Abstract
Certain fungi thrive in highly radioactive environments including the defunct Chernobyl nuclear reactor. Cryptococcus neoformans (C. neoformans), which uses L-3,4-dihydroxyphenylalanine (L-DOPA) to produce melanin, was used here to investigate how gamma radiation under aqueous aerobic conditions affects the properties of melanin, with the aim of gaining insight into its radioprotective role. Exposure of melanized fungal cell in aqueous suspensions to doses of γ-radiation capable of killing 50 to 80% of the cells did not lead to a detectable loss of melanin integrity according to EPR spectra of melanin radicals. Moreover, upon UV-visible (Xe-lamp) illumination of melanized cells, the increase in radical population was unchanged after γ-irradiation. Gamma-irradiation of frozen cell suspensions and storage of samples for several days at 77 K however, produced melanin modification noted by a reduced radical population and reduced photoresponse. More direct evidence for structural modification of melanin came from the detection of soluble products with absorbance maxima near 260 nm in supernatants collected after γ-irradiation of cells and cell-free melanin. These products, which include thiobarbituric acid (TBA)-reactive aldehydes, were also generated by Fenton reagent treatment of cells and cell-free melanin. In an assay of melanin integrity based on the metal (Bi+3) binding capacity of cells, no detectable loss in binding was detected after γ-irradiation. Our results show that melanin in C. neoformans cells is susceptible to some damage by hydroxyl radical formed in lethal radioactive aqueous environments and serves a protective role in melanized fungi that involves sacrificial breakdown.
Highlights
Melanins are complex polymers, formed by oxidation of various precursors, including tyrosine, tryptophan, and L-3,4-dihydroxyphenylalanine (L-DOPA) and are found in cells of all biological kingdoms
Cryptococcus neoformans cell growth The strain used for these experiments was cap67, an acapsular, avirulent strain of C. neoformans derived from the serotype D strain B3501 [18]
The yields increased with increasing concentration of iron and hydrogen peroxide. These results strongly suggest that the soluble products generated by c-irradiation of melanized cells include some from hydroxyl radical damage to melanin, since OHN is the reactive species common to both the c-irradiation and the Fenton reagent protocols
Summary
Melanins are complex polymers, formed by oxidation of various precursors, including tyrosine, tryptophan, and L-3,4-dihydroxyphenylalanine (L-DOPA) and are found in cells of all biological kingdoms. Recent evidence from one of our laboratories suggested that melanin in live Cryptococcus neoformans (C. neoformans) cells can function both in energy transduction [8] and as a radioprotectant [9]. This fungus is of special interest because melanized microbial species are found in highly radioactive environments such as the cooling pools of nuclear reactors, in the stratosphere, in space stations, and inside the damaged nuclear reactor at Chernobyl (reviewed in [10]). The physics of interaction of ionizing radiation with synthetic eumelanins and pheomelanins (sulfur-containing) was described based on a variety of physicochemical techniques including electron paramagnetic resonance (EPR) spectroscopy [9,11].
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