| Summary: | 博士 === 國立成功大學 === 環境工程學系碩博士班 === 97 === The dioxin-like compounds are one of the most hazardous material groups in the world. Traditional chemical analysis methods (e.g., HRGC/HRMS and HRGC/LRMS) are time-consuming and costly for these compounds. Thus, the major aim of this study is to build a rapid and less-cost bioassay method. The Xenobiotic Detection Systems - chemical activated luciferase gene expression (CALUX) bioassay was thus employed in this study for detecting dioxin-like compunds.
The dissertation is mainly concerned with three issues: 1) a review of the bioassay literature; 2) building the bioassay method for detecting dioxin-like compounds in milk and relative quality assurance/ quality control (QA/QC); and 3) discussing the effects of eight metal ions (Hg2+, Ag+, Cu2+, Fe2+, As5+, Cr6+, Cd2+, and Pb2+) on the bioassay method.
The results of part one show that the main dioxin bioassay methods are immunoassay, the biological metabolic reaction of dioxins (including the DNA recombinant cell), and biosensors. The methods that based on the first and second principles have been accepted as standard analysis methods by U.S. EPA, and thus they are stable and accurate enough for use with real samples. Although biosensors are not a standard analysis method used by the U.S. EPA, however, they are important because they offer real-time on-site monitoring.
The results of part two show that the analyzed data suggest that the CALUX bioassay criteria have: (1) control chart for quality control (QC) standards within the μ ± 2σ range; and (2) a recovery efficiency (i.e., relative standard deviation (RSD) of 20.7%. This bioassay method was utilized on 28 commercially available pasteurized milk samples, and the results illustrate that CALUX is a powerful approach for screening a large number of such samples.
Finally, the results of part three show further study found that the bioassay cell-line was sensitive to particular metals, which might be a potential interfering factor. A two-level factorial-designed experiment was undertaken to identify the statistically significant factors from eight metal ions (Hg2+, Ag+, Cu2+, Fe2+, As5+, Cr6+, Cd2+, and Pb2+), and the results revealed that Hg2+, Cu2+, and Cr6+ had significant effects on the luciferase activity expression of the H1L6.1 cells. In addition, the correlation between luciferase activity and the relative survival ratio of H1L6.1 cells with metal concentrations was not comparable. This reveals the inhibiting effect of metals on luciferase activity may not only be caused by the reduction of H1L6.1 cells, but may also be caused by the other mechanisms, such as a reduction of luciferase enzyme production or the inhibiting effect of metal ions on CYP1A activation.
The study illustrates that the bioassay method it presents is a rapid and less-costly approach for detecting dioxin-like compounds. However, potential interference factors, (specifically heavy metal ions) for the bioassay should be eliminated in the pretreatment process to maintain the stability of analysis. The proposed bioassay method with metabolite assay could be emplyeed in risk assessment by using the physiologically based pharmacokinetic model; in addition, the biosensors could be used as an on-site monitoring tool when combined with portable instruments.
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