Effect of americium-241 on luminous bacteria. Role of peroxides

Effect of americium-241on luminous bacteria.Role of peroxidesM.Alexandrova a ,*,T.Rozhko a ,G.Vydryakova b ,N.Kudryasheva a ,ba Siberian Federal University,Svobodny 79,660041Krasnoyarsk,RussiabInstitute of Biophysics SB RAS,Akademgorodok 50,660036Krasnoyarsk,Russiaa r t i c l e i n f oArticle history:Received 25October 2010Received in revised form 12February 2011Accepted 13February 2011Available online 8March 2011Keywords:Luminous bacteria Radiotoxicity Am-241Hormesis Peroxidea b s t r a c tThe effect of americium-241(241Am),an alpha-emitting radionuclide of high speci fic activity,on lumi-nous bacteria Photobacterium phosphoreum was studied.Traces of 241Am in nutrient media (0.16e 6.67kBq/L)suppressed the growth of bacteria,but enhanced luminescence intensity and quantum yield at room temperature.Lower temperature (4 C)increased the time of bacterial luminescence and revealed a stage of bioluminescence inhibition after 150h of bioluminescence registration start.The role of conditions of exposure the bacterial cells to the 241Am is discussed.The effect of 241Am on luminous bacteria was attributed to peroxide compounds generated in water solutions as secondary products of radioactive decay.Increase of peroxide concentration in 241Am solutions was demonstrated;and the similarity of 241Am and hydrogen peroxide effects on bacterial luminescence was revealed.The study provides a scienti fic basis for elaboration of bioluminescence-based assay to monitor radiotoxicity of alpha-emitting radionuclides in aquatic solutions.Ó2011Elsevier Ltd.All rights reserved.1.IntroductionRecent years have seen a change in the approach in radio-ecological studies:biota in toto might be considered 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.Microorganisms are the simplest and perhaps most basic part of the biosphere,and their physiological indices can serve as an indi-cator of the state of the biosphere on the whole.Hence,microor-ganisms can be used as bioassays to monitor environmental radiotoxicity.Generally,the main feature of all bioassays is an integral response that accounts for non-additivity of the effects of numerous environmental pollutants and natural components.In this case,the bioassays indicate media toxicity,considering that toxic effect of a sum of compounds (including radioactive ones)can be higher or lower than the sum of the effects of these compounds.Radiometric analyses per se do not characterize medium hazard to living organisms since they do not consider the integral effect of all substances and difference in sensitivity of various organisms.It is supposed (Kudryasheva et al.,1998)that only a combination ofradiometric,chemical and biological methods can provide complete information on ecological state of a medium.Assay systems based on luminous marine bacteria are good candidates for radiotoxicity monitoring.Bacterial bioluminescent assays are widely used to monitor environmental toxicity for more than forty years (Girotti et al.,2008;Ivask et al.,2009;Kudryasheva et al.,2003;Roda et al.,2009;Thomas et al.,2009),and now they are the traditional and important biotechnological applications of bioluminescence phenomenon.The tested parameter here is the luminescence intensity of bacteria that can be easily measured instrumentally.The advantages of bioluminescent assays are their high sensitivity,simplicity,rapidity (1e 3min),and availability of the devices for toxicity registration (Gitelson and Kratasyuk,2002).Bacterial bioluminescent assays are humane,as they do not apply high organisms.In 1990,the bioluminescent system of enzymatic coupled reactions was suggested as a toxicity assay (Kratasyuk,1990);its applications were developed (Esimbekova et al.,2007,2009).Mechanisms of exogenous compounds interactions with enzyme systems were reviewed in (Kudryasheva,2006a,b ).For the first time,bacterial bioluminescence was used to monitor radiation toxicity in Min et al.(2003).In this work,the effect of gamma-ray irradiation on a recombinant Escherichia coli strain was studied.In our previous works (Rozhko et al.,2007,2008),we studied sensitivity of bioluminescent assay systems to americium-241(241Am),an alpha-emitting radionuclide,the product of pluto-nium radioactive decay.It is known to be increasingly accumulated by the environment.Observations in the waters of the Chernobyl*Corresponding author.Tel.:þ7(391)2494242;fax:þ7(391)2433400.E-mail address:maka-alexandrova@rambler.ru (M.Alexandrova).Contents lists available at ScienceDirectJournal of Environmental Radioactivityjou rnal homepage:www.e lse /locate/jenvrad0265-931X/$e see front matter Ó2011Elsevier Ltd.All rights reserved.doi:10.1016/j.jenvrad.2011.02.011Journal of Environmental Radioactivity 102(2011)407e 411zone contaminated with radioactive fallout demonstrated that aquatic plants accumulate241Am in their biomass in a species-specific manner(Gudkov,2002).Accumulation of241Am by sedi-ments and aquatic plants in the Siberian River Yenisey is currently under study(Bolsunovsky,2010;Zotina et al.,2010).We found that bioluminescent assay systems in vivo and in vitro were shown to be sensitive to241Am in the activity range 0.1e6kBq/L(Rozhko et al.,2007,2008).The addition of241Am to bacterial assay systems was found to result in a50-h activation of bioluminescence intensity,which was followed by inhibition of bioluminescence in all samples of different activity concentrations. The doses of0.25e1.5Gy were delivered to bacterial cells by the time of bioluminescence inhibition.The adaptation of bacterial bioluminescent systems for bioassay purposes requires further basic investigations on the bacteria properties under various irradiation conditions,as well as mecha-nisms of the radionuclide impact on bacterial luminescence as a physiological function of the microorganism.The current paper provides additional data on the influence of alpha radiation on luminous bacteria.We consider chronic effect of 241Am on bacterial growth and bioluminescence.Bioluminescent kinetics at20 C and4 C were analyzed,and relative biolumi-nescence quantum yields were calculated.Accumulation of241Am in cells and DNA was determined.The question of our interest deals with the molecular mecha-nism of bioluminescence activation and inhibition under exposure to radiation.Bioluminescence of bacteria can be considered as a simple model process for understanding activation and inhibition of physiological functions of higher organisms.Radiolysis of water is a known process occurring under ionizing radiation(Fridovich,1998;Halliwell,2003).Secondary products of ionizing radiation(ions and 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 metabolic reactions,mainly,oxidation of organic compounds. Depending on concentrations,they can either activate or inhibit the metabolic processes(Kislenko and Berlin,1991;Kudryashov,2004). We hypothesize that peroxide compounds can be responsible for activation and inhibition of the bioluminescence function of lumi-nous bacteria.In our experiments,we monitor peroxides in241Am solutions and compare the effects of hydrogen peroxide and241Am on luminous bacteria P.phosphoreum,thus elucidating the role of peroxides in activation and inhibition of bioluminescence.2.Materials and methodsLuminous bacteria P.phosphoreum from the Collection of the Institute of Biophysics SB RAS(CCIBSO863),strain1883IBSO,were applied.Nutrient media used for bacterial growth included:1L H2O,30g NaCl,1g KH2PO4$12H2O,0.5g(NH4)2HPO4,0.2g MgSO4$7H2O,6g Na2HPO4,3ml glycerol,and5g peptone.Reagents included potassium ferricyanide,3%H2O2solution and luminol (Molecular probes Inc.).Solutions of241Am(NO3)3were used as the source of ionizing radiation.Bacteria were grown in30mL nutrient media with241Am(NO3)3 involved;media activity was0.16,0.67,1.7,3.3,6.7kBq/L.Control (without Am)and tested(with Am)bacterial suspensions were examined and compared.Optical density(D)of suspensions was used to monitor bacterial growth.Number of bacterial cells(N)was calculated as:N¼KD. Here,K is a coefficient of linear dependence calculated in prelimi-nary microscopic experiments with thin-layer suspensions by method of direct enumeration(Bianchi and Giuliano,1996;Kepner and Pratt,1994).Cells were visualized withfluorochrome DAPI.Fig.1presents the examples of bacterial growth in control and radioactive nutrient media.Bioluminescence intensity was measured in bacterial suspen-sions sampled at two growth stages:in15,and22h,Fig.1.The samples were placed into microplates;the time-course of their bioluminescence intensity was measured in solutions of3%NaCl at þ20 C.The samples were kept atþ20 C orþ4 C.The volume ratio of bacterial suspension and NaCl solution in microplate samples was1:30.Specific bioluminescence intensity,I s,was calculated as: I s¼I=NHere,I is a bioluminescent intensity of radioactive sample,N is a number of cells in the sample.Values of I s were plotted vs.time of light emission.Relative quantum yields of the samples were calculated as a ratio of areas restricted by bioluminescence kinetics curves in the presence and absence of241Am.Bioluminescent measurements were carried out in four repli-cations;SD values were calculated.Fig.2presents the examples of time-course of I s values in control and radioactive nutrient media.Relative bioluminescence intensity,I rel s,was calculated as I rel s¼I s=I s contr,where I s is a specific bioluminescent intensity in radioactive solution,I s contr is a specific bioluminescent intensity in a control sample without241Am.To assess the influence of H2O2on the bacteria,the effect of H2O2 on bioluminescence was investigated using the following reaction mixture:50m L bacterial suspension,50m L H2O2in3%NaCl. Concentration of H2O2in the mixture varied up to10À3M.Relative bioluminescence intensity,I rel,was calculated as I rel¼I/I contr,where I is a bioluminescent intensity in H2O2solution,I contr is a biolumi-nescent intensity in a control sample without H2O2.Peroxide component in solutions of241Am(NO3)3was deter-mined by the chemiluminescent method(Komagoe and Katsu, 2006).Luminol chemiluminescence was triggered by addition of potassium ferricyanide.Bioluminescence and chemiluminescence intensity was measured by TriStar Multimode Microplate Reader LB941,Berthold Technologies.Optical density of bacterial suspension was registered by colorimeter KFK-2MP,Russia.The number of cells was counted byfluorescence microscope Zeiss Axioskop40with a Filter Set02, Zeiss,Germany.The DNA was extracted with the IsoCode Paper according to manufacturer instructions(Gupta et al.,2010;Lee et al.,2010). Briefly,from8to10m L aliquots of bacterial cultures werespotted Fig.1.Time-course of bacterial growth in a control sample and in the presence of 241Am,1.7kBq/L.M.Alexandrova et al./Journal of Environmental Radioactivity102(2011)407e411 408directly onto the corner of paper and DNA was eluted in a 100m L volume of sterile water,with 10m L used for electrophoresis.Elec-trophoresis was performed in 1%agarose gel in 1ÂTAE buffer.The s oncentration of DNA was determined by a spectrophotometric method (Sagi et al.,2009)with the UVIKON 943double-beam spectrophotometer,Italy.The 70m g samples of DNA were prepared,their radioactivity was analyzed.To determine the accumulation of 241Am in bacterial cells,the bacterial suspensions were centrifuged (6000Âg ,4 C,15min),bacterial and supernatant fractions were separated.The radioac-tivity of 241Am in bacterial,supernatant and DNA samples was measured by gamma-counter Wallac Wizard 1480,PerkinElmer,Finland.Sensitivity of the counter to 241Am is 0.05Bq per sample.The activity concentrations of 241Am were calculated in nutrient solutions (kBq/L),bacterial fraction (%),and DNA (mBq/m g).3.Results and discussion 3.1.Effect of241Am on luminous bacteriaKinetic curves for bacterial growth in a control sample and in the presence of 241Am,1.7kBq/L,are compared in Fig.1.Fig.3presents a number of bacteria in nutrient media of different radioactivity after 22-h bacterial growth.As is seen,the exposure to 241Am suppresses bacterial growth.Bacterial suspensions were sampled for bioluminescence measurements at two stages of bacterial growth e 15and 22h (shown in Fig.1with vertical arrows).The time-courses of biolu-minescent intensity in the control sample and in the presence of 241Am,1.7kBq/L,at room temperature are presented in Fig.2.Fig.4demonstrates the same result in terms of relative bioluminescent intensity,I rel .It is seen that I rel >1during the whole period of light registration.The radioactive sample demonstrated an increase of luminescent intensity (bioluminescence activation)during the whole period of light registration,as compared to that of the control sample.Similar results were obtained in solutions of different activity concentrations.Luminescence quantum yields were calculated for all radioac-tive solutions;they exceeded that of control sample (Fig.5).The time-courses and quantum yield dependences of 15-h and 22-h samples were similar.To increase time of exposure of the bacteria to the 241Am,we prolonged their luminescence lifetime up to 300h (Fig.6)by keeping the bacterial microplate samples at lower temperature.The Figure demonstrates the time-course of relative biolumines-cent intensity of the radioactive sample kept at 4 C.These condi-tions revealed bioluminescence activation (I rel >1)at t <80h being followed by bioluminescence inhibition (I rel <1)at t >150h in the radioactive sample.The change in bioluminescence time-course under exposure to 241Am shown in Fig.6is similar to those described previously (Rozhko et al.,2007).However,the difference occurs under conditions of the current experiment when the bioluminescence inhibition was observed only at lower temperature after 150h of exposure to 241Am (Fig.3),while in the previous paper (Rozhko et al.,2007)the bioluminescence inhibition took place at room temperature after 50h of exposure to 241Am.Most probably,this difference is related to conditions of exposure to 241Am:in this work the bacteria were grown in radioactive nutrient media,while in Rozhko et al.(2007)the 241Am was added to 15-h bacterial suspension according to standard technique of the bioluminescent assay.This difference is probably a demonstration of bacterial adaption ability to the unfavorable stress-factor.Its relation to genetic ‘adaptive response ’(Volkert,1988;Yu et al.,2006)might be considered in further experiments.Hence,the data demonstrate that the onset time of biolumi-nescence inhibition by 241Am depends on conditions of growing of the bacteria (radioactive nutrient media or not),and temperature.Activation of bioluminescence in Figs 2and 4e 6can be ascribed to the hormesis phenomenon (Calabrese and Baldwin,2001;Kaiser,Fig.2.Effect of 241Am on bacterial luminescence at 20 C,15-h bacterial culture:time-course of speci fic bioluminescence intensity (I s )in control and radioactive media,1.7kBq/L.Fig.3.Number of bacteria vs.241Am activity concentrations,A(Am),kBq/L,in 22-hbacterialculture.Fig.4.Effect of Am-241on bacterial luminescence at 20 C,15-h bacterial culture:time-course of relative bioluminescence intensity (I rel s)in radioactive media,1.7kBq/L.M.Alexandrova et al./Journal of Environmental Radioactivity 102(2011)407e 4114092003;Heinz et al.,2010).Activation of the vital functions of various organisms is a well-known effect;it is a general property of all living organisms.Hormesis is attributed to triggering of cell defenseresponse under the influence of low concentrations of toxic compounds,low dose radiation,and other stressors(Yu et al.,2006; Calabrese and Baldwin,2001).Examples of hormesis are nonspe-cific adaptive syndrome of plants(Pakhomova,1995)and stress reaction in animals(Selye,1980).The molecular mechanism of radiation hormesis is a question of high interest.Accumulation of241Am by bacterial cells was evaluated.It was found that the bacteria accumulated60e70%of the radionuclide from any22h suspension irrespective of its activity.The DNA electrophoretic diagram of all samples showed no visible changes. Radioactivity(1.7mBq/m g)was found only in DNA isolated from the sample of maximal activity.3.2.Role of peroxides in effects on luminous bacteriaTo verify the hypothesis on involvement of peroxides into radionuclide effect on the bacterial cells,we compared the effects of hydrogen peroxide(H2O2)and241Am as model compounds on bacterial luminescence(Fig.7and Fig.6,respectively).Fig.7gives the dependence of bioluminescent intensity on H2O2concentration. It is seen that lower concentrations of the peroxide(210À7e10À4M) activate bacterial luminescence,whereas higher concentrations (>10À4M)inhibit it.Activation reaches w300%at C(H2O2)¼1.710À6M.Bioluminescence activation by H2O2is supposed to deal with the increase of rates of metabolic oxidative reactions.One of them is the bioluminescence reaction which includes a peroxide compound (peroxyhemiacetal)as an intermediate(Nemtseva and Kudryasheva, 2007).The excess amount of reaction components is known to inhibit all the reactions and quench electron-excited states offluo-rofores(Lakowicz,2006),thus inhibiting bioluminescence and other metabolic processes.Previously in Remmel et al.(2003),activation and inhibition of bacterial luminescence by hydrogen peroxide was also reported,with Vibrio harveyi strain as an example.The luminol chemiluminescent method was applied to deter-mine peroxides in water solutions of241Am(NO3)3.Fig.8presents the dependence of peroxide concentration on activity of41Am in water solutions.One can see that the increase of peroxide concentration follows the rise of241Am activity.It was found that at A(241Am)¼5kBq/L,the concentration of peroxides exceeds their background concentration up to18times.It is possible that the increase of peroxide concentration takes place in solutions of all alpha-radionuclides of high specific activity.Hence,the peroxides produced in the solution of241Am can be a reason of the processes shown in Figs.2e5.Thus,the datapresentedFig.6.Effect of Am-241on bacterial luminescence at4 C,22-h bacterial culture:time-course of relative bioluminescence intensity(I rel s)in radioactive media,2kBq/L.Fig.7.Relative bioluminescent intensity of P.phosphoreum(I re l)at different concen-trations of H2O2C(H2O2),mM.Fig.5.Relative bioluminescence quantum yields vs.activity concentration of media,A(Am),kBq/L.Fig.8.Concentration of peroxides,C(H2O2),mM,at different activities of241Am inwater solutions,A(Am),kBq/L.M.Alexandrova et al./Journal of Environmental Radioactivity102(2011)407e411410in Figs.6e8support our hypothesis on the involvement of peroxides, secondary products of alpha-decay of241Am,in water solutions,into functioning of bioluminescent system of luminous bacteria.However,the comparison of H2O2concentrations and241Am radioactivity that either activates or inhibits bioluminescence, suggests that peroxides could not be the sole moiety affecting the bacteria.The argument is supported by the data of our last paper (Alexandrova et al.,2010)demonstrating bioluminescence activa-tion by beta-emitting radionuclide,tritium,which does not produce peroxides in water solutions.Probably,another type of particle,hydrated electrons,can 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 exposed to ionizing radiation.The results under consideration call for broad investigations of elementary mechanisms of ionizing radiation impact on living organisms.4.ConclusionWe showed that the presence of241Am in nutrient media (0.16e6.7kBq/L)suppressed the growth of bacteria and activated their luminescence at room temperature.Lower temperature increased the time of bacterial luminescence and revealed a stage of bioluminescence inhibition after150h of bioluminescence registration start.Analysis of241Am effects in the current and previous(Rozhko et al.,2007)experiments brought about conclu-sion that bioluminescence inhibition start depends on conditions of bacterial growth(radioactive nutrient media or not),and temper-ature of bioluminescence registration.The results were explained in terms of adaptive response and hormesis-effect in the bacteria exposed to241Am as to a stress-factor.It was found that bacteria accumulated up to60e70%of the 241Am from the nutrient solutions.We related the effects of241Am on luminous bacteria to peroxide compounds which are generated as products of water radiolysis. 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