汉黄芩素通过DNA氧化应激的抑制和OGG1表达的调节来衰减依托泊苷诱导的氧化DNA的损伤和凋亡

Wogonin attenuates etoposide-induced oxidative DNA damage and apoptosis via suppression of oxidative DNA stress and modulation of OGG1

expression

Sabry M.Attia a ,c ,?,Sheikh Fayaz Ahmad a ,Gamaleldin I.Harisa b ,d ,Ahmed M.Mansour c ,El Sayed M.El Sayed c ,Saleh A.Bakheet a

a

Department of Pharmacology and Toxicology,College of Pharmacy,King Saud University,Riyadh,Saudi Arabia b

Department of Pharmaceutics,College of Pharmacy,King Saud University,Riyadh,Saudi Arabia c

Department of Pharmacology and Toxicology,College of Pharmacy,Al-Azhar University,Cairo,Egypt d

Department of Biochemistry,College of Pharmacy,Al-Azhar University,Cairo,Egypt

a r t i c l e i n f o Article history:

Received 20February 2013Accepted 10July 2013

Available online 17July 2013Keywords:

DNA damage/repair Apoptosis

Oxidative DNA stress Secondary tumors

a b s t r a c t

Damage to DNA can lead to many different acute and chronic pathophysiological conditions,ranging from cancer to endothelial damage.The current study has been initiated to determine whether the ?avo-noid wogonin can attenuate etoposide-induced oxidative DNA damage and apoptosis in mouse bone marrow cells.We found that oral administration of wogonin before etoposide injection signi?cantly attenuates etoposide-induced oxidative DNA damage and apoptosis in a dose dependent manner.Etopo-side induced a signi?cant down-regulation of mRNA expression of the OGG1repair gene and marked bio-chemical alterations characteristic of oxidative DNA stress,including increased 8-OHdG,enhanced lipid peroxidation and reduction in reduced glutathione.Prior administration of wogonin ahead of etoposide challenge restored these altered parameters.Importantly,wogonin had no antagonizing effect on etopo-side-induce topoisomerase-II inhibition.Conclusively,our study indicates that wogonin has a protective role in the abatement of etoposide-induced oxidative DNA damage and apoptosis in the bone marrow cells of mice via suppression of oxidative DNA stress and enhancing DNA repair through modulation of OGG1repair gene expression.Therefore,wogonin can be a promising chemoprotective agent and might be useful to avert secondary leukemia and other drug-related cancers in cured cancer patients and med-ical personnel exposing to the potent carcinogen etoposide.

ó2013Elsevier Ltd.All rights reserved.

1.Introduction

Etoposide (VP-16),a topoisomerase II inhibitor,is one of the most popular chemotherapeutic drugs used in the treatment of various malignancies (Hande,1998).However,its utilisation is associated with the development of acquired drug resistance by the cancer cells and various side effects in the host including geno-mic damages and apoptosis (Attia,2008a ).The genotoxic potential of VP-16has become of great interest because of its serious effects on the chromosomes of non-tumour cells.In fact,after application of VP-16,damage to DNA may result as DNA fragmentation,chro-mosomal breaks and micronucleus formation causing genomic instability and may lead to mutagenesis,carcinogenesis or ?nally to apoptotic cell death (Seiter,2005;Ng et al.,2006).Apoptotic cell death plays a role not only in anticancer effects but also in adverse reactions,such as bone marrow suppression.In animals,VP-16is a somatic and germ-cell genotoxic agent capable of inducing both numerical and structural chromosome aberrations (Ashby et al.,1994;Turner et al.,2001;Attia et al.,2002,2003).Moreover,fol-low-up studies of patients who received VP-16therapy revealed an increased incidence of acute myeloid leukaemia characterised by site-speci?c rearrangements in the mixed multiple leukaemia gene on chromosome 11q23(Kitazawa et al.,2001;Felix et al.,2006).

The ?avonoid wogonin (WOG)is one of the active ?avonoids in Scutellaria baicalensis Georgi radix and presents extensive pharma-cological actions such as antioxidant,antiviral,anti-thrombotic,anti-in?ammatory,neuroprotective as well as various immuno-modulatory effects (Tai et al.,2005).Recently,more studies focused on its anti-proliferative and apoptosis inducing activity in various human tumor cells (Chen et al.,2002;Li-Weber,2009).In addition,it’s differentiation inducing effect and potential effect

0278-6915/$-see front matter ó2013Elsevier Ltd.All rights reserved.https://www.360docs.net/doc/f3616020.html,/10.1016/j.fct.2013.07.022

?Corresponding author at:Department of Pharmacology and Toxicology,College of Pharmacy,King Saud University,Riyadh,Saudi Arabia.Tel.:+966542927708;fax:+96614677200.

E-mail addresses:attiasm@https://www.360docs.net/doc/f3616020.html,.sa ,attiasm@https://www.360docs.net/doc/f3616020.html, (S.M.Attia).

of overcoming multidrug resistance were reported(Lee et al., 2009).The?avonoid WOG has been shown to markedly potenti-ates the anti-proliferative effect of VP-16in various tumor cells (Lee et al.,2009;Enomoto et al.,2011).The potentiating effect of WOG involves multiple mechanisms.For example,in a functional assay for P-glycoprotein(P-gp),WOG suppressed excretion of cal-cein,a substrate for P-gp,in tumor cells.Moreover,WOG decreased the excretion of radiolabeled VP-16and accordingly increased intracellular content of VP-16in the cancer cells.Thus,P-gp inhibi-tion and accumulation of VP-16in cancer cells seem to be part of the mechanism of potentiation of the action of VP-16by WOG (Enomoto et al.,2011).

In various cancer cells,WOG has been shown to potentiates VP-16-Induced apoptosis in these cells.In contrast,WOG has been shown to protect bone marrow cells and thymocytes from VP-16-induced apoptotic cell death(Lee et al.,2007,2009;Enomoto et al.,2011).These contradictory actions of WOG on apoptosis are distinguished by cancer or normal cell types.Similarly,Fas et al.(2006)reported that WOG sensitizes TNF a-resistant leuke-mia cells to TNF a-induced apoptosis and does not affect the viabil-ity of normal T-cells.The authors accounted for its mechanism by production of reactive oxygen species(ROS)and the difference in the oxidation–reduction status of cancer and normal cells.ROS accumulation may be partly involved in the potentiating effect of WOG because VP-16is known to increase ROS generation in cancer cells(Kurosu et al.,2003).Since the pharmacological properties of WOG may be distinguished by the cell type,it may enhance the pharmacologic actions of VP-16and ameliorate its adverse effects. Therefore,the combination of VP-16with WOG might be of thera-peutic bene?t.

The concept of providing protection against genotoxicity in non-tumor tissues will represent a promising approach of prevent-ing the unavoidable toxicity from cytotoxic chemotherapy;this will allow the safe use of increased drug doses for the bene?t of fu-ture cancer patients.Considering the widespread use of VP-16in clinical oncology and the ability of WOG to improve the therapeu-tic outcome from VP-16(Enomoto et al.,2011),prompted us to investigate whether the non-toxic doses of WOG in combination with VP-16can ameliorate VP-16-induced oxidative DNA damage and apoptosis in mice bone marrow cells.The unrepaired DNA damage was assessed by the bone marrow micronucleus test and the kinetics of repair of oxidative DNA damage was assessed by co-met assay.Apoptosis was analyzed by the occurrence of a hypodip-loid DNA peak and the activity of caspase-3in mouse bone marrow cells.In addition,quantitative analysis of mRNA expression of the DNA repair-related gene8-oxoguanine glycosylase(OGG1)was performed by real-time reverse polymerase chain reaction(RT-PCR).The biochemical alterations characteristic of oxidative DNA damage induced by VP-16such as8-hydroxy-20-deoxyguanosine (8-OHdG),lipid peroxidation and reduced glutathione(GSH)levels have been evaluated.Further,the catalytic activity of topoisomer-ase II was evaluated in the presence of WOG and/or VP-16.

2.Material and methods

2.1.Animals

Adult male Swiss albino mice aged10–14weeks and weighing25–30g were obtained from Experimental Animal Care Center,College of Pharmacy,King Saud University.The animals were maintained in an air-conditioned animal house at a temperature of25–28°C,relative humidity at$50%and photo-cycle of12:12h light and dark periods.The animals were provided with Purina rodent’s chow pel-lets and water ad libitum.All experiments were carried out according to the Guide-lines of the Animal Care and Use Committee at King Saud University.Each treatment group and vehicle control group consisted of?ve randomly assigned ani-mals.The total number of animals in this study was260treated mice and45vehicle control.2.2.Drugs and chemicals

VP-16and WOG were purchased from Sigma–Aldrich(St Louis,MO,USA).The drugs were dissolved in5%dimethylsulfoxide(DMSO)in sterile distilled H2O, mixed on a magnetic stirrer for at least30min prior to administration.VP-16 was administered by intraperitonial injection within1h following preparation. The doses of VP-16were selected on the basis of its effectiveness in inducing chro-mosomal damage(Ashby et al.,1994;Turner et al.,2001;Attia et al.,2009)and the selected doses are within the dose range used for human chemotherapy.WOG was administered through oral intubations and the gavage administrations were made 24h and1h prior to the VP-16injection.Control animals were received equal amounts of5%DMSO in sterile distilled H2O and the administrated volumes were 0.01ml per1g body weight.All other chemicals were of the?nest analytical grade.

2.3.Measurement of unrepaired DNA damage by micronucleus test

In the micronucleus test,the animals were treated with10or20mg/kg body weight WOG before0.5,1or10mg/kg body weight VP-16treatment.Cyclophos-phamide was used at a concentration of40mg/kg body weight as a positive control mutagen(Attia,2007;Attia et al.,2009).Mice were sacri?ced at24h after VP-16or cyclophosphamide treatment and femurs bone marrow cells were collected in tubes containing foetal calf serum.Bone marrow smears were prepared and stained with May-Gruenwald/Giemsa as described previously(Attia et al.,2005;Attia, 2007).At least two sides were made for each animal and allowed to dry overnight. Per animal,2000polychromatic erythrocytes(PCE)of coded slides were scored for the presence of micronuclei(MN)(Attia et al.,2009).The frequencies of PCE were determined in slide areas where1000normochromatic erythrocytes(NCE)could be counted to determine a shift in erythroblast proliferation.The values were ex-pressed as%PCE of the total erythrocyte counts to determine a reduction of eryth-roblast proliferation.

2.4.Alkaline comet assay

The alkaline comet assay was used to evaluate the kinetics of repair of DNA strand breaks in animals treated with10mg/kg body weight VP-16and/or10or 20mg/kg body weight WOG.Animals were sacri?ced by cervical dislocation at1, 3or24h after the exposure to10mg/kg body weight VP-16.At speci?c time,bone marrow cells from one femora were collected and the alkaline comet assay was per-formed as described elsewhere(Attia et al.,2010).The slides were stained with ethidium bromide then studied by using a?uorescent microscope equipped with appropriate?lter.Images from100cells per animal were randomly selected for analysis using TriTek CometScore version1.5software and the olive tail moment [(Tail mean–Head mean)?Tail%DNA/100]was calculated.

2.5.Modi?ed version of comet assay

For detecting oxidative DNA damage,the modi?ed comet assay using lesion-speci?c enzymes were also used on parallel slides to detect oxidized purines and pyrimidines.The modi?ed comet assay was performed as described previously by Attia et al.(2013).Brie?y,the mouse bone marrow cells were embedded in agarose on microscope slides and stored in the lysis buffer as previously described(Attia et al.,2010).After lysing,the slides were washed three times for5min each with an enzyme buffer(40mM HEPES,0.1M KCl,0.5mM EDTA,0.2mg/ml bovine serum albumin,with the solution adjusted to pH8.0with KOH).After washing,excess li-quid was removed,and the following solutions were applied to slides prepared from each treatment group:50l l of enzyme buffer alone(control),50l l of enzyme buffer containing1l g/ml of formamidopyrimidine glycosylase(Fpg,which cataly-ses excision of oxidized purines)or50l l of enzyme buffer containing1l g/ml of endonuclease III(Endo III,which catalyses excision on oxidized pyrimidines).The slides were kept in a moist box and incubated at37°C for30–45min.After the en-zyme treatment,the slides were placed in a horizontal electrophoresis chamber to perform DNA unwinding then electrophoresis and slide staining were the same as mentioned previously(Attia et al.,2010).Thereafter,slides were analyzed as de-scribed above for the alkaline comet assay.

2.6.Detection of apoptosis

To study the in?uence of WOG on the VP-16-induced apoptosis in mouse bone marrow cells,6groups of15mice each were treated as in the alkaline comet assay and?ve mice from each group were sacri?ced by cervical dislocation at1,3or24h after the exposure to VP-16.At speci?c time,bone marrow cells were collected and the apoptotic cells were quanti?ed with propidium iodide as mentioned earlier (Bakheet et al.,2011).The stained cells were then analyzed by?ow cytometry using a FACSCalibur cytometer and cells containing hypodiploid DNA were considered as apoptotic.The percentage of apoptotic cells were calculated using a computer sys-tem CellQuest(BD Biosciences).Moreover,the activity of caspase-3in signal trans-duction was investigated in mouse bone marrow cells at24h sampling regimen by use of the DEVD-p NA cleavage assay as described elsewhere(Attia,2012a).

S.M.Attia et al./Food and Chemical Toxicology59(2013)724–730725

2.7.Expression analyses

Animal treatment was the same as in the alkaline comet assay.The animals were killed by cervical dislocation at24h after VP-16treatment to estimate the gene expression levels of OGG1.Total RNA from bone marrow cells were isolated using TRIzol reagent(Invitrogenò)according to the manufacturer’s instructions then the quantity and quality of the RNA were determined with a NanoDrop 8000.Thereafter,?rst-strand cDNA synthesis was performed using the High-Capac-ity cDNA reverse transcription kit(Applied Biosystems),according to the manufac-turer’s instructions.Brie?y,1.5l g of total RNA from each sample was added to a mixture of 2.0l l of10?reverse transcriptase buffer,0.8l l of25?dNTP mix (100mM),2.0l l of10?reverse transcriptase random primers,1.0l l of Multi-Scribe reverse transcriptase,and3.2l l of nuclease-free water.The?nal reaction mixture was kept at25°C for10min,heated to37°C for120min,heated for 85°C for5s,and?nally cooled to4°C.

Quantitative analysis of mRNA expression were performed by RT-PCR by sub-jecting the resulting cDNA to PCR ampli?cation using96-well optical reaction plates in the ABI Prism7500System(Applied Biosystemsò).The25-l l reaction mix-ture contained0.1l l of10l M forward primer and0.1l l of10l M reverse primer (40nM?nal concentration of each primer),12.5l l of SYBR Green Universal Master-mix,11.05l l of nuclease-free water,and1.25l l of cDNA sample.The primers used in the current study were as follows:OGG1forward primer50-GAT TGG ACA GTG CCG TAA-30,reverse primer50-GGA AGT GGG AGT CTA CAG-30and Cyclophilin A: forward primer50-TGG TCA ACC CCA CCG TGT TCT TCG-30,reverse primer50-TCC AGC ATT TGC CAT GGA CAA GA-30(GenScript,Piscataway,NJ).The RT-PCR data were analyzed using the relative gene expression(i.e.,DD CT)method,as described in Applied Biosystems User Bulletin.The data are presented as the fold change in gene expression normalized to the endogenous reference gene(Cyclophilin A) and relative to untreated calibrator group(Livak and Schmittgen,2001).

2.8.Measurement of oxidative DNA stress markers

To study the effect of WOG on the oxidative DNA stress induced by VP-16treat-ment,animal were treated as in the alkaline comet assay and peripheral blood sam-ples were collected from the heart at24h after VP-16treatment for estimation of serum8-OHdG,lipid peroxidation and GSH levels.The amount of8-OHdG was determined using the Bioxytech8-OHdG-ELISA Kit(OXIS Health Products,Portland, OR,USA)according to the manufacturer’s instructions as described previously(At-tia,2012a)and the concentration of8-OHdG was interpolated from a standard curve drawn with the assistance of logarithmic transformation.Reported values are the average of triplicate determinations and levels of8-OHdG are expressed as ng/ml.The extent of lipid peroxidation was assayed by measuring one of the end products of this process,the thiobarbituric acid-reactive substances(TBARS) by the method of Ohkawa et al.(1979)with some modi?cations as described else-where(Attia,2008b).The concentration of TBARS was calculated using standard curves of increasing1,1,3,3-tetramethoxypropane concentrations,and expressed as nmol/ml.GSH was assayed with5,50-dithiobis(2-nitrobenzoic acid)according to the protocol described by Ellman(1959)as described previously(Attia,2008b). The concentration of GSH was calculated from standard curve that was obtained from freshly prepared standard solutions of GSH and expressed as l g/ml.

2.9.Topoisomerase II inhibition assay

To determine if WOG would has an antagonizing effect on VP-16-induced topo-isomerase II inhibition,topoisomerase II-a activity was measured by inhibition of decatenation of kinetoplast DNA(kDNA)by100l M VP-16and/or100l M of WOG using a TopoGen(Columbus,OH,USA)assay kit as described previously(Attia, 2012b).

2.10.Statistical analysis

Results were expressed as means±SD of the mean.Data of micronucleus test, comet assay and propidium iodide staining were analyzed by the non-parametric tests,the Mann–Whitney U-test and Kruskal–Wallis test followed by Dunn’s for multiple comparisons.Data on oxidative DNA stress parameters,caspase-3activity and expression analyses were?rstly tested for homogeneity of variance and nor-mality and then analyzed by employing parametric tests,Student t-test and one-way ANOVA followed by Tukey–Kramer for multiple comparisons by using software computer program(GraphPad InStat;DATASET1.ISD).Results were considered sig-ni?cantly different if the P-value was<0.05.

3.Results and discussion

Flavonoids have become active medicine clinically and exami-nation of their anti-genotoxic and anti-apoptotic effects has re-ceived increasing attention in recent years.As mentioned in Introduction,WOG has various pharmacological and therapeutic actions.In spite of its powerful ef?cacies,WOG shows minor or al-most no toxicity on normal cells.The oral LD50value of WOG in mice was found to be greater than3g/kg(Hui et al.,2002).Since WOG is safe and readily available natural product,one needs to examine its interaction with drugs.Thus,the intention of the cur-rent study was to determine whether WOG can attenuate etopo-side-induced DNA damage and apoptosis in mouse bone marrow cells.The current study demonstrates that WOG was neither geno-toxic nor apoptogenic in mouse bone marrow at the doses tested. Moreover,it is able to protect mouse bone marrow cells against the VP-16-induced oxidative DNA damage and produce a decline in cell proliferation as observed by the reduction in MN frequen-cies,DNA strand breaks as well as increases in mitotic activities, respectively.The apoptogenic effect of VP-16was also reduced by WOG pre-treatment.These effects,however,are dose depen-dent,been detected clearly at the higher dose of WOG.

3.1.Effect of WOG on VP-16-induced MNPCE and bone marrow suppression

The levels of unrepaired DNA damage as detected by the micro-nucleus test are shown in Table1.The positive control mutagen cyclophosphamide was used and this compound produced the ex-pected responses and the results of this compound were in the same range as those of earlier studies(Attia,2007;Attia et al., 2009).These data con?rmed the sensitivity of the experimental protocol followed in the detection of genotoxic effects.VP-16 caused signi?cant increases in MN induction at all doses tested. However,an inverse dose response was found between1and 10mg/kg body weight.In the micronucleus test,comparison of the PCE/NCE ratio in bone marrow of treated animals to vehicle-control animals provides an indication of cytotoxicity.When eryth-roblast proliferation is depressed,the ratio will decrease,when proliferation is increased to repopulate the PCE pool,the ratio is in-creased.Some cytotoxicity can provide a valuable indication of bone-marrow exposure by the test agent,but excessive toxicity is undesirable.The decline of the MN yields with increasing doses was accompanied by a signi?cant reduction of PCE frequencies so that at low PCE rates hardly any MNPCE could be seen.These re-sults are in accord with the previous data published by Ashby Table1

Frequencies of MNPCE and PCE in bone marrow of mice after treatment with the indicated doses of wogonin(WOG)and/or etoposide(VP-16).

Chemical(mg/kg body weight)%MNPCE(mean±SD)%PCE A(mean±SD)

Control0.30±0.0749.4±1.34

WOG(10)0.34±0.1148.4±2.30

WOG(20)0.28±0.1048.8±1.09

VP-16(0.5) 1.58±0.31**46.4±2.30

WOG(10)+VP-16(0.5)0.98±0.32a47.2±2.77

WOG(20)+VP-16(0.5)0.78±0.31b47.4±2.30

VP-16(1) 2.78±0.44**44.4±4.27

WOG(10)+VP-16(1) 2.08±0.3148.2±2.68

WOG(20)+VP-16(1)0.92±0.30b47.6±2.30

VP-16(10) 1.68±0.38**40.0±3.0**

WOG(10)+VP-16(10) 1.12±0.21a44.4±2.70

WOG(20)+VP-16(10)0.66±0.15b46.2±2.48a

Cyclophosphamide(40) 1.76±0.35#41.6±4.21#

MNPCE=Micronucleated polychromatic erythrocytes;PCE=polychromatic eryth-rocytes;WOG=wogonin;VP-16=etoposide.

A Numbers of PCE were counted in microscopic?elds which contained1000 normochromatic erythrocytes(NCE);%PCE were calculated as%PCE=[PCE/ (PCE+NCE)]?100.

**P<0.01compared with the solvent control(Kruskal–Wallis test followed by Dunn’s multiple comparisons test).

a P<0.05.

b P<0.01compared with corresponding VP-16alone(Mann–Whitney U-test). #P<0.01compared with control(Mann–Whitney U-test).

726S.M.Attia et al./Food and Chemical Toxicology59(2013)724–730

1.Effects of wogonin[WOG(mg/kg body weight)]on etoposide[VP-16(mg/kg body weight)]-induced DNA strand breaks in mouse bone marrow cells.The were subjected to comet assay after1,3and24h of exposure to VP-16,with without enzyme digestion after the lysis.Cells without enzyme treatment were

exposed to enzyme buffer alone(A)cells treated with endonuclease III(Endo were digested for45min(B)and those treated with formamidopyrimidine glycosylase(Fpg)were digested for30min(C).?P<0.05and??P<0.01compared the corresponding solvent control(Kruskal–Wallis test followed by Dunn’s multiple comparisons test).a P<0.05and b P<0.01compared with the correspond-VP-16alone(Mann–Whitney U-test).

2.Percentage of apoptotic cells in bone marrow of mice1,3or24h after treatment with wogonin[WOG(mg/kg body weight)]and/or etoposide[VP-16(mg/ body weight)].%Apoptotic cells denote the percentage of cells with hypodiploid DNA content.??P<0.01compared with the corresponding solvent control(Kruskal–Wallis test followed by Dunn’s multiple comparisons test).a P<0.05and b P<0.01 compared with the corresponding VP-16alone(Mann–Whitney U-test).

S.M.Attia et al./Food and Chemical Toxicology59(2013)724–730727

and also inhibited lipopolysaccharide and cytokine-induced C6 glial cell apoptosis(Kim et al.,2001).

As shown in Fig.3,no apparent difference could be observed in caspase-3activity between the control group and the WOG-treated group.The hydrolytic enzyme activity of caspase-3towards DEVD p NA was signi?cantly elevated in animals treated with VP-16in comparison with the control.Pre-treatment of mice with WOG sig-ni?cantly reduced the elevated caspase-3activity relative to the value obtained after treatment with VP-16alone(P<0.01).In con-trast,WOG has been reported to activate caspase-3and induce apoptotic cell death in some cancer cells(Chen et al.,2002).Thus, the effect of WOG on apoptotic cell death differs according to the type of cells.

3.3.Mechanisms by which WOG protected against VP-16-induced DNA damage and apoptosis

It has been reported that VP-16generates phenoxyl radical or quinone intermediates in the redox reaction(Haim et al.,1986).

Accumulation of these radicals may cause damage to cellular gen-ome and other critical biomolecules,ultimately inducing genotox-icity and leukemia(Attia,2010).Moreover,it is believed that accumulation of lipid peroxyl radicals induced by VP-16during their oxidation may cause damage to cell membrane leading to li-pid peroxidation(Ladner et al.,1989).The exact mechanism by which WOG protected against VP-16-induced genomic damage and apoptosis is not well known.Possible explanations for this pro-tection is that pre-treatment with WOG would enhancing DNA re-pair and allow interception of free radicals generated by VP-16 before they reach DNA to induce DNA damage and apoptosis.

To ensure the role of increase oxidative stress in the induction of oxidative DNA damage,modi?ed comet assay was performed in parallel with the alkaline comet assay.Alkaline comet assay is generally considered as a reliable method of DNA strand breaks assessment due to its versatility and sensitivity.Inclusion of the enzyme Fpg and Endo-III makes this assay more sensitive towards the detection of oxidative stress induced DNA damage(Collins et al.,1993).As shown in Fig.1,the VP-16treatment led to signif-icant increase in the DNA strand breaks+Endo-III(B),and DNA strand breaks+Fpg(C)in olive tail moment as compared to control groups at all sampling times with the highest value at1h after VP-16treatment.WOG pre-treatment followed by VP-16signi?cantly reduces the DNA strand breaks+Endo-III and strand breaks+Fpg values as opposed to the group receiving VP-16only.Thus,oxida-tive DNA damage induced by VP-16may be one of the most impor-tant factors responsible for the bone marrow toxicity and the protective effect of WOG resides,at least in part,in its radical scav-enger activity.

Among the various DNA oxidative lesions,8-OHdG is the most abundant oxidized base generated in vivo by various types of ROS.This aberrant base is a pre-mutagenic lesion,inducing G-C to T-A transversions.A substantial body of evidence indicates that 8-OHdG is possibly an important biomarker of carcinogenesis (Nishigori et al.,2004).In mammalian cells,this oxidized base is speci?cally recognized and excised by the OGG1enzyme.This en-zyme initiates the highly conserved base excision repair of small chemical alterations in DNA.As shown in Fig.4,the expression of OGG1gene in mice exposed to VP-16was signi?cantly down regulated relative to the untreated control.On the other hand, when pre-treatment of different doses of WOG was given prior to VP-16treatment,a signi?cant restoration of mRNA expression of the OGG1gene was observed and the higher dose of WOG gave the more effective restoration of OGG1gene.

The biochemical alterations characteristic of oxidative DNA stress activity induced by VP-16such as8-OHdG,lipid peroxida-tion and GSH have been also measured after treatment with VP-16and/or WOG.The present study demonstrates that WOG pre-treatment reduced the VP-16induced accumulation of8-OHdG and lipid peroxidation and prevented the reduction in GSH signif-icantly(Table2).The increased GSH level suggests that protection by WOG may be mediated through the modulation of cellular anti-oxidant levels.These observations con?rm earlier studies in which WOG were reported to scavenge free radicals and lipid peroxides

3.Effects of wogonin[WOG(mg/kg body weight)]and/or etoposide[VP-16 (mg/kg body weight)]on caspase-3activity in the bone marrow cells of (mean±SD).??P<0.01versus control,b P<0.01versus VP-16alone(one-way ANOVA and post hoc Tukey–Kramer multiple comparison test).

4.Effects of wogonin[WOG(mg/kg body weight)]and/or etoposide[VP-16 (mg/kg body weight)]on8-oxoguanine-DNAglycosylase(OGG1)mRNA expression the bone marrow cells of mice(mean±SD).??P<0.01versus control,a P<

b P<0.01versus VP-16alone(one-way ANOVA and post ho

c Tukey–Kramer multiple comparison test).

Table2

Effects of wogonin(WOG)and/or etoposide(VP-16)on the bone marrow contents of 8-OHdG,lipid peroxidation(TBARS)and GSH in mice(mean±SD).

Treatment group

(mg/kg body weight)

8-OHdG

(ng/ml)

TBARS

(nmol/ml)

GSH

(l g/ml) Control 3.21±0.82 2.92±0.79 3.74±0.56 WOG(10) 3.91±0.87 3.58±0.43 4.76±0.67 WOG(20) 3.34±0.89 3.44±0.71 5.22±1.10 VP-16(10)11.3±2.7**9.41±2.2** 1.46±0.4** WOG(10)+VP-16(10) 6.62±1.5b 6.14±2.1a 2.56±0.9# WOG(20)+VP-16(10) 4.46±1.2b 4.40±0.9b 3.61±0.9b WOG=Wogonin,VP-16=etoposide,8-OHdG=8-hydroxy-20-deoxyguanosine, TBARS=thiobarbituric acid-reactive substances,GSH=reduced glutathione.

**P<0.01compared with the solvent control.

a P<0.05.

b P<0.01compared with VP-16alone(one-way ANOVA followed by Tukey–Kramer multiple comparisons test).

#P<0.05compared with VP-16alone(Student t-test).

(Shieh et al.,2000;Lim,2003;Chang et al.,2007)and this?nding is also in harmony with the positive responses in which WOG atten-uated excitotoxic and oxidative stress-induced neuronal damage in primary cultured rat cortical cells(Cho and Lee,2004).

An important consideration of co-administration of VP-16and WOG is how it will possibly affect the anti-tumor treatment ef?-cacy;there are,however,important differences between these two processes,which suggest that a reduction in side effects does not necessarily go hand-in-hand with a reduction in the anti-tu-mor effects.To determine whether WOG antagonizes VP-16-in-duced topoisomerase II inhibition,the activity of topoisomerase II-a was measured by inhibition of decatenation of kDNA by WOG and/or VP-16.Fig.5shows the fully catenated kDNA(Lane 1)cannot enter the gel due to its large size;however,following incubation with puri?ed topoisomerase II-a(Lanes2–5),two decatenation products could be shown:nicked monomer kDNA (upper band)and covalently closed circular DNA(lower band;re-laxed).As seen in Lanes2and3,the solvent control and WOG (100l M)do not affect decatenation of kDNA,while100l M VP-16totally block the reaction(Lane4).Importantly,the addition of WOG could not reverse VP-16-induce topoisomerase II inhibi-tion(Lane5).Consequently,WOG should not diminish the anti-tu-mor activity of VP-16through its ability to prevent the formation of free radical intermediates.

3.4.Conclusion

The present study demonstrated that WOG was neither geno-toxic nor apoptogenic for the mouse bone marrow cells at the se-lected doses.Moreover,WOG is able to attenuate VP-16-induced oxidative DNA damage and apoptosis in mouse bone marrow cells. VP-16induced a signi?cant alteration of mRNA expression of OGG1gene and marked biochemical alterations characteristic of oxidative DNA stress,including increased8-OHdG and lipid perox-idation generation and reduction in GSH.Prior administration of WOG ahead of VP-16challenge ameliorated these altered parame-ters.VP-16has a direct inhibitory effect on topoisomerase II,an important component of its anti-tumor activity,and this will be unchanged by any manipulations that alter the redox reaction. The improvement in mitotic activity of bone marrow cells of ani-mals pre-treated with WOG in VP-16toxicity may focus attention on the bene?cial effect of WOG to overcome the haematological toxicity induced by VP-16.The antioxidant properties of WOG have already been described(Shieh et al.,2000;Lim,2003;Chang et al., 2007).For the?rst time,however,we demonstrated that these properties are also effective on a potent human carcinogen,such as VP-16.Therefore,WOG can be a promising chemoprotective agent and might be useful to avert secondary acute myeloid leukemia and other drug-related cancers in cured cancer patients and medical personnel exposing to VP-16.

Con?ict of Interest

The authors declare that there are no con?icts of interest. Acknowledgements

The authors extend their appreciation to the Deanship of Scien-ti?c Research at King Saud University for funding the work through the research group Project No.RGP-VPP-120.

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