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pISSN 2287-7991, eISSN 2287-8009
J.Prev.Vet.Med.Vol.40,No.1:1-6,March 2016
http://dx.doi.org/10.13041/jpvm.2016.40.1.1
Biological effects of indirect contact with QELB
Y
®
powder on
nonmacrophagic and macrophage-derived cell lines
Hyung Tae Lee
1
, Dalmuri Han
1
, June Bong Lee
1
, GunWoong Bahng
2
, Jong Doo Lee
3
, and Jang Won Yoon
1
†
1College of Veterinary Medicine & Institute of Veterinary Science, Kangwon National University, Chuncheon 24341, Republic of Korea
2QELBY Research Institute and Department of Mechanical Engineering, The State University of New York, Korea (SUNY Korea),
Incheon 21985, Republic of Korea
3Quantum Energy Research Center, Yongin 17039, Republic of Korea
1)
A
bstract
:
Bioenergetics has been defined as the biology of energy transformations and energy exchanges within and between
living organisms and their environment; this field now includes the concept of bioenergetic medicine, e.g., therapeutic
approaches involving biophotons. QELBY
®
powder is a patented quantum energy-radiating material (patent No. 10-1172018),
to be precise, a biologically active silicon dioxide-containing mineral powder that radiates reductive energy in infrared
wavelength. In this study, we examined possible biological effects of indirect contact with QELBY
®
powder on various
mammalian cell lines derived from macrophagic (MØ) and nonmacrophagic cells, including Raw 264.7 (mouse-derived MØ cell
line), HD11 (chicken-derived MØ cell line), and HeLa (human cervical cancer cell line). Our comparison among the cells with
and without indirect contact with QELBY
®
powder showed that this indirect contact significantly (i) increased the mitochondrial
membrane potential (up to 1.36-fold) regardless of the cell type (p < 0.05), (ii) decreased the intracellular concentration of ATP
in HeLa cells but not in the MØ-derived cells (p < 0.05), and (iii) promoted repair of damaged DNA and/or protected DNA
from damage during oxidative stress according to a standard comet assay (single-cell alkaline gel electrophoresis). Taken
together, these results imply that indirect contact with QELBY
®
powder can make cells more metabolically active by increasing
the mitochondrial membrane potential and by alleviating DNA damage caused by oxidative stress.
Key words:
QELBY
®
, indirect contact, biological activity, mitochondrial membrane potential, oxidative stress
INTRODUCTION
Bioenergetics has been defined as the biology of energy
transformations and energy exchanges within and between
living organisms and their environment [14]; this field now
includes the concept of bioenergetic medicine, for exam ple,
therapeutic approaches involving biophotons [3]. QELBY
®
powder is a patented quantum energy-radiating material
(patent No. 10-1172018); to be precise, it is a biologically
active silicon dioxide-containing mineral powder that
radiates reductive energy.
Both physical and electrical properties of QELBY
®
powder have been determined [4]. This powder consists
of particles ~40 nm in size, is functionally stable at high
temperatures (e.g., 1,000
℃
), and has excellent electrical
conductivity in wet form ; zeta potentials were found to
be -37.74 mV [4]. Notably, a recent study revealed a
Received 15 March 2016, Revised, Accepted 22 March 2016
†
Corresponding Author. Jang Won Yoon, Tel: +82-33-250-8791, Fax: +82-33-259-5625,
E-mail: jwy706@kangwon.ac.kr
Copyright © 2016 The Korean Society of Preventive Veterinary Medicine.
The full text is freely available on the web at http://www.jpvm.kr/.
significant anti-inflammatory activity of QELBY
®
powder
in an experimental model based on lipopolysaccharide
(LPS)-stimulated murine Raw 264.7 macrophagic cells. For
example, coculture of the LPS-stimulated Raw 264.7 cells
for 48 h with filtered liquid extract of QELBY
®
powder in
Dulbecco’s Modified Eagle’s Medium (DMEM) enhances cell
proliferation but decreases protein levels of proinflammatory
cytokines such as TNF-
α
and expression of inducible nitric
oxide synthetase (iNOS), resulting in a decrease in nitric
oxide (NO) production in a dose-dependent manner [5].
Although the mechanism(s) behind such an anti-inflammatory
activity is unclear, we can suppose that both direct and
indirect mechanisms are involved in these biological
activities. For example, this treatment may affect cells
directly by means of certain components present in
QELBY
®
powder or indirectly via energy transformation
in Q ELBY
®
powder and transfer into the cells.
In this study, we hypothesized that indirect contact with
QELBY
®
powder can modulate cellular activities via
transformation of reductive quantum energy and its
transfer into the cells. To this end, we studied possible
2 Hyung Tae Lee, Dalmuri Han, June Bong Lee, GunWoong Bahng, Jong Doo Lee, and Jang Won Yoon
Cell line Origin Cell type Growth media
*
Reference
HD11 Chicken Macrophage RPMI 1640, 8% (v/v) FBS, 1% (v/v) PS [2]
Raw 264.7 Mouse Macrophage RPMI 1640, 10% (v/v) FBS, 1% (v/v) PS [9]
HeLa Human Cervical cancer Low glucose DMEM, 10% (v/v) FBS, 1% (v/v) PS [13]
*
Abbreviations: DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, Fetal bovine serum; PS, Penicillin-Streptomycin.
Table 1. Cell lines and growth media in this study
alterations in the mitochondrial membrane potential (
Δψ
m), in production of intracellular ATP, and in the cellular
proliferation rate as standard indicators of cell function;
we also assessed changes in DNA damage/repair activity
under oxidative stress after indirect contact with QELBY
®
powder of three mammalian cell lines derived from MØ
or non-MØ cells: Raw 264.7 (mouse-derived MØ cell line),
HD11 (chicken-derived MØ cell line), and HeLa (human
cervical cancer cell line; Table 1).
MATERIALS AND METHODS
Cell lines, growth media, and culture conditions
Cell lines and growth media that were used in this study
are listed in Table 1. All the listed growth media and
supplements were purchased from Gibco-BRL (Gibco-BRL,
Grand Island, NY, USA). In general, cells were grown at 37
℃
in a humidified incubator at 5% CO
2
. To determine biological
effects of indirect contact with QELBY
®
powder on the
various cell lines shown in Table 1, we grew the cells in
a sterile polystyrene container filled or not filled with 30
g of QELBY
®
powder (Quantum Energy, Inc., Korea).
Measurement of
Δψ
m
Cells were seeded at approximately 10
3
/well in a
96-well black clear-bottom culture plate (Corning Inc.,
Corning, NY, USA) and grown at 37
℃
in a humidified
incubator at 5% CO
2
for 48
–
72 h with or without indirect
contact with QELBY
®
powder as described above.
Δψ
m of
the cells was then measured using the TMRE
Δψ
m Assay
Kit (Abcam, Cambridge, UK) and the microplate reader
SpectraMax M2 (Molecular Devices, Sunnyvale, CA, USA).
Determination of intracellular ATP concentrations
Cells were plated at ~5.0 × 10
5
/well in a 6-well culture
plate (SPL Life Science, Korea) and grown with or without
the indirect contact with QELBY
®
powder as described
above. The cells were collected using a sterile cell scraper
(SPL Life Science) and washed twice with sterile
phosphate-buffered saline (PBS). Intracellular ATP levels
were measured by means of the EnzyLight
TM
ATP Assay
Kit (BioAssay Systems, Hayward, CA, USA) on the
luminometer GloMax 20/20 (Promega, Madison, WI, USA).
The cell proliferation assay
The cellular proliferation rates were evaluated by means
of a premix WST-1 Cell Proliferation Assay Kit (Takara,
Shiga, Japan). In brief, cells were seeded at approximately
10
3
/well in a 96-well culture plate (Corning Inc.) and
precultured for 24 h as mentioned above until application
of indirect contact with QELBY
®
powder. After incubation
for 24
–
72 h with or without the indirect contact with
QELBY
®
powder, the culture medium was removed, and
the cells were washed twice with PBS. Next, the culture
medium with the WST-1 reagent was added to each well,
and the cells were incubated at 37
℃
in the humidified
incubator at 5% CO
2
for 2 h. Cellular proliferation rates
were measured at 450 nm on a microplate reader (Model
680; Bio-Rad Laboratories, Hercules, CA, USA).
The comet assay (single-cell alkaline gel electrophoresis)
To determine the effects of the indirect contact with
QELBY
®
powder on DNA damage and repair induced by
oxidative stress, a standard comet assay was performed.
In brief, HeLa cells were seeded at ~10
6
/plate in 10-cm
2
plates and grown for 24 h. After the cells were incubated
with 100
M hydrogen peroxide (H
2
O
2
) for 5 or 20 min
at 4
℃
, the culture supernatants were removed, and the
cells were washed twice with PBS. After that, the cells were
incubated with or without indirect contact with QELBY
®
powder for up to 4 h under the same conditions as above.
The cells were then collected using a sterile cell scraper
(SPL Life Science), and the comet assay was carried out
using a COMET SCGE Assay Kit (Enzo Life Sciences Inc.,
Farmingdale, NY, USA) and the Olympus IX71 fluorescence
microscope (Olympus, Tokyo, Japan). Tail migration
Biological activity of QELBY
®
powders 3
Fig. 1. Measurement of the mitochondrial membrane potential (
Δψ
m) in various cell lines with or without indirect contact with QELBY
®
powder. Macrophage (MØ)-derived (HD11 and Raw 264.7) and non-MØ cell lines (HeLa) were cultured with or without indirect
contact with QELBY
®
powder for 48 and 72 h (see Materials and Methods). At the indicated time points, 48 h (A) or 72 h (B),
Δψ
m was measured using the TMRE Mitochondrial Membrane Potential Assay Kit. The values of fluorescence intensity (reflecting
Δψ
m
)
are
p
resented as mean ± SD of at least three inde
p
endent ex
p
eriments
(
***
p
< 0.01; *
p
< 0.05; NS, not si
g
nificant
)
.
distance was measured from the comet head to the end
of the migrating tail as previously described [6]. At least
five independent experiments were conducted for
statistical significance.
Statistical analysis
All the data were analyzed in the OriginPro 8 software
(Origin-Lab Corporation, Northampton, MA, USA) and
expressed as mean ± SD of at least three independent
experiments. Statistical significance was analyzed by the
Mann-Whitney test or Student’s
t
test where differences
with a p value <0.05 were considered significant.
RESULTS
Δψ
m was increased by the indirect contact with
QELBY
®
powder regardless of the cell type
Because it is known that
Δψ
m is an effective indicator
of overall cellular metabolic status [3, 7], we examined
Δψ
m in various cell lines with or without the indirect contact
with QELBY
®
powder. As shown in Fig. 1,
Δψ
m values were
significantly increased by the indirect contact with QELBY
®
powder in all the cell types analyzed. To be precise,
Δψ
m
values increased up to 1.36-fold in HD11 and HeLa cells
after the indirect contact with QELBY
®
powder for 48 or
72 h, as compared to the corresponding untreated cells
(Fig. 1 and Table 2, p < 0.01). Although no significant
differences in
Δψ
m of Raw 264.7 cells after 72 h were
observed between the control and the indirect-contact
groups, this parameter was increased at 48 h in the
indirect-contact group (p < 0.01). This result was similar
to that observed in HD11 and HeLa cells (Fig. 1 and Table
2). These data indicate that indirect contact with QELBY
®
powder can increase
Δψ
m regardless of the cell type.
The indirect contact with QELBY
®
powder slightly
decreased the intracellular ATP levels in the
cancer-derived cells (HeLa) but not in MØ-derived
cells (HD11 and Raw 264.7)
Because the indirect contact with QELBY
®
powder
increased
Δψ
m in all the cell types tested, we hypothesized
that this increased
Δψ
m would affect the cellular
proliferation rate. To test this hypothesis, we compared
the proliferation rates of the cells with or without indirect
contact with QELBY
®
powder. The results showed no
changes in the proliferation rate regardless of the cell type
(data not shown and Table 2).
One of the essential functions of mitochondria is the
synthesis of ATP during electron transport [7]. Because the
cellular proliferation rates were not affected by the indirect
contact with QELBY
®
powder, we decided to measure
intracellular ATP concentrations in cells with or without
4 Hyung Tae Lee, Dalmuri Han, June Bong Lee, GunWoong Bahng, Jong Doo Lee, and Jang Won Yoon
Cell line MMP (
Δψ
m)
a
Intracellular [ATP]
b
Cell proliferation rate
48 hrs
c
72 hrs 48 hrs 72 hrs 48 hrs 72 hrs
HD11 +
d
+nc
d
nc nc nc
Raw 264.7 ++
d
nc nc -
d
nc nc
HeLa + ++ nc nc nc nc
a
Mitochondrial membrane potential.
b
Concentration of ATP.
c
Incubation time with indirect contact of the QELBY
®
powders.
d
Symbols: +, significant increase less than 1.3-folds; ++, significant increase more than 1.3-folds; -, significant decrease; nc, not changed
when compared to those of the negative control groups (without indirect contact).
Table 2. Summary of the biological activities of the QELBY
®
powder-mediated indirect contact in various cell lines
the indirect contact with QELBY
®
powder. No significant
changes were observed in the MØ-derived cell lines: HD11
and Raw 264.7 (data not shown and Table 2). In contrast,
the intracellular concentration of ATP was slightly
decreased in HeLa cells after the indirect contact with
QELBY
®
powder for 72 h (p < 0.05; Fig. 2). Collectively,
these findings indicated that the indirect contact with
QELBY
®
powder slightly reduced the intracellular ATP
levels in the cancer-derived cells (HeLa) but not in
MØ-derived cells (HD11 and Raw 264.7).
Fig. 2. Intracellular ATP concentrations of HeLa cells with or without
the indirect contact with QELBY
®
powder. HeLa cells were
grown with or without indirect contact with QELBY
®
powder
for 48 and 72 h (see Materials and Methods). At the
indicated time points, the intracellular ATP concentrations
were quantitated using the EnzyLight
TM
ATP assay. The
values of luminescence intensity (which reflect intracellular
ATP concentrations) are presented as mean ± SD of at least
four inde
p
endent ex
p
eriments
(
*
p
< 0.05
)
.
The indirect contact with QELBY
®
powder promoted
repair of damaged DNA and/or protected DNA from
damage induced by H
2
O
2
-mediated oxidative stress
To determine whether the indirect contact with QELBY
®
powder activates the DNA damage repair system, the
cellular DNA damage and repair were quantitated at the
single-cell level using a standard comet assay (see
Materials and Methods
). The results revealed that
immediately after exposure to H
2
O
2
-mediated oxidative
stress, nuclear DNA was more condensed in the cells that
were in indirect contact with QELBY
®
powder than in the
control cells (without this indirect contact; Fig. 3). In line
with this observation, the average distance from the comet
head to its tail (reflecting DNA damage) was much shorter
for the cells in indirect contact with QELBY
®
powder than
for the control cells (Fig. 3). Even after 2 h of this indirect
contact, the length of the comet tails for the cells in indirect
contact with QELBY
®
powder was almost the same as for
the cells not exposed to oxidative stress (Fig. 3). These
results show that indirect contact with QELBY
®
powder
can promote repair of DNA and/or protect DNA from
damage during oxidative stress, in particular that induced
by H
2
O
2
.
DISCUSSION
A growing body of evidence suggests that certain energy
transformations or energy exchanges within and between
living organisms and their environment may affect
biological system s via direct and/or indirect contact.
QELBY
®
powder is a quantum energy-radiating material,
and it was reported that a filtered suspension of QELBY
®
powder in a liquid medium exerts anti-inflammatory action
on LPS-stimulated Raw 264.7 cells [5], implying that
Biological activity of QELBY
®
powders 5
Fig. 3. Inhibition of DNA damage by indirect contact with QELBY
®
powder in HeLa cells under oxidative stress. HeLa cells
were incubated with 100
M H
2
O
2
for 20 min at 4
℃
. In the
cells with or without indirect contact with QELBY
®
powder
for up to 4 h, the extent of DNA damage was evaluated by
a standard comet assay. At least 50 cells per sample were
analyzed; the cells were examined at 200× magnification.
The numbers inside each picture show the relative distance
from the comet head to the migrating tail in a single cell.
QELBY
®
powder likely contains certain biologically active
ingredients that have not yet been characterized. Our
previous characterization of QELBY
®
powder revealed that
it possesses unique physical and electrical properties [4].
This material (i) has excellent electrical conductivity in wet
form, (ii) yields the zeta potential of -37.74 mV, and (iii)
decreases the redox potential of water via indirect contact.
Prompted by these findings, the present study was carried
out to test the hypothesis that the indirect contact with
QELBY
®
powder has some biological effects when applied
to eukaryotic cells owing to a kind of energy transformation.
In this study, we demonstrated that the indirect contact
with QELBY
®
pow der significantly (i) increased
Δψ
m (up
to 1.36-fold) regardless of the cell type, (ii) decreased the
intracellular concentration of ATP in HeLa cells but not
in the MØ-derived cells, and (iii) promoted repair of
damaged DNA and/or protected DNA from damage during
oxidative stress. To the best of our knowledge, this is the
first study to show that indirect contact with QELBY
®
powder makes cells more metabolically active by increasing
Δψ
m and by decreasing DNA damage during oxidative
stress. Similar results were reported in a recent study on
cellular effects of biophotons [3], defined as biologically
active light energy. In swine peripheral blood mononuclear
cells, irradiation by means of a biophoton energy projector
enhanced various cellular activities, such as antigen uptake
and
Δψ
m, and promoted production of a pathogen-specific
IgG when this treatment was used after vaccination with
AR-XTM, a commercially available vaccine for atrophic
rhinitis caused by
Bordetella bronchiseptica
(ChoongAng
Vaccine Laboratory, Inc., Korea) [3].
Mitochondria are subcellular organelles that participate
in a variety of processes in eukaryotic cells, for example,
ATP production, cellular proliferation, metabolic pathways
(e.g., the tricarboxylic acid cycle), and apoptosis [3, 7, 8,
11, 12, 15]. Therefore, measurement of
Δψ
m is useful for
assessing the cellular metabolic status in general.
In our study, the indirect contact with QELBY
®
powder
did increase
Δψ
m regardless of the cell type, but no increase
in intracellular concentrations of ATP was observed.
Although we cannot explain this discrepancy between
Δψ
m
and intracellular ATP levels, it should be noted that the
intracellular concentration of ATP decreased in the
cancer-derived cells (HeLa) but not in the MØ-derived cells.
Further research is needed to determine whether the
observed reduction in intracellular concentration of ATP in
HeLa cells is a general phenomenon among various
cancer-derived cells or cell lines.
It is well known that there are various reactive oxygen
species in cells, and these compounds cause severe damage
to macromolecules such as DNA [1], thus leading to cell
death. Our results revealed that the indirect contact with
QELBY
®
powder protects DNA from damage during
oxidative stress, in particular stress induced by H
2
O
2
.
Because oxidative stress is known to be associated with
carcinogenesis and inflamm atory diseases in humans and
animals [10], it would be interesting to assess possible
anticancer activity of QELBY
®
powder and its molecular
mechanism(s) in an appropriate experimental animal model.
In summary, our experiments showed biological effects
of indirect contact with QELBY
®
powder on MØ-derived
and non-MØ cell lines. The indirect contact with QELBY
®
powder may not only increase
Δψ
m but also decrease DNA
damage during oxidative stress, thus making the cells more
metabolically active.
ACKNOWLEDGEMENTS
This study was supported by a grant from Quantum
Energy, Inc. (No. C1012202-01-01). J.W.Y. was supported
by the 2014 Research Grant from Kangwon National
University (No. C1011129-01-01).
6 Hyung Tae Lee, Dalmuri Han, June Bong Lee, GunWoong Bahng, Jong Doo Lee, and Jang Won Yoon
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