1990;50:2068-2074.Cancer Res Vernon E. Steele, Gary J. Kelloff, Betty P. Wilkinson, et al. Cells by Potential Chemopreventive AgentsInhibition of Transformation in Cultured Rat Tracheal Epithelial  

  

Updated Version http://cancerres.aacrjournals.org/content/50/7/2068

Access the most recent version of this article at:

  

Citing Articles http://cancerres.aacrjournals.org/content/50/7/2068#related-urls

This article has been cited by 16 HighWire-hosted articles. Access the articles at:

  

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

SubscriptionsReprints and

.pubs@aacr.orgDepartment atTo order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions.permissions@aacr.orgDepartment at

To request permission to re-use all or part of this article, contact the AACR Publications

American Association for Cancer Research Copyright © 1990 on October 1, 2012cancerres.aacrjournals.orgDownloaded from

[CANCER RESEARCH 50. 2068-2074. April 1. 1990|

Inhibition of Transformation in Cultured Rat TrachéalEpithelial Cells byPotential Chemopreventive Agents1

Vernon E. Steele, Gary J. Kelloff, Betty P. Wilkinson, and Julia T. Arnold2

Environmental Sciences Division. .\Sl Technology Services Corporation, Research Triangle Park, ,\orth Carolina 27709 (V. E. S., B. P. W., J. T. A.], and Division ofCancer Prevention and Control, \ational Cancer Institute, Betnesda, Maryland 20892 fG. J. K¡

ABSTRACr

Twenty-eight compounds »erescreened for chtmopreventive activityby using a rat trachea! epithelial cell transformation inhibition assay. Inthis ne»assay, chemicals »eretested for their ability to inhibit theformation of transformed rat trachea! epithelial cell colonies which arisefollowing exposure to the carcinogen benzo(a)pyrene. "I'he 15 positive

compounds were /V-acetylcysteine, bismuththiol. calcium glucarate, (±)catechin, diallyl disulfide, glycaric acid, i>-glucaro-l,4-lactone. \-(4-hydro\yphenyl)retinamide, n-limonene, mesna, retinole acid, rutin, quer-cetin, silymarin, and taurine. In examining the nature of compounds thatinhibited rat trachea! epithelial cell transformation, several possiblechemoprevcntivc mechanisms appeared to be predominant: compoundsthat »erepositive (a) increased glutathione levels or enhanced conjugation; (b) increased cytochrome P-450 activity; (c) displayed nucleophilicactivity; or (d) induced differentiation.

Thirteen compounds were negative in the rat trachea! epithelial transformation inhibition assay: crocetin. difluoromethylornithine, ellagic acid,esculetin, enoxalone. ibuprofen, levamisole. nordihydroguaiaretic acid, i -2-oxothia*olidine-4-carboxyIate, piroxicam, sodium butyrate, i>-.. n>-copherol acetate, and polyethylene glycol 400. It »asevident from theseresults that this assay would not detect compounds that »ere(a) anti-promoting in nature; (b) glutathione inhibitors; (c) differentiation inhibitors; (d) O6-methylguanine inhibitors; (<')organ specific; or (/) inactive.

The rat trachea! epithelial cell transformation inhibition assay appeared to identify chemopreventive compounds that act at early stages ofthe carcinogenic process.

INTRODUCTION

A significant amount of research data has been collectedsuggesting that a number of chemical compounds may have theability to inhibit, retard, or reverse one or more stages ofcarcinogenesis (for review see Refs. 1 and 2). Since cancerusually evolves in a prolonged, multistage manner, agents thatinhibit or retard one or more of these stages could affect theoverall cancer incidence. Antineoplastic agents such as antiox-idants, protease inhibitors, mixed function oxidase inhibitors,prostaglandin synthesis inhibitors, tumor growth factor inhibitors, and antiinflammatory agents could specifically inhibit oneor more phases of cancer induction.

Although whole animal test systems can evaluate a limitednumber of compounds for chemopreventive activity each year,short-term screens (usually in vitro systems) offer the ability totest many more compounds per year at a much lower cost. Thisscreening process provides data to (a) help select and prioritizepotential chemopreventive chemicals for whole animal tests; (b)accelerate the rate of chemical evaluation; and (c provide dataon the possible mechanisms of action.

While cancer risk continues to increase, as in the case of lungcancer in the United States and elsewhere, compounds most

Received 6/21/89: revised 11/22/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

' Supported by the Division of Cancer Prevention and Control. NationalCancer Institute, under Contract N01-CN55503-03.

2To whom requests for reprints should be addressed, at NSI TechnologyServices Corporation. P. O. Box 12313. Research Triangle Park, NC 27709.

likely to combat it are those that suppress the evolution of theneoplastic process. The most extensively studied suppressingagents are the retinoids (3-6). Research on the inhibition ofrespiratory tract cancer in rats and hamsters by high doses ofretinoids has not been encouraging (7-10); however, Saffiottiet al. (11) did inhibit hamster respiratory tract tumors withvitamin A. Recently, several investigators were able to inhibitmorphological transformation of carcinogen-treated RTE1 cellswith retinoid exposure (12, 13).4

In recent studies, we have evaluated the tumorigenicity ofvarious types of morphologically transformed RTE cell coloniesderived from carcinogen-exposed RTE cells (14). An assay todetect chemopreventive agents was developed with this system(15), and in this report we evaluate the responsiveness of thissystem to agents with various proposed chemopreventive mechanisms.

MATERIALS AND METHODS

Chemicals. Bismuththiol. catechin, enoxalone (if-glycyrrhetinic acid),nordihydroguaiaretic acid, mesna (2-mercaptoethanesulfonic acid), silymarin group, and taurine were purchased from Aldrich Chemical Co.(Milwaukee, WI). JV-Acetylcysteine, benzo(a)pyrene, calcium glucarate,crocetin, ellagic acid, esculetin, glucaric acid, n-glucaro-1,4-lactone,ibuprofen, levamisole, d-limonene, 2-oxo-4-thiazolidine-4-carboxylicacid, piroxicam, polyethylene glycol 400, quercetin, retinoic acid, rutin,and sodium butyrate were purchased from Sigma Chemical Co. (St.Louis, MO). Diallyl disulfide was purchased from Crescent ChemicalCo. (Hauppauge, NY). Difluoromethyl ornithine was obtained fromMerrell Dow Pharmaceuticals, Inc. (Cincinnati, OH). The agent iV-(4-hydroxyphenyl)retinamide was received as a gift from McNeil Pharmaceutical Co. (Spring House, PA). n-n-Tocopherol acetate was purchased from Henkel Chemical Co. (Minneapolis, MN).

Compound Solubility. Each chemical was tested initially for its solubility in RTE cell culture media. If the compound was insoluble orslightly soluble in RTE cell culture media, then DMSO was used as asolubilizer. Once solubilized in DMSO, the compound was added toculture medium such that the concentration of DMSO did not exceed0.2%.

TrachéalCell Isolation. To isolate RTE cells, tracheas were asepti-cally excised from 8- to 12-week-old rats, cannulated with sterilepolyethylene tubing, and rinsed with Joklik's modified minimal essen

tial medium. Details of this method have been published previously(16, 17). The tracheas then were filled with a Pronase solution (typeXIV, Sigma) and incubated overnight in a refrigerator (4 ±3°C).Thefollowing day, the liuin.-n.ilepithelial cells were rinsed from the tracheas

with DM EM plus 10% fetal bovine serum. The cells were filtered byusing a 28-jim pore size Nitex filter to remove clumps, and then werecentrifuged, resuspended in DMEM plus 10% FBS, and counted todetermine cell concentration. Cell viability was determined by trypanblue exclusion. For all tests 20,000 cells were plated into RTE culturemedium consisting of Hani's F-12 (GIBCO) mixed 1:1 with 3T3-

-1The abbreviations used are: RTE, rat trachéal epithelial; B(a)P,benzo(o)pyrene; DMSO. dimethyl sulfoxide: RA, all-frans-retinoic acid; CFC,colony-forming cells: DMEM, Dulbecco's modified Eagle's medium; DMBA,7.12-dimethvlbenz(a)anthracene: DFMO. difluoromethyl ornithine; 4-HPR, N-(4-hydroxyphcnyl)retinamide; FBS. fetal bovine serum.

4 M. J. Mass. Retinoid benzoic acids (arotenoids) and other retinoids inhibit

in vitro transformation of rat trachéalepithelial cells, manuscript in preparation.

2068

American Association for Cancer Research Copyright © 1990 on October 1, 2012cancerres.aacrjournals.orgDownloaded from

TRANSFORMATION INHIBITION IN RATTRACHEAL CELLS

conditioned DMEM containing 2% FBS plus the following growthfactors: 10 ßg/m\insulin (Sigma), 1 ><Mhydrocortisone (Sigma), 5 ^g/ml transferrin (Sigma), and 0.2% bovine hypothalamus extract (18).The 3T3-conditioned medium was prepared by incubating DMEM plus2% FBS with mouse 3T3 cells for the last 3 days of log phase growth.The culture dishes were coated with a film of 600 //g of collagen(Vitrogen 100. Collagen Corp., Palo Alto, CA) per dish. At Day 14 theculture medium was changed to a modified RTE medium containing a3:1 mix of F-12 and 3T3-conditioned medium.

Cytotoxicity Testing. To interpret any reductions in transformationfrequencies as true inhibition of the transformation process and notmerely a reduction due to overt cellular cytotoxicity, we conducted botha range-finding cytotoxicity test and another cytotoxicity test at thetime of the actual transformation inhibition assay. In the range-findingcytotoxicity test, 20,000 viable cells/60-mm dish were exposed to thehighest soluble concentration in medium (up to 1 mg/ml), plus 4 logdilutions of that concentration. A concentration of chemopreventivetest agent was chosen from these data such that the colony-formingefficiency was not reduced below 80% that of control levels (i.e., a 20%reduction compared to controls). That concentration plus 4 half-logdilutions were used in the transformation inhibition assay. The resultsof the second cytotoxicity assay were used to determine the number ofCFCs per dish. The number of CFCs was used to determine thetransformation frequency.

Assay for Transformation Inhibition. Ten experimental groups wereused for each assay: medium control, solvent control, B(a)P alone,B(a)P plus RA (positive control), RA alone, and B(a)P plus chemopreventive agent (at five concentrations).

Twenty-four 60-mm dishes/group were plated with 20,000 cells eachat the beginning of each experiment. Appropriate cultures were exposedto 10 ng of B(a)P/ml on Day 1 for 24 h. The chemical was removed,and the cultures were rinsed once with Joklik's minimum essential

medium and rehydrated with RTE culture medium. All cultures weremaintained in a 5% CO: in air incubator at 37°C.Fresh chemopreven

tive agent (or 30 ng/ml RA) was added with each biweekly mediumchange thereafter. Three of these dishes from each group were stainedfor cytotoxicity determinations on approximately Day 5 and the remainder were cultured until Day 30. At Day 30, the remaining cultureswere fixed, stained with méthylèneblue, and scored for transformedcolonies. Previous studies (15) have shown that all Class III transformedcolonies or foci with greater than 2500 cells/mm2 progress to form

tumors when injected into nude mice. A very high percentage of ClassII foci (>1300-2500 cells/mm2) also became tumorigenic. Class I foci(<1300 cells/mm2) usually do not progress to tumorigenicity: therefore,

only Class II and III foci were considered as evidence of transformationin this assay.

Analysis of Data. The number of foci per dish was calculated byadding all of the Class II and III foci in each dish and dividing by thenumber of scored dishes. The transformation frequency was calculatedby dividing the foci per dish by the number of CFC per dish. Thisprocedure corrects for any cytotoxicity of the chemopreventive agent.This transformation frequency was corrected for background by subtracting the DMSO control transformation frequency. Next, the percentage of B(a)P transformation was calculated by dividing each transformation frequency by that seen in B(a)P-only-treated cultures. If theinhibitory activity at all nontoxic doses was less than 20%, a negativesign is shown in the Efficacy column (Table 1). If the inhibitory activitywas greater than 20%, then the result is positive. The inhibition resultswere questionable in instances where the test concentration was cyto-toxic (<80% control colony-forming efficiency).

All culture materials and actual test cultures were routinely screenedfor bacteria, fungus, yeast, and Mycoplasma. If contamination wasfound, the media, media components, or cultures were not used in theexperiments.

RESULTS AND DISCUSSION

The 28 chemopreventive agents tested are listed in Table 1in alphabetical order. Many of these compounds were chosenbecause of the availability of whole animal data. The concentra

tion that induced the maximum transformation inhibition isshown in the second column. If transformation was not inhibited then the highest concentration tested is shown. The concentrations chosen for testing were from nontoxic to marginallytoxic concentrations. The percentage of B(a)P transformationshown in the final column of numbers shows the inhibitoryability of the agent. Negative numbers results when transformation frequencies were below background. The most effectivecompound to inhibit RTE transformation was RA. Concentrations of RA as low as 0.33 MM inhibited the formation oftransformed foci. This is in agreement with previously published data (12). Silymarin, bismuththiol, and 4HPR also wereeffective at low concentrations. Fig. 1 shows typical concentration response curves for o-«-tocopherol acetate, quercetin, calcium glucarate, and 4HPR. All results shown in Table 1 arefrom tests with valid positive and negative controls. A widevariation in control values was observed between tests. Suchvariability is common in primary epithelial cell culture systems.Current efforts seek to reduce background variation. The compounds that were tested fall into several groups that possiblyoperate by common mechanisms.

The first group are compounds that enhance biochemicaldefense mechanisms against DNA damage. A/-Acetylcysteine isa precursor of intracellular glutathione and is often administered to respiratory patients who have previously inhaled toxicagents (i.e., cigarette smoke or occupational toxins). Such exposures are expected to deplete intracellular glutathione inresponse to the insult. Previous studies have shown that N-acetylcysteine induced a significant increase in oxidized glutathione in rat liver preparations and a reduction in mutagenicityby carcinogens (19). In this same study, /V-acetylcysteine markedly inhibited urethane-induced lung tumor formation. Themechanism is thought to be that /V-acetylcysteine increases theintracellular levels of glutathione that bind the electrophilicmetabolites before they can interact with the DNA. Otherstudies have shown that A/-acetylcysteine is a positive modifierof A/-methyI-/V'-nitro-/V-nitrosoguanidine-induced mutagenic

ity in hamster V79 cells (20). In the RTE cell studies reportedhere, AAacetylcysteine was tested at concentrations rangingfrom 61.3 to 6130 fiM (Table 1). A slight, but not significant,cytotoxicity was noted at the highest dose (6130 ¿/M).Thelowest three concentrations all induced higher transformationfrequencies than B(a)P alone. A greater than 98% inhibition oftransformation was seen at 1839 ¿IM,and a similar inhibitionoccurred at 6130 ^M; the agent, therefore, was consideredpositive.

L-2-OxothiazoIidine-4-carboxylate is a stable derivative ofcysteine and can increase glutathione levels in rat liver, butdecrease levels in kidney (21). Increases in glutathione levelshave been found to protect cells against many xenotoxins. whiledecreases could enhance the carcinogenic process. L-2-Oxothia-zolidine-4-carboxylate, however, was negative in this assay. Infact, all five concentrations tested seemed to enhance the transformation frequency of B(a)P-treated RTE cells. It may be thatin RTE cells, this compound decreases glutathione levels similar to the activity seen in the kidney.

Bismuththiol is a thiazole with two sulfhydryls, and thereforepossibly acts to bind electrophilic metabolites (22). Bismuththiol was positive at all but one of the concentrations tested.The concentration range tested was 0.67 to 67 ^M, and nocytotoxicity was apparent over this range. Thus, thiazole couldact as a chemopreventive agent by binding the electrophilicintermediates of B(a)P.

Taurine is a component of bile secretions and conjugates bile2069

American Association for Cancer Research Copyright © 1990 on October 1, 2012cancerres.aacrjournals.orgDownloaded from

TRANSFORMATION INHIBITION IN RATTRACHEALCELLS

Table l In vitro screening of chemoprerentire agents with the rat trachéalepithelial transformation inhibition assay

CompoundA'-AcetylcysteineBismuththiolCalcium

glucarateCatechinCrocetinDiallvl

disulfideDFMOEllagic

acidEnoxaloneEsculetinGlucaric

acidD-Glucarolactone4HPRIbuprofenLevamisoleD-LimoneneMesnaN

DCA''2-OxothiazolidinePiroxicamPEG

400QuercetinRetinoic

acidRutinSilymarinSodium

butyrateTaurineD-n-Tocopherol„Foci/dishCFC/dish4(C;

agent + B(a)PConcentration(ilM)1839.06.73440.011.39.2204.04.21.06.45.64760.01428.00.26145.5415.021.9609.03.32039.0301.875.010.00.33451.06.2272.479.9630.0transformation)No.

ofdishes1920IX202015161520161621191421172021171692021191821IX16CFC/dish192177199IOS5586965160306203169243283165116249335589115184103533349193207160245182%ofcontrolCFC9811413470866261901721159914313788951001119194978082106101831088694Foci/dish±SD0.26

±0.441.2±5.217.7±9.50.2

±0.477.8±7.84.4±3.632.1

±6.954.3+4.854.3+9.268.2±4.317.3

±4.857.8±4.12.2

+2.849.6±3.840.9±8.70.82±7.80.05±0.262.5±5.149.9±5.330.1

±5.231.3±6.43.4±3.12.7

±2.618.9±8.07.7+4.947.8±4.60.56+0.749.2±5.3%

Agent +B(o)Ptransformation±SD°0.14

±0.20.68±0.38.88±4.80.19

±0.414.0±1.86.38±5.249.5±10.833.9±5.817.8+3.133.7+5.310.2±3.023.8

±2.50.78±1.030.0±3.135.2±7.70.33±0.30.01

5±0.110.6±1.343.5+6.416.3

±3.130.3+13.70.64

±0.60.77±0.79.80±4.33.72±2.429.9±3.60.23±0.327.0±7.2%

B(a)Ptransformation±SD2.83

±3.314.8±3.926.3+4.014.8

±3.98.38±0.818.4±5.745.7

±8.336.9±4.117.3

+6.536.9±4.136.9±4.165.1

±5.817.3+6.530.3

±4.125.8+2.97.03±2.60.07

±0.18.38±0.825.8±2.914.2

±7.118.4±5.78.38

±0.810.8+6.014.2+7.17.03±2.626.4±4.00.69

±0.130.3±4.1%

solventtransformation±SD0.11

±0.21.70±1.310.1±1.91.70±1.31.64±1.44.10±1.79.44±3.53.00±1.43.66±2.13.00+

1.43.00±1.41.92

±0.93.66±2.18.58±2.410.8±5.60.08±0.20.00±0.01.64±1.410.8±5.69.30±2.65.58±2.21.64+

1.40.70±0.89.30±2.60.06

±0.110.0±1.90.019

±0.18.58±2.4Corrected

%ofB(a)Ptransfor

mation*1.10-7.79-7.54-11.54182.7815.98110.3691.15103.5990.5621.2434.63-21.1098.62162.073.6021.43132.94217.11143.44193.28-14.840.6910.2552.51122.2531.4584.81Efficacy'++-I-+_4---——++-f-—++——__++++—+--

'", solvent transformation

% B(a)P transformation —¿�ri solvent transformation

' The efficacy was scored positive if the percentage of B(a)P transformation was less than 80%.* NDGA. nordihydroguaiaretic acid; PEG 400, polyethylene glycol 400; Mesna. 2-mercaptoethane sulfonic acid.

Fig. 1. RTE cell transformation inhibitionassay concentration response curves for (A) D-«-tocopherol acetate; (B) quercetin; (C) calcium glucarate; and (£>) iY-(4-hydroxy-phenyDretinamide.

Concentrano,, (mM)

63 0

120

0.33 099 3.3 9.9 33.010"' x Concentration (mM )

10 34

Concentration (mM)

103 077 26 0.77 2610J x Concentration (mM)

7.6«

acid. In a recent study, tauromustine, a taurine-based nitrosou-rea, has shown antitumor activity against Lewis lung, colon,and other carcinomas (23). Taurine was positive at all concentrations tested. There was no apparent cytotoxicity over theconcentration range tested. The possible mechanism for thispositive result could be that taurine detoxifies B(a)P by aconjugation process.

Calcium glucarate has been shown to reduce the frequency

of DMBA-induced rat mammary tumors, possibly through amechanism of increasing the clearance of carcinogens as glu-curonide conjugates (24). In that study, calcium glucarate actedsynergistically with 4HPR to suppress tumor formation. Calcium glucarate also reduced the increase of oncofetal protein inrodents fed carcinogens that can undergo glucuronidation (25).In the RTE cell transformation inhibition assay, calcium glucarate was positive and effectively inhibited B(a)P-induced

2070

American Association for Cancer Research Copyright © 1990 on October 1, 2012cancerres.aacrjournals.orgDownloaded from

TRANSFORMATION INHIBITION IN RATTRACHEAL CELLS

transformation of respiratory epithelial cells. This result agreeswith the inhibition of tumorigenesis of in vivo studies citedabove.

Glucaric acid is the end product of the glucuronidation pathway and is a metabolite produced by inducible enzyme systems(26). Because of its nature, one might expect it also to inducethe conjugation of xenobiotics such as B(a)P and to detoxifythem. In the RTE cell assay, glucaric acid inhibited the transformation of these respiratory epithelial cells following exposure to B(fl)P at the two highest doses.

D-Glucaro-l,4-lactone is an anticarcinogen which may act toclear reactive xenobiotics through the glucuronide pathway(27). D-Glucaro-l,4-lactone was positive at all concentrationsin the RTE assay. While the effect was not totally inhibitory, itdid reduce transformation frequencies by 24 to 35%, comparedto B(a)P alone. These results agree with its proposed activity.

D-Limonene is isolated from various ethereal oils, particularly from peels of lemons and oranges. Limonene, like manyterpenoids, induces carcinogen-metabolizing enzymes, especially the P-450IIB subfamily (27). Because of this induction,limonene was effective in reducing the frequency of DMBA-induced rat mammary tumors (28). In a recent report, limoneneinhibited forestomach tumors and reduced pulmonary adenomas in mice treated with /V-nitrosodiethylamine (29). Limoneneeffectively inhibited the transformation of RTE cells by B(a)Pand is therefore positive. The percentage of inhibition rangedfrom 65 to 96%. The mechanism of reduction of B(a)P-inducedtransformation of limonene in RTE cells may be its ability tostimulate xenobiotic metabolizing enzymes and inactivateB(a)P.

Diallyl disiiliuli\ a component of garlic oil, has been shownto inhibit 1,2-dimethylhydrazine-induced colon and liver cancer(30, 31), /V-nitrosodiethyamine-induced forestomach cancer(29), and /V-nitrosomethylbenzylamine-induced esophagealcancer (32). Brady et al. (33) showed that diallyl disulfideinhibited the enzyme P-450IIE1, a carcinogen-activating enzyme. In the RTE cell assay, diallyl disulfide was positive forinhibiting B(a)P-induced transformation at all but one concentration tested. If diallyl disulfide did inhibit cytochrome P-450enzyme activity in the RTE cells, the amount of activated B(a)Pwould be reduced and a lower transformation rate would result.

A second common mechanism suggested by the results is thatperoxidase inhibitors of free radical scavengers inhibit RTEtransformation in this assay. Catechin or cyanidanol is knownto inhibit lipid peroxidation by cisplatin possibly by acting as afree radical scavenger (34). Catechin was positive in the RTEcell assay. Catechin was positive at 3.4 /IM and reached 100%inhibition at 10.2 UM.No cytotoxicity was apparent in the doserange tested. As a free radical scavenger or antioxidant, catechinmight be expected to reduce the transformation frequency.

Mesna is a mercaptoethylsulfonate-related compound thathas been effective in reducing cyclophosphamide-induced urinary bladder carcinogenesis in rats (35). Its properties as ananticarcinogen may be related to its antioxidant-nucleophiliccharacteristics. Mesna also has been reported to enhance thelevel of intracellular glutathione in murine lymphocyte cells(36). Mesna effectively inhibited transformation at all concentrations and was positive in this assay. No evidence of cytotoxicity was present at any concentration. Mesna may have inhibited B(a)P-induced transformation in RTE cells by trappingelectrophilic reactive metabolites of B(a)P.

Rutin is a flavonoid found in many plants such as buckwheat,tobacco, forsythia, and pansies. Rutin has been investigated asa scavenger of Superoxide anions (37). Rutin was positive only

at the highest concentration tested, 451 /¿M.Transformationwas reduced by almost 90% in the absence of cytotoxicity.Rutin is similar to other antioxidants that have been mostlypositive in the RTE cell assay, and also may have nucleophilicproperties.

Quercetin, like rutin, is a flavonoid and is found in barks andrinds such as rhododendron, clover blossoms, and ragweed.Quercetin has been shown to be a peroxidase inhibitor of humancells (38). Quercetin was positive at the three highest concentrations (3.3, 10, and 33 UM). No cytotoxicity was apparentover the concentration range tested. The mechanism of actionof this flavonoid may be similar to that of rutin.

Silymarin is a group of three isomers (silybin, silidianin, andsilicristin) isolated from the fruit of Silybum marianum. Silymarin is a flavonolignan that has antihepatotoxic activity (39)and induces mitochondria! calcium release (40). Silymarin waspositive over the entire concentration range tested in the RTEcell assay. The two highest concentrations were cytotoxic whentested during the actual assay; however, the three lower non-

toxic concentrations inhibited transformation by 38 to 47%.This flavonolignan may act by a mechanism(s) similar to thatof other flavonoids.

D-a-Tocopherol acetate (vitamin E) acts as a free radicalscavenger and antioxidant by protecting the polyunsaturatedfatty acid components on membranes from peroxidation (41).Vitamin E has been reported to act as a chemopreventive agentin mice (42) and for gastric cancer in rats (43). In humanstudies, fecal mutagens are reduced in patients given vitaminsC and E (44). However, D-«-tocopherol acetate was negative inthe RTE cell assay at all nontoxic concentrations tested. Thisnegative result disagrees with that of most other free radicalscavengers, possibly because it may not have been completelysolubilized in the culture medium and therefore not availableto the cells.

The RTE cell assay gave mixed results for compounds whichact as differentiation inhibitors or inducers. The agent 4HPR,a vitamin A analogue that was received as a gift from McNeillPharmaceutical Co., has been shown to suppress mammarytumor induction in DMBA-treated rats (24). The mechanismof action is probably similar to that of RA in that terminaldifferentiation of the epithelial cells is induced and thereforefew initiated cells survive to form tumors. 4HPR was positivefor chemopreventive activity at the two highest concentrations,0.26 and 0.8 /IM. No cytotoxicity was present in the testconcentration range of 0.008 to 0.8 ¿IM.4HPR was perhaps themost interesting compound that inhibited transformation. Atthe higher concentrations, we observed a shift from the typicalClass II and III foci (progenitors of malignant cells) to thebenign Class I foci. This observation was not seen for other testagents. This compound also was the most powerful inhibitor oftransformation (100% inhibition at 0.26 ^M). The results of thepresent studies agree with the in vivo results and show thatvitamin A-like effects (possibly differentiation inducing) by4HPR can inhibit can inhibit early stages of the RTE transformation process.

The positive control used for all of the compounds studiedwas RA. Our studies and previous studies using a similar system(12, 13) have shown that RA is an effective inhibitor of RTEtransformation induced either by B(a)P or /V-methyl-/V'-nitro-

./V-nitrosoguanidine. Range-finding cytotoxicity studies haveshown that RA can reduce reproductive survival of RTE cellsat concentrations of 1 pM or greater (data not shown). In theRTE assay system described here, RA reduce the transformation by 100% at concentrations of 0.1 ¿IMor greater.

2071

American Association for Cancer Research Copyright © 1990 on October 1, 2012cancerres.aacrjournals.orgDownloaded from

TRANSFORMATION INHIBITION IN RATTRACHEAL CELLS

¿odium butyrate is the salt of a fatty acid that inducesalterations in growth, morphology, and gene expression similarto that seen in cellular differentiation (45). These effects mayresult from histone hyperacetylation and structural chromatinalterations (46). Sodium butyrate has been used in two clinicalstudies: one, a partial remission of acute myelogenous leukemia(47), and a second where no response was seen in nine patientswith acute leukemia (48). Sodium butyrate was negative in theRTE transformation inhibition assay at all concentrations.Agents that induce differentiation in hemopoietic cells mayhave no effect on trachea! epithelial cells.

Esculetin is an inhibitor of the lipoxygenase pathway and hasbeen shown to inhibit the differentiation of the human mono-cyte cell line, U937 (49). In the RTE assay, esculetin wasconsidered negative for inhibiting B(a)P-induced transformation. Although a marginal inhibitory effect was seen at thelowest concentration (0.056 ¿¿M),this effect was not seen athigher nontoxic concentrations. Inhibitors of differentiationmay not be expected to inhibit epithelial cancer, and our resultsreflect this possibility.

Two compounds that increase oxygen diffusion into cells didnot inhibit transformation in RTE cells. Crocetin, a carotenoidcompound, increases oxygen diffusion (50), binds strongly toplasma albumin (51), and has a small effect of retarding thedevelopment of skin tumors on nude mice pretreated withDMBA and croton oil (52). Crocetin was negative in this assay.All five concentrations induced transformation frequencies similar to, or in some cases higher than, that seen with B(a)P alone.The highest concentration (30.5 ^M) appeared cytotoxic in theactual assay, yet induced a transformation frequency comparable to that of B(a)P alone. While these results conflict withits reported anticancer properties, it is possible that crocetinmay act to retard later stages of tumor promotion that wouldnot be evident in this 30-day assay.

Nordihydroguaiaretic acid, like esculetin, is an inhibitor oflipid peroxidation by lipoxygenase, and could have anticarcin-ogenic activity by blocking free radical peroxidation (53). Nordihydroguaiaretic acid did not inhibit transformation at any ofthe concentration tested, and therefore was negative. The transformation frequencies were very similar to that seen with B(a)P-alone exposure. The negative result may suggest that free radical peroxidation does not play an important role in RTE celltransformation as measured by focus formation.

Several compounds were tested that may have activity anti-promoters. DFMO is reported to be a potent irreversible inhibitor of ornithine decarboxylase (54). Also, DFMO has beenshown to inhibit chemically induced and transplantable tumorsin mice and rats (55, 56). DFMO has been shown to modulatethe effects of anticancer drugs in vitro (e.g., Ref. 57), and inPhase II clinical trials is a marginal inhibitor of small cellcarcinoma (58). DFMO was tested at concentrations rangingfrom 4.2 to 420 n\i, but the cytotoxicity was moderate at thehigher doses. The transformation frequencies of groups treatedwith DFMO and B(a)P were comparable to that seen withB(a)P-alone treatment. Since the transformation frequencyclosely paralleled the cytotoxicity, the compound was considered negative. The negative results in the RTE cell assay arenot contradictory with in vivo results, since the mechanism ofthe actions of DFMO are presumably involved in later stagesof tumor promotion.

Enoxalone, or 18-fi-glycyrrhetinic acid, has shown strongantipromoter activity in DMBA-initiated teleocidin-promotedmouse skin tumorigenesis in vivo (59). Enoxalone is a terpenoidderivative and was tested at concentrations ranging from 0.64

to 64 ßMin the RTE assay. It was negative at the nontoxicconcentrations of 0.64, 2.1, and 6.4 ^M. Enoxalone was toxicat concentrations of 21 and 64 ^M, and no transformed fociwere seen. Since inhibitors of tumor promotion do not appearto be effective in this short-term assay, the results may agreewith in vivo data.

Ibuprofen is a nonsteroidal antiinflammatory drug and apropionic acid derivative (60). While this compound does lessenthe activity of meptazinol (an opioid) (61), it probably has littlechemopreventive effect. Ibuprofen was negative at all nontoxicdoses tested in the RTE cell assay. Transformation was reducedto background levels at the highest concentration, but thecytotoxicity was very high. However, the antiinflammatoryproperties of ibuprofen may have antipromotional activity andmay prove active in initiation-promotion tests.

Piroxicam is a nonsteroidal antiinflammatory agent that hasbeen shown to inhibit the growth of innoculated adenocarci-noma cells in rats (62). Piroxicam appeared negative at allnontoxic concentrations. The highest concentration (905.5 /¿M)was considered toxic since it reduced the number of CFC byabout two-thirds. The highest concentration also reduced thetransformation frequency below background levels. Since antiinflammatory agents most likely act by inhibiting the inflammatory activity of tumor promoters, negative results are expected in the RTE cell assay.

One compound that may block the methylation of DNA byalkylating agents is ellagic acid. Ellagic acid is a naturallyoccurring plant phenol that has been shown to reduce methyl-benzylnitrosamine-induced esophageal carcinomas in rats (63).More recent results by Barch and Fox (64) show that ellagicacid selectively reduces the formation of O6-methylguanine inthe DNA of esophageal cells compared to 7-methylguanine.This study suggests that ellagic acid blocks the methylation ofthe O6 position of guanine. Four of five concentrations of ellagic

acid appeared to stimulate transformation of RTE cells byB(a)P. The highest dose, 3.3 //M, did effectively inhibit thetransformation by 95%, but was highly cytotoxic; therefore, theagent was negative. Since the mechanism of B(a)P-inducedtransformation probably does not involve the O6-methylation

process, the negative results agree with the proposed mechanism.

Levamisole is a nonsteroidal antirheumatic drug and is animmune-modulating drug (65). There is little or no evidence oflevamisole being active as an chemopreventive agent. Levamisole did not inhibit B(a)P-induced transformation of RTE cells

at any concentration tested in the assay. The percentage oftransformation was actually higher in all groups compared toB(a)P-alone-treated cells.

Polyethylene glycol 400 is a polymer of low toxicity used asointment of suppository bases. Polyethylene glycol 400 is notreported to have chemopreventive activity, and in the RTE cellassay, was negative at all doses tested.

In summary, the RTE cell transformation inhibition assaywas used to screen 28 compounds for chemopreventive activity.Several possible chemopreventive mechanisms appeared to bepredominant for compounds that were positive. Positive compounds might (a) induce differentiation; (b) increase glutathionelevels or enhance conjugation; (c) display nucleophilic activity;or (d) increase cytochrome P-450 activity. It is also evidentfrom the results that this assay will not detect compounds thatare (a) antipromoters in nature; (b) glutathione level inhibitors;(c) differentiation inhibitors; (d) O'-methylguanine inhibitors

(this compound might be positive if a methylating carcinogenwas used instead of B(a)P; (e) organ specific; or (/) inactive.

2072

American Association for Cancer Research Copyright © 1990 on October 1, 2012cancerres.aacrjournals.orgDownloaded from

TRANSFORMATION INHIBITION IN RATTRACHEAL CELLS

Therefore, the results of the RTE cell transformation inhibitionassay appear to identify those chemopreventive compounds thatact at early stages of the carcinogenic process.

ACKNOWLEDGMENTS

The authors would like to acknowledge Dr. Marc Mass, UnitedStates Environmental Protection Agency, for helpful discussion.

REFERENCES

1. Wattenberg, L. W. Chemoprevention of cancer. Cancer Res.. 45: 1-8, 1985.2. Bertram. J. S.. Kolonel, N. N., and Meyskens, F. L.. Jr. Rationale and

strategies for chemoprevention of cancer in humans. Cancer Res., 47: 3012-3031, 1987.

3. Sporn. M. B., and Newton, D. L. Retinoids and chemoprevention of cancer.In: M. S. Zedeck and M. Lipkin (eds.). Inhibition of Tumor Induction andDevelopment, pp. 71-100. New York: Plenum Publishing Corp.. 1981.

4. Sporn, M. B. Retinoids and suppression of carcinogenesis. Hosp. Pract., 18:83-98, 1983.

5. Sporn, M. B., and Roberts, A. B. The role of retinoids in differentiation andcarcinogenesis. Cancer Res., 43: 3034-3040. 1983.

6. Moon, R. O. and Itri. L. M. Retinoids and cancer. In: M. B. Sporn, A. B.Roberts, and D. S. Goodman (eds.). The Retinoids, pp. 327-371. Orlando,FL: Academic Press, 1984.

7. Smith. D. M., Rodgers, A. E., and Newberne, P. M. Vitamin A andbenzo(a)pyrene carcinogenesis in the respiratory tract of hamsters fed asemisynthetic diet. Cancer Res., 35: 1485-1488, 1975.

8. Smith, D. M., Rodgers, A. E., Herndon, B. J.. and Newborne. P. M. VitaminA uvciml acetate) and benzo(a)pyrene induced respiratory tract carcinogenesis in hamsters fed a commercial diet. Cancer Res., 35: 11-16. 1975.

9. Stinson, S. F.. Resnik. G.. and Donahoe, R. The effect of 3 retinoids ontrachéalcarcinogenesis with A'-methyl-A'-nitrosourea in hamsters. J. Nati.Cancer Inst., 66: 947-951, 1981.

10. Varita. T. P.. Nettesheim. P., and Mitchell. T. J. Failure of two retinoids toinhibit trachea! carcinogenesis in hamsters. Carcinogenesis (Lond.). /: 255-262, 1980.

11. Saffiotti, U., Montesano, R., Sellakumar. A. R., and Borg. S. A. Experimentalcancer of the lung: inhibition by vitamin A of tracheobronchial squamousmetaplasia and squamous cell tumors. Cancer (Phila.). 20: 857-864, 1976.

12. Mass, M. J., Nettesheim, P., Beeman, D. K., and Barrett. J. C. Inhibition oftransformation of rat trachea! epithelial cells by retinole acid. Cancer Res.,«.•5688-5691.1984.

13. Fitzgerald, J. D., Barrett, J. C.. and Nettesheim, P. Changing responsivenessto all rram retinole acid of rat trachea! epithelial cells at different stages ofneoplastic transformation. Carcinogenesis (Lond.), 7: 1715-1721, 1986.

14. Steele, V. E., Arnold. J. T., and Mass. M. 3. In n'roand in vitro characteristics

of early carcinogen-induced premalignant phenotypes in cultured rat trachéalepithelial cells. Carcinogenesis (Lond.). 9: 1121-1127, 1988.

15. Steele, V. E., Wilkinson, B. P., and Arnold. J. T. Inhibition ofbenzo(a)pyrene-induccd malignant transformation in cultured trachea! epithelial cells by retinole acid. In Vitro Cell Dev. Bio!., 24: 14A, 1988.

16. Steele. V. E.. Arnold, J. T.. Van Arnold, J.. and Mass, M. J. Evaluation ofa rat trachéalepithelial cell culture assay system to identify respiratorycarcinogens. Environ. Mol. Mutagen, 14: 48-54, 1989.

17. Steele, V. E., and Mass, M. J. A rat trachéaltransformation system forassessment of environmental agents as carcinogens and promoters. Environ.Int.,//: 323-329. 1985.

18. Maciag. T., Cerundolo. J.. Ilsley, S.. Kelley, P. R.. and Forand. R. Anendothelial cell growth factor from bovine hypothalamus: identification andpartial characterization. Proc. Nati. Acad. Sci. USA, 76: 5674-5678. 1979.

19. De Flora, S., Rossi. G. A., and De Flora, A. Metabolic, demutagenic. andcarcinogenic effects of A'-acelylcysteine. Respiration. 50 (Suppl. 1): 43-49,

1986.20. Romert, L., and Jenssen. D. Mechanism of A'-acetylcysteine (NAC) and other

thiols as both positive and negative modifiers of MNNG-induced mutagen-icity in V79 Chinese hamster cells. Carcinogenesis (Lond.). 8: 1531-1535,1987.

21. Bauman, P. F., Smith, T. K., and Bray, T. M. Effect of dietary proteindeficiency and L-2-oxythiazolidine-4-carboxylate on the diurnal rhythm ofhepatic glutathione in the rat. J. Nutr.. 8: 1049-1054, 1988.

22. Kusterer. K.. and Szabo. S. Gastric mucosal protection by acetazolamidederivatives: role of carbonic anhydrase and sulfhydryls. Eur. J. Pharmacol../<</.•7-13, 1987.

23. Hartley-Asp, B., Christensson, P. I., Gunnarsson. K.. and Gunnersson, P. O.Anti-tumor, lexicological, and pharmacokinetic properties of a novel taurine-based nitrosourea (TCNU). Invest. New Drugs, 6: 19-30, 1988.

24. Abou-lssa, H. M., Duruibe, V. A., Minton, J. P., Larroya, S., el al. Putativemetabolites derived from dietary combinations of calcium glucarate and N-(4-hydroxyphenyl)retinimide act synergistically to inhibit the induction of ratmammary tumors by 7,12-dimethylbenz[a)anthracene. Proc. Nati. Acad. Sci.USA. «5:4181-4184, 1988.

25. Walaszek, Z.. Hanausek-Walaszek. M.. and Webb, T. E. Repression bysustained-release beta-glucuronidase inhibitors of chemical carcinogen-me-

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

dialed induction of a marker oncofetal protein in rodents. J. Toxicol. Environ.Health. 23: 15-27, 1988.Brewster, M. A. Biomarkers of xenobiotic exposures. Ann. Clin. Lab. Sci..18: 306-317, 1988.Austin. C. A.. Shepard, E. A.. Pike. S. F.. Rabin. B. R.. and Phillips. I. R.The effect of terpenoid compounds on cytochrome P-450 levels in rat liver.Biochem. Pharmacol., 37: 2223-2229. 1988.Elson. C. E.. Maltzman. T. H.. Boston, J. L., Tanner, M. A., and Gould, M.N. Anti-carcinogenic activity of D-limonene during the initiation and promotion/progression stages of DMBA-induccd rat mammary carcinogenesis.Carcinogenesis (Lond.). 9: 331-332. 1988.Wattenburg, L. N., Sparnins, V. L., and Barany. G. Inhibition of iV-nitroso-dicthylamine carcinogenesis in mice by naturally occurring organosulfurcompounds and monoterpenes. Cancer Res.. 49: 2689-2692, 1989.Wargovich. M. J. Diallyl sulfide. a flavor component of garlic (A/Humsatirum). inhibits dimethylhydrazine-induced colon cancer. Carcinogenesis(Lond.). A:487-489. 1987.Hayes, M. A.. Rushmore. T. H.. and Goldberg, M. T. Inhibition of hepato-carcinogenic responses to 1,2-dimethylhydrazine by diallyl sulfide. a component of garlic oil. Carcinogenesis (Lond.), 8: 1155-1157. 1987.Wargovich, M. J.. Woods. C.. Eng, V. W. S.. Stevens. L. C., and Gray. K.Chemoprevention of A'-nitrosomethylbenzylamine-induced esophageal cancer in rats by the naturally occurring thioether, diallyl sulfide. Cancer Res..«.•6872-6875,1988.Brady. J. F., Li, D., Ishizaki, H.. and Yang, C. S. Effect of diallyl sulfide onrat liver microsomal nitrosamine metabolism and other monooxygenaseactivities. Cancer Res.. 48: 5937-5940. 1988.Hanneman. J.. and Baumann. K. Cisplatin-induced lipid pcroxidation anddecrease of gluconeogenesis in rat kidney cortex: different effects of antioxi-dants and radical scavengers. Toxicology, 51: 119-132. 1988.Schmahl. D.. and Habs. M. R. Prevention of cyclophosphamide-inducedcarcinogenesis in the urinary bladder of rats by administration of mesna.Cancer Treat. Rev.. 10: 57-61. 1983.I idi-In,. R. K. The generation of oxygen radicals: a positive signal forlymphocyte activation. Cell Immunol.. /5: 175-182. 1988.Robak. J.. and Gryglewski, R. J. Flavonoids are scavengers of Superoxideanions. Biochem. Pharmacol.. 37: 837-841. 1988.Princemail, J.. Deby. C.. Thirion. A., de Bruyn-Dister, M., and Goutier. R.Human myeloperoxidase activity is inhibited in vitro by quercetin. Comparison with three related compounds. Experimentia (Basel). 44:450-453. 1988.Campos. R., Garrido, A., Guerra. R.. and Valenzuela, A. Acetominophenhepatotoxicity in rats is attenuated by silybin dihcmisuccinatc. Prog. Clin.Biol. Res., 280: 375-378. 1988.Chavez, E., and Bravo, C. Silymarin-induced mitochondrial Ca!* release.Life Sci., «.-975-981. 1988.Leung. H., Vang, M. J.. and Mavis, R. D. The cooperative interactionbetween vitamin E and vitamin C in suppression of peroxidation of membranephospholipids. Biochim. Biophys. Acta. 664: 266-272, 1981.Chan, J. T., and Black, H. S. The mitigating effect of dietary antioxidantson chemically-induced carcinogenesis. Experimentia (Basel). 34: 110-111,1977.Weisburger, J. H. Vitamin C. vitamin E, and the prevention of gastric cancer:discussion. Ann. NY Acad. Sci., 335: 278-279, 1980.Dion. P. W.. Bright-See, E. B., Smith, C. C., and Bruce, W. R. The effect ofdietary ascorbic acid and «-tocopherol on fecal mutagenicity. Mutât.Res..102: 27-37, 1983.Kruh, J. Effects of sodium butyrate, a new pharmacological agent, on cellsin culture. Mol. Cell. Biochem.. 42: 65-82. 1982.Chalkey. R.. and Shires, A. The isolation of HTC variant cells which canreplicate in butyrate. Changes in histone acetylation and tyrosine aminotrans-ferase induction. J. Biol. Chem., 260: 7698-7704, 1985.Novogrodsky, A., Ravid. A., Shkolnik. T., Stenzel. K. H.. Rubin, A. L., andZiavov, R. Effect of polar organic compounds on Icukcmic cells: butyrate-induced partial remission of acute myelogeneous leukemia in a child. Cancer(Phila.), 51:9-14, 1983.Miller, A. A., Kurschel, E., Schmidt. C. G.. and Osieka. R. Clinical andpharmacological study of sodium butyrate in patients with acute leukemia.Proc. Am. Assoc. Cancer Res., 26: 1046, 1985.Bomalaski, J. S., Freundlich. B.. Steiner, S., and Clark, M. A. The role offatty acid metabolites in the differentiation of the human monocyte-like cellline U937. J. Leukocyte Biol.. 44: 51-57, 1988.DiLuccia. R. C.. and Gainer, J. L. Increasing alveolar oxygen transport.Aviat. Space Environ. Med., 51: 18-20, 1980.Miller, T. L., Willett, S. L., Moss, M. E., Miller. J.. and Beiinka. B. A., Jr.Binding of crocetin to plasma albumin. J. Pharm. Sci., 71: 173-177. 1982.Matthews-Roth, M. M. Effect of crocetin on experimental skin tumors inhairless mice. Oncology (Basel). 39: 362-364. 1982.Abisogun. A. O.. Daphna-Iken, D.. Reich. R., Kransfelder, D., and Tsafriri,A. Modulatory role of eicosanoids in vascular changes during the preovula-tory period in the rat. Biol. Reprod.. 38: 756-762. 1988.Zhang. S. Z., Luk. G. D., and Hamilton, S. R. n-Difluoromethyl ornithine-induced inhibition of growth of autochthonous experimental colonie tumorsproduced by azoxymcthane in male F344 rats. Cancer Res.. 48: 6498-6503,1988.Kingsnorth, A. N., King, W. W., Diekema, K. A., McCann, P. P., Ross, J.S., and Malt, R. A. Inhibition of ornithine decarboxylase with 2-difluoro-methylornithine: reduced incidence of dimethylhydrazine-induced colon tumors in mice. Cancer Res.. 43: 2545-2549. 1983.

2073

American Association for Cancer Research Copyright © 1990 on October 1, 2012cancerres.aacrjournals.orgDownloaded from

TRANSFORMATION INHIBITION IN RATTRACHEAL CELLS

56. Nigro. N. D., Bull, A. W., and Boyd, M. E. Inhibition of intestinal carcino- 60. Needs, C. J., and Brooks, P. M. Antirheumatic medication during lactation,genesis in rats: effect of difluoromelhylornithine with piroxicam or fish oil. Br. J. Rheumatol., 24: 291-297, 1985.J. Nati. Cancer Inst., 77: 1309-1313, 1986. 61. Stevens. R. J. Evidence for a pharmacokinetic interaction between ibuprofen

57. Sunkara. P. S.. Fowler. S. K., Nishioka, K.. and Rao. P. N. Inhibition of and meptazinol in the mouse. J. Pharm. Pharmacol., 36: 779-781, 1984.polyamine biosynthesis by n-difluoromethylornithine potentiates the cyto- 62. Ross, D. S.. Blitzer, D.. Roy, T., and Murphy. J. E. Piroxicam inhibits thetoxic effects of arabinosylcytosine in HeLa cells. Biochem. Biophys. Res. growth of an adenocarcinoma isograft in Fischer rats. J. Surg. Res.. 45: 249-Commun.. 95: 423-430, Ì980. 253, 1988.

58. Abeloff, M. D., Rosen, S. T.. Luk, G. D., Baylin, S. B.. Zeltzman. M.. and 63. Mandel, S., Shivapurkar, N., Superczynski. M.. and Stoner, G. Inhibition ofSjoerdsma, A. Phase II trials of n-difluoromethylornilhine. an inhibitor of methylbenzylnitrosamine-induced esophageal tumors in rats by ellagic acid,polyamine synthesis in advanced small cell lung and colon cancer. Cancer Proc. Am. Assoc. Cancer Res.. 27: 124, 1986.Treat Rep., 70:834-845. 1986. 64. Barch. D. H.. and Fox. C. C. Selective inhibition of methylbenzylnitrosamine-

59. Nishino. H.. kitagawa. K.. and Iwashima. A. Antitumor-promoting activity induced formation of esophageal 06-methylguanine by dietary ellagic acid inof glycyrrhetic acid in mouse skin tumor formation induced by 7,12-dimeth- rats. Cancer Res.. 48: 7088-7092, 1988.ylbenz[fl(anthracene plus teleocidin. Carcinogenesis (Lond.). 5: 1529-1530. 65. Drews, J. The experimental and clinical use of immune-modulating drugs in1984. the prophylaxis and treatment of infections. Infection. 13: 241-250, 1985.

2074

American Association for Cancer Research Copyright © 1990 on October 1, 2012cancerres.aacrjournals.orgDownloaded from