Biochemical impacts of transition metals in the king bolete (Boletus edulis)
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- Institutt for kjemi 
The present thesis investigates the reciprocal relationship between metals and wild-growing macromycetes. An inter-diciplinary approach was needed in order to reveal, on the molecular level, how these organisms respond to, and are affected by, potentially toxic metals. This was accomplished by applying a selection of techniques from the fields of analytical chemistry and molecular biology. Samples were collected in the years 1998, 2000, and 2002 near two metal smelters: the still active Outokumpu Norzink zinc (Zn) smelter at Odda, S.W. Norway and the former copper (Cu) smelter at Sulitjelma, N. Norway. In the 1998 collection round, reference samples were collected several km from the emission sources. For subsequent rounds of sampling, reference samples were collected from rural areas in Central Norway. Atomic absorption spectrophotometry (AAS, flame mode) and high resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) were used to determine cadmium (Cd), Zn, and Cu in soil and fruiting body tissues. Mercury (Hg) was determined in samples collected at Odda and the reference areas in Central Norway, by cold vapour atomic fluorescence spectrometry (CVAFS). Cytosolic compounds were separated by size exclusion chromatography (SEC, =gel permeation chromatography) and anion exchange chromatography, and distribution of endogenous metals was investigated by AAS and CVAFS. Parallel runs of samples spiked with the radiotracer 109Cd were performed to further study binding of Cd to cytosolic components. Cd-binding proteins from the fly agaric (Amanita muscaria) were analyzed with respect to amino acid composition, whereas the N terminal amino acid sequence was determined for a Cd-binding protein from the king bolete (penny bun, Boletus edulis). Cd-binding components from cytosolic extracts, separated by SEC, were further separated and characterized by highperformance liquid chromatography-mass spectrometry (HPLC-MS) Cytosolic Cd-binding capacity (CCBC) was determined, to investigate whether increased synthesis of metal-binding compounds is part of the direct defense against metal toxicity in macromycetes. Furthermore, cytosolic extracts of B. edulis were analyzed with respect to activities of superoxide dismutase (SOD) and catalase (CAT), two enzymes important in the cellular defense against free radicals. In the same species, heat shock protein 70 kDa (HSP70, a molecular chaperone), as well as concentrations of glutathione (GSHTOT=GSH + 0.5 GSSG) and free proline were determined. Two markers of oxidative damage to cellular molecules were determined in B. edulis, in order to make possible conclusions about the effectiveness of the defense systems expressed in this species in handling the increased metal toxicity. These markers were frequency of apurinic/apyrimidinic (AP) sites in DNA and concentration of lipid hydroperoxides. The results showed elevated concentrations of Cd, Zn, and Cu in surface soil close to both smelters. Hg was elevated near the Outokumpu Norzink Zn smelter at Odda (not determined at Sulitjelma, see above). Metal concentrations in fruiting body tissues were more dependent on species-specific accumulation potentials for metals than on metal concentrations in substrate. Determinations of amino acid composition of Cd-binding proteins of A. muscaria, revealed that these compounds do not belong to the metallothionein (MT) family. The same applies to a Cd-binding protein from B. edulis. HPLC-MS demonstrated the presence of phytochelatins (PCs) in B. edulis growing near the Zn smelter at Odda. This is the first report of PCs in a macromycete. CCBC correlated quite well with exposure to Cd, Zn, Hg, and Cu in 42 samples of B. edulis (Spearman’s p<0.001 for all four metals). Furthermore, the difference in CCBC between the exposed group (growing near the Zn smelter at Odda) and the reference group was also statistically significant (Mann Whitney’s p<0.001), indicating the induction of Cd-binding cytosolic compounds as a response to metal exposure in this species. In 48 samples of B. edulis, expressions of SOD, CAT, and HSP70 were significantly positively correlated to the degree of exposure (Spearman’s p<0.01 for the association between Cd and SOD, p<0.001 for all other correlations between SOD, CAT, or HSP70 on one side and Cd, Zn, Hg, or Cu on the other). These results indicate that toxic effects arising from metal exposure, are not effectively alleviated by direct defense mechanisms (e.g. PCs) alone. GSHTOT correlated negatively with exposure, suggesting that the intracellular pool of GSH is reduced as a result of PC synthesis. Determination of frequency of AP sites and concentration of lipid hydroperoxides in 31 fruiting bodies of B. edulis showed that both damage parameters were significantly elevated in the exposed group (growing near the Zn smelter at Odda) relative to the reference group (p<0.001). Furthermore, the two damage parameters correlated significantly positively with exposure to the four metals (p<0.05) and with each other (p<0.001). From this, it is concluded that although a host of direct and indirect defense mechanisms is induced against metal toxicity in B. edulis, damage to vital cellular components do occur.