Journal of Environmental Radioactivity 142 (2015) 68e77

Contents lists available at ScienceDirect

Journal of Environmental Radioactivity

journal homepage: www.elsevier.com/locate/jenvrad

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-235238, 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 elds.
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. Benets 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, avin mononucleotide;
DRIFT, diffuse reectance infrared Fourier transform.
* Corresponding author. Tel.: 7 391 2494242; fax: 7 391 2433400.
E-mail address: n_qdr@yahoo.com (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 inuence e from primary physicochemical processes under
ionization conditions in water solutions to macromolecular transformation and cellular membrane processes. Regularities of
ionizing radiation inuence 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 specic 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.

N.S. Kudryasheva, T.V. Rozhko / Journal of Environmental Radioactivity 142 (2015) 68e77

Scope of application of the bioluminescent bacteria-based assay

should be clearly specied. It is classied 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, sh, 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
nonspecicity (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 modied by different researchers
and adapted for their specic 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 I1 =Icontr
Here, Icontr and I1 are bioluminescent intensities in the absence
and presence of exogenous compounds, respectively. Value of

69

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 rst time in 1990 (Kratasyuk, 1990). It can be
considered as simplied luminescent function of the bacteria,
characterizing changes of rates of biochemical reactions under the
inuence 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 denite 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 rst one, catalyzed by NADH:FMNoxidoreductase, is a reduction of FMN by NADH:
NADH:FMNoxidoreductase

NADH FMN ! FMN$H NAD

(1)

In the second reaction, catalyzed by bacterial luciferase, the

reduced avin (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 RCHO O2 !FMN RCOO H2 O hn

(2)

Peculiarity of the enzymatic assay system is its specicity 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 classication 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
classication suggested.
3. Effects of alpha- and beta-emitting radionuclides on
luminous marine bacteria
For the rst 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 reies:
changes of kinetic parameters of bioluminescence of fungi exposed

70

N.S. Kudryasheva, T.V. Rozhko / Journal of Environmental Radioactivity 142 (2015) 68e77

to X-ray irradiation were reported in (Kobzeva et al., 2014); effects

of a proton beam from a cyclotron on kinetics of rey 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(235238), 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 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 specic 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 . 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 inuence

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
specic radioactivity was not found under the conditions of the
experiment at all times of exposure. Fig. 2B demonstrates bioluminescence intensity vs. HTO specic 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
specic 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 specic
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).

N.S. Kudryasheva, T.V. Rozhko / Journal of Environmental Radioactivity 142 (2015) 68e77

71

Fig. 2. Relative bioluminescence intensity of lyophilized bacteria, Irel , in HTO, 2 MBq/L. Dependence on (A) time and (B) specic 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
specic 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 m-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 specic 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 unied, 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 () and

Fig. 3. Dependence of relative bioluminescent intensity of luminous bacteria P.phosphoreum, Irel , on time of exposure to 3 ( 10 Bq/L) and 241Am ( 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-(235238) were chosen as the radionuclides of high and low
specic 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 specic 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 bers 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 .
phosphoreum in solution of Am-241 (3000 Bq L1), redistribution of
Am-241 in cell culture and damage of cells exposed to the

72

N.S. Kudryasheva, T.V. Rozhko / Journal of Environmental Radioactivity 142 (2015) 68e77

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 .
Corresponding specic 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) xed from HTO (A 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 unied.

Since the term toxicity is dened 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 inuence of low
concentrations of toxic compounds, low dose radiation, and
other stressors. Examples of this phenomenon are: nonspecic
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 rst and second stages of exposure to HTO
(A 10 MBq/L) and Am-241 (A 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 rst 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.

N.S. Kudryasheva, T.V. Rozhko / Journal of Environmental Radioactivity 142 (2015) 68e77

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 rst entered to the
scientic 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 rst 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
intensication of the charge transfer processes in bioluminescent
systems, resulting in increase of rates of active ion transport across

73

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
xed 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 specic radioactivity of solutions can be
considered as an evidence of specic 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 specic 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 specic radioactivity, A. Time of exposure
e 18 min. Enzyme system (Selivanova et al., 2013).

74

N.S. Kudryasheva, T.V. Rozhko / Journal of Environmental Radioactivity 142 (2015) 68e77

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 specic 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 Reectance 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

specic 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
signicant 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 specic 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
scientic eld.
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.

N.S. Kudryasheva, T.V. Rozhko / Journal of Environmental Radioactivity 142 (2015) 68e77

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. Radioeol. 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 uorescence 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. Articial 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.

75

Deprezc, K., Robbensd, J., Nobelsb, I., Vanparysb, C., Vanermena, G., Tireza, K.,
Michielsc, L., Weltens, R., 2012. DISCRISET: a battery of tests for fast waste classication e application of tests on waste extracts. Waste Manag. 32, 2218e2228.
Deryabin, D.G., Aleshina, E.S., 2008. Natural and recombinant luminescent microorganisms in biotoxicity testing of mineral waters. Appl. Biochem. Microbiol.
44, 378e381.
Deryabin, D.G., Karimov, I.F., 2010. Characteristics of the response of natural and
recombinant luminescent microorganisms in the presence of Fe(2) ions. Appl.
Biochem. Microbiol. 46, 28e32.
Devyatkov, N.D., Golant, M.B., Betskiy, O.V., 1991. Millimeter Waves and their Role in
the Processes of Vital Activity. Radios and Connection, Moscow, 168 pp. (in
Russian).
Donnelly, K., Chen, J., Huebner, H., Brown, K., 1997. Utility of four strains of white-rot
fungi for the detoxication of 2,4,6-trinitrotoluene in liquid culture. Environ.
Toxic. Chem. 16, 1105e1110.
Esimbekova, E.N., Torgashina, I.G., Kratasyuk, V.A., 2009. Comparative study of
immobilized and soluble NADH: FMN-oxidoreductase-luciferase coupled
enzyme system. Biochem. Mosc. 74, 695e700.
Esimbekova, E.N., Kondik, A.M., Kratasyuk, V.A., 2013. Bioluminescent enzymatic
rapid assay of water integral toxicity. Environ. Monit. Assess. 185, 5909e5916.
Esimbekova, E., Kratasyuk, V., Shimomura, O., 2014. Application of enzyme bioluminescence in ecology. Adv. Biochem. Eng. Biotechnol. 144, 67e109. http://
dx.doi.org/10.1007/978-3-662-43385-0_3.
Evans, E.A., 1974. Tritium and its Compounds. John Wiley&Sons Inc., New York,
pp. 663e672.
Fedorova, E., Kudryasheva, N., Kuznetsov, A., Mogilnaya, O., Stom, D., 2007. Bioluminescent monitoring of detoxication processes: activity of humic substances
in quinone solutions. J. Photochem. Photobiol. B 88, 131e136.
Feinendegen, L.E., 2005. Evidence for benecial low level radiation effects and radiation hormesis. Br. J. Radiol. 78, 3e7.
Feinendegen, L.E., Pollycove, M., Neumann, R.D., 2007. Whole-body responses to
low-level radiation exposure: new concepts in mammalian radiobiology. Exp.
Hematol. 35, 37e46.
Fridovich, I., 1998. Oxygen toxicity: a radical explanation. J. Exp. Biol. 201,
1203e1209.
hlich, H., 1968. Long-range coherence and energy storage in biological systems.
Fro
Int. J. Quant. Chem. 2, 641e649.
Gerasimova, M.A., Kudryasheva, N.S., 2002. Effects of potassium halides on bacterial
bioluminescence. J. Photochem. Photobiol. 66 (3), 218e222.
Girotti, S., Ferri, E., Fumo, M., Maiolini, E., 2008. Monitoring of environmental pollutants by bioluminescent bacteria. Anal. Chim. Acta 608, 2e21.
Goldberg, Z., Rocke, D.M., Schwietert, C., Berglund, S.R., Santana, A., Jones, A.,
Lehmann, A., Stern, R., Lu, R., Siantar, C.H., 2006. Human in vivo dose-response
to controlled, low-dose low linear energy transfer ionizing radiation exposure.
Clin. Cancer Res. 12, 3723e3729.
Grabert, E., Kossler, F., 1997. About the effects of nutrients on the luminescent
bacteria test. In: Hastings, J.W., Kricka, L.J., Stanley, P.E. (Eds.), Bioluminescence
and Chemiluminescence. John Wiley & Sons, Chichester, pp. 291e294.
Gudkov, D.I., Zub, L.N., Derevets, V.V., Kuz'menko, M.I., Nazarov, A.B., Kaglian, A.E.,
Savitskii, A.L., 2002. 90Sr, 137Cs, 238Pu, 239240Pu, and 241Am radionuclides in
macrophytes within the Krasnensky ood plain: species specicity of concentration and distribution in phytocenosis components. Radiat. Biol. Radioecol. 42,
419e428.
Guseynov, O., Selivanova, M., Litvinova, I., Karpenok, P., Guseynova, V., Petrova, A.,
Kudryasheva, N., 2014. Effect of radioisotope tritium on bioluminescence and
mutations in luminous bacteria P. phosphoreum 1883 IBSO. Luminescence 29,
57e58.
Halliwell, B., Gutteridge, J.M.C., 2007. Free Radicals in Biology and Medicine. Oxford
University Press, New York, p. 704.
Hastings, J.W., 2012. Bioluminescence. Chapter 52 in Cell Physiology Sourcebook.
Elsevier, pp. 925e947.
Heinz, G.H., Hoffman, D.J., Klimstra, J.D., Stebbins, K.R., 2010. Enhanced reproduction in mallards fed a low level of methylmercury: an apparent case of hormesis. Environ. Toxicol. Chem. 29, 650e653.
, N., Fang, W.H., 2014. Understanding bacterial bioluminesHou, C., Liu, Y.J., Ferre
cence: a theoretical study of the entire process, from reduced avin to light
emission. Chem. A Eur. J. 20, 7979e7986.
Ilyin, L.A., Kutsenko, S.A., Savateev, N.V., Sofronov, G.A., Tiunov, L.A., 1990. Toxicological problems in mitigation strategies of chemical industries. J. All-Union
Mendeleev Chem. Soc. 35, 440e447.
Ivask, A., Rolova, T., Kahru, A., 2009. A suite of recombinant luminescent bacterial
strains for the quantication of bioavailable heavy metals and toxicity testing.
BMC Biotechnol. 9 (1), 41.
Jo, E.R., Jung, P.M., Choi, J., Lee, J.W., 2012. Radiation sensitivity of bacteria and virus
in porcine xenoskin for dressing agent. Radiat. Phys. Chem. 81, 1259e1262.
Kaiser, J., 2003. Hormesis: sipping from a poisoned chalice. Science 302, 376e379.
Kamnev, A.A., Sadovnikova, J.N., Tarantilis, P.A., Polissiou, M.G., Antonyuk, L.P., 2008.
Responses of Azospirillum brasilense to nitrogen deciency and to wheat lectin:
a diffuse reectance infrared Fourier transform (DRIFT) spectroscopic study.
Microb. Ecol. 56, 615e624.
Kamnev, A.A., Tugarova, A.V., Alexandrova, M.A., Tarantilis, P.A., Polissiou, M.G.,
Kudryasheva, N.S., 2011. Effects of a- and b-emitting radionuclides on Photobacterium phosphoreum: bioluminescence and diffuse reectance FTIR spectroscopic studies. In: Proc. Colloquium Spectroscopicum Internationale XXXVII
(Aug. 28eSept. 02, 2011, Buzios, Rio de Janeiro, Brazil).

76

N.S. Kudryasheva, T.V. Rozhko / Journal of Environmental Radioactivity 142 (2015) 68e77

Kamnev, A.A., Tugarova, A.V., Tarantilis, P.A., Gardiner, P.H.E., Polissiou, M.G., 2012.
Comparing poly-3-hydroxybutyrate accumulation in Azospirillum brasilense
strains Sp7 and Sp245: the effects of copper(II). Appl. Soil Ecol. 61, 213e216.
Kamnev, A.A., Tugarova, A.V., Selivanova, M.A., Tarantilis, P.A., Polissiou, M.G.,
Kudryasheva, N.S., 2013. Effects of americium-241 and humic substances on
Photobacterium phosphoreum: bioluminescence and diffuse reectance FTIR
spectroscopic studies. Spectrochim. Acta A Mol. Biomol. Spectrosc. 100,
171e175.
Kirillova, T.N., Kudryasheva, N.S., 2007. Effect of heavy atom in bioluminescent reactions. Anal. Bioanal. Chem. 387, 2009e2016.
Kirillova, T.N., Gerasimova, M.A., Nemtseva, E.V., Kudryasheva, N.S., 2011. Effect of
halogenated uorescent compounds on bioluminescent reactions. Anal. Bioanal. Chem. 400, 343e351.
Kislenko, V.N., Berlin, A.A., 1991. Kinetics and mechanism of hydrogen peroxide
oxidation of organic substances. Russ. Chem. Rev. 60, 949e981.
Kobzeva, T.V., Melnikov, A.R., Karogodina, T.Y., Zikirin, S.B., Stass, D.V., Molin, Y.N.,
Rodicheva, E.K., Medvedeva, S.E., Puzyr, A.P., Burov, A.A., Bondar, V.S.,
Gitelson, J.I., 2014. Stimulation of luminescence of mycelium of luminous fungus Neonothopanus nambi by ionizing radiation. Luminescence 29, 703e710.
Kratasyuk, V.A., 1990. Principles of luciferase biotesting. In: Biological Luminescence. World Scientic, Singapore, pp. 550e558.
Kratasyuk, V.A., Esimbekova, E.N., Gladyshev, M.I., Khromichek, E.B.,
Kuznetsov, A.M., Ivanova, E.A., 2001. The use of bioluminescent biotests for
study of natural and laboratory aquatic ecosystems. Chemosphere 42, 909e915.
Kudryasheva, N.S., 1999. Mechanisms of the effect of xenobiotics on bacterial
bioluminescence. Luminescence 14, 199e200.
Kudryasheva, N.S., 2006. Bioluminescence and exogenous compounds. Physicochemical basis for bioluminescent assay. J. Photochem. Photobiol. B 83, 77e86.
Kudryasheva, N.S., Tarasova, A.S., 2015. Pollutant toxicity and detoxication by
humic substances: mechanisms and quantitative assessment via luminescent
biomonitoring. Environ. Sci. Pollut. Res. 22, 155e167. http://dx.doi.org/10.1007/
s11356-014-3459-6.
Kudryasheva, N.S., Zuzikova, E.V., Gutnyk, T.V., Kuznetsov, A.M., 1996. Metallic salts
action on bacterial bioluminescent systems of different complexity. Biozika 41,
1264e1269.
Kudryasheva, N.S., Kratasyuk, V.A., Esimbekova, E.N., Vetrova, E.V., Kudinova, I.Y.,
Nemtseva, E.V., 1998. Development of the bioluminescent bioindicators for
analyses of pollutions. Field Anal. Chem. Technol. 5, 277e280.
Kudryasheva, N.S., Kudinova, I.Y., Esimbekova, E.N., Kratasyuk, V.A., Stom, D.I., 1999.
Effects of quinones and phenols on the NAD(H)-dependent triple systems.
Chemosphere 38, 751e758.
Kudryasheva, N.S., Esimbekova, E.N., Remmel, N.N., Kratasyuk, V.A., Visser, A.J.W.G.,
van Hoek, A., 2003a. Effect of quinones and phenols on the triple-enzyme
bioluminescent system with protease. Luminescence 18, 224e228.
Kudryasheva, N.S., Nemtseva, E.V., Visser, A.J.W.G., van Hoek, A., 2003b. Interaction
of aromatic compounds with Photobacterium leiognathi luciferase: uorescence
anisotropy study. Luminescence 18, 156e161.
Kudryasheva, N.S., Nemtseva, E.V., Kirillova, T.N., 2004. Exogenous compounds in
studying the mechanism of electron-excited state formation in bioluminescence. Biopolymers 74, 100e104.
Kudryasheva, N.S., Selivanova, M.A., Petrova, A.S., Rozhko, T.V., Tugarova, A.V.,
Kamnev, A.A., Devyatlovskaya, A.N., 2014. Bioluminescence as a tool for
studying mechanisms of radiation hormesis and radiation toxicity. Luminescence 29, 26e27.
Kudryashov, Y.B., 2004. Radiation Biophysics (Ionizing Radiation). Fizmatlit, Moscow, 448 pp. (in Russian).
Kurvet, I., Ivask, A., Bondarenko, O., Sihtm
ae, M., Kahru, A., 2011. LuxCDABEe
transformed constitutively bioluminescent Escherichia coli for toxicity
screening: comparison with naturally luminous Vibrio scheri. Sensors 11,
7865e7878.
Leippe, D.M., Nguyen, D., Zhou, M., Good, T., Kirkland, T.A., Scurria, M., Bernad, L.,
Ugo, T., Vidugiriene, J., Cali, J.J., Klaubert, D.H., O'Brien, M.A., 2011. A bioluminescent
assay for the sensitive detection of proteases. Biotechniques 50, 105e110.
Lenhart, J.J., Cabaniss, S.E., MacCarthy, P., Honeyman, B.D., 2000. Uranium (VI)
complexation with citric, humic and fulvic acids. Radiochim. Acta 88, 345e353.
Lenskii, L.A., 1981. Physics and Chemistry of Tritium. Energoizdat (in Russian),
Moscow.
Lloyd, D.C., Edvards, A.A., Leonard, A., Deknut, G.L., Verschaeve, L., Natarajan, A.T.,
Darrudi, F., Obe, G., Palitti, F., Tanzarella, A., Tawn, E.J., 1992. Chromosomal aberrations in human lymphocytes induced in vitro by very low doses of X-rays.
Int. J. Radiat. Biol. 61, 335e343.
Luckey, T.D., 1980. Hormesis with Ionizing Radiation. FL. CRC Press, Inc., Boca Raton,
225 pp.
Ma, X.Y., Wang, X.C., Ngo, H.H., Guo, W., Wu, M.N., Wang, N., 2014. Bioassay based
on luminescent bacteria: interferences, improvements, and applications. Sci.
Total Environ. 468e469, 1e11.
Matsumoto, H., Hamada, N., Takahashi, A., Kobayashi, Y., Ohnishi, T.J., 2007. Vanguards of paradigm shift in radiation biology: radiation-induced adaptive and
bystander responses. J. Radiat. Res. 48, 97e106.
Medvedeva, S.E., 1999. Transfer of xenobiotics through cell membranes of luminous
bacteria. Luminescence 14, 267e270.
~ ar, G., Loureiro, J., Coutinho, A.P., Trova
~o, J., Nunes, I.,
Mesquita, N., Portugal, A., Pin
Botelho, M.L., Freitas, H., 2013. Flow cytometry as a tool to assess the effects of
gamma radiation on the viability, growth and metabolic activity of fungal
spores. Int. Biodeterior. Biodegrad. 84, 250e257.

Min, V.J., Lee, C.W., Gu, M.B., 2003. Gamma-radiation dose-rate effects on DNA
damage and toxicity in bacterial cells. Radiat. Environ. Biophys. 42, 189e192.
Morrisseya, R., Hill, C., Begley, M., 2013. Shining light on food microbiology; applications of lux-tagged microorganisms in the food industry. Trends Food Sci.
Technol. 32, 4e15.
Mossman, J.L., 2001. Deconstructing radiation hormesis. Health Phys. 80, 263e269.
Mothersill, C., Seymour, C., 2014. Implications for human and environmental health
of low doses of ionising radiation. J. Environ. Radioact. 133, 5e9.
Natecz-Jawecki, G., Rudz, B., Sawicki, J., 1997. Evaluation of toxicity of medical devices using spirotox and microtox tests: I. Toxicity of selected toxicants in
various diluents. J. Biomed. Mater. Res. 35, 101e105.
Nemtseva, E.V., Kudryasheva, N.S., 2007. The mechanism of electronic excitation in
bacterial bioluminescent reaction. Uspekhi Khimii 76, 101e112.
Pakhomova, V.M., 1995. The main provisions of the modern theory of stress and
nonspecic adaptation syndrome in plants. Tsytologya 37, 66e91.
Paul, J., Kadam, A.A., Govindwar, S.P., Kumar, P., Varshney, L., 2013. An insight into
the inuence of low dose irradiation pretreatment on the microbial decolouration and degradation of reactive red-120 dye. Chemosphere 90, 1348e1358.
Petukhov, V.N., Fomchenkov, V.M., Chugunov, V.A., Kholodenko, V.P., 2000. Plant
biotests for soil and water contaminated with oil and oil products. Appl. Biochem. Microbiol. 36, 564e567.
Popp, F.A., Yan, Y., 2002. Delayed luminescence of biological systems in terms of
coherent states. Phys. Lett. A 293, 93e97.
Qua, R., Wanga, X., Liub, Z., Yanb, Z., Wang, Z., 2013. Development of a model to
predict the effect of water chemistry on the acute toxicity of cadmium to
Photobacterium phosphoreum. J. Hazard Mater. 262, 288e296.
Rana, D., Matsuura, T., Kassim, M.A., Ismail, A.F., 2013. Radioactive decontamination
of water by membrane processes. Desalination 321, 77e92.
Ranjan, R., Rastogi, N.K., Thakur, M.S., 2012. Development of immobilized biophotonic beads consisting of Photobacterium leiognathi for the detection of
heavy metals and pesticide. J. Hazard. Mater. 225e226, 114e123.
Ray, S.D., Farris, F.F., Hartmann, A.C., 2014. Encyclopedia of Toxicology. Elsevier,
pp. 944e948.
Remmel, N.N., Titova, N.M., Kratasyuk, V.A., 2003. Oxidative stress monitoring in
biological samples by bioluminescent method. Bull. Exp. Biol. Med. 136,
209e211.
Rizzo, L., 2011. Bioassays as a tool for evaluating advanced oxidation processes in
water and wastewater treatment. Water Res. 45, 4311e4340.
Roda, A., Pasini, P., Mirasoni, M., Michchelini, E., Guardigli, M., 2004. Biotechnological application of bioluminescence and chemiluminescence. Trends Biotechnol. 22, 295e303.
Roda, A., Guardigli, M., Michelini, E., Mirasoni, M., 2009. Bioluminescence in
analytical chemistry and in vivo imaging. Trac-Trends Anal. Chem. 28, 307e322.
Rozhko, T.V., Kudryasheva, N.S., Kuznetsov, A.M., Vydryakova, G.A., Bondareva, L.G.,
Bolsunovsky, A.Y., 2007. Effect of low-level a-radiation on bioluminescent assay
systems of various complexity. Photochem. Photobiol. Sci. 6, 67e70.
Rozhko, T.V., Kudryasheva, N.S., Aleksandrova, M.A., Bondareva, L.G.,
Bolsunovsky, A.Y., Vydryakova, G.V., 2008. Comparison of effects of uranium
and americium on bioluminescent bacteria. J. Sib. Fed. Univ. Biol. 1, 60e64.
Rozhko, T.V., Bondareva, L.G., Mogilnaya, O.A., Vydryakova, G.A., Bolsunovsky, A.Y.,
Stom, D.I., Kudryasheva, N.S., 2011. Detoxication of Am-241 solutions by humic
substances: bioluminescent monitoring. Anal. Bioanal. Chem. 400, 329e333.
Sakuragi, T., Sawa, S., Sato, S., Kozaki1, T., Mitsugashira, T., Hara, M., Suzuki, Y., 2004.
Complexation of americium(III) with humic acid by cation exchange and solvent extraction. Radioanal. Nucl. Chem. 26, 309e314.
Sanotskiy, I.V., 1970. Methods for Determining the Toxicity and Hazards of Chemicals. Medicine, Moscow.
Saran, M., Bors, W., 1997. Radiation chemistry of physiological saline reinvestigated:
evidence that chloride-derived intermediates play a key role in cytotoxicity.
Radiat. Res. 147, 70e77.
Saran, M., Bertram, H., Bors, W., Czapski, G., 1993. On the cytotoxicity of irradiated
media. To what extent are stable products of radical chain reactions in physiological saline responsible for cell death? Int. J. Radiat. Biol. 64, 311e318.
Selivanova, M.A., Mogilnaya, O.A., Badun, G.A., Vydryakova, G.A., Kuznetsov, A.M.,
Kudryasheva, N.S., 2013. Effect of tritium on luminous marine bacteria and
enzyme reactions. J. Environ. Radioact. 120, 19e25.
Selivanova, M.A., Rozhko, T.V., Devyatlovskaya, A.S., Kudryasheva, N.S., 2014. Comparison of chronic low-dose effects of alpha- and beta-emitting radionuclides
on marine bacteria. Cent. Eur. J. Biol. 9, 951e959.
Selye, H., 1980. Changing distress into eustress: Hans Selye voices theories on stress.
Tex. Med. 76, 78e80.
~ a-Valle, M., Aguilar-Moreno, M., Balc
Serment-Guerrero, J., Bren
azar, M., 2012. Evidence of DNA double strand breaks formation in Escherichia coli bacteria
exposed to alpha particles of different LET assessed by the SOS response. Appl.
Radiat. Isot. 71, 66e70.
Shao, Y., Wu, L.L., Gao, H.W., Wang, F., 2012. Effect of soluble sulde on the activity
of luminescent bacteria. Molecules 17, 6046e6055.
Silva, J.C.G.E., Machado, A.A.S.C., Oliveira, C.J.S., 1996. Study of the Interaction of a
soil fulvic acid with UO2
2 by self-modeling mixture analysis of synchronous
molecular uorescence spectra. Analyst 121, 373e379.
Silva, J.C.G.E., Machado, A.A.S.C., Oliveira, C.J.S., Pinto, M.S.S.D.S., 1998. Fluorescence
quenching of anthropogenic fulvic acids by Cu(II),Fe(III) and UO2
2 . Talanta 45,
1155e1165.
Snigireva, G.P., Khaimovich, T.I., Bogomazova, A.N., Gorbunova, I.N., Nagiba, V.I.,
Nikanorova, E.A., Novitskaia, N.N., Khazins, E.D., 2009. Cytogenetic examination

N.S. Kudryasheva, T.V. Rozhko / Journal of Environmental Radioactivity 142 (2015) 68e77
of nuclear specialists exposed to chronic beta-radiation of tritium. Radiat. Biol.
Radioecol. 49, 60e66.
Southam, C.M., Ehrlich, J., 1943. Effects of extracts of western red-cedar heartwood
on certain wood-decaying fungi in culture. Phytopathology 33, 517e524.
Staunton, S., Dumat, C., Zsolnay, A., 2002. Possible role of organic matter in radiocaesium adsorption in soils. J. Environ. Radioact. 58, 163e173.
Stom, D.I., Geel, T.A., Balayan, A.E., Kuznetsov, A.M., Medvedeva, S.E., 1992. Bioluminescent method in studying the complex effect of sewage components. Arch.
Environ. Contam. Toxicol. 22, 203e208.
Szent-Gyorgyi, A., 1957. Bioenergetics. Acad. Press, New York.
Tarasova, A.S., Stom, D.I., Kudryasheva, N.S., 2011. Effect of humic substances on
toxicity of inorganic oxidizer. Bioluminescent monitoring. Environ. Toxicol.
Chem. 30, 1013e1017.
Tarasova, A.S., Kislan, S.L., Fedorova, E.S., Kuznetsov, A.M., Mogilnaya, O.A.,
Stom, D.I., Kudryasheva, N.S., 2012. Bioluminescence as a tool for studying
detoxication processes in metal salt solutions involving humic substances.
J. Photochem. Photobiol. 117, 164e170.
Thakur, M.S., Ragavan, K.V., 2013. Biosensors in food processing. J. Food Sci. Technol.
50, 625e641.
Thomas, D.J.L., Tyrrel, S.F., Smith, R., Farrow, S., 2009. Bioassays for the evaluation of
landll leachate toxicity. J. Toxicol. Environ. Health 12, 83e105.
Tigini, V., Giansanti, P., Mangiavillano, A., Pannocchia, A., Varese, G.C., 2011. Evaluation of toxicity, genotoxicity and environmental risk of simulated textile and
tannery wastewaters with a battery of biotests. Ecotoxicol. Environ. Saf. 74,
866e873.
Tomac, A., Mascheroni, R.H., Yeannes, M., 2013. Modelling the effect of gamma
irradiation on the inactivation and growth kinetics of psychrotrophic bacteria in
squid rings during refrigerated storage. Shelf-life predictions. J. Food Eng. 117,
211e216.
Vetrova, E.V., Kudryasheva, N.S., Visser, A.J., van Hoek, A., 2005. Characteristics of
enclogenous avin uorescence of Photobacterium leiognathi luciferase and
Vibrio scheri NAD(P)H: FMN-oxidoreductase. Luminescence 20, 205e209.
Vetrova, E.V., Kudryasheva, N.S., Kratasyuk, V.A., 2007. Redox compounds inuence
on the NAD(P)H: FMN-oxidoreductase e luciferase bioluminescent system.
Photochem. Photobiol. Sci. 6, 35e40.

77

Vetrova, E.V., Kudryasheva, N.S., Cheng, K.H., 2009. Effect of quinone on the uorescence decay dynamics of endogenous avin bound to bacterial luciferase.
J. Biophys. Chem. 141, 59e65.
Wang, W., Nykamp, J., Huang, X.D., Gerhardt, K., Dixon, D.G., Greenberg, B.M., 2009.
Examination of the mechanism of phenanthrenequinone toxicity to Vibrio
scheri: evidence for a reactive oxygen species-mediated toxicity mechanism.
Environ. Toxicol. Chem. 28, 1655e1662.
Wang, C.R., Tian, Y., Wang, X.R., Yu, H.X., Lu, X.W., Wang, C., Wang, H., 2010.
Hormesis effects and implicative application in assessment of leadcontaminated soils in roots of Vicia faba seedlings. Chemosphere 80, 965e971.
Xavier, M.P., Dauber, C., Mussio, P., Delgado, E., Maquieira, A., Soria, A., Curuchet, A.,
rquez, R., Me
ndez, C., Lo
pez, T., 2014. Use of mild irradiation doses to control
Ma
pathogenic bacteria on meat trimmings for production of patties aiming at
provoking minimal changes in quality attributes. Meat Sci. 98, 383e391.
Xu, T., Close, D., Smartt, A., Ripp, S., Sayler, G., 2014. Detection of organic compounds
with whole-cell bioluminescent bioassays. Adv. Biochem. Eng. Biotechnol. 144,
111e151.
Yea, Z., Zhao, Q., Zhang, M., Gao, Y., 2011. Acute toxicity evaluation of explosive
wastewater by bacterial bioluminescence assays using a freshwater luminescent bacterium, Vibrio qinghaiensis sp. Nov. J. Hazard. Mater. 186, 1351e1354.
Zaka, R., Chenal, C., Misset, M.T., 2002. Study of external low irradiation dose effects
on induction of chromosome aberrations in Pisum sativum root tip meristem.
Mutat. Res. 517, 87e99.
Zakhvataev, V.E., Khlebopros, R.G., 2012. The KupershtokheMedvedev electrostrictive instability as possible mechanism of initiation of phase transitions,
domains and pores in lipid membranes and inuence of microwave irradiation
on cell. Biophysics 57, 61e67.
Zotina, T.A., Bolsunovsky, A.Y., Bondareva, L.G., 2010. Accumulation of Am-241 by
suspended matter, diatoms and aquatic weeds of the Yenisei River. J. Environ.
Radioact. 101, 148e152.
Zotina, T.A., Kalacheva, G.S., Bolsunovsky, A.Y., 2011. Biochemical fractionation and
cellular distribution of americium and plutonium in the biomass of freshwater
macrophytes. J. Radioanal. Nucl. Chem. 290, 447e451.