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(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{(%)"
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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,"!",„.,,
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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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