Effect of low-dose ionizing radiation on luminous marine bacteria: radiation hormesis and toxicity

Journal of Environmental Radioactivity 142 (2015) 68e77

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Review

Effect of low-dose ionizing radiation on luminous marine bacteria: radiation hormesis and toxicity N.S. Kudryasheva a, b, *, T.V. Rozhko b, c a

Institute of Biophysics SB RAS, Akademgorodok 50, 660036, Krasnoyarsk, Russia Siberian Federal University, Svobodny 79, 660041, Krasnoyarsk, Russia c Krasnoyarsk State Medical Academy, P. Zheleznyaka 1, 660022, Krasnoyarsk, Russia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 November 2014 Received in revised form 12 January 2015 Accepted 12 January 2015 Available online 30 January 2015

The paper summarizes studies of effects of alpha- and beta-emitting radionuclides (americium-241, uranium-235þ238, and tritium) on marine microorganisms under conditions of chronic low-dose irradiation in aqueous media. Luminous marine bacteria were chosen as an example of these microorganisms; bioluminescent intensity was used as a tested physiological parameter. Non-linear dose-effect dependence was demonstrated. Three successive stages in the bioluminescent response to americium241 and tritium were found: 1 e absence of effects (stress recognition), 2 e activation (adaptive response), and 3 e inhibition (suppression of physiological function, i.e. radiation toxicity). The effects were attributed to radiation hormesis phenomenon. Biological role of reactive oxygen species, secondary products of the radioactive decay, is discussed. The study suggests an approach to evaluation of non-toxic and toxic stages under conditions of chronic radioactive exposure. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Marine bacteria Low-dose effects Radiation hormesis Radiotoxicity Reactive oxygen species

1. Introduction Risk of radioactive contamination of the World Ocean is an important problem of modern ecology. Evaluation of radiation toxicity for marine organisms under conditions of low-dose irradiation and understanding the mechanisms of toxic effects is a challenge for researchers working in related fields. Microorganisms are simplest and basic part of marine ecosystems and their physiological indices can serve as indicators of the state of the ecosystems on the whole. Hence, microorganisms can be used to monitor environmental toxicity, including radiotoxicity. Marine luminous bacteria are good candidates for such investigations. Glowing of the bacteria is not only an attractive natural phenomenon; it is a bacterial physiological function applicable in ecological investigations. The luminous marine bacteria are used as bioassays to monitor toxicity of aquatic media for several decades (Girotti et al., 2008). Luminescence intensity is main tested physiological parameter here; it can be easily measured instrumentally. Benefits of the luminous bacteria-based assay are high rate, simplicity, and sensitivity. Abbreviation: ROS, reactive oxygen species; H-3, tritium; HTO, tritiated water; NADH, nicotinamide adenine dinucleotide reduced; FMN, flavin mononucleotide; DRIFT, diffuse reflectance infrared Fourier transform. * Corresponding author. Tel.: þ7 391 2494242; fax: þ7 391 2433400. E-mail address: [email protected] (N.S. Kudryasheva). http://dx.doi.org/10.1016/j.jenvrad.2015.01.012 0265-931X/© 2015 Elsevier Ltd. All rights reserved.

On the other part, the marine bacteria can be considered as simple model living objects for studying mechanisms of radionuclide influence e from primary physicochemical processes under ionization conditions in water solutions to macromolecular transformation and cellular membrane processes. Regularities of ionizing radiation influence on the microorganisms can be applied to analyze the radioactive impact on higher organisms. Hence, luminous marine bacteria are good candidates for (1) radiotoxicity monitoring, and (2) molecular mechanism investigations. The latest years have seen a development in studying the biological effects of low-dose radiation on luminous marine bacteria. The necessity arises to review and colligate the data obtained. The question is: how to relate characteristics of radioactive media (types of radionuclides and specific radioactivity of their solutions) with rates of physicochemical and biochemical processes in bacterial cells. Self-defense processes and toxic suppression of physiological functions are questions of special interest. 2. Luminescence of marine bacteria and environmental toxicity monitoring This section discusses a background for application of marine bacteria as a bioassay in radioecological investigations.

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Scope of application of the bioluminescent bacteria-based assay should be clearly specified. It is classified as a biological assay. It is generally accepted that biological assays, along with chemical and radiological ones, are a basis of ecological investigations. In general, the term “toxicity” is of biological origin; it means a suppression of organism's physiological functions (Ilyin et al., 1990; Sanotskiy, 1970). Classic bioassays involve the use of mice, frogs, microorganisms, fish, algae, crustaceans, plants, higher organisms, etc. (Tigini et al., 2011; Rizzo, 2011; Petukhov et al., 2000; Donnelly et al., 1997). The examples of their physiological functions monitored during bioassay procedure are life-time, the rates of growth, movement, and respiration, as well as bioluminescent intensity. The main features of all classic bioassays are integral response and nonspecificity (Kudryasheva and Tarasova, 2015). As a result, the current approach to ecological investigations bases on the combination of chemical, radiometric, and biological methods that can provide complete information on the ecological state of a medium (Ma et al., 2014; Deprezc et al., 2012; Tigini et al., 2011; Kudryasheva et al., 1998). Bioluminescence of marine bacteria is highly sensitive to the presence of toxic compounds; this is why the marine bacteria have been widely used to assess environmental toxicity for more than forty years. Now, the biological assay systems involving luminous marine bacteria are the traditional biotechnological application of the bioluminescence phenomenon (Xu et al., 2014; Thakur and Ragavan, 2013; Ranjan et al., 2012; Roda et al., 2009, 2004; Thomas et al., 2009; Ivask et al., 2009; Girotti et al., 2008; Kudryasheva et al., 1998). The advantages of the bacteria-based bioluminescent assays are high sensitivity, simplicity, rapidity (1e3 min), and availability of devices for toxicity registration. High rates of the bioluminescent assay provide a large number of measurements under comparable conditions, proper for statistical treatment. These advantages account for intensive research of the functions of luminous bacteria and their sensitivity to exogenous compounds (Hastings, 2012; Deryabin and Karimov, 2010; Deryabin and Aleshina, 2008; Girotti et al., 2008), as well as mechanisms of light emitting (Hou et al., 2014; Nemtseva and Kudryasheva, 2007). The bioassay based on bioluminescent bacteria was described in 1969 by Kossler as reported in (Grabert and Kossler, 1997). In the late 1980s the test was standardized in Germany as a method to detect pollutants. Later, it was modified by different researchers and adapted for their specific purposes (Tarasova et al., 2012; Ranjan et al., 2012; Rozhko et al., 2007; Kratasyuk et al., 2001; Natecz-Jawecki et al., 1997; Stom et al., 1992; Bulish and Isenberg, 1981). Now the luminous bacteria are widely used as a bioassay to monitor toxicity of water solutions (Shao et al., 2012; Girotti et al., 2008), e.g., acute toxicity of wastewaters contaminated with metals (Qua et al., 2013), organic oxidizers (Wang et al., 2009), or compositions of explosive nitrated organic compounds (Yea et al., 2011). Genes of bacterial enzymes responsible for the generation of light (lux genes) can be cloned from a bioluminescent microorganism into organism that is not naturally bioluminescent; light output can be monitored to provide information on the metabolic state, quantity of cells, and toxicity of the environment (Morrisseya et al., 2013; Kurvet et al., 2011; Roda et al., 2009, 2004; Girotti et al., 2008). Integral toxicity of solutions can be evaluated by relative bioluminescent intensity, Irel:

I rel ¼ I1 =Icontr Here, Icontr and I1 are bioluminescent intensities in the absence and presence of exogenous compounds, respectively. Value of

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Irel < 1 shows suppression of the luminescent function of the bacteria by exogenous compounds (i.e. the solution is toxic), while Irel > 1 shows activation of the luminescent function. The bacterial bioluminescent assays can base on biological systems of different complexity e bacteria or their enzymes (Ma et al., 2014; Selivanova et al., 2013; Fedorova et al., 2007; Rozhko et al., 2007; Kudryasheva et al., 1996), thus providing studying the effects of toxic compounds at cellular or biochemical levels, respectively. Bacterial bioluminescent enzyme system was suggested as a bioassay for the first time in 1990 (Kratasyuk, 1990). It can be considered as simplified luminescent function of the bacteria, characterizing changes of rates of biochemical reactions under the influence of toxicants. Enzymatic assays avoid the problem of the classic whole-organism-based assays, namely, the difference in sensitivities of various assay organisms. Possibility to change sensitivity to definite toxic compounds by varying the component concentrations and constructing the polyenzymatic coupled systems is an advantage of the enzymatic assays (Leippe et al., 2011; Kudryasheva et al., 2003a, 1999, Kudryasheva, 1999). Bioluminescent enzymatic reagent immobilized into starch gel was developed in (Esimbekova et al., 2013, 2009). The technological applications of the bioluminescent enzymatic system were reviewed in (Esimbekova et al., 2014). The conventional bioluminescent enzymatic assay is based on coupled bacterial bioluminescent enzyme system; it involves two enzymatic reactions. The first one, catalyzed by NADH:FMNoxidoreductase, is a reduction of FMN by NADH: NADH:FMNoxidoreductase

NADH þ FMN ƒƒƒƒƒƒƒƒƒƒƒƒ! FMN$H þ NADþ

(1)

In the second reaction, catalyzed by bacterial luciferase, the reduced flavin (ionized form) and the long-chain aldehyde are oxidized by molecular oxygen to yield the corresponding acid, H2O, FMN, and a quantum of light (lmax about 500 nm): luciferase

FMN,H þ RCHO þ O2 ƒƒƒƒƒ!FMN þ RCOO þ H2 O þ hn

(2)

Peculiarity of the enzymatic assay system is its specificity to oxidizers (Vetrova et al., 2007; Fedorova et al., 2007; Tarasova et al., 2011, 2012). Mechanisms of interactions of exogenous compounds with bioluminescent enzyme systems were intensively studied. Basing on a broad investigation of effects of model toxic exogenous compounds, a classification of the effects on the bioluminescent enzymatic assay system was suggested (Kudryasheva, 2006) and developed. The effects of different groups of exogenous compounds e organic dyes (Nemtseva and Kudryasheva, 2007; Kudryasheva et al., 2004, 2003b), oxidizers (Vetrova et al., 2009, 2007, 2005), halogen-substituted molecules (Kirillova et al., 2011; Kirillova and Kudryasheva, 2007; Gerasimova and Kudryasheva, 2002), salts of stable and radioactive metals (Selivanova et al., 2013; Tarasova et al., 2011, 2012; Alexandrova et al., 2011; Rozhko et al., 2007; Kudryasheva et al., 1999, 1996), are discussed according to the classification suggested. 3. Effects of alpha- and beta-emitting radionuclides on luminous marine bacteria For the first time, bioluminescence of bacteria was used to monitor radiation toxicity in (Min et al., 2003). This work considered the effect of gamma-ray irradiation on recombinant Escherichia coli. Few works were concerned with effects of ionizing radiation on non-marine luminous organisms e fungi and fireflies: changes of kinetic parameters of bioluminescence of fungi exposed

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to X-ray irradiation were reported in (Kobzeva et al., 2014); effects of a proton beam from a cyclotron on kinetics of firefly luciferase reaction were revealed and explained by elimination of dissolved oxygen in aqueous solutions (Berovic et al., 2008). Effects of alpha- and beta-emitting radionuclides (Am-241, U(235þ238), and H-3) on luminous marine bacteria Photobacterium phosphoreum were reported in a series of works (Rozhko et al., 2007, 2008; Alexandrova et al., 2010, 2011; Selivanova et al., 2013, 2014). The detailed investigation of the effects was conducted using tritium and Am-241 as examples. Brief description of radioactive properties and effects of these radionuclides follows below. Tritium (H-3), beta-emitting radionuclide, is permanently generated by space radiation at the rate of 1200 atoms s1 m2 in the top layers of Earth's atmosphere (Lenskii, 1981). In the Earth Ocean, it is mostly presented as a component of tritiated water, HTO. Until the 1950s, the tritium concentration in natural waters was low e one tritium per 1018 hydrogen atoms. However after atmosphere nuclear tests it increased 1000-fold. Since a half-life of tritium is 12.32 years, its concentration eventually decreased, though local rise of tritium content took place around nuclear power plants. Local nuclear incidents increase tritium concentration dramatically as tritium is a by-product of a lot of radiochemical reactions. In the future, controlled fusion reactors can bring an additional threat of contamination with tritium. Tritium is considered to be one of the less dangerous isotopes. Maximal energy of its beta-particles is low (18.59 keV) and the maximal range of their path is short (5.8 mm at 20 С in vacuum). Beta-particles are entirely absorbed by the skin's surface layers; this is why tritium is not dangerous outside an organism. However, as an internal source of irradiation it can be unsafe. Tritium is capable of penetrating into cell organelles and can substitute hydrogen (protium) atoms in organic molecules. This is why tritium irradiation can produce a local damage. Tritium specific ionization ability (2.2  106 ions per cm) exceeds that of other beta-emitting radionuclides. Hence, tritium toxicity is a challenging problem for researchers (Evans, 1974; Snigireva et al., 2009). Additionally, tritium presents a convenient subject for studying a protective response of organisms to low-dose radiation due to its low energy beta-decay. The effect of tritium-labeled amino acid valine (0.3e1.0 MBq/ mL) on luminous bacteria Р. phosphoreum was studied in (Alexandrova et al., 2010). The amino acid was used as a component of nutrient bacterial medium here. Tritium was found to suppress bacterial growth, but stimulate luminescence: luminescent intensity, quantum yield and time of light emitting increased. The studies (Selivanova et al., 2013, 2014) aimed at the effects of tritium taken as component of tritiated water, HTO, on bacterial bioluminescence. Paper (Selivanova et al., 2013) considers influence

of tritium (0.0002e200 MBq/L) on three bioluminescent assay systems of different complexity e bacteria-based assays (intact and lyophilized preparations of P. phosphoreum) and enzyme-based assay (reactions 1,2). Bioluminescent intensity, bacterial growth, cell damage, and tritium accumulation were under investigation. Tritium initiated three stages in bacterial bioluminescence kinetics: threshold effect, activation and inhibition (Fig. 1). Fig. 1a demonstrates absolute values of bioluminescent intensity of control and radioactive sample, and Fig. 1b e values of normalized bioluminescent intensity. Similar results were reported (Selivanova et al., 2013) for lyophilized preparation of marine bacteria. Fig. 2A presents three stages of bioluminescent kinetics of lyophilized bacteria in HTO. It reveals the same three stages, similar to living bacteria. The dependence of bacterial bioluminescence intensity on HTO specific radioactivity was not found under the conditions of the experiment at all times of exposure. Fig. 2B demonstrates bioluminescence intensity vs. HTO specific radioactivity at two exposure times e 21 h (activation stage, as seen from Fig. 2A) and 53 h (inhibition stage, as seen from Fig. 2A). Lyophilized bacteria were taken here as an example. The effects of HTO on bioluminescent kinetics were similar for intact and lyophilized bacteria as seen from Fig. 1B and 2A. However, the stages durations were different: threshold and activation stages were shorter for lyophilized preparation of bacteria under similar experimental conditions. It is known that lyophilized bacterial preparation includes more damaged cells than intact bacteria (Medvedeva, 1999), and this damage might reduce bioluminescence threshold and activation stages. Hence, damaged bacteria are more sensitive to radiation of tritium, and toxic effect begins earlier, as compared to non-damaged cells. Tritium increases bioluminescence intensity up to 230% if the bacteria were grown in the nutrient media involving tritiated water, HTO (Selivanova et al., 2013), and 500% e if the bacteria were grown in the nutrient media involving tritium-labeled amino acid valine (Alexandrova et al., 2010). Rises of the overall bioluminescence quantum yields were to up to 50 and 500%, respectively. Americium-241 (241Am), alpha-emitting radionuclide of high specific radioactivity, is a by-product of plutonium radioactive decay. Its half-life is 432, 8 Years. It is known to be increasingly accumulated by the environment due to its ability to be bound by organic compounds, to concentrate on the surface of cells and penetrate through the cellular membrane by means of specific cellular proteins, siderophores (Choppin et al., 1971, 1997). For example, observations in the waters of the Chernobyl zone contaminated with radioactive fallout demonstrated that aquatic plants accumulate Am-241 in their biomass (Gudkov et al., 2002).

Fig. 1. Bioluminescent intensity of Photobacterium phosphoreum vs. time of exposure to HTO, 2 MBq/L: A e absolute values, I, (Selivanova et al., 2013); B e relative values, Irel (Selivanova et al., 2014).

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Fig. 2. Relative bioluminescence intensity of lyophilized bacteria, Irel , in HTO, 2 MBq/L. Dependence on (A) time and (B) specific radioactivity, A, at 21 h (:) and 53 h (A) exposure time (Selivanova et al., 2013).

Accumulation of Am-241 by sediments and aquatic plants in the Siberian river Yenisei is currently under study (Bolsunovsky, 2010; Zotina et al., 2010, 2011). In (Rozhko et al., 2007), the sensitivity of bioluminescent assay systems to americium-241 was investigated. Similar to paper (Selivanova et al., 2013), this study addressed the effects on bioluminescent assay systems of different levels of organization e bacteria or enzyme reactions. Three bioluminescent assay systems were used: intact bacteria P. phosphoreum, lyophilized bacteria P. phosphoreum, and enzyme-based system (reactions 1, 2). Solutions of 241Am(NO3)3 were applied as a source of alpha-radiation at specific radioactivities 0.1e6.7 kBq/L (ca. 3$1012e2$1010 M). Similar to tritium (Fig. 1A, B and Fig. 2A), activation and inhibition stages were found in bioluminescent kinetics of the systems. It was shown that activation processes predominate in all three bioluminescent assay systems subjected to short-term exposure (less than 20e55 h), and inhibition processes e to longer-term exposure to radiation. Increase of the bioluminescence intensity of bacteria reached to 400%. Bacterial systems were found to be more sensitive to Аm-241 (up to 1012 M) than the enzymatic system. The paper (Selivanova et al., 2014) compares effects of Am-241, alpha-emitting radionuclide of high specific activity, and tritium (H-3), beta-emitting radionuclide, on luminous bacteria under chronic low-dose irradiation. It was shown that the bacterial response to the alpha- and beta-emitting radionuclides was unified, including three successive stages mentioned before: (1) absence of the effect, (2) activation, and (3) inhibition (Fig. 3). However, time characteristics of the stages in solutions of 241Am241 and H-3 were different under similar radiation doses delivered to the bacteria. Times of bioluminescence activation (ТВА) and

Fig. 3. Dependence of relative bioluminescent intensity of luminous bacteria P.phosphoreum, Irel , on time of exposure to 3Н (А ¼ 10 МBq/L) and 241Am (А ¼ 0.3 kBq/L) (Selivanova et al., 2014).

inhibition (TBI) were suggested as parameters to characterize the results of chronic low-dose irradiation of microorganisms. The values of TBA and TBI of Am-241 were shorter than those of H-3 (Fig. 3), revealing higher toxic impact of the alpha-irradiation. The paper (Rozhko et al., 2008) compares effects of alphaemitting radionuclides on P. phosphoreum. The Am-241 and U-(235þ238) were chosen as the radionuclides of high and low specific radioactivities, respectively. Solutions of nitrates of these metals, Am(NO3)3 and UO2(NO3)2, were studied. The effect of uranium was found at higher concentrations (>107 M or >0.3 Bq/L) than that of americium (>1011 M or >300 Bq/L). Additionally, only bioluminescent inhibition was observed in the presence of uranium (Fig. 4A), while americium produced three stages in the bioluminescence kinetics, as discussed above. Effect of uranium was compared to that of europium (Eu), stable heavy metal, chemical analog of Am-241 (Rozhko et al., 2008). The effect of UO2(NO3)2 appeared to be similar to this of Eu(NO3)3 (Fig. 4B), as well as to the salts of other stable metals (Kudryasheva et al., 1996; Gerasimova et al., 2002). It was concluded that the contributions of radiation and chemical components to the effect of the radionuclides depend on radionuclide specific radioactivity. The effect of uranium on the bacteria is conditioned by its chemical, but not radioactive properties. The effect of americium-241 was due to its radioactive component only. Damage of bacterial cells in HTO was visualized by electron microscopy (Selivanova et al., 2013) (Fig. 5). Under chronic exposure to HTO, visible cell ultrastructure damages were registered in all bacterial cells. In contrast to the control sample, the cytoplasm contained condensed DNA fibers and ribosomes, as well as irregular electron dense sites localized near the cell poles. Similar changes of bacterial structure were observed under exposure to Am-241 (Rozhko et al., 2011). Radiotoxicity of water solutions is affected by water-soluble organic compounds which are always present in natural water bodies. Humic substances, products of oxidative transformation of organic compounds in soil and bottom sediments, polyfunctional macromolecules, can form complexes with actinides (Antunes et al., 2007; Sakuragi et al., 2004; Staunton et al., 2002; Lenhart et al., 2000; Silva et al., 1998, 1996), thereby shielding water microorganisms from alpha-particles and secondary products of alpha-decay of the actinides. This accounts for radiotoxicity decrease in the presence of humic substances. The work (Rozhko et al., 2011) investigated the effect of watersoluble humic substances on bioluminescence intensity of Р. phosphoreum in solution of Am-241 (3000 Bq L1), redistribution of Am-241 in cell culture and damage of cells exposed to the

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Fig. 4. Bioluminescent intensity (Irel) vs time of exposure in solutions of Eu(NO3)3 (A), and UO2(NO3)2 (B). Concentrations of the salts: 1e103, 2e104, 3e105, 4e107, 5e1011 М. Corresponding specific radioactivity of the solutions of UO2(NO3): 1e3000, 2e300, 3e30, 4e0.3, 5e3,105 Bq/L (Rozhko et al., 2008).

Fig. 5. Ultrastructure of P. phosphoreum cells: (A) control sample, (B) fixed from HTO (A ¼ 100 MBq/L) (Selivanova et al., 2013).

radionuclide. It was demonstrated that humic substances reduce the effect of Am-241 on bioluminescence intensity, change the distribution of Am-241 between bacterial cells and intercellular media, and decrease the damage to the cells. Therefore, it was demonstrated that water-soluble humic substances can serve as protecting agents for water microorganisms exposed to alpharadionuclides. Decrease of HTO effects in the presence of humic substances was reported in (Alexandrova et al., 2012).

4. On molecular mechanisms of radiation hormesis and toxicity As shown in the previous section, the responses of luminous bacteria to alpha- (Alexandrova et al., 2011; Rozhko et al., 2007, 2011) and beta- (Selivanova et al., 2013; Alexandrova et al., 2012) emitting radionuclides were similar (Figs. 1B, 2A and 3), Three bioluminescent kinetic stages in solutions of Am-241 and H-3 (alpha- and beta-emitting radionuclides, respectively) were: absence of effect, activation and inhibition of bioluminescence. The bacterial response can be interpreted in terms of standard reaction of organisms to stress-factor; it includes the following successive stages: (1) stress recognition or a “threshold” for the effect, (2) adaptive response/syndrome, and (3) suppression of the physiological function. Hence, the response of bacterial cells to alpha and beta low-intensive emission is unified.

Since the term “toxicity” is defined as a suppression of biological functions of organisms, the third stage can be attributed to radiation toxicity. Activation of vital functions of various organisms is a wellknown effect, common to all living organisms. It is attributed to triggering of cell defense response under the influence of low concentrations of toxic compounds, low dose radiation, and other stressors. Examples of this phenomenon are: nonspecific adaptive syndrome of plants (Pakhomova, 1995) and stress reaction of animals (Selye, 1980). Additionally, it is known that low doses of bioactive substances serve as effective drugs (Calabrese and Blain, 2011; Halliwell and Gutteridge, 2007; Burlakova et al., 2004; Wang et al., 2010). In our experiments, the doses that could be delivered to bacterial biomass after first and second stages of exposure to HTO (A ¼ 10 MBq/L) and Am-241 (A ¼ 0.3 kBq/L) (Fig. 3) were evaluated and compared in (Selivanova et al., 2014). They were estimated as 0.0005 and 0.0015 Gy for HTO, as well as 0.0004 and 0.001 Gy for Am-241. These values are much lower than a tentative limit of a low-dose interval, 0.2 Gy (Goldberg et al., 2006; Matsumoto et al., 2007; Burlakova et al., 2004). Hence, the first and the second stages in bioluminescence kinetics (threshold effect and bioluminescence activation, Figs. 1e3) might be attributed to low-dose effects of the radionuclides on marine bacteria. The low-dose conditions were provided in experiments with the luminous marine bacteria in (Rozhko at al., 2007, 2008; Alexandrova et al., 2011; Selivanova et al., 2013) as well.

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Nonlinear dependencies presented in Figs 1e3 can be ascribed to the hormesis phenomenon (Calabrese and Baldwin, 2001; Kaiser, 2003; Heinz et al., 2010; Calabrese, 2013, 2014). A brief description of hormesis phenomenon is presented below. Generally, hormesis is a term for generally favorable biological responses to low exposures to toxins and other stressors, with the ionizing radiation involved. In toxicology, hormesis is a dose response phenomenon characterized by a low-dose stimulation, higher dose inhibition, resulting in either a J-shaped or an inverted U-shaped dependence. The term ‘hormesis’ first entered to the scientific lexicon in 1943 (Southam and Ehrlich, 1943) based on observations that extracts from the Red Cedar tree enhanced the metabolism of fungal species. The rapid and extensive exponential growth of hormesis citations in the biomedical community takes place for the last two decades (Calabrese, 2013). Evidence emerged that hormesis is highly generalizable, independent on biological model or endpoint measured, inducing agent and level of biological organization (e.g. cell, organ, organism). The hormesis mechanisms are not understood yet. Theoretical investigations of biological low-dose effects have been carried out since the 60s of the 20th century. Researches are concerned with properties of biological structures (cells, macromolecules, and lowmolecular substrates) and hydrogen-bond network of water molecules (Szent-Gyorgyi, 1957; Popp and Yan, 2002). A model of coherent acoustic electric waves (Devyatkov et al., 1991), theory of €hlich, 1968), and theory coherent long-range order oscillations (Fro of solitons in biomolecules (Davydov, 1973) considered polar structures in biomembranes and biomacromolecules. Problem of oscillation decay in viscous cellular cytosol is under consideration in (Zakhvataev and Khlebopros, 2012). The first detailed book on hormesis, with an exclusive focus on radiation, was provided by Luckey (1980). Radiobiological effects of low doses have been studied since the 70s, last century (Burlakova et al., 2004; Feinendegen, 2005; Feinendegen et al., 2007; Ray at al., 2014; Mothersill and Seymour, 2014), including effects on microorganisms (Paul et al., 2013; Tomac et al., 2013: Jo et al., 2012; Mesquita et al., 2013; Xavier et al., 2014). There exist two models explaining mechanism of radiation hormesis; they consider the adaptive response as related with DNA damage or cell membrane processes (Albers, 1967; Lloyd et al., 1992; Zaka et al., 2002; Mossman, 2001; Serment-Guerrero et al., 2012; Rana at al., 2013; Mothersill et al., 2014; Jo et al., 2012). As follows from the previous section, luminous bacteria revealed complex responses to chronic effect of Am-241 and H-3 (Rozhko et al., 2007; Alexandrova et al., 2011; Selivanova et al., 2013, 2014; Kudryasheva et al., 2014) (Figs. 1e3) in the range of radioactivities 1.0e6.7 kBq/L and 0.0002e200 MBq/L, respectively. The responses were not linear; monotonic dependencies of bioluminescence intensity on exposure time and radioactivity were not found. Hence, this result is in accordance with a nonlinear doseeffect relationship under low-dose irradiation and can be interpreted in terms of hormesis phenomenon. Experimental studies elucidating the molecular mechanisms of radiation hormesis and radiation toxicity in bacterial cells are of high interest. Mechanisms of radiation hormesis and toxicity of beta-emitting radionuclides were discussed with tritium, H-3, taken as an example in (Selivanova et al., 2013). It was suggested that molecules of tritiated water, HTO, can easily penetrate through cell membranes; this is why electrons and helium-3 cations, primary products of tritium radioactive decay, can affect all bacterial structures e from the bacterial cell walls to enzymes and their substrates inside the cells. Stimulatory effects of HTO might be accounted for by intensification of the charge transfer processes in bioluminescent systems, resulting in increase of rates of active ion transport across

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the cell membrane (Albers, 1967), rates of redox enzyme reactions, etc. The same processes at higher times of exposure can inhibit bioluminescent function of bacteria (Figs. 1e3) and damage bacterial cells (Fig. 5) (Selivanova et al., 2013; Rozhko et al., 2011). Independency of bacterial response on HTO radioactivity at fixed times of exposure (as shown in Fig. 2B) was observed for intact and lyophilized bacteria in a wide radioactivity interval: 104 e 200 MBq/L (Selivanova et al., 2013). As can be concluded from Figs. 1e3, the electron-induced processes in cells appeared to be time-dependent, but not radioactivity-dependent, under the conditions of the experiment. Bioluminescence activation and independency on the specific radioactivity of solutions can be considered as an evidence of specific compensatory intracellular mechanisms that form a basis for protective response of the cell as a whole at low-dose exposures. Due to low energy of beta-particles, tritium presents a convenient tool for studying the compensatory cellular mechanisms. To evaluate details of the protective mechanisms, the effects of HTO on the bioluminescent system of enzyme reactions (reactions 1, 2) catalyzed by the bacterial enzymes were studied and compared to those on bacterial cell (Selivanova et al., 2013). In contrast to the bacterial cells, the enzyme system revealed the dependence of bioluminescence intensity on HTO specific radioactivity (compare Figs. 2B and 6). Probably, additional compensatory mechanisms in bacterial cells moderate the response to chronic irradiation and make it three-staged universal. It was concluded in (Selivanova et al., 2013) that increase of biological system complexity (from enzymes to cells) enhanced stability against HTO. In (Alexandrova et al., 2011) the effects of Am-241 on luminous bacteria were related to Reactive Oxygen Species (ROS) which are generated as products of water radiolysis in the presence of molecular oxygen. Radiolysis of water is a known process occurring under ionizing radiation (Fridovich, 1998; Halliwell and Gutteridge, 2007). Secondary products of ionizing radiation (peroxides, radicals, cationradicals, and anion-radicals) affect water ecosystems and their inhabitants. Peroxide compounds are an important group among the secondary products of ionizing radiation. With regard to metabolic processes, peroxides are intermediates in a series of oxidative metabolic reactions, including bioluminescent reactions (Nemtseva and Kudryasheva, 2007). Depending on concentrations, they can either activate or inhibit the metabolic processes (Kislenko and Berlin, 1991; Kudryashov, 2004).

Fig. 6. Bioluminescent intensity, Irel , vs. HTO specific radioactivity, A. Time of exposure e 18 min. Enzyme system (Selivanova et al., 2013).

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It was hypothesized (Alexandrova et al., 2011) that peroxide compounds can be responsible for activation and inhibition of the bioluminescence function of the luminous bacteria. The study compared the effects of hydrogen peroxide and Am-241 on P. phosphoreum. Hydrogen peroxide was shown to activate or inhibit bacterial luminescence, depending on concentration (Fig. 7) and time of exposure. In Remmel et al. (2003), activation and inhibition of bacterial luminescence by hydrogen peroxide was also reported, with Vibrio harveyi strain as an example. Hence, hydrogen peroxide, similar to Am-241, produced activation and inhibition stages in bioluminescent kinetics of the bacteria. Peroxide compounds were found in solutions of Am-241 (Alexandrova et al., 2011). Hence, the role of peroxides (as secondary products of ionizing radiation in Am-241 solution) in activation and inhibition of bioluminescence was elucidated. Paper (Selivanova et al., 2014) compares oxidative properties of 241 Am solutions and tritiated water, HTO. Increase of peroxide concentrations in 241Am solutions was demonstrated, however, peroxides were not found under exposure to HTO (Fig. 8). Additionally, rates of NADH (i.e. endogenous reducer, component of bacterial bioluminescent enzyme system, reaction 2) oxidation under exposure to Am-241 and HTO were determined (Selivanova et al., 2014). The rates in Am-241 solutions appeared to be higher. The results attribute the biological effects of Am-241, alphaemitting radionuclide of high specific radioactivite, to ROS generated in water solutions as secondary products of radioactive decay, however, the effects of beta-emitting radionuclide, tritium, are not concerned with the peroxide formation in water solutions. Another type of particles, hydrated electrons or other charged particles, must be responsible for the change of bioluminescence intensity, too. They can be involved into electron transfer chain of coupled metabolic redox reactions in organisms, thus increasing or decreasing the rates of metabolic processes. In saline chloride-containing solutions, the hypochlorite and other active chlorine derivatives of oxidative nature are formed as products of radiolysis; they can also contribute to suppression of physiological functions of organisms (Czapski et al., 1992; Saran et al., 1993, 1997). Diffuse Reflectance Infrared Fourier Transform (DRIFT) spectroscopy was used to control possible metabolic responses of the bacteria to radioactive stress. All DRIFT spectra of bacterial cells both exposed and non-exposed to Am-241 were very similar showing a low content of intracellular poly-3-hydroxybutyrate (at

Fig. 7. Bacterial bioluminescent intensity, Irel, at different concentrations of H2O2 (Alexandrova et al., 2011).

Fig. 8. Comparison of peroxide concentrations in solutions of Am(NO3)3, and HTO. A e specific radioactivity (Selivanova et al., 2014).

the level of a few percent of dry biomass) and no or negligible spectroscopic changes (Kamnev et al., 2013). However, preliminary similar DRIFT studies of bacterial cells exposed to tritiated water, HTO (Kamnev et al., 2011), demonstrated a slight but statistically significant shift of the amide-I band of cellular proteins. As was earlier stated in (Kamnev et al., 2008) such a shift shows a stressinduced increase in the content of beta-structured proteins and, in the case of HTO, it can be interpreted as a specific response of bacterial cells to low-dose chronic radioactivity. Some similar responses to heavy-metal stress were observed in rhizobacteria (Kamnev et al., 2012). Mutagenic effect of HTO or tritium labeled amino acid valine, 100 MBq/L, was investigated using restriction analysis of marker amplicons (Guseynov et al., 2014). Mutations were not found in bacterial DNA. Role of membrane processes of bacteria in biological responses to alpha- and beta-emitting radionuclides, possible genetic mutations, changes in composition of intracellular proteins and lowmolecular components, is a question of further interest in this scientific field. 5. Conclusions Recent years have seen a change in the approach in radioecological studies: biota in toto is considered now as a target of radiation impact, with the human included as a part of biota and integrated into the biosphere by a multiplicity of functional interrelations. As microorganisms are the most basic and numerous part of biosphere, their physiological indices are useful for monitoring the state of the biosphere on the whole. The paper summarizes studies of effects of alpha- and betaemitting radionuclides on marine microorganisms under conditions of low-dose irradiation. Luminous marine bacteria were chosen as an example of these microorganisms since they are known to be sensitive to toxic compounds. Toxic effects of nonradioactive compounds on the marine bacteria are well-studied for several decades; however the radioactive impact has come to attention of researchers not long ago. Bioluminescent intensity was used here as main testing physiological parameter of the bacteria. Non-linear dose-effect response of the bacteria to the chronic radioactive exposure was demonstrated. Three successive stages in the bioluminescent response to Am-241 and H-3 were found under conditions of low-dose irradiation: (1) absence of effects, (2) activation, and (3) inhibition. They were interpreted in terms of bacterial response to stress factor as stress recognition, adaptive response/syndrome, and suppression of physiological function (i.e.

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radiation toxicity), respectively. The effects were attributed to radiation hormesis phenomenon. In Am-241 aquatic solutions, an increase of peroxide concentration and NADH (endogenous organic reducer) oxidation rates were demonstrated. The results reveal a biological role of reactive oxygen species generated in water solutions as secondary products of the radioactive decay. The study aimed at understanding the effects of low-dose irradiation on marine microorganisms. Additionally, it suggests an approach to evaluation of non-toxic and toxic stages under conditions of chronic radiation exposure. The time when bioluminescent intensity reverses from activation to inhibition can be considered as a beginning of a toxic stage. This paper demonstrates advantages of luminous marine bacteria for monitoring the microorganism’ response to alpha- and beta-emitting radionuclides under conditions of low-dose irradiation. Additionally, the advanced experience in effects of exogenous compounds on luminous bacteria suggests that the bacteria is a convenient object to study physico-chemical mechanisms of toxic and hormetic effects of the radionuclides. Bioluminescence of bacteria can be considered as a simple model process for understanding activation and inhibition of physiological functions of higher organisms. Acknowledgments This work was supported by the Russian Foundation for Basic Research, Grant No.13-04-01305a, the Program “Molecular and Cellular Biology” of the Russian Academy of Sciences, project VI 57.1.1. The part of the work (review of effects of americium-241) was supported by the Russian Science Foundation, Grant No. 1414-00076. References Albers, R.W., 1967. Biochemical aspects of active transport. Biochemistry 36, 727e756. Alexandrova, M.A., Rozhko, T.V., Badun, G.A., Bondareva, L.G., Vydryakova, G.A., Kudryasheva, N.S., 2010. Effect of tritium on growth and bioluminescence of bacteria P. Phosphoreum. Radiat. Biol. Radioeсol. 6, 613e618. Alexandrova, M., Rozhko, T., Vydryakova, G., Kudryasheva, N., 2011. Effect of americium-241 on luminous bacteria. role of peroxides. J. Environ. Radioact. 102, 407e411. Alexandrova, M.A., Badun, G.A., Kudryasheva, N.S., 2012. Effect of tritium on bioluminescent systems. Luminescence 27, 95e96. Antunes, M.C.G., Pereira, C.C.C., Silva, J.C.G.E., 2007. MCR of the quenching of the EEM of fluorescence of dissolved organic matter by metal ions. Anal. Chim. Acta 595, 9e18. Berovic, N., Parker, D.J., Smith, M.D., 2008. An investigation of the reaction kinetics of luciferase and the effect of ionizing radiation on the reaction rate. Eur. Biophys. J. 38 (4), 427e435. Bolsunovsky, A., 2010. Artificial radionuclides in sediment of the Yenisei River. Chem. Ecol. 26, 401e409. Bulish, A.A., Isenberg, D.L., 1981. Use of the luminescent bacterial system for rapid assessment of aquatic toxicity. ISA Trans. 20, 29e33. Burlakova, E.B., Konradov, A.A., Maltseva, E.X., 2004. Effect of extremely weak chemical and physical stimuli on biological systems. Biophys. Mosc. 49, 522e534. Calabrese, E.J., 2013. Hormetic mechanisms. Crit. Rev. Toxicol. 43, 580e606. Calabrese, E.J., 2014. Hormesis: a fundamental concept in biology. Microb. Cell. 1, 145e149. Calabrese, E.J., Baldwin, L.A., 2001. The frequency of U-shaped dose responses in the toxicological literature. Toxicol. Sci. 62, 330e338. Calabrese, E.J., Blain, R.B., 2011. The hormesis database: the occurrence of hormetic dose responses in the toxicological literature. Reg. Toxicol. Pharmacol. 61, 73e81. Choppin, G.R., 1971. Structure and thermodynamics of lanthanide and actinide complexes in solution. Pure Appl. Chem. 27, 23. Choppin, G.R., Labonne-Wal, N., 1997. Comparison of two models for metal-humic interactions. J. Radioanal. Nucl. Chem. 221, 67e80. Czapski, G., Goldstein, S., Andorn, N., Aronovitch, J., 1992. Radiation-induced generation of chlorine derivatives in nitrous oxide-saturated phosphate buffered saline: toxic effects on Escherichia coli cells. Free Radic. Biol. Med. 12, 353e364. Davydov, A.S., 1973. The theory of contraction of proteins under their excitation. J. Theor. Biol. 38, 559e569.

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