Determination of intracellular levels of reactive oxygen species using

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Reactive oxygen species (ROS) are reactive molecules which are produced ... DA) assay is used to determine intracellular levels of ROS in cancer cells. In.
Progress in Reaction Kinetics and Mechanism, 2014, 39(3), 281 – 291 doi:10.3184/146867814X14043731662945

RESEARCH PAPER

Determination of intracellular levels of reactive oxygen species using the 2,7-dichlorofluorescein diacetate assay by kinetic Monte Carlo simulation Hadis Bashiri*, Hamed Moradmand Jalali and Hossein Rasa Department of Physical Chemistry, Faculty of Chemistry, University of Kashan, Kashan, Iran *E-mail: [email protected]; [email protected]

ABSTRACT Reactive oxygen species (ROS) are reactive molecules which are produced normally by metabolic reactions. 2,7-Dichlorofluorescein diacetate (DCHFDA) assay is used to determine intracellular levels of ROS in cancer cells. In the present study, the mechanism and kinetic parameters of this determination are obtained by kinetic Monte Carlo simulation. The values of the rate constants for the suggested mechanism were obtained by simulation. Then, the effect of DCHF‑DA concentration on the rate of reaction was studied. According to the results, the product concentration should increase on increasing the DCHF‑DA concentration. In addition, the effect of ROS concentration on the rate of reaction was determined.

KEYWORDS:

reactive oxygen species, enzymatic reaction, 2,7-dichlorofluorescein diacetate, kinetic Monte Carlo simulation, rate constant, esterase

1. INTRODUCTION Reactive oxygen species (ROS) are reactive molecules including oxygen such as singlet oxygen, the superoxide and hydroxyl radicals, and hydrogen peroxide [1]. The ROSs are produced normally by one-electron reduction of molecular oxygen in the mitochondrial electron transport chain [2,3]. Lipid oxidation by ROS such as superoxide anion, hydroxyl radicals and hydrogen peroxide causes a decrease in the nutritional value of lipids, their safety and appearance. These radicals are very unstable and react rapidly with other groups or substances in the body, leading to cell or tissue damage. Uncontrolled generation of ROS can 281

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cause damage to cellular components such as lipids [4, 5], proteins [6, 7] and nucleic acids [8, 9] and may lead to ageing, inflammation, neurodegenerative disorders and cancer [10]. In order to measure intracellular levels of ROS, some groups have applied the 2,7-dichlorofluorescein diacetate (DCHF-DA) assay [11 – 16]. DCHF‑DA is a cell-permeable probe for the detection of intracellular ROS; the DCHF‑DA is lipophilic and readily diffuses across cell membranes. The DCHF‑DA undergoes deacetylation by intracellular esterase enzymes and produces the 2,7-dichlorofluorescein (DCHF). Esterase, k DCHF - DA  → DCHF

(1)

In the presence of hydrogen peroxide (H2O2) and other ROS, DCHF is oxidised to highly fluorescent DCF [17, 18]. k ′, fast → DCF DCHF + ROS 

(2)

The DCF fluorescence intensity can be easily determined and is the basis of the cellular assay for detecting intracellular levels of ROS. Loetchutinat et al. [16] have been used the DCHF‑DA assay to measure ROS produced in drugsensitive (K562) human erythromyelogenous leukaemia cell lines. The purpose of this study was determination of the mechanism of enzymatic reaction 1, which is used to determinate the amount of ROS in K562 cell line, by kinetic Monte Carlo (kMC) simulation. According to the results obtained, the effects of the initial concentrations of ROS and DCHF‑DA on the rate of reaction were to be studied.

2. SIMULATION METHOD For simulation of the experimental data, we used the kMC method developed by Gillespie [19]. Kinetic simulations were carried out with the help of the Chemical Kinetic Simulator software, version 1.01 (from IBM, Almaden Research Center, http://www. almaden.ibm.com/st/msim/) [20]. In the simulation algorithm, the reaction mechanism is considered as a collection of several steps:

nN + mM + ... → products

(3)

The input data for the simulation are the steps, their rate constants (ki) and the number of molecules (Ci). The algorithm of this simulation is based on the reaction probability density function, P(τ,i), as obtained by the Master equation. A random value of τ (time of each step) can be generated by clearly drawing www.prkm.co.uk

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a random number (r1) from the uniform distribution in the unit interval and taking (4) τ =  (1/a)ln[1/r1] where we have in summary: ai = kiCi (i =  1,2,…,M) M

M

i =1

i =1

a = ∑ ai = ∑ k i C i

(5) (6)

Then, a random integer i may be created by drawing another random number (r2) from the uniform distribution in the unit interval by taking i to be that integer for which, 2

(7)

In this method, two random numbers r1 and r2 are created to calculate τ and i by Eqns (4) and (7). The simulation is extended by repeatedly selecting at random among the probability-weighted steps in the mechanism, and updating the reactant’s and product’s populations in accord with the stoichiometry of the selected step, system state variables, and reaction rates. The results are a set of concentration versus time curves. This stochastic numerical simulation method has been used to simulate several chemical reactions [21 – 23]. In the present project, kMC simulation has been used to effect a kinetic study of the determination of intracellular levels of ROS in cancer cells by the DCHF-DA assay.

3. RESULTS AND DISCUSSION An experimental enzymatic reaction was chosen [16] to be studied by kMC. Loetchutinat et al. [16] have studied an enzymatic reaction for determination of ROS by DCHF‑DA in the K562 cell line. In this process, DCHF‑DA is hydrolysed by esterase enzyme in K562 cells to produce 2,7-dichlorofluorescein (DCHF). Then DCHF is oxidised by ROS to be converted to DCF. The DCF obtained shows strong fluorescence and can thus be determined versus time. For modelling of this enzymatic reaction in the process of conversion of DCHF‑DA to DCHF, we used kMC simulation. The input data for the simulations are temperature (310 K), initial concentration of DCHF‑DA ([DCHF-DA]0 =  1.0 × 10 – 7 M), initial concentration of ROS ([ROS]0 =  6.6 × 10 – 5) [16], the steps of mechanism and the rate constants of the various steps. This reaction was simulated in 1 × 105 K562 cell lines. Reactions 1 and 2 were as www.prkm.co.uk

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suggested by Loetchutinat et al. [16] as the mechanism of determination of ROS in cancer cells by the DCHF‑DA assay. By using Monte Carlo simulation, different mechanisms that have been reported for enzymatic reactions were examined to determine the best fit with experimental data. The mechanism that fits well with experimental data is the Michaelis – Menten mechanism: (8)

(9) (10) In the above mechanism, E represents the esterase enzyme. In this mechanism, the combination of esterase with DCHF‑DA (E.DCHF-DA) is formed by an equilibrium reaction (8). k1 and k2 in reaction (8) are rate constants of the forward and reverse reactions, respectively. The E.DCHF‑DA converts to DCHF via reaction 9 and rate constant of this step is k3. Finally, DCHF is oxidised by ROS to produce DCF (reaction 10). By comparing the mechanism obtained and the reported mechanism of Loetchutinat et al. [16], it is concluded that the last step of the suggested mechanism is the same, i.e. reaction (2). The mechanism of reaction 1 was determined as reactions (8) and (9). The rate-determining step (RDS) of the Michaelis – Menten mechanism is reaction (9). So, the rate of reaction (1) can be obtained from the following equation: R =  k3 [E.DCHF-DA]

(11)

The concentration of E.DCHF-DA can be obtained by the steady-state approximation as:

(12) From Eqn (12), the value of E.DCHF-DA is obtained as: (13) www.prkm.co.uk

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By substitution of Eqn (13) in Eqn (11), one arrives at: (14) Loetchutinat et al. [16] have obtained a rate coefficient for reaction (1) in one K562 cell as kexp = 6.1 × 10 – 9 (cell – 1 s – 1). With respect to this result, the value of kexp for different numbers of K562 cells will be changed. With respect to Eqn (14), the rate coefficient of reaction (1) is obtained as: (15) From Eqn  (15) and kexp, the rate constants of the proposed mechanism were obtained as adjustable parameters by simulation. The values of the rate constants were listed in Table 1. It was shown in Table 1 that reaction (9) is the RDS. Loetchutinat et al. [16] have reported the rate coefficient for reaction (2) in one K562 cell as k2 [ROS]0 =  5.2 × 10 – 7 (cell – 1 s – 1). With respect to the initial concentration of ROS ([ROS]0 =  6.6 × 10 – 5 mol L – 1), k2 in one K562 cell is obtained as k2 =  7.90 × 10 – 3(L mol – 1 cell – 1 s – 1). Finally, the value of k2 in 1 × 105 K562 cells is obtained as k2 =  7.90 × 102 (L mol – 1 s – 1). Figure 1 shows the experimental and simulated values of DCF concentration as a function of time in 1 × 105 K562 cells. As seen in this Figure, there is a good agreement between the experimental [16] and simulated data. This agreement indicates that the suggested mechanism can be suitable to study this enzymatic reaction. The mechanism obtained has been used for this reaction for different cell numbers, i.e. at 2 × 105 and 4 × 105 K562 cell lines. Concentrations of DCF versus time have been obtained by simulation and the results are shown in Figure 1. There is very good conformity between the simulated and experimental data [16]. It is to be expected that the value of the rate coefficient of reactions (1) and (2) depend on the number of K562 cells. Loetchutinat et al. [16] have found that the value of kexp is constant in one K562 cell. The values of k1, k2, k3 and k4 should Table 1 Rate constants of mechanism of enzymatic reaction obtained by simulation No. of K562 cells

k1(L mol – 1 s – 1)

k2(s – 1)

k3(s – 1)

k 4(L mol – 1 s – 1)

1 × 105 2 × 105 4 × 105

1.00 1.52 2.00

1.00 1.00 1.00

6.10 × 10 – 4 8.00 × 10 – 4 12.20 × 10 – 4

7.90 × 102 1.58 × 103 3.16 × 103

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Figure 1 Kinetics of DCHF‑DA oxidation by ROS at different concentrations of K562 cells: (∆) 1 × 105, (◊) 2 × 105, (○) 4 × 105, experimental data (open circles) and simulated data (solid line).

be changed by changing the number of K562 cells. The rate constants of the mechanism at 2 × 105 and 4 × 105 K562 cells are given in Table 1. By increasing the number of cancer cells, the values of k1, k3 and k4 increase. As seen in Table 1, while the values obtained for k2 are constant at different numbers of cell lines, those of k1, k3 and k4 depend on the number of K562 cells. It is plausible that the changes of k2 with the number of K562 cells is negligible. As seen in Figure  1, the suggested mechanism can be used to study the kinetics of this enzymatic reaction for different numbers of K562 cells. It can be concluded that the mechanism obtained is suitable to study the kinetics of the determination of intracellular levels of ROS in cancer cells by DCHF-DA. This mechanism can be used to predict the rate of this reaction under different conditions. Thus, the mechanism and kinetic parameters have been used to obtain the effect of the initial concentrations of DCHF‑DA and ROS on the rate of DCF production. 3.1 Effect of initial concentration of DCHF‑DA on the rate of DCF production To study the effect of initial DCHF‑DA concentration on the rate of DCF production, different initial concentrations of DCHF‑DA were chosen, namely 1.00 × 10 – 8, 5.00 × 10 – 8, 1.00 × 10 – 7, 5.00 × 10 – 7 and 1.00 × 10 – 6 M. The input data for the simulations were temperature (310 K), initial concentrations of DCHFDA, initial concentration of ROS ([ROS]0 =  6.6 × 10 – 5 M) [16], the steps of the mechanism obtained and the rate constants (Table 1). www.prkm.co.uk

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Figure 2 Kinetics of DCF production for different initial concentrations of DCHF‑DA performed in (a) 1 × 105 K562 cells, (b) 2 × 105 K562 cells and (c) 4 × 105 K562 cells.

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Figure  2(a) shows the effect of initial concentrations of DCHF‑DA on the concentration of DCF obtained in 1 × 105 K562 cells. Evidently, the rate of DCF production increases on increasing the initial concentration of DCHFDA. In 1 × 105 K562 cells, the DCF concentration from oxidation of 1.00 × 10 – 8, 5.00 × 10 – 8, 1.00 × 10 – 7, 5.00 × 10 – 7 and 1.00 × 10 – 6 M of DCHF‑DA after 450 s are obtained as 1.26 × 10 – 9, 6.12 × 10 – 9, 1.25 × 10 – 8, 6.25 × 10 – 8 and 1.25 × 10 – 7 M, respectively. There is thus a direct relation between DCF concentration and initial concentration of DCHF‑DA ([DCF] ∝ [DCHF-DA]0). Figures 2(b) and 2(c) show the effect of initial concentrations of DCHF‑DA on the concentration of DCF obtained in 2 × 105 and 4 × 105 K562 cells. Evidently, the rate of DCF production increases on increasing the initial concentration of DCHF-DA. With further numbers of cell lines, it was shown once more that there is a direct relation between DCF concentration and initial concentration of DCHF‑DA ([DCF] ∝ [DCHF-DA]0). In 2 × 105 K562 cells, the DCF concentrations obtained from oxidation of 1.00 × 10 – 8, 5.00 × 10 – 8, 1.00 × 10 – 7, 5.00 × 10 – 7 and 1.00 × 10 – 6 M DCHF‑DA after 450 s are 1.98 × 10 – 9, 9.74 × 10 – 9, 1.98 × 10 – 8, 9.54 × 10 – 8 and 1.98 × 10 – 7 M, respectively. On doubling the number of cell lines, the concentrations of DCF after 450 s were obtained about 1.57 times the concentration of DCF obtained in 1 × 105 K562 cells (i.e. 2[DCF] ∝ 1.57[K562 cell]0). For 4 × 105 K562 cells, the DCF concentration from oxidation of different initial concentrations of DCHF‑DA after 450 s were obtained as 3.09 × 10 – 9, 1.54 × 10 – 8, 3.08 × 10 – 8, 1.55 × 10 – 7 and 3.08 × 10 – 7 M, respectively. It was shown that by doubling the number of cell lines, the concentration of DCF after 450 s obtained was about 1.57 times that of the concentration of DCF obtained in 2 × 105 K562 cells (2[DCF] ∝ 1.57[K562 cell]0). 3.2 Effect of initial concentration of ROS on rate of DCF production By using the mechanism and kinetic parameters obtained, the simulation has been done for different initial concentrations of ROS to determine the effect of its concentration on the rate of reaction. To study the effect of ROS initial concentration on the rate of DCF production, different initial concentrations of ROS were chosen. The input data for the simulations were temperature (310 K), initial concentrations of ROS, initial concentration of DCHF‑DA ([DCHFDA]0 =  1.0 × 10 – 7 M) [16], the steps of the mechanism and rate constants obtained (Table 1). Figure  3(a) shows the effect of initial concentrations of ROS on the concentrations of DCF obtained in 1 × 105 K562 cells. In 1 × 105 K562 cells, the DCF concentrations from oxidation of DCHF‑DA after 450 s were obtained as 2.06 × 10 – 9, 7.10 × 10 – 9, 9.46 × 10 – 9, 1.25 × 10 – 8, 1.28 × 10 – 8 and 1.27 × 10 – 8 M, respectively. www.prkm.co.uk

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Figure 3 Kinetics of DCF production for different initial concentrations of ROS performed for various concentrations of K562 cells: (a) 1 × 105, (b) 2 × 105, (c) 4 × 105.

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As seen from Figure 3(a), the rate of DCF production increases by increasing the initial concentration of ROS. However, for initial ROS concentrations more than 6.6 × 10 – 5 M, further increase of ROS concentration was without effect on the rate of DCF production. Figures 3(b) and 3(c) show the effect of initial concentrations of ROS on the concentrations of DCF obtained for 2 × 105 and 4 × 105 K562 cells. As seen from Figure 3, for an initial ROS concentration of less than 6.6 × 10 – 5 M, the rate of DCF production increases on increasing the initial concentration of ROS. According to Figure 3, the maximum concentration of ROS, which can be attained by a solution of DCHF‑DA with concentration 1.0 × 10 – 7 M, is 6.6 × 10 – 5 M and it is not dependent on the number of K562 cells. In 2 × 105 K562 cells, the DCF concentrations from oxidation of different initial concentrations of ROS after 450 s were obtained as 3.19 × 10 – 9, 1.09 × 10 – 8, 1.48 × 10 – 8, 1.88 × 10 – 8, 1.98 × 10 – 8 and 1.96 × 10 – 8 M, respectively. It was shown that by doubling the number of cell lines, the concentration of DCF after 450 s was obtained at about 1.55 times that obtained for the concentration of DCF in 1 × 105 K562 cells (2[DCF] ∝ 1.55[K562 cell]0). In 4 × 105 K562 cells, the DCF concentration from oxidation of different initial concentrations of ROS after 450 s were obtained as 5.20 × 10 – 9, 1.72 × 10 – 8, 2.31 × 10 – 8, 2.99 × 10 – 8, 3.06 × 10 – 8 and 3.08 × 10 – 8 M, respectively. It was shown that by doubling the number of cell lines, the concentration of DCF after 450 s was obtained at about 1.58 times that of the figure for DCF in 2 × 105 K562 cells (2[DCF] ∝ 1.58[K562 cell]0).

4. CONCLUSION By applying kMC simulation, the mechanism of deacetylation of DCHF‑DA by cellular esterase enzymes has been found. The results obtained, which have been obtained by using this mechanism, are in perfect agreement with experimental data. The mechanism obtained has been used for experimental data at different numbers of K562 cell lines. The mechanism and kinetic parameters obtained have been used to determine the effect of initial concentrations of DCHF‑DA and ROS on the rate of DCF production at different cell numbers. For different cell line numbers, it was shown that there is a direct relation between DCF concentration and initial concentration of DCHF‑DA ([DCF] ∝ [DCHF-DA]0). It was shown the maximum concentration of ROS, which can be determined by a solution of DCHF‑DA with concentration 1.0 × 10 – 7 M, is 6.6 × 10 – 5 M and this is not dependent on the number of K562 cells. It was shown when the number of cell lines is doubled; the concentration of DCF obtained is multiplied in 1.57. www.prkm.co.uk

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5. ACKNOWLEDGEMENT The authors are grateful to University of Kashan for supporting this work through grant no. 256750/I.

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