TOXICOLOGICAL SCIENCES 87(2), 529–536 (2005)
doi:10.1093/toxsci/kfi260
Advance Access publication July 20, 2005
Coplanar Polychlorinated Biphenyls Activate the Aryl Hydrocarbon
Receptor in Developing Tissues of Two TCDD-Responsive
lacZ Mouse Lines
Jeffrey C. Bemis,* Daniel A. Nazarenko,† and Thomas A. Gasiewicz*,1
*Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642;
†Alkermes Inc., Cambridge, Massachusetts 02139
Received July 4, 2005; accepted July 13, 2005
In utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)
can have an immediate impact on developmental processes that
then lead to long-term deficits in function. To define the specific
tissues affected by TCDD during development, we developed
a lacZ-reporter gene mouse model driven by activation of the aryl
hydrocarbon receptor (AhR). Exposure to TCDD on gestational day
(GD) 14 results in strong activation of the lacZ transgene in
numerous tissues including fore and hind paws, ear, and genital
tubercle. Experiments were conducted to examine the ability of
alternative AhR ligands to activate our model system. The coplanar
polychlorinated biphenyl congeners 3,4,5,3#,4#-pentachlorobiphenyl
(PCB126) and 3,4,3#,4#-tetrachlorobiphenyl (PCB77) both induced
staining in fetal tissues identical to that observed following TCDD
exposure. Exposure of fetuses to the PCB mixture Aroclor 1254
and the non-coplanar congener 2,3,6,2#,5#-pentachlorobiphenyl
(PCB95) did not result in any activation of the lacZ transgene.
In addition to the testing of alternative ligands, another line of
reporter mice was generated to determine the potential influence
of the site of insertion of the lacZ transgene on the reported
observations. Both TCDD and the coplanar PCBs induced a similar
pattern of staining in the new line as compared to that observed in
the original lacZ reporter mouse line. The ability of AhR ligands,
other than TCDD, to activate the AhR-mediated transgene, in
combination with the insertion-site independence of the response,
strengthens the data previously derived from this model and
increases the utility of this system for investigations examining
AhR-mediated events during development.
Key Words: aryl hydrocarbon receptor; polychlorinated biphenyls; dioxin; lacZ.
INTRODUCTION
The aryl hydrocarbon receptor (AhR) is an important
cellular target for environmental contaminants such as
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD and other
1
To whom correspondence should be addressed at Department of Environmental Medicine, University of Rochester School of Medicine and Dentistry,
601 Elmwood Avenue, Box EHSC, Rochester, NY 14642. Fax: 585–256–2591.
E-mail: [email protected].
related halogenated aromatic hydrocarbons (HAHs) bind to the
AhR and initiate a process involving translocation of the
receptor to the nucleus, association of AhR with other proteins,
binding of the complex to dioxin responsive elements (DREs)
on DNA, and finally transcription of selected genes. The AhR
signaling pathway and its resultant gene products are known to
influence both developmental and cell-signaling processes, as
well as metabolism of endogenous and exogenous compounds
(Hankinson, 1995).
A great deal of our understanding of AhR function comes
from investigating the effects of exposure to TCDD and other
HAHs. The responses to AhR ligands varies with dose and time
of exposure, but they include effects such as immunotoxicity,
neurotoxicity, and alterations in developmental processes
(Birnbaum and Tuomisto, 2000). Targeted deletion of AhR
results in abnormalities in growth (Gonzalez and FernandezSalguero, 1998; Lahvis and Bradfield, 1998), supporting a role
of the AhR in development. Experiments designed to investigate the interaction of AhR with other signaling pathways
(Hayashida et al., 2004; Puga et al., 2000) and the identification
of potential endogenous ligands (Song et al., 2002) are beginning to shed light on additional aspects of AhR function. Based
on these studies, there is growing evidence that the AhR plays
a critical role in the regulation of growth and differentiation.
As a means of examining AhR activation in specific cells and
tissues following in vivo exposure to AhR ligands, our laboratory developed a transgenic mouse model containing a bacterial
b-galactosidase reporter gene under the exclusive control of
several DREs (Willey et al., 1998). AhR-mediated transcription of the b-gal enzyme, and subsequent treatment of the
tissues to reveal b-galactosidase-mediated X-gal staining, can
identify tissues where the AhR pathway is activated by either
endogenous or exogenous agents. In our initial characterization
of this model, 24 h exposure to TCDD, beginning at gestational
day (GD) 13, resulted in X-gal staining in numerous regions
including craniofacial structures, ear, palate, paws, and genital
tubercle. Control animals exposed to vehicle revealed only
faint, but detectable X-gal staining in genital tubercle, strongly
suggesting activation of the AhR by an endogenous compound
Ó The Author 2005. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.
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BEMIS, NAZARENKO, AND GASIEWICZ
or process (Willey et al., 1998). Thus, the DRE-lacZ reporter
mouse represents a model that can examine the activation of
AhR via potential endogenous ligands, as well as target the
sites of action of exogenous AhR ligands.
In addition to TCDD, other compounds can activate AhRmediated transcription. Polychlorinated biphenyls (PCBs) are
a family of ubiquitous environmental contaminants that
possess widely varying toxic potential dependent primarily
on chlorination pattern and density (Tilson and Kodavanti,
1998). This group of toxicants can be separated into two groups
designated by the chlorine substitution of the ortho positions of
the biphenyl ring. Polychlorinated biphenyl congeners without
chlorines in the ortho positions are called ‘‘coplanar’’ because
the two phenyl rings can assume a planar state. Chlorination of
the ortho positions does not favor alignment of the phenyl
rings; therefore the other class of PCB congeners is termed
‘‘ortho-substituted’’ or ‘‘non-coplanar.’’
Non-ortho chlorine-substituted, or coplanar PCBs, have been
implicated as developmental and immune system toxicants and
are suspected carcinogens (Tilson and Kodavanti, 1998). By
virtue of their ability to assume a planar state, which structurally
resembles TCDD, coplanar PCBs are believed to elicit many of
their effects through the AhR. Given these observations, we
hypothesized that the DRE-lacZ reporter mouse would respond
to coplanar PCBs in a manner similar to TCDD and should have
little or no reactivity to ortho-substituted congeners.
In addition to testing the efficacy of coplanar PCBs in the
original DRE-lacZ line, we tested another line of DRE-lacZ
reporter mice generated in our laboratory. A potential limitation of the observations made using the original DRE-lacZ
mouse line is that the responses we observed after TCDD
exposure could be dependent on factors particular to the site of
insertion of the lacZ transgene into the mouse genome. Thus, it
is possible that the lacZ induction observed in particular tissues
was not a direct response of AhR signal transduction, but rather
a consequence of positional effects caused by endogenous
regulators, mutations or recombinations, or concatemermediated effects (Pinkert, 2002). It is also possible that lacZ
induction was not observed in certain tissues because that
particular site of insertion was obstructed by tissue-specific
factors that restricted AhR access and transactivation. A new
line was created to confirm that the AhR ligand-mediated
effects on the lacZ reporter gene are reproducible after random
insertion, and thus the activity of the transgene is not as likely
to be dependent on the site of insertion into the mouse genome.
In addition to the well-characterized TCDD response, the new
line (referred to as the W-line) was also tested with PCBs to
determine its response to other AhR agonists.
MATERIALS AND METHODS
Selection and preparation of toxicants. 3,4,5,3#,4#-Pentachlorobiphenyl
(PCB126), 3,4,3#,4#-tetrachlorobiphenyl (PCB77), 2,3,6,2#,5#-pentachlorobiphenyl (PCB95), and Aroclor 1254 (A1254; Lot # 124–191, >99% purity) were
obtained from Accustandard (New Haven, CT). PCB126 and PCB77 were
selected for their respective potencies in activating the AhR (Hestermann et al.,
2000) and their use in numerous in vivo toxicity studies (Crofton et al., 2000;
Geller et al., 2000). PCB95 was chosen to maintain the same number of
chlorines as PCB126, in addition to the significant activity of PCB95 in several
model systems (Gafni et al., 2004; Schantz et al., 1997). Finally, A1254 is one
of the more well-studied technical PCB mixtures that was used in industry and
subsequently released into the environment (Erickson, 1997). Thus, when
compared to single PCB congeners, A1254 represents a more environmentally
relevant toxicant mixture to study in terms of its potential impact on human
health. We selected the 1 mg/kg concentration as the highest dose of any of the
PCBs used in this study based on the study of Hesterman et al. (2000), which
reports that PCB77 has about 10003 less affinity for the AhR and about 5003
less relative potency for the receptor as compared to TCDD. The Aroclor
mixture and PCB95 were also used at this concentration to provide a measure of
dose equivalency. It is possible that considerably higher doses of the Aroclor
1254 mixture would activate our model, but because of potential general toxic
effects in the mother and relevancy to other studies, such high concentrations
were not examined.
TCDD serves as our positive control for activation of the lacZ transgene.
Exposure of mice to 5 lg/kg TCDD po will reliably produce b-galactosidase
activity in the previously mentioned tissues, and staining has been observed
after exposure to concentrations as low as 1 lg/kg po (personal observation).
However, we selected the dose of 30 lg/kg because this concentration had been
used in previous experiments, and it has been well demonstrated that this dose
maximally elicits lacZ reporter gene activity in this murine model. Individual
congeners were dissolved in dioxane to create concentrated stock solutions.
Aliquots of the stock solutions were taken to dryness under a gentle nitrogen
stream. The toxicants were then dissolved in olive oil via thorough mixing
for a minimum of 12 h. A1254 was dissolved directly in olive oil in an identical manner.
Animal handling and dosing. Timed-pregnant DRE-lacZ dams were
generated by overnight pairing, followed by removal of the male and
examination for a sperm plug. Successful matings were further verified by
steady weight gain and gentle palpation for fetuses. For the purposes of
gestational dating, the first day after pairing was termed gestational day (GD) 0.
Dams were dosed at GD 14 via oral gavage of olive oil control or olive oil
containing TCDD or PCBs. A minimum of three dams, with at least three pups
per dam, were used for each exposure condition.
Twenty-four hours after dosing, the dams were sacrificed by CO2 overdose,
and the entire uterus was removed to cold PBS. The condition and position of
each fetus in the uterus was recorded, then each fetus was removed and placed
in PBS. The fetuses were fixed in 10% neutral buffered formalin for 20 min at
room temperature, followed by extensive rinsing in PBS.
X-gal staining. Based on a modification of Cheng et al. (1993), bgalactosidase activity was visualized by incubation of the intact fetus in
a solution containing 5 mM K3Fe(CN)3, 5 mM K4Fe(CN)6, 2 mM MgCl2,
0.02% Nonidet P-40, 0.01% sodium deoxycholate, 1 mg/ml 5-bromo-4-chloro3-indoyl-b-D-galactoside (X-Gal) in PBS (pH 7.9–8.0). Fetuses were maintained in this solution for 5 h at 37°C, and then rinsed thoroughly in PBS.
Presence of the blue dibromo-dichloro indigo X-gal precipitate formed via the
cleavage of the X-gal substrate by b-galactosidase was recorded with an
Olympus SZX12 stereomicroscope (Melville, NY) and Image Pro Plus
software (Media Cybernetics, Silver Spring, MD). After X-gal staining,
individual fetuses were tested via polymerase chain reaction (PCR) analysis
to verify the presence of the lacZ reporter gene (Willey et al., 1998).
Comparative assessment of X-gal staining in fetuses. Activation of AhR
in the DRE-lacZ mouse model results in a recognizable pattern of staining
throughout the fetus (Willey et al., 1998). The X-gal staining pattern in the
whole fetuses reported here was most strongly visualized in the ears, fore and
hind paws, and genital tubercle (GT). However, these tissues do not represent
the entire staining profile observed in the fetus after TCDD exposure (see
Willey et al., 1998). For the purposes of this study, these sites were selected
PCB ACTIVATION OF AhR/LacZ REPORTER MOUSE
531
because of their intense, reproducible staining, ease of identification, and ability
to serve as a means to rapidly screen and compare AhR activation across
treatment conditions. All results are based on observations made from at least
three dams per treatment condition, with a range of 3–10 fetuses per dam.
In an attempt to characterize the staining observed in the fetuses, we
developed a scale of staining intensity for the respective tissues examined in
this study. Olive oil–treated controls provided us with the background level of
staining; thus, no staining was observed in either ear or paw, and minimal
staining was seen in genital tubercle only (data not shown; however, the
background staining pattern of the controls matched that of PCB95-treated
fetuses, Fig. 3). Wild-type (WT) controls exposed to vehicle or TCDD showed
no staining in any regions examined. Using tissues from fetuses exposed to 30
lg/kg TCDD as our maximal response (for example, see Fig. 7), we visually
graded the fetuses based on the amount and intensity of the X-gal precipitate in
the selected tissues. Any tissue showing either no staining or background
staining was scored – or ‘‘background,’’ respectively; any visible staining, þ;
moderate staining, þþ; and maximal staining, þþþ. Our effort to apply
a semi-quantitative approach to characterizing the staining observed in the
fetuses is based purely on extensive observation of our model and serves as
a means for comparisons within these experiments only.
Histological examination of X-gal staining. To more readily compare the
X-gal staining profile of mice exposed to PCBs with those administered TCDD,
an examination of the tissue-specific distribution of b-galactosidase activity
was performed. Whole fetuses were processed according to the above X-gal
method and then embedded in Tissue-Tek O.C.T. (Sakura Finetek USA,
Torrance, CA) and cut into 12-lm sections using a Microm HM 500 OM
cryostat (Richard-Allen Scientific, Kalamazoo, MI). The sections were thawed
onto slides, counter-stained with eosin, and mounted with GVA mounting
medium (Zymed, San Francisco, CA). Images were captured using an Olympus
BH2 microscope and Image Pro Plus software.
RESULTS
Coplanar PCBs Induce AhR-Mediated Transcription
of b-Galactosidase
Microscopic examination of whole fetuses harvested from
timed-pregnant dams dosed with 50–500 lg/kg PCB126 po
revealed staining of ears, paws, and GT (Fig. 1). Application of
the grading scale of intensity and character of the staining
suggests a dose-dependent relationship between the concentration of PCB126 administered and the activation of AhRinduced transcription (Table 1). Exposure of fetuses to PCB77
also induced staining in the ear, paw, and GT (Fig. 2), albeit at
doses much higher (1000 lg/kg, po) than those required to
observe staining after exposure to PCB126.
Neither PCB95 (Fig. 3) nor the A1254 mixture (data not
shown) elicited any response of the AhR-mediated lacZ
reporter gene. Exposure of the fetuses to concentrations as
high as 1000 lg/kg failed to result in any staining of the
selected tissues above background levels. The concentrations
of PCBs used here did not appear to adversely affect the dam,
and no weight loss was observed following the 24 h exposure
period (data not shown).
Histological examination of X-gal staining in tissue sections
of fetuses treated with PCB126, as compared to fetuses
exposed to TCDD further substantiated the similarities between the staining profiles of these two AhR ligands. X-gal
FIG. 1. Gestational day 15 fetus exposed to 150 lg/kg 3,4,5,3#,4#pentachlorobiphenyl (PCB126) for 24 h via maternal gavage. A. Anterior
fetus showing X-gal staining in tissues of the external ear. B. Posterior fetus
showing X-gal staining in the plantar surface of the hind paw and genital
tubercle (GT). Inset: Ventral aspect of GT showing X, glans; Y, preputial
swelling; and Z, urethral seam and proximal urethra opening.
staining in the GT was generally localized to the ventral regions
of the structure and was most predominant in the glans and
urethral seam, ending around the proximal urethral opening
(Fig. 1A and Fig. 4). In the paw, X-gal staining was closely
associated with the footpads of both the fore and hind limbs.
Staining was localized to the metatarsal and tarsal pads of the
hind limb (Fig. 1B) and their analogous pads on the forelimb.
Curiously, the tarsal pads showed a more heavily focused
staining pattern (Fig. 5 A–D), whereas the metatarsal pads displayed a more ring-like staining profile (Fig. 1B and Fig. 7B).
No staining was observed in the digital pads of any paws. In
addition to the staining localized to the pads, the junctions
between the digits showed strong reporter gene signaling. The
X-gal staining in the ear was localized to a region of the outer
ear termed the ‘‘external acoustic meatus’’ (Fig. 6). These
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BEMIS, NAZARENKO, AND GASIEWICZ
TABLE 1
b–Galactosidase Staining Intensity in Fetal Tissues Following
Exposure to PCBs or TCDD
Agent (lg/kg, po)
TCDD (30)
PCB126 (500)
(150)
(50)
(15)
PCB77 (1000)
(500)
A1254 (1000)
PCB95 (1000)
Ear
Paw
GT
þþþ
þþ/þ
þ
þ
þ
þ
þþþ
þþ
þþ
þ
þ
þþþ
þþ/þþþ
þþ
þ
þ
þ
bkgd.
bkgd.
bkgd.
Note. Staining intensity: , no visible stain; þ, visible stain; þþ, moderate
stain; þþþ, maximal stain.
PCB: polychlorinated biphenyls; TCDD: 2,3,7,8-tetrachlorodibenzo-p-dioxin;
PCB126: 3,4,5,3#,4#-pentachlorobiphenyl; PCB77: 3,4,3#,4#-tetrachlorobiphenyl;
A1254: aroclor 1254; PCB95: 2,3,6,2#,5#-pentachlorobiphenyl; GT: genital
tubercle; bkgd: background.
regions of PCB126-induced AhR activity correspond well to
those associated with TCDD exposure.
New DRE-lacZ Line Responds to Both TCDD and PCBs
Exposure of fetuses derived from the W-line to 30 lg/kg
TCDD produced considerable staining in the ear, the fore and
hind paws, and GT (Fig. 7). This line was similarly responsive
to PCB126 and PCB77 at the same doses as employed with the
established DRE-lacZ mouse line (data not shown). As
observed in the original line of DRE-lacZ mice, the W-line
fetuses did not respond to administration of A1254 or C95
(data not shown).
FIG. 2. Gestational day 15 fetus exposed to 1000 lg/kg 3,4,3#,4#tetrachlorobiphenyl (PCB77) for 24 h via maternal gavage. A. Anterior fetus
showing X-gal staining in tissues of the external ear. B. Posterior fetus showing
X-gal stain in hind paw (arrows) and GT.
DISCUSSION
The goal of this study was to examine the response of an
established DRE-lacZ mouse line to known AhR ligands other
than TCDD. In addition, we sought to determine the potential
influence of the site of integration of the transgene on the
activity of the reporter gene by testing the response of another
mouse line, generated using similar techniques, to known AhR
ligands.
Exposure of pregnant dams to PCB126 and PCB77 resulted
in activation of the AhR/DRE-driven pathway, leading to
transcription of the lacZ transgene in specific tissues of the
developing fetus. Figures 1 and 2 demonstrate that the tissuespecific profile of PCB-induced staining of the fetus is
comparable to that observed after exposure to TCDD (Fig. 7).
Additionally, cryotome sectioning of stained fetuses treated
with PCB126 or TCDD revealed a similar pattern of bgalactosidase–positive cells at the cellular level (Figs. 4–6).
Although in certain cases the staining patterns didn’t exactly
overlap, or the intensity of staining varied, the general
pattern and characteristics of the staining observed between
the TCDD- and PCB126-treated fetuses was quite similar.
Some of these differences may be attributed to slightly
different orientation of the tissue during cryotome slicing, as
well as to inter-individual variations in staining and the
differences in activity of the ligands at the AhR.
Identification of the exact cellular localization and specific
cell type(s) with which the X-gal signal is associated in the
various tissues was not attempted in this study. Our intention
was to compare the activity of AhR ligands other than TCDD,
and to confirm the pattern of AhR activation. Further studies
can now be initiated, in specific tissues, using co-localization of
the reporter gene signal with other cellular markers to
positively identify cell types affected by TCDD or PCB
exposure during development.
PCB ACTIVATION OF AhR/LacZ REPORTER MOUSE
533
FIG. 4. 12-lm sagittal sections through the genital tubercle (GT) of
a gestational day 15 fetus exposed for 24 h to 30 lg/kg tetrachlorodibenzo-pdioxin (A,C) or 150 lg/kg 3,4,5,3#,4#-pentachlorobiphenyl (PCB126) (B,D)
via maternal gavage. Sections A & B are from more lateral regions of the GT,
whereas C & D are closer to the midline.
Hayward, 1999). In terms of cumulative dose, the concentrations of PCB126 used in the present studies are well within
those used in the Rice studies and, in fact, are in the lower range
of concentrations. However, the effects of acute versus chronic
dosing may not necessarily be similar. With respect to the
concentrations of coplanar congeners employed here, our data
demonstrate the transplacental potency of these compounds for
the AhR and the sensitivity of the DRE-lacZ mouse model to
AhR ligands.
FIG. 3. Gestational day 15 fetus exposed to 1000 lg/kg 2,3,6,2#,5#pentachlorobiphenyl (PCB95) for 24 h via maternal gavage. A. Anterior fetus
showing lack of X-gal staining in tissues of the external ear. B. Posterior fetus
showing lack of X-gal staining in hind paw and background levels of X-gal
in GT.
The presence of AhR and its accessory proteins in mouse
embryos has been reported as early as GD 13.5 (Jain et al.,
1998) in tissues such as palate, lung, liver, urogenital sinus, and
GT. Thus our reported activation of the lacZ reporter gene
corresponds to several previously identified sites of expression
of AhR. This observation, in addition to the similarity in
staining profile between PCB126 and TCDD, suggests that
in utero exposure to PCB126 initiates activation of pathways
potentially involved in AhR-mediated alterations in neonatal
development.
PCB126 has previously been employed to examine the
effects of developmental exposure to a coplanar PCB congener
that is a known AhR agonist. Rice and colleagues employed
a dosing regime of 0.25 or 1 lg/kg/day for 5 days a week,
beginning 35 days prior to mating and continuing through
gestation and lactation (Crofton and Rice, 1999; Rice and
FIG. 5. 12-lm sections through the hind paw of GD 15 fetus exposed for
24 h to 30 lg/kg tetrachlorodibenzo-p-dioxin (A,C) or 150 lg/kg 3,4,5,3#,4#pentachlorobiphenyl (PCB126) (B,D). Sections A and B are 43 images of
varying orientations of the paw, and C and D are the corresponding 203 images
indicated by the boxes. Locations of digits and footpads are indicated to provide
the orientation of the sections.
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BEMIS, NAZARENKO, AND GASIEWICZ
FIG. 6. 12 lm sagittal sections through the ear of a GD 15 fetus exposed
for 24 h to 30 lg/kg tetrachlorodibenzo-p-dioxin (A,C) or 150 lg/kg
3,4,5,3#,4#-pentachlorobiphenyl (PCB126) (B,D). Sections A and B are 43
images of the internal structures of the pinna of the ear (dorsal up, nose to right)
and C and D are the corresponding 203 images indicated by the boxes.
The observation that higher doses of PCB77 are required to
elicit a similar intensity of b-galactosidase staining as that seen
following PCB126 (1000 lg/kg versus 50 lg/kg) exposure
maintains the structure–activity relationships (SARs) established for the effects of PCBs on the AhR. Hesterman et al.
(2000) demonstrated a considerably higher EC50 value for
PCB77 than for PCB126 in EROD induction (0.73 versus
0.029 nM, respectively) and a 1000-fold increase in relative
potency of induction of Cytochrome P-450 1A (CYP1A) protein in Poeciliopsis lucida hepatocellular carcinoma (PLHC-1)
cells when comparing PCB126 to PCB77. In the whole animal,
some tenfold higher concentrations of PCB77 were required to
elicit similar alterations in sex-based behavior after exposure to
PCB126 (Amin et al., 2000). The fact that our data support
established PCB SARs further strengthens our confidence that
the DRE-lacZ mouse models in vivo AhR function.
The intense staining observed in GT after TCDD exposure
suggests significant activation of AhR-mediated events in this
developing tissue. Willey et al. (1998) reviewed the effects of
TCDD on phallic development and reproduction, supporting
the observation that early disruption of differentiation processes by TCDD exposure leads to long-term functional
deficits. More recent experiments describe the effects of TCDD
on the development of the closely associated urogenital sinus
(UGS) and its impact on prostate gland formation. TCDD
exposure at GD 13 acts specifically on the UGS to reduce
budding, and this reduction eventually leads to alterations in
duct formation of the prostate (Ko et al., 2004a; Lin et al.,
2004). This effect is mediated by TCDD-induced activation of
the AhR in ventral UGS mesenchymal cells (Ko et al., 2004a),
and it does not involve AhR-mediated disruption of normal
androgen signaling (Ko et al., 2004b). It is interesting to note
FIG. 7. Gestational day 15 fetus, derived from the newly generated DRElacZ line (W-line), exposed to 30 lg/kg tetrachlorodibenzo-p-dioxin (TCDD) for
24 h via maternal gavage. A. Anterior fetus showing X-gal staining in tissues of
the external ear. B. Posterior fetus showing X-gal staining in hind paw and GT.
that the region of staining in the genital tubercle in our model
exposed to TCDD or PCBs was also predominantly in the
ventral mesenchymal tissues. The observations from Peterson’s
group, combined with ours, illustrate the influence that TCDD
may have on the developing reproductive system. Our present
data predict that other exogenous AhR ligands, such as the
coplanar PCBs used here, would induce similar effects during
UGS differentiation.
The creation of a new line of DRE-lacZ mice was prompted
by the potential influence of intrinsic regulatory regions
surrounding the reporter gene transcript. Weis et al. (1991)
demonstrated that regulatory regions, intrinsic to the site of
insertion of the transgene, influenced the activity of a lacZ
reporter gene, driven by regulatory elements from a major
histocompatability complex. By creating another mouse line,
the W-line, under the assumption that the chances of insertion
into the same site in the genome as in the original DRE-lacZ
PCB ACTIVATION OF AhR/LacZ REPORTER MOUSE
line are miniscule, we tested whether the lacZ response was
integration-site dependent. After exposure to both TCDD and
coplanar PCBs, the distribution of staining in fetuses derived
from the W-line was identical to that seen in the original DRElacZ mouse model. While this observation cannot entirely
eliminate integration-site dependence, it does serve to significantly minimize the chances that any positional effects are
driving the response of the transgene, and it confirms the utility
of this mouse model in investigations of AhR-mediated toxicity
and the potential function of the AhR in normal development.
Our use of the DRE-lacZ mouse model has extended beyond
experiments that examine the general reporter gene activity
into investigations of tissue specific responses to TCDD.
Williamson et al. (2005) used the reported transcriptional
activation of AhR in immature cerebellar granule cells after
acute TCDD exposure in early postnatal mice. We have also
collaborated with Dr. Richard Peterson’s group at the University of Wisconsin to examine localization of TCDD-mediated
AhR activity in the developing prostate (Kelman et al., 2005).
The increasing application of this mouse model in investigations examining TCDD-mediated toxicity necessitates studies
such as those described here.
In summary, these data confirm that the activation of bgalactosidase observed is dependent on signaling through the
AhR and is not some phenomenon derived from TCDD
exposure alone. Additionally, generation of a new line of
DRE-lacZ mice indicates that the effects of both TCDD and
PCBs on the DRE-driven lacZ reporter gene are most likely
insertion-site independent. Both of these investigations serve to
validate the DRE-lacZ mouse as a suitable model for TCDDinduced toxicity and strengthen the observations we have
previously reported from this mouse model.
ACKNOWLEDGMENTS
We thank Nancy Proper and Jen Laruffa for their work in maintaining and
genotyping the lacZ mice. This work was supported by Center Grant ES01247,
NIH Training Grant ES07026, and Grant ES09430. Conflict of interest: none
declared.
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