Mutagenic/recombinogenic effects of four lipid peroxidation products in Drosophila

Food and Chemical Toxicology 53 (2013) 221–227

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Food and Chemical Toxicology

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Mutagenic/recombinogenic effects of four lipid peroxidation products in Drosophila

Es�ref Demir a,⇑, Fatma Turna a, Bülent Kaya a, Amadeu Creus b,c, Ricard Marcos b,c

a Akdeniz University, Faculty of Sciences, Department of Biology, 07058 Campus, Antalya, Turkeyb Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Biociències, Universitat Autònoma de Barcelona, Campus de Bellaterra,08193 Cerdanyola del Vallès, Spainc CIBER Epidemiología y Salud Pública, Instituto de Salud Carlos III, Spain

a r t i c l e i n f o

Article history:Received 31 October 2012Accepted 29 November 2012Available online 10 December 2012

Keywords:Somatic mutation and recombination test(SMART)Wing spot testAcroleinCrotonaldehyde4-Hydroxy-hexenal4-Oxo-2 nonenal

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⇑ Corresponding author. Tel.: +90 242 310 22 43; faE-mail address: [email protected] (E. Dem

a b s t r a c t

The human diet is an important factor in the development of different diseases. Lipid peroxidation duringfrying in edible vegetable liquid oils of food components is a mechanism leading to the formation of freeradicals. Such radicals induce tissue damage and are implicated in diverse pathological conditions,including aging, atherosclerosis, brain disorders, cancer, lung disorders and various liver disorders. Inthe present study, we decided to investigate the genotoxic effects of four lipid peroxidation productsin the in vivo Drosophila wing somatic mutation and recombination test. In this test, point mutation, chro-mosome breakage and mitotic recombination produce single spots; while twin spots are produced onlyby mitotic recombination. Drosophila is a suitable eukaryotic organism for mutagenicity studies and alsoits metabolism is quite similar to that of mammalians. Since conflicting data exist on the possible risk ofseveral lipid peroxidation products for humans, we have selected four of them, namely acrolein, croton-aldehyde, 4-hydroxy-hexenal (4-HHE) and 4-oxo-2-nonenal (4-ONE). Especially at the highest concentra-tions tested all exert both mutagenic and recombinogenic effects in the Drosophila SMART assay, showinga direct dose–effect relationship. This is the first study reporting genotoxicity data in Drosophila for thesecompounds.

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1. Introduction

The main goal of the genetic toxicology is the evaluation of theDNA damage caused by the wide variety of environmental agents.Human populations are exposed to a large number of xenobioticcompounds, due to environmental and/or occupational sources aswell as to other factors related with diet, life style, habits and med-ical procedures. Epidemiological studies clearly evidenced that thehuman diet is an important factor in the development of severalcancer diseases (Block et al., 1992; Carr and Frei, 1999) and differentDNA adducts (Abraham et al., 2011). Thus, it is important to inves-tigate how safe are the natural and processed components of diet.

During various processes, such as thermal treatment of food-stuffs, many chemical reactions occur, including oxidation, hydro-lysis, polymerization, cyclization and isomerization reactions;leading to the formation of undesirable secondary products (Gertz,2000). Lipid peroxidation, resulting from free radical damage topolyunsaturated fatty acids, also generates cytotoxic alkanals likeacrolein, crotonaldehyde, 4-hydroxy-hexenal (4-HHE) and 4-oxo-2-nonenal (4-ONE) (Indart et al., 2002, 2007; Srivastava et al.,2010) and highly reactive compounds have formed during frying

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and cooking. Lipid peroxidation products, such as a,b-unsaturatedaldehydes, are able to covalently modify the DNA bases (Kawaiet al., 2004) and can cause tissue damage (Wacker et al., 2001),congenital malformations (Refsgaard et al., 2000) and variouschronic and degenerative (such as cancer, aging and human ath-erosclerosis) (Osada, 2002) and neurodegenerative diseases (Volkelet al., 2006; Schneider et al., 2008). Therefore, these products rep-resent a potential genotoxic risk to humans.

Several papers have been published on the toxicity and geno-toxicity of oxidized oils and the formation of toxic and genotoxiccompounds from such oxidized oils (Billek, 2000; Edem, 2002).Health aspects of thermally oxidized oils and fats have been re-viewed evaluating about 60 years of investigations performed toanswer the question if heated fats are detrimental to health (Billek,2000). Thermal stressing is produced during the frying process,resulting in the formation of a high number of new compoundsthrough thermal, oxidative and hydrolytic reactions. An importantroute for formation of new compounds is breakdown of hydroper-oxides giving rise to volatiles and short-chain compounds attachedto the glyceridic backbone forming part of non-volatile molecules.Whereas the volatiles are largely removed from the oil during fry-ing and have implications in the flavour of both the frying oil andthe fried food, the non-volatile compounds remain in the frying oiland are absorbed by the food modifying the oil nutritional and

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222 E. Demir et al. / Food and Chemical Toxicology 53 (2013) 221–227

physiological properties. These compounds, such as core alde-hydes, are easily absorbed in the intestinal tract after hydrolysisby pancreatic lipase. Furthermore, some reports on 9-oxononanoicacid, the major short-chain glycerol-bound compound originallybound to the triacylglycerol molecule in used frying fats and oils,indicate that it could induce lipid peroxidation and affect hepaticmetabolism (Minamoto et al., 1988; Kanazawa and Ashida,1991). From hydroperoxides, a wide range of secondary oxidationproducts are formed amongst which polymeric compounds standout, showing more or less high toxicity (Esterbauer, 1993).

The food-derived lipid peroxidation products are formed duringthe frying of foods with oil at high temperatures. Both food muta-gens and their metabolites can cause different types of DNA dam-age. Although several studies dealing with the genotoxic propertiesof oxidized oils and their secondary products have been reported,no studies on their genotoxic potential in Drosophila melanogasterimaginal wing disc cells have been conducted until now. For thisreason, we planned to carry out a genotoxicity study by using D.melanogaster as a test system.

The somatic mutation and recombination test (SMART) in D.melanogaster have been designed to detect a wide variety of geno-toxic effects in a rapid an inexpensive way in only one generation.The relevance of such SMART assays is to be in vivo procedures andthat the metabolic machinery of Drosophila is similar to that ofmammals (Vogel, 1987). However, >60% of genes associated withhuman diseases have Drosophila orthologues (Bernards and Harih-aran, 2001). The development of SMART assays has provided rapid,sensitive, versatile and also cheap ways to investigate both themutagenic and recombinogenic effects of chemicals. It must benoted that the quantitation of the recombinogenic activity of acompound is of primary importance for genotoxicity screening(Demir et al., 2008, 2011a).

Since the four lipid peroxidation products (acrolein, crotonalde-hyde, 4-HHE and 4-ONE) are widely formed during making friedfood in edible vegetable oils, there is a need for more genotoxicitydata in order to assess their potential health hazards for humanbeings.

2. Materials and methods

2.1. Chemicals

4-hydoxy-hexenal (4-HHE, P98% purity; CAS No. 160708-91-8) and 4-oxo-2-nonenal (4-ONE, P98% purity; CAS No. 103560-62-9) were purchased from CaymanChemical Company (Ann Arbor, MI, USA). Acrolein (P99% purity; CAS No. 107-02-8), crotonaldehyde (P99.5% purity; CAS No. 4170-30-3) and ethyl methanesulfo-nate (EMS, 99% purity; CAS No. 62-50-0) were obtained from Sigma–Aldrich ChemieGmbH (Steinheim, Germany). Prior to use, acrolein, crotonaldehyde and EMS weredissolved in distilled water, while 4-HHE and 4-ONE were dissolved in ethanol andmethyl acetate, respectively.

2.2. Drosophila strains

Two D. melanogaster strains were used: the multiple wing hairs strain with thegenetic constitution mwh/mwh, and the flare-3 strain with the genetic constitutionflr3/In (3LR) TM3, BdS. More detailed information on the genetic symbols and descrip-tions can be found in Lindsley and Zimm (1992).

2.3. Experimental procedure

The wing spot test is based on the loss of heterozygosity in somatic cells of lar-vae (Graf et al., 1984). The trans-heterozygous larvae were obtained by parentalcrosses between flr-3 virgin females and mwh males. Eggs from this cross were col-lected during 8 h periods in culture bottles containing standard growth medium.After 72 ± 4 h treatment, the larvae were floated off with tap water and then trans-ferred into plastic vials containing 1.5 g dry Drosophila Instant Medium (CarolinaBiological Supply Company Burlington, NC, USA) rehydrated with 9 mL of thefreshly prepared test solutions. The larvae were fed with different concentrationsof the test compounds. Feeding ended with pupation of the surviving larvae; thismeans that treatment last for about 48 h. All experiments were performed at25 ± 1 �C and a relative humidity of approximately 65%. The concentrations of the

lipid peroxidation products: 5, 10 and 25 mM for acrolein; 10, 25 and 50 mM forcrotonaldehyde; 0.1, 0.5 and 1 mM for 4-hydroxy-hexenal (4-HHE) and 4-oxo-2-nonenal (4-ONE). The distilled water, ethanol (1%) and methyl acetate (1.7%) servedas a negative control; 1 mM concentration of EMS was used as a positive control.

2.4. Preparation and microscopic analysis of wings

After metamorphosis, all surviving flies were collected from the treatment vialsand stored in a 70% ethanol solution at +4 �C. Afterwards, the wings were carefullyremoved, under an Olympus SZH model stereo microscope, and mounted on slidesin Faure’s solution (30 g gum arabic, 30 mL glycerol, 50 g chloral hydrate and 50 mLdistilled water). The wings were scored at 400� magnification using a Leitz Labor-lux S model light microscope, for the presence of clones of cells showing malformedwing hairs. Such somatic spots appeared as single spots, showing either the multi-ple wing hairs (mwh) or the flare (flr3) phenotype, and twin spots showing adjacentmwh and flr3 areas. Three categories of spots were recorded: small single spots (1–2cells), large single spots (>2 cells) and twin spots (Demir et al., 2011a; Vales et al., inpress). In each series, 80 wings (40 individuals) were examined. The scoring of fliesand data evaluation were performed following the standard procedures for thewing spot assay, as used in recent investigations (Demir et al., 2011a; Vales et al.,in press).

2.5. Statistical analysis

The conditional binomial test of Kastenbaum and Bowman (1970) was appliedto assess differences between the frequencies of each type of spot in treated and theconcurrent negative control, with significant levels of a = b = 0.05. The multipledecision procedure proposed by Frei and Würgler (1988) was applied to judgethe overall response of an agent as positive, weakly positive, negative, or inconclu-sive. As recommended, we consider the treatment as positive if the frequency ofmutant spots in the treated series is at least m (multiplication factor) times greaterthan in the control series. Since small single spots and total spots have a compara-tively high spontaneous frequency, m is fixed at a value of 2 (testing for a doublingof the spontaneous frequency). For the large single spots and the twin spots, whichhave a low spontaneous frequency, m = 5 is used. The frequency of clone formationwas calculated, without size correction, by dividing the number of mwh clones perwing by 24,400, which is the approximate number of cells inspected per wing(Alonso-Moraga and Graf, 1989). The percentage of recombination induced by thetreatments with crotonaldehyde, 4-HHE and 4-ONE were calculated according toFrei and Würgler (1995).

3. Results

In this study we report the genotoxic activity of acrolein,crotonaldehyde, 4-HHE and 4-ONE, in the wing spot test ofDrosophila. From our study we can conclude that all selectedcompound have mutagenic and recombinogenic potential in thesomatic cells of Drosophila, being the first reported data using thisassay of Drosophila.

The results obtained in the Drosophila wing spot test after larvaeexposure to acrolein, crotonaldehyde, 4-HHE and 4-ONE are shownin Tables 1–4. As expected, EMS (at only 1 mM), which was used asa positive control, induced all kinds of spots and confirmed itsstrong mutagenic and recombinogenic activity (Graf et al., 1984).

Preliminary toxicity studies were carried out to define the rangeof doses to be tested in the genotoxicity studies. Toxicity was mea-sured as the increase in the percentage of treated larvae that doesnot reach the adult stage. The selected highest doses induced amortality lower than 75% in comparison with control. Moreover,the criteria to choose the final selected doses were based on tworeasons: First, a reduction in the percentage of developing treatedlarvae is a clear indication that the compounds affected the larvaeand, in addition, the number of emerging adults must be high en-ough to perform the genotoxicity experiments.

Table 1 shows the results obtained with the transheterozygouslarvae treated with the different doses of acrolein. As indicated,this chemical was administered to 3-day-old (third instar) larvaeat doses ranging from 5 to 25 mM. The treatment was given tothe larvae until they completed development (almost 48 h). The re-sults obtained from trans-heterozygous wings (mwh/flr3) demon-strated either negative or inconclusive results for all categories ofspots at 5 mM exposure concentration, while demonstrated clearly

Table 1Summary of the results obtained in the Drosophila wing spot test after treatments with acrolein.

Test compounds andconc. (mM)

Number ofwings (N)

Small singlespots (1–2 cells)(m = 2)

Large singlespots (>2 cells)(m = 5)

Twin spots(m = 5)

Total mwh spots(m = 2)

Total spots(m = 2)

Frequency of cloneformation per 105 cells

No Fr D No Fr D No Fr D No Fr D No Fr D

Marker heterozygous wings (mwh/flr3)Distilled water 80 14 (0.18) 7 (0.09) 0 (0.00) 20 (0.25) 21 (0.26) 1.025 80 23 (0.29) i 1 (0.01) � 0 (0.00) i 24 (0.30) i 24 (0.30) � 1.2310 80 31 (0.39) + 7 (0.09) � 1 (0.01) i 39 (0.49) + 39 (0.49) + 2.0025 80 38 (0.48) + 3 (0.04) � 1 (0.01) i 42 (0.53) + 42 (0.53) + 2.151 mM EMS 80 218 (2.73) + 82 (1.03) + 18 (0.23) + 310 (3.88) + 318 (3.98) + 15.88

Balancer heterozygous wings (mwh/TM3)Distilled water 80 19 (0.24) 2 (0.03) 21 (0.26) 21 (0.26) 1.085 80 18 (0.23) � 4 (0.05) i 22 (0.28) � 22 (0.28) � 1.1310 80 33 (0.41) + 12 (0.15) + 45 (0.56) + 45 (0.56) + 2.3125 80 37 (0.46) + 3 (0.04) i 40 (0.50) + 40 (0.50) + 2.051 mM EMS 80 63 (0.79) + 30 (0.38) + 85 (1.06) + 93 (1.16) + 4.35

Conc: concentration, No: number, Fr: frequency, D: statistical diagnosis according to Frei and Würgler (1988), +: positive, �: negative,i: inconclusive; m: multiplication factor; probability levels: a = b = 0.05.

Table 2Summary of the results obtained in the Drosophila wing spot test after treatments with crotonaldehyde.


Number ofwings (N)



Twin spots(m = 5)


Total spots(m = 2)

Frequency of cloneformation per 105 cell


Marker heterozygous wings (mwh/flr3)Distilled water 80 14 (0.18) 1 (0.01) 1 (0.01) 16 (0.20) 16 (0.20) 0.8210 80 22 (0.28) i 3 (0.04) i 2 (0.03) i 27 (0.34) i 27 (0.34) i 1.3825 80 32 (0.40) + 2 (0.03) i 3 (0.04) i 37 (0.46) + 37 (0.46) + 1.9050 80 35 (0.44) + 6 (0.08) i 1 (0.01) i 42 (0.53) + 42 (0.53) + 2.151 mM EMS 80 136 (1.70) + 80 (1.00) + 34 (0.43) + 240 (3.00) + 250 (3.13) + 12.30

Balancer heterozygous wings (mwh/TM3)Distilled water 80 18 (0.23) 5 (0.06) 23 (0.29) 23 (0.29) 1.1810 80 16 (0.20) � 4 (0.05) � 20 (0.25) � 20 (0.25) � 1.0225 80 24 (0.30) i 9 (0.11) i 33 (0.41) i 33 (0.41) i 1.6950 80 16 (0.20) � 7 (0.09) � 23 (0.29) � 23 (0.29) � 1.181 mM EMS 80 77 (0.96) + 25 (0.31) + 100 (1.25) + 102 (1.28) + 5.12


Table 3Summary of the results obtained in the Drosophila wing spot test after treatments with4-Hydroxy-hexenal (4-HHE).


Number ofwings (N)



Twin spots(m = 5)


Total spots(m = 2)

Frequency of cloneformation per 105 cells


Marker Heterozygous wings (mwh/flr3)Distilled water 80 19 (0.24) 0 (0.00) 0 (0.00) 19 (0.24) 19 (0.24) 0.97Ethanol (1%) 80 23 (0.29) i 2 (0.03) i 1 (0.01) i 26 (0.33) i 26 (0.33) i 1.330.1 80 22 (0.28) i 4 (0.05) i 0 (0.00) i 26 (0.33) i 26 (0.33) i 1.330.5 80 31 (0.39) i 8 (0.10) + 1 (0.01) i 40 (0.50) + 40 (0.50) + 2.051 80 71 (0.89) + 21 (0.26) + 9 (0.11) + 98 (1.23) + 101 (1.26) + 5.021 mM EMS 80 167 (2.09) + 83 (1.04) + 31 (0.39) + 275 (3.44) + 281 (3.51) + 14.09

Balancer heterozygous wings (mwh/TM3)Distilled water 80 16 (0.20) 5 (0.06) 21 (0.26) 21 (0.26) 1.08Ethanol (1%) 80 14 (0.18) � 7 (0.09) � 21 (0.26) � 21 (0.26) � 1.080.1 80 12 (0.15) � 14 (0.18) + 26 (0.33) i 26 (0.33) i 1.330.5 80 14 (0.18) � 13 (0.16) + 27 (0.34) i 27 (0.34) i 1.381 80 28 (0.35) + 15 (0.19) + 43 (0.54) + 43 (0.54) + 2.201 mM EMS 80 74 (0.93) + 35 (0.44) + 108 (1.35) + 109 (1.36) + 5.53


E. Demir et al. / Food and Chemical Toxicology 53 (2013) 221–227 223

Table 4Summary of the results obtained in the Drosophila wing spot test after treatments with 4-oxo-2-nonenal (4-ONE).

Test compounds and conc.(mM)

Number of wings(N)

Small singlespots (1–2cells) (m = 2)


Twin spots(m = 5)

Total mwhspots (m = 2)

Total spots(m = 2)

Frequency of cloneformation per 105

cells


Marker Heterozygous wings (mwh/flr3)Distilled water 80 18 (0.23) 3 (0.04) 3 (0.04) 24 (0.30) 24 (0.30) 1.23Methyl acetate (1.7%) 80 20 (0.25) i 1 (0.01) - 0 (0.00) � 21 (0.26) � 21 (0.26) � 1.080.1 80 19 (0.24) � 5 (0.06) i 1 (0.01) � 25 (0.31) � 25 (0.31) � 1.280.5 80 21 (0.26) i 3 (0.04) i 0 (0.00) � 24 (0.30) � 24 (0.30) � 1.231 80 44 (0.55) + 4 (0.05) i 2 (0.03) � 50 (0.63) + 50 (0.63) + 2.561 mM EMS 80 57 (0.71) + 24 (0.30) + 10 (0.13) + 90 (1.13) + 91 (1.14) + 4.61

Balancer heterozygous wings (mwh/TM3)Distilled water 80 18 (0.23) 1 (0.01) 19 (0.24) 19 (0.24) 0.97Methyl acetate (1.7%) 80 17 (0.21) � 3 (0.04) i 20 (0.25) � 20 (0.25) � 1.020.1 80 19 (0.24) � 3 (0.04) i 22 (0.28) i 22 (0.28) i 1.130.5 80 20 (0.25) i 1 (0.01) i 21 (0.26) � 21 (0.26) � 1.081 80 19 (0.24) � 5 (0.06) i 24 (0.30) i 24 (0.30) i 1.231 mM EMS 80 50 (0.63) + 4 (0.05) i 54 (0.68) + 54 (0.68) + 2.77



positive results at 10 and 25 mM for small single spots, total mwhspots and total spots. On the contrary, the data obtained from bal-ancer-heterozygous wings (mwh/TM3) after exposure to 10 and25 mM of acrolein demonstrated clearly positive results for smallsingle spots, total mwh spots and total spots. In other words, 10and 25 mM concentrations of acrolein showed mutagenic effectsin Drosophila, with a direct dose–response relationship, but with-out the induction of recombinogenic activity.

For crotonaldehyde, the results obtained from trans-heterozy-gous wings (mwh/flr3) demonstrated inconclusive results for allspots categories at 10 mM, while demonstrated clearly positive re-sults exposure at 25 and 50 mM concentrations for small singlespots, total mwh spots and total spots (Table 2). In balancer-hetero-zygous wings, recombination is suppressed and only clones in-duced by somatic mutation can be observed. The results obtainedfrom mwh/TM3 exposure to 10, 25 and 50 mM concentrations ofcrotonaldehyde are either negative or inconclusive. This wouldindicate that crotonaldehyde exerts its genotoxic effect mainly bymitotic recombination. From a comparison of the results obtainedin trans-heterozygous and balancer-heterozygous flies, we deter-mined that the fraction of the mutants in trans-heterozygous fliesdue to recombination increased 12.2% and 82.8% at 25 and 50 mMof crotonaldehyde, respectively (Table 2).

The results obtained from the testing of 4-HHE demonstratedpositive results for the different spots categories at 1 mM, in bothtypes of individuals, being the effects more pronounced in the mar-ker heterozygous wings (mwh/flr3). 4-HHE was mutagenic at 1 mMand also showed recombinogenic activity (47.1%) at 0.5 mM con-centration (Table 3).

Finally, in Table 4 there are the results obtained in the genotox-icity testing of 4-ONE. Looking at the results obtained with 4-ONE,the data from mwh/flr3 demonstrated either negative or inconclu-sive results for all categories of spots at 0.1 and 0.5 mM concentra-tions, while reflected clearly positive results at 1 mM for smallsingle spots, total mwh spots and total spots (Table 4). On the con-trary, the results obtained from mwh/TM3 exposure to all concen-trations of 4-ONE demonstrated negative or inconclusive results.According to the results obtained from 4-ONE showed a recombi-nogenic activity (110%) at 1 mM concentration (Table 4).

As a summary of the results obtained in this work, we can statethat the relative genotoxic potencies according to the inducedclone formation for the highest concentration are as follows: 4-HHE > 4-ONE > acrolein > crotonaldehyde.

4. Discussion

This study contributes to show the usefulness of Drosophila as aeukaryotic model for detecting the genotoxic potential of acrolein,crotonaldehyde, 4-HHE and 4-ONE. Our results, showing that lar-vae exposure to these compounds induces mutation and somaticrecombination in the wing imaginal disc cells, support not onlyDrosophila as a suitable biosystem but also reinforce the usefulnessof the wing spot test as an easy method to detect in vivo genotox-icity (Demir et al., 2008, 2011a, 2012). In this context, it must beremembered that the quantification of the recombinagenic activityof a compound is of primary importance for genotoxicity screening(Graf and Würgler, 1996), since this event is strongly linked to car-cinogenesis process (Sengstag, 1994; Lupski, 2007).

Previously we have investigated the effects of acrolein, croton-aldehyde, 4-HHE and 4-ONE on in vitro conditions in the mouselymphoma assay using the L5178Y/Tk+/� �3.7.2C mouse lym-phoma cell line. Our results showed that the four compounds aremutagenic in this test (Demir et al., 2011b). The confirmation ofthese positive effects in the in vivo Drosophila wing spot test isinteresting in terms of health risk because Drosophila can be con-sidered as a good health model system, since over 60% of humandisease genes have fly homologues, indicating that the fly responseto physiological insults is comparable to humans (Schneider, 2000;Koh et al., 2006; Marsh and Thompson, 2006). This would reinforcethe use of Drosophila in a first tier in vivo test for genotoxicevaluation.

Previous studies on the genotoxicity of lipid peroxidation prod-ucts show quite different results, depending on the system used.Lipid peroxidation-derived aldehydes, generated during the pro-cess of lipid peroxidation, can easily diffuse into cells and this al-lowed the original initiation site to be attacked. Therefore, it hasbeen suggested that they are not only end products of lipid perox-idation processes, but also act as mediators of oxidative stress(Uchida et al., 1999). These aldehydes can particularly react withnucleotides, lipoproteins, proteins, glutathione and DNA (Refsg-aard et al., 2000).

Acrolein, an a, b-unsaturated carbonyl compound, is foundwidely in the air polluted from cigarette smoking (O’Brien et al.,2005) and it is formed in the cells via lipid peroxidation (Chunget al., 1999). Acrolein initiates urinary bladder carcinogenesis inrats (Cohen et al., 1992), it is clastogenic for cultured Chinese ham-ster ovary (CHO) cells (Irwin, 2006) while mutagenic for bacteria


(Parent et al., 1996) and cultured human peripheral blood lympho-cytes (Smith et al., 1990; Dittberner et al., 1995). Feng et al. (2006)have reported formation of acrolein-DNA adducts at lung cancermutational hotspots in the p53 tumour suppressor gene in humanbronchial epithelial cells and lung fibroblasts. Results from Sunet al., (2006) demonstrated that acrolein is a mitochondrial toxinand that mitochondrial dysfunction caused by acrolein may playan important role in acrolein toxicity such as hepatotoxicity andalso smoking-related diseases. Acrolein is genotoxic and cytotoxicin human lymphocytes (Wilmer et al., 1986; Esterbauer et al.,1991). Sierra et al. (1991) investigated the genotoxicity of acroleinusing 2 different Drosophila SMART assays and 2 germinal tests.The results indicated that acrolein is mutagenic in the sex linkedrecessive lethal test when injected and had genotoxic effects inboth SMART assays, obtaining several indications that acrolein ismetabolized into secondary genotoxic products. As expected, wehave confirmed the mutagenic effects of acrolein in the Drosophilawing spot assay.

Crotonaldehyde is used primarily for the manufacture of sorbicacid and other organic chemicals. It is found in tobacco smoke andis a combustion product of diesel engines and wood but also occursnaturally in meat, fish, many fruits (apples, grapes, tomatoes andstrawberries) and vegetables (Brussel sprouts, cabbage, carrotsand cauliflower), cheese, milk, bread, beer, wine, and liquors (IARC,1995; HSDB, 1998). It has been detected in drinking water, wastewater and human milk and in expired air from non-smokers.

No consistent human data were located describing carcinogenic-ity associated to crotonaldehyde exposure. The U.S. EnvironmentalProtection Agency (USEPA) classified crotonaldehyde as in group C(a possible human carcinogen) based on limited animal data (Chunget al. 1986; USEPA, 1998). The International Agency for Research onCancer (IARC) concluded that there was inadequate evidence forhumans and in experimental animals to establish the carcinogenic-ity of crotonaldehyde and placed it in group 3, not classifiable as toits carcinogenicity to humans (IARC, 1995). However, it seems thatcrotonaldehyde in food may contribute to a general cancer risk(Eder and Budiawan, 2001). Crotonaldehyde is an important indus-trial chemical, an environmental pollutant and a by-product of lipidperoxidation (Marnett, 1994; Pan and Chung, 2002). It was provedto induce micronuclei, sister chromatid exchanges and chromo-some aberrations in vitro in human blood peripheral lymphocytes(Dittberner et al., 1995; Czerny et al., 1998; Jha et al., 2007),mutagenic effects in the Ames test (Schuhmacher, 1990; Eder andHoffmann, 1992) and induces liver tumours in rodents (Chunget al., 1986). Crotonaldehyde was clastogenic in D. melanogasterand significantly increased the frequency of sex-linked recessivelethals, reciprocal translocations and chromosome breakage(Woodruff et al., 1985). Our results indicate that 25 and 50 mMcrotonaldehyde concentrations induce somatic recombination, asfound in the wing spot test in D. melanogaster.

4-hydroxy-hexenal (4-HHE) is the major cytotoxic aldehydewith high biological activity derived from the peroxidation processof polyunsaturated fatty acids. 4-HHE, a natural flavour compound,originates from phospholipid-bound omega-3 unsaturated fattyacids such as eicosapentaenoic and docosahexaenoic acids. 4-HHEis present as a natural flavouring compound in many fruits and veg-etables (De Vincenzi et al., 1989), and it is widely used as a foodadditive. 4-HHE reacts directly with DNA, forms deoxyguanosineadducts and it has shown to be mutagenic in bacteria. 4-HHE wasapproved for food use by the US Food and Drug Administrationand the Council of Europe (1981) included it in the list of artificialflavouring substances. However, the extent to which 4-HHE is usedas natural food additive in the European Community is not knownat present. Predominantly, 4-HHE and other C6 aldehydes areformed by enzymatic lipid peroxidation from C18 acids after dis-ruption of cells (Hatanaka et al., 1986). In addition, 4-HHE is also

generated by autoxidation processes (Esterbauer et al., 1990). 4-HHE exhibits cytopathological effects such as mitochondrial dys-function and induction of apoptosis (Kristal et al., 1996; Choudharyet al., 2002) and mutagenic effects in the Ames test (Schuhmacher,1990; Eder and Hoffmann, 1992). Gölzer et al. (1996) showed that4-HHE forms DNA adducts and induces DNA damage in humanlymphoblastoid cells and in colon mucosa cells from rats and hu-mans. Moreover, 4-HHE has shown induction of micronuclei, sisterchromatid exchanges and numerical aberrations in Namalva cells, ahuman lymphoblastoid cell line, and in human blood lymphocytes(Dittberner et al., 1995). Some studies have demonstrated the pres-ence of 4-HHE adducts in atherosclerotic lesions (Yamada et al.,2004) and diabetic retinas (Bacot et al., 2007). In the present work4-HHE showed recombinogenic and mutagenic activity in the wingimaginal disc cells at 0.5 and 1 mM concentrations, respectively.

4-oxo-2-nonenal (4-ONE) was identified as a highly reactiveelectrophilic alkenal, endogenously produced by decompositionof lipid hydroperoxides (Lee and Blair, 2000; Spiteller et al.,2001) and is cytotoxic (Shibata et al., 2006; Lee et al., 2009).4-ONE, as a a,b-unsaturated aldehyde, is produced from theomega-6 series of fats, such as linoleic acid triglyceride. Polyunsat-urated lipids in lipoproteins and membranes are particularly sus-ceptible to oxidative stress damage, leading to the production ofsome reactive aldehydes such as 4-ONE (Lee and Blair, 2000),which are capable of modifying proteins (Sayre et al., 2001,2006) and DNA (Marnett et al., 2003). These adductions in proteinsand DNA are thought to be involved in the pathogenesis of severalhuman diseases such as atherosclerosis (Rosenfeld et al., 1990),diabetes mellitus (Yamanouchi et al., 2000) and carcinogenesis(Kawai et al., 2002). 4-ONE is causally involved in a series of broadpathophysiological effects, associated with oxidative stress (Westet al., 2004; Kovacic, 2006; Shibata et al., 2006). It would be capa-ble of inducing apoptosis in endothelial cells. Moreover, a studyhas demonstrated that 4-ONE can induce apoptosis in colorectalcancer cells with potency similar to that of 4-hydroxy-2-nonenal(West et al., 2004). 4-ONE also induces cell death in a wide varietyof cell types, partly by modulating intracellular signaling path-ways. Shibata et al. (2006) showed that the DNA alkylation pro-duced by 4-ONE strongly activates p53 in human neuroblastomaSH-SY5Y cells. Sakuma et al. (2010) showed that 4-ONE has the po-tential to induce rat liver cell death via xanthine oxidase-derivedROS generation. Picklo et al. (2011) evidenced that 4-ONE potentlyalters human mitochondrial function. In our investigation, 4-ONEwas able to increase the frequency of small and total spots onlyat the high concentration tested of 1 mM in the marker heterozy-gous wings. In other words, 1 mM concentration of 4-ONE showedrecombinogenic effect.

In summary, our results show that acrolein, crotonaldehyde, 4-HHE and 4-ONE are genotoxic in the Drosophila wing spot test.Chemicals used were ranked in decreasing order according to theirinduced mutant frequency as 4-HHE, 4-ONE, acrolein and crotonal-dehyde at the highest concentration tested in SMART assay. Finally,our results are of interest because they contribute with new data tothe understanding on how particular lipid peroxidation productsact as potentially genotoxic agents.

5. Conflict of Interest

The authors declare that there are no conflicts of interest.

Acknowledgements

Es�ref Demir is supported by a doctorate fellowship from theAkdeniz University, Antalya (Turkey) and the Council of HigherEducation (YÖK), Ankara (Turkey). This investigation was


supported in part by the Management Unit of Research Project ofAkdeniz University (Project ID: 2009.03.0121.004), Antalya (Tur-key) and by the Generalitat de Catalunya (CIRIT, 2009SGR-725),Barcelona (Spain).

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Mutagenic/recombinogenic effects of four lipid peroxidation products in Drosophila