In Vitro Formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in Calf Thymus DNA upon Treatment of 2'-deoxyguanosine with Propyl Gallate and 2,6-di-tert-butyl-p-benzoquinone

Oxidative DNA damage caused by propyl gallate (PG) and 2,6-ditert-butyl-p-benzoquinone (BHT-quinone, a metabolite of butylated hydroxytoluene (BHT)) was a nalyzed from the 8-hydroxy-2 ′-deoxyguanosine (8-OHdG) formation in calf thymus DNA and DNA base, 2 ′-deoxyguanosine (dG). PG in the presence of CuCl 2 increased the 8OHdG formation in calf thymus DNA by around 9.17 ti mes as compared to the control (untreated DNA). In the presence of CuCl 2 at 1.28×10 -5 M, the 8-OHdG per dG ratio resulting from the reac tion of dG with PG at various concentrations (20–150 ppm) ranged from 75.50 to 31 2.06 8-OHdG per 10 5 dG. The 8-OHdG formation increased when the PG concentration was increased from 20 ppm to 80 ppm, and then, it began to plateau around 80 ppm. On the other hand, BHT-quinone increased the formation of 8-OHdG in the presence of CuCl 2 by 0.05 times as compared to the control (untreated DNA). LC-MS/MS analysis was used to identify the molecular structure of 8-OHdG, which had a base peak (M + 1) at m/z = 284 and two main fragments at m/z = 1 67.9 and m/z = 139.9.


Introduction
Propyl gallate (PG) and butylated hydroxytoluene (BHT) are synthetic antioxidants that are widely used as food additives to prevent food deterioration due to oxidation. PG and BHT have been investigated as chemopreventive agents in several animal experiments [1][2][3]. Although their use has been approved as food additives in many countries, the safety of these antioxidants is a controversial issue because several research reports have indicated that they have some adverse health effects [4][5][6][7][8][9][10].
In contrast to these protective effects, PG was shown to induce apoptosis due to DNA fragmentation in hepatocytes [4]. Jacobi et al. [5] reported that PG induced single strand breaks in the presence of Cu (II). Gallic acid and its alkylesters caused strand scission in the presence of Cu (II) and 8-hydroxy-2′deoxyguanosine  was the best-characterized base adduct formed via the action of the reactive oxygen species (ROS) [6]. Ito et al. reported that BHT can act as tumor promoter in rat urinary bladder and thyroid [7]. BHT is known to be extensively metabolized in its target tissues, and the toxic as well as tumor-promoting activities of BHT are thought to be mediated by metabolites of the parent compound [8]. One of the BHT metabolites, 2,6-di-tertbutil-p-benzoquinone (BHT-quinone), has been reported to cause cleavage of supercoiled DNA [9]. Oikawa et al. [10] reported that BHT metabolites, i.e., BHT-quinone and 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-2,5-cyclohexadienone (BHT-OOH), induced oxidative DNA damage.
Because of the widespread use of PG and BHT and the adverse effect of these antioxidants on DNA, it is necessary to investigate their interaction with DNA.
In this study, the binding or interaction of PG and BHTquinone with calf thymus DNA was observed using a UV-Vis spectrophotometer. Furthermore, to determine the interaction of PG and BHT-quinone with DNA bases, we examined the covalent addition products, known as "DNA adducts" (i.e., 8-OHdG), which served as biomarkers for oxidative DNA damage in calf thymus DNA at the DNA base, 2′-deoxyguanosine (dG). This adduct formation was mediated by cupric chloride and was detected with HPLC equipped with UV-Vis detectors. Finally, the molecular structures of these adducts were identified using LC-MS/MS.

Analysis of formation of 8-OHdG in calf thymus
DNA by PG and BHT-quinone in the presence of CuCl 2 . Calf thymus DNA was treated with PG in the presence of CuCl 2 in 50 mM Tris HCl (pH 7.4). The reaction mixture of 100 µL was incubated at 37 °C for 1 h. Then, the reaction mixture containing 2.67 mg/mL DNA, 4.27×10 -5 M CuCl 2 , and 40 ppm PG was hydrolyzed to deoxyribonuleosides with a mixture of micrococcal nuclease (2 mU/µg DNA) and phosphodiesterase II (0.04 mU/µg DNA) in 0.1 M sodium succinate and 0.05 M CaCl 2 buffer (pH 6). The mixture was incubated at 37 °C for 3 h. The same procedure was applied to 200 ppm BHT-quinone. Then, the hydrolyzed DNA was analyzed by HPLC equipped with UV-Vis detectors using an octadecylsilane column (XBridge C18, 4.6 x 250 mm, 10 µm (Waters)) with 10 mM NaH 2 PO 4˙H2 O/15% methanol at a flow rate of 0.1 ml/min. The hydrolyzed DNA was also analyzed by LC-MS/MS with a TQ Detector (Waters); the column used was a reversed-phase column (Atlantis T3, 4.6 x 50 mm, 5 µm (Waters)). Gradient elution was performed using 0.1% formic acid in water (solvent A) and 0.1% formic acid in methanol (solvent B). The flow rate was set to 0.5 mL/min. The gradient elution applied for this analysis is summarized in Table 1.
Mass spectrometric detection was performed on a triple quadrupole tandem mass spectrometer coupled with an electrospray ionization source. The optimized ESI source parameters are listed in Table 2. The determination of 8-OHdG formation was performed under the multiple reaction monitoring (MRM) mode.

Results and Discussion
UV-Vis spectrophotometer analysis of interaction of PG with DNA. Binding or interaction of a compound with DNA causes adsorption spectra changes [11]. On the other hand, the interaction between BHTquinone and DNA was not analyzed using the UV-Vis spectrophotometer, because BHT-quinone had maximum absorption at around 260.40 nm, which was close to the absorption peak of calf thymus DNA at 259 nm (not shown); thus, the spectra were not selected for analyzing the interaction between BHT-quinone and DNA.
Formation of 8-OHdG in calf thymus DNA. The HPLC chromatogram of the control (untreated DNA) shown in Figure 2 indicates that the DNA was hydrolyzed into its four nucleosides. The nucleosides were separated from one another. The retention time for dG was 6.938 min. The HPLC analysis revealed approximately 49.27% and 52.87% decrease in the peak area of dG following treatment with PG and BHTquinone, respectively, in the presence of CuCl 2 .
It is suggested that the treatment of DNA with PG and BHT-quinone in the presence of CuCl 2 destroyed dG. Beach and Gupta [12] had shown that guanine bases in DNA are the predominant sites to be attacked by chemical On the other hand, LC-MS/MS analysis of the control (untreated DNA) and the DNA treated with PG and BHT-quinone in the presence of CuCl 2 detected 8-OHdG, which had a base peak (M +. + 1) at m/z = 284 and two main fragments at m/z = 167.9 and m/z = 139.9 ( Figures  3, 4, and 5, respectively). This is due to the lower limit of detection of LC-MS/MS as compared to that of HPLC.
The formation of 8-OHdG in calf thymus DNA in PG mediated with CuCl 2 was 9.17 times higher than that in the control (untreated DNA). On the other hand, the formation of 8-OHdG in calf thymus DNA in BHTquinone mediated with CuCl 2 was 0.05 times higher than that in the control (untreated DNA).  On the other hand, HPLC analysis of the reaction mixture of dG and BHT-quinone at various concentrations (160-360 ppm) in the presence of 1.28×10 -5 M CuCl 2 did not detect 8-OHdG formation (data not shown), but LC-MS/MS analysis identified 8-OHdG formation ( Figure  9). This is due to the lower limit of detection of LC-MS/MS as compared to that of HPLC.

Formation of 8-OHdG resulting from the reaction of dG with PG and BHT-quinone in the presence of
The present study showed that PG and BHT-quinone in the presence of CuCl 2 increased the formation of 8-OHdG in calf thymus DNA and dG. The formation of 8- OHdG is an indicator of oxidative stress and is known to be mutagenic in mammalian cells [13]. Thus, 8-OHdG is known to cause DNA misreplication, leading to mutation or cancer [14].
The results of the experiments showed that the formation of 8-OHdG induced by PG and BHT-quinone was stimulated by CuCl 2 . Copper is found in the body, and the normal level of copper in various organs is approximately 0.85-3.60 µg/mL [15]. Free Cu(II) ions are found in the nucleus, and they have a high affinity for DNA [16]. Copper ions are known to mediate oxidative damage to DNA, which is detected as 8-OHdG formation [17].
An oxidative DNA lesion is assumed to be caused by the hydroxyl radical generated from hydrogen peroxide (H 2 O 2 ) and copper through the Fenton reaction [18]. The formation of 8-OHdG induced by gallic acid may be due to the hydroxyl radical (˙OH) produced depending on the transition metals [6]. The possible mechanisms of metalmediated hydroxyl radical formation induced by PG are shown in Figure 10.
Metal-mediated auto-oxidation of PG generates the semiquinone radical. The Cu (II) ion is reduced by gallate to form Cu(I), which reduces molecular O 2 to the superoxide anion (O2˙ˉ) [6]. Then, O2˙ˉ may undergo a dismutation reaction, represented by Equation (1) The hydroxyl radical (˙OH) is a highly reactive ROS that can react with DNA adducts, such as 8-OHdG, as an oxidative DNA damage marker [22]. Although all DNA bases are susceptible to damage, guanine is the most prone to oxidative modification [23]. The mechanism of 8-OHdG formation results from the reaction of 2'-deoxyguanosine (dG) with hydroxyl radicals (˙OH), as shown in Figure 11. The results showed increasing formation of 8-OHdG induced by PG mediated with CuCl 2 , which is suggested to occur because of the increase in the hydroxyl radicals.  concentrations of PG. These phenomena can be attributed to the ability of compounds to participate in more than one reaction. In this situation, a compound could act as a pro-oxidant at low concentrations and stimulate the formation of radicals. However, at higher concentrations, these compounds may scavenge free radicals. This is the case for compounds that are strong reducing agents, such as ascorbic acid and gallic acid [24].

2'deoxyguanosine
The results of this study showed that PG, a gallic acid derivative, could act as a pro-oxidant at low concentrations in the presence of metal ions. The pro-oxidant effect of such compounds is thought to be responsible for DNA damage, as demonstrated by researchers [4][5][6].
On the other hand, BHT-quinone is a quinone compound and one of the metabolites of BHT antioxidant. The addition of CuCl 2 to 200 ppm BHT-quinone induced a slight increase in 8-OHdG levels (0.05 times as compared to the control). Studies conducted by Oikawa et al. [10] showed that BHT-quinone caused DNA damage not in the presence of Cu(II) but in the presence of NADH and Cu(II).

Conclusions
From the results of this study, it was concluded that PG induced the formation of 8-OHdG, as an indicator of oxidative DNA damage, in calf thymus DNA and 2'deoxyguanosine (dG) in the presence of CuCl 2 . PG in the presence of CuCl 2 increased the 8-OHdG formation in calf thymus DNA by around 9.17 times as compared to the control (untreated DNA). On the other hand, BHT-quinone (BHT metabolite) at high concentration, in the presence of CuCl 2 , had a slight effect on 8-OHdG formation. Further in vivo studies on the formation of 8-OHdG induced by PG and BHT-quinone in the presence of CuCl 2 are required to observe additional details as to whether 8-OHdG formation is dose-dependent and to consider the dynamic process of repair and mutagenicity of the adduct target that can ultimately lead to cancer formation.