Characterization Of Mercury-Binding Metallothioneins (MTs) And Measurement Of Lead Isotopic Ratios With Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

This dissertation consists of two parts, which are the characterization of multimetal-binding metallothioneins (MTs) in rat tissues after oral intake of cinnabar and HgCli (from chapter 1 to 4), and the measurement of lead isotopic ratios with inductively coupled plasma mass spectrometry (ICP-MS) (from chapter 5 to 8). Cinnabar, made up of natural mineral, is one of the widely used components in Chinese traditional medicines (TCM), and has been proved to have prominent curative effects in the clinic for more than two thousand years. But the safety and toxicity of the drug has been under debate all through in clinic. MTs, which abundantly exists in -SH groups, containing about 30% of cysteine in the molecule, and playing a detoxification role against heavy metals, are used as a biomarker to assess the mercurial toxicity in rats fed orally with cinnabar. The principle knowledge of MTs, such as the structural features and the biological functions, and the characterizing methods of MTs, including the traditional determination and modern hyphenated techniques, are summarized in chapter 1 through the review of more than 100 published references. In chapter 2, the distribution of heavy metals (including Hg, Cu and Zn) and MTs, measured with ICP-MS and a modified mercury saturation assay respectively, is investigated in tissues including liver, kidney, heart, brain, testicle and blood of rats fed orally with cinnabar and HgCl2. The relationship between heavy metal levels and MT concentrations is discussed. The results indicate that the amounts of Hg increasingly accumulate in the rat tissues, especially in liver, kidney and testicle. The poison of cinnabar is noticeable or even fatal if the dose is excessive or the drug is taken for a long period at anormal dosage. A positively high correlation of MT concentrations and Hg levels in both of liver and kidney validates that MTs can be used as a biomarker for the toxicological study of cinnabar.Based on the hyphenated technique of size-exclusion chromatography (SEC) with inductively coupled plasma mass spectrometry (ICP-MS), the multimetal-binding MT fractions in rat tissues after oral intake of cinnabar and HgCl2 are investigated in chapter 3. The results indicate that the amount of Hg-binding MTs in rat tissues of all cinnabar groups is much lower comparing with that of HgCl2 group. With the increase of both the feeding time and dosage of cinnabar, the amounts of Hg-binding MT fractions in rat kidney of cinnabar groups increase significantly comparing with the control group. Meanwhile, more Cu-binding MTs are synthesized in rat kidney, which validates that MTs play an important detoxification role for nephritic mercury. The Hg-binding MT fractions in rat liver of all cinnabar groups are almost independent of the cinnabar dosage (2.5-5.0 g/Kgbw) and feeding time (2-4 weeks), but are significantly more than that of the control group. The amounts of Hg-MTs in other rat tissues of cinnabar groups, the HgCl2 group and the control group are compared and discussed.In Chapter 4, the Hg-binding MT polymorphism of rat is investigated by the established hyphenated techniques, such as rp-HPLC coupling with ICP-MS and ESI-MS, respectively. After being purified with size-exclusion column (Sephadex G-75) and dialysis (M<1000 Da), the extracts of rat kidney and liver are separated with narrow-bore reverse phase column and eluted by increasing methanol at gradient mode. The MT polymorphism with different binding-metal (including Hg, Cu and Zn) species is obtained with the detectionof ICP-MS. The chromatograms of UV and TIC of ESI-MS are obtained through the analysis of rpHPLC-ESI-MS. Because of the little data reported on the identification of Hg-binding MT polymorphism with ESI-MS and the unavailable standards of Hg-MTs, the tentative formulas of Hg-MT isoforms and subisoforms are only interpreted by the measured masses at the vicinity of apexes in TIC of ESI-MS and the apo-MTs reported. The results indicate that there is some Hg-binding MT species in kidney of rat a...