of MDBK cells to diplonine. Collectively, these results
indicate that mitochondrial damage could be central in the toxicity of the two S. maydis
metabolites and possibly in the development of diplodiosis.
