Genotoxic effects of camphorquinone and DMT on human oral and intestinal cells

Highlights

  • Camphorquinone (CQ) induced DNA lesions in OKF6/TERT2 and Caco-2 cells.

  • This DNA damage is mainly caused by the generation of 8-oxoguanine.

  • The antioxidant glutathione prevented CQ-associated DNA damage in Caco-2 cells.

  • Recovery following CQ-treatment significantly reduced DNA damage in Caco-2 cells.

Abstract

Objective

Released components of oral biomaterials can leach into the oral cavity and may subsequently reach the gastrointestinal tract. Camphorquinone (CQ) is the most common used photoinitiator in resinous restorative materials and is often combined with the co-initiator N , N -dimethyl- p -toluidine (DMT). It has been shown that CQ exerts cytotoxic effects, at least partially due to the generation of reactive oxygen species (ROS). Objective of this study was to examine the cytotoxic and genotoxic potential of CQ in human oral keratinocytes (OKF6/TERT2) and immortalized epithelial colorectal adenocarcinoma cells (Caco-2). Furthermore, the effects of visible-light irradiation and the co-initiator DMT were investigated as well as the generation of ROS, the potential protective effect of glutathione (GSH) and a recovery period of CQ-treated Caco-2 cells.

Methods

The alkaline comet assay was used to determine DNA damage. Additionally, an enzyme modified comet assay was applied, which detects 7,8-dihydro-8-oxoguanine (8-oxoguanine), a reliable marker for oxidative stress.

Results

Our data revealed that high concentrations of CQ induced DNA lesions in OKF6/TERT2 cells. This DNA damage is at least partly caused by the generation of 8-oxoguanine. In addition, CQ and DMT increased ROS formation and induced DNA damage in Caco-2 cells. CQ-treatment resulted in generation of 8-oxoguanine. The antioxidant GSH efficiently prevented CQ-associated DNA damage. Furthermore, a recovery following CQ-treatment significantly reduced DNA damage.

Significance

We conclude that CQ-induced DNA damage is caused by oxidative stress in oral and intestinal cells. These lesions can be prevented and possibly repaired by GSH-treatment and recovery of cells after the photoinitiator is removed from cultures.

Introduction

High amounts of various organic compounds may leach from resinous restorative materials in the first days after application. Subsequently lower quantities of these components are continuously released into the oral cavity due to degradation or erosion over time . Leached substances, which are diluted by saliva, will end up in the gastrointestinal tract . To date, no data is available in dental/medical literature about interactions of resinous compounds with intestinal cells. Camphorquinone (CQ) is the most important photoinitiator in dental composites which is always combined with a co-initiator, such as N , N -dimethyl- p -toluidine (DMT) . It has been calculated that CQ-concentrations up to 14 mM can potentially leach from light-curing resinous filling material . Low concentrations of isolated CQ as well as in combination with DMT cause cytotoxic effects in cell cultures and generate reactive oxygen species (ROS). It was documented that irradiated CQ and other photoinitiators can significantly increase ROS concentrations in various human cell types, which is associated with an enhanced toxicity . However, a marked increase of ROS was also caused by CQ without light irradiation . Intracellularly, elevated levels of ROS may result in alterations or damage of lipids, proteins and nucleic acids, which finally will cause cellular dysfunction . Cells respond to oxidative stress with redox-regulating molecules and antioxidants such as glutathione (GSH) and enzymes like glutathione peroxidase, glutathione reductase, catalase, and superoxide dismutase . It was found that antioxidants like GSH, ascorbic acid, and N -acetyl- l -cysteine (NAC) reduce high ROS concentrations, which were generated by CQ and other initiators .

It has been documented that CQ is also genotoxic. CQ was mutagenic in the bacterial umu test, and genotoxic in the DNA synthesis inhibition test at concentrations of 5 to 20 mM . Single and double strand breaks in plasmid DNA were observed as well as the formation of micronuclei in chinese hamster ovarian (CHO) cells exposed to CQ . Nomura et al. investigated genotoxicity using a bacterial assay. However, their results were inconclusive due to cytotoxic effects. Recently, Volk et al. showed genotoxicity associated with elevated ROS levels for CQ concentrations up to 2.5 mM in human gingival fibroblasts using the comet assay.

The complex mechanisms of the CQ-induced genotoxicity are not yet fully understood. Therefore, it was objective of our study to analyze the effects of irradiated and non-irradiated CQ on human oral keratinocytes (OKF6/TERT2), which are one of the first target cells of released resinous substances, and immortalized epithelial colorectal adenocarcinoma cells (Caco-2). Goal of our experiments was to find out, whether CQ causes DNA strand breaks and oxidative DNA modifications due to elevated ROS levels in keratinocytes . The hypothesis, which we set forth, was that non-irradiated CQ without and with DMT significantly elevate ROS levels and is genotoxic in cultures of immortalized human adenocarcinoma cells (Caco-2). Furthermore, we hypothesized that antioxidant GSH will prevent or reduce these effects.

The alkaline comet assay was used to determine DNA strand breaks and alkali labile sites. This method prove DNA damage, detecting 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 breaks under the alkaline conditions of the assay . The hOGG1-modified comet assay, which reveals oxidative DNA modifications and is an indicator of oxidative stress , was applied to analyze the formation of oxidized 7,8-dihydro-8-oxoguanine (8-oxoguanine) .

Materials and methods

Cell cultures

The immortalized human oral keratinocyte cell line OKF6/TERT2 was provided by Dr. J. Rheinwald (Harvard University). The keratinocytes were immortalized by transfection to express hTERT, the telomerase catalytic subunit, yet retain normal growth and differentiation characteristics . OKF6/TERT2 cells were cultured in accordance with the protocols described by Dickson et al. in a keratinocyte serum-free medium (ker-sfm no. 17005-042) containing 25 μg/mL bovine pituitary extract (BPE), 0.2 ng/mL epidermal growth factor (EGF) (all from GIBCO/Invitrogen, Darmstadt, Germany), 0.4 mM CaCl 2 , and penicillin (100 U/mL)/streptomycin (100 mg/mL) (all from Biochrom KG, Berlin, Germany). For passaging, a 0.125% trypsin/0.01% EDTA solution (Sigma, Deisenhofen, Germany) in PBS and Dulbecco’s modified Eagle medium/F-12 medium (DMEM/F-12, Biochrom KG, Berlin, Germany) including 10% fetal bovine serum (FBS, Lonza, Verviers, Belgium) was used. Exponentially growing cultures (5–8 d old) were used as source of cells for the next passage. For both experimental and control groups, cells were grown in a medium containing higher concentrations of nutrients (DF-K medium), mixed (1:1) of GIBCO ker-sfm and a DMEM/F-12 medium containing calcium-free, glutamine-free DMEM (#21068-028) with Ham’s F-12 supplemented with 0.2 ng/mL EGF, 25 μg/mL BPE (#11765-054, all GIBCO/Invitrogen, Darmstadt, Germany), 1.5 mM/L glutamine, 2.5 μg/mL amphotericin and penicillin (100 U/mL)/streptomycin (100 mg/mL) (all from Biochrom KG, Berlin, Germany).

Caco-2 cells (clone C2BBe1), provided by Prof. Dr. U. Seidler (Hannover Medical School), were cultured in accordance with the protocols obtained from ATCC (American Type Culture Collection, Rockville, MD, USA). The colon carcinoma cell line was used as an in vitro model of the intestinal epithelium. For the experiments Caco-2 cells were cultured in Advanced MEM (Gibco BRL, Life Technologies, Eggenstein, Germany) containing 2 mM/L glutamine, 2.5 μg/mL amphotericin, penicillin (100 U/mL)/streptomycin (100 mg/mL), supplemented with 10% FBS. Cells were passaged by a short treatment with 0.25% trypsin/0.02% EDTA at regular intervals.

Both cell lines were maintained as monolayer cultures at 37 °C in a humidified atmosphere of 5% CO 2 . Cell viability (95–98%) was estimated by using trypan blue dye and the TC10 automated cell counter before plating for experiments (all Bio-Rad Laboratories, Hercules, CA, USA). 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 dental materials and the antioxidant GSH

OKF6/TERT2 cells were pre-cultured for 24 h followed by a treatment with different CQ concentrations (2.5–5 mM) in the dark for 6 h. For visible light (VL-) activation of CQ, cells were irradiated for 20 s directly after addition of CQ using a dental curing light (800 mW/cm 2 ; Elipar II, Espe, Seefeld, Germany).

Caco-2 cells were allowed to grow for 24 h and were subsequently treated with CQ (1 mM and 2.5 mM), DMT (2.5 mM) or a combination of both substances (1 mM CQ + 2.5 mM DMT) for 6 h. We selected this combination, because of the documented mixing ratio of CQ: DMT in resin materials . Furthermore, this concentration generated an amount of ROS, which is comparable to 2.5 mM CQ alone . In an additional experiment, the treatment solutions containing the compounds were removed after 6 h and substituted by fresh growth medium. After a recovery time of 18 h, cells were used for further experiments. For antioxidant treatment, Caco-2 cells were exposed to 2.5 mM CQ combined with GSH (2.5 mM or 5 mM) for 6 h.

Stock solutions of CQ (VOCO, Cuxhaven, Germany) and DMT (Merck, Darmstadt, Germany) were prepared in ethanol (Baker, Taufkirchen, Germany) and were freshly diluted in medium prior to each experiment. The final concentration of ethanol did not exceed 0.25% in order to avoid toxic effects due to the solvent. Cells incubated with medium containing 0.25% ethanol and cells grown in medium alone served as solvent control (c1) and negative control (c2). The preparation of the CQ solutions and the treatment of cells were performed under dimmed room light to avoid the photoactivation of CQ.

Reactive oxygen species (ROS)

ROS-formation in Caco-2 cells was evaluated using the oxidation-sensitive dye 2,7-dichlorofluorescin diacetate (DCFH-DA, Sigma, Deisenhofen, Germany). Cells were seeded in 96-well plates at a density of 2 × 10 4 cells/well. After growing for 48 h, cells were loaded with DCFH-DA in Advance MEM for 20 min in the dark and then washed with Hanks’ salt solution (HBSS, Biochrom KG, Berlin, Germany). Cells were treated with CQ (0.5–2.5 mM), DMT (1–2.5 mM), or a combination of 1.0 mM CQ with DMT (1.0 mM, 2.5 mM). Fluorescence was read in the fluorescence reader FLx 800 (BioTek, Bad Friedrichshall, Germany) for 90 min directly after adding the substances and then every 15 min at Excitation ( Ex )/Emission ( Em ) = 485 nm/528 nm. The maximum rate of fluorescence increase ( v max ) was calculated.

Propidium iodide (PI) assay

The PI assay was used to evaluate cell viability. Cells seeded in 96-well plates (OKF6/TERT2: 2 × 10 4 cells/well, Caco-2: 1 × 10 4 cells/well) were grown for 24 h and then treated with the materials with and without VL-irradiation. After treatment, 55 μM propidium iodide (Sigma, Taufkirchen, Germany) was added and incubated for 20 min in the dark at room temperature. Fluorescence ( F PI ) was read at Ex / Em = 530 nm/645 nm. Background measurements (blank) were obtained from cell-free wells containing PBS and PI. Subsequently, the surfactant Nonident P-40 (Fluka, Seelze, Germany) was added for 20 min at room temperature in the dark, to lyse all vital cells. Fluorescence measurements were repeated at the same wavelengths to obtain F max , a function of total cell number (PI is membrane impermeable and stains only non-vital cells ). Percentage of viability was calculated as 100 − ( F PI − blank/ F PI(max) − blank) × 100, where F PI is the measured PI fluorescence.

Comet assay

The alkaline comet assay and an enzyme-modified alkaline comet assay were used to detect DNA damage and oxidative DNA modifications.

The alkaline comet assay was performed according to Tice et al. with minor modifications. Cells were grown in 6-well plates (OKF6/TERT2: 1.5 × 10 5 cells/well, Caco-2: 7 × 10 4 cells/well) for 24 h and then treated with the materials with or without irradiation. Cultures incubated with 0.5 μL/mL ethylmethanesulphonate (EMS) (Sigma, Taufkirchen, Germany) for 1 h served as positive control (c3). After treatment, cells were detached from the culture plate by a brief trypsin/EDTA treatment. Trypsin activity was stopped by adding 800 μL FBS-containing 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 at 54 × g and supernatants were removed. Then, cells were re-suspended in 90 μL of 0.75% (w/v) pre-heated low melting agarose (LMA, peqlab biotechnologies GmbH, Erlangen, Germany) and transferred to fully frosted slides (Menzel-Gläser, Braunschweig, Germany), which were pre-coated with 0.5% (w/v) normal melting agarose (peqlab biotechnologies GmbH, Erlangen, 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 each agarose layer. After gelation, the coverslips were removed and slides were incubated in lysis solution (2.5 M NaCl, 100 mM Na 2 EDTA, 10 mM Tris–HCL, 8 g/L NaOH (all Roth, Karlsruhe, Germany), 1% Triton-X100, 10% DMSO (both Sigma, Taufkirchen, Germany)) over night at 4 °C. After lysis, slides were placed on a cooled electrophoresis platform (peqlab biotechnologies GmbH, Erlangen, Germany) containing pre-cooled electrophoresis buffer (300 mM NaOH, 1 mM Na 2 EDTA, pH > 13). DNA was allowed to unwind for 20 min followed by electrophoresis for 20 min at 30 V/320 mA. Then, slides were 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, Merk, Darmstadt, Germany) or GelRed™ (dilution 1:5000; BioTrend, Cologne, Germany). Slides were analyzed using a fluorescence microscope (BX 60, Olympus, Hamburg, Germany) and the Comet 6.0 software (Andor Technology, Belfast, Northern Ireland). One hundred cells were evaluated per slide and tail moment (TM = (Tail mean − Head mean) × Tail%DNA/100) was used for data analysis. Tail moments are becoming higher equivalent to increasing DNA damage .

The enzyme-modified comet assay was performed according to Smith et al. . It is based on the alkaline comet assay, but after cell lysis, gels were rinsed three times with enzyme buffer: 40 mM HEPES (Biochrom, Berlin, Germany), 100 mM KCl (Merk, Darmstadt, Germany), 0.5 mM Na 2 EDTA, 0.2 mg/mL bovine serum albumin (Sigma, Taufkirchen, Germany), diluted in HPLC water (Roth, Karlsruhe, Germany) (pH 8). Then gels were incubated for 10 min with 0.32 U/gel of human 8-hydroxy-guanine DNA-glycosylase 1 (hOGG1, New England Biolabs, Frankfurt am Main, Germany) at 37 °C. For this, gels were covered with 100 μL of buffer or hOGG1 in buffer and placed in quadriPERM dishes (Greiner Bio-One, Frickenhausen, Germany). This was followed by DNA-unwinding, electrophoresis and staining according to the alkaline comet protocol (see above). Potassium bromate (KBrO 3 , treatment time: 1 h, concentration: 1 mM for OKF6/TERT2, 2 mM for Caco-2, c4) was used as positive control in the hOGG1-modified alkaline comet assay.

Statistical analysis

Experiments were run three times with six replicates each (ROS formation, PI assay) or two replicates (comet assay). Measured fluorescence values were converted into % of untreated control cells (c2 = 100%) in experiments using microtiter plates. Data were expressed as means ± standard deviation (SD). One-way ANOVA followed by a Bonferroni posttest (Prism 5.04, GraphPad Software, La Jolla, USA) were used for statistical analysis of ROS formation, PI readings and comet assays. Statistical differences of the same concentrations with and without enzyme treatment, GSH treatment and additional recovery time, were determined with the paired t -test. P -values < 0.05 were considered significant.

Materials and methods

Cell cultures

The immortalized human oral keratinocyte cell line OKF6/TERT2 was provided by Dr. J. Rheinwald (Harvard University). The keratinocytes were immortalized by transfection to express hTERT, the telomerase catalytic subunit, yet retain normal growth and differentiation characteristics . OKF6/TERT2 cells were cultured in accordance with the protocols described by Dickson et al. in a keratinocyte serum-free medium (ker-sfm no. 17005-042) containing 25 μg/mL bovine pituitary extract (BPE), 0.2 ng/mL epidermal growth factor (EGF) (all from GIBCO/Invitrogen, Darmstadt, Germany), 0.4 mM CaCl 2 , and penicillin (100 U/mL)/streptomycin (100 mg/mL) (all from Biochrom KG, Berlin, Germany). For passaging, a 0.125% trypsin/0.01% EDTA solution (Sigma, Deisenhofen, Germany) in PBS and Dulbecco’s modified Eagle medium/F-12 medium (DMEM/F-12, Biochrom KG, Berlin, Germany) including 10% fetal bovine serum (FBS, Lonza, Verviers, Belgium) was used. Exponentially growing cultures (5–8 d old) were used as source of cells for the next passage. For both experimental and control groups, cells were grown in a medium containing higher concentrations of nutrients (DF-K medium), mixed (1:1) of GIBCO ker-sfm and a DMEM/F-12 medium containing calcium-free, glutamine-free DMEM (#21068-028) with Ham’s F-12 supplemented with 0.2 ng/mL EGF, 25 μg/mL BPE (#11765-054, all GIBCO/Invitrogen, Darmstadt, Germany), 1.5 mM/L glutamine, 2.5 μg/mL amphotericin and penicillin (100 U/mL)/streptomycin (100 mg/mL) (all from Biochrom KG, Berlin, Germany).

Caco-2 cells (clone C2BBe1), provided by Prof. Dr. U. Seidler (Hannover Medical School), were cultured in accordance with the protocols obtained from ATCC (American Type Culture Collection, Rockville, MD, USA). The colon carcinoma cell line was used as an in vitro model of the intestinal epithelium. For the experiments Caco-2 cells were cultured in Advanced MEM (Gibco BRL, Life Technologies, Eggenstein, Germany) containing 2 mM/L glutamine, 2.5 μg/mL amphotericin, penicillin (100 U/mL)/streptomycin (100 mg/mL), supplemented with 10% FBS. Cells were passaged by a short treatment with 0.25% trypsin/0.02% EDTA at regular intervals.

Both cell lines were maintained as monolayer cultures at 37 °C in a humidified atmosphere of 5% CO 2 . Cell viability (95–98%) was estimated by using trypan blue dye and the TC10 automated cell counter before plating for experiments (all Bio-Rad Laboratories, Hercules, CA, USA). 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 dental materials and the antioxidant GSH

OKF6/TERT2 cells were pre-cultured for 24 h followed by a treatment with different CQ concentrations (2.5–5 mM) in the dark for 6 h. For visible light (VL-) activation of CQ, cells were irradiated for 20 s directly after addition of CQ using a dental curing light (800 mW/cm 2 ; Elipar II, Espe, Seefeld, Germany).

Caco-2 cells were allowed to grow for 24 h and were subsequently treated with CQ (1 mM and 2.5 mM), DMT (2.5 mM) or a combination of both substances (1 mM CQ + 2.5 mM DMT) for 6 h. We selected this combination, because of the documented mixing ratio of CQ: DMT in resin materials . Furthermore, this concentration generated an amount of ROS, which is comparable to 2.5 mM CQ alone . In an additional experiment, the treatment solutions containing the compounds were removed after 6 h and substituted by fresh growth medium. After a recovery time of 18 h, cells were used for further experiments. For antioxidant treatment, Caco-2 cells were exposed to 2.5 mM CQ combined with GSH (2.5 mM or 5 mM) for 6 h.

Stock solutions of CQ (VOCO, Cuxhaven, Germany) and DMT (Merck, Darmstadt, Germany) were prepared in ethanol (Baker, Taufkirchen, Germany) and were freshly diluted in medium prior to each experiment. The final concentration of ethanol did not exceed 0.25% in order to avoid toxic effects due to the solvent. Cells incubated with medium containing 0.25% ethanol and cells grown in medium alone served as solvent control (c1) and negative control (c2). The preparation of the CQ solutions and the treatment of cells were performed under dimmed room light to avoid the photoactivation of CQ.

Reactive oxygen species (ROS)

ROS-formation in Caco-2 cells was evaluated using the oxidation-sensitive dye 2,7-dichlorofluorescin diacetate (DCFH-DA, Sigma, Deisenhofen, Germany). Cells were seeded in 96-well plates at a density of 2 × 10 4 cells/well. After growing for 48 h, cells were loaded with DCFH-DA in Advance MEM for 20 min in the dark and then washed with Hanks’ salt solution (HBSS, Biochrom KG, Berlin, Germany). Cells were treated with CQ (0.5–2.5 mM), DMT (1–2.5 mM), or a combination of 1.0 mM CQ with DMT (1.0 mM, 2.5 mM). Fluorescence was read in the fluorescence reader FLx 800 (BioTek, Bad Friedrichshall, Germany) for 90 min directly after adding the substances and then every 15 min at Excitation ( Ex )/Emission ( Em ) = 485 nm/528 nm. The maximum rate of fluorescence increase ( v max ) was calculated.

Propidium iodide (PI) assay

The PI assay was used to evaluate cell viability. Cells seeded in 96-well plates (OKF6/TERT2: 2 × 10 4 cells/well, Caco-2: 1 × 10 4 cells/well) were grown for 24 h and then treated with the materials with and without VL-irradiation. After treatment, 55 μM propidium iodide (Sigma, Taufkirchen, Germany) was added and incubated for 20 min in the dark at room temperature. Fluorescence ( F PI ) was read at Ex / Em = 530 nm/645 nm. Background measurements (blank) were obtained from cell-free wells containing PBS and PI. Subsequently, the surfactant Nonident P-40 (Fluka, Seelze, Germany) was added for 20 min at room temperature in the dark, to lyse all vital cells. Fluorescence measurements were repeated at the same wavelengths to obtain F max , a function of total cell number (PI is membrane impermeable and stains only non-vital cells ). Percentage of viability was calculated as 100 − ( F PI − blank/ F PI(max) − blank) × 100, where F PI is the measured PI fluorescence.

Comet assay

The alkaline comet assay and an enzyme-modified alkaline comet assay were used to detect DNA damage and oxidative DNA modifications.

The alkaline comet assay was performed according to Tice et al. with minor modifications. Cells were grown in 6-well plates (OKF6/TERT2: 1.5 × 10 5 cells/well, Caco-2: 7 × 10 4 cells/well) for 24 h and then treated with the materials with or without irradiation. Cultures incubated with 0.5 μL/mL ethylmethanesulphonate (EMS) (Sigma, Taufkirchen, Germany) for 1 h served as positive control (c3). After treatment, cells were detached from the culture plate by a brief trypsin/EDTA treatment. Trypsin activity was stopped by adding 800 μL FBS-containing 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 at 54 × g and supernatants were removed. Then, cells were re-suspended in 90 μL of 0.75% (w/v) pre-heated low melting agarose (LMA, peqlab biotechnologies GmbH, Erlangen, Germany) and transferred to fully frosted slides (Menzel-Gläser, Braunschweig, Germany), which were pre-coated with 0.5% (w/v) normal melting agarose (peqlab biotechnologies GmbH, Erlangen, 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 each agarose layer. After gelation, the coverslips were removed and slides were incubated in lysis solution (2.5 M NaCl, 100 mM Na 2 EDTA, 10 mM Tris–HCL, 8 g/L NaOH (all Roth, Karlsruhe, Germany), 1% Triton-X100, 10% DMSO (both Sigma, Taufkirchen, Germany)) over night at 4 °C. After lysis, slides were placed on a cooled electrophoresis platform (peqlab biotechnologies GmbH, Erlangen, Germany) containing pre-cooled electrophoresis buffer (300 mM NaOH, 1 mM Na 2 EDTA, pH > 13). DNA was allowed to unwind for 20 min followed by electrophoresis for 20 min at 30 V/320 mA. Then, slides were 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, Merk, Darmstadt, Germany) or GelRed™ (dilution 1:5000; BioTrend, Cologne, Germany). Slides were analyzed using a fluorescence microscope (BX 60, Olympus, Hamburg, Germany) and the Comet 6.0 software (Andor Technology, Belfast, Northern Ireland). One hundred cells were evaluated per slide and tail moment (TM = (Tail mean − Head mean) × Tail%DNA/100) was used for data analysis. Tail moments are becoming higher equivalent to increasing DNA damage .

The enzyme-modified comet assay was performed according to Smith et al. . It is based on the alkaline comet assay, but after cell lysis, gels were rinsed three times with enzyme buffer: 40 mM HEPES (Biochrom, Berlin, Germany), 100 mM KCl (Merk, Darmstadt, Germany), 0.5 mM Na 2 EDTA, 0.2 mg/mL bovine serum albumin (Sigma, Taufkirchen, Germany), diluted in HPLC water (Roth, Karlsruhe, Germany) (pH 8). Then gels were incubated for 10 min with 0.32 U/gel of human 8-hydroxy-guanine DNA-glycosylase 1 (hOGG1, New England Biolabs, Frankfurt am Main, Germany) at 37 °C. For this, gels were covered with 100 μL of buffer or hOGG1 in buffer and placed in quadriPERM dishes (Greiner Bio-One, Frickenhausen, Germany). This was followed by DNA-unwinding, electrophoresis and staining according to the alkaline comet protocol (see above). Potassium bromate (KBrO 3 , treatment time: 1 h, concentration: 1 mM for OKF6/TERT2, 2 mM for Caco-2, c4) was used as positive control in the hOGG1-modified alkaline comet assay.

Statistical analysis

Experiments were run three times with six replicates each (ROS formation, PI assay) or two replicates (comet assay). Measured fluorescence values were converted into % of untreated control cells (c2 = 100%) in experiments using microtiter plates. Data were expressed as means ± standard deviation (SD). One-way ANOVA followed by a Bonferroni posttest (Prism 5.04, GraphPad Software, La Jolla, USA) were used for statistical analysis of ROS formation, PI readings and comet assays. Statistical differences of the same concentrations with and without enzyme treatment, GSH treatment and additional recovery time, were determined with the paired t -test. P -values < 0.05 were considered significant.

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Nov 23, 2017 | Posted by in Dental Materials | Comments Off on Genotoxic effects of camphorquinone and DMT on human oral and intestinal cells
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