The Ah receptor (AHR) mediates the metabolic adaptation to a number of planar aromatic chemicals. Essential steps in this adaptive mechanism include AHR binding of ligand in the cytosol, translocation of the receptor to the nucleus, dimerization with the Ah receptor nuclear translocator, and binding of this heterodimeric transcription factor to dioxin-responsive elements (DREs) upstream of promoters that regulate the expression of genes involved in xenobiotic metabolism. The AHR is also involved in other aspects of mammalian biology, such as the toxicity of molecules like 2,3,7,8-tetrachlorodibenzo-p-dioxin as well as regulation of normal liver development. In an effort to test whether these additional AHR-mediated processes require a nuclear event, such as DRE binding, we used homologous recombination to generate mice with a mutation in the AHR nuclear localization/DRE binding domain. These Ahr nls mice were found to be resistant to all 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxic responses that we examined, including hepatomegaly, thymic involution, and cleft palate formation. Moreover, aberrations in liver development observed in these mice were identical to that observed in mice harboring a null allele at the Ahr locus. Taken in sum, these data support a model where most, if not all, of AHR-regulated biology requires nuclear localization. The aryl hydrocarbon receptor (AHR)1 regulates an adaptive metabolic response to a variety of planar aromatic chemicals that are widely dispersed in the environment. Over the last 20 years, the mechanistic details of this adaptive signaling pathway have been well characterized (1-4). The AHR is a basic helix-loop-helix-PAS (bHLH-PAS) transcription factor. Upon binding agonists, the AHR translocates from the cytoplasm to the nucleus, where it forms a heterodimer with another bHLH-PAS protein known as the aryl hydrocarbon nuclear translocator (ARNT). This heterodimeric complex interacts with dioxin-responsive elements (DREs) within the genome and upregulates the transcription of a battery of xenobiotic metabolizing enzymes (XMEs). These regulated XMEs include the cytochrome P450s Cyp1a1, Cyp1b1, and Cyp1a2 and the phase II enzymes Gst-a1 and Ugt1-06 (reviewed in Refs. 2 and 3).In addition to regulating an adaptive metabolic response, the AHR also mediates toxic responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and plays an important role in normal development. Early genetic and pharmacological experiments provided evidence that the AHR mediates toxic responses to TCDD and related pollutants (5). Highly reproducible toxic endpoints in rodent species include thymic involution, hepatomegaly, epithelial hyperplasia, and teratogenesis. More recently, generation of null alleles at the Ahr locus in mice revealed that the AHR also plays an important role in normal mammalian development (6 -9). Across laboratories, the most reproducible phenotype associated with the homozygous null allele is a smaller liver. We have proposed that smaller liver size is the result of the pers...

Mice harboring mutations in the Ahr locus display a patent ductus venosus and smaller livers throughout life. We tested the hypothesis that these hepatic aberrations are secondary to a developmental defect in hepatovascular blood flow by performing a detailed analysis of hepatic development in wild-type and Ahr Ϫ/Ϫ mice. This study revealed necrotic lesions in the peripheries of Ahr Ϫ/Ϫ fetal livers as early as embryonic day 15.5, with an increasing incidence up to postnatal day 1 and resolution by 2 weeks post partum. To visualize perfusion of fetal livers, we injected fluorescein isothiocyanate-labeled dextran into the cranial artery and monitored hepatic fluorescence by microscopy. The peripheries of the median and left lobes displayed decreased perfusion in regions corresponding to those regions that displayed necrosis at later developmental times. An examination of adult Ahr Ϫ/Ϫ animals revealed that smaller livers are predominantly due to decreased sizes of the left and right lobes, corresponding to regions of decreased perfusion and hepatic necrosis observed in fetal livers. Histological aberrations in the portal vein also support a model in which perfusion is compromised in the Ahr Ϫ/Ϫ liver. Taken in sum, these results indicate that the Ahr locus is required for normal perfusion of the developing liver and that disruption of the AHR signaling pathway gives rise to fetal hepatic necrosis and consequent liver deformation which persists through adulthood.

The pollutant, 2,3,7,8-tetrachlorodibenzo-p-dioxin ("dioxin"), has been implicated in the etiology of a wide variety of human birth defects. In an effort to identify pharmacological blockers of dioxin-induced terata, we performed a histological and microscopic analysis of the developing murine palate that had been exposed to dioxin. In both in vivo and in vitro model systems, we observed that dioxin exposure leads to a reduction in the number of filopodial extensions at the medial epithelial edge of the developing palate. Given that this filopodial aberration is similar to the phenotype observed in Tgf␤3 null mice, a mutant known to display a 100% incidence of cleft palate, we examined the interaction between TGF␤3 and dioxin in palatal fusion. We found that that the addition of TGF␤3 to an in vitro palate culture model prevented the dioxin-induced reduction in filopodial density. Moreover, TGF␤3 exposure completely prevented the dioxin-induced block of palatal fusion in this system. Although these data do not point to a direct cellular or molecular mechanism for TGF␤3 dioxin antagonism, these results do suggest that TGF␤3 or stimulators of this signaling pathway hold potential as antidotes for dioxin-induced terata and that this opposing pharmacology may extend to additional toxicological endpoints.Halogenated dioxins and related halogenated dibenzofurans are persistent chemicals that are widely dispersed in the global environment (1, 2). These compounds are introduced as byproducts of certain industrial processes as the result of the municipal handling of waste materials or because of their trace contamination of certain commercial products.1 In addition to chronic sources of exposure, there have been several environmental accidents in which human populations have been exposed to higher concentrations of these contaminants. These include the exposure of citizens and soldiers during the Vietnam War to dioxin 2 contamination of the defoliant known as "Agent Orange" (3-6), the exposure of populations in both China and Japan when halogenated dioxins and halogenated dibenzofurans were accidentally introduced into cooking oil (7-9), and the exposure of citizens of Seveso, Italy after an explosion at a nearby chlorinated-phenol plant (10 -12).Epidemiological studies performed after many of these high exposure incidents suggest that halogenated dioxins and halogenated dibenzofurans are linked to human toxicity and increases in congenital anomalies (13,14). Putative responses include changes in menses, low sperm count, and delayed time to pregnancy (15,16). Evidence of human teratogenicity from halogenated dioxins and halogenated dibenzofurans include the following: 1) reports of hyperpigmentation, gingival hyperplasia, hyperkeratosis, skull calcification, and perinatal teeth (17); 2) reports of abnormalities in musculoskeletal development (18 -20); 3) reports of low birth weight and length (18); 4) reports of fragile teeth and finger and toenail abnormalities (18); and 5) reports of delayed cognitive and behavioral de...

The aryl hydrocarbon receptor (encoded by the Ahr locus) is a ligand-activated transcription factor that mediates the toxicology and teratology of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin). In an effort to understand the role of the maternal compartment in dioxin teratology, we designed a breeding strategy that allowed us to compare the teratogenic response in embryos from Ahr ؊/؊ (null) and Ahr ؉/؉ (wild-type) dams. Using this strategy, we demonstrate that embryos from the Ahr ؊/؊ dams are 5-fold more sensitive to dioxin-induced cleft palate and hydronephrosis as compared with embryos from an Ahr ؉/؉ dam. Moreover, this increased teratogenic sensitivity extends beyond dioxin, because embryos from Ahr ؊/؊ dams exhibited a 9-fold increase in their sensitivity to the fetotoxic effects of the glucocorticoid, dexamethasone. In searching for an explanation for this increased sensitivity, we found that more dioxin and dexamethasone reached the embryos from Ahr ؊/؊ dams as compared with embryos from Ahr ؉/؉ dams. We propose that increased deposition of teratogens/fetotoxicants to the embryonic compartment is the result of porto-systemic shunting and/or blocked P4501A induction in Ahr ؊/؊ dams. In addition to demonstrating the importance of maternal AHR in teratogenesis, these data may have implications that reach beyond the mechanism of action of dioxin. In this regard, the Ahr ؊/؊ mouse may provide a system that allows pharmacological agents and toxicants to be more easily studied in a model where first pass clearance is a significant obstacle.Chemical teratogens can perturb normal mammalian development through direct action on the embryo, indirectly through maternal toxicity, or as the result of a combination of both of these mechanisms. The maternal compartment can afford protection by functioning as a metabolic or physical barrier that reduces embryonic exposure to a given agent (1). The maternal compartment also holds the potential to serve as a site of teratogen bioactivation and may serve to increase the concentrations of active metabolites that directly perturb normal embryonic development (2, 3). Understanding the relative contributions of maternal and embryonic physiology for a given teratogen can be difficult, especially when chemical agents induce a variety of teratogenic end points or display remarkable pharmacological potency.One approach to understanding the relative contributions of the maternal and embryonic physiology is to define these two compartments via their respective genotypes. Such an approach is particularly powerful when working with null alleles that have been generated via gene targeting. In such cases, the dominance order is clear, as is the functional relationship between one or two copies of the wild-type allele. Through appropriate genetic crosses one can then generate informative combinations of maternal and embryonic genotypes that can help identify when maternally or embryonically expressed loci play a differential role in the teratogenic response to a specific chemical (4).Based...

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