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Ochratoxin A (OTA) is a widespread mycotoxin produced by several fungal species, including Aspergillus ochraceus and Penicillium verrucosum. OTA has been reported to cause nephrotoxicity. The contamination of OTA in food is an ongoing global concern; many international organizations have conducted risk assessments of OTA and established limits on OTA in foodstuffs and calculated tolerable human intakes of OTA. In this thesis research, we evaluated the toxicological effects of OTA to Caenorhabditis elegans based on multiple endpoints, including growth, reproduction, and locomotion, using high-throughput assays.


Locomotion assay. Age-synchronized L4 stage nematodes were washed off the K-agar plates with K-medium, where they were counted in 10 µL and adjusted to obtain concentration of 1 nematode per µL. Approximately 900 nematodes were dispensed into a 24-well plate, such that each designated well contained 900 µL of K-medium. The 900 synchronized C. elegans per well were assessed by the wMicroTracker. Before the 24-hour experiment, the nematodes were allowed to settle for an hour and their basal motility was measured, using the wMicroTracker, for 60 min to obtain a reference point for motility prior to treatment. The operation temperature was 20ºC. After the basal recoding, 100 µL of 10 × test solutions containing OP50 were added to each well to a final volume of 1000 µL. Eight concentrations of the test solutions were used: 0, 1, 5, 10, 20, 40, 80, and 160 µM. The motility of nematodes was measured by the wMicroTracker every hour for 24 hours continuously. Three experiments were conducted, and each concentration group had three replicate wells.


The result of the locomotion experiment for exposure of OTA to C. elegans displayed a time-dependent decrease in the motility of worms, but, as time went on, a concentration-dependent increase in the motility of worms was found as compared to the untreated control (Fig. 4.5). These findings indicated that toxicological evaluations of OTA in C. elegans presented similar results in other studies of mycotoxins in C. elegans. So far, no study has reported the toxic effects of OTA in C. elegans. This thesis research suggested that C. elegans model can serve as a good model organism for evaluation of toxic effects on OTA and mechanistic studies of OTA-induced adverse health effects.

Figure 4.5 The graph represented the mean motility of the nematodes over time in three replicate wells of each concentration. The vertical axis shows values of the motility, while the first measurement is for the basal motility. The horizontal axis shows the time for the measurement. The L4 synchronized nematodes and the 24-well plate were used in the experiments. 900μl worm suspension (900 worms), containing OTA with different concentrations of 0 µM (blue), 1 µM (red), 5 µM (gray), 10 µM (yellow), 20 µM (aqua), 40 µM (green), 80 µM (deep blue), and 160 µM (brown), were transferred to separate wells respectively. The motility was measured by the wMicroTracker every hour for 24 hours.


Joonsu Jang under the Direction of Jia-Sheng Wang.