2.1

Chemicals

Methacrylic acid (MA), methacrylic acid methylester (MAME), phosphate buffer solution, NADPH, cyclohexene oxide, glutathione, 2,3-epoxymethacrylicacid methylester (2,3-EMME) and Diazald ^® (N-methyl-N-nitroso-4-toluolsulfonic acid amide) for the production of diazomethane were purchased from Sigma–Aldrich (Steinheim, Germany). Purified pooled human liver microsomes (HLM) were purchased from Sigma–Aldrich. All chemicals were of the highest quality commercially available. They were used without further purification. 2,3-EMA was synthesized by oxidation of MA with hydrogen peroxide/hydrogen carbonate according to the method described by Yao and Richardson .

Stock solutions were prepared immediately prior to each incubation by diluting 2,3-EMME and 2,3-EMA from stem solutions in sodium phosphate buffer (0.1 M, pH 7.4) to a final concentration of 100 μmol/L. HLM were stored at −80 °C. For control experiments HLM were inactivated by heating at 100 °C for 30 min.

2.2

Cell cultures and preparations

Human gingiva fibroblasts (HGF) were obtained from Oligene (Berlin, Germany, Cat. No. 1110412). Human pulp fibroblasts (HPF) were obtained from biopsies of healthy premolar and molar teeth which were obtained from the “Department of Operative Dentistry and Periodontology”, LMU, Munich.

HGF and HPF were grown on 75 or 175 cm ² cell culture flasks to approximately 70% confluence in a 5% CO ₂ atmosphere, 96% humidity and 37 °C. The medium used was Quantum 333 (PAA Laboratories, Pasching, Austria) with l -glutamine supplemented with 50 IU/mL penicillin, 50 μg/mL streptomycin, and 50 μg/mL amphotericine . After reaching confluence cells were washed with Dulbecco’s Phosphate Buffered Saline (PBS), trypsinized and seeded into a headspace vial at a density of 2 × 10 ⁴ cells/vial in 3 mL growth media and were subsequently incubated for 48 h to reach confluence as described above.

Human tumor buccal kerotinocytes (SqCC/Y1-cells) were obtained from Prof. R. Lotan (University of Texas, M. D. Anderson Cancer Center, Houston, TX). They were cultured as described for HGF/HPF. Chinese hamster V79 lung fibroblasts (V79-cells) and a cDNA-derived human CYP2E1 expressing V79 cell line (V79-CYP2E1) were cultivated in DMEM, supplemented with 1 mM sodium pyruvate, 4 mM l -glutamine, 10% fetal calf serum, 100 units penicillin/mL and 100 μg/mL streptomycin, at 37 °C, 7% CO ₂ and 90% saturated atmospheric .

All chemicals were purchased from PAA Laboratories, Cölbe, Germany.

2.3

Total RNA isolation and RT-PCR analysis

RNA was isolated from subconfluent cell cultures using a RNeasy mini kit (Qiagen, Hilden, Gemany) and transcribed using random primers p(dN)6 with Avian Myeloblastosis Virus AMV reverse transcriptase (Roche 1st strand cDNA synthesis kit, Roche, Mannheim, Germany) according the manufacturers instructions. PCR was performed in a PCR Express Thermocycler (Thermo Life Sciences, Egelsbach, Germany) using 0.5 μM of forward primer ATT CCC AAG TCC TTC ACC CG and reverse primer TGT GGC TTC CAG GCA AGT AGT G in a total volume of 25 μL. Conditions were: 1.5 mM Mg ²⁺ , 25 mM Tris–(hydroxymethyl)-methylamino-propane sulfonic acid, pH 9.3, 50 mM KCl, 1 mM mercaptoethanol, 0.2 mM dNTP each, and 0.5 units of Thermus Icelandicus (Red Hot thermostable) DNA polymerase (AB Technologies, Surrey, GB) in a cycler program of 3 min 94 °C; 35 cycles 1 min 94 °C, 1 min 60 °C, 1 min 72 °C, 10 min 72 °C, followed by 5 min at 4 °C. 5 μL of each reaction mixture was separated on a 1% agarose/TBE gel. A band of 540 bp is characteristic for the expression of human CYP450-2E1. Human skin keratinocytes and human hepatocellular carcinoma cell line Hep G 2 have a well documented expression of CYP450-2E1 . Therefore human primary keratinocytes from a healthy, nonatopic male individual and the human hepatocellular carcinoma cell line Hep G 2 were used as biological positive controls for the expression of CYP450-2E1 in the RT-PCR analysis.

2.4

Incubation procedure for human liver microsomes (HLM)

The incubation procedure for HLM followed the procedure described by Cotrell et al. . In a 2 mL headspace vial phosphate buffer (1 mL, 0.1 M, pH 7.4), HLM (2.3 mg/mL final protein concentration in the same buffer), cyclohexene oxide (14 mM) as an epoxide hydrolases inhibitor and the substrate (final concentration 100 μmol/L) were added. The system was equilibrated at 37 °C for 15 min. Reactions were initiated by the addition of NADPH (1 mmol final concentration in sodium phosphate 0.1 M, pH 7.4). The reaction mixture was incubated for 60 min on a shaker at 100 rpm at 37 °C. Control incubations using heat-inactivated HLM were performed in order to ensure that 2,3-EMA did not originate from another source than metabolic activation.

The reaction was terminated by the addition of a saturating amount of NaCl followed by addition of cycloheptanone (2 μL, 17 mM) as an internal standard. Samples for analysis (100 μL) were taken with a syringe, extracted into hexane, dried over anhydrous magnesium sulphate and methylated as described in Section 2.5 . All experiments were repeated three times. Pyrazole (100 μM) served as a selective inhibitor of the expression of CYP450-2E1 .

2.5

Derivatization procedure

The analyte 2,3-EMA is a carboxylic acid which is deprotonated at physiological pH (7.4). In order to increase its vapor pressure and facilitate the analysis by GC/MS it was reacted into its corresponding methyl ester 2,3-EMME by methylation. For methylation a solution of diazomethane in ice-cooled ether (3 mL) was freshly prepared by the reaction of 500 mg Diazald ^® with 0.8 ml 6 N NaOH. From this ready-to-use solution aliquots were taken and added drop wise to the dried hexane extract until the yellow color of diazomethane did not disappear anymore. This solution was stored in darkness for further 15 min to ensure that the reaction was completely finished.

2.6

GC/MS analysis of the incubation extracts

For the headspace analysis SPME fibers (50/30 μm DVB/Carboxen/PDMS Stable Flex for Manual Holder, Supelco) were brought into contact with the gas phase of the vial for 2 min.

After enrichment of the analytes they were thermically desorbed from the SPME fiber surface for 3 min in the injector of the gas chromatograph. The extract was analyzed using a Finnigan MAT 4200 mass spectrometer (Finnigan, Bremen, Germany) coupled to a Chrompack CP 9001 gas chromatograph (Varian, Darmstadt, Germany) equipped with a DB-5 capillary column (30 m, 0.25 mm i.d., 1 μm film; Phenomenex, Aschaffenburg, Germany) for separation. The column was held at 100 °C for 3 min following desorption, raised at 4°/min to 150 °C and held for 2 min, then raised at 10°/min to 250 °C and held for 5 min. For separation helium 5.0 was used as carrier gas at a flow rate of 1 mL/min. The mass spectrometer operated in the electron ionization mode (EI, 70 eV) and scanned over the range m / z 35–350 at a rate of 1 scan/s. The mass spectrometer was operated in “full-scan” mode for identification and in “single ion monitoring” mode at m / z 101 for quantification of 2,3-EMME.

2.1

Chemicals

Methacrylic acid (MA), methacrylic acid methylester (MAME), phosphate buffer solution, NADPH, cyclohexene oxide, glutathione, 2,3-epoxymethacrylicacid methylester (2,3-EMME) and Diazald ^® (N-methyl-N-nitroso-4-toluolsulfonic acid amide) for the production of diazomethane were purchased from Sigma–Aldrich (Steinheim, Germany). Purified pooled human liver microsomes (HLM) were purchased from Sigma–Aldrich. All chemicals were of the highest quality commercially available. They were used without further purification. 2,3-EMA was synthesized by oxidation of MA with hydrogen peroxide/hydrogen carbonate according to the method described by Yao and Richardson .

Stock solutions were prepared immediately prior to each incubation by diluting 2,3-EMME and 2,3-EMA from stem solutions in sodium phosphate buffer (0.1 M, pH 7.4) to a final concentration of 100 μmol/L. HLM were stored at −80 °C. For control experiments HLM were inactivated by heating at 100 °C for 30 min.

2.2

Cell cultures and preparations

Human gingiva fibroblasts (HGF) were obtained from Oligene (Berlin, Germany, Cat. No. 1110412). Human pulp fibroblasts (HPF) were obtained from biopsies of healthy premolar and molar teeth which were obtained from the “Department of Operative Dentistry and Periodontology”, LMU, Munich.

HGF and HPF were grown on 75 or 175 cm ² cell culture flasks to approximately 70% confluence in a 5% CO ₂ atmosphere, 96% humidity and 37 °C. The medium used was Quantum 333 (PAA Laboratories, Pasching, Austria) with l -glutamine supplemented with 50 IU/mL penicillin, 50 μg/mL streptomycin, and 50 μg/mL amphotericine . After reaching confluence cells were washed with Dulbecco’s Phosphate Buffered Saline (PBS), trypsinized and seeded into a headspace vial at a density of 2 × 10 ⁴ cells/vial in 3 mL growth media and were subsequently incubated for 48 h to reach confluence as described above.

Human tumor buccal kerotinocytes (SqCC/Y1-cells) were obtained from Prof. R. Lotan (University of Texas, M. D. Anderson Cancer Center, Houston, TX). They were cultured as described for HGF/HPF. Chinese hamster V79 lung fibroblasts (V79-cells) and a cDNA-derived human CYP2E1 expressing V79 cell line (V79-CYP2E1) were cultivated in DMEM, supplemented with 1 mM sodium pyruvate, 4 mM l -glutamine, 10% fetal calf serum, 100 units penicillin/mL and 100 μg/mL streptomycin, at 37 °C, 7% CO ₂ and 90% saturated atmospheric .

All chemicals were purchased from PAA Laboratories, Cölbe, Germany.

2.3

Total RNA isolation and RT-PCR analysis

RNA was isolated from subconfluent cell cultures using a RNeasy mini kit (Qiagen, Hilden, Gemany) and transcribed using random primers p(dN)6 with Avian Myeloblastosis Virus AMV reverse transcriptase (Roche 1st strand cDNA synthesis kit, Roche, Mannheim, Germany) according the manufacturers instructions. PCR was performed in a PCR Express Thermocycler (Thermo Life Sciences, Egelsbach, Germany) using 0.5 μM of forward primer ATT CCC AAG TCC TTC ACC CG and reverse primer TGT GGC TTC CAG GCA AGT AGT G in a total volume of 25 μL. Conditions were: 1.5 mM Mg ²⁺ , 25 mM Tris–(hydroxymethyl)-methylamino-propane sulfonic acid, pH 9.3, 50 mM KCl, 1 mM mercaptoethanol, 0.2 mM dNTP each, and 0.5 units of Thermus Icelandicus (Red Hot thermostable) DNA polymerase (AB Technologies, Surrey, GB) in a cycler program of 3 min 94 °C; 35 cycles 1 min 94 °C, 1 min 60 °C, 1 min 72 °C, 10 min 72 °C, followed by 5 min at 4 °C. 5 μL of each reaction mixture was separated on a 1% agarose/TBE gel. A band of 540 bp is characteristic for the expression of human CYP450-2E1. Human skin keratinocytes and human hepatocellular carcinoma cell line Hep G 2 have a well documented expression of CYP450-2E1 . Therefore human primary keratinocytes from a healthy, nonatopic male individual and the human hepatocellular carcinoma cell line Hep G 2 were used as biological positive controls for the expression of CYP450-2E1 in the RT-PCR analysis.

2.4

Incubation procedure for human liver microsomes (HLM)

The incubation procedure for HLM followed the procedure described by Cotrell et al. . In a 2 mL headspace vial phosphate buffer (1 mL, 0.1 M, pH 7.4), HLM (2.3 mg/mL final protein concentration in the same buffer), cyclohexene oxide (14 mM) as an epoxide hydrolases inhibitor and the substrate (final concentration 100 μmol/L) were added. The system was equilibrated at 37 °C for 15 min. Reactions were initiated by the addition of NADPH (1 mmol final concentration in sodium phosphate 0.1 M, pH 7.4). The reaction mixture was incubated for 60 min on a shaker at 100 rpm at 37 °C. Control incubations using heat-inactivated HLM were performed in order to ensure that 2,3-EMA did not originate from another source than metabolic activation.

The reaction was terminated by the addition of a saturating amount of NaCl followed by addition of cycloheptanone (2 μL, 17 mM) as an internal standard. Samples for analysis (100 μL) were taken with a syringe, extracted into hexane, dried over anhydrous magnesium sulphate and methylated as described in Section 2.5 . All experiments were repeated three times. Pyrazole (100 μM) served as a selective inhibitor of the expression of CYP450-2E1 .

2.5

Derivatization procedure

The analyte 2,3-EMA is a carboxylic acid which is deprotonated at physiological pH (7.4). In order to increase its vapor pressure and facilitate the analysis by GC/MS it was reacted into its corresponding methyl ester 2,3-EMME by methylation. For methylation a solution of diazomethane in ice-cooled ether (3 mL) was freshly prepared by the reaction of 500 mg Diazald ^® with 0.8 ml 6 N NaOH. From this ready-to-use solution aliquots were taken and added drop wise to the dried hexane extract until the yellow color of diazomethane did not disappear anymore. This solution was stored in darkness for further 15 min to ensure that the reaction was completely finished.

2.6

GC/MS analysis of the incubation extracts

For the headspace analysis SPME fibers (50/30 μm DVB/Carboxen/PDMS Stable Flex for Manual Holder, Supelco) were brought into contact with the gas phase of the vial for 2 min.

After enrichment of the analytes they were thermically desorbed from the SPME fiber surface for 3 min in the injector of the gas chromatograph. The extract was analyzed using a Finnigan MAT 4200 mass spectrometer (Finnigan, Bremen, Germany) coupled to a Chrompack CP 9001 gas chromatograph (Varian, Darmstadt, Germany) equipped with a DB-5 capillary column (30 m, 0.25 mm i.d., 1 μm film; Phenomenex, Aschaffenburg, Germany) for separation. The column was held at 100 °C for 3 min following desorption, raised at 4°/min to 150 °C and held for 2 min, then raised at 10°/min to 250 °C and held for 5 min. For separation helium 5.0 was used as carrier gas at a flow rate of 1 mL/min. The mass spectrometer operated in the electron ionization mode (EI, 70 eV) and scanned over the range m / z 35–350 at a rate of 1 scan/s. The mass spectrometer was operated in “full-scan” mode for identification and in “single ion monitoring” mode at m / z 101 for quantification of 2,3-EMME.