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Toxicology 249 (2008) 97101

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Toxicology

j o u rna l h om epag e : www.e l s ev i e r . co m/ lo ca t e / t o x i c o l

-Tocopherol, MDAHNE and 8-OHdG levels in liver and heart mitochondriaof adriamycin-treated rats fed with alcohol-free beer

Victoria Valls-Belles a , , Ma del Carmen Torres a , Laura Boix a ,Pilar Mu niz b , Maria Luisa Gonzalez-SanJose b , Pilar Codo ner-Franch aa Departamento de Pediatra, Ginecologa y Obstetricia, Facultad de Medicina, Universidad de Valencia, Spainb Departamento de Biotecnologa y Ciencia de los Alimentos, Universidad de Burgos, Spain

a r t i c l e i n f o

Article history:Received 12 February 2008Received in revised form 8 April 2008Accepted 11 April 2008Available online 22 April 2008

Keywords:Alcohol-free beerAdriamycinOxidative stressAntioxidants

a b s t r a c t

Different studies indicate that oxidative stress and mitochondrial damage are key factors in differentpathogenic process. The aim of this study was to investigate the possible protective role of alcohol-freebeer on adriamycin-induced (ADR) heart and liver toxicity using biomarkers of oxidative stress. Thiseffect was compared with the effect of alcohol beer intake and with a control group. Rats were randomlydivided into six groups. The rst group received no adriamycin, was fed with water and was regarded asthecontrol group;the secondgroupwas injected with a ADR(two cycles of 5 mg/kg); the third and fourthgroups were fed with alcohol-free and beer for 21 days, respectively and the fth and sixth groups werefed with alcohol-free and beer beginning 7 days before the administration of a rst dose of ADR. Beerwas administrated intragastrically and ADR (two cycles of 5mg/kg) was intraperitoneally. The levels of MDA+ 4HNE (malondialdehyde and 4-hydroxynonenal) in heart mitochondria was higher in the grouptreated with ADR alone than in the control groups, and it was lower in the groups treated with ADR that drank beer than in the ADR group alone. However, no difference was observed in liver mitochondriabetween thegrouptreated with ADRand thegrouptreated with ADRthat drank beer.Signicant decreasein the levels of heart and liver -tocopherol was observed in the ADR group when compared to thecontrol groups, and thisdecreasewas normalizedby beertreatment.Interestingly,the levelsof antioxidant

-tocopherol in liver were signicantly higher in rats that consumed alcohol-free beer than in thosethat consumed alcohol beer. Intake of alcohol-free beer showed a DNA protective effect to decreasessignicantly the levels of 8-OHdG levels in heart and liver increased by the ADR-treatment.

In conclusion, this study clearly indicated that alcohol-free beer consumption signicantly reduces theadriamycin-induced oxidative stress.

2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Oxidative stress is involved in the pathology of many diseases,suchas atheroclerosis, diabetes, neurodegenerative diseases, aging,cancer, etc. Dietary antioxidants may afford protection againstoxidative stress-related diseases. Beverages as the beer, coffee,wine, etc. contributed in the dietary antioxidant intake in theMediterranean diet.

Beer is made of malt, hop, water and yeast and is rich in bothnutrient and non-nutrient components including carbohydrates,amino acids, vitamins and phenolic compounds ( Bamforth, 2002;Gasowskiet al., 2004; Rivero etal., 2005 ). Among these compounds,phenols are well-documented antioxidants. Phenolic constituents

Corresponding author. Tel.: +34 963983215.E-mail address: Victoria.Valls@uv.es (V. Valls-Belles).

of beer include benzoic- and cinnamic acid derivates, coumarins,catechins, di, tri and oligomeric proanthocyanidins, chalcones andavonoids ( Gorinstein et al., 2000; Bartolome et al., 2000; Nardiniet al., 2006; Lermusieau et al., 2001 ). Several parameters duringbrewing such as variety of barley and malting process; tempera-ture and pH during mashing, sparging and boiling; variety of hopsadded during wort boiling; and yeast fermentation inuence beertype and quality ( Woffenden et al., 2002; Andersen et al., 2000;Lugasi and Hovari, 2003 ).

The benecial effects of moderate alcohol beer consumptionhave been studied through in-vitro and in-vivo studies ( Ghiselli etal., 2000; van der Gaag et al., 2000; Vinson et al., 2003; Lugasiand Hovari, 2003; Nozawa et al., 2004; Gorinstein et al., 2007;Addolorato et al., 2008 ). Most of the studies were carried out withbeers containing alcohol. However, consumption of alcohol-freebeer is increasing nowadays, and there are progressively more rec-ommendations on consumption of alcohol-free beverages aimed

0300-483X/$ see front matter 2008 Elsevier Ireland Ltd. All rights reserved.

doi: 10.1016/j.tox.2008.04.010

http://www.sciencedirect.com/science/journal/0300483Xmailto:Victoria.Valls@uv.eshttp://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.tox.2008.04.010http://localhost/var/www/apps/conversion/tmp/scratch_8/dx.doi.org/10.1016/j.tox.2008.04.010mailto:Victoria.Valls@uv.eshttp://www.sciencedirect.com/science/journal/0300483X

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at both youthand people with hazardous employments. In a previ-ous study we have determined the total phenolic compounds andantioxidant activities of alcohol-free beer and compared it withthose of beers containing alcohol ( Rivero et al., 2005 ). Althoughthese results showed a high antioxidant capacity of the beerstudied, such effect could not be extrapolated to in-vivo studies.Therefore, the aim of the present study was to evaluate the inu-

ence of moderate amount of free alcohol beer in rats submitted toadriamycin.Adriamycin is a well-characterized drug for the study of the

mechanisms of oxidative stress. It is widely accepted thatoxidativestress and the production of free radicals are involved in adri-amycin action ( Davies andDoroshow,1986; Valls et al.,1994;Singalet al., 2000; Quiles et al., 2002; Berthiaume and Wallace, 2007 ).Thesefree radicals induce membrane lipidperoxidation, DNA dam-age, oxidative phosphorylation, decreased mitochondrial ATPaseactivity, etc. Polyphenols can be considered as potential protec-tors against toxicity caused by adriamycin due to their antioxidantcapacity (radical-scavenging or chelating). In the present study,rats were treated with adriamycin, which is a well-characterizeddrug for the study of the mechanisms of heart and liver oxidativedamage. The biomolecules toxicity showed by adriamycin is welldocumented in previous studies, where it was observed that adri-amycin induced oxidative damage in biomolecules such as lipidsand DNA ( Mu niz et al., 1995; Valls-Belles et al., 2006 ). On the otherhand, dietary antioxidants such as phenolic compounds showedprotective effect against adriamycin toxicity ( Quiles et al., 2002;Valls-Belles et al., 2006; Qin et al., 2007 ). Our aim was to deneif the consumption of alcohol-free beer shows protective effect onoxidative stress in liver and heart of rats and compare it with ratsthat consumption alcohol beer.

Toinvestigate the effects of alcohol-free beer on oxidative stressinduced by adriamycin, we evaluate the effect of the beer intake,on the inhibition of oxidative damage to lipids and DNA and theprotective effect on the levels of -tocopherol in liver and heartfrom rats.

2. Materials and methods

2.1. Reagents

Reagents used in the experiments were as follows: Protease A type VIII, EGTA,HEPES, 8-hydroxydeoxyguanosine, 4-2-2 -azinobis-(3-ethylbensothiazoline)-6-sulfonic acid (ABTS; Sigma Chemical Company, St. Louis, MO, USA), adriamycin(Pharmacia-Upjohn; Milan, Italy). The kits used were: 8-OHdG and MDA+ HAE(malondialdehyde and 4-hydroxynonenal) (Oxis Health Products Inc., 6040 NCutter Circle, Suite 317, Portland, OR, USA). Other reagents were obtained from forScharlau (Barcelona, Spain) and Merck (Darmstadt, Germany). All reagents wereprepared using water passed through Milipore Mili Q water purication systems(Milipore Corp., Bedford, MA, USA).

2.2. Beer samples

Two kinds of beer were used in this study. Alcohol-free beer (alcohol levelslower than 0.5%) and alcohol beer (clear beer with a 5.4% of alcohol content) andwere obtained from commercial sources in Spain. Beers were refrigerated and usedimmediately upon opening to prevent loss of phenols through oxidation.

2.3. Animals and treatment

Wistar male rats weighing between 250 and 300 g were housed in individualcages; maintained under controlled conditions of light cycle (12 h of light and 12hof darkness) and temperature (22 C); and fed on a standard diet (IPM-20; Letica,Barcelona, Spain).

The rats were divided into six groups of eight rats each. ADR was intraperi-toneally (i.p.) injected in two doses of 5 mg/kg. The beer was administeredintragastrically using a probe in an amount equivalent to moderate consume(400mL/d)in humansweighing70 kg.In that sense, 1.5mL ofbeerwasadministeredto rats of 300 g of weight.

Rats of group 1 (control group, C) received water and two doses of isotonic

saline solution (i.p. injected on the 7th and 14th days of the study) for 21 days.

Rats of group 2 (ADR group) drank water and received two doses of adriamycininjectionadministered on the7th and14thdaysof thestudy. Rats of groups3 and4(control+ Beergroups)alcohol-free beeror alcohol beerduring 21days andreceivedtwo doses of saline solution. Rats of groups 5 and 6 (ADR + Beer) were treated withtwodosesof adriamycinon the7th and14thdaysof thestudy,anddrank alcohol-freeor alcohol beer during 21 days.

2.4. Preparation of heart and liver mitochondria

Liver and heart mitochondria were isolated using the method described bySantos etal. (2002) . L iverand heart tissueswere excised,mincedin ice-coldmediumcontaining 250 mM sucrose, 1 mM EGTA, and 5 mM HEPESKOH (pH 7.4), andhomogenized with Potter-Elvehjen homogenizer; homogenate protease was addedin heart. Homogenates were centrifuged at 900 g for 10min, low-speed super-natant was discarded, and the pellet was resuspended in isolation medium andcentrifuged at 9000 g for 10min. Finalmitochondrial pellets were resuspended inisolation medium.

2.5. Quantication of MDA4HNE (malondialdehyde4-hydroxynonenal)

MDA and 4HNE levels of rat liver and heart mitochondria were measuredby the kit HAE-586 (Bioxytech, OxisResearch), which is based on the reac-tion of a chromogenic reagent ( N -methyl-2-phenylindole, NMPI) with MDA and4-hydroxyalkenals at 45 C. The product of the reaction was recorded spectropho-tometrically at 586 nm.

2.6. Quantication of 8-OHdG levels in DNA from rat liver and heart

Rat heart and liver DNA was isolated as described by Gupta (1984) . IsolatedDNA was washed twice with 70% ethanol, dried, and dissolved in 200 L of 10mMTrisHCl(pH 7.0) forits enzymaticdigestion. Briey,0.5 g DNA/ L ofsampleswereincubated with 100 units of DNase I in 40 L 10 mM TrisHCl at 37 C for 1 h. ThepH of the reaction mixture was then lowered with 15 L of 0.5 M sodium acetate(pH5.1), 10 L of nuclease P1 (ve units)and incubatedfor 1 h. Readjustment of pHwith 100 L of0.4 M TrisHCl (pH7.8) wasfollowed bythe addition ofthree unitsof alkaline phosphatase; samples were incubated for longer than an hour. Analysis of 8-OHdG was determined by ELISA using the kit 8-OH-dG-EIA-Biotech (Oxis HealthProducts Inc., Portland, OR, USA).

2.7. Assay for -tocopherol

-Tocopherol was analysed using HPLC UV 291 nm detector ( Arnaud etal., 1991 ). Mitochondria with a concentration of 1 mg/mL were mixed with2 mL of ethanol/hexan (2/5, v/v), sonicated and centrifuged at 3000rpm for3 min. The organic layer was evaporated with N 2 and reconstituted with200 L of mobile phase. 20 L of this solution was injected in the HPLC usingdiclorometane/acetonitrile/methanol (20/70/10) as mobile phase.

2.8. Protein measurement

Total protein concentration was evaluated using bovine albumin as a standard,following Lowry method modied by Markwell et al. (1978) .

2.9. Statistical analysis

Statistical analysisof thedata wascarriedout usingone-way analysisof variance(ANOVA) and a paired t -Students was used for statistical evaluation of signicantdifferencesbetweenthe twogroups.The level ofcondence requiredfor signicancewas selected at p < 0.05. Statistical analyses were performed using the StatgraphicsPlus4.0 statistical softwarepackage forMS Windows(StatgraphicsPlus forWindows4.0; Users Guide; Manugistics Inc., Rockville, MD, USA; 1999).

3. Results

The study was carried out in heart and liver mitochondria of differentgroups of rats, control group (C), rats which drank alcoholbeer or alcohol-free beer (C + Beer), rats treated with adriamycin(ADR) and rats that were treated with adriamycin and drank beer(ADR + Beer).

Damage to lipids was measured as MDA + 4HNE; its results areshownin Fig.1. Theintakeofbothtypesofbeer(C+Beer)inducesnochanges in thelevels of lipid peroxide of heart or liver.MDA+ 4HNElevels in heart of the ADR-treated rats (ADR), are signicantly ele-vated( p < 0.005) with values 1.7 times higherwhen compared withthecontrolgroup (C). In the groupof rats ADR-treated and that beer

intake(ADR + Beer),thelipidperoxidelevels(MDA + 4HNE)in heart,

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Fig. 1. MDA + 4HNE levels in liver and heart mitochondria in the four groups of ratstudied. C: control group; ADR: adriamycin group; C + Beer: group of rats whichconsumed beer; ADR+ Beer: rats which consumed beer and were also treated withadriamycin. Values aremeans S.D. (n = 8).Valueswith differentalphabeticallettersare signicantly different ( p

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4. Discussion

The lack of signicant difference in heart and liver in the oxida-tive stress biomarkers (MDA + 4HNE; -tocopherol and 8-OHdG)between control groups (C) and rats that consume beer (C + Beer),showsthatthe intakeof beer does not modifythe tissuebiomarkersof oxidative stress. Difference in the -tocopherol levels between

the group of rats that consume alcohol-free and alcohol beer inliver but not in heart was observed. This difference between tis-sues could be due to the alcohol metabolism. In fact, the 90% of theethanol intake is biotransformed in the liver, and only 10% is bio-transformed in other tissues such as heart, brain, etc. The alcoholbeerconsumptionresulted ina signicantdecreaseof -tocopherollevels of liver mitochondria when compared with the group thatconsumes alcohol-free beer or with the control group. This maybe due to the toxic effect of ethanol where a subliminal oxidativestress took place ( Gasbarrini et al., 1998 ).

Beer intake seems to have inuence on the oxidative stressbiomarkers in mitochondria the liver and heart of rats treated withadriamycin. Adriamycin is a well-documented drug that it is usedto induce oxidative stress.

In agreement with previous studies using adriamycin ( Quileset al., 2002; Yilmaz et al., 2006 ), our results showed that adri-amycin gives rise a high MDA + 4HNE levels and a decrease of

-tocopherol levels in mitochondria of heart and liver rat. It iswidely accepted that oxidative stress and the production of freeradicals are involved in ADR action, in relation to toxicity effects.Thus, it has been reported that ADR leads to direct oxidative injuryto lipid ( Quiles et al., 2002 ) and different studies reporting amelio-ration of adriamycin toxicity by vitamin E through neutralization of lipid peroxidation due to its antioxidant effect ( Quiles et al., 2002;Kalenderet al.,2005 ). -Tocopherol vitaminis considered themajorchain-breaking antioxidant, especially in lypophilic moieties suchas membranes ( Halliwell and Gutteridge, 1999 ).

The decrease of MDA + 4HNE and increase of -tocopherol lev-els in heart mitochondria of rat treated with adriamycin and fedwith both type of beer, ensured that in our experimental con-ditions the beer consumption induces an antioxidant effect. Thebeer consumption (independent of the presence of alcohol) actto prevent the oxidative damage generated by the adriamycin, isthe cause of cell toxicity. However, in the liver mitochondria, theprotective effect on lipid peroxidation was minor and it was notstatisticallysignicant whencomparedwith thegrouptreatedadri-amycin alone. These results are in agreement with studies of otherauthors, which did not observe signicant protective effect on lipidperoxidation in liver from rats that consume beer ( Gasbarrini et al.,1998 ).

Thisinhibition of peroxidationandthe recuperationof the levelsof -tocopherol, in rats treated with ADR and that drank beer, maybe due to the presence in the beer of compounds with antioxidant

capacity. Among thesecompounds, are the polyphenols thatdue totheir radical-scavenging and iron-chelating properties, can be con-sidered as potential protectors against chronic cardiotoxicity andhepatotoxicity caused by doxorubicin ( van Acker et al., 2001; Valls-Belles et al., 2006; Bast et al., 2007; V aclav kov a et al., 2008 ). Thus,they can interact withmembrane phospholipids throughhydrogenbonding to the phospholipids polar head groups to help to main-tain the integrity of the membranes by preventing the access of deleterious molecules to the hydrophobic region of the bilayers ormaintaining reduced -tocopherol.

Other biomarker studied in this work was the base modied 8-OHdG, whose levels arecorrelativewith diseases suchas cancer andaging ( Zwart et al., 1999; Grifths et al., 2002 ). It is known that theadriamycin-induced DNA injuries included oxidized pyrimidines

and 8-hydroxyguanine. In a previous in-vitro study, we observed

the capacity of beer to protect the DNA of 8-OHdG generation. Fur-thermore, DNA damage induced by adriamycin appears to play animportant early role in lethal cardiac myocyte injury through apathway involving p53 and mitochondria ( LEcuyer et al., 2006 ).The intake of the two types of beer in rats treated with adriamycinshowed a protective effect against oxidative damage, decreasingthe levels of 8-OHdG in heart and liver DNA. These results showed

that the inhibition of oxidative DNA damage after administrationof adriamycin suggests a possible mechanism through which beermay inhibit the diseases related to DNA oxidation. Several in-vitroand in-vivo reports demonstrated that compounds isolated fromsuch beers decreased oxidative DNA damage ( Arimoto et al., 1999;Nozawa et al., 2004; Rivero et al., 2005 ).

In conclusion, our results showed that a moderate consumptionof alcohol-free beer could be protect against liver and heart dam-age induced by free radical production. In this sense, the bioactivecompounds present in beer appeared to be able to counteractsometoxic action exerted by oxidative stress.

Acknowledgments

The authors thank the nancial support of Centro de Infor-maci on Cerveza y Salud and to Miss Yolanda Fernand ez for herhelp with the English revision.

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