Mutation Research 629 (2007) 148–160

Evaluation of chemopreventive agents for genotoxic activity

Rupa S. Doppalapudi a,∗, Edward S. Riccio a, Linda L. Rausch a, Julie A. Shimon a,Pam S. Lee a, Kristien E. Mortelmans a, Izet M. Kapetanovic b,

James A. Crowell b, Jon C. Mirsalis a

a SRI International, Biosciences Division, Menlo Park, CA 94025, USAb National Cancer Institute, Division of Cancer Prevention, Bethesda, MD 20892, USA

Received 19 August 2006; received in revised form 12 February 2007; accepted 14 February 2007Available online 25 February 2007

Abstract

We conducted genetic toxicity evaluations of 11 candidate chemopreventive agents with the potential for inhibiting carcinogenesisin humans at increased risk of cancer. The compounds were evaluated for bacterial mutagenesis in the Salmonella-E. coli assay,for mammalian mutagenesis in mouse lymphoma cells, for chromosome aberrations in Chinese Hamster Ovary (CHO) cells, andfor micronucleus induction in mouse bone marrow. Tested agents were indole 3-carbinol (I3C), bowman-birk inhibitor concentrate(BBIC), black tea polyphenols (BTP), farnesol, geraniol, l-Se-methylselenocysteine (SeMC), 5,6-dihydro-4H-cyclopenta[1,2]-dithiol-3-thione(DC-D3T), 4′-bromoflavone, 2,5,7,8-tetramethyl-(2R-[4R,8R,12-trimethyltridecyl] chroman-6-yloxy) acetic acid(alpha-TEA), SR13668 (2,10-dicarbethoxy-6-methoxy-5,7-dihydro-indolo[2,3-b] carbazole and SR16157 (3-O-sulfamoyloxy-7�-methyl-21-(2-N,N-diethylaminoethoxy)-19-norpregna-1,3,5(10)-triene). All these agents, except I3C and BTP, were negative inthe Salmonella-E. coli assay in the presence and absence of metabolic activation (S9). I3C and BTP induced a weak mutagenicresponse in the presence and absence of S9 with strains TA100 and TA98, respectively. Of the three compounds tested in the mouse

lymphoma assay (I3C, BBIC, and BTP), only BTP was mutagenic in the presence of S9. In the chromosomal aberration assay, of the8 compounds that were tested, 4′-bromoflavone elicited a positive response in the absence of S9 only, while SR16157 was positivein the presence of S9. The results with geraniol remain inconclusive. I3C, BBIC and BTP were not tested in the chromosomalaberration assay. None of the 11 agents induced micronuclei in mouse bone marrow erythrocytes.© 2007 Elsevier B.V. All rights reserved.

rrations

Keywords: Chemopreventive agents; Mutagenesis; Chromosomal abe

1. Introduction

Chemoprevention is a novel approach that focuseson preventing or delaying carcinogenesis through phar-macologic, biologic, and nutritional intervention [1].

∗ Corresponding author at: Biosciences Division, PN 171, SRI Inter-national, Menlo Park, CA 94025, USA. Tel.: +1 650 859 6457;fax: +1 650 859 2889.

E-mail address: rupa.doppalapudi@sri.com (R.S. Doppalapudi).

1383-5718/$ – see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.mrgentox.2007.02.004

; Micronuclei

The National Cancer Institute’s (NCI) Division of Can-cer Prevention, Chemopreventive Agent DevelopmentResearch Group has initiated an investigational agentdevelopment program for chemopreventive agents withpotential for inhibiting the process of carcinogenesis.The purpose is to obtain new agents suitable for clinicaltrials in humans who do not have cancer but may be at

increased risk for cancer. Agents being developed by theChemopreventive Agent Development Research Groupmust be tested for toxicity; all toxicological data and asummary of toxicity observed in animals treated with

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he agent must be submitted to the U.S. Food and Drugdministration (FDA) as part of the Investigational Newrug (IND) application for human clinical trials. The

dentification of chemopreventive agents is conductedy the systematic review of epidemiological and experi-ental carcinogenesis literature; collaborations with the

harmaceutical, nutritional and biotechnology indus-ries; and the Rapid Access to Preventive Interventionevelopment (RAPID) Program [1–3]. Potential chemo-reventive agents are then tested in preclinical toxicitynd pharmacokinetics studies [4]. Finally, compoundshat show promise in animal studies and an acceptableafety profile are tested as chemopreventive agents ineople at high risk for cancer because of a precancerousondition, a family history of cancer, lifestyle factorsuch as smoking, or other factors [1].

Cancer is characterized by genetic instability, whichas often been examined at the single base mutationevel but is also evidenced by gross chromosomal abnor-

alities. These consist of abnormalities of chromosomeumber, gross deletions, translocations, and gene ampli-cations. Molecular and cytogenetic evidence indicates

hat the induction of structural and numerical chromo-ome aberrations in cells play an important role in theeoplastic development of certain tumors [5]. Recenttudies have shown that increases in chromosomal aber-ations in humans indicate increased cancer risk, and thathe frequency of chromosomal aberrations in peripherallood lymphocytes is a relevant biomarker for cancerisk in humans [6–8]. Genetic toxicology tests are initro or in vivo assays designed to detect compoundshat induce genetic damage directly or indirectly. Fixa-ion of DNA damage can result in gene mutations, loss ofeterozygosity, chromosomal loss or gain, and chromo-ome aberrations. These events may play an importantole in many malignancies and may also induce heri-able effects leading to birth defects. Thus, identifyingenotoxic effects of chemopreventive agents is impor-ant for the risk/benefit assessment of their potentialse in humans, in particular when proposed for humanst increased risk for developing cancer, but who areisease-free at the time of drug administration.

The aim of the present study was to examine theenotoxic potential of chemopreventive agents usinghe in vitro Salmonella-E. coli mutagenicity assay,he in vitro mouse lymphoma assay or the chro-

osomal aberration assay in Chinese hamster ovaryells (CHO-CA), and the in vivo mouse bone mar-

ow micronucleus assay. These assays are currentlyecommended by the International Conference on Har-onization (ICH) [9], Food and Drug Administration

nd other regulatory agencies [10]. The chemicals tested

esearch 629 (2007) 148–160 149

were: (1) indole-3-carbinol (I3C), a breakdown prod-uct of the glucosinolate glucobrassicin (glucosinolatesare beta-thioglucoside N-hydroxysulfates, which areprimarily found in cruciferous vegetables); (2) bowman-birk inhibitor concentrate (BBIC), a soybean-derivedserine protease inhibitor with demonstrated anticar-cinogenic activity; (3) black tea polyphenol (BTP), amixture in tea leaves that is a natural plant antiox-idant and thus belongs to a class of chemicals thathave been shown to prevent damage to DNA and othermolecules caused by free radicals; (4) farnesol, partof the squalene-cholesterol biosynthetic pathway andwith a capacity to anchor proteins to lipid bilayers;(5) geraniol, both farnesol and geraniol of are iso-prenoid alcohols found at low levels in several foods andessential oils; (6) l-Se-methylselenocysteine (SeMC),a naturally occurring form of selenium produced inplants such as garlic, onions, leeks, and broccoli; (7)5,6-dihydro-4H-cyclopenta[1,2]dithiole-3-thione (DC-D3T), a phase II enzyme inducer that increases therate of detoxification of chemical carcinogens; (8) 4′-bromoflavone, an inducer of phase II detoxificationenzymes; (9) alpha-TEA is a synthetic analog of thenaturally occurring vitamin E; (10) SR13668, a syn-thetic analog of I3C; and (11) SR16157, an anti-estrogenagent.

2. Materials and methods

2.1. Chemicals

All eleven chemopreventive agents, I3C (CAS No. 700-06-1), BBIC, BTP, farnesol (CAS No 4602-84-0), geraniol (CASNo. 106-24-1), SeMC, DC-D3T, 4′-bromoflavone, alpha-TEA,SR13668, SR16157 were provided through the NCI’s Divi-sion of Cancer Prevention (DPC) repository. The positivecontrols sodium azide (CAS No. 26628-22-8) was obtainedfrom Sigma Chemical Co. (St. Louis, MO); 9-aminoacridinehydrochloride (CAS No.52417-22-8), 2-nitrofluorene (CASNo. 607-57-8), and 4-nitroquinoline N-oxide (CAS No. 56-57-5) were obtained from Aldrich Chemical Co (Milwaukee,WI); 2-aminoanthracene (CAS No. 613-13-8) obtained fromSigma–Aldrich Corp. (St. Louis, MO); methyl methanesul-fonate (MMS, CAS No. 66-27-3) and ethyl methanesulfonate(EMS, CAS No. 1675-54-3) obtained from Sigma–AldrichCo., (St. Louis, MO); 20-Methylcholanthrene (MCA, CAS No.766-40-5) obtained from Aldrich Chemical Co. (Milwaukee,WI); cyclophosphamide (CAS No.6055-19-2) and urethane(CAS No. 51-79-6) was obtained from Sigma Chemical Co. (St.

Louis, MO), and S9 was obtained from Molecular ToxicologyInc., (Boone, NC).

Using these chemicals, bacterial mutation assay, mouselymphoma assay or CHO chromosomal aberration assay and invivo mouse bone marrow micronucleus assays were conducted

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in compliance with Good Laboratory Practice (GLP) and theInternational Conference on Harmonisaiton (ICH) Guidelines.

2.2. Bacterial mutation assay

Salmonella typhimurium LT2 strains (TA1535, TA1537,TA98, and TA100) were obtained from Dr. Bruce Ames(University of California, Berkeley), and E. coli strain WP2(uvrA) was obtained from the National Collection of Indus-trial and Marine Bacteria (Aberdeen, Scotland). Strains werekept frozen in nutrient broth supplemented with 10% ster-ile glycerol at −80 ◦C. Experiments with Salmonella strainswere performed as described previously [11]. The procedureused with the E. coli strain was as described earlier [12]. Astandard plate incorporation procedure or the preincubationmodification to the standard plate incorporation procedure wasused. A dose range-finding experiment was conducted withSalmonella strain TA100 in the presence and absence of arat liver metabolic activation (S9) system to determine a suit-able dose range. S9 consisted of Aroclor 1254-induced ratliver activation prepared at a concentration of 5% or 10%.The test article was tested over a wide range of dose levels(at least five), with the highest dose level (depending on solu-bility or cytotoxicity) being 5000 �g/plate. The test article wastwice evaluated for mutagenicity using five tester strains, withthree plates per dose level, both with and without S9 mixture.Unless a positive response was obtained in the first assay with5% S9 mix, the second assay was performed using a 10% S9mix. The positive controls in the absence of S9 were sodiumazide (TA1535 and TA100), 9-aminoacridine hydrochloride(TA1537), 2-nitrofluorene (TA98), and 4-nitroquinoline N-oxide [WP2 (uvrA)]. In the presence of S9 for all strains, thepositive control was 2-aminoanthracene.

Statistical analysis was performed using: (1) Levene’s test[13] to determine if a difference exists among treatment vari-ances; (2) one-tailed Dunnett’s t-test [14] for comparison oftreatments with solvent controls and within-levels pooled vari-ance; and (3) evaluation of dose-relatedness by regressionanalysis [15], using a t-statistic to test the significance of theregression. Results were considered positive if reproducibleand statistically significant (p < 0.01) increases in revertantswere observed at one or more dose levels; negative if values forthe dose levels were not reproducible or significant; or equiv-ocal if results cannot be clearly identified as being positive ornegative.

2.3. Mouse lymphoma cell tk+/− → tk−/− gene mutationassay

For this assay, L5178Y cells, clone 3.7.2C, heterozygous atthe tk locus, were originally obtained from Dr. Donald Clive

of former Burroughs Wellcome Co., Research Triangle Park,NC. The stock cells were grown as a suspension culture underexponential growth conditions and treated with methotrexate(0.1 �g/ml) in the presence of 3 �g/ml thymidine, 5 �g/mlhypoxanthine, and 7.5 �g/ml glycine (THG) prior to mutage-

esearch 629 (2007) 148–160

nesis experiments to eliminate cells lacking thymidine kinaseactivity. Experiments were conducted and data were collectedusing a standard procedure [16,17]. Methyl methanesulfonate(MMS) or ethyl methanesulfonate (EMS) were used in theabsence of S9, 20-Methylcholanthrene (MCA) was used inthe presence of S9 for positive controls. S9 consisted of Aro-clor 1254-induced rat liver metabolic activation prepared at aconcentration of 1%. An initial cytotoxicity assay was con-ducted using a wide range of dose levels (at least 10–15), withthe highest dose level being 5000 �g/ml unless problems wereencountered with solubility. Exposure times were 4 and 24 hwithout S9 and 4 h with S9. Based on the cytotoxicity results,the dose levels were selected and mutagenicity was evaluatedtwice. In the initial experiment, cells were treated with the testarticle for 4 h with and without S9. In the repeat experiment,cells were treated for 4 h with S9 and for 24 h without S9 if theresults in the initial experiment were negative.

Statistical analysis was performed using Microsoft Excel2000 and statistical software (JMP version 5.0 by SASInstitute) on a Windows-based computer. A Trend test was cal-culated as the linear regression of the log-transformed mutationfrequency of TFTr cells in order of the concentrations (i.e., 1for solvent control, 2 for the lowest concentration, etc.). Thesignificance of the trend test was determined from a two-sidedtest of the significance of the slope in the linear regression. Inaddition, in a test using the error term from the one-way anal-ysis of variance (ANOVA), the mean of the log-transformedTFTr cell mutation frequencies of cultures treated with eachtest article concentration was compared to the solvent con-trol mean by Dunnett’s test, with significance assessed at the0.05 confidence level. The mean mutation frequency of TFTr

cells for positive controls was compared to the solvent con-trol mean, after log transformation, by a one-sided t-test, withsignificance assessed at the 0.01 and 0.05 confidence level. Atest article was considered positive when it met the followingcriteria: (1) a significant (p < 0.05) dose-related increase in themutation frequency (MF) occurred, (2) the mean MF of a set ofduplicate cultures treated with one or more of the three highestacceptable concentrations of the test article was statisticallysignificant (p < 0.05), (3) at least one concentration induced anaverage absolute increase in MF (net over mean solvent con-trol MF in this study) greater than 40 per 106 cells, and (4) theresults were reproducible in a repeat experiment. A test articlewas considered a negative when a reproducible, statisticallysignificant response was not induced according to the abovecriteria.

2.4. CHO chromosome aberration assay

CHO cells (ATCC CCL 61 CHO-K1, proline-requiring)were obtained from the American Type Culture Collection(Rockville, MD). Cells were grown in an atmosphere of 5%

CO2 at 37 ◦C in F-12 medium with 10% fetal bovine serum(FBS), 2 mM GlutaMAX, and 1% penicillin-streptomycinsolution to maintain exponential growth. Cells were grownin this medium during exposure to dose formulations withoutS9. F-12 medium with 2.5% FBS (containing GlutaMAX and

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enicillin-streptomycin in the above concentrations) was usedo grow cells exposed to dose formulations with S9. S9 con-isted of Aroclor 1254-induced rat liver metabolic activationrepared at a concentration of 10%. Based on the solubilityf the test article, 6 final concentrations (5 test article con-entrations plus negative controls), usually ranging from 0 to000 �g/ml, were tested. The exposure periods were 3 and 21 hithout MA and 3 h with S9. MMS was used in the absence of9 and cyclophosphamide in the presence of S9. At the end of

he exposure period, cells were either harvested or rinsed andllowed to incubate in fresh media until harvest at 3 or 21 h afterxposure. The standard procedure was used for harvesting cul-ures [18]. For each dose level, 1000 cells for mitotic index and00 cells for structural chromosome aberrations were analyzedsing standard criteria [19] except for slides containing highevels of damage (i.e., ≥25% structurally aberrant cells). Fortatistical analysis, the numbers of cells with structural chro-osome damage observed in the test article and the positive

ontrol treatment groups were compared with those for theoncurrent solvent control group by using Fisher’s Exact testsignificance level of p < 0.05, one-tailed) [FISHEX.PL.XLT. 1.0 macro run on MS Excel v. 5.0]. A test article was consid-red positive if there was: (1) a statistically significant increasep < 0.05) in the frequency of cells with structural chromoso-al damage at one or more dose levels, (2) this increase was

ose related and reproducible. A test article was consideredegative if none of the criteria for a positive response wereet. It was called inconclusive when the results could not be

dentified clearly as positive or negative.

.5. Mouse bone marrow micronucleus assay

Male and female Swiss-Webster mice were obtained fromharles River Laboratories (Kingston, NY; Portage, MI) andere approximately 7 weeks of age at the time of dosing.nimals were maintained in clear polycarbonate cages withardwood chip bedding and were provided Purina Rodenthow and tap water ad libitum. All animal work was approvedy SRI’s Institutional Animal Care and Use CommitteeIACUC) in full compliance with all regulations of the NIHffice of Laboratory Animal Welfare (OLAW). Animals wereiven a single oral administration of vehicle or test articleince it is the route of human exposure. The dose range find-ng experiment was performed using 3 males and 3 femaleser treatment group, with one sacrifice time (48 h). Treatmentroups included one vehicle control group and five test article-reated groups with a highest dose lvel of 2000 mg/kg whichs widely recommended as the top dose level for genotoxicityssays. Based on the animal death, compound related toxic-ty and PCE/RBC ratio in the range finding experiment, threeose levels were chosen for the micronucleus experiment. Five

ales and 5 females were treated for each dose level and at

ach time-point. At 24 and 48 h after dose administration, miceere euthanized, bone marrow was harvested, and smears wererepared on microscope slides [20,21] and stained using acri-ine orange [22]. Vehicle control and positive control groups

esearch 629 (2007) 148–160 151

were maintained simultaneously. Urethane (300 mg/kg) wasused as a positive control and were sacrificed at 24 h. Fivemales and 5 females were scored for micronucleus analysis ineach treatement group and at each time-point. For each animal,200 cells were scored to determine the ratio of polychromaticerythrocytes (PCE) to red blood cells (RBC), and 2000 PCEwere scored for micronuclei (MN). Cochran-Armitage trendtest and the normal test for equality of binomial proportions[23] were used for statistical analysis. A test article was consid-ered positive if there was: (1) a statistically significant increase(p < 0.05) in micronucleated PCE; (2) the increase was dose-related; and (3) the micronucleated PCE frequency was greaterthan the mean historical micronucleus frequency ±2 standarddeviations (S.D.). The test article was considered negative ifnone of the criteria for a positive response were met, and itwas considered equivocal if a statistically significant increasein micronucleated PCE frequency was observed, but it did notmeet (2) or (3) of the positive criteria.

2.6. Solvent/vehicle controls

The following solvents were used for the in vitro stud-ies. Dimethyl sulfoxide (DMSO) obtained from Mallinkrodt(Phillipsburg, NJ) was the solvent for I3C, farnesol, geran-iol, DC-D3T, 4′-bromoflavone, alpha-TEA, SR13668, andSR16157. Sterile water (Baxter Healthcare Corp, Deerfield,IL) was used for BTP and BBIC. For BBIC formula-tions, 100 mg/ml was suspended in water and centrifugedat ∼4,400 rpm for ∼15 min. The supernatant was pooledand retained for further dilutions. For the in vivo mousebone marrow micronucleus assay, methyl cellulose, 400 CPSUSP (Sigma–Aldrich Inc, St. Louis, MO) was used for I3C,DC-D3T, 4′-bromoflavone, SR13668, and SR16157; corn oil(Sigma–Aldrich, St. Louis, MO) was used for farnesol andgeraniol, and sterile water was used for BBIC and BTP. Alpha-TEA was dissolved in DMSO and then suspended in peanutoil (Spectrum Chemical, Gardena, CA).

3. Results and discussion

It has been reported that I3C is a promising agentagainst breast and cervical cancers [24]. Anticarcino-genic activity of BBIC [25,26] and antiproliferativeactivity of geraniol [27,28] have been reported usingvarious assays. 4′-Bromoflavone significantly inhibitedthe incidence and multiplicity of mammary tumorsand greatly increased tumor latency [29]. Alpha-TEAinduces a dose-dependent DNA synthesis arrest andapoptosis; inhibits colony formation, and significantlyreduces tumor cell proliferation [30]. Recent studies withalpha-TEA indicate that it is a potent inducer of apop-

tosis in a wide variety of epithelial cancer cell types incell culture, including breast, prostate, lung, colon, ovar-ian, cervical, and endometrial cells [31,32]. SR13668exhibits antitumor activity against breast, prostate and

152 R.S. Doppalapudi et al. / Mutation Research 629 (2007) 148–160

Table 1Overview on the genotoxicity of candidate chemopreventive agents

Compound Test

Salmonella/E.coli MOLY CHO/CA MN

I3C Weak, mutagenic responsewith strain TA100 (+/−S9)

Negative Not done Negative

BBIC Negative Negative Not done NegativeBTP Weak, mutagenic response

with strain TA98 (+/− S9)Negative without S9; Positive with S9 Not done Negative

Farnesol Negative Not done Negative NegativeGeraniol Negative Not done Inconclusive NegativeSeMC Negative Not done Negative NegativeDC-D3T Negative Not done Negative Negative4′-Bromoflavone Negative Not done Positive without S9; Negative with S9 NegativeAlpha-TEA Negative Not done Negative NegativeSR13668 Negative Not done Negative Negative

study in

SR16157 Negative Not done

MOLY, Mouse lymphoma assay; CHO/CA, Chromosomal aberrationMetabolic activation.

ovarian tumors in experimental animals [33]. SR16157has potential for treatment of breast cancer. Little, ifany information, on the genotoxic potential of theseagents has been reported in the literature. In the presentstudy, the genotoxic potential of chemopreventive agentswas evaluated using the Salmonella-E. coli bacterialmutagenesis assay, the mouse lymphoma assay or thechromosomal aberration assay with CHO cells, and amouse bone marrow micronucleus assay. An overviewon the genotoxicity of the chemopreventive agents eval-uated in this study is given in Table 1.

3.1. I3C

The Salmonella-E. coli assay (Ames test) was per-formed in a dose range between 39.1 and 2,500 �g/plate.The results are presented in Table 2. A weak muta-genic response was observed in the Ames test withstrain TA100, in the presence and in the absence of S9.Statistically significant responses (p < 0.01) were seenin the first experiment at 625 �g/plate in the absenceof S9 and at doses ≥156.2 �g/plate in the presenceof S9. In all instances there was a dose dependentincrease by regression analysis in the number of revertantcolonies. In the confirmatory experiment, the significantincrease in revertant colonies was observed at dose lev-els ≥312.5 �g/plate in the presence and absence of S9.In the mouse lymphoma assay, based on cytotoxicityresults, I3C was tested over doses from 77 to 325 �g/ml

without S9 and from 2.3 to 30 �g/ml with S9. There wasno statistically significant increase in MF with or withoutS9. It has been reported that I3C had little or no effect onthe inhibition of mutagenesis induced by nitropyrine and

Negative without S9; Positive with S9 Negative

Chinese Hamster Ovary cells; MN, Micronucleus study in mice; S9,

I3C itself was not an antimutagen against aflatoxin B1[34,35]. I3C was not tested in the CHO/CA assay. How-ever, it was reported in the literature that it did induceDNA adducts in CHO cells [36].

In the mouse micronucleus assay, I3C was tested inmice at 375, 750, or 1500 mg/kg. No statistically sig-nificant increases in the frequency of micronucleatedPCE were found at any dose level in the male micesacrificed at either time point or in the female mice sacri-ficed at 48 h. However, statistically significant increasesin the incidence of micronucleated PCE were seen at24 h in female mice administered 750 mg/kg (0.35%)and 1500 mg/kg (0.32%) of I3C. The Cochran-Armitagetrend test showed a statistically significant, dose-relatedincrease in the frequency of micronuclei in female miceat 24 h after treatment. However, these micronucleusfrequencies were within two standard deviations of thehistorical control value (0.46%); therefore this responsewas not considered to be a positive result in this assay. Ina similar study [37], I3C also did not induce micronucleiin mouse bone marrow and peripheral blood lympho-cytes.

3.2. BBIC

The microbial mutagenicity assay was performedover doses ranging from 3.12 to 200 �l per plate ofextract of BBIC that was prepared by adding 100 mgof the compound to 1 ml of water. After centrifugation,

the supernatant was used as the test compound and wastested both in the presence and absence of S9. BBICwas negative at the dose levels tested. In the mouselymphoma assay, mutagenesis experiments were con-

R.S. Doppalapudi et al. / Mutation Research 629 (2007) 148–160 153

Table 2Effect of I3C on bacterial strains in the Salmonella-E.coli plate-incorporation assay

Dose/Plate �g Mean ± standard deviation revertants/plate

TA1535 TA1537 TA98 TA100 WP2uvrA

Experiment 1Without S9

0 18 ± 3 11 ± 4 19 ± 6 131 ± 8 24 ± 539.1 14 ± 2 12 ± 4 30 ± 4 135 ± 21 25 ± 478.1 15 ± 3 10 ± 4 19 ± 7 147 ± 2 21 ± 1

156.2 13 ± 1 7 ± 1 25 ± 4 175 ± 16 26 ± 6312.5 12 ± 4 11 ± 5 25 ± 3 182 ± 14 26 ± 7625 15 ± 4 7 ± 2 24 ± 3 239 ± 27* 23 ± 4

1250 8 ± 4 Toxic 18 ± 2 153 ± 33 23 ± 3

With S9 (5%)0 16 ± 3 13 ± 4 29 ± 3 145 ± 17 27 ± 3

39.1 15 ± 3 12 ± 1 30 ± 2 137 ± 21 26 ± 978.1 11 ± 3 8 ± 4 27 ± 11 167 ± 4 21 ± 6

156.2 11 ± 3 14 ± 1 27 ± 2 186 ± 13* 30 ± 6312.5 17 ± 3 14 ± 1 32 ± 4 204 ± 21* 28 ± 9625 12 ± 4 11 ± 3 32 ± 1 253 ± 3* 35 ± 3

1250 11 ± 4 10 ± 3 26 ± 4 227 ± 10* 30 ± 7

Experiment 2Without S9

0 15 ± 2 9 ± 3 23 ± 3 133 ± 16 26 ± 639.1 14 ± 6 6 ± 1 18 ± 5 131 ± 14 22 ± 478.1 13 ± 4 4 ± 1 20 ± 2 151 ± 9 29 ± 1

156.2 14 ± 3 11 ± 3 20 ± 6 150 ± 12 23 ± 8312.5 17 ± 4 8 ± 4 25 ± 4 197 ± 3* 21 ± 3625 10 ± 8 8 ± 1 22 ± 7 248 ± 24* 26 ± 3937.5 NT NT NT 280 ± 18* NT

1250 10 ± 5 Toxic 23 ± 4 198 ± 15* 19 ± 62500 Toxic Toxic Toxic NT 19 ± 5

With S9 (5%)0 12 ± 4 11 ± 3 25 ± 5 137 ± 12 23 ± 7

39.1 12 ± 4 6 ± 1 22 ± 4 158 ± 10 30 ± 478.1 11 ± 4 8 ± 4 35 ± 5 174 ± 25 21 ± 4

156.2 13 ± 2 6 ± 2 32 ± 6 194 ± 9 26 ± 2312.5 10 ± 4 10 ± 4 36 ± 3* 229 ± 27* 24 ± 6625 15 ± 1 7 ± 1 36 ± 4 292 ± 15* 25 ± 6937.5 NT NT NT 317 ± 54* NT

1250 9 ± 2 6 ± 2 27 ± 3 274 ± 31* 21 ± 6

doiai(ettil

2500 Toxic Toxic

* = p < 0.01 (Dunnett’s test); NT, not tested.

ucted at doses ranging from 3.13 to100 �l/ml of extractf BBIC (100 mg/ml of water). No statistically signif-cant (p < 0.05) increase in MF was observed withoutctivation (4 h exposure). However, a statistically signif-cant increase in MF was seen at the highest dose level100 �l/ml) and it was a dose-related increase for the 24 hxposure, but this response had a high standard devia-

ion associated with the average MF. It is unlikely thathis response would be reproducible because the tox-city of this dose level is already near the maximumimit (27% relative total growth). Therefore, BBIC was

14 ± 2 NT 23 ± 6

evaluated as negative without S9. No statistically sig-nificant (p < 0.05 by Dunnett’s analysis) increases inMF were observed in the mutagenesis experiments withactivation.

In the mouse bone marrow micronucleus experi-ment, BBIC was tested at 500, 1000, or 2000 mg/kg.No statistically significant increase in the frequency

of micronucleated PCE was observed and there wasno significant dose-related increase in the frequency ofmicronuclei. No cytogenetic tests of BBIC have previ-ously been reported.

154 R.S. Doppalapudi et al. / Mutation Research 629 (2007) 148–160

Table 3Effect of BTP on bacterial strains in the Salmonella-E.coli plate-incorporation assay

Dose/Plate �g Mean ± standard deviation revertants/plate

TA1535 TA1537 TA98 TA100 WP2uvr A

Experiment 1Without S9

0 12 ± 4 7 ± 2 24 ± 4 140 ± 19 29 ± 3156.2 12 ± 3 6 ± 2 27 ± 5 112 ± 3 23 ± 5312.5 11 ± 5 8 ± 2 23 ± 5 118 ± 6 25 ± 5625 16 ± 2 7 ± 1 34 ± 6 122 ± 11 23 ± 31250 13 ± 5 8 ± 1 40 ± 4* 132 ± 8 26 ± 52500 10 ± 4 6 ± 3 32 ± 5 122 ± 21 15 ± 25000 11 ± 3 3 ± 1 31 ± 3 122 ± 6 17 ± 2

With S9 (5%)0 18 ± 1 9 ± 3 29 ± 3 143 ± 8 34 ± 7156.2 11 ± 3 10 ± 3 27 ± 7 134 ± 17 26 ± 8312.5 12 ± 4 9 ± 3 34 ± 4 124 ± 21 30 ± 11625 16 ± 4 8 ± 1 45 ± 5 139 ± 11 25 ± 61250 14 ± 3 8 ± 1 50 ± 8* 147 ± 1 18 ± 62500 11 ± 4 9 ± 4 46 ± 5* 137 ± 6 24 ± 35000 12 ± 3 9 ± 4 38 ± 9 120 ± 16 13 ± 1

Experiment 2Without S9

0 13 ± 0 8 ± 1 30 ± 3 136 ± 10 28 ± 1156.2 11 ± 2 5 ± 1 34 ± 7 119 ± 10 28 ± 8312.5 9 ± 3 8 ± 4 39 ± 1 121 ± 9 28 ± 8625 10 ± 4 6 ± 2 48 ± 9* 123 ± 17 31 ± 11250 13 ± 1 8 ± 1 49 ± 5* 131 ± 9 19 ± 22500 11 ± 2 11 ± 2 45 ± 7 128 ± 16 17 ± 55000 11 ± 2 11 ± 2 46 ± 7 136 ± 20 15 ± 1

With S9 (!0%)0 16 ± 2 11 ± 2 36 ± 7 126 ± 5 30 ± 9156.2 14 ± 2 8 ± 2 36 ± 6 128 ± 21 31 ± 7312.5 11 ± 3 8 ± 2 34 ± 7 131 ± 6 29 ± 4625 11 ± 3 9 ± 3 42 ± 2 137 ± 13 28 ± 41250 14 ± 1 12 ± 2 61 ± 5* 156 ± 6 24 ± 42500 14 ± 2 11 ± 2 62 ± 3* 131 ± 13 15 ± 2

5000 11 ± 2 13 ± 1

* =Significant (p < 0.01) by Dunnett’s test.

3.3. BTP

The dose levels tested in the microbial mutagenic-ity assay were 156.2–5000 �g/plate in the presence andabsence of S9. BTP was found to elicit a weak, but repro-ducible, mutagenic response with strain TA98 in thepresence and absence of S9. No other strain appearedto demonstrate a mutagenic response. The results arepresented in Table 3.

Based on cytotoxicity results, BTP was tested in the

mouse lymphoma assay at doses ranging from 99 to1000 �g/ml with S9 and 82–490 �g/ml without S9 for 4hr exposure, and at doses ranging from 92 to 256 �g/mlwithout S9 for 24 h exposure. A statistically significant

43 ± 1 125 ± 12 12 ± 4

and dose-related (p < 0.05) increase in MF was seen incultures treated with≥343 �g/ml with S9. The frequencyof small colonies was increased relative to that in thesolvent control cultures. There was no statistically sig-nificant increase in MF in the absence of S9. It is apparentthat BTP requires S9 to show a genotoxic effect. Theresults are presented in Table 4.

In the mouse micronucleus assay, BTP was tested at375, 750, and 1500 mg/kg. Neither a significant increasein micronuclei nor a dose-related increase in the fre-

quency of micronuclei was found in either male or femalemice. In the literature, it has been reported that whenBTP was coincubated with benzo(a)pyrene (BP) andcyclophosphamide (CP), there was a dose-dependent

R.S. Doppalapudi et al. / Mutation Research 629 (2007) 148–160 155

Table 4Mutagenicity of BTP in L5178Y mouse lymphoma cells

Dose (�g/ml) RSG (%) RCE (%) RTG (%) MF ±S.D.

4 Hr Exposure Without S9Control 100 100 100 25 482 92 95 87 26 7118 89 113 100 19 2168 86 92 79 18 1240 70 111 77 35 5343 30 103 31 52* 9490 13 78 10 64* 11MMS, 5 94 94 88 80** 5EMS, 200 92 83 76 363** 6

4 Hr Exposure With S9Control 100 100 100 19 7168 78 76 59 26 7240 63 95 60 28 3343 35 99 34 61* 9490 33 92 30 76* 9700 21 79 17 103* 81000 8. 58 4.6 117b 15MCA, 5 15 36 5.5 388** 19

24 Hr Exposure Without S9Control 100 100 100 64 1192 57 117 58 46 7154 35 98 30 63 39256 21 63 9.6 89 14320 11 Not cloned400 7.5 Not cloned500 5.4 Not clonedMMS, 5 78 63 48 319** 62EMS,100 74 62 44 772** 114

4 Hr Exposure With S9Control 100 100 100 29 299 97 110 107 29 9165 107 86 92 32 6274 85 95 80 44 6392 61 101 62 77* 4560 21 Not cloned800 11 Not clonedMCA, 5 19 75 14 206** 25

R lvent coc ll frequm tt’s ana

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SG, Growth of cells during the expression period relative to that of soells; RTG, Relative total growth (RSG × RCE/100); MF, Mutant ceethansulfonate; MCA, 3-Methylcholanthrene; * = p < 0.05 by Dunne

eduction in BP- and CP-induced chromosomal aberra-ions, micronuclei and sister chromatid exchanges [38].

.4. Farnesol

The microbial mutagenicity assay was conducted atose levels of 0.61–19.5 �g/plate in the absence of S9,

nd at 4.88–156.2 �g/plate in the presence of S9. Notatistically significant increase in the number of rever-ants was observed. Similarly, an analog of farnesol,rednisolone farnesylate when tested with and without

ntrol cells; RCE, Cloning efficiency relative to that of solvent controlency (per 106 cells); MMS, Methyl methanesulfonate; EMS, Ethyllysis; ** = p < 0.01 by Student’s t-test.

S9 in a dose range of 312–5000 �g/plate, showed nomutagenicity in the Ames Salmonella test [39].

Induction of chromosome aberrations in CHO cellswas evaluated at doses from 4.9 to 19.5 �g/ml for a3 h exposure, from 1.2 to 4.9 �g/ml for a 21 h expo-sure without S9, and from 19.5, to 78.1 �g/ml with S9for s 3 h exposure. There was no statistically significant

increase in the number of cells with structural aberra-tions compared to control. No increases in polyploidywere observed in the presence or absence of S9. Ourresults are in contrast to published results with pred-

156 R.S. Doppalapudi et al. / Mutation R

Fig. 1. Induction of chromosomal aberrations in CHO cells treatedwith geraniol.

nisolone farnesylate which revealed a slight increase inthe incidence of structural chromosomal aberrations inCHO cells at the 1500 �g/ml dose level [39].

In the mouse micronucleus assay, farnesol was testedat 500, 1000, and 2000 mg/kg. No statistically signifi-cant increases in the frequency of micronucleated PCEwere seen at any dose level. The Cochran-Armitage trendtest did not show a statistically significant dose-relatedincrease in the frequency of micronuclei in either maleor female mice treated with farnesol. Our results arein agreement with published data which also revealedthat there were no significant increases in micronucle-ated PCE in mice following exposure to prednisolonefarnesylate [39].

3.5. Geraniol

The microbial mutagenicity assay was conducted atdose levels of 9.76–12.5 �g/plate in the absence of S9and 19.5–625 �g/plate in the presence of S9. No statis-tically significant increases in the number of revertantswere observed.

The induction of chromosomal aberrations in CHOcells was evaluated at doses from 39.1 to 156.3 �g/mlwith and without S9 for 3 h exposure, and from 9.8 to39.1 for 21 h exposure without S9. In the absence ofS9, there was no statistically significant increase in thenumber of cells with structural aberrations comparedwith controls. However, in the presence of S9, therewas a significant increase in the number of cells withstructural aberrations at the 78.1 and 156.3 �g/ml doselevels. Fig. 1 shows the total aberrations with and with-out S9. Chromatid exchanges (2–2.5%) were observedat all dose levels. These findings were not reproducible

in the second chromosome aberration experiment at thehighest dose level (156.3 �g/ml). Furthermore, chro-matid exchanges, which are rare events, were observedin treated cultures in both experiments (0.5–2.5%), and

esearch 629 (2007) 148–160

none were observed in controls. The levels were not sta-tistically significant and no dose-response relationshipwas found. No increases in polyploidy were observed inthe presence or absence of S9.

In the micronucleus experiment, geraniol was testedat 375, 750, and 1500 mg/kg. No statistically significantincreases in the frequency of micronucleated PCE wereseen at any dose level in either male or female mice atthe 24 or 48 h time point, and there was no dose-relatedincrease in the frequency of micronuclei. The literaturelacks cytogenetic results on geraniol.

3.6. SeMC

The microbial mutagenicity assay was conductedat doses from 156.2 to 5000 �g/plate in the pres-ence and absence of S9. Cytotoxicity was observed at5000 �g/plate in the presence and absence of S9 for allthe Salmonella tester strains. No significant increase inrevertants was observed. Selenium, in combination withgreen tea, has been reported to have a co-antimutageniceffect in vitro to protect against heterocyclic amine-induced mutagenesis and carcinogenesis [40].

In the chromosome aberration experiments, CHOcells were exposed for 3 h to SeMC at dose levelsof 1250–5000 �g/ml without S9 and 19.5–156.3 �g/mlwith S9, and for 21 h at 19.5–156.3 �g/ml in the absenceof S9. No statistically significant increases comparedwith controls were found in structural chromosomalaberrations in the presence or absence of S9. Noincreases in polyploidy were observed in the presenceor absence of S9.

In the mouse micronucleus assay, animals weretreated with a single administration of 4, 8, or 16 mg/kgSeMC. No statistically significant increase in the fre-quency of micronucleated PCE was seen at any doselevel in either male or female mice at the 24 or 48 h timepoint, and there were no dose-related increases in thefrequency of micronuclei in any dose group except thefemales treated with 16 mg/kg and sacrificed at the 24 htime point. All the frequencies were within the historicalcontrol range.

3.7. DC-D3T

Doses ranging from 125 to 2000 �g/plate based onsolubility were tested for microbial mutagenicity in thepresence and absence of S9. No statistically significant

increase in the number of revertants was observed.

The induction of chromosomal aberrations was eval-uated at 12.5 to 50 �g/ml dose levels with and withoutS9 for 3 h and at 50 to 200 �g/ml for 21 h exposure in

R.S. Doppalapudi et al. / Mutation R

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ig. 2. Induction of chromosomal aberrations in CHO cells treatedith 4′-bromoflavone.

he absence of S9. There was no statistically significantncrease in the number of cells with structural aberra-ions compared to controls in the presence or absencef S9. No increases in polyploidy were observed in theresence or absence of S9.

In the mouse micronucleus experiment, DC-D3T wasested at 250, 500, and 1000 mg/kg. No statistically sig-ificant increase in the frequency of micronucleated PCEas observed at any dose level in either male or femaleice at the 24 or 48 h time point, and there was no

ose-related increase in the frequency of micronuclein any dose group. No genetoxic results on DC-D3T arevailable in the literature.

.8. 4′-Bromoflavone

In the microbial mutagenicity assay, doses rangingrom 23.4 to 750 �g/plate, based on the cytotoxicityesults, were tested in the presence and absence of S9.o increase in the number of revertants was observed.In the chromosomal aberration study, tests at 75,

50, and 300 �g/ml for 3 h in the absence of S9howed a statistically significant increase in structuralberrations. CHO cells exposed for 21 h showed a dose-ependent increase in structural aberrations comparedith controls, but this increase was significant only

t the 300 �g/ml dose level. In contrast, in the pres-nce of S9, there was no significant increase in cellsith structural aberrations at any dose level (37.5, 75,50, or 300 �g/ml) compared with controls (Fig. 2). Noncreases in polyploidy were observed in the presence orbsence of S9.

Mice were exposed to a single administration of′-bromoflavone at dose levels of 250, 500, and

000 mg/kg. No statistically significant increase in therequency of micronucleated PCE was observed at anyose level in either male or female mice at the 24 or 48 hime point. Evaluation by Cochran–Armitage trend test

esearch 629 (2007) 148–160 157

revealed no statistically significant dose-related increasein the frequency of micronuclei in any dose group. Geno-toxic studies on this chemical have not been reported inthe literature.

3.9. Alpha-TEA

The microbial mutagenicity assay was conducted atdoses ranging from 39.1 to 2500 �g/plate, in the presenceand absence of S9. No statistically significant increasein the number of revertants was observed.

CHO cells were exposed to alpha-TEA for 3 h at doselevels of 9.75, to 156 �g/ml in the presence of S9 and2.4–39 �g/ml in the absence of S9. For 21 h exposure,cells were treated with a dose range of 0.3–2.4 �g/ml.No statistically significant increase in the number ofcells with structural aberrations was observed at any doselevel compared with controls. There was no significantincrease in polyploidy.

In the mouse micronucleus experiment, alpha-TEAwas tested at 250, 500, or 1000 mg/kg. There wasno statistically significant increase in the frequency ofmicronucleated PCE at any dose level in either maleor female mice at the 24 or 48 h time point. Evaluationby the Cochran-Armitage trend test revealed no statisti-cally significant dose-related increase in the frequencyof micronuclei in any dose group. Genotoxicity studieson this agent have not been previously reported.

3.10. SR13668

The microbial mutagenicity assay was conductedover doses ranging from 78.1 to 2500 �g/plate, in thepresence and absence of S9. No statistically significantincrease in the number of revertants was observed.

CHO cells were exposed for 3 h at dose levels of19.6, to 312.5 �g/ml in the presence of S9 and at0.6–9.75 �g/ml in the absence of S9. In neither the pres-ence nor the absence of S9 was there a statisticallysignificant increase in the number of cells with structuralaberrations at any dose level compared with controls. Noincreases in polyploidy were observed in the presence orabsence of S9.

Mice were exposed to a single administration ofSR13668 at dose levels of 500, 1000, and 2000 mg/kg,and sacrificed 24 or 48 h later. No statistically significantincrease in the frequency of micronucleated PCE wasseen at any dose level in either male or female mice at the

24 or 48 h time point, with one exception: in male micegiven 1000 mg/kg SR13668 and sacrificed at 48 h, therewas a significant increase in micronucleus frequency(0.24%) compared with controls (0.12%). However,

158 R.S. Doppalapudi et al. / Mutation R

Fig. 3. Induction of chromosomal aberrations in CHO cells treatedwith SR16157.

this increase was within two standard deviations of themean historical control value (mean + 2 S.D. = 0.48%),evaluation by Cochran-Armitage trend test revealed nostatistically significant dose-related increase in the fre-quency of micronuclei, and an increase was not observedat the higher dose level (2000 mg/kg). Based on theseobservations, the micronucleus response was classifiedas negative for SR13668. Cytogenetic studies with thisdrug have not been previously reported.

3.11. SR16157

Based on cytotoxicity, a microbial mutagenicity assaywas conducted over doses of 2.44–56.3 �g/plate in thepresence and absence of S9. No statistically significantincrease in the number of revertants was observed.

In the chromosomal aberration experiment, CHOcells were exposed at dose levels of 2.4–9.75 �g/ml for3 h and at 1.2–4.9 �g/ml for 21 h in the absence of S9.No increase in chromosomal aberrations was observedat any dose level. In the presence of S9, exposure for 3 hto 19.5, 39, and 78 �g/ml resulted in statistically signif-icant increase at the 78 �g/ml dose level. The results arepresented in Fig. 3. There was no dose-related increasein polyploidy in the presence or absence of S9.

In the mouse micronucleus experiment, 500, 1000,1500, and 2000 mg/kg dose levels were tested. Therewas no statistically significant increase in the frequencyof micronucleated PCE at any dose level in eithermale or female mice at the 24 or 48 h time pointwith one exception: in male mice given 2000 mg/kgSR16157 and sacrificed at 24 h, there was a sig-nificant increase in micronucleus frequency (0.23%)compared with controls (0.12%). However, this increasewas within 2 S.D. of the mean historical control

value (mean + 2 S.D. = 0.40%). Also, evaluation by theCochran-Armitage trend test revealed no statisticallysignificant dose-related increase in the frequency ofmicronuclei in any dose group; therefore, SR16157 is

esearch 629 (2007) 148–160

considered to be negative in the bone marrow micronu-cleus assay.

4. Conclusions

I3C and BTP induced a weak mutagenic responsein the Salmonella mutagenesis assay; BTP was alsomutagenic in the mouse lymphoma assay. SR16157 and4′-bromoflavone elicited positive responses in the CHOchromosome aberration assay while results with geran-iol were considered inconclusive. All of the responsesin these in vitro assays were relatively weak, generallyoccurred only at the highest concentration which wasnear the limit of cytotoxicity, and were not always repro-ducible between experiments or between cultures. TheCHO chromosome aberration test has a significant rate offalse positive results with some test chemicals that appar-ently do not react with DNA [41]. It has been reportedthat there are many non-DNA targets with which acompound may interact, that could indirectly result inpositive responses in this assay [42,43].

In contrast, none of the candidate chemopreventiveagents induced micronuclei in the mouse bone mar-row assay which suggest that the in vivo risk of thesecompounds to humans is relatively small. This micron-culeus assay satisfies the requisite for in vivo genetictoxicology testing because the bone marrow is a well-perfused tissue, and levels of drug-related materials inbone marrow are often similar to those observed in bloodor plasma. Pharmacokinetics data are available on all ofthe compounds reported here, and these compounds, allbeing developed as oral chemopreventive agents, haveadequate bioavailability that should result in reasonablelevels in the bone marrow. Since the in vivo micronu-cleus assay was negative in mice at high dose levelsof geraniol, 4′-bromoflavone, and SR16157, the incon-clusive and positive results in the CHO chromosomalaberration assay may not be biologically relevant. Also,the positive results of these test agents in vitro occurred atconcentrations thousands of times higher than the plasmalevels expected in human clinical use.

In conclusion, we have used a battery of in vitrogenotoxicity tests to assess the potential mutagenic riskof candidate chemopreventive agents to human popula-tions in clinical trials. While weak positive results wereobserved for a few compounds in in vitro assays, theconsistent negative results observed in the more rele-vant in vivo micronucleus assay, even at extremely high

doses (typically over 1000 mg/kg) suggests that thesecompounds are of minimal genotoxic risk to patientsundergoing clinical trials. Additional mechanistic stud-ies to better characterize the in vitro positive results for

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3C, BTP, SR16157, and 4′-bromoflavone may be usefuln understanding the results reported here.

cknowledgement

This work was supported by National Cancer Insti-ute, National Institutes of Health, under contracts01-CN-15010 (WS #60) and N01-CN-95033 (WS49).

ppendix A. Supplementary data

Supplementary data associated with this article can beound, in the online version, at doi:10.1016/j.mrgentox.007.02.004.

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