2013

University of Toronto

[RADIOACTIVITY AND FUNGI,
THE ROLE OF MELANIN]

Introduction
Melanin has long been identified as a key pigment molecule in almost all organisms. It
has a many different biological functions, from its use as a defense mechanism against predators,
to aiding the ability of cells to protect against harmful UV radiation. As a molecule, it exists in
two forms, primarily Eumelanin, a dark brown pigment made of 5,6-dihydroxyindole (DHI) and
5,6-dihydroxyindole-2-carboxylic acid monomers, with relatively low sulfur content (<1%), and
Pheomelanin, a reddish-brown pigment made of Benzothiazine/Benzothiazole subunits, with
relatively high sulfur content (10-12%) (Ito S, 1985). The slightly different structure of these two
melanin molecules conveys slightly different chemical properties, but is typically used in the
same way in organisms theyre found in. As well, the widespread use of Melanin can also be
attributed to the ease at which its synthesized (Ito S., 1985).
Many fungi actively synthesize Melanin, and its two known roles so far are its uses as a
virulence factor (Wheeler & Bell A., 1988), and as a method to protect the cells in harsh
environments. From the extreme cold (Robinson, 2001) to the high levels of radioactivity of the
Chernobyl reactors and coolant water, (Zhdanova et al. 2001), melanin has been identified in
almost all fungal organisms living in these harsh environments. Studies looking into Fungal
fossils have identified that almost all fungi that survived through the period in the past dubbed
Magnetic Zero, where the Earth lost its ability to protect against cosmic radiation, were
melanized (Hulot et al, 2003). In addition, lab results suggest melanin has a role on
radioprotectivity (Mirchink et al. 1972). However, further analysis showed that a fungicidal dose
for non-melanized radiation is 1.710
4
Gray, which are orders of magnitude higher than levels
found in Chernobyl (Saleh et al, 1988).
This suggested a new role of melanin in Fungi under ionizing radiation. Since pigments
like Chlorophyll have long been known to be able to harvest light energy at very specific
wavelengths, scientists have brought forth a new theory: that ionizing radiation changes the
properties of melanin, possibly allowing fungus to use it as a method of harvesting energy
(Dadachova et al., 2007).
Radiotrophic Fungi Isolated from Chernobyl Reactors
Radiotrophic fungi are fungi that are believed to be able to use external radiation as a
source of energy. The origins of this were first discovered in 1991, when Zhdanova et al.
discovered and extracted 5 samples of fungi, including Cladosporium cladosporioides and
Penicillium roseo-purpureum, from soils surrounding the Chernobyl reactor (Zhdanova et al.,
1991). When grown in the presence of radioactive particles (from the Chernobyl Reactor), it was
seen fungi actively grew towards and into these particles, completely breaking them down over
50-100 days (Zhdanova et al, 1994).
In a more recent study conducted by Zhdanova et al, where they used external beams of
beta and gamma radiation (in the forms of
32
P and
109
Cd respectively) to control for confounding
factors, researchers showed two-thirds of fungi actively grew towards radioactive sources
(Zhdoanova et al, 2004). In total, they showed that two-thirds of all fungi tested showed
significant directional growth towards the particles giving off gamma radiation. While not
significant, there was still directional growth towards the sources of beta-radiation. From these
studies, the authors concluded that fungi actively grew towards ionizing radiation, and proposed
that Melanin might act in a similar manner to Chlorophyll, except with the ability to absorb a
much wider wavelength.
Radiotrophic Fungi on the Russian Orbital Station, Mir
One unlikely source of Radiotrophic fungi was found on both the former Russian space
station Mir, as well as the current International Space Station (Novikova et al, 2006). While it
did exist as a microgravity research station, the fungal species isolated were not from the
scientific experiments, implicating contamination by flight materials, the crew, or possibly other
experiments. It was shown that, as long as some level of humidity was maintained, even in the
presence of little or no other biological material, these fungi were able to survive, and even thrive
( Novikova et al). All fungi found on the station were intrinsically melanized, implicating
melanin in aiding the survival of the fungi.
Another interesting phenomena, regarding the role of fungi in space, was discovered in
the Biorisk experiment (Novikova et al., 2007), conducted to identify if any fungal or bacterial
organisms would undergo morphological changes that would become dangerous in space. This
experiment showed a slew of morphological changes in the fungi tested (Penicillium expansum
and Aspergillus versicolor), including increased melanin and increased mitochondrial production
(Novikova et al., 2007) under nutrient-poor conditions. Considering that the level of background
radiation, 4 centi-Gray per year, is nowhere near fungicidal, this also implies that the role of
melanin is not acting as a shield against radiation.
Fungal Growth and Radiation
Another source of Fungi that appeared to thrive off some form of radiation was found at
the Evolution Canyon of Israel. This canyon is characterized by the two slopes, with the
southern-facing slope receiving up to 8-times more sunlight than the northern facing slope. As
such, it was found that melanin levels differed between the northern-facing slopes compared to
the southern facing slope, even when comparing the exact same species. Specifically, the
melanized fungi Aspergillus niger found on the northern-facing slopes were found to have
melanin levels 3-fold higher compared to their southern counterparts (Singaravelan et al., 2008).
In addition, their study found that the northern facing slopes survived much better under the
presence of UVA radiation, and researchers proposed that melanin was induced in A. niger as an
adaptive trait.
However, when 11 fungi from the northern facing slope and similar counterparts were
isolated from the southern facing slope, it was found that, up to 400 Gray of Gamma radiation,
the growth rate of Fungi isolated from southern slope showed significantly higher growth
compared to that found on the northern slope (explained by higher melanin levels), but when
radiation was increased to 4,000 gray, the rate of growth of the fungi found on the Northern
slope matched up to that found on the Southern slope. Upon 10,000 Gray of radiation, growth
rate for both fungi slowed to a stop at about the same time. This suggests that the effect of
radiation on growth is affected by the amount of melanin present in the fungi itself.
Another study, conducted by Dadachova et al. (2007), looked at the effects of radiation
on the growth of 3 separate fungal strains (Cryptococcus neoformans, Cladosporium
sphaerospermum, and Wangiella dermatitidis. When they measured the fungis ability to grow
under ionizing radiation, they showed that mealanized fungi they studied showed increased
growth under radiation (2.4 fold increase in C. neoformans, 1.6 fold increase in C.
sphaerospermum, and 1.3 fold increase in W. dermatitidis).
However, they while they showed that melanized cells grew better in radiation, they also
showed that non-irradiated non-melanized cells grew better than their melanized counterparts in
C. neoformans. As well, while there was a large increase in colony-forming units of melanized
fungal cells under radiation, these colonies were a lot more immature compared to their non-
melanized counterparts, and had a lot smaller radii (8-26 fold less mass per colony). When the
dry weight was measured for each of the 3 fungi studied, it showed only a 7-9% increase in dry
weight under ionizing radiation. This suggested that the process of making melanin had negative
effects on fitness for fungi that didnt intrinsically make melanin, likely due to toxic
intermediates in the process of making melanin.
Mechanisms for the Metabolism of Radiation
Knowing that fungi showed increased growth under the presence of radiation, several hypothesis
were proposed about the precise mechanisms of this action. One such study looked at the ability
of radiation to affect the ability of Melanin to act as an electron donor (Gan et al., 1976).
Irradiated melanin showed a 4-fold increase in ability to reduce NADH under the presence of
melanin(Dadochova et al, 2007). However, what was curious was that all radiation, regardless
including thermal, light, and ultraviolet, caused this increase in rate of reduction, showing that
this rate was not affected by the level of electron energies, but rather the presence of the energy.
Furthermore, they found that exposure to radiation caused an increased ability to metabolize, as
measured an XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)
and MTT ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, which is
typically used to measure cell viability (Mossman et al., 1983). Because of the inability of XTT
to cross the cell membrane, this assay was also used to identify the localization of the increased
metabolic activity, which showed that increased metabolism was localized to where melanin was
located (Dadochova et al., 2007). However, melanized cells also showed a slight increase in
metabolic activity, which was theorized to be caused by the melanins ability to shrink cellular
pores, likely causing the cell to increase cellular metabolism in order to survive.
A more recent study looked at the effect of ionizing radiation on the ATP levels of a
fungal cell. This experiment showed that, in melanized cells under all types of radiation
(Gamma, Ultraviolet, and Sunlight), exposure actually decreased ATP levels (Dadochova et al,
2011). They proposed numerous mechanisms for this, one of which being that Fungus utilize the
energy from the EM radiation in a way similar to photosynthesis, which causes ATP
consumption but increases the amount of NADH in cells. This is supported by previous studies
(Dadochova et al, 2007) (Zhdanova et al, 2008) which all show that melanized cells undergo
better growth when exposed to ionizing radiation under limited nutrient conditions.
Conclusion
Overall, only one thing could be said about the role of radioactivity in melanin: that more
study is required. Melanin polymers are still poorly understood, and their role in increasing cell
size can only be speculated upon as of now, but we can say that the one property of melanin that
makes it an amazing pigment is its capability to absorb all types of electromagnetic radiation.
This confers the known ability to shield, but a still relatively unknown process in energy
transduction. Extreme environments push the boundaries of evolution, some of which we are still
discovering now, such as radiotrophic fungi: organisms that use the extremely dangerous
radiation to promote their own growth.

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