Genetic Differences in Sensitivity to Alterations of Mandible Structure Caused by the Teratogen 2,3,7,8-Tetrachlorodibenzo- p -Dioxin

Abstract

The contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental pollutant and teratogen that has been shown to alter craniofacial development. Differences in sensitivity to TCDD are attributed primarily to differences in alleles at the Ahr locus coding for the aryl–hydrocarbon receptor (AHR) that binds TCDD and mediates its effects by altering gene expression. The authors used geometric morphometric methods to evaluate differences in the effects of small in utero exposures of TCDD on adult mandible size and shape in five different inbred mouse strains with the same Ahr alleles. Because of the known effects of this toxicant on bone and craniofacial structures, the authors hypothesized that TCDD would decrease mandible size and alter mandible shape, but that the effects of TCDD exposure would differ among the inbred strains. The authors found that TCDD did alter mandible size and shape, but these effects were limited to specific strains and also differed between the sexes. The relative sensitivity to TCDD’s effects on mandibles did not correspond with the previously reported sensitivity to TCDD’s effects on molars. The authors hypothesize that beyond Ahr-related effects, variation in response to TCDD reflects differences in the genetic architecture controlling the trait being evaluated, thus explaining the species, strain, and trait specificity of TCDD.

Introduction

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an industrial by-product that accumulates in the food chain and acts as a developmental toxicant and teratogen (Birnbaum 1994). In mammals, TCDD is passed via placental transfer and lactation to the offspring of exposed mothers where, due to its lipophilicity, it freely enters the cells of the developing organism. Upon entering the cells, TCDD binds to the cytoplasmic aryl–hydrocarbon receptor (AHR), which then enters the nucleus and alters gene transcription (Birnbaum 1994). This altered transcription is presumably responsible for TCDD’s teratogenic effects, which include altered craniofacial morphology in fish (Hill, Howard, and Cossins 2004) and mice (Allen and Leamy 2001) and increased incidences of cleft palate in laboratory rodents (Birnbaum 1994).

Differences in sensitivity to TCDD within species have been attributed primarily to Ahr alleles that produce AHRs with altered functions (Poland and Glover 1980; Simanainen et al. 2003; Miettinen et al. 2005). However, other studies have suggested that the genetic basis of TCDD sensitivity is likely to be complex. For example, non-Ahr loci are known to alter the sensitivity to TCDD-induced cleft palate (Thomae et al. 2006), hepatic porphyria (Robinson et al. 2002), and mouse molar size (Keller, Allen, et al. 2007). Furthermore, we recently completed studies on TCDD’s effect on molar development in five different inbred mouse strains that, based on their functionally similar Ahr ^b alleles, would be expected to exhibit similar sensitivity to TCDD. We found that they differed in their level of third molar agenesis (Keller, Huet-Hudson, and Leamy 2007) and molar shape alteration (Keller, Huet-Hudson, and Leamy 2008), with the C3H/HeJ and CBA/J strains being most susceptible to these effects of TCDD (Keller, Huet-Hudson, and Leamy 2007, 2008).

Mandible development is controlled by a large number of genes (Klingenberg et al. 2001), and exposure to TCDD has been shown to alter craniofacial morphology (Allen and Leamy 2001; Hill, Howard, and Cossins 2004; Keller, Huang, et al. 2007). We therefore were interested to know whether the differences in TCDD sensitivity that we noted previously would extend to mandible traits and if we would detect greater sensitivity in the same two inbred strains previously showing the greatest response in molars (Keller, Huet-Hudson, and Leamy 2007, 2008). We hypothesized that low environmentally relevant doses of TCDD would significantly reduce the size of the mandibles and also alter their shape. Furthermore, since environmental stressors such as TCDD can differentially alter pleiotropic (Pavlicev et al. 2007) and epistatic effects of different alleles (Keller, Allen et al. 2007), we hypothesized that the impact of TCDD on mandible size and shape would differ among inbred strains even though they all possess Ahr ^b alleles.

Materials And Methods

Population

Six inbred mouse strains (C57BL/6J, BALB/cByJ, A/J, CBA/J, C3H/HeJ, and C57BL/10J) were purchased from Jackson Laboratories (Bar Harbor, Maine). All mice were at least 20 g (approximately six to ten weeks old) at the time of breeding. All strains were maintained and bred separately in the University of North Carolina at Charlotte vivarium under standard housing conditions and provided Purina Mouse Chow (Formula Number 8604 or Formula Number 2014 for pregnant and nursing females, Harlan Teklad, Indianapolis, Indiana) and water ad libitum. The strains used in this study were selected because they all contained the sensitive b allele at the Ahr locus that mediates TCDD’s effects (Poland and Glover 1980). The study population was composed of the offspring of pregnant females that were orally dosed with different levels of TCDD on gestation day 13 (GD13, where the presence of a vaginal plug was designated GD0). Mothers from treatment group 1 (T1) received a dose of 0.01 μg TCDD/kg body weight, treatment group 2 (T2) received 0.1 μg TCDD/kg body weight, treatment group 3 (T3) received 1.0 μg TCDD/kg body weight, and mothers of the control group were given equivalent volumes of corn oil without TCDD. Further details are provided in Keller, Huet-Hudson, and Leamy (2007).

The levels of TCDD used in this study were kept intentionally low (compared to typical mouse studies) in order to identify subtle differences in sensitivity among strains with high susceptibility to TCDD’s effects and because the highest dose initially used (10 μg TCDD/kg body weight) resulted in extensive cannibalization of the offspring by their mothers. Nonetheless, the lowest dose used in this study is still probably near the current yearly dioxin toxic equivalent exposure in the United States (Schecter et al. 2001), although higher exposures occur with certain diets, in more contaminated regions, and for certain occupations (Birnbaum 1994). Although the dosing regimen was originally chosen primarily to assess TCDD’s effects on odontogenesis (see Keller, Huet-Hudson, and Leamy 2007), it also appeared appropriate for assessing the effects of stress on mandible development, because doses as low as 0.5 μg/kg have been shown to alter mandible size and shape (Allen and Leamy 2001). Additionally, the timing of exposure (GD13) coincides with the formation of Meckel’s cartilage (a major signal center) in the mouse mandible, and intramembranous bone formation follows shortly thereafter on gestation day 15 (GD15) (Frommer and Margolies 1971).

We attempted to rear a minimum twenty-five offspring in each of the twenty-four Strain × Treatment groups, but difficulties rearing A/J mice lead us to abandon that strain completely. The mice belonging to the 20 remaining Strain × Treatment groups were euthanized at seventy days of age, weighed to the nearest tenth of a gram, and skeletonized using dermestid beetles. After eliminating those mandibles that were broken and unusable or were determined to be outliers, a total of 486 mice were available for the analysis. All Strain × Treatment groups were composed of individuals from at least four litters, the specific numbers of litters in each of the four treatment groups in each strain being C57BL/6J (4,4,5,5); BALB/cBYJ (5,5,5,8); CBA/J (7,8,6,8); C3H/HeJ (6,6,7,6); and C57BL/10J (5,6,5,5). All procedures involving animals were approved by the Institutional Animal Care and Use Committee at the University of North Carolina at Charlotte.

Calculation of Mandible Size and Shape

After skeletonization, the right and left mandibles of each mouse were separated at the mandibular symphysis. The lateral view of each mandible was magnified with an Olympus SZ60 microscope (Olympus America Inc., Melville, New York) attached to an Olympus Q-color 3 digital camera (Olympus America, Melville, New York) for digitization. Fourteen landmark points around the perimeter of the mandible, two internal landmark points, and two curves (each defined by twenty equally spaced semilandmarks) were chosen for digitizing (see Figure 1). For each left and right mandible, we used software (tpsDig version 1.40, freely downloadable from http://life.bio.sunysb.edu/morph/) to obtain 112 x and y coordinates from the landmark and semilandmark positions. Using this software, a single operator manually selected the sixteen landmark points on the image of each mandible. The operator then selected twenty temporary landmarks along each curve and used the software to place the landmarks in their final, evenly spaced positions. Because the amount of curvature in these two regions is constant and not overly sharp, the landmark density created by twenty equally spaced was sufficient to accurately approximate the curve (Zelditch et al. 2004).

The 112 digitized x and y values were used to calculate the centroid size and 112 Procrustes shape coordinates for each mandible. To obtain mandible (centroid) size, we calculated the square root of the sum of the squared distances from each landmark to the mandible’s centroid (the mean of all x and y values for that mandible). For shape, one of the two sides of each mandible was first reflected so that the two sides could be superimposed. Then, the 112 shape coordinates were calculated using generalized Procrustes analysis, which translates, rotates, and rescales all of the mandibles to minimize differences among mandibles and remove all variation unrelated to shape (Dryden and Mardia 1998). Four degrees of freedom are lost in this superimposition process. In addition, because the spacing of semilandmarks along the curve is arbitrary, which adds another nuisance parameter to remove from the analysis of shape, each semilandmark has only one degree of freedom rather than the two normally associated with each x, y coordinate. Thus, shape had a total of sixty-eight degrees of freedom in our analysis. The means of the left and right sides for mandible centroid size and all 112 shape coordinates of each individual were calculated for use in assessing mandible size and shape differences. Additionally, a subset of mandibles from seventy mice was digitized a second time by the same individual to assess measurement error, which was low (<1% for mandible size and approximately 6% for mandible shape).

Statistical Analyses of Size and Shape

Preliminary analyses indicated that there were significant sex and sex interaction responses to TCDD in this population, and we therefore treated males and females separately throughout all subsequent analyses. For each sex, the factors affecting mandible size were evaluated using a two-way ANOVA, while a Procrustes ANOVA (Klingenberg and McIntyre 1998) of the same design was used for mandible shape. In these analyses, treatment was a fixed effect that measured differences among TCDD exposures, and strain was a fixed effect used to evaluate differences among inbred strains. The Treatment × Strain interaction was a measure of whether the effect of TCDD varied among inbred strains, and litter was a random factor nested within the Treatment × Strain interaction that represented non-genetic maternal effects. Treatment, strain, and the Treatment × Strain interaction were tested over litter, and litter was tested over the error. We hypothesized that significant Treatment × Strain interactions would be present and, where so, used planned comparisons to test for differences between the control group and each treatment group within each strain. Because these three comparisons within each strain were nonorthogonal, we evaluated their significance using the sequential Bonferroni procedure (Rice 1989).

In addition to these ANOVAs, we used ANCOVA to test the specific hypotheses that with increasing TCDD exposure over the combined strains, mandible size would decrease and the Procrustes distance (see below) would increase. Strains served as a classification factor in these analyses, and treatment was used as the regression covariate. Since the hypothesis in each case was either (for Procrustes distance) that the regression coefficient would be greater than zero (positive in sign) or (for mandible size) that the regression coefficient would be less than zero (negative in sign), we treated these as one-tailed tests and halved the probability associated with the regression when assessing its significance.

Depiction of TCDD Effects on Mandible Shape

Due to the multivariate nature of shape, differences in mandible shape between groups could not be depicted simply by graphing the means of each group, as could be done for mandible size. To accurately depict the changes in mandible shape caused by TCDD, we calculated the differences between the means of the Procrustes shape coordinates at each landmark in the control and the T3 group for each strain. These differences were visualized as lines with the same orientation and relative magnitude running from the mean position of each landmark in the control group to the mean position of the same landmark in the treatment group.

Because it can be difficult to assess visually whether one shape change is greater than another, we also calculated Procrustes distances between the control group and the corresponding treatment groups for each strain. The Procrustes distances were calculated as the square root of the sum of the squared differences between the means of the superimposed shape coordinates in the control group and each treatment group (Dryden and Mardia 1998). Standard errors for these values were obtained from the standard deviations of one thousand Procrustes distances generated from bootstrap resamplings of the data within each Strain × Treatment group. Although this measure of shape does not provide any indication of the direction of change, it allowed us to readily compare the overall magnitude of shape change between the different groups and to present them in a manner similar to the univariate traits. We tested our hypothesis that Procrustes distances would increase with TCDD exposure using a one-tailed ANCOVA as described above.

Results

Two-way ANOVAs for survival and ten-week body weight of the in utero exposed mice indicated that there were no significant differences due to treatment or the Strain × Treatment interaction (all p >.05). Therefore, the doses of TCDD used in this study did not appear to affect offspring survival or ten-week body weight for any of the inbred strains.

Mandible Size

The two-way ANOVA for mandible size in males indicated that there were significant differences among strains (F = 85.76; df = 4, 82; p <.0001) and litters (F = 2.70, df = 82, 140; p <.0001), but not among treatments (F = 2.03; df = 3, 82; p = 0.12) or for the Strain × Treatment interaction (F = 1.06; df = 12, 82; p =.41). Similarly, strains (F = 106.64; df = 4, 80; p <.0001) and litters (F = 3.04; df = 4, 80; p <.0001) had highly significant effects on mandible size in female mice, while treatment (F = 0.38; df = 3, 80; p =.77) and the Strain × Treatment interaction (F = 0.82; df = 12, 80; p =.63) did not. For both sexes, therefore, the ANOVAs showed that mandible size varied among strains but apparently was not affected by TCDD.

The results of the ANCOVAs for mandible size were somewhat different. For males, the significant strain effect was confirmed (F = 23.95; df = 4, 232; p <.0001), but there also was a significant Strain × Treatment interaction (F = 3.56; df = 4, 232; p =.0077) as well. We therefore calculated separate regressions of mandible size on TCDD treatment for each of the five strains, and for one strain, C3H/HeJ, the regression was significant and also negative in sign (b = –26.1; df = 1, 232; p =.0033). The regression for the CBA/J mice also was negative, but did not reach statistical significance. Females showed a significant strain effect as well (F = 36.50; df = 4, 234; p <.0001), but not a significant Strain × Treatment interaction. These results therefore suggest that TCDD does act to decrease mandible size as hypothesized, but only in the male mice from the C3H/HeJ strain.

The means for mandible centroid size among males and females of each Strain × Treatment group are displayed in Figure 2. For both sexes in all strains, the mean mandible size was generally similar across all treatment groups. In support of the preliminary analysis, male mandibles were consistently larger than female mandibles, with the mean mandible size for males of a particular strain exceeding the mean mandible size of females of the same strain by approximately 0.5 mm (for C57BL/10J mice) to 0.8 mm (for C3H/HeJ). The relative mandible size among the strains was similar for males and females, with BALB/cByJ mice possessing the largest mandibles (mean = 49.5 mm for males and 49.0 mm for females), while CBA/J mice (mean = 44.9 mm for males and 44.4 mm for females) had the smallest mandibles. For males, the (significant) downward trend for mandible size throughout the treatment groups in the C3H mice is apparent. C3H females also show a similar decline, but the slight increase in the T2 dosage group probably prevented the regression from reaching significance.

Mandible Shape

The results of the Procrustes ANOVA for mandible shape in males indicated that there were significant differences due to strains (F = 49.09; df = 272, 5576; p <.0001), treatments (F = 2.09; df = 204, 5576; p <.0001), litters (F = 3.06; df = 5576, 9520; p <.0001), and the Strain × Treatment interaction F ( = 1.22; df = 816, 5576; p <.0001). For females, there were also significant differences in mandible shape due to strains (F = 61.77; df = 272, 5440; p <.0001), treatments (F = 1.90; df = 204, 5440; p <.0001), and litters (F = 2.84; df = 5440, 9792; p <.0001), but there was no significant Strain × Treatment interaction (F = 0.97; df = 816, 5440; p =.69).

As hypothesized, there was a significant Strain × Treatment interaction in males, so we evaluated the effects of the different levels of TCDD on mandible shape via nonorthogonal contrasts that compared the control group to each of the three TCDD treatment groups in each strain. The results indicated that for males of the C57BL/6J, C3H/HeJ, and C57BL/10J strains, but not the BALB/cByJ or the CBA/J strains, the control and the T3 groups differ significantly in mandible shape (all p <.01). Furthermore, the control versus T2 contrast was significant for the C57BL/6J males (p <.01), and the control versus T1 contrast was significant for the C3H/HeJ males (p <.01). An additional contrast comparing the control group to all treatment groups was significant only for the C3H/HeJ mice (p <.0001). Thus for males, the C3H/HeJ (especially) and C57BL/6J mice appear to be more sensitive to TCDD than the BALB/cByJ or CBA/J mice, with C57BL/10J exhibiting intermediate sensitivity.

The trends in the magnitude of mandible shape change due to TCDD exposure for males of each strain are depicted in terms of Procrustes distances from the control in Figure 3A. In agreement with the results of the Procrustes ANOVA, the C3H/HeJ mice exhibited the largest response to the highest dose of TCDD, followed by the C57BL/6J mice. The mean Procrustes distances across all doses were lower in the three remaining strains, particularly the C57BL/10J strain. The Procrustes distances from the control in the CBA/J and BALB/cByJ mice remained relatively constant at all TCDD exposures, but the Procrustes distance increased with increasing TCDD in the C57BL/10J male mice so that it was slightly greater than that of the CBA/J and BALB/cByJ at the highest dose. The differences in these trends probably account for the significant Strain × Treatment interaction in the two-way ANOVA.

C3H/HeJ and C57BL/6J females also exhibited the largest change in Procrustes distance with TCDD exposure (Figure 3B). The significant treatment effect in female mice is apparent as a general trend for increased Procrustes distance with increased TCDD exposure. This trend was confirmed by a significant positive regression for Procrustes distance on treatment across all strains (b = 0.0013, p =.029). So although our hypothesis that we would detect a significant Strain × Treatment interaction was not supported for female mice, the results did support our hypothesis that shape change would increase with TCDD exposure.

Figures 4A (males) and 4B (females) show the relative change in landmark position from each control group to its respective T3 group and thus depict the effects of the highest dose of TCDD on mandible shape in each strain. It is apparent that the changes in mandible shape due to TCDD were quite variable in both direction and magnitude. In agreement with trends in the Procrustes distances, BALB/cByJ mice exhibited relatively little change, and C3H/HeJ mice, particularly males, changed a great deal. Changes in landmark position were relatively evenly dispersed throughout the entire mandible in all affected groups, but the direction of landmark shifts varied considerably between the strains that were affected. For example, the shift in landmarks located in the posterior portion of the ramus of C3H/HeJ males appears to indicate a compression of this region, while the same region in C57BL/6J males exhibits a shortening of the condyloid process relative to the angular process. In most cases, females appear to be less affected than males of the same strain. In those cases for which significant changes were observed, however, the direction of the change in landmark position was quite similar in males and females.

Discussion

TCDD Effects on Mandible Size and Shape

Our results indicated that for males, but not females, the effects of TCDD on mandible shape differed among the strains. Specifically, C3H/HeJ and C57BL/6J mice were most affected by TCDD, and the BALB/cByJ and CBA/J mice were the least affected. In their studies on molar morphology, Keller, Huet-Hudson, and Leamy (2007, 2008) showed that C3H/HeJ and CBA/J mice both were quite sensitive to TCDD, so this toxicant appears to affect both tooth and mandible morphology in C3H/HeJ mice but mandible traits only in C57BL/6J mice. Keller, Huang, et al. (2007) also found that C57BL/6J congenic mice exhibited TCDD-induced changes in mandible morphology. Effects of TCDD therefore appear to be trait specific, as noted previously (Thomae et al. 2006; Simanainen et al. 2003; Poland and Glover 1980).

Despite TCDD’s clear effect on mandible shape over multiple strains, differences in mandible size were detectable only in C3H/HeJ male mice. This result was somewhat surprising since Allen and Leamy (2001) found that TCDD did alter mandible size in an F₂ intercross population of mice formed from two paprental inbred strains, one of which possessed the Ahr ^d allele associated with resistance to TCDD. However, the mice in that study were dosed on GD9, so it is possible that earlier prenatal exposure to TCDD might have had a greater effect on the mandible. The anterior portion of Meckel’s cartilage, which delineates the rostral end of the mandible and appears to regulate mandible growth (Ramaesh and Bard 2003), has formed and is already close to its final position on GD13 but has not yet started to form on GD9 (Frommer and Margolies 1971). The seemingly narrow window of opportunity for TCDD-induced reduction in mandible size suggests that it may be productive to focus on identifying genes that are involved in the developmental processes occurring between GD9 and GD13.

It was interesting that we detected significant Sex × Strain × Treatment interaction effects in our analysis. A similar result was obtained in a previous study of the effects of TCDD on the molars and mandibles of congenic and C57BL/6J mice (Keller, Huang, et al. 2007). In that study, the two strains of mice differed only at the Ahr locus, so it was suggested that the AHR was responsible for the differential effects of TCDD on males and females via its interaction with the estrogen signaling pathway (Keller, Huang, et al. 2007). This hypothesis is supported by studies showing direct estrogen receptor-AHR interactions that alter transcription (Beischlag and Perdew 2005; Boverhof et al. 2006). However, all strains possess the same AHR in this study, so the differential responses to TCDD among sexes and strains may be due to genetic differences in the estrogen pathway.

Differential Responses to TCDD

Our results indicate that genes other than those at the Ahr locus influence sensitivity to TCDD and, given the diversity of its effects and variability of response across species and strains (Birnbaum 1994), it seems likely that TCDD sensitivity actually involves many genes. It is probable that some of the loci influencing TCDD sensitivity are those specifically involved in metabolism and elimination of xenobiotic chemicals, particularly those such as Cyp1a1, Cyp1a2, and glutathione-S-transferase that are downstream of AHR and have been shown to be upregulated by exposure to TCDD (Birnbaum 1994). However, our results confirm those of previous studies (Thomae et al. 2006; Simanainen et al. 2003; Poland and Glover 1980; Keller, Huet-Hudson, and Leamy 2007, 2008), suggesting that TCDD sensitivity is trait specific as well as strain specific. This may be because many of the genes that influence sensitivity to TCDD-induced teratogenesis are specific to the development of the particular trait being evaluated rather than involved in xenobiotic (TCDD) metabolism.

Greater sensitivity to TCDD-induced mandible alterations could be due to allelic variants of genes that control mandible development that are simply more sensitive to perturbations in the environment. For example, alleles at these loci may differ in the rate at which their products are transcribed or degraded, making them more variable in expression (Raser and O’Shea 2004; Pedraza and Paulsson 2008) so that their effect on morphogenesis is more likely to change in the presence of a stressor. Similarly, alleles with marginal function may exhibit inadequate activity to produce the genetically programmed phenotype in the presence of a stressor, while alleles with more optimal function remain sufficiently efficient to produce the phenotype. There is evidence for the existence of abundant genetic variation influencing mandible development (Klingenberg et al. 2001) that could explain the differential response to TCDD’s effects in our mouse population without requiring polymorphisms in genes specifically involved in attenuating the effects of toxins.

Candidate Genes

We searched the Mouse Phenome Database (http://www.jax.org/phenome) for genes affecting skeletal, particularly craniofacial, phenotypes that possess exons with single nucleotide polymorphisms (SNPs) that are known to be polymorphic between the most susceptible (C3H/HeJ and C57BL/6J) and the most resistant (BALB/cByJ) strains. These genes and their known effect on skeletal phenotype are listed on Table 1. As shown, the functions of the various candidate genes are quite varied and include cell cycle control and apoptosis, regulation of signal transduction and transcription, metabolism of endogenous and exogenous compounds, cell adhesion and extracellular matrix production, and DNA repair. While these candidate genes may be helpful in generating new hypotheses to test, this list is intended to be representative rather than exhaustive. These genes represent only those with polymorphisms in exons, so any relevant genes with alleles that segregate in the appropriate manner but differ only in the regulatory regions would not have been identified in our search. Given the AHR’s role in transcription regulation, genes with polymorphic regulatory regions would also be candidates for causing differential sensitivity to TCDD.

In addition to the genes listed on Table 1, our database search revealed several genes involved in xenobiotic metabolism with different alleles in the sensitive and resistant strains. These include several cytochrome P450 genes (Cyp2a12, Cyp2a4, Cyp2c54, and Cyp2c50) and glutathione-S-transferase family members (Gstm3 and Gstm6) as well as Arnt2, which codes for the AHR’s dimerization partner. A search of the Dioxin Responsive Gene Database (http://www.nies.go.jp/health/drgdb/drgdb-top/TOP.htm) revealed that three of the xenobiotic metabolizing genes (Cyp2a12, Cyp2c50, and Gstm6) contain the dioxin responsive elements to which the TCDD-AHR-ARNT complex binds to initiate transcription. Therefore, polymorphisms in these genes could be responsible for the differences in sensitivity to TCDD. However, one would expect that if these genes were solely responsible, the same strains that were sensitive to TCDD’s effects on mandible development would have also been sensitive to its effects on molar development.

Conclusions

We were able to detect differences in sensitivity to the disruptive effect of TCDD on mandible development in inbred strains of mice that possess similar Ahr alleles. Our results suggest that such differences depend on genes beyond the Ahr locus, and these genes may include those involved in xenobiotic metabolism and the estrogen pathway, but are also likely to include genes involved in the development of the trait being evaluated. The use of multiple strains with the same Ahr allele is therefore warranted in studies of the teratogenic effects of TCDD.