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Mutagenesis, Vol. 14, No. 3, 335-338, May 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press

Effects of oleic acid, docosahexaenoic acid and eicosapentaenoic acid on background and genotoxin-induced frequencies of SCEs in Indian muntjac fibroblasts

Siobhán Higgins, M. Helena Vasconcelos1 and Nora M.O'Brien2

Nutritional Sciences, Department of Food Science and Technology, University College, Cork, Republic of Ireland


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Muntjac cells were cultured at 5x105 cells/10 cm Petri dish for 24 h prior to addition of fatty acids (50 µM) which were delivered to the cells complexed with 2% bovine serum albumin (fatty acid-free) and incubated for a further 24 h. Parallel dishes were processed for lipid extraction and GC analysis. This analysis showed highly significant (P < 0.01) uptake by the cells of each fatty acid. Genotoxins (75 µM hydrogen peroxide, 20 µM t-butylhydroperoxide and 2.4 µM mitomycin C) were added to the cells for 1 h prior to the end of the 24 h fatty acid incubation period. Control (no genotoxin or fatty acid) treatments were included. No difference was observed in background frequencies of SCEs between controls and fatty acid treatments, thus indicating that these fatty acids per se do not cause DNA damage. The cells incubated with the genotoxins showed increased (P < 0.05) frequencies of SCEs when compared with control frequencies. Cells incubated with genotoxins in the presence of fatty acids also showed significantly higher (P < 0.05) levels of SCEs when compared with control frequencies. When cells supplemented with genotoxins in the presence of fatty acids were compared with cells treated with genotoxins alone, higher levels of SCEs were observed in the former, suggesting that the fatty acids exacerbate DNA damage caused by these genotoxins.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Numerous studies have associated dietary parameters with cancer risk. Particular attention has been given to the influence of dietary fat. Epidemiological and experimental evidence suggests that diets rich in {omega}-3 fatty acids reduce cancer risk. The incidence of cancer is low in Eskimos on their traditional `fish-rich' diet. Recently, high fish intake was reported to be protective against colorectal cancer in a group of South African fishermen (Schloss et al., 1997Go). These authors suggested that the low incidence of colorectal carcinoma in fishermen might be explained by the protective effects of fish ({omega}-3) oils, but not by other `protective' dietary items such as fibre, antioxidant vitamins or calcium. Studies conducted in animals indicate that high dietary intake of fish oils increased tissue lipid oxidation and it has been suggested that peroxidation products of {omega}-3 fatty acids may exert cytotoxic effects and modulate the rate of tumour cell proliferation (Gonzalez et al., 1993Go; Gonzalez, 1995Go; Shao et al., 1995Go; Ramesh and Das, 1998Go). Further evidence is provided by in vitro studies suggesting that {omega}-3 fatty acids have cytotoxic action against tumour cells (Begin et al., 1986; Chajes et al., 1995Go; Kumar and Das, 1995Go) and that this activity may be due to their ability to enhance free radical generation and lipid peroxidation in these cells. The peroxidation of unsaturated lipids is known to generate a variety of products that possess DNA damaging potential (Burcham, 1998Go). The mechanisms of free radical oxidation of unsaturated lipids and the products formed have been extensively studied (Porter et al., 1995Go). However, while active research is on-going, the types and toxicological significance of DNA damage induced by lipid oxidation products is not well understood at present (Burcham, 1998Go).

In vitro work has shown that squalene, a natural fish oil, inhibits sodium arsenite-induced sister chromatid exchanges (SCEs) (Fan et al., 1996Go). Methyl mercury chloride-induced SCEs are inhibited by an {omega}-6 fatty acid, {gamma}-linoleic acid (Chandrika Seethala Bala et al., 1993Go). On the other hand, oxidized metabolites of arachidonic acid were found to cause DNA damage by inducing SCEs in Chinese hamster ovary cells in a dose-dependent manner (Weitberg, 1990Go). To the best of our knowledge no one has examined the modulatory effects of {omega}-3 fatty acids on chemical induction of SCEs in vitro. The SCE assay monitors DNA damage in a sensitive and reproducible way by detecting the exchanges of DNA replication products (Latt and Schreck, 1980Go).

The genotoxins used in our study were hydrogen peroxide (H2O2), t-butylhydroperoxide (TBH) and mitomycin C (MMC). H2O2 can give rise to hydroxyl radicals (·OH) spontaneously and also in the presence of iron or copper. These ·OH radicals are thought to be ultimately responsible for the ability of H2O2 to induce SCEs and damage DNA (Larramendy et al., 1987Go). Further evidence is provided by the reduction in SCE induction by H2O2 in the presence of the antioxidant enzyme catalase (Speit et al., 1982Go) or the iron chelator o-phenanthroline (Larramendy et al., 1987Go). Minnunni et al. (1992) reported that TBH can generate active oxygen species and this potential is reduced in the presence of an antioxidant mixture consisting of ascorbic acid, {alpha}-tocopherol and lecithin. MMC is a powerful inducer of SCEs, possibly by the formation of DNA–protein crosslinks (Ishii, 1981Go). However, Wolff (1978) speculated that alkylation at the O6 position of guanine residues might be one of the major lesions by which MMC induces SCEs.

The aim of this study, therefore, was, firstly, to investigate if the unsaturated fatty acids oleic acid, docosahexaenoic acid and eicosapentaenoic acid have any effect per se on background frequencies of SCEs in Indian muntjac fibroblasts and, secondly, to elucidate whether they modulate genotoxin-induced SCE frequencies. Our results show that the fatty acids studied have no effect per se on background frequencies of SCEs. However, they exacerbate the DNA damage caused by H2O2, TBH and MMC.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals and reagents
cis-9-octadecenoic acid (oleic acid, OA, 99% purity), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA, 99% purity), cis-5,8,11,14,17-eicosapentaenoic acid (EPA, 99% purity), H2O2, TBH, MMC, bovine serum albumin (BSA, fatty acid-free), 5-bromo-2'-deoxyuridine (BrdU), Dulbecco's phosphate-buffered saline (PBS), Giemsa stain, bisbenzimide (Hoechst 33258) and fluorescein diacetate were purchased from Sigma Chemical Co. (Poole, UK). Colcemid was obtained from Harlan Sera-Lab Ltd (Crawley Down, UK). Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FCS) and Gurr's buffer tablets were obtained from Gibco BRL-Life Technologies (Paisley, UK). Cell culture plastics were purchased from Brownes (Foxrock, Dublin, Ireland). Solvents used were high performance liquid chromatography grade.

Fatty acid preparation
Fatty acid preparation was performed under anaerobic conditions and by avoiding exposure to light, using a method adapted by Joulain et al. (1994). Fatty acids (OA, EPA and DHA) were dissolved in ethanol under nitrogen flow. Aliquots of the ethanolic solutions were stored at –80°C and a fresh aliquot was used in each experiment. For experiments, the fatty acids were diluted with experimental growth medium such that the final ethanol concentration was <0.2%.

Cells
Indian muntjac (IM) fibroblasts were kindly provided by Drs S.Musk and I.Johnson (Institute of Food Research, Norwich, UK). IM fibroblasts were used for the SCE assay because of their small number of chromosomes (7) and their short cell cycle (15–20 h). The cells were cultured in DMEM medium supplemented with 20% FCS and 2 mM L-glutamine. They were subcultured every 5–7 days and were grown in the absence of antibiotics in a humidified atmosphere with 5% CO2 at 37°C. Cells were screened for mycoplasma by the Hoechst staining method (Mowles, 1990Go).

Cell treatments
For all experiments cells were cultured at 5x105 cells/10 cm Petri dish for 24 h prior to addition of fatty acids. Both fatty acids and genotoxins were delivered to and incubated with the cells in darkness. Fatty acids (50 µM) were delivered to the cells complexed with 2% BSA (fatty acid-free) and incubated for 24 h. In the experiments with genotoxins (H2O2, TBH and MMC), these were added to the cells for 1 h prior to the end of the 24 h incubation period. The genotoxins were prepared in sterile PBS immediately before use. Blanks were cultured in growth medium alone. Controls were cultured in growth medium plus the same quantity of ethanol and BSA used to solubilize the fatty acids in the experimental treatment groups.

For the fatty acid uptake experiments, cells were washed twice with PBS at the end of the treatment period, scraped into PBS and sonicated for 3x15 s. Lipid was extracted according to the method of Lepage and Roy (1986) and the fatty acid methyl esters were quantified by gas chromatography (Varian 3800 GC) using a DB-Wax capillary column (30 mx0.32 mm i.d.) and nitrogen as carrier gas. Fatty acids were identified by retention time comparisons with a mix of standard fatty acid methyl esters.

For SCE analysis, following the incubation period with fatty acids, with or without genotoxin, cells were washed twice with PBS, refed fresh medium and incubated with 16 µM BrdU for a further 48 h in darkness. Cells were treated with 40 nM colcemid for 3 h to arrest the cells in metaphase, harvested by gentle scraping and incubated with 0.075 M KCl for 10 min (37°C). The samples were then fixed three times in freshly prepared ice-cold Carnoy's fixative (methanol:acetic acid 3:1) before being dropped onto methanol-cleaned microscope slides. Visualization of the SCEs was possible by staining the different chromatids with fluorescent plus Giemsa stain (Perry and Wolff, 1974Go). Briefly, slides were incubated in Hoechst 33258 solution (5 µg/ml), made up in PBS, for 25 min in the dark. Slides were washed twice with PBS and then exposed to UV light at 366 nm for 2.5 h. Following a wash in diluted PBS they were further stained for 3 min in 3% (v/v) Giemsa in Gurr's buffer. The frequency of SCE was scored as the number of exchanges in 20 metaphases/slide (majority second cycle) and expressed as number of SCEs/chromosome.

The slides prepared for SCE assay were also scored for mitotic indices (MI). Two-hundred nuclei were counted and the number of mitoses expressed as a percentage of the total number of nuclei. Cells were assessed for viability throughout the experiment using the fluorescein diacetate/ethidium bromide assay (Strauss, 1991Go).

Statistical analysis
The results are expressed as means ± SEM for percentage of total fatty acids, means ± SEM for SCEs/chromosome and means ± SEM for MI of at least three independent experiments. Overall comparison of treatment groups was performed by one-way analysis of variance (one-way ANOVA). Thereafter, significance of paired differences was analysed by the least significant difference test (LSD); P values of <0.01 and <0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Preincubation of IM fibroblast cells for 24 h with 50 µM OA, DHA or EPA in the presence of BSA resulted in a 10–12% increase in uptake of each fatty acid when compared with control values (Table IGo). Using one-way ANOVA followed by LSD, the increases were found to be highly significant for all three fatty acid treatments (P < 0.01).


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  		Table I. Uptake of the fatty acids (50 µM) OA (C18:1n-9), EPA (C20:5n-3) and DHA (C22:6n-3) by Indian muntjac fibroblast cells
 
The SCE assay was carried out after incubation for 24 h with the fatty acids. No difference was observed in the background frequency of SCEs between controls, blanks and fatty acid treatments (Table IIGo), thus indicating that these fatty acids do not cause DNA damage per se under the conditions of the assay.


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  		Table II. Background frequencies of SCEs and mitotic index [MI (%)] in Indian muntjac fibroblast cells treated with the fatty acids (50 µM) OA, EPA and DHA
 
IM cells were incubated with increasing concentrations of H2O2, TBH or MMC to determine a suitable concentration of genotoxin to use in all subsequent experiments. From the dose–response data, 75 µM H2O2, 20 µM TBH and 2.4 µM MMC were selected. Cells incubated with each of the three genotoxins (H2O2, TBH or MMC) showed increased (P < 0.05) frequencies of SCEs when compared with blank/control frequencies (Table IIIGo). Cells incubated with genotoxins in the presence of fatty acids also showed significantly higher (P < 0.05) levels of SCEs when compared with blank/control frequencies (Table IIIGo). When cells supplemented with genotoxins in the presence of fatty acids were compared with cells treated with genotoxins alone, higher levels of SCEs were observed in the former (Table IIIGo). OA enhanced induction of SCEs by 9, 33 and 23%, EPA caused an increase in SCEs by 20, 40 and 27% and DHA caused an increase in SCEs by 8, 32 and 29% when incubated with H2O2, TBH and MMC, respectively.


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  		Table III. Background frequencies of SCEs and mitotic index [MI (%)] in Indian muntjac fibroblast cells treated with the genotoxins H2O2 (75 µM), TBH (20 µM) or MMC (2.4 µM) with or without fatty acids (50 µM) OA, EPA and DHA
 
No significant differences (one-way ANOVA, LSD) were found in MI data (Tables II and IIIGoGo), which indicates that the number of mitoses did not vary between the treatment conditions. The viability of the cells was >70% in all experiments, as indicated by the fluorescein diacetate/ethidium bromide assay.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The SCE assay detects DNA interchanges in metaphase cells between sister chromatids at homologous loci (Latt and Schreck, 1980Go). It is generally thought that an increase in SCEs is an extremely sensitive indicator of genetic damage and its repair in mammalian cells, but the actual mechanism of SCE formation remains to be elucidated.

Our results demonstrate that pre-treatment of IM fibroblasts with OA, EPA or DHA for 24 h induced no increase in SCE frequencies above control levels in unsupplemented cells, indicating that these fatty acids by themselves are not genotoxic. Similarly, de Kok et al. (1994) reported that linoleic acid did not induce SCEs above control levels in isolated human lymphocytes. However, when IM fibroblast cells pre-incubated with OA, EPA or DHA for 24 h were subsequently exposed to genotoxic agents, the fatty acids were found to exacerbate the DNA damage caused by the genotoxins (Table IIIGo). A 10–12% enrichment of OA, EPA or DHA within the cellular membranes occurred following the 24 h pre-incubation period (Table IGo). Both in vivo and in vitro studies have shown that dietary or media lipid changes quickly alter the polyunsaturated fatty acid composition of the membrane phospholipids (Hatala et al., 1994Go; Williams and Maunder, 1994Go; Calviello et al., 1998Go). It has been postulated that changes in the fatty acid profiles of the membranes might modify the physico-chemical environment of the cell sufficiently to affect such functions as receptor activity, enzyme activity or permeability to pharmacological agents (Cave and Erickson-Lucas, 1982Go; Carroll and Parenteau, 1991Go; Brown and Subbaiah, 1994Go). Dietary supplementation with EPA and DHA inhibited the growth of Morris hepatocarcinoma 3924A in rats (Calviello et al., 1998Go). The authors concluded that the anti-tumoral effect of EPA was related mainly to its inhibition of cell proliferation, whereas that of DHA corresponded with induction of apoptosis. The alterations in tumor cell membrane fatty acid composition induced by EPA and DHA appear to be factors underlying their differential actions on cell proliferation and apoptosis.

Lipid oxidation gives rise to a range of products some of which are potentially genotoxic (Burcham, 1998Go). Oxidized metabolites of unsaturated fatty acids are known to cause DNA damage in vitro (Weitberg, 1990Go). Some in vitro studies which have shown this effect have used a metal such as iron (McNeill and Wills, 1985Go) or an enzyme (de Kok et al., 1994Go) to oxidize the fatty acids prior to incubating with cells. In addition, antioxidants (superoxide dismutase, catalase, glutathione peroxidase and vitamin E) have been shown to be effective in reducing the amount of fatty acid hydroperoxide-induced genetic damage (Weitberg, 1990Go; Kumar and Das, 1995Go; Ramesh and Das, 1998Go). The genotoxins used in the present study included H2O2 and TBH, both of which are well-known oxidizing agents (Speit et al., 1982Go; Larramendy et al., 1987Go; Minnunni et al., 1992Go). Therefore, it is quite plausible that these genotoxins could induce oxidation of the fatty acids in pre-treated cells and result in DNA damage. However, MMC, which is a bifunctional alkylating agent and not a traditional oxidizing agent (Ishii, 1981Go), also appeared to have the same effect.

The amplifying effect of the fatty acids on the genotoxicity of H2O2, TBH and MMC appear to be unrelated to their degree of unsaturation, since incubation with DHA did not produce more SCEs than incubation with oleic acid.

In summary, the present findings show that the unsaturated fatty acids which were used in these experiments did not show any genotoxic tendencies when supplemented on their own. However, when supplemented in combination with genotoxins, the fatty acids enhanced genotoxic action. Future work will focus on the mechanism underlying this fatty acid-induced enhancement of genotoxic action and, in particular, on the possibility that changes in fatty acid profile of cell membranes may affect genotoxin uptake by cells.


   Acknowledgments
 
S.H. and M.H.V. were supported by EU project FAIR-CT-95-0085.


   Notes
 
1 Present address: Instituto de Patologia e Imunologia Molecular da Universidade do Porto (IPATIMUP), Rua Dr Roberto Frias, 4200 Porto, Portugal Back

2 To whom correspondence should be addressed. Tel: +353 21 902884; Fax: +353 21 270244; Email: nob{at}ucc.ie Back


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 Results
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Received on November 12, 1998; accepted on January 18, 1999.


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