| Summary: | 博士 === 國立臺灣大學 === 職業醫學與工業衛生研究所 === 105 === Maleic anhydride is an anthropogenic organic chemical reagent used in an array of industrial and personal care products. The purposeful adulteration of maleic anhydride by foods manufacturers into starch-based delicacies to improve texture, elasticity and prolong storage time instigated food safety concerns in Taiwan. This unapproved use of maleic anhydride as a food additive, combined with the continuous risk of exposure from packaging migration, underscore the need to investigate the potential risks upon ingestion.
Despite prior knowledge indicating maleic anhydride can lead to varying degrees of nephrotoxicity in animals, its mechanisms of toxicity remain elusive. This dissertation began to develop a new analytical method using liquid chromatography tandem-mass spectrometry (LC-MS/MS) to determine maleic acid concentrations in biofluids. Subsequently, the kinetic fate and bioavailability of ingested maleic acid in serum and urine in both male and female Sprague-Dawley rats were studied using the aforementioned analytical protocol. Pharmacokinetic results confirmed that maleic acid is absorbed, metabolized and eliminated rapidly, along with low bioavailability.
The next portion of this thesis examined the association between maleic acid consumption and the occurrence of oxidant attacks and molecular changes. A rapid and highly sensitive off-line LC-MS/MS method was developed to simultaneously quantify multiple biomarkers indicative of oxidative stress, with elevations associated with increases in risks of various diseases. Application of the abovementioned method demonstrated that oral single-dose exposure to maleic acid induced oxidative DNA damage and lipid peroxidation, as demonstrated by the statistically significant increases in urinary levels of 8-NO2Gua, 8-OHdG, and 8-isoPGF2α, particularly in high-dosed male rats within 12 h of consumption. Our findings also demonstrated that increases in concentration of these biomarkers persist for days after consumption; male rats appear to be more sensitive to oxidative burden compared to their counterparts.
Finally, a subchronic toxicity study employing both LC-MS/MS and proton nuclear magnetic resonance spectroscopy (1H NMR) spectroscopy was conducted to elucidate the molecular events; organs were collected at necropsy and underwent histology analysis. Our results demonstrated that maleic acid increases urinary concentrations of 8-OHdG, 8-NO2Gua and 8-isoPGF2α; analysis of acetoacetate, hippurate, alanine, and acetate exhibited time- and dose-dependent variations in the treatment groups. Physio-morphological alterations were evident in body weights, relative kidney weights; histology analysis revealed cellular changes in the kidneys and liver. Our findings suggest that maleic acid consumption escalates oxidative burden, triggers membrane lipid destruction, and disrupts energy metabolism.
The analytical methods demonstrated high sensitivity, specificity and stability capable of detecting maleic acid in biofluids and multiple urinary biomarkers at parts-per-billion concentrations. Our results demonstrated that with rapid absorption and elimination, as well as absence of clinical manifestations, both single and repeat exposures to maleic acid trigger cytotoxicity. The experimental approaches can be applied to clarify toxicity of other food contaminants or environmental toxicants for risk assessment. Additional studies targeting inhalation exposure and dermal absorption may further increase the merit of risk assessment.
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