(CANCER RESEARCH 49, 2703-2708, May 15, 1989]

2,3,7,8-Tetrachlorodibenzo-p-dioxin Enhancement of TV-Methyl-W-nitro-W-

nitrosoguanidine-induced Transformation of Rat TrachéalEpithelial

Cells in CultureNoriho Tanaka,1 Paul Nettesheim, Thomas Gray, Karen Nelson, and J. Carl Barrett2

National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

ABSTRACT

The abilities of various dioxins to induce toxicity or transformation ofrat trachea! epithelial cells in culture were examined. 2,3,7,8-Tctrafhlo-rodibenzo-/7-dioxin (TCDD) was not cytotoxic and did not induce transformation as measured by the induction of growth-altered, preneoplasticcells (termed enhanced growth variants). However, TCDD enhanced thetransformation of TV-methyl-W-nitroWV-nitrosoguamdine (MNNG)-ini-tiated rat trachea! epithelial cells. Other dioxin congeners with 0 to 3chloro substitutions were inactive in enhancing MNNG transformation.TCDD was most effective at a concentration of 0.3 nMand when treatmentwas administered immediately after MNNG exposure. The dose-responsecurves for enhancement of MNNG-induced transformation and inductionof aryl hydrocarbon hydroxylase activity by TCDD were similar. Theseresults are consistent with the hypothesis that the enhancement of celltransformation by TCDD is mediated through the TCDD receptor. TCDDalso enhanced transformation when the cells were treated before MNNGtreatment. The ability of 12-0-tetradecanoylphorbol-13-acetate (TPA)to enhance MNNG-induced rat trachea! epithelial transformation wasalso examined. In contrast to the findings with TCDD, the number oftransformed colonies was increased only by pretreatment with TPAfollowed by MNNG. TPA-pretreatment enhanced equally the number ofnormal cells forming colonies and the total number of transformedcolonies after selection; therefore, the transformation frequency (transformants per total surviving colonies) was unchanged by TPA. In contrast,TCDD treatment enhanced the transformation frequency in MNNG-exposed cultures since the number of transformed colonies increasedwhile the number of total colonies remained constant. Thus, TCDDappears to act by a different mechanism than TPA. TCDD enhancementof MNNG-induced transformation may be attributed to a promotionaleffect, a comutagenic action, or a modulation of cell proliferation and/ordifferentiation mediated through the TCDD receptor.

INTRODUCTION

2,3,7,8-Tetrachlorodibenzo-p-dioxin is the most potent member of the chlorinated dibenzo-/7-dioxins, a class of toxic andcarcinogenic environmental contaminants found in herbicidesand fungicides (1-5). The toxicity of dioxins to animals ishighly variable among different species (6-9). It has been proposed that certain toxic responses produced by TCDD3 in mice

depend on the presence of the binding protein encoded by theAh locus (11, 12). This protein is the putative receptor formultiple inducers of AHH activity and other enzymes (6, 11,12). TCDD is carcinogenic in rats and mice producing a varietyof tumors (1-5). TCDD is also a potent tumor promoter in ratliver and mouse skin (13, 14).

Received 10/14/88; revised 2/14/89; accepted 2/17/89.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.

1Present address: Department of Cell Biology, Hatano Research Institute,Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257, Japan.

2To whom requests for reprints should be addressed.'The abbreviations used are: RTE, rat trachea! epithelial; TCDD, 2,3,7,8-

tetrachlorodibenzo-p-dioxin; MNNG, /V-methyl-Ar'-nitro-A'-nitrosoguanidine;TPA, 12-O-tetradecanoylphorbol-13-acetate; AHH, aryl hydrocarbon hydroxylase; DD, dibenzo-p-dioxin; 2,7-DCDD, 2,7-dichlorodibenzo-p-dioxin; 2,3,7-TCDD*, 2,3,7-trichlorodibenzo-p-dioxin; PBS, phosphate-buffered saline; CFE,colony-forming efficiency; EGV, enhanced growth variant.

Limited studies of the mechanism of action of TCDD at thecellular level have been performed. In contrast to its highlytoxic nature in vivo, TCDD is not toxic to a variety of cells inculture (15, 16). TCDD has been extensively examined formutagenic activity with generally negative results (17-23).Abernethy et al. (24) showed that while TCDD was a potentpromoter of MNNG-initiated C3H/10T1/! cell transformation,

it was inactive as an initiator or a complete carcinogen in thiscell transformation model. In the present study we have examined the activity of TCDD and related dioxins in the rat trachéalepithelial cell transformation system (25-29) and have alsoobserved that TCDD enhances MNNG-initiated transformation of these epithelial cells.

MATERIALS AND METHODS

Chemicals. Various dioxins including DD, 2,7-DCDD, 2,3,7-TCDD*, TCDD, and MNNG were obtained from Chemical Repository

(National Cancer Institute, Bethesda, MD). TPA was obtained fromChemical Carcinogenesis (Eden Prairie, MN). Stock solutions of dioxincompounds (0.01-0.1 mg/ml) were prepared in dimethyl sulfoxide andTPA (1 mg/ml) was dissolved in acetone.

RTE Cell Culture. RTE cells were obtained from the tracheas of 9-to 15-week-old male Fischer 344 rats by published methods (26, 27),using 1% promise (type XIV; Sigma) treatment with slight modifications. Harvested cells were plated onto an irradiated feeder layer of 3T3cells (4 x 105/60-mm dish) in Ham's F-12 medium supplemented with

5% fetal bovine serum (GIBCO), 1 Mg/ml insulin, 0.1 Mg/ml hydrocortisone, and 1% penicillin/streptomycin (GIBCO). The number of trachea! cells plated for colony forming assays and transformation assayswere 2 x IO3 and IO4 cells per dish, respectively. For colony-forming

assays five dishes per experimental group were used and for transformation assays 15 dishes per group.

Colony Forming Assay. The day after plating, RTE cells were treatedwith dioxin analogues for 2-7 days. In experiments involving MNNG,cells were allowed to attach overnight and then treated with 0.2 Mg/m'MNNG for 4 h in 20 mM 4-(2-hydroxyethyl)-l-piperazineethanesul-fonic acid-buffered F-12 medium (pH 6.8) without serum. After MNNGtreatment, the cells were washed with PBS (Ca2+- and Mg2+-free). The

cultures were then treated with medium containing various doses ofdioxins or TPA in growth medium for 2-7 days. The toxicity of thetreatments was determined from the number of colonies surviving at 7days after plating. The CFE at day 7 was calculated as

Number of colonies in treated culturesNumber of colonies in control cultures

x 100%

Transformation Assay. The cells were exposed to indicated chemicals(e.g., MNNG plus TCDD) and were allowed to express carcinogen-

induced transformation by growing on the 3T3 feeder layer for 7 days.Seven days after the beginning of chemical treatment, feeder cells wereremoved with 0.002% EDTA by vigorous pipetting as described (26).The cultures were continued for an additional 4 weeks with weeklychanges of the medium. At 5 weeks after the start of exposure, thedishes were fixed with methanol and stained with 10% aqueous Giemsa.In previous studies (28, 29), we describe the development of 4 different

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DIOXIN ENHANCEMENT OF TRACHEAL CELL TRANSFORMATION

morphological types of colonies seen during early stages of clonaltransformation of RTE cells. Two of the colony types, designated typesI and II, were considered to be nontransformed or minimally transformed because of their small size and because they were composedmostly of large, seemingly inactive cells. Two classes of colonies,designated types III and IV, were considered to be transformed becausethey were large, obviously expanding, and were composed of small,dark-staining cells. Cultures were scored for EGV colonies by thecriteria described (28, 29). The transformation frequency was calculatedas the number of type HI and type IV EGV colonies per culture at 5weeks divided by the surviving colony-forming cells per culture determined from the CFE measured on day 7.

AHH Assay. Induction of AHH activity by TCDD was assayed asfollows. RTE cells (2.5 x IO5 cells/ 100-mm dish) were plated into 15dishes for each group using fresh F-12 medium mixed 1:1 with conditioned medium from 3T3 cultures. Five days after plating, cultureswere treated with various doses of TCDD. After treatment for 2 days,cells were washed twice with cold PBS, harvested by scraping with arubber policeman in cold PBS, and centrifuged at 2500 rpm for 10 minat 4°C.The pellets were resuspended in 0.5 ml of buffer containing 25

mM Tris-HCl (pH 7.4)-0.25 M sucrose, and then sonicated three timesfor 10s in an ice bath. A part of the homogenate was used to measureprotein by Lowry assay with a Bio-Rad kit (Bio-Rad). The homogenatewas centrifuged for 10 min at 10,000 x g and recentrifuged for 40 minat 100,000 x g. The resultant microsome fraction was assayed for AHHactivity by the method of Jansing and Shain (30).

Statistical Methods. Transformation induction by single compoundswas tested against the control by means of a i statistic, based onassuming binomial distributions for the number of transformed EGVs.Enhancement of transformation induction by the dioxin analogues wastested by reference to Finney's null model of "simple independentaction" (31). When, as here, the response of interest is rare, this is

approximately equivalent to a null model of response additivity. Thusit is assumed that under no synergy the difference in transformationfrequency induced by a test compound in the presence of MNNGshould be the same as the difference in the absence of MNNG. Underthe alternative hypothesis that there is enhancement of effect, thecorresponding difference would be greater in the presence of MNNG.For each 2x2 table of transformation frequencies being compared,the interaction parameter describing departure from the no-synergymodel was fitted and tested by using standard maximum likelihoodtechniques, under the assumption that for each treatment combinationthe number of transformed EGVs is binomially distributed. Model fitswere accomplished using the GLIM (32) statistical software. All P

values cited are based on one-sided tests, i.e., testing for enhancementand not for inhibition of effect.

RESULTS

Enhancement of Transformation Induction by Dioxin Analogues in RTE Cells. We examined the toxicity and transformation of RTE cells treated with various dioxin analoguesalone or in combination with MNNG. Relative survivals weredetermined by comparing the CFE of dioxin-treated groups tothat of the control group. When the RTE cells were treatedwith each dioxin alone at high doses (300-900 nvi), there wasno significant cytotoxic effect of any of the analogues at thedoses used (Table 1). Treatment of the cells for 4 h with 0.2itg/ml MNNG reduced the relative survival to 60% of the cellstreated with solvent only. Posttreatment of the MNNG-treatedcells with any of the four dioxin derivatives did not furtherreduce the CFE of the cells (Table 1).

Treatment of the cells with any of the four dioxin derivativesalone (0.3-0.9 /¿M)failed to increase the transformation frequency (i.e., number of EGV colonies per total colonies) abovethe spontaneous frequency (which ranged from 4-fold) the MNNG-induced transformation. Posttreatmentof MNNG-treated cultures with the other dioxin derivatives(the nonchlorinated derivative, DD, or the di- or trichlorinatedderivatives, 2,7-DCCD or 2,3,7-TCDD*) failed to enhancesignificantly the MNNG-induced transformation frequency(Table 1).

Effects of Varying Dose of TCDD on Enhancement of MNNG-induced Transformation and AHH Activity Inductions. The effectof various doses of TCDD on enhancement of MNNG-inducedtransformation of RTE cells is shown in Table 2. TCDD alonefailed to induce transformation except possibly at the highestdose (300 HM). This small effect observed in this experimentwith TCDD was statistically significant (P < 0.02) relative to

Table 1 Effects of different dioxin analogues on transformation of RTE cells by MNNG

CFERelativeChemicalControlDD2,7,-DCDD2,3,7,-TCDD«2,3,7,8-TCDDDose(%)"survivalo

:0.9MM :!.8

1.0!.81.00.9

MM 3.21.10.9MM 2.70.90.3MM 3.11.1MNNG

0.2MB/ml+DD+2+2+2,7,-DCDD,3,7,-TCDD«,3,7,8-TCDD0.9MM0.9MM0.9

MM0.3

MM.7

0.6.50.5.90.7.50.5.9

0.7Total

no. ofsurvivingcolonies3614420044803975488023802310280521902820Total

no. oftransformedEGVsc2131491116845Tf(%)"0.06O.O/O.O/o.otfO.Otf0.38*0.48*0.57*0.37*1.6'Relative

increase ofMNNG-

inducedtransformationNA'NANANANA1.01.31.51.04.2

" Measured at day 7.'' Total number of surviving colonies calculated from average colonies per dish at day 7 multiplied by number of dishes per experimental group.c EGVs scored as previously described (26-29). The total number of EGV colonies per experimental group (15 dishes) is given.

*Tf, transformation frequency = Total number EGV coloniesTotal surviving colonies at day 7 '

' NA, not applicable.^Not significantly different from control.1 Significantly different (P < 0.01) from control.* Not significantly different from expected additive effects of TCDD and MNNG.' Significantly different from expected additive effects of TCDD and MNNG (P < 0.0001 ).

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DIOXIN ENHANCEMENT OF TRACHEAL CELL TRANSFORMATION

Table 2 Dose dependency ofTCDD enhancement of transformation

ChemicalControlTCDD

alone0.03nM0.3nM3nM30nM300

nMMNNG

alone (0.2Kg/ml)MNNG(0.2 f.g/ml) 4-TCDD0.03

nM0.3nM3nM30nM300

nMCFE(%)'4.84.24.84.14.44.73.33.12.52.92.73.3Relativesurvival1.00.91.00.90.91.00.70.70.50.60.60.7Total

no.ofsurvivingcolonies720063007200615066007050462046503750435040504950Totalno.oftransformedEGVsc001115101753514645T{(%)"

DIOXIN ENHANCEMENT OF TRACHEAL CELL TRANSFORMATION

Table 3 Dependency ofTCDD enhancement of transformation on timing of exposure

Chemical"ControlTCDDTCDDTCDDTCDDMNNG+TCDD+TCDD+TCDD+TCDD+TCDDTimeof exposureCFE(day)

(%)»-10247i

iiiiii i 6.6eOr^Tj

6.6n;,»-,;»/,i6.31

unuiamman7.1-!

4.4323

8,"!",„.,,

DIOXIN ENHANCEMENT OF TRACHEAL CELL TRANSFORMATION

transformation can be considered. TCDD is a tumor promoterin vivo (13, 14) and may possibly act as a tumor promoter inthe RTE in vitro model. However, enhancement of MNNG-induced transformation was most effective when the dioxin wasadministered before or soon after MNNG treatment. Delayingtreatment for 4 days after MNNG treatment resulted in areduced effect. This is not consistent with classical tumorpromoter effects on mouse skin where promoter treatment canbe delayed for extended time after initiator treatment (35). Inthe lOT'/z transformation model, TCDD treatment of these

fibroblasts could be delayed for 5 days after MNNG and anenhancing effect was still observed. This is consistent with apromotional effect but different from our findings with RTEcells.

In the RTE system, TCDD enhanced the number of transformants without affecting the CFE of the treated cells; therefore, the transformation frequency of MNNG-treated cultureswas enhanced by TCDD. TPA, the most active tumor promoterin mouse skin (35), was relatively ineffective in enhancingMNNG-initiated RTE cell transformation when added afterMNNG. Pretreatment of the RTE cells with TPA increased theCFE of the cells as previously reported (27,33). A proportionateincrease in the transformed colonies resulted if TPA treatmentwas followed by MNNG treatment. The transformation frequency was the same in the MNNG only and TPA-MNNGtreatment groups even though the latter had nearly 7 times thenumber of EGV colonies. This finding is similar to the mouseskin experiments of Pound (36) who showed that tumor promoter treatment in vivo prior to initiator enhanced the tumoryield. In the RTE cell culture model, TCDD was effective whentreatment was given immediately before or soon after MNNGwhich is another difference in the effects of TPA versus TCDD.Thus, TCDD and TPA appear to act by different mechanismsin this model.

The effects of TCDD in our experiments are also consistentwith a cocarcinogenic effect. Experiments are in progress totest whether TCDD enhances transformation by other carcinogens and whether it exerts a comutagenic activity withMNNG. Since MNNG does not require metabolic activation,the induction of AHH is not likely to play a role in the TCDDeffect on cell transformation.

TCDD may act to modulate the response of the cells toMNNG by unknown nonmutagenic mechanisms. TCDD isknown to affect proliferation, differentiation, and responses togrowth factors of a number of epithelial cells in culture (37-45). These effects may play a role in the expression of thetransformed phenotype by MNNG-treated RTE cells. Mc-Kinney and others have reported that TCDD is structurallyrelated to thyroid hormones and binds to thyroid-binding proteins (46-48). Furthermore, thyroid hormones modulateTCDD-mediated myelotoxicity (49) and TCDD and thyroxinederivatives cause thymic involution in TCDD-responsive butnot TCDD-nonresponsive strains of mice (50). Since certaincell transformation systems have shown a critical role of thyroidhormone for transformation by MNNG and other carcinogens(51-53), this finding raises the interesting possibility thatTCDD exerts its toxic and carcinogenic activities through thethyroid hormone effector pathways. Experiments to test thispossibility in the RTE system are in progress.

In summary, we have observed that TCDD is not a completecarcinogen in the RTE transformation system. TCDD enhancesMNNG-induced transformation of the cells but TCDD treatment immediately after MNNG treatment is most effective,unlike classical tumor promoter effects in vivo. Also, the action

of the tumor promoter TPA in this system differed from TCDD.TCDD may act as a tumor promoter or as a cocarcinogenicagent in this model. Further studies to elucidate the mechanismof action of TCDD in this system may yield new insightsconcerning this important environmental substance.

ACKNOWLEDGMENTS

We would like to thank Sandy Sandberg for her expert typing andediting of this manuscript. The statistical analyses of these studies wereperformed by Dr. Clarice R. Weinberg of the National Institute ofEnvironmental Health Sciences and are gratefully acknowledged.

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