Mutagenesis vol. 18 no. 5 pp. 465-470,
September 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
,ß-Unsaturated carbonyl compounds: induction of oxidative DNA damage in mammalian cells
Department of Chemistry, Division of Food Chemistry and Environmental Toxicology, University of Kaiserlautern, 67663 Kaiserslautern, Germany and 1Zentrallaboratorium Deutscher Apotheker Forschungs gGmbH, 65760 Eschborn, Germany
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Abstract |
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,ß-Unsaturated carbonyl compounds occur in food and other environmental media. Due to their reactivity with cellular nucleophiles (e.g. Michael adduct formation with DNA bases and with glutathione) they might represent a potential health risk. In this study, induction of oxidative DNA damage was investigated in mammalian cells, as a consequence of glutathione depletion induced by selected food relevant 2-alkenals, including E-(2)-hexenal (HEX), (2E,4E)-2,4-hexadienal (HEXDI) and (E)-2-cinnamaldehyde (CA) and the cyclic analogue 2-cyclohexen-1-one (CHX). Oxidative DNA breakage was monitored with the Comet assay, using treatment with formamidopyrimidine-DNA glycosylase (FPG). Total cellular glutathione (tGSH) was determined in a kinetic, photometric assay. After 1 h incubation of V79 cells with HEX (100 µM) and CHX (300 µM), HEXDI and CA (300 µM each), tGSH was depleted down to <20% of control (viability >85%). Under these conditions, FPG-sensitive sites were not observed; moderate direct DNA breakage, however, was detectable. During 3 h post-incubation (without test compound) distinct oxidative DNA breakage occurred in HEX- and CA-, but not in CHX- and HEXDI-pretreated cells. Direct DNA breakage was markedly diminished, most probably by repair processes, and tGSH concentrations were observed to increase again within 3 h post-treatment. The results give strong evidence for alkenal-mediated oxidative stress contributing to cytotoxic/genotoxic cell damage. The extent of oxidative stress appears to be influenced by structure-specific properties of the alkenals. |
Introduction |
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,ß-Unsaturated carbonyl compounds are widely distributed in food (Götz-Schmidt et al., 1986
; Boelens and van Gemert, 1987; Feron et al., 1991). They are naturally formed in fruits and vegetables as a result of enzymatic and/or spontaneous lipid oxidation. Some representatives, such as (E)-2-hexenal (HEX), (2E,4Z)-2,4-hexadienal (HEXDI) and cinnamaldehyde (CA) are used as flavouring agents (Figure 1) (Council of Europe, 2000). Others have been identified as food contaminants; 2-cyclohexen-1one (CHX) (Figure 1), for example, was detected in artificially sweetened soft drinks (Hahn, 1996). Because of their ,ß-unsaturated carbonyl structure the compounds are highly reactive towards cellular nucleophiles, which results in DNA damage, formation of DNA and protein adducts, mutagenicity and enzyme inhibition (Burgl et al., 1967; Chung et al., 1988; Williams et al., 1989; Kautiainen, 1992; Gölzer et al., 1996; Dittberner et al., 1997; Eder et al., 1999; Glaab et al., 2001; Ichihashi et al., 2001). Additionally, a distinct formation of glutathione (reduced form, GSH) adducts has been described for 2-alkenals and CHX in vitro and in rodents (Boyland and Chasseaud, 1967; Berhane et al., 1994; Eisenbrand et al., 1995; Janzowski et al., 2000a). GSH plays an important role in different physiological processes, including antioxidant defence (Halliwell and Gutteridge, 1999; Kelly, 1999; Pocernich et al., 2001). As a consequence of GSH depletion, the effects of reactive oxygen species (ROS) can prevail, leading to increased cytotoxic and genotoxic cell damage (Breen and Murphy, 1995).
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In a previous study, we showed that 2-alkenal/CHX exposure results in DNA damage and glutathione depletion in V79 and Caco-2 cells after 1 h incubation (Glaab et al., 2001
). In the present study we have extended these findings to elucidate whether oxidative DNA damage is induced in a post-incubation period. V79 cells were incubated with selected 2-alkenals (HEX, HEXDI and CA) and CHX in concentrations which are not cytotoxic but sufficient to strongly deplete cellular glutathione (Janzowski et al., 2000b; Glaab et al., 2001). Oxidative DNA damage (FPG-sensitive sites/oxidative purine modifications) and glutathione depletion were comparatively monitored during a 3 h post-incubation with alkenal-free medium. In some experiments the human colon cell line Caco-2 was also included. |
Materials and methods |
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Chemicals
2-Alkenals, CHX (purity
95%), GSH, glutathione reductase and NADPH were obtained from Sigma-Aldrich Chemie GmbH (Steinheim, Germany). N-methyl-N'-nitronitrosoguanidine was provided by Fluka (Neu-Ulm, Germany). Low and normal melting point agarose was obtained from Serva Electrophoresis GmbH (Heidelberg, Germany). Organic solvents and all other chemicals were of analytical grade or complied with the standards needed for cell culture experiments.
Cells and media
V79 cells were kindly provided from Prof. Dr J. Doehmer (Technische Universität, München, Germany). Caco-2 cells were obtained from the German Cancer Research Center (Heidelberg, Germany). Dulbecco’s modified Eagle’s medium (DMEM), DMEM/Nutrient Mix F12, foetal calf serum (FCS), sodium pyruvate and penicillin/streptomycin were obtained from Life Technologies GmbH (Eggenstein, Germany).
Cell culture, incubation with test compound and post-incubation
V79 cells (Chinese hamster lung fibroblasts) (Ford and Yerganian, 1958) were maintained in DMEM, supplemented with 10% FCS and 1 mM sodium pyruvate.
Caco-2 cells (originating from human colon adenocarcinoma) (Fogh et al., 1977) were cultivated in DMEM/Nutrient Mix F12 supplemented with 20% FCS. All cells were cultured with 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C, 5% CO2 and 95% saturated atmospheric humidity.
Cell suspensions were obtained from the monolayer by treatment with trypsin (0.5%) after washing with phosphate-buffered saline (PBS). Aliquots of 1 ml of suspension of V79 (2 x 106 cells/ml) and Caco-2 cells (2.5 x 105 cells/ml) were incubated with test compound (dissolved in 1% DMSO) for 1 h at 37°C in incubation medium (culture medium without FCS). In post-incubation experiments, cells were incubated for 1, 2 or 3 h in 1 ml of culture medium after elimination of test compounds (centrifugation for 10 min at 800 g).
Cytotoxicity
Following (post-)incubation, the cell suspension (50 µl) was mixed with trypan blue solution (50 µl, 0.5% in PBS) and microscopically checked for membrane integrity. Viability was expressed as a percentage of total cells (absolute viability in per cent). Only cell suspensions with viabilities >80% were used for determination of glutathione and DNA damage, to avoid artefacts resulting from cell death.
Glutathione depletion
Total glutathione [tGSH = reduced glutathione (GSH) + oxidized glutathione (GSSG)] was determined in V79 cells in a kinetic assay by photometric determination of 5-thio-2-nitrobenzoate (TNB), according to Gallagher et al. (1994) with slight modifications. Cell suspensions (1 ml) were centrifuged and resuspended in 430 µl of phosphate buffer (125 mM sodium phosphate containing 6.3 mM EDTA, pH 7.5). A 50 µl aliquot of this suspension was submitted to photometric protein determination using bis-chinolic carbonic acid (Pierce, Rockford, IL). To a 300 µl aliquot of the cell suspension was added 5-sulfosalicylic acid (300 µl, 10%) to precipitate proteins (5 min, 4°C). After centrifugation (12 300 g), 20 µl of the supernatant or glutathione standard solution (0.5–10 µM) were added to the NADPH/5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) solution (100 µl of 6 mM DTNB solution, 20 µl of 20 mM NADPH, 700 µl of phosphate buffer, 150 µl of bidistilled water) in a cuvette. After addition of glutathione reductase (10 µl, 0.5 U), TNB formation rate (
E/10 min) was monitored at 412 nm and compared with standards. Cellular tGSH concentrations (nmol/mg protein) were expressed as a percentage of the solvent control (0 h post-incubation). Means and ranges or SD were obtained from two or more independent experiments, each performed in duplicate.
Alkaline single cell gel electrophoresis (SCGE, Comet assay) and evaluation of (oxidative) DNA breakage
Alkaline SCGE was performed according to Singh et al. (1988), with slight modifications. Aliquots of cell suspensions (0.5–1 x 105 cells) were centrifuged (10 min, 800 g), mixed with 65 µl of low melting point agarose, distributed onto a glass microscope slide pre-coated with a layer of normal melting point agarose, covered with a coverslip and kept at 4°C to allow solidification of agarose. After removing the coverslip, slides were immersed in lysis solution for 1 h at 4°C. After lysis, slides were washed three times in enzyme buffer, drained and covered with 50 µl of either enzyme buffer or formamidopyrimidine-DNA glycosylase (FPG) enzyme, sealed with a coverslip and incubated for 30 min at 37°C, as described (Collins et al., 1996).
DNA was allowed to unwind (pH > 13, 20 min, 4°C) and horizontal gel electrophoresis (Bio-Rad Sub Cell GT) was conducted at 4°C for 20 min (25 V, 300 mA) using a Bio-Rad 300 power supply. Then slides were washed three times with 0.4 M Tris, pH 7.5, stained with ethidium bromide (40 µl, 10 µg/ml) and viewed microscopically with a Zeiss Axioskop 20, equipped with filter set 15 (excitation, BP 546/12; emission, LP 590). Slides were analysed by computerized image analysis (Perceptive Instruments, Haverhill, UK), scoring 2 x 50 images per slide (2 gels/slide). DNA migration is directly expressed as mean tail intensity (TI%) from one slide.
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Results |
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Glutathione depletion
HEX (30 µM, 100 µM; Figure 2A), CHX (100 µM, 300 µM; Figure 2B), HEXDI (100 µM, 300 µM; Figure 3A) and CA (300 µM; Figure 3B) induced a strong depletion of glutathione in V79 cells down to <20% of the control during a 1 h incubation (0 h post-incubation). CA (100 µM; Figure 3B) was found to be less effective. tGSH concentration of control cells was 15.5 nmol/mg protein (SD 4.8, n = 20). During 3 h post-incubation (without test compound) a distinct increase in tGSH (>30% of control) was observed in cells pretreated with HEX (30 µM), HEXDI and CA (both 100 and 300 µM) (Figures 2A and 3A and B). In contrast, in HEX (100 µM) and CHX (100 and 300 µM) pretreated cells, tGSH levels stayed below 25% (Figure 2A and B).
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Cytotoxicity (loss of membrane integrity) of cells was <15% in all incubation/post-incubation experiments.
Direct DNA breakage and FPG-sensitive sites
After 1 h incubation of V79 cells with HEX (100 µM; Figure 4A), CHX (300 µM; Figure 4B), HEXDI (300 µM; Figure 4C) and CA (300 µM; Figure 4D) a distinct direct DNA breakage (TI% > 20) was observed, whereas the lower concentrations tested were only weakly DNA damaging (100 µM CHX and CA, TI% 5–7%) or not effective (30 µM HEX and 100 µM HEXDI, TI% < 5%). Additional oxidative DNA breakage (FPG-sensitive sites) was not observed at this time point.
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During 3 h post-incubation (without test compound), direct DNA breakage was diminished from TI% >20 to <8% (Figure 4A–D). FPG-sensitive sites only became apparent in HEX- (100 µM) and CA-treated (300 µM) cells (Figure 4A and D, columns 11 and 12).
For HEX and CHX the time course of FPG-sensitive site formation (0–3 h post-incubation) was monitored (Figure 5). In HEX-treated (100 µM) cells a time-dependent increase in FPG-sensitive sites occurred after 2 h post-incubation, whereas 30 µM HEX and 300 µM CHX did not induce oxidative DNA breakage within 3 h.
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HEX was similarly tested in the human colon cell line Caco-2 (Figure 6). Basal levels of DNA breakage (control ± FPG treatment) were found to be slightly higher, compared with V79 cells. At 0 h post-incubation with 100 µM HEX at best marginal effects were observed; during 3 h post-incubation, however, both direct DNA breakage and FPG-sensitive sites were found to be markedly increased.
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Viability (membrane integrity) of cells was >85% in all incubation/post-incubation experiments.
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Discussion |
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Glutathione, when depleted by
,ß unsaturated carbonyl compounds down to <20% (Glaab et al., 2001), was found to be replenished to varying extents during subsequent post-incubation in the absence of 2-alkenals and CHX. This might be due to either de novo synthesis of GSH or to decomposition of the alkenal–GSH adduct, which has been found to dissociate back to alkenal and GSH following a kinetic equilibrium mainly influenced by GSH concentrations (Eisenbrand et al., 1995). The tGSH replenishment rate appeared to be primarily influenced by the nature of the respective compound and its concentration, with CHX and HEX treatments (100 µM each) leading to nearly no reappearance of tGSH after 3 h (300 µM HEXDI > 300 µM CA > 100 µM CHX 
100 µM HEX). Since viability of the cells was at least 85% in all experiments, glutathione loss from membrane leakage can be excluded.
The extent of direct DNA damage resulting from 1 h incubation with HEX (100 µM), CHX, HEXDI or CA (300 µM each) was found to decrease in the course of post-incubation. FPG-sensitive sites were only observed with HEX (100 µM) and CA (300 µM). Unexpectedly, CHX-treated cells which remained at low glutathione levels during post-incubation did not undergo oxidative DNA damage. This is also apparent from low HEX treatment (30 µM) strongly depleting glutathione, but allowing for its reconstitution during post-incubation. It may thus be concluded that effective and persistent glutathione depletion is a prerequisite for 2-alkenal-mediated generation of ROS and that the nature and the concentration of the respective alkenal is of similar relevance for DNA damage to be induced. In addition to formation of DNA adducts, 2-alkenals may act as enzyme inhibitors, including enzymes of cellular antioxidant defence. For example, inhibition of glutathione reductase by CA in a cell-free system has been reported (Vander Jagt et al., 1997). Microsomal glucose 6-phosphatase has been found to be inhibited by certain unsaturated aliphatic aldehydes, e.g. HEX and other SH group-binding compounds (Jorgensen et al., 1992).
In the human colon cell line Caco-2, HEX induced less direct DNA damage compared with V79 (1 h incubation), as described previously (Glaab et al., 2001). However, after 3 h post-incubation, direct DNA damage in Caco-2 cells had increased, whereas in V79 cells it was markedly reduced. Whether this reflects differential repair of direct DNA damage in V79 as compared with Caco-2 cells is the subject of our further investigations. Of note, in both cell lines HEX showed a similar potential to induce oxidative DNA damage.
In conclusion, ,ß-unsaturated carbonyl compounds induce oxidative purine modifications (FPG-sensitive sites) in mammalian cells, in addition to direct DNA breakage. For induction of FPG-sensitive sites, alkenal-mediated secondary ROS generation might play a role. Since this oxidative damage becomes apparent after post-incubation (for up to 3 h) it appears to be a consequence of GSH depletion, of additional processes interfering with further antioxidant defence systems. This cellular imbalance of pro- and antioxidative reactions might result in increased endogenous ROS production. Formation of promutagenic oxidized bases, such as 8-hydroxyguanine, might well contribute to the described genotoxic potential of these compounds that have been described to generate cyclic propano adducts of DNA bases. Moreover, enhanced ROS generation might initiate lipid peroxidation and other processes, leading to enhanced cytotoxic/genotoxic cell damage.
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Acknowledgements |
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We thank M.Lorez and N.Fiedler for assistance in cell culture and determination of glutathione and DNA damage and A.R.Collins for critically reading the manuscript.
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Notes |
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2To whom correspondence should be addressed. Tel: +49 631 205 2532; Fax: +49 631 205 3085; Email: janzo{at}rhrk.uni-kl.de
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Received on March 31, 2003; revised on May 29, 2003; revised on June 19, 2003; accepted on June 23, 2003.
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