Caution. The following chemicals are hazardous and should be handled carefully: 2,6-DMA, 2,6-DMAP, N-OH-2,6-DMA, 3,5-DMA, 3,5-DMAP, and N-OH-3,5-DMA.
Sources. Reagents and cell culture materials were purchased from the following sources: cell culture materials, Lonza (Walkersville, MD); fetal bovine serum (FBS), Atlanta Biological; 2,6-DMA, 8-azaadenine (8-AA), 6-thioguanine (6-TG), dimethyl sulfoxide (DMSO), NADP, and DL-isocitric acid, N-acetyl cysteine (NAC), Sigma Chemical Co., St Louis, MO; and 3,5-DMA, Acros Organics (Geel, Belgium).
Synthesis of N-hydroxyl metabolites
. N-OH-2,6-DMA was synthesized in 50% yield by reduction of 1,3-dimethyl-2-nitrobenzene with zinc dust in aqueous NH4
Cl according to a published procedure (Kamm, 1941
). The product was obtained as light yellow needles and characterized by mass spectro metry (calcd. for [C8
, 138.0913; found, 138.0913) and 1
H-NMR (300 MHz, DMSO-d6
, 25°C): δ 2.27 (s, 6H), 6.84 (dd, 7.5 Hz, 1H), 6.92 (d, 7.5 Hz, 2H), 7.16 (d, 2.2 Hz, 1H), 8.08 (d, 2.2 Hz, 1H). N-OH-3,5-DMA was similarly synthesized in 70% yield from 5-nitro-m
-xylene to give a crystalline product of light yellow needles. MS: calcd. for [C8
, 138.0913; found, 138.0913. 1
H-NMR: δ 2.17 (s, 6H), 6.37 (bs, 1H), 6.44 (m, 2H), 8.10 (d, 2.2 Hz, 1H), 8.19 (d, 2.2 Hz, 1H).
Synthesis of aminophenols
. 2,6-DMAP was synthesized in two steps by coupling 3,5-dimethylphenol with the diazonium ion formed by diazotization of sulfanilic acid and reduction of the azo dye with sodium hydrosulfite as described previously (Gan et al., 2001
) following the procedure of Albert (1954)
with minor modifications. The same approach was used to generate 3,5-DMAP: a mixture of sulfanilic acid (1.73g, 10 mmol), Na2
(0.53g, 5 mmol), and H2
O (10ml) was heated to 60°C with stirring. After all the sulfanilic acid was dissolved, the solution was cooled in an ice bath to 15°C. A solution of sodium nitrite (0.74g, 10.7 mmol) in H2
O (2ml) was then added drop wise. The resulting solution was poured at once into aqueous hydrochloric acid (14.1ml, 5.6% wt/vol) in an ice bath and the mixture was allowed to stand for 15min. It was then added to a solution of 2,6-dimethylphenol (1.22g, 10 mmol) in aqueous NaOH (2.2g, 55 mmol in 20ml H2
O) at 5°C. The dark red reaction mixture was stirred well and allowed to stand for 1h at room temperature. It was then heated to 60°C and to it was gradually added aqueous sodium hydrosulfite (1% wt/vol) until yellow crystals precipitated from colorless solution. After standing 15min at 50°C, the yellow suspension was cooled to 20°C and filtered. The filtrate was washed with 1% sodium hydrosulfite aqueous solution and dried under vacuum. Yield: 82%. MS: calculated for [C8
, 138.0913; found 138.0910. 1
H-NMR (500 MHz, DMSO-d6
, 25°C): δ 7.17 (s, 1H), 6.15 (m, 2H), 4.3 (s, 2H), 2.02 (s, 6H).
Cell survival and mutation at the
. AA8 and UV5 CHO cells were purchased from ATCC. Two derivative cell lines 5P3NAT2 and 5P3NAT2R9, which are functionally heterozygous at the aprt
locus, were generously provided by Dr J. S. Felton (Lawrence Livermore National Laboratory). The repair-deficient (RD) 5P3NAT2 and -proficient (RP) 5P3NAT2R9 cells both express the cDNAs of the mouse CYP1A2
and human NAT2
genes but differ in repair capability. Details concerning the construction and characterization of these cell lines were described previously (Wu et al., 2003
). Prior to each experiment, cells were incubated for 2 days in medium containing CAAT (10µM cytidine, 100µM adenine, 1µM aminopterin, and 17.5µM thymidine) followed by 2–5 days in TAC (thymidine, adenine, and cytidine) medium to reduce the background aprt
mutant frequency. All cells were routinely maintained by monolayer culture in α-Minimal Essential Medium (MEM) containing L-glutamine supplemented with penicillin 100 units/ml, streptomycin 100 µg/ml, and 10% heat-inactivated FBS (complete MEM) in a humidified atmosphere with 5% CO2
The parental amines, 2,6- and 3,5-DMA, and their N-hydroxyl metabolites dissolved in DMSO were added to exponentially growing cells in 100-mm tissue culture dishes containing 0.5×106
cells in 10ml of complete MEM. The cell lines expressing NAT2
(both 5P3NAT2 and 5P3NAT2R9) were exposed to 0–1000μM of parent compounds for 48h in complete MEM or N-hydroxyl metabolites for 1h in serum-free (SF) MEM. Control cultures were treated with the same volume of vehicle (0.1% DMSO) for 48h or 1h. Concentrations of 2,6- and 3,5-DMA and their metabolites used for mutagenicity experiments were established based on MTT cytotoxicity assays (data not shown). Following treatment, cells were allowed to recover for 24h before determining survival by trypan blue exclusion and maintained in complete MEM thereafter. Previous studies showed that a relative survival of about 30% after chemical exposure facilitated optimum estimates of mutant frequency (Thilly, 1985
). Triplicate cultures were exposed to determine mutagenic potencies of 2,6- and 3,5-DMA and their N
-hydroxyl metabolites. Seven days after treatment, 6×105
cells from each group were placed in 100ml selective medium containing 80 µg/ml 8-AA and plated at 6×104
cells/10ml/100-mm dish for determination of mutagenicity after 14 days. For determining plating efficiency, dishes were seeded with 200 cells/10ml/100-mm dish in triplicate and incubated for 14 days in the absence of selecting agent.
Cell survival and mutation at the aprt locus with and without ascorbic acid or NAC. AA8 and UV5 cells were adapted to Ham’s F-12 medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated FBS (complete Ham’s) medium without ascorbate. The cells were seeded at 1×106 per well of six-well plates and incubated overnight in complete Ham’s medium prior to the treatment. Cells were exposed to N-OH-2,6-DMA (5, 10, 25, 50, 100, and 250μM) or 2,6-DMAP (5, 10, 25, 50, and 100μM) ± 5mM NAC, and N-OH-3,5-DMA (5, 10, 25, 50, 100, and 250μM) or 3,5-DMAP (5, 10, 25, 50, and 100 μM) ± ROS scavengers (5mM NAC or 50 μg/ml ascorbate) for 1h. After treatment, the cells were washed twice with SF Ham’s medium and incubated in complete Ham’s medium for additional 24h prior to determining cell survival. Mutation assay was performed as described above after 7 days of phenotypic expression.
Cell survival and gpt mutagenesis in AS52 cells. CHO AS52 cells, kindly provided by Dr Helga Stopper (University of Würzburg, Germany), were cultured in Ham’s medium supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated FBS (complete Ham’s) in a humidified atmosphere with 5% CO2 at 37°C. The medium was changed routinely, and cells were subcultured when confluence reached about 90%. Cultures were cleansed of pre-existing gpt mutants by culturing in MPA medium (10 µg/ml mycophenolic acid [MPA], 250 μg/ml xanthine, 22 μg/ml adenine, 11 μg/ml thymidine, and 1.2 μg/ml aminopterin) for 7 days followed by recovery medium enriched with xanthine (11.5 μg/ml), adenine (3 μg/ml), and thymidine (1.2 μg/ml) for 3 days.
AS52 cells were placed in six-well plates at a density of 0.5×106 cells per well the day before treatment. Cells were cultured for 5h at 37°C in SF Ham’s medium containing 2,6-DMA or 3,5-DMA (0–1000μM), with or without 5mM NAC, and a human liver S9 (BD Gentest) preparation comprising 16 μl S9 (440 μg S9 protein) and 65 μl sterile-filtered core mixture (25mg/ml NADP, 45mg/ml DL-isocitric acid) per milliliter of SF Ham’s medium. At the end of the treatment period, cells were washed and placed in complete Ham’s medium. For dosing with metabolites of 2,6- and 3,5-DMA, cells were seeded at 1×106 per well of six-well plates and incubated overnight in complete Ham’s medium prior to treatment. The cells were washed two times with SF Ham’s medium and exposed to N-OH-2,6-DMA (5,10, 25, 50, 100, and 250μM) or 2,6-DMAP (5, 10, 25, 50, and 100μM), and N-OH-3,5-DMA (5, 10, 25, 50, 100, and 250μM) or 3,5-DMAP (5, 10, 25, 50, and 100μM) ± 5mM NAC for 1h in SF Ham’s. After the treatment, the cells were processed as above.
AS52 cell viability was determined 24h after the treatments with trypan blue exclusion, and the cells were maintained in complete Ham’s medium for 7 days for phenotypic expression. The level of cell survival was normalized to the negative control and presented as percentage of control. For the gpt mutagenicity measurement, after 7 days incubation, 5×105 cells from each group were placed in 100ml complete Ham’s medium containing selection agent 6-TG (10µM) and plated at 5×104 cells/10ml/100-mm dish for determination of mutagenicity. For plating efficiency analysis, 2500 cells in 50ml complete Ham’s medium from each dose were seeded in 100-mm dishes at a density of 500 cells/10ml. After 14 days incubation, colonies were stained with 0.5% crystal violet in 50% methanol/water for 5 mins, rinsed, and counted. The spontaneous mutant frequency was determined using the negative control (DMSO treated).
DNA extraction, PCR amplification, and molecular analysis of gpt mutants. Single unstained gpt mutant colonies were identified and transferred to 24-well plates and grown to approximately 2×106 mutant cells. Genomic DNA was extracted from each mutant using GenElute mammalian genomic DNA miniprep kit (Sigma). Amplification of the genomic DNA was performed in two rounds of nested PCR in a PTC-200 DNA Engine Thermal Cycler (Bio-Rad, Hercules, CA). One microgram of template DNA was used to run the first round with 10 µl 10×PCR buffer, 2 µl dNTP mix, 0.5 µl taq polymerase, 73.5 µl sterile water, 0.2 µl each 25mM forward (bases −199 to −181; 5′-AAGCTTGGACACAAGACAG-3′) and reverse (bases 520 to 540; 5′-CCAGAATACTTACTGGAAAC-3′) primers (IDT) and amplified with a PCR profile of 94°C: 1min, 30 cycles of 94°C: 1min, 47°C: 1min, 72°C: 1min, and a final extension of 72°C for 7min. The product from this reaction was filtered using a Centricon 50 concentrator and resuspended in 100 µl sterile water to avoid nonspecific binding with remaining primers, and a 10 µl aliquot was used as template in the second round of PCR using nested primers (bases −23 to −4; 5′-ATAAACAGGCTGGGACACTT-3′ and bases 460 to 470; 5′-AGTGCCAGGCGTTGAAAAGA-3′). The PCR conditions were the same in the second round as in the first round reaction, except that the annealing temperature was 52°C. The quantity of gpt gene amplification was analyzed by electrophoresis on 0.8% agarose gels stained with ethidium bromide. The 0.5kb PCR product was cut out and purified for DNA sequence analysis using the QIAquick Gel Extraction Kit (Qiagen). DNA sequencing was carried out by the Dana-Farber/Harvard Cancer Center DNA Resource using the primer sets: 5′-ATAAACAGGCTGGGACACTT-3′ and 5′-AGTGCCAGGCGTTGAAAAGA-3′.
Quantification of intracellular ROS generation. Intracellular ROS detection studies were performed using a Cm-H2DCFDA ROS detection kit (Molecular Probes/Invitrogen). AS52 cells were placed in six-well plates at a density of 1×106 cells per well the day before treatment, washed two times with SF Ham’s medium, and exposed for 1h to various concentrations of N-OH-2,6-DMA, 2,6-DMAP, N-OH-3,5-DMA, or 3,5-DMAP in the presence or absence of 5mM NAC in SF Ham’s medium, wells were washed two times with SF Ham’s medium. Cells were then incubated in complete Ham’s medium for 24h at 37°C, after which they were washed with PBS, treated with trypsin-versene for 5min and suspended in 1ml/well SF Ham’s medium. Aliquots of 100 μl cell suspension from each dose were pipetted into 96-well plates and mixed with 10 μl Hank’s Buffered Salt Solution (HBSS) containing Cm-H2DCFDA (final concentration 25μM) activated by preincubation at 37°C for 30min. ROS generation was measured immediately with an HTS 7000 Plus Bio Assay micro-reader (485nm excitation, 530nm emission; PerkinElmer Life Sciences). ROS levels generated by 1×106 viable treated cells were expressed as percentage of ROS produced by an equal number of viable negative control cells.
CometChip and alkaline comet assay
. The alkaline comet assay, used to detect total DNA strand breaks, was performed on the CometChip using the protocol described by Wood et al. (2010)
. Molten 1% normal melting point agarose (Invitrogen) was poured on top of a sheet of GelBond film (Lonza). The polydimethylsiloxane mold with microposts used to form the microwells was placed into the agarose and removed after the agarose gelled. The gelled agarose with microwells was sandwiched between a glass substrate and a bottomless 96-well plate (Greiner BioOne) and sealed with mechanical force to create the multiwall version of the comet platform, the CometChip.
AS52 cells placed in 100-mm dishes at a density of 1×107
cells were exposed to N-OH-2,6-DMA (50 and 100μM), 2,6-DMAP (10, 25, and 50μM), N-OH-3,5-DMA (50 and 100μM), or 3,5-DMAP (10, 25, and 50μM) ± 5mM NAC using the method described above. This set of treatments was repeated on two additional occasions to provide a set of three independent experiments. After 24 h incubation, 100 μl of cells (106
cells/ml) were pipetted into each of the agarose 96 wells. The bottomless 96-well plate form was then removed, and the gel was covered with 1% low melting point agarose (Invitrogen). After overnight lysis, the CometChips were placed into an electrophoresis chamber filled with alkaline unwinding buffer (0.3M NaOH and 1mM Na2
EDTA) for 40min at 4°C. Electrophoresis was performed at the same temperature with the same buffer for 30min at 1V/cm and a current of 300 mA. The chips were then neutralized twice for 15min in fresh buffer (0.4M Tris-HCl at pH 7.5) at 4°C. After neutralization, the CometChips were stained with SYBR Gold (Invitrogen) according to the manufacturer’s instructions for the fluorescence imaging. Graphs were captured using a Nikon 80i upright microscope coupled with an automatic scanning stage and analyzed using the Guicometanalyzer, custom software written in MATLAB (The Mathworks) by Wood et al. (2010)
. The results generated by the software showed percentage of tail DNA, which represented the level of DNA damage. The cells treated with 100μM H2
are the positive control.
In each treatment group, 50–150 comet images were collected and analyzed. However, cells treated with 50μM 3,5-DMAP and 100μM N-OH-3,5-DMA were highly damaged, and viability was less than 30% as indicated in . Hence, fewer comets were collected (20–70 comets per experiment) due to the constraint of fewer viable cells.
FIG. 1. Survival and mutagenicity of AS52 cells treated with 3,5-DMAP, N-OH-3,5-DMA, and 3,5-DMA + S9. Protection against cell killing by ascorbate and NAC is readily apparent when cells were treated with aminophenol (upper left panel) or N-hydroxylamine (lower (more ...)
Statistical analysis. Linear regression analysis was used to evaluate the relationship between dose and ROS production in treated cells as measured by fluorescence intensity. Student’s t-test was used for analysis of differences observed in the Comet assays. Statistical analysis of gpt mutants was performed using Fisher’s exact method.