Abstract
Lipid peroxidation is considered as one of the manifestations of cellular damage in the toxicity of ochratoxin A (OA). OA; its three natural analogs, OB, OC, and Oalpha; and four synthetic analogs, d-OA, the ethylamide of OA (OE-OA), O-methylated OA (OM-OA), and the lactone-opened OA (OP-OA) were used to study free radical generation in bacteria with Bacillus brevis as a model system. The uptake of the different ochratoxins by B. brevis varied substantially depending on the molecular structures. Electron paramagnetic resonance spectroscopy using alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone as a spin trapping agent showed an enhanced free radical generation due to the addition of OA and most of the analogs. The EPR signals could be further enhanced by the addition of Ca2+, a calcium ionophore and an ATPase uncoupler, whereas they were eliminated by incubating the growing cells with vitamin E. The spin adduct hyperfine splitting constants indicate the presence of alpha-hydroxyethyl radicals resulting from generated hydroxyl radicals, which are trapped by alpha-(4-pyridyl-1-oxide)-N-tert-butyl nitrone. The results further suggest that OA induces free radical production in this model system by enhancing the permeability of the cellular membrane to Ca2+.
Highlights
Ochratoxins, of which ochratoxin A (OA)1 is the most prevalent, are secondary fungal metabolites of some toxigenic species of Aspergillus and Penicillium (Cole and Cox, 1981)
B. brevis was used as a model organism for this EPR study as it is highly sensitive to OA (Madhyastha et al, 1994) and produces a reproducible and sustained signal when monitored in an EPR spectrometer
Ochratoxins and Chemicals—OA, OB, and O␣ were isolated from a culture of Aspergillus ochraceus NRRL 3174 (Xiao et al, 1995). d-OA, OE-OA, O-methylated OA (OM-OA), and OP-OA were synthesized according to Xiao et al (1995, 1996); OC was synthesized according to van der Merwe et al (1965a, 1965b)
Summary
Ochratoxins and Chemicals—OA, OB, and O␣ were isolated from a culture of Aspergillus ochraceus NRRL 3174 (Xiao et al, 1995). d-OA, OE-OA, OM-OA, and OP-OA were synthesized according to Xiao et al (1995, 1996); OC was synthesized according to van der Merwe et al (1965a, 1965b). The toxins were dissolved in dimethyl sulfoxide. Medium, and Growth Conditions—B. brevis was obtained from American Type Culture Collection (ATCC), Rockville, MD. For the EPR experiments, the bacteria were cultured for 24 h in Erlenmeyer flasks and the protein concentration was determined according to Bradford (1976). Prior to the EPR experiments, the late logarithmic cultures were centrifuged at 500 ϫ g for 15 min and the supernatant was discarded. The samples could be stored for up to 8 h at 4 °C without loss of EPR signals They were incubated at room temperature 15 min prior to the initiation of the EPR experiments.
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