EPA/600/D-90/006
March,1990
ANALYSIS OF 2,3,7,8-TCDD TUMOR PROMOTION ACTIVITY
AND ITS RELATIONSHIP TO CANCER
James W. Holder, Ph.D.
Human Health Assessment Group (RD-689)
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC 20460
Heinrich Menzel
Hessische Landesanstalt fur Umwelt
Federal Republic of Germany
Presented at
The International Conference on Dioxins
Umea, Sweden
August 1988
Proceedings Published in
Chemosphere 19(1-6), 861-868
The views expressed in this paper are those of the authors and do
not necessarily represent the views or policies of the U.S.
Environmental Protection Agency or Hessische Landesanstalt fur
Umwelt.
-------
NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
-------
EPA/600/D-90/006
March 1990
ANALYSIS OF 2.3.7.8-TCDD TUMOR PROMOTION ACTIVITY
AND ITS RELATIONSHIP TO CANCER
JAMES W. HOLDER " and HEINRICH M, MENZEL b
Genetic Toxicology Assessment Branch, Office of Health ind
Environmental Assessment, Office of Research and Development,
United States Environmental Protection Agency, USA;
Hessische I. andesans ta 11 fur Unuelt, Federal Republic of Germany,
ABSTRACT
2,3,7,8-tetrachlorodibenzo-p-diojun (hereifter referred to as TCDD) has a high estimated cancer potency in
animals which has been reasoned to imply that TCDD might be carcinogenic to man. The animal cancer data show
that TCDD can act in a solitary manner causing tumors without the participation of other known factors.
However, there exists animal cancer data indicating that TCDD can act as a tumor promoting compound. This
analysis examines which type of carcinogen and which mechanism best characterizes TCDD cancer activiiy. It
is suggested that TCDD acts by a hormonal mechanism to cause cancer in a solitary manner, at low doses, in two
species, and in a number of different organs, including rare sites. These observations in iota characterize TCDD
as a complete carcinogen, which by definition encompasses both initiation and promotion carcinogenic activities.
KEYWORDS
Total carcinogen; tumor promoter; tumor initiator; hormonal carcinogen.
INTRODUCTION
In order to best assess the hazards to human life from chemical carcinogens, it is necessary to incorporate as much
understanding of the mechanism of action of animal and human carcinogens into the hazard assessment process
as possible. The carcinogenic response to TCDD in mice and rats seems to indicate adichotomous mechanism: one
mechanism proceeding by complete carcinogenesis and the other by tumor promotion. The first mechanism
indicates that TCDD can act as a solitary carcinogen needing no other factors to cause cancer. The second
mechanism refers to a type of incomplete carcinogenesis where the compound completes the initial,
subcarcinogenic insult of a total carcinogen, e.g. from another source, so as to cause tumorigenesis.
We consider, in the delineation of the cancer mechanism, that it is important to qualitatively model what type
of mechanism is likely to be operative in TCDD-exposed man. Quantitative models used to explain cancer
dosimetry must of necessity be based on the best available realistic qualitative mechanistic model.
This analysis shall proceed in turn through the extant TCDD tumor promotion data, complete carcinogenesis
data, hormonal-related mechanisms to TCDD, and finally through a summary analysis of the mechanism which
best characterizes TCDD carcinogenicity.
EVIDENCE FOR THE TUMOR PROMOTION EFFECTS OF TCDD
a. in vivo Support for TCDD-Related Tumor Promotion (Dirgct BioassaM)
Pilot et al, (1980) have presented data on the promotion characteristics of TCDD in female Charles River rats
(Fig. 1). The primary purpose of this study was to demonstrate tumor promoter-related quantitative liver enzyme
focus formation. Positively staining foci for 7 -glutamyl transpeptidase (GGT) was used as a liver tumor-
promotion marker for liver cells disposed to preneoplasia by TCDD. These foci were markedly increased
compared to controls (see protocol, Fig. 1).
The Pitot study also enumerated the number of female rats with hepatocellular carcinoma among the dose groups
(4-7 rats/TCDD group, FIg.l), No liver cancers were found in any control group, none in the low-dose TCDD
group (10 ng/kg/day given s.c, in corn oil), but 5/7 rats had liver cancers at the high-dose TCDD group (100 ng
The views .expressed in this presentation *re those of the authors and not necessarily those of our respective
countries or
-------
TCDD/kg b.w./day). A positive control group was tested also with phenobarbital (0,05% in diet) which showed
8/10 rats had liver cancers. Although few animals were tested giving a lowered statistical inference of the tumor
results (compared to a standard bioassay with SO animals/scx/dose group), it is clear that sufficient fields of cells
were scanned to determine the TCDD-related histochemical effects of TCDD and phenobarbital in the rat liver.
Pitot et fl/.(198Q) have suggested that, because of the positive tumor promotion results, which are associated with
particular enzyme foci formation in the liver, it is reasonable to hypothesize that all the tumors associated with
persistent TCDD exposure arise from the tumor promoting effects of TCDD on "already initiated* cells in the
test animal. This further implies that there is no primary initiating effect of TCDD on these female rat cells
(cf. DISCUSSION section).
A study in HRS/J mouse skin is presented in Fig. 2 (Poland et a/,,1982). When hairless mice (hr/hr) were first
exposed to 0.2 nmol of DMBA (initiation) and then exposed to repetitive exposures of either SO ng TCDD/mouse
or 2000 ng TPA/mouse (a known skin tumor promoter), papillomas of the epithelium were formed in both cases.
It is notable that TCDD alone in this experiment caused no increases in skin papillomas. But when a subthreshold
dose of a complete carcinogen was applied to skin, then subsequent TCDD or TPA exposure caused tumor
promotion in hairless mice. TCDD is about 40 times more potent as a tumor promoter in this system than is TPA.
In congeneic mice which are genetically (hr/+), no tumors can be promoted by repetitive exposures to TCDD,
thereby suggesting that a single recessive trait (hr) may be involved in the TCDD-mediated tumor promotion in
HRS/J mice. However, the possibility of other genetic loci involvement is not ruled out.
A tumor promotion bioassay was done for the U.S. National Toxicology Program (U.S.NTP, 19S2i) in mice.
TCDD was applied dermally onto the dorsal region of skin (Fig. 3). Swiss-Webster mice showed marginal increases
in fibrosircomas upon TCDD exposure in either the male TCDD or DMBA/TCDD dose groups which are not
viewed as statistically increased cancer responses; however, definite increases in fibrosarcomas were observed
in the female TCDD and DMBA/TCDD dose groups. These increases, which were about the same (30% and 28%,
Fig. 3), showed that TCDD alone increased the fibrosarcomas, but prior initiation with DMBA was not further
enhanced by repetitive exposure to TCDD. This suggests that TCDD is acting as a total carcinogen. It is
interesting that fibrosarcomas were caused by TCDD and not cancers of the mouse epithelium, onto which the
three times/week applications in 0.1 ml. acetone were applied. It is known, for instance, that TPA and certain
other phorbol esters do promote tumors in that same epithelium (Felling and Slaga, 1985, and references therein).
Other results (Fig. 4) found in "normal" wild-type mice (presumably +/+ at the hr locus) are also negative for
TCDD skin tumor promotion (Berry el a/,, 1978). The results in all of the mice strains tested for tumor promotion
taken together suggest that HRS/J hairless mice may be unique in showing skin tumor promotion responses as
a result of repetitive TCDD exposures to the shaved and depilatated mouse dorsal skin epithelium.
b.in vitro Support for TCDD-Related Tumor Promotion Effects
TCDD has little transforming ability in 10 T 1/2 cells alone, but was a promoter of transformation following
MNNG((N-MethyI-N*-nitro-N-nitrosoguanidine), a known tumor initiator (Abernethy et at., 1985; Abernethy and
Boreiko, 1987). Following initiating (low) levels of MNNG in this assay, TCDD was a very potent transforming
agent, since it has a high transformation efficiency: 29%-39% @ 4 picomolar.
Tumor Promotion In Charles River Bats.
Top Panel. Protocol is presented as a time line.
Initiation is by intragastric intubation (IX) of
diethylnitrosamine; subcarcinogenic dose of
DEN * 10 mg/kg. After a rest period, tumor
promotion was effected by persistent subcutaneous
injections of TCDD 8t either 140 ng or 1400 ng
TCDD per kg body weight per dose. A promotion
dose was given twice/week. Dosing periods; DEN,
TCDD, or phenobarbital alone, 28 wks; as tumor
promoters, TCDD for 14 wks or phenobarbital
for 28 wks.
Bottom Panel. Indicates the incidence of cancer
in the rat liver identified as hepatocellular
carcinomas. Note the short period of TCDD
treatment for TCDD as a promoter while causing
about the same incidence as for phenobarbital
as a promoter at twice the treatment TCDD
period (14 versus 28 wks). Hence, at doses
given by Pitot and his collaborators, TCDD
tumors in the liver develop faster.
ran i MM HIM n ewuus inn HATS •»
imnummMm mrunoi • i,!.7,i-rca> nm
ut» i> PITOT. H. , IT «i. <19«0>
CMC*> glaum* *4 !Uf - Ilia
IRC LIKE
mill: i • IBITIATM, DirrnTMiirmiiuniii IBIIO
» « TO** »mniiii, 'Z,3,?,J.TCM
FH • rURTlAL MIMrKTQMY (HI^LtCArieMt
icon rm u»««
GTT CILL fOCI
TUC TQ
1400 M
""«»
111
10 TIUTMIHTI/llfH
•UlCMTAlllOVkir /K&. «,H.
cs fluUTiOH or TCOO fip«$
•^^B jcom ro* fi«a
I
,»«
• I ••
TtMTHMT
CM • DEN
PN • TOO
PH *
mENOUUMITU.
BATS No. fOCl/enl
Vie*
Kt - SEN 10
• PHENOeAfttlTM. •
}«
25
5«
171
ill
LWKUJSCIiilKE
0/« « 0%
»f! * 0*
»// » 71%
1/19 > SOt
-------
Fig. 3
Fig. 2
I
TWO* PROMOTION BY 2,3,7,4-TOO U HAIRLESS MICE
I
INITIATOR
QMBA
(0,2 NMOL
ON84-
(Q.2)
OMB 4
10.2)
MNNG
15 HOL
MNNG
' MNNC
HMNS
ACETONE
BEFEBIKCE
PROMOTES TUMOR INCICEUCE TUMOB MULTIPLICITY
ACETONE i \ o.i
i
TPA
(2000 NG/MSI.) 56 \ 1.9
TCDD • 98 % 2.0
(50 io/Mst.)
TCDD (3.8 MQ/HSE.) 5 % 0.05
)
TCDO (7.5 NG/MS£.) 55 % 0.7
TCDO (IS NO/ME.) 77 1 1.5
TCDD (30 NS/MSE.) 100 * 4.0
TCCD '(30 NC/Mit.) 0 % 0
POLAND, PALIN, 1 GLOVIS (1982) 3JO. (Nev.) 271 - 273
Fig. 4
TUWfi PROHOTIN6 AJILITY IN CD-I NICE 1Y 2,3,7.8-TCOO
PROTOCOL
IN CO-1 MOUSE SKIN IOORJ»L)
TREATED TWICE/MEEK ro« 30 UIIKS
100 S4/«BPL!C*TION IN 0.2 ML.
ACITONE
INITIATOR PROMOTER QflS£ IMCIPIMCI TUMORS/HOUSE
OMBA 0 0
(200NHOI.)
OMIA
OHBA
TPA 2,000 N&
TPA 10.000 HC
I
92
0 .03
8.1
TCOD
TCDO
100 NG
100 NG
REFERENCE:
BERRY, DZ£JOV»MNI, JUCHAU, IHACKEN, GLCASOH, SI.»G«
RESE40CM COMMUNICATICNS IN CMEHIC«L P*THCLOGV
«NB PHAHMACOLOST 2J}- (1> 101 - 108 (19781.
NATIMtti. TOIICauXY ftOfUH - 198J DERHAL aWSURE TO TCOO
(JWIJI - MMTtl MICK)
TREATMENT
SO uc.
OMBA
TCDD
DOSE
RATE
TOTAL
DOSE
DURATION
S
I
1
T
MALE DOSE
one TINE SKIN
• tMTITXVI SKIN
THIiTHtNTS (O.lKL)
1 NC' 025 KC I.W.
3K/UECK
17.1 M/«/«Y
2 ve»BS
TREATMENT
CONTROL
TCDD
DHBA/TCDO
CONTROL
- TCDO
OMBA/TCDD
FIBROSARCOMA
INCIDENCE
MALES
3/42
6/28
S/30
F E N A L £ S
2/41
8/27
8/29
FEMALE DOSE *
ONI TINE SKIN
APPLICATION (0 IML!
KEPITXTIVt *KIN
T«f ATWftlTS (O.lm.)
• 5 us/. 025 KG t.u.
3X/MEEH
85.7 KG/KG/OAT
2 »f*BS
PERCENT
7
21
17
5
30
28
P-VALUE
0.08
0.18
0.007
0,01
rATISTICAL
HFERENCE: ITCWJ INCIDENCE! »eK*us tDMBA/TCOO INCIDENCE]
1ALES, P e 0.50, FEMALES, P = 0.55 -— MO DIFFEflENCES
««ro«E, TCCD TREATMENT APPARENTLY NOT DIFFERENT FROM
TMI OMBA/TCDD TREAMENT,
Fig. S
-
-ss-S^itjiial^'S^fe::* f: :^ ' ^ -
KOCI1A 119781 SAVA6E STUDY *--^ - -
-&••'**H»
CANCER INCIDENCE IN SHUSUE-DAMUY RATS
Jf| [EVIDENCE FOR INDUCED CAKEK FROM 2.3,7,8-TCDO]
.; *j|i.: i-j-i-; ^^^^•ff^g^^r^^^^gg^^^y^.-^-,!:jft»;iy>--
CANCER
TYPE/SEX
M
HARD
PALATE
F
M
2,
0
(PER
0/85
(0)
0/86
(0)
0/85
' TONGUE (0)
F 1/86
M-
LIVER
F
M
LUNG
F
(1.1)
2/85
(2.3)
1/86
(1.1)
5/65
(5.8)
0/86
(0)
3,7,8-TCDD
1.0
e • * T
0/50
(OS
0/10
(0)
1/50
(2!
0/50
(Q!
0/50
(0)
0/50
(0)
0/50
(0)
0/50
(0)
DOSE-RATE
10
I N C I D f
'O/SQ
(0)
1/50
1/50
(2)
0/50
(0!
0/50
(0)
2 '50
(ii
• 0/50
(0)
0/50
(0!
(NG/KG/OAY)
100
NCI)
4/50«
(8)
4/49*
(8)
3/SO«
(6)
2/49
(41
1/50
(2)
11/49.
(22)
1/50
' (21
7/49.
114)
RESULT
* INCREASE
«• INCREASE
(P =0.016>
* INCREASE
(P s .048)
NO EFFECT
NO EFFECT
. INCREASE
-------
The degree of cellular malignancy associated with the in vitro focus formation was likely significant because type
III cells [defined originally by Reznikoff et a/., 1973] were scored. Since transformation is viewed to be an
essential series of cellular conversions in the development of cancer, these observations in 10 T 1/2 cells indicate
this subset of cellular transformations is caused by TCDD. The connection between in vitro transformation to
tumor promotion is not well understood as of yet, however, and can only be surmised as being part of the process.
Although TCDD facilitates cell transformation like a tumor promoter, TCDD does not inhibit intercellular
communication - as many tumor promoters have been observed to do (Williams, 1980;Troskoe/a/., 1981) measured
by the 3H-Uridine cellular exchange, or by movement of Lucifer yellow dye among cells in intercellular transfer
assays (Boreikoe/a/,, 1986). This lack of interference in cell-to-cell communication suggests that TCDD is either
not a lumor promoter, or that TCDD is a tumor promoter acting by a different mechanism than a classic promoter
such as TPA.
Two of the major re-programming cellular effects caused by tumor promoters are increased mitosis in some cell
subpopulationsand increased terminal cellular differentiation in other cell populations (Willey J.C., et al., 1984).
For example, TCDD has been found to increase keratinizadon in skin (Knutspn and Poland, 1980). Hyperplasia
from TCDD exposure is seen in vitro in cultured human keratinocytes, in exposures to human epidermis, and in
animal systems (Milstone and LaVigne, 1984; Gianotti, 1977; Poland and Knutson, 1982). Both properties of
increased cellularity and maturation are descriptive of tumor promotion activity but are also manifest by
complete carcinogens, which possess both initiation and promotion activities.
EVIDENCE OF TCDD ACTING ALONE AS A TOTAL CARCINOGEN
Sprague-Dawley rats were gavgged with average daily TCDD doses of 0,1,10, and lOOng/kg/day (Kocibi.ef a/.,
1978). The summary of the Kociba study is presented in Fig. 5. Increases in malignant tumors were observed in
four different organs: hard palate (including nasal turbina te cancers), tongue, liver, and lung. This tumor activity
is carcinogenic activity since TCDD caused malignant tumors in these organs (Kociba, 1984). Mechanistic
activities of tumor initiation, promotion, and progression are all indicated by these rat malignant responses to
TCDD oral exposure.
It is interesting to note that the cancer responses were positive at the 100 ng/kg/day dose and not at 10 ng/kg/day
(Fig.5). The same pattern of response was seen (100 + and 10 •) in the Pilot tumor promotion experiment where
TCDD was administered to rats subcutaneously (Pilot et a/., 1980). The observations that different routes of
TCDD exposure show the same break in the cancer response curve (at the same dose level shows a null response),
in radically different protocols, and in different rat strains, all suggest that this dose region may be one that
observationally defines (not proves) an apparent cancer threshold for TCDD in the rat.
It is also interesting to note (in the same experiment): not only were tumors increased but also tumors were
decreased by TCDD. These tumor decreases are shown in Fig. 6 and Fig. 7. We note one exception to tumor
incidence decreases, i.e. an increase in benign tumors of the adrenal gland cortex. However, decreases were
observed at the same time in adrenal pheochromocytomas (Fig, 7), The tissues affected by TCDD-related cancer
decreases are all endocrine organs, thereby strongly suggesting the mechanism of action of 2,3,7,8-
tetrachlorodibenzo-p-dioxin is linked to these hormonal systems. This "protection effect" of TCDD has not been
adequately discussed or factored into any complete hazard evaluation or risk assessment of TCDD as of yet. This
dichotomous tumor pattern imposes an additional complexity when ascribing the "estimated" carcinogenicity
hazard from TCDD exposure and how this relates to human carcinogenicity risk.
Later work on TCDD carcinogenicity has been reported by the NTP in Osborrie-Mendel rats and B6C3F1 mice
(U.S.NTP, 1982b). Hepatocellular carcinomas are the dominant cancer response in the Osborne-Mendel rats with
females and males demonstrating carcinogenicity increases only at the highest dose tested (Fig, 8). A mostly
benign response to TCDD was also observed in the follicular thyroid at the highest dose and in both sexes. It is
notable that tongue, hard palate, and lung tumors did not appear increased in the Osborne-Mendel rat in this NTP
study as in the above Kociba study in Sprague-Dawley rats (cf, Fl|. 5). However, the liver repeated as being a
cancerous site between the two studies as well as increases in an endocrine organ (thyroid), whereas decreases
were observed in Sprague-Dawley rat endocrine organs (pituitary, pancreas, thyroid, uterus, mammae, and
adrenals) with only benign tumors increased in the adrenal cortex Sprague-Dawley rat.
A similar carcinogenicity response in the NTP study was observed inB6C3Fl mice(Fl§, 9). That is, in the mouse
just as in the rat, liver and thyroid tumors were also increased by TCDD exposure.
DISCUSSION
It is clear that repetitive TCDD exposure, after a subcircinogenic initial exposure of a carcinogen, can bring
about or promote tumors. This activity of tumor promotion seems to be manifest in Charles River rats and HRS/J
-------
Fig. 6
KOCISA [1978! GAVA6E CANCER IHCIDDCC IN SPiASUE-OAMLElf RATS
[EVIDENCE FOR DECREASED TIMOR RESPONSE FROM 2. 3, 7.8-TCDO]
Fig. 7
KDCIBA [1978] SAVAGE CANCER INCIDENCE IN SPRA&UE-OAKLEY RATS
IEVIDEKCE FOR OECtEASED TUMOR RESPONSE FSOK 2.3,7,8-TCDO
ORGAN SITE /
MALIGNANCY
STATUS
S = SCN1CN
PITUITARY M
In » B)
F
PANCREAS
Ac I MB N
(M * 8>
' ISLIT F
(M • B)
THYROID K
Iw * B)
f
0
E
26/85
(30)
49/86
(17)
28/85
(33»
5/86
(5.81
11/85
(13)
18/66
(21)
NOTE:' • INDICATES
8-TCOD* DOSE-RATE
1.0 10.0
BCtuT INC
6/50*
(12)
18/50
(3$)
6/10.
112)
3/50
16)
0/50.
(0)
3/50.
P-VALUE
(NS/KG/DAY
100
I 9 I N C
11/30 13/50
(221 (26)
13/50 14/49.
(36) (28)
12/50
1/50
(2)
1/50»
(2)
2/SO.
(4>
LESS THAN
9/50-
(18)
0/49
(0)
7/50
(14)
6/49
(12)
O.US
RESULT
I)
LOU DOSE I
DECREASE ¥
ONLY HID
OOSE I
DECREASE V
LOU 1 HID
DOS! !
DECREASE V
ORGAN SITE/
MALIGNANCY
STATUS
M - MALIGNANT
S = ICKlGN
UTIRUS F
(B ONLY.)
MAMMAE F
(I 9NIY)
ADRENAL
IB OKLY)
F
ADRENAL
M
(B ONLY)
F
2,3,7,8-TCDO DOSE-
0 1-0 10
C 0 N T I N
28/86 12/50
(331 (241
73/86 35/50
(85) (70)
U * T I
11/50
(22)
36/59
(72)
RATE IN;
100
0 N OF
7/49-
(14)
24/49.
(49)
ADBINAL FHIOCHMMOCYYOMAS
28/85 6/50. 10/50 4/50.
(33) (12) (20) <8>
7/86 2/50 1/50 3/49
(8) (4) 12) 16)
U»1CM*L
0/85 0/50
(01 (0)
9/86 6/50
110! (12)
C01TII
2/SO
(4)
2/50
(4)
5/SO*
(10)
5/49
(10)
K5/OAY )
KOCIIA STUOY
HIGH
OOSE !
DECREASE V
HIGH
DOSE I
DECREASE V
LQM i HIGH
OOSE
DECREASE \
NO EFFECT
(EXCEPT
HIGH
DOSE
INCREASE
NO EFFECT
ON)
NOTE: • INDICATES F-VALUE LISS THAN 0.05.
Fig. 8
::?~: i'.'•"?. f '-Kit •&&%'*. i-3'ffiiElRSIfjJJH'lBBS
. : U.S. NATIONAL TOXICOUWY
I!',! C19821 6AVAiE STUDY m THE RAT
'•'• RESULTS r» OS«O««E-HiKOEL R*TJ (DutcmoiMS * AotMM*$>
SSSiHEIj S!
Fig. 9
U.S. KATIONAL TOXICOLD6T PRD6RAM
[19821 SAVAGE STUDY (N THE HOUSE
RESULTS m B6C3F1 Mice (CARCINOMAS * ABENOWAS!
ORSAN
SITE
0
LIVER
« 0/124
(0!
F 5/124
(4)
2,3,7,8-TCDD DOSE-RATE
3,0 7.0
S * C C N T INCIB
0/50
(0)
1/49
(2
THi'RQ'f.O' - FOUICULAH,
M 6/118 »/4«'
(5) (10)
' F 5/122
(4)
2/45
(4)
0/50
(0)
3/50
MOSTLY ADENOMAS
8/50*
(16)
f * .02
1/49
(2)
(N6/K6/B«Y)
70 RESULT
C N C t)
4/50*
(8)
15/49.
(31)
P • 5
11/SO.
(22)
P •
6/47.
(13)
P s
INCREASE
006
INCREASE
x 10-*
INCREASE
.002
INCREASE
.05
ORGAN
SITE
2,3.7,8-TCDO DOSE-RATE (NO/W/DAY)
0 3.0 7.0 70
(PERCENT INCIBtN'CE)
LIVER
H
31/123
(25)
12/49
(24)
4/123 8/SO.
(3) (12)
P = .03
13/49 27/50. INCREASE
(27) (44)
P = 3 x 10'4
5/48. 11/47. INCREASE
(13) (23)
f s ,03 f = 2 » 10"*
THYROID -
M
F
FQLLICULAD
0/114
(0)
0/136
(0)
P
BENISN,
3/48
(6)
3/50.
(6)
« ,02
ONLY
0/48
(0)
1/47
(2)
0/49 NO EFFECT
(0)
5/46- INCREASE
(11) ,
P * 8 x 1C'4
-------
hairless mice (hr/hr), but not in some "normal" mice strains (Swiss-Webster or CD-I) which are presumed to be
wild type at the hr locus. ID the hairless mouse cross experiments mice which were heterozygous (hr/+) at the hr
locus also did not show tumor promotion activity with TCDD suggesting the hr trait may be a accessary element
for mouse skin tumorigenicity .
It appears, then, that tumor promotion can be observed in some test strains, and not in others. Furthermore, it
is noted (Fig. 3) in Swiss-Webster mice that, although TCDD skin tumor promotion is not observed in DMBA
treated mice, TCDD by itself caused significant increases in fibrosarcomas in females (p=0.007) and a borderline
response in males (p-0,08). These results suggest that although TCDD did not promote or enhance tumors in this
test system it can cause carcinogenesis. This points to a total carcinogen mechanism in this system for TCDD. In
a single report TCDD showed tumor initiation activity in the mouse (Digiovanni, 1977), Consequently, we surmise
from all the TCDD tumor promotion data that TCDD has tumor promotion characteristics only in certain strains
tested in the two-stage carcinogenesis system.
The cancers observed by Kociba and his collaborators in Sprague-Dawley rats were tumors in liver and squamous-
cell carcinomas of the lung, the later being an uncommon tumor type occurring spontaneously in the iung. Also,
the tongue and hard palate responded with carcinomas both of which are direct-route sites and are rare tumors.
The occurrence of tumors is revealing as to the carcinogenicity of TCDD: (1) it strengthens the causal relationship
between chemical exposure and cancerous outcome because the outcome is improbable due to random chance, and,
(2) it indicates the probable effect of TCDD is'likely direct on these tissues to cause cancer rather than
"promoting" cryptic initiation lesions Jhypothetieally caused by prior events] in these normally cancer-free
tissues. Were this latter point not true, then it would be expected that the theoretical initiation lesions, if really
present, might nonspecif ically interact with other promotion agents, such as exist in the diet, to cause cancer. But,
cancer is only rarely observed in the hard palate and tongue of the rat.
So it is reasoned that analogous to a complete cancer-causing agent in man, such as tobacco smoke which contains
known initiation and promotion agents and is carcinogenic to the human tongue, hard palate, and lung, TCDD
can also be carcinogenic to these same tissues in the Sprague Dawley rtt thereby indicating that TCDD is a
complete carcinogenic agent, at least in the rat. It remains to be proven whether TCDD is also carcinogenic to
these tissues in TCDD-exposed humans. Since no other known agent participated in the rare cancer formation
in the Kociba TCDD rat study, then it is reasoned that TCDD is a complete carcinogen possessing both initiation
and promotion properties, although the proportional degree of each is not yet known; The relative proportion of
the initiation/promotion mechanisms need not be the same among compounds testing positive for cancer in test
species and may not necessarily correlate with the same proportion of these mechanism types in man.
Tumor promotion is not a new concept. Inception of this etiology took place in the 1920's by investigators who
suggested a noncarcinogenic effect such as wound healing could evoke cancer in some cases, called traumatic
cancer (Deelman, 1924, 1927). Later, the seminal work of Peyton Rous showed that certain chemical treatments
could "seed* apparently normal tissue with persistent cells, in which later in time could be stimulated or enhanced
by other noncarcinnogenic chemicals (or even by mechanical stimulus) to form tumors in the seeded tissues (Rous
and Kidd, 1939; MacKensie and Rous, 1941). These authors referred to the latter stimulus which caused cell
growth leading to tumors as "extraneous encouragement agents", and it was later defined as promoting agents
(Fried wald and Rous, 1944). In this work the term initiating agents was also given to the irreversible carcinogenic
action of chemicals which would start the process. With such concepts of stages of effects in sequence started the
conceptualization of the commonly accepted initiation/promotion hypothesis of carcinogenesis which still is
considered a valuable working model today.
Other investigators pursued the initiation/promotion concept referring to it as "specific cellular
reaction'/developing factor (Mottram, 1945), and initiation process/promotion process (Berenblum and Shubik,
1949). These studies, and other genetic studies, provided support for the two stage hypothesis (Olinos, et aL, 1951).
The operative concept was also supported by the evolving somatic mutation hypothesis (Berenblum and Shubik,
1949): cancer was determined to arise from a somatic mutation of a few cells [there being variation among these
few mutated cells as to the type of lesion (Shubik, 1950)]. Some of these cells, in time, could be developed by a
number of diverse agents to unscheduled cellular growth which proceeded in greater abundance [presumably]
from mutated cells to hyperplastic regions, tumors, and finally in some cases to cancers which disseminated
throughout the body. These developing agents, which by all authors were referred to as noncarcinogens, enhance
the chances of cancer in initiated tissues and were referred to as tumor promoters.
This concept of tumor promotion has been studied by a number of investigators (Boutwell, 1964; Van Duuren,
1969; Berenblum, 1969; Shubik, 1984; Felling and Slaga, l985;Schulte-Herman, 1985) since the foundation works
discussed above. The concept of tumor promotion in our opinion may be generally defined as:
Promotion is a reversible set of cellular processes which can cause unscheduled and/or mis-controlled cellular
growth allowing for relative enrichment of initiated cells compared to the normal field of cells in which these
initiated cells reside. Promotion leads to expanded clones, as well as. allows for reversible qualitative changes
in the normal cellular differentiation process. Cancer can result from this promotion process if further changes
in genetic expression {progression} are imposed on the exposed field of cells,
(
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For other discussion of tumor promotion definition refer to U.S. EPA, 1987.
It is instructive to note that (1) tumor promotion does not take place in the absence of initiation, i.e. initiation
precedes promotion which in turn precedes progression into malignancy, and (2) tumor promoters are not
carcinogens. We reason that TCDD produces cancers (malignancy) alone without any known prior tissue .initiation
which indicates TCDD is a complete carcinogen needing no assistance in causing cancer. If TCDD were iust a
tumor promoter as has been suggested (Shu et al., 1987), then all the responding tissues (liver, lung, hard palate,
tongue, skin, and thyroid) would of necessity have already been initiated at the time of TCDD exposure. We view
this alternative as unlikely since the responses occur in a number of species/strains, and in a number of organs,
including palate and tongue which are rare sites, These occurrences are not indicative of promoters studied to
date which usually affect one organ, and only if that tissue has been initiated previously. We know of no
published example where just a promoter exposure alone caused a dose-response of malignant tumors at a rare
site. Lastly, even though it may be argued that the tongue and hard palate may have extraordinarily high TCDD
exposures, this does not necessarily account for the rare carcinogenic response of these tissues any more than
other carcinogens which are similarly administered by gavage. We conclude there must have been some specificity
for the tongue and hard palate in the Sprague Dawley rat in the Kociba study. However, since this response was
from a single study, it will be necessary to replicate these results in these tissues in order to increase certainty
of the hard palate, tongue, squamous lung cancer responses.
A control often done in earlier work was to test whether a chemical could promote itself (e.g. Boutwell, 1964),
This control started the process by a subcarcinogenic dose of the suspected carcinogen. If no other treatment is
given, no tumors result; but if repetitive isoquantal doses of the same test chemical are applied, tumors dj. result
if the chemical is a carcinogen. This is interpreted to mean that processes of initiation and promotion occur, i.e.
a total carcinogen can promote itself. We view TCDD as having promotion activity, but this does not preclude
that TCDD is a total carcinogen capable of promoting it's own initiation process. It should not be a surprise, then,
that at the right dose-rate total carcinogens wilt act as tumor prompters in tumor promoter assays. We view TCDD
acting this way in the tumor promotion assays reviewed here (Pilot el at., 1980; Poland and Glover, 1982).
It remains then to address what kind of carcinogen is TCDD. The present genotoxicity information on TCDD is
mostly negative although positives do occur (U.S. EPA, 1985). The mostly negative genotoxicity results suggest
that TCDD is not generally positive by current methodology, and the true genotoxicity may not be known since
the appropriate end-point may not have tested yet. Some authors have taken the position that TCDD must act
upon the genetic material [but by an unknown mechanism] (Giri, 1986). The classical mutation mechanism
whereby covalent-binding mutagens alter the DNA content may not be operating in the case of TCDD. TCDD
may alter the genetic informational flow by a here-to-fore undescribed carcinogenic mechanism.
Due to the extended residence times of TCDD in the body {(t1/2), «• 1-4 mo. in rodents and 6-10 yrs.in man],
TCDD could interact with functional chromatin, cell generation after cell generation, so as to alter genetic
expression by persistent phenotypic changes. TCDD likely interacts by noncovalent binding in the rat since DNA
measured covaknt binding s 1 TCDD molecule/1011 nucleotides (Poland and Glover, 1979). However, assuming
2 x 109 base pairs/cell, the estimated number (maximum) of hits in the rat are:
1 molecule TCDD x 4»10g nuclootides ss j molecule of fGDP
10" nucleotid«B on« «ukaryotic call 25 calls
This hit frequency of TCDD covalent linkage to DNA is very low in rats being less than one hit per cell and
4-6 orders of magnitude less than most chemical rat carcinogens (Poland and Knutson, 1982a). However, in long
term human TCDD exposure over 50% of fecal label has been reported to be TCDD-metabolites (Wendling et.al,,
1988), thereby not ruling out the possibility of tumor initiators being generated [over long periods] in man.
The relationship of TCDD cellular actions is known to be related to a specific cytosolic receptor protein Ah,
which binds tightly to TCDD, Kd » 0,3 * 10"f M~l. The receptor-ligand complex is thought to initiate and
promulgate many of the pleiotropic cellular effects of TCDD including wasting of some tissues and hyperplasia
in others (Poland and Knutson, 1982). One of the most characteristic TCDD-induced biochemical events is a
rapid increase in P-450 enzymes. This event leads to an important observation: pretreatment of animals with
TCDD can abrogate subsequent cancer responses from known carcinogens (DiGiovanni et al,, 1980). Also
interesting, and perhaps related, is the TCDD-related suppression of tumors in the pituitary, pancreas, thyroid,
uterus, mammae, and adrenals. Such tumor incidence suppressions are likely to be related to TCDD ** hormonal
influences in these tissues. Neither of these negative carcinogenic influences caused by TCDD have been factored
into the hazard evaluation of TCDD in any risk assessment to date.
Evidence is continuing to build from a number of laboratories that TCDD affects the adrenals and thymus
(Greenlee et at., 1985), thyroid (Rozman et a/., 1984; Henry arid Gasiewicz, 1987; Romkes, et al., 1987), estradiol-
(Umbriet et al., 1988) glucocorticoid and cholesterol-producing systems, as well as epidermal growth factor
cellular activity (cf. Greenlee ei at., 1987, and references therein). It has been suggested that (1) there is a
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relationship of the TCDD Ah receptor protein function to the steroid and thyroid receptor protein functions, and
(2) the relationship may be due (in part) to sequence homology among these receptors (Evans, 1988). There are
no cross-bindings of these ligands among the other systems'reccptors. The sharing of a common motif among the
essential receptor proteins indicates a superfamily of receptors which coordinate major cellular systems (Green
and Chambon, 1986). TCDD may cause its pleiotropic effects by perturbing the superfamily gene expression by
interaction with each of the various hormone-receptor reaction sequelae at their respective functional sites in
chromatin.
Given the hormonal relationships of TCDD to cancer (at least in animal test systems), we postulate that TCDD
is a hormonal carcinogen. TCDD may be unique in its close association with so many essential organismic control
systems, and might be expected to act in a hormonal-like fashion. Moreover, the tight hormone-like binding to
the Ah receptor may relate to the supreme cancer potency estimate for TCDD. As a comparative example,
estimated cancer potency [to humans, units are in (mg/kg/day)"1] based on rodent positive tests are: TCDD
(156,000), aflatoxia BI (2,900), ethylene dibromide (41), benz[a]pyrene (11.5), bis-(2-chloroethyl)-ether (1.1),
DDT/DDE (0.34), vinyl chloride (0.017), and methylene chloride (0.008). Part of this extreme potency is no doubt
due to the accumulation of this mctabolically stable compound. Lastly, the carcinogcnicity effects of TCDD in
hormonal organs are also expected to be influenced by the interplay hormonally among the interactive target
systems, i.e. direct TCDD effects in one target affect a second target [in part] by disturbance of homeostasis
between the targets.
We conclude: the bioassay data designate TCDD as a total carcinogen, which can participate in tumor promotion
assays so as to complete the carcinogcnicity action started by other carcinogens. TCDD is unique in its supreme
cancer potency and close functional relationships with a number of key hormonal regulation systems. Inference
to other hormonal actions suggests that TCDD may also mechanistically show a threshold, which would suggest
that the traditional assumption made with all carcinogens that there is a finite positive chance of cancer from
even at one molecule of exposure (some 15 orders of magnitude lower than total body burden of TCDD) may not
be the correct assumption to make in the case of TCDD. The existence of a threshold has not yet been
demonstrated and further work on TCDD dosimetry needs to be done to properly assess the carcinogenic hazard.
We think the uniqueness of this compound offers an excellent investigative tool for hormonal carcioogenicity
mechanisms. The derivation of risk from human exposure, using quantitative modeling should take into account
the qualitative biological concepts discussed here: complete carcinogenicity, presumptive nongenotoxicity or
mutation, probable dose-rate limitation at low doses, and hormonal systems interactions.
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TECHNICAL REPORT DATA
(Please read Instructions ,6'n Mf reverse before completing}
1. REPORT NO.
EPA/600/D-90/Q06
4. T.ITJLE AND SUBTITLE
2,
Analysis of 2,3,7,8-TCDD Tumor Promotion Activity
and its Relationship to Cancer
6, ee«FgR(YHNG ORGANIZATION (ODDE
E'PA/bod/021"
3,
J
§, .B.E.P.QPT'PATE
March:. 1990
7. A,yT,HQR.
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