Infrared laser pulse triggers increased singlet oxygen production in
tumour cells
S.G. Sokolovski1*, S.A. Zolotovskaya1, A. Goltsov2, C. Pourreyron3, A.P. South3, and E.U. Rafailov1
1Photonics
and Nanoscience Group, School of Engineering, Physics and Mathematics, University of
Dundee, Dundee DD1 4HN, UK;
2
Centre for Research in Informatics and Systems Pathology (CRISP), University of Abertay Dundee, DD1
1HG, UK;
3
Division of Cancer Research, Medical Research Institute, Ninewells Hospital and Medical School,
University of Dundee, Dundee, DD1 9SY, UK
*To whom correspondence should be addressed. E-mail: [email protected]
Supplementary Materials
Description of computational model. The model of laser-induced ROS-generation considers
processes of ROS production and scavenging in the cytosol and includes the following ordinary
differential equations which describe kinetics of the primary and secondary ROS (R1 and R2,
respectively) 1, hydrogen peroxidase (H2O2), and the proteins involved in cellular antioxidant
systems: peroxidase (Px) and antioxidant protein containing thiol group (PSH):.
dR1
 Vo  VRO ,1  VH2O2  Vlaser
dt
dH 2O2
 VH2O2  VPx  VR2  VRO ,2
dt
dPx
 VPx  VPSH
dt
dPSH
 VRed  VPSH
dt
dR2
 VR 2  Vdeg
dt
(1)
(2)
(3)
(4)
(5)
Eq. (1) describes kinetics of primary ROS pool (O2‾ and 1O2), R1, which is determined by the
following processes: endogenous production of superoxide anion O2‾ (reaction rate Vo) and singlet
oxygen 1O2 by laser pulse (reaction rate Vlaser); oxidation of cellular species by O2‾ and 1O2 (VRO) and
conversion of O2‾ to H2O2 by superoxide dismutase, SOD (reaction rate VH2O2).
This model only considers ROS generation and scavenging in the cytosol not taking into account
mitochondrial ROS production and their degradation by mitochondrial reductase thioredoxin, TrxR
(TrxR2) and hydrogen peroxide catalase. The ROS production rate in cytosol, Vo, depends on
superoxide anion generation by membrane-associated NADPH oxidase (NOX), leakage of H2O2 and
e− from mitochondria into cytosol, and production of superoxide anion by xanthine oxidase. The
value of Vo was estimated based on the production rate of H2O2 in normal and tumour cells 2 and
finally calibrated to describe low rest level of H2O2 (H2O2o) in normal cells and its increased level in
tumour cells (Table S1).
VRO,1 describes the reaction rate of ROS scavenging through oxidation of cellular proteins and free
amino acids, P 3. VRO,1 is given in mass action approximation:
VRO ,1  k RO  P  R1 .
.
(6)
Not considering a change in a balance of reduced and oxidized cellular proteins (P is constant) we
introduce the rate constant kPO=kRO P and rewrite Eq. 6 in the form of a one order reaction
VRO ,1  kPO R1
(7)
Although reactivity of superoxide anion relating to amino acid oxidation is low, however the impact
of this process is high because of high overall intracellular concentration of amino acids (>0.1 M3) .
VH2O2 determining generation rate of H2O2 by SOD from O2‾ was represented as Mechaelis-Menten
equation:
VH2O2 
Vmax,SOD  R1
,
R1  K SOD
(8)
where Vmax,SOD=kSODSOD, kSOD (2.4 min-1 μM [9]) and SOD are SOD catalytic rate and its
concentration, respectively.
Here we ignore non-enzymatic of O2‾ to H2O2 conversion due to its low rate (knonenz =310-5
µM/min4) in comparison with enzymatic reaction (8).
Vlaser describes laser-induced 1O2 generation rate (see dashed lines in Figs. 3B, C and 4A) and was
given in the form
Vlaser

t  t0
 Vlaser ,o 1 



2b
1

 .


(9)
We determined the parameters of Eq (9) so that to model experimental shape of laser impulse of 3
min duration and switched on at time t0=4.5 min after the start of the experiment . Parameter =1.4
and b=4. We suggested that laser generates singlet oxygen with constant rate, Vlaser,o depending on
irradiation dose. Value of Vlaser,o was defined as a result of model fitting against experimental data on
oxygen radical kinetics at different irradiation doses (see Fig. 1C).
Eq. (2) describing kinetics of H2O2 takes in main roots of its processing in the cell: production
from primary ROS (VH2O2); conversion into H2O by the enzymatic antioxidative system (Px/PSH)
with the rate VPx) 1,5; scavenging through oxidation of cellular species (VRO,2); production of
secondary radicals by H2O2 through Fenton reactions, VR2 (e.g. hydroxyl radical, OH, peroxyl
radical ROO, lipid peroxyl radical LOO, lipid alkoxyl radical LO, and others 1).
VPx describes conversion of H2O2 into H2O by peroxidase, Px (Tpx and Gpx) involving oxidation
of PSH proteins, PSHox (Trx and GSH) with the rate VpSH 5-7.. We modelled catalytic cycle of Px
enzyme by two connected reactions catalysed by Px (Tpx and Gpx) and PSH (Trx and GSH). Rate
equations VPx and VPSH were approximated by mass action equations:
VPx  kPx  Px  H 2O2 ,
(10)
VPSH  kPSH  PSH  Pxox .
(11)
VR2 defines OH production rate through oxidation of free Fe2+ by H2O2 well known as Fenton
reaction 1. Rate equation of VR2 is given in mass action kinetics8:
VR 2  k R 2  H 2O2  Fe 2 .
(12)]
VRO,2 designates the H2O2 degradation rate oxidising of proteins and free amino acids, P 9. Rate
equation VRO,2 is given in mass action approximation like VRO,1 with the same rate constant kPO (Eq
(7)):
VRO ,2  k PO  R2 .
(13)
Eq. (3) describes kinetics of the reduced peroxidase, Pp: oxidation of Px (Tpx and Gpx) by H2O2 and
its reduction by PSH (Trx and GSH) with the rate equations (10) and (11), respectively.
Eq. (4) describes catalytic cycle of reductase, Red (TR and GR): reduction of PSHox (Trxox and
GSS) by reductase Red and reduction of Red by NADPH. Rate equation VRed was given in
Mechaelis-Menten form for two-substrate reaction:
Vmax,Red  PSH ox  NADPH
VRed 
,
(14)
K PSH  K NADPH  1  PSH ox / K PSH  NADPH / K NADPH 
where Vmax,Red  kRed  Red , kRed (1500 min-1 10) and Red are caralytic rate of reducrase, Red, and its
concentration, respectively. As the parameters of the Px/PSH/Red antioxidant system (Eqs. 10, 11,
and 14), the kinetic parameters of human Tpx/Trx/TR antioxidant system were used in the model (see
Table S1 and S2).
Eq. (7) phenomenologically models the secondary ROS kinetics: production of the secondary radicals
by H2O2, VR2 (Eq (14)) and their degradation with the rate
Vdeg  k ged  R2 .
(15)
The parameters of the model are listed in Table S1 and S2.
Table S1. Parameters of the model. (fp denotes free parameters estimated as a result of model fitting
against experimental data, cp – changeable parameters estimated on the basis of fitting procedure and
experimental data available from literature; fxp – fixed parameter in the model calibration).
Parameter Description
Vo
Production rate of ROS in
normal and cancer cells
Value in model
0.2 µM/min in normal
cells (cp);
7 µM/min in cancer cells
(cp)
kPO
Rate constant of protein
oxidation by H2O2
Generation rate of 1O2 by laser
irradiation at different dose
radiation
0.1 min-1 (fp)
SOD maximal rate
Mechaelis-Menten constant of
SOD
Px rate constant (substrate
H2O2)
PSH rate constant
120 µM min-1 (cp)
1 µM (cp)
2.4 min-1 11
60 µM 11
2.4 103 µM-1 min-1 (fxp)
Tpx, 2.4 103 µM-1min-1 12
1.2 102 µM-1 min-1 (fxp)
Trx, 1.2 102 µM-1 min-1
Vlaser,o
Vmax,SOD
KSOD
kPx
kPSH
Literature data
0.19-0.45 µM/min,
production rate of H2O2
in normal cells 8;
4.5 – 8.3 µM/min,
production rate of H2O2
in tumour cells 8
1) 4.5 µM/min at 47.7
J/cm2 in HaCaT, HeLa
(fp);
2) 5.2 µM/min at
71.6 J/cm2 in HaCaT cells
(fp);
12
KPSH
KNADPH
Vmax,Red
kR2
Fe2+
kdeg
Mechaelis-Menten constant of
reductase, Red (substrate
PSHox)
Mechaelis-Menten constant of
reductase, Red (substrate
NADPH)
Reductase maximum rate
Rate constant of Fenton
reaction
Concentration of free Fe2+ in
cells
Rate constant of secondary
ROS degradation
10 µM (cp)
TR, 1.4-34 µM 10
88 µM (cp)
TR, 88 µM 10
300 µM min-1 (cp)
0.01 µM/min (cp)
1.1 10-2 µM/min 8
10 µM (cp)
5-500 µM 8
0.07 min-1 (fp)
Table S2. Initial concentration of enzymes and metabolites in the model
Species
Description
Value in model
Literature data
H2O2o
Basal level of H2O2 in
normal and cancer cells
0.01 µM in normal cells (cp);
0.3 µM in cancer cells (cp)
Px
Peroxidase concentration
in normal cells
Antioxidant protein
concentration in normal
cells
NADPH concentration
2 µM (cp);
<1 (10-3-0.7) µM in
normal cells 8;
0.2 µM in tumour cells 8
Tpx, 19 µM 12
4 µM (cp);
Trx, 0.4 µM 12
0.3 µM (fxp)
0.3 µM
PSH
NADPH
12
Figures:
Figure S1. Representative single channel currents of HaCaT cells pre-incubated with 10 μM α–
tocopherol recorded at –100 mV holding voltage at cell-attached configuration before, during, and
after 1268 nm laser irradiation of 47.7 J/cm2 (n=4).
Figure S2. Effect of higher temperature (RT+3ºC) on the DHOE fluorescence in HaCaT and HeLa
cell lines (mean ± SE).
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