Clin Oral Invest DOI 10.1007/s00784-013-1178-x
SHORT COMMUNICATION
Oxidative stress is responsible for genotoxicity of camphorquinone in primary human gingival fibroblasts Miriam Wessels & Gabriele Leyhausen & Joachim Volk & Werner Geurtsen
Received: 29 July 2013 / Accepted: 26 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract Objectives The photoinitiator camphorquinone (CQ), used in dental restorative materials, was found to be cytotoxic in cell cultures. Previously, we have shown that CQ induces alkali labile sites and DNA strand breaks in human gingival fibroblasts (HGF) associated with an increase of intracellular reactive oxygen species (ROS). Therefore, the objective of our study was to evaluate if DNA damage in HGF cells is caused by the generation of ROS. Material and methods HGF cells were treated with different concentrations (0.5–2.5 mM) of CQ. The cell viability was assessed using propidium iodide (PI) assay. Oxidative DNA damage was evaluated by an enzyme-modified comet assay using human 8-hydroxyguanine DNA-glycosylase 1 (hOGG1), which converts oxidized 7,8-dihydro-8oxoguanine (8-oxoguanine) into DNA strand breaks and functions as a marker for oxidative modified DNA. Results The results showed that CQ induced DNA damage in HGF cells without cytotoxic effects for the chosen treatment time. CQ treatment led to the generation of 8-oxoguanine in DNA, which can be shown by a significant increase in tail moment after CQ treatment by the enzyme-modified comet assay. Conclusion It may be concluded that DNA damage due to CQ is caused by oxidative stress in gingival fibroblasts. Clinical relevance A more detailed insight into genotoxic mechanisms in oral cells can be of great importance for a better understanding of the biocompatibility of CQ. Keywords Camphorquinone . Reactive oxygen species . Oxidative DNA damage . Comet assay M. Wessels (*) : G. Leyhausen : J. Volk : W. Geurtsen Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, 30625 Hannover, Germany e-mail: [email protected]
Introduction Camphorquinone (CQ) is the most important photoinitiator used in dental adhesives and resin composites [1]. It has an αdicarbonyl site, which absorbs light at a wavelength of 468 nm. Visible light irradiation of CQ triggers the formation of free radicals and initiates an efficient polymerization of (co)monomers [2–4]. Since CQ is not incorporated in the polymer network during and after polymerization, it may leach into the oral cavity [5, 6]. Calculations according to Noda et al. [7] resulted in CQ concentration up to 14 mM that can potentially leach into the oral cavity, considering CQ concentrations in light-curing dental resins of approximately 0.2–1.0 % (w/w). Taira et al. [8] found CQ levels ranging between 0.06 and nearly 0.1 % (w/w) in eluates of various composite resins which are equivalent to concentrations of 3– 5 mM. Subsequently, it may interact with metabolic pathways of oral cells. Many resin composites are found to be cytotoxic in cell cultures. CQ was also identified as a cytotoxin [5]. However, the basic mechanisms are not yet fully investigated. It has been shown that CQ caused elevated levels of reactive oxygen species (ROS) in various oral cell lines [9–12]. ROS can cause cellular damage and cell lysis in oral cells (reviewed in [13]). ROS are also known to cause alterations in nucleic acids, e.g., base modifications, base lesions, and strand breaks [14, 15]. These alterations are associated with numerous diseases (e.g., cancer and inflammation) and might also play a potential role in periodontal diseases [13]. DNA damage was also investigated in conjunction with CQ treatment. Pagoria et al. [16] reported single- and double-strand breaks in plasmid DNA in a cell-free system, whereas Heil et al. [17] showed CQ-induced genotoxic and mutagenic effects in prokaryotic cells using the umu test. Li et al. [18] demonstrated the formation of micronuclei in CHO cells exposed to CQ. Recently, we found that CQ induces alkali labile sites and
Clin Oral Invest
DNA strand breaks in human gingival fibroblasts (HGF) connected with a rapid increase of intracellular ROS formation [12]. Therefore, objective of the present study was to evaluate whether the genotoxic potential of CQ was caused by intracellular ROS using the comet assay. For the investigation of oxidative DNA damage, an enzyme-modified alkaline comet assay was performed. In this assay, human 8-hydroxyguanine DNA-glycosylase 1 (hOGG1) is used. This enzyme converts 7,8-dihydro-8-oxoguanine (8-oxoguanine), a marker of oxidative damage to DNA [19], into DNA strand breaks [20].
Materials and methods Cell cultures Primary HGF were cultured from biopsies of the healthy gingiva of a permanent molar. Informed consent was obtained from the tissue donor according to the guidelines of the Institutional Review Board. Outgrown cells were cultured in Dulbecco’s modified Eagle’s medium with 4.5 g/L glucose, 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, 3.7 g/L NaHCO3, 100 U/mL penicillin, and 100 mg/ mL streptomycin (all from Biochrom KG, Berlin, Germany), supplemented with 10 % fetal calf serum (FCS, Lonza, Verviers, Belgium) at 37 °C and 10 % CO2 in a humidified atmosphere. Confluent monolayers were detached by trypsin/ ethylenediaminetetraacetic acid (EDTA) (0.25 % trypsin, 0.02 % EDTA) and seeded in new culture flasks. Cell viability (95–98 %) was always analyzed before plating for experiments using trypan blue dye (Sigma, Taufkirchen, Germany) exclusion test. All cultures were routinely tested for mycoplasma contamination by means of the mycoplasma detection kit Venor GeM (Minerva Biolabs, Berlin, Germany).
Treatment of cells with CQ HGF cells from passages 5–10 were precultured for 24 h followed by treatment with different CQ concentrations (0.5–2.5 mM) in the dark. Stock solutions of CQ (VOCO, Cuxhaven, Germany) were prepared in ethanol (Baker, Taufkirchen, Germany) and were freshly diluted in DMEM prior to each experiment. The final concentration of ethanol did not exceed 0.5 % to avoid cytotoxic effects. The complete preparation of the used CQ solutions was done under dim green safe light, and the cells were treated under dim room light to avoid the photoactivation of CQ. Cells incubated with DMEM containing 0.5 % ethanol and cells incubated with fresh DMEM alone served as solvent control (c1) and negative control (c2).
Propidium iodide assay Cells were seeded in 96-well plates (1×104 cells/well) for 24 h and then treated for 3 h with CQ. After treatment, 55 μM propidium iodide (Sigma, Taufkirchen, Germany) was added and incubated for 20 min in the dark at room temperature. Fluorescence (FPI) was read in a fluorescence reader FLx 800 (BioTek, Bad Friedrichshall, Germany) at an excitation wavelength (Ex) of 530 nm and an emission wavelength (Em) of 645 nm. Background measurements (blank) were obtained from cell-free wells containing media and PI. This procedure was followed by addition of the surfactant Nonident P-40 (Fluka, Seelze, Germany) for 20 min at room temperature in the dark to lyse all vital cells. The fluorescence measurements were repeated at the same wavelengths to obtain Fmax, a function of total cell number (PI stains only non-vital cells [21]). Percentage of viability was calculated as 100−(FPI − blank/FPI(max) −blank)×100, where FPI is the measured PI fluorescence. Comet assay DNA damage and oxidative DNA lesions were detected using the alkaline comet assay and an enzyme-modified alkaline comet assay. The alkaline (pH 13) comet assay detects single- and double-strand breaks, cross-links, incomplete excision repair sites, as well as apurinic or apyrimidinic sites, which are alkali labile and therefore appear as strand breaks under the alkaline conditions of the assay [22]. The assay was conducted according to Tice et al. [23] with minor modifications. HGF cells were grown in six-well plates (1.5×105 cells/ well) for 24 h and then treated with CQ (0.5–2.5 mM) or 0.5 μL ml−1 ethyl methanesulphonate (EMS, positive control, c3; Sigma, Taufkirchen, Germany) for 3 h. After treatment, cells were detached from the culture plate by a brief trypsin/ EDTA treatment. Trypsin activity was stopped by adding 50 μL Trypsin neutralizing solution (PromoCell, Heidelberg, Germany) and 800 μL FCS-containing DMEM media. Cells were re-suspended and transferred to reaction cups. The following steps were conducted under red light to avoid additional unspecific DNA damage. Cell suspensions were centrifuged, and supernatants were removed. Cells were again resuspended in 80 μL of 0.75 % (w/v) pre-heated low melting agarose (LMA, Sigma, Taufkirchen, Germany) and transferred to fully frosted slides (Menzel-Gläser, Braunschweig, Germany), which were pre-coated with 0.5 % (w/v) normal melting agarose (Sigma, Taufkirchen, Germany) and chilled at 4 °C. Afterwards, one additional layer of 100 μL of 0.75 % LMA per gel was applied. A coverslip was used to flatten out each agarose layer. After gelation, the coverslips were removed and slides were incubated in lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, 8 g L−1 NaOH (all Roth, Karlsruhe, Germany), 1 % Triton-X100, 10 %
Clin Oral Invest
Discussion Recently, we have reported that the photoinitiator CQ caused DNA damage in primary human gingival fibroblasts using the alkaline comet assay [12]. In this study, we further examined this DNA damage in HGF cells using an enzyme-modified comet assay, measuring the generation of 8-oxoguanine. We showed that CQ-induced genotoxicity is caused at least in part by oxidative DNA damage. Human gingival fibroblasts are one of the first target cells in the oral cavity for dental materials. CQ is not incorporated into the polymer network, and several studies have shown that 150
100
50
total cells viable cells
Cytotoxicity was determined after a 3 h treatment period of HGF with 0.5 to 2.5 mM CQ using the PI assay. No cytotoxicity was observable at this time point for all concentrations used. Results are expressed as total and viable cells in
5 2.
m M 25
m M
C
C
Q
Q C m M 5
0.
Cell viability
Q
0 c2
Results
The genotoxic potency of CQ was determined by the enzymemodified alkaline comet assay showing an increase of tail moment for all CQ concentrations used (Fig. 2). Using the Bonferroni posttest, only the cells treated with 2.5 mM CQ and hOOG1 enzyme showed a significant increase of the tail moment (ANOVA, Bonferroni posttest p 0.01). By testing differences within the same concentration with and without enzyme treatment, a significant increase of tail moment in enzyme-treated cells for all CQ concentrations can be shown. We observed an elevation of tail moment from 1.17±0.63 to 2.15±0.35 for the lowest concentration (p=0.025), 1.45±0.34 to 3.15±0.37 for 1.25 mM CQ (p=0.0024), and 3.05±0.37 to 5.93±0.43 for the highest concentration 2.5 mM CQ (p= 0.0009).
1.
Experiments were run at least three times with each six replicates (PI assay) or two replicates (comet assay). Data were expressed as mean±SD. Statistical analysis of the comet assay was performed by one-way ANOVA followed by a Bonferroni posttest for comparison of CQ-treated cells with the controls. For differences within the same concentration with and without enzyme treatment, paired t test was applied; p values13). DNA was allowed to unwind for 20 min followed by electrophoresis for 20 min at 24 V/ 300 mA. Slides were then neutralized by rinsing with neutralizing buffer (0.4 M Tris–HCl, pH 7.4) and stained with 80 μL ethidium bromide solution (20 μg mL−1, Merck, Darmstadt, Germany). Slides were analyzed using a fluorescence microscope (Axioskop, Zeiss, Göttingen, Germany) and the Comet III software (Perceptive Instruments, Haverhill, UK). One hundred cells were scored per slide, and tail moment was used for data analysis. Tail moment was defined by the percentage of DNA in the tail multiplied by the length between the center of the head and tail. The enzyme-modified comet assay was done according to Smith et al. [20]. It is based on the alkaline comet assay, but after cell lysis, gels were rinsed with enzyme buffer (40 mM HEPES (Biochrom, Berlin, Germany), 100 mM KCl (Merck, Darmstadt, Germany), 0.5 mM Na2 EDTA, 0.2 mg mL− 1 bovine serum albumin (Sigma, Taufkirchen, Germany), pH 8) at room temperature followed by incubation with 0.32 U/gel of hOGG1 (New England Biolabs, Frankfurt am Main, Germany) at 37 °C. For incubation, gels were covered with 100 μL of either buffer or hOGG1 in buffer and placed in quadriPERM dishes (Greiner Bio-One, Frickenhausen, Germany). This step was followed by DNA unwinding, electrophoresis, and staining, which is processed the same way as described in the alkaline comet protocol. Potassium bromate (KBrO3, 1 mM) was used as a positive control of the hOGG1-modified alkaline comet assay.
treatment
Fig. 1 Cytotoxicity of CQ in HGF. Relative number of viable and total cells (% control) as determined by the PI assay after incubating HGF for 3 h with CQ (0.5–2.5 mM). Values are mean±SD, c1 solvent control (0.5 % ethanol), c2 growth medium control (negative control)
Clin Oral Invest 40
a,b
30
tail moment
20 a,b
6
4
b b
2
CQ m
2. 5
M
M
CQ
CQ m 1. 25
0. 5
m
M
c4
c3
c1
c2
0
Fig. 2 Genotoxicity of CQ in HGF. HGF were treated for 3 h with CQ (0.5–2.5 mM) in the dark. Tail moment was used as quantitative measure for DNA damage. White bars represent DNA damage detected by alkaline comet assay. Gray bars show the effect of hOGG1. Data represent mean±SD, c1 solvent control (0.5 % ethanol), c2 growth medium control, c3 0.5 μL mL−1 ethyl methanesulfonate (EMS, negative control for oxidative DNA damage), c4 0.5 mM potassium bromate (KBrO3, positive control for oxidative DNA damage). Letters above bars represent significant differences (p
SHORT COMMUNICATION
Oxidative stress is responsible for genotoxicity of camphorquinone in primary human gingival fibroblasts Miriam Wessels & Gabriele Leyhausen & Joachim Volk & Werner Geurtsen
Received: 29 July 2013 / Accepted: 26 December 2013 # Springer-Verlag Berlin Heidelberg 2014
Abstract Objectives The photoinitiator camphorquinone (CQ), used in dental restorative materials, was found to be cytotoxic in cell cultures. Previously, we have shown that CQ induces alkali labile sites and DNA strand breaks in human gingival fibroblasts (HGF) associated with an increase of intracellular reactive oxygen species (ROS). Therefore, the objective of our study was to evaluate if DNA damage in HGF cells is caused by the generation of ROS. Material and methods HGF cells were treated with different concentrations (0.5–2.5 mM) of CQ. The cell viability was assessed using propidium iodide (PI) assay. Oxidative DNA damage was evaluated by an enzyme-modified comet assay using human 8-hydroxyguanine DNA-glycosylase 1 (hOGG1), which converts oxidized 7,8-dihydro-8oxoguanine (8-oxoguanine) into DNA strand breaks and functions as a marker for oxidative modified DNA. Results The results showed that CQ induced DNA damage in HGF cells without cytotoxic effects for the chosen treatment time. CQ treatment led to the generation of 8-oxoguanine in DNA, which can be shown by a significant increase in tail moment after CQ treatment by the enzyme-modified comet assay. Conclusion It may be concluded that DNA damage due to CQ is caused by oxidative stress in gingival fibroblasts. Clinical relevance A more detailed insight into genotoxic mechanisms in oral cells can be of great importance for a better understanding of the biocompatibility of CQ. Keywords Camphorquinone . Reactive oxygen species . Oxidative DNA damage . Comet assay M. Wessels (*) : G. Leyhausen : J. Volk : W. Geurtsen Department of Conservative Dentistry, Periodontology and Preventive Dentistry, Hannover Medical School, 30625 Hannover, Germany e-mail: [email protected]
Introduction Camphorquinone (CQ) is the most important photoinitiator used in dental adhesives and resin composites [1]. It has an αdicarbonyl site, which absorbs light at a wavelength of 468 nm. Visible light irradiation of CQ triggers the formation of free radicals and initiates an efficient polymerization of (co)monomers [2–4]. Since CQ is not incorporated in the polymer network during and after polymerization, it may leach into the oral cavity [5, 6]. Calculations according to Noda et al. [7] resulted in CQ concentration up to 14 mM that can potentially leach into the oral cavity, considering CQ concentrations in light-curing dental resins of approximately 0.2–1.0 % (w/w). Taira et al. [8] found CQ levels ranging between 0.06 and nearly 0.1 % (w/w) in eluates of various composite resins which are equivalent to concentrations of 3– 5 mM. Subsequently, it may interact with metabolic pathways of oral cells. Many resin composites are found to be cytotoxic in cell cultures. CQ was also identified as a cytotoxin [5]. However, the basic mechanisms are not yet fully investigated. It has been shown that CQ caused elevated levels of reactive oxygen species (ROS) in various oral cell lines [9–12]. ROS can cause cellular damage and cell lysis in oral cells (reviewed in [13]). ROS are also known to cause alterations in nucleic acids, e.g., base modifications, base lesions, and strand breaks [14, 15]. These alterations are associated with numerous diseases (e.g., cancer and inflammation) and might also play a potential role in periodontal diseases [13]. DNA damage was also investigated in conjunction with CQ treatment. Pagoria et al. [16] reported single- and double-strand breaks in plasmid DNA in a cell-free system, whereas Heil et al. [17] showed CQ-induced genotoxic and mutagenic effects in prokaryotic cells using the umu test. Li et al. [18] demonstrated the formation of micronuclei in CHO cells exposed to CQ. Recently, we found that CQ induces alkali labile sites and
Clin Oral Invest
DNA strand breaks in human gingival fibroblasts (HGF) connected with a rapid increase of intracellular ROS formation [12]. Therefore, objective of the present study was to evaluate whether the genotoxic potential of CQ was caused by intracellular ROS using the comet assay. For the investigation of oxidative DNA damage, an enzyme-modified alkaline comet assay was performed. In this assay, human 8-hydroxyguanine DNA-glycosylase 1 (hOGG1) is used. This enzyme converts 7,8-dihydro-8-oxoguanine (8-oxoguanine), a marker of oxidative damage to DNA [19], into DNA strand breaks [20].
Materials and methods Cell cultures Primary HGF were cultured from biopsies of the healthy gingiva of a permanent molar. Informed consent was obtained from the tissue donor according to the guidelines of the Institutional Review Board. Outgrown cells were cultured in Dulbecco’s modified Eagle’s medium with 4.5 g/L glucose, 10 mM 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, 3.7 g/L NaHCO3, 100 U/mL penicillin, and 100 mg/ mL streptomycin (all from Biochrom KG, Berlin, Germany), supplemented with 10 % fetal calf serum (FCS, Lonza, Verviers, Belgium) at 37 °C and 10 % CO2 in a humidified atmosphere. Confluent monolayers were detached by trypsin/ ethylenediaminetetraacetic acid (EDTA) (0.25 % trypsin, 0.02 % EDTA) and seeded in new culture flasks. Cell viability (95–98 %) was always analyzed before plating for experiments using trypan blue dye (Sigma, Taufkirchen, Germany) exclusion test. All cultures were routinely tested for mycoplasma contamination by means of the mycoplasma detection kit Venor GeM (Minerva Biolabs, Berlin, Germany).
Treatment of cells with CQ HGF cells from passages 5–10 were precultured for 24 h followed by treatment with different CQ concentrations (0.5–2.5 mM) in the dark. Stock solutions of CQ (VOCO, Cuxhaven, Germany) were prepared in ethanol (Baker, Taufkirchen, Germany) and were freshly diluted in DMEM prior to each experiment. The final concentration of ethanol did not exceed 0.5 % to avoid cytotoxic effects. The complete preparation of the used CQ solutions was done under dim green safe light, and the cells were treated under dim room light to avoid the photoactivation of CQ. Cells incubated with DMEM containing 0.5 % ethanol and cells incubated with fresh DMEM alone served as solvent control (c1) and negative control (c2).
Propidium iodide assay Cells were seeded in 96-well plates (1×104 cells/well) for 24 h and then treated for 3 h with CQ. After treatment, 55 μM propidium iodide (Sigma, Taufkirchen, Germany) was added and incubated for 20 min in the dark at room temperature. Fluorescence (FPI) was read in a fluorescence reader FLx 800 (BioTek, Bad Friedrichshall, Germany) at an excitation wavelength (Ex) of 530 nm and an emission wavelength (Em) of 645 nm. Background measurements (blank) were obtained from cell-free wells containing media and PI. This procedure was followed by addition of the surfactant Nonident P-40 (Fluka, Seelze, Germany) for 20 min at room temperature in the dark to lyse all vital cells. The fluorescence measurements were repeated at the same wavelengths to obtain Fmax, a function of total cell number (PI stains only non-vital cells [21]). Percentage of viability was calculated as 100−(FPI − blank/FPI(max) −blank)×100, where FPI is the measured PI fluorescence. Comet assay DNA damage and oxidative DNA lesions were detected using the alkaline comet assay and an enzyme-modified alkaline comet assay. The alkaline (pH 13) comet assay detects single- and double-strand breaks, cross-links, incomplete excision repair sites, as well as apurinic or apyrimidinic sites, which are alkali labile and therefore appear as strand breaks under the alkaline conditions of the assay [22]. The assay was conducted according to Tice et al. [23] with minor modifications. HGF cells were grown in six-well plates (1.5×105 cells/ well) for 24 h and then treated with CQ (0.5–2.5 mM) or 0.5 μL ml−1 ethyl methanesulphonate (EMS, positive control, c3; Sigma, Taufkirchen, Germany) for 3 h. After treatment, cells were detached from the culture plate by a brief trypsin/ EDTA treatment. Trypsin activity was stopped by adding 50 μL Trypsin neutralizing solution (PromoCell, Heidelberg, Germany) and 800 μL FCS-containing DMEM media. Cells were re-suspended and transferred to reaction cups. The following steps were conducted under red light to avoid additional unspecific DNA damage. Cell suspensions were centrifuged, and supernatants were removed. Cells were again resuspended in 80 μL of 0.75 % (w/v) pre-heated low melting agarose (LMA, Sigma, Taufkirchen, Germany) and transferred to fully frosted slides (Menzel-Gläser, Braunschweig, Germany), which were pre-coated with 0.5 % (w/v) normal melting agarose (Sigma, Taufkirchen, Germany) and chilled at 4 °C. Afterwards, one additional layer of 100 μL of 0.75 % LMA per gel was applied. A coverslip was used to flatten out each agarose layer. After gelation, the coverslips were removed and slides were incubated in lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, 8 g L−1 NaOH (all Roth, Karlsruhe, Germany), 1 % Triton-X100, 10 %
Clin Oral Invest
Discussion Recently, we have reported that the photoinitiator CQ caused DNA damage in primary human gingival fibroblasts using the alkaline comet assay [12]. In this study, we further examined this DNA damage in HGF cells using an enzyme-modified comet assay, measuring the generation of 8-oxoguanine. We showed that CQ-induced genotoxicity is caused at least in part by oxidative DNA damage. Human gingival fibroblasts are one of the first target cells in the oral cavity for dental materials. CQ is not incorporated into the polymer network, and several studies have shown that 150
100
50
total cells viable cells
Cytotoxicity was determined after a 3 h treatment period of HGF with 0.5 to 2.5 mM CQ using the PI assay. No cytotoxicity was observable at this time point for all concentrations used. Results are expressed as total and viable cells in
5 2.
m M 25
m M
C
C
Q
Q C m M 5
0.
Cell viability
Q
0 c2
Results
The genotoxic potency of CQ was determined by the enzymemodified alkaline comet assay showing an increase of tail moment for all CQ concentrations used (Fig. 2). Using the Bonferroni posttest, only the cells treated with 2.5 mM CQ and hOOG1 enzyme showed a significant increase of the tail moment (ANOVA, Bonferroni posttest p 0.01). By testing differences within the same concentration with and without enzyme treatment, a significant increase of tail moment in enzyme-treated cells for all CQ concentrations can be shown. We observed an elevation of tail moment from 1.17±0.63 to 2.15±0.35 for the lowest concentration (p=0.025), 1.45±0.34 to 3.15±0.37 for 1.25 mM CQ (p=0.0024), and 3.05±0.37 to 5.93±0.43 for the highest concentration 2.5 mM CQ (p= 0.0009).
1.
Experiments were run at least three times with each six replicates (PI assay) or two replicates (comet assay). Data were expressed as mean±SD. Statistical analysis of the comet assay was performed by one-way ANOVA followed by a Bonferroni posttest for comparison of CQ-treated cells with the controls. For differences within the same concentration with and without enzyme treatment, paired t test was applied; p values13). DNA was allowed to unwind for 20 min followed by electrophoresis for 20 min at 24 V/ 300 mA. Slides were then neutralized by rinsing with neutralizing buffer (0.4 M Tris–HCl, pH 7.4) and stained with 80 μL ethidium bromide solution (20 μg mL−1, Merck, Darmstadt, Germany). Slides were analyzed using a fluorescence microscope (Axioskop, Zeiss, Göttingen, Germany) and the Comet III software (Perceptive Instruments, Haverhill, UK). One hundred cells were scored per slide, and tail moment was used for data analysis. Tail moment was defined by the percentage of DNA in the tail multiplied by the length between the center of the head and tail. The enzyme-modified comet assay was done according to Smith et al. [20]. It is based on the alkaline comet assay, but after cell lysis, gels were rinsed with enzyme buffer (40 mM HEPES (Biochrom, Berlin, Germany), 100 mM KCl (Merck, Darmstadt, Germany), 0.5 mM Na2 EDTA, 0.2 mg mL− 1 bovine serum albumin (Sigma, Taufkirchen, Germany), pH 8) at room temperature followed by incubation with 0.32 U/gel of hOGG1 (New England Biolabs, Frankfurt am Main, Germany) at 37 °C. For incubation, gels were covered with 100 μL of either buffer or hOGG1 in buffer and placed in quadriPERM dishes (Greiner Bio-One, Frickenhausen, Germany). This step was followed by DNA unwinding, electrophoresis, and staining, which is processed the same way as described in the alkaline comet protocol. Potassium bromate (KBrO3, 1 mM) was used as a positive control of the hOGG1-modified alkaline comet assay.
treatment
Fig. 1 Cytotoxicity of CQ in HGF. Relative number of viable and total cells (% control) as determined by the PI assay after incubating HGF for 3 h with CQ (0.5–2.5 mM). Values are mean±SD, c1 solvent control (0.5 % ethanol), c2 growth medium control (negative control)
Clin Oral Invest 40
a,b
30
tail moment
20 a,b
6
4
b b
2
CQ m
2. 5
M
M
CQ
CQ m 1. 25
0. 5
m
M
c4
c3
c1
c2
0
Fig. 2 Genotoxicity of CQ in HGF. HGF were treated for 3 h with CQ (0.5–2.5 mM) in the dark. Tail moment was used as quantitative measure for DNA damage. White bars represent DNA damage detected by alkaline comet assay. Gray bars show the effect of hOGG1. Data represent mean±SD, c1 solvent control (0.5 % ethanol), c2 growth medium control, c3 0.5 μL mL−1 ethyl methanesulfonate (EMS, negative control for oxidative DNA damage), c4 0.5 mM potassium bromate (KBrO3, positive control for oxidative DNA damage). Letters above bars represent significant differences (p
