Human adenoma cells are highly susceptible to the genotoxic action of 4-hydroxy-2-nonenal

Mutation Research 526 (2003) 19–32

Human adenoma cells are highly susceptible to thegenotoxic action of 4-hydroxy-2-nonenal

Anja Schaeferhenricha, Gabriele Beyer-Sehlmeyera, Grit Festaga, Alma Kuechlerb,e,Natja Haaga, Anja Weiseb, Thomas Liehrb, Uwe Claussenb, Brigitte Marianc,

Wolfgang Sendtd, Johannes Scheeled, Beatrice Louise Pool-Zobela,∗a Department of Nutritional Toxicology, Institute for Nutrition, Friedrich Schiller University Jena,

Dornburger Strasse 25, D-07743 Jena, Germanyb Institute for Human Genetics and Anthropology, Friedrich Schiller University Jena, Kollegiengasse 10, D-07743 Jena, Germany

c Institute of Cancer Research, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austriad Department of Surgery, Friedrich Schiller University Jena, Bachstrasse 18, D-07743 Jena, Germany

e Department of Radiotherapy, Friedrich Schiller University Jena, Bachstrasse 18, D-07743 Jena, Germany

Received 30 October 2002; received in revised form 6 January 2003; accepted 10 January 2003

Abstract

Oxidative stress and resulting lipid peroxidation are important risk factors for dietary-associated colon cancer. To get a betterunderstanding of the underlying molecular mechanisms, we need to characterise the risk potential of the key compounds,which cause DNA damage in cancer-relevant genes and especially in human target cells. Here, we investigated the genotoxiceffects of 4-hydroxy-2-nonenal (HNE) and hydrogen peroxide (H2O2) in human colon cells (LT97). LT97 is a recentlyestablished cell line from a differentiated microadenoma and represents cells from frequent preneoplastic lesions of the colon.The genomic characterisation of LT97 was performed with 24-colour FISH. Genotoxicity was determined with single cellmicrogelelectrophoresis (Comet assay). Comet FISH was used to study the sensitivity ofTP53—a crucial target gene forthe transition of adenoma to carcinoma—towards HNE. Expression of glutathione S-transferases (GST), which deactivatesHNE, was determined as GST activity and GSTP1 protein levels. LT97 cells were compared to primary human colon cellsand to a differentiated clone of HT29. Karyotyping revealed that the LT97 cell line had a stable karyotype with only twoclones, each containing a translocation t(7;17) and one aberrant chromosome 1. The Comet assay experiments showed thatboth HNE and H2O2 were clearly genotoxic in the different human colon cells. HNE was more genotoxic in LT97 than inHT29clone19A and primary human colon cells. After HNE incubation,TP53 migrated more efficiently into the comet tailthan the global DNA, which suggests a higher susceptibility of theTP53 gene to HNE. GST expression was significantlylower in LT97 than in HT29clone19A cells, which could explain the higher genotoxicity of HNE in the colon adenoma cells.In conclusion, the LT97 is a relevant model for studying genotoxicity of colon cancer risk factors since colon adenoma arecommon preneoplastic lesions occurring in advanced age.© 2003 Elsevier Science B.V. All rights reserved.

Keywords: TP53; Microadenoma cells; Human colon; Genotoxicity; 4-Hydroxy-2-nonenal

Abbreviations: APC, adenomatous polyposis coli gene; FISH, fluorescence in situ hybridisation; GSH, glutathione; GST, glutathioneS-transferase; H2O2, hydrogen peroxide; HBSS, Hank’s balanced salt solution; HNE, 4-hydroxy-2-nonenal; Ki-ras, Kirsten rat sarcoma;PBS, phosphate buffered saline; SSC, saline sodium citrate;TP53, tumour protein p53 gene

∗ Corresponding author. Tel.:+49-3641-949670; fax:+49-3641-949672.E-mail address: [email protected] (B.L. Pool-Zobel).

0027-5107/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0027-5107(03)00012-5

20 A. Schaeferhenrich et al. / Mutation Research 526 (2003) 19–32

1. Introduction

Our growing knowledge of the impact of nutritionon human health is showing that a dietary imbalanceis likely to enhance the risk of developing colorectalcancer. Epidemiological evidence suggests that highmeat and alcohol intake increase this risk[1], probablyby enhancing exposure to food-derived carcinogens orby favouring endogenous formation of risk compounds[2,3]. Especially human faeces contain a large varietyof toxic and genotoxic factors[4], some of which aremodulated by diet[5,6].

Risk factors enhance carcinogenesis by acting asmutagens[7], a mechanism that is in line with sev-eral key alterations identified in colon tumours[8].Tumour suppressor genes, especially the adenoma-tous polyposis coli (APC) gene andSMAD4/2 arecommonly inactivated by deletions[9,10]. Mutationor amplification of the Kirsten rat sarcoma (Ki-ras)proto-oncogene is another genetic event that occurs inapproximately 50% of colorectal carcinoma and largeadenoma[11–13]. A high number of mutations wereidentified in tumour protein p53 (TP53) gene of colontumours[14], some of which were probably crucialfor the transition of adenoma to carcinoma.

Only a minor proportion of genetic lesions found intumours are due to inheritance. Environmental factorsare much more likely to cause the alterations[15,16].However, to date we can only assume that certain com-pounds in the diet are causative factors[4]. This ismainly based on studies showing that individual riskcompounds are carcinogenic in animal experimentsand genotoxic in short-term assays of genetic toxicol-ogy [3,17,18]. In particular, it was almost impossibleto study early changes affecting the normal colonicepithelial cells due to the lack of manageable culturemethods for those cells. Normal human cells lose theirin vivo-like metabolic capacity after only minutes orhours in culture and it is not possible to maintain themin culture for more than a few days[19].

There has been little research on normal humancolon cells in culture[20]. The authors recentlydemonstrated the validity of using intact primary cellsincubated in vitro for 0–60 min as models to deter-mine genotoxic effects, including specific damage ofTP53 in primary human colon cells[18,21,22]. Wehave also reported novel findings on the develop-ment of short-term cell cultures using normal colonic

epithelial cells from rats[23] and humans. In the fol-lowing, we have combined our approaches to assesshow a newly established cell line (LT97), consistingof epithelial cells representing an early premalignantgenotype, serves as a target to investigate the impactof risk factors. The LT97 cells have typical genetictraits of adenoma, such as loss of bothAPC tumoursuppressor gene alleles and mutated Ki-ras allele, butnormalTP53 [20].

We have investigated the genotype of LT97 using24-colour fluorescence in situ hybridisation (FISH)[24] and further characterised their epithelial naturewith specific antibodies. Their susceptibility to thegenotoxic impact of 4-hydroxy-2-nonenal (HNE) wasdetermined, since this endogenously formed com-pound (a product of lipid peroxidation) is consideredto be a putative cancer causing agent after its epoxi-dation[25] and may be dietary related[26]. In humancolon tumour cells, HNE is genotoxic and its activ-ity can be effectively abolished by butyrate-inducedexpression of glutathione S-transferases (GST). Es-pecially GSTP1, which is available in high amountsin colon cells[27], and GSTA4-4, which is specificfor HNE [28], are effective in conjugating oxidationproducts with glutathione (GSH). Therefore, we alsoassessed GST polymorphisms, GST activity, levelsof GSH, and the expression of GSTP1 protein as afirst selection of markers for susceptibility to HNE.We used single cell microgelelectrophoresis (Cometassay)[29] to determine the global DNA damageinduced by HNE and measured the damage causedto TP53 by Comet FISH[30]. We also measured ge-nomic damage in primary human colon cells[18] andin the differentiated colon cell line HT29clone19A[31], thus allowing for a direct comparison of the sen-sitivity of microadenoma derived cells to other modelcolon cells used in vitro[22]. Finally, the genotoxic-ity of hydrogen peroxide (H2O2), a source of reactiveoxygen species via the Fenton reaction[32], was alsomeasured to gain more general knowledge about thesensitivity of LT97 to products of oxidative stress.

2. Materials and methods

2.1. Cell lines and culture conditions

The human colon adenoma cell line LT97 wasestablished from colon microadenomas of a patient

A. Schaeferhenrich et al. / Mutation Research 526 (2003) 19–32 21

with familial adenomatous polyposis coli[20]. LT97was maintained in a culture medium (MCDB 302)containing 20% L15 Leibovitz medium, 2% fe-tal calf serum (FCS), 1% penicillin/streptomycin,0.2 nM triiodo-l-thyronine, 1�g/ml hydrocortisone(302 basic medium) supplemented with 10�g/ml in-sulin, 2�g/ml transferrin, 5 nM sodium selenite and30 ng/ml epidermal growth factor (EGF).

HT29clone19A is a permanently differentiatedsub-clone derived from the carcinoma cell line HT29treated with sodium butyrate[31]. HT29clone19A wasmaintained in Dulbecco’s modified Eagle medium(DMEM) supplemented with 10% FCS and 1%penicillin/streptomycin[27]. Under given laboratoryconditions LT97 cells doubled their number within72–96 h and HT29clone19A cells within 24 h. Pas-sages 50–60 and 20–40, respectively, were used forthe experiments.

The epithelial nature of the cells was recon-firmed using the epithelial-specific Ber-EP4 anti-body (1�g/ml; DAKO, Hamburg, Germany) and thefibroblast-specific FIB1 antibody AS02 (1�g/ml;Dianova, Hamburg, Germany) as primary andCyTM3-conjugated goat anti-mouse IgG (7�g/ml;Dianova, Hamburg, Germany) as secondary antibody.The evaluation was performed by fluorescence mi-croscopy (Zeiss Axiovert 100M, Jena, Germany)[20].Cytokeratin was visualised with avidin–peroxidasereagents (Vektor Laboratories, Burlingham, CA).

2.2. Isolation of primary human colon cells

Colon tissue was obtained from patients who hadgiven their informed consent after having been ad-mitted to hospital for surgery of colorectal tumours,diverticulitis, and colon polyps[22]. The average age(±S.D.) of all donors was 62± 12 years (n = 36):41% of the donors were male and 59% female. TheEthical Committee of the Friedrich Schiller Uni-versity Jena approved the study. Non-tumour tissuewas stored in Hank’s balanced salt solution (HBSS;8.0 g/l NaCl, 0.4 g/l KCl, 0.06 g/l Na2HPO4·2H2O,0.06 g/l K2HPO4, 1 g/l glucose, 0.35 g/l NaHCO3,4.8 g/l HEPES, pH 7.2), it was transported to thelaboratory on ice within 1 h and worked up imme-diately. The human colon epithelium was separatedfrom the tissue by perfusion-supported mechanicaldisaggregation. Epithelial stripes were minced and

incubated in HBSS supplemented with 2 mg/ml pro-teinase K (Sigma, Steinheim, Germany) and 1 mg/mlcollagenase P (Boehringer, Mannheim, Germany) for60 min at 37◦C. The suspensions of primary humancolon cells were diluted with HBSS, centrifuged andre-suspended in RPMI or phosphate buffered saline(PBS; 8 g/l NaCl, 1.44 g/l Na2HPO4, 0.2 g/l KCl,0.2 g/l KH2PO4, pH 7.3). Viability and cell yield weredetermined with trypan blue and the cell number wasadjusted to 2× 106 cells/ml.

2.3. GST polymorphisms and associatedbiochemical parameters

DNA extraction succeeded with QIAamp mini kit(QIAGEN GmbH, Hilden, Germany). Multiplex PCRdetectedGSTM1 andGSTT1 deletion polymorphisms,using the original procedures[33,34]. Sequence poly-morphisms of GSTP1 and GSTM3 were detectedusing the restriction fragment length polymorphism(RFLP)-PCR technique[35,36]. Activity of GST wasdetermined spectrophotometrically at 340 nm (30◦C)using 1 mM 1-chloro-2,4-dinitrobenzene and 1 mMGSH as substrates[37]. Alkaline phosphatase activ-ity was quantified by measuring the hydrolysis ofp-nitrophenylphosphate (5 mM) using 50�l cytosoland 150�l Tris–HCl buffer (50 mM, pH 10) at 37◦Cwith p-nitrophenol (0–200�M) as standard.Table 1shows enzyme activities expressed in units and spe-cific activities as units per mg protein or per cellnumber, respectively[38]. For this, we determined thetotal protein content using the method by Bradford[39]. The detection of GSTP1 protein with Westernblotting proceeded according to laboratory stan-dards[59]. Intracellular GSH levels were determinedusing a colorimetric assay (glutathione assay kit;Calbiochem-Novabiochem, Schwalbach, Germany).Three parallels were performed for each of these as-says. The mean values of at least five independentexperiments are shown.

2.4. Determination of the karyotype of LT97 cells(24-colour FISH)

Metaphase chromosomes from LT97 cells were pre-pared according to standard protocols for 24-colourFISH. Slides were pre-treated with RNAse and pepsinto diminish plasma background signals[40]. Further


Table 1Comparison of the basal levels of GST/GSH specific parameters and the alkaline phosphatase (AP) activity in human colon cells ofdifferent transition states

Cells GST activity GSTP1 protein GSH (nmolper 106 cells)

Protein (�gper 106 cells)

AP activity(�mol/minper 106 cells)nmol/min per

106 cellsnmol/min permg protein

ng per 106

cellsng per mgprotein

Primary 27.1± 11(n = 10)

139 ± 9a 194 ± 93(n = 15)

1330± 170a n.d. 85.6± 41.7(n = 5)

0.085± 0.02(n = 5)

LT97 49.5± 7.4(n = 6)

350 ± 160(n = 6)

425 ± 209(n = 13)

2775± 1313(n = 13)

1.91 ± 0.23(n = 5)

154.0± 35.0(n = 13)

0.105± 0.041(n = 9)

HT29clone19A 34.8± 2.2(n = 26)

407 ± 64(n = 26)

734 ± 209(n = 19)

5555± 1245(n = 19)

6.29 ± 1.1(n = 15)

90.8± 5.4(n = 21)

0.192± 0.016(n = 15)

The specific enzyme activities were determined as described under materials and methods and expressed in units per mg protein or per106 cells, respectively. The data are expressed as mean± S.D.. n, number of independent determinations, or in the case of primary coloncells, number of individual patients; n.d., not determined.

a Data from[58].

steps of the 24-colour FISH assay were done as de-scribed[41]. Microdissection-derived whole chromo-some paints for all human chromosomes were labelledwith five different fluorochromes or ligands (R110,Texas-Red, Spectrum-Orange, biotin and digoxi-genin) according to an established labelling scheme[42] using a modified degenerate oligonucleotideprimer-polymerase chain reaction (DOP-PCR). Afterhybridisation for 72 h in a humid chamber at 37◦C anda washing series in 2× saline sodium citrate (SSC)containing 50% formamide (three times for 5 min at42◦C) and in 2× SSC (three times for 5 min at 37◦C),the biotinylated and digoxigenated probes were de-tected with Cy5-avidin and anti-digoxigenin-Cy5.5(Amersham, Buckinghamshire, UK), respectively.The 24-colour FISH slides were counterstainedwith 4′-6-diamino-2-phenylindole-dihydrochloride(DAPI), which results in an R-banding-like patternalong the chromosomes, thus allowing the identifi-cation of chromosomal sub-bands. The metaphaseswere evaluated on an Axioplan 2 imaging fluores-cence microscope (Zeiss, Jena, Germany) equippedwith “ISIS3” digital FISH imaging system (Meta-Systems, Altlussheim, Germany) using a PCO VC45CCD camera with on-chip integration (PCO, Kehl,Germany). Eighteen metaphases of the cell linewere evaluated. For further breakpoint character-isation of the aberrant chromosomes, multicolourbanding (MCB) was performed according to Liehret al. [43]. This technique is based on overlapping,region-specific microdissection libraries, which are

labelled in different colours (proceedings are the sameas applied in 24-colour FISH). This leads to changingfluorescence intensity ratios along the chromosomes,which the MetaSystems software converts to assigndifferent pseudo-colours to specific chromosomalregions.

2.5. Treatment of the cells with chemicals

Stock solutions of HNE (CAS 75899-68-2) inethanol and of H2O2 (CAS 7722-84-1) in PBS wereadded to cell suspensions containing 2×106 cells/ml.Final concentrations were 100–250�M for HNE and18.8–150�M for H2O2. HNE suspensions were in-cubated for 30 min in a shaking water bath at 37◦C.Those containing H2O2 were incubated for 5 min at4◦C to avoid repair of the induced oxidative DNAdamage[44]. The substances were removed by cen-trifugation and the viability of the cells was testedby trypan blue exclusion. The cell pellets were takenup in agarose, distributed onto slides, and then pro-cessed according to the protocol for the microgelelec-trophoresis as described below.

2.6. Determination of genetic damage(Comet assay)

Cell pellets were mixed with 0.7% low melting pointagarose and 50�l of the cell suspensions were dis-tributed onto microscope slides pre-coated with 200�lof 0.5% normal melting agarose. After the agarose


solidified, slides were immersed in a lysis solution(10 mM Tris–HCl, 100 mM Na2EDTA, 2.5 M NaCl,10% dimethylsulfoxide (DMSO), 1% Triton X-100,pH 10) for at least 60 min. Slides were placed inan electrophoresis chamber containing alkaline buffer(1 mM Na2EDTA, 300 mM NaOH, pH 13) for DNAunwinding. After 20 min, the current was switchedon and electrophoresis was carried out at 1.25 V/cm,300 mA for 20 min. The slides were removed from theelectrophoresis chamber and washed three times for5 min each with neutralisation buffer (4.2 M Tris–HCl,0.08 M Tris–base, pH 7.2). Slides were stained withSYBR-Green (1�l/ml; 30�l per slide) or they weredehydrated in ethanol and used later for Comet FISH.All steps of the Comet assay were conducted underred light. Each experiment was reproduced indepen-dently at least three times.

Microscopic evaluation of the images was quanti-fied using the image analysis system of Kinetic Imag-ing (Liverpool, UK). Fifty cells were evaluated perslide and the percentage of fluorescence in the tail (tailintensity (TI)) was scored. The means of three to fourreplicates were used to calculate the means of at leastthree independently reproduced experiments.

2.7. Determination of TP53 migration(Comet FISH)

For the Comet FISH experiments, LT97 microade-noma cells and primary human colon cells were used.HT29clone19A cells could not be used for CometFISH because of their high genetic variability with64–69 chromosomes per cell[45].

Digoxigenin-labelledTP53 probes were obtainedfrom Oncor (Gaithersburg, UK). TheTP53 DNAprobe (about 80 kb) was a mixture of digoxigenin-labelled probes specific for genomic sequences in-cluding theTP53 locus (17p13.1).

The dehydrated slides from the Comet assay werere-hydrated (double-distilled H2O for 10 min), thetarget DNA was denatured (0.5 M NaOH for 30 min),the gels were neutralised (PBS, 1 min) and dehydratedthrough an ethanol series (70, 80 and 95% for 5 mineach). The slides were dried at room temperature andeach gel was covered with 30�l Hybrisol VI (On-cor, Gaithersburg, UK). The hybridisation mixture(30�l per slide), containing the digoxigenin-labelledTP53 probes (Oncor, Gaithersburg, UK), was dena-

turised at 37◦C for 5 min and added to the slides.The slides were covered with plastic cover slips(24 mm × 24 mm) and incubated in hybridisationchambers for 24 h at 37◦C. The slides were washed for5 min in 2× SSC (0.3 M NaCl, 0.03 M sodium citrate,pH 7.2) at 72◦C followed by 5 min washing in 1×PBD (phosphate buffered detergent; Oncor, Gaithers-burg, UK) at room temperature. For detection ofdigoxigenin-labelled probes, anti-digoxigenin-AP-Fabfragments (variable sequence of immunoglobulin)and 2-hydroxy-3-naphthoicacid-2′-phenylanilide-pho-sphate (HNPP) fluorescent detection set were used(Roche, Mannheim, Germany). The enzymatic sig-nal amplification was performed according to themanufacturer’s instructions. SYBR-Green (1�l/ml;30�l per slide) was used to counterstain the HNPP-detected probes.

Comet FISH experiments were evaluated by firstdetermining the total number ofTP53 signals per cell.The expected number of spots in a normal metaphaseor interphase nucleus is two. On the slides hybridisedwith theTP53 probes cells without, with only one sig-nal and with two signals were counted and the hy-bridisation efficiency was determined. For the furtherComet FISH evaluation, only cells with two fluores-cent spots were used and the localisation of theTP53signals in the comet head or tail was recorded. Cellnumbers scored for Comet FISH ranged from 100 to130 cells per slide.

3. Results

3.1. Properties of LT97, growth, andepithelial nature

The LT97 microadenoma colon cells are of epithe-lial origin staining positive for cytokeratins (Fig. 1A)and for epithelial cell-specific antibody Ber-EP4(DAKO, Hamburg, Germany), while LT97 cells werenegative for fibroblast-specific antibody FIB1 AS02(Dianova, Hamburg, Germany) (Fig. 1B). LT97 cellshave a premalignant genotype with characteristicalterations inAPC and Ki-ras [20]. About 10% ofthe cells in these cultures are in the S-phase and themajority of the cell population is in G1, resulting inan overall doubling time of 72–96 h[20], which wasreconfirmed here.


Fig. 1. Characterisation of the epithelial origin of the LT97 cell line. (A) Cytokeratin staining: LT97 cultures were fixed with methanol–acetone(1:1) and then stained with a polyclonal pan-cytokeratin antibody to confirm the epithelial nature of the cells. The control stains receivednormal rabbit serum. (B) Visualisation of the epithelial and fibroblast-specific antigens in LT97, HT29clone19A and colonic fibroblasts byimmunofluorescent staining. Therefore, cells fixed on glass cover slips were stained using monoclonal Ber-EP4 antibodies or FIB1 AS02,respectively, and imaged via CyTM3-labelled IgG using Zeiss Axiovert 100M at 400× magnification.

3.2. GST polymorphisms and associatedbiochemical parameters

Table 2shows the polymorphic character of GSTsin LT97 in comparison to HT29clone19A. Genotyp-ing of cellular DNA from LT97 revealed the presenceof the GSTT1∗1 gene, whereas noGSTM1 gene wasdetectable. Furthermore, we detected both∗A wildtype allele and the three base pair deletion containing∗B allele of GSTM3. For GSTP1, we identified thewild type allele GSTP1∗A as well as the heterozy-gous sequence polymorphism at nucleotide+313(Alw26I restriction site) present in theGSTP1∗1B andGSTP1∗1C alleles.Table 1shows the relative levels of

Table 2Patterns of genetic polymorphisms for LT97 adenoma cells incomparison to gene patterns of the HT29clone19A cell line

Cells GSTM1 GSTM3 GSTT1 GSTP1

LT97 0 AB Positive A∗B/CHT29clone19A B A Positive A∗B/C

The polymorphisms forGSTM1 and GSTT1 were determined si-multaneously by multiplex PCR, and the sequence polymorphismsof GSTM3 and GSTP1 were assessed by RFLP-PCR techniquedescribed in materials and methods.

GST activities, GSH levels, GSTP1 protein amounts,and the total protein contents for LT97, in compari-son to primary and transformed human colon cells.The expression of GSTP1 protein in LT97 cells isapproximately only one-half and GSH is one-third ofthe levels detected for the clone of HT29. There is nocorrelation between GST activity and GSTP1 proteinalthough this protein is the most abundant protein. Itis difficult to compare the values of these cell lines toprimary human cells due to the heterogeneity of thefreshly isolated populations of colon cells. However,for those samples, which were worked up during theexperimental period of this set of experiments, thevalues are lower than those obtained for LT97. Ineach case the values were highest in the transformedtumour-derived cells and low in the primary cells.LT97 takes an intermediate place in this order, whichcorresponds to its relative status of cell transforma-tion. The differentiation parameter, activity of alka-line phosphatase, also reflects this sequence of order(Table 1). Interestingly, the protein content was higherin LT97 than both average values for the heteroge-neous primary cells as well as for HT29clone19A.This may be a reflection of the specific transitionalstage of these cells and of their high metabolicactivity.


3.3. Karyotype of LT97 (24-colour FISH)

The 24-colour FISH karyotyping revealed thatthe LT97 cell line consists of two different clones,in a relation 1:1. Both clones share one aberra-tion, i.e. a translocation between the chromosomes7 and 17, indicating monoclonal origin. Further-more, aberrant chromosomes 1 were present inboth clones: one clone contained an isochromosome1q, the other clone a dicentric translocation of thelong arm of chromosomes 1 and 18. Further char-acterisation by multicolour banding specified theaberration breakpoints (data not shown). Accord-ing to ISCN 1995[46], the karyotype illustrated inFig. 2 was determined to be: 46–50, XX,+i(1)(q10),+der(7)t(7;17)(q31.3;q21.3)[cp15]/44–46, XX,+dic(1;18)t(q10:qter;q10:qter), der(7)t(7;17)(q31.3;q21.3),−18[cp14].

3.4. Genetic damage (Comet assay)

HNE and H2O2 were clearly genotoxic without anyindication of concomitant cytotoxicity. Cell viabilitydetermined by trypan blue exclusion test was always>80%. Thus, the Comet assay results, given below,mainly reflect the direct genotoxic effects of the testedcompounds.

Fig. 3 compares the impacts of HNE and H2O2 onthe global DNA damage in human colon cells repre-senting three different stages of cell transformation.The global DNA damage is determined as percentageof DNA in the comet tail (tail intensity (%)). H2O2 wassignificantly genotoxic at 75�M in HT29clone19Aand LT97 cells, or beginning from at 37.5�M in pri-mary human colon cells. The comparison of the effectsof individual concentrations in the three cell lines doesnot reveal any significant differences. HNE was alsogenotoxic in all cell types beginning from 150�M.For this compound, however, the relative sensitivitiesof the different cell types were significantly different.HNE had a significantly higher impact on LT97 thanon HT29clone19A and primary colon cells. A moredirect comparison of the relative sensitivities of thetwo cell lines (HT29clone19A and LT97) is possiblewhen analysing each compound in both cell types onthe same day and in the same electrophoresis run. Acorresponding subset of for three individually repro-duced experiments is shown inFig. 4. HNE again

shows a higher impact in the LT97 cells than in theHT29clone19A cells, whereas the impact of H2O2 wasnot different in the two cell lines.

3.5. Migration of TP53 (Comet FISH)

Based on the results of the genomic karyotyping,it was technically feasible to use LT97 cells for de-termining specific migration properties of DNA se-quences related to the tumour suppressor geneTP53.The incubation without DNA probes (negative con-trols) resulted in a lack of signals. The hybridisationefficiency was determined to be 77.0 ± 7.7% for theprimary colon cells and 83.3±0.5% for the LT97 cells.For the further Comet FISH evaluation, only cells withtwo fluorescent spots were used.

Fig. 5compares the migration of the global DNA tothe migration ofTP53-specific signals in primary hu-man colon cells and LT97 microadenoma cells, whichhad been treated with 100–250�M HNE. TP53 mi-grates to a higher proportion into the comet tail thanglobal DNA. This difference in migration is significantfor both primary and LT97 colon cells after treatmentwith 200 and 250�M HNE.

4. Discussion

The continuous exposure of the human colon toa complex mixture of compounds of dietary origin,or resulting from digestive, microbial, and excretoryprocesses, may pose a special type of risk. The impactof risk factors from the gut lumen on the epithelialcells depends on different factors, including geno-toxic potency of the compounds and the ability of themucosal cells to deactivate the compounds, to repairtoxic damage, or to remove damaged cells entirely(apoptosis). HNE, the major product of linoleic acidoxidation, is one of the suggested putative risk factors[47]. In the presence of peroxides and reactive oxygenspecies, it readily converts to its epoxide 2,3-epoxy-4-hydroxynonanal[25]. The reaction of the epoxidewith the DNA base guanine leads to the formation of1,N2-etheno-dG[48], a promutagenic lesion which in-duces several types of base pair substitution mutationsand−1 and−2 base frame shifts[49]. Included in thisspectrum is also the G→ A transition type of muta-tion that was found in mammalian DNA[50] and that

Fig. 2. The 24-colour FISH results of the two clones of LT97 (pseudo-colour representations). Both clones of LT97 are present inan equal proportion. According to the ISCN 1995[46], the karyotype of LT97 can be described as follows: 46–50, XX,+i(1)(q10),+der(7)t(7;17)(q31.3;q21.3)[cp15]/44–46, XX,+dic(1;18)t(q10:qter;q10:qter), der(7)t(7;17)(q31.3;q21.3),−18[cp14]. (A) Karyogram ofclone 1 containing an additional isochromosome 1q and an additional translocation chromosome 7 der(7)t(7;17) apart from two normalchromosomes 1 and 7, respectively (one normal chromosome 4 is overlaid by a normal chromosome 7). (B) Karyogram of clone 2characterised by an additional dicentric chromosome dic(1;18) and the translocation chromosome 7 der(7)t(7;17). (Note that two normalchromosomes 1 but only one normal chromosomes 7 and 18 are present; one chromosome 3 is overlaid by a chromosome 19.) (C)Pseudo-colours for each individual chromosome.Abbreviations: p = short arms of the acrocentric chromosomes 13–15, 21 and 22 consistingof repetitive DNA, pseudo-coloured differently; h= heterochromatic DNA, which is polymorphic and can be present at chromosomes 1,9 and 16. Images were captured with the ISIS3 digital FISH imaging system (MetaSystems, Altlussheim, Germany) using a PCO VC45CCD camera (PCO, Kehl, Germany) on an Axioplan 2 microscope (Zeiss, Jena, Germany).


Fig. 3. Genotoxic effects induced by (A) 18.8–150�M H2O2 (5 min at 4◦C) and (B) 100–250�M HNE (30 min at 37◦C) in primaryhuman colon cells, LT97 cells and HT29clone19A cells. The figure shows tail intensities (mean± S.D.) from independent experiments(n = 5–7), each mean was calculated from three to four parallel slides, 50 cells were evaluated per slide. The significance of individualcompound-induced effects was calculated by one-way analysis of variance (ANOVA), including Dunnett’s multiple comparison test(∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001: significantly different from solvent control). The differences between primary human colon cells,LT97 cells and HT29clone19A cells were calculated by ANOVA including Bonferroni post-test for comparing selected pairs of columns(∗∗P < 0.01, ∗∗∗P < 0.001: significant difference between two columns). The results for the H2O2-damaged primary human colon cellsand for the HT29clone19A cells have been reported previously[22].


Fig. 4. Genotoxic effects induced by (A) 18.8–150�M H2O2 (5 min at 4◦C) and (B) 100–250�M HNE (30 min at 37◦C) in LT97 and inHT29clone19A cells. This subset of results was obtained from simultaneous assessments of each compound in both cell types during thesame experimental run (n = 3). The figure shows tail intensities (mean±S.D.) calculated from three parallel slides, 50 cells were evaluatedper slide. The significance of individual compound-induced effects was calculated by one-way analysis of variance (ANOVA), includingDunnett’s multiple comparison test (∗P < 0.05, ∗∗P < 0.01: significantly different from solvent control). The differences between LT97and HT29clone19A cells were calculated by ANOVA including Bonferroni post-test for comparing selected pairs of columns (∗P < 0.05,∗∗P < 0.01: significant difference between two columns).


Fig. 5. Direct comparison of migrated DNA representing global cellular DNA andTP53 signals in the tail, following the treatment of(A) primary human colon cells and (B) LT97 cells with 100–250�M HNE (30 min at 37◦C) (n = 3). The significant differences fromsolvent control were calculated by one-way analysis of variance (ANOVA), including Dunnett’s multiple comparison test (∗P < 0.05,∗∗∗P < 0.001). The differences between global cellular DNA andTP53 signals in the tail were calculated by one-way analysis of variance,including Bonferroni post-test for comparing selected pairs of columns (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001: significant differencebetween two columns).

is a typical alteration inTP53 from colon carcinoma.In our study, we have been able to show that HNE andH2O2 are genotoxic in human colon cells representingdifferent stages of cell transformation. The first signif-icant genotoxic concentration of HNE in all cell typeswas 150�M. Interestingly, LT97 microadenoma cellswere more susceptible to HNE than primary or trans-formed colon cells. The significantly higher sensitivityof the adenoma cells compared to the HT29clone19Acells could be due to the relatively lower activity andexpression of the GST/GSH system. An increased rateof DNA damage is induced in cells which have beendepleted of GSH[51] and concomitant with the in-duction of various GSTs[59], genotoxicity of HNE isabolished[27]. GSTs effectively inactivate HNE, espe-cially GSTA4-4[52], and in LT97, both GSH and GSTare less active or expressed than in HT29clone19Acells. Also the LT97 cells lackGSTM1, which has highsubstrate specificity for 1-chloro-2,4-dinitrobenzene(CDNB), the compound used for determining GST ac-tivity. Accordingly, genetic polymorphisms and gene

expression levels together are crucial for the suscepti-bility of LT97 to HNE.

Another interesting result was that the tumour sup-pressor geneTP53 was especially sensitive towardHNE. Comparing the migration of global DNA to themigration of TP53, the latter is more susceptible tothe damaging activity of the tested compound. Thiseffect is significant for primary human colon cells andLT97 microadenoma cells after treatment with 200 and250�M HNE.

This is important since alterations of theTP53 geneare frequently encountered genetic events in colorec-tal cancer and they are late events occurring during thetransition of adenoma to malignant carcinoma[53,54].Several pathways may lead to inactivation ofTP53,such as missense and nonsense mutations or deletionsof the gene, of which 11,000 mutations are described.Two hundred and nine mutations were detected in tis-sues from colon cancer, in comparison to only six mu-tations in adenoma. G:C→ A:T transitions, mostly inCpG sites, account for more than 60% of all recorded


point mutations, with mutational hot spots occurringat codons 175, 248 and 273. These could result fromdeamination of 5-methylcytosine[14] or from etheno-adducts, which may also be formed from the epoxida-tion product of HNE[55].

Now it is important to determine whether an alteredmigration ofTP53 DNA is actually associated with anenhanced mutation rate inTP53 of LT97 and how in-creasing HNE concentrations modulate the mutationalspectrum and offer a selective growth advantage to thecells in comparison to H2O2, for example. Since LT97cells are one of the rare colon cell lines which have astable karyotype as well as a non-mutatedTP53, theywill offer a unique possibility to study these associa-tions. The studies are of importance since our findingsimply that carriers of microadenomas/adenomas are athigh risk from dietary factors which enhance levels oflipid peroxidation. This is due to the special suscepti-bility of the microadenoma cells to HNE and also tothe sensitivity ofTP53, which is a crucial target genein this preneoplastic tissue. A conservative estimationhas been that adenoma can be detected in approxi-mately 20% of the population of seniors aged 50–60years and that the incidence is associated with dietaryfactors[56,57]. It may well be that earlier lesions oc-cur in even a higher proportion of the general pop-ulation, meaning that the “healthy” gut of an elderlyperson has a high probability of harbouring groups ofcells undergoing the first stages of cell transformationadjacent to normal cells. Thus, the elderly could es-pecially benefit from dietary strategies which reducelipid peroxidation and the formation of HNE.

In conclusion, here we present studies utilising anovel approach to investigate the impact of putativerisk factors in human colon cells. The LT97 cells, re-cently established from microadenomas, are relevantmodels of preneoplastic lesions that are quite com-mon, especially in advanced age. The Comet assay isa useful method to determine genetic damage in so-matic cells regardless of proliferate activity[29,44].In the Comet assay, HNE is more genotoxic in LT97than in transformed HT29clone19A cells. The imme-diate effects of risk factors on migration ofTP53 [30],which is the relevant gene that is damaged when ade-noma develop into carcinoma, can be determined bythe method of Comet FISH. The pronounced geno-toxic effect in theTP53 DNA sequences could meanthat TP53 has a special susceptibility to mutation-

induction by HNE, an implication which needs fur-ther analysis. However, the findings on the sensitivityof human colon cells to HNE and H2O2 underline thenecessity to prevent excessive lipid peroxidation as itmay arise from diet.

Acknowledgements

Parts of this work were supported by the DeutscheKrebshilfe (Grant Number 10-1572-Po-1), theBMELF (99-HS-093), the Deutsche Forschungs-gemeinschaft (DFG, PO284/6-1), and the WilhelmSander Stiftung (99.105.1). We thank M. Wacker, P.Wanek and Prof. Dr. E. Eder (Institute for Pharma-cology and Toxicology, Julius Maximilians Univer-sity Würzburg, Germany) for supplying HNE-DMA(dimethylacetal).

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