EGF and TGF-{alpha} Expression Influence the Developmental Toxicity of TCDD: Dose Response and AhR Phenotype in EGF, TGF-{alpha}, and EGF + TGF-{alpha} Knockout Mice

Barbara D. Abbott*,1, Angela R. Buckalew*, Michael J. DeVito{dagger}, David Ross{dagger}, P. Lamont Bryant{ddagger} and Judith E. Schmid*

* Reproductive Toxicology Division and {dagger} Environmental Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and {ddagger} The Procter & Gamble Co., Health Care Research Center, 8700 Mason-Montgomery Road, Mason, Ohio 45040-9642

Received August 8, 2002; accepted October 10, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces cleft palate (CP) and hydronephrosis (HN) in mice. The etiology of these defects involves hyperproliferation of epithelial cells of the secondary palatal shelf and ureter, respectively. These effects correlate with altered expression of the epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor-{alpha} (TGF-{alpha}). In this study, the developmental toxicity of TCDD was examined in EGF, TGF-{alpha}, and double EGF + TGF-{alpha} knockout (–/–) and wild type (WT) mice. The influence of background genetics in responsiveness to TCDD was examined using liver 7-ethoxyresorufin-O-deethylase (EROD) activity. Animals were dosed by gavage with 0, 0.2, 1, 5, 24, 50, 100, or 150 µg TCDD/kg (5 ml/kg) body weight on gestation day 12. The mixed genetic background of WT, EGF (–/–), and EGF + TGF-{alpha} (–/–) made these mice less responsive to TCDD relative to C57BL/6J and TGF-{alpha} (–/–), which have a C57BL background. These results show that EGF and TGF-{alpha} are not required for response to TCDD; however, the specific ligand available to bind EGFR affects the responsiveness to TCDD. EGF (–/–) mice are less responsive for CP, but more sensitive to HN. TGF-{alpha} (–/–) mice were similar to WT in sensitivity for induction of CP and HN. The responses of EGF + TGF-{alpha} (–/–) mice were like the WT except at higher doses where sensitivity to CP increased, suggesting that the responses may be mediated by alternative ligands for EGFR that are not functional equivalents of EGF or TGF-{alpha}. In conclusion, the EGFR pathway is mechanistically important in responses of the embryo to TCDD. Specific ligands confer sensitivity or resistance that are target tissue-dependent.

Key Words: hydronephrosis; cleft palate; EGF; TGF-{alpha}; EGF receptor; TCDD.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Studies in mice of the mechanism through which the environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces cleft palate and hydronephrosis revealed correlations between effects on epithelial cell proliferation and changes in the expression of the epidermal growth factor receptor (EGFR), epidermal growth factor (EGF), and transforming growth factor-{alpha} (TGF-{alpha}; Abbott, 1997Go; Abbott and Birnbaum, 1998Go). The EGFR signaling pathway is a complex signaling pathway that includes multiple receptors and ligands and is a key regulator of cellular proliferation and differentiation and is critical to normal embryonic development. EGFR (–/–) mice have multiple abnormalities, including cleft palate, and rarely survive postnatally (Miettinen et al., 1995Go, 1999Go; Sibilia and Wagner, 1995Go; Threadgill et al., 1995Go). Ligands that bind to EGFR and mediate signal transduction include EGF, TGF-{alpha}, amphiregulin (AR), epiregulin (ER), heparin-binding EGF (HB-EGF), and betacellulin (BTC). EGFR and its ligands have roles in implantation, growth, and survival of the conceptus and specific spatial and temporal patterns of expression are required during morphogenesis of the palate, kidney, heart, lung, and mammary gland (Giudice, 1999Go; Johnson et al., 1994Go; Luetteke et al., 1999Go; Miettinen et al., 1999Go; Sakurai et al., 1997Go; Schuger et al., 1996Go; Toyoda et al., 1995Go; Wang et al., 2000Go; Wiley et al., 1992Go).

Disruption of the EGFR pathway may be a key mediator of the developmental toxicity of TCDD. In the mouse, exposure in utero to TCDD altered the proliferation and differentiation of epithelial cells in the secondary palatal shelves and in the developing ureter, leading to failure of palatal shelf fusion and occlusion of the ureteric lumen, respectively (Abbott, 1997Go; Bryant et al., 1997Go). However, the sensitivity for induction of these developmental defects differs between the target tissues (Birnbaum, 1995Go). Hydronephrosis could be induced at lower exposures relative to the induction of cleft palate. There was also a difference between these tissues in the expression patterns of EGFR, EGF, and TGF-{alpha} during normal morphogenesis and following exposure to TCDD (Bryant et al., 1997Go). In the palate, the expression of TGF-{alpha} declines during palatogenesis and EGFR expression and proliferation cease in the medial epithelial cells prior to fusion (Abbott, 1997Go). In the ureteric epithelial cells, EGF and TGF-{alpha} have similar temporal expression patterns and are present from early stages of ureteric bud outgrowth through the later gestational stages (Bryant et al., 1997Go). After exposure of the palate to TCDD, EGFR, EGF, and TGF-{alpha} increased while in the ureter only EGF increased.

Based on the differences in ureteric and palatal expression patterns and responses to TCDD, it was hypothesized that the responses in the target tissues were influenced by the specific ligand(s) playing a major role in regulating developmental events. Although the EGFR ligands are similar in sequence and compete for binding to the receptor, their binding is not identical and the responses of the cells to each ligand can be very different (Puddicombe et al., 1996Go; Solic and Davies, 1997Go). In tissues such as the ureter, which expressed multiple ligands, normal morphogenesis may depend on maintaining a balance between the expression of these ligands and disturbing that balance could alter the regulation of cellular proliferation or differentiation.

A better understanding of the role of the EGFR pathway in developmental toxicity apparently requires unraveling of the divergent roles of EGF and TGF-{alpha} in the target tissues. In the present study, we used knockout (–/–) mice in which EGF or TGF-{alpha} or both were not expressed. The EGF (–/–) mice are healthy and fertile, and the adult mice display no adverse phenotypic effects (Luetteke et al., 1999Go). The TGF-{alpha} (–/–) mice have a moderate, variable phenotype consisting of wavy hair and whiskers (Dlugosz et al., 1995Go; Mann et al., 1993Go) and eye defects (Luetteke et al., 1993Go). We studied the dose-response of the developmental toxicity of TCDD in wild type and EGF and TGF-{alpha} (–/–) mice. This study also addresses issues related to the background genetics of the different strains. When using genetically manipulated mice, there needs to be attention to the potentially divergent background genetics of mice that were originally produced and/or maintained in different facilities. This is particularly important when dosing with TCDD, as there are strain-dependent sensitivities related to polymorphisms of the AhR (Poland and Knutson, 1982Go). The AhR affinity for TCDD in sensitive strains, such as C57BL/6N or C57BL/6J mice, is approximately six times higher than in the less sensitive strains, such as DBA mice (Ema et al., 1994Go). We therefore used the liver ethoxyresorufin-o-deethylase (EROD) activity of these mice to characterize their AhR phenotype and assign appropriate wild type strains as controls. The results of the dose-response study clarify the roles of EGF and TGF-{alpha} in mediating the developmental toxicity of TCDD in the palate and urinary tract.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Several breeding pairs of EGF and EGF + TGF-{alpha} (–/–) mice were obtained from David Lee, University of North Carolina at Chapel Hill (Chapel Hill, NC). These mice were derived from 129 and C57BL/6J strains (Lee et al., 1995Go; Luetteke et al., 1993Go, 1999Go) and were of mixed genetic background and thus appropriate wild type (WT) mice with 129 x C57BL/6J background were also provided by David Lee’s laboratory. Two breeding pairs of TGF-{alpha} (–/–) mice (Tgfatm1Ard, developed by A. R. Dunn, Ludwig Institute for Cancer Research; Mann et al., 1993Go) were obtained from Jackson Laboratory (Bar Harbor, ME). The TGF-{alpha} (–/–) were backcrossed over 10 generations into the C57BL/6J background by Jackson Laboratory and thus the appropriate wild type for comparison would be the C57BL/6J strain. Colonies of WT, EGF (–/–), EGF + TGF-{alpha} (–/–), and TGF (–/–) were established in the animal facility at the U.S. Environmental Protection Agency (Research Triangle Park, NC). Food (Agway rat, mouse, and hamster 3000) and distilled water were provided ad libitum and all animals were housed under controlled conditions of temperature (72 ± 2°F) and light (12/12-h light/dark cycle, 0600 to 1800 h). All of the homozygous knockout strains of mice were normal and maintained good health throughout the study period. The EGF (–/–) do not display any observable phenotype and the EGF + TGF-{alpha} (–/–) and TGF-{alpha} (–/–) displayed only a pronounced waviness in the coat and whiskers, consistent with the reported phenotype associated with knockout of TGF-{alpha} (Lee et al., 1995Go; Luetteke et al., 1993Go, 1999Go; Mann et al., 1993Go). Male and female mice of the same genetic background were housed together overnight from 1630 to 0730 h. Females were checked for vaginal plugs and weighed at 0730 h the next morning, which was designated as gestation day (GD) 0, and plug-positive females were weighed.

Timed-pregnant female C57BL/6J mice were obtained from Jackson Laboratory (Bar Harbor, ME). The 8–10-week-old virgin female mice were housed overnight with proven breeder C57BL/6J males and checked the next morning for evidence of mating (sperm plug positive, GD 0). Animals were shipped during the first week of pregnancy to the United States Environmental Protection Agency animal facility in Research Triangle Park, North Carolina. The pregnant C57BL/6J mice were maintained in the colony, as described above, until necropsy on GD 17.5.

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). Pregnant females were weighed on GD 12 and dosed at 1300 h by gavage with vehicle or 0.2, 1, 5, 24, 50, 100, or 150 µg TCDD/kg body weight at 5 ml/kg. The number of pregnant females (litters) for each dose and genotype are presented in Tables 1–4GoGoGoGo. There were at least 5 pregnant females in each of the treatment groups, except for the highest exposure (150 µg TCDD/kg) for WT, EGF (–/–), and EGF + TGF-{alpha} (–/–), each of which had 3 pregnant females.


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TABLE 1 Pregnant Wild Type, EGF (–/–), and EGF + TGF-{alpha} (–/–) Female Body and Liver Weights (Means ± SE) with Oral Administration of Vehicle or TCDD (1, 24, 50, 100, or 150 µg/kg) on GD 12
 

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TABLE 2 Pregnant C57BL/6J and TGF-{alpha} (–/–) Female Body and Liver Weights (Means ± SE) with Oral Administration of Vehicle or TCDD (0.2, 1, 5, or 24 µg/kg) on GD 12
 

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TABLE 3 Wild Type, EGF (–/–), and EGF + TGF-{alpha} (–/–) Hydronephrosis with Oral Administration of Vehicle or TCDD (1, 24, 50, 100, or 150 µg/kg) on GD 12
 

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TABLE 4 C57BL/6J and TGF-{alpha} (–/–) Hydronephrosis after Oral Administration of Vehicle or TCDD (0.2, 1, 5, or 24 µg/kg) on GD 12
 
This dose-response study required a prolonged period to complete and there were limited numbers of animals of each strain available each week of the study. The initial stage of the study included dosing at 24 µg TCDD/kg, a level known to produce a high incidence of clefting in the C57BL/6 background strain. Based on the responses to that dose, additional dose levels were selected for the different strains to clarify apparent sensitivity or resistance. The responses to 24 µg TCDD/kg were reported in Bryant et al.(2001)Go and are presented in this final dose response report. Although previously reported, that dose group and its contiguous controls constitutes an integral part of the overall study. As these animals are a scarce resource and in keeping with the policy to reduce the number of animals used, additional animals were not dosed at that level for the present report. However, it should be noted that in addition to providing additional exposure levels, the present report also includes the responses of C57BL/6J (not represented in the initial report), and the characterization of EROD responses that reveal the appropriate background strains for comparison. Concurrent controls were included throughout the duration of the dose-response experiments for all of the strains.

The developmental stage at the time of dosing (GD 12) was compared for each strain and found comparable (data not shown). Our laboratory has been performing palatal organ culture with these strains of wild type and knockout mice over the same period as the dose-response study. That protocol requires collection of embryos on GD 12 for dissection of the craniofacial tissues. A large number of litters have been collected from each strain and observations at necropsy indicated that all the strains are at the same developmental stage on the morning of GD 12 (corresponding to stages exposed in the in vivo study). The observations are further documented by photographs of intact embryos and dissected craniofacial views of the palatal shelves from at least three embryos of each of three litters from each strain (data not presented).

EROD assay.
Liver was collected from adult male and female mice of each strain, frozen, and stored at –80°C until all of the specimens were obtained. Livers were collected from a total of 9 WT, 10 EGF (–/–), 13 EGF + TGF-{alpha} (–/–), 12 C57BL/6J, and 13 TGF-{alpha} (–/–). Treatment groups across the genotypes were as follows: C57BL/6J were dosed with 0 (n = 6) or 1 µg TCDD/kg (n = 6); TGF-{alpha} (–/–) were dosed with 0 (n = 3), 0.2 (n = 5), or 1 µg TCDD/kg (n = 5); WT were dosed at 0 (n = 4) or 1 µg TCDD/kg (n = 5); EGF (–/–) was dosed at 0 (n = 4), 1 (n = 3), 50 (n = 2), or 100 µg TCDD/kg (n = 1); and EGF + TGF-{alpha} (–/–) mice were dose at 0 (n = 5), 1 (n = 5), or 100 µg TCDD/kg (n = 3). Microsomal protein concentrations were determined using a protein assay kit (Bio-Rad, Richmond, CA) with bovine serum albumin as the standard. Hepatic microsomal EROD was determined using a kinetic assay (De Vito et al., 1993Go). The reaction mixture consisted of a 0.05 M Tris-buffer at pH 8.0 containing 0.5–1 mg/ml, 4.5 µM ethoxyresorufin. A total of 210 µl were placed in an opaque 96-well plate. The sample plate was incubated for 3 to 5 min in the plate reader at 37°C. The reaction was initiated by adding 25 µl of NADPH (11.2 mg/ml) and production of resorufin was determined spectrofluorometrically (Molecular Devices Gemini XS Microplate) for 5 min at 37°C with an excitation wavelength of 544 and an emission wavelength of 590. Data points were recorded at 36 s intervals. Final results were calculated as pmol of resorufin per mg of protein per min. All chemicals used in this assay were purchased from Sigma Chemical Co. (St. Louis, MO) and were of the highest grade commercially available.

Statistical analysis of EROD data.
Group means and SEs were calculated by SAS Proc Means ( SAS Institute, 1989Go). The data were log transformed to improve homogeneity of variance across groups, and ANOVAs were performed by SAS Proc GLM on the log transformed data. C57BL/6J and TGF-{alpha} (–/–) data were analyzed separately from the WT, EGF (–/–), and EGF + TGF-{alpha} (–/–) data set. Two-way ANOVA was used to examine the effects of genotype, dose and the genotype by dose interaction on log(EROD). If the interaction was not significant it was removed from the model. Pairwise t-tests were used to test the significance of differences between individual groups.

Necropsy.
On GD 17.5, pregnant females were weighed, anesthetized by CO2 inhalation, and killed 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, which stained the hemosiderin pigment of each implantation site blue-black (Narotsky et al., 1997Go). The number of live and dead pups, as well as the number of early and late resorptions, were recorded. Fetuses were decapitated and the mandible was removed to examine the secondary palate and record a fused or cleft palate. The kidneys and ureters were exposed by removing the gastrointestinal tract and the dissected fetal head and body were then submerged in Bodian’s fixative (2% formaldehyde, 5% acetic acid, 72% ethanol, 21% water; Narotsky et al., 1997Go) for storage at room temperature. The fixed kidneys were bisected under a dissecting microscope to evaluate the presence and severity of hydronephrosis. Two independent observers evaluated each kidney and assigned hydronephrotic severity scores utilizing the scoring system described in Bryant et al. (2001Go; see below).

Parameters evaluated.
For each genotype and treatment, maternal parameters included body weight on GD 0, body weight on GD 17.5 adjusted for gravid uterine weight (weight on GD 17.5 minus the weight of intact uterus), weight gain (adjusted GD 17.5 body weight minus the weight on GD 0), liver weight, and relative liver weight ([liver weight/GD 17.5 adjusted weight] x 100). The GD 0 body weight was not available for the C57BL/6J pregnant females (these animals were bred at Jackson Laboratory and were not received in our animal facility until later in the first week of pregnancy). Thus, C57BL/6J body weight gain could not be calculated; however, adjusted body weight on GD 17.5 was calculated and used to calculate relative liver weight for these animals. The parameters evaluated for each litter included the total number of implantation sites, the number of live fetuses (no dead fetuses were found in any of the treatment groups), and the proportion of early and late resorptions, (number of resorptions/total number of implantations). Fetal parameters were evaluated for individual fetuses and litter means were the basis of all comparisons across treatments/genotypes. Teratogenic responses evaluated included the incidence of cleft palate (number of fetuses with cleft palate/total number of live fetuses) and the incidence and severity of hydronephrosis. As shown in Figure 1Go (previously published in Bryant et al. [2001Go] and adapted from Woo and Hoar [1972Go]), a value of 0 was assigned when the papilla totally filled the renal pelvic space; + 1 was assigned for a slight dilation of the renal pelvis; + 2 for reduction in papilla size and noticeable dilation of the pelvic space (considered hydronephrotic); + 3 was assigned if there was a very short papillae and compressed renal tissue; + 4 kidneys displayed virtually no papilla and a thin renal wall. The incidence of hydronephrosis was based on the number of fetuses with a score of >= 2/total number live fetuses and was determined for left, right, and as an overall incidence for each litter (severity scores >= 2 on either left or right/total live fetuses). Incidence and severity were evaluated for the left and right kidney and a combined score was calculated from the mean of the left and right scores.



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FIG. 1. The kidneys of each fetus were examined for presence of hydronephrosis and a score was assigned to quantify the degree of dilation of the renal pelvis. Scores ranged from 0 to 4 and a value equal to or higher than 2 was considered hydronephrotic. The bisected kidneys of fetuses shown in A–E illustrate the morphological features associated with scores 0 to 4, respectively. This scoring system was previously published by our laboratory in Bryant et al.(2001)Go.

 
Statistical analysis.
All analyses were done using SAS Proc Means and Proc Glm ( SAS Institute, 1989Go). For the pup data, the litter means were used as the unit of analysis. Means and SEs were calculated for each genotype by dose group. For each outcome variable, a one-way ANOVA was performed for each genotype to look for differences in outcome among the dose groups. (An arcsin transformation was used for all proportions to stabilize the variance.) If the ANOVA F-test was significant, a pairwise t-test was calculated between each dose and the control group. A linear regression was also run for each genotype for each outcome, and a significant slope was interpreted as a significant trend across the dose range. For each wild type-knockout pair of genotypes (WT-EGF, WT-E + T, and C57-TGF-{alpha}), a two-way ANOVA was run to look for any interaction between dose group and genotype. Where the interaction term was significant, contrasts were used to test the interaction at each dose. A regression model was also used for each genotype pair to test for differences in the slope across the dose range between the two genotypes.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
EROD Assay
EROD activity was evaluated in microsomal preparations of livers from mice with a mixed 129 x C57BL/6J genetic background [WT, EGF (–/–), EGF + TGF-{alpha} (–/–)], and from wild type C57BL/6J mice and TGF-{alpha} (–/–) mice having a C57BL/6J genetic background. EROD activity (expressed as pmol/mg protein/min) increased with exposure to TCDD in all of the strains, however the C57BL/6J background conferred greater sensitivity for induction of EROD activity. C57BL/6J and TGF-{alpha} (–/–) showed a significant dose-related increase for induction of EROD activity (Fig. 2Go; p < 0.001). Exposure of TGF (–/–) to 0.2 µg TCDD/kg also induced EROD significantly (23.9 ± 3.1, 222 ±72, control and 0.2 µg TCDD, respectively; p < 0.001). The responses of the C57BL/6J and TGF-{alpha} (–/–) strains were not significantly different. WT, EGF (–/–), and EGF + TGF-{alpha} (–/–) mice with a 129 x C57BL/6J mixed genetic background exhibited only a slight or no induction of EROD activity after exposure to 1 µg TCDD/kg (EGF (–/–) p < 0.01, WT and EGF + TGF-{alpha} (–/–) not significantly altered). However, these strains have elevated EROD activities relative to controls after exposures to 50 and 100 µg TCDD/kg [EGF (–/–), p < 0.001, 578 ± 84 and 554 ± 0 at 50 and 100 µg TCDD/kg, respectively; EGF + TGF-{alpha} (–/–) p < 0.001, 802 ± 289 at 100 µg TCDD/kg]. These values are similar to those attained in the strains with a C57BL/6J background after exposure to 1 µg TCDD/kg (512 ± 26). ANOVA did not detect any influence of the sex of the animals on EROD activity and data for males and females of the same genotype and dose group were combined.



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FIG. 2. Liver EROD activity (pmol/mg protein/min) increased with TCDD exposure in wild type and knockout adult mice. The mice having a C57BL/6J genetic background [C57BL/6J and TGF-{alpha} (–/–)] show significant induction of enzyme activity after exposure to 1 µg TCDD/kg (p < 0.001). Mice with a mixed 129 x C57BL/6J genetic background [WT, EGF (–/–), EGF + TGF-{alpha} (–/–)] have little or no induction after exposure to 1 µg TCDD/kg, although the slight increase for EGF (–/–) was significant (p < 0.01). a = p < 0.01, b = p < 0.001, relative to appropriate control group [WT vs. EGF (–/–) or EGF + TGF-{alpha} (–/–); C57BL/6J vs. TGF-{alpha} (–/–)].

 
Maternal Data
Maternal body weight on GD 0, adjusted body weight on GD 17.5, GD 0–17.5 body weight gain, liver weight, and liver-to-body-weight ratio are presented for WT, EGF (–/–), and EGF + TGF-{alpha} (–/–) in Table 1Go and for C57BL/6J and TGF-{alpha} (–/–) in Table 2Go. In general, there were no differences in maternal body weights at the beginning of pregnancy (GD 0), in adjusted body weight or weight gains on GD 17.5 across either genotype or treatment group. Random differences in GD 0 body weight were noted at GD 0 for the WT 24 µg/kg group and the EGF (–/–) 1 µg/kg group (Table 1Go). Interactions were detected between EGF and WT (across all dose groups) for initial and adjusted (not including uterine contents) body weight and between TGF-{alpha} (–/–) and C57BL/6J for adjusted body weight (Tables 1 and 2GoGo). These significant body weight differences do not represent a dose or genotype-related patterns and these variations were likely due to random differences in a few animals in each of these groups. This is particularly likely for the GD 0 body weights, as treatment was administered on GD 12. In all genotypes, the absolute and relative liver weights increased in a dose-related manner and these trends were significant (p values 0.05 and 0.01 as shown in Tables 1 and 2GoGo). Several dose groups within each genotype were significantly elevated relative to their respective genotype control (p < 0.05, 0.01, 0.001 as shown). A notable exception was the EGF + TGF-{alpha} (–/–) group of mice in which no significant increase in absolute or relative liver weight was detected, either as a dose-related trend or for comparisons of specific dose groups to their control group.

Litter Data
The WT, EGF (–/–), EGF + TGF-{alpha} (–/–), C58BL/6J, and TGF-{alpha} (–/–) litters did not have significant differences in the number of early or late resorptions per litter or live fetuses per litter. For all the genotypes and dose groups, there were few in utero deaths and no pups died just prior to GD 17.5. Based on the small size of the early resorptions and the relatively few larger or late resorptions, it appears that most of the deaths occurred prior to the GD 12 treatment. There was no significant effect of treatment or genotype on survival in utero. The number of embryos implanting in C57BL/6J and TGF-{alpha} (–/–) females was not significantly different. There was a significant increase in numbers of embryos implanting in the uterus in WT litters dosed with 1, 24, and 150 µg TCDD/kg compared to control litters, (9.4 ± 0.9, p < 0.5; 10.3 ± 0.6, p < 0.01; 10.7 ± 0.3, p < 0.01) versus 7.6 ± 0.4 in control litters, respectively). However as implantation occurs well before treatment on GD 12, these differences are considered random and may be related to the unusually low mean for the WT control group. The overall mean (across all dose groups) of implanted embryos did not differ between the C57BL/6N and TGF-{alpha} (–/–) groups, but was significantly lower in WT (n = 35 litters, 8.8 ± 0.4 implantations per litter, p < 0.05) and EGF (–/–) (n = 35, 8.5 ± 0.4, p < 0.01) relative to EGF + TGF-{alpha} (–/–) (n = 32, 10.0 ± 0.4), suggesting that the double knockout results in greater numbers of implanted embryos.

Cleft Palate
Each fetus was evaluated for the presence of cleft palate, and the incidence across genotypes and treatments is presented in Figure 3Go. The WT, EGF (–/–), and EGF + TGF-{alpha} (–/–) showed a TCDD dose-related trend for induction of cleft palate (p < 0.05; Fig. 3AGo). Although the incidence of cleft palate was significantly (p < 0.05) increased in WT at 24 µg TCDD/kg, the EGF (–/–) and EGF + TGF-{alpha} (–/–) showed no significant increase relative to control. Even at 50 µg TCDD/kg, the EGF (–/–) litters did not have a significant increase in the incidence of cleft palate. The differences between the treated and control rates of cleft palate induction in the EGF (–/–) litters (magnitude of response) after treatment with 1, 24, and 50 µg TCDD/kg were significantly lower compared to the WT (p < 0.05), suggesting that the EGF (–/–) fetuses were less sensitive. In contrast, the magnitude of response in the EGF + TGF-{alpha} (–/–) litters was significantly higher than in the WT, when the responses at 100 µg TCDD/kg (p < 0.01) are compared, suggesting that the double knockout may be more responsive than WT at the higher exposure levels. In the C57BL/6J and TGF-{alpha} (–/–) mice exposure to 24 µg TCDD/kg produced cleft palate in all fetuses (p < 0.001; Fig. 3BGo). Exposure to 5 µg TCDD/kg significantly increased the incidence of cleft palate only in the TGF-{alpha} (–/–) fetuses (p < 0.05) and not in the C57BL/6J, and there was a significant difference between the strains (p < 0.05).



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FIG. 3. Cleft palate incidence is shown on a per litter basis by dose for mice with mixed C57BL/6J x 129 genetic background in (A) WT, EGF (–/–), and EGF + TGF-{alpha} (–/–), and in (B) mice having C57BL/6J genetic background, C57BL/6J and TGF-{alpha} (–/–). The incidence of cleft palate increased with dose in all strains and significant differences relative to the control are noted as a = p < 0.05, b = p < 0.01, c = p < 0.001. The magnitude of response (treated rate corrected for control level) was significantly lower in the EGF (–/–) litters (p < 0.05). However, the EGF + TGF-{alpha} (–/–) were more sensitive relative to WT after 100 µg TCDD/kg (p < 0.01). TGF-{alpha} (–/–) were similar to C57BL/6J for induction of cleft palate, except at the 5 µg TCDD/kg exposure.

 
Hydronephrosis
The severity and incidence of hydronephrosis was evaluated for each fetus. There was a significant dose related increase in severity and incidence of hydronephrosis in every genotype (Figs. 4 and 5GoGo). In WT, EGF (–/–), and EGF + TGF-{alpha} (–/–) the incidence and severity of hydronephrosis was significantly (p < 0.001) increased relative to controls at doses of 24 µg TCDD/kg or higher (Fig. 4Go). However, only the EGF (–/–) fetuses had significantly (p < 0.001) increased hydronephrosis incidence and severity scores after exposure to 1 µg TCDD/kg.



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FIG. 4. Hydronephrosis severity (A) and incidence (B) increased with dose in the WT, EGF (–/–), and EGF + TGF-{alpha} (–/–) litters (mean ± SEM, combined right and left scores on a litter basis). After exposure to 24 µg TCDD/kg or higher all strains exhibited significant increases in severity and incidence relative to their respective controls (a = p < 0.001). Only the EGF (–/–) group had significant responses after exposure to 1 µg TCDD/kg. With respect to overall incidence of hydronephrosis, the magnitude of the response (scores corrected for control level) was significantly higher for the EGF (–/–) litters compared to WT (a = p < 0.05). The magnitude of the severity of hydronephrosis was greater in EGF (–/–) litters relative to EGF + TGF-{alpha} (–/–) (p < 0.05).

 


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FIG. 5. Hydronephrosis severity (A) and incidence (B) in C57BL/6J and TGF-{alpha} (–/–) increased with dose and the responses were similar for the two strains of mice (mean ± SEM, combined right and left scores on a litter basis). Treated compared to control: a = p < 0.01, b = p < 0.001.

 
The C57BL/6N and TGF-{alpha} (–/–) litters were similar in their dose-related responses to TCDD (Fig. 5Go). Both strains responded to 0.2 µg TCDD/kg with significant increases in both incidence and severity (p < 0.01) and to 1 µg TCDD/kg or higher doses with near maximal responses (severity scores > 3, 97–100% of pups affected in all litters, p < 0.001).

The degree of dilation of the control renal pelvis on GD 18 appeared strain dependent and may have been affected by lack of EGF and/or TGF expression. The dilation resulted in elevated control scores for severity and incidence of hydronephrosis in some of the strains (Figs. 4 and 5GoGo). In order to compare the responses of the different strains to TCDD, an adjustment was made to take into account the control renal dilation and associated increased scores. For each dose group, the severity and incidence of hydronephrosis were adjusted by subtracting the control scores of that strain. This provided a measure of the magnitude of the response to TCDD for overall severity and incidence of hydronephrosis. The adjusted magnitude of response to TCDD was greater in the EGF (–/–) than in the WT (across all dose groups, p < 0.05) with significant differences for comparisons of groups dosed with 1, 24, and 50 µg/kg (p < 0.05). The magnitude of response for overall severity of hydronephrosis (adjusting for control scores and comparing across genotypes) was greater in the EGF (–/–) than in the EGF + TGF-{alpha} (–/–) at doses of 1 µg/kg (p < 0.05), 24 and 50 µg/kg (p < 0.01), 100 and 150 µg/kg (p < 0.05). The magnitude of responses did not differ when C57BL/6J and TGF-{alpha} (–/–) were compared to each other for severity or incidence.

When considering the responses to treatment for left and right kidneys separately, the responses to treatment were generally similar with significant dose-related increases in both incidence and severity in left and right kidneys regardless of genotype (Tables 3 and 4GoGo). When evaluating hydronephrotic severity, the magnitude of response (treated group rate adjusted for control rate) in the left kidney in the EGF (–/–) group was significantly greater than the EGF + TGF-{alpha} (–/–) response at 1 µg/kg (p < 0.05), 24 and 50 µg/kg (p < 0.001), 100 and 150 µg/kg (p < 0.01). A similar increased responsiveness in EGF (–/–) compared to EGF + TGF-{alpha} (–/–) was observed in severity scores for the right kidney at doses of 1 µg/kg (p < 0.05), 24 and 50 µg/kg (p < 0.001) and 100 µg/kg (p < 0.057) and 150 (NS, p < 0.097). Comparisons of the magnitude of response in WT and EGF (–/–) revealed stronger responses for hydronephrotic severity in the EGF (–/–) (p = 0.06 overall dose groups, p = 0.07 at 24 µg/kg, p < 0.01 at 50 and 100 µg/kg and p < 0.06 at 150 µg/kg). The EGF (–/–) response was marginally increased relative to WT when the incidence was compared (adjusting for control rate and comparing the magnitude of response across doses, p = 0.076 on the right kidney and p = 0.082 on the left; p < 0.05 at 1 µg/kg both right and left, p = 0.08 at 24 µg/kg and p = 0.05 at 50 µg/kg on the left. Analysis of the effects of exposure on the right kidney versus the left showed a general increase in severity and incidence of hydronephrosis on the right side and this was significant for the WT and EGF + TGF genotypes. For the C57BL/6J and TGF (–/–) fetuses, TCDD produced a dose related increase in severity and incidence of hydronephrosis in both right and left kidneys. Comparison of the responses on the right versus the left sides did not reveal a significant difference due to side.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our studies demonstrate that EGF expression clearly influences the responsiveness of the embryo to TCDD. Mice that do not express EGF are less responsive to TCDD for induction of cleft palate and more sensitive to TCDD for induction of hydronephrosis. Supporting the importance of EGF expression in the response to TCDD is the observation that exposure of wild type C57BL/6N mice to TCDD increased EGF expression in both palatal and urinary tract epithelial cells (Abbott, 1997Go; Bryant et al., 1997Go). Further support is present in the responses of the TGF-{alpha} (–/–), as their embryos are expected to express EGF and they have a robust response to TCDD. The inverse interpretation is also interesting as EGF (–/–) mice are expected to express TGF-{alpha} but not EGF, and it appears that TGF-{alpha} in the absence of EGF is not sufficient to mediate responses in the palate. However, TGF-{alpha} when expressed in the ureter in the absence of EGF provided an enhanced response to TCDD. This may be related to normal patterns of growth factor expression during palatogenesis, as EGF expression is relatively robust and increases during formation of the secondary palate, whereas TGF-{alpha} expression is low during palatogenesis (Abbott, 1997Go). At this stage of urinary tract development, the ureteric epithelial cells express both EGF and TGF-{alpha} (Bryant et al., 1997Go). Thus, in the ureter, expression of TGF-{alpha} in the absence of EGF confers increased sensitivity for hydronephrosis, while expression of EGF alone mediates a C57BL/6J-like response in this tissue.

Knockout of both EGF and TGF-{alpha} did not alter responsiveness to TCDD with the exception of an increased incidence of cleft palate at the higher exposure levels (100 and 150 µg/kg). The responses of these double-knockout fetuses make it clear that neither growth factor is absolutely required for mediating the induction of cleft palate and hydronephrosis by TCDD. The lack of expression of both EGF and TGF-{alpha} does not impair normal development and the knockouts are similar to wild types in numbers of live, normal fetuses at term (Lee et al., 1995Go; Luetteke et al., 1993Go, 1999Go; Mann et al., 1993Go). It is likely that one or more of the other ligands for the EGF receptor is able to substitute for EGF and TGF-{alpha} in normal development and in response to toxicants, although the specific responses may differ.

Cleft palate was observed in a dose-related manner in all strains of mice. However, in the absence of EGF expression, significant induction of cleft palate was only observed at doses of 100 or 150 µg TCDD. The lack of a significant response in EGF (–/–) was previously reported for a lower exposure (Bryant et al., 2001Go) and the present dose-response extends and confirms the importance of EGF expression in mediating TCDD-induced cleft palate. In contrast, absence of TGF-{alpha} did not inhibit responsiveness to TCDD, although there was a suggestion of increased sensitivity at the 5 µg TCDD/kg dose. However, a maximal response at all doses above 5 µg/kg makes this difficult to interpret. Further exposures in a lower range or increasing the numbers examined at lower doses might reveal whether this is a true increase in sensitivity at lower exposures.

In the absence of both EGF and TGF-{alpha}, the induction of cleft palate is markedly increased at exposure above 50 µg/kg, compared to the WT or single knockout of EGF. Since in these double knockout animals, the induction of cleft palate cannot be mediated by either EGF or TGF-{alpha} binding to the EGF receptor, there is a possibility that one of the other ligands for the receptor (e.g., BTC, AR, HB-EGF, or ER) could be mediating the response. Since craniofacial development in the EGF + TGF (–/–) embryos appears normal, it is probable that regulation of palatogenesis proceeds via one or more of the alternate ligands as well. There is a potential for alterations in EGFR expression and activity due to the absence of these ligands as well. The importance of the EGFR pathway in craniofacial development is supported by observations of cranial and mandibular malformations and cleft palate in newborn EGFR (–/–) mice (Miettinen et al., 1999Go). The expression patterns of the EGFR ligands BTC, HB-EGF, AR, and ER have not been characterized in the developing palate or urinary tract. AR is known to be involved in branching morphogeneis in the lung and ductal outgrowth in the mammary gland (Luetteke et al., 1999Go; Schuger et al., 1996Go). The role of AR in lung and mammary gland suggests a possible similar role in the development of the urinary ductal system. Expression of EGF and TGF-{alpha} have been more extensively characterized in the embryo.

The lack of sensitivity of the EGF (–/–) embryo to induction of cleft palate while exhibiting increased sensitivity to hydronephrosis is probably related to the dependence of each tissue on specific patterns of growth factor expression during morphogenesis. Palatal epithelial cells express EGF and the EGF receptor, however expression of TGF-{alpha} protein is relatively low and decreases during palate formation in C57BL/6N embryos (Abbott, 1997Go). Expression of both EGF and TGF-{alpha} protein and mRNA increase in palate after exposure to TCDD. Thus, it would appear that upregulation of EGF by TCDD is a critical step in mediation of cleft palate. In the EGF (–/–) and EGF + TGF (–/–) this cannot occur. This also suggests that ligands other than EGF are poor mediators of the response or possibly that their expression must be upregulated to become effective (possibly this upregulation only occurs at the higher exposures). There is also a possibility that alternative, non-EGFR pathways become invoked at the higher exposures. Further research is clearly required to determine expression patterns of the other EGFR ligands to clarify mechanisms involved in response to TCDD in the absence of EGF and/or TGF-{alpha}.

In contrast to the palate, in the ureter both EGF and TGF-{alpha} are expressed during development and absence of either appears to affect renal maturation. The increased responsiveness in the EGF and TGF-{alpha} (–/–) embryos may be related to the apparent immaturity or abnormal morphology of the kidney on GD 18, reflected in increased size and dilation scores of control kidneys. Although there were no abnormalities reported in adult mice during initial characterization of the knockout mice (Luetteke et al., 1993Go, 1999Go; Mann et al., 1993Go) the fetal urinary tract appears affected by the loss of these growth factors. It is also of interest that the EGFR (–/–) on a CD-1 genetic background (lethal perinatally) had renal malfunction (Threadgill et al., 1995Go). Elevated blood urea nitrogen correlated with cystic dilation of the collecting ducts and alterations of the ductal epithelia. While the EGFR pathway is not required for initial morphogenesis of the urinary tract, it appears that it has a major role in differentiation of the final structures. EGF (–/–) fetuses may reflect this dependence with initial urinary immaturity, which resolves by adulthood. The present dose-response study confirms our previous report of increased severity and incidence of hydronephrosis in the EGF (–/–) embryos (Bryant et al., 2001Go) and extends the range at which that occurs to as low as 1 µg TCDD/kg. The C57Bl/6J and TGF-{alpha} (–/–) mice, which have the higher affinity AhR, responded at 0.2 µg TCDD/kg with significant elevations in severity and incidence of hydronephrosis. Our previous study compared the responses in the TGF-{alpha} (–/–) to the mixed genetic background WT and reported a significant difference (Bryant et al., 2001Go). In the present study, the TGF-{alpha} (–/–) fetal responses were compared to C57BL/6J wild type. Both groups of mice were highly sensitive, exhibiting dose-related increases in hydronephrosis that were not significantly different. The enhanced responses of the TGF-{alpha} (–/–) when compared to observations in mice with a mixed 129 x C57BL background (WT, EGF, and EGF + TGF-{alpha}) can now be attributed to differential sensitivity associated with strain-dependent AhR polymorphisms.

Maternal and fetal toxicity were limited to observations of increased liver weight in the pregnant female, even though some strains were exposed to doses as high as 150 µg TCDD/kg. Maternal and fetal survival were not affected in any of the strains or knockouts at any of the TCDD exposures. These observations are consistent with published reports (Birnbaum et al., 1985Go; Poland and Knutson, 1982Go). The increased liver weight is a typical murine response to TCDD exposure and has been reported previously in mice, guinea pigs and rats (Courtney and Moore, 1971Go; Hruska and Olson, 1989Go; Neubert and Dillmann, 1972Go; Poland and Knutson, 1982Go; Schwetz et al., 1973Go). In our previous study (Bryant et al., 2001Go), we reported an increased degree of response for liver weight in TGF-{alpha} (–/–) compared to WT. That observation reflects the differences in sensitivity that are related to strain-dependent AhR polymorphisms, as revealed in the EROD activities of the present study. When the TGF-{alpha} (–/–) is compared to wild type C57BL/6J, both have significant trends for liver weight increase but there is no significant difference in the degree of response.

There is an obvious and strong influence of genetic background on sensitivity to TCDD that must be considered in the outcome and interpretation of this study. The wild type and knockout mice used in this study differ in their overall background genetics and this directly influenced their sensitivity to TCDD. The WT, EGF (–/–), and EGF + TGF-{alpha} (–/–) mice required higher levels of exposure to TCDD to produce responses when compared with the C57BL/6J wild type and TGF-{alpha} (–/–) mice. These differences can now be attributed to strain specific AhR polymorphisms that affect ligand binding. The TGF-{alpha} (–/–) mice, which were backcrossed for more than 10 generations with C57BL/6J mice (Jackson Laboratories, www.jaxmice.jax.org) and the C57BL/6J wild type mice were expected to have the high affinity allele and this was confirmed by the EROD activity in livers from these strains. The TGF-{alpha} (–/–) showed comparable sensitivity to TCDD-induced EROD activity relative to the C57BL/6J wild type and it is concluded that the animals have that genetic background. The WT, EGF (–/–), and EGF + TGF-{alpha} (–/–) mice were derived, as are most knockout mice, in a manner that produced a mixed genetic background from both the 129 and C57BL strains. The 129 strain of mice expresses an AhR allele of low binding affinity for TCDD relative to the C57BL strain. Consistent with this genetic background, induction of EROD activity required exposure to 50–100 times as much TCDD as that required in the C57BL/6J background. This grouping of strains into high and low AhR affinity categories explains the need for different dose response ranges and clarifies the selection of appropriate wild type animals for comparison to each knockout strain.

These mechanisms of response to environmental agents are highly relevant to human development. The EGFR its ligands are expressed in developing human palatal shelves and expression of EGFR, EGF, and TGF-{alpha} are perturbed by exposure of these tissues to TCDD in culture (Abbott et al., 1999aGo,bGo). Epidemiological studies associate polymorphisms of the TGF-{alpha} gene with increased risk for nonsyndromic cleft lip and palate (Ardinger et al., 1989Go; Bianchi et al., 2000Go; Machida et al., 1999Go; Mitchell, 1997Go; Shiang et al., 1993Go; Stoll et al., 1992Go; Tanabe et al., 2000Go). There is a possibility that variants of the growth factor genes for the EGFR pathway could elicit either protective effects or enhance sensitivity to toxicants. As demonstrated in the present study, the specific ligand available to bind to the EGFR can dramatically affect the responses and sensitivities of target tissues.

In conclusion, studies in the knockout mice demonstrate that EGF expression influences the responsiveness of the embryo to TCDD. TGF-{alpha} is not sufficient to mediate responses to TCDD in the palatal cells, but sustains a robust response in the urinary tract. Expression of TGF-{alpha} in the absence of EGF confers increased sensitivity for hydronephrosis. EGF and TGF-{alpha} are not absolutely required for responses to TCDD; however, modifying expression patterns of these ligands modulates responsiveness in both ureter and palate. This study supports the conclusion that the EGFR pathway is mechanistically important in the responses of the embryo to TCDD. Further, it appears that there are specific roles for each growth factor in response to toxicants and that modulation of expression patterns leads to morphological consequences.


    ACKNOWLEDGMENTS
 
David C. Lee and the members of his laboratory deserve special recognition for developing and providing the EGF and EGF + TGF-{alpha} (–/–) mice used in this study and for providing the WT background mice. The stock solution of TCDD was prepared by Janet Diliberto, Experimental Toxicology Division, NHEERL, ORD, U.S. Environmental Protection Agency, Research Triangle Park, NC.


    NOTES
 
1 To whom correspondence should be addressed at NHEERL Building (Room 2109), U.S. Environmental Protection Agency, 2525 East Highway 54, Durham, NC 27713. Fax: (919) 541-4017. E-mail: abbott.barbara{at}epa.gov. Back

This paper has been reviewed by the National Health and Environmental Effects Research Laboratory, U.S. EPA. Mention of trade names of commercial products does not constitute endorsement/recommendation for use.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Abbott, B. D. (1997). Developmental toxicity of dioxin: Searching for the cellular and molecular basis of morphological responses. In Handbook of Experimental Pharmacology: Section III: Pathogenesis and Mechanisms of Drug Toxicity in Development, Vol. 124 (R. Kavlock and G. Daston, Eds.), pp. 407–433. Springer-Verlag, New York.

Abbott, B. D., and Birnbaum, L. S. (1998). Dioxins and teratogenesis. In Molecular Biology of the Toxic Response (A. Puga and K. Wallace, Eds.), pp. 439–447. Taylor and Francis, Washington, DC.

Abbott, B. D., Held, G. A., Wood, C. R., Buckalew, A. R., Brown, J. G., and Schmid, J. (1999a). AhR, ARNT, and CYP1A1 mRNA quantitation in cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in culture. Toxicol. Sci. 47, 62–75.[Abstract]

Abbott, B. D., Schmid, J. E., Brown, J. G., Wood, C. R., White, R. D., Buckalew, A. R., and Held, G. A. (1999b). RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone. Toxicol. Sci. 47,76–85.[Abstract]

Ardinger, H. H., Buetow, K. H., Bell, G. I., Bardach, J., VanDemark, D. R., and Murray, J. C. (1989). Association of genetic variation of the transforming growth factor-alpha gene with cleft lip and palate. Am. J. Hum. Genet. 45, 348–353.[ISI][Medline]

Bianchi, F., Calzolari, E., Ciulli, L., Cordier, S., Gualandi, F., Pierini, A., and Mossey, P. (2000). [Environment and genetics in the etiology of cleft lip and cleft palate with reference to the role of folic acid]. Epidemiol. Prev. 24, 21–27.[Medline]

Birnbaum, L. S. (1995). Developmental effects of dioxins. Environ. Health Perspect. 103(Suppl. 7), 89–94.

Birnbaum, L. S., Weber, H., Harris, M. W., Lamb, J. C., and McKinney, J. D. (1985). Toxic interaction f specific polychlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin: Increased incidence of cleft palate in mice. Toxicol. Appl. Pharmacol.77, 292–302.[ISI][Medline]

Bryant, P. L., Clark, G. C., Probst, M. R., and Abbott, B. D. (1997). Effects of TCDD on Ah receptor, ARNT, EGF, and TGF-alpha expression in embryonic mouse urinary tract. Teratology 55, 326–337.[ISI][Medline]

Bryant, P. L., Schmid, J. E., Fenton, S. E., Buckalew, A. R., and Abbott, B. D. (2001). Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice lacking the expression of EGF and/or TGF-{alpha}. Toxicol. Sci. 62, 103–114.[Abstract/Free Full Text]

Courtney, K. D., and Moore, J. A. (1971). Teratology studies with 2,4,5-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol. Appl. Pharmacol.20, 396–403.[ISI][Medline]

De Vito, M. J., Maier, W. E., Diliberto, J. J., and Birnbaum, L. S. (1993). Comparative ability of various PCBs, PCDFs, and TCDD to induce cytochrome P450 1A1 and 1A2 activity following 4 weeks of treatment. Fundam. Appl. Toxicol.20, 125–130.[ISI][Medline]

Dlugosz, A. A., Cheng, C., Williams, E. K., Darwiche, N., Dempsey, P. J., Mann, B., Dunn, A. R., Coffey, R. J., Jr., and Yuspa, S. H. (1995). Autocrine transforming growth factor alpha is dispensible for v-rasHa-induced epidermal neoplasia: Potential involvement of alternate epidermal growth factor receptor ligands. Cancer Res. 55, 1883–1893.[Abstract]

Ema, M., Ohe, N., Suzuki, M., Mimura, J., Sogawa, K., Ikawa, S., and Fujii-Kuriyama, Y. (1994). Dioxin binding activities of polymorphic forms of mouse and human arylhydrocarbon receptors. J. Biol. Chem. 269, 27337–27343.[Abstract/Free Full Text]

Giudice, L. C. (1999). Genes associated with embryonic attachment and implantation and the role of progesterone. J. Reprod. Med. 44, 165–171.[ISI][Medline]

Hruska, R. E., and Olson, J. R. (1989). Species differences in estrogen receptors and in the response to 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure. Toxicol. Lett.48, 289–299.[ISI][Medline]

Johnson, S. E., Rothstein, J. L., and Knowles, B. B. (1994). Expression of epidermal growth factor family gene members in early mouse development. Dev. Dyn. 201, 216–226.[ISI][Medline]

Lee, D. C., Fenton, S. E., Berkowitz, E. A., and Hissong, M. A. (1995). Transforming growth factor alpha: expression, regulation, and biological activities. Pharmacol. Rev. 47, 51–85.[ISI][Medline]

Luetteke, N. C., Qiu, T. H., Fenton, S. E., Troyer, K. L., Riedel, R. F., Chang, A., and Lee, D. C. (1999). Targeted inactivation of the EGF and amphiregulin genes reveals distinct roles for EGF receptor ligands in mouse mammary gland development. Development 126, 2739–2750.[Abstract/Free Full Text]

Luetteke, N. C., Qiu, T. H., Peiffer, R. L., Oliver, P., Smithies, O., and Lee, D. C. (1993). TGF-{alpha} deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice. Cell 73, 263–278.[ISI][Medline]

Machida, J., Yoshiura, K., Funkhauser, C. D., Natsume, N., Kawai, T., and Murray, J. C. (1999). Transforming growth factor {alpha} (TGF-{alpha}): Genomic structure, boundary sequences, and mutation analysis in nonsyndromic cleft lip/palate and cleft palate only. Genomics 61, 237–242.[ISI][Medline]

Mann, G. B., Fowler, K. J., Gabriel, A., Nice, E. C., Williams, R. L., and Dunn, A. R. (1993). Mice with a null mutation of the TGF alpha gene have abnormal skin architecture, wavy hair, and curly whiskers and often develop corneal inflammation. Cell 73, 249–261.[ISI][Medline]

Miettinen, P. J., Berger, J. E., Meneses, J., Phung, Y., Pedersen, R. A., Werb, Z., and Derynck, R. (1995). Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor. Nature 376, 337–341.[ISI][Medline]

Miettinen, P. J., Chin, J. R., Shum, L., Slavkin, H. C., Shuler, C. F., Derynck, R., and Werb, Z. (1999). Epidermal growth factor receptor function is necessary for normal craniofacial development and palate closure. Nat. Genet. 22, 69–73.[ISI][Medline]

Mitchell, L. E. (1997). Transforming growth factor {alpha} locus and nonsyndromic cleft lip with or without cleft palate: A reappraisal. Genet. Epidemiol. 14, 231–240.[ISI][Medline]

Narotsky, M. G., Brownie, C. F., and Kavlock, R. J. (1997). Critical period of carbon tetrachloride-induced pregnancy loss in Fischer-344 rats, with insights into the detection of resorption sites by ammonium sulfide staining. Teratology 56, 252–261.[ISI][Medline]

Neubert, D., and Dillmann, I. (1972). Embryotoxic effects in mice treated with 2,4,5-trichlorophenoxyacetic acid and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Naunyn Schmiedebergs Arch. Pharmacol.272, 243–264.[Medline]

Poland, A., and Knutson, J. C. (1982).2,3,7,8-Tetrachlorodibenzo-p-dioxin and related halogenated aromatic hydrocarbons: Examination of the mechanism of toxicity. Annu. Rev. Pharmacol. Toxicol.22, 517–554.[ISI][Medline]

Puddicombe, S. M., Chamberlin, S. G., MacGarvie, J., Richter, A., Drummond, D. R., Collins, J., Wood, L., and Davies, D. E. (1996). The significance of valine 33 as a ligand-specific epitope of transforming growth factor alpha. J. Biol. Chem.271, 15367–15372.[Abstract/Free Full Text]

Sakurai, H., Tsukamoto, T., Kjelsberg, C. A., Cantley, L. G., and Nigam, S. K. (1997). EGF receptor ligands are a large fraction of in vitro branching morphogens secreted by embryonic kidney. Am. J. Physiol. 273, F463–F472.[Abstract/Free Full Text]

SAS Institute. (1989). SAS/STAT Users Guide, Version 6, 4th ed. SAS Institute, Cary, NC.

Schuger, L., Johnson, G. R., Gilbride, K., Plowman, G. D., and Mandel, R. (1996). Amphiregulin in lung branching morphogenesis: Interaction with heparan sulfate proteoglycan modulates cell proliferation. Development 122, 1759–1767.[Abstract/Free Full Text]

Schwetz, B. A., Norris, J. M., Sparschu, G. L., Rowe, U. K., Gehring, P. J., Emerson, J. L., and Gerbig, C. G. (1973). Toxicology of chlorinated dibenzo-p-dioxins. Environ. Health Perspect. 5, 87–99.[Medline]

Shiang, R., Lidral, A. C., Ardinger, H. H., Buetow, K. H., Romitti, P. A., Munger, R. G., and Murray, J. C. (1993). Association of transforming growth factor {alpha} gene polymorphisms with nonsyndromic cleft palate only (CPO). Am. J. Hum. Genet. 53, 836–843.[Medline]

Sibilia, M., and Wagner, E. F. (1995). Strain-dependent epithelial defects in mice lacking the EGF receptor. Science 269, 234–238.[ISI][Medline]

Solic, N., and Davies, D. E. (1997). Differential effects of EGF and amphiregulin on adhesion molecule expression and migration of colon carcinoma cells. Exp. Cell Res. 234, 465–476.[ISI][Medline]

Stoll, C., Qian, J. F., Feingold, J., Sauvage, P., and May, E. (1992). Genetic variation in transforming growth factor {alpha}: Possible association of BamHI polymorphism with bilateral sporadic cleft lip and palate. Am. J. Hum. Genet. 50, 870–871.[ISI][Medline]

Tanabe, A., Taketani, S., Endo-Ichikawa, Y., Tokunaga, R., Ogawa, Y., and Hiramoto, M. (2000). Analysis of the candidate genes responsible for non-syndromic cleft lip and palate in Japanese people. Clin. Sci. (Lond.) 99, 105–111.[ISI][Medline]

Threadgill, D. W., Dlugosz, A. A., Hansen, L. A., Tennenbaum, T., Lichti, U., Yee, D., LaMantia, C., Mourton, T., Herrup, K., Harris, R. C., and et al. (1995). Targeted disruption of mouse EGF receptor: effect of genetic background on mutant phenotype. Science 269, 230–234.[ISI][Medline]

Toyoda, H., Komurasaki, T., Ikeda, Y., Yoshimoto, M., and Morimoto, S. (1995). Molecular cloning of mouse epiregulin, a novel epidermal growth factor-related protein, expressed in the early stage of development. FEBS Lett. 377, 403–407.[ISI][Medline]

Wang, J., Mayernik, L., Schultz, J. F., and Armant, D. R. (2000). Acceleration of trophoblast differentiation by heparin-binding EGF-like growth factor is dependent on the stage-specific activation of calcium influx by ErbB receptors in developing mouse blastocysts. Development 127, 33–44.[Abstract/Free Full Text]

Wiley, L. M., Wu, J. X., Harari, I., and Adamson, E. D. (1992). Epidermal growth factor receptor mRNA and protein increase after the four-cell preimplantation stage in murine development. Dev. Biol. 149, 247–260.[ISI][Medline]

Woo, D. C., and Hoar, R. M. (1972). Apparent hydronephrosis as a normal aspect of renal development in late gestation of rats: The effect of methyl salicylate. Teratology 6, 191–196.[ISI][Medline]