Oxidative stress promotes the development of transformation: involvement of a potent mutagenic lipid peroxidation product, acrolein

Wakako Takabe, Etsuo Niki, Koji Uchida1,, Satoshi Yamada2,, Kimihiko Satoh3, and Noriko Noguchi4,

Research Center for Advanced Science and Technology, The University of Tokyo, Komaba, Meguro, Tokyo 153-8904,
1 Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Nagoya 464-8601,
2 Tsukuba Research Laboratory, NOF Co., Tsukuba 300-2635,
3 Second Department of Biochemistry, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The effect of intracellular oxidative stress on the development of cell transformation was studied. Mouse embryo C3H/10T1/2 fibroblasts pre-treated with benzo[a] pyrene, developed transformed foci on exposure to free radical generators, such as 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and 3-morpholinosydnonimine hydrochloride (SIN-1). These compounds generate peroxyl radicals and peroxynitrite, respectively. Neither AAPH nor SIN-1 alone induced transformation. The level of intracellular antioxidants, such as {alpha}-tocopherol and glutathione (GSH), decreased with time of exposure to the free radical generators, whereas the addition of exogenous {alpha}-tocopherol, GSH and ebselen showed a reduction in the frequency of transformation. An early event during exposure to AAPH and SIN-1 was the generation of acrolein, a highly mutagenic lipid peroxidation product, which was suppressed by the addition of {alpha}-tocopherol. Furthermore, it was confirmed that acrolein induced the transformation of cells which were pre-treated with benzo[a]pyrene but not of the untreated cells. These results suggest that acrolein may act as an important mediator of cell transformation under oxidative stress.

Abbreviations: AAPH, 2,2'-azobis(amidinopropane) dihydrochloride; BP, bensol[a]pyrene; D-MEM, Dulbecco's modified Eagle medium; DMSO, dimethyl sulfoxide; D-PBS, Dulbecco's-phosphate buffered saline; FBS, fetal bovine serum; FDP-lysine, N{varepsilon}-(3-formyl-3,4-dehydropiperidino) lysine; GSH, glutathione; H2O2, hydrogen peroxide; HNE, 4-hydroxy-2-nonenal; HO , hydroxyl radical; 1O2, singlet oxygen; O2 –, superoxide; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; PMA, phorbol 12-myristate 13-acetate; RNS, reactive nitrogen species; ROS, reactive oxygen species; SIN-1, 3-morpholinosydnonimine hydrochloride; SOD, superoxide dismutase; SOS, sodium 1-octansulfonate.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Reactive oxygen species (ROS) and more recently reactive nitrogen species (RNS), such as superoxide (O2 –), hydroxyl radical (HO), singlet oxygen (1O2), hydrogen peroxide (H2O2) and peroxynitrite are thought to play an important role in carcinogenesis (15). In support of this concept, 8-hydroxy-2'-deoxyguanosine (8-OHdG), one of the major products of DNA base modification, has been found to increase in human carcinoma cells (610). These data imply that convergent mechanisms dependent on ROS and RNS may explain a role in the process of carcinogenesis. One hypothesis is that mediators of the effects of ROS and RNS may lie in the initiation of the formation of oxidation products including those derived from lipid peroxidation. For example, the generation of 4-hydroxy-2-nonenal (HNE), which is one of the final products of lipid peroxidation, has been observed in human colorectal carcinoma (10) and in an experimental animal model of renal carcinoma (11,12). These findings and the observation that antioxidants elicit responses consistent with an anti-cancer effect in experimental animals (5,13) and in in vitro cell culture systems (14,15) also support the involvement of oxidative stress in carcinogenesis. However, few specific mediators derived from cellular lipid peroxidation have been identified.

Carcinogenesis is a multistep process (16), but model systems, in which cultured cells are treated with a mutagen and/or carcinogen and develop transformed foci, have been established (17). It has been reported that O2 – promotes the transformation of the benzo[a]pyrene pre-treated C3H/10T1/2 mouse embryo fibroblast, which was suppressed by superoxide dismutase (SOD) and/or catalase (18). In the present study, the potency of peroxyl radical and peroxynitrite to promote the transformation of cells was investigated. Peroxynitrite is known to initiate the lipid peroxidation of the phosphatidylcholine liposomes and low density lipoprotein (1921). The peroxyl radical plays a pivotal role as a chain carrying species for lipid peroxidation independent of the chain initiating species resulting in generation of the oxidation products. Acrolein is a terminal product of lipid peroxidation and is also highly reactive toward proteins and DNA (22,23). In the present study, we followed the generation of acrolein in the early stages of the exposure of ROS and RNS to cells and present evidence that implicates acrolein as a promoter of transformation.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
{alpha}-Tocopherol was kindly supplied by Eisai (Tokyo, Japan). Ebselen was a kind gift from Daiichi Pharmaceutical (Tokyo, Japan). Benzo[a]pyrene and 2,2'-azobis(amidinopropane) dihydrochloride (AAPH) were obtained from Wako (Osaka, Japan). 3-Morpholynosydnonimine hydrochloride (SIN-1) was obtained from Dojindo (Kumanato, Japan). Phorbol 12-myristate 13-acetate (PMA), sodium 1-octansulfonate (SOS), dimyristoyl phosphatidylcholine and glutathione (GSH) were obtained from Sigma (St Louis, MO). Dulbecco's modified Eagle medium (D-MEM) and calcium free Dulbecco's-phosphate buffered saline (D-PBS) were obtained from GIBCO BRL, Life Technologies, Inc (Rockville, MD). Fetal bovine serum (FBS) was obtained from JRH Biosciences (Tokyo, Japan).

Cell culture and transformation assay
Mouse embryo C3H/10T1/2 fibroblasts were cultured in D-MEM containing 10% FBS in a 100 mm dish in an atmosphere of 5% CO2 in air at 37°C. We followed the experimental protocol and criteria for the transformation described in the literature (18). Details of the experimental protocol are shown in Scheme 1.Go



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Scheme 1. Time schedule of experiment of cell transformation. After 48 h treatment of cells with 0.5 µM benzo[a]pyrene (BP), the medium was removed and cells were washed with D-PBS. AAPH and SIN-1 were added every 4 days and daily, respectively, for 3 weeks. The antioxidants were added 5 h before the addition of the free radical generators. To exclude non-specific effects of antioxidants the cells pre-treated with BP were treated with each antioxidant without the addition of the free radical generators. After the period for chemical exposure, the medium was changed every 4 days. The control cells were treated with the appropriate vehicle and the medium changed according to the same protocol for reagent exposure.

 
Treatment of cells with antioxidants and free radical generators
The concentrations of AAPH and SIN-1 used in the study were determined by testing their cytotoxicity in cell culture and by measuring their potential to cause lipid peroxidation in soybean phosphatidylcholine liposomes. The effect of the antioxidants on the radical generating capacity of AAPH and SIN-1 was determined in a separate series of experiments. The oxygen consumption due to oxygen radical generation by the thermal decomposition of AAPH and SIN-1 was measured in a dimyristoyl phosphatidylcholine liposome suspension in PBS (pH 7.4) with an oxygen monitor equipped with an oxygen electrode (Model YSI 5300; Yellow Springs Instrument, Yellow Springs, OH, USA). GSH was added into the aqueous phase whereas the lipophilic antioxidants, such as {alpha}-tocopherol and ebselen, were added as a methanol or acetonitrile solution.

The cytotoxicity of the radical generators was judged from the effect on the viability of cells with trypan blue staining. AAPH and SIN-1 were added in a solution of sterile PBS to the culture medium. {alpha}-Tocopherol and ebselen were added as a dimethyl sulfoxide (DMSO: final concentration, 0.1%) solution. The time schedule for the transformation experiment is shown in Scheme 1.Go Forty-eight hours after treatment with benzo[a]pyrene which was added as a DMSO (final concentration, 0.1%) solution, the medium was removed. After the cells were washed three times with D-PBS, they were treated with the free radical generators. Antioxidants were added for a period of 5 h before the addition of the free radical generator. Only the vehicle for the antioxidant was added to the control cells. AAPH and SIN-1 decompose to give peroxyl radical (24) and both O2 – and NO, respectively. In the case of SIN-1 a competition then ensues for cellular superoxide dismutase and NO resulting in the formation of peroxynitrite (25,26). Since the half-lives of AAPH and SIN-1 are about 6 days (24) and 52 min (unpublished data), respectively, AAPH was added every 4 days and SIN-1 was added daily as a PBS solution for 3 weeks. To investigate the ability of acrolein to induce transformation of cells, acrolein was added daily as a DMSO solution to the cells for 3 weeks. The treatment of cells with the chemicals was performed in a CO2 incubator at 37°C.

Preparation for analysis of intracellular antioxidants
Aliquots of cultured cells were used for analysis. At an appropriate time after treatment with free radical generators, cells were trypsinized and centrifuged at 100 g for 3 min.

Analysis of {alpha}-tocopherol
The cell pellet was dissolved in 50 µl 10 mM EDTA aqueous solution and was extracted by mixing with 125 µl methanol and 250 µl hexane for 5 min, followed by centrifugation at 13 000 g for 3 min at 4°C. Aliquots of the hexane layer were injected onto an HPLC. {alpha}-Tocopherol was analyzed by electrochemical detector (2005 EC, Shiseido) set at 600 mV using LC-8 column (5 µm particle size, 250x4.6 mm, Supelco) and 50 mM NaClO4 in methanol as a mobile phase.

Analysis of GSH
Cells were homogenized in 100 µl 5% trichloroacetic acid and centrifuged at 13 000 g for 3 min at 4°C. The supernatant was filtered (0.2 µm pore size, Millipore) and injected onto an HPLC. GSH was determined by measuring the oxidative current over a range of electrode potentials by coulometric detection (Coulochem II, ESA) with a guard cell and an analytical cell, which were set at 550 and 700 mV, respectively. LC-18 column (5 µm, 250x4.6 mm, Supelco) in column oven set at 15°C was used and 2% acetonitrile aqueous solution (pH 2.7) containing 75.4 mM sodium phosphate and 147 µM sodium 1-octansulfonate (SOS) was used as the mobile phase.

Determination of acrolein
Acrolein in cell homogenates prepared by using a microsonicator in 50 mM phosphate buffer (pH 7.2) was assayed by either competitive or direct ELISA using a monoclonal antibody raised against acrolein–lysine adducts as previously reported (20).

Protein assay
Protein concentration was determined by protein assay reagent (BioRad, Hercules, CA) using serum albumin as a standard.

Statistical analyses
Groups of test data (mean ± SD) were compared for significant differences using a Students t-test for unpaired observations.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Induction of transformation by free radical generators
The potency of peroxyl radical and peroxynitrite as transformation inducers was investigated using mouse embryo C3H/10T1/2 cells. AAPH and SIN-1 were used as generators of peroxyl radical and peroxynitrite, respectively. The ratio of the concentrations of AAPH and SIN-1 that induced lipid peroxidation in the phosphatidylcholine liposomal membranes to a similar extent was 5:1 (data not shown). Neither AAPH (200 µM) nor SIN-1 (40 µM) at the concentration used in the present study affected the viability of the cells.

Cells treated with benzo[a]pyrene alone developed transformed foci to some extent, whereas treatment with AAPH or SIN-1, after benzo[a]pyrene pre-treatment, significantly enhanced the number and size of foci (Figure 1Go). Figure 2Go shows the quantification of these data over a number of experiments. Treatment with either AAPH or SIN-1 alone did not induce transformation of cells. Treatment of cells with PMA following a 48 h incubation of the cells with AAPH or SIN-1 did not induce transformation (data not shown). These results suggest that both peroxyl radical and peroxynitrite can act as inducers of cell transformation after benzo[a]pyrene pre-treatment in these two-step carcinogenesis experiments.



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Fig. 1. Transformation of C3H10T1/2 induced by treatment with 0.5 µM benzo[a]pyrene and free radical generators. The concentrations of AAPH and SIN-1 were 200 and 40 µM, respectively.

 


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Fig. 2. Transformation of benzo[a]pyrene-treated C3H/10T1/2 cells by reactive oxygen and nitrogen species. The treatment of cells was performed according to Scheme 1.Go BP represents treatment of cells with 0.5 µM benzo[a]pyrene for 48 h. AAPH (200 µM) or SIN-1 (40 µM) was added to cells every 4 days or daily, respectively, for 3 weeks with and without BP treatment. *Statistical significance versus NONE (P < 0.005); **statistical significance versus BP (P < 0.005).

 
Change in the intracellular level of antioxidants during exposure to free radicals
Changes in the intracellular level of the antioxidants, such as {alpha}-tocopherol and GSH, during exposure of the cells to free radicals were examined. {alpha}-Tocopherol is one of the most abundant lipophilic antioxidants in vivo, whereas GSH is a hydrophilic antioxidant and antioxidant enzyme co-factor present in millimolar concentrations. These two endogenous antioxidants have distinct but complementary activity as scavengers. Glutathione reacts directly with peroxynitrite and in conjunction with enzymes detoxifies lipid peroxidation products but not peroxyl radicals. In contrast, {alpha}-tocopherol is an excellent scavenger of lipid peroxyl radicals in the cell and also reacts with peroxynitrite to form tocopherol quinone. The culture medium was not changed for 96 h during the experiment. AAPH was added once at time 0 and SIN-1 was added every 24 h. As shown in Figure 3aGo, {alpha}-tocopherol decreased with time in both treatments. The extent of depletion of {alpha}-tocopherol was more remarkable during the SIN-1 treatment and, after 72 h of incubation, >90% of the {alpha}-tocopherol was depleted. In contrast, about 50% of {alpha}-tocopherol still remained after 96 h of incubation with AAPH. The change in intracellular GSH was modest and did not achieve statistical significance (Figure 3bGo). This is possible because both enzymatic regeneration and de-novo synthesis were able to compensate for consumption on exposure to ROS and RNS. These results suggest that both inducers of oxidative and nitrosative stress, AAPH and SIN-1, targeted membranes for modification and this process was occurring in the cells during the transformation process.



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Fig. 3. Changes in intracellular antioxidants by free radical exposure. Cells were treated with either 200 µM AAPH or 40 µM SIN-1 for 96 h in culture medium in 100-mm dish. AAPH was added once at time 0 and SIN-1 was added four times every 24 h. At appropriate times, cells were removed from the dish and (a) {alpha}-tocopherol and (b) GSH were measured. {circ}, none; {blacktriangleup}, AAPH; {blacksquare}, SIN-1.

 
Inhibition of free radical-induced transformation by antioxidants
To confirm that ROS/RNS play a role in the observed transformation, the effects of the simultaneous addition of antioxidants were studied. In this experiment, in addition to exogenous {alpha}-tocopherol and GSH, ebselen, a selenium-containing synthetic antioxidant, which is known to have GSH-peroxidase-like activity and reduces peroxynitrite, was used. Since ebselen is a lipophilic antioxidant, it partitions to the cell membranes or lipoprotein in the culture medium.

In the first series of experiments indirect effects of the antioxidants on the ROS/RNS generating systems were determined. This was approached by measuring oxygen consumption in a lipid phase system to model the membrane but composed of saturated fatty acids and thus incapable of supporting lipid peroxidation. This design removes the effects of the antioxidants on the initiating oxidants or propagation phase as would occur in the plasma membrane of the cell. The rate of oxygen consumption induced by 20 mM AAPH was 11.2 ± 0.10 nM/s, which was not changed by the addition of GSH (2 mM), {alpha}-tocopherol (100 µM) or ebselen (100 µM). The rate of oxygen consumption induced by 200 µM SIN-1 in the presence of the saturated liposome was 12.2 ± 0.02 nM/s. In the presence of the highest concentration of ebselen (100 µM) the rate of oxygen consumption was 10.4 ± 0.03 nM/s. These data indicate that the antioxidants used at the concentrations in the cell experiments are unable to significantly change the generation of ROS/RNS from AAPH and SIN-1.

Supplementing the culture medium with {alpha}-tocopherol (100 µM) produced about a 1.5-fold increase of intracellular {alpha}-tocopherol after 3 h of incubation, whereas addition of GSH (2 mM) into the medium did not change the concentration of intracellular GSH (data not shown). As reported in Table IGo, GSH showed the most significant inhibition in both the AAPH- and SIN-1-induced transformations. The fact that {alpha}-tocopherol also inhibited the frequency of transformation induced by AAPH and SIN-1 to 39 and 13%, respectively, of the positive control suggests that lipid peroxidation in the cell membranes is involved in the development of transformation.


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Table I. Inhibition of free radical-induced transformation of C3H/10T1/2 cells by antioxidants
 
Ebselen was more effective against SIN-1-induced transformation than AAPH most likely because of its decomposing activity towards peroxynitrite. It should be noted that ebselen, which does not act directly as a peroxyl radical scavenger, showed an inhibitory effect against the AAPH-induced transformation consistent with a mechanism involving downstream products of lipid peroxidation rather than peroxyl radicals per se. In a series of control experiments it was found that none of the antioxidants used in the present study inhibited BP-initiated transformation (Figure 4Go).



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Fig. 4. Effect of antioxidants on BP-induced transformation. The treatment of cells was performed according to Scheme 1.Go BP represents treatment of cells with 0.5 µM benzo[a]pyrene for 48 h. {alpha}-Tocopherol (100 µM), ebselen (20 µM) and GSH (2 mM) were added to BP-treated cells every 4 days for 3 weeks.

 
Generation of acrolein within cells by exposure to ROS/RNS
To test the concept, suggested by the previous data, that lipid peroxidation produced in the cells contributes to the development of transformation, the formation of acrolein was measured. This lipid peroxidation product has also been shown to form stable adducts with proteins and so could contribute to transformation. Both AAPH and SIN-1 induced a remarkable increase in the intracellular amount of acrolein, which was quantified by ELISA with a monoclonal antibody raised against the acrolein–lysine adduct (Figure 5Go). Acrolein was generated more slowly in the cells in response to AAPH-treated cells compared with SIN-1. Acrolein generated within the cells decreased with time, suggesting that secondary reactions and/or turnover of the modified proteins were also occurring. The generation of acrolein induced by AAPH and SIN-1 was inhibited by pre-incubation of the cells with {alpha}-tocopherol (Figure 6a and bGo). Acrolein was not increased in the cells treated with benzo[a]pyrene alone or in combination with {alpha}-tocopherol.



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Fig. 5. Generation of acrolein within cells by exposure to reactive oxygen and nitrogen species. Cells were treated as in Figure 3Go and acrolein was measured by ELISA using a monoclonal antibody raised against acrolein–lysine adduct. {blacktriangleup}, AAPH; {blacksquare}, SIN-1.

 


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Fig. 6. Inhibition of generation of acrolein in free radical generator-treated cells by {alpha}-tocopherol. Cells were treated with 0.5 µM benzo[a]pyrene (BP) for 48 h and incubated with 100 µM {alpha}-tocopherol (Toc) for 5 h. After treatment with either (a) 200 µM AAPH or (b) 40 µM SIN-1 for 3 and 1.5 h, respectively, acrolein was measured by ELISA using a monoclonal antibody. *Statistical significance (P < 0.005).

 
Induction of transformation by acrolein
In order to confirm the ability of acrolein to induce transformation of cells, C3H/10T1/2 cells were treated with acrolein following pre-treatment with benzo[a]pyrene. The concentration of acrolein for this experiment was estimated from the results with AAPH and SIN-1 (Figure 5Go). However, since the increase in acrolein was transient it was added to the cells daily. Acrolein significantly induced transformation of benzo[a]pyrene-treated cells but not control untreated samples (Figure 7Go), suggesting that acrolein is capable of mediating transformation induced by reactive oxygen and nitrogen species. These concentrations of acrolein did not increase the rate of cell transformation in a dose-dependent manner due to cytotoxic effects.



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Fig. 7. Transformation of benzo[a]pyrene-treated C3H/10T1/2 cells by acrolein. The treatment of cells was performed according to Scheme 1.Go BP represents treatment of cells with 0.5 µM benzo[a]pyrene for 48 h. Acrolein was added to cells daily for 3 weeks with and without BP treatment. *Statistical significance versus NONE (P < 0.005); **statistical significance versus BP (P < 0.005).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The promotion effect of PMA or O2 – could be demonstrated for mouse embryo C3H/10T1/2 cells that had been initiated by benzo[a]pyrene (18,27), X-rays (28) or ultraviolet light (29). Although carcinogenesis is a complex multistep process, this method is useful for evaluating the potency of chemicals in carcinogenesis. In the present study, it was shown that the direct addition of peroxyl radicals or secondary lipid peroxidation initiated by the simultaneous formation of NO and O2 was capable of acting as promoter in cells pre-treated by benzo[a]pyrene.

The AAPH and SIN-1-dependent transformation was suppressed by the antioxidants used in this study. The hydrophilic antioxidant, GSH, was the most effective for inhibiting the development of transformation induced by both AAPH and SIN-1, suggesting that the ROS and RNS generated from AAPH and SIN-1 can be neutralized effectively by this compound. This is consistent with the known ability of GSH to scavenge peroxynitrite and to decompose lipid peroxides or terminate the aldehydic mediators we have identified in the present study. It has recently been reported that in a cellular setting the balance between NO and O2 –, and hence peroxynitrite formation, from SIN-1 can be altered through the cell providing alternate electron acceptors than oxygen (30). However, even though the yield of peroxynitrite will be altered by these reactions it has been shown that this oxidant is formed in substantial amounts in a cellular setting (31). The effects of the lipophilic antioxidants even at the higher concentration on the generation of ROS and RNS from AAPH and SIN-1 is minor and cannot explain inhibition of transformation by these compounds. The effective inhibition of the SIN-1-induced transformation by ebselen is most likely due to the decomposition of peroxynitrite by ebselen (32). Interestingly, ebselen showed a moderate inhibition against the AAPH-induced transformation. It is known that ebselen does not scavenge peroxyl radicals but reduces the hydroperoxide to the corresponding alcohol (33,34). This would also decrease formation of aldehydes generated via the lipid peroxide as an intermediate. The observation of the inhibitory effect of ebselen on the AAPH-induced transformation is also consistent with the hypothesis that decomposition products of lipid peroxides, including aldehydes, are formed within the cells and are involved in the development of transformation. These aldehydes represent the end products of lipid peroxidation but are still highly reactive to various biomolecules such as proteins and DNA, which are thought to contribute to the pathogenesis of a number of diseases including cancer (10,35).

Alkylating agents, including acrolein, are versatile mutagens and/or carcinogens. Among all the {alpha},ß-unsaturated aldehydes, acrolein is by far the strongest electrophile and shows the highest reactivity with the nucleophilic, functional groups present in proteins and DNA (36). Like other {alpha},ß-unsaturated aldehydes, acrolein selectively reacts with cysteine, histidine and lysine residues of proteins. Of these, lysine is reported to generate the most stable product, N{varepsilon}-(3-formyl-3,4-dehydropiperidino) lysine (FDP-lysine), which is recognized by the monoclonal antibody (mAb5F6) (22) used in this study. In addition to the reactions with proteins, acrolein reacts with a variety of nucleophilic sites in DNA, forming cyclic propano and other adducts with DNA bases (37,38). Indeed, acrolein is known to cause chromosomal aberrations, sister chromatid exchanges, point mutations and reduce the colony-forming efficiency of mammalian cells (39). Moreover, acrolein has been suggested to initiate bladder cancer in rats under certain conditions (40). Consistent with its potential in mutagenesis and carcinogenesis, site-specific mutagenesis studies have shown that acrolein based adducts are likely to induce mutations (41,42). Here it is shown that acrolein was formed during the exposure of cells to both ROS and RNS and that acrolein induced transformation of the cells. The involvement of acrolein and its adducts in the development of transformation is then a potentially convergent route for this process on exposure of cells to oxidative or nitrosative stress.


    Notes
 
4 To whom correspondence should be addressed Email: nonoriko{at}oxygen.rcast.u-tokyo.ac.jp Back


    Acknowledgments
 
We are grateful Dr Victor Darley-Usmar (Alabama University, USA) for helpful discussions. This work was supported in part by Grant-in-Aid for Cancer Research (10-4) from the Ministry of Health and Welfare.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 21, 2000; revised February 13, 2001; accepted February 15, 2001.