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Cytotoxicity of four trichothecenes evaluated by three colorimetric bioassays
J Widestrand1, T Lundh, H Pettersson
1Department of Animal Nutrition and Management, Swedish University of Agricultural Sciences, Uppsala, Sweden. Johan.Widestrand@huv.slu.se
Abstract:
The application of cell culture technique for screening of low concentrations of Fusarium mycotoxins was examined. Three colorimetric bioassays were used to determine the cytotoxicity of the trichothecenes T-2 toxin (T-2), HT-2 toxin (HT-2), deoxynivalenol (DON) and nivalenol (NIV) to 3T3 mouse fibroblasts (3T3 cells). The bioassays assess DNA synthesis (incorporation of 5-bromo-2'-deoxyuridine; BrdU), metabolic activity (cleavage of 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide; MTT) and cell membrane damage (release of lactate dehydrogenase; LDH), respectively. The BrdU bioassay was the most sensitive and the IC50 values (50% response compared to untreated cells) of T-2, HT-2, DON and NIV were 4.6, 13, 263 and 365 ng/ml, respectively. At the same toxin concentrations used in the BrdU bioassay, only T-2 and HT-2 were toxic enough to obtain IC50 values using the MTT bioassay. The IC50 values for T-2 and HT-2 were 12 and 68 ng/ml, respectively. When determined by the LDH bioassay, the IC50 values of T-2 and HT-2 were 18 and 42 ng/ml, respectively. At the tested concentrations, DON and NIV had a minor effect on the 3T3 cells when evaluated by the MTT and LDH bioassays. The BrdU bioassay in combination with 3T3 cells was found to be a suitable method for determination of trichothecene-induced toxicity at low concentrations.
Insights
The BrdU cell culture assay effectively detects low concentrations of Fusarium mycotoxins, specifically trichothecenes like T-2 toxin and HT-2 toxin. This method offers a sensitive approach for assessing mycotoxin-induced toxicity in 3T3 mouse fibroblasts.
Area of Science:
- Toxicology
- Cell Biology
- Mycotoxicology
Background:
- Fusarium mycotoxins pose a significant threat to human and animal health.
- Accurate detection of low mycotoxin concentrations is crucial for risk assessment.
- Cell culture techniques offer a controlled environment for toxicological studies.
Purpose of the Study:
- To evaluate cell culture-based bioassays for screening low concentrations of Fusarium mycotoxins.
- To compare the sensitivity of three different bioassays in detecting trichothecene cytotoxicity.
- To determine the suitability of 3T3 mouse fibroblasts for mycotoxin toxicity assessment.
Main Methods:
- Three colorimetric bioassays were employed: BrdU (DNA synthesis), MTT (metabolic activity), and LDH (cell membrane damage).
- Cytotoxicity of T-2 toxin, HT-2 toxin, deoxynivalenol, and nivalenol was assessed using 3T3 mouse fibroblasts.
- IC50 values were determined for each toxin and bioassay combination.
Main Results:
- The BrdU bioassay demonstrated the highest sensitivity, with IC50 values for T-2, HT-2, DON, and NIV at 4.6, 13, 263, and 365 ng/ml, respectively.
- MTT and LDH bioassays showed significant toxicity only for T-2 and HT-2 toxins at the tested concentrations.
- DON and NIV exhibited minimal effects on 3T3 cells when assessed by MTT and LDH assays.
Conclusions:
- The BrdU bioassay combined with 3T3 cells is a suitable and sensitive method for detecting low concentrations of trichothecene mycotoxins.
- This approach aids in the screening and toxicological evaluation of Fusarium mycotoxins.
- Cell-based assays provide valuable insights into the cytotoxic effects of mycotoxins.