Enzymatic Responses to Low-Intensity Radiation of Tritium.

The present study considers a possible role of enzymatic reactions in the adaptive response of cells to the beta-emitting radionuclide tritium under conditions of low-dose exposures. Effects of tritiated water (HTO) on the reactions of bacterial luciferase and NAD(P)H:FMN-oxidoreductase, as well as a coupled system of these two reactions, were studied at radioactivity concentrations ≤ 200 MBq/L. Additionally, one of the simplest enzymatic reactions, photobiochemical proton transfer in Coelenteramide-containing Fluorescent Protein (CLM-FP), was also investigated. We found that HTO increased the activity of NAD(P)H:FMN-oxidoreductase at the initial stage of its reaction (by up to 230%); however, a rise of luciferase activity was moderate (<20%). The CLM-FP samples did not show any increase in the rate of the photobiochemical proton transfer under the exposure to HTO. The responses of the enzyme systems were compared to the 'hormetic' response of luminous marine bacterial cells studied earlier. We conclude that (1) the oxidoreductase reaction contributes significantly to the activation of the coupled enzyme system and bacterial cells by tritium, and (2) an increase in the organization level of biological systems promotes the hormesis phenomenon.

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 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.

Fungi outcompete bacteria under increased uranium concentration in culture media.

As a key part of water management at the Ranger Uranium Mine (Northern Territory, Australia), stockpile (ore and waste) runoff water was applied to natural woodland on the mine lease in accordance with regulatory requirements. Consequently, the soil in these Land Application Areas (LAAs) presents a range of uranium concentrations. Soil samples were collected from LAAs with different concentrations of uranium and extracts were plated onto LB media containing no (0 ppm), low (3 ppm), medium (250 ppm), high (600 ppm) and very high (1500 ppm) uranium concentrations. These concentrations were similar to the range of measured uranium concentrations in the LAAs soils. Bacteria grew on all plates except for the very high uranium concentrations, where only fungi were recovered. Identifications based on bacterial 16S rRNA sequence analysis showed that the dominant cultivable bacteria belonged to the genus Bacillus. Members of the genera Paenibacillus, Lysinibacillus, Klebsiella, Microbacterium and Chryseobacterium were also isolated from the LAAs soil samples. Fungi were identified by sequence analysis of the intergenic spacer region, and members of the genera Aspergillus, Cryptococcus, Penicillium and Curvularia were dominant on plates with very high uranium concentrations. Members of the Paecilomyces and Alternaria were also present but in lower numbers. These findings indicate that fungi can tolerate very high concentrations of uranium and are more resistant than bacteria. Bacteria and fungi isolated at the Ranger LAAs from soils with high concentrations of uranium may have uranium binding capability and hence the potential for uranium bioremediation.

Differential proteome analysis of a selected bacterial strain isolated from a high background radiation area in response to radium stress.

The present study describes the response of a bacterial strain, isolated from a hot spring in an area with the highest levels of natural radiation, under radium ((226)Ra) stress. The bacterium has been characterized as a novel and efficient radium biosorbent and identified as a variant of Serratia marcescens by biochemical tests and molecular recognition. In order to gain insights into key cellular events that allow this strain to survive and undergo (226)Ra adaptation and biosorption, the strain was tested under two experimental conditions of 1000 and 6000 Bq (226)Ra stress. A proteomic approach involving two-dimensional polyacrylamide gel electrophoresis and mass spectrometry was used to identify the differentially expressed proteins under (226)Ra stress. Functional assessment of identified proteins with significantly altered expression levels revealed several mechanisms thought to be involved in (226)Ra adaptation and conferring resistant phenotype to the isolate, including general stress adaptation, anti-oxidative stress, protein and nucleic acid synthesis, energy metabolism, efflux and transport proteins. It suggests that this strain through evolution is particularly well adapted to the high background radiation environment and could represent an alternative source to remove (226)Ra from such areas as well as industrial radionuclide polluted wastewaters.

Normal axial skeleton structure in common roach Rutilus rutilus (Actinopterygii: Cyprinidae) and malformations due to radiation contamination in the area of the Mayak (Chelyabinsk Province, Russia) nuclear plant.

This study was designed to describe normal axial skeletal structure in common roach Rutilus rutilus from putative unaffected environmental conditions, and the occurrence of skeletal malformations in the fish from an area under radiation contamination. Specimens were collected from water bodies of the Techa Cascade Reservoirs located near the Mayak atomic industry plant in the River Ob' drainage, Chelyabinsk Province, Russia. One sample was collected from Lake Irtyash, a reservoir of drinkable water, supplying the town of Ozersk, and the other one from a technical reservoir which is a storage of liquid radioactive waste from Mayak and characterized by high radioactive contamination (mostly (90)Sr and (137)Cs). A comparison was made with historical material collected from the River Ob' before the middle of the 20th century, i.e. before the environment became affected by radioactive contamination. A high number of abnormalities of the axial skeleton were detected in both Mayak samples, in 94 and 97% of examined specimens, in contrast to about 20% in the historical specimens. The abnormalities were in both the unpaired fins and the vertebral column, including the caudal complex and included supernumerary elements, fusions, deformities and displacement of the elements. Most axial skeleton abnormalities, however, were minor, such as splitting, shortening or deformation of spines. Severe defects, such as extensive scolioses, lordoses and kyphoses, were not found. The causes of the abnormalities were not identified in this study, but the high incidence of malformations may be attributed to genetically determined imbalance during development. The almost equal distribution of abnormalities among the fish from non-contaminated and radioactive contaminated reservoirs may be explained by either recent gene flow within the population of R. rutilus in the River Techa system or the effect of unknown unfavourable environmental factors such as chemical pollution.

[Study on biosorption of uranium by Rhodotorula glutinis].

Characteristic of uranium biosorption in water solution by Rhodotorula glutinis was investigated in the present study and the optimal pH for uranium adsorption was found to be 6-7. At the same time, maximum adsorption capacity of 149.4 mgU/(g dry cell) was identified, and Langmuir adsorption models can be used to simulate the isothermal biosorption process with high correlation coefficient of 0.99. According to Fourier transform infrared spectra, a new peak at wave number of 904 cm(-1), which can be assigned to the stretch vibration of UO2, was detected in the cell which was contacted by the uranium, indicated that uranium was really absorbed by Rhodotorula glutinis. Changes in the uranium-exposed yeast biomass were in the stretching vibrations of amino or hydroxyl groups, which shift from 3309 to 3287 cm(-1), and in the stretching vibrations of C--O band, which shift from 1068 to 1080 cm(-1), and these are all attributed to the important role that they may played in the binding of uranium. Hardly any changes can be found in the characteristic IR adsorbing peaks of protein at wave numbers of 1653, 1540 and 1237 cm(-1) before and after uranium adsorption, making it clear that the major component and the structure of the biomass remained intact. 96% of the absorbed uranium can be easily desorbed by 0.1 mol x L(-1) NaHCO3. Obviously, the application potential of this yeast in the uranium wastewater treatment was very wide and expansive, and more more work should be done to realize its industrial use.

Caulobacter crescentus as a whole-cell uranium biosensor.

We engineered a strain of the bacterium Caulobacter crescentus to fluoresce in the presence of micromolar levels of uranium at ambient temperatures when it is exposed to a hand-held UV lamp. Previous microarray experiments revealed that several Caulobacter genes are significantly upregulated in response to uranium but not in response to other heavy metals. We designated one of these genes urcA (for uranium response in caulobacter). We constructed a reporter that utilizes the urcA promoter to produce a UV-excitable green fluorescent protein in the presence of the uranyl cation, a soluble form of uranium. This reporter is specific for uranium and has little cross specificity for nitrate (<400 microM), lead (<150 microM), cadmium (<48 microM), or chromium (<41.6 microM). The uranium reporter construct was effective for discriminating contaminated groundwater samples (4.2 microM uranium) from uncontaminated groundwater samples (<0.1 microM uranium) collected at the Oak Ridge Field Research Center. In contrast to other uranium detection methodologies, the Caulobacter reporter strain can provide on-demand usability in the field; it requires minimal sample processing and no equipment other than a hand-held UV lamp, and it may be sprayed directly on soil, groundwater, or industrial surfaces.

Physicochemical and mineralogical characterization of soil-saprolite cores from a field research site, Tennessee.

Site characterization is an essential initial step in determining the feasibility of remedial alternatives at hazardous waste sites. Physicochemical and mineralogical characterization of U-contaminated soils in deeply weathered saprolite at Area 2 of the DOE Field Research Center (FRC) site, Oak Ridge, TN, was accomplished to examine the feasibility of bioremediation. Concentrations of U in soil-saprolite (up to 291 mg kg(-1) in oxalate-extractable U(o)) were closely related to low pH (ca. 4-5), high effective cation exchange capacity without Ca (64.7-83.2 cmol(c) kg(-1)), amorphous Mn content (up to 9910 mg kg(-1)), and the decreased presence of relative clay mineral contents in the bulk samples (i.e., illite 2.5-12 wt. %, average 32 wt. %). The pH of the fill material ranged from 7.0 to 10.5, whereas the pH of the saprolite ranged from 4.5 to 8. Uranium concentration was highest (about 300 mg kg(-1)) at around 6 m below land surface near the saprolite-fill interface. The pH of ground water at Area 2 tended to be between 6 and 7 with U concentrations of about 0.9 to 1.7 mg L(-1). These site specific characteristics of Area 2, which has lower U and nitrate contamination levels and more neutral ground water pH compared with FRC Areas 1 and 3 (ca. 5.5 and <4, respectively), indicate that with appropriate addition of electron donors and nutrients bioremediation of U by metal reducing microorganisms may be possible.