ISSN 16076729, Doklady Biochemistry and Biophysics, 2015, Vol. 460, pp. 30–33. © Pleiades Publishing, Ltd., 2015. Original Russian Text © V.S. Bondar, A.P. Puzyr, A.E. Burov, S.E. Medvedeva, E.K. Rodicheva, T.V. Kobzeva, A.R. Melnikov, T.Y. Karogodina, S.B. Zikirin, D.V. Stass, Yu.N. Molin, J.I. Gitelson, 2015, published in Doklady Akademii Nauk, 2015, Vol. 460, No. 4, pp. 468–471.
BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY
Effect of Ionizing Radiation on the Luminescence of Mycelium of Luminous Fungus Neonothopanus nambi V. S. Bondara, b, A. P. Puzyra, b, A. E. Burovb, c, S. E. Medvedevaa, b, E. K. Rodichevaa, b, T. V. Kobzevad, A. R. Melnikovd, e, T. Y. Karogodinad, S. B. Zikirind, e, D. V. Stassd, e, Academician Yu. N. Molind, e, and Academician J. I. Gitelsona, b Received July 18, 2014
DOI: 10.1134/S1607672915010093
Currently, the luminescent systems and the mech anisms of luminescence of many living organisms are well studied: the enzymes (luciferases) catalyzing the lightemission reactions and their substrates (luciferins) of these organisms were isolated and char acterized [1]. However, this problem for higher lumi nous fungi remains unsolved, and the molecular orga nization of their luminescent system is still poorly understood. First of all, it remains unclear which enzyme (or enzyme complex) performs the function of luciferase in fungi and what is the structure of luciferin, the substrate of lightemitting reaction. In the early 1990s, it was assumed that the mechanism of fungal luminescence involve reactive oxygen species (ROS) and enzymes with oxidase function [1, 2].
oxidase complex and cytochrome P450 system) can catalyze the oxidation of organic substrates (including luciferin) with the involvement of ROS. Thus, the study of the relationship between the for mation and transformation of reactive oxygen radicals and the luminescence of higher fungi is important for understanding the mechanisms of luminescence. It is well known [6, 7] that the production of ROS in bio logical objects can be stimulated by exposure to phys ical, chemical, and biological factors. In particular, the activation of ROS generation under the influence of ionizing radiation and, as a result, the stimulation of superweak chemiluminescence of plant and animal cells was shown [8, 9]. However, in available literature we found no similar papers describing the stimulation of luminescence of luminous fungi. In the present study, we investigated the effect of ionizing radiation on the luminescence of the fungus N. nambi. Experiments were performed with the mycelium of N. nambi inhabiting the tropical forests of South Viet nam [10]. The culture of the fungus for research was kindly provided by Vietnamese researcher Dao Thi Van (private collection of strains of the BIOLUMI Co., Ltd. company, Vietnam). Samples of luminous mycelium in the form of films and globules were obtained using the technologies of stationary and sub merged cultivation of the fungus, which were devel oped by us earlier [3, 5]. To remove the components of the culture medium and exometabolites that can inhibit light emission, the grown mycelium was washed with deionized water (DW) obtained using the MilliQ system (Millipore, United States). For this purpose, the film mycelium was incubated in DW for 12–14 h. The globular mycelium was also placed in DW and aerated for the same period of time. It was previously shown [4, 5] that washing with water signif icantly increases N. nambi mycelium luminescence. The effect of ionizing radiation on the intensity and spectra of light emission of mycelium was evaluated using the instrument developed by us earlier and the design of which was described in detail in [11]. The
The results of our recent studies of the luminous fungus Neonothopanus nambi also indicate that ROS and oxidases are involved in the mechanism of its luminescence and indicate the relationship of the fungal luminescent system with membrane structures [3–5]. On the basis of these data, it was assumed that the fol lowing membranebound enzyme systems can be involved in the lightemitting reaction: oxidases (including peroxidases) of the lignindegrading com plex, cytochrome P450 system, and enzymes of the mitochondrial respiratory chain. These enzyme sys tems can produce ROS, and two of them (ligninolytic
a
Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Akademgorodok, Krasnoyarsk, 660036 Russia b Siberian Federal University, Svobodnyi pr. 79, Krasnoyarsk, 660041 Russia c Special DesignTechnology Bureau “Nauka,” Krasnoyarsk Scientific Center, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk, Russia d Voevodskii Institute of Chemical Kinetics and Combustion, Siberian Branch, Russian Academy of Sciences, ul. Institutskaya 3, Novosibirsk, 630090 Russia e Novosibirsk State University, ul. Pirogova 2, Novosibirsk, 630090 Russia email: [email protected] 30
EFFECT OF IONIZING RADIATION ON THE LUMINESCENCE Luminescence intensity, abr. units 80
31
Light emission, arb. units 16 14 12
2
60 10
1 8 40
6 4 2
20
0 200 0
1
2
3
4
5
6
7
8
9 10 11 12 13 Time, h
Fig. 1. Typical luminescence kinetics of N. nambi myce lium samples placed in the measuring cuvette of the recording device.
device consists of a stationary Xray tube for irradiat ing the study object and a photodetector (PMT) with a system of lenses and a grating monochromator. This device allows measuring the lightemission intensity and recording the luminescence spectrum of an object before, during, and after irradiation. The sample (several globules or a film mycelium fragment (5 × 5 mm) was placed in a polystyrene cuvette 8 mm in diameter and 8 mm high, filled with DW. The cuvette was placed in the device, and the intensity and spectrum of the mycelium luminescence was recorded. During recording, the sample for differ ent periods of time was irradiated with a full range of bremsstrahlung at an accelerating voltage of 40 kV and an estimated dose rate of 85 krad/h. The luminescent signal was detected in the direction perpendicular to the incident Xray beam. The correction of the spec trum relative to the curve of the spectral sensitivity of the photomultiplier was not performed. It was shown that the luminescent signals recorded from the N. nambi mycelium samples placed in the measuring cuvette had the same type of kinetics (Fig. 1). The differences were observed only in the light emission intensity of different mycelium samples. At first, a high level of luminescence was observed; later, it decreased and reached a steadystate level, which was retained for a long time (several hours) (Fig. 1). The elevated level of luminescence detected at the initial period of time was apparently due to the mechanical action exerted on the fungal mycelium during the preparation of samples and their transfer into the mea suring cuvette. In the case of the globular mycelium, the lightemission intensity decreased to the steady DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 460
300
400
500
600 700 Wavelength, nm
Fig. 2. Light emission spectra of N. nambi mycelium sam ples: (1) control sample (without irradiation) and (2) experi mental sample (at constant Xray irradiation). The arrow indicates the band (λmax = 325 nm) of the radiation induced fluorescence of the measurement cuvette made of polystyrene.
state level more rapidly (from several to tens of min utes) compared to the film mycelium fragments (sev eral hours). This could be due to the fact that, during the preparation of the film mycelium samples, the fun gus experienced a much greater mechanical stress (trauma), and its recovery (relaxation) required more time. The experiments showed that Xray radiation does not change the lightemission spectrum of the fungus N. nambi. As can be seen from the data presented in Fig. 2, the luminescence spectra of the control (nonir radiated) and experimental (constantly exposed to Xray radiation) mycelium samples were identical and had a maximum at 525 nm. The presence of an addi tional band with a maximum at 325 nm in the experi mental samples (Fig. 2) was due to the radiation induced fluorescence of the material (polystyrene) from which the measuring cuvette was made. It was shown that the same band with a maximum at 325 nm was recorded when an empty cuvette was used. Our studies showed that a longterm (2–3 h) expo sure to Xray radiation stimulated the luminescence of the fungus N. nambi (Fig. 3). Our results show that irradiation of the mycelium for approximately 20 min led to an increase in its lightemission intensity. This increase was characterized by a slow kinetics: the luminescent signal reached a maximum level within several hours. The maximum values of the radiation stimulated light emission of the mycelium can exceed its initial luminescence level at 5–7 times and more (Fig. 3). The radiationstimulated luminescence was characterized by a slow (for several hours) decay kinet ics. It was shown that this step was insensitive to Xray 2015
32
BONDAR et al.
Luminescence intensity, abr. units 400
Luminescence intensity, abr. units 120 100 1
200
3
2
80 60 1
2 40 20
0 0
1
2
3
4
5
6
7
8
9 10 11 12 13 Time, h
0 0
1
2
3
4
5
6
7
8
9 10 11 12 13 Time, h
Fig. 3. Kinetics of luminescence of N. nambi mycelium under a longterm (2–3 h) exposure to irradiation. The arrows indicate the moments of switching on (1) and off (2) the Xray irradiation.
Fig. 4. Kinetics of luminescence of N. nambi mycelium at repetitive shortterm (10 min) exposure to irradiation. The arrows show the moments of switching on the Xray irradi ation.
radiation, because switching it off did not change the kinetics of the luminescent signal decay (Fig. 3). As can be seen from these data, after decrease in the lumi nescence intensity to a certain level significant changes in the luminescence kinetics of the mycelium were observed. A significant decrease in its light emis sion was recorded for a relatively short (approximately 30 min) period of time. Then, the mycelium com pletely lost the ability to emit light: the values of recorded signals coincided with the background noise of the measuring system. The observed effect of Xray radiation is typical for this fungal species. Similar radi ationinduced changes in the light emission were recorded using different samples (globules and films) of N. nambi mycelium obtained at periodic cultivation of the fungus for six months. In the next series of experiments (Fig. 4) we showed that, at a fixed radiation power, a decrease in the time of Xray exposure of N. nambi mycelium was accom panied by a reduced stimulatory effect on its lumines cence. As can be seen from the data, shortterm irra diations of mycelium were not accompanied by the loss of its luminescence. Moreover, periodic short term (10 min) irradiation of the mycelium against the background of attenuation of its radiationactivated luminescence resulted not only in new stimulation of luminescence but also in an increase in its level (Fig. 4). Probably, it may indicate a dosedependent effect of radiation on the light emission by the fungus. Thus, we demonstrated the stimulation of lumines cence of N. nambi mycelium exposed to ionizing radi ation. It is shown that the magnitude of the observed effect and the kinetics of luminescence depended on
the Xray radiation dose. Longterm exposure led to a significant increase in the light emission of the fungus with subsequent loss of its luminescence. Shortterm irradiations each time were accompanied by a lesser stimulation of luminescence; however, the fungus did not lose the ability to emit light. The irreversible loss of luminescence cannot be associated with the thermal inactivation of mycelium under a longterm exposure. Calculations showed that, at the heat capacity of water a 75.35 J/mol K and the radiation power of 85 krad/h, the temperature of the sample increased at a rate of not more than 0.2°C/h. On the basis of this fact, it can be assumed that the stimulation of luminescence radia tion is mediated by the generation of radicals in the aqueous medium surrounding and filling the myce lium. The presence of the latent period (approxi mately 20 min) before the phase of light signal devel opment, the increase and subsequent decrease in the luminescence intensity may reflect the amount of oxy gen radicals present in the system and the functional activity of the enzymes (or enzyme complexes) that ensure luminescence with the involvement of ROS. Under a longterm exposure, the amount of oxygen radicals generated in the sample can be sufficiently large. It is known [12] that the radiation yield of radicals in water is approximately 5/100 eV. On the basis of cal culations of the amount of water contained in a myce lium sample at a radiation power of 85 krad/h and exposure duration of 2 h, the amount of generated rad icals may reach approximately 1.5 × 1016. In turn, the high level of ROS induced by ionizing radiation will be accompanied by the development chain oxidation
DOKLADY BIOCHEMISTRY AND BIOPHYSICS
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2015
EFFECT OF IONIZING RADIATION ON THE LUMINESCENCE
reactions in the mycelium (for example, by the activa tion of lipid peroxidation). The activation of this pro cess by ionizing radiation is well known [13, 14]. In this case, the sharp decrease in the luminescent signal observed at the final stage of irradiation and the com plete loss of luminescence by the fungus can be associ ated with the disturbance of its enzyme complexes resulting from the oxidative damage. On the other hand, taken together, the abovementioned facts sug gest that the luminescence of higher fungi is an addi tional mechanism protecting them from the damaging action of ROS. This assumption seems plausible because the hypothesis that the light emission of living organisms is aimed at protecting against the damage caused by reactive oxygen radicals was expressed as early as the middle of the last century [15]. ACKNOWLEDGMENTS This work was supported in part by the Support Program for Interdisciplinary Projects of the Siberian Branch of the Russian Academy of Sciences (project no. 71) and the Program of the President of the Rus sian Federation for Support of Leading Scientific Schools (project no. NSh5744.2014.3). REFERENCES
2. Shimomura, O., Exp. Bot., 1992, vol. 43, pp. 1519– 1525. 3. Bondar, V.S., Puzyr, A.P., Purtov, K.V., et al., Dokl. Bio chem. Biophys., 2011, vol. 438, pp. 138–140. 4. Bondar, V.S., Shimomura, O., and Gitelson, J.I., J. Sib. Fed. Univ. Biol., 2012, vol. 5, no. 2, pp. 331–351. 5. Bondar, V.S., Rodicheva, E.K., Medvedeva, S.E., et al., Dokl. Biochem. Biophys., 2013, vol. 449, pp. 80–83. 6. Vladimirov, Y.A., in: Free Radicals in the Environment, Medicine and Toxicology, London: Richelieu Press, 1994, pp. 345–373. 7. Halliwell, B. and Gutteridge, J.M.C., Free Radicals in Biology and Medicine, Oxford: Oxford Press, 1999. 8. Mantel, J., Freidin, M., Bulich, A.A., et al., Phys. Med. Biol., 1983, vol. 28, pp. 599–602. 9. Asada, K., Ann. Rev. Plant Physiol. Plant. Mol. Biol., 1999, vol. 50, pp. 601–639. 10. Vydryakova, G.A., Dao, T.Van., Shoukouhi, P., et al., Mycology, 2011, vol. 3, pp. 89–99. 11. Kalneus, E.V., Melnikov, A.R., Korolev, V.V., et al., Appl. Magn. Resonance, 2013, vol. 44, pp. 81–96. 12. Spinks, J.W.T. and Woods, R.J., An Introduction to Radiation Chemistry, New York: Wiley, 1976. 13. Yin, H., Xu, L., and Porter, N.A., Chem. Rev., 2011, vol. 111, pp. 5944–5972. 14. Khalil, A. and Fulop, T., Can. J. Physiol. Pharmacol., 2001, vol. 79, pp. 114–121. 15. McElroy, W.D. and Strehler, B.L., Arch. Biochem., 1949, vol. 22, pp. 420–433.
1. Shimomura, O., Bioluminescence: Chemical Principles and Methods, Singapore: World Sci., 2006.
DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 460
33
Translated by M. Batrukova
2015
BIOCHEMISTRY, BIOPHYSICS AND MOLECULAR BIOLOGY
Effect of Ionizing Radiation on the Luminescence of Mycelium of Luminous Fungus Neonothopanus nambi V. S. Bondara, b, A. P. Puzyra, b, A. E. Burovb, c, S. E. Medvedevaa, b, E. K. Rodichevaa, b, T. V. Kobzevad, A. R. Melnikovd, e, T. Y. Karogodinad, S. B. Zikirind, e, D. V. Stassd, e, Academician Yu. N. Molind, e, and Academician J. I. Gitelsona, b Received July 18, 2014
DOI: 10.1134/S1607672915010093
Currently, the luminescent systems and the mech anisms of luminescence of many living organisms are well studied: the enzymes (luciferases) catalyzing the lightemission reactions and their substrates (luciferins) of these organisms were isolated and char acterized [1]. However, this problem for higher lumi nous fungi remains unsolved, and the molecular orga nization of their luminescent system is still poorly understood. First of all, it remains unclear which enzyme (or enzyme complex) performs the function of luciferase in fungi and what is the structure of luciferin, the substrate of lightemitting reaction. In the early 1990s, it was assumed that the mechanism of fungal luminescence involve reactive oxygen species (ROS) and enzymes with oxidase function [1, 2].
oxidase complex and cytochrome P450 system) can catalyze the oxidation of organic substrates (including luciferin) with the involvement of ROS. Thus, the study of the relationship between the for mation and transformation of reactive oxygen radicals and the luminescence of higher fungi is important for understanding the mechanisms of luminescence. It is well known [6, 7] that the production of ROS in bio logical objects can be stimulated by exposure to phys ical, chemical, and biological factors. In particular, the activation of ROS generation under the influence of ionizing radiation and, as a result, the stimulation of superweak chemiluminescence of plant and animal cells was shown [8, 9]. However, in available literature we found no similar papers describing the stimulation of luminescence of luminous fungi. In the present study, we investigated the effect of ionizing radiation on the luminescence of the fungus N. nambi. Experiments were performed with the mycelium of N. nambi inhabiting the tropical forests of South Viet nam [10]. The culture of the fungus for research was kindly provided by Vietnamese researcher Dao Thi Van (private collection of strains of the BIOLUMI Co., Ltd. company, Vietnam). Samples of luminous mycelium in the form of films and globules were obtained using the technologies of stationary and sub merged cultivation of the fungus, which were devel oped by us earlier [3, 5]. To remove the components of the culture medium and exometabolites that can inhibit light emission, the grown mycelium was washed with deionized water (DW) obtained using the MilliQ system (Millipore, United States). For this purpose, the film mycelium was incubated in DW for 12–14 h. The globular mycelium was also placed in DW and aerated for the same period of time. It was previously shown [4, 5] that washing with water signif icantly increases N. nambi mycelium luminescence. The effect of ionizing radiation on the intensity and spectra of light emission of mycelium was evaluated using the instrument developed by us earlier and the design of which was described in detail in [11]. The
The results of our recent studies of the luminous fungus Neonothopanus nambi also indicate that ROS and oxidases are involved in the mechanism of its luminescence and indicate the relationship of the fungal luminescent system with membrane structures [3–5]. On the basis of these data, it was assumed that the fol lowing membranebound enzyme systems can be involved in the lightemitting reaction: oxidases (including peroxidases) of the lignindegrading com plex, cytochrome P450 system, and enzymes of the mitochondrial respiratory chain. These enzyme sys tems can produce ROS, and two of them (ligninolytic
a
Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Akademgorodok, Krasnoyarsk, 660036 Russia b Siberian Federal University, Svobodnyi pr. 79, Krasnoyarsk, 660041 Russia c Special DesignTechnology Bureau “Nauka,” Krasnoyarsk Scientific Center, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk, Russia d Voevodskii Institute of Chemical Kinetics and Combustion, Siberian Branch, Russian Academy of Sciences, ul. Institutskaya 3, Novosibirsk, 630090 Russia e Novosibirsk State University, ul. Pirogova 2, Novosibirsk, 630090 Russia email: [email protected] 30
EFFECT OF IONIZING RADIATION ON THE LUMINESCENCE Luminescence intensity, abr. units 80
31
Light emission, arb. units 16 14 12
2
60 10
1 8 40
6 4 2
20
0 200 0
1
2
3
4
5
6
7
8
9 10 11 12 13 Time, h
Fig. 1. Typical luminescence kinetics of N. nambi myce lium samples placed in the measuring cuvette of the recording device.
device consists of a stationary Xray tube for irradiat ing the study object and a photodetector (PMT) with a system of lenses and a grating monochromator. This device allows measuring the lightemission intensity and recording the luminescence spectrum of an object before, during, and after irradiation. The sample (several globules or a film mycelium fragment (5 × 5 mm) was placed in a polystyrene cuvette 8 mm in diameter and 8 mm high, filled with DW. The cuvette was placed in the device, and the intensity and spectrum of the mycelium luminescence was recorded. During recording, the sample for differ ent periods of time was irradiated with a full range of bremsstrahlung at an accelerating voltage of 40 kV and an estimated dose rate of 85 krad/h. The luminescent signal was detected in the direction perpendicular to the incident Xray beam. The correction of the spec trum relative to the curve of the spectral sensitivity of the photomultiplier was not performed. It was shown that the luminescent signals recorded from the N. nambi mycelium samples placed in the measuring cuvette had the same type of kinetics (Fig. 1). The differences were observed only in the light emission intensity of different mycelium samples. At first, a high level of luminescence was observed; later, it decreased and reached a steadystate level, which was retained for a long time (several hours) (Fig. 1). The elevated level of luminescence detected at the initial period of time was apparently due to the mechanical action exerted on the fungal mycelium during the preparation of samples and their transfer into the mea suring cuvette. In the case of the globular mycelium, the lightemission intensity decreased to the steady DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 460
300
400
500
600 700 Wavelength, nm
Fig. 2. Light emission spectra of N. nambi mycelium sam ples: (1) control sample (without irradiation) and (2) experi mental sample (at constant Xray irradiation). The arrow indicates the band (λmax = 325 nm) of the radiation induced fluorescence of the measurement cuvette made of polystyrene.
state level more rapidly (from several to tens of min utes) compared to the film mycelium fragments (sev eral hours). This could be due to the fact that, during the preparation of the film mycelium samples, the fun gus experienced a much greater mechanical stress (trauma), and its recovery (relaxation) required more time. The experiments showed that Xray radiation does not change the lightemission spectrum of the fungus N. nambi. As can be seen from the data presented in Fig. 2, the luminescence spectra of the control (nonir radiated) and experimental (constantly exposed to Xray radiation) mycelium samples were identical and had a maximum at 525 nm. The presence of an addi tional band with a maximum at 325 nm in the experi mental samples (Fig. 2) was due to the radiation induced fluorescence of the material (polystyrene) from which the measuring cuvette was made. It was shown that the same band with a maximum at 325 nm was recorded when an empty cuvette was used. Our studies showed that a longterm (2–3 h) expo sure to Xray radiation stimulated the luminescence of the fungus N. nambi (Fig. 3). Our results show that irradiation of the mycelium for approximately 20 min led to an increase in its lightemission intensity. This increase was characterized by a slow kinetics: the luminescent signal reached a maximum level within several hours. The maximum values of the radiation stimulated light emission of the mycelium can exceed its initial luminescence level at 5–7 times and more (Fig. 3). The radiationstimulated luminescence was characterized by a slow (for several hours) decay kinet ics. It was shown that this step was insensitive to Xray 2015
32
BONDAR et al.
Luminescence intensity, abr. units 400
Luminescence intensity, abr. units 120 100 1
200
3
2
80 60 1
2 40 20
0 0
1
2
3
4
5
6
7
8
9 10 11 12 13 Time, h
0 0
1
2
3
4
5
6
7
8
9 10 11 12 13 Time, h
Fig. 3. Kinetics of luminescence of N. nambi mycelium under a longterm (2–3 h) exposure to irradiation. The arrows indicate the moments of switching on (1) and off (2) the Xray irradiation.
Fig. 4. Kinetics of luminescence of N. nambi mycelium at repetitive shortterm (10 min) exposure to irradiation. The arrows show the moments of switching on the Xray irradi ation.
radiation, because switching it off did not change the kinetics of the luminescent signal decay (Fig. 3). As can be seen from these data, after decrease in the lumi nescence intensity to a certain level significant changes in the luminescence kinetics of the mycelium were observed. A significant decrease in its light emis sion was recorded for a relatively short (approximately 30 min) period of time. Then, the mycelium com pletely lost the ability to emit light: the values of recorded signals coincided with the background noise of the measuring system. The observed effect of Xray radiation is typical for this fungal species. Similar radi ationinduced changes in the light emission were recorded using different samples (globules and films) of N. nambi mycelium obtained at periodic cultivation of the fungus for six months. In the next series of experiments (Fig. 4) we showed that, at a fixed radiation power, a decrease in the time of Xray exposure of N. nambi mycelium was accom panied by a reduced stimulatory effect on its lumines cence. As can be seen from the data, shortterm irra diations of mycelium were not accompanied by the loss of its luminescence. Moreover, periodic short term (10 min) irradiation of the mycelium against the background of attenuation of its radiationactivated luminescence resulted not only in new stimulation of luminescence but also in an increase in its level (Fig. 4). Probably, it may indicate a dosedependent effect of radiation on the light emission by the fungus. Thus, we demonstrated the stimulation of lumines cence of N. nambi mycelium exposed to ionizing radi ation. It is shown that the magnitude of the observed effect and the kinetics of luminescence depended on
the Xray radiation dose. Longterm exposure led to a significant increase in the light emission of the fungus with subsequent loss of its luminescence. Shortterm irradiations each time were accompanied by a lesser stimulation of luminescence; however, the fungus did not lose the ability to emit light. The irreversible loss of luminescence cannot be associated with the thermal inactivation of mycelium under a longterm exposure. Calculations showed that, at the heat capacity of water a 75.35 J/mol K and the radiation power of 85 krad/h, the temperature of the sample increased at a rate of not more than 0.2°C/h. On the basis of this fact, it can be assumed that the stimulation of luminescence radia tion is mediated by the generation of radicals in the aqueous medium surrounding and filling the myce lium. The presence of the latent period (approxi mately 20 min) before the phase of light signal devel opment, the increase and subsequent decrease in the luminescence intensity may reflect the amount of oxy gen radicals present in the system and the functional activity of the enzymes (or enzyme complexes) that ensure luminescence with the involvement of ROS. Under a longterm exposure, the amount of oxygen radicals generated in the sample can be sufficiently large. It is known [12] that the radiation yield of radicals in water is approximately 5/100 eV. On the basis of cal culations of the amount of water contained in a myce lium sample at a radiation power of 85 krad/h and exposure duration of 2 h, the amount of generated rad icals may reach approximately 1.5 × 1016. In turn, the high level of ROS induced by ionizing radiation will be accompanied by the development chain oxidation
DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 460
2015
EFFECT OF IONIZING RADIATION ON THE LUMINESCENCE
reactions in the mycelium (for example, by the activa tion of lipid peroxidation). The activation of this pro cess by ionizing radiation is well known [13, 14]. In this case, the sharp decrease in the luminescent signal observed at the final stage of irradiation and the com plete loss of luminescence by the fungus can be associ ated with the disturbance of its enzyme complexes resulting from the oxidative damage. On the other hand, taken together, the abovementioned facts sug gest that the luminescence of higher fungi is an addi tional mechanism protecting them from the damaging action of ROS. This assumption seems plausible because the hypothesis that the light emission of living organisms is aimed at protecting against the damage caused by reactive oxygen radicals was expressed as early as the middle of the last century [15]. ACKNOWLEDGMENTS This work was supported in part by the Support Program for Interdisciplinary Projects of the Siberian Branch of the Russian Academy of Sciences (project no. 71) and the Program of the President of the Rus sian Federation for Support of Leading Scientific Schools (project no. NSh5744.2014.3). REFERENCES
2. Shimomura, O., Exp. Bot., 1992, vol. 43, pp. 1519– 1525. 3. Bondar, V.S., Puzyr, A.P., Purtov, K.V., et al., Dokl. Bio chem. Biophys., 2011, vol. 438, pp. 138–140. 4. Bondar, V.S., Shimomura, O., and Gitelson, J.I., J. Sib. Fed. Univ. Biol., 2012, vol. 5, no. 2, pp. 331–351. 5. Bondar, V.S., Rodicheva, E.K., Medvedeva, S.E., et al., Dokl. Biochem. Biophys., 2013, vol. 449, pp. 80–83. 6. Vladimirov, Y.A., in: Free Radicals in the Environment, Medicine and Toxicology, London: Richelieu Press, 1994, pp. 345–373. 7. Halliwell, B. and Gutteridge, J.M.C., Free Radicals in Biology and Medicine, Oxford: Oxford Press, 1999. 8. Mantel, J., Freidin, M., Bulich, A.A., et al., Phys. Med. Biol., 1983, vol. 28, pp. 599–602. 9. Asada, K., Ann. Rev. Plant Physiol. Plant. Mol. Biol., 1999, vol. 50, pp. 601–639. 10. Vydryakova, G.A., Dao, T.Van., Shoukouhi, P., et al., Mycology, 2011, vol. 3, pp. 89–99. 11. Kalneus, E.V., Melnikov, A.R., Korolev, V.V., et al., Appl. Magn. Resonance, 2013, vol. 44, pp. 81–96. 12. Spinks, J.W.T. and Woods, R.J., An Introduction to Radiation Chemistry, New York: Wiley, 1976. 13. Yin, H., Xu, L., and Porter, N.A., Chem. Rev., 2011, vol. 111, pp. 5944–5972. 14. Khalil, A. and Fulop, T., Can. J. Physiol. Pharmacol., 2001, vol. 79, pp. 114–121. 15. McElroy, W.D. and Strehler, B.L., Arch. Biochem., 1949, vol. 22, pp. 420–433.
1. Shimomura, O., Bioluminescence: Chemical Principles and Methods, Singapore: World Sci., 2006.
DOKLADY BIOCHEMISTRY AND BIOPHYSICS
Vol. 460
33
Translated by M. Batrukova
2015
