Effect of terahertz radiation on drug activity in bacterial cells

doi:10.21203/rs.3.rs-4102529/v1

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Effect of terahertz radiation on drug activity in bacterial cells

https://doi.org/10.21203/rs.3.rs-4102529/v1

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The biological effects of terahertz (THz) waves have been increasingly studied in recent years with the development of THz wave generation and detection technology. THz waves have been reported to change membrane permeability and induce conformational changes in protein molecules. Drugs action on cells involves membrane permeability, and we therefore investigated the effect of THz waves on the activity of the cytotoxic drug bleomycin on Escherichia coli. 0.46 THz radiation with an average power of 2.5 W/cm², pulse duration of 10 ms, and a repetition frequency of several Hz was noncytotoxic to E. coli cells. However, 0.46 THz radiation enhanced the cytotoxic activity of bleomycin in E. coli cells, and the drug-enhancing effect depended on the power density of the THz waves. The effect of THz radiation on drug uptake into cells was investigated based on the activity of the drug remaining in the culture medium after THz radiation or non-radiation. The activity of the drug remaining in the culture medium after THz radiation did not differ from that remaining after non-radiation. This indicates that THz radiation does not affect the bacterial cell-membrane permeability to bleomycin. Thus, this study suggests that 0.46 THz radiation enhances the cytotoxicity of bleomycin towards E. coli cells and may influence the mechanism of bleomycin action within cells rather than affecting drug uptake.

Terahertz radiation

bacterial cell

DNA cleavage agent

bleomycin

The technology of terahertz (THz) generation and detection has progressed in recent decades, and has potential for use in fields such as security imaging, nondestructive device testing, and medical imaging of cancer and skin burns [1–5]. Furthermore, in high-speed wireless communications, THz waves are expected to be used as a future 6th generation mobile communications system [2].

As the applications of THz waves expand in various fields, the opportunities for the human body to be exposed to THz waves will also increase. Therefore, the biological effect of THz waves should be clarified, and an increasing number of reports have investigated the effects of THz waves on living organisms and biomolecules in recent decades. Sergeeva et al. reported that 2.3 THz waves are neither mutagenic nor genotoxic to bacterial cells such as Escherichia coli, and Salmonella typhimurium, unlike X-rays [6]. In mammalian cells, in vitro experiments have shown that THz radiation has no effect cell adhesion, proliferation, differentiation, or morphology [7, 8].

However, several researchers have reported that THz radiation can cause transcriptional changes in human and mouse cells [8–10]. Bogomazava et al. reported that the transcription of approximately 1% of genes in human embryonic stem cells increased following THz irradiation. Bock et al. also showed that 89% of genes in mouse stem cells did not respond to THz radiation, whereas the remaining genes were activated or repressed at the transcriptional level.

THz waves have been reported to change membrane permeability [11, 12] and induce conformational changes in protein molecules [13–16]. Cherkasova et al. measured the characteristic bands in UV absorption and circular dichroism spectra of albumin exposed to THz waves and found that THz can change the conformation of this protein and that the changes depend on the exposure duration and radiation power [13]. Yamazaki et al. reported that THz irradiation enhances the polymerization of actin in vitro and changes actin filament morphology in aqueous solutions via shockwaves in vivo [14–16].

The mechanism behind these results is unknown, but the THz interaction process may occur because the vibration frequency of the hydrogen bonds in biomolecules is in the THz range [17–21]. That is, THz radiation is suggested to vibrate hydrogen bonds in DNA or protein-DNA molecules, resulting in transcriptional changes in genes and conformational changes in protein molecules. Furthermore, the three-dimensional structure and reactions of biological molecules such as proteins and DNA are controlled by systems via hydrogen-bond interactions with surrounding molecules, so many biological molecules could be affected by THz waves.

We previously have studied the biological effects of commercial frequency, and 60 Hz electromagnetic fields and their medical applications, although these frequencies are quite low compared with THz frequencies. We found that 60 Hz magnetic fields can enhance the effect of antimicrobial drugs on E. coli cells and the activity of anticancer drugs in human cancer cells in vitro [22, 23]. Related to this mechanism, we also obtained data suggesting that magnetic fields change the cell membrane potential and the conformation of cell membrane proteins [24]. The results suggested that 60 Hz magnetic fields can affect drug transporters in the cell membrane and drug uptake, causing enhanced drug activity. Therefore, magnetic field exposure specifically at a tumor site may enable effective target therapy, thereby reducing drug dosage and limiting side effects. However, 60 Hz magnetic fields are spread over a wide area and cannot be specifically applied to the tumor target site, as can be achieved with light. Several reports indicate that both 60 Hz and THz fields can affect the conformation and function of biomolecules although their powers are significantly different. Then we used THz waves with non-thermal intensity generated by a gyrotron (FU CW GVIB [25]) and investigated whether these waves could affect the drug activity in bacterial cells.

2.1 THz Radiation

Figure 1 shows an overview of the gyrotron (FU CW GVIB) used to generate 0.46 THz radiation and the experimental setup in this study. The output from the gyrotron was transmitted through a waveguide and radiated from above the sample. The distance from the waveguide exit to the sample was 10 mm, with a Gaussian power distribution on the irradiation surface. Waves with a power density of 0.25 or 2.5 W/cm² were generated and irradiated to the samples containing bacterial cells with or without drug. The pulse duration was set 20 ms for the power density of 0.25 W/cm², and 10 ms for the power density of 2.5 W/cm². During the experiments, the repetition frequency of THz radiation pulses was controlled in the range from 1 to 3 Hz, and the temperatures of the non-radiation and THz radiation groups was kept at the same temperature, 27℃ for 0.25 W/cm² and 33℃ for 2.5 W/cm².

2.2 Drug and Cell Culture

Bleomycin was purchased from Nippon Kayaku Co., Ltd. Bleomycin is a cytotoxic drug often used clinically as an antitumor antibiotic because this can cause DNA strand breaks in bacterial and human cancer cells.

The bacterial cell line E. coli JE5595 was obtained from the National BioResource Project of the National Institute of Genetics, Japan.

2.3 Measurements of Bleomycin Activity

The cytotoxic activities of bleomycin under THz radiation or non-radiation were measured via colony assay of bacterial cell viability. Bleomycin was added to an E. coli culture in logarithmic phase in LB medium to a final concentration of 1 µM; the culture was divided into wells on 24 well plates to a 2-mm deep aqueous solution for application of THz radiation. The sample plates were then covered with breathable polyurethane films, and temperature sensors were installed in the sample cultures to monitor the change in temperature during the experiment (Fig. 1b). One plate was placed in the THz radiation area, and another plate was placed in the non-radiation area on a heating device. The difference in temperature between the radiation and non-radiation groups was within 0.5 ℃ by controlling the repetition frequency of THz radiation. Cells from the culture of each group were then spread on LB agar and incubated until colonies formed to demonstrate the number of viable cells (as colony forming units) in the sample following treatment with THz radiation and bleomycin or with bleomycin alone (non-radiation).

2.4 Bleomycin Activity Remaining in the Culture Supernatant

After THz radiation or non-radiation, each E. coli culture was centrifuged to separate E. coli cells and culture supernatants containing any drug remaining in the medium. The supernatants were transferred into a new E. coli culture and the activities were measured by colony assay.

3.1 Viability of E. coli cells by THz radiation

First, we investigated whether 0.46 THz radiation was cytotoxic towards E. coli cells.

E. coli cultures of the logarithmic phase were divided into wells on 24-well plates, and one plate was placed under 0.46 THz radiation with 2.5 W/cm² (Fig. 1b), another one was placed on a heating device under non-radiation. Figure 2 shows the temperature of E. coli cultures at 0.46 THz, 2.5 W/cm² radiation or non-radiation for 2 h. The temperature difference between each group was within 0.5 ℃ either with or without radiation.

The viable cell numbers of the THz radiation and non-radiation groups was determined via colony assay. As shown in Fig. 3, the cell number and proliferation between the two groups did not significantly differ. The results indicated that 0.46 THz waves with an average power of 2.5 W/cm² for 2 h were noncytotoxic to E. coli cells. Our result is consistent with the finding of Williams et al. who showed that human epithelial and embryonic stem cells were unaffected by THz radiation [7].

3.2 Bleomycin activity in E. coli cells under THz radiation

To investigate the effect of THz waves on bleomycin activity, E. coli cultures in the presence of bleomycin (final concentration 1 µM) were treated with or without 0.46 THz radiation for 2 h. The respective temperatures of these E. coli cultures including bleomycin treated with 0.46 THz, 0.25 W/cm² or 2.5 W/cm² radiation and non-radiation were almost the same, similar to Fig. 2.

The viable cell numbers before and after each treatment for 2 h are shown in Fig. 4.

Bleomycin, a cytotoxic drug, causes DNA strand breaks, and the number of viable cells in the group treated with bleomycin decreased to one tenth of that before the treatment (BLM + non-radiation in Fig. 4a, c). The number of viable cells treated with bleomycin plus THz radiation decreased more than that treated with bleomycin in the absence of THz waves. The relative ratio of viable cells in the bleomycin plus THz radiation group (with 0.25 W/cm²) was 0.52 and was significantly different compared with that in the bleomycin only group. At the stronger 2.5 W/cm² THz radiation, the viability of cells treated with bleomycin plus THz wave decreased to 0.30-fold that of bleomycin treatment in the absence of THz wave (BLM + THz radiation, Fig. 4b, d). These results suggest that 0.46 THz radiation enhances the cytotoxicity of bleomycin against bacterial cells and that the enhancing effect depends on the power density of the THz wave.

The results were similar to the enhancement of antibacterial and anticancer drugs by 60 Hz magnetic fields shown in previous studies [22, 23].

3.3 Effect of THz radiation on membrane permeability to Bleomycin

Generally, when cytotoxic drugs are added to the culture medium, portion of the drugs are taken into the cells, while some remains in the medium. Our previous results suggested that magnetic fields (60 Hz) change the permeability of the cell membrane and influence drug intake in bacterial cells. Other studies have also shown that THz radiation can affect membrane permeability [11, 12]. Therefore, we measured the activity of the remaining drugs in the culture medium after THz radiation at 2.5 W/cm² or non-radiation. Figure 5 shows the relative ratio of cell viability by treatment with the remaining drug in the culture medium after THz radiation to that after non-radiation. No significant differences were found in the drug activity that remained in the culture supernatants after THz radiation or non-radiation. The result indicated that THz radiation does not affect the bacterial cell membrane permeability to bleomycin.

Using a similar experimental method, we had previously investigated the effect of 60 Hz magnetic fields on the membrane permeability of drugs. These results suggest that 60 Hz magnetic fields can affect membrane permeability and increase drug uptake into cells. Therefore, the enhancing mechanisms of drug activity by THz radiation and 60 Hz magnetic fields appear to differ.

The present study suggests that THz radiation increased the activity of bleomycin but did not affect cell permeability to the drug. Bleomycin has been reported to have sequence-selective DNA- degradation properties and requires redox-active metal ions such as Fe2⁺ and Cu2⁺ and dioxygen to generate radicals in the DNA degradation process [26]. Therefore, THz radiation may affect the intracellular mechanism of action of bleomycin in DNA degradation.

The effects of THz radiation on cell viability and the drug activity of bleomycin to E. coli cells were investigated in the present study. 0.46 THz radiation at 2.5 W/cm² had no cytotoxicity to E. coli cells. However, 0.46 THz radiation enhanced the cytotoxicity of bleomycin to E. coli cells, and the enhancing effect depended on the power density of THz wave. Although the enhancing mechanism of THz radiation on bleomycin activity is currently unclear, our results suggest that 0.46 THz may affect the mechanisms of action of bleomycin within cells rather than via drug uptake.

Funding This work was supported by Grant-in Aid for Scientific Research (C) under Grants 22K04214 and 22K12763 from MEXT in Japan.

Data Availability Not applicable

Conflict of interest The authors declare no conflicts of interest associated with this manuscript.

Author Contribution

Kakikawa wrote the main manuscript text and All authors prepared figures 1-5, and reviewed the manuscript.

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No competing interests reported.

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Effect of terahertz radiation on drug activity in bacterial cells

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Abstract

Figures

1 Introduction

2 Materials and Methods

2.1 THz Radiation

2.2 Drug and Cell Culture

2.3 Measurements of Bleomycin Activity

2.4 Bleomycin Activity Remaining in the Culture Supernatant

3 Results and Discussions

3.1 Viability of E. coli cells by THz radiation

3.2 Bleomycin activity in E. coli cells under THz radiation

3.3 Effect of THz radiation on membrane permeability to Bleomycin

4 Conclusions

Declarations

Author Contribution

References

Additional Declarations

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