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Physico-chemical evaluation of rationally designed melanins as novel nature-inspired radioprotectors.

Schweitzer AD, Howell RC, Jiang Z, Bryan RA, Gerfen G, Chen CC, Mah D, Cahill S, Casadevall A, Dadachova E - PLoS ONE (2009)

Bottom Line: There was no change in the quality or quantity of the stable free radicals after high-dose (30,000 cGy), high-energy ((137)Cs, 661.6 keV) irradiation, indicating a high degree of radical stability as well as a robust resistance to the ionizing effects of gamma irradiation.We propose that due to melanin's numerous aromatic oligomers containing multiple pi-electron system, a generated Compton recoil electron gradually loses energy while passing through the pigment, until its energy is sufficiently low that it can be trapped by stable free radicals present in the pigment.Controlled dissipation of high-energy recoil electrons by melanin prevents secondary ionizations and the generation of damaging free radical species.

View Article: PubMed Central - PubMed

Affiliation: Department of Nuclear Medicine, Albert Einstein College of Medicine, New York, New York, United States of America.

ABSTRACT

Background: Melanin, a high-molecular weight pigment that is ubiquitous in nature, protects melanized microorganisms against high doses of ionizing radiation. However, the physics of melanin interaction with ionizing radiation is unknown.

Methodology/principal findings: We rationally designed melanins from either 5-S-cysteinyl-DOPA, L-cysteine/L-DOPA, or L-DOPA with diverse structures as shown by elemental analysis and HPLC. Sulfur-containing melanins had higher predicted attenuation coefficients than non-sulfur-containing melanins. All synthetic melanins displayed strong electron paramagnetic resonance (2.14.10(18), 7.09.10(18), and 9.05.10(17) spins/g, respectively), with sulfur-containing melanins demonstrating more complex spectra and higher numbers of stable free radicals. There was no change in the quality or quantity of the stable free radicals after high-dose (30,000 cGy), high-energy ((137)Cs, 661.6 keV) irradiation, indicating a high degree of radical stability as well as a robust resistance to the ionizing effects of gamma irradiation. The rationally designed melanins protected mammalian cells against ionizing radiation of different energies.

Conclusions/significance: We propose that due to melanin's numerous aromatic oligomers containing multiple pi-electron system, a generated Compton recoil electron gradually loses energy while passing through the pigment, until its energy is sufficiently low that it can be trapped by stable free radicals present in the pigment. Controlled dissipation of high-energy recoil electrons by melanin prevents secondary ionizations and the generation of damaging free radical species.

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Survival of CHO cells after a 600 cGy dose with synthetic melanins compared to controls (without melanin or with melanin ‘ghosts’ from C. neoformans).Images of plates irradiated at (a) 85 keV (200 kVp) and (c) 113 keV (320 kVp) with each row of 3 wells corresponding to either no melanin, C. neoformans ghosts, MEL1, MEL4, MEL2, or MEL3b (from top left to bottom right). Clonogenic survival plots are shown for (b) 85 keV and (d) 113 keV. The clonogenic survival of irradiated cells was determined by crystal violet staining. Error bars show the standard deviations.
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pone-0007229-g004: Survival of CHO cells after a 600 cGy dose with synthetic melanins compared to controls (without melanin or with melanin ‘ghosts’ from C. neoformans).Images of plates irradiated at (a) 85 keV (200 kVp) and (c) 113 keV (320 kVp) with each row of 3 wells corresponding to either no melanin, C. neoformans ghosts, MEL1, MEL4, MEL2, or MEL3b (from top left to bottom right). Clonogenic survival plots are shown for (b) 85 keV and (d) 113 keV. The clonogenic survival of irradiated cells was determined by crystal violet staining. Error bars show the standard deviations.

Mentions: We then investigated the radioprotective properties of the synthetic melanins in a biological system in presence or absence of melanins by irradiating Chinese hamster ovary (CHO) cells – a cell line extensively used in radiobiology – with doses of X-rays of different energies that are lethal for humans (experimental set-up, Fig. 3). The clonogenic survival of irradiated cells was determined by crystal violet staining. To investigate whether fungal melanins conferred radioprotection of CHO cells, we also mixed CHO cells with C. neoformans fungal melanin shells (dubbed “ghosts” in [2]), which conferred a protective effect towards non-melanized fungal cells in our previous studies [11]. MEL2, MEL3b, and MEL4 were found to be equally radioprotective in spite of having very different numbers of stable free radicals as per EPR: CHO cells incubated with either MEL2, MEL3b, or MEL4 manifested 23, 21, and 21% increases, respectively, in survival after 600 cGy irradiation at 85 keV (200 kVp) when compared to controls with no melanin (P<0.05); and 7, 10, and 9% increases, respectively, in survival at 113 keV (320 kVp) (P<0.05) (Fig. 4). In contrast, the presence in cell culture of MEL1 – the melanin with the lowest attenuation coefficient of all melanins tested for radioprotection and with intermediate number of stable radicals - produced no dramatic increases in cell survival, providing an internal control for the association of the radioprotective effect with only certain types of melanin under given experimental conditions. The radioprotection by melanins was also dose-dependent – Fig. 5 shows the data for irradiation of CHO cells with 661 keV 137Cs radiation in presence of 20 or 100 µg/mL MEL2 with the higher concentration of melanin being significantly more radioprotective than the lower one.


Physico-chemical evaluation of rationally designed melanins as novel nature-inspired radioprotectors.

Schweitzer AD, Howell RC, Jiang Z, Bryan RA, Gerfen G, Chen CC, Mah D, Cahill S, Casadevall A, Dadachova E - PLoS ONE (2009)

Survival of CHO cells after a 600 cGy dose with synthetic melanins compared to controls (without melanin or with melanin ‘ghosts’ from C. neoformans).Images of plates irradiated at (a) 85 keV (200 kVp) and (c) 113 keV (320 kVp) with each row of 3 wells corresponding to either no melanin, C. neoformans ghosts, MEL1, MEL4, MEL2, or MEL3b (from top left to bottom right). Clonogenic survival plots are shown for (b) 85 keV and (d) 113 keV. The clonogenic survival of irradiated cells was determined by crystal violet staining. Error bars show the standard deviations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749938&req=5

pone-0007229-g004: Survival of CHO cells after a 600 cGy dose with synthetic melanins compared to controls (without melanin or with melanin ‘ghosts’ from C. neoformans).Images of plates irradiated at (a) 85 keV (200 kVp) and (c) 113 keV (320 kVp) with each row of 3 wells corresponding to either no melanin, C. neoformans ghosts, MEL1, MEL4, MEL2, or MEL3b (from top left to bottom right). Clonogenic survival plots are shown for (b) 85 keV and (d) 113 keV. The clonogenic survival of irradiated cells was determined by crystal violet staining. Error bars show the standard deviations.
Mentions: We then investigated the radioprotective properties of the synthetic melanins in a biological system in presence or absence of melanins by irradiating Chinese hamster ovary (CHO) cells – a cell line extensively used in radiobiology – with doses of X-rays of different energies that are lethal for humans (experimental set-up, Fig. 3). The clonogenic survival of irradiated cells was determined by crystal violet staining. To investigate whether fungal melanins conferred radioprotection of CHO cells, we also mixed CHO cells with C. neoformans fungal melanin shells (dubbed “ghosts” in [2]), which conferred a protective effect towards non-melanized fungal cells in our previous studies [11]. MEL2, MEL3b, and MEL4 were found to be equally radioprotective in spite of having very different numbers of stable free radicals as per EPR: CHO cells incubated with either MEL2, MEL3b, or MEL4 manifested 23, 21, and 21% increases, respectively, in survival after 600 cGy irradiation at 85 keV (200 kVp) when compared to controls with no melanin (P<0.05); and 7, 10, and 9% increases, respectively, in survival at 113 keV (320 kVp) (P<0.05) (Fig. 4). In contrast, the presence in cell culture of MEL1 – the melanin with the lowest attenuation coefficient of all melanins tested for radioprotection and with intermediate number of stable radicals - produced no dramatic increases in cell survival, providing an internal control for the association of the radioprotective effect with only certain types of melanin under given experimental conditions. The radioprotection by melanins was also dose-dependent – Fig. 5 shows the data for irradiation of CHO cells with 661 keV 137Cs radiation in presence of 20 or 100 µg/mL MEL2 with the higher concentration of melanin being significantly more radioprotective than the lower one.

Bottom Line: There was no change in the quality or quantity of the stable free radicals after high-dose (30,000 cGy), high-energy ((137)Cs, 661.6 keV) irradiation, indicating a high degree of radical stability as well as a robust resistance to the ionizing effects of gamma irradiation.We propose that due to melanin's numerous aromatic oligomers containing multiple pi-electron system, a generated Compton recoil electron gradually loses energy while passing through the pigment, until its energy is sufficiently low that it can be trapped by stable free radicals present in the pigment.Controlled dissipation of high-energy recoil electrons by melanin prevents secondary ionizations and the generation of damaging free radical species.

View Article: PubMed Central - PubMed

Affiliation: Department of Nuclear Medicine, Albert Einstein College of Medicine, New York, New York, United States of America.

ABSTRACT

Background: Melanin, a high-molecular weight pigment that is ubiquitous in nature, protects melanized microorganisms against high doses of ionizing radiation. However, the physics of melanin interaction with ionizing radiation is unknown.

Methodology/principal findings: We rationally designed melanins from either 5-S-cysteinyl-DOPA, L-cysteine/L-DOPA, or L-DOPA with diverse structures as shown by elemental analysis and HPLC. Sulfur-containing melanins had higher predicted attenuation coefficients than non-sulfur-containing melanins. All synthetic melanins displayed strong electron paramagnetic resonance (2.14.10(18), 7.09.10(18), and 9.05.10(17) spins/g, respectively), with sulfur-containing melanins demonstrating more complex spectra and higher numbers of stable free radicals. There was no change in the quality or quantity of the stable free radicals after high-dose (30,000 cGy), high-energy ((137)Cs, 661.6 keV) irradiation, indicating a high degree of radical stability as well as a robust resistance to the ionizing effects of gamma irradiation. The rationally designed melanins protected mammalian cells against ionizing radiation of different energies.

Conclusions/significance: We propose that due to melanin's numerous aromatic oligomers containing multiple pi-electron system, a generated Compton recoil electron gradually loses energy while passing through the pigment, until its energy is sufficiently low that it can be trapped by stable free radicals present in the pigment. Controlled dissipation of high-energy recoil electrons by melanin prevents secondary ionizations and the generation of damaging free radical species.

Show MeSH