On mechanism of antioxidant effect of fullerenols.

Fullerenols are nanosized water-soluble polyhydroxylated derivatives of fullerenes, specific allotropic form of carbon, bioactive compounds and perspective pharmaceutical agents. Antioxidant activity of fullerenols was studied in model solutions of organic and inorganic toxicants of oxidative type - 1,4-benzoquinone and potassium ferricyanide. Two fullerenol preparations were tested: С60О2-4(ОН)20-24 and mixture of two types of fullerenols С60О2-4(ОН)20-24+С70О2-4(ОН)20-24. Bacteria-based and enzyme-based bioluminescent assays were used to evaluate a decrease in cellular and biochemical toxicities, respectively. Additionally, the enzyme-based assay was used for the direct monitoring of efficiency of the oxidative enzymatic processes. The bacteria-based and enzyme-based assays showed similar peculiarities of the detoxification processes: (1) ultralow concentrations of fullerenols were active (ca 10-17-10-4 and 10-17-10-5 g/L, respectively), (2) no monotonic dependence of detoxification efficiency on fullerenol concentrations was observed, and (3) detoxification of organic oxidizer solutions was more effective than that of the inorganic oxidizer. The antioxidant effect of highly diluted fullerenol solutions on bacterial cells was attributed to hormesis phenomenon; the detoxification was concerned with stimulation of adaptive cellular response under low-dose exposures. Sequence analysis of 16S ribosomal RNA was carried out; it did not reveal mutations in bacterial DNA. The suggestion was made that hydrophobic membrane-dependent processes are involved to the detoxifying mechanism. Catalytic activity of fullerenol (10-8 g/L) in NADH-dependent enzymatic reactions was demonstrated and supposed to contribute to adaptive bacterial response.

Is bacterial luminescence response to low-dose radiation associated with mutagenicity?

Luminous marine bacteria are widely used in bioassays with luminescence intensity being a physiological parameter tested. The purpose of the study was to determine whether bacterial genetic alteration is responsible for bioluminescence kinetics change under low-dose radiation exposure. The alpha-emitting radionuclide 241Am and beta-emitting radionuclide 3H were used as the sources of low-dose ionizing radiation. Changes of bioluminescence kinetics of Photobacterium phosphoreum in solutions of 241Am(NO3)3, 7 kBq/L, and tritiated water, 100 MBq/L, were studied; bioluminescence kinetics stages (absence of effect, activation, and inhibition) were determined. Bacterial suspension was sampled at different stages of the bioluminescent kinetics; the doses accumulated by the samples were close or a little higher than a tentative limit of a low-dose interval: 0.10 and 0.85 Gy for 241Am, or 0.11 and 0.18 Gy for 3H. Sequence analysis of the 16S ribosomal RNA gene did not reveal a mutagenic effect of low-dose alpha and beta radiation in the bacterial samples. Previous results on bacterial DNA exposed to low-dose gamma radiation (0.25 Gy) were analyzed and compared to those for alpha and beta irradiation. It is concluded that bioluminescence activation and/or inhibition under the applied conditions of low-dose alpha, beta and gamma radioactive exposure is not associated with DNA mutations in the gene sequences tested.

Exposure of luminous marine bacteria to low-dose gamma-radiation.

The study addresses biological effects of low-dose gamma-radiation. Radioactive 137Cs-containing particles were used as model sources of gamma-radiation. Luminous marine bacterium Photobacterium phosphoreum was used as a bioassay with the bioluminescent intensity as the physiological parameter tested. To investigate the sensitivity of the bacteria to the low-dose gamma-radiation exposure (≤250 mGy), the irradiation conditions were varied as follows: bioluminescence intensity was measured at 5, 10, and 20°Ð¡ for 175, 100, and 47 h, respectively, at different dose rates (up to 4100 µGy/h). There was no noticeable effect of gamma-radiation at 5 and 10°Ð¡, while the 20°Ð¡ exposure revealed authentic bioluminescence inhibition. The 20°Ð¡ results of gamma-radiation exposure were compared to those for low-dose alpha- and beta-radiation exposures studied previously under comparable experimental conditions. In contrast to ionizing radiation of alpha and beta types, gamma-emission did not initiate bacterial bioluminescence activation (adaptive response). As with alpha- and beta-radiation, gamma-emission did not demonstrate monotonic dose-effect dependencies; the bioluminescence inhibition efficiency was found to be related to the exposure time, while no dose rate dependence was found. The sequence analysis of 16S ribosomal RNA gene did not reveal a mutagenic effect of low-dose gamma radiation. The exposure time that caused 50% bioluminescence inhibition was suggested as a test parameter for radiotoxicity evaluation under conditions of chronic low-dose gamma irradiation.

On the mechanism of biological activation by tritium.

The mechanism of biological activation by beta-emitting radionuclide tritium was studied. Luminous marine bacteria were used as a bioassay to monitor the biological effect of tritium with luminescence intensity as the physiological parameter tested. Two different types of tritium sources were used: HTO molecules distributed regularly in the surrounding aqueous medium, and a solid source with tritium atoms fixed on its surface (tritium-labeled films, 0.11, 0.28, 0.91, and 2.36 MBq/cm(2)). When using the tritium-labeled films, tritium penetration into the cells was prevented. The both types of tritium sources revealed similar changes in the bacterial luminescence kinetics: a delay period followed by bioluminescence activation. No monotonic dependences of bioluminescence activation efficiency on specific radioactivities of the films were found. A 15-day exposure to tritiated water (100 MBq/L) did not reveal mutations in bacterial DNA. The results obtained give preference to a "non-genomic" mechanism of bioluminescence activation by tritium. An activation of the intracellular bioluminescence process develops without penetration of tritium atoms into the cells and can be caused by intensification of trans-membrane cellular processes stimulated by ionization and radiolysis of aqueous media.

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

The paper summarizes studies of effects of alpha- and beta-emitting radionuclides (americium-241, uranium-235+238, 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 americium-241 and tritium were found: 1--absence of effects (stress recognition), 2--activation (adaptive response), and 3--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.

Antioxidant activity of humic substances via bioluminescent monitoring in vitro.

This work considers antioxidant properties of natural detoxifying agents-humic substances (HS) in solutions of model inorganic and organic compounds of oxidative nature-complex salt K3[Fe(СN)6] and 1,4-benzoquinone. Bioluminescent system of coupled enzymatic reactions catalyzed by NAD(P)H: FMN-oxidoreductase and bacterial luciferase was used as a bioassay in vitro to monitor toxicity of the oxidizer solutions. Toxicities of general and oxidative types were evaluated using bioluminescent kinetic parameters-bioluminescence intensity and induction period, respectively. Antioxidant activity of HS was attributed to their ability to decrease both general and oxidative toxicities; the HS antioxidant efficiency was characterized with detoxification coefficients D GT and D OxT, respectively. Dependencies of D GT and D OxT on HS concentration and time of preliminary incubation of the oxidizers with HS were demonstrated. The optimal conditions for detoxification of the oxidizers were >20-min incubation time and 0.5 × 10(-4) to 2 × 10(-4) M of HS concentration. The present study promotes application of the enzymatic luminescent bioassay to monitor toxicity of pollutants of oxidative nature in environmental and waste waters in remediation procedures.

Pollutant toxicity and detoxification by humic substances: mechanisms and quantitative assessment via luminescent biomonitoring.

The paper considers mechanisms of detoxification of pollutant solutions by water-soluble humic substances (HSs), natural detoxifying agents. The problems and perspectives of bioassay application for toxicity monitoring of complex solutions are discussed from ecological point of view. Bioluminescence assays based on marine bacteria and their enzymes are of special attention here; they were shown to be convenient tools to study the detoxifying effects on cellular and biochemical levels. The advantages of bioluminescent enzymatic assay for monitoring both integral and oxidative toxicities in complex solutions of model pollutants and HS were demonstrated. The efficiencies of detoxification of the solutions of organic oxidizers and salts of metals (including radioactive ones) by HS were analyzed. The dependencies of detoxification efficiency on time of exposure to HS and HS concentrations were demonstrated. Antioxidant properties of HS were considered in detail. The detoxifying effects of HS were shown to be complex and regarded as 'external' (binding and redox processes in solutions outside the organisms) and/or 'internal' organismal processes. The paper demonstrates that the HS can stimulate a protective response of bacterial cells as a result of (1) changes of rates of biochemical reactions and (2) stabilization of mucous layers outside the cell walls. Acceleration of auto-oxidation of NADH, endogenous reducer, by HS was suggested as a reason for toxicity increase in the presence of HS due to abatement of reduction ability of intracellular media.

Effect of tritium on luminous marine bacteria and enzyme reactions.

The paper studies chronic effect of tritiated water, HTO, (0.0002-200 MBq/L) on bioluminescent assay systems: marine bacteria Photobacterium phosphoreum (intact and lyophilized) and coupled enzyme reactions. Bioluminescence intensity serves as a marker of physiological activity. Linear dependencies of bioluminescent intensity on exposure time or radioactivity were not revealed. Three successive stages in bacterial bioluminescence response to HTO were found: (1) absence of the effect, (2) activation, and (3) inhibition. They were interpreted in terms of reaction of organisms to stress-factor i.e. stress recognition, adaptive response/syndrome, and suppression of physiological function. In enzyme system, in contrast, the kinetic stages mentioned above were not revealed, but the dependence of bioluminescence intensity on HTO specific radioactivity was found. Damage of bacteria cells in HTO (100 MBq/L) was visualized by electron microscopy. Time of bioluminescence inhibition is suggested as a parameter to evaluate the bacterial sensitivity to ionizing radiation.

Bioluminescence as a tool for studying detoxification processes in metal salt solutions involving humic substances.

The paper considers effects of humic substances (HS), as natural attenuators of toxicity, on solutions of model inorganic pollutants, metal salts - Pb(NO(3))(2), СоСl(2), CuSO(4), Eu(NO(3))(3), СrСl(3), and K(3)[Fe(СN)(6)]. Luminous bacteria Photobacterium phosphoreum and bioluminescent system of coupled enzymatic reactions were used as bioassays to monitor toxicity of salt solutions. The ability of HS to decrease or increase toxicity was demonstrated. Detoxifying concentrations of HS were determined; detoxification coefficients were calculated at different times of exposure of salt solutions to HS. To study the combined effects of HS and salts on bioluminescent assay systems, the rates of biochemical reactions and bacterial ultrastructure were analyzed. The detoxifying effects were explained by: (1) decrease of free metal content in water solutions under metal-HS binding; (2) increase of biochemical reaction rates in a bioluminescent assay system under HS effect; (3) enhancement of mucous layers on cell surface as a response to unfavorable impact of toxicants. Detoxifying mechanisms (2) and (3) reveal the active role of bioassay systems in detoxification processes.

Redox compounds influence on the NAD(P)H:FMN-oxidoreductase-luciferase bioluminescent system.

A review of the mechanisms of the exogenous redox compounds influence on the bacterial coupled enzyme system: NAD(P)H:FMN-oxidoreductase-luciferase has been done. A series of quinones has been used as model organic oxidants. The three mechanisms of the quinones' effects on bioluminescence were suggested: (1) inhibition of the NADH-dependent redox reactions; (2) interactions between the compounds and the enzymes of the coupled enzyme system; and (3) intermolecular energy migration. The correlation between the kinetic parameters of bioluminescence and the standard redox potential of the quinones proved that the inhibition of redox reactions was the key mechanism by which the quinones decrease the light emission intensity. The changes in the fluorescence anisotropy decay of the endogenous flavin of the enzyme preparations showed the direct interaction between quinones and enzymes. It has been demonstrated that the intermolecular energy migration mechanism played a minor role in the effect of quinones on the bioluminescence. A comparative analysis of the effect of quinones, phenols and inorganic redox compounds on bioluminescent coupled enzyme systems has been carried out.

Characteristics of endogenous flavin fluorescence of Photobacterium leiognathi luciferase and Vibrio fischeri NAD(P)H:FMN-oxidoreductase.

The bioluminescent bacterial enzyme system NAD(P)H:FMN-oxidoreductase-luciferase has been used as a test system for ecological monitoring. One of the modes to quench bioluminescence is the interaction of xenobiotics with the enzymes, which inhibit their activity. The use of endogenous flavin fluorescence for investigation of the interactions of non-fluorescent compounds with the bacterial luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri has been proposed. Fluorescence spectroscopy methods have been used to study characteristics of endogenous flavin fluorescence (fluorophore lifetime, the rotational correlation time). The fluorescence anisotropy behaviour of FMN has been analysed and compared to that of the enzyme-bound flavin. The fluorescence characteristics of endogenous flavin of luciferase and NAD(P)H:FMN-oxidoreductase have been shown to be applicable in studying enzymes' interactions with non-fluorescent compounds.

Exogenous compounds in studying the mechanism of electron-excited state formation in bioluminescence.

Using a series of exogenous fluorescent molecules as potential energy acceptors, the hypothesis on the activity of the upper electron-excited states in bioluminescence was tested. The results in bacterial and firefly bioluminescent enzyme systems were compared. Similar activity to the energetic precursor in bacterial bioluminescence was not proven in the case of the firefly system, the result of a very efficient intramolecular energy transfer in the emitter of the firefly bioluminescence. The influence of a number of metallic salts on a bacterial bioluminescent enzyme system was studied. Bioluminescence inhibition coefficients were compared to the free energies of electron withdrawing of cations. The correlation shows that inhibition and activation of luminescence intensity result from the effects of cations on electron transfer in the bioluminescent system.

Effect of quinones and phenols on the triple-enzyme bioluminescent system with protease.

The study addressed the effects of redox-active compounds on trypsin activity. Series of organic oxidizers (quinones) and reducers (phenols) were chosen as model redox-active compounds. Trypsin activity was quantified by bioluminescent technique. Interactions of these compounds with trypsin were studied by fluorescent and light absorption methods. Luminescence intensity decay constants in the reduced nicotinamidadeninedinucleotide (NADH): flavinmononucleotide (FMN)-oxidoreductase (R)-luciferase (L)-trypsin (T) (R + L + T) triple-enzyme system were calculated and compared in the presence of different concentrations of quinones and phenols. The triple-enzyme system was shown to be sensitive to quinones and not sensitive to phenols. It has been found that the effects produced by quinones on the coupled enzyme system (R + L) and on the trypsin molecule (T) are not related. The conclusions were extrapolated to the properties of other proteases and antiproteases.

Interaction of aromatic compounds with Photobacterium leiognathi luciferase: fluorescence anisotropy study.

The time-resolved and steady-state fluorescence techniques were employed to elucidate possible interactions of four aromatic compounds (anthracene, POPOP, MSB and 1,4-naphthalendiol) with bacterial luciferase. Fluorescence spectra and fluorescence anisotropy decays of these compounds were studied in ethanol, water-ethanol solutions and in the presence of bacterial luciferase. Shifts of fluorescent spectra and differences in rotational correlation times are interpreted in terms of weak (hydrophobic) interactions of the molecules with the enzyme. These interactions suggest the feasibility of intermolecular energy transfer by an exchange resonance mechanism with a collision-interaction radius as a way of excitation of these compounds in the reaction catalysed by bacterial luciferase.

Estimation of energy of the upper electron-excited states of the bacterial bioluminescent emitter.

The hypothesis of activity of the upper electron-excited states of the bacterial bioluminescent emitter was verified using dye molecules as foreign energy acceptors. Six compounds were selected having fluorescent state energies ranging from 25,700 to 32,000 cm(-1) (anthracene, pyrene, 1.4-bis(5-phenyloxasol-2-yl)benzene (POPOP), p-bis(o-methylstyryl)benzene (MSB), 2-methoxy-naphtalene, p-terphenyl), exceeding that of the bioluminescent emitter (22,000 cm(-1)). Their absorption spectra do not overlap with the bioluminescence spectrum; the trivial light absorption and the intermolecular resonance S-S energy transfer were excluded. Bacterial bioluminescent spectra of the coupled enzyme system NADH:FMN-oxidoreductase-luciferase in the presence of MSB were presented as an example. The weak sensitized fluorescence of MSB was registered. The results obtained have confirmed the activity of the energetic precursor in the bacterial bioluminescence. Its energy can be located in the interval of 26,000-27,000 cm(-1).

Effects of potassium halides on bacterial bioluminescence.

The effects of potassium halides KCl, KBr and KI on NADH:FMN-oxidoreductase-luciferase bioluminescent coupled enzyme system were studied. The influence of salt additions on bioluminescence intensity and bioluminescence light yield was investigated. The inhibition and activation parameters of the salts were calculated using their dependencies on concentration of the salts. The correlation between the inhibition of bioluminescence intensity and the halide mass was demonstrated: the inhibiting ability of the salts increases with the increase of atomic weight of the anions. The inhibition parameters increase and the activation parameters decrease, accordingly.

Upper electron-excited states in bioluminescence: experimental indication.

The involvement of upper electron-excited states in bacterial bioluminescence process was studied with excitation energy-accepting molecules. The fluorescent aromatic compounds, anthracene and 1.4-bis(5-phenyloxazol-2-yl)benzene, were chosen. Energies of their lowest excited singlet states are higher than the energy of the analogous state of the bioluminescence emitter; their absorption spectra and bioluminescence do not overlap. Hence, the excitation of these molecules by singlet-singlet energy transfer or by light absorption is excluded. Sensitized fluorescence of these compounds in the bioluminescence systems has been recorded, indicating the activity of upper electron-excited states in the bioluminescent process.

Bioluminescent water quality monitoring of salt lake Shira.

The coupled bioluminescent enzyme system luciferase-NADH:FMN-oxidoreductase was used as a biotest in ecological monitoring of the health resort salt lake Shira (South Siberia, Russia). The technique was adapted to saltwater conditions. Bioluminescence kinetic parameters sensitive to pollutants were determined. Conditions for the use of bacterial bioluminescence biotests in salty environmental media were established.

Mechanisms of the effect of xenobiotics on bacterial bioluminescence.

The influence of xenobiotics on the bioluminescent enzyme system is considered in terms of molecular action on the primary physicochemical processes-energy, electron and hydrogen (e(-) + H(+)) transduction. Dyes, non-fluorescent chemically inert organic compounds, redox-active organic compounds and metallic salts were investigated. The influence of the different xenobiotics depends in a complex way on physicochemical characteristics of the xenobiotic molecules (spectral-luminescent characteristics, electron-acceptation energy and redox potential).