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Laboratory of Toxicology, National Public Health Institute, FIN-70701 Kuopio, Finland [M. V., J. T. T., M. U., R. P., J. M-P., J. T.]; Institute of Environmental Medicine, Karolinska Institutet, S-171 77 Stockholm, Sweden [Y. B., G. S., S. F., L. W.]; AstraZeneca, R&D Södertälje, Safety Assessment, S-15185 Södertälje, Sweden [Y. B., L. W.]; Department of Pharmacology and Toxicology, University of Kuopio, FIN-70701 Kuopio, Finland [M. U.]; National Food and Veterinary Research Institute, Regional Laboratory of Kuopio, FIN-70701 Kuopio, Finland [R. P.]; Department of Food and Environmental Hygiene, Faculty of Veterinary Medicine, University of Helsinki, FIN-00014 Helsinki, Finland [R. P.]; Department of Pathology and Forensic Medicine, University of Kuopio and Kuopio University Hospital, FIN-70211 Kuopio, Finland [V-M. K.]; Laboratory of Chemistry, National Public Health Institute, FIN-70701 Kuopio, Finland [T. V.]; and Institute of Ecological Chemistry, GSF-National Research Center, D-85758 Neuherberg, Germany [C. K., K-W. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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100-fold)
sensitivity difference between L-E and H/W rats. The same sensitivity
difference but 10-fold less foci were observed between
nonhepatectomized/noninitiated L-E and H/W rats. Induction of AHF was
related to hepatotoxicity but not to cytochrome P4501A1 activity in the
liver. Liver TCDD concentrations were similar in both strains. H/W rats
are exceptionally resistant to induction of AHF by TCDD, and the
resistance is associated with an altered transactivation domain of the
aryl hydrocarbon receptor. Genetic differences may account for
significant interindividual/intraspecies sensitivity differences in
dioxin-induced carcinogenesis. Understanding the role of
transactivation domain of the aryl hydrocarbon receptor in
carcinogenesis is therefore likely to improve dioxin risk assessment. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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TCDD induces a variety of biological responses ranging from induction of cytochrome P-450 1A (CYP1A) to reproductive and developmental defects, immunotoxicity, thymus atrophy, epithelial disorders, liver damage, wasting syndrome, and cancer (reviewed in Ref. 3 ). In a recent re-evaluation of the carcinogenicity of TCDD, the IARC has upgraded the classification from possible human carcinogen (group 2B; Ref. 4 ) to human carcinogen (group 1; Ref. 5 ). The evidence for carcinogenicity of TCDD was considered limited in humans and sufficient in experimental animals. In lifetime bioassays, TCDD was found to be a multisite carcinogen in both genders of all animal species studied (rats, mice, and hamsters), causing several tumor types at sites distant from the point of administration. On the other hand, TCDD does not interact with DNA, and despite a few conflicting reports, it is not considered directly genotoxic (reviewed in Ref. 5 ). Accordingly, in two-stage initiation-promotion models, TCDD seems to lack tumor-initiating activity. There is, however, an overflow of data indicating that TCDD is a potent tumor promoter in rat and mouse liver and lung, as well as in mouse skin (reviewed in Ref. 5 ; see examples in Refs. 6, 7, 8, 9, 10, 11 ). TCDD has also proved to be positive in cell transformation assays measuring tumor-promoting activity in cultured rodent or human cells in vitro (12 , 13) .
The current risk assessment of dioxins has mainly been based on a 2-year carcinogenicity bioassay with TCDD in Sprague Dawley rats (14) . It uses the incidences of liver neoplasms in females as the critical end point of toxicity. Nevertheless, the mechanisms of TCDD-induced carcinogenesis are incompletely understood. In the absence of direct genotoxicity, several possible mechanisms have been proposed. Specific binding of TCDD to AHR and the subsequent induction of gene expression seems to have an important role in mediating a variety of toxic effects of TCDD (15, 16, 17, 18) . Activation of AHR may also be involved in the carcinogenicity of TCDD, but the details of its role in different stages of carcinogenesis remain unclear (5) . In rats (but not in other rodent species), females seem to be more sensitive to the hepatocarcinogenicity of TCDD. Ovarian hormones are likely to be involved, because ovariectomy has been shown to inhibit promotion of TCDD-induced preneoplastic foci and liver tumors (8) . Indirect genotoxicity attributable to TCDD-induced CYP1A2- or CYP1B1-mediated metabolism of estradiol to catechol estrogens and subsequently increased formation of reactive oxygen species has been suggested (19 , 20) . Possible epigenetic mechanisms of TCDD-induced hepatocarcinogenesis include selectively increased cell proliferation and, more importantly, reduced apoptosis in focal cell populations, resulting in net growth of AHF (21) , down-regulation of epidermal growth factor receptor in liver (22) , altered gap-junctional communication (23 , 24) , and increased expression of the proto-oncogene product ras p21 protein (25) . Furthermore, there appears to be a distinct correlation between hepatotoxicity and the development of hepatocellular neoplasms (14 , 26) .
A characteristic feature in the toxicity of dioxins is exceptionally large sensitivity differences among animal species or even strains of the same species. These differences, although best illustrated for acute lethality, together with lack of understanding of the critical mechanisms, highly complicate the risk assessment of dioxins. An animal model based on >1000-fold sensitivity difference in acute lethality of TCDD between two rat strains has been established in our laboratory. L-E (Turku/AB) rats are highly sensitive, having an LD50 of 10 µg/kg. H/W (Kuopio) rats, on the other hand, are the most TCDD-resistant mammals known with an LD50 of >9600 µg/kg (27 , 28) . The toxicokinetics of TCDD is nearly similar in these strains (29) . No substantial differences between these rat strains could be detected in the amount of hepatic AHR, binding affinity of TCDD to AHR, or specific binding of the activated AHR to DNA (17 , 30) . However, the AHR of H/W rats was shown recently to harbor a point mutation, which results in an insertion/deletion type alteration at the 3' end of the coding region of cDNA (16) . At the protein level, the molecular mass of the receptor is smaller because of a loss of amino acids from the transactivation domain. Although this deviant AHR seems to account for the tremendous resistance of H/W rats to acute lethality and intermediate resistance to liver toxicity (18) , H/W rats and L-E rats show nearly similar sensitivity to induction of CYP1A1 activity, thymic atrophy, embryotoxicity, or decreases in serum thyroxine and melatonin levels (reviewed in Ref. 3 ). This animal model is therefore highly useful for studying the significance of intraspecies sensitivity differences in end points critical for risk assessment of dioxins. The objective of the present study was to compare the sensitivity of L-E and H/W rats to the liver tumor-promoting activity of TCDD in relation to liver TCDD concentrations and other end points of toxicity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Animals.
Inbred female L-E (Turku/AB) and outbred female H/W
(Kuopio) rats were obtained from the breeding colony of the
National Public Health Institute (Kuopio, Finland), kept in an specific
pathogen-free barrier unit. The animals are regularly subjected to
health surveys consisting of serological and bacteriological screening
as suggested by the Federation of European Laboratory Animal Science
Associations (FELASA) (31)
. These surveys indicated
that the animals were free of typical rodent pathogens. The rats were 5
weeks of age and weighed 70.1 ± 7.8 g (L-E;
mean ± SD) or 81.7 ± 3.9 g (H/W)
at hepatectomy. They were housed in stainless steel, wire-bottomed
cages 5 rats/cage and given standard pelleted R36 feed (Ewos,
Södertälje, Sweden), and tap water ad libitum.
The room was artificially illuminated from 7 a.m. to 7 p.m.,
the ambient temperature was 21.5 ± 1°C, and relative
humidity was 55 ± 10%.
Experimental Design.
Experimental groups and dosages are shown in Table 1
. In addition to the main study, a
separate experiment (satellite groups) of high-dose levels was carried
out on the resistant H/W rats. In this experiment, H/W rats were
exposed to total doses of 170 µg/kg (the same as the highest dose of
the main study) or 1700 µg/kg (given either as weekly doses or as a
single dose).
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At termination, the rats were anesthetized with CO2/O2 (70/30%). Blood samples were drawn from the left ventricle, and the rats were exsanguinated by cutting the aorta. The rats were subjected to cross necropsy tissue sampling for preneoplastic hepatic foci and histopathology. The weights of liver and thymus were recorded.
The study protocol was approved by the Animal Experiment Committee of the University of Kuopio and the Kuopio Provincial Government, and it was in accord with institution guidelines.
Analysis of AHF.
Liver samples were fixed in ice-cold acetone, embedded in paraffin, cut
to the thickness of 4 µm, and immunohistochemically stained for
GST-P, the placental isoform of GST. GST-P-positive foci were
quantitated using a Leica Aristoplan microscope connected to a
Quantimet 570 Image Processing and Analysis system (Leica Cambridge
Ltd., Cambridge, United Kingdom; Refs. 33
and
34
).
Biochemical Assays.
Plasma samples for activities of ALAT, ASAT, and GGT were analyzed
according to the guidelines of the Committee on Enzymes of the
Scandinavian Society for Clinical Chemistry and Clinical Physiology
(1974 and 1979) using a selective chemistry analyzer (Kone Specific;
Kone Instruments, Espoo, Finland).
Hepatic CYP1A1 activity was measured as O-dealkylation of 7-ethoxyresorufin (EROD) in S9 fraction using a Shimadzu RF-5000 spectrofluorometer (33 , 35) .
Histopathology.
Liver samples were preserved in 10% neutral buffered formalin,
dehydrated, embedded in paraffin wax, and cut to the thickness of 5
µm. The tissue slices were mounted on glass slides, stained with
Mayer’s H&E, and examined using light microscope by one pathologist
(V-M. K.). A semiquantitative scoring of lesions (+, a mild response;
++, a moderate response; and +++, an extensive response) was used where
appropriate.
Analysis of Micronucleated Erythrocytes.
In the main study, MNPCE and MNRET frequencies were studied in 5 -/-
and in 5 or 10 +/+ rats of both strains. In addition, micronuclei were
analyzed in PCEs and in RETs of +/+ rats of the satellite groups. For
the analysis of MNPCEs, femurs of the animals were dissected out at
necropsy and processed according to Schmid (36)
. For the
analysis of MNRETs, blood samples were collected by cardiac puncture in
anesthetized animals at necropsy. The smear slides were fixed in
methanol, air dried, and stained with acridine orange as described by
Hayashi et al. (37)
. One thousand bone marrow
PCEs and one thousand RETs per animal were analyzed for micronuclei by
fluorescent microscopy. The proportion of PCEs to normochromatic cells
in the total erythrocyte population of bone marrow was scored by
counting the cells until the score for one cell type reached one
thousand. The frequency of immature erythrocytes in peripheral blood
(RETs) was analyzed in 500 (main study) or in 200 (satellite groups)
erythrocytes per rat. The analyses were done on coded slides by one
microscopist (J. M-P.).
Analysis of Liver TCDD Concentrations.
Liver TCDD concentrations for the main study were determined in five
rats/each dosage group. In addition, background liver concentrations of
CDDs and polychlorinated dibenzofurans at the end of the study were
determined in untreated control and vehicle control animals. About
0.2 g of each lyophilized rat liver was ground with 5 g of
Na2SO4 and brought onto a
column previously filled from the bottom to the top with 5 g of
Na2SO4, 2.5 g of
SiO2, 5 g of SiO2/44%
H2SO4, and 2.5 g of
SiO2/33% NaOH. After addition of the
13C-labeled standards for quantification, the
column was eluted with 140 ml of cyclohexane:dichloromethane (50:50,
v/v). After reduction to a small volume, the eluate was chromatographed
on a glass column containing 4 g of activated
Al2O3 with
hexane:dichloromethane of different ratios. Just before GC/MS analysis,
1,2,3,4-13C12-TCDD was
added as recovery standard. Separation of the CDDs and quantification
were carried out by capillary GC-MS with a high resolution mass
spectrometer (Finnigan MAT 95 s, Argenteuil, France) in
EI-/SIM-mode at a resolution of r = 10,000.
Operating conditions for GC (HP 6890 Series) were according to Schramm
et al. (38)
. The quality of all solvents was
for residue analysis (Promochem, Wesel, Germany), and the other
chemicals were of the highest purity available.
Liver TCDD concentrations for the satellite groups were determined for two to eight rats/treatment group at the National Public Health Institute (Kuopio, Finland). The weighted samples were spiked with 840 pg of 13C-labeled TCDD/F standard (Cambridge Isotope Laboratories, Inc., Woburn, MA), mixed with anhydrous Na2SO4, dried in an oven at 80°C, put at the top of the acidic silica gel column, and cleaned and analyzed according to Kiviranta et al. (39) .
Statistics.
For comparisons between groups of the main study the data of continuous
variables with presumed normal distribution were tested for homogeneity
of group variances using the Bartlett’s test. If the variances were
homogeneous as such or after appropriate transformation (at the level
of 0.01), comparisons between treatment groups were performed using the
one-way ANOVA, followed by the Least Significant Difference test. If
the variances were heterogeneous, comparisons were made using the
nonparametric Kruskal-Wallis ANOVA, followed by the Mann-Whitney
U test. Incidences of histopathological findings were
analyzed using Fisher’s exact test. Frequencies of micronucleated
cells and immature erythrocytes were analyzed by the Mann-Whitney
U test. All tests were two-sided.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. +/+ rats
gained more weight than -/- rats, and this difference was more
conspicuous in L-E rats. In +/+ L-E rats, the increase in body weight
during the TCDD treatment period (from week 5 to week 25 of the study)
was 78.3% (P < 0.001) and 44.4%
(P < 0.001) of controls at 1.7 or 17
µg/kg, respectively, whereas in the -/- L-E rats, the difference
was significant only at 17 µg/kg (63.3% of controls;
P < 0.001). In H/W rats, the increase in
body weight at 17 and 170 µg/kg was 81.4 (P < 0.05) and 56.3% (P < 0.001) of
controls for +/+ animals and 82.6 and 49.9% (P < 0.001) of controls for -/- animals, respectively. Body weight
development of the H/W satellite groups receiving 170 or 1700 µg/kg
TCDD was also markedly below controls. The magnitude of the effect
observed in L-E rats at 1.7 and 17 µg/kg was comparable with that in
H/W rats at 17 and 170 µg/kg, respectively. This indicates about a
10-fold sensitivity difference in body weight development between the
strains.
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.
This rat showed a characteristic manifestation of TCDD intoxication
with signs of wasting syndrome (a body weight loss by 26%, loss of
body fat) and gastrointestinal hemorrhagia. In the satellite groups,
there were 16 deaths, all of which were also considered treatment
related. These results suggest that H/W rats are less resistant to the
long-term than to acute (42-day) mortality of TCDD (27
, 28)
. In general, TCDD-related mortality was higher in rats
assigned to PH/NDEA than in -/- rats.
Quantitative Stereology of GST-P-positive Hepatic Foci.
Results from the stereological evaluation of altered foci are shown in
Fig. 1
. No foci were detected in
untreated -/- control rats of either strain, and the values were very
low for -/- vehicle control rats (mean volume fraction, 0.006 and
0.01%, and mean number of foci/cm3
, 26 and 46 in
H/W and L-E rats, respectively). The volume fraction of foci was
significantly (P < 0.001; t test)
higher in both untreated and vehicle-treated +/+ control L-E rats than
in similarly treated H/W rats (3.58 and 2.14% in untreated and
vehicle-treated L-E rats, respectively, and 0.55 and 0.42% in
untreated and vehicle-treated H/W rats, respectively). However, +/+
controls did not show any strain differences in the mean number of
foci. Thus, PH/NDEA without TCDD resulted in a significantly larger
volume of GST-P-positive foci in L-E rats than in H/W rats.
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. Comparison of the dose-response curves reveals that
L-E rats were 100-fold more sensitive to the promotion of foci by
TCDD than H/W rats. In L-E rats, the volume fraction of foci and the
number of foci/cm3
were significantly
(P < 0.001) and dose dependently increased
at 1.7 and 17 µg/kg, but in H/W rats only at 170 µg/kg
(P < 0.01 for -/- rats and
P < 0.001 for +/+ rats). The highest volume
fraction of foci was observed in +/+ H/W satellites at 1700 µg/kg
(18.5% of the liver volume occupied by foci; Fig. 1B
). The
number of foci, however, was rather low in this group (Fig. 1D)
. Also, the values for -/- H/W satellites were low
(Fig. 1, A and C)
. In general, the focus
parameters were about 10–20 times higher in +/+ than in -/- rats,
but otherwise PH/NDEA had no influence on the dose-response
characteristics or sensitivity to TCDD. The higher background incidence
of foci in +/+ L-E rats did not affect their sensitivity to
TCDD-induced tumor promotion, because -/- rats responded similarly at
the same dose levels. Accordingly, the sensitivity difference between
L-E and H/W rats was the same (100-fold) with and without PH/NDEA.
Liver Histopathology.
A summary of histopathological findings is shown in Tables 3
and 4
.
Altered hepatic foci were observed in -/- L-E rats only at 1.7 and 17
µg/kg and in -/- H/W rats only at 170 and 1700 µg/kg. However, in
+/+ rats, altered foci were present also in control and low-dosage
groups. In +/+ H/W rats, there was a dose-related increase in the
incidence of foci, and the severity of response (in terms of the
abundance of foci per liver) was increased at 170 µg/kg and above. In
+/+ L-E rats, on the other hand, all examined rats had foci, but the
severity of the response was increased at 1.7 and 17 µg/kg.
Hepatocellular adenoma/foci was observed in one (of 9 examined) +/+ L-E
rats at 17 µg/kg and in 4 (of 9) and 3 (of 8) +/+ H/W satellite rats
at 170 and 1700 µg/kg, respectively. A cholangiocarcinoma was found
in one -/- H/W rat (of 8) that had received a single dose of TCDD at
1700 µg/kg. No other hepatic malignancies were observed.
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Plasma Enzyme Activities.
Increased activities of ALAT, ASAT, and GGT in plasma are indicators of
liver damage. Activities of these enzymes in plasma are shown in Fig. 2
. In general, L-E rats were more
sensitive than H/W rats exhibiting dose dependently increased enzyme
activities in plasma at 1.7 (in most cases) and 17 µg/kg, whereas H/W
rats typically responded only at 170 µg/kg and higher dose levels
(satellites). Plasma ALAT activities were elevated only at 17 µg/kg
in L-E rats (P < 0.001). In satellites, the
highest elevation was observed at the 1700 µg/kg single-dose group.
ASAT activities were significantly elevated at 1.7 and 17 µg/kg in
L-E rats and at 170 µg/kg in H/W rats. The elevations of both ALAT
and ASAT activities were more pronounced in -/- than in +/+ groups.
In contrast, plasma GGT activities showed elevations only in +/+
groups. The elevations were significant (P < 0.001) at 1.7 and 17 µg/kg in L-E rats and at 170 µg/kg in H/W
rats. In linear regression analysis, volume fractions of AHF were best
(P < 0.001) correlated with plasma ASAT
activities (r = 0.63, 0.71, 0.71, and 0.87
for +/+ L-E, -/- L-E, +/+ H/W, and -/- H/W rats, respectively),
indicating an association between induction of foci and hepatotoxicity.
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). The
maximum EROD activity was measured at 1.7 µg/kg TCDD in L-E rats and
at 17 µg/kg TCDD in H/W rats. At higher dose levels, the activity
decreased, most likely because of overt toxicity and liver toxicity
observed at 17 µg/kg in L-E rats and at 170 µg/kg and above in H/W
rats.
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. The increases
were significant at dose levels of 1.7 µg/kg and above in L-E rats
and at 17 µg/kg and above in H/W rats. Thus, L-E rats were 10-fold
more sensitive, but this difference may reflect a more profound
decrease in body weight gain in this strain. The maximum increase
occurred at 170 µg/kg. At the highest dose levels, the increases were
slightly more prominent in the PH/NDEA groups.
TCDD treatment resulted in dose dependently decreased body
weight-related thymus weights in H/W and L-E rats (thymus weight in
untreated L-E rats is only about one-third of that in untreated H/W
rats; Fig. 3, E and F
). Both strains showed
similar sensitivity by responding at the same dose levels. The decrease
became statistically significant at 17 µg/kg, but it was more
profound in L-E rats (19.8 and 24.4% of the control value in -/- and
+/+ L-E rats, respectively) than in H/W rats (63.6 and 72.6% of the
control value in -/- and +/+ H/W rats, respectively).
Micronucleated Erythrocytes in Bone Marrow and Peripheral Blood.
Frequencies of MNPCEs in bone marrow and MNRETs in peripheral blood are
shown in Fig. 4, A–D
. The
background amount of MNPCEs in bone marrow was 2–3-fold higher in L-E
rats than in H/W rats. There was a tendency to a slight increase in
MNPCEs at the highest doses, but the increase was significant only in
-/- H/W rats at 170 µg/kg (P < 0.05). In
peripheral blood, slight increases in MNRETs were observed in +/+ H/W
rats (including satellites) at 170 µg/kg and in -/- L-E rats at 17
µg/kg. The proportion of young erythrocytes (PCEs in bone marrow and
RETs in peripheral blood) in the erythrocyte population is considered
as an indicator of bone marrow toxicity, and decreased proportion of
PCEs generally reflects suppression of erythropoiesis. In this study,
however, TCDD treatment resulted in an increased proportion of PCEs and
RETs, indicating stimulation of erythropoiesis (Fig. 4, E–H)
. In bone marrow, H/W rats showed more clear increases,
especially in -/- groups (P < 0.01 at 170
µg/kg). The increases were more uniform in peripheral blood,
achieving statistical significance at 17 µg/kg in L-E rats and at 170
µg/kg in H/W rats.
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Liver TCDD Concentrations.
Background liver concentrations of CDDs and polychlorinated
dibenzofurans in untreated controls and vehicle controls at the end of
the study were very low (range, 0.003–0.03 ng of WHO-TEq/g dry weight;
WHO-TEq is TCDD equivalent quantity according to WHO), indicating lack
of contamination in the animal room. Liver TCDD concentrations at the
end of the study reflected accurately the doses administered (Fig. 5)
. There were no consistent differences
between L-E and H/W rats or attributable to PH/NDEA. Analytical data
make it possible to relate the tumorigenic effects with liver TCDD
concentrations. For the induction of AHF, the LOAEL in L-E rats is 10
ng/kg/day (total dose, 1.7 µg/kg), corresponding to the liver TCDD
concentration of 7.97 ± 0.38 (mean ± SE) ng/g dry weight (about 2.11 ng/g wet weight). The NOEL for the same
response is 1 ng/kg/day (total dose, 0.17 µg/kg) and the
corresponding liver TCDD concentration 0.61 ± 0.04 ng/g
dry weight (0.16 ng/g wet weight). For H/W rats, the LOAEL is 1000
ng/kg/day (total dose, 170 µg/kg) and the liver TCDD concentration
626 ± 41 ng/g dry weight (166 ng/g wet weight), and
NOEL 100 ng/kg/day (total dose 17 µg/kg) and liver TCDD concentration
78.1 ± 4.7 ng/g dry weight (20.7 ng/g wet weight).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This study demonstrated a remarkable (100-fold) sensitivity
difference between L-E and H/W rats in TCDD-induced development of AHF.
This difference is essentially attributable to the exceptional
resistance of H/W rats to the focus development, because the
sensitivity of L-E rats did not differ from that reported earlier for
female Sprague Dawley, Fischer 344, and Wistar rats (reviewed in Ref.
5
). Furthermore, in agreement with the present L-E data,
our previous studies with initiated Sprague Dawley rats using the same
treatment protocol revealed increased induction of GGT- or
GST-P-positive foci at the total dose levels of 1.1–24 µg/kg
(maintenance dose, 6.3–143 ng/kg/day; Refs. 9
, 24
, 33,
and 43
). Thus, LOAEL for induction of AHF is in general
10 ng/kg/day (or slightly below), and NOEL is 1 ng/kg/day.
Moreover, dose responses for AHF in two-stage tumor promotion studies
and development of hepatocellular adenomas in 2-year carcinogenicity
bioassays are also very similar (44)
. It can be concluded
that all other rat strains studied thus far, except H/W rats (LOAEL,
1000 ng/kg/day; NOEL, 100 ng/kg/day), are quite similar in sensitivity
to the induction of AHF.
Toxicokinetic factors do not account for the resistance of H/W rats, because no strain differences in liver TCDD concentrations were detected. Liver TCDD concentrations in L-E and H/W rats were also similar to the steady-state concentrations reported earlier for Sprague Dawley rats at comparable dose levels (9 , 10) .
In previous studies, diverging sensitivity of C57BL/6 and DBA/2 mice to
TCDD have been used in attempts to clarify the mechanism of
TCDD-induced tumorigenicity. Lower binding affinity of the DBA/2 mouse
AHR for TCDD is reflected in their 10-fold higher resistance to a
variety of characteristic TCDD-induced effects compared with the
sensitive C57BL/6 mice. There is some indirect evidence that the AHR
would have a role in promotion of skin tumors by TCDD, but the role of
AHR in liver carcinogenesis has been less clear (5)
. An
attempt to establish a rank order of sensitivity for liver tumor
promotion among C57BL/6 and DBA/2 mice and their crosses
(B6D2F1 mice) using a single dose level of TCDD
(daily dose, 7.14 ng/kg/day) was not successful (45)
. Our
results suggest an association between the exceptional resistance to
TCDD-induced liver tumorigenesis and the altered transactivation domain
of the AHR. This is in accord with our previous data, indicating that
the resistance to TCDD-induced toxic effects (lethality, body weight
loss, and hepatotoxicity) of H/W rats segregates with the altered
Ahr allele (18)
.
Comparison of the outcome of carcinogenicity bioassays in rats and mice reveals no marked species differences in sensitivity to TCDD-induced liver carcinogenesis (reviewed in Ref. 5 ). On the other hand, hamsters that are very resistant to the lethality of TCDD did not develop liver tumors at total dose levels of up to 600 µg/kg (46) . Squamous cell carcinomas of the facial skin, however, were observed at the highest dose level only. It is interesting to note that of all of the rodents studied, only H/W rats and hamsters share the relative resistance to the tumorigenic effect of TCDD. Furthermore, another common feature is the altered structure of the transactivation domain in both H/W rat and hamster AHR. In H/W rat AHR, there is a loss of amino acids near the COOH-terminal end (16) , whereas in the case of hamster AHR, the functionally essential Q-rich subdomain is substantially expanded (47) .
In the present study, TCDD was found to dose dependently induce a small
amount of GST-P-positive foci also in noninitiated L-E and H/W rats. It
is noteworthy that the same (100-fold) sensitivity difference was
detected also between noninitiated L-E and H/W rats, and the effect was
observed at the same dose levels in initiated and noninitiated rats.
Previous studies did not pay much attention to induction of foci by
TCDD in noninitiated animals, apparently because of the lower response
compared with that in initiated animals. There are only a few reports
about increased generation of foci with some dose dependence
(10)
and time dependence (25
, 48)
in
noninitiated rats. These foci were suggested to result from promotion
of "spontaneously" initiated cells by TCDD. Furthermore, recent
modeling of the experimental data have raised suggestions that in
addition to its promoting activity, TCDD would have some initiating
activity, possibly by an indirect mechanism (49
, 50)
.
Taken together, TCDD is able to induce a tumorigenic response in
noninitiated animals with a potency similar to that observed with
initiation.
According to the current view, TCDD is not a genotoxic compound (5) . Nevertheless, increased frequencies of micronucleated erythrocytes observed at the highest dose levels of the present study indicate occurrence of some chromosomal damage. These dose levels caused overt toxicity and stimulation of erythropoiesis, as indicated by increased frequency of PCEs in bone marrow and reticulocytes in peripheral blood. In addition, the increase in micronuclei occurred at higher dose levels than the induction of AHF in L-E rats, suggesting that these phenomena are not interrelated. Therefore, the slight induction of micronuclei does not necessarily represent a specific genotoxic effect but may rather be secondary to other toxic effects. It should still be noted that contrary to all of the negative genotoxicity data, there are a few studies from one laboratory reporting dose-dependent induction of micronuclei and sister chromatid exchanges in human lymphocytes treated with relatively low concentrations of TCDD in vitro (51, 52, 53) . Unfortunately, inaccurate reporting and the fact that the frequencies of micronuclei also in TCDD-treated lymphocytes were well within the normal baseline frequency (54) render the significance of these findings questionable.
Development of hepatocellular neoplasms and AHF appear to be correlated
with hepatotoxicity in TCDD-treated rats (9
, 10
, 14 , 26)
.
Our data provide further support for this concept: (a)
increased plasma ASAT, ALAT, and GGT activities were observed at the
same dose levels that induced AHF, showing significant correlations;
and (b) the same (100-fold) sensitivity difference
between L-E and H/W rats was observed for indicators of hepatotoxicity
and induction of foci.
Dose responses for induction of liver EROD activity were similar in L-E and H/W rats. These results are in agreement with our previous data after a single dose of TCDD (30 , 55) and confirm that the AHR-mediated induction of CYP1A1 activity is normal also in H/W rats. The results also clearly indicate that enzyme induction and tumor promotion are not interrelated: (a) they follow different dose responses, as reported earlier (10 , 56) ; and (b) H/W rats are as sensitive as L-E rats to CYP1A1 induction but 100-fold more resistant to the induction of AHF.
Thymus atrophy is one of the characteristic end points of dioxin
toxicity. A decrease in relative thymus weights was parallel in both
strains indicating similar potency of TCDD for this effect.
Nevertheless, the magnitude of the decrease was clearly smaller in H/W
than in L-E rats. These findings are in accordance with our earlier
observations after a single dose of TCDD (57
, 58)
and
indicate that thymus atrophy, which is an AHR-mediated phenomenon
(15)
, develops also in H/W rats despite their deviant AHR.
Increased relative liver weight is also a typical response elicited by
TCDD and reflects enzyme induction-associated proliferation of
endoplasmic reticulum in liver and a decreased amount of adipose tissue
at higher dose levels. L-E rats were 10-fold more sensitive, mainly
because of more severe body weight loss.
In conclusion, a remarkable sensitivity difference of two orders of magnitude in induction of AHF by TCDD was found between L-E and H/W rats. The exceptional resistance of H/W rats is associated with an altered transactivation domain of the AHR. A similar sensitivity difference was observed in hepatotoxicity and long-term mortality, whereas the two strains were equally sensitive to AHR-mediated CYP1A1 induction and thymic atrophy. Our results imply that genetic differences may account for significant interindividual/intraspecies sensitivity differences in dioxin-induced carcinogenesis. Understanding the role of the AHR transactivation domain in carcinogenesis is therefore expected to result in improved risk assessment of dioxins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
|---|
1 This study was supported by Contract
ENV4-CT96-0336 from the European Commission, DG XII, and Grant
5410/4011/89 from the Academy of Finland, Research Council for
Environmental Sciences. 
2 To whom requests for reprints should be
addressed, at National Public Health Institute, Laboratory of
Toxicology, P. O. Box 95, FIN-70701 Kuopio, Finland. Phone:
358-17-201329; Fax: 358-17-201265; E-mail: matti.viluksela{at}ktl.fi
3 The abbreviations used are: TCDD,
2,3,7,8-tetrachlorodibenzo-p-dioxin; CDD, chlorinated
dibenzo-p-dioxin; AHR, aryl hydrocarbon receptor; AHF,
altered hepatic foci; L-E, Long-Evans; H/W, Han/Wistar; NDEA,
nitrosodiethylamine; PH, partial hepatectomy; GST-P, glutathione
S-transferase, placental form; ALAT, alanine
aminotransferase; ASAT, aspartate aminotransferase; GGT, 
-glutamyl
transpeptidase; EROD, ethoxyresorufin
O-deethylase; MNPCE, micronucleated bone marrow
polychromatic erythrocyte; MNRET, micronucleated peripheral blood
reticulocyte; GC-MS, gas chromatography-mass spectrometry; LOAEL,
lowest observable adverse effect level; NOEL, no-effect level.
Received 6/ 6/00. Accepted 10/16/00.
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REFERENCES |
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