Assessing the Involvement of Free Radicals in Fungicide Toxicity Using α-Tocopherol Analogs

Lipid peroxidation has been implicated as the mode of action of aromatic hydrocarbon and dicarboximide fungicides. Evidence to support this hypothesis includes the antagonism of fungicide toxicity by the free radical scavenger α-tocopherol. The I₅₀ for the aromatic hydrocarbon fungicide tolclophos-methyl is 0.3 μ/ml for Ustilago maydis grown in liquid shaking cultures. Growth of cultures was inhibited by at least 80% when treated with 0.6 μg/ml of the fungicide. In the presence of 232 μM α-tocopherol and 0.6 μg/ml tolclophos-methyl, only 45% inhibition of growth was observed, indicating antagonism of fungicide toxicity by this compound. However, methyl ether tocopherol, which has no antioxidant activity, was just as effective in antagonizing toxicity. This can be attributed to the hydrophobic nature of α-tocopherol rather than its antioxidant activity. Using HPLC to quantify tolclophos-methyl, we determined that it is more soluble in α-tocopherol. Therefore, protection from these fungicides by α-toxopherol is most likely due to preferential partitioning of these compounds into the α-tocopherol. This was further confirmed by the finding that a compound unrelated to α-tocopherol but sharing its hydrophobic properties, phosphatidylcholine (dipalmitoyl), was even more effective in antagonizing toxicity. In addition, the protective role observed with α-tocopherol was not observed with the water soluble analog Trolox C. These data do not support the hypothesis that α-tocopherol protects U. maydis from the fungicides due to its antioxidant properties, leaving open to question the role of lipid peroxidation as the mode of action of these fungicides.