(CANCER RESEARCH 55, 537-543, February 1. 1995) Evaluation of Chemopreventive Agents in Different Mechanistic Classes Using a Rat TrachéalEpithelialCell Culture Transformation Assay1

Julia T. Arnold,2 Betty P. Wilkinson, Sheela Sharma,3 and Vernon E. Steele

Cellular and Molecular Toxicology Program, ManTech Environmental Technology. Research Triangle Park. North Carolina 27709 ¡J. T. A., B. P. W.. S. S.I. and Chemoprevention Branch, Division of Cancer Prevention and Control, National Cancer Institute, NIH, Bethesda, Maryland 20892 ¡V.E. S.]

ABSTRACT progress to human clinical trials (1). Chemopreventive agents already undergoing clinical trials include retinoic acid, ß-carotene, The rat trachea! epithelial (RTE) cell focus inhibition assay was used to yV-(4-hydroxyphenyl) retinamide, vitamins C and E, piroxicam, cal identify potential Chemopreventive agents. Ninety-nine agents were eval cium, ibuprofen, oltipraz, difluoromethylornithine, 18-ß-glycyrrhet- uated for their ability to inhibit benzo[a]pyrene-induced transformation inic acid and /V-acetyl-L-cysteine (2, 3). of RTE cells. Freshly isolated RTE cells were exposed to benzo[a]pyrene alone or in combination with a Chemopreventive agent. After 30 days in The multistage nature of the process of cancer development in culture, transformed foci were scored and inhibition was quantitated. In cludes perturbations in the normal functioning within cells and the these studies, foci formation was inhibited mainly by agents which mod genome of the organism over a period of many years. It is a cyclical ulate the initiation of carcinogenesis by altering drug-metabolizing en process of DNA damage, proliferation, clonal selection, and progres zymes, inhibiting the binding of benzo[a]pyrene to DNA, enhancing de sion. This process could potentially be modulated by chemicals that toxification of activated carcinogens, or by inducing epithelial cell effect cellular enzyme systems, gene expression, signal transduction differentiation. Such agents include antioxidants, free radical scavengers, glutathione S-transferase enhancers, vitamins, retinoids, and sulfhydryl pathways, differentiation, or interactions with surrounding cells and compounds. Agents which inhibit ornithine decarboxylase and arachi- extracellular matrices. Many chemical compounds may have the abil donic acid metabolism were not as effective. The RTE assay provides ity to inhibit, retard, or reverse one or more stages of carcinogenesis important data for agent selection prior to whole animal-screening assays and thus could affect the overall cancer incidence (4). in the development of chemoprevention drugs. A wide range of compounds has shown the ability to inhibit carcinogenesis in vivo (5). The most extensively studied suppressing agents are the retinoids (6, 7). Saffiotti et al. (8) found inhibition of INTRODUCTION hamster respiratory tract tumors with vitamin A, and Mass el al. (9) Chemoprevention is an important defense strategy against human were able to inhibit the transformation of carcinogen-treated primary cancer since it is highly unlikely for one to avoid all carcinogenic trachéalepithelial cells with retinoid exposure. Other inhibitory agents insults. The objective of chemoprevention is to administer one or occur naturally in allium and cruciferous vegetables (10-12). There is more chemical agents, naturally occurring or synthetic, which may a large variety of chemical classes that can protect against cancer, have multiple biological mechanisms to inhibit various stages of including phenols, Õndoles,aromatic isothiocyanates, methylated fla- carcinogenesis. vones, coumarins, terpenes, dithiolthiones, plant sterols, protease in Target populations for chemoprevention include those who by hibitors, selenium salts, ascorbic acid, tocopherols, and retinol (13). genetic background or previous occupational exposure are at higher These inhibitors could be classified as blockers or suppressors by the risk for developing cancer, those who have had a primary cancer and stage in the carcinogenic process at which they exert their inhibitory seek to reduce recurrence, and the general population with unknown effects: (a) preventing metabolic activation of carcinogen; (b) block risks. Candidate Chemopreventive agents are identified by epidemio- ing reactive metabolites from cellular target sites; or (c) suppressing logical surveys, experimental research findings, clinical observations, promotion or progression of neoplasia. Antineoplastic agents such as or structural homology with known Chemopreventive agents. antioxidants, modifiers of mixed-function oxidases, free radical scav A rigorous and systematic evaluation of the efficiency of these engers, or inducers of glutathione S-transferase could inhibit the natural and synthetic agents is necessary before their usefulness in metabolism and binding of the carcinogen in the initiation phase, cancer prevention can be evaluated in clinical trials. Because of the while anti-inflammatory agents, protease inhibitors, or inhibitors of large number of potential agents, rapid and cost-efficient means of prostaglandin synthesis, ornithine decarboxylase, or protein kinases screening them are needed. In vitro assays such as the rat trachéal could suppress the promotion phase of carcinogenesis. Many of the epithelial cell focus inhibition assay (referred to as the RTE4 assay) agents tested in this study are multimechanistic and may have several are relatively inexpensive and have been developed to evaluate the of the above mentioned activities. effects of various agents on inhibiting cell transformation. These In this RTE assay, primary trachea! epithelial cells are treated with short-term in vitro systems provide data for the selection and ranking B[a]P for 24 h in the presence of the test Chemopreventive agent. The of potential Chemopreventive chemicals for whole animal tests, ac carcinogen is removed and the cells are cultured for 30 days with the celerate the rate of chemical evaluation, and provide data on possible test agent. This allows the agent to be present in the early stages of mechanisms of action. Agents found positive in the whole animal tests initiating events. Normal cells differentiate after 2-3 weeks, whereas B[íí]P-inducedcellscontinue to grow to form colonies or foci. At 30 Received 8/8/94; accepted 12/1/94. days, foci of morphologically transformed, preneoplastic cells are The costs of publication of this article were defrayed in part by the payment of page identified, and inhibition is scored as a decrease in the number of these charges. This article must therefore be hereby marked advertisement in accordance with foci compared to the B[a]P alone-treated cultures. 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the National Cancer Institute Contracts N01-CN-55503-05, N01- The RTE assay has been shown to be sensitive to several classes of CN-95172-02, and NOI-CN-95172-06. 2 Present address: Department of Pathology, CB #7525, University of North Carolina, Chemopreventive agents (14-19). Of the 99 compounds tested in this Chapel Hill, NC 27599-7525. study, it was generally found that morphological transformation was 3 To whom requests for reprints should be addressed, at ManTech Environmental inhibited or reduced by agents that are more effective in protecting Technology, P.O. Box 12313, Research Triangle Park, NC 27709. 4 The abbreviations used are: RTE, rat trachéalepithelial; B[a]P, benzo|«]pyrene; against DNA damage than those that may be antiproliferative. The ODC, ornithine decarboxylase; CFE, colony-forming efficiency. compounds that tend to be positive in the assay are those that prevent 537

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1995 American Association for Cancer Research. CHEMOPREVENTIVE MECHANISMS IN EPITHELIAL CELLS or block DNA damage by: (a) altering drug-metabolizing enzymes Compound Solubility. For each chemopreventive test agent, the highest (antioxidants, modifiers of mixed-function oxidases); (b) inhibiting soluble concentration up to 1 mM was tested for solubility in RTE cell culture binding of B[a]P to DNA (inducers of glutathione 5-transferase and media (see below). If insoluble in media, one of the following solvents was free radical scavengers); (c) detoxifying B[a]P by increasing gluta used: DMSO; ethanol; acetone; or tetrahydrofuran, with the final concentration on the cells not exceeding 0.2, 0.5, 0.3, or 1.0% respectively. thione levels; or (d) inducing differentiation (retinoids). Other posi tive classes of compounds include antihistamines, immunomodula- Assay for Inhibition of Transformation. Trachea! cell isolation methods, culture media requirements, assay protocols, and data analysis have been tors, vitamins, flavonoids, and sulfhydryl compounds. The RTE assay described previously (16), and are summarized briefly here. An initial range- has been found to be a good predictor of in vivo activity of chemo- finding assay was performed for each chemopreventive test agent over a wide preventive agents. It has a predictive value for the hamster lung range of concentrations to determine nontoxic concentrations for the focus model, correctly identifying 15 of 23 agents, and was especially inhibition assay. Concentrations were considered nontoxic if the mean CFE helpful in identifying the negative agents (20). was within 20% of control values, since such values were statistically com Compounds that affect the later stages of cell proliferation and parable to control. The highest nontoxic dose plus four half-log dilutions were progression are not as frequently positive in the RTE assay. The short used for the focus inhibition assay, which included the following nine exper imental groups: medium control, solvent control, B[a]P alone at 10 fig/ml, duration of the RTE assay may not be adequate to identify agents with B[o]P plus all-/rans-retinoic acid at 30 nM (positive control), and B[a]P plus mechanisms associated with progression, such as inhibitors of orni- test chemopreventive agent (five groups at half-log concentrations). thine decarboxylase activity, prostaglandin synthesis, protein kinase Primary rat trachéalcells were isolated. All experimental groups except activity, or anti-inflammatory agents. This assay, in conjunction with media and solvent controls were exposed to 10 /xg/ml B[a]P and the test agent a battery of other in vitro assays that are able to detect inhibition at or retinoic acid on day 1 for 24 h. On day 2 the carcinogen was rinsed from the later stages of cell transformation, should provide an effective screen cells which were then cultured until day 30, refeeding twice/week. The test to identify cancer-preventing compounds. groups received fresh chemopreventive agent, while the B[a]P and solvent controls received culture media with appropriate solvent concentrations. Par allel dishes were set up for testing of cytotoxicity under actual assay conditions MATERIALS AND METHODS and were scored on day 6 for CFE. On day 15, the media were reduced in serum and growth factors to increase selection for transformed foci. On day 30, Chemicals. The following test agents were obtained from Aldrich Chem the cultures were fixed, stained, and scored for morphologically transformed ical Co. (Milwaukee, WI): /V-(6-Aminohexyl)-5-chloro-l-naphthalene sulfon- colonies or foci. Previous studies have shown that Class III transformed colonies or foci that have greater than 2500 cells/mm2 can progress to form amide; apigenin; arginine HC1; benzyl isothiocyanate; carnosine; chlorogenic acid; curcumin; dehydroepiandrosterone; dimethyl fumerate; ethylvanillin; fu tumors when injected into nude mice (21). A very high percentage of Class II foci (1300-2500 cells/mm2) also became tumorigenic. Class I foci (<1300 marie acid; sodium molybdate; propyl gallate; purpurin; simethicone; sodium cells/mm2) usually do not progress to tumorigenicity. Therefore, in this assay thiosulfate; ursolic acid; vanillin; and vitamin Kv Folie acid, quinacrine HC1, and uric acid were purchased from Chemical only Class II and III foci were considered as morphological evidence of early Dynamics Corp. (South Plainfield, NY). The garlic-derived compounds allyl transformation events. Numbers of foci from experimental groups were aver methyl disulfide, diallyl sulfide, and diallyl trisulfide were supplied by Co aged, solvent control background was subtracted, and the results were com lumbia Organic Chemical, Inc. (Camden, SC). Anethole trithione was obtained pared to the group treated with B[a)P alone to calculate the percentage of from Solvay Pharma LTM, Suresnes, France. inhibition. The data was analyzed and considered positive if the agent inhibited The following agents were obtained through the National Cancer Institute carcinogen-induced foci formation by 20% or more over the control of B[o]P Division of Cancer Prevention and Control Repository, (Bethesda, MD): alone at nontoxic concentrations. BASF 47848; BASF 47850; BASF 47851; BASF 51328; carbenoxolone; All culture materials and actual test cultures were tested routinely for etoperidone; ß-glycyrrhetinic acid; oltipraz; retinoyl-D,L-leucine; RO16-9100; contamination by bacteria, fungus, yeast, and Mycoplasma. If contamination and RO19-2968. was found, the media, media components, or cultures were not used in the Other agent suppliers include: allyl methyl trisulfide, Oxford Chemicals, experiments. Ltd. (Bloomfield, NJ); p-aminobenzoic acid, Spectrum Chemical Manufactur ing (Gardner, CA); p-bromophenacyl bromide and DMSO, Pierce (Rockford, RESULTS AND DISCUSSION IL); cromolyn sodium, Interchem Corp. (Paramus, NJ); 5,8,11,14-eicosatet- raynoic acid, Biomolecular Research Labs, Inc., (Plymouth Meeting, PA); In this study, 99 natural or synthetic compounds were screened for lovastatin, Merck, Sharp and Dohme (West Point, PA); Maharishi Amrit chemopreventive activity using the RTE assay. Out of the 99 potential Kalash 4 and 5, Maharishi Ayurveda Products (Lancaster, MA); myricetin, chemopreventive agents tested, 58 were found to be positive and 41 Fluka Biochemika (Ronkonkoma, NY); phenethylisothiocyanate, Eastman were negative for inhibiting B[a]P-induced morphological transfor Kodak Co. (Hartford, CT); phloretin, Transworld Chemical, Inc. (Rockville, mation. The concentration ranges for testing the agents in the RTE MD); sodium suramin, FBA Pharmaceuticals (West Haven, CT); thiolutin, Pfizer (Doreville, GA); a-tocopherol succinate-polyethylene glycol-1000, assay were chosen to be the highest nontoxic concentrations as de termined by the initial range-finding assay. A cytotoxicity assay was Eastman Chemical Products Company (Kingsport, TN). Ajoene was obtained from Dr. Eric Block of Albany State University. Chlorophyll was obtained performed concurrently with the transformation inhibition assay to from American Tokyo Kasei, Inc. (Atlanta, GA). Ascorbyl palmitate, glycerol assess the cytotoxicity at the actual test concentrations. If cytotoxicity monooleate, lanosterol, propylene glycol, and riboflavin-5'-phosphate were was noted during the actual test, the data for that concentration was obtained from Pfaltz and Bauer, Inc. (Waterbury, CT). not considered. Compounds were scored positive if they reduced the The remaining compounds were purchased from Sigma Chemical Co. transformation frequency by >20% compared to control (B[a]P (St. Louis, MO): acetylsalicylic acid; amiloride HC1; antineoplaston AIO; exposure alone). benzo[a]pyrene; ß-carotene; caffeic acid; cysteamine HC1; diphenhydra- Results of all agents tested are given in Table 1 where the 99 mine; ferulic acid; glycine; a-glycyrrhetinic acid; hydrochlorothiazide; compounds are listed in alphabetical order in column 1. In column 2, inositol hexaphosphate sodium; indomethacin; D-mannitol; meclizine; meclofenamate sodium; melatonin; D-L-methionine; /»-methoxyphenol; the concentration that induced maximum inhibition of transformation méthylèneblue; miconazole; morin; nicotinic acid; D-L-palmitoylcarnitine is given in (UM.If transformation was not inhibited, then the highest Cl; polyvinylpyrrolidone; praziquantel; promethazine; all-/rans-retinoic concentration tested is shown. The third column indicates the maxi acid; rhapontin; sodium selenite; ß-sistosterol; steviol; sulfasalazine; su- mum percentage of inhibition of B[a]P-induced foci, compared to lindac; tetracycline; transforming growth factor /3; thioctic acid; 2-thioxo- B[a]P alone controls. If some of the test concentrations were cytotoxic 4-thiazolidone; triprolidine; and verapamil. (with less than 80% of control colony-forming efficiency) those 538

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Table 1 In vitro screening of potential chemopreventive agents using the rat trachéalepithelial cell focus inhibition assav Primary rat trachea! epithelial cells were treated with B[fl]P for 24 h in the presence of the test chemopreventive agent. The carcinogen was removed and the cells were cultured for 30 days with the tesi agent. Foci of morphologically transformed, preneoplastic cells were identified, and inhibition was scored as a decrease in the number of these foci compared to the B[a]P-alone treated cultures.

AgentAcetylsalicylic (fAM)"5550.30.00251.9740.3752.1880.124121.811.1043.77.2290.2640.3132.760.20113216.65.2513265.58828.2156558.60.27126.410.40.2630.560.2133.9180.52.410.00115567.986.180.840133216.376.370.11.383.87.030.250.3concentration inhibition(%)ME*NE25NENE963923NENE98NE1007610058NENENE4954NENE709110090NE53NE10087434010056NE27NE7341NE78NENE1006476NE1001004857NENENENE60NENE98292962100NE97NENE5621100NENE100NE100NE4347Resultr--+--•f++--•f-++++-_-•f+--++++-+-•f++++•f_+-+•f-+--•f++-++•f+----+--+++++-+--+++--+-+-•f+ acidAjoeneAllyl

disulfideAllylmethyl trisulfideAmiloride/j-Aminobenzoicmethyl

acidAnethole tritinoneAntineoplaston AIOApigeninArginine

HC1Ascorbyl palmitateBASF 47848BASF 47850BASF-47851BASF

51328Benzyl isothiocyanate/7-Bromophenacyl BrCaffeic acidCarbenoxoloneCarnosineß-CaroteneChlorogenic

acidChlorophyllCromolyn,

NaCurcuminCysteamine

HC1DHEADiallyl

sulfideDiallyl trisulfideDimethyl fumerateDiphenhydramineEthylvanillinEtoperidoneETYAFerulic

acidFolie acidFumarie acidGlycerol monooleateGlycinea-Glycyrrhetinic

acidß-Glycyrrhetinic acidHydrochlorothiazideIMPIndomethacinLanosterolLovastatinMAK-4MAK-5D-MannitolMeclizineMeclofenamate,

tig/ml10 fig/ml54890.65218842.620.124.10.0802.0914579.91.08.121330.00690.0180.10941000.320.09414.1131420.1170.00062.382382092.620.0030.578102.4112.3Maximum

NaMelatoninD.L-Methionine/7-MethoxyphenolMéthylène

blueMiconazoleMolybdate,

NaMorinMyricetinNicotinic

acidOltiprazD.L-Palmitoylcarnitine

HC1Phenethylisothiocy anatePhloretinPolyvinylpyrrolidonePraziquantelPromethazinePropyl

gallatePropylene glycolPurpurinQuinacrine

HC1Retinoyl-D,L-leucineRhapontinRiboflavin-5'-phosphateRO16-9100RO

19-2968Selenite, NaSimethiconeß-SistosterolSteviolInhibitory

539

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Table 1 Cimunued inhibitionResult'NENE100 (%)* AgentSulfasalazineSulindacSuramin, concentration(/¿M)"75.328.1216.80.4

NaTetracyclineTGF-ßThioctic +64 +90 nM48.50.0001319.022.50.0660.31817.90.66065.70.610.100.2220.080Maximum+NE69 acidThiolutinThiosulfate, +NE85 Na2-Thioxo-4-thiazolidonea-Tocopherol +52 1000TriprolidineUricsuccinate PEG- +33 +23 acidUrsolic +NENE57 acidVanillinVerapamilVitamin

+100 AVitamin +55 K,W-7Inhibitory +NE

" The concentration that induced maximum inhibition of transformation is given in /IM. If transformation was not inhibited, then the highest concentration tested is shown. '' Maximum percentage of inhibition of B[«]P-induced foci, compared to B[u]P alone controls. Agents with less than 20% inhibition are listed as not effective. ' If the inhibitory activity at a nontoxic dose was greater than 20%, a positive sign (+) is shown in the result column. If less than 20% then the result is negative (-). '' NE, not effective; DHEA, dehydroepiandrosterone; ETYA. 5.8,11,14-eicosatetraynoic acid; IHP, inositol hexaphosphate, sodium; MAK-4 and MAK-5. Maharishi Amrit Kalash, formula 4 and 5, aqueous extract used; TGF-ß,transforming growth factor ß;a-tocopherol succinate PEG, ot-tocopherol succinate polyethylene glycol 1000; W-7, W-(6-aminohexyl)- 5-chloro-l-naphthalene sulfonamide. inhibition results were discarded and only nontoxic results are shown. agents found to have these activities in other systems, 22 were found Agents with less than 20% inhibition are listed as not effective. The to be positive in the RTE assay. Table 2 lists these agents starting with last column indicates a positive or negative response in the RTE assay. the most effective at inhibiting B[a]P-induced transformation. Anti If the inhibitory activity at a nontoxic dose was greater than 20%, a oxidants block initiation by scavenging free radicals that are involved positive sign (+) is shown in the result column. If the inhibitory in the activation of carcinogens. Antioxidants may produce changes in activity was less than 20% then the result is negative (-). All results the metabolite profile of B[a]P, contributing to increased detoxifica shown are from single trials due to the screening nature of the study. tion or decreased activation of B[a]P. They can also act by altering Tests are considered valid if: (a) the retinoic acid-positive control inhibits carcinogen-metabolizing enzymes in microsomes (uridine 5'-diphos- transformation by 20% or more; (b) B[a]P induces transformation at least phoglucuronyltransferase, aniline hydroxylase) or in the cytosol (glu- twice that of background levels; and (c) there are at least 8 dishes scored cose-6-phosphate dehydrogenase, UDP-glucose dehydrogenase, glu- in critical control or treatment groups. Transformation frequencies of tathione 5-transferase, and epoxide hydratase) (22). Beyond initiation, B[a]P-treated cultures averaged 3-6 times over background. free radical scavengers may be effective by preventing the formation These chemopreventive test agents have been reported to exhibit of tumor-promoting reactive electrophiles, by preventing lipid peroxi- various biological activities and mechanisms of chemoprevention. An dation, or by gene or enzyme activation or deactivation. Compounds analysis of the mechanisms of the positive agents in this assay gives with these activities could block the neoplastic process in various insight into what mechanisms of chemoprevention may be involved in stages. the inhibition of cell transformation in the RTE assay. The most common One of the effective free radical scavengers was sodium selenite. mechanistic categories of the test agents are listed: (a) antioxidants and Selenium is a necessary cofactor for the enzyme glutathione free radical scavengers; (b) retinoid derivatives; (c) enhancers of gluta- peroxidase, which catalyzes the reduction of hydrogen peroxide and thione 5-transferase; (d) antihistamines; (e) anti-inflammatory agents; (/) hydroperoxides within the cell. Selenium-dependent glutathione inhibitors of arachidonic acid metabolism and prostaglandin synthesis; (g) inhibitors of ODC; and (h) inhibitors of protein kinase C. These mechanisms can be grouped as anti-initiating or antipromot- ing. A potential chemopreventive agent may have an inhibitory effect anywhere in the cancer process. Some agents act to block metabolism Antioxidants/FRS |326 of the carcinogen or increase detoxification pathways in the cell. Retinoids |85|93 These mechanisms would be considered anti-initators since they GSH Enhancers would inhibit carcinogen-DNA adduci formation. Other agents may AntihistaminesAntiinflammatory¡31 inhibit signal transduction pathways, ornithine decarboxylase, or pros taglandin synthesis that are regulators of cellular proliferation. These agents are called antiproliferators. Many agents have been found to |79 have multiple biological activities, and may be represented in several AAInhibitorsODC 188|16lj4 | mechanistic classes. While it was beyond the scope of this study to Inhibitors determine the exact mechanisms involved in inhibiting B[o]P-induced PKC Inhibitors22 transformation in the RTE assay, it is helpful to gain insight into those classes of agents that are detected by the RTE assay to aid in future O 20 40 60 80 100 120 selection of test agents. Fig. 1 indicates the percentage of positive Percent Positive Agents agents in each mechanistic class and gives the number of positive Fig. 1. Response of various mechanistic classes in the RTE Assay. Most common agents out of the total number in that category. mechanistic classes are represented, expressing the percentage of positive agents. For each class, actual numbers of positive agents (expressed within ham) is given out of total Two mechanisms strongly represented by the agents positive in the numbers (expressed outside bars). FRS, free radical scavengers; GSH, glutathione; A4, RTE assay are the antioxidants and free radical scavengers. Out of 32 arachidonic acid; PKC, protein kinase C. 540

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1995 American Association for Cancer Research. CÕIEMOPREVENTIVE MECHANISMS IN EPITHELIAL CELLS peroxidase may lower the level of potentially damaging peroxide Table 3 Mechanistic classes of agents weakly detected hy the RTE assay radicals generated from B[a]P metabolism. Selenium affects the me AgentArachidonic (%)"10091787364Ml412929NENENI tabolism and DNA binding of polcyclic hydrocarbons such as 7,12- inhibitorsMeclofenamatc,acid dimethylbenz(a)anthracene (23). Selenium was positive in the RTE NaCurcuminIMPa-Glycyrrhetinic assay, completely suppressing transformation (100%) at 0.578 ¿IM. Agents that induce glutathione 5-transferase are found to be posi acidTelracyclineMorinß-Glycyrrhetinic tive in the RTE assay. Five of nine agents with this mechanism tested positive (Table 2). These agents also contain a sulfhydryl group. acidETYAOuinacrine Increasing cellular levels of glutathione can protect against initiation events by directly scavenging or trapping electrophilic carcinogenic HC1Acetylsalicylic metabolites such as B[o]P-7,8-diol-9,10 epoxide, thus detoxifying the acidAjoeneApigenin/>Bromophenacyl carcinogen (24). The most effective agent in this group was oltipraz, a synthetic dithiolthione which provided 98% inhibition at 133 /AM. BrCaffeic NI;NENENENENE917360575647412VNENENENF.N acidHydrochlorothiazideMyricetinPropyl

gallateW-7Ornithinc Table 2 Mechanistic Classes of agents effectively detected by the RTE assay AgentAntioxidants/frec inhibitorsCurucumina-Glycyrrhetinicdecarboxylase

scavengersFerulicradicai acidMorinVerapamilPurpurinSteviolß-Glycyrrhetinic acidSelenite, NaMeclofenamate, NaAscorbyl palmitatePromethazine/j-Aminobenzoic acidD.L-Palmitoylcarnitine acidCurcuminEthylvanillinIHF'MAK-5TetracyclineMAK-4PhloretinMorinFolie HC1ApigeninArginine

HC1/»-Bromophenacyl BrGlycineIndomcthacinMyricetinPropyl

HNL;NI;NE29NENENE100NI;NENENENI;NE

gallateW-7Prolein

acidVitamin inhibitorsD,L-Palmitoylcarnitinekinase C K,ß-Carotenea-Tocopherol HCIAmilorideRiboflavin-5

1000CarnosineETYAUricsuccinate-PEG '-PhosphateThiosulfate, NaAnti-inflammatory agentsMeclofenamate. acidDimethyl NaAcetylsalicylic (umeraleApigeninChlorophyllFumarie acidCarbenoxoloneIndomethacinu-MannitolSulfasalazineUrsolic

acidCaffeic acidMéthylène blue/)-MethoxyphenolMyricetinPropylene AcidInhibition " Maximum percentage of inhibition of B[n]P-induced foci, compared to B[fl]P alone controls. Agents with less than 20% inhibition arc listed as noi effective. Abbreviations: glycolPropyl 1HP, inositol hexaphosphate, sodium: ETYA, 5,8,11,14-eicosaletraynoic acid: NE, not gallaleChlorogenic effective (<20% inhibition): W-7, ¿V-(6-aminohexyl)-5-chloro-l -naphthalene sulfonamide. acidGlutathione enhanchersOltiprazBenzyl

isothiocyanateDiallyl trisulfideAnethole Oltipraz is an antischistosomal drug which causes induction of the trithioneAllyl glutathione 5-transferase liver enzymes, and has been found recently disulfideDiallylmethyl to induce another protein, alfatoxin B, aldehyde reducíase,in livers of sulfideAjoeneAllyl oltipraz-treated animals and is used as a biomarker for oltipraz effect.5 trisulfidePraziquantclRetinoidmethyl The retinoids are highly effective as chemopreventive agents and some, such as 4-hydroxyphenol retinamide (tested previously, Ref. derivativesBASF 51328Retinoyl-D-L-leucineR016-9100Vilamin 16), are currently in use in clinical trials to prevent recurrence of breast cancers (25). The retinoid analogues tested in this study were ABASF synthesized to provide chemoprotection with less toxicity. In the RTE 47850BASF-47851RO assay 6 of 9 retinoids were positive at nontoxic doses, providing 19-2968BASF complete inhibition in nearly all assays (Table 2). The retinoids have 47848AntihistaminesDiphenhydramineCromolyn. anti-initiating mechanisms as well as the ability to induce differenti ation (26). NaTriprolidineInhibition(%)"100UHI10098"7%4187787h64f>4h260565554524')292323NENENENENENENENENENEW58533925NENENENEUH)10010010010076NENE1007033All of the three agents known to be antihistamines gave positive results in this assay (Table 2). How they may function to inhibit " Maximum percentage of inhibition of B[a)P-induced foci, compared to B[a]P alone B[a]P- induced transformation in an in vitro system is unclear. controls. Agents with less than 20% inhibition are listed as not effective. * NE, not effective (<20% inhibition); IHP, Inositol hexaphosphate, sodium; ETYA, 5,8,11,14-eicosatetraynoic acid; MAK-4 and MAK-5, Maharishi Amrit Kalash, formula 4 5 S. Sharma, G. P. Wyatt, L. N. Anderson, V. E. Steele, and G. J. Kelloff, Character and 5, aqueous extract used; a-tocopherol succinate PEG 1000, a-tocopherol succinate ization of a 38 Kdalton prolein that is highly induced by oltipraz, manuscript in polyethylene glycol 1000. preparation. 541

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Diphenhydramine, the active agent in nonprescription antihistamines, helps to quickly define agents with chemopreventive potential for was the most effective agent in this group with 100% inhibition at further development in animal studies and clinical trials. 34 /UM. Agents classified as antiproliferators are those that inhibit the ACKNOWLEDGMENTS progression of an initiated, preneoplastic cell. This group would include agents that inhibit ODC activity, inhibit the metabolism of We acknowledge CCS Associates, Inc. (Mountain View, CA) for informa arachidonic acid to prostaglandins, and inhibit protein kinase activity. tion on agent mechanisms. This information was provided to National Cancer Institute under contract No. NO1-CN-25417. We are also grateful to Gail P. These activities are characteristics of proliferating cells that when modulated, appear to inhibit cancer progression. ODC is the rate- Wyatt and Kyle Garris for technical support. limiting enzyme in the synthesis of polyamines, which are involved in proliferation and differentiation (27). Arachidonic acid metabolites REFERENCES serve as second messengers in proliferating cells (28). Protein kinases 1. Boone, C. W., Steele, V. E., and Kelloff, G. J. Screening for chemopreventive (anticarcinogenic) compounds in rodents. Mutât.Res., 267: 251-255, 1992. are involved in signal transduction, and protein kinase C is the 2. Boone, C. W., Kelloff, G. J., and Malone, W. E. Identification of candidate cancer putative receptor for the phorbol ester, 12-O-tetradecanoylphorbol chemopreventive agents and their evaluation in animal models and human clinical 13-acetate (29). Agents which act to inhibit these mechanisms did not trials: a review. Cancer Res., 50: 2-9, 1990. 3. Kelloff, G. J., Boone, C. W., Malone, W. F., and Steele, V. E. 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26. Lotan, R. Suppression of squamous cell carcinoma growth and differentiation by 29. O'Brian, C. A., Ward, N. E., loannides, C. G., and Dong, Z. Potential strategies retinoids. Cancer Res., 54 (Suppl.): 1987s-1990s, 1994. of chemoprevention through modulation of protein kinase C activiiy. In: V. E. 27. Verma, A. K. Ornithine decarboxylase, a possible target for human cancer prevention. In: Steele, G. D. Stoner, C. W. Boone, and G. J. Kelloff (eds.). Cellular and V. E. Steele, G. D. Sloner, C. W. Boone, and G. J. Kelloff (eds.), Cellular and Molecular Molecular Targets for Chemoprevention, pp. 161-172. Boca Raion, FL: CRC Targets for Chemoprevention, pp. 207-224. Boca Raton, FL: CRC Press, 1992. Press, 1992. 28. Zenser, T. V. and Davis, B. B. Arachidonic acid metabolism. In: V. E. Steele, G. D. 30. Sharma, S., Stutzman, J. D., Kelloff, G. J., and Steele, V. E. Screening of potential Stoner, C. W. Boone, and G. J. Kelloff (eds.), Cellular and Molecular Targets for chemopreventive agents using biochemical markers of carcinogenesis. Cancer Res., Chemoprevention, pp. 225-245. Boca Raton, FL: CRC Press, 1992. 54: 5848-5855, 1994.

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Julia T. Arnold, Betty P. Wilkinson, Sheela Sharma, et al.

Cancer Res 1995;55:537-543.

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