Download Dose-Response Examination of UDP

TOXKOLOGICAL SCIENCES 46, 31-37 (1998)
ARTICLE NO. TX982510
Dose-Response Examination of UDP-Glucuronosyltransferase Inducers
and Their Ability to Increase both TGF-/3 Expression and Thyroid
Follicular Cell Apoptosis1
Kyle L. Kolaja2 and Curtis D. Klaassen3
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160
Received January 7, 1998; accepted May 29, 1998
Certain hepatic microsomal-inducing chemicals that also
induce hepatic UDP-glucuronosyltransferase (UDP-GT) can
cause thyroid follicular cell hyperplasia and, ultimately, thyroid adenomas (Hill et al, 1989). Induction of UDP-GT may
cause thyroid proliferation by reducing serum thyroid horExposure to certain microsomal enzyme inducers that increase mones (triiodothyronine, T3, and thyroxine, T4) leading to
UDP-glucuronosyltransferase (UDP-GT) activity decreases thy- feedback secretion of thyrotropin (TSH) (Hill et al, 1989). In
roid hormone levels, which may lead to a subsequent Increase in fact, the UDP-GT inducers phenobarbital (PB) and pregthyroid-stimulating hormone (TSH). This elevation of serum TSH nenolone-16a-carbonitrile (PCN) decrease thyroid hormone
has many effects on the thyroid, including increasing thyroid levels (Baiter and Klaassen, 1994). Because serum thyroid
follicular cell proliferation, leading to hyperplasia. While induchormones are regulated by the hypothalamus-pituitary-thyroid
tion of UDP-GT activity decreases thyroid hormone levels by
enhancing biotransformation and subsequent biliary secretion, axis, reduction in serum thyroid hormone levels causes a
only certain UDP-GT inducers exhibit the ability to increase concomitant TSH release (Hill et al, 1989). TSH then stimuserum TSH levels. For example, phenobarbital (PB) and preg- lates the thyroid gland to increase thyroid hormone synthesis
nenolone-16a-carbonitrile (PCN) increase serum levels of TSH, and secretion, as well as producing follicular cell hyperplasia
while 3-methylcholanthrene (3MC) and Aroclor 1254 (PCB) do and hypertrophy. Indeed, treatment with UDP-GT inducers,
not. Increased serum TSH concentration also enhances thyroid PCN and PB, increased serum levels of TSH (Liu et al, 1995)
gland expression of TGF-/3,, an anti-proliferative, pro-apoptotic and increased thyroid weight, thyroid follicular cell proliferaprotein. In a previous study in our laboratory, rats were treated for tion, and apoptosis (Hood et al, 1995; Kolaja et al, 1997).
various times (up to 90 days) with PB and PCN, which increased
In contrast to PCN and PB, other UDP-GT inducers do not
TGF-/3, protein and apoptosis. The present study was designed to
increase TSH despite a dramatic reduction of serum T4 levels
examine the dose-response effect of TSH-increasing (PB and
PCN) and nonincreasing (3MC and PCB) UDP-GT inducers on (Barter and Klaassen, 1994). Aroclor 1254 (PCB), a polyapoptosis and TGF-/3,. PB and PCN, UDP-GT inducing com- chlorinated biphenol mixture, and 3-methylcholanthrene inpounds which increase serum TSH, increased the percentage of duce UDP-GT activity and decrease T 4 levels, yet no subseTGF-/3,-positive follicular cells and increased apoptosis. In con- quent increase in TSH is observed (Liu et al, 1995). In
trast, UDP-GT inducers that did not increase TSH (3MC and addition, these compounds that do not increase serum levels of
PCB) did not alter cell death or TGF-0 production. These data TSH and also do not increase thyroid follicular cell proliferasuggest that the increase of TGF-0 by TSH may serve to regulate tion (Hood et al, 1995). The exact mechanism of this discrepthe growth of hyperplastic thyroid, e IWS sodaj of Toufcotoc.
ancy is not understood. The effects on the thyroid gland of PB,
Key Words: apoptosis; thyroid; phenobarbital (PB); pregnenolo- PCN, PCB, and 3-methylcholanthrene (3MC) are summarized
ne-16a-carbonitrile (PCN); 3-methylcholanthrene (3-MC); Aro- in the legend to Table 1 (Barter and Klaassen, 1995; Hood
clor 1254 (PCB); and TGF-0,.
et al, 1995; Liu et al, 1995).
TSH enhances the expression of TGF-/3,, a potent growth
1
inhibitor of many epithelial cell types, in thyroid follicular cells
This work was supported in part by USPHS Grant ES-03192.
2
This author was supported in part by USPHS Training Grant ES-07079,
(Rogers, 1996). Propylthiouracil (PTU) and methimazole
NRSA ES-05812, and the Kansas Health Foundation.
(MM1) treatments, both of which increase serum TSH levels,
3
To whom correspondence and reprint requests should be addressed at
also
increase thyroid follicular cell TGF-/3, mRNA and protein
Department of Pharmacology, Toxicology, and Therapeutics, University of
(Logan
et al, 1994; Morosini et al, 1996). In addition, TSH
Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS, 66160infusion stimulates thyroid gland production of TGF-/3, (Mo7417. Fax: (913) 588-7501; E-mail: [email protected].
Dose-Response Examination of UDP-Glucuronosyltransferase
Inducers and Their Ability to Increase both TGF-/J Expression
and Thyroid Follicular Cell Apoptosis. Kolaja, K. L., and Klaassen, C. D. (1998). Toxicol. Sci. 46, 31-37.
31
1096-6080/98 $25.00
Copyright C 1998 by the Society of Toxicology.
All rights of reproduction in any form reserved.
32
KOLAJA AND KLAASSEN
rosini et al., 1996). The apparent paradoxical increased expression of TGF-/3!, a negative growth factor, may help blunt the
proliferative stimulus of excess TSH and stabilize the growth
of the thyroid (Morosini et al., 1996), possibly by inducing
apoptosis.
In this study, we hypothesize that the UDP-GT inducers
PCN and PB, which have been shown to increase serum TSH
(Barter and Klaassen, 1994; Liu et al., 1995), will also increase
TGF-/3, expression, whereas 3MC and Aroclor 1254, which do
not increase TSH, will not increase TGF-/3, expression. Because TGF-/3, induces apoptosis in a variety of tissues, including thyroid follicular cells, we speculate that in treatment
groups where increased TGF-/3, is observed, an induction of
apoptosis will be observed. The immunohistochemical detection of TGF-/3J and the histologic presence of apoptosis will be
used to examine the above parameters. This study will provide
histologic insight for the possible growth regulating mechanisms of the increase of TGF-/3, by TSH in thyroid follicular
cells.
MATERIALS AND METHODS
Chemicals. PB was purchased from Spectrum Chemical Manufacturing
Corp. (Gardena, CA). PCN was prepared from 16-dehydropregnenolone (Pfaltaz & Bauer, Inc., Watbury, CT). 3MC was purchased from Sigma Chemical
Co. (St. Louis, MO). Aroclor 1254 was kindly provided by Dr. Steven Aust
(Utah State University, Logan, UT).
Animals and experimental design. One hundred and fifty (150) pathogenfree male Sprague-Dawley rats, aged-matched (between 225 and 250 g: Sasco,
Omaha, NE), were divided randomly into groups of six rats, housed in
polypropylene cages, and maintained at 70°F on a 12-h light/dark cycle.
Throughout the entire study, all rats were maintained in an AAALAC-accredited animal facility according to the NTH Guide for the Care and Use of
Laboratory Animals. During a 1-week acclimation period, rats were administered both tap water and standard laboratory rodent chow (Ralston-Purina,
Chicago, IL) ad libitum. After the acclimation period, rats were randomly
separated into one of the 17 dose groups outlined in Table 1.
Five rats per group per time point were euthanized after 14 days of dietary
treatment. Rats were asphyxiated with CO 2 , weighed, and necropsied. Thyroid
glands were removed, weighed, Formalin-fixed (>24 h), paraffin-embedded,
sectioned (5 /im), and mounted on glass slides for histologic analysis.
Apoptosis. Microscopic evaluation of hematoxylin and eosin-stained
slides was used to quantify the incidence of apoptosis in the thyroid.
Apoptosis was defined by morphologic characteristics similar to those
described in other tissues (Bursch et al., 1984, 1985; Kolaja et al., 1996),
which include increased cytoplasmic eosinophilia, increased nuclear condensation, fragmentation of DNA, and separation from surrounding follicular cells. At least 5000 thyroid follicular cells per slide were examined for
the histologic presence of apoptosis. In addition, the morphologic presence
of apoptosis was verified by immunohistochemical detection of fragmented
DNA (Apoptag kit, ONCOR, Gaithersburg, MD). Apoptotic index was
determined by dividing the number of apoptotic follicular cells by the total
number of follicular nuclei observed.
TGF-P, immunohistochemistry.
TGF-0, immunohistochemistry was
performed on Formalin-fixed, paraffin-embedded tissue was previously described with minor modifications (Logan et al, 1994). Briefly, slides were
incubated with primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA)
for 48 h (4°C) at a dilution of 1:25. An avidin-biotin complex kit (Santa Cruz
TABLE 1
Group
Group name
1
2
3
4
5
Control
300 PB
600 PB
1200 PB
2400 PB
200 PCN
400 PCN
800 PCN
1600 PCN
25 3MC
50 3MC
100 3MC
200 3MC
25PCB
50PCB
100 PCB
200 PCB
6
7
8
9
10
11
12
13
14
15
16
17
Treatment
Control diet
300 mg Phenobarbital/kg of diet
600 mg Phenobarbital/kg of diet
1200 mg Phenobarbital/kg of diet
2400 mg Phenobarbital/kg of diet
200 mg Pregnenolone-16a-carbonitrile/kg of diet
400 mg Pregnenolone-16a-carbonitrile/kg of diet
800 mg Pregnenolone-16a-carbonitrile/kg of diet
1600 mg Pregnenolone-16a-carbonitrile/kg of diet
25 mg 3-Methylcholanthrene/kg of diet
50 mg 3-Methylcholanthrene/kg of diet
100 mg 3-Methylcholanthrene/kg of diet
200 mg 3-Methylcholanthrene/kg of diet
25 mg Aroclor 1254/kg of diet
50 mg Aroclor 1254/kg of diet
100 mg Aroclor 1254/kg of diet
200 mg Aroclor 1254/kg of diet
Note. Previously, we have shown that certain doses of UDP-GT inducers
used in this study will alter thyroid hormone status and TSH levels (Liu et al.,
1995; Hood et al., 1996). For instance, 7-day treatment with PB (600 and 1200
ppm) decreased T 4 levels and increased serum TSH. Similarly, PCN treatment
with 200 ppm and above decreased T 4 levels and increased serum TSH. In
contrast, 3MC and PCB at doses examined (PCB:50-400 ppm; 3MC 0-200
ppm) did not alter serum TSH values, yet PCB (25 ppm and greater) and 3MC
(100 ppm and greater) decreased thyroid hormone. Importantly, increased
serum TSH was observed in PB- and PCN-treated rats, but not 3MC- and
PCB-treated rats (Hood et al., 1995).
Biotechnology) was used according to the manufacturer's instructions for
secondary, tertiary, and diaminobenzidine chromagen reagents. TGF-/3, was
visualized by the presence of brown pigment in TGF-/3,-positive cells compared to the unstained, nonlabeled cells. TGF-P, labeling index was determined by dividing the number of follicular cells with TGF-B,-positive nuclei
by the total number of follicular nuclei observed. At least 3000 thyroid
follicular cells per slide were scored for the immunohistochemical presence of
TGF-8,.
Statistics. Statistical difference (p < 0.05) from the control group was
determined by ANOVA, followed by Duncan's multiple-range post-hoc test
(Gad and Weil, 1986).
RESULTS
Apoptosis
Apoptosis, detected by morphologic criteria (see Fig. 1;
Bursch et al, 1984, 1985), was validated by immunohistochemical expression of fragmented DNA (Kolaja et al., 1996).
Figure 2 shows the incidence of apoptotic follicular thyroid
cells after treatment with PB, PCN, 3MC, or PCB. As shown
in Fig. 2A, PB treatment of doses of 300, 600, and 1200 ppm
lead to an increase in the incidence of apoptosis. Interestingly,
2400 ppm PB did not increase apoptosis (Fig. 2A) or increase
TSH (Hood et al., 1995). All doses of PCN examined (200,
400, 800, and 1600 ppm) increased the percentage of apoptotic
33
THYROID APOPTOSIS AND TGF-/3 EXPRESSION
FIG. 1. (A) An H&E-stained control rat thyroid (200x). Note the large colloidal spaces that contain thyroglobulin. Both 3MC- and PCB-treated thyroids
had a similar morphologic appearance. (B) An H&E staining of a hyperplastic rat thyroid (200 X). Both PB and PCN treatment led to thyroid follicular cell
hyperplasia. A representative example shown here is treated with PCN. Note the lack of colloidal space as well as an apoptotic thyrocyte (denoted by an arrow).
thyroid follicular cells (Fig. 2B). In contrast, neither PCB nor
3MC, at any dose examined, effected apoptosis in the thyroid
gland (Figs. 2C and 2D).
Immunohistochemical Detection of TGF-fi,
The immunohistochemical expression of TGF-8, was detected on sectioned thyroid tissue treated with PB, PCN, 3MC,
and PCB (Fig. 3 and 4). PB treatment at doses of 300, 600, and
1200 ppm lead to an increase in TGF-0, immunohistochemical
expression in thyroid follicular cells (Fig. 4A). These same
doses of PB also increased apoptosis (Fig. 4A). PCN treatment
increased the percentage of thyroid follicular cells that express
TGF-/3, (Fig. 4B). Similar to PB, the same doses of PCN that
increased TGF-0, also increased apoptosis (Figs. 2B and 4B).
3MC (Fig. 4C) and PCB (Fig. 4D) did not affect TGF-/3,
expression in thyroid follicular cells.
DISCUSSION
Treatment with the microsomal enzyme inducers that
induce UDP-GT activity, PCN and PB, increased the inci-
KOLAJA AND KLAASSEN
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FIG. 2. Incidence of apoptotic thyroid follicular cells of rats treated with 300, 600, 1200, and 2400 mg phenobarbital/kg of diet (PB), 200, 400, 800, and
1600 ppm pregnenolone-16a-carbonitrile (PCN)/kg of diet, 50, 100, 200, and 400 3-methylcholanthrene (3-MQ/kg of diet, and 25, 50, 100, and 200 mg 1254
Aroclor (PCB)/kg of diet. Values represent the mean ±SE of 5-6 rats per group. Statistical significance (»p < 0.05) was determined by ANOVA followed by
Duncan's post-hoc test
dence of apoptosis. In contrast, no change in thyroid follic- els of TSH and increased observation of apoptosis in thyroid
ular cell apoptosis in 3MC- and PCB-treated rats was ob- follicular cells suggest a modulating mechanism is also inserved. This increase in programmed cell death was volved. The enhanced production of TGF-/3, by TSH offers an
associated with increased immunohistochemical detection attractive solution.
The induction of apoptosis in hyperplastic, proliferating
of the anti-proliferative, pro-apoptotic growth factor TGF/3j. Of importance, compounds that elevate serum TSH also thyroid glands, at first, appears to be paradoxical. The inincrease TGF-/3, and apoptosis. These data suggest that creased apoptosis in thyroid follicular cells occurs, however, at
TSH's ability to increase TGF-0, may lead to apoptosis of a 10-fold lower rate than the proliferation (Hood et al, 1995).
thyroid follicular cells.
The increased apoptosis may serve as a mechanism to elimiOur laboratory has previously characterized the effects of nate the older cells as the thyroid struggles to maintain cellular
UDP-GT treatment on thyroid hormone economy (Hood et al, equilibrium, as seen in involuting tissue (Bursch et al, 1984,
1995; Liu et al, 1995). After 7 days of treatment, free T 4 was 1985).
decreased in PB (1200 ppm; 50% of control), PCN (1600 ppm;
TGF-0,, a 25-kDa peptide, is a cytokine growth factor
46%), 3MC (400 ppm; 45%), and PCB (200 ppm; 18%). This that is involved in regulating both proliferation and cell
decrease in T 4 was a dose-response phenomenon (Hood et al., death (Sporn and Roberts, 1985). TGF-/3, has been sug1995). TSH serum concentrations, however, were increased gested to be the initiating stimulus for apoptotic cell death
only in rats treated with PB (140% of control) and PCN (Oberhammer et al, 1993). TGF-/3, treatment to cultured
(185%). This increase in serum TSH leads to increased thyroid feline goiters, cultured porcine thyrocytes, and a rodent
proliferation (Hood et al., 1995).
thyroid cell line, FRTL-5 cells, lead to cell death (Asmis et
An increase in serum TSH also stimulates the production al, 1993, 1996). Several thyroid diseases such as adenomas,
and secretion of TGF-0,. Interestingly, TGF-0 stimulates a carcinomas, Hashimoto's disease, and Graves' Disease exfurther release of TGF-0, (Islam etal, 1997). In contrast to the hibit increased TGF-/3, and increased apoptosis in the redata presented, TSH inhibits Fas-antigen-mediated apoptosis in spective thyroid follicular cells (Okanayu et al, 1995).
thyroid follicular cells (Kawakami et al, 1996). Elevated lev- Recent evidence indicates that TGF-/3, increased the forma-
THYROID APOPTOSIS AND TGF-0 EXPRESSION
35
FIG. 3. Immunohistochemical staining for TGF-/3, in (A) control rat thyroid and (B) the thyroid from a PCN-treated rat (800 ppm). Note the positive staining
of the C cells (parafollicular cells) in control thyroid (denoted by arrow). In the thyroid of the PCN-treated rat (as well as PB), the TGF-0,-positive thyroid
follicular cells tended to be in clusters or focal areas.
tion of reactive oxygen species, known inducers of apoptosis (Islam et ai, 1997). Taken together, these data suggest
that a relationship among elevated TSH, elevated TGF-/3,,
and apoptosis in hyperplastic thyroid exists.
The lack of direct dose-related increases in TGF-0, expression and apoptosis are not cause for alarm. Measurement of TGF-/3,-positive thyroid follicular cells does not
indicate the quantitative amount of protein that is present,
but rather, a qualitative assessment of the expression of
TGF-0, in UDP-GT inducer-treated rodent thyroid. In addition to method-based concerns, the nonlinear dose-response relationship may also be explained by physiologic
mechanisms. Although nonlinear, both responses are parallel and these data presented indicate that a relationship
between increased TSH and TGF-/3, and apoptosis are indicated.
Taken together, the present data suggest that there is an
association between the induction of TSH by PB and PCN and
an increase in apoptosis in thyroid follicular cells. This conclusion is supported by the fact that no increase in apoptosis
was observed in thyroid follicular cells from 3MC- and Aroclor
1254-treated rats. The increased cell death may be due, in part,
to an increased production of the anti-proliferative, pro-apoptotic protein TGF-0,.
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FIG. 4. Immunohistochemical expression of TGF-/3, in thyroid follicular cells (% of total follicular cell population) treated with 300, 600, 1200, and 2400
mg PB/kg of diet, 200, 400, 800 and 1600 ppm PCN/kg of diet, 50, 100, 200, and 400 3-MC/kg of diet, and 25, 50, 100, and 200 mg PCB/kg of diet. Values
represent the mean ±SE of 5-6 rats per group. Statistical significance (*p < 0.05) was determined by ANOVA followed by Duncan's post-hoc test.
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