Exposure of melanin to ionizing radiation, and possibly other forms of electromagnetic radiation, changes its electronic properties. Melanized fungal cells manifested increased growth relative to non-melanized cells after exposure to ionizing radiation, raising intriguing questions about a potential role for melanin in energy capture and utilization.

Ionizing irradiation changed the electron spin resonance (ESR) signal of melanin, consistent with changes in electronic structure. Irradiated melanin manifested a 4-fold increase in its capacity to reduce NADH relative to non-irradiated melanin. HPLC analysis of melanin from fungi grown on different substrates revealed chemical complexity, dependence of melanin composition on the growth substrate and possible influence of melanin composition on its interaction with ionizing radiation. XTT/MTT assays showed increased metabolic activity of melanized C. neoformans cells relative to non-melanized cells, and exposure to ionizing radiation enhanced the electron-transfer properties of melanin in melanized cells. Melanized Wangiella dermatitidis and Cryptococcus neoformans cells exposed to ionizing radiation approximately 500 times higher than background grew significantly faster as indicated by higher CFUs, more dry weight biomass and 3-fold greater incorporation of 14 C-acetate than non-irradiated melanized cells or irradiated albino mutants. In addition, radiation enhanced the growth of melanized Cladosporium sphaerospermum cells under limited nutrients conditions.

Melanin pigments are ubiquitous in nature. Melanized microorganisms are often the dominating species in certain extreme environments, such as soils contaminated with radionuclides, suggesting that the presence of melanin is beneficial in their life cycle. We hypothesized that ionizing radiation could change the electronic properties of melanin and might enhance the growth of melanized microorganisms.

Copyright: © 2007 Dadachova et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Despite the high prevalence of melanotic microorganisms in radioactive environments, it is unlikely that melanin is synthesized solely for the purposes of protection (shielding) from ionizing radiation. For example, in high elevation regions inhabited by melanotic fungi the background radiation levels are approximately 500–1,000 higher than at sea level, which amounts to a dose of 0.50–1.0 Gy/year. Since the overwhelming majority of fungi, melanized or not, can withstand doses up to 1.7×10 4 Gy [9] , there is no apparent requirement for melanin as a radiation protector. On the other hand, biological pigments play a major role in photosynthesis by converting the energy of light into chemical energy. Chlorophylls and carotenoids absorb light of certain wavelengths and help convert photonic energy into chemical energy during photosynthesis. Given that melanins can absorb visible and UV light of all wavelengths [16] , we hypothesized that exposure to ionizing radiation would change the electronic properties of melanin and affect the growth of melanized microorganisms. Here we report the results of physico-chemical investigations of melanin electronic properties after radiation exposure and the enhanced growth of melanized fungi under conditions of radiation flux.

The role of melanin in microorganisms living in high electromagnetic radiation fluxes is even more intriguing when the pigment is considered from a paleobiological perspective. Many fungal fossils appear to be melanized [10] , [11] . Melanized fungal spores are common in the sediment layers of the early Cretaceous period when many species of animals and plants died out which coincides with the Earth's crossing the “magnetic zero” resulting in the loss of its : “shield” against cosmic radiation [12] . Additionally, radiation from a putative passing star called Nemesis has been suggested as a cause of extinction events [13] . The proliferation of melanotic fungi may even have contributed to the mass extinctions at the end of Cretaceous period [14] . A symbiotic association of plants and a melanotic fungus that allows for extreme thermotolerance has been attributed to heat dissipating properties of melanin [15] . Melanotic fungi inhabit the extraordinarly harsh climate of Antarctica [5] . Hence, melanins are ancient pigments that have probably been selected because they enhance the survival of melanized fungi in diverse environments and, perhaps incidentally, in various hosts. The emergence of melanin as a non-specific bioprotective material may be a result of the relative ease with which these complicated aromatic structures can be synthesized from a great variety of precursors [2] , [4] , [5] , [16] – [23] .

The term “melanin” originates from melanos - a Greek word for black. Melanin is a high molecular weight pigment, ubiquitous in nature, with a variety of biological functions [1] . Many fungi constitutively synthesize melanin [2] , which is likely to confer a survival advantage in the environment [3] by protecting against UV and solar radiation [reviewed in 4] . Melanized microorganisms inhabit some remarkably extreme environments including high altitude, Arctic and Antarctic regions [5] . Most dramatically, melanized fungal species colonize the walls of the highly radioactive damaged reactor at Chernobyl [6] and surrounding soils [7] . These findings, and the laboratory observations of the resistance of melanized fungi to ionizing radiation [8] , [9] , suggest a role for this pigment in radioprotection.

Results

Metabolic activity of melanized and non-melanized cells in the presence of electromagnetic radiation We investigated whether the changes in electron transfer properties of melanin post exposure to ionizing radiation (high-energy photons, see Table 2) may also be observed in melanized cells exposed to ionizing radiation. The metabolic activity of C. neoformans cells was evaluated with 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT assay) [28] and 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT assay). The use of XTT and MTT assays in parallel can help to define the location of the melanin-mediated electron transfer in the cells since positively charged MTT is taken into the cells via the plasma membrane potential and is reduced intracellularly; while the negatively charged XTT is largely cell-impermeable and its reduction occurs extracellularly, at the cell surface [29]. The melanized and non-melanized C. neoformans cells were exposed to ionizing radiation in the dark at 22°C overnight. The irradiation was performed in a constant field of 0.05 mGy/hr, a non-fungicidal radiation dose that is comparable to the doses inside the Chernobyl reactor [6]. Following exposure to radiation, the XTT or MTT reagents were added to the samples and absorbance was measured at 492 or 550 nm, respectively. The XTT assay showed significant increase in electron-transfer events in the irradiated melanized cells in comparison with non-irradiated melanized or irradiated non-melanized cells (Fig. 5a). Increased absorbance at 492 nm was also observed for dead (heat killed) melanized cells in comparison to non-melanized ones, showing that melanin can reduce XTT reagent by itself (Fig 5a). Irradiation of dead cells caused significant increase in the XTT reduction, thus confirming our hypothesis that radiation enhances electron-transfer properties of melanin. In contrast, there was no difference between the irradiated and non-irradiated melanized and non-melanized cells subjected to MTT assay (Fig. 5b). The difference between the MTT and XTT assays may be explained by the occurrence of radiation-related melanin-mediated electron transfer events near cell wall where melanin is located that led to higher XTT reduction in irradiated melanized samples. Interestingly, both irradiated and non-irradiated melanized cells showed higher activity by MTT assay than non-melanized cells (Fig. 5a, b). Given that melanization is associated with reduced pore size that could reduce passive nutrient uptake [25] and that melanin is synthesized from highly reactive cytotoxic intermediates of the oxidation of L-Dopa - it is possible that melanization requires a higher metabolism for cell survival. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. The influence of ionizing radiation or heat on the metabolic activity of melanized and non-melanized C. neoformans cells. a, b) irradiated and non-irradiated cells: a) XTT; b) MTT. c) XTT of cells grown at room temperature (22°C) or at 30°C. The cells were kept in the dark while being exposed to ionizing radiation or different temperatures. Following the exposure, XTT or MTT reagents were added to the samples and absorbance was measured at 492 or 550 nm for XTT and MTT, respectively. https://doi.org/10.1371/journal.pone.0000457.g005 In another series of experiments, the melanized and non-melanized cells were grown overnight in the dark at room temperature (22°C) or 30°C. Melanized cells demonstrated increased XTT reduction activity at both temperatures in comparison with non-melanized controls (Fig. 5c), and increasing the temperature to 30°C caused a statistically significant increase in XTT reduction in melanized cells (P<0.05) while a small decrease was observed for non-melanized cells. Overall, these experiments showed the increase in electron transfer properties of melanin in melanized cells post exposure to ionizing radiation and to less extent - to heat.

Ionizing radiation enhances growth and 14C-acetate uptake of melanized C. neoformans cells To expand the observations of the influence of irradiated melanin on the growth of melanized cells, we measured the growth of melanized and non-melanized C. neoformans cells supplied with limited nutrients and placed into the radiation flux. To maintain a steady population of melanized cells, we used the same H99 strain of C. neoformans as in XTT/MTT experiments since it is capable of making melanin when maintained with L-Dopa while its laccase disrupted [Lac(-)] mutant is incapable of melanization [30]. The cells were grown into stationary phase up to 30 hr. There were significantly more (P = 0.006) CFUs for irradiated melanized wild type H99 samples at 18, 23 and 30 hr than for non-irradiated samples (Fig. 6a), while the difference in CFUs at 18 hr between irradiated and non-irradiated Lac(-) mutant was not significant (Fig. 6b). Lac(-) without radiation in the presence of L-dopa grows better than wild type H99 (Fig. 6a and b). There was also a slight increase in the CFU's of irradiated Lac(-) cells at 23 and 30 hr. However, the crucial difference between the wild type H99 and Lac(-) cells is that the exposure to ionizing radiation produced approximately 2.5 times more CFUs in irradiated melanized cells than in non-irradiated melanized controls, while irradiation of Lac(-) cells resulted only in a 1.1-fold increase in CFUs (Fig. 6e). The dry weight measurements performed at 20 hr showed a consistent and significant 6.5% increase for irradiated melanized samples (P = 0.02) while there was no difference in weight for the mutant strain after irradiation. The relatively small yet significant increase in dry weight of the melanized cells is a result of the high percentage of immature cells, with smaller capsules synthesized de novo in the dividing melanized irradiated cell culture. In this regard, a cell diameter that is one-half to one-third of that for a mature cell results in a 8- and 26-fold decrease in cell mass, respectively. Quantification of whole cell sizes using India ink stained cells showed that proximately 50% of melanized irradiated cells had volumes 2 times smaller than those in the irradiated Lac(-) mutant population (results not shown), accounting for the relatively small increase in the dry weight of the melanized H99 samples in comparison to their larger increase in CFUs. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. Growth and incorporation of 14C-acetate by melanized C. neoformans H99 cells and non-melanized Lac(-) H99 cells lacking the laccase enzyme under conditions of limited nutrients supply in a radiation field of 0.05 mGy/hr or at background radiation level. a) growth of melanized H99 cells; b) growth of non-melanized Lac(-) H99 cells; c) incorporation of 14C-acetate into melanized H99 cells; d) incorporation of 14C-acetate into non-melanized Lac(-) H99 cells; e) ratio of irradiated to non-irradiated cells CFUs and cpms ratios (normalized CFUs and cpms) for melanized H99 and non-melanized Lac(-) H99 cells. https://doi.org/10.1371/journal.pone.0000457.g006 To obtain additional evidence that exposure to ionizing radiation enhanced melanized cell growth, we measured the incorporation of a 14C-labeled carbon source (acetate) into melanized and non-melanized C. neoformans cells with and without radiation flux. In the photosynthesis field the incorporation of 14C-acetate in bacteria subjected to visible light is considered to be indicative of their photoheterotrophic capabilities [31]. We measured a lower absolute uptake of 14C-acetate by wild type H99 compared to Lac(-) cells (Fig. 6c,d). There was no incorporation of 14C-acetate into heat killed melanized or non-melanized cells, which excludes the possibility that radiation promoted the passive absorption of 14C-acetate on melanin. Importantly, when melanized and non-melanized Lac(-) H99 cells were incubated with 14C-acetate with and without radiation – there was almost 3 times more incorporation of 14C-acetate into irradiated melanized cells than into non-irradiated melanized cells, while the ratio of 14C-acetate incorporation into irradiated to non-irradiated Lac(-) cells was only slightly higher than 1 (Fig. 6c,d and e). Overall, these results demonstrate that the presence of melanin contributes to the enhancement of cellular growth upon exposure to ionizing radiation in conditions of limited nutrients.