* Environmental Sciences and Engineering Department, School of Public Health, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599;
Biostatistics and Research Support Staff, National Health and Environmental Effects Research Laboratory, Research Triangle Park, North Carolina 27711; and
Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711
Received January 11, 2001; accepted March 19, 2001
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ABSTRACT |
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Key Words: TCDD; hydronephrosis; cleft palate; EGF; TGF-; EGF receptor..
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INTRODUCTION |
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TCDD is a reproductive and developmental toxicant in wildlife and laboratory animals, including rats, hamsters, and mice. The primary teratogenic abnormalities produced in mice are hydronephrosis and cleft palate (Moore, 1973). In the C57BL/6N mouse, these responses are seen at doses that do not result in maternal or fetal mortality (Birnbaum et al., 1985
). The sensitive period for induction of hydronephrosis by TCDD extends throughout gestation and into the postnatal period, as neonatal mice exposed lactationally for up to 4 days after birth develop hydronephrosis (Couture-Haws et al., 1991a
,b
).
TCDD's toxic effects are generally believed to be mediated via the aryl hydrocarbon receptor (AhR). Upon binding ligand, the receptor heterodimerizes with its nuclear translocator and this complex translocates to the nucleus where it regulates gene expression by binding to the dioxin response element (Neubert et al., 1993; Peterson et al., 1993
). TCDD also alters growth factor expression and disrupts regulation of differentiation and proliferation of epithelial cells in adult tissues and during embryo/fetal development. In normal development, cell proliferation and differentiation are controlled by families of growth factors and their receptors, including the epidermal growth factor receptor (EGFR) that binds epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-
) (Dohr et al., 1994
). Both EGF and TGF-
bind to the EGFR with similar affinity. Binding of EGF results in rapid internalization of the receptor and degradation, however after TGF-
binds to the receptor the internalized complex can escape degradation and return to the cell surface, allowing sustained signaling through the receptor pathway (Decker, 1990
; Lax et al., 1988
). During fetal development TGF-
and EGF are expressed in numerous organs including the palate, kidney, brain, pituitary, skin, salivary gland, spleen, and lung (Abbott et al., 1987
; Bryant et al., 1997
; Ebner and Derynck, 1991
; Kudlow et al., 1988
; Madtes et al., 1988
; Rall et al., 1985
; Snead et al., 1989
; Wilcox and Derynck, 1988b
).
There is a pattern of specific spatial and temporal localization of TGF- and EGF in the epithelial cells of the developing urinary tract and palate, suggesting that they play a role in formation of these tissues (Abbott and Birnbaum, 1990b
; Bryant et al., 1997
). Exposure to TCDD stimulates the epithelial cells that line the lumen of the ureter to proliferate abnormally, which results in occlusion of the lumen and produces hydronephrosis (Abbott et al., 1987
). Exposure to TCDD on GD 10 decreases expression of EGF in the ureteric epithelia on GD 13 and 14, while expression of TGF-
is unchanged on GD 14 (Bryant et al., 1997
). Based on these observations, it was suggested that hyperplasia of the epithelial cells could be due to a TCDD-induced imbalance in the relative expression levels of EGF and TGF-
. In the present study, we exposed pregnant mice to TCDD on GD 12, when the kidney is forming from a ureteric bud as an outgrowth of the mesonephric duct with an associated, aggregated cap of mesenchymal cells. The branching ureter invades the mesenchyme and induces a mesenchymal-to-epithelial conversion to form structures of the kidney such as the glomerulus and proximal tubules. Subsequent morphogenesis and differentiation of the tubular epithelium result in the establishment of a functional nephron (Grobstein, 1955
; Kispert et al., 1998
; Saxen and Sariola, 1987
). The elongation of the ureter and development of an unobstructed lumen during the GD 1216 period is critical to normal urinary function as the kidney matures and releases urine to the bladder. Regulation of these events by growth factors is critical and disruption of growth factor expression by TCDD appears to be a major factor in the teratogenic response.
The palate, which is the other major teratogenic site in TCDD-exposed mice, is also in early stages of morphogenesis on GD 12. During normal palatogenesis in the rodent, the shelves of the palate grow vertically on either side of the tongue. By GD 14 the shelves elevate above the tongue, come into contact, and fuse. In C57BL/6N embryos exposed to TCDD on GD 12, the shelves come into contact, but hyperplasia of the medial epithelial cells prevented adherence and fusion (Abbott and Birnbaum, 1990b; Pratt et al., 1984
). As was the case for the urinary tract, the etiology of TCDD-induced cleft palate also appears to involve dysregulation of growth factors. TCDD exposure increased EGF and EGFR expression in the palatal epithelia (Abbott et al., 1987
).
In the present study, we utilize knockout EGF (/), TGF- (/), and EGF + TGF-
(/) mice to examine the role of this signal transduction system in the induction of hydronephrosis and cleft palate following embryonic exposure to TCDD. The use of these animals revealed that TCDD-induced hydronephrosis was most pronounced in the EGF (/) and TGF-
(/), but the severity of hydronephrosis in EGF + TGF-
(/) fetuses was similar to that of WT. In the palate, EGF plays a key role in TCDD-induced teratogenicity, as this exposure regimen did not induce cleft palate in the EGF (/) or EGF + TGF-
(/). In contrast, the absence of TGF-
did not reduce the palatal response to TCDD. This study provides substantial support for the hypothesis that this growth factor pathway has a critical role in the induction of teratogenic responses by TCDD.
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MATERIALS AND METHODS |
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TGF- (/) (Tgfatm1Ard) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The Tgfatm1Ard (TGF-
) mutant strain was developed by Dr. Ashley Dunn at the Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch, using the 129-derived E14 ES cell line (Mann et al., 1993
). Mice carrying the TGF-
mutation were backcrossed 5 or more generations to C57BL/6J mice and this makes it very likely that the genetic background of these (/) will differ from that of the EGF (/) and EGF + TGF-
(/). Mice homozygous for the TGF-
mutation develop normally, are healthy and of normal size and weight. The TGF-
(/) display a pronounced waviness in the coat and whiskers and dramatic derangement of the hair follicles, and aged homozygous mice occasionally show some corneal inflammation that may be the result of a defect in wound healing (Dlugosz et al., 1995
).
The EGF (/) mice were produced by the Lee laboratory by replacing EGF exon 20 with the neomycin resistant cassette using a targeting vector (Luetteke et al., 1999). Deletion of that exon eliminated the first 2 EGF disulfide loops, critical in activation of the EGFR. The EGF (/) mice were fertile, and survival and growth was not significantly different from wild types. To produce the EGF + TGF-
(/) mice, the Lee laboratory utilized TGF-
(/) mice produced in their laboratory and mated them to EGF (/) mice to obtain double heterozygotes (Lee et al., 1995
; Luetteke et al., 1993
, 1999
). The double (+/) mice were then intercrossed to obtain homozygous EGF + TGF-
(/). These animals retained the wavy coat phenotype and survived to maturity. They displayed an increase in eye defects as well as accelerated hair loss, dermatitis, and skin ulceration with aging. We have confirmed the genotypes of the mice in our studies by Southern blot, as described in Leutteke et al. (1999). The TGF-
(/) line from Jackson Laboratory that was used in our study, and the knockout line produced by the Lee laboratory have similar phenotypes with waviness in the coat and whiskers, and derangement of hair follicles, and no alterations in fertility or survival.
Chemical and dosing.
A stock solution of TCDD (100 µg/ml), chemical purity 98% by gas chromatography/mass spectroscopy, (Radian Corporation, Austin, TX), was prepared by dissolving the compound in acetone, adding corn oil, and removing the acetone under vacuum (Savant Speed Vac, Savant Instruments, Inc., Farmingdale, NY). Dosing solution was prepared by diluting the stock solution in corn oil to a final concentration of 4.8 µg/ml. Pregnant females were weighed on GD 12 and dosed by gavage with either corn oil or TCDD at 24 µg/kg, 5 ml/kg.
Necropsy.
On GD 17.5, pregnant females were weighed, anesthetized by CO2 inhalation, and sacrificed by cervical dislocation. The maternal liver and intact uterus were removed and weighed. The fetuses were removed from the uterus and placed on ice. To determine the total number of implantation sites, the uteri were placed in a 10% ammonium sulfide solution that stained the hemosiderin pigment of each implantation site blue-black (Narotsky et al., 1997; Narotsky and Kavlock, 1995
). The number of live and dead pups, as well as the number of early and late resorptions, was recorded.
Each fetus and its placenta were weighed separately. Fetuses were subsequently decapitated and the mandible was removed to allow for assessment of cleft palate. The palates were classified as normal (no cleft palate), cleft (secondary palate not fused), or posterior cleft (palate fused in the anterior hard palate, but not in the posterior soft palate). The gastrointestinal tract and liver were removed to expose the urogenital system. Fetal livers were weighed and the gender was recorded. Observations of hydroureter were recorded for left and right ureters. The fetal head and body were submerged and stored at room temperature in Bodian's fixative (2% formaldehyde, 5% acetic acid, 72% ethanol, 21% water) (Narotsky et al., 1995).
To assess the presence and severity of hydronephrosis, each fetus was removed from the Bodian's fixative. Leaving the organs in situ each kidney was cut in half in the transverse plane and evaluated under a dissecting microscope (Leica, WILD M3Z, Heerbrugg, Switzerland). Two independent observers evaluated each kidney and assigned hydronephrotic severity scores utilizing a scoring system adopted from Woo and Hoar (1972). As shown in Figure 1, a value of 0 was assigned to kidneys in which the papilla totally filled the renal pelvic space; +1 was assigned to kidneys that had a slight dilation of the renal pelvis (i.e., small gap between the papilla and the renal wall); the value +2 was given to kidneys with reduced papilla size and noticeable dilation of the pelvic space. All kidneys that received a severity score of +2 or higher were considered hydronephrotic and the incidence of the defect was based on this criterion. A value of + 3 was assigned to hydronephrotic kidneys that had very short papillae and compressed renal tissue, creating a larger gap or marked dilation of the renal space. The most severely hydronephrotic kidneys were assigned a score of +4 and displayed virtually no papilla and a thin renal wall.
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For each maternal parameter a two-way ANOVA was performed to determine if there was an association between these variables and genotype or treatment, or if there was any interaction between genotype and treatment. The significance levels were generated from the F-tests for main effects or interaction terms, or from t-tests for contrasts among the groups. For the proportions of resorptions, an arcsin transformation was used to stabilize the variance (Kleinbaum et al., 1988).
For the litter and fetal continuous parameters, a linear model was run that included treatment, genotype, the treatment-genotype interaction, litter (nested within treatment and genotype), and gender. For the dichotomous variables (cleft palate, incidence of hydronephrosis) Fisher's exact test and Cochran-Mantel-Haenszel test were used to determine if there was an association between gender and these variables in all pups or across treatment groups and/or genotypes. As there was no evidence of any association with gender in any of the variables (p > 0.05 in all tests), litter means were used in analyzing these variables, and two-way ANOVAs were performed as described above, using an arcsin transformation to stabilize the variance (Kleinbaum et al., 1988). Similarly, for the hydronephrotic severity scores, litter means were calculated and analyzed with two-way ANOVA. The difference between the left and right sides for the incidence and severity of hydronephrosis was examined and the litter mean for each was also analyzed using a two-way ANOVA. All analyses were done using SAS software (SAS Institute, 1989
).
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RESULTS |
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Litter Data
As shown in Table 2, there was no significant effect of genotype on the number of embryos implanting in the uterus or between genotypic groups for survival in early stages of development prior to treatment on GD 12 (early resorptions). There was no significant effect of TCDD exposure on fetal survival, as there were no significant increases in late resorptions and no dead fetuses on GD 17.5. The gender distribution was similar across genotypes and treatments, indicating that there was no gender-related bias for survival in response to TCDD exposure. Also, the knockout of TGF-
, EGF, or both growth factors did not render either male or female fetuses more vulnerable to death in utero as there were no significant differences in the number of live male and female fetuses at the time of collection.
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TCDD treatment reduced placenta weight in the EGF + TGF- (/) and WT groups relative to their respective control litters (p < 0.05), but EGF (/) and TGF-
(/) placenta weights were not significantly changed. There were no significant differences in placenta weights across genotypes when comparing control litters. Across all genotypes and treatments, the male fetuses had significantly (p < 0.001) heavier placentas, 131.7 ± 1.5 compared to 129.0 ± 1.7 mg for female fetuses.
Cleft Palate
Each fetus was evaluated for the presence of cleft palate, and the incidence across genotypes and treatments is presented in Table 4. The controls of each genotype were not significantly different. No cleft palate occurred in the 43 WT controls (Fig. 2A
) and rates were low for EGF (/) (1 out of 63), TGF-
(/) (1 out of 44), and EGF + TGF-
(/) (1 out of 42). Exposure to TCDD significantly increased the incidence of cleft palate in WT (p < 0.05, 11 affected out of 58) (Fig. 2B
) and in the TGF-
(/) group, in which all of the 41 fetuses had cleft palate (Fig. 2D
) (p < 0.001). The degree of change relative to the control within genotype was significantly greater for TGF-
(/) group (p < 0.001) compared to WT. There was 1 cleft palate among 63 control EGF (/) fetuses and that was a full cleft similar to that shown for TCDD-treated WT (Fig. 2B
). TCDD did not significantly increase the incidence of cleft palate in the EGF (/) fetuses. In the EGF (/) group, the 3 clefts that occurred (46 fetuses from 6 litters) were only in the posterior, soft palate (Fig. 2C
). This phenotype is in contrast to the full cleft palate observed in the treated WT and the TGF-
fetuses (Figs. 2B and 2D
, respectively). In the EGF + TGF-
(/) group, frequencies of cleft palate were similar in the control (1 out of 42 fetuses) and treated (3 out of 43), and there was no significant effect of treatment. The palates of the EGF + TGF-
(/) were generally not morphologically different from WT controls (Figs. 2E and 2F
).
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The effect of EGF and/or TGF- gene knockout on kidney development and background level of hydronephrosis can be examined by comparing the severity scores of controls of each genotype. When comparisons are made between controls of the genotypic groups, WT severity scores were significantly lower (p < 0.05) than any of the other genotypes, right, left or both kidneys considered together. For the right kidney and average scores, TGF-
(/) kidneys were significantly more dilated than the other 3 genotypes (p < 0.05). For the left kidney, TGF-
(/) scores were significantly higher than EGF (/) and WT (p < 0.05). Across all genotypes, the severity on the right was higher than that on the left (p < 0.01).
All kidneys that received a severity score of +2 or higher were classified as hydrodronephrotic and litter means are presented in Table 4. TCDD exposure caused a significant increase in the incidence of hydronephrosis for all genotypes on left, right, and in the overall assessment (p < 0.001). The highest response rate was observed in the TGF-
(/) group, in which all of the fetuses developed hydronephrosis. Statistical comparisons of the genotypes did not detect a significant difference in the degree of increase in response to TCDD between WT and the knockout groups (except where 100% incidence was reached in TGF-
for left and right sides, so no further increase could be realized). The knockout of TGF-
(/) appeared to adversely affect kidney development and comparisons across controls of the genotypes revealed increased hydronephrosis in TGF-
(/) fetuses (p < 0.01 vs. WT, EGF [/], and EGF + TGF-
[/], on left, right, and overall). The incidence in the EGF + TGF-
(/) control litters also exceeded that observed in WT for right side and overall measures (p < 0.001). EGF (/) controls also showed higher incidence than WT (p < 0.05), but only in the overall scores. The incidence of hydronephrosis was also compared between left and right kidneys and the right kidney was affected more frequently than the left (across genotypes and treatments) (p < 0.001).
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DISCUSSION |
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EGF and TGF- expression were not required for the fetal urinary tract to respond to TCDD, as embryos that did not express EGF + TGF-
(/) responded to TCDD exposure with a severity and incidence of hydronephrosis comparable to that of WT. The response in either EGF (/) or TGF-
(/) was greater than that of either the WT or the EGF + TGF (/). Based on these outcomes, we hypothesize that the WT-like response of the EGF + TGF-
(/) is due to occupancy of the receptor by alternative EGFR ligands, and that the increased response of EGF (/) or TGF-
(/) fetuses is a consequence of disturbing the competition between EGF and TGF-
for binding to the EGFR. Since EGF and TGF-
bind the EGFR with similar affinity (Ebner and Derynck, 1991
), the relative abundance and timing of the availability of these ligands could be critical to regulating the proliferation and differentiation of the ureteric epithelial cells. The relative expression or balance between the levels of these growth factors may also impact the responses of the cells to TCDD. This hypothesis is supported by studies showing that binding of EGF and TGF-
produce different responses in cultured human keratinocytes. In those cultures, TGF-
stimulated colony expansion with a 2-fold greater increase in radius relative to nonstimulated cultures, when compared with EGF (Barrandon and Green, 1987
). Additionally, in an in vivo study, TGF-
was a more potent mediator of angiogenesis in the hamster cheek pouch compared to EGF (Schreiber et al., 1986
). TGF-
is a more potent mitogen than EGF in a number of tissues, such as the brain, pituitary, and liver (Brenner et al., 1989
; Kudlow et al., 1988
; Wilcox and Derynck, 1988a
). When compared to EGF, TGF-
stimulated cellular proliferation for a longer time in murine L cells and human keratinocyte cell lines. It was also observed in that study, that the processing of the ligand- (EGF or TGF-
) EGFR complex by the cells was dependent on the specific ligand bound to the EGFR (Ebner and Derynck, 1991
). EGF caused a complete down-regulation and degradation of membrane EGFR, resulting in a refractory period. In contrast, after internalization, less of the TGF-
/receptor complex is degraded and TGF-
rapidly releases from the receptor and both TGF-
and the receptor are immediately recycled to the cell surface. This would allow for continued responsiveness and sustained cellular proliferation in the presence of TGF-
.
TCDD exposure of C57BL/6N embryos on GD 10 was previously shown to decrease the expression of EGF in the fetal urinary tract on GD 13 and 14, but TCDD had no effect on the expression of TGF- on GD 14 (Bryant et al., 1997
). Thus, in the TCDD-treated C57BL/6N and the EGF (/) fetuses, TGF-
has less competition (or no competition) from EGF for binding to the EGFR. Thus, TCDD-exposed ureteric epithelial cells could experience prolonged proliferative stimulation when the competition for EGFR shifts to TGF-
. The expression of EGF in the absence of TGF-
(TGF-
/), also increased the responsiveness of fetal urinary tract to TCDD, further suggesting that both ligands play a role in regulation of appropriate cellular responses and that balanced or competitive binding is needed. In the EGF + TGF-
(/) fetuses, 1 or more of the other ligands that bind to EGFR (amphiregulin [Shoyab et al., 1989
], heparin-binding EGF [HB-EGF] [Harding et al., 1996
], betacellulin [Shing et al., 1993
], or epiregulin [Luetteke et al., 1999
]), apparently compensated for the absence of EGF and TGF-
and permitted embryonic development and survival. EGF, TGF-
, HB-EGF, and betacellulin mRNA are expressed in adult kidney, but epiregulin and amphiregullin mRNA are not detected using RT-PCR analysis (Fenton, unpublished data). It is not clear if all of these ligands for EGFR have similar affinities. However, all of the ligands stimulate renal epithelial cell proliferation in culture (Sakurai et al., 1997
). Shoyab et al. (1989) reported that amphiregulin was not as potent as EGF or TGF-
, based on combined data from competition assays and a direct comparison of the growth stimulatory properties of EGF and amphiregulin in cultured, normal rat kidney cells. It was also found that exogenous TGF-
or EGF aided in renal recovery after injury, but exogenous HB-EGF failed to reproduce the decrease in apoptosis in cultured renal epithelial cells (Takemura et al., 1997
).
The present study also provides evidence that these growth factors are essential for normal kidney development, as all of the control fetuses that did not express EGF and/or TGF- displayed dilated pelvic renal spaces. The absence of hydroureter in these control fetuses suggests that the dilated renal pelvic space, small papilla, and reduced blastema thickness are due to retarded or abnormal kidney development. Absence of hydroureter, which is a consequence of occlusion of the ureteric lumen, implies that the lumen is unobstructed and not involved in the renal dilation.
In contrast to the urinary tract responses, in the developing palate there were dramatically different responses to TCDD associated with specific growth factor availability. The induction of cleft palate by TCDD appears highly dependent on expression of EGF. With the dosing regimen of the present study, EGF (/) fetuses did not develop full cleft palate, and only a few of the fetuses exhibited soft palatal clefts. Similar outcomes were noted in the EGF + TGF- (/) fetuses. (Preliminary data from our laboratory indicate that the EGF [/] fetuses will develop full cleft palate after exposure to 100 µg/kg on GD 12. The response can be produced in absence of EGF, but only at substantially higher exposures than required for WT. [Abbott, unpublished data.]) The role of EGF in response to TCDD should also be considered in light of the expression of these growth factors and their receptor in the fetal palate (Abbott and Birnbaum, 1989
, 1990b
; Abbott and Pratt, 1987
). Abbott and Birnbaum (1990a) localized EGFR, EGF, and TGF-
protein immunohistochemically in C57BL/6N fetal palatal shelves. EGFR was expressed strongly in palatal epithelial cells, but decreased in medial epithelial cells just prior to fusion. In treated palates, the expression of EGFR was maintained in this region and hyperproliferation was observed. EGF was observed to increase with gestational age in the palatal cells and expression declined in response to TCDD, while TGF-
was detected only at low levels and was not affected by TCDD exposure.
Although some of the teratogenic responses in EGF and/or TGF- (/) fetuses differed from WT, many of the well-characterized responses remained intact. For example, the "classic" response of increased liver weight was present in pregnant females and fetuses regardless of growth factor status. TCDD induced a significant increase in maternal and fetal absolute and relative liver weights, regardless of growth factor status, confirming that these (/) mice responded to TCDD as expected for these endpoints. The increase in liver weight in TCDD-exposed animals has been reported for several species including mice, guinea pigs, and rats (Abbott et al., 1987
; Birnbaum et al., 1985
; Courtney and Moore, 1971
; Hruska and Olson, 1989
; Neubert and Dillmann, 1972
; Schwetz et al., 1973
). In the TGF-
(/), the degree of response in treated maternal and fetal livers exceeded that observed for WT. TGF-
is the primary ligand for EGFR in the liver (Rall et al., 1985
), but studies that characterized the phenotypes of TGF-
(/) mice revealed no apparent alterations in postnatal liver structure or function (Luetteke et al., 1993
).
Another response typically observed after treatment of pregnant mice with TCDD is the absence of significant effects on maternal weight gain, maternal or fetal survival, or mean fetal weight (Birnbaum et al., 1985; Poland and Knutson, 1982
). Similar outcomes were observed in the present study and the deletion of either EGF or TGF-
or both did not affect maternal or fetal survival or body weights in either controls or TCDD-exposed groups. However, fetuses with both growth factors inactivated exhibited the lowest body weights of all groups in the present study. This prenatal growth retardation may be resolved postnatally as Luetteke et al. (1993) reported similar weights for wild type and EGF (/) pups on postnatal days 7, 14, and 21.
Also, in agreement with previous reports (Birnbaum et al., 1985; Couture et al., 1990
; Moore, 1973
), the hydronephrosis induced by TCDD was more severe and frequent for the right kidney, compared to the left side. This was true regardless of growth factor status. Additionally, the gender distribution was similar across genotypes and treatments, indicating that there was no gender-related bias for survival due to the deletion of EGF or TGF-
, or in response to TCDD exposure.
In conclusion, this study demonstrated that hydronephrosis was induced by TCDD in the EGF + TGF- (/) fetuses at a level comparable to the WT, but the incidence and severity of hydronephrosis was substantially greater in the EGF (/) and TGF-
(/). Thus, although not a requirement for the ureteric epithelial hyperplastic response to TCDD, EGF and TGF-
substantially influence the incidence and severity of the hydronephrotic response. EGF and TGF-
are also not required for normal palatal development. EGF appears to be a major mediator of TCDD-induced cleft palate, as palates without EGF expression were substantially less responsive to TCDD. However, the absence of TGF-
does not protect against dioxin-induced palatal teratogenicity as clefts were induced in all TGF-
(/) fetuses. The TCDD-induced disruption in the EGFR signal transduction pathway, particularly in the relative expression of EGF and TGF-
, appears to be a major factor in the teratogenicity produced by this compound. The present study demonstrates that in the absence of EGF and/or TGF-
, other EGFR ligands can mediate the response to TCDD. Further study is needed to define the potential roles of amphiregullin, HB-EGF, betacellulin or epiregulin in normal development and in response to xenobiotic insult.
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ACKNOWLEDGMENTS |
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NOTES |
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1 Current address: The Proctor & Gamble Co., Health Care Research Center, 8700 Mason-Montgomery Road, Mason, OH 450409642.
2 To whom correspondence should be addressed at Reproductive Toxicology Division (MD 67), National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711. Fax: (919) 541-4017. E-mail: abbott.barbara{at}epa.gov.
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