**3. Health**

Melanin's unique properties allow it to have diverse applications in the field of human health, whether in a pharmaceutical, medical device, or antimicrobial. One of the most heavily studied health applications of melanin involves protection from radiation, given that fungal melanin is known for its radioabsorptive properties.

For example, in an experiment where mice were fed black mushrooms *Auricularia auricila-judae* and soon after irradiated, they tended to exhibit improved survival than the control over the course of 45 days [51]. Researchers postulated that melanin's ability to dissipate Compton electron energy and scavenge free radicals would shield the mice's gastrointestinal (GI) tract, preventing cellular apoptosis. When mouse GI tissue was examined 24 h post-irradiation, researchers found fewer apoptotic cells in the tissue of mice that had been fed black mushrooms. In addition, mice fed white mushrooms supplemented with melanin had the same outcome as those who were given melanized mushrooms to begin with, pointing to the role of melanin in the increased survival rates. However, because melanin is insoluble, its protective effects remained mostly limited to the gastrointestinal tract [51]. Melanin extract from *Auricularia auricula* also showed promise in reducing oxidative stress and enhancing survival in liver cells exposed to high doses of ethanol, providing a theoretical basis for the substance's ability to treat alcoholic liver disease. The investigators associated their results with the liver cell's activation of the antioxidase Nrf2 and the inhibition of the cytochrome CYP2E1, which produces ROS as it metabolizes ethanol. Additional investigations would further elucidate the pathway producing this phenomenon [52].

In a separate mice study, those given melanin from the fungus *Gliocephalotrichum simplex* not only experienced better survival from irradiation, but also showed improvement in spleen parameters, reduced oxidative stress in the liver, and reduced production of inflammatory cytokines [53]. The authors suggested that a key mechanism of melanin's protective property in the study was its ability to reverse the decrease in phosphorylation of the transducing protein ERK that is commonly seen upon radiation exposure [53]. Radioprotective technologies are needed for the protection of multiple vulnerable demographics. For example, radiation can have a harmful effect on patients receiving it for diagnostic or therapeutic purposes [51]. Cardiac diagnostic procedures alone account for about one-fifth of the radiation exposure per person per year in the US [54]. In addition, some occupations receive high levels of radiation exposure, including healthcare professionals [54] and military personnel [55].

Currently, melanin has been proposed as a potential vehicle to protect astronauts from space radiation [56]. Particulate radiation is of particular concern in space travel, especially as it can generate secondary radiation upon interaction with spacesuits or spacecraft components [56]. The idea of fungal melanins as a potential material to protect against dangerous radiation in space originated from a series of studies demonstrating the ability of fungal melanin to attenuate and protect against different types of ionizing radiation [4,57–59]. A comparison of melanized and non-melanized forms of *Cryptococcus neoformans* and *Cryomyces antarcticus* found in both cases that the melanized cells were more resistant to a deuteron dose nearly 300,000 times higher than the dose lethal for humans [4]. In addition, samples of *C. antarcticus* were exposed for over a year on the International Space Station (ISS) to the radiation conditions in low Earth orbit. The samples not only survived, but also maintained a mutational load below 5% and sustained metabolic activity [60]. The organism's thick layers of melanin have been implicated as a possible explanation for its survival [60]. Given melanin's ability to protect microorganisms from both ionizing and particulate radiation, it may be a favorable material for protection in manned space travel [56]. For example, a recent preprint concluded that a melanotic *Cladosporium sphaerospermum* plate on the International Space Station produced attenuated radiation levels compared to a control plate [61]. However, more research about the radioprotective properties of melanin is needed to confirm that the application of melanin to space materials is possible and beneficial.

Allomelanin has recently been investigated for both its porosity and its ability to absorb nerve gas stimulants in solution [62]. Given fungal melanin's natural ability to absorb harmful materials while allowing essential cellular materials such as water and nutrients to pass through, the material's intrinsic porosity is a valid possibility. In addition, melanin's extensive binding capacity can permit it to bind to many possibly harmful materials. Researchers worked with both synthetic allomelanin derived from 1,8-DHN and natural melanin ghosts derived from fungal cells. While the melanin ghosts provided a higher binding capacity for paraoxon and diazinon than some synthetic allomelanins, they were not as porous as the synthetic analogues [62]. In addition, investigators noted allomelanin's potential in protective, breathable fabrics by applying synthetic melanin on nylon-cotton fabric. Researchers also noted that the obtained melanin ghosts contained other polysaccharides such as chitin and glucan, making it more difficult to examine the properties of natural allomelanin [62].

Although not specifically for radioprotection, the melanized medicinal mushroom *Inonotus obliquus* has shown in vitro effects against tumor growth and diabetes mellitus [63]. In one investigation, B16-F10 melanoma cells that were exposed to aqueous extracts of *I. obliquus* for 48 h exhibited decreasing, dose-dependent viability [64]. The study also noted that the antitumor properties performed in vivo as well; mice that received the extract through an intraperitoneal route showed inhibited tumor mass growth. These effects were associated with G0/G1 arrest through the down-regulation of p53, pRb, and p27 proteins, although the investigators acknowledged the need for further research to elucidate a complete mechanism of action [64]. Thus, it is possible that melanin may not be a contributor to the extract's antitumor properties. However, melanin's presence in *I. obliquus* may still have health benefits. Water-soluble melanin complexes from the organism have shown insulin-sensitizing activity [65]. When the melanin complex was extracted via filtration and rotary evaporation, it reduced adiposity in high-fat obese mice [65].

Fungal melanin has also been implicated to have potential use in the functional biointerfaces used for stem cell manipulation [66]. Eumelanins were proposed for this purpose due to their antioxidant and electrical properties. However, eumelanin can be vulnerable to degradation from alkaline or oxidative stress. Manini et al. propose the alternative use of fungal allomelanins, citing the material's resistance to degradation and smoothness [66]. Such a mycomelanin film was able to encourage the differentiation of stem cells towards an endodermal lineage [66].
