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. 2020 Aug 19;5(34):21796–21804. doi: 10.1021/acsomega.0c02843

Protective Effect of Thymidine on DNA Damage Induced by Hydrogen Peroxide in Human Hepatocellular Cancer Cells

Yan Li 1, Jianru Guo 1, Huixia Zhang 1, Christopher Wai Kei Lam 1, Wendi Luo 1, Hua Zhou 1,*, Wei Zhang 1,*
PMCID: PMC7469367  PMID: 32905386

Abstract

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Intracellular ribonucleotide (RN) and deoxyribonucleotide (dRN) pool sizes are critical for the fidelity of DNA synthesis. They are likely to be severely perturbed by many factors which disrupt the integrity and stability of DNA, leading to DNA damage. Exogenously supplied nucleosides are able to increase the deoxynucleoside triphosphate pools, then reverse the DNA damage, and decrease the oncogene-induced transformation dramatically. In this study, the impact of thymidine on the hydrogen peroxide (H2O2)-induced DNA damage was investigated in HepG2 liver cancer cells. From the result of the comet assay, the tail length of cells in the thymidine 600 μM + H2O2 1.0 mM group was dramatically decreased from 42.1 ± 10.8 to 21.9 ± 2.4 μm compared to that exposed with 1.0 mM H2O2 (p < 0.05), suggesting that pretreatment of thymidine reduced the DNA damage of HepG2 cells. Although the RN and dRN contents decreased in the damage group, most of them presented increasing tendency when pretreated with thymidine, especially the key metabolites dCTP, which was mainly related with the decline in the rate of DNA synthesis. The restoration also showed a significant G0/G1 phase arrest of cell cycle progression from 44.6 ± 2.2 to 56.6 ± 0.4% after pretreated with thymidine (p < 0.05). In conclusion, our data demonstrated that the pretreatment with thymidine had a potential protective ability against oxidative damage for DNA in HepG2 cells through the perturbation of RN and dRN pools as well as cell cycle arrest, which should provide new insights into the molecular basis of preventing H2O2-induced oxidative DNA damage in mammalian cells.

1. Introduction

Maintaining an appropriate level and balance of ribonucleotides (RNs) and deoxyribonucleotides (dRNs) is vital for DNA integrity and stability because they are building blocks of RNA and DNA synthesis.1 Endogenous RNs and dRNs are essential metabolites in cell function, playing important roles in the regulation and modulation of key cellular processes involved in energy metabolism as well as biochemical signaling pathways.2 However, DNA is considered to be a specifically sensitive cellular target with the potential of being subjected to numerous alterations in its chemical and physical properties.3 Inevitably, endogenous RN and dRN pool sizes can be severely perturbed by the internal and external factors, which disrupt the integrity and stability of DNA, leading to DNA damage. At cellular level, unbalanced changes of RNs and dRNs are associated with many processes including replication and transcription, cell cycle arrest, genetic mutations, and cell death.4

In general, DNA damage can be induced on the basis of several chemical reactive species and physical agents while the intrinsic instability of chemical bonds in DNA may occur spontaneously as well.5,6 Among them, oxidative DNA damage is regarded as the most common insult. Under conditions of cellular oxidative stress, mainly characterized by increased levels of hydrogen peroxide (H2O2), the induced DNA damage can cause genomic instability, which may also involve attack to free bases in the cellular and mitochondrial deoxynucleoside triphosphate (dNTP) pools because the free dNTP precursor pool is more susceptible to be damaged than bases in duplex DNA.7−9 The dNTP levels in bacteria, yeast, and mammalian cells presented different variations after induction of DNA damage.10−12 To cope with the harmful consequences of DNA damage, cells have evolved a variety of strategies to rescue the replication stress and formed a complex network of DNA repair mechanisms. Substantial efforts have been made in recent years to investigate the protective effect against DNA damage.3 In yeast, the dNTP pool sizes increased drastically in order to response DNA damage, which is responsible for the improved survival.13 Our previous study demonstrated that dNTP pools in HepG2 liver cancer cells elevated more than LO2 normal liver cells after DNA damage, which showed more efficient DNA repair as well as improved survival probability.14 In addition, exogenously supplied nucleosides could increase the dNTP pools, then reverse the DNA damage, and decrease the oncogene-induced transformation dramatically.15 Furthermore, nucleoside mono- and diphosphates are also involved in many physiological processes such as signal transduction and nutrient metabolism.16 However, little information regarding the alterations in mono- and diphosphate nucleotides is univocally elucidated during the DNA damage process. Hence, in order to understand the exact mechanism of the protective effect against DNA damage, it is critical to elucidate the disturbances on RN and dRN pool sizes during the DNA damage and repair.

Thymidine is a commonly used agent of a DNA replication inhibitor. Excess thymidine could cause an accumulation of cells that slowly traverse the S phase and synchronize cells in the G1/early S phase.17 This agent has been often used to obtain populations of synchronized cells because it is a constituent of normal cell components and there is no danger of the side effects, which follow the incorporation of structural analogues into the cellular macromolecules.18,19 Additionally, because thymine is the only base unique to DNA, a study of the synthesis of thymidine triphosphate (TTP) might lead to an understanding of molecular events during DNA damage and repair. However, little information is available about this research field. Herein, the present study was designed to investigate whether thymidine is capable of reducing DNA damage in HepG2 cell line and characterize the underlying mechanism at the same time. H2O2 was used as a DNA damaging agent. First, the cytotoxicity of thymidine and H2O2 in HepG2 cell line was examined by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Subsequently, the comet assay was performed to assess the observed influence of thymidine on H2O2-induced DNA damage. After that, the underlying mechanism was investigated by the determination of cellular RNs and dRNs as well as cell cycle analysis. The results may provide new insights into the molecular mechanism of preventing H2O2-induced oxidative DNA damage in mammalian cells.

2. Results

2.1. Cytotoxic Activity by the MTT Viability Assay

At the outset of this study, the cytotoxicity of thymidine and H2O2 in HepG2 cell line was examined. As shown in Figure 1a, more than 85% cells survived after treatment with thymidine from 50 to 1600 μM for different time points, indicating that there was no obvious cytotoxic effect of thymidine on this cell line. The MTT assay also demonstrated the inhibition of the growth of cells after treatment with H2O2 in a time-dependent manner (Figure 1b). Comparatively, the cell number decreased moderately as the concentration of H2O2 increased in 0.5 h of incubation. At this time, H2O2 from 0.125 to 1.0 mM showed barely cytotoxic effect in HepG2 cells in comparison to control. After that, 1.0 mM H2O2 was used to investigate the cytotoxicity of combined H2O2–thymidine pretreatment. The cells were pretreated with the indicated concentrations of thymidine (300, 600, and 1200 μM) for 4 h and then cultured in the presence of 1.0 mM H2O2 for 0.5 (Figure 1c), 1 (Figure 1d), and 2 (Figure 1d) h. As shown in Figure 1c–e, the cell viabilities were increased in the combined groups, indicating the reduction of cytotoxicity. Therefore, three different concentrations of thymidine (300, 600, and 1200 μM) were selected for investigating its effect on H2O2-induced DNA damage.

Figure 1.

Figure 1

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Cell viability of HepG2 cells as measured by the MTT assay. Cells were treated with thymidine at various concentrations (up to 1600 μM) for 5, 10, and 20 h (a); cells were treated with H2O2 at various concentrations (up to 8 mM) for 0.5, 1, and 2 h (b); cells were pretreated with the indicated concentrations of thymidine (300, 600, and 1200 μM) for 4 h and then cultured in the presence of 1.0 mM H2O2 for 0.5 (c), 1 (d), and 2 h (e). The cell viability of untreated and 1.0 mM H2O2-treated cells for different incubation times is shown for comparison. Note: *p < 0.05 and **p < 0.01, compared with the control group; #p < 0.05, compared with the H2O2 1.0 mM group.

2.2. DNA Damage Test

In order to assess the influence of the selected concentrations of thymidine on H2O2-induced DNA damage, the comet assay of HepG2 cell line upon different agent treatments was performed. This assay has been often used as a potential tool for adequately detecting the degree of DNA damage. Photographs of DNA comets are shown in Figure S1. For control and thymidine-alone groups (Figure S1a–d), samples appeared nearly circular because there was no DNA damage. After being exposed to H2O2 1.0 mM for 0.5 h, obvious comets were observed, indicating the DNA damage in this group (Figure S1e). However, the degree of DNA damage in three combined groups presented decreasing tendency with the increased concentration of thymidine pretreatment. For the sake of assessing the head and tail of comet intensity in response to different groups, quantification of comets was carried out on the basis of image analysis. The mean values of tail length, % DNA in tail, and tail moment of comets are presented in Figure 2. From the results, the tail length changed significantly (p < 0.05) between the H2O2 group and the three combined groups with the mean values of 42.1 ± 10.8, 31.7 ± 7.5, 21.9 ± 2.4, and 20.3 ± 4.2 μm, respectively. The mean values of % DNA in tail of samples in the three combined groups were significantly lower than that of the H2O2 group (p < 0.05), suggesting that less DNA fragmentation occurred and these fragments migrated shorter during the electrophoresis of the comet assay. Moreover, for the samples in the thymidine 1200 μM + H2O2 1.0 mM group, which showed only slight tails, the % DNA in tail and tail moment were similar to control and thymidine-alone groups. Besides, after the DNA repair in the absence and presence of thymidine for 2, 4, and 10 h, unrepaired DNA fragments were also measured. From the results of the photographs of DNA comets, partial repair was presented after 2 h (Figure S1i–l), and cells were completely repaired until 10 h (Figure S1q–t). However, the addition of thymidine has no obvious effect on the process of repair, and it was verified based on the quantification of comets in Figure 3. Thus, the results demonstrated that thymidine is likely to have the effect on reducing H2O2-induced DNA damage. Moreover, thymidine with high concentration could present more influence on this type of DNA damage.

Figure 2.

Figure 2

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Image analysis of the comet assay. Tail length (a), % DNA in tail (b), and tail moment (c) of 1.0 mM H2O2-treated cells for 0.5 h with or without pretreatment with the indicated concentrations of thymidine. Note: *p < 0.05, compared with the control group; #p < 0.05, compared with the H2O2 1.0 mM group.

Figure 3.

Figure 3

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Image analysis of the comet assay. Cells were allowed to repair in fresh medium following exposure to 1.0 mM H2O2 for 0.5 h with or without pretreatment with thymidine at different concentrations. Tail length (a), % DNA in tail (b), and tail moment (c) of cells after 2 h of repair; tail length (d), % DNA in tail (e), and tail moment (f) of cells after 4 h of repair; tail length (g), % DNA in tail (h), and tail moment (i) of cells after 10 h of repair. Note: *p < 0.05 and **p < 0.01, compared with control group; #p < 0.05 and ##p < 0.01, compared with the H2O2 1.0 mM group.

2.3. Intracellular Metabolic Perturbation of RN and dRN Pools

For gaining insights into the detail intracellular metabolic perturbation in response to DNA damage, the determination of RN and dRN pools was performed. The amounts of intracellular RN and dRN pools as expressed in pmol/106 cells are shown in Tables 1 and 2. The dNTP levels are presented in Figure 4. These intracellular metabolic levels varied significantly among different groups. For the 1.0 mM H2O2 group, the contents of RN pools decreased remarkably (p < 0.05 or p < 0.01) than those of the control group except AMP, GTP, and GMP. Similarly, cells in this group also contained a significant less contents of dRN pools in addition to dAMP, dGDP, dGMP, and dTDP (p < 0.05 or p < 0.01). Totally, most of the RN and dRN profiles in cells presented decreasing tendency after being exposed to 1.0 mM H2O2. Besides, although the concentration of thymidine employed in this study showed seldom cytotoxic effect on the HepG2 cell line, there was still some influence on the metabolic perturbation in the thymidine-alone group, especially for dRN pools. At the same time, the energy charge decreased extremely (p < 0.01) when compared with the control group (Table 3).

Table 1. RN Pools in HepG2 Cells of Different Groups (pmol/106 Cells)a.

  control H2O2 1.0 mM thymidine 600 μM thymidine 600 μM + H2O2 1.0 mM
ATP 12,827.97 ± 1235.10 7337.74 ± 632.19** 10,540.90 ± 2928.40 14,971.50 ± 534.25*##
ADP 1345.18 ± 119.22 780.61 ± 182.28* 1929.40 ± 457.70 2135.53 ± 122.76**##
AMP 192.57 ± 31.90 156.28 ± 62.48 449.15 ± 78.75** 356.11 ± 28.75**##
CTP 886.84 ± 104.24 351.34 ± 48.35** 1002.78 ± 320.96 1509.03 ± 86.21**##
CDP 199.61 ± 39.15 42.88 ± 6.04** 445.84 ± 85.59* 507.53 ± 24.86**##
CMP 127.65 ± 2.79 96.70 ± 15.78* 207.17 ± 57.93 209.53 ± 18.10**##
GTP 2598.13 ± 198.55 2462.88 ± 247.51 2401.30 ± 707.72 3698.19 ± 352.85**##Δ
GDP 538.77 ± 64.01 432.02 ± 77.57* 1053.14 ± 267.98* 1111.22 ± 130.13**##
GMP 20.91 ± 2.82 27.80 ± 15.21** 50.27 ± 7.70** 48.07 ± 3.54**
UTP 10,109.00 ± 776.89 3519.84 ± 298.32** 8919.08 ± 2423.15 13,819.12 ± 1350.83*##Δ
UDP 569.61 ± 115.80 152.56 ± 32.52** 1535.74 ± 345.18** 1637.57 ± 184.15**##
UMP 52.76 ± 5.70 25.48 ± 10.54* 103.75 ± 17.46** 89.70 ± 6.45**##
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a

*p < 0.05 and **p < 0.01, compared with the control group; #p < 0.05 and ##p < 0.01, compared with the H2O2 1.0 mM group; and Δp < 0.05 and ΔΔp < 0.01, compared with the thymidine 600 μM group.

Table 2. dRN Pools in HepG2 Cells of Different Groups (pmol/106 Cells)a.

  control H2O2 1.0 mM thymidine 600 μM thymidine 600 μM + H2O2 1.0 mM
dATP 27.95 ± 2.09 21.47 ± 2.74* 44.32 ± 12.50 53.50 ± 4.04**##
dADP 0.02 ± 0.00 0.01 ± 0.00** 0.07 ± 0.01** 0.06 ± 0.00**##
dAMP UDL UDL 0.01 ± 0.00 0.01 ± 0.00
dCTP 25.19 ± 0.85 8.02 ± 1.36** 7.25 ± 2.01** 14.86 ± 0.92**##ΔΔ
dCDP 0.04 ± 0.01 0.01 ± 0.00** 0.01 ± 0.00** 0.02 ± 0.00**##Δ
dCMP 0.01 ± 0.00 UDL 0.01 ± 0.00 0.01 ± 0.00
dGTP 13.27 ± 0.94 7.70 ± 0.73** 47.77 ± 5.95** 47.93 ± 6.50**##
dGDP 2.45 ± 0.29 1.45 ± 0.63 28.30 ± 4.39** 19.32 ± 0.88**##Δ
dGMP 0.87 ± 0.27 0.68 ± 0.15 15.61 ± 3.49** 6.25 ± 0.21**##ΔΔ
dTTP 110.26 ± 10.53 31.42 ± 2.51** 672.97 ± 170.43** 922.27 ± 47.90**##
dTDP 2.14 ± 0.25 3.41 ± 0.31** 18.17 ± 3.56** 16.89 ± 1.03**##
dTMP 0.08 ± 0.01 0.02 ± 0.01** 0.42 ± 0.09** 0.45 ± 0.06**##
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a

UDL, under detected limit of assay; *p < 0.05 and **p < 0.01, compared with the control group; #p < 0.05 and ##p < 0.01, compared with the H2O2 1.0 mM group; and Δp < 0.05 and ΔΔp < 0.01, compared with the thymidine 600 μM group.

Figure 4.

Figure 4

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Levels of dNTP pools after thymidine and H2O2 treatments. Data are means ± standard deviation (SD) from three independent experiments. Note: *p < 0.05 and **p < 0.01, compared with the control group; #p < 0.05 and ##p < 0.01, compared with the H2O2 1.0 mM group; Δp < 0.05 and Δp < 0.01, compared with the thymidine 600 μM group. The black column indicates the control group; the blue column indicates the H2O2 1.0 mM group; the green column represents the thymidine-alone group; and the red column expresses the combined group.

Table 3. General Properties of HepG2 Cells in Each Groupa.

  control H2O2 1.0 mM thymidine 600 μM thymidine 600 μM + H2O2 1.0 mM
ATP/ADP 9.62 ± 1.37 9.75 ± 1.53 5.43 ± 0.46** 7.02 ± 0.28*#ΔΔ
ATP/dATP 458.24 ± 12.12 343.88 ± 17.73** 238.26 ± 4.11** 281.84 ± 28.09**#
CTP/dCTP 35.10 ± 3.04 44.08 ± 1.62* 136.89 ± 5.27** 101.59 ± 1.32**##ΔΔ
GTP/dGTP 197.60 ± 27.03 325.44 ± 60.61* 51.37 ± 17.90** 77.56 ± 3.03**##
Energy charge 0.94 ± 0.01 0.94 ± 0.01 0.89 ± 0.01** 0.92 ± 0.00Δ
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a

*p < 0.05 and **p < 0.01, compared with the control group; #p < 0.05 and ##p < 0.01, compared with the H2O2 1.0 mM group; and Δp < 0.05 and ΔΔp < 0.01, compared with the thymidine 600 μM group.

Additionally, the contents of RN and dRN pools of samples in the thymidine 600 μM + H2O2 1.0 mM group were significantly higher than those of the control group, except dCTP, dCDP, and dCMP. By contrast, the samples in combined group also showed a significant increase of all the RN and dRN pools when compared with the damage group. However, following the combined treatment on the basis of thymidine and H2O2 in HepG2 cells, there were similar amounts among most of the tested metabolites between this group and the thymidine-alone group apart from GTP, UTP, dCTP, dCDP, dGDP, and dGMP, which exhibited distinctly increasing tendency in the first four metabolites and decreased obviously in the other two dRN levels. Interestingly, the energy charge in the combined group was significantly (p < 0.05) higher than that of the thymidine 600 μM group, indicating that the severely perturbed energy balance may change back to the normal level gradually after the combination of thymidine 600 μM and H2O2 1.0 mM. In other words, H2O2 could give rise to HepG2 cells with DNA damage and stimulate the perturbation of RN and dRN pools. Further, it also seems to imply that the integration of thymidine and H2O2 was likely to diminish the impact of H2O2 on cells according to the change in the RN and dRN levels, which may be the reason for thymidine to exert the protective effect on oxidative DNA damage induced by H2O2.

2.4. Cell Cycle Arrest Analysis

With regard to the response of DNA damage, cell division is arrested in many eukaryotic cells.20 In order to evaluate the effect of thymidine on H2O2-induced cell cycle phase distribution, cell cycle analysis was performed. Results in Figure 5 showed that there were no obvious differences between the control and 1.0 mM H2O2 groups. However, when cell samples were cultured with different concentrations of thymidine (300, 600, and 1200 μM) for 4 h before being exposed to 1.0 mM H2O2, they were observed arresting at the G0/G1 phase with the percentages of 49.0 ± 0.8, 56.6 ± 0.4, and 61.3 ± 0.7%, respectively. The increase of cell population in the G0/G1 phase was accompanied by a concomitant reduction in the G2/M phase, which showed significant difference compared with control and 1.0 mM H2O2 groups. In particular, the high concentration groups presented similar results. Our in vitro data indicated that treatment of thymidine could result in a significant G0/G1 phase arrest of cell cycle progression to reveal the protective effect on oxidative DNA damage of HepG2 cells. It is suggested that one of the mechanisms by which thymidine may act is through the influence on cell cycle. Further, thymidine with high concentration exhibited more severe arrest in the G0/G1 phase than that of the lower one.

Figure 5.

Figure 5

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Statistical analysis of cell cycle distribution in HepG2 cells upon different treatments. Note: *p < 0.05 and **p < 0.01, compared with the control group; #p < 0.05 and ##p < 0.01, compared with the H2O2 1.0 mM group. The black column indicates the control group; the blue column indicates the H2O2 1.0 mM group; the green column represents the thymidine 300 μM + H2O2 1.0 mM group; the red column shows the thymidine 600 μM + H2O2 1.0 mM group; and the pink column indicates the thymidine 1200 μM + H2O2 1.0 mM group.

3. Discussion

Generally, compared with normal cells, cancer cells constitutively produce large but nonlethal amounts of reactive oxygen species that function as signaling molecules to constantly activate redox-sensitive transcription factors and responsive genes, which are involved in the survival as well as the proliferation of cancer cells.21 Hence, decreases in oxidative stress may inhibit the proliferation of tumor cells.22 In this study, the putative protective effect of thymidine on H2O2-induced DNA damage was investigated in HepG2 cell line using the comet assay. Earlier studies have shown that H2O2 could give rise to DNA strand breakage on account of generating hydroxyl radicals close to the DNA molecule.23 Therefore, the protective effect was assessed as the decreased induction of DNA breaks, which was verified by some parameters of the comet assay. Tail length was often used in many studies. However, tail length can be useful at low levels of DNA damage; once the tail of comet is established, its length tends not to change. On the other hand, the % DNA in tail is known as the most useful parameter because it is linearly related to DNA break frequency over a wide range of damages.24 The calculation of tail moment covers both of % DNA in tail and tail length. Therefore, in this study, tail length, % DNA in tail, and tail moment were used to interpret the comet. All the values of these three parameters in the thymidine + H2O2 group were lower than that of the H2O2 group, suggesting that exposure of thymidine was not toxic to HepG2 cells but it could protect DNA to H2O2-induced DNA strand breaks.

On the other hand, the integrity of DNA is crucial for cell division, and oxidative alteration can perturb DNA transcription, translation, and replication and cause the mutations, cell senescence, and death.25 With respect to successfully and accurately performed DNA synthesis and the subsequent cell replication, cells have a critical requirement for a rapid and balanced supply of intracellular dNTP pools. dNTP pools are known as the substrates for DNA synthesis, and their intracellular concentrations are precisely controlled by the complex allosteric regulation of ribonucleotide reductase (RNR) as well as other enzymes of purine and pyrimidine dRN metabolism. In this study, although the treatment of thymidine had no significant cytotoxicity to HepG2 cells, the RNs and dRNs were perturbed, leading to the considerable decrease of the rate of DNA synthesis under this situation. Briefly, as thymidine is phosphorylated to TTP through the pyrimidine salvage pathway in cells, excess thymidine treatment could result in striking expansions of the cellular TTP level, which is an allosteric inhibitor of the reduction of CDP by RNR (Figure 6).26 Then, the dGTP and dATP levels are also increased and the intracellular concentrations of dCDP and dCTP pools are drastically decreased in accordance with the stimulating reduction of a single substrate. As demonstrated by Bjursell and Reichard,27 DNA synthesis required a critical size of the dCTP pool, which was one of the essential precursors of DNA, and this pool may have a regulatory function for the rate of DNA synthesis. Therefore, the decrease of dCTP pool was mainly related to the decline in the rate of DNA synthesis. In other words, thymidine exerted its effect on the decline of DNA synthesis on the basis of inhibiting the reduction of CDP to dCDP by RNR, hence inhibiting the formation of dCTP. These mechanisms are also consistent with the previous study reported by Wilkinson and McKenna.28 Additionally, ATP is a universal energy source of cells in many biological processes. A large decrease in ATP level is inherent to decreased cell viability.29 ATP/ADP ratio is a more important parameter to reveal the energy status of cells. At the same time of the inhibition of dCTP, the energy charge decreased extremely, suggesting that the utilization of ATP was inhibited. In this case, it may also cause the decrease of dCDP and dCTP levels because the binding of ATP at the substrate specificity site was concerned with the enzyme to promote the reduction of CDP to dCDP and then affect the dCTP pool.30

Figure 6.

Figure 6

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Pathways of dNTP synthesis in mammalian cells. Note: red plus symbols indicate positive effects and blue minus symbols indicate inhibition of RNR.

In addition, all the dNTP pools showed obvious decreasing tendency after being exposed to H2O2, and the TTP and dCTP levels were the most pronounced. However, when combined with thymidine treatment, the dNTP levels were increased significantly compared with the damage group. According to the variability of the four dNTP pools, TTP level presented the most obvious increasing tendency and dGTP and dATP levels also increased significantly with the variation of TTP via the allosteric regulation of RNR. Interestingly, when HepG2 cells were exposed to H2O2 after treatment with thymidine for 4 h, dCTP showed an increasing tendency, which was contrary to the effect of the allosteric regulation of RNR. Based on a previous study, intracellular dNTP pools for DNA replication and repair arise mainly through the tightly regulated pathway of RN reduction by RNR, although cells are capable of deoxynucleosides salvage as well.31 Hence, it indicated that the expansion of dCTP pool in the combined group may be attributed to the salvage pathway in order to rescue the decrease of the cellular dCTP and DNA synthesis may restore to the normal level. At this time, the energy charges were restored gradually, suggesting that the utilization of ATP was stimulated. Therefore, the dNTP pools become balanced gradually, which may be the mechanism of reducing oxidative DNA damage.

The cellular processes such as mitosis, DNA replication, and cell growth must be coordinated during cell cycle progression. In the present study, pretreatment of thymidine could interfere with the cell cycle and induce arrest at G0/G1 phase to decrease the DNA fragmentation and exhibit the protective effect on oxidative DNA damage of HepG2 cells. Normally, when cells arrest in the G0/G1 phase, they may die or may be repaired and re-enter into the next phase of the cell cycle.32−34 In addition, mammalian cells are programmed to cycle checkpoints, which enable DNA damage repair according to slowing or arresting cell cycle progression, thereby preventing the transmission of damaged chromosomes.35−37 We therefore suspect that thymidine pretreatment arrested cells in the G0/G1 phase to retard the process of DNA replication and enable the DNA repair by expanding the corresponding time for the sake of reducing oxidative DNA damage development. The increased dCTP pool was likely to be relative with this reparative process.

Moreover, DNA damage can be induced by a kind of chemical agents or physical factors including methyl methanesulfonate, H2O2, 4-nitroquinoline-N-oxide (4-NQO), nucleoside analogue, UV radiation, and so on. A number of efforts have been made in recent years in order to investigate the protective effect against DNA damage. For example, Arumugam and Ramesh found that pretreatment of sesame oil can effectively protect against DNA damage and lipid peroxidation induced by 4-NQO.38 Lemon balm extract was proved to be considered as a candidate for the development of oral/topical photoprotective ingredients against UVB-induced DNA damage.39 Erol et al. demonstrated that the pretreatment of gallic acid could significantly reduce both nDNA and mtDNA damages occurred with H2O2 exposure.40 However, there is no study of the effect of thymidine on these types of DNA damages. Furthermore, oxidative DNA damage is regarded as the most common insult. Under conditions of cellular oxidative stress, which generated endogenously by metabolic reactions or exogenously (cigarette smoke and other air pollutants), H2O2 could be produced at a high level that caused DNA damage.41 The protection of oxidative DNA damage becomes a crucial issue. Hence, in this study, the ability of thymidine to protect against H2O2-induced oxidative DNA damage could be recommended as an effective supplementary in the relevant field.

4. Conclusions

In conclusion, we have presented evidence that thymidine was capable of protecting against H2O2-induced DNA damage in the HepG2 liver cancer cell line via perturbation of RNs and dRNs as well as cell cycle arrest. dCTP is the key factor of the whole process. The allosteric regulation of RNR as well as the salvage pathway played an important role in the whole process. In addition, thymidine can be used to protect against oxidative damage associated with elevated H2O2 production. Furthermore, the unique features of thymidine also provided new insights into the molecular basis of preventing H2O2-induced oxidative DNA damage in mammalian cells.

5. Materials and Methods

5.1. Reagents and Chemicals

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin–streptomycin solution, and 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) solution were acquired from GIBCO (Grand Island, NY, USA). Phosphate-buffered saline (PBS) and SYBR safe DNA gel stain were obtained from Invitrogen Co. (Carlsbad, CA, USA). In addition, dimethyl sulfoxide (DMSO) and MTT were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). A Cell Cycle Analysis Kit used in this study was bought from KeyGen Biotech. Co. (Nanjing, Jiangsu, China), while hydrogen peroxide (H2O2) 30% was obtained from Fuyu Chemical Co., Ltd. (Tianjin, China). Triton X-100 and EDTA disodium salt dehydrate (EDTA-2Na) were acquired from MP Biomedicals, LLC., Illkirch, France. Moreover, microscope slides (clear glass ground edges, 25.4 × 76.2 mm), microscope cover glass (24 × 24 mm), and low-melting-point agarose were purchased from Weijia Co. (Guangzhou, Guangdong, China). The reagents used for liquid chromatography–mass spectrometry (LC–MS) analysis including methanol (LC–MS grade), acetic acid, and acetonitrile were bought from Anaqua Chemical Supply (Houston, TX, USA). Thymidine (≥99% pure), the stable isotope-labeled adenosine-13C10,15N5-triphosphate (ATP13C,15N), trichloroacetic acid (TCA), diethylamine, hexylamine, trioctylamine, and 1,1,2-trichlorotrifluoroethane were also purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Ultrapure water was obtained based on a Milli-Q Gradient water system (Millipore, Bedford, MA, USA). Other reagents were of analytical grade.

5.2. Cell Line and Measurement of Cell Viability

The HepG2 cell line was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in DMEM medium supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37 °C under 5% CO2/95% air.

In this study, the cytotoxicity of thymidine, H2O2 treatment, and the combined H2O2–thymidine pretreatment was assessed by using the MTT method.34 At first, growing HepG2 cells were harvested, seeded into 96-well plates, and incubated at 37 °C and 5% CO2 for 24 h. Then, cells were treated with different agents at various concentrations. For thymidine group, this agent was added to cells with increasing concentrations ranging from 50 to 1600 μM at 5, 10, and 20 h, respectively. In addition, cells in the H2O2 group were treated with H2O2 from 0.125 to 8 mM at 0.5, 1, and 2 h, respectively. Moreover, we focused on the preventing H2O2-induced oxidative DNA damage in mammalian cells. Hence, the pretreatment of thymidine was conducted for this study. In the combined group, cells were cultured with thymidine at 300, 600, and 1200 μM for 4 h, respectively. Subsequently, cells were exposed to 1.0 mM H2O2 for 0.5, 1, and 2 h. In regard to the control group, cells were incubated without any treatment. After the incubation, cells in each well were treated with 10 μL MTT solution, respectively, and further incubated for 4 h. Afterward, the medium was removed, and DMSO was used to dissolve the formazan crystals formed. Each tested plate was read at 570 nm (the reference wavelength was 650 nm) on a microplate UV–vis spectrophotometer (Infinite M200 PRO, Tecan Austria).

5.3. Comet Assay Analysis

HepG2 cells were plated in six-well plates (4.5 × 105 cells/well) for 24 h and then cultured with thymidine at the concentrations of 300, 600, and 1200 μM for 4 h. Subsequently, they were exposed to H2O2 with a concentration of 1.0 mM for 0.5 h. Meanwhile, cells with or without pretreatment with the indicated concentrations of thymidine were also used. Then, cells were collected to investigate DNA damage according to the comet assay under alkaline conditions based on the technique reported by Olive and Banáth.42 At the same time, the kinetics of repair of H2O2-induced breaks was also investigated in the absence and presence of thymidine at the concentrations of 300, 600, and 1200 μM for 2, 4, and 10 h, respectively, which was also analyzed by the comet assay. After all the treatments, the cells were washed and resuspended with ice-cold PBS. Then, the cell pellets were mixed with 100 μL of low melting point agarose at 37 °C, placed on a microscope slide with a coverslip, and kept for 10 min at 4 °C to solidify. After the coverslips were removed, all the slides were immersed into a lysis solution for 2 h at 4 °C in a dark place. After that, they were denaturized in an alkaline buffer solution for 20 min. Electrophoresis was performed in the chilled denaturation buffer for 20 min via a voltage of 25 V. At last, the slides were stained with an SYBR safe DNA gel stain. A fluorescent microscope (Leica Microsystems Ltd., Wetzlar, Germany) combined with a charge-coupled device camera connected to a personal computer was used to detect the comets. Observed comets were evaluated by image analysis by using TriTek Comet Score software (version 1.5) for linking the comet parameters to estimate the degree of DNA damage. The interpretation of comets was expressed by tail length, percentage tail DNA, and tail moment. These parameters are defined as follows:

  • Tail length: length of the comet tail measured from the right border of head area to the end of tail.

  • Percentage tail DNA: percent of DNA in the comet tail.

  • Tail moment: tail length × % DNA in tail.

5.4. LC–MS/MS Analysis

The monolayer cells were also divided into different groups including the control group, H2O2 group, thymidine-alone group, and combination group. Then, the tested cell samples were washed and resuspended with ice-cold PBS. The number of cells in each group was counted before centrifugation at 1200 rpm for 5 min. The cells were then washed and centrifuged again and processed with 150 μL of 15% TCA containing 7.5 μL of 20.0 μM internal standard (ATP13C,15N). Afterward, the processed samples were ice-bathed for 10 min and centrifuged at 13,000 rpm for 15 min. The supernatant was separated and neutralized twice with an 80 μL mixture containing trioctylamine and 1,1,2-trichlorotrifluoroethane (45:55; v/v). Eventually, the final samples were analyzed by the established LC–MS/MS method to determine the cellular RN and dRN pools.43

5.5. Cell Cycle Distribution Analysis

HepG2 cells were also cultured as mentioned in the “comet assay analysis” part. After the indicated treatments, all the samples were harvested and washed with ice-cold PBS, trypsinized, and centrifuged at 1200 rpm for 5 min. The cell pellet was suspended to be fixed with cold 70% (v/v) ethanol and stored overnight at −20 °C. Subsequently, the fixed cells were collected, resuspended in cold PBS, and incubated with propidium iodide (Sigma-Aldrich) containing 0.05% RNase A (Sigma-Aldrich) at room temperature for 30 min in the dark. At last, the cell cycle distribution profile after the staining treatment was assessed by a flow cytometer (Muse cell analyzer, Merck Millipore, Darmstadt, Germany). The percentages of cells in G0/G1, S and G2/M phases were analyzed by using MODFIT software (Verity Software House, USA).

5.6. Statistical Analysis

All the data were acquired from at least triplicate experiments in a parallel manner and expressed as mean ± SD of individual values. The statistical significance of any difference among different groups was analyzed by one-way analysis of variance, while the comparison between two groups was evaluated according to Student’s t-test. The level of significance was defined as 95% (p < 0.05), while p < 0.01 indicates very significant.

Acknowledgments

This work was supported by the Macao Science and Technology Development Fund (project no. 0052/2018/A2 and 0061/2019/AGJ).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.0c02843.

  • Photographs of cells analyzed by the comet assay (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao0c02843_si_001.pdf (394.4KB, pdf)

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