Tomato oleoresin inhibits DNA damage but not diethylnitrosamine-induced rat hepatocarcinogenesis

ARTICLE IN PRESS

0940-2993/$ - se

doi:10.1016/j.et

�CorrespondE-mail addr

Experimental and Toxicologic Pathology 60 (2008) 59–68

www.elsevier.de/etp

Tomato oleoresin inhibits DNA damage but not

diethylnitrosamine-induced rat hepatocarcinogenesis

Clarissa Scolasticia, Gisele A.D. Lopesb, Luıs F. Barbisanb,�, Daisy M.F. Salvadoria

aDepartment of Pathology, Faculty of Medicine, UNESP Sao Paulo State University, Botucatu, SP 18618-000, BrazilbDepartment of Morphology, Institute of Biosciences, UNESP Sao Paulo State University, Botucatu, SP 18618-000, Brazil

Received 5 September 2007; accepted 15 January 2008

Abstract

Various studies have shown that lycopene, a non-provitamin A carotenoid, exerts antioxidant, antimutagenic andanticarcinogenic activities in different in vitro and in vivo systems. However, the results concerning its chemopreventivepotential on rat hepatocarcinogenesis are ambiguous. The aim of the present study was to investigate the antigenotoxicand anticarcinogenic effects of dietary tomato oleoresin adjusted to lycopene concentration at 30, 100 or 300 ppm(administered 2 weeks before and during or 8 weeks after carcinogen exposure) on liver of male Wistar rats treatedwith a single intraperitoneal dose of 20 or 100mg/kg of diethylnitrosamine (DEN), respectively. The level of DNAdamage in liver cells and the development of putative preneoplastic single hepatocytes, minifoci and foci of alteredhepatocytes (FHA) positive for glutathione S-transferase (GST-P) were used as endpoints. Significant reduction ofDNA damage was detected when the highest lycopene concentration was administered before and during the DENexposure (20mg/kg). However, the results also showed that lycopene consumption did not reduce cell proliferation innormal hepatocytes or the growth of initiated hepatocytes into minifoci positive for GST-P during early regenerativeresponse after 70% partial hepatectomy, or the number and area of GST-P positive FHA induced by DEN (100mg/kg) at the end of week 10. Taken together, the data suggest a chemopreventive effect of tomato oleoresin against DNAdamage induced by DEN but no clear effectiveness in initiating or promoting phases of rat hepatocarcinogenesis.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Chemoprevention; Diethylnitrosamine; DNA damage; GST-P liver foci; Hepatocarcinogenesis; Tomato oleoresin;

Lycopene

Introduction

Many components from medicinal or dietary plantshave been identified as potential chemopreventive agentsable to inhibit DNA damage, and even retard or reversethe multi-step carcinogenesis process (Kelloff, 2000;Knasmuller and Verhagen, 2002; Surh, 2002; Aggarwal

e front matter r 2008 Elsevier GmbH. All rights reserved.

p.2008.01.010

ing author. Tel./fax: +55 14 38116264.

ess: barbisan@ibb.unesp.br (L.F. Barbisan).

and Shishodia, 2006). Among these agents, the carote-noids have been viewed as the most promising candi-dates for cancer preventive agents (Slattery et al., 2000;Nishino et al., 2002).

Lycopene, a non-provitamin A carotenoid, is synthe-sized by microorganisms and plants, especially bytomatoes, and it is one of the most potent antioxidantsamong the dietary carotenoids due mainly to itsmany conjugated double bounds (Rao and Agarwal,2000; Bhuvaneswari and Nagini, 2005; Rao et al., 2006).

ARTICLE IN PRESSC. Scolastici et al. / Experimental and Toxicologic Pathology 60 (2008) 59–6860

The antioxidant activity of lycopene is mainly dependenton its scavenging properties of singlet oxygen andperoxyl radicals. Besides antioxidant activity, non-oxidative mechanisms have been proposed for the roleof lycopene in the prevention of cancer, such asregulation of intercellular gap junction communication,hormonal and immune systems, and metabolic path-ways of xenobiotics (Rao and Agarwal, 2000; Bhuva-neswari and Nagini, 2005; Rao et al., 2006). In fact,tomato consumption and serum lycopene seem to beassociated with the lower risk for several cancers,particularly those of prostate, gastrointestinal tract,lung and urinary bladder (Bhuvaneswari and Nagini,2005; Rao et al., 2006).

Some studies have shown cancer chemopreventiveproperties of lycopene in mouse lung, rat mammarygland, liver, colon and urinary bladder in differentchemically induced carcinogenesis models (Cohen,2002). However, regarding rat hepatocarcinogenesis,the results have proven ambiguous. Absence of protec-tive effects of this carotenoid (300mg/kg diet) adminis-tered 2 weeks before and during aflatoxin B1 doses inmale Wistar rats and for 70 weeks in male Long-EvansCinnamon (LEC) rats have not reduce the developmentof preneoplastic lesions or spontaneous liver tumors,respectively (Gradelet et al., 1998; Watanabe et al.,2001). Contrarily, Astorg et al. (1997) showed thatlycopene consumption (300mg/kg diet) 2 weeks beforeand during a single dose of 100mg/kg of diethylnitro-samine (DEN) significantly reduced the size and volumebut not the number of preneoplastic liver lesions.Besides, lycopene (70mg/kg body weight (b.w.)) admi-nistered continually (i.e., before, during and after DEN/2-acethylaminofluorene (2-AAF) treatments) to maleWistar rats reduced the development of preneoplasticliver lesions (Toledo et al., 2003).

Ito et al., 1988, 1996; Moore et al., 1999). This 8-weekmedium-term rat liver assay uses the development ofputative preneoplastic foci of altered hepatocytes (FHA)that express the placental form of the enzyme glutathioneS-transferase (GST-P) as the endpoint (Ito et al., 1988).GST-P expression is a useful marker for most FHA andliver tumors in rat hepatocarcinogenesis (Satoh et al.,1989; Ito et al., 1988). DEN is a potent genotoxiccarcinogen that has been used as an initiating agent indifferent rat hepatocarcinogenesis models (Ito et al.,1988; Dragan et al., 1991). It has been reported that afterits biotransformation, DEN produces the promutagenicadducts O6-ethyldeoxyguanosine and O4- and O6-ethyl-deoxythymidine and 8-hydroxyguanine (8-OHG) thatmay initiate liver carcinogenesis (Verna et al., 1996;Nakae et al., 1997). Consequently, the analysis of liverDNA damage may be relevant to evaluating themodifying influences of chemopreventive agents on theinitiation phase of rat hepatocarcinogenesis (Moore et al.,1999). The comet assay is a simple and fast methodology

for detecting in vitro and in vivo genotoxicity (Tice et al.,1991; Burlinson et al., 2007). The simple version of thealkaline comet assay may detect DNA strand breaks,alkaline labile sites and transient repair sites (Tice et al.,1991; Burlinson et al., 2007).

Using a medium-term liver bioassay, the present studywas designed to evaluate the potential protective actionof lycopene on the initiation and promotion phases ofrat hepatocarcinogenesis. Chemically induced primaryDNA damage (comet assay), cell proliferation anddevelopment of GST-P positive FHA were used asendpoints.

Material and methods

Animals

Six-week-old male Wistar rats were obtained from theMultidisciplinary Center for Biological Investigation(CEMIB, UNICAMP Campinas-SP, Brazil) and housedin an experimental room under controlled conditions oftemperature (2272 1C), humidity (50710%) and 12 hlight/dark cycle, with ad libitum access to drinking waterand basal diet (NUVILAB – Nuvital, Curitiba-PR,Brazil). Body weight, water and food consumption weremeasured three times a week throughout the experi-mental period. The University Ethical Committee forAnimal Research approved the protocols used in thisstudy (Protocol number 386/2004).

Chemicals

DEN (CAS 7756; Sigma-Aldrich Co., St. Louis MO,USA) was used in order to induce FHA. Thishepatocarcinogen was administered at a single intraper-itoneal (i.p.) dose of 20 (analysis of DNA damage) or100mg/kg b.w. (analysis of single cells, minifoci andFHA positive for GST-P).

Immunohistochemical staining for detecting singlehepatocytes, minifoci and FHA positive for GST-P, andproliferating hepatocytes was performed using anti-ratGST-P polyclonal antibody (clone 311) and anti-mouseproliferating cell nuclear antigen (PCNA, clone PC10)purchased from Medical and Biological LaboratoriesCo. (MBL, Tokyo, Japan) and Dako A/S (Glostrup,Denmark), respectively. Biotinilated anti-rabbit andanti-mouse IgG secondary antibody and avidin–biotinperoxidase complex kit from Vector Laboratories Inc.(Burlingame, CA, USA) were also used. 3,30-diamino-benzidine tetrahydrochroride (DAB) was purchasedfrom Sigma-Aldrich Co.

Tomato oleoresin (dewaxed Lyc-O-MatoTM 6%) wasdonated by LycoRed Natural Products Industries (Beer-Sheva, Israel). Of the total lycopene (6%), 93% was in

ARTICLE IN PRESSC. Scolastici et al. / Experimental and Toxicologic Pathology 60 (2008) 59–68 61

the all-trans form and 7% consisted of cis-lycopeneisomers. Lyc-o-MatoTM was mixed and homogenizedwith a powdered basal diet at concentrations of 30, 100and 300 ppm of lycopene (marker) and stored at 4 1C inthe dark until used. Basal diet or the basal dietscontaining tomato oleoresin were given ad libitum tothe animals from different groups.

In the comet assay, ethidium bromide, NaCl, EDTA,Tris, sodium N-lauryl sarcosine, Triton X-100, dimethylsulfoxide and low-melting-point agarose were purchasedfrom Sigma-Aldrich Co. whereas normal agarose andHanks balanced salt solution (HBSS) were provided byInvitrogen (Grand Island, NY, USA).

Experimental design

Animals were randomly distributed into nine groups(35–40 rats in each) (Fig. 1): Group 1, negative control,received a single i.p. dose of 0.9% NaCl at the secondweek, and basal diet throughout the experimental period(10 weeks); Group 2 (a,b) positive control, was treatedwith a single i.p. dose of 20mg/kg b.w. (a) or 100mg/kgb.w. (b) of DEN at the second week, and basal dietthrough all experimental period; Groups 3 (a,b), 4 (a,b)and 5 (a,b) were fed basal diet containing tomatooleoresin adjusted to lycopene concentration at 30, 100or 300 ppm, respectively, for the first 2 weeks, and

Fig. 1. Experime

received a single i.p. dose of 20mg/kg b.w. (a) or100mg/kg b.w. (b) of DEN at the end of the secondweek, when they started receiving basal diet until theend of the experimental period; Groups 6 (b), 7 (b) and 8(b), were initially fed basal diet (during the first 2 weeks),received a single i.p. dose of 100mg/kg b.w. (b) of DENat the end of the second week and, then were fed basaldiet containing tomato oleoresin adjusted to lycopeneconcentration at 30, 100, or 300 ppm of lycopene,respectively, until the end of the experimental period;Group 9, was fed basal diet containing tomato oleoresinadjusted to lycopene concentration at 300 ppm lycopenethroughout the experimental period (10 weeks), andreceived a single i.p. injection of 0.9% NaCl at thesecond week. Seventy percent PH was performed atweek 5 of the experiment. The animals were killed 4 hafter DEN administration for the comet assay, or 48, 72or 120 h after PH for the single cells and minifocipositive for GST-P and cell proliferation analysis, or 8weeks after DEN administration for GST-P positiveFHA analysis.

Immunohistochemical staining for GST-P and

PCNA

At sacrifice, the liver was removed and weighed, andsmall fragments were fixed in 10% phosphate-buffered

ntal design.

ARTICLE IN PRESSC. Scolastici et al. / Experimental and Toxicologic Pathology 60 (2008) 59–6862

formalin solution, embedded in paraffin and sectioned(5 mm thickness) for hematoxylin and eosin (H&E)staining or immunohistochemical demonstration ofplacental form of GST-P and PCNA expression. Briefly,deparaffinized 5-mm-thick serial liver sections on poly-L-lysine coated slides were treated with 3% H2O2 inphosphate-buffered saline for 15min, nonfat milk for60min, anti-rabbit GST-P (1:1000 dilution) or anti-mouse PCNA (1:200 dilution) antibodies for 120min,biotinylated anti-rabbit or anti-mouse IgG antibodies(1:200 dilution) for 60min, and avidin–biotin peroxidasesolution (1:1:50 dilution) for 45min. Chromogencolor development was accomplished with DAB asthe substrate to demonstrate the sites of peroxidasebinding. The slides were counterstained with Harris’hematoxylin.

Analysis of single cells, minifoci and FHA positive

for GST-P, and PCNA rates.

Single hepatocytes and minifoci (2–15 hepatocytes)positive for GST-P (analyzed 48, 72 or 120 h after PHand GST-P positive FHA (450 hepatocytes) (analyzed8 weeks after DEN injection, 100mg/kg) were measuredusing a Nikon photomicroscope (Microphot-FXA)connected to the KS-300 apparatus (Kontron Elektro-nic, Germany). The liver areas were analyzed in a specialMacro-Stand device (support with Canon TV zoom lensV6� 16/16-100mm plus a Canon 58mm close-up 240lens connected to a CCD black-and-white video cameramodule with a Sony DC-777 camera unit) connected tothe KS-300 system. Data were expressed as number ofsingle hepatocytes, minifoci or FHA per cm2 and as area(mm2/cm2) (FHA).

PCNA S-phase labeling indices (PCNA LI%) weredetermined as the percentage of labeled hepatocytenuclei among the total number of cells scored (�2000hepatocytes). PCNA LI% were analyzed in animalskilled 48, 72 or 120 h after PH.

Comet assay

DNA damage was evaluated in liver cells sampledfrom rats treated with a low dose rather than a high doseof DEN to eliminate DNA fragmentation associatedwith the necrotic/apoptotic death process (necrogeniceffects) (Agner et al., 2004). The liver was excised,washed in saline solution, and a small fragment of theleft lobule was transferred to a Petri dish kept on ice.The fragment was washed, minced, and suspended into1ml of HBSS, supplemented with 20mM EDTA and10% DMSO. Liver tissue was minced again and thesuspension containing isolated cells was transferred to atube maintained on ice until the preparation of the slides(Agner et al., 2004).

DNA damage was measured using the comet assayunder alkaline conditions (Tice et al., 1991). Volumes of10 ml of liver cell suspensions, were mixed with 120 ml of0.5% low-melting-point agarose (37 1C), layered ontopre-coated slides with normal-melting-point agarose,placed under a coverslip, and maintained at 4 1C, for5min, for agarose solidification. The coverslip wasgently removed, and slides submersed into a cold lysingsolution (2.5M NaCl, 100mM EDTA, 10mM Tris 10%DMSO and 1% Triton X-100) for 24 h. After lysing, theslides were briefly washed in PBS and placed onto ahorizontal electrophoresis unit filled with fresh electro-phoresis alkaline buffer (300mM NaOH and 1mMEDTA, pH413), for 20min, at 4 1C to allow DNAunwinding and expression of alkali-labile sites. Electro-phoresis was conducted in the same solution at 4 1C, for20min, at 25V and 300mA. After electrophoresis, theslides were neutralized (0.4M Tris at pH 7.5), dehy-drated in absolute ethanol, and dried at room tempera-ture. Before analysis, the slides were stained with 50 mlethidium bromide (20 ml/ml). Fifty randomly selectedcells (nucleoids) per animal were examined at 400�magnification in a fluorescence microscope, using anautomated image analysis system (Comet Assay II,Perceptive Instrument, Suffolk, UK). Two parameterswere adopted as indicators of DNA damage: tailintensity (% tail DNA, in % pixels), and tail moment(product of tail DNA/total DNA divided by the tailcenter of gravity, in arbitrary units).

Statistical analysis

Data were compared using one-way ANOVA (bodyand liver weights, and food consumption), and Krus-kal–Wallis test (GST-P, comet assay and PCNA data).The contrasts among the groups were analyzed by theTukey or Student-Newman-Keuls methods. Significantdifferences was assumed when Po0.05.

Results

A total of 18 rats (5 rats in Group 1, 4 rats in Group2, 5 in Group 8 and 4 in Group 9) died during PHproceeding.

Fig. 2 shows the DNA damage levels (tail intensityand tail moment parameters) in liver cells of rats fedtomato oleoresin containing lycopene as a marker andtreated or not with DEN (20mg/kg). Besides thestatistical difference between positive and negativecontrols (G2 group vs. G1 and G9 groups, Po0.05),the results showed a significant reduction of DEN-induced DNA damage in liver cells of animals fedlycopene at the highest concentration (300 ppm) (G2group vs. G5 group, Po 0.05).

ARTICLE IN PRESS

Fig. 2. Effect of dietary tomato oleoresin on DNA damage

induced by a single low dose of diethylnitrosamine (DEN).

Tail moment and tail intensity were measured using the Comet

assay on liver cells isolated 4 h after DEN administration.

G1 ¼ 0.9% NaCl, G2 ¼ DEN, G3 ¼ LYC 30ppm+DEN,

G4 ¼ LYC 100 ppm+DEN, G5 ¼ LYC 300 ppm+DEN,

G9 ¼ 0.9% NaCl+LYC 300 ppm. DEN: diethylnitrosamine

(20mg/kg b.w.), LYC 1, LYC 2 and LYC 3: tomato oleoresin

adjusted to lycopene concentration at 30, 100 or 300 ppm,

values are means7S.D. (five rats/group). *Statistically differ-

ent from groups G1 and G9 and **statistically different from

group G2, Po 0.05.

C. Scolastici et al. / Experimental and Toxicologic Pathology 60 (2008) 59–68 63

Table 1 presents the data of final body weight, body-weight gain, relative liver weight, food and tomatooleoresin consumption from animals killed 8 weeks afterDEN administration (100mg/kg). The mean dailyestimated lycopene intakes were 2.5, 8.7 and 25.2mg/kg b.w, for basal diets containing 30, 100 and 300 ppm,respectively. A reduction in the relative liver weight wasobserved in the group treated with tomato oleoresin,when compared to the respective positive control group(G2 group vs. G6, G7 and G8 groups, Po0.01). Thisfinding was not accompanied by any histologicalchanges.

Tables 2 and 3 present the mean values of relativeliver weight, PCNA labeling indices and number ofinitiated hepatocytes and minifoci positive for GST-P inliver from animals killed 48, 72 and 120 h after PH. Anincrease in relative liver weight simultaneous with adecrease in PCNA LI% (Fig. 2A–C) was observed indifferent groups between 48 and 120 h after the PH(Po0.05) (Table 2). Also, the results showed a decreasein number of single hepatocytes positive for GST-Psimultaneous with an increase in number of minifoci,related to the time of sacrifice after PH (Po0.05)(Table 3). However, tomato oleoresin consumption didnot change relative liver weights, cell proliferation ratesor the growth of initiated hepatocytes into minifocipositive for GST-P (Fig. 3D) at 48, 72 or 120 h after PH.

Table 4 presents the mean values of number and area ofFHA positive for GST-P (Fig. 3E) from animals killed 8weeks after DEN administration (100mg/kg). A signifi-cant increase of development of GST-P positive FHA(number and area) was observed in the animals treatedwith DEN when compared to those treated only withvehicle (negative control) or fed the highest concentrationof lycopene (300ppm) (G1 and G9 groups vs. G2 to G8groups, Po0.01). However, no protective effect oflycopene on development of GST-P positive FHA wasdetected in the DEN-initiated animals when tomatooleoresin was administered either during the initiation orpromoting phases of rat hepatocarcinogenesis.

Discussion

Lycopene is the major carotenoid in human plasma,tissue and diet. It is unique among the carotenoids inthat it has one major food source: tomatoes and tomatoproducts (Bhuvaneswari and Nagini, 2005; Rao et al.,2006). Epidemiological and experimental studies havesuggested a strong association between lycopene intakeand reduced risk for a variety of cancers (Bhuvaneswariand Nagini, 2005; Rao et al., 2006). However, noindependent effect of lycopene on hepatocellular carci-noma risk was observed in middle-aged and olderChinese men (Yuan et al., 2006) and with regard torat hepatocarcinogenesis the results have proven ambig-uous. Thus, the present study aimed to evaluate thechemopreventive effect of lycopene in rats submitted toa well-established medium-term liver carcinogenesisassay.

Mellert et al. (2002) reported that a 13-week treatmentwith a synthetic crystalline lycopene, at dose levels of500, 1500 and 3000mg/kg b.w./day, did not inducesignificant toxicological changes in male and femaleWistar rats. However, Breinholt et al. (2003) haveshown that 10-week treatment with 500 ppm of lycopene(tomato oleoresin) in food increased both the oxidativeDNA damage levels in peripheral lymphocytes and thenumber of spontaneous GST-P positive liver FHA whencompared to non-treated animals, suggesting a pro-oxidant effect. Our results showed a reduction in relativeliver weight values in initiated and lycopene-supplemen-ted groups, compared to the positive control group atthe end of the 10th week, although no abnormalitiesindicative of toxicity were detected in the liver afterhistological analysis.

On the other hand, we and others have demonstratedthat lycopene presents an in vivo and in vitro protectiveeffect on DNA damage induced by various xenobiotics(Gradelet et al., 1998; Toledo et al., 2003; Scolasticiet al., 2007; Ferreira et al., 2007). Recently, we haveshown that lycopene (10, 25, or 50 mM) protected

ARTICLE IN PRESS

Table 1. Body weight, body-weight gain, relative liver weight, food and lycopene consumptions of rats submitted to the DEN-PH

model and fed tomato oleoresina

Treatmentb/group Final body

weight (g)cBody-weight

gain (g)

Relative liver

weight (%)

Food consumption

(g/rat/day)

Lycopene intake

(g/rat/day)

NaCl (G1, n ¼ 8) 433.0722.6 148.3717.3 3.0670.19 26.572.61 –

LYC3 (G9, n ¼ 10) 401.9728.6 130.8724.6 2.9570.15 26.672.60 23.673.7

DEN (G2, n ¼ 10) 410.8766.2 133.17 22.1 3.2170.30 25.972.50 –

Pre-initiation

LYC1+DEN

(G3, n ¼ 10)

393.2741.1 122.2718.2 3.0370.18 26.672.51 3.0570.35

LYC2+DEN

(G4, n ¼ 10)

397.3726.4 131.2722.2 3.1270.17 27.872.71 10.8571.34

LYC3+DEN

(G5, n ¼ 10)

385.8733.0 118.2720.9 3.0070.21 25.272.50 30.2273.57

Post-initiation

DEN+LYC1

(G6, n ¼ 10)

387.3727.1 119.2721.6 2.9470.26� 26.272.53 2.2570.31

DEN+LYC2

(G7, n ¼ 10)

396.8730.4 127.7723.5 2.9370.15� 26.472.57 7.6870.96

DEN+LYC3

(G8, n ¼ 10)

396.1734.8 126.5726.1 2.9070.13� 26.572.59 22.6373.30

aValues are means7S.D.bDEN: diethylnitrosamine (100mg/kg b.w.), LYC1, LYC2, LYC3: tomato oleoresin adjusted to lycopene concentration at 30, 100 or 300 ppm,

respectively, n ¼ number of rats.cAt the end of the 10th week.�Statistically different from Group G2, Po0.01.

Table 2. Relative liver weight (RLW %) and hepatic PCNA labeling index (PCNA LI %) of rats submitted to the DEN-PH model

and fed tomato oleoresina

Treatmentb/group RLW % and PCNA LI% after PH

48hc 72 h 120 h

RLM % PCNA LI% RLM % PCNA LI% RLM % PCNA LI%

NaCl (G1, n ¼ 22) 2.7470.17 4.8771.99 3.0070.16� 1.5970.55� 3.1370.22� 0.7170.29�

DEN (G2, n ¼ 21) 2.5570.21 3.3071.71 2.9170.17� 1.4070.52� 3.2370.13�,�� 0.7470.21�

DEN+LYC3 (G8, n ¼ 20) 2.5270.11 4.9071.91 2.8670.16� 1.7170.61� 3.1570.32�,�� 0.9070.31�

LYC3 (G9, n ¼ 21) 2.6470.15 3.3271.45 3.0870.35� 1.8470.74� 3.1170.30� 0.8470.18�

aValue are means7S.D.bDEN: diethylnitrosamine (100mg/kg b.w.), LYC3 ¼ tomato oleoresin adjusted to lycopene concentration at 300 ppm, n ¼ number of rats.cSacrifice after 70% partial hepatectomy (PH).�Statistically different from 48h after PH (Po0.05).��Statistically different from 72 h after PH (Po0.05).

C. Scolastici et al. / Experimental and Toxicologic Pathology 60 (2008) 59–6864

against DEN-induced primary DNA damage (cometassay) in HepG2 cells when this carotenoid was addedbefore or simultaneously with this mutagen/carcinogen(Scolastici et al., 2008). Also, the present study showed ahepatoprotective effect of lycopene (300 ppm) againstprimary DNA damage in liver cells induced by DEN(20mg/kg). Enzymes of the cytochrome P-450 2E1(CYP2E1) subfamily play a role in the biotransforma-tion of a range of compounds, including DEN (Vernaet al., 1996). It has been reported that DEN biotrans-

formation produces the promutagenic DNA lesionswhich play an important role in DNA damage andinduction of initiated hepatocytes (Verna et al., 1996;Nakae et al., 1997). Therefore, lycopene feeding couldhave prevented DNA damage by modulating themetabolism of this carcinogen through inhibition ofthe CYP2E1 enzymatic complex, induction detoxifica-tion pathways and/or or free radical scavenger propri-eties, thus reducing oxidative DNA damage (Astorget al., 1997; Gradelet et al., 1998; Rao and Agarwal,

ARTICLE IN PRESS

Table 3. Number of single cells and minifoci positive for GST-P of rats submitted to the DEN-PH model and fed tomato

oleoresina

Treatmentb/group GST-P positive single hepatocytes GST-P positive minifoci

48 hc 72 h 120 h 48 h 72 h 120 h

DEN (G2, n ¼ 21) 128.2713.87 58.079.68� 46.4710.12� 0 8.376.76� 10.675.01�

DEN+LYC3 (G8, n ¼ 20) 120.8 735.77 50.3712.12� 47.576.94** 0 6.773.08 15.277.59�

aValues are mean7S.D.bDEN: diethylnitrosamine (100mg/kg b.w.), LYC3: tomato oleoresin adjusted to lycopene concentration at 300 ppm, n ¼ number of rats.cSacrifice: 48, 72 and 120 h after the 70% partial hepatectomy (PH).�Statistically different from 48 h after PH (Po0.05).

Fig. 3. (A–C) PCNA positive hepatocytes in liver of control animals at 48, 72 and 120 h after 70% partial hepatectomy

(40� objective); (D) single cells and minifoci positive for GST-P (40� objective) and (E) foci of altered hepatocytes positive for

GST-P (20� objective).

C. Scolastici et al. / Experimental and Toxicologic Pathology 60 (2008) 59–68 65

ARTICLE IN PRESS

Table 4. Number and area of GST-P positive FHA of rats submitted to the DEN-PH model and fed tomato oleoresina

Treatmentb/group Number of rats GST-P positive foci

Number (N/cm2) Area (mm2/cm2)

NaCl (G1) 8 0.4170.53� 0.00770.01�

LYC3 (G9) 10 0.2370.29 � 0.00170.001�

DEN (G2) 10 8.9372.27 0.0770.02

Pre-initiation

LYC1+DEN (G3) 10 10.1674.98 0.0970.06

LYC2+DEN (G4) 10 10.7173.72 0.1070.04

LYC3+DEN (G5) 10 8.7872.63 0.0770.03

Post-initiation

DEN+LYC1 (G6) 10 8.2374.23 0.0970.04

DEN+LYC2 (G7) 10 10.2774.14 0.1070.06

DEN+LYC3 (G8) 10 9.1873.25 0.0970.04

aValues are mean7S.D.bDEN: diethylnitrosamine (100mg/kg b.w.), LYC1, LYC2, LYC3: tomato oleoresin adjusted to lycopene concentration at 30, 100 and 300 ppm,

respectively.�Statistically different from Groups G2 to G8, Po 0.01.

C. Scolastici et al. / Experimental and Toxicologic Pathology 60 (2008) 59–6866

1999). It should be emphasized that, besides lycopene,other substances (g-tocopherol, a-tocopherol, b-caro-tene, phytofluene and phytoene) present in the tomato-oleoresin supplementation used herein (Richelle et al.,2002) may contribute to explaining the current results.

Since initiation is a rare event, affecting only a fewhepatocytes, the numbers of initiated hepatocytes andFHA are a major determinant for the risk of developingliver cancer since single hepatocytes and minifocipositive for GST-P are detected very early in carcino-gen-treated rats (Moore et al., 1987 Satoh et al., 1989).The lycopene feeding before and during the treatmentwith 100mg/kg of DEN did not alter the number ofpreneoplastic GST-P positive FHA analyzed at the endof the 10th week. The apparent discrepancy concerningthe chemopreventive effect of lycopene on DNA damageand the absence of an anti-initiating activity against rathepatocarcinogenesis is conflicting. The probable me-chanism that explains the discrepancy can be in partassociated with the different carcinogen doses used here.

Some works have shown that the mutagenic/carcino-genic effects detected in high DEN doses cannot bequantitatively extrapolated to low doses (Williams et al.,1993, 1996). Astorg et al. (1997) described an anti-initiating activity of lycopene (300 ppm in diet) against asingle i.p. dose of 100mg/kg b.w. in male Wistar rats.Their findings are based on a reduction in the size andvolume but not the number of g-glutamyltranspeptidase(g-GT) and GST-P positive FHA. However, the numberrather than size (area) and volume of FAH is a suitableparameter to evaluate the extension of liver initiation(Hendrich et al., 1987). Thus, an effective influence oflycopene on the initiation phase of rat hepatocarcino-genesis should continue to be investigated.

Lycopene intake did not present an anti-promotingeffect against the development of GST-P positive FHAinduced by DEN. This carotenoid did not inhibit cellproliferation of normal hepatocytes or the growth ofinitiated hepatocytes into minifoci after the proliferativestimulus induced by 70% PH, nor did it inhibitdevelopment of GST-P positive FHA during the post-initiation period. The non-protective effect can be inpart explained by the lycopene supplementation doses,which may be insufficient to inhibit the growth anddevelopment of putative preneoplastic hepatocellularlesions (Watanabe et al., 2001).

Several in vitro studies have demonstrated an anti-proliferative action of lycopene on different tumor celllines. Amir et al. (1999) described a concentration-dependent reduction of HL-60 promyelocytic leukemiacell growth after treatment with lycopene. In another setof studies, the growth stimulation of MCF-7 mammarycancer cells by insulin-like growth factor I (IGF-I) wasmarkedly reduced by lycopene (Karas et al., 2000). InLNCaP human prostate cancer cells, after 6, 24 and 48 hof incubation with lycopene, cell proliferation wasobserved to be inhibited by up to 33% (Hwang andBowen, 2004, 2005); in Hep3B human hepatoma cellline, lycopene inhibited cell growth in a dose-dependentmanner (Park et al., 2005). Similar results were observedin SK-Hep1 human hepatoma cells, i.e., lycopeneinhibited cell growth at rates of 5% (0.1 mM) and 40%(50 mM), after 24-h incubation, and at rates of 30%(5 mM) and 40% (10 mM), after 48 h incubation (Hwangand Lee, 2006). Besides in vitro assays, in vivo studieshave also shown inhibition of cell proliferation incolonic crypt of male Sprague–Dawley rats treatedwith tomato juice (2.0% in the drinking water)

ARTICLE IN PRESSC. Scolastici et al. / Experimental and Toxicologic Pathology 60 (2008) 59–68 67

(Sengupta et al., 2003) or in normal urothelial epithe-lium and in transitional cell carcinoma induced byN-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in maleFischer rats orally treated with lycopene (0.0025% in thedrinking water) (Okajima et al., 1998). To our knowl-edge, no report exists on cell proliferation in the liver oflycopene-supplemented rats.

The effect of other carotenoids on cell proliferationhas also been described. Vitamin A, b-carotene and all-trans and 9-cis retinoic acid have strongly inhibitedcell proliferation when administered during the progres-sion phase of rat hepatocarcinogenesis in the ‘‘resistanthepatocyte’’ model (Silveira et al., 2001, Morenoet al., 2002). Our findings indicate that lycopene didnot alter early phases but the possibility exists thatlycopene concentrations used herein could inhibit theprogression phase of chemically induced rat hepatocar-cinogenesis.

In conclusion, our results suggest a chemopreventiveeffect of tomato oleoresin against DNA damage inducedby a low dose of DEN but no clear effectiveness ininitiating or promoting phases of rat hepatocarcinogen-esis by a high dose of this hepatocarcinogen.

Acknowledgments

This study was supported by the Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq)and Coordenacao de Aperfeicoamento de Pessoal deNıvel Superior (CAPES). The authors also thankDr. Ana Lucia dos Anjos Ferreira (Faculty of Medicine,Department of Internal Medicine, UNESP Botucatu,SP, Brazil) for her valuable suggestions in reviewing andediting this manuscript and Dr. Zohar Nir (LycoRedNatural Products Industries, Ltd., Beer-Sheeva, Israel)for the lycopene donation. Scolastici, C. and Lopes,G.A.D. were recipients of fellowships from CAPES andFundacao de Amparo a Pesquisa do Estado de SaoPaulo (FAPESP, 05/57180-4), respectively.

References

Aggarwal BB, Shishodia S. Molecular targets of dietary agents

for prevention and therapy of cancer. Biochem Pharmacol

2006;71:1397–421.

Agner AR, Barbisan LF, Scolastici C, Salvadori DM. Absence

of carcinogenic and anticarcinogenic effects of annatto in

the rat liver medium-term assay. Food Chem Toxicol

2004;42:1687–93.

Amir H, Karas M, Giat J, Danilenko M, Levy R, Yermiahu T,

et al. Lycopene and 1,25-dihydroxyvitamin D3 cooperate in

the inhibition of cell cycle progression and induction of

differentiation in HL-60 leukemic cells. Nutr Cancer 1999;

33:105–12.

Astorg P, Gradelet S, Berges R, Suschetet M. Dietary lycopene

decreases the initiation of liver preneoplastic foci by

diethylnitrosamine in the rat. Nutr Cancer 1997;29:60–8.

Bhuvaneswari V, Nagini S. Lycopene: a review of its potential

as an anticancer agent. Curr Med Chem Anticancer Agents

2005;5:627–35.

Breinholt VM, Molck AM, Svendsen GW, Daneshvar B,

Vinggaard AM, Poulsen M, et al. Effects of dietary

antioxidants and 2-amino-3-methylimidazo[4,5-f]-quinoline

(IQ) on preneoplastic lesions and on oxidative damage,

hormonal status, and detoxification capacity in the rat.

Food Chem Toxicol 2003;41:1315–23.

Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab

SY, Collins AR, et al. In vivo comet assay workgroup, part

of the fourth international workgroup on genotoxicity

testing. Fourth international workgroup on genotoxicity

testing: results of the in vivo comet assay workgroup.

Mutat Res 2007;627:31–5.

Cohen LA. A review of animal model studies of tomato

carotenoids, lycopene, and cancer chemoprevention. Exp

Biol Med 2002;227:864–8.

Dragan YP, Rizui T, Xu YH, Hully JR, Bawa N, Campbell

HA, et al. An initiation-promotion assay in rat liver as a

potential complement to the 2-year carcinogenesis bioassay.

Fundam Appl Toxicol 1991;16:525–47.

Ferreira AL, Salvadori DM, Nascimento MC, Rocha NS,

Correa CR, Pereira EJ, et al. Tomato-oleoresin supplement

prevents doxorubicin-induced cardiac myocyte oxidative

DNA damage in rats. Mutat Res 2007;631:35.

Gradelet S, Le Bon AM, Berges R, Suschelet M, Astorg

P. Dietary carotenoids inhibit aflatoxin B1-induced liver

preneoplastic foci and DNA damage in the rat: role of the

modulation of aflatoxin B1 metabolism. Carcinogenesis

1998;19:403–11.

Hendrich S, Campbell HA, Pitot HC. Quantitative stereo-

logical evaluation of four histochemical markers of altered

foci in multistage hepatocarcinogenesis in the rat. Carci-

nogenesis 1987;8:1245–50.

Hwang ES, Bowen PE. Cell cycle arrest and induction of

apoptosis by lycopene in LNCaP human prostate cancer

cells. J Med Food 2004;7:284–9.

Hwang ES, Bowen PE. Effects of lycopene and tomato paste

extracts on DNA and lipid oxidation in LNCaP human

prostate cancer cells. Biofactors 2005;23:97–105.

Hwang ES, Lee HJ. Inhibitory effects of lycopene on the

adhesion, invasion, and migration of SK-Hep1 human

hepatoma cells. Exp Biol Med 2006;231:322–7.

Ito N, Tsuda H, Tatematsu M, Inoue T, Tagawa Y, Aoki T,

et al. Enhancing effect of various hepatocarcinogens on

induction of preneoplastic glutathione S-transferase placen-

tal form positive foci in rats: an approach for a new medium-

term bioassay system. Carcinogenesis 1988;9:387–94.

Ito N, Hasegawa R, Imaida K, Hirose M, Shirai T. Medium-

term liver and multi-organ carcinogenesis bioassay for

carcinogens and chemopreventive agents. Exp Toxicol

Pathol 1996;48:113–9.

Karas M, Amir H, Fishman D, Danilenko M, Segal S, Nahum

A, et al. Lycopene interferes with cell cycle progression and

insulin-like growth factor I signaling in mammary cancer

cells. Nutr Cancer 2000;36:101–11.

ARTICLE IN PRESSC. Scolastici et al. / Experimental and Toxicologic Pathology 60 (2008) 59–6868

Kelloff GJ. Perspectives on cancer chemoprevention research

and drug development. Adv Cancer Res 2000;78:199–334.

Knasmuller S, Verhagen H. Impact of dietary factors on

cancer causes and DNA integrity: new trends and aspects.

Food Chem Toxicol 2002;8:1047–50.

Mellert W, Deckardt K, Gembardt C, Schulte S, Van

Ravenzwaay B, Slesinski R. Thirteen-week oral toxicity

study of synthetic lycopene products in rats. Food Chem

Toxicol 2002;40:1581–8.

Moore MA, Nakagawa K, Satoh K, Ishikawa T, Sato

K. Single GST-P positive liver cells-putative initiated

hepatocytes. Carcinogenesis 1987;8:483–6.

Moore MA, Tsuda H, Tamano S, Haguwara A, Imaida K,

Shirai T, et al. Marriage of a medium-term liver model to

surrogate markers—a practical approach for risk and

benefit assessment. Toxicol Pathol 1999;27:237–42.

Moreno FS, S-Wu T, Naves MMV, Silveira ER, Oloris SC, da

Costa MA, et al. The inhibitory effects of b-carotene and

vitamin A during the progression phase of hepatocarcino-

genesis involve inhibition of cell proliferation but not

alterations in DNA methylation. Nutr Cancer 2002;

44:80–8.

Nakae D, Kobayashi Y, Akai H, Andoh N, Satoh H, Ohashi

K, et al. Involvement of 8-hydroxyguanine formation in the

initiation of rat liver carcinogenesis by low dose levels of

N-nitrosodiethylamine. Cancer Res 1997;57:1281–7.

Nishino H, Murakoshi M, Ii T, Takemura M, Kuchide M,

Kanazawa M, et al. Carotenoids in cancer chemopreven-

tion. Cancer Metastasis Rev 2002;21:257–64.

Okajima E, Tsutsumi M, Ozono S, Akai H, Denda A, Nishino

H, et al. Inhibitory effect of tomato juice on rat urinary

bladder carcinogenesis after N-butyl-N-(4-hydroxybutyl)ni-

trosamine initiation. Jpn J Cancer Res 1998;89:22–6.

Park YO, Hwang ES, Moon TW. The effect of lycopene on

cell growth and oxidative DNA damage of Hep3B human

hepatoma cells. Biofactors 2005;23:129–39.

Rao AV, Agarwal S. Role of lycopene as antioxidation

carotenoid in the prevention of chronic diseases: a review.

Nutr Res 1999;19:305–23.

Rao AV, Agarwal S. Role of antioxidant lycopene in cancer

and heart disease. J Am Coll Nutr 2000;19:563–9.

Rao AV, Ray MR, Rao LG. Lycopene. Adv Food Nutr Res

2006;51:99–164.

Richelle M, Bortlik K, Liardet S, Hager C, Lambelet P, Baur

M, et al. A food-based formulation provides lycopene with

the same bioavailability to humans as that from tomato

paste. J Nutr 2002;132:404–8.

Satoh K, Hatayama I, Takeoka N, Tamai K, Shimizu T,

Tatematsu M, et al. Transient induction of single GST-P

positive hepatocytes by DEN. Carcinogenesis 1989;10:

2107–11.

Scolastici C, Alves de Lima RO, Barbisan LF, Ferreira AL,

Ribeiro DA, Salvadori DM. Lycopene activity against

chemically induced DNA damage in Chinese hamster ovary

cells. Toxicol In Vitro 2007;21:840–5.

Scolastici C, Alves de Lima RO, Barbisan LF, Ferreira AL,

Ribeiro DA, Salvadori DM. Antigenotoxicity and anti-

mutagenicity of lycopene in HepG2 cell line evaluated by

the comet assay and micronucleus test. Toxicol In Vitro

2008;22:510–4.

Sengupta A, Ghosh S, Das S. Tomato and garlic modulate

azomymethane-induced colon carcinogenesis in rats. Eur

J Cancer Prev 2003;12:195–200.

Silveira ER, Naves MMV, Vannucchi H, Jordao Jr AA, Dagli

ML, Moreno FS. Vitamin A and all-trans and 9-cis retinoic

acids inhibit cell proliferation during the progression phase

of hepatocarcinogenesis in Wistar rats. Nutr Cancer

2001;40:244–51.

Slattery ML, Benson J, Curtin K, Ma K-N, Schaeffer D,

Potter JD. Carotenoids and colon cancer. Am J Clin Nutr

2000;71:575–82.

Surh YJ. Anti-tumor promoting potential of selected spice

ingredients with antioxidative and anti-inflammatory

activities: a short review. Food Chem Toxicol 2002;40:

1091–7.

Tice RR, Andrews PW, Hirai O, Singh NP. The single cell gel

(SCG) assay: an electrophoretic technique for the detection

of DNA damage in individual cells. Adv Exp Med Biol

1991;283:157–64.

Toledo LP, Ong TP, Pinho ALG, Jordao Jr AA, Vanucchi H,

Moreno FS. Inhibitory effects of lutein and lycopene on

placental glutathione S-transferase positive preneoplastic

lesions and DNA strand breakage induced in Wistar rats by

the resistant hepatocyte model of hepatocarcinogenesis.

Nutr Cancer 2003;47:62–9.

Verna L, Whysner J, Williams GM. N-Nitrosodiethylamine

mechanistic data and risk assessment: bioactivation, DNA-

adduct formation, mutagenicity, and tumor initiation.

Pharmacol Ther 1996;71:57–81.

Watanabe S, Kitade Y, Masaki T, Nishioka M, Satoh K,

Nishino H. Effects of lycopene and Sho-saiko-to on

hepatocarcinogenesis in a rat model of spontaneous liver

cancer. Nutr Cancer 2001;39:96–101.

Williams GM, Gebhardt R, Sirma H, Stenback F. Non-

linearity of neoplastic conversion induced in rat liver by low

exposures to diethylnitrosamine. Carcinogenesis 1993;14:

2149–56.

Williams GM, Iatropoulos MJ, Wang CX, Ali N, Rivenson A,

Peterson LA, et al. Diethylnitrosamine exposure-responses

for DNA damage, centrilobular citotoxicity, cell prolifera-

tion and carcinogenesis in rat liver exhibit some non-

linearities. Carcinogenesis 1996;17:2253–8.

Yuan J-M, Gao Y-T, Ong C-N, Ross RK, Yu MC.

Prediagnostic level of serum retinol in relation to reduced

risk of hepatocellular carcinoma. J Natl Cancer Inst 2006;

98:482–90.