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. 2020 Aug 16;29(11):1531–1539. doi: 10.1007/s10068-020-00804-9

Comparison of gamma irradiation and heating treatment on cytotoxicity, insulinotropic activity, and molecular structure change of mistletoe viscothionin

Jong-Heum Park 1, Jae-Kyung Kim 1, Beom-Seok Song 1,✉
PMCID: PMC7561639  PMID: 33088602

Abstract

Mistletoe offers health-promoting effects; however, it has toxicity, requiring careful application. Viscothionin is a polypeptide of mistletoe that while contributing to toxicity also demonstrates anti-cancer and anti-diabetic activities. The aim of this study was to evaluate whether gamma irradiation or heating treatment could selectively reduce viscothionin-mediated cytotoxicity. Gamma irradiation effectively inhibited viscothionin-induced cytotoxicity to RIN5mF cells, but heating treatment did not affect its cytotoxicity. Both heating and gamma irradiation further increased the insulinotropic activity of viscothionin, whereas the effect of gamma irradiation was dose-dependent and diminished above 20 kGy. Structural analysis showed that gamma irradiation significantly altered the ordered structure of viscothionin, unlike heating treatment, resulting in a change of its molecular properties, which could be linked to the observed changes in the cytotoxicity and insulinotropic activity of the polypeptide. These results suggest gamma irradiation as an alternative method for minimizing viscothionin toxicity without interfering with anti-diabetic effect.

Keywords: Polypeptide of mistletoe, Toxicity, Gamma irradiation, Heating treatment, Structural analysis

Introduction

Plant-derived traditional herbal medicines are increasingly applied in modern medicine for the treatment of several conditions as alternatives to chemically synthesized drugs in both developed and developing countries, and now frequently observed in pharmacies as well as food stores (Ekor, 2014). Mistletoe has long been widely used as a complementary health supplement or an herbal folk medicine for the prevention and treatment of various diseases in Europe and East Asia owing to a variety of biological activities, including immunomodulatory properties and anti-cancer, anti-diabetes, and anti-hypertension effects (Büssing, 2000a; Drozdoff et al., 2018). The major constituents of mistletoe are lectin, alkaloids, polysaccharides, and viscothionin (Pfüller, 2000). Viscothionin is a polypeptide mixture of approximately 6 kDa comprising 46 highly conserved amino acids with reported anti-cancer activity (Giudici et al., 2003; Tabiasco et al., 2002). However, there is minimal information on its biological effects or therapeutic efficacy in other contexts. Stein et al. (1999) have found that viscothionin activates human granulocytes, and Huber et al. (2006) reported that viscothionin-rich mistletoe extracts influence cellular immune responses in human peripheral blood mononuclear cells, such as monocytes and macrophages. Furthermore, recent studies have revealed that viscothionin is an active ingredient contributing to the anti-diabetic and hepatic steatosis-protecting activities of mistletoe (Kim et al., 2014; Park et al., 2019). However, since most of the components containing viscothionin are also known to be closely related to mistletoe-mediated toxicity (Park et al., 1999), care is required in their applications. The toxicity of mistletoe is generally mitigated by physio-chemical and biological methods such as alcohol extraction, heating treatment, and fermentation with lactic acid bacteria (Büssing, 2000b). Moreover, in the Far East, it is common to infuse mistletoe into boiling water for several hours preparing it as a safe decoction (Park et al., 2019).

Ionizing irradiation is another effective food sanitary technology that can inactivate foodborne pathogens in foods and extend their shelf stability (Farkas, 2006). Ionizing irradiation has also been used to reduce the allergenicity of milk, eggs, and shrimp (Jeon et al., 2002; Lee et al., 2001; Seo et al., 2007), and to diminish the toxicity of lipopolysaccharide (Nerkar and Bandekar, 1986). In addition, this technology has been effectively applied to alleviate the cardiotoxicity of the anti-cancer drug doxorubicin without affecting its anti-cancer activity (Kim et al., 2009).

Therefore, the present study aimed to evaluate the ability of traditional heat treatment and ionizing radiation to the toxicity of viscothionin and to evaluate the consequences of the treatment on its hypoglycemic properties. Here, the evidence for an effective method to mitigate the toxic effects of the mistletoe polypeptide viscothionin without compromising its beneficial pharmacological activity was provided.

Materials and methods

Preparation of mistletoe powder

Fresh mistletoe (Viscum album var. coloratum Ohwi) leaves and branches were purchased from the Dong Nam herbal medicine shop (Seoul, Korea). The plants were thoroughly washed with distilled water and freeze-dried. The mistletoe was then homogenized (HMF-3100S; Hanil, Seoul, Korea) into powder and strained with a 500-μm mesh sieve. The powder was kept at − 80 °C prior to use.

Purification of mistletoe viscothionin

The purification of mistletoe viscothionin was performed in accordance with our previously described method (Kim et al., 2014; Kim et al., 2015; Park et al., 2019). Briefly, 50 g of the mistletoe powder was dissolved in 750 mL of 2% acetic acid and stirred overnight. The homogenate was centrifuged at 4000×g for 30 min, and the resulting supernatant was lyophilized. The dried material was then dialyzed in 25 mM sodium acetate buffer (pH 4.8) using membrane tubing (molecular weight cut-off: 3500). The viscothionin in the dialysate was subsequently purified using CM Sepharose cation exchange column (20 × 4.6 cm) and Sephadex G-50 gel filtration column (70 × 1.4 cm). All the processes related to extraction and purification of viscothionin from mistletoe powder were performed at 4 °C. The final purified viscothionin content was about 2.7 mg/g dried mistletoe powder. The viscothionin was dissolved in distilled water at 6 mg/mL for both the gamma irradiation and heating treatment.

Gamma irradiation

Five-milliliter aliquots of the extracted viscothionin samples were irradiated with the doses of 0, 5, 10, 20, 30, and 50 kGy in a 60CO gamma irradiator (AECL, R-79, MDS Nordion Inc., Canada) at Korea Atomic Energy Research Institute (Jeongeup, Korea). The source strength was approximately 11.1 PBq with a dose rate of 10 kGy/h, and the absorbed doses were measured using the alanine-EPR dosimetry system (Bruker Instruments, Rheinstetten, Germany). The actual doses were within 2% of the target doses.

Heating treatment

Five-milliliter aliquots of viscothionin samples were heat-treated in boiling water for 3, 6, 9, or 12 h. The samples were then immediately cooled in ice water for subsequent testing.

SDS-PAGE

PAGE of irradiated and heat-treated viscothionin samples (10 μg) was carried out with precast 12% NuPAGE Bis–Tris gels (Invitrogen, San Diego, CA, USA) at 100 V for 1 h in a NuPAGE 2-(N-morpholino) ethanesulfonic acid (MES) SDS running buffer (Invitrogen) according to the manufacturer’s instructions. A SeeBlue Plus2 pre-stained standard protein marker (Invitrogen) was used to determine the molecular mass of the migrating viscothionin band. The gel was visualized by Coomassie Brilliant Blue R-250 staining.

Cytotoxicity test

Rat insulinoma RINm5F cells (Korean Cell Line Bank No. 40071; Seoul, Korea) were used for cytotoxicity assessment of the viscothionin samples. The cells were maintained in a Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal bovine serum and 100 U/mL of penicillin and streptomycin (all from Invitrogen) in 5% CO2 at 37 °C. Two types of assays were used to assess viscothionin-mediated cytotoxicity. For MTT assay, RINm5F cells were plated in 96-well plates at 2 × 104/well and incubated at 37 °C with 5% CO2 for 24 h. The cells were then treated with different concentrations (0–120 μg/mL) of viscothionin samples for an additional 23 h. After incubation, MTT solution (Sigma-Aldrich, St. Louis, MO, USA; 5 mg/mL in phosphate buffered saline) was added, and the plate was further incubated for 1 h, followed by detection at 595 nm using an enzyme-linked immunosorbent assay (ELISA) reader (Varioskan Flash; Thermo Fisher Scientific, Ratastie, Finland). Cytotoxicity was also assessed with the trypan blue dye exclusion assay. RINm5F cells were plated in 48-well plates at 5 × 105 cells/well and incubated at 37 °C in 5% CO2 for 24 h. The cells were washed three times with Krebs–Ringer bicarbonate (KRB) buffer (115 mM NaCl, 4.7 mM KCl, 1.28 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 24 mM NaHCO3, 10 mM 4-2-hydroxyethyl-1-piperazineethanesulfonic acid, 1 g/L bovine serum albumin, and 1.1 mM d-glucose, pH 7.4), and further incubated for 1 h in the KRB buffer containing 5.4 mM d-glucose and different concentrations (0–50 μg/mL) of irradiated and heat-treated viscothionin samples. Finally, the cells were harvested, and mixed with 0.4% trypan blue stain solution (Invitrogen). Then, the viable (unstained) and dead (stained) cells in the mixture were separately counted using a hemocytometer (Sigma-Aldrich).

Insulinotropic activity test

Insulin secretion by rat insulinoma RINm5F cells exposed to irradiated and heat-treated viscothionin samples was assayed according to a previously described method (Park et al., 2019). Briefly, RINm5F cells were seeded in 48-well plates at 5 × 105 cells/well in 5% CO2 at 37 °C. After 24 h incubation, the cells washed three times with KRB buffer, and treated with the KRB buffer containing 5.4 mM d-glucose and different concentrations (0–50 μg/mL) of viscothionin samples for another 1 h. As a positive control, the cells were treated with 5 μg/mL of glibenclamide (Sigma-Aldrich), a hypoglycemic agent used in the treatment of diabetes (Kalra et al., 2015). The insulin content released in the buffer was analyzed using ultrasensitive rat insulin ELISA kit (Shibayagi Co., Ltd, Japan).

UV absorption spectroscopic analysis

UV spectra of irradiated or heat-treated viscothionin samples were monitored on a spectrophotometer (UV-1601PC; Shimadzu, Japan). The samples were diluted with distilled water to achieve a concentration of 1 mg/mL, transferred to a quartz cuvette with a 1-mm path length, and scanned with UV–visible spectrophotometer. The scanning range was recorded at between 200 and 400 nm.

CD analysis

The CD spectrum analysis of irradiated or heat-treated viscothionin samples was performed using a Jasco J-715 CD spectropolarimeter (Jasco Corp., MD, USA) fitted with a 150-W xenon lamp at the Korea Basic Science Institute (Ochang, Korea). Far UV spectra were monitored in the range of 190–250 nm. The diluted viscothionin samples (1 mg/mL) were transferred to a quartz cuvette with a 1-mm path length. Triplicate scans of the CD spectra obtained from the samples were averaged and the spectrum for the background of distilled water was subtracted. The CD spectra are represented in terms of the mean residue ellipticity (degrees × square centimeters per decimole).

Protein secondary structure analysis

The secondary structure analysis of irradiated or heat-treated viscothionin was performed using CDNN software (Böhm, 1997) with the help of the Korea Basic Science Institute (Ochang, Korea).

Statistical analysis

All data were analyzed using the two-tailed Student’s t-test and the Excel software of the Microsoft Office package (Microsoft, USA, 2016). The data are represented as the mean ± the standard error of the mean (SEM), per group. Statistical significant results (non-treated versus treated groups) are presented as * (p < 0.05), ** (p < 0.005) or *** (p < 0.001).

Results and discussion

Molecular weight and concentration of viscothionin is changed by gamma irradiation

The effect of gamma irradiation and heating treatment on the molecular weight change of the polypeptide viscothionin was examined by SDS-PAGE analysis. The viscothionin isolated from mistletoe was observed as a single band with an estimated size of about 6 kDa (Fig. 1A, lane 2; B, lane 2). The SDS-PAGE profile of the viscothionin samples gamma-irradiated at different doses (0, 5, 10, 20, 30, and 50 kGy) showed a decreased band intensity, and this effect became gradually stronger with an increase in the irradiation dose (Fig. 1A). In particular, the band was absent for the sample irradiated at 50 kGy (lane 7), indicating complete denaturation of the polypeptide at this dose. Besides, the increase of the irradiation dose was accompanied by an increase in the background color intensity of the high molecular weight region (above 10 kDa), suggesting that the crosslinking of the polypeptide probably occurred (Fig. 1A, lanes 3–7). However, heating treatment did not appear to influence the band intensity, and heating for more than 9 h induced only a slight change in the viscothionin content, suggesting that the polypeptide is structurally stable to long-term heating (Fig. 1B). This result was consistent with a previous report by Kuttan et al. (1988), which observed that viscothionin is heat-resistant. Conversely, the molecular weight of viscothionin marginally decreased with an increase in the heating time (Fig. 1B, lanes 5 and 6).

Fig. 1.

Fig. 1

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SDS-PAGE pattern of the polypeptide viscothionin isolated from mistletoe and changes in the band intensity following gamma-irradiation and heating treatment. (A) Lane 1, molecular weight marker; lane 2, non-irradiated viscothionin; lane 3, irradiated with 5 kGy; lane 4, irradiated with 10 kGy; lane 5, irradiated with 20 kGy; lane 6, irradiated with 30 kGy; lane 7, irradiated with 50 kGy. (B) Lane 1, molecular weight marker; lane 2, non-treated viscothionin; lane 3, heat-treated for 3 h; lane 4, heat-treated for 6 h; lane 5, heat-treated for 9 h; lane 6, heat-treated for 12 h

SDS-PAGE analysis showed that gamma irradiation, unlike heating treatment, significantly caused viscothionin molecular degradation and the consequent reduction of viscothionin concentration. Typically, irradiation causes physico-chemical changes to proteins, including molecular weight reduction, or aggregation through chemical modifications of amino acids, breakages of peptide bonds, breakdowns of disulfide bridges, and crosslinking (Cieśla et al., 2000). These changes eventually lead to irreversible alterations that not only modify the tertiary structures but also affect the natural biological functions (Lee et al., 2001; Seo et al., 2007).

Cytotoxicity of viscothionin is reduced by gamma irradiation

The results of the MTT assay showed that gamma irradiation gradually decreased the polypeptide-mediated cytotoxicity in a dose-dependent manner (Fig. 2A). However, heating treatment did not substantially reduce the cytotoxicity of viscothionin compared to that of the control (Fig. 2C). In addition, there was a distinct difference in the half-maximal inhibitory concentration (IC50) values of the irradiated and heat-treated viscothionin according to irradiation dose and heating time, respectively. Specifically, gamma irradiation up to 50 kGy increased the IC50 values of the polypeptide from 3 to 114 μg/mL, suggesting an approximately 40-fold reduction in viscothionin-mediated cytotoxicity with a direct relationship between the changes in the cytotoxicity and applied irradiation dose (R2 = 0.9; Fig. 2B). Although heating treatment up to 12 h also increased the IC50 values of the polypeptide by 1.7-fold from 3 μg/mL to 5.6 μg/mL (R2 = 0.8468; Fig. 2D), the effect was considered negligible compared with the influence of gamma irradiation. On the other hand, the results of the trypan blue dye exclusion assay showed that neither the irradiated nor heat-treated polypeptide induced cytotoxicity to RIN5mf cells within 1 h (Fig. 3A and B).

Fig. 2.

Fig. 2

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Cytotoxic effects of the gamma-irradiated or heat-treated polypeptide viscothionin on rat insulinoma RINm5F cells. (A) Irradiated viscothionin. (B) Relationship between the IC50 values of irradiated viscothionin and irradiation dose. (C) Heated-treated viscothionin. (D) Relationship between the IC50 values of heat-treated viscothionin and heating time

Fig. 3.

Fig. 3

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Insulinotropic enhancing effect of the (A) gamma-irradiated or (B) heat-treated mistletoe polypeptide viscothionin on rat insulinoma RINm5F cells

Insulinotropic activity of viscothionin is enhanced by gamma irradiation up to 20 kGy

Viscothionin induced insulin secretion by RIN5mF cells in a concentration-dependent manner. In addition, gamma irradiation further promoted the polypeptide-induced insulin secretion by RIN5mF cells (1.5- to 2- fold higher, compared to the basal condition) up to a dose of 20 kGy; of note, the greatest effect was observed when a dose of 10 kGy was used (Fig. 3A). Remarkably, while non-irradiated viscothionin at the lowest concentration tested (5 μg) did not induced insulin secretion by RIN5mF cells, gamma irradiation up to a dose of 20 kGy led to a significant insulinotropic effect caused by the polypeptide at the same concentration (Fig. 3A). Moreover, such an effect was almost equal to or higher than that of the same concentration of glibenclamide, used as the positive control (Fig. 3A). Although viscothionin is the major active component behind the hypoglycemic effect of mistletoe (Park et al., 2019), this polypeptide showed cytotoxicity, as per the MTT assay even in the context of low viscothionin concentration (4 μg), which may be a counter-indication of its application/use (Fig. 2A). However, gamma irradiation up to a dose of 20 kGy significantly reduced or completely abrogated the cytotoxicity of more than 20 μg of viscothionin (Fig. 2A). This strongly indicates that gamma irradiation can effectively reduce the toxicity of viscothionin without affecting its insulinotropic activity. Of note, the reported effects of gamma irradiation were completely lost when doses of 30 and 50 kGy were used, and the original activity of the viscothionin was also diminished with the higher-dose irradiation. This result suggests that it was related to the structural denaturation of the polypeptide by irradiation as shown in Fig. 1A. Heating treatment also promoted the insulinotropic activity of viscothionin by 1.6-fold (Fig. 3B), which was comparable with the effect of gamma irradiation at a dose of 20 kGy (Fig. 3A). However, unlike gamma irradiation, heating treatment did not reduce the insulinotropic activity at any time point.

UV absorption spectrum of viscothionin is changed by gamma irradiation

To explore the mechanism contributing to the irradiation- or heat-induced effects on viscothionin-mediated cytotoxicity and insulinotropic activity, UV absorption analysis was performed. After irradiation treatment, the UV absorption intensity of the polypeptide gradually increased in the range of 200–400 nm in an irradiation dose-dependent manner (Fig. 4A). In particular, there was an apparent peak of absorbance near 240 nm. In general, UV absorption spectroscopy reflects a conformational change of the side chains of aromatic amino acid residues such as tryptophan, phenylalanine, and tyrosine present in a protein, which have major absorption bands in the wavelength region between 200 and 300 nm. Among these, tyrosine is the only aromatic amino acid with an ionizable side chain, the phenolic hydroxyl group (Radi, 2013), and deprotonation such as hydrogen atom abstraction from the phenolic hydroxyl side chain of the amino acid increases its UV absorption intensity near 240 nm (Asher et al., 1991; Close et al., 2000). Irradiation is also known to induce the same chemical reaction to various organic molecules by active radicals generated from water radiolysis (Besic et al., 2001; Tamba and Torreggiani, 2004).

Fig. 4.

Fig. 4

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UV absorption spectra of the (A) gamma-irradiated or (B) heat-treated polypeptide viscothionin

Our recent data obtained from the partial N-terminal sequencing analysis of isolated viscothionin showed that at least one tyrosine residue is present in the polypeptide (Park et al., 2019), and the whole sequence of viscothionin demonstrated more than two tyrosine residues in the polypeptide (Giudici et al., 2003). Therefore, the observed UV spectral change of viscothionin might be due to molecular modification of its tyrosine residues by gamma irradiation. Similar results have been obtained on tyrosine residues in the protein models of the bacteriorhodopsin of Halobacterium salinarium and in the peptide models of angiotensin II (Balashov et al., 1991; Muccio and Cassim, 1979).

Far UV-CD spectrum of viscothionin is changed by gamma irradiation

CD spectroscopy measures the differences in the absorption between left-handed and right-handed polarized light and can be used to determine a protein’s secondary structure in the far UV spectral region (190–250 nm) (Sung et al., 2011; Sreerama and Woody, 2004). At these wavelengths, a chromophore is a peptide bond, and distinct signals arise when it is located in a native, folded environment, α-helix, β-sheet, and random-coil structure, which can provide information on the characteristic shape and magnitude of a CD spectrum (Sung et al., 2011; Sreerama and Woody, 2004). For example, two negative peaks emerging near 208 nm and 220 nm and a positive peak near 193 nm are characteristic of the α-helix of proteins, whereas a negative peak at 214 nm is characteristic of a β-sheet of proteins (Sung et al., 2011). Gamma irradiation affected the CD spectrum of viscothionin, suggesting significant conformational modifications on its secondary and tertiary structures (Fig. 5A). Specifically, an increase in the irradiation dose decreased the positive maximum ellipticity value of the polypeptide at 196 nm and its negative minimum values at 207 nm and 221 nm. This result indicated that gamma irradiation caused a decrease in the α-helical structure, with a concomitant increase in the random coil structure of the polypeptide.

Fig. 5.

Fig. 5

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Far-UV CD spectra of the (A) gamma-irradiated or (B) heat-treated polypeptide viscothionin

The far UV-CD curves of irradiated mistletoe viscothionin were then converted into the secondary structural contents using CDNN software, which confirmed that gamma irradiation decreased the α-helical structural content of viscothionin in an irradiation dose-dependent manner (Fig. 6A). Concurrently, the random coil structural content increased dramatically. Such changes in the CD spectra of irradiated viscothionin can be closely related to the irradiation-mediated water radiolytic effect. Generally, irradiation generates various forms of active radicals such as hydrogen peroxide, hydroxyl radical, and superoxide anion radical in the presence of water molecules (Lee et al., 2012), and the active radicals formed can subsequently oxidize amino acid residues in proteins or cleave their covalent bonds (Cho and Song, 2000). Therefore, these reactions can ultimately induce changes in the secondary and tertiary structure of the proteins.

Fig. 6.

Fig. 6

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Changes of the secondary structure of the (A) gamma-irradiated or (B) heat-treated polypeptide viscothionin

In contrast, although heating treatment also affected the UV absorption spectrum, far UV-CD spectrum, and secondary structural content of the polypeptide in a heating time-dependent manner, the effect was very marginal compared with that of gamma irradiation (Figs. 4B, 5B, and 6B). This suggests that the ordered structure of the polypeptide remained reasonably stable despite heating for up to 12 h.

In conclusion, our data show that gamma irradiation up to 20 kGy could increase the insulinotropic activity of the mistletoe polypeptide viscothionin, while significantly reducing its cytotoxicity. Heating treatment also promoted the anti-diabetic activity of the polypeptide but did not alleviate its cytotoxicity. In addition, SDS-PAGE, UV absorption, and far UV-CD spectra analyses revealed that the irradiation, unlike heating treatment, altered the ordered structure of the polypeptide, resulting in a change of its molecular properties, which contributed to the reduction in the cytotoxicity and promotion in the insulinotropic activity of viscothionin. These results suggest that gamma irradiation might be a more effective method than heating treatment in selectively mitigating the toxicities of mistletoe extracts or their components, without compromising their beneficial effects.

Acknowledgements

This work was supported by the National Basic Research Program of the Korea Atomic Energy Research Institute funded by the Korea Government. Park JH and Kim JK were responsible for experimental test. Park JH performed the purification of viscothionin from mistletoe. Park JH and Song BS designed the concept of the work and prepared the manuscript. We would like to thank the Korea Basic Science Institute for the analysis of the protein secondary structure of viscothionin and Editage (www.editage.co.kr) for English language editing.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Footnotes

Publisher's Note

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Contributor Information

Jong-Heum Park, Email: jhpark21@kaeri.re.kr.

Jae-Kyung Kim, Email: jkim@kaeri.re.ekr.

Beom-Seok Song, Email: sbs0110@kaeri.re.kr.

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