Mutation Research 578 (2005) 15–22

Protection against aflatoxin-B1-induced liver mutagenesisby Scutellaria baicalensis

Johan G. de Boera,∗, Brendan Quineya, Patrick B. Waltera, Cynthia Thomasa,Kimberley Hodgsona, Susan J. Murchb, Praveen K. Saxenac

a Centre for Biomedical Research, University of Victoria, P.O. Box 3020 STN CSC, Victoria, BC, Canada V8W 3N5b National Tropical Botanical Garden, 3530 Papalina Road, Kalaheo, Kauai, HI 96741, USA

c Department of Plant Agriculture, University of Guelph, Guelph, Ont., Canada N1G 2W1

Received 26 April 2004; received in revised form 27 July 2004; accepted 9 January 2005Available online 8 April 2005

Abstract

We have measured the inhibition of the mutagenicity of the mycotoxin aflatoxin-B1 in the liver of the rat by plant materialof Scutellaria baicalensis, or Huang-qin. The addition of one percent dried Huang-qin to the feed of the animals reduced themutant frequency of a subsequent administration of aflatoxin-B1 by approximately 60 and 77%, respectively, for two differentbatches of the plant material. The addition of Huang-qin also increased the expression of the gene for glutathioneS-transferaseA5 subunit by 2.5–3.0-fold, and decreased expression of P450 cytochrome 3A2 by 1.8–2.0-fold. The greater increase of theexpression of the GST gene may result in the protection shown by Huang-qin. The sensitivity of the hepatic mitochondria tos ontainingH uang-qinm mbinationo for betterp©

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welling, a measure of the mitochondrial permeability transition, is increased significantly in animals that are on a diet cuang-qin. This may lead to increased sensitivity to apoptosis on treatment with toxic compounds. The two batches of Haterial show differences in both chemical composition and preventive potential. This study demonstrates how a cof generating and analysis of plant varieties together with a mammalian assay for efficacy may improve the searchlant-based prevention of cancer initiation.2005 Elsevier B.V. All rights reserved.

eywords: Mutagenicity; Plants;Scutellaria; Chemoprevention; Transgenic;lacI

. Introduction

More than 25% of the pharmaceuticals commonlyvailable by prescription are derived from leafy plants

∗ Corresponding author. Tel.: +1 250 472 4067;ax: +1 250 472 4075.

E-mail address: jdboer@uvic.ca (J.G. de Boer).

while numerous other whole plant preparationsused for the treatment of human diseases throughodeveloped and developing world[1]. However, herbapreparations are mixtures of various plants, and cohundreds of potentially active ingredients[2]. This maypresent a problem as most ingredients are not wellacterized and some are potentially toxic. This is explified by several instances of reported organ fail

027-5107/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.mrfmmm.2005.01.016

16 J.G. de Boer et al. / Mutation Research 578 (2005) 15–22

and even fatal toxicity[3,4], as well as many instancesof other adverse effects. An additional concern is thepossibility of interactions between herbal componentsand pharmaceutical drugs[5]. Nevertheless, the bene-ficial potential of many herbs is undeniable and charac-terization of their beneficial as well as toxic propertiesis urgent and important.

The Chinese herbScutellaria baicalensis Georgi(Huang-qin) is a popular and widely used herb in tra-ditional Chinese medicine. It has anti-inflammatoryand anti-pyretic effects, and is used in mixtures forskin disorders, jaundice, hepatitis, and inflammatorydiseases[6,7]. In addition, some secondary metabo-lites of S. baicalensis, including baicalin and wogo-nin have been shown to have apoptotic and anti-tumorproperties[8]. Extracts from the roots (Radix) ofS.baicalensis have protective effects against the genotox-icity of various chemicals in bacterial assays, includingN-methyl-N′-nitro-N-nitrosoguanidine and aflatoxin-B1 [9]. Flavonoids fromS. baicalensis were recentlyshown to inhibit proliferation of various human hep-atoma cell lines[7].

Plants have the potential to provide us with manybeneficial compounds. In the current study the potentialantimutagenic effects ofS. baicalensis were assessedin an animal model system. We provided rats with feedcontaining 1% of one of two mixtures of ground upS. baicalensis, with different compositions of relevantsecondary metabolites. Subsequently the animals wereexposed to aflatoxin-B1 by oral gavage. The inductiono rgeto ity,bg itionts xinm ep-a

2

2

qin( d,O con-t nin

determined (Murch et al., in press, in Plant Science).Huang-qin lines were obtained from clonally propa-gated de novo shoots regenerated from wild-harvestedseedlings[10,11]. Mixtures of roots and shoots of sev-eral lines were combined to obtain enough materialfor the experiment. Plant tissue was frozen in liquidN2, freeze dried to complete dryness for 24 h (Lab-conco, Caltec Scientific Ltd., Toronto, Ont., Canada)and ground in a coffee mill (Braun) to a fine powder.The average dry weight of the plantlets was 11.6%.

The contents of relevant chemicals in the materialof two mixtures used in this study are mentioned in theSection3. Aflatoxin-B1 was obtained from Sigma (St.Louis, MO, USA). All reagents used during packagingand plating of phage were supplied by Stratagene (LaJolla, CA, USA).

2.2. Animal treatment

F344lacI transgenic BigBlue® transgenic rats wereobtained from Stratagene (La Jolla, CA) and bred toproduce sufficient numbers of female animals for thisstudy. The rats were housed individually and fed AIN-93G diet throughout the study. Seven- to ten-week-old female rats were randomly assigned into severalgroups (control, aflatoxin, aflatoxin-Huang qin A, andaflatoxin-Huang qin B). Aflatoxin was administered at0.5 mg/kg body weight (suspended in corn oil) by oralgavage. Control animals received corn oil only. Thebody weight of the animals was measured at the time ofa ng-q /kgf deda rted3 ntils ooda

2

dc Bi uidn si obe rer’si for

f mutation was assessed in the liver, the primary targan for aflatoxin mutagenicity and carcinogenicy using a rat model with the retrievable bacteriallacIene that acts as a target gene for mutation. In add

o mutation, we measured the effects ofS. baicalen-is on the expression of genes involved in aflatoetabolism, and the effects on the sensitivity of htic mitochondria to the permeability transition.

. Materials and methods

.1. Chemicals and plants

Several optimized germplasm lines of Huang-Scutellaria baicalensis; Richter’s Herbs, Goodwoont., Canada) were previously developed and the

ents of baicalin, baicalein, wogonin, and melato

flatoxin administration. Dried and powdered Huain material was provided in the feed as 10 gm

eed (powdered feed with plant material, water adnd dried into cookies). Preventive treatments staweeks prior to gavage with aflatoxin and lasted u

acrifice, 2 weeks after the aflatoxin treatment. Fnd drinking water were supplied ad libitum.

.3. Mutant recovery

All rats were killed by CO2 asphyxiation anervical dislocation 2 weeks after the aflatoxin1

njection. The liver was removed, flash frozen in liqitrogen and stored at−80◦C. Genomic DNA wa

solated using a dialysis method[12] and packaged intacteriophage particles with TranspackTM packagingxtract (Stratagene) according to the manufactu

nstructions[12,13]. Phage particles were screened

J.G. de Boer et al. / Mutation Research 578 (2005) 15–22 17

the presence of a mutatedlacI gene on 25 cm× 25 cmplates onE. coli SCS-8 with X-gal following standardprotocols [14]. Statistical analysis of the mutantfrequencies was done with the Generalized CochranArmitage test[15]. p-values are two-tailed.

2.4. Analysis of mitochondrial swelling

Mitochondria were isolated from liver tissue by aconventional differential centrifugation method[16].The final mitochondrial pellet was suspended in about1 ml of buffer (containing 210 mM mannitol, 70 mMsucrose, and 5 mM Hepes, pH 7.35) to give a final con-centration of 80–100 mg of mitochondrial protein/ml.Mitochondrial suspensions were stored on ice and usedwithin 3–6 h after isolation. The protein content of thefinal mitochondrial suspension was determined by thebiuret method using bovine serum albumin as standard.

A standard incubation was made using 2 mg mito-chondrial protein/ml and 60�M Ca2+, in 2 ml MSHbuffer with 5 mM succinate as substrate. The effect ofaflatoxin and plant extracts A and B in the diet on mito-chondrial swelling was measured by performing this in-cubation with and without 2 mM inorganic phosphate.The incubations stood for 60 s, to allow for Ca2+ up-take, before adding the phosphate. Swelling of the mi-tochondria was measured by a decrease in absorbanceat 540 nm. Data points are an average of three measure-ments. Error bars indicate standard error of the mean.

2

s-i Aw rip-tr aqD gp5t 33 g-3 r-G res-c ncei TheG renceg

tions were performed in quadruplicate and at five dif-ferent template concentrations for each sample. Datawas checked for linear concentration dependence andanalyzed using the REST software developed by Pfaffland Horgan[19] to obtain changes in the ratio of tar-get gene to the GAPDH gene, using the�-�-Ct method[20]. Two animals each from the aflatoxin and the afla-toxin with Huang-qin mix B were analyzed for com-parison.

3. Results

Previous analysis of the plant material showed thatthe mixtures contained 3.13, 1.5, and 0.021�g/g dryweight for baicalin, baicalein, and wogonin, respec-tively, and 65.3 nmole/g dry weight melatonin formix A, and 0.94, 0.41, and 0.003�g/g dry weightfor baicalin, baicalein, and wogonin, respectively, and1176 nmole/g dry weight melatonin for mix B (Murchet al., in press, in Plant Science). The amount of theplant material needed for a 50% reduction in freeradical formation using 1,1-diphenyl-2-picrylhydrazyl(DPPH)[21] was calculated as 674 and 202�g for mixA and B, respectively (lower number indicates higherantioxidant potential).

The addition of plant material to the feed did notalter the weight of the animals. Body weight was mea-sured at the time of aflatoxin administration and foundto be nearly identical for all animals (aflatoxin group22 -e s oft xin-B ing,a redf upso toc ntf ionsf perg ,000ps eousb ap-p ntfi bya 2

.5. Analysis of gene expression

Total RNA was isolated from liver tissue ung the RNeasy Mini Kit (Qiagen) and cDNas prepared with Omniscript Reverse Transc

ase (Qiagen), using 1�M oligo-dT primers. Theesulting cDNA is amplified using Platinum TNA Polymerase (InVitrogen), with the followinrimers [17]. GAPDH: 5′-cacacccatcacaaacat-3′ and′-ttcaacggcacagtcaag-3′, 239 basepairs; GST-A5: 5′-ccacaatgcctgggtccat-3′ and 5′-atgggagtttgatgtttgaa-′88 base pairs; P450-2A3: 5′-tactacaagggcttaggga′ and 5′-cttgcctgtctccgcctctt-3′ 348 base pairs. Sybreen (Molecular Probes) was used as the fluoent detection dye, with ROX as an internal referen a Stratagene MX4000 real time PCR cycler.APDH housekeeping gene was used as the refeene for normalization purposes[17,18]. Amplifica-

20± 7.6 g; mix A group 218± 9.4 g; mix B group15± 3.5 g). Mutants in thelacI gene were recovred from the liver tissues of all treatment group

he rats, 2 weeks after the treatment with aflato1. Mutant frequencies were determined by platnd approximately 1.5 million plaques were recove

or each group. DNA sequencing of selected grof mutants did not indicate significant outliers duelonal expansion.Table 1shows the obtained mutarequencies. We followed statistical recommendator this type of study and included four to five ratsroups and recovered approximately 200,000–350laque-forming units per tissue sample[22]. Previoustudies have shown repeatedly that the spontanackground mutant frequency in the rat liver isroximately 4× 10−5 [13], consistent with our currending. Aflatoxin increased the mutant frequencypproximately 33× 10−5 to a final frequency of 37.

18 J.G. de Boer et al. / Mutation Research 578 (2005) 15–22

Table 1Total plaque forming units (pfu) screened, total mutant plaques observed and mutant frequencies for liver tissues from Big Blue rats

Grouping/rat # pfu Screened Mutant plaques Mutant frequency Average (±S.D.)

HQD-1 222,580 11 4.9× 10−5

HQC-1 267,200 106 39.7× 10−5

HQC-2 358,260 159 44.4× 10−5

HQC-3 226,900 80 35.3× 10−5

HQC-4 282,740 77 27.3× 10−5 37.2 (±6.9)

HQA-1 226,940 41 18.1× 10−5

HQA-2 207,780 36 17.3× 10−5

HQA-3 221,200 38 17.2× 10−5

HQA-4 202,340 35 17.3× 10−5

HQA-5 225,920 44 19.5× 10−5 17.9* (±0.9)

HQB-1 218,360 26 11.9× 10−5

HQB-2 345,060 40 11.6× 10−5

HQB-3 203,300 25 12.3× 10−5

HQB-4 240,400 29 12.1× 10−5

HQB-5 244,980 31 12.7× 10−5 12.1* (±0.4)

With aflatoxin treatment: HQA, Huang-qin strain A; HQB, Huang-qin strain B; HQC, no Huang-qin. Without aflatoxin treatment: HQD,background mutation. Significantly different from aflatoxin only group (* p < 0.01).

(±6.9)× 10−5, or a 7.5-fold increase. The mutationspectrum of mutants recovered after aflatoxin treatmenthas been determined in other studies[23,24]. GC→ TAtransversions constitute the dominant fraction of thetreatment-induced mutants, characteristic for aflatoxin-B1, both in animal systems as well as in thep53 genein aflatoxin-induced liver tumors. The inclusion ofthe plant material in the diet significantly reduced theaflatoxin-induced mutant frequency. In the group re-ceiving Huang-qin mix A, aflatoxin exposure resultedin an average mutant frequency of 17.9 (±0.9)× 10−5,while in the mix B group aflatoxin yielded a frequencyof 12.1 (±0.4)× 10−5. Both reductions are significantat p < 0.01. Mix B is significantly more effective thanmix A (p = 0.003 for comparison between mix A andB).

The potential for swelling of mitochondria, isolatedfrom the liver tissues, was measured (Fig. 1). Twoweeks after administration of aflatoxin B1 the inducedswelling between aflatoxin-treated rats and a controlrat was indistinguishable (control curve is identical toaflatoxin alone). The addition of Huang-qin to the feed,however, resulted in a sensitization of the mitochondriato swelling, with mix A resulting in significantly moreswelling than with mix B. In the presence of 2 mMPi, both mixes resulted in curves showing a high sen-sitivity to swelling, similar to that seen for mix A in

Fig. 1. Measurements of mitochondrial swelling at 540 nm: (A)aflatoxin-treated and given Huang-qin mix A; (B) aflatoxin-treatedand given Huang-qin mix B; (C) aflatoxin only. Each data point isan average of three measurements. Error bars indicate standard er-ror of the mean. The no-aflatoxin control is indistinguishable fromaflatoxin alone (curve C) and is not shown.

Fig. 1, but display an even steeper response (data notshown).

We measured the changes in the levels of mRNAof glutathioneS-transferase A5 and of the cytochromeP4503A2 after the addition of Huang-qin to the feed(Table 2). Real time measurements by quantitative PCRrevealed that the relative levels of mRNA for GST-A5,normalized with the expression of the housekeeping

J.G. de Boer et al. / Mutation Research 578 (2005) 15–22 19

Table 2Ratio of change in expression in aflatoxin/HQB vs. aflatoxin alone,using the housekeeping GAPDH gene as an internal reference gene

GST-A5(AF-mixB)/(AF)

P450-3A2(AF-mixB)/(AF)

Animal set 1 1.4-fold up 2.6-fold downAnimal set 2 2.2-fold up 4.0-fold down

gene GAPDH, increased by a small factor (1.4- and2.2-fold in two animal sets, respectively (p < 0.001)).The levels of P4503A2, on the other hand, decreasedby 2.6- and 4.0-fold, respectively (p < 0.04).

4. Discussion

Our study has shown thatS. baicalensis providessignificant protection against the genotoxicity of thehepatocarcinogen aflatoxin B1. The plant family ofScutellariae includes many individual species, includ-ing baicalensis andbarbata, both of which have nu-merous medicinal uses.S. baicalensis (or Huang-qin)is one of several herbals used in Yin Zhi Huang, a mix-ture used to treat neonatal jaundice in Asia, and in PC-SPES, a contemporary mixture of eight herbs (includ-ing seven Chinese herbs) that is used as a form of alter-native medicine for the treatment of prostate cancer.S.baicalensis suppresses the in vitro growth of LNCaPcells and lowers PSA levels[25]. Scutellaria root mate-rial (Ogen in Japanese traditional medicine) is also oneof several herbals that are found in mixtures called Sho-saiko-to and San’o-shashin-to. The active componentsof Scutellaria are thought to include the flavonoidsbaicalein, baicalin, and wogonin[26]. Baicalin, a glu-curonated form of baicalein, inhibits the proliferationof prostate cancer cells lines[27], dependent on theparticular cell line, and is associated with increaseda elsob hoied ids,a opto-s ax.B n bye -i

recently showed that Huang-qin contains high levelsof melatonin, also a powerful antioxidant and a humanneurohormone.

The herbal extracts ofScutellaria (as 2% offeed) have inhibitory properties against azoxymethane-induced aberrant crypt foci in rat colons[31]. An-timutagenic activity was shown as baicalein and ex-tracts ofS. baicalensis reduce the mutagenicity andchromosome aberrations caused by aflatoxin-B1 in theSalmonella typhimurium TA98 and TA100 bacterialreversion assay[32]. Our current study demonstratesthat the powdered form of the plant in a 1:1 ratioof root and shoot tissues, mixed in with the feed, isable to reduce the aflatoxin-induced mutation levelsin the liver of rats. The daily intake of baicalin andbaicalein combined in this diet is approximately 2.0and 0.6 mg/day/kg bodyweight, for mix A and mix B,respectively. Baicalin is absorbed poorly in the rat in-testinal tract; however, it is hydrolyzed to baicalein bythe gastric content. Baicalein is readily absorbed andsubsequently converted back to baicalin in the body[33].

Increases in mutant frequency as a result of afla-toxin exposure are the result of induced G:C to A:Ttransversion mutations. This specificity has been ob-served in other studies in thelacI gene[23,24,34]. Suchtransversions are also recovered at codon 249 in thep53 gene in tumors of the liver of individuals in areasat high risk for aflatoxin contamination of the daily diet[35]. Previously we have shown that the administrationo ta-gA ivee edb theei bya isfi s ofhf els.A thera i-c theat c-t s

poptosis[27]. All three compounds deplete the levf glutathione in the cell[7]. The flavones found inS.aicalensis have powerful antioxidant properties. Ct al. [28] demonstrated protection against H2O2 oxi-ation in neuronal HT-22 cells by all three flavonond protection against subsequent cell death by apis by increasing the levels of Bcl-2 and reducing Baicalein scavenges hydroxyl radicals, as showlectron spin resonance[28], and inhibites lipid perox

dation in rat liver microsomes[29]. Murch et al.[30]

f the dioxin TCDD reduces aflatoxin-induced muenicity in the female rat liver, but not in male rats[36].flatoxin is metabolized in the rat liver to a reactpoxide (AFBO) by mainly P450-3A2, and detoxifiy glutathioneS-transferase (GST). TCDD inducesxpression cytochrome P450 genes[37]. A reduction

n aflatoxin-induced genotoxicity can be achieveddministration of oltipraz or 1,2-dithiol-3-thione. Thnding is utilized in chemoprevention trials in areaigh risk of aflatoxin-induced liver cancer[38]. The ef-

ect is believed to result from an increase in GST levhigh constitutive level of GST is thought to be

eason that mice are resistant to aflatoxin[24]. Kim etl. [39] suggested from their results with rat liver mrosomes that baicalein inhibited the formation offlatoxin metabolites AFBO and AFBM1, most likely

hrough inhibition of CYP1A1 and 1A2. The reduion in mutagenicity in thelacI gene of the rat liver i

20 J.G. de Boer et al. / Mutation Research 578 (2005) 15–22

consistent with a lowering of metabolic activation ofaflatoxin or an increase in detoxification.

In order to determine alterations in the expressionof genes for relevant proteins, we measured changesin the mRNA levels of genes that are important for themetabolism of aflatoxin in the rat. Huang-qin decreasedthe expression of the cytochrome P4503A2 gene, rel-ative to the GAPDH housekeeping gene, several fold.This enzyme is the major form involved in activationto the reactive epoxide form of aflatoxin. The decreaseof the expression of this gene is consistent with the re-duction in mutagenicity in the animals fed Huang-qin.At the same time, the level of the A5 unit of glutathioneS-transferase increased in these cells[40]. The balanceof the change favors an increase in the removal of thereactive epoxide form of aflatoxin.

Interestingly, the mix that conferred the highest de-gree of protection has the lowest concentrations of thethree flavonoids. On the other hand, the level of antiox-idant is highest in this mixture, in particular of mela-tonin, which is present in approximately 18-fold higherconcentration in the B mixture. Meki et al.[41] recentlydemonstrated that melatonin may enhance the aflatoxindetoxification pathways in rats, in particular GST en-zyme activity, and suggested that melatonin may be atherapy against aflatoxin toxicity.

We also demonstrated that the addition of Huang-qin to the feed increased the sensitivity of the mito-chondria to swelling, a marker for the mitochondrialpermeability transition (MPT). This mitochondrial per-m ingp ownt go-n drialmt ucet pase3 par-a pa-r ra-t orem wasm is,m anda veb eta ein,b es-

sion in human hepatoma cell lines, albeit the pointof inhibition depended on the cell line and the com-pound, in addition to causing a decrease in mitochon-drial membrane potential. Similar results were obtainedby Matsuzaki et al.[45] who observed apoptosis andnecrosis, again depending on the cell line, of hepato-cellular carcinoma lines. Our results therefore indicatethat the feeding with Huang-qin resulted in a sensi-tization of the mitochondria in the liver, after whichtreatment with aflatoxin results in cell death. Afla-toxin alone has also been shown to cause an acute in-crease in mitochondrial swelling[46]. We measuredswelling 2 weeks after aflatoxin exposure. At thattime there was no difference between control and tis-sue from aflatoxin-treated rats. However, continuousfeeding with Huang-qin may protect against aflatoxin-induced damage and reduce the mutant frequency byelimination of those cells most heavily damaged byaflatoxin. This differential may be because of unevenhistological distribution of aflatoxin in the liver tissue[47].

A large number of natural and chemically inducedS. baicalensis varieties exist (Murch et al., in press).Analysis of secondary metabolites revealed large dif-ferences in the concentration of important compounds,including baicalin, baicalein, wogonin, and melatonin,as well as in antioxidant potential. This study hasdemonstrated the potential for use of the Big Bluemodel system for assessment of the impact of suchmedicinal plant preparations on mammalian physiol-o areai ida-t andm ucel logi-c loitt pre-v ed tob lians

A

andE cials icala

eability transition is considered one of the initiatathways in apoptosis. Recently, it has been sh

hat all three flavonoids (baicalein, baicalin and woin) dose dependently cause the loss of mitochonembrane potential, a prelude to MPT[42,43]. Fur-

hermore, baicalein and baicalin were shown to indhe release of cytochrome c and activation of cas, both signs of imminent apoptosis. Our resultsllel this well, with higher doses of flavonoids (preation A) causing more rapid swelling than prepaion B. Interestingly preparation B, which has melatonin, and was a more effective antimutagen,oderate in its induction of swelling. In support of thelatonin has recently been shown to inhibit MPTpoptosis[44], thus this effect of melatonin could haeen inhibitory to swelling in preparation B. Changl. [7] have shown that all three flavonoids, baicalaicalin, and wogonin, inhibited the cell cycle progr

gy. One ongoing challenge for research in thiss the lack of sufficient research systems for elucion of the complex interactions between plantsammals. Plants have an amazing ability to prod

arge numbers of compounds that have physioal and pharmaceutical effects. In order to exphis potential for beneficial uses in treatment andention of human diseases, these compounds nee recognized, identified, and tested in mammaystems.

cknowledgements

The authors wish to thank the National Sciencengineering Research Council of Canada for finanupport. James Holcroft provided invaluable technssistance.

J.G. de Boer et al. / Mutation Research 578 (2005) 15–22 21

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