Cigarette smoke induces direct DNA damage in the human B-lymphoid cell line Raji

Q. Yang, M. Hergenhahn1, A. Weninger and H. Bartsch

Division of Toxicology and Cancer Risk Factors, Deutsches Krebsforschungszentrum, Heidelberg, Germany


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
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Human lymphoid cells (Raji) were exposed to water-soluble compounds from cigarette smoke (CS) generated in a smoking machine. DNA damage, as detected by alkaline single-cell microelectrophoresis (COMET assay), was induced in a time- and concentration-dependent manner in the cells. Most of the rapidly induced DNA damage was attributable to direct-acting compounds since cytochrome P450-related metabolic activities (ethoxy- and pentoxyresorufin-O-deethylases and coumarin-7-hydroxylase) were absent or very low. In addition, induction of DNA damage could be inhibited only slightly by ß-naphthoflavone and coumarin. Vitamin C enhanced DNA damage in Raji cells probably by redox cycling of catechol and hydroquinone present in CS implicating reactive oxygen intermediates as another source of DNA damage. N-acetylcysteine, a radical scavenger and glutathione precursor, reduced DNA damage in Raji cells when exposure to CS was followed by 2 h post-incubation in culture medium. Unrepaired DNA damage caused by CS persisted longer than {gamma}-irradiation-induced DNA damage. Among the CS constituents, acrolein, but not formaldehyde and acetaldehyde, induced DNA damage although less intensely than CS itself. At 50 and 100 µM concentrations, acrolein also inhibited repair of {gamma}- irradiation-induced DNA damage in the COMET assay. Inhibition of DNA synthesis by acrolein at 50 µM was demonstrated by an immunochemical assay for bromo-deoxyuridine incorporation; however, inhibition of a representative repair enzyme, 8-oxoguanosine hydrolase, by either CS or acrolein was not observed. The present results further confirm the presence of direct-acting genotoxic components and inhibitors of DNA repair in the gas phase of tobacco smoke, that may contribute to DNA damage and smoking-associated cancers of the upper aerodigestive tract.

Abbreviations: BrdU, bromodeoxyuridine; CS, cigarette smoke (aqueous solution); hOGG1, human 8-oxoguanosine glycosylase/lyase; HU, hydroxyurea; NAC, N-acetyl-cysteine; ß-NF, ß-naphthoflavone; PBSG, phosphate-buffered saline–0.45% glucose.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Worldwide, tobacco smoking is the major risk factor for several types of human cancer (reviewed in 1,2). Tobacco smoke contains major classes of carcinogens that include polycyclic aromatic hydrocarbons, aromatic amines (1) and tobacco-specific nitrosamines (2,3). In addition, toxic compounds such as formaldehyde, acetaldehyde, acrolein (46), short-lived radicals and reactive oxygen intermediates generated by redox cycling from catechol and hydroquinone (7,8) and NOx (9,10) may also contribute to the toxic and carcinogenic effects of tobacco smoke. Direct DNA-damaging compounds present in cigarette smoke (CS) have previously been reported to include reactive oxygen intermediates (7,8), peroxynitrite (10), ethylating agents (11,12) and unidentified compounds (13). So far, no clear cause–effect relationships have been established between individual or classes of compounds from tobacco smoke, and their effects on induction of genetic damage in human cells though smoking-related DNA damage was found at enhanced levels in target tissues and oral mucosa of smokers (e.g. 14–16). A recent analysis of genotoxic effects induced by CS suggested that the pattern of induced mutations in Salmonella may be due to some prevailing compounds such as polycyclic aromatic hydrocarbons and aromatic amines (17). We report here on DNA damage and repair inhibition induced by compounds in aqueous extracts of CS in a B-lymphoid cell line (Raji) containing Epstein–Barr virus episomes. The cell line was chosen for two reasons: (i) Raji cells have low cytochrome P450-related metabolizing activity which would allow determination of the levels of directly induced DNA damage, and (ii) we recently demonstrated that exposure of Raji cells to CS at low concentrations induced DNA damage in bulk cellular DNA and in a gene-specific fashion also in Epstein–Barr virus episomes (18).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
If not stated otherwise, chemicals and enzymes were purchased from Sigma (Munich, Germany), Boehringer Ingelheim (Heidelberg, Germany) and Boehringer Mannheim (Mannheim, Germany). Raji cells were a kind gift of Dr A.Polack (GSF Neuherberg, Germany).

Test for CYP1A1- and 2B-related metabolism in Raji cells: use of cytochrome P450 inhibitors
To exclude metabolism of polycyclic aromatic hydrocarbons and tobacco-specific nitrosamines (19), the possible activities of ethoxyresorufin-O-deethylase, pentoxyresorufin-O-deethylase and coumarin-7-hydroxylase (CYP2A6) metabolism in Raji cells were tested according to Reiners et al. (20). In brief, cells were permeabilized by freezing overnight at –80°C and thawing. To stimulate maximal metabolism they were incubated in the presence of optimal concentrations of co-factors, an NADPH-generating system and the substrates ethoxy-resorufin, pentoxy-resorufin and coumarin, respectively. Metabolic products were measured in aliquots of the cell supernatants by fluorescence measurement and compared with standard curves obtained under the same conditions but without cellular proteins.

To exclude further cytochrome P450 metabolism in activation of some DNA-damaging agents from CS, cells were exposed to the metabolic inhibitors ß-naphthoflavone (ß-NF) (21) and coumarin (22,23) at relatively high concentrations of 0.5 and 1 mM, respectively. Cells were treated by preincubation for 10 min with 5 ml of 0.5 mM ß-NF plus 1 mM coumarin in phosphate-buffered saline–0.45% glucose (PBSG), then pelleted and treated with 5 ml CS 1:10 or 1:30 in PBSG.

Treatment of cells
Raji cells were kept in RPMI-1640/10% FBS, under 5% CO2 at 37°C.
Treatment by {gamma}-irradiation.
Where indicated, cells were {gamma}-irradiated in a 60Co-irradiation apparatus (Atomic Energy, Canada), at the dose levels indicated, then used either immediately for the COMET assay, or after 2 h in medium at 37°C in the incubator to allow DNA repair to take place.

Treatment with CS solutions.
Fifteen filter cigarettes (a mixture of different commercial brands of filter cigarettes) were smoked in a smoke machine (Borgwaldt Technik GmbH, Hamburg, Germany) with some modifications (see ref. 3) drawing volumes of 35 ml in 2 s, equivalent to a single inhalation. Water-soluble compounds of the smoke were retained by a series of three wash bottles each filled with 80 ml of PBSG. The CS from the first bottle was used; some water-insoluble tar components precipitated out on the walls of the wash bottles. Thus, a major part of the water-soluble compounds from the smoke of a single cigarette was contained in ~5 ml CS. Cells (7.5x105 per treatment group) were washed with medium, then with PBSG; they were subsequently exposed either to the levels of {gamma}-irradiation indicated, at room temperature, or to 5 ml CS solutions at the concentrations indicated, at 37°C in a CO2 incubator for the time periods given.

CS toxicity on Raji cells
In brief, Raji cells were exposed for 10 min at room temperature to the following CS dilutions in PBSG: 1:3, 1:10, 1:30 and 1:100. Subsequent to incubation, cells were washed in PBSG, and resupended in medium; cells were then transferred to 12-well plates, and incubated for 1–3 days. Control cells were treated in the same way except that smoke solutions were replaced by PBSG. Aliquots of the cells were removed from the wells and counted by a cell counter (Schärfe Systems, Reutlingen, Germany). The whole procedure was performed four times, with similar results (Figure 1Go).



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Fig. 1. Growth curves of Raji cells treated with different concentrations (refer to key) of CS for 10 min. n = 4 experiments, duplicates. Cells were counted as described in Materials and methods, results are given as means ± SD of four experiments.

 
Determination of aldehydes in CS
Aldehyde concentrations in CS solutions were determined as follows. In two experiments, two 10 ml aliquots of CS were mixed with 2 ml dinitrophenylhydrazine reagent (saturated solution in 1 M phosphoric acid) and 2 ml ethanol. The aldehydes were reacted for 2 h at room temperature; hydrazones were extracted into chloroform. Chloroform solutions were evaporated to dryness and redissolved in 10 ml CHCl3. Identification and quantitative determination of aldehyde hydrazones were performed by HPLC using RP18 columns, solvent systems of ethanol:water 52:48 and acetonitrile:water 55:45, respectively, and UV detection at 337 nm. Reference compounds were prepared from authentic aldehydes and recrystallized from boiling ethanol/water; standard curves were used to quantify aldehyde hydrazones from CS.

Mean aldehyde concentrations in CS were (experiments 1 and 2, respectively) formaldehyde 80 µM (100 and 60 µM), acetaldehyde 1400 µM (1500 and 1300 µM); and acrolein 350 µM (500 and 200 µM).

COMET assays
Raji cells in growth phase were washed twice with PBSG, and three aliquots of 2.5x105 cells each were subjected to the COMET assay as described previously (24,25).

DNA was stained by ethidium bromide; for evaluation of COMETS, DNA migration was measured for 40 cells each on two to three microscope slides carrying identically treated cells, under a fluorescence microscope (Laborlux 11; Leitz, Wetzlar, Germany) from one side of the COMET to the other (overall COMET length) and evaluated by a computer program (Perceptive Instruments, Essex, UK). Three size classes were used, normal cells <35 µm; moderately damaged cells 35–70 µm; and severely damaged cells >70 µm (24).

For time-course studies of repair, cells were exposed for 10 min to CS, aldehyde and acrolein solutions, respectively, then washed twice with PBSG and analysed; cells to be analysed after 2 h were incubated in medium at 37°C in the CO2-incubator followed by PBSG washing and COMET assay.

Inhibition of bromodeoxyuridine (BrdU) incorporation by acrolein
Cells in plateau phase were placed into fresh medium; after 24 h, aliquots of cells were treated with 10 mM hydroxyurea (HU) for 1 h at 37°C in the CO2-incubator; control cells were not preincubated with HU. Cells were washed in PBS, resuspended in serum-free medium with or without 50 µM acrolein, and incubated for 10 min in the incubator. Thereafter, all cell batches were washed with full medium and incubated in full medium containing 10 µM BrdU for 30 min at 37°C in the CO2 incubator. BrdU incorporation in cells was determined with the BrdU Labelling and Detection Kit I (Boehringer Mannheim) following the supplier's instructions (26). In brief, after BrdU labelling, cells were centrifuged onto poly-L-lysine-coated slides, and stained by indirect immunofluorescence with an FITC-labelled second antibody. Fluorescent labelling was recorded by a video camera attached to a fluorescence microscope; data were analysed by Leica Quantimet 500 software (Leica, Bensheim, Germany). The number of labelled and total nuclei, respectively, were counted (sum of two experiments): 284 labelled nuclei/936 nuclei (30% labelled) for control cells, 241/1008 (24% labelled) for HU-treated cells, 5/423 (1% labelled) for acrolein-treated cells; practically no labelling was recorded for HU + acrolein-treated cells. Results are expressed as relative intensity versus relative area of fluorescent signals, which reflect the levels of DNA replication and repair activity.

Lack of inhibition of human 8-oxoguanosine glycosylase/lyase (hOGG1) activity by acrolein and CS
hOGG1 activity in Raji cell extracts was determined as described by Nash et al. (27) with minor modifications. Briefly, ~3.8x108 Raji cells were collected by centrifugation; a cell extract was prepared as described by Sopta et al. (28). In a total volume of 40 µl per assay, 20 µl of the cell extract and 1.5 µl of 32P-5'-end-labelled double-stranded 25mer (25mer in ref. 27) carrying an 8-oxoguanosine in the middle (position 13) were incubated for 2 h at 30°C. For determination of the effects of CS and acrolein on the repair reaction, the cell extract was preincubated briefly with 4 and 8 µl, respectively, undiluted CS, or 8 µl of 1.25 mM and 250 µM acrolein solutions, such that the final concentrations were CS 1:10 and 1:5, and acrolein 250 and 50 µM, respectively. For borohydride trapping, sodium cyanoborohydride was added at a final concentration of 50 mM. After addition of 20 µl Laemmli sample buffer, the samples were boiled and 20 µl each were electrophoresed on 6–13% gradient or 20% polyacrylamide gels; reference lanes contained double-stranded oligonucleotide either untreated or cleaved at the 8-oxoguanine residue by 1 M piperidin for 1 h at 90°C. Products were visualized by autoradiography after drying of the gel. A strong and a weaker band of hOGG1 were visible at ~45 kDa when NaCNBH4 was present during the reaction, as described (27); cleavage of the substrate by hOGG1 was evident from the appearance of a new band below the substrate band. (Pre)incubation with either CS or acrolein did not change the intensity of the band of the cleavage product in at least two assays.


    Results
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 Materials and methods
 Results
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Growth of Raji cells after exposure to CS
Exposure of Raji cells was lethal at dilutions of 1:3 and 1:10 of CS, and growth retarding at 1:30 as shown by growth curves of Raji cells after exposure to CS (Figure 1Go).

Lack of CYP1A1- and 2B-dependent metabolism in Raji cells
In order to verify that Raji cells do not metabolize carcinogens like benzo[a]pyrene, aromatic amines and nitrosamines, the method of Reiners et al. (20) was used to determine CYP1A1- and 2B-catalyzed activities. No metabolism related to these enzymes was found in several preparations of permeabilized Raji cells. Since CYP2A6 has been reported to metabolize NNK (23), the possible presence of CYP2A6 was also assayed for. In the presence of coumarin, only a small time-dependent increase of fluorescence was found in some batches of cells (data not shown).

COMET formation induced by CS
Freshly prepared CS was used for all subsequent experiments. Figure 2A and BGo shows concentration dependence and time course of CS on induction of DNA damage. Significantly increased numbers of severely damaged cells were induced by treatment with undiluted CS and CS at dilutions of 1:3, 1:10 and 1:30 for 10 min (Figure 2AGo). When the time dependence of damage induction was tested, different intervals of exposure toward CS 1:30 resulted in increasing levels of severe damage after 10, 15, 30 and 60 min. There were no severely damaged cells in the control (Figure 2BGo).




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Fig. 2. COMET assay: DNA damage in Raji cells induced by CS. (A) Concentration dependence: different dilutions of CS applied for 10 min. (B) CS 1:30 applied for different time intervals. n = 2 experiments, COMET measurements in duplicate or triplicate. Results are given as means ± SD of two experiments. Open bars, <35 µm, normal cells; hatched bars, 35–70 µm, moderate damage; closed bars, >70 µm, severe damage.

 
Minor inhibition of COMET formation by two cytochrome P450 inhibitors
To exclude further participation of cytochrome P450 metabolism in activation of DNA-damaging agents, cells were exposed to a mixture of the broad-range metabolic inhibitors ß-NF (mainly for CYP1A-dependent metabolism) (21) and coumarin (for CYP2A6-dependent metabolism) (22) in addition to cigarette smoke. A statistically significant decrease was noted for induction of severe DNA damage by CS 1:10 with a corresponding increase in the percentage of moderately damaged cells; this suggests either a low level of metabolism in Raji cells resulting in the generation of DNA-damaging metabolites (Table IGo), or a direct interaction of inhibitors with reactive CS compounds. Inhibition was not observed at CS 1:30.


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Table I. COMET assay: low inhibitory effect of two P450 inhibitors on CS-induced DNA damage in Raji cells
 
Enhancing effect of vitamin C and protective effect of NAC on DNA damage
Exposure of Raji cells to CS in the presence of ascorbic acid (1 mM) or after preincubation of the cells for 10 min with the vitamin increased the percentage of cells with moderate DNA damage immediately after exposure or after 2 h repair time; however, the percentage of severely damaged cells was slightly decreased (Table IIGo). No protective effect of 1 mM NAC was obtained when cells were exposed to CS for 10 min and processed immediately thereafter; however, a protective effect against severe DNA damage became evident when post-exposure (repair) time was prolonged to 2 h. Under these conditions, NAC was also protective against acrolein-induced moderate and severe DNA damage.


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Table II. COMET assay: pro- and anti-damaging effects of vitamin C and NAC, respectively, on CS-induced DNA damage in Raji cells
 
DNA damage and inhibition of DNA repair by aldehydes in CS
Previous studies on biological effects of aldehydes and of cigarette smoke had demonstrated that formaldehyde, acetaldehyde and acrolein present in CS can have profound toxic effects on cells in culture (46). Aldehyde concentrations obtained under the present conditions were in the lower mM range in CS (see Materials and methods). Cells exposed to a mixture of these aldehydes (at the concentrations found in CS), exhibited a lower level of damage than if exposed to CS (Figure 3AGo). When the undiluted mixture of aldehydes at their concentrations determined in CS (formaldehyde 80 µM, acetaldehyde 1400 µM and acrolein 500 µM), and dilutions 1:5, 1:10 and 1:30 of this mixture were tested in comparison with acrolein alone, both the aldehyde mixture (undiluted and at dilution 1:5) and acrolein alone (500 and 100 µM) induced the same low levels of damage in comparison with the severe damage induced by CS (Figure 3AGo). This suggests that the effect of the aldehyde mixture was weakly genotoxic, and was mainly due to acrolein.




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Fig. 3. COMET assay: DNA damage in Raji cells induced by 10 min exposure to CS, the mixture of aldehydes in CS (formaldehyde 80 µM, acetaldehyde 1400 µM and acrolein 500 µM), and acrolein (500 µM) and dilutions 1:5, 1:10 and 1:30 of these solutions. Three control groups were exposed to PBSG only. (A) Damage measured immediately after exposure. (B) Damage measured 2 h after exposure. n = 2 experiments, COMET measurements in duplicate or triplicate. Results are given as means ± SD of two experiments. Open bars, acrolein; hatched bars, mixture of aldehydes; closed bars, CS.

 
Repair inhibition by CS and acrolein in the COMET assay
When the effect of CS and its aldehyde components was tested on repair of DNA damage, severe and moderate CS-induced damage was found to increase rather than to be repaired in 2 h (as evident from Figures 3B and 4GoGo versus Figure 3AGo). The same was true for moderate damage induced by the undiluted mixture of aldehydes and acrolein (at their concentrations in CS) and by dilutions 1:5 and 1:10. Since the mixture of aldehydes behaved similarly to acrolein alone, the main effect on DNA damage and repair inhibition appeared to be due to acrolein. Acrolein at 50 µM (which is near the concentration in CS 1:10) did not induce DNA damage, whereas 100 µM induced moderate damage in some cells (data not shown). However, both concentrations strongly inhibited repair of {gamma}-irradiation-induced DNA damage during a 2 h period (Figure 4Go).



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Fig. 4. COMET assay: Acrolein and CS effects on DNA damage induced by 22.4 Gy of {gamma}-irradiation and its repair during 2 h. Open bars, <35 µm, normal cells; hatched bars, 35–70 µm, moderate damage; closed bars, >70 µm, severe damage. n = 2 experiments, COMET measurements in duplicate or triplicate. Results are given as means ± SD of two experiments.

 
Inhibition of BrdU incorporation by acrolein
Repair of {gamma}-irradiation-induced DNA damage as evidenced by the COMET assay mainly reflects joining of single-strand breaks following removal of modified bases and degradation products of deoxyribose residues, and reinsertion of intact deoxynucleotides (29).

To study the possible inhibition of nucleotide incorporation due to acrolein at the DNA synthesis/repair step, we employed an immunofluorescent assay which detects BrdU incorporation (see Materials and methods) (26). After a 10 min preincubation of cells with 50 µM acrolein, incorporation of BrdU (replication plus repair) was determined during a period of 30 min; in parallel cell samples, DNA synthesis was inhibited by a 1 h preincubation with 10 mM HU which resulted in less intense labelling (Figure 5AGo). Consistently, preincubation with 50 µM acrolein inhibited incorporation of BrdU totally in both control (see Figure 5AGo for a representative experiment, and 5B for histograms) and HU-pretreated cells (no positive nuclei; data not shown). Thus, inhibition of DNA (re)synthesis by acrolein was evident.



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Fig. 5. Inhibition of BrdU incorporation into Raji cells. (A) BrdU-associated fluorescence in control cells (left panel), HU-treated cells (middle panel) and cells treated with 50 µM acrolein (right panel); data from a representative experiment. (B) Histograms of relative light intensity over spots versus relative area of fluorescent spots, pooled data from two experiments with five microscopic fields in each. Left panel, 284 labelled nuclei/936 nuclei (30% labelled); middle panel, 241 labelled nuclei/1008 nuclei (24% labelled); right panel, five labelled nuclei/423 nuclei (1% labelled).

 
Lack of inhibition of hOGG1 by either acrolein or CS
In addition to other types of damage, {gamma}-irradiation is known to induce 8-oxo-guanosine residues in DNA (30) which are excised by the DNA glycosylase/lyase activity of hOGG1, a representative member of the DNA glycosylase/lyase family of repair enzymes (27,31). We therefore hypothesized whether CS or acrolein would inhibit this enzyme at the catalytic lysine-249 function (27), since acrolein is known to modify the {varepsilon}-amino function of lysine residues (32). To test this hypothesis, borohydride-induced covalent binding (`borohydride trapping') of this enzyme to a radiolabelled duplex containing 8-oxoguanosine (27) was employed to identify the enzyme. In the presence of 50 mM Na CNBH3, a double band of OGG1/2 at ~45 kDa was readily revealed as described previously (27). The 5'-end-labelled 25mer duplex was cleaved partially by repair extracts from Raji cells; however, no evidence was obtained for inhibition of OGG1/2 by either CS or acrolein pretreatment of the extracts.


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 Materials and methods
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 References
 
The strong epidemiological relationship between tobacco smoking and increased risk of several types of cancer, together with experimental studies, suggest that carcinogenic nitrosamines, polycyclic aromatic hydrocarbons, aromatic amines and additional toxic compounds in CS reach target cells to exert their carcinogenic and cocarcinogenic actions (1,2). In the present experiments, DNA damage by aqueous solutions of CS was revealed by the COMET assay. As shown, compounds washed out from the gas phase of cigarette smoke rapidly induced severe DNA damage in Raji cells (Figures 1–3GoGoGo). Direct-acting compounds are mostly implicated as Raji cells were found to possess only low levels of several cytochrome P450 activities. Accordingly, relatively high concentrations of the broad range cytochrome P450-inhibitors ß-NF and coumarin could only slightly decrease DNA damage induced by CS at the highest concentration (CS 1:10). The occurrence of direct-acting compounds in CS was reported previously by others. Müller et al. (10) demonstrated the formation of peroxynitrite, a known DNA-damaging agent, in `smoke-bubbled PBS'. Matsukura et al. (33) reported on direct cytotoxic and mutagenic effects of CS condensate, while Prevost and Shuker (11) described a direct-acting ethylating agent in tobacco smoke that could explain the relatively high levels of 3-ethyl-adenine excreted in the urine of smokers (11,12).

We confirmed the presence of short-lived radicals and reactive oxygen intermediate-generating compounds (7,8) in the CS used here by a chemiluminescence assay (not shown). Since reactive oxygen intermediates derived from these agents may also cause DNA damage, the effects of the radical scavengers vitamin C and NAC were investigated. Vitamin C under certain conditions is able to induce mutations and DNA damage (3436). A slight enhancing effect of vitamin C on CS-induced moderate DNA damage in Raji cells was demonstrated by the COMET assay (Table IIGo). Our data confirm that radicals generated through redox cycling may contribute to initial DNA damage. This is also supported by the induction of cell transformation, mutations and other genetic damage by catechol and hydroquinone at 1–30 µM concentration in Syrian hamster embryo cells (37). NAC, a chemopreventive agent, was shown to reduce DNA adduct formation from cigarette smoke in the rat trachea (38). There was a lack of protective effects of NAC on initial CS-induced DNA damage in Raji cells (Table IIGo); however, when Raji cells were left for 2 h to repair before the COMET assay, the protective effect of NAC against CS- and acrolein-induced damage was evident suggesting that NAC might block the toxic effects of acrolein and other CS components (Table IIGo).

In the present study, DNA damage induced by CS persisted longer when compared with {gamma}-irradiation-induced damage which was completely repaired within 2 h (Table IIGo; Figure 4Go). This may be due to genotoxic compounds from CS taken up by the cells which cause delayed DNA damage, and/or it may be due to slower or inhibited DNA repair kinetics. Known constituents of CS, such as the mixture of the aldehydes formaldehyde, acetaldehyde and acrolein can also damage DNA (46,39); in this study, acrolein had the strongest DNA-damaging effect of the three aldehydes but was still less effective than CS. Acrolein at a 7-fold lower concentration (50 µM) than present in CS did not induce DNA damage per se in the COMET assay (Figure 3AGo) but substantially inhibited repair of {gamma}-irradiation-induced damage as shown by persistence of damaged DNA; this indicates that DNA ligation or a preceding step in DNA repair was inhibited. In an assay for BrdU incorporation into DNA, treatment of Raji cells with acrolein inhibited BrdU incorporation in control cells almost totally (Figure 5Go) as well as in HU-pretreated cells (virtually all nuclei negative for BrdU incorporation; data not shown). Since nucleotide incorporation following depletion of deoxynucleotides by a 1 h preincubation with HU is thought to reflect repair (40,41), 10 mM HU-treated Raji cells appear to repair DNA damage actively, and this is inhibited by acrolein; alternatively, BrdU incorporation under the present conditions indicates repair plus residual DNA replication which are both inhibited by acrolein (5). With respect to the consequences of such inhibition, recent studies demonstrated an increased number of chromosomal aberrations following inhibition of DNA synthesis (42,43).

Our data do not exclude inhibition of DNA repair by acrolein also at an earlier step in the DNA repair process, i.e. damage recognition and excision. For example, Dypbukt et al. (5) reported previously that cigarette smoke inhibits the DNA repair enzyme O6-methyl-guanosine-DNA methyltransferase. During the present study, no evidence was found that another repair enzyme, hOGG1 which employs a catalytic lysine function in repair of 8-oxo-deoxyguanosine-substituted DNA (27), was inhibited substantially by either CS or acrolein at different concentrations (data not shown). Acrolein–DNA adducts were described recently as possible biomarkers of deleterious effects of smoking in oral mucosa (39), and acrolein–protein adducts, either of endogenous origin or from environmental exposure, were detected in arteriosclerotic plaques (32). Thus, the inhibition of DNA synthesis and/or repair by acrolein may have a relevant role in fixation of DNA damage due to the ubiquitous occurrence of acrolein in car exhausts, tobacco smoke and heat decomposition products of oils and fats (39), and possibly also from endogenous sources through lipid peroxidation (44).


    Acknowledgments
 
We wish to thank Dr F.Kuchenmeister for introducing us to the COMET assay, Mrs K.Klimo for excellent assistance for the cell growth and chemiluminescence assays, and Mrs U.Schmitt for help in preparing the repair extracts.


    Notes
 
1 To whom correspondence should be addressed Email: m.hergenhahn{at}dkfz-heidelberg.de Back


    References
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 

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Received August 7, 1998; revised June 9, 1999; accepted June 10, 1999.