Effect of low-level α-radiation on bioluminescent assay systems of various complexity

Abstract

This study addresses the effects of low-level α-radiation on bioluminescent assay systems of different levels of organization: in vivo and in vitro. Three bioluminescent assay systems are used: intact bacteria, lyophilized bacteria, and bioluminescent system of coupled enzyme reactions. Solutions of ²⁴¹Am(NO₃)₃ are used as a source of α-radiation. It has been shown that activation processes predominate in all the three bioluminescent assay systems subjected to short-term exposure (20–55 h) and inhibition processes in the systems subjected to longer-term exposure to radiation. It has been found that these effects are caused by the radiation component of ²⁴¹Am³⁺ impact. The intensity of the ²⁴¹Am³⁺ effect on the bioluminescent assay systems has been shown to depend on the ²⁴¹Am³⁺ concentration, level of organization and integrity of the bioluminescent assay system. The bioluminescent assay systems in vivo have been found to be highly sensitive to ²⁴¹Am³⁺ (up to 10⁻¹⁷ M).

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Introduction

For several decades, the effect of ionizing radiation on living organisms has been intensively studied. Numerous experimental results show that in the cell, irreparable damage is done to the most important macromolecules—DNA and proteins.^1,2,3 There are a great number of hypotheses on the mechanisms of radioactive impact, and none of them has become universally accepted.^4,5 A large part of the studies addressed the effect of high-level radiation on biological objects. However, increasing radioactive contamination of the environment makes the question about effects of low and super low radiation on living organisms very important. The problem of low and super low dose radiation is related to the radiobiological paradox—an insignificant amount of absorbed ionizing energy causes an extremely pronounced biological effect.^1–3,6–9 For example, a biological effect of α-particles on cell cultures is registered even under the impact of 0.05–0.3 particle per cell.^10,11

Detailed investigations of the effect of low dose radiation on living organisms are conducted using simple assay systems such as microorganisms, bacteria, plant and mammal cells, tissues of laboratory mice, drosophila flies, freshwater crustaceans, etc.^12–16 Assay systems based on luminous bacteria can be good candidates for such investigations. Bacterial bioluminescent assays are widely used to monitor environmental toxicity.^18,19 The tested parameter is the luminescence intensity of bacteria, which can be easily measured instrumentally. The advantages of the bioluminescent assays are their rapidity, sensitivity, simplicity, and availability of the devices for toxicity registration.^19,20

Bioluminescent bacteria have been used as a bioassay for almost a half of a century. The bioassay was described in its current form in 1969.²¹ Later, it was modified by different researchers and adapted for their specific purposes; numerous applications of the bacterial bioassay are known.^21–30 In 1990 the in vitro bioluminescent system of enzymatic coupled reactions was suggested as a toxicity assay;³¹ its applications were developed.^32–34 Now, the bioluminescent ecological assay is a traditional and important biotechnological application of the bioluminescence phenomenon.^20–23,35

General radiotoxicity was already investigated using bioluminescence of recombinant Escherichia coli strains under γ-ray irradiation.³⁶ However, bioluminescent bioassays have not been used yet to study the effect of low-level α-ray irradiation. In their current forms they are not sensitive to a low-level α-ray irradiation; hence, the bioluminescent technique should be modified: bioluminescent reagents should be incubated with radionuclides.

A number of researchers,^4,11,12 assumed that there is a relationship between the level of organization of a biological object and its response to low-level radiation exposure, which can be of both theoretical and practical interest. Bioluminescent assay systems can be conveniently used to verify this assumption as they can be based on biological objects of different levels of organization, bacteria-based and enzyme-based bioassays (in vivo and in vitro), providing comparison of the effect of low-level α-ray irradiation on enzymes and bacterial cells.

Thus, the purpose of this study was to investigate the effect of low-level α-radiation on bioluminescent assay systems of various complexity (in vivo and in vitro).

Materials and methods

Objects

Three bioluminescent assay systems (AS) were applied: AS(1)—cell suspension of 16 h P. phosphoreum 1883 IBSO culture from the Collection of Luminous Bacteria CCIBSO-836, AS(2)—a microbiotest 677F preparation i.e. lyophilized luminous bacterial cells of P. phosphoreum 1883 IBSO, and AS(3)—a kit of reagents for analytical bioluminescence, which includes lyophilized preparations of luciferase (0.5 mg ml⁻¹) and NADH:FMN-oxidoreductase (0.15 units of activity). All the ASs were produced in the Institute of Biophysics SB RAS, Krasnoyarsk, Russia.

As the source of ionizing radiation we used solutions of ²⁴¹Am(NO₃)₃ of concentrations 10⁻¹⁵, 5 × 10⁻¹⁶, and 10⁻¹⁷ M in 1.1 M NaNO₃ (pH 7.0). Activities of these solutions were 12000, 6000, and 1000 Bq l⁻¹, respectively.

To investigate the effect of low-level α-radiation, the radioactive and control samples were prepared as follows.

Radioactive samples: solutions of ²⁴¹Am(NO₃)₃ in 1.1 M NaNO₃ were added to AS(1), AS(2), or AS(3).

Control samples: a 1.1 M NaNO₃ solution was added to AS(1), AS(2), or AS(3).

Radioactive and control samples were maintained at constant temperature: +4 °C for AS(1), and −5 °C for AS(2) and AS(3). In some time-periods (0.5 h at the beginning of exposure to radiation and up to 10 h at the end of the exposure) the test samples (radioactive and control) were prepared and the bioluminescent intensity was measured at 20 °C. Measuring of bioluminescent intensity was carried out using standard procedure;^17–20 it went on until the bioluminescent intensity in the control samples decreased to 20% of the maximal one. Time-courses of the bioluminescent intensity in the control and radioactive samples are presented in Fig. 1 taking AS(1) as an example.

Fig. 1 Bioluminescent intensity (I) versus time (t).

The composition of the test samples and the procedure of radiotoxicity evaluation are described below.

Compositions of the test samples

(a) Radioactive (or control) test samples based on AS(1) or AS(2): 50 µl of radioactive (or control) samples based on AS(1) or AS(2), 500 µl of the 3% NaCl solution.

(b) Radioactive (or control) test samples based on AS(3): 10 µl of radioactive (or control) samples based on AS(3), 50 µl of the 0.0025% tetradecanal solution, 200 µl of the 0.05 M potassium phosphate buffer, pH 6.8, 50 µl of 5 × 10⁻⁴ M FMN solution, and 200 µl of 4 × 10⁻⁴ M NADH solution. The reaction was initiated by addition of NADH solution.

Evaluation of radiotoxicity of the test samples

Kinetics of bioluminescence signal was measured using a CL3606 Biochemiluminometer (SEDD “Nauka”, Russia). The relative bioluminescent intensity I^rel was calculated according to:
where: I_rad is the bioluminescence intensity in the radioactive test sample and I_contr is the bioluminescence intensity in the control test sample.

Values of I^rel were plotted vs. time of exposure to ²⁴¹Am³⁺. To evaluate the radiotoxicity of the samples, the maximal and the minimal relative bioluminescent intensities (I^rel_max and I^rel_min) were determined. Values of I^rel_max correspond to the bioluminescence activation in the early stage of exposure; I^rel_min values characterize the bioluminescence inhibition in the final stage of exposure.

Results and discussion

The following characteristics of the three ASs were under consideration: AS(1) contained whole bacterial cells; in AS(2) some of the cell walls were considerably damaged by lyophilization (freezing and rehydration)¹⁷ and penetration of low-molecular substances into the cells was facilitated;¹⁸ AS(3) included NADH:FMN-oxidoreductase and luciferase-enzymes catalyzing bioluminescent system of coupled reactions (1) and (2):

Using AS(1) and AS(3), one can compare radionuclide effects on the whole cells and enzymes. Hence, it is a way to evaluate the significance of the system's level of organization in its response to radiation exposure. A comparison of responses of AS(1) and AS(2) allows one to determine the role of cell integrity in this process.

It was found that the bioluminescence intensity in AS(1), AS(2), or AS(3) depended upon the time of their exposure to ²⁴¹Am³⁺. Fig. 2 shows the relationship of I^rel to exposure time for AS(1) at the highest ²⁴¹Am³⁺ concentration used (C = 10⁻¹⁵ M). The I^rel_max and I^rel_min are indicated in the figure.

Fig. 2 Relative bioluminescent intensity I^relvs. time of exposure (t/h) to: 1. Am³⁺: C_Am = 10⁻¹⁵ M, AS(1) and 2. Eu³⁺: C_Eu = 10⁻¹⁵ M, AS(1).

It can be seen that, first, irradiation activates the bioluminescence (I^rel > 1) during 55 h of exposure, and, second, during the final stage of exposure, 55–85 h, the bioluminescence is inhibited (I^rel < 1).

Similar relationships between bioluminescence intensity and exposure time were obtained for AS(2) and AS(3). Activation predominated for 35 and 20 h of the exposure in AS(2), and AS(3), respectively. Values of I^rel_max were found at 6, 4, and 2 h, and values of I^rel_min at approximately 85, 50 and 35 h of exposure in AS(1), AS(2), and AS(3), respectively.

Fig. 3 shows I^rel_max and I^rel_min for all the three systems: AS(1), AS(2), and AS(3) at three concentrations of ²⁴¹Am³⁺.

Fig. 3 Effect of different concentrations of ²⁴¹Am³⁺ on AS(1), AS(2) and AS(3); (a) bioluminescence activation, (b) bioluminescence inhibition.

It can be seen in this graph that, the I^rel_max value decreases and the I^rel_min value increases as ²⁴¹Am³⁺ concentration gets lower in every system. The most pronounced effects (I^rel_max = 4; I^rel_min = 0.4) were registered in AS(1) at the highest ²⁴¹Am³⁺ concentration (C = 10⁻¹⁵ M). I^rel_max values decrease from AS(1) to AS(3) for all the three ²⁴¹Am³⁺ concentrations (Fig. 3(a)), while the I^rel_min values change insignificantly from AS(1) to AS(3) (Fig. 3(b))

Thus, a comparison of the ²⁴¹Am³⁺ effect on the bioluminescent ASs suggests that there are two kinds of effects: activation and inhibition. The more complex and the less damaged is the system, the more strongly pronounced is the activation.

Differences in the response of AS(1) and AS(2) can be accounted for by differences in the state of the cells in them (whole cells and damaged ones). It should be noted that bioluminescent activation was observed even in the enzymatic reactions—AS(3).

The impact of ²⁴¹Am³⁺ on the bioluminescent assay systems can include a radiation component and a chemical one. To isolate the radiation component, we compared effects of ²⁴¹Am³⁺ with that of trivalent nonradioactive metals Eu³⁺ and Fe³⁺. According to the periodic table, europium is the closest analog of americium in its electronic structure and, hence, chemical properties.

Model solutions of europium(III) nitrate and iron(III) chloride were used to estimate the chemical component. Within the concentration range from 10⁻⁷ to 10⁻¹⁷ M, neither europium nitrate nor iron chloride affected AS(1), AS(2) or AS(3). The absence of the effect of europium nitrate on AS(1) is demonstrated in Fig. 2 as an example. Hence, all the effects described above can be ascribed to the radiation component of the ²⁴¹Am³⁺ impact.

Activation of the vital function of various organisms is a well-known phenomenon. As suggested by some researchers,^37–43 the activation can be accounted for by unfavorable influence triggering cell defence responses. As soon as recovery processes in the system become active, radioactivity impact decreases.^44,45 In our study we showed experimentally that ionizing radiation, within a certain range of low intensities, stimulates the bioluminescence intensity. (The bioluminescence intensity is supposed to be the main vital function of bacteria.^21–25) These data are consistent with the results obtained for other organisms.^{15,16,44–51}

The molecular mechanism of a change in bioluminescent intensity can be explained as follows: activation of bioluminescence must be caused by formation of radicals under the impact of ionizing radiation, and inhibition by their excess amount. A similar explanation for activation and inhibition of bacterial bioluminescence under the impact of UV irradiation was given by Czyz and colleagues.⁵²

It was also reported in ref. 52 that the death rate of UV-irradiated bacterial cells with a luminescent system was lower than that of their dark variants. It is quite possible that the luminescent system takes part in neutralizing free radicals. Additionally, the authors speculated that one of the possible roles of bacterial luminescence is efficient DNA repair in a photoreactivation-like reaction. The same assumption can be made in the case of ionizing radiation. However, the noticeable response of the enzyme system to the ionizing radiation (AS(3) in Fig. 3(a),(b)), when there cannot be any DNA repair, is indicative of the complex mechanism of the cell defence responses.

The results obtained for the simplest assays can be taken into consideration for predicting effects of low-level α-radiation on complex organisms, including humans.

Conclusions

(1) There were two effects in bioluminescence dynamics (activation and inhibition), which corresponded to different times of exposure to ²⁴¹Am³⁺. Irradiation activates bioluminescence during 20–55 h of exposure, and inhibits it during the final stage of exposure. (2) The intensity of the ²⁴¹Am³⁺ effect on a bioluminescent assay system depended on: (i) ²⁴¹Am³⁺ concentration (higher values of I^rel_max and lower values of I^rel_min were observed under higher concentrations of ²⁴¹Am³⁺.); and (ii) level of organization of the bioluminescent systems and cell integrity (the most pronounced effect was observed in the intact bacteria, and the least in the enzyme reaction). (3) Bioluminescent assay systems in vivo are highly sensitive to ²⁴¹Am³⁺ (up to 10⁻¹⁷ M). (4) The radiation component of the ²⁴¹Am³⁺ effect contributed for the most part to activation and inhibition of bioluminescence.

Acknowledgements

The work was supported by a joint grant of the Krasnoyarsk Regional Scientific Foundation and Russian Foundation for Basic Research N05-03-97701-R_Yenisei; Award No. RUX0-002-KR-06 of the U.S. Civilian Research & Development Foundation (CRDF) and RF Ministry of Education and Science, BRHE program; grant of the “Molecular and Cellular Biology” program of the Russian Academy of Science.

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